WO2018208846A1

WO2018208846A1 - Compositions, systems, and methods for assessing and improving vascular health and treatments involving the same

Info

Publication number: WO2018208846A1
Application number: PCT/US2018/031686
Authority: WO
Inventors: Robert Maxfield LONG; Hans Vink
Original assignee: Microvascular Health Solutions, LLC
Priority date: 2017-05-08
Filing date: 2018-05-08
Publication date: 2018-11-15

Abstract

Compositions, systems, and methods for assessing or improving vascular health, treatments or treatment methods involving the same, systems and methods for diagnosing vascular health or impairment, and compositions and methods for use in treating endothelial glycocalyx to improving vascular health or treat a (vascular health-associated) disease or other condition, in human and non-human mammals are disclosed. Computer systems, hardware storage devices, and methods used to determine glycocalyx impairment using a dynamically extrapolated characterization of a plurality of microvascular vessels, diagnose a subject as suffering from glycocalyx dysfunction or impairment, and assess or determine the health, functionality, or impairment of the glycocalyx as a function of blood flow. Compositions comprising glucosamine, hyaluronan, fucoidan, and antioxidants improve vascular health by enhance synthesis of new glycocalyx, protecting existing glycocalyx against damage, and enhancing repair of damaged glycocalyx.

Description

COMPOSITIONS, SYSTEMS, AND METHODS FOR ASSESSING AND IMPROVING VASCULAR HEALTH AND TREATMENTS INVOLVING THE SAME BACKGROUND

1. Technical Field

The present disclosure relates to compositions, systems, and methods for assessing and/or improving vascular health, and to treatments or treatment methods involving the same. Specifically, the present disclosure relates to systems and methods for diagnosing vascular impairment and to compositions and methods for use in treating endothelial glycocalyx to improving vascular health and/or treat (vascular health-associated) disease(s) or other condition(s), in human and non-human mammals.

2. Relevant Technology

The glycocalyx is a polysaccharide-rich layer found on the luminal surface of epithelial cells lining mammalian organs and tissues. In the case of the vascular system, the glycocalyx coats the luminal surface of the endothelium - the vascular endothelial cells lining the inside of all blood vessels. As depicted in Figure 1, for example, in vivo imaging of a capillary blood vessel illustrates that red blood cells (RBC) flowing through the lumen of the blood vessel do not contact the vessel wall endothelium. Figure 2 illustrates a detailed view of an electron micrograph image capturing a cross-section of a capillary. As depicted, the dense glycocalyx extends from the endothelial cells into the lumen of the blood vessel, forming a micro-thin, gel-like layer.

Until recently, the role of the endothelial glycocalyx had not been well understood. In theory, however, the glycocalyx may act as a protective barrier for the vascular wall or may provide a micro-environment for certain vascular processes. Molecules that associate with the glycocalyx may dynamically interact with the endothelial cells to play a role in orchestrating a variety of functions in the circulatory system. The circulatory system, in turn, plays a role in regulating adequate organ perfusion and in the distribution and exchange of oxygen, nutrients, and hormones within tissues. Furthermore, microcirculation controls tissue hydration and organizes the defense against pathogens.

As illustrated in Figures 2A-2D, endothelial glycocalyx can be observed in varying degrees of thickness and/or density; indications of the "health" of the endothelial glycocalyx. Figure 2A, for example, depicts an electron micrograph image of a "healthy" endothelial glycocalyx, while Figure 2D depicts a severely damaged or perturbed "unhealthy" endothelial glycocalyx. Figure 2B and Figure 2C illustrate, respectively, intermediate states of endothelial glycocalyx health (e.g., as indicated visually by the thickness and/or density thereof). The cause(s) of such structural damage and/or depletion of the endothelial glycocalyx remain largely unknown.

Impairment of the glycocalyx barrier through structural damage or depletion, functional deficiency, or other mechanism may be a contributing cause of microvascular endothelial dysfunction, including inflammatory and coagulatory endothelial activation, vascular leakage of fluid, proteins, and other substances (e.g., cholesterol), failure to properly modulate perfused blood vessel density, and other deleterious conditions, leading to general and specific negative vascular health indicators. As depicted in Figure 3B, for example, an unhealthy endothelial glycocalyx is associated with a "leaky" endothelium, as evidenced by (1) the presence (or "leakage") of cholesterol (or other substances, such as fluids, proteins, etc.) in (or into) the subendothelial space, and (2) a constricted lumen, which may reduce blood flow or perfusion into distal capillaries, muscles, organs, etc., increase blood pressure, and so forth. As illustrated in Figure 3A, however, a healthy (thick and/or dense) endothelial glycocalyx is associated with a well-formed endothelium and healthy blood vessel structural configuration.

Accordingly, there is a need for products and processes for treating (e.g., supporting and/or maintaining) endothelial glycocalyx.

BRIEF SUMMARY

Embodiments of the present disclosure address one or more of the foregoing or other problems in the art with compositions, systems, and methods for assessing and/or improving vascular health, and with treatments or treatment methods involving the same. Some embodiments relates to systems and methods for diagnosing vascular impairment or health. Some embodiments relates to compositions and methods for use in treating endothelial glycocalyx, preferably to improving vascular health and/or treat (vascular health-associated) disease(s) or other condition(s), in human and non-human mammals.

Some embodiments of the present disclosure can include systems and/or methods for determining or diagnosing vascular impairment or health in a human or non-human mammalian patient or subject. In some embodiments include optimizations for determining or diagnosing glycocalyx impairment using a dynamically extrapolated characterization of a plurality of microvascular vessels. Some embodiments can include computer systems, hardware storage devices, and/or methods for determining or diagnosing glycocalyx impairment using a dynamically extrapolated characterization of a plurality of microvascular vessels. In particular, a microscopy image of a plurality of microvascular vessels can be captured using a camera component of a (computer) system. Image data corresponding to at least some of the microvascular vessels captured within the microscopy image can be segmented into a plurality of segmented data portions. A profile can be generated for each of the segmented data portions. The profiles can be compiled together and an extrapolated characterization of the compiled profiles can be created. The extrapolated characterization can be compared against a predetermined threshold value in order to select a target characterization level from within the extrapolated characterization. A particularized set of rules can be applied to the target characterization level to generate a glycocalyx impairment determination. The glycocalyx impairment determination can displayed on a user interface, compiled into a physical report or electronic file, and/or conveyed to the patient as a diagnosis of vascular impairment.

Some embodiments can incorporate or include diagnosing a human or non-human mammalian patient or subject as suffering from glycocalyx dysfunction or impairment in response to the generation of a glycocalyx impairment determination. Some embodiments can incorporate or include assessing and/or determining the health, functionality, and/or impairment of the glycocalyx as a function of blood flow, in a human or non-human mammalian patient or subject.

Some embodiments can include providing a treatment plan or prescription for improving vascular (or glycocalyx) health and/or functionality. In some embodiments, the treatment plan or prescription can include administering or receiving (a dosage of) a composition or composition of matter (e.g., a nutritional and/or health supplement, pharmaceutical, nutraceutical, etc.). Some embodiments (e.g., compositions) can include nutritional building blocks required to support and maintain a healthy endothelial glycocalyx, antioxidants to help prevent damage to the glycocalyx, and/or glycocalyx mimetics for acute repair of damaged endothelial glycocalyx. In at least one embodiment, the building blocks comprise glucosamine, the antioxidants comprise superoxide dismutase, catalase, and/or one or more polyphenols, and the glycocalyx mimetics comprise hyaluronan and/or fucoidan. The components can be combined in an orally administrable form.

Components of compositions can act synergistically and/or beneficially to improve vascular health by supporting a healthy endothelial glycocalyx. In some embodiments, the combination of components included in the compositions can provide an effect on glycocalyx maintenance that is greater than taking any single component alone or the sum of reported effects of the individual components. For instance, the nutritional building blocks can aid not only in the synthesis of new glycocalyx, but also in acute repair of damaged glycocalyx and/or protecting the glycocalyx against structural damage. Similarly, the antioxidants can not only help to prevent damage to the glycocalyx, but also aid in acute repair of damaged glycocalyx and/or synthesis of new glycocalyx. Likewise, the glycocalyx mimetics can not only induce acute repair of damaged glycocalyx, but also aid in the synthesis of new glycocalyx and/or help to prevent damage to the glycocalyx.

Compositions can improve vascular health by enhance synthesis of new glycocalyx by providing and increasing production of glycocalyx precursors, protecting existing glycocalyx against damage, such as by oxidation degradation, by providing and increasing production of antioxidants, some of which associate with endothelial glycocalyx, and enhancing repair of damaged glycocalyx by providing glycocalyx mimetics and increasing the prevalence of glycocalyx scaffold for association and incorporation thereof. Compositions can include or comprise glucosamine, hyaluronan, fucoidan, and/or one or more antioxidants, such as superoxide dismutase, catalase, and/or polyphenol(s).

An embodiment of the present disclosure can include a composition for treating endothelial glycocalyx, the composition comprising glucosamine, hyaluronan and/or fucoidan, and one or more antioxidants, such as superoxide dismutase, catalase, and/or polyphenol. The composition can exhibit a synergistic, therapeutic effect on the endothelial glycocalyx when administered to a mammal.

Another aspect of the present disclosure includes repairing damaged endothelial glycocalyx by providing and/or administering exogenous (nonsulfated) glycosaminoglycan and/or (sulfated) polysaccharides, such as hyaluronan and/or fucoidan, that associate with existing glycocalyx structures at sites of glycocalyx perturbation.

A further aspect of the present disclosure includes stimulating endothelial glycocalyx synthesis by providing and/or administering glycocalyx precursor and/or substrate, such as glucosamine, for local production and incorporation of glycocalyx constituents.

Yet another aspect of the present disclosure includes reducing damage (e.g., (oxidative) degradation) of endothelial glycocalyx and/or constituents thereof by providing antioxidant enzymes, such as superoxide dismutase and/or catalase, and/or antioxidant compounds, such as polyphenols, that reduce (excessive, local) levels or concentrations of reactive oxygen species.

Additional features, aspects, and advantages of exemplary embodiments of the present disclosure will be set forth in the description which follows and, in part, will be apparent from the description or may be learned by the practice of such exemplary embodiments. The features and advantages of such embodiments may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. These and other features, aspects, and advantages will become more fully apparent to those of skill in the art from the following description and appended claims, or may be learned by the practice of such exemplary embodiments as set forth hereinafter.

BRIEF DESCRIPTON OF THE DRAWINGS

In order to further clarify the above and other advantages and features of the present disclosure, and to describe the manner in which the above-recited and other advantages and features of the present disclosure can be obtained, a more particular description of various embodiments of the present disclosure will now be rendered with reference to the appended drawings, in which the exemplary embodiments are illustrated. It is appreciated that these drawings depict only typical embodiments of the present disclosure and are not, therefore, to be considered limiting of its scope. The present disclosure will, therefore, be described and explained with additional specificity and detail through the use of the accompanying drawings in which:

Figure 1A is an in vivo image of a capillary blood vessel;

Figure IB is a detailed view of an electron micrograph image capturing a cross-section of a capillary blood vessel;

Figures 2A-2D illustrate diminishing health of endothelial glycocalyx;

Figure 3A illustrates an electron micrograph image capturing a cross-section of a blood vessel having a generally healthy glycocalyx;

Figure 3B illustrates an electron micrograph image capturing a cross-section of a blood vessel having a generally unhealthy glycocalyx;

Figure 4A is a cartoon depicting aspects of a healthy glycocalyx under physiological conditions;

Figure 4B is a cartoon depicting aspects of a perturbed glycocalyx;

Figure 5A is a cartoon depicting additional aspects of a healthy glycocalyx under physiological conditions;

Figure 5B is a cartoon depicting additional aspects of a perturbed glycocalyx;

Figure 6A illustrates an example image of sublingual microvascular structures from a video recording;

Figure 6B illustrates software identified microvessels and small vascular segments in the image of Figure 6 A;

Figure 6C depicts example radial intensity profiles graphs illustrating red blood cell column widths for each vascular segment in the image of Figure 6B; Figure 7 illustrates RBC width (cumulative RBC width distribution) of an individual vascular segment in the image of Figure 6B;

Figure 8 illustrates average PBR profiles, as a function of median RBC column width, in a group of 15 young healthy controls;

Figure 9 illustrates the reproducibility of PBR score calculations between measurements;

Figure 10 illustrates the effect of age and disease on PBR;

Figure 11 illustrates average calculated feed vessel flow in low flow and high flow states for healthy controls and VCI patients;

Figure 12 illustrates the intraperson change in capillary density between the low flow and high flow states for healthy controls and VCI patients;

Figure 13 A illustrates change in capillary density with increase in flow in healthy controls;

Figure 13B illustrates change in capillary density with increase in flow in VCI patients; Figure 14 illustrates the ratio of change in capillary density to change in feed vessel flow in healthy controls and VCI patients;

Figure 15 illustrates an introduction to a computing system;

Figure 16 illustrates an exemplary computer system that can dynamically determine an extrapolated characterization of a plurality of microvascular vessels.

Figure 17 is a plot of flow-PBR values of numerous blood vessels in an individual;

Figure 18 illustrates increase in perfused capillary density (estimated from relative capillaryVRBC velocity) with increase in feed vessel RBC velocity in a user interface environment;

Figure 19 is a graph of data illustrated in Figure 18;

Figure 20 illustrates an exemplary user interface;

Figure 21 illustrates an exemplary method;

Figure 22 is a cartoon depicting additional aspects of healthy and perturbed glycocalyx;

Figure 23 illustrates the change in PBR following nitroglycerine challenge in MVA patients and control groups;

Figure 24 illustrates a flowchart depicting synergistic interactions and effects of components of a synergistic glycocalyx treatment composition administered to a subject in need thereof in according to an embodiment of the present disclosure;

Figure 25 illustrates the change in various endothelial glycocalyx health indicators in subject humans treated with a synergistic glycocalyx treatment composition according to an embodiment of the present disclosure; Figures 26A-26B illustrate change in capillary density with increase in flow in healthy young controls; and

Figures 27A-27B illustrate change in capillary density with increase in flow in Type 2 diabetes patients.

DETAILED DESCRIPTON

Before describing various embodiments of the present disclosure in further detail, it is to be understood that this disclosure is not limited only to the specific parameters, verbiage, and description of the particularly exemplified systems, methods, and/or products that may vary from one embodiment to the next. Thus, while certain embodiments of the present disclosure will be described in detail, with reference to specific features (e.g., configurations, parameters, properties, steps, components, ingredients, members, elements, parts, and/or portions, etc.), the descriptions are illustrative and are not to be construed as limiting the scope of the present disclosure and/or the claimed invention. In addition, the terminology used herein is for the purpose of describing the embodiments, and is not necessarily intended to limit the scope of the present disclosure and/or the claimed invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains. Weight- or mass-based measurements provided herein (e.g., milligrams (mg)) are generally provided as measurements on a dry weight or dry mass basis.

Various aspects of the present disclosure, including systems, methods, and/or products may be illustrated with reference to one or more embodiments, which are exemplary in nature. As used herein, the terms "embodiment" means serving as an example, instance, or illustration, and should not necessarily be construed as preferred or advantageous over other aspects disclosed herein. In addition, reference to an "embodiment" of the present disclosure or invention is intended to provide an illustrative example thereof without limiting the scope of the claimed invention.

As used in this specification and the appended claims, the singular forms "a," "an" and "the" each contemplate, include, and specifically disclose both the singular and plural referents, unless the context clearly dictates otherwise. For example, reference to an "antioxidant" contemplates and specifically discloses one, as well as a plurality of (e.g., two or more, three or more, etc.) antioxidants. Similarly, use of a plural referent does not necessarily require a plurality of such referents, but contemplates, includes, specifically discloses, and/or provides support for a single, as well as a plurality of such referents, unless the context clearly dictates otherwise. As used throughout the present disclosure, the words "can" and "may" are used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). Additionally, the terms "including," "having," "involving," "containing," "characterized by," as well as variants thereof (e.g., "includes," "has," and "involves," "contains," etc.), and similar terms as used herein, including the claims, shall be inclusive and/or open-ended, shall have the same meaning as the word "comprising" and variants thereof (e.g., "comprise" and "comprises"), and do not exclude additional, un-recited elements or method steps, illustratively.

For the sake of brevity, the present disclosure may recite a list or range of numerical values. It will be appreciated, however, that where such a list or range of numerical values (e.g., greater than, less than, up to, at least, and/or about a certain value, and/or between two recited values) is disclosed or recited, any specific value or range of values falling within the disclosed values or list or range of values is likewise specifically disclosed and contemplated herein. By way of illustrative example, disclosure of "up to 1,000 mg" of a particular ingredient or component includes a specific disclosure of: (i) any value greater than zero and less than or equal to 1,000 milligrams, including but not limited to 0.01 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, 750 mg, 990 mg, and 1,000 mg; and/or (ii) any range of values from or between greater than zero and less than or equal to 1,000 milligrams, including but not limited to 0.01-1,000 mg, 1 mg - 990 mg, 5 mg - 750 mg, 10 mg - 500 mg, and 50 mg - 100 mg.

To facilitate understanding, like references (i.e., like naming of components and/or elements) have been used, where possible, to designate like elements common to different embodiments of the present disclosure. Similarly, like components, or components with like functions, will be provided with similar reference designations, where possible. Specific language will be used herein to describe the exemplary embodiments. Nevertheless it will be understood that no limitation of the scope of the disclosure is thereby intended. Rather, it is to be understood that the language used to describe the exemplary embodiments is illustrative only and is not to be construed as limiting the scope of the disclosure (unless such language is expressly described herein as essential).

While the detailed description is separated into sections, the section headers and contents within each section are for organizational purposes only and are not intended to be self- contained descriptions and embodiments or to limit the scope of the description or the claims. Rather, the contents of each section within the detailed description are intended to be read and understood as a collective whole, where elements of one section may pertain to and/or inform other sections. Accordingly, embodiments specifically disclosed within one section may also relate to and/or serve as additional and/or alternative embodiments in another section having the same and/or similar products, methods, and/or terminology.

Abbreviated list of defined terms

To assist in understanding the scope and content of the foregoing and forthcoming written description and appended claims, a select few terms are defined directly below.

The term "condition" refers to any disorder, disease, injury, or illness, as understood by those skilled in the art, that is manifested or anticipated in a patient. Manifestation of such a condition can be an early, middle, or late stage manifestation, as known in the art, including pre-condition symptoms, signs, or markers. Anticipation of such a condition can be or include the predicted, expected, envisioned, presumed, supposed, and/or speculated occurrence of the same, whether founded in scientific or medical evidence, risk assessment, or mere apprehension or trepidation.

The term "patient," as used herein, is synonymous with the term "subject" and generally refers to any animal under the care of a medical professional, as that term is defined herein, with particular reference to (i) humans (under the care of a doctor, nurse, or medical assistant or volunteer) and (ii) non-human animals, such as non-human mammals (under the care of a veterinarian or other veterinary professional, assistant, or volunteer).

The terms "medical professional" as used herein, generally refers to any individual or entity that is responsible for or participates in providing health care to an animal, including human and non-human animals, such as non-human mammals, with particular emphasis on licensed health care providers or unlicensed providers, such as assistants, technicians, and/or volunteers, particularly those covered under the (blanket) license or insurance of a health care provider. This term may, when contextually appropriate, include an oncologist, a surgeon, a physician's assistant, a nurse, a phlebotomist, a veterinarian, etc.

The term "cancer" refers to an abnormal, typically uncontrolled, growth of cells. A "cancerous cell" as used herein comprises a malignant cell having an abnormal, typically uncontrolled, growth. As such, the term cancer is an umbrella term encompassing a plurality of different distinctive diseases characterized by malignant cells growing in a typically uncontrolled manner.

The term "co-administration" and similar terms refer to concurrent, sequential, and/or combined administration of two or more components. For instance, two components can be co-administered by administering each component in a separate dosage concurrently, simultaneously, or sequentially (e.g., distinct administrations separated by a period of time). The period of time can be very small (e.g., substantially, immediately following a first administration) or longer (e.g., 1-60 seconds, 1-60 minutes, 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, and so forth, or any value or range of values therebetween). Concurrent or simultaneous administration can include overlapping administration timeframes for the two or more components or administration of a combination product comprising a mixture of the two or more components.

"CAD" means coronary artery disease.

"CMR" means cardiovascular magnetic resonance.

"ΜΡΓ means myocardial perfusion index.

"MPR" means myocardial perfusion reserve.

"MPRI" means myocardial perfusion reserve index.

"MVA" means microvascular angina.

"PBR" means perfused boundary region.

"VCI" means vascular cognition impairment.

General Description of Illustrative Embodiments

Various embodiments of the present disclosure include compositions, systems, and methods for assessing and/or improving vascular health, and/or treatments or treatment methods involving the same. Some embodiments relates to systems and methods for diagnosing vascular impairment or health. Some embodiments relates to compositions and methods for use in treating endothelial glycocalyx, preferably to improving vascular health and/or treat (vascular health-associated) disease(s) or other condition(s), in human and non- human mammals.

Systems and methods for assessing vascular health

Some embodiments include novel solutions for the (clinical) assessment of vascular health, using a novel and inventive system and/or method. Certain embodiments include novel solutions for on-line quantification of the glycocalyx barrier properties of the microcirculation in a subject, using a novel and inventive system and/or method. In some embodiments, the method can be performed by a computer system, one or more computer processors, and/or computer software (e.g., GlycoCheck™ Glycocalyx Measurement Software and/or System).

The glycocalyx is the gel-like layer lining the luminal side of the endothelium in blood vessels which acts as a protective barrier for the vascular wall; impairment of the glycocalyx barrier is regarded as a primary step in microvascular dysfunction. Inventive software has been developed for automated analysis of video recordings of the (micro)vascular (micro)circulation (e.g., sublingual microcirculation). The microvascular microcirculation can be representative of circulation elsewhere in the subject. The captured videos and/or (pixel-containing) images or frames thereof can be provided by a range of commercially available clinical videomicroscopic cameras that can be positioned non-invasively (e.g., under the tongue) in a patient friendly manner.

The software automatically controls the quality of the videorecordings with respect to tissue motion, light intensity and image focus, resulting in an acceptable recording time of 1 - 2 minutes. Next, the software automatically identifies all available microvessels in the recording and calculates in them the perfused boundary region (PBR) as a measure of glycocalyx barrier properties; the analysis of over 3000 vascular segments takes about 3 min per patient, allowing for online monitoring of a patient's glycocalyx quality. Results using this methodology so far indicate excellent reproducibility of the PBR measurement in healthy young subjects.

Moreover, robust increases in PBR, indicative of a compromised glycocalyx barrier, have been observed in small groups of patients with diabetes and during septic shock, providing a tool for online glycocalyx monitoring and assessment of acute vascular risk in individual patients.

The microcirculation serves major functions in the body: it regulates adequate organ perfusion and the distribution and exchange of oxygen, nutrients, and hormones within tissues. Furthermore, it controls tissue hydration and organizes the defense against pathogens. The endothelial glycocalyx has been demonstrated to be central in orchestrating these functions. Accordingly, the endothelial glycocalyx is central to microvascular function.

The glycocalyx, a gel-like layer lining the endothelial cells at the luminal side, provides a micro-environment for many important vascular processes. The glycocalyx has been shown to limit the accessibility of lipids and proteins and to form a barrier for adhesion of platelets and inflammatory cells to the vascular wall; further, the glycocalyx is involved in mechanosensing and transduction of hemodynamic stimuli to the endothelium, thereby regulating the production of amongst others nitric oxide (Figure 4A). Moreover, glycocalyx play important roles in endothelial function - shear induced NO-synthesis, superoxide dismutation, permeability - 'sieving' barrier, coagulation - inhibition of platelet adherence, coagulation regulatory factors, and inflammation - prevention of leukocyte adhesion.

Consequently, damage to the glycocalyx has been associated with impaired endothelial function, including inflammatory and coagulatory endothelial activation, vascular leakage of fluid and proteins and a diminished NO bioavailability (Figure 4B). Damaged or impaired glycocalyx exhibit and/or are associated with reduced nitric oxide (NO) availability, increased oxidative stress, increased leakage of macromolecules, increased platelet adherence, increased thrombin generation, increased leukocyte adhesion and diapedesis.

In line with its important functions, the microcirculation is in recent years increasingly recognized as locus of early pathophysiological processes associated with the onset of cardiovascular disease in e.g. diabetes and hyperlipidemia, as well as with circulatory failure in critically ill patients in the ICU. Noticeably, microvascular failure in critically ill patients is nowadays regarded as the most sensitive indicator of circulatory failure associated with adverse outcome. While novel bedside techniques allowing evaluation of the microcirculation within a patient have been introduced in recent years, their potential applicability for functional hemodynamic monitoring in daily clinical practice has been hindered by the current lack of robust and on-line quantification of microvascular function. To meet this need, GlycoCheck™ has been dedicated in developing a software solution which permits online assessment of the glycocalyx barrier properties of the microcirculation in an individual patient; this solution is described here.

At least in part, (on-line) monitoring of the perfused boundary region (PBR) provides an indication of glycocalyx state. The perfused boundary region (PBR) is the main readout parameter calculated by GlycoCheck™ Glycocalyx Measurement Software. The PBR in microvessels is the cell-poor layer which results from the phase separation between the flowing red blood cells (RBC) and plasma. The PBR includes the most luminal part of glycocalyx that does allow cell penetration. The outer edge of PBR is defined by the protective part of the glycocalyx that does not allow cell penetration and which shields the endothelial surface from direct contact with circulating cells. Loss of glycocalyx integrity allows for deeper penetration by the outer edge of the RBC-perfused lumen and thereby increases PBR, resulting in increased vulnerability of the endothelium.

Figures 5A and 5B portray the perfused boundary region (PBR) in a healthy microvessel (Figure 5A) and in a microvessel at risk (Figure 5B). Deterioration of the cell-impermeable glycocalyx barrier in a vessel results in an outward movement of the outer edge of the RBC- perfused lumen resulting in an increased PBR in that vessel.

GlycoCheck™ Glycocalyx Measurement Software complies with current commercially available cameras made for on-line microscopic observations of the sublingual microcirculation. The actual measurement covers two stages:

1) recording of an adequate number of high-quality movie frames. The software guarantees the recording of a sufficient number of high-quality videos by online monitoring of a) tissue motion, b) illumination intensity, and c) focus level of the sublingual microvasculature. Video acquisition automatically starts when image quality is within acceptable range and automatically stops when a sufficient number of vascular segments (> 3000) have been collected for analysis (see below). Data acquisition does not take more than 1-2 minutes.

2) image analysis resulting in calculation of the PBR throughout the monitored microcirculation.

The analysis automatically starts when images during the recording stage have been collected (Figure 6A). The software automatically identifies all available microvessels and defines small vascular segments every 10 μπι along the length of the detected vasculature (Figure 6B). For each vascular segment 840 radial intensity profiles are obtained, which are tested for the presence of RBCs and signal quality, and red blood cell column widths are determined from these intensity profiles (Figure 6C). Thus, for each vascular segment, a calculation of PBR from cumulative distribution is obtained (e.g., by calculation of RBC column width for series of, for example, 40 consecutive frames in each vascular segment). It will be appreciated that alternative methods can include between 5 and 500 frames, preferably between 10 and 100 frames, or any suitable number of frames.

This results in a RBC width distribution of each individual vascular segment from which the median RBC width (RBCW) as well as the outer edge of the RBC perfused lumen (Dperf) is determined (see Figure 7). The distance of the median RBC width value to the position of the outer edge of the RBC perfused lumen is measured and defined as the RBC Perfused Boundary Region (PBR), i.e. PBR = (Dperf - RBCW) / 2. The calculated PBR values of a given patient are classified according to their corresponding median RBC column width (Figure 8) and an average PBR in the diameter range of 5 - 25 microns is calculated to provide a single PBR value for each patient. Figure 8 illustrates PBR values (mean ± SEM, n=15 subjects) for incremental bin classes of median RBC column width in healthy young male volunteers (age: 20-29 years). PBR values of a group of healthy young individuals is demonstrated to be reproducible (Figure 9). Figure 9 illustrates the difference in PBR between two consecutive measurements in these subjects using GlycoCheck™ Glycocalyx Measurement Software.

Moreover, pilot data show increases in PBR, indicative of an impaired glycocalyx barrier, in diabetics and septic shock patients (Figure 10). In particular, Figure 10 illustrates PBR values obtained with GlycoCheck™ Glycocalyx Measurement Software in healthy young volunteers on two consecutive visits (see Figure 9), in healthy old subjects (age: 60-83 years), in type 2 diabetics, and in critically ill patients during septic shock. Groups consist of n=l l- 16 patients.

Thus, GlycoCheck™ Glycocalyx Measurement Software allows prompt and easy quantification of the perfused boundary region within the sublingual microvasculature of a person, thereby providing critical information about the properties of the glycocalyx barrier and vascular health. The measurement is non-invasive and is patient friendly. The measurement is highly reproducible, and preliminary data indicate that the measurement is capable of identifying microvascular glycocalyx deterioration in people with diabetes and in critically ill patients during septic shock conditions allowing identification of individuals that are at acute vascular risk for organ complications.

Furthermore, application of the Glycocalyx Measurement Software in clinical cohort studies can contribute to defining early prognostic markers of microvascular deterioration and early onset of vascular complications. The Glycocalyx Measurement Software opens new opportunities for online functional hemodynamic monitoring of (micro)vascular health, and allows for personalized monitoring of therapy efficacy which might be particularly useful in critically ill patients.

Uncoupling of Microvascular Blood Flow and Perfused Capillary Density in Patients with Early Cogitation Impairment

Increased vascular risk may be associated with impaired cognition. Recent studies are, however, inconclusive whether microvascular blood flow is actually reduced in early stages of cognitive decline. Although the lack of correlation between MRI based measures of cerebral microvascular flow and cognition scores may be due to technical limitations to detect small reductions in microvascular blood flow, it may also be possible that early cognitive decline is the result of impaired nutrient delivery to brain cells due to alterations at the capillary level that may precede reductions in microvascular blood flow.

Brain tissue demand for oxygen and glucose is relatively high and changes dynamically with local increases in metabolic activity. To match nutrient delivery at the cellular level to temporal changes in local metabolic need, it is essential that increases in microvascular blood flow are coupled to increases in the number of blood perfused capillary blood vessels. Changes in perfused capillary density are essential to adjust microvascular surface area and nutrient diffusion distances to local metabolic need. Uncoupling of local capillary exchange conditions from metabolic changes in microvascular flow might contribute to cellular damage at sites most distant from nutritive capillaries. In the present embodiment, sublingual microvascular blood flow and perfused capillary density were measured in healthy controls and patients with early decline of cognitive function (i.e., vascular cognition impaired (VCI) patients) to test the hypothesis that uncoupling of microvascular blood flow and perfused capillary density contributes to decline of cognitive function despite apparently normal levels of microvascular blood flow.

In the present embodiment, microvascular measurements were performed according to the following protocol in healthy controls (15 males) and VCI patients (15 males). Cognition was tested by using the obtained scores.

Imaging of sublingual microvessels

Intravital microscopic recordings of sublingual microvessels were collected with a digital clinical video camera (KK Technology Ltd) connected to a laptop based automated video acquisition and image analysis system (GlycoCheck BV). A series of 10 - 20 short video recordings (< 2 seconds long) were collected for each individual in order to obtain at least 3000 vascular segments ranging from 5 to 25 microns in diameter. Vessels were automatically grouped in diameter classes and vessels with diameters between 4.5 - 5.5 microns were defined as capillaries, while vessels with diameters 8.5 - 9.5 microns were defined as feed vessels.

Blood flow measurements

For analysis of the effect of microvascular blood flow on capillary density, spontaneous variability of flow during the data recording allowed for selection of 2 videos per individual with low and high blood flow, respectively. Feed vessel blood flow was measured in each video recording by adding the flows of all the individual feed vessels of that video recording. Blood flow of a given feed vessel was calculated by multiplying feed vessel red blood cell velocity times feed vessel red cell content. Microvascular blood flow per video was divided by video surface area to normalized blood flow to tissue surface area.

Microvascular blood flow as measured in a low flow video and a high flow video of each individual in the group of healthy controls and in the group of VCI patients with early decline of cognition (see Figure 11). Average microvascular blood flow (689 +/- 509 vs 705 +/- 417, Con vs. VCI) as well as the difference between intra-individual low flow (330 +/- 364 vs 394 +/- 324, Con vs. VCI) and high flow (1049 +/- 780 vs. 1016 +/- 676, Con vs. VCI, P < 0.001 High vs Low flows in both Controls and VCI) are similar for healthy controls and patients with early cognition impairment.

Capillary density Automatically detected red cell containing capillaries were counted in the low- and high- flow video recording of each individual in order to test for the effect of changes in microvascular blood flow on the number of red cell containing capillaries. In addition, to compare the level of red cell perfused capillaries between individuals, average flow per individual was calculated as well as the ratio of feed vessel RBC velocity over capillary RBC velocity. The rational for this approach is that if the number of blood perfused capillaries increases when microvascular blood flow increases, the relative increase in capillary red cell velocity will be smaller than the relative increase in feed vessel red cell velocity. An additional advantage of this strategy is that capillaries containing stagnant red blood cells are not taken into account as these capillaries do not contribute to nutrient exchange.

Density of red cell containing capillaries were determined in a low flow video and a high flow video for each individual (see Figure 12). Capillary density was significantly higher in the high flow video in the healthy controls (12.3 +/- 11.3 vs 21.5 +/- 14.5, low flow density vs. high flow density, P < 0.001). Capillary density was identical in the low flow - and high flow video of the patients with early cognition impairment (16.6 +/- 21.7 vs. 16.0+/- 22.3, low flow density vs. high flow density).

Coupling of microvascular blood flow and perfused capillary density

Perfused capillary density increases proportionally with individual microvascular blood flow in healthy controls (P < 0.001) (see Figure 13 A), but this is completely absent in patients with early cognition impairment (see Figure 13B). These data reveal uncoupling of microvascular blood flow and perfused capillary density in individuals with declined cognition.

Without being bound to any theory, in healthy control patients, the glycocalyx functions to properly regulate vascular recruitment as metabolic and vascular needs change in the individual. For instance, as feed vessel blood flow increases, additional vessels (e.g., capillaries) are recruited, becoming perfused with blood, thereby opening additional routes of blood passage and maintaining a constant and/or heathy capillary blood velocity. Were it not so, the increasing in feed vessel flow through a constant number of capillaries would result in tissue starvation as nutrients and blood components are rushed through the vascular system without adequate exposure time or contact with vascular structures. Accordingly, the increase in capillary density in response to increased flow in healthy individuals helps to maintain vascular health.

Similarly, dysfunction or inefficiency in increasing capillary density in response to increased blood flow is a hallmark of vascular dysfunction and/or glycocalyx impairment, or risk thereof. Because the glycocalyx functions to properly regulate vascular recruitment as metabolic and vascular needs change, patients with impaired glycocalyx function exhibit the inability or reduced / impaired ability to recruit or increase capillary density in response to increased blood flow. For this reason, assessing vascular health in glycocalyx impaired patients has proven challenging. Using previous methods, for example, some vascular impaired patients appeared to have healthier, more robust vascular function in certain (e.g., low flow) states of vascular activity. In reality, however, this is an artifact of serious vascular dysfunction (as described above).

Embodiments of the present disclosure can account for the lack of flow dependent glycocalyx response, recruitment of capillaries, and/or changes in PBR. In particular, certain embodiments can include methods of accurately assessing vascular health and/or diagnosing vascular impairment. Embodiments can include methods for measuring and calculating one or more parameters that accurately reflect true vascular health.

Correlation of capillary recruitment capacity with cognition score Per individual, the change in capillary density per flow change was calculated. The results demonstrated a significant difference between controls and VCI patients.

Sublingual microvascular blood flow and capillary densities do not necessarily reflect brain microvascular function, but the current study does reveal a systemic difference at the level of capillary blood flow control in patients with impaired cognition. This is a finding consistent with data demonstrating that sublingual assessment of microvascular function allows identification of systemic microvascular vulnerability in many different patient groups with, for example, diabetes, kidney disease, heart disease, stroke, systemic inflammatory challenges (such as sepsis), and the like. Additional studies may test whether the observed sublingual uncoupling of microvascular blood flow and perfused capillary density also extend to the brain, but the results are in line with findings that individuals with systemic microvascular complications are at higher risk of cognitive decline. See Figure 14.

Mechanism of capillary recruitment

The exact mechanism that controls the number of blood perfused capillaries during changes in microvascular blood flow is unknown. In the present embodiment, we also determined the effect of microvascular blood flow on the interaction of red blood cells with the endothelial glycocalyx, a voluminous protective gel-like compartment that lines the inner surface of the microvascular system and that limits capillary red cell content. Consistent with the flow dependent recruitment of red cell perfused capillary we also found a strong flow dependency of the interaction of red blood cells with the endothelial glycocalyx in healthy controls. Patients with impaired cognition display a strong reduction in the flow dependent interaction of red cells with the endothelial glycocalyx, suggesting that reduced glycocalyx volume or impaired flow dependent modulation of glycocalyx volume may contribute to the observed uncoupling of microvascular blood flow and capillary density. Systemic damage of the endothelial glycocalyx is found in many other patients that suffer from microvascular disease and may contribute to the established link between microvascular complications and cognition.

Despite similar levels and ranges of sublingual microvascular flow, capillary density in patients with impaired cognition did not change during spontaneous changes in microvascular blood flow or in relation to interperson flow differences. Capillary density in healthy individuals increases proportionally with changes of microvascular flow and perfused capillary density is higher in individuals with higher microvascular flow. Uncoupling of capillary density from microvascular blood flow in individuals with cognitive decline may cause failure of the microvascular network to deliver sufficient amounts of oxygen and nutrients to tissue cells during increases in metabolic demand.

Loss of capillary recruitment capacity independently of control of microvascular blood flow may explain why measurements of microvascular blood flow fail to demonstrate impaired microvascular function in relation to reduced cognition. Average capillary density and average microvascular blood flow appeared to be similar in healthy controls and cognition impaired patients under baseline conditions, which suggests that angiogenic rescue mechanisms at the capillary level are able to maintain a sufficient number of blood perfused capillaries that provide adequate levels of oxygen and nutrients to tissue cells under resting conditions. However, this passive network of a limited number of capillaries fails to optimize exchange conditions when microvascular blood flow is increased during a metabolic challenge. Uncoupling of capillary exchange capacity from microvascular blood flow control may cause starvation of small tissue regions that are most distant from the small and static number of available capillaries.

The uncoupling of microvascular blood flow and perfused capillary density in individuals with declined cognition suggests that a similar uncoupling may present in patient with other conditions associated with vascular dysfunction. Because it may not be possible to determine in advance which subjects may exhibit uncoupling of microvascular blood flow and perfused capillary density, a method for accurately assessing vascular health (e.g., by measuring perfused capillary density, calculating PBR, etc.) independent of changes in blood flow may be necessary in some embodiments. Certain embodiments of the present disclosure can account for or factor in the potential for uncoupling of microvascular blood flow and perfused capillary density with systems and/or methods for (accurately) diagnosing vascular impairment or health.

Systems and methods for accurately diagnosing vascular impairment or health

Some embodiments of the present disclosure can include systems and/or methods for diagnosing vascular impairment or health in a human or non-human mammalian patient or subject. Some embodiments can include computer systems, hardware storage devices, and/or methods for determining glycocalyx impairment using a dynamically extrapolated characterization of a plurality of microvascular vessels. In particular, a microscopy image of a plurality of microvascular vessels can be captured using a camera component of a (computer) system. Image data corresponding to at least some of the microvascular vessels captured within the microscopy image can be segmented into a plurality of segmented data portions. A profile can be generated for each of the segmented data portions. The profiles can be compiled together and an extrapolated characterization of the compiled profiles can be created. The extrapolated characterization can be compared against a predetermined threshold value in order to select a target characterization level from within the extrapolated characterization. A particularized set of rules can be applied to the target characterization level to generate a glycocalyx impairment determination. The glycocalyx impairment determination can displayed on a user interface and/or conveyed as a diagnosis to vascular impairment.

Embodiments can significantly improve the conventional technology by accurately, reliably, and robustly differentiating between healthy glycocalyces and unhealthy glycocalyces. In particular, the embodiments improve the conventional technology because they eliminate the need for human approximations and determinations when evaluating the health level of a glycocalyx. Some embodiments can provide a standardized representation and/or approximations of vascular health. For instance, some embodiments can provide a single metric, or a plurality of metrics, for estimating the overall health of the glycocalyx. For instance, some embodiments can provide a (single) microvascular health score (MVHS) that approximates and/or represents the overall health level of a glycocalyx. Similarly, some embodiments can provide a (single) flow dependent capillary recruitment capacity that approximates and/or represents the overall health level of a glycocalyx. In some embodiments, a single metric can operate to standardize health determination for glycocalyces. In some embodiments, two or more metrics can provide a more detailed understanding of vascular health. In either case, standardization can significantly improve the technology because it eliminates the possibility of confusion and misinterpretation for doctors, nurse, and other medical practitioners.

The current embodiments also operate to improve the underlying functionality and operations of the computer system by reducing the amount of computing processes that are required to determine a glycocalyx' s health levels. Indeed, instead of collecting and processing an extremely large amount of data that spans a large range, the embodiments are able to extrapolate a subset of data to generate projected trends. Using these trends, the embodiments are able to provide reliable, accurate, and robust glycocalyx estimations. Consequently, fewer computing resources are required in order to generate these results.

To achieve these and other benefits, the embodiments capture a microscopy image of a plurality of microvascular vessels using a computer system's camera. Then, image data corresponding to at least some of the microvascular vessels captured within the microscopy image is segmented into a plurality of segmented data portions. Subsequently, a profile is generated for each of these segmented data portions. These profiles are then compiled together. Once the profiles are compiled together, then an extrapolated characterization of the compiled profiles is created. Next, the extrapolated characterization is compared against a predetermined threshold value in order to select a target characterization level from within the extrapolated characterization. Then, a particularized set of rules is applied to the target characterization level to generate a glycocalyx impairment determination. Finally, this glycocalyx impairment determination is displayed on a user interface.

Having just described various high-level benefits and features of the present embodiments, attention will now be directed to Figures 15-16, which illustrate exemplary computing system. Following that discussion, various architectures and supporting illustrations will be detailed with respect to Figures 17-20. Subsequently, various diagrams and methods will be discussed with respect to Figure 21. The application also includes subject matter on compositions and methods for diagnosing and treating glycocalyx impairment. This subject matter is appended at the latter end of this disclosure.

Illustrative Computer System As illustrated in Figure 15, in its most basic configuration, the computer system 100 includes various different components. For example, Figure 15 shows that computer system 100 includes at least one hardware processing unit 105, input/output (I/O) interfaces 110, graphics rendering engines 115, one or more sensors 120, and storage 125.

The storage 125 may be physical system memory, which may be volatile, non-volatile, or some combination of the two. The term "memory" may also be used herein to refer to nonvolatile mass storage such as physical storage media. If the computing system 100 is distributed, the processing, memory, and/or storage capability may be distributed as well. As used herein, the term "executable module," "executable component," or even "component" can refer to software objects, routines, or methods that may be executed on the computing system 100. The different components, modules, engines, and services described herein may be implemented as objects or processors that execute on the computing system 100 (e.g. as separate threads).

The disclosed embodiments may comprise or utilize a special-purpose or general -purpose computer including computer hardware, such as, for example, one or more processors (such as processor 105) and system memory (such as storage 125), as discussed in greater detail below. Embodiments also include physical and other computer-readable media for carrying or storing computer-executable instructions and/or data structures. Such computer-readable media can be any available media that can be accessed by a general-purpose or special- purpose computer system. Computer-readable media that store computer-executable instructions in the form of data are physical computer storage media. Computer-readable media that carry computer-executable instructions are transmission media. Thus, by way of example and not limitation, the current embodiments can comprise at least two distinctly different kinds of computer-readable media: computer storage media and transmission media.

Computer storage media are hardware storage devices, such as RAM, ROM, EEPROM,

CD-ROM, solid state drives (SSDs) that are based on RAM, Flash memory, phase-change memory (PCM), or other types of memory, or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store desired program code means in the form of computer-executable instructions, data, or data structures and that can be accessed by a general -purpose or special-purpose computer.

The computer system 100 may also be connected (via a wired or wireless connection) to external sensors 130 (e.g., one or more remote cameras, accelerometers, gyroscopes, acoustic sensors, magnetometers, etc.). Further, the computer system 100 may also be connected through one or more wired or wireless networks 135 to remote systems(s) 140 that are configured to perform any of the processing described with regard to computer system 100.

During use, a user of the computer system 100 is able to perceive information through a display screen that is included among the I/O interface(s) 110 and that is visible to the user. The I/O interface(s) 110 and sensors 120/130 also include gesture detection devices (e.g., a mouse) and/or other movement detecting components (e.g., cameras) that are able to detect user input.

The graphics rendering engine 115 is configured, with the processor(s) 105, to generate and display a user interface for displaying information and receiving user input.

A "network," like the network 135 shown in Figure 15, is defined as one or more data links and/or data switches that enable the transport of electronic data between computer systems, modules, and/or other electronic devices. When information is transferred, or provided, over a network (either hardwired, wireless, or a combination of hardwired and wireless) to a computer, the computer properly views the connection as a transmission medium. The computer system 100 will include one or more communication channels that are used to communicate with the network 135. Transmissions media include a network that can be used to carry data or desired program code means in the form of computer-executable instructions or in the form of data structures. Further, these computer-executable instructions can be accessed by a general-purpose or special-purpose computer. Combinations of the above should also be included within the scope of computer-readable media.

Upon reaching various computer system components, program code means in the form of computer-executable instructions or data structures can be transferred automatically from transmission media to computer storage media (or vice versa). For example, computer- executable instructions or data structures received over a network or data link can be buffered in RAM within a network interface module (e.g., a network interface card or "NIC") and then eventually transferred to computer system RAM and/or to less volatile computer storage media at a computer system. Thus, it should be understood that computer storage media can be included in computer system components that also (or even primarily) utilize transmission media.

Computer-executable (or computer-interpretable) instructions comprise, for example, instructions that cause a general-purpose computer, special-purpose computer, or special- purpose processing device to perform a certain function or group of functions. The computer- executable instructions may be, for example, binaries, intermediate format instructions such as assembly language, or even source code. Although the subject matter has been described in language specific to structural features and/or methodological acts, it is to be understood that the subject matter defined in the appended claims is not necessarily limited to the described features or acts described above. Rather, the described features and acts are disclosed as example forms of implementing the claims.

Those skilled in the art will appreciate that the embodiments may be practiced in network computing environments with many types of computer system configurations, including personal computers, desktop computers, laptop computers, message processors, hand-held devices, multi-processor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, mobile telephones, PDAs, pagers, routers, switches, and the like. The embodiments may also be practiced in distributed system environments where local and remote computer systems that are linked (either by hardwired data links, wireless data links, or by a combination of hardwired and wireless data links) through a network each perform tasks (e.g. cloud computing, cloud services and the like). In a distributed system environment, program modules may be located in both local and remote memory storage devices.

As discussed above, computer systems are able to provide a broad variety of different functions. One such function includes determining glycocalyx impairment using a dynamically extrapolated characterization of a plurality of microvascular vessels. Accordingly, attention will now be directed to Figure 16, which illustrates an exemplary computer system for performing these functions.

Identifying, Determining, Diagnosing Glycocalyx Impairment

Figure 16 illustrates an exemplary computer system 200 for determining glycocalyx impairment using a dynamically extrapolated characterization of a plurality of microvascular vessels. Computer system 200 includes all of the features, functionalities, and abilities that were discussed in connection with computer system 100 of Figure 15.

As shown, computer system 200 includes a plurality of components. These components include a camera component 205, a segment component 210, a profile component 215, a characterization component 220, and a user interface (UI) component 225. The computer system 200 also includes storage 230. Storage 230 is analogous to the storage 125 shown in Figure 15. Figure 16 shows that storage 230 may include a plethora of information. By way of example, storage 230 includes rules 235. Although storage 230 is shown as having only rules 235, it will be appreciated that the storage 230 is able to store any manner of digital content and any amount of digital content. Accordingly, the embodiments should not be limited simply to the illustration shown in Figure 16. The camera component 205 is configured to interact with an actual camera unit in order to collect image data of a real -world object. The camera may be any type of camera suitable for capturing microscopic image data. By way of example and not limitation, the camera may be a Charge-Coupled Device (CCD) camera. Regardless of what type of camera is used, the camera is able to acquire image data relating to a human's microvascular vessels. In other words, the camera is able to view a human's microvascular system and is even able to view the individual red blood cells that are passing through the vessels.

In some instances, the camera uses sublingual (i.e. below the tongue) microscopy to acquire image data for the microvascular vessels located underneath a person's tongue. At this particular location, the vessels are pronounced and more easily viewable by the camera. As a result, some embodiments use a sublingual microscopy camera to generate microscopy images of a plurality of microvascular vessels.

The camera component 205 ensures that a sufficient number of high-quality images are captured by the camera. To ensure that the images are of high enough quality, the camera component 205 is able to monitor tissue motion, illumination intensity, and focus level of the microvascular vessels.

After the images of the microvascular vessels have been acquired, the computer system 200 is able to perform segmenting operations on that data. To clarify, the segment component 210 is able to analyze the image data to actually identify the various microvascular vessels.

Here, it is worthwhile to note that the vessels may be identified in a variety of ways. For example, the vessels may be identified by identifying the flow of red blood cells through the vessels. In other words, the progression of the individual red blood cells are viewable in the image data and the segment component 210 is able to identify this progression.

In other instances, the vessels may be identified by monitoring a vessel (i.e. a branching vessel) that branches into multiple sub-vessels. When this branching vessel is monitored, the segment component 210 is able to determine the branching vessel's blood flow. Once this blood flow is determined, then the embodiments are able to compare that blood flow to the blood flow of the multiple-sub vessels. The flow of the branching vessels correlates with the flow of the multiple sub-vessels.

Here, an example will be helpful. Suppose a branching vessel is branching into three sub- vessels. From the image data, however only two of those sub-vessels are immediately recognizable. By analyzing the flow of the branching vessel and the flows of the two sub- vessels, the embodiments are able to determine that there must be at least one additional sub- vessel. As a result, the embodiments are able to identify the microvascular vessels even if those vessels are not immediately apparent from the image data.

Once the microvascular vessels are identified, then the segment component 210 segments those vessels into a plurality of portions. Here, it will be appreciated that the vessels themselves are not being segmented; instead, the image data corresponding to the vessels is being segmented. By way of example and not limitation, suppose a first vessel is identified in the image data. Once it is identified, then the segment component 210 will act on the image data to break (i.e. segment) that vessel into a plurality of portions. For example, vessel might be segmented into two, three, four, or more different portions based on a determined, or rather preselected, length that is desired for each vessel. Stated differently, the image data might show that the vessel has a length of "x." Further, the preselected length might be "y." As a result, the vessel will be divided into a plurality of portions, where each divided portion has a length of "y" until the entire length "x" is achieved. Accordingly, the segment component 210 operates on the image data to segment the image data corresponding to the microvascular vessels into a plurality of portions. Here, it is worthwhile to note that any number of vessels might be included in the captured image data. Consequently, the segment component 210 is able to segment all of the identified vessels in the image data into a plurality of portions. Some vessels might have more portions than other vessels because some vessels are longer than other vessels.

The segment length may be any length. By way of example, the segment length may be

9.0 micrometers, 9.5 micrometers, 10 micrometers, 10.5 micrometer, 11.0 micrometers, etc.

After the image data is operated on to generate segmented portions for each of the identified vessels, the profile component 215 generates a profile for each of those portions. This profile may include a plethora of information.

To illustrate, the profile for each portion may include that portion's (1) determined blood flow, (2) information corresponding to the vessel's perfused boundary region (PBR) (i.e. the permeable portion of the vessel's glycocalyx) at that portion, (3) information corresponding to the vessel's endothelium at that portion, (4) information corresponding to the impermeable portion of that vessel's glycocalyx, or even (5) information the accessibility of lipids, proteins, plasma proteins, plasma proteoglycans, platelets, and monocytes passing through that portion of the vessel. Accordingly, the profile may include a wide variety of information. It is advantageous that the profile include as much information about the glycocalyx as possible. Furthermore, the profile may include information corresponding to the vessel's (1) endothelial functions, (2) permeability, (3) coagulation, (4) or inflammation, (5) leakage, (6) platelet adherence, and/or (7) other barrier properties of the glycocalyx.

After the profiles for each segment are generated, the computer system then compiles these profiles together in order to identify various trends in the data. Here, the characterization component 220 is able to compile all of the profiles together. By "compiling," it is generally meant that similar data from each profile is collected and analyzed. The compiled information may be stored and analyzed in the form of a database. The data is then analyzed to identify one or more actual trends present in the compiled information.

Turning briefly to Figure 17, Figure 17 illustrates such a compilation of the data. Figure

17 illustrates a graphical representation of profile information for a plurality of vessels. Notably, these vessels were identified as having a diameter of 9 microns (e.g., the title "D09" indicates that the diameter is 9 microns). The "x" axis is representative of blood flow while the "y" axis is representative of PBR. This information was included in the profiles. By compiling the profile data, the graph in Figure 17 can be generated.

As illustrated in Figure 17, when the blood flow is low, then the PBR varies greatly. Indeed, the characteristics of the glycocalyx are such that when blood flow is low, the glycocalyx is more permeable, resulting in an increased PBR values (i.e. a higher value on the y axis) while when the blood flow is high, the glycocalyx is less permeable, resulting in lower PBR values.

It will be appreciated that the characterization component 220 is able to compile the profile data in order to generate a representation such as the graph shown in Figure 17. Notably, however, the characterization component 220 is also able to identify one or more actual trends that are present in the profile data. By way of example, Figure 17 shows that a fitted curve (see dashed line) has been generated to match the general trend of the plotted profile data. Here, the fitted curve is represented by the following equation:

y = 1.6806e-° ⁰⁴³

It will be appreciated that this is an example equation only. Indeed, each compiled profile data will have its own unique equation trend. As a result, the embodiments should not be limited solely to that which is illustrated in Figure 17.

Here, it is worthwhile to note that the characterization component 220 is able to extrapolate the data beyond just the actual trend shown in Figure 17. For example, as shown in Figure 17, the actual trend ends at approximately the "14" point on the x axis. Notably, however, the characterization component 220 is able to extrapolate this trend even further so that it includes one or more "projected" trends. Here, the projected trend is illustrated by the solid line in Figure 17.

Research has shown that by extrapolating the profile characterizations so that the trends are projected into high blow flow situations creates much more reliable, accurate, and robust glycocalyx impairment estimations. Furthermore, by extrapolating the trend to the higher blood flows, the embodiments are able to accurately distinguish between healthy glycocalyces and non-healthy glycocalyces. Accordingly, the embodiments generate a projected trend using the compiled profile information.

Once the projected trend is determined, the characterization component 220 then compares the extrapolated characterization (including the projected trend data) against a predetermined threshold value to select a target characterization level. Using Figure 17 as an example, the embodiments may select the predetermined threshold value of a blood flow rate of 20. By comparing this threshold value to the extrapolated characterization data, the target level may be determined. In Figure 17, if the threshold level were 20, then the target characterization level would be a PBR value of approximately 0.7.

Then, the characterization component 220 evaluates a set of rules against this target characterization level in order to generate a glycocalyx impairment determination. In particular this set of rules includes a correlation between the number of blood vessels whose profiles were included in the extrapolated characterization and the red blood cell content for those blood vessels. This correlation is then compared against the target characterization level. This comparison generates the glycocalyx impairment determination. In some embodiments, the equation shown below may illustrate this relationship:

(# blood vessels) * (RBC content of blood vessels)

Glycocalyx Impairment = ; : : ; ;

target characterization level

Accordingly, embodiments of the present disclosure are able to generate one or more impairment values using advanced techniques of capturing image data, segmenting that image data, generating profiles for those segments, compiling those profiles, and then generating an extrapolated characterization. This extrapolated characterization includes a projected trend of the profile data and can be used to accurately distinguish between health and non-healthy glycocalyces.

Attention will now be focused on Figures 18 and 19, which illustrate (estimated) perfused capillary density as function of feed vessel RBC velocity, thirteen different snapshots of RBC velocity. Each dot illustrates a mean value and there are thirteen different mean values. This data is useful when determining blood flow. Figure 18 illustrates the data in a computer system user interface environment. In particular, Figure 18 shows (estimated) perfused capillary density as function of feed vessel RBC velocity. Figure 19 illustrates the same data in an enlarged, detail report.

Attention will now be turned Figure 20 which shows an exemplary user interface that can be used to generate and display glycocalyx impairment values. As shown, the user interface can be used to illustrate the glycocalyx determination in an effort to estimate the overall health of a person. This user interface include the target characterization level discussed earlier (shown as the "PBR high flow" value).

Using this interface, the embodiments are also able to receive user input. For example, if the user were to press the "Generate MVHS Report," then the embodiments will generate a report details the various operations that were discussed earlier. Further, the report will include data corresponding to a person's glycocalyx impairment determination.

Illustrative Methods

The following discussion now refers to a number of methods and method acts that may be performed. Although the method acts may be discussed in a certain order or illustrated in a flow chart as occurring in a particular order, no particular ordering is required unless specifically stated, or required because an act is dependent on another act being completed prior to the act being performed. These methods are implemented by one or more processors of a computer system (e.g., the computer system 200 of Figure 20). By way of example, a computer system includes one or more computer-readable hardware storage media that store computer-executable code. This computer-executable code is executable by the one or more processors to cause the computer system to perform these methods.

Figure 21 shows an exemplary method 700 that can be used to differentiate between healthy and non-health glycocalyces. Various illustrative methods can include one or more steps outlined in method 700 of Figure 21, namely:

Capture, Using A Camera Of The Computer System, A Microscopy Image Of The Plurality Of Microvascular Vessels;

For Image Data Corresponding To At Least Some Microvascular Vessels Captured Within The Microscopy Image, Segment The Image Data Corresponding To Each Of The At Least Some Microvascular Vessels Into A Plurality Of Segmented Data Portions;

Generate A Profile For Each Of The Plurality Of Segmented Data Portions;

After Compiling The Profiles For Each Of The Plurality Of Portions, Generate An Extrapolated Characterization Of The Compiled Profiles; Compare The Extrapolated Characterization Against A Predetermined Threshold Value To Select A Target Characterization Level;

Evaluate A Particularized Set Of Rules Against The Target Characterization Level To Generate A Glycocalyx Impairment Determination; and/or

Display The Glycocalyx Impairment Determination On A User Interface Of The

Computer System.

It will be appreciated that some embodiments of the present disclosure need not include each of the foregoing steps, while other embodiments may include all of the foregoing steps.

Some embodiments include a method of accurately assessing vascular health or function. For instance, some embodiments include accurately assessing, determining, or calculating PBR in a subject. In some embodiments, a method of accurately assessing, determining, or calculating PBR in a subject can include measuring PBR in a low flow state and extrapolating (from the low flow PBR data) to determine PBR in a high flow state.

In some embodiments, a method of accurately assessing, determining, or calculating PBR in a subject can include one or more step, preferably selected from the group consisting of: capturing one or more images (e.g., videos) of vascular structures (e.g., blood vessels), the images preferably including pixels representative of one or more vascular components or structures (e.g., vessel walls, RBC, etc.); determining median RBC widths (p50) and PBR values of one or more (e.g., all) individual measurement sites (e.g., blood vessels); grouping vessels in diameter classes based on their p50 values; measuring RBC velocity (Vrbc) in (all) vessels; multiplying Vrbc with RBCfilling% to obtain estimate of Frbc (RBC flow) for (all) vessels; for each diameter class (in range of 5 - 25 microns), constructing Frbc - PBR plots; for each diameter class, fitting Frbc-PBR data with appropriate curve (e.g. exponential fit); estimating PBR at high Frbc (PBR high); estmating PBR at Frbc = 0 (PBR low); using fitted curve parameters (e.g. exponential decay parameter) to describe flow dependency of PBR of each diameter class; optionally or alternatively, calculating PBR-low - PBR high for each diameter class; constructing diameter - PBR-high curve; and calculating mean PBR high avg by averaging of PBR-high scores of diameter classes 5 - 25 microns.

Some embodiments include accurately assessing, determining, or calculating RBC filling% in a subject. An illustrative method of accurately assessing, determining, or calculating RBCfilling% in a subject can include each of the above-recited steps, but replacing "PBR" with "RBCfilling%" and/or using linear fit instead of exponential fit. Embodiments can, therefore, include estimating RBCfilling% at high Frbc (RBCfilling%_high) for each diameter class and/or calculating RBCfilling%_high_avg by averaging RBCfilling%_high scores of diameter classes 5 - 25 microns.

Some embodiments include accurately assessing, determining, or calculating valid density in a subject. An illustrative method of accurately assessing, determining, or calculating valid density in a subject can include one or more step, preferably selected from the group consisting of: capturing one or more images (e.g., videos) of vascular structures (e.g., blood vessels), the images preferably including pixels representative of one or more vascular components or structures (e.g., vessel walls, RBC, etc.); for each video, determining flow level by adding all flow of feed vessels (see previous definition of feed vessels and how to measure flow); for each video, obtaining valid density (Density) for each diameter class; using all videos, constructing Flow-Density plots for each diameter class; using (linear) fit to obtain Density high for each diameter class at high flow; and adding all Density high estimates for all diameter classes 5 - 25 microns to Density high total.

Some embodiments include accurately assessing, determining, or calculating the ratio of D Valid / D Total in a subject. An illustrative method of accurately assessing, determining, or calculating the ratio of D Valid / D Total in a subject can include one or more step, preferably selected from the group consisting of: capturing one or more images (e.g., videos) of vascular structures (e.g., blood vessels), the images preferably including pixels representative of one or more vascular components or structures (e.g., vessel walls, RBC, etc.); for each video: obtaining ratio of Density valid / Density total (Ratio); using all videos: construct Flow-Ratio plot; and using (linear) fit to estimate Ratio high at high Frbc.

Some embodiments include accurately assessing, determining, or calculating a microvascular health score (MVHS) in a subject. An illustrative method of accurately assessing, determining, or calculating MVHS in a subject can include: (i) determining, calculating, or estimating: Density high total; Ratio high; RBCfilling%_high_avg; and PBR high avg; and (ii) calculating MVHS using the following formula:

MVHS = Density high total x Ratio high x RBCfilling%_high_avg / PBR high avg The microcirculation has increasingly been recognized to be at the center of several pathophysiological processes. However, accessible insight into microvascular function in a patient has been limited by a current lack of real-time quantification tools. Embodiments of the present invention provide novel and inventive solutions for accurately assessing (micro)vascular health and function, preferably in real-time and/or at the bed side.

Non-invasive assessment of microvascular dysfunction in patients with MVA The present embodiment evaluated the microvascular function in patients with microvascular angina (MVA) by assessing 1) the endothelial glycocalyx barrier properties using sublingual microscopy, and 2) the myocardial perfusion reserve (MPR) using cardiovascular magnetic resonance (CMR) imaging. Sublingual microscopy was performed in 13 MVA patients (angina pectoris, ST-depression on treadmill testing, normal coronary angiogram) and compared with 2 control groups of 13 volunteers and 14 patients with known obstructive coronary artery disease (CAD). To test the glycocalyx -mediated microvascular responsiveness, the erythrocyte perfused boundary region (PBR) was assessed at baseline and after nitroglycerin challenge. The baseline PBR of MVA patients was similar to controls with CAD (p=0.72), and larger than in volunteers (p=0.02). Only the volunteers demonstrated a significant increase in PBR after nitroglycerin (p=0.03). In the 13 MVA patients, adenosine stress CMR perfusion imaging was performed. Although a significant increase in myocardial perfusion was observed in both the sub endocardium and subepicardium during stress, the subendocardial perfusion reserve was significantly lower (p=0.02). The PBR responsiveness of the sublingual microvasculature showed a strong correlation with the transmural MPR (r=0.86,p<0.001). Thus, patients with MVA can be characterized by microvascular glycocalyx dysfunction using sublingual microscopy. The strong correlation between sublingual PBR responsiveness and MPR suggests that the glycocalyx may play an important role in the regulation of microvascular volume for myocardial perfusion and supports the concept of impaired glycocalyx barrier properties in MVA.

Patients with typical angina pectoris on exertion with corresponding ST-depression on electrocardiography, despite normal or near-normal coronary arteries on invasive coronary angiography remain a diagnostic challenge for physicians. Although there is currently no consensus on the underlying pathophysiology, this entity has been described as microvascular angina (MVA) as it is proposed that coronary microvascular dysfunction plays an important role. In contrast to the epicardial coronary vasculature, direct assessment of coronary microvascular dysfunction remains challenging. Cardiovascular magnetic resonance (CMR) perfusion imaging has the ability to detect myocardial ischemia non-invasively with a good spatial resolution. However, previous studies using CMR perfusion imaging to detect myocardial ischemia as a marker for coronary microvascular dysfunction in patients with MVA have reported conflicting results. Alternatively, the microcirculation can be directly assessed in easily accessible regions (e.g. the sublingual circulation) by novel non-invasive imaging techniques. Experimental as well as clinical studies using intravital microscopy of the sublingual microvasculature have shown that damage to the endothelial glycocalyx may reflect microvascular dysfunction. The endothelial glycocalyx is a cell-hindering layer on the luminal side of blood vessels that contributes significantly to the protection of the vascular wall against atherogenic stimuli.

Figure 22 portraits the endothelial glycocalyx and its relation to the perfused boundary region (PBR) in a microvessel. In panel Ά', for example, healthy glycocalyx limits the accessibility of blood-borne lipids and proteins and forms a barrier for adhesion of platelets and inflammatory cells to the vascular wall. It is also involved in mechanosensing and transduction of hemodynamic stimuli to the endothelium, thereby regulating the production of amongst others nitric oxide. In the center and right are schematically illustrated, the perfused boundary region (PBR) in relation to the glycocalyx barrier properties in a blood vessel. The PBR is the cell-poor layer which results from the phase separation between the flowing erythrocytes and plasma. It covers the cell-permeable part of the glycocalyx, to which erythrocytes have limited access, while the cell-impermeable part cannot be accessed at all. In a healthy vessel, outward movement of the erythrocytes under baseline conditions is restricted by the relatively thick cell-impermeable glycocalyx barrier, resulting in a small PBR. Nitroglycerin, however, is anticipated to robustly increase the PBR in this vessel by impairing this barrier. The PBR is the main readout parameter of the sublingual imaging, and calculated from the median erythrocyte column width (P50) and outer diameter of erythrocyte perfused lumen (Dperf); this is further depicted and explained in Figures 5A-9 and the corresponding text.

Perturbation of this protective layer allows deeper cell penetration towards the endothelium, leading to an increase in the erythrocyte perfused boundary region (PBR). A thick endothelial glycocalyx (i.e. a healthy state with a low PBR) is associated with efficient perfusion of the microvascular bed while a thin glycocalyx (i.e. a high risk state with an increased PBR) reflects a perturbed microvascular perfusion.

In panel 'B' of Figure 22, damage to the glycocalyx has been associated with all features of a malfunctioning microcirculation: endothelial activation, vascular leakage and a diminished NO bioavailability. A damaged glycocalyx is associated with a reduction of the cell-impermeable glycocalyx part allowing the outer edge of the erythrocyte perfused lumen to move in sideward direction towards the endothelium, resulting in an increase in PBR at baseline already and the absence of a PBR response to nitroglycerin (center and right).

Some embodiments of the present disclosure are directed to methods of investigating the microvascular function of patients with MVA using these non-invasive imaging techniques. Therefore, we assessed the glycocalyx-mediated microvascular function using sublingual microscopy in a well described homogeneous population of MVA patients in comparison with a control group of healthy volunteers. Additionally, we evaluated the MPR of the MVA patients with high spatial resolution adenosine stress CMR perfusion imaging at 3.0 Tesla.

Population

Consecutive patients with MVA were prospectively enrolled at our institution. The MVA patients had typical exercise-induced angina pectoris and corresponding ischemic ST-changes on electrocardiography (defined as >0.1 mV horizontal or down sloping ST-segment depression 80 ms after the J point). They all had previously undergone invasive coronary angiography, showing normal coronary arteries (n = 8) or minimal vessel wall irregularities (i.e. <25% stenosis, n = 5). Patients younger than 18 years and patients with contraindications for either CMR (e.g. metallic implants, pacemaker) or adenosine (e.g. atrioventricular conduction abnormalities, severe asthma) were excluded. Of the 16 eligible MVA patients, 13 gave informed consent and completed the entire study protocol.

In addition to these 13 MVA patients, 13 volunteers without a history of chest pain, documented coronary artery disease or myocardial infarction and 14 patients with known obstructive CAD (examined approximately one hour before their scheduled percutaneous revascularization) of similar age and sex distribution were recruited during the study period to serve as control groups for the sublingual microscopy. Subjects (i.e. MVA patients, volunteers, or CAD patients) with a history of (medically) controlled mild hypertension or hypercholesterolemia were not excluded. Our local Institutional Review Board only approved adenosine stress CMR perfusion imaging in the patients with MVA, since there was no myocardial ischemia suspected in the volunteers, and already known obstructive CAD with ischemia in the patients with CAD.

Sublingual microvasculature imaging

All 40 study subjects (i.e. 13 MVA patients, 13 volunteers, and 14 CAD patients) underwent imaging of the sublingual microvasculature using a handheld sidestream darkfield microscan videomicroscope (MicroVision Medical Inc., Wallingford, PA). Analyses of glycocalyx barrier properties were performed by calculating the PBR using GlycoCheck Glycocalyx Measurement Software (GlycoCheck, Maastricht, the Netherlands). The measurements were performed after an overnight fast, during which the study subjects were also asked to refrain from smoking. Prescribed medication was continued. Each subject underwent 2 baseline measurements and 2 measurements performed starting 2 minutes after sublingual administration of nitroglycerin (0.4mg spray dose). Under physiological conditions, nitroglycerin is anticipated to rapidly increase the PBR by modulating the barrier properties of the glycocalyx, but this effect is diminished in case of glycocalyx degradation in diseased states. Thus, an increased baseline PBR as well as an impaired PBR response to nitroglycerin were considered to reflect microvascular dysfunction as a result of perturbation of the endothelial glycocalyx.

The techniques and reproducibility of imaging and analysis of the glycocalyx barrier properties have been described previously. The parameters of interest for the analysis are schematically depicted in Figure 5A, while a detailed description of both the sublingual imaging technique and the calculation of the PBR are described at least in relation to Figures 5 A-9 and the corresponding text.

The sublingual microvasculature was imaged with the subject in a supine position using a handheld sidestream darkfield microscan videomicroscope (MicroVision Medical Inc., Wallingford, PA). This microscope is equipped with a 5X magnifying objective lens system- containing probe (numerical aperture: 0.2), imaging the erythrocytes in the tissue-embedded microcirculation using green pulsed LED ring illumination. The region chosen for measurement was the central sublingual area; pressure on the tissue was avoided to ensure normal flow.

Automated image recording and analysis were performed using GlycoCheck Glycocalyx Measurement Software (GlycoCheck, Maastricht, the Netherlands). By online monitoring of tissue motion, illumination intensity, and focus level of the sublingual microvasculature, the recording of a sufficient number of high-quality videos was guaranteed by the software. Sublingual images were acquired at a frame rate of 23 per second with a final on-screen magnification of 325x. Video acquisition automatically started when image quality was within an acceptable range and automatically stopped when a sufficient number of videos had been collected for analysis; per measurement approximately 10 movies of 40 consecutive frames were recorded, giving a total recording time of -2-5 min. Image analysis involved four steps which in the end resulted in the determination of the PBR at multiple sites along the microvasculature. Analysis automatically started during the recording stage {step 1). The first frame was used to automatically identify all available microvessels with a diameter between 5 and 25 μπι. Every 10 μπι along the length of the detected vasculature marker lines were placed perpendicular to the vessel direction {step 2). Each line represented a single vessel segment, for which 21 parallel intensity profiles (every ± 0.5 μπι) were obtained; within each intensity profile the erythrocyte column width was calculated from the full width half maximum. This was done for all 40 consecutive frames in a movie {step 3; note that just 9 representative RBC column width tracings and not the total number of 21 for a particular vessel segment are shown). In this way, a total of 840 erythrocyte column width measurements per vessel segment were generated, for which a cumulative distribution was calculated {step 4). From this distribution, the median erythrocyte column width (P50) was taken while the outer edges of the erythrocyte perfused lumen (Dperf) were derived using linear extrapolation between the 25th and 75th percentile values {step 4). Subsequently, the PBR was calculated from the distance of the median erythrocyte column width to the outer edge of the erythrocyte perfused lumen; i.e. PBR = (Dperf - P50) / 2. See also Figures 6A-7.

Figure 23 presents the results of sublingual perfused boundary region (PBR) measurements in patients with microvascular angina (MVA), volunteers, and controls with obstructive coronary artery disease (CAD). Baseline PBR measurements (panel A) in MVA patients were significantly higher than in volunteers. After nitroglycerin challenge (panel B), all groups had a comparable PBR. Panel C represents the PBR response (i.e. the difference between panel A and B). Only the volunteers demonstrated a significant increase in PBR after nitroglycerin challenge {p = 0.03), which was a significantly larger PBR response than observed in the MVA patients (Panel C).

For each measurement in a subject, the calculated PBR values were classified according to their P50 (range 5-25μπι, interval Ι μπι), providing a median PBR per bin of median erythrocyte column width, from which the average PBR was calculated to provide a single PBR value per subject per measurement. The baseline PBR was taken as the average of both baseline measurements, while for the nitroglycerin PBR the highest PBR value in either one of the two measurements after nitroglycerin challenge was taken. The PBR response was calculated by subtracting the baseline PBR from the nitroglycerin PBR.

Cardiovascular magnetic resonance imaging

Following the sublingual microvascular imaging, the 13 MVA patients subsequently underwent CMR imaging on a 3.0 Tesla MR system (Achieva, Philips Healthcare, Best, the Netherlands) equipped with a cardiac software package and a SENSE 6 element cardiac array coil. These patients were asked to refrain from caffeine and beta-blockers the morning of the study. The median time from invasive coronary angiography to CMR imaging was 4 weeks (range 0-19 weeks).

All images were acquired with electrocardiographic triggering and during expiratory breath hold. The protocol included standard cine (steady-state free precession) and late gadolinium enhancement imaging. Stress perfusion imaging was performed during intravenous adenosine at a dose of 140μg/kg/min during heart rate and blood pressure monitoring. The pulse-sequence used for perfusion imaging has been described in detail previously. Perfusion data were acquired in 3 slices in short-axis view using a saturation recovery gradient echo pulse sequence accelerated with k-t SENSE (TR/TE 3.2/1.54ms, flip angle 15°, saturation prepulse delay 110ms, acquisition window 140ms, slice thickness 8mm, k-t factor of 5 with 11 k-t interleaved training profiles, effective acceleration 3.8). After 4 minutes of adenosine infusion, a bolus of 0.05mmol/kg body weight gadobutrol (Gadovist, Bayer-Schering, Germany) was administered. Rest perfusion was performed after approximately 15 minutes, using the same bolus technique.

Two independent observers analyzed the CMR perfusion images qualitatively, using commercially available software (CAAS MRV 3.0, Pie Medical Imaging, Maastricht, the Netherlands). Any discrepancies were resolved in consensus. The image quality was scored as 0 = poor, 1 = average, and 2 = excellent. Perfusion defects were deemed present if a subendocardial or transmural delay of contrast enhancement was seen relative to a remote region and persisted for more than 5 frames. Initial delay of subendocardial contrast enhancement, which disappeared after approximately 5 heartbeats, was considered an artifact.7, 9 For semi-quantitative analysis, the endocardial and epicardial contours were traced manually and automatically divided into the 16 segments of the basal, mid, and apical slices according to the AHA 17-segment model. These segments were further subdivided into subendocardial and subepicardial segments (i.e. the inner and outer 50% of each segment, respectively). Additionally, a region of interest was drawn in the cavity of the left ventricle to record an arterial input function. Quantitative analysis of the tissue enhancement curves was performed using Matlab (The MathWorks Inc., Natick, MA). Signal intensity was converted to signal enhancement by dividing by the mean pre-contrast signal. Perfusion was quantified using Patlak Plot analysis, which assumes that there is no contrast efflux from the tissue into the vasculature. As such, it assumes a linear relationship between enhancement in the myocardium (Ctis) and the time integral of the enhancement in the blood pool (Cbl) (equation 1). The slope of the curve Ktr can be used as a measure for erfusion of the myocardium.

To improve the goodness of fit of Ktr, the enhancement curves were manually selected to include the start of the arrival of the contrast agent in the left ventricle up to the maximum enhancement in the myocardium before fitting the model. The MPR was calculated by dividing the Ktr of the stress perfusion by the Ktr of the perfusion in rest.

Statistical analysis Continuous variables with normally distributed data are expressed as mean ± standard deviation (SD), otherwise as median with the interquartile range (IQR). Categorical data are expressed as frequencies with percentages. Differences in baseline characteristics between the study groups were evaluated using one-way ANOVA (for continuous data) and the Chi- square or Fisher's exact test (for categorical data). For comparisons within subjects (e.g. rest versus stress) the Wilcoxon test for paired samples was used. The Mann Whitney U test was used to compare data between study groups. The Spearman correlation coefficient was used to evaluate the correlation between the sublingual PBR measurements and the CMR derived MPRI ratio. SPSS version 17.0 (SPSS Inc., Chicago, IL) was used for all statistical analyses. A two-tailed value of p<0.05 was considered statistically significant.

Baseline characteristics

Characteristics of the study subjects are presented in Table 1. The three study groups demonstrated similar distributions of age and sex. The MVA patients and the control subjects with CAD more often had cardiovascular risk factors, especially smoking or a family history of CAD, than the volunteers.

Table 1. Baseline characteristics of the study population.

MVA Controls P Value

Volunteers CAD

N = 13 N = 13 N = 14

Age, years 65 ± 9 63 ± 10 68 ± 8 0.38

Male (%) 6 (46) 9 (69) 9 (64) 0.52

BMI, kg/m² 26 ± 3 25 ± 3 28 ± 3 0.02

Diabetes mellitus (%) 2 (15) 0 (0) 3 (21) 0.34

History of smoking (%) 10 (77) 4 (31) 12 (86) <0.01

Current (%) 0 (0) 1 (8) 2 (14) 1.00

Hypertension (%) 5 (38) 5 (38) 7 (50) 0.78

Hypercholesterolemia (%) 6 (46) 3 (23) 8 (57) 0.16

Family history of CAD (%) 8 (62) 2 (15) 8 (57) 0.04

Values are presented as mean ± standard deviation or proportions (%), when appropriate.

BMI = body mass index; CAD = coronary artery disease; MVA = microvascular angina.

Sublingual microvasculature imaging

The median baseline PBR of patients with MVA was similar to that in the controls with CAD (2.02μιη [IQR 1.95μπι-2.14μπι] and 2.02μιη [IQR 1.73μιη-2.18μιη], respectively, p = 0.72), but significantly larger compared with the volunteers (1.90μπι [IQR 1.77μπι-2.02μιη], p = 0.02, Figure 23, panel Ά'). This suggests that, compared with volunteers, both MVA and CAD patients showed a deeper erythrocyte penetration into the glycocalyx because of a decrease in its integrity. Figure 23, panel 'B' shows that the maximum PBR after NTG challenge did not differ significantly between any of the groups (2.09μπι [IQR 1.85μηι- 2.16μιη], 1.96μπι [IQR 1.85μπι-2.14μπι], and 2.12μιη [IQR 2.01 μm-2.33μm] for MVA patients, volunteers, and controls with CAD, respectively). Hence, the nitroglycerin challenge caused only a minimal increase in median PBR in MVA patients and control subjects with CAD (p = 0.70 and p = 0.18, respectively), whereas the volunteers demonstrated a significant increase in their PBR upon nitroglycerin challenge. Comparisons between groups revealed that the median PBR response to nitroglycerin was significantly smaller in MVA patients than in the volunteers (p = 0.03, Figure 23, panel 'C').

A major challenge in the assessment of coronary microvascular dysfunction and its role in occurrence of myocardial ischemia in MVA is that the direct evaluation of the structure and function of small coronary vessels is cumbersome. Intravital microscopy has emerged as a promising non-invasive tool to assess the microcirculation directly in easily accessible regions. To our knowledge, this is the first study to investigate the glycocalyx-mediated microvascular responsiveness in both patients with MVA and obstructive CAD. Our results are in line with previous clinical studies that have reported evidence of endothelial glycocalyx loss in patients with accelerated vascular disease (e.g. patients with type 1 diabetes, dialysis patients, and lacunar stroke patients with white matter lesions). The underlying mechanisms for endothelial glycocalyx perturbation during these conditions of increased cardiovascular risk are currently not fully understood. Since the endothelial glycocalyx is anticipated to act as a protective layer which shields the vessel wall from atherogenic stimuli, impairment of this protective barrier is considered a primary step in microvascular dysfunction. Moreover, it has been demonstrated in animal studies that an intact endothelial glycocalyx exhibits an increase in blood-perfused microvascular volume after nitroglycerin or adenosine challenge by increasing glycocalyx accessibility. These findings imply that the glycocalyx also plays an important role in the regulation of microvascular volume for perfusion. The strong correlation between the sublingual PBR- response and the CMR-derived MPRI measurements seems to support this role, and may indicate a generalized glycocalyx loss in patients with MVA.

CMR perfusion imaging in patients with microvascular angina

Over the last decade, several studies have explored the ability of CMR perfusion imaging to detect myocardial ischemia in patients with MVA. However, these studies differed considerably in study design and reported conflicting results. Panting et al. used adenosine stress perfusion CMR at 1.5 Tesla and reported evidence of hypoperfusion in the sub endocardium in patients with MVA. In an attempt to reproduce these data Vermeltfoort et al., however, found that MVA patients have a similar increase in myocardial perfusion during adenosine in both the sub endocardium and subepicardium. The fact that Panting et al. included only patients with ST-segment depression on exercise electrocardiography whereas Vermeltfoort et al. also included patients with an abnormal perfusion single-photon emission computed tomography (and only 25% with ST-depression), suggests that the study populations of these 2 studies are not directly comparable. A study by Karamitsos et al. investigated 18 MVA patients, all demonstrating ST-depression on exercise electrocardiography, using CMR imaging for absolute quantification of the myocardial blood flow at 3.0 Tesla. Although they found no evidence of transmural hypoperfusion in these MVA patients, no comparison was made between subendocardial and subepicardial myocardial blood flow. As a result, subendocardial hypoperfusion may not have been detected. The most recent study by Thomson et al. investigated a cohort of 118 women with MVA and reported a lower global, subendocardial and subepicardial MPRI during pharmacologic stress in patients with MVA compared with controls.

Here, all 13 MVA patients undergoing CMR imaging had a normal systolic left ventricular function without regional wall motion abnormalities on cine images. None of the patients had evidence of hyperenhancement on late gadolinium enhancement images. During adenosine vasodilator stress, the 13 MVA patients undergoing CMR perfusion imaging demonstrated an increase in mean heart rate from 69±12 to 97±16 beats per minute. At peak stress, both the mean systolic and diastolic blood pressure decreased slightly from 138±14 to 130±17mmHg and 78±9 to 73±7mmHg, respectively. No relevant side effects were observed during adenosine infusion, although some patients reported transient chest pain (N = 5), flushing (N = 5), or dyspnea (N = 1).

Four patients had excellent, 8 had average, and 1 had poor image quality due to inadequate breath holding. Of the 12 patients with interpretable images, none had evidence of perfusion defects on visual perfusion analysis. Semi-quantitative perfusion analysis showed that the myocardial perfusion index (MPI) for the entire transmural extent increased significantly during adenosine stress (0.05 [IQR 0.04-0.06] to 0.06 [IQR 0.06-0.10], p = 0.006), with an MPRI of 1.35 (IQR 1.09-1.78). Similarly, the MPI increased in subendocardial segments from 0.05 (IQR 0.04-0.07) to 0.06 (IQR 0.06-0.10), p = 0.050, and in subepicardial segments from 0.04 (IQR 0.04-0.06) to 0.07 (IQR 0.06-0.09), p = 0.002. However, the MPR of subendocardial segments was significantly lower than that of subepicardial segments (i.e. an MPRI of 1.22 [IQR 0.97-1.66] vs. 1.49 [IQR 1.22-2.04], p = 0.03).

A value of <0.72 has been proposed in a previous publication (performed at 1.5 Tesla using gadopentate dimeglumine contrast medium) as a possible cut-off value for the ratio of sub endocardial -to- subepicardial MPRI to distinguish between MVA patients and healthy controls. However, in the current study, we found a wide range of sub endocardial -to- subepicardial MPRI ratio (median 0.80 [IQR 0.70-0.91]), and only 3 patients demonstrated a ratio of <0.72 (i.e. 0.64, 0.65, and 0.68).

Here, the MPI increased significantly during adenosine stress in both subendocardial and subepicardial segments. However, we observed that the MPI reserve of the sub endocardium (i.e. the subendocardial MPRI) was significantly lower as compared with the subepicardial MPRI. While glycocalyx-mediated microvascular dysfunction is expected to affect the myocardium diffusely, the subendocardium seems also in other forms of microvascular disease (e.g. cardiac transplant arteriopathy and systemic inflammation) to be more vulnerable to hypoperfusion than the subepicardium. This subendocardial vulnerability could be due to a slightly higher metabolic demand and a lower blood supply. In addition, Algranati et al. found that this subendocardial vulnerability to hypoperfusion stems from the combined effects of cardiac contraction, vascular pressure-dependent compliance, and potential transmural differences in vessel anatomy. Thus, diffuse microvascular glycocalyx perturbation which also can be considered an alteration of the vessel wall properties may therefore result in perfusion disturbances most prominent in subendocardial layers.

It has been suggested by Panting et al. that the more pronounced difference in this sub endocardial -to- subepicardial MPRI ratio in MVA patients can be used to distinguish between MVA patients and healthy controls. In the current study, performed at a higher magnetic field strength, however, our homogeneous group of MVA patients demonstrated a considerable range of subendocardial -to-subepicardial MPRI's and only 23% of the MVA patients had a subendocardial-to-subepicardial MPRI ratio of <0.72. A difference in subendocardial and subepicardial MPRI is to some extent the result of normal physiological mechanisms and, thus, a single cut-off value for MPRI in all patients may be debatable.

Correlation between sublingual microvascular function and MPR

To explore the correlation between the sublingual assessment of glycocalyx-associated microvascular function and myocardial perfusion measurements by CMR, we performed a correlation analysis for the PBR-response and the transmural MPRI. It revealed a strong correlation between PBR-increase and MPRI (r = 0.86, p <0.001). It has been proposed that microvascular dysfunction plays an important role in MVA, but direct evidence is lacking. In the current e, sublingual microscopy demonstrated that patients with MVA can be characterized by glycocalyx-mediated microvascular dysfunction in comparison with volunteer control subjects. The MVA patients exhibit a larger PBR at baseline, reflecting deeper penetration of circulating erythrocytes towards the endothelium, indicating a decrease in glycocalyx integrity. Additionally, the physiological increase in PBR upon nitroglycerin challenge that was observed in the volunteers was absent in patients with MVA. The patients with obstructive CAD had a similar large median PBR at baseline as the MVA patients and also failed to show a significant increase upon nitroglycerin challenge. However, it should be noted that a considerably wider variation in PBR's was observed in the CAD patients. This may indicate that there is heterogeneity in the contribution of microvascular disease in patients with obstructive CAD, and possibly even absence of microvascular dysfunction in a proportion of these patients.

Adenosine stress CMR perfusion imaging in our MVA patients showed no evidence of visual perfusion defects, but revealed that the perfusion reserve was significantly lower in the sub endocardium as compared with the subepicardium. The strong correlation that was found between the sublingual PBR-response and the CMR-derived MPRI measurements in MVA patients supports the concept that the glycocalyx may play an important role in the regulation of microvascular volume for myocardial perfusion and indicates a glycocalyx loss in MVA patients. Sublingual PBR measurements may therefore be a useful tool for non-invasively characterizing microvascular dysfunction in MVA.

The characteristics of patients included in previous MVA studies are highly variable. The current study therefore used a strict definition of MVA aimed at including a homogeneous group of patients, albeit with a modest sample size. Controversy remains on the inclusion of patients with cardiovascular risk factors (e.g. hypertension or hypercholesterolemia) as they may form underlying causes of microvascular dysfunction. We designed our inclusion criteria in accordance with the current recommendations and guidelines and did not exclude MVA patients or control subjects that had a history of (medically) controlled mild hypertension or hypercholesterolemia. As a result, our study groups showed similar prevalence of these cardiovascular risk factors, implying that the observed differences in the glycocalyx-mediated microvascular responsiveness between the 3 study groups cannot be attributed to these risk factors. Due to regulations by our local Institutional Review Board, we were only able to perform myocardial perfusion imaging in the MVA patients. As a result, direct comparisons between myocardial perfusion measurements between all three study groups could not be performed. In addition, mild CAD such as calcification or plaque might have been present in the group of volunteers. However, sublingual microvascular function was clearly different between volunteers and patients with obstructive CAD.

The prognostic consequences of MVA are not well described and the treatment of patients with MVA currently consists predominantly of risk factor modification and symptom control. However, MVA patients frequently have persisting or even worsening symptoms over time, are often referred to further and repeated (non-)invasive testing and the quality of life is impaired. Although novel drugs may offer beneficial effects, the optimal therapeutic strategy in MVA patients currently remains unknown. The results of the current study show that sublingual microscopy is a promising diagnostic tool to characterize MVA patients by glycocalyx-mediated microvascular dysfunction. Sublingual PBR-responsiveness correlated strongly to the CMR-derived MPR. Thus, the glycocalyx may play an important role in the regulation of microvascular volume for myocardial perfusion. Therefore, MVA patients might benefit from therapeutic interventions aimed at restoring the endothelial function by limiting the degradation of the endothelial glycocalyx as well as supplementation of a damaged glycocalyx.

Illustrative Compositions and Uses Thereof

Certain embodiments of the present disclosure address and/or provide solutions to the problem of glycocalyx deficiency, dysfunction, degradation, vascular health-related problems resulting therefrom, and/or one or more of the foregoing or other problems in the art with novel, effective, and/or synergistic compositions and methods for treating endothelial glycocalyx. As used herein the term "synergistic" refers to the phenomenon where the combination of two or more components (e.g., therapeutic agents, ingredients, etc.) provides an effect that is greater than the effect of the individual components or the sum of the components acting alone. Thus, embodiments of the present disclosure can include a combination or mixture of components in an amount effective to produce or exhibit a (physiological) effect (when administered to a subject or patient in need thereof) that is greater than the effect of any single component or the sum of the effects of each component acting alone.

One or more embodiments of the present disclosure include products, compositions, systems, and/or methods of manufacturing and/or using the products, compositions, systems. Some embodiments include Current Good Manufacturing Practice (cGMP)-grade products, compositions, and/or ingredients. Some embodiments can include one or more nutritional building blocks (e.g., glycocalyx precursor or substrate molecule) adapted to support and maintain a healthy endothelial glycocalyx (e.g., by increasing (natural) production thereof). Such glycocalyx building blocks, precursors, and/or substrate molecules can include, for example, glucosamine (e.g., glucosamine sulfate). Some embodiments can include one or more antioxidant (e.g., enzyme or compound) to help prevent damage to the glycocalyx. Such antioxidant can include, for example, superoxide dismutase (SOD) (e.g., extracellular SOD, SOD3, copper- or zinc- conjugated SOD, etc.), catalase (e.g., iron(III)- or iron(IV)-conjugated catalase), and/or one or more polyphenols). Some embodiments can include one or more glycocalyx mimetics, glycocalyx -binding compound, or glycocalyx-associating compounds for acute repair of damaged glycocalyx or sites of glycocalyx damage (e.g., degradation, alteration, thinning, etc.). Such glycocalyx mimetics, glycocalyx-binding compound, or glycocalyx-associating compounds can include, for example, hyaluronan (e.g., sodium hyaluronate) and/or fucoidan (e.g., fucoidan sulfate). The components of the compositions can act synergistically to improve vascular health by supporting a healthy endothelial glycocalyx, such that the combination of components can provide an effect on glycocalyx maintenance that is greater than taking any single component alone or the sum of reported effects of the individual components. In at least one embodiment, a composition for use in treating endothelial glycocalyx is provided.

Some embodiments of the present disclosure include one or more methods of

(synergistically) treating (e.g., supporting, maintaining, protecting, repairing, patching, and/or enhancing production of) endothelial glycocalyx, methods of improving vascular health, methods of restoring healthy endothelial glycocalyx, etc. Embodiments can also achieve a desired result (e.g., physiological reaction, biological response, etc.), such as increased endothelial glycocalyx density or thickness, reduced endothelial glycocalyx damage or degradation, improved vascular shear stress signaling, reduced blood pressure, increased blood nitric oxide levels and/or production, increased oxygen exchange, decreased endothelial permeability (e.g., to large plasma molecules, thereby decreasing leakage thereof into or across the endothelium), reduced accumulation of cholesterol in sub-endothelial space, increased size or volume of perfused boundary region, improved blood perfusion or distribution (e.g., to capillaries, organs, muscles, etc.), microvascular blood volume, and so forth.

Embodiments can include providing or administering to a subject or patient (e.g., mammal, human, etc.) an effective amount of a composition, or a composition in an amount effective, to (synergistically) treat endothelial glycocalyx, improve vascular health, restore healthy endothelial glycocalyx, etc. The composition can include an effective amount of one or more nutritional building blocks that can aid not only in the synthesis of new glycocalyx, but also in acute repair of damaged glycocalyx and/or protecting the glycocalyx against structural damage. The composition can also include an effective amount of one or more antioxidants that can not only help to prevent damage to the glycocalyx, but also aid in acute repair of damaged glycocalyx and/or synthesis of new glycocalyx. The composition can also include an effective amount of one or more glycocalyx mimetics that can not only induce acute repair of damaged glycocalyx, but also aid in the synthesis of new glycocalyx and/or help to prevent damage to the glycocalyx.

By repairing, protecting, and enhancing synthesis of endothelial glycocalyx, embodiments of the present disclosure can treat (e.g., bolster, maintain, support, etc.) the glycocalyx, (e.g., ensuring structural and functional integrity thereof). Such structural and functional integrity can be associated with overall vascular (e.g., microvascular) health and function, including proper regulation and/or modulation of perfused blood vessel density, blood pressure, vascular barrier properties, blood flow or perfusion into distal capillaries, muscles, organs, etc., inflammatory and/or coagulatory response, and so forth.

Illustrative components (and combinations thereof) useful in the formation, production, and/or manufacture of certain embodiments of the present disclosure, and illustrative methods (and steps thereof) involving the same, will be discussed in further detail below. For organizational purposes only, such components have been grouped into three categories based on their direct and/or primary function or mode of action in treating (e.g., supporting or maintaining) a (healthy) endothelial glycocalyx. Such modes of actions include (1) synthesizing (or producing new) endothelial glycocalyx (e.g., through supporting natural pathways and/or processes with glycocalyx precursors or building blocks), (2) repairing (or patching) damaged or perturbed glycocalyx (structural features) (e.g., with glycocalyx- mimetics), and (3) protecting (or defending) existing endothelial glycocalyx (e.g., with antioxidants).

Synthesizing Endothelial Glycocalyx - Glycocalyx Precursors Glucosamine

Glucosamine is an amino sugar and a precursor in the biochemical synthesis of glycosylated proteins and lipids. Although there is a substantial amount of data about the health benefits of glucosamine, its exact role in glycocalyx synthesis has not been previously recognized or documented. For example, it has not been reported or recognized that (soluble and/or exogenous) glucosamine (e.g., glucosamine sulfate) can be introduced (orally, intravenously, etc.) to stimulate synthesis of endothelial glycocalyx (through natural biochemical pathways), as contemplated by the present disclosure.

Indeed, glucosamine can increase synthesis of two main constituents of the endothelial glycocalyx, namely heparan sulfate and hyaluronan. Moreover, labeled glucosamine administered to cultured endothelial cells is incorporated in the glycocalyx. Thus, the addition and/or administration of glucosamine can support endothelial glycocalyx production by providing a (necessary and/or rate-limiting) component in the synthesis thereof. Accordingly, glucosamine can be or comprise a molecular precursor of endothelial glycocalyx.

One or more embodiments of the present disclosure can include glucosamine (e.g., in an amount effective to increase vascular production of endothelial glycocalyx). In some embodiments the glucosamine can be included (or provided) as glucosamine sulfate and/or other form of (D-)glucosamine. The glucosamine can be natural or synthetic. In a preferred embodiment, the glucosamine can be or comprise glucosamine (sulfate) extracted and/or purified from a (non-GMO) plant source, such as vegetable(s), (e.g., corn). In at least one embodiment, the glucosamine (sulfate) (extract) can have a purity greater than or equal to about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

The glucosamine can be included in compositions of the present disclosure in an amount of at least about 50 mg, 100 mg, 200 mg, 250 mg, 300 mg, 350 mg, 375 mg, 400 mg, 500 mg, 600 mg, 700 mg, 750 mg, 800 mg, 900 mg, 1000 mg, 1200 mg, 1500 mg, 2000 mg, 2500 mg, 3000 mg, or more. In a preferred embodiment, glucosamine can be provided in an amount sufficient to result in the administration of about 1500 mg of glucosamine (or glucosamine extract) per day. per day. Accordingly, certain embodiments (e.g., a daily dose) can include about 1500 mg of (corn) glucosamine (sulfate) (extract).

Repairing Endothelial Glycocalyx - Glycocalyx-mimetics

Hyaluronan

Hyaluronan, or hyaluronic acid, is a polysaccharide with a structure similar to heparan sulfate and heparin; that is, it includes repeats of the monosaccharides glucosamine and glucuronic acid. Unlike heparan sulfate, however, hyaluronan is a nonsulfated glycosaminoglycan (that lacks sulfation) and is not linked to a core protein. Endogenous hyaluronan also associates with the glycocalyx, becoming incorporated into the endothelial membrane by its synthesizing enzyme hyaluronan synthase ("HAS") or via binding to CD44 or other hyaluronan binding proteins. Although reports of some of the benefits of hyaluronan are known, especially in patients with fibromyalgia, it has not been previously recognized that hyaluronan is a part of glycocalyx repair. In particular, although hyaluronan is a building block of the glycocalyx, it has not been previously known or recognized that hyaluronan can be introduced to repair damaged glycocalyx (at sites of glycocalyx damage or perturbation), as contemplated by the present disclosure. Furthermore, due to its high molecular weight (e.g., reaching long lengths of up to several microns), it is also unclear to what degree (orally) administered (exogenous) hyaluronan can or does enter the circulatory system (e.g., by means of the lymphatic system) intact. For instance, without being bound to any theory, the half-life of hyaluronan in the circulatory system may be very short (e.g., 5 minutes or less). While existing theory and/or accepted wisdom attributes the short half-life of hyaluronan to enzymatic or other degradation (e.g., by oxidants, such as free radicals), the present disclosure reports the (direct) association of hyaluronan with and/or binding of hyaluronan to existing endothelial glycocalyx (e.g., resulting in the rapid decrease of free, circulating hyaluronan after administration.

Indeed, hyaluronan can be, act as, and/or provide a glycocalyx -mimetic (or patch) that associates with existing glycocalyx structures at sites of glycocalyx perturbation. Introduction and/or administration of (exogenous) hyaluronan causes thickening of endothelial glycocalyx at sites of damage and/or thinning of the glycocalyx. Thus, in addition to (naturally) produced hyaluronan, which is synthesized and incorporated into the endothelium, exogenous or administered hyaluronan can (directly) associate with existing glycocalyx and/or glycocalyx - sparse regions of the endothelium.

One or more embodiments of the present disclosure can include hyaluronan (e.g., in an amount effective to increase the density of endothelial glycocalyx). In some embodiments the hyaluronan can be included (or provided) as sodium hyaluronate. The hyaluronan can be natural or synthetic. In a preferred embodiment, the hyaluronan can be or comprise hyaluronan (sodium salt) extracted and/or purified from a (non-GMO) microbial (e.g., bacterial) source, such as Streptoccoccus, (e.g., Streptoccoccus equi subsp. Zooepidemicus). In at least one embodiment, the hyaluronan (sodium salt) (extract) can have a purity greater than or equal to about 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%.

The hyaluronan can be included in compositions of the present disclosure in an amount of at least about 5 mg, 10 mg, 12 mg, 15 mg, 17.5 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, or more. In a preferred embodiment, hyaluronan can be provided in an amount sufficient to result in the administration of about 70 mg of hyaluronan (or hyaluronan extract) per day. Accordingly, certain embodiments (e.g., a daily dose) can include about 425 mg of {Streptoccoccus (equi subsp. Zooepidemicus)) hyaluronan (or sodium hyaluronate) (extract).

Hyaluronan can also be found in a wide variety of polymerization states and/or molecular weight (MW) sizes. Embodiments of the present disclosure can include so-called high molecular weight (HMW) hyaluronan, having a MW of greater than about 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 1200 kDa, 1500 kDa, 1800 kDa, or more and/or less than about 3000 kDa, 3200 kDa, 3500 kDa, 3800 kDa, 4000 kDa, 4200 kDa, 4500 kDa, 4800 kDa, or 5000 kD. At least one embodiment can include hyaluronan having a MW of between about 100-5000 kDa, between about 500-4500 kDa, between about 1000-4000 kDa, between about 1200-3800 kDa, between about 1500- 3500 kDa, or between about 1800-3000 kDa.

Fucoidan

Fucoidans are sulfated fucosylated (polysaccharide) polymers that exhibit some heparin/heparan sulfate-like properties, which are components of the glycocalyx matrix structure. Fucoidans have been isolated and studied for various biological activities, but have not been shown or recognized as contributing to glycocalyx repair (at sites of glycocalyx damage or perturbation), as contemplated by the present disclosure. Thus, although some benefits of fucoidan are known, it has not been previously known or contemplated that fucoidan can be introduced (exogenously) to repair damaged glycocalyx. Moreover, similar to hyaluronan, it is also unclear to what degree (orally) administered (exogenous) fucoidan can or does enter the circulatory system intact.

Indeed, fucoidan can be, act as, and/or provide a glycocalyx mimetic (or patch) that helps repair and maintain the backbone of the glycocalyx by (directly) associating with existing glycocalyx and/or glycocalyx-sparse regions of the endothelium. Thus, in addition to (naturally) produced heparin/heparan sulfate, which are synthesized and incorporated into the endothelium, exogenous or administered fucoidan can (directly) associate with existing glycocalyx and/or glycocalyx-sparse regions thereof. Introduction and/or administration of (exogenous) fucoidan causes thickening of endothelial glycocalyx at sites of damage and/or thinning of the glycocalyx. Certain fucoidans can also have antioxidant properties. Accordingly, introduction and/or administration of fucoidan can provide site-directed antioxidant activity at the endothelial glycocalyx and/or regions of the glycocalyx being repaired. In some embodiments, fucoidan may inhibit breakdown of glycocalyx by inhibiting glycocalyx breakdown enzymes such as heparinase. One or more embodiments of the present disclosure can include fucoidan (e.g., in an amount effective to increase the density of endothelial glycocalyx). In some embodiments the fucoidan can be included (or provided) as fucoidan sulfate. The fucoidan can be natural or synthetic. In a preferred embodiment, the fucoidan can be or comprise fucoidan (sulfate) extracted and/or purified from a (non-GMO) plant, preferably (brown, green, or red) seaweed or algae, more preferably Laminaria, such as Laminaria japonica. In at least one embodiment, the fucoidan (sulfate) (extract) can have a purity greater than or equal to about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%. Other genus and/or species of seaweed, including but not limited to Monostroma, such as Monostroma nitidum, are also contemplated herein.

The fucoidan can be included in compositions of the present disclosure in an amount of at least about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 106.25 mg, 125 mg, 150 mg, 200 mg, 212.5 mg, 250 mg, 300 mg, 350 mg, 400 mg, 425 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1200 mg, or more. In a preferred embodiment, fucoidan can be provided in an amount sufficient to result in the administration of about 425 mg of fucoidan (or fucoidan extract) per day. Accordingly, certain embodiments (e.g., a daily dose) can include about 425 mg of (Laminaria japonica) fucoidan (sulfate) (extract).

Protecting Endothelial Glycocalyx - Antioxidants

Superoxide dismutase and catalase

Superoxide dismutase (SOD) (e.g., extracellular superoxide dismutase (ecSOD), or SOD3) and catalase are enzymes found in most living organisms exposed to oxygen, including animals, (green) plants, and most bacteria. SOD "dismutases" superoxide anions (0₂ ^") to oxygen (0₂), or hydrogen peroxide (H₂0₂), which can be further decomposed (e.g., partially reduced) to hydroxyl radical (HO^"), or (fully reduced) to water (H₂0) and 0₂ by catalase. Thus, SOD and catalase enzymes act as antioxidants capable of helping to prevent cellular and molecular damage caused by free radicals, such as reactive oxygen species / oxygen radicals, through "scavenging" the oxidants.

Glycocalyx can become damaged, perturbed, and/or destroyed by the action of oxidants, such as reactive oxygen species / oxygen radicals (e.g., 0₂ ^"). Although supplemental forms of SOD and catalase have been reported to provide health benefits, there are no reports of either SOD or catalase having a potential impact on glycocalyx protection or defense, as contemplated by the present disclosure. Thus, although some benefits of SOD and catalase are known, it has not been previously recognized or contemplated that SOD and/or catalase can be introduced orally to protect or defend endothelial glycocalyx against (oxidative) damage (by free radicals). Moreover, it is also unclear to what degree (orally) administered (exogenous) SOD and catalase can or does enter the circulatory system intact.

Indeed, SOD and catalase can help protect and maintain the backbone of the endothelial glycocalyx. Reducing the degradation of glycocalyx constituents caused by excessive local levels of oxygen radicals can be one benefit of providing the anti-oxidant enzymes SOD and catalase, which bind to the glycocalyx and/or lower local concentrations of oxygen species, which in turn reduce oxidative damage to the glycocalyx and, thereby, preserve endothelial function.

For instance, exogenous, labeled SOD administered (orally and/or intravenously) to mice co-localizes (binds (directly) to) the endothelial glycocalyx. By so doing, the local concentration of SOD at the endothelial glycocalyx is increased, providing added or enhanced (site-directed) protection against oxygen radicals at the endothelial glycocalyx. Thus, in addition to (naturally) produced SOD, which may be synthesized and bound to the endothelium, exogenous or administered SOD can (directly) associate with (bind to) endothelial glycocalyx. Accordingly, introduction and/or administration of SOD can provide site-directed antioxidant activity at the endothelial glycocalyx.

Moreover, (oral and/or intravenous) administration of catalase to mice increases catalase concentrations in blood vessels. Thus, in addition to (naturally) produced SOD, which may be produced and secreted into the blood stream, exogenous or administered catalase can be absorbed into the blood stream and be available for oxidant scavenging at or adjacent to the endothelial glycocalyx (and SOD bound thereto). Accordingly, introduction and/or administration of catalase can also provide antioxidant activity in blood vessels and/or at the endothelial glycocalyx.

One or more embodiments of the present disclosure can include SOD (e.g., in an amount effective to increase scavenging of oxidants, such as free radicals, particularly reactive oxygen species / oxygen radicals (e.g., 0₂ ^") at or adjacent to the endothelial glycocalyx). In some embodiments the SOD can be included (or provided) as ecSOD, or SOD3, preferably bound to copper or zinc and/or as copper- or zinc-conjugated SOD. The SOD can be natural or synthetic. In a preferred embodiment, the SOD can be or comprise (ec)SOD(3) extracted and/or purified from (or be included as an extract of or from) a (non-GMO) plant (part), preferably one or more fruit or vegetable plant parts, more preferably Momordica charantia (a.k.a. bitter melon) (fruit). Other plant parts, including, for example, olive (fruit), artichoke (leaf), white grape (fruit), and/or red grape (fruit), are also contemplated herein. One or more embodiments of the present disclosure can include catalase (e.g., in an amount effective to increase scavenging of oxidants, such as reactive oxygen species- / oxygen radical -precursors (e.g., H2O2), illustratively at or adjacent to the endothelial glycocalyx). In some embodiments the catalase can be included (or provided) bound to iron, such as iron(III) or iron(IV) and/or as iron(III)- or iron(IV)-conjugated catalase. The catalase can be natural or synthetic. In a preferred embodiment, the catalase can be extracted and/or purified from (or be included as an extract of or from) a (non-GMO) plant (part), preferably one or more fruit or vegetable plant parts, more preferably Momordica charantia (a.k.a. bitter melon) (fruit). Other plant parts, including, for example, olive (fruit), artichoke (leaf), white grape (fruit), and/or red grape (fruit), are also contemplated herein.

Antioxidants, such as SOD and/or catalase, can be included in compositions of the present disclosure (e.g., as Momordica charantia (a.k.a. bitter melon) (fruit) extract) in an amount of at least about 5 mg, 7.5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 100 mg, 150 mg, 200 mg, or more. In a preferred embodiment, SOD and/or catalase can be provided in an amount sufficient to result in the administration of about 30 mg of SOD and/or catalase (or SOD and/or catalase extract) per day. Accordingly, certain embodiments (e.g., a daily dose) can include about 30 mg of ((bitter) melon (fruit)) antioxidant (e.g., SOD and/or catalase) (extract).

Other naturally occurring antioxidants - polyphenols

Antioxidants are potent scavengers of free radicals and other oxidants and serve as inhibitors of neoplastic processes and other cellular and/or molecular degrading, damaging, or destroying activities thereof. Naturally occurring antioxidants, widely available in fruits, vegetables, nuts, flowers and bark, possess a broad spectrum of biological and therapeutic properties against free radicals and oxidative stress. It has not, however, been shown that particular antioxidant compounds, such as polyphenols, can lower the concentration of oxygen radicals or other oxidants at or adjacent to the endothelial glycocalyx and/or and reduce oxidative damage thereto, as contemplated by the present disclosure. Thus, although some benefits of antioxidant compounds, such as polyphenols, are known, it has not been previously known or contemplated that such antioxidants can be introduced (exogenously) to protect and defend endothelial glycocalyx. Moreover, it is also unclear to what degree (orally) administered (exogenous) antioxidants, such as polyphenols, can or do enter the circulatory system intact.

Indeed, polyphenols can help protect and maintain the backbone of the endothelial glycocalyx by reducing the prevalence of oxidants in the blood stream. For instance, polyphenols extracted from olive, artichoke, and (white and/or red) grapes extracts can lower the concentration of oxygen radicals (e.g., in blood vessels) and thereby reduce damage of the glycocalyx.

In a preferred embodiment, antioxidants, such as polyphenols, can be extracted and/or purified from (or be included as an extract of or from) (non-GMO) plant(s), preferably (fruits and vegetables, including olive (fruit), artichoke (leaf), and white grape (fruit), and/or red grape (fruit). Antioxidants, such as polyphenols, can be included in compositions of the present disclosure in an amount of at least about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 120 mg, 150 mg, 200 mg, 240 mg, 300 mg, 350 mg, 400 mg, 450 mg, 480 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, or more. In a preferred embodiment, polyphenols (e.g., derived and/or extracted from olive (fruit), artichoke (leaf), and white grape (fruit), and/or red grape (fruit)) can be provided in an amount sufficient to result in the administration of about 450 mg of (olive (fruit), artichoke (leaf), and white grape (fruit), and/or red grape (fruit)) polyphenol (extract) per day. Accordingly, certain embodiments (e.g., a daily dose) can include about 450 mg of (olive (fruit), artichoke (leaf), and white grape (fruit), and/or red grape (fruit)) polyphenol (extract).

In certain embodiments, (a mixture of (enzymatic and/or molecular)) antioxidants can be obtained as a (non-GMO) plant or plant-based extract. For instance, one or more fruit(s), vegetable(s), and/or other plant(s) or plant part(s) can be processed so as to extract, isolate, purify, and/or concentrate antioxidant(s), such as SOD, catalase, and/or polyphenol(s) therefrom. Extract(s) from a plurality of such plants or plant parts can be mixed together and/or included as an antioxidant component of (compositions of) the present disclosure. In at least one embodiment, for instance, the antioxidant mixture can comprise extracts, isolates, and/or concentrates of or from olive (fruit), artichoke (leaf), and white grape (fruit), red grape (fruit), and/or melon (fruit).

A mixture of (enzymatic and/or molecular) antioxidants, including, for example, SOD, catalase, and/or polyphenol(s) can be included in compositions of the present disclosure in an amount of at least about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 120 mg, 150 mg, 200 mg, 240 mg, 300 mg, 350 mg, 400 mg, 450 mg, 480 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, or more. In a preferred embodiment, the antioxidant mixture can be provided in an amount sufficient to result in the administration of about 480 mg of the antioxidant (extract) mixture per day. Accordingly, certain embodiments (e.g., a daily dose) can include about 480 mg of (olive (fruit), artichoke (leaf), and white grape (fruit), red grape (fruit), and/or (bitter) melon (fruit)) polyphenol (extract). Excipients and non-active components

Various components (e.g., therapeutic agents or (active) ingredients) of the composition (as described above) may be combined with one or more excipients, such as microcrystalline cellulose and/or silicon dioxide, and then encapsulated using techniques known in the art. For example, the therapeutic agents may be combined with the excipients and dry blended. The resulting dry blend may then be encapsulated by hand or by machine. The microcrystalline cellulose and silicon dioxide act as excipients to allow the dry blend to flow smoothly into the capsules. Other known excipients may be added or substituted as necessary as determined by those having skill in the art. In addition, such excipients may be added in varying amounts without necessarily departing from the scope of this disclosure. The capsules may be any known in the art such as softgels, gelatin, and/or vegetarian capsules. The composition can also be provided as a tablet, pill, powder, dry blend, tincture, solution, suspension, (flavored or unflavored) drinks or drink mixes, aerosols, or other suitable form of matter.

In at least one embodiment, components of the present disclosure (e.g., therapeutic agents or (active) ingredients of the composition) can be coated and/or encapsulated to protect the therapeutic agents or (active) ingredients from degradation in the acidic conditions of the mammalian gut. For instance, in some embodiments, a protective coating can be formed around a dry blend of the components and the coated dry blended components optionally encapsulated (in a protective capsule) for oral administration. Alternatively, the coated dry blended components can be provide for and/or prepared as an oral slurry, solution, and/or suspension, such as a drink or tincture.

EXAMPLE 1

An illustrative embodiment of (an encapsulated) composition of the present disclosure is provided in the table below:

Composition of a 750.75 mg capsule

Ingredient Amount (mg/capsule)

Fucoidan (85%) 106.25 mg

Antioxidants (SOD, catalase, polyphenols) 120.00 mg

Glucosamine Sulfate 375.00 mg

Hyaluronic Acid (1800-3000 kDa) 17.50 mg

Microcrystalline Cellulose 130.00 mg

Silicon Dioxide 2.00 mg Total mg per capsule 750.75 mg

The composition provided in Example 1 can be obtained commercially from Microvascular Health Solutions, L.L.C., a Delaware limited-liability company, under the trademark E DOCALYX™.

Embodiments of the present disclosure (e.g., one or more capsules having a composition of components according to Example 1) can be administered one or more times per day, and preferably up to four times per day. For example, four capsules can be administered once per day, two capsules can be administered two times per day, or one capsule can be administered four times per day. Alternatively, double-dose capsules comprising twice the above amounts can be provided. Such double-dose capsules can be provided, for example, as two capsules that can be administered one time per day or one capsule that can be administered two times per day. Other dosages, regimen, treatment (schedules), and/or formulations are also contemplated herein.

A preferred embodiment of the present disclosure comprises a daily dosage form of a composition comprising up to, at least, and/or about 1500 mg of glucosamine (e.g., glucosamine sulfate), 480 mg of a mixture of antioxidants (e.g., superoxide dismutase, catalase, and/or polyphenols), 425 mg of fucoidan (e.g., fucoidan sulfate), and 70 mg of hyaluronan (e.g., sodium hyaluronate). Such a daily dosage form can comprise a single dose, two one-half doses, three one-third doses, four one-fourth doses, and so forth. In addition, the dosage form can be provided as one, two, three, four, or more capsules, tablets, pills, or other dosage forms, such as a volume of liquid dose (e.g., drink), weight of a powdered or other dry dose (e.g., powdered drink mix), etc.

The embodiment described in Example 1 can represent a one-fourth daily dosage for a human or other (large) mammal. It will be appreciated, that dosage amounts of one or more (e.g., each) of the components (e.g., active ingredients and/or therapeutic agents) can be adjusted and/or modified, preferably while maintaining a similar ratio between such components. Accordingly, in at least one embodiment, a mammalian dosage form of the present disclosure can comprise a composition having a ratio of components according to the formula 375: 120: 106.25: 17.50 (glucosamine (e.g., glucosamine sulfate): mixture of antioxidants (e.g., superoxide dismutase, catalase, and/or polyphenols):fucoidan (e.g., fucoidan sulfate): hyaluronan (e.g., sodium hyaluronate)), by weight, volume, or molar. Other ratios, for example, alternative ratios of up to about +/- 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%), 45%), or 50%> of one or more of the foregoing components (or ratios thereof) are also contemplated herein.

Synergistic effects of active ingredients

As indicated herein, embodiments (e.g., compositions and/or methods) of the present disclosure can improve (micro)vascular health, not only through direct modes of action (as described above), but by exhibiting beneficial synergistic effects on endothelial glycocalyx (e.g., over the benefits of taking any single component (of the composition) alone or the sum of reported effects of the individual components. Complimentary or synergistic benefits of such components (or compositions (e.g., nutritional supplements) including the same) on the endothelial glycocalyx (and overall vascular health) include further enhancing the repair of glycocalyx damage, further stimulating synthesis of new glycocalyx (e.g., by enhancing production of glycocalyx precursors or building blocks), and/or further protecting glycocalyx (and constituents / components thereof) from (oxidative and/or other forms of degradation (e.g., enzymatic)).

Figure 24 is an illustrative synergistic flowchart illustrating one or more complimentary, synergistic, and/or overlapping modes of activity, leading to further enhanced or improved health of endothelial glycocalyx. As depicted in Figure 24, a healthy and/or improved glycocalyx (GX) 1 can be achieved by three general, separate, distinct, overlapping, intersecting, and/or interrelated modes of action and/or activity, indicated as: A. repairing damaged GX; B. protecting against damage to GX; and C. synthesizing new GX.

As described herein, the addition and/or administration of one or more glycocalyx- mimetics 10, such as hyaluronan (HA) and/or fucoidan(s) (FS), can result in the (direct) incorporation 12 of such compounds (polysaccharides and/or glycosaminoglycans) at sites of GX damage and/or perturbation. Incorporation 12 can, therefore, repair (or patch) sites where the GX has been damaged, degraded, and/or destroyed, leading to healthy and/or improved GX 1. One consequence of healthy and/or improved GX 1 (e.g., from incorporation 12) can be an increase in the ability of the endothelium to function in shear stress (SS) signaling pathways 14. In particular, a healthy GX is associated with proper levels and/or modulation of SS signaling.

Proper and/or increased SS signaling 14 can result in a corresponding increase in the expression and/or activity of hyaluronan synthase (HAS) 18 and/or one or more (other) SS sensitive genes 16. The addition and/or administration of one or more GX precursors and/or building blocks 20, such as glucosamine, can feed one or more of activities 16 and/or 18, resulting, respectively, in an increase in the production of one or more GX polysaccharides 22, such as heparin sulfates, and/or increase production of one or more (nonsulfated) glycosaminoglycans 24, hyaluronic acid (HA). Increased production 22 and/or 24 can result in, correspond with, and/or be accompanied by incorporation 12 of such polysaccharides and/or glycosaminoglycans as new endothelial GX, further and/or synergistically improving, enhancing, and/or supporting healthy GX 1.

Proper and/or increased SS signaling 14 can also result in increased nitric oxide production 26. Increased nitric oxide production 26 can lead to decreased expression of one or more GX-degrading enzymes 28, such as heparinase, which can enzymatically digested and/or degrade heparin and/or heparin-like compounds (heparinoids), such as those described herein. Such degradation can cause (substantial and/or significant) damage to endothelial GX. Accordingly, decreased expression of GX degrading enzymes 28, such as heparinase, can cause and/or result in an (overall) decrease in endothelial GX damage 30, further and/or synergistically improving, enhancing, and/or supporting healthy GX 1.

Increased nitric oxide production 26 can also lead to improved and/or increased oxygen radical scavenging 32, which can further decrease endothelial GX damage 30, resulting in further and/or synergistically improved, enhanced, and/or healthy GX 1. Healthy and/or improved GX 1 also provides an increase in antioxidant (e.g., enzyme), such as superoxide dismutase (SOD), specifically, extracellular superoxide dismutase (ecSOD), or SOD3, binding sites 34. The addition and/or administration of one or more antioxidants 38, such as SOD, catalase, and/or polyphenols, can result in a corresponding increase in SOD at the GX 36, a corresponding increase in catalase at the GX 40 and/or a corresponding increase in polyphenols at the GX 42. One or more of increased antioxidants 36, 40, 42, can also result in improved and/or increased oxygen radical scavenging 32, which can further decrease endothelial GX damage 30, resulting in further and/or synergistically improved, enhanced, and/or healthy GX 1.

The increase in SOD at the GX 36 from the addition and/or administration of SOD at 38, in particular, coupled with the increase in antioxidant-binding sites 34 (e.g., for enzymes, such as SOD) provided by healthy and/or improved GX resulting from the addition and/or administration of one or more glycocalyx-mimetics 10, such as hyaluronan (HA) and/or fucoidan(s) (FS) and/or one or more GX precursors and/or building blocks 20, such as glucosamine, as described above, can enhance and/or increase oxygen radical scavenging 32, which can further decrease endothelial GX damage 30, resulting in further and/or synergistically improved, enhanced, and/or healthy GX 1. This healthy and/or improved GX 1 further enhances SS signaling 14, thereby enhancing nitric oxide production 26 and expression of relevant genes 16 and downstream products 18 and, coupled with the addition and/or administration of glucosamine 20 results in further production of GX constituents 22 and 24, which incorporate as GX 12, even further and/or synergistically improving, enhancing, and/or supporting healthy GX 1, which further protects against damage thereto, as described above.

Accordingly, embodiments of the present disclosure, including compositions comprising one or more components described herein, can produce synergistic (beneficial) outcomes, effects, and/or results on the structural and/or functional state and/or health of endothelial glycocalyx. Such components, while individually and/or directly impacting the health of endothelial glycocalyx through one of (A.) repairing damaged glycocalyx, (B.) protecting against damage to glycocalyx, or (C.) synthesizing new glycocalyx, can also synergistically enhance the health of endothelial glycocalyx by feeding, enhancing, or otherwise affecting one or more additional pathways and/or modes of action.

Specifically, inducing and/or effectuating repair (or patching) of damaged glycocalyx (e.g., through the addition and/or administration of one or more glycocalyx-mimetics, such as hyaluronan (HA) and/or fucoidan(s) (FS)), can also protect and/or defend against damage, degradation, and/or perturbation to the glycocalyx and/or increase (natural) synthesis of new glycocalyx. Similarly, protecting and/or defending against damage, degradation, and/or perturbation to the glycocalyx (e.g., through the addition and/or administration of one or more antioxidants, such as superoxide dismutase, catalase, and/or one or more polyphenols), can also enhance repair (or patching) of damaged glycocalyx and/or increase (natural) synthesis of new glycocalyx. Likewise, increasing (natural) synthesis of new glycocalyx (e.g., through the addition and/or administration of one or more glycocalyx precursors and/or building blocks, such as glucosamine), can also protect and/or defend against damage, degradation, and/or perturbation to the glycocalyx and/or enhance repair (or patching) of damaged glycocalyx.

Based on the foregoing description, the combined effect of certain, individual therapeutic agents or ingredients can be greater than the literature reported effects of each therapeutic agent or ingredient alone on glycocalyx repair, synthesis, and/or protection. Thus, although available literature shows an impact of the foregoing individual therapeutic agents on vascular and/or overall health, there is no report or suggestion of any one of them demonstrating an impact as an (exogenous and/or oral) supplement having an effect on glycocalyx repair and/or synthesis, let alone a synergistic effect.

Methods A preferred method of administration comprises administering (to a patient or subject (e.g., a mammal, such as a human), in need thereof) an effective amount of one or more compositions described herein (e.g., in an acceptable pharmaceutical dose and/or dosage form, such as one, two, three, four, or more capsules, tablets, pills, etc., or a corresponding amount (e.g., by weight, volume, or molar) of powder, gel, granules, solution, suspension, tincture, etc.). Preferably, the compositions are administered orally, but the compositions may also be formulated for sublingual, rectal, vaginal, intravenous, subcutaneous, intramuscular, and transdermal administration as well. Thus, the compositions may be administered by various methods of delivery, such as but not limited to, conventional oral dosage forms, prepared drinks, (flavored or unflavored) drink mixes, aerosols, and intravenous drips. The compositions may be made using conventional techniques, such as by mixing the active agents with suitable excipients, such as but not limited to, binders, fillers, preservatives, disintegrators, flow regulators, plasticizers, dispersants, emulsifiers, etc. Suitable food-grade additives, such as but not limited to, sweeteners, colors, and flavoring agents may also be incorporated to encourage consumption.

Embodiments of the present disclosure are not limited to administration to humans, and may be administered to any animal, but are preferably administered to mammals. For example, one use aspect of the present disclosure comprises the administration of the compositions of the present disclosure to humans. While another use aspect of the present disclosure comprises the administration of the compositions of the present disclosure to mammals in the form of a veterinary composition that may be administered to bovines, equines, ovines, caprines, canines, felines, and other domestic animal species.

Thus, an embodiment of the present disclosure comprises administering a composition to a mammal comprising a (therapeutically-) effective amount of one or more of the components (e.g., active ingredients and/or therapeutic agents) described herein. For example, an oral dosage form of the compositions may be administered one or more times a day to achieve the desired physiological reaction or biological response.

As described in greater detail herein, each therapeutic agent is independently helpful for supporting and maintaining a healthy endothelial glycocalyx, thereby preserving or improving vascular health. For example, components such as glucosamine can be limiting reagents (precursor and/or building blocks) in a pathway for the synthesis of two main constituents of the endothelial glycocalyx - heparan sulfate and hyaluronan. Accordingly, the addition of available components such as glucosamine can enhance and support production of new glycocalyx, and can also support activities that protect and/or defend the glycocalyx against structural and/or functional damage, degradation, destruction, perturbation, etc. and/or repair (e.g., patch) endothelial glycocalyx at sites of glycocalyx damage, perturbation, etc.

Components such as hyaluronan and fucoidan can be and/or function as glycocalyx- mimetics that can repair (e.g., patch) endothelial glycocalyx at sites of glycocalyx damage, perturbation, etc. Accordingly, the addition of available components such as hyaluronan and fucoidan can repair damaged glycocalyx, and can also support activities that protect and/or defend the glycocalyx against structural and/or functional damage, degradation, destruction, perturbation, etc. and/or synthesize and incorporate new glycocalyx at the endothelium.

Component such as superoxide dismutase, catalase, and/or polyphenols can protect and/or defend endothelial glycocalyx against damage and/or degradation caused by enzymes and/or oxidants. Accordingly, the addition of components such as superoxide dismutase, catalase, and/or polyphenols can protect and/or defend endothelial glycocalyx against damage and/or degradation, and can also support activities that synthesize and incorporate new glycocalyx at the endothelium and/or repair (e.g., patch) endothelial glycocalyx at sites of glycocalyx damage, perturbation, etc.

Such components can be combined in any suitable manner or fashion, including various amounts (by weight, volume, or molar) and/or dosage forms to produce one or more compositions of the present disclosure. The compositions disclosed herein can be made into any dosage form and administered to a mammal, and more preferably to a human being. Preferably, an effective amount of one or more of the active agents can be mixed with appropriate excipients into the compositions disclosed herein and administered orally as an acceptable pharmaceutical dosage form, such as a capsule or tablet, though any suitable dosage form can be used in some embodiments. An oral dosage form of the compositions may be administered one or more times a day to achieve the desired physiological reaction or biological response. Optionally, the amount of each therapeutic agent or ingredient may be adjusted in each individual dosage form, and taken as needed to maintain the desired level of effectiveness.

Vascular health, particularly health of the endothelial glycocalyx, can be assessed via suitable detection or modulation of the endothelial glycocalyx. Methods of such detection and suitable biosensor devices are described in U.S. Patent No. 8,759,095, the entirety of which is incorporated by reference herein. One suitable method of detection includes the use of the GLYCOCHECK® Microvascular Health Monitor available from Microvascular Health Solutions, which is a complete imaging solution for screening a subject's or patient's perfused boundary region ("PBR") by accurately measuring and monitoring changes in the PBR in real time. The PBR in microvessels is the cell-poor layer which results from phase separation between the flowing red blood cells ("RBC") and plasma, and represents the most luminal part of the endothelial glycocalyx that allows cell penetration. Loss of endothelial glycocalyx integrity allows for deeper penetration by the outer edge of the RBC-perfused lumen, thereby increasing PBR, resulting in increased vulnerability of the endothelium.

PBR is thus a measure for the depth of penetration of red blood cells in the glycocalyx (or into the region where healthy glycocalyx should be found). Low values of PBR indicate a mechanically stable glycocalyx that protects the vessel wall against damage by circulating blood cells and other constituents, molecules or reagents circulating in the blood. The PBR is the main readout parameter calculated by the GLYCOCHECK® software. Calculation of further qualitative and/or quantitative (e.g., scores or numeric) measurements or representations can be performed manually or automatically (e.g., by the GLYCOCHECK® software). One such parameter - the Micro- Vascular Health Score (MVHS) - is discussed in further detail below.

The measurement may be performed non-invasively with a digital camera placed under the patient's tongue, under-arm area, vagina, rectum, or other (highly) vascular area. It is noted that such measurements, while providing a local read of blood vessel structural features is highly indicative of an overall and/or systemic vascular landscape. For instance, measurements taken from one of the aforementioned locations can be confirmed (as accurate and representative of systemic vascular landscape) by measurement at other locations.

Other measurable indicators include (blood) volume, width and dimension of the glycocalyx, vessel density, the number of perfused vessels per tissue surface versus total number of vessels, RBC filling percentage, capillary volume reserve, enzyme activity, and presence or absence of glycocalyx contributing constituents, nitric oxide concentration in the blood, etc. Changes in any of the foregoing parameters, alone or in combination, are useful indicators for assessing vascular health.

Glycocalyx health may also be assessed via biological samples. For example, the status, volume or dimension of the glycocalyx and/or the activity of one or more enzymes of glycocalyx metabolism, may be performed via in vitro assays on a biological sample removed from a subject. Such in vitro assays are generally easy to perform and amenable to high- throughput analysis via techniques known in the art. Suitable samples include, but are not limited to, samples of whole blood, plasma or serum obtained from a subject. In vivo, such fluids directly contact the vascular endothelial tissue and are responsive to glycocalyx perturbation. Some glycocalyx indicators, for example glycocalyx associated lectin-like proteins, may also be detectable in urine.

Thus, profiles of lectin-like proteins that normally associate with the glycocalyx, as determined in the foregoing samples can provide suitable information about glycocalyx volume or dimension and/or molecular accessibility. Glycocalyx perturbation may also be diagnosed by detecting the presence and/or concentrations of glycocalyx derived molecules, such as but not limited to: oligo- or poly-saccharides, glycosaminoglycans, hyaluronan, heparan sulfate or proteoglycans; enzymes that catalyze glycocalyx anabolism or catabolism, such as hyaluronidase and/or heparinase; and/or endogenous or exogenous substances that can become incorporated or otherwise associated with the glycocalyx.

More invasive techniques for assessing vascular health include invasive microscopic visualization techniques which comprise the injection of fluorescent labels attached to glycocalyx -bound proteins or glycocalyx permeating tracer molecules, and are contemplated herein.

As described above, the GLYCOCHECK® system, or other suitable computer system, can be used to measure PBR in subjects and/or patients. A PBR score can be calculated therefrom, providing an indication of the structural stability of the endothelial glycocalyx. In addition, (the in vivo camera of) the system can be used to measure capillary / blood vessel density in subjects and/or patients, providing an indication of overall number of capillary blood vessels that are visible (and red blood cell-perfused) in the captured region. A capillary red blood cell filling percentage can also be measured by the system in subjects and/or patients, providing an indication of the amount of red blood cells per blood vessel. Taken together, these indicators can be used to calculate a microvascular health score (MVHS) of the subjects and/or patients (e.g., population), which is directly proportional to blood vessel density, directly proportional to the capillary red blood cell filling percentage, and inversely proportional to PBR score.

The following results were obtained in a population of self-proclaimed "healthy" humans (with no known disease state or underlying health concerns) using the system. A pretreatment baseline (BL) measurement was taken for each of the following vascular / glycocalyx health indicators: PBR; blood vessel density; and red blood cell filling percentage, and/or score(s) or measurements calculated therefrom. MVHS was also calculated from the foregoing indicators. The population averages of each indicator were normalized to 100% and plotted in Figure 25 as a pretreatment baseline measurement of vascular / glycocalyx health (BL). The population was then orally administered a daily (4x) oral dosage of the composition described in Example 1 over a treatment course of 4 months, with the foregoing indicators measured and/or calculated after one, two, three, and four months, respectively. The (4x) dosage included about 1500 mg of glucosamine sulfate per day, about 425 mg of fucoidan sulfate per day, about 70 mg of sodium hyaluronate per day, and about 480 mg of a mixture of antioxidants (superoxide dismutase, catalase, and polyphenols), per day.

As illustrated in Figure 25, over the course of treatment, the population (on average) demonstrated an overall decrease in the PBR score (e.g., reflecting a more stable glycocalyx), an overall increase in the RBC filling percentage, indicating a per capita increase in the amount of red blood cells per blood vessel, and a significant increase in the number of capillaries visible and red blood cell-perfused. This blood vessel density increase can be seen as early as one month into the course of treatment and continues to improve month after month.

Accordingly, compositions of the present disclosure can substantially improve blood vessel density (i.e., the number of capillaries (e.g., perfused with red blood cells)), can increase the red blood cell filling percentage of such blood vessels, and can improve the stability of endothelial glycocalyx (as evidenced by a decrease in perfused boundary region). The MVHS based on these measurements and/or calculations improved substantially (e.g., by about 50%) over the course of treatment, beginning at the first month (e.g., about 5-10% improvement), to the second month (e.g., about 10-15%) improvement), to the third month (e.g., about 30%> improvement), and so forth. Accordingly, embodiments of the present disclosure can produce a substantial and/or significant (e.g., between about 5% -50%) improvement in microvascular health (over a one-month, two-month, three-month, and/or four-month treatment (comprising daily doses of the composition described in Example 1)).

These results indicate a surprising and unexpected improvement in the overall (micro)vascular health and specific indications described above following treatment with composition according to embodiments of the present disclosure.

It will also be appreciated that, based on the results presented in Figure 25, further improvement in microvascular health is also contemplated herein. Indeed, because the microvascular health indicators have not plateaued by the fourth month of treatment, one of ordinary skill in the art would expect the level of such health indicators to continue improving over an additional course of treatment (e.g., five-months, six-months, seven- months, eight-months, nine-months, ten-months, eleven-months, twelve-months, or more).

In addition to the foregoing, patients or subjects treated with compositions of the present disclosure show a decrease in blood pressure and/or an increase in blood (plasma) nitric oxide levels, consistent with the proposed role of endothelial (microvascular)) glycocalyx and/or the flow chart depicted in Figure 24.

Without being bound to any theory, new medical science has revealed the importance of the glycocalyx; a transparent, microthin, gel-like layer lining your blood vessels that protects your entire circulatory system. The glycocalyx maintains a healthy capillary network— comprising 99% of your circulatory system— that nourishes your organs. Aging, poor diet, lack of exercise, genetics, stress, smoking— and even conditions such as diabetes and high blood pressure can cause the glycocalyx to become compromised. Organs starve. Health declines. You may look— and even feel— healthy on the outside, but inside your microvascular system, a completely different situation could be developing. Organ starvation is one reason that diseases begin in the body. When the capillaries begin to lose their function, vital organs don't receive the nutrients needed to be healthy... the silent spiral of health decline begins. For example,

1. Over time, aging, poor diet, lack of exercise, genetics, stress, smoking— and even conditions such as diabetes and high blood pressure— combined with other risk factors, degrade the gel-like lining of the blood vessels.

2. Damaged microvessels (capillaries) become leaky, lose function and the number of capillaries decreases. Early warning signs begin.

3. Vital delivery of nutrients, hormones, and oxygen and removal of waste and carbon dioxide is compromised.

4. Organ starvation begins, weakening vital processes in the heart, kidneys, lungs and brain.

5. Diseases can set in, including:

Heart & Kidney Disease

Lung Disease

Stroke & Dementia

Septic Shock

Inflammatory Disorders

Cancer Metastasis

6. Complications develop throughout the body.

7. Critical organs fail.

8. Death.

Your organs are healthy when they are nourished with vital nutrients and oxygen, while waste and carbon dioxide are removed. Every heartbeat is an opportunity for replenishment. This replenishment— an exchange of nutrients and waste removal— takes place in the capillaries of the microvascular system. For instance, with every beat, the heart pumps blood through the microvascular system, which nourishes the trillions of cells that make up your organs. Deterioration in the microvascular system— comprising more than 99% of the contact area between the blood and the organs— has long been overlooked as the starting point of several health conditions, which lead to life-threatening diseases. Only recently, have we been able to look at the 99%. In the past, science has focused on the visible 1% of the total vascular system— the larger blood vessels. Without a continuous delivery of nutrients and removal of waste, organs starve and you are at greater risk of heart disease, stroke, high blood pressure, diabetes, kidney disease, dementia, inflammatory disorders and cancer metastasis.

Several Diseases and Conditions are Linked to Vascular Health. For example:

Diabetes (high blood glucose level). Vascular links:

A healthy microvascular system is important for transport of glucose from blood to organs.

High blood glucose damages (micro) vascular system and causes blindness, kidney failure, heart attack and stroke.

Hypertension (blood pressure is higher than accepted level). Vascular links:

Hypertension is associated with loss of microvascular density.

Hypertension increases cardiovascular risk (heart attack, stroke, kidney failure).

Heart Disease (loss of pump function of heart). Vascular links:

Loss of microvascular density causes heart attack.

Insufficient number of capillaries per heart muscle fiber impairs heart pump function (heart failure).

Kidney Disease (impaired production of urine causing increased blood volume and hypertension). Vascular link:

Damage of microvessels causes leakage of blood proteins into urinary space, damage of renal filtration units and kidney failure.

Stroke (blood clot in brain artery causing brain damage). Vascular links:

Damage to vascular wall causes blood clots.

Microvascular damage causes White Matter Lesions with poor neurological prognosis.

Dementia (early cognition impairment: neurological complication). Vascular link:

Healthy microvascular system is essential to maintain intact neuro-vascular unit and support normal neurological function. Septic Shock (loss of circulation blood volume causes drop in blood pressure, impaired organ blood flow resulting in acute kidney failure, reduced lung function, heart attack, stroke and brain damage). Vascular links:

Leaky microvessels result in loss of blood plasma volume to tissue space.

Damaged vascular wall causes increased blood clotting and inflammation.

Inflammatory Disorders (rheumatoid arthritis, vasculitis, allergies, glomerulonephritis, autoimmune diseases, scleroderma and atherosclerosis). Vascular links:

Attack of microvascular system by inflammatory cells results in loss of capillaries. Increased capillary permeability causes tissue edema.

Cancer (uncontrolled growth of tumors). Vascular link:

Leaky microvessels allow tumor cells to enter the vascular system and redistribute to different parts of our body, causing tumor metastasis (secondary tumors).

Research into multiple diseases reveals how organ starvation is related to the importance of a robust microvascular system. Embodiments of the present disclosure allows researchers and scientists to study and better understand the critical role of the capillaries. In the past, blood vessels were thought to be hollow tubes. With today's high resolution video microscopes, a discovery reveals that the microvascular system is coated with a transparent, gel-like lining. This lining protects the inside walls of the capillaries and enables the transfer of nutrients and waste removal from vital organs. This gel-like lining of the capillaries and all other blood vessels is called the glycocalyx. Its integrity is essential to the healthy function of all cells, organs and body systems.

When this glycocalyx lining is healthy, so are the blood vessels, which are essential for good health and vitality. Over time, the glycocalyx wears down and develops gaps or holes. This happens because of aging, poor diet, lack of exercise, genetics, stress, smoking— and even conditions such as diabetes and high blood pressure. Nutrient delivery and waste removal falter. Organs suffer and starve. Non-invasive test (in a healthcare provider's office), which records videos from under your tongue, calculates your on-the-spot systemic Microvascular Health Score.

Fortunately, you can reverse the effects of aging, lack of exercise, offset genetics, stress and lifestyles, and restore the vitality of your organs. Compositions of the present disclosure, and their methods of use, for example, can promote healthy organs associated with vascular health, including the heart, brain, kidneys, lung, muscle, skin and eyes.

Some embodiments can include a method of treating a disease or condition (e.g., in a human or non-human mammal). In some embodiments, the disease or condition can be associated with impaired vascular health or function. Some embodiments can include, for example, administering (a therapeutically-effective amount of) a composition (to a mammal). The composition can include or comprise, for example, one or more molecular precursor of endothelial glycocalyx, one or more antioxidant that associates with endothelial glycocalyx; and/or one or more glycocalyx mimetic. In some embodiments, administering can include one or more oral administrations. In some embodiments, administering can include one or more sublingual, intravenous, subcutaneous, intramuscular, and/or transdermal administrations.

In some embodiments, the disease or condition (associated with impaired vascular health or function) can be, include, or be selected from the group consisting of, for example, type-2 diabetes, hypertension or high blood pressure, heart disease, kidney disease, liver disease, stroke, myocardial infarction or heart attack, sepsis or septic shock, cancer or metastatic cancer, inflammation, inflammatory disorders, erectile dysfunction, head aches or migraine headaches, severe premenstrual syndrome, memory loss, hearing loss, loss of mental focus, fatigue or chronic fatigue syndrome, thinning hair or balding, leg cramps, cold hands, cold feet, and eczema or skin conditions. In particular, similar to the VCI patients described above, dysfunction or inefficiency in increasing capillary density in response to increased blood flow is found in patients with type-2 diabetes, hypertension or high blood pressure, heart disease, kidney disease, liver disease, stroke, myocardial infarction or heart attack, sepsis or septic shock, cancer or metastatic cancer, inflammation, inflammatory disorders, erectile dysfunction, head aches or migraine headaches, severe premenstrual syndrome, memory loss, hearing loss, loss of mental focus, fatigue or chronic fatigue syndrome, thinning hair or balding, leg cramps, cold hands, cold feet, and eczema or skin conditions. Glycocalyx impairment may be a (primary) cause of these patients exhibiting the inability or reduced / impaired ability to recruit or increase capillary density, whether in number and/or size) in response to increased blood flow.

By way of example, impaired flow dependent recruitment of capillary density in type 2 diabetes patients (compared with healthy controls) is illustrated in Figures 26A-27B. As illustrated in Figures 26A and 26B, capillary density increases with increasing blood flow in healthy young control subjects. On the contrary, Figures 27A and 27B illustrate that capillary density does not significantly increase with increasing blood flow in type 2 diabetes patients. These results, and the corresponding study parameters, are similar to those described (above) for VCI patients and for low blood flow and high blood flow changes in feed vessel (>=10 microns in diameter) red blood cell velocity and capillary density (expressed as the ratio of red cell velocity in feed vessels / capillary vessels = V>=10 / V4-7).

In the healthy controls (Figures 26 A and 26B):

Low flow RBC velocity in feed vessels (V>=10) = 0.93 pixels/frame

High flow RBC velocity in feed vessels (V>=10) = 2.59 pixels/frame

Low flow capillary density (V>=10/V4-7) = 1.08

High flow capillary density (V>=10/V4-7) = 1.59

In Type 2 diabetics (Figures 27 A and 27B):

Low flow RBC velocity in feed vessels (V>=10) = 1.19 pixels/frame

High flow RBC velocity in feed vessels (V>=10) = 2.86 pixels/frame

Low flow capillary density (V>=10/V4-7) = 1.21

High flow capillary density (V>=10/V4-7) = 1.28

Embodiments of the present disclosure can account for the lack of flow dependent glycocalyx response, recruitment of capillaries, and/or changes in PBR in such patients. In addition, some embodiments of the present disclosure can provide a risk assessment, advanced warning, or pre-diagnosis of one or more of the foregoing conditions by diagnosing glycocalyx impairment. Some embodiments can include compositions (or methods) for treating or prophylactically treating one or more of the foregoing conditions. Compositions can include components and formulations as described in the present disclosure. Methods can include administering compositions of the present disclosure to a patient (e.g., human or non- human mammal) with or diagnosed with glycocalyx impairment and/or one or more of the foregoing conditions. In some embodiments, treating the disease or condition can include, correspond with, or be accomplished by means or treating the (endothelial) glycocalyx and/or improving vascular health (thereby).

Conclusion

The foregoing embodiments and examples are illustrative in nature and non-restrictive. The present disclosure may be embodied in other specific compositions without departing from the spirit, scope or attributes thereof. Thus, it will be readily apparent to those skilled in the art that modifications, derivations and improvements may be made without departing from the scope of the disclosure, and such modifications, derivations and improvements are intended to fall within the full scope and protection of the appended claims. Other equivalents to the specific embodiments disclosed herein may be recognizable to those skilled in the art and are also intended to fall within the full scope and protection of the appended claims. While the foregoing detailed description makes reference to specific exemplary embodiments, the present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the described embodiments are to be considered in all respects only as illustrative and not restrictive. For instance, various substitutions, alterations, changes, and/or modifications of the inventive features described and/or illustrated herein, and additional applications of the principles described and/or illustrated herein, which would occur to one skilled in the relevant art and having possession of this disclosure, can be made to the described and/or illustrated embodiments without departing from the spirit and scope of the disclosure as defined by the appended claims. Such substitutions, alterations, and/or modifications are to be considered within the scope of this disclosure.

The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. The limitations recited in the claims are to be interpreted broadly based on the language employed in the claims and not limited to specific examples described in the foregoing detailed description, which examples are to be construed as non-exclusive and non-exhaustive. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

It will also be appreciated that various features or aspects of certain embodiments can be compatible with, combined with, included in, and/or incorporated into other embodiments of the present disclosure. For instance, systems, methods, and/or products according to certain embodiments of the present disclosure may include, incorporate, or otherwise comprise features described in other embodiments disclosed and/or described herein. Thus, disclosure of certain features relative to a specific embodiment of the present disclosure should not be construed as limiting application or inclusion of said features to the specific embodiment.

In addition, unless a feature is described as being requiring in a particular embodiment, features described in the various embodiments can be optional and may not be included in other embodiments of the present disclosure. Moreover, unless a feature is described as requiring another feature in combination therewith, any feature herein may be combined with any other feature of a same or different embodiment disclosed herein. It will be appreciated that while features may be optional in certain embodiments, when features are included in such embodiments, they can be required to have a specific configuration as described in the present disclosure.

It will also be appreciated that changes may be made in the function and arrangement of elements discussed, described, and/or disclosed without departing from the spirit and scope of the disclosure. Various embodiments may omit, substitute, or add other procedures or components as appropriate. For instance, the methods described may be performed in an order different from that described, and various steps may be added, omitted, or combined. Likewise, any steps recited in any method or process described herein and/or recited in the claims can be executed in any suitable order and are not necessarily limited to the order described and/or recited, unless otherwise stated (explicitly or implicitly). Such steps can, however, also be required to be performed in a specific order or any suitable order in certain embodiments of the present disclosure.

Furthermore, while various aspects of various embodiments have been disclosed herein, other aspects are contemplated. Some well-known aspects of illustrative systems, methods, products, and the like are not described herein in particular detail in order to avoid obscuring aspects of the example embodiments. While a number of aspects similar or equivalent to those described herein can be used to practice embodiments of the present disclosure, only certain aspects are described herein. Such aspects are, however, also contemplated herein.

Each of the appended claims, as well as the recited elements thereof, is intended to be combinable with any other claim(s) and/or element(s) in any suitable combination or dependency without regard to the dependency in which said claims are presented. While certain embodiments and details have been included herein and in the attached disclosure for purposes of illustrating embodiments of the present disclosure, it will be apparent to those skilled in the art that various changes in the methods and apparatus disclosed herein may be made without departing from the scope of the invention, which is defined in the appended claims. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

CLAIMS What is claimed is:

1. A computer system comprising:

a camera;

one or more processors; and

one or more computer-readable hardware storage devices having stored thereon computer-executable instructions, the computer-executable instructions being executable by the one or more processors to cause the computer system to:

generate a profile for each of the plurality of segmented data portions;

after compiling the profiles for each of the plurality of portions, generate an extrapolated characterization of the compiled profiles;

compare the extrapolated characterization against a predetermined threshold value to select a target characterization level;

evaluate a particularized set of rules against the target characterization level to generate a glycocalyx impairment determination or diagnosis; and

display the glycocalyx impairment determination or diagnosis on a user interface of the computer system and/or producing a file or report, in physical or electronic form, that displays the glycocalyx impairment determination or diagnosis.

2. The computer system of claim 1, wherein the plurality of microvascular vessels are located at a sublingual area of a user of the computer system.

3. The computer system of claim 1 or 2, wherein a quality of the microscopy image is automatically controlled by determining one or more of the following: (1) a tissue motion associated with the plurality of microvascular vessels, (2) a light intensity associated with the camera, or (3) an image focus of the camera, and wherein the tissue motion is compared against a tissue motion threshold, the light intensity is compared against a light intensity threshold, and the image focus is compared against an image focus threshold.

4. The computer system of any one of claims 1-3, wherein the at least some of the microvascular vessels that were captured within the microscopy image and that include corresponding image data are identified within the microscopy image by determining a blood flow velocity for each of the at least some of the microvascular vessels.

5. The computer system of any one of claims 1-4, wherein the profile for each of the plurality of segmented data portions includes a red blood cell velocity determination.

6. The computer system of any one of claims 1-5, wherein the profile for each of the plurality of segmented data portions includes ratio determination between blood flow and perfused boundary region (PBR) for each segmented data portion.

7. The computer system of claim 6, wherein the extrapolated characterization is generated by identifying one or more trends formed by the compiling of the profiles for each of the plurality of portions.

8. The computer system of claim 7, wherein the one or more trends are identified using a fitted curve that tracks a progression of compiled profiles.

9. The computer system of any one of claims 1-8, wherein the extrapolated characterization is generated by identifying one or more actual trends of the compiled profiles, the one or more actual trends being extended to include one or more projected trends, whereby the extrapolated characterization includes the one or more actual trends and the one or more projected trends.

10. The computer system of claim 9, wherein the target characterization level is included in the one or more projected trends of the extrapolated characterization such that the target characterization level is a projected level.

11. One or more hardware storage devices having stored thereon computer-executable instructions, the computer-executable instructions being executable by one or more processors of a computer system to cause the computer system to:

generate a profile for each of the plurality of segmented data portions;

compare the extrapolated characterization against a predetermined threshold value to select a target characterization level; evaluate a particularized set of rules against the target characterization level to generate a glycocalyx impairment determination or diagnosis; and

12. A method for determining or diagnosing glycocalyx impairment using a dynamically extrapolated characterization of a plurality of microvascular vessels, the method being performed by one or more processors of a computer system, the method comprising:

capturing, using a camera of the computer system, a microscopy image of the plurality of microvascular vessels;

for image data corresponding to at least some microvascular vessels captured within the microscopy image, segmenting the image data corresponding to each of the at least some microvascular vessels into a plurality of segmented data portions;

generating a profile for each of the plurality of segmented data portions;

after compiling the profiles for each of the plurality of portions, generating an extrapolated characterization of the compiled profiles;

comparing the extrapolated characterization against a predetermined threshold value to select a target characterization level;

evaluating a particularized set of rules against the target characterization level to generate a glycocalyx impairment determination or diagnosis; and

displaying the glycocalyx impairment determination or diagnosis on a user interface of the computer system and/or producing a file or report, in physical or electronic form, that displays the glycocalyx impairment determination or diagnosis.

13. A computer system for use in performing the method of claim 12, the computer system comprising:

a camera;

one or more processors; and

capture, using a camera of the computer system, a microscopy image of the plurality of microvascular vessels; for image data corresponding to at least some microvascular vessels captured within the microscopy image, segment the image data corresponding to each of the at least some microvascular vessels into a plurality of segmented data portions;

generate a profile for each of the plurality of segmented data portions;

evaluate a particularized set of rules against the target characterization level to generate a glycocalyx impairment determination; and

display the glycocalyx impairment determination on a user interface of the computer system and/or produce a file or report, in physical or electronic form, that displays the glycocalyx impairment determination or diagnosis.

14. A method for improving vascular health in a mammal, the method comprising:

(i) diagnosing endothelial glycocalyx impairment in the mammal; and

(ii) treating endothelial glycocalyx by administering to the mammal a composition comprising:

one or more molecular precursor of endothelial glycocalyx, preferably in an amount effective to increase production of endothelial glycocalyx;

one or more antioxidant that associates with endothelial glycocalyx, preferably in an amount effective to at least partially protect existing endothelial glycocalyx against damage caused by enzyme and/or oxidant; and

one or more glycocalyx mimetic, preferably in an amount effective to increase the density of endothelial glycocalyx at a site of endothelial glycocalyx damage.

15. The method of claim 14, wherein the one or more molecular precursor of endothelial glycocalyx comprise glucosamine, or a suitable salt or derivative thereof, preferably glucosamine sulfate.

16. The method of claim 14 or 15, wherein the one or more antioxidant that associates with endothelial glycocalyx comprise superoxide dismutase.

17. The method of any one of claims 14-16, wherein the one or more glycocalyx mimetic are selected from the group consisting of:

(i) nonsulfated glycosaminoglycans, preferably hyaluronan, or a suitable salt or derivative thereof, more preferably sodium hyaluronate; (ii) sulfated polysaccharides, preferably fucoidan, or a suitable salt or derivative thereof, more preferably fucoidan sulfate; and

(iii) combinations thereof.

18. The method of any one of claims 14-17, wherein:

the one or more molecular precursor of endothelial glycocalyx comprise glucosamine, or a suitable salt or derivative thereof, preferably glucosamine sulfate;

the one or more antioxidant that associates with endothelial glycocalyx comprise superoxide dismutase; and

the one or more glycocalyx mimetic comprise:

(i) nonsulfated glycosaminoglycans, preferably hyaluronan, or a suitable salt or derivative thereof, more preferably sodium hyaluronate; and

(ii) sulfated polysaccharides, preferably fucoidan, or a suitable salt or derivative thereof, more preferably fucoidan sulfate.

19. The method of any one of claims 14-18, wherein the composition further comprises one or more additional antioxidants selected from the group consisting of:

catalase, or a suitable transition metal conjugate or derivative thereof, preferably iron(III)-conjugated catalase or iron(IV)-conjugated catalase; and

one or more polyphenols.

20. The method of claim 18 or 19, wherein:

the glucosamine is include as glucosamine sulfate in an amount of at least about 375 mg, preferably at least about 1500 mg,

the fucoidan is included as fucoidan sulfate in an amount of at least about 106.25 mg, preferably at least about 425 mg,

the hyaluronan is include as sodium hyaluronate in an amount of at least about 17.5 mg, preferably at least about 70 mg, and/or

total antioxidants are included as a mixture of:

(i) superoxide dismutase,

(ii) catalase, or a suitable transition metal conjugate or derivative thereof, preferably iron(III)-conjugated catalase or iron(IV)-conjugated catalase, and/or

(iii) one or more polyphenols, preferably a mixture of polyphenols, the total antioxidants included in an amount of at least about 120 mg, preferably at least about 480 mg.

21. The method of any one of claims 14-20, wherein the composition is configured to be safe and effective for oral administration.

22. The method of any one of claims 14-21, wherein diagnosing glycocalyx impairment comprises:

capturing, using a camera of a computer system, a microscopy image of a plurality of microvascular vessels;

for image data corresponding to at least some microvascular vessels captured within the microscopy image, segmenting image data corresponding to each of the at least some microvascular vessels into a plurality of segmented data portions;

generating a profile for each of the plurality of segmented data portions;

23. The method of claim 22, wherein the plurality of microvascular vessels are located at a sublingual area of a user of the computer system.

24. The method of claim 22 or 23, wherein a quality of the microscopy image is automatically controlled by determining one or more of the following: (1) a tissue motion associated with the plurality of microvascular vessels, (2) a light intensity associated with the camera, or (3) an image focus of the camera, and wherein the tissue motion is compared against a tissue motion threshold, the light intensity is compared against a light intensity threshold, and the image focus is compared against an image focus threshold.

25. The method of any one of claims 22-24, wherein the at least some of the microvascular vessels that were captured within the microscopy image and that include corresponding image data are identified within the microscopy image by determining a blood flow velocity for each of the at least some of the microvascular vessels.

26. The method of any one of claims 22-25, wherein the profile for each of the plurality of segmented data portions includes a red blood cell velocity determination.

27. The method of any one of claims 22-26, wherein the profile for each of the plurality of segmented data portions includes ratio determination between blood flow and perfused boundary region (PBR) for each segmented data portion.

28. The method of claim 27, wherein the extrapolated characterization is generated by identifying one or more trends formed by the compiling of the profiles for each of the plurality of portions.

29. The method of claim 28, wherein the one or more trends are identified using a fitted curve that tracks a progression of compiled profiles.

30. The method of any one of claims 22-29, wherein the extrapolated characterization is generated by identifying one or more actual trends of the compiled profiles, the one or more actual trends being extended to include one or more projected trends, whereby the extrapolated characterization includes the one or more actual trends and the one or more projected trends.

31. The method of claim 30, wherein the target characterization level is included in the one or more projected trends of the extrapolated characterization such that the target characterization level is a projected level.

32. A method of treating a disease or condition associated with impaired vascular health or function in a mammal, the method comprising administering to the mammal a therapeutically-effective amount of a composition, the composition comprising:

one or more molecular precursor of endothelial glycocalyx;

one or more antioxidant that associates with endothelial glycocalyx; and

one or more glycocalyx mimetic.

33. The method of claim 32, wherein the one or more molecular precursor of endothelial glycocalyx comprise glucosamine, or a suitable salt or derivative thereof, preferably glucosamine sulfate.

34. The method of claim 32 or 33, wherein the one or more antioxidant that associates with endothelial glycocalyx comprise superoxide dismutase.

35. The method of any one of claims 32-34, wherein the one or more glycocalyx mimetic are selected from the group consisting of:

(i) nonsulfated glycosaminoglycans, preferably hyaluronan, or a suitable salt or derivative thereof, more preferably sodium hyaluronate;

(ii) sulfated polysaccharides, preferably fucoidan, or a suitable salt or derivative thereof, more preferably fucoidan sulfate; and

(iii) combinations thereof.

36. The method of any one of claims 32-35, wherein:

the one or more molecular precursor of endothelial glycocalyx comprise glucosamine, or a suitable salt or derivative thereof, preferably glucosamine sulfate; the one or more antioxidant that associates with endothelial glycocalyx comprise superoxide dismutase; and

the one or more glycocalyx mimetic comprise:

37. The method of any one of claims 32-36, wherein the composition further comprises one or more additional antioxidants selected from the group consisting of:

one or more polyphenols, preferably a mixture of polyphenols.

38. The method of claim 36 or 37, wherein:

total antioxidants are included as a mixture of:

(i) superoxide dismutase,

39. The method of any one of claims 32-38, wherein administering comprises one or more oral administrations.

40. The method of any one of claims 32-38, wherein administering comprises one or more sublingual, intravenous, subcutaneous, intramuscular, and/or transdermal administrations.

41. The method of any one of claims 32-40, wherein administration of the composition exhibits a therapeutic effect on the endothelial glycocalyx of the mammal, the one or more molecular precursor enhancing synthesis of new glycocalyx, enhancing repair of damaged glycocalyx, and protecting existing glycocalyx against damage, the one or more antioxidant protecting existing glycocalyx against damage, enhancing synthesis of new glycocalyx, and enhancing repair of damaged glycocalyx, and

the one or more glycocalyx mimetic enhancing repair of damaged glycocalyx, enhancing synthesis of new glycocalyx, and protecting existing glycocalyx against damage.

42. The method of any one of claims 32-41, wherein:

the one or more molecular precursor of endothelial glycocalyx are included in the composition in an amount effective to increase production of endothelial glycocalyx;

the one or more antioxidant that associates with endothelial glycocalyx are included in the composition in an amount effective to at least partially protect existing endothelial glycocalyx against damage caused by enzyme and/or oxidant; and

the one or more glycocalyx mimetic are included in the composition in an amount effective to increase the density of endothelial glycocalyx at a site of endothelial glycocalyx damage.

43. The method of any one of claims 32-42, wherein the disease or condition associated with impaired vascular health or function is selected from the group consisting of type-2 diabetes, hypertension or high blood pressure, heart disease, kidney disease, liver disease, stroke, myocardial infarction or heart attack, sepsis or septic shock, cancer or metastatic cancer, inflammation, inflammatory disorders, erectile dysfunction, head aches or migraine headaches, severe premenstrual syndrome, memory loss, hearing loss, loss of mental focus, fatigue or chronic fatigue syndrome, thinning hair or balding, leg cramps, cold hands, cold feet, and eczema or skin conditions.

44. A method of enhancing vascular function in a mammal, the method comprising administering to the mammal a therapeutically-effective amount of a composition, the composition comprising:

45. A method of assessing vascular health in a mammal, the method comprising: determining, calculating, or estimating:

(i) Density high total;

(ii) Ratio high;

(iii) RBCfilling%_high_avg; and

(iv) PBR high avg; and

calculating a microvascular health score (MVHS) for the mammal using the following formula:

MVHS = Density high total x Ratio high x RBCfilling%_high_avg / PBR high avg.

46. The method of claim 45, wherein:

determining, calculating, or estimating Density high total comprises one or more step, preferably selected from the group consisting of: capturing one or more images (e.g., videos) of vascular structures (e.g., blood vessels), the images preferably including pixels representative of one or more vascular components or structures (e.g., vessel walls, RBC, etc.); for each video, determining flow level by adding all flow of feed vessels (see previous definition of feed vessels and how to measure flow); for each video, obtaining valid density (Density) for each diameter class; using all videos, constructing Flow-Density plots for each diameter class; using curve fit to obtain Density high for each diameter class at high flow; and adding all Density high estimates for all diameter classes 5 - 25 microns to Density_high_total;

determining, calculating, or estimating Ratio high comprises one or more step, preferably selected from the group consisting of: capturing one or more images (e.g., videos) of vascular structures (e.g., blood vessels), the images preferably including pixels representative of one or more vascular components or structures (e.g., vessel walls, RBC, etc.); for each video: obtaining ratio of Density valid / Density total (Ratio); using all videos: construct Flow-Ratio plot; and using curve fit to estimate Ratio high at high Frbc; determining, calculating, or estimating RBCfilling%_high_avg comprises one or more step, preferably selected from the group consisting of: capturing one or more images (e.g., videos) of vascular structures (e.g., blood vessels), the images preferably including pixels representative of one or more vascular components or structures (e.g., vessel walls, RBC, etc.); determining median RBC widths (p50) and RBCfilling% of one or more (e.g., all) individual measurement sites (e.g., blood vessels); grouping vessels in diameter classes based on their p50 values; measuring RBC velocity (Vrbc) in (all) vessels; multiplying Vrbc with RBCfilling% to obtain estimate of Frbc (RBC flow) for (all) vessels; for each diameter class (in range of 5 - 25 microns), constructing Frbc - RBCfilling% plots; for each diameter class, fitting Frbc- RBCfilling% data with appropriate curve fit; estimating RBCfilling% at high Frbc (RBCfilling%_high); estmating RBCfilling% at Frbc = 0 (RBCfilling%_low); using fitted curve parameters to describe flow dependency of RBCfilling% of each diameter class; optionally or alternatively, calculating RBCfilling%-low - RBCfilling%_high for each diameter class; constructing diameter - RBCfilling%-high curve; and calculating mean RBCfilling%_high_avg by averaging of RBCfilling%-high scores of diameter classes 5 - 25 microns;

determining, calculating, or estimating PBR high avg comprises one or more step, preferably selected from the group consisting of: capturing one or more images (e.g., videos) of vascular structures (e.g., blood vessels), the images preferably including pixels representative of one or more vascular components or structures (e.g., vessel walls, RBC, etc.); determining median RBC widths (p50) and PBR values of one or more (e.g., all) individual measurement sites (e.g., blood vessels); grouping vessels in diameter classes based on their p50 values; measuring RBC velocity (Vrbc) in (all) vessels; multiplying Vrbc with RBCfilling% to obtain estimate of Frbc (RBC flow) for (all) vessels; for each diameter class (in range of 5 - 25 microns), constructing Frbc - PBR plots; for each diameter class, fitting Frbc-PBR data with appropriate curve fit; estimating PBR at high Frbc (PBR high); estmating PBR at Frbc = 0 (PBR low); using fitted curve parameters to describe flow dependency of PBR of each diameter class; optionally or alternatively, calculating PBR-low - PBR high for each diameter class; constructing diameter - PBR-high curve; and calculating mean PBR high avg by averaging of PBR-high scores of diameter classes 5 - 25 microns.

47. A composition for use in improving vascular health, enhancing vascular function, or treating a disease or condition associated with impaired vascular health or function in a mammal, the composition comprising: