EP1561104A1 - Cellules endotheliales comme instrument de diagnostic de maladies cardiovasculaires - Google Patents

Cellules endotheliales comme instrument de diagnostic de maladies cardiovasculaires

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Publication number
EP1561104A1
EP1561104A1 EP03779961A EP03779961A EP1561104A1 EP 1561104 A1 EP1561104 A1 EP 1561104A1 EP 03779961 A EP03779961 A EP 03779961A EP 03779961 A EP03779961 A EP 03779961A EP 1561104 A1 EP1561104 A1 EP 1561104A1
Authority
EP
European Patent Office
Prior art keywords
endothelial cells
endothelial
cells
group
marker
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03779961A
Other languages
German (de)
English (en)
Inventor
Andreas M. Zeiher
Stefanie Dimmeler
Lothar RÖSSIG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Goethe Universitaet Frankfurt am Main
Original Assignee
Goethe Universitaet Frankfurt am Main
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Goethe Universitaet Frankfurt am Main filed Critical Goethe Universitaet Frankfurt am Main
Publication of EP1561104A1 publication Critical patent/EP1561104A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Definitions

  • Endothelial cells as a diagnostic tool for cardiovascular diseases
  • the invention relates to a method for the immunocytological determination of apoptotic endothelial cells and / or endothelial Norrun cells in samples such as e.g. from peripheral blood, e.g. Flow cytometry or a solid phase immunoassay, as a diagnostic tool in patients who are either manifest or at risk for developing cardiovascular disease, and as a tool to improve the prevention and therapeutic management of such a disease.
  • plaques which is expressed by acute arterial occlusion.
  • Such events including myocardial infarction and stroke, occur when a pre-existing atherosclerotic plaque is destabilized and removed or even torn off.
  • stable stages of atherosclerosis had preceded it for years, which did not necessarily lead to subjective complaints or clinically noticeable symptoms.
  • Established procedures to identify patients with incipient or progressive vascular abnormalities who are at risk for developing unstable plaques include documentation of changes in the exercise ECG and ultrasound scans of the peripheral arteries.
  • these examinations only detect larger plaques, which lead to a significant stenosis and impaired blood flow, and generally only lead to clinical symptoms under physical stress.
  • Endothelial cell damage is generally regarded as the key event in triggering atherosclerosis (Ross RN Engl. J. Med. 1999; 340: 115-126).
  • the rate of renewal of EC is increased in those areas of the vessels that are particularly susceptible to the later development of atherosclerotic lesions (Caplan BA, Schwartz CJ Atherosclerosis. 1973; 17: 401-417). This suggests that before the development of the Lesions of numerous ECs die off.
  • Programmed cell death apoptosis
  • pro-atherogenic stimuli which include traditional cardiovascular risk factors such as oxidative stress caused by smoking or dysproteinemia, induce EC apoptosis in vitro.
  • the shear forces caused by the laminar flow of the blood have a clearly protective effect on the survival of EC by completely suppressing the pro-apoptotic activation by atherogenic stimuli (Gimbrone MA, Nagel T., Topper JN J Clin. Invest. 1997; 99: 1809-1813; Dimmeier S., Haendeler J., Rippmann V., Nehls M., Zeiher AM FEBS Lett. 1996; 399: 71-74).
  • microparticles which can also be released after pro-inflammatory activation of the endothelium, does not allow discrimination between inflammatory activation and apoptosis of the EC, a dependence on the activity of the disease has been demonstrated (Mallat Z., Benamer H., Hügel B., Benessiano J., Steg PG, Freyssinet JM, Tedgui A. Circulation. 2000; 101: 841-843).
  • pro-inflammatory processes are the main mechanism underlying endothelial activation and the progression of cardiovascular disorder
  • pathogenesis of atherosclerosis as well as that of systemic heart failure have properties that they share with inflammatory symptoms of other diseases.
  • this includes the increase in pro-inflammatory cytokines and other plasma proteins.
  • detectable plasma vascular activation indicators lack diagnostic specificity and are therefore of limited clinical use for stratifying cardiovascular risk.
  • Endothelial dysfunction of this kind is a generally recognized characteristic that indicates the later manifestation of atherosclerosis at an early stage.
  • the histopathological analysis of atherosclerotic plaques in the human carotid artery showed EC apoptosis in the downstream part of the plaques (Tricot O., Mallat Z., Heymes C, Belmin J., Lesche G., Tedgui A. Circulation. 2000; 101: 2450-2453).
  • Plaque erosion is characterized by the lack of endothelium, with only slight inflammation of the exposed intima (Färb A., Burke AP, Tang AL, Liang TY, Mannan P., Smialek J., Virmani R. Circulation.
  • plaque erosion is responsible for approximately 40% of cases of thrombosis-induced sudden death (Färb A., Burke AP, Tang AL, Liang TY, Mannan P., Smialek J., Virmani R. Circulation. 1996 ; 93: 1354-1363).
  • plaque erosions are quite common in diabetics or pre-menopausal women, and they also occur increasingly in people with elevated CRP (C-reactive protein) - serum levels (Burke AP, Tracy RP, Kolodgie F., Malcom GT, Zieske A., Kutys R., Pestaner J., Smialek J., Virmani R. Circulation. 2002; 105: 2019-2023).
  • plaque instability and thus its potential to trigger a life-threatening cardiovascular event does not depend on the extent of the plaque. Instead, unstable plaques, which tend to tear off or erode, are characterized by a destabilization of the plaque structure.
  • the clinical pictures of an acute transformation of the vascular lesions are accompanied by an increased EC apotosis, which is a marker for plaque instability.
  • Pro-inflammatory activation and increased apoptosis of the endothelium are also widespread in non-atherosclerotic heart failure.
  • a quantification of apoptosis by analyzing endothelial cells that are rejected into the circulation (shedding) could determine the extent of the disease before the clinical manifestation begins.
  • This object of the present invention is achieved by a method for the identification and / or quantification of endothelial cells associated with cardiovascular diseases in a sample.
  • the method according to the invention comprises the steps of: (a) obtaining a sample to be examined and containing endothelial cells; (b) incubating the sample with one or more molecules that specifically bind to one or more of the following marker molecules of the endothelial cells: i) endothelial cell markers and / or apotptose markers; or ii) endothelial cell markers and / or endothelial progenitor cell markers; (c) identification and / or quantification of the endothelial cells based on the bound molecules using immunocytological methods; and (d) comparing the result obtained for the sample to be examined with the result of a reference sample.
  • Endothelial progenitor cells are mobilized from the bone marrow in patients with myocardial infarction (Shintani S., Murohara T., Ikeda H., Ueno T., Honma T., Katoh A., Sasaki K., Shimada T., Oike Y., Imaizumi T Circulation. 2001; 103: 2776-2779). Since recent experimental studies show that endothelial progenitor cells can contribute to regeneration of the stripped arterial sections (Walter DH, Rittig K., Bahlmann F., Kirchmair R., Silver M.
  • a method according to the invention is preferred, the endothelial cells originating from a mammal, in particular from humans.
  • a method according to the invention is further preferred, the endothelial cells to be analyzed, identified and / or quantified being selected from the group consisting of apoptotic endothelial cells, endothelial progenitor cells and mature endothelial cells.
  • the sample to be analyzed can be any sample which contains endothelial cells.
  • the sample can be pretreated with peripheral blood e.g. an anticoagulant, in particular heparin, is added, lysis of the erythrocytes between steps (a) and (b) can also be carried out or the sample can be used directly.
  • peripheral blood e.g. an anticoagulant, in particular heparin
  • lysis of the erythrocytes between steps (a) and (b) can also be carried out or the sample can be used directly.
  • a method according to the invention is preferred in which the sample to be examined is selected from the group consisting of peripheral blood, cell culture suspensions and suspensions which contain cells mechanically, chemically and / or enzymatically detached from a vessel wall. Such enzymatic detachment can e.g. can be obtained by collagenase.
  • a method according to the invention is particularly preferred, the sample to be examined being peripheral blood.
  • An essential aspect of the method according to the invention is the incubation of the sample with one or more molecules that bind specifically to one or more of the marker molecules of the endothelial cells.
  • These molecules can be selected from a very large variety of molecules specific for endothelial cells. It is preferred that the molecules binding to a marker are selected from the group consisting of antibodies or parts or fragments thereof and receptor ligands or parts thereof. A very large number of peptides, proteins and small molecules, such as hormones or the like. These specifically bound molecules are then used as the basis for the further analysis, identification and / or quantification of the endothelial cells.
  • a method according to the invention is particularly preferred, the antibodies, parts or fragments thereof comprising polyclonal antibodies, monoclonal antibodies, Fab fragments, single C z ⁇ antibodies and diabodies.
  • components of the method can be bound to a solid phase, so that the molecules binding to a marker can be present in solution or matrix immobilized.
  • matrices such as, for example, resin matrices and / or conventional column matrices.
  • a method according to the invention is particularly preferred in which the molecules binding to a marker are coupled to one or more detection molecules from the group consisting of fluorescein thioisocyanate, phycoerythrin, enzymes (for example horseradish peroxidase) and magnetic bead.
  • the molecules binding to a marker can be detected with an antibody to which one or more detection molecules are coupled. It is therefore an indirect detection of the binding of the molecule to the respective endothelial cell marker.
  • two-stage detections are well known to the person skilled in the art, for example from anti-antibody detection technology.
  • an essential aspect of the method according to the invention are the endothelial cell markers themselves, to which the above-mentioned molecules bind. These markers can be selected from all markers specific for endothelial cells. It is preferred that the endothelial cell marker is selected from the group consisting of CD 146, von Willebrandt factor (vWF) and vasular endothelial growth F ct ⁇ r receptor 1 (VEGF receptor 1); the apoptosis marker is selected from the group consisting of Annexin V and PD-ECGF, and the markers of endothelial progenitor cells are selected from the group consisting of CD 133 and CD 34. However, these are only examples of markers from which those skilled in the art can easily understand can determine and apply others.
  • vWF von Willebrandt factor
  • VEGF receptor 1 vasular endothelial growth F ct ⁇ r receptor 1
  • the apoptosis marker is selected from the group consisting of Annexin V and PD-ECGF
  • At least one further marker which characterizes non-endothelial cells can also be detected, such as CD45. This serves to delimit the endothelial cells to be identified and / or quantified from other cells contained in the sample.
  • immunocytological methods can be used to identify and / or quantify the endothelial cells. All methods that allow a specific determination based on the marker / molecule interaction are suitable. Preferred methods are those selected from the group consisting of flow cytometry and solid phase immunoassays. So-called “cell sorters” can also be used.
  • the determined data from the examination (s) of the endothelial cells from the sample are usually compared with a reference sample.
  • Which sample can serve as a reference sample will depend in particular on the type of sample examined and the medical history of the individual from whom the sample to be examined comes.
  • a method according to the invention is preferred in which the reference sample originates from one or the mean of several mammals in which / in which a cardiovascular disease was excluded. However, this does not necessarily have to be the case if e.g. the progression of a disease should be determined, an "old" sample from the same patient can also be used as a reference sample. It is obvious to the person skilled in the art which samples are suitable as reference sample for the method according to the invention.
  • the result for apoptotic endothelial cells is related to the result for the total of endothelial cells, and / or the result for apoptotic endothelial cells is related to the result for endothelial cells.
  • the cardiovascular diseases to be diagnosed and / or prognosticated and / or their therapy can be selected from the group consisting of stable and unstable angina, myocardial infarction, current heart syndrome, coronary artery disease and heart failure.
  • stable and unstable angina myocardial infarction
  • current heart syndrome current heart syndrome
  • coronary artery disease coronary artery disease
  • heart failure current heart syndrome
  • the invention preferably provides a flow cytometric method for determining rejected, circulating EC in peripheral blood.
  • a sensitive and highly specific new method is made available, with which damaged endothelium, which precedes plaque development, can be detected and the transition from stable to unstable atherosclerotic phenotype can be quantified.
  • the method not only enables a higher sensitivity compared to previous methods, but also offers the possibility of further characterizing the cells with regard to their survival and their degree of differentiation and their origin within the vascular system.
  • Such a detailed profile of the number and properties of apoptotic, circulating EC provides information about the condition of the endothelium, which is superior to all currently available markers.
  • the flow cytometry method can also be adapted for use as a solid-state immunosorbant assay, thereby making a simplified, practical application of the same principle available.
  • kits comprising means for carrying out the method according to the invention, possibly together with other components and / or auxiliaries.
  • Such means are preferably at least one antibody for the detection of at least one endothelial marker and means for the subsequent identification and / or quantification of the endothelial cells.
  • the kit may also include other components and / or enzymes for performing the methods of the present invention, e.g. Instructions for use to interpret the results of the test in relation to the patient's risk profile and appropriate countermeasures and therapy suggestions.
  • Another aspect of the present invention thus relates to the use of the method according to the invention for the diagnosis and / or prognosis of cardiovascular diseases and / or for monitoring their therapy.
  • This is done through the quantitative and critical determination of rejected, floating and circulating endothelial cells and / or endothelial progenitor cells, which are an indicator of the damaged endothelium.
  • suitable countermeasures can then be implemented by the attending physician in order to positively influence the patient and to prevent the adverse event or at least reduce the severity of the affected patient.
  • such therapy can include the administration of lipid-lowering agents, selected from the group consisting of appreciation, in particular atorvastatin.
  • other possible therapies are known to the person skilled in the art to treat cardiovascular diseases, which can be carried out according to conventional schemes.
  • Figure 1 Flow cytometry measurement of circulating EC.
  • Circulating cells from peripheral blood were analyzed after erythrocyte lysis.
  • Figure 4 Relationship between circulating apoptotic EC and AC133 / KDR + endothelial progenitor cells in patients with CAD.
  • SA stable angina
  • U unstable angina
  • MI acute myocardial infarction
  • Serum for the determination of the concentrations of CRP and hsCRP (turbidimetric test, Boehringer Mannheim or Ultrasensitive N Latex CRP Monotest, Behring) and the serum lipid fractions (Boehringer Mannheim) were also collected in all patients at the time of the examination.
  • Erythrocyte lysis in 10 ml of peripheral venous heparin blood was carried out using a commercially available lysis solution (Becton Dickinson, BD).
  • the circulating EC were then analyzed using a suitably modified protocol according to the Mancuso et al. and Monestiroli et al. (Mancuso P., Burlini A., Pruneri G., Goldhirsch A., Martinelli G., Bertolini F. Blood. 2001; 97: 3658-3661; Monestiroli S., Mancuso P., Burlini A., Pruneri G ., Dell'Agnola C, Gobbi A., Martinelli G., Bertolini F. Cancer Res.
  • CD45 + signals identified with a directly perCP-conjugated monoclonal antibody against human CD45 (BD) was quantified in order to report each measured cell population with regard to the total number of leukocytes in relation to the WBC number .
  • CD45 " cells were further analyzed, namely by double staining with antibodies against the EC-specific epitopes CD146 (Mel-CAM, MUC18, S-Endo-1; Shih IMJ Patol.
  • endothelial progenitor cells were carried out in a subset of the patients, as described earlier (Vasa M., Fichtischerer S., Aicher A., Adler K., Urbich C, Martin H., Zeiher AM, Dimmeler S. Circ. Res. 2001; 89: El-7).
  • 100 ul peripheral blood were incubated with FITC or perCP conjugated monoclonal antibodies against human CD34 (BD).
  • FITC or perCP conjugated monoclonal antibodies against human CD34 BD
  • As a set of secondary markers that recognize more immature progenitor cells blood samples with monoclonal antibodies against human CD 133 (Milteny; PE-conjugated) and against human KDR (Sigma) followed by a FITC-conjugated secondary antibody were immunostained. Isotype-identical antibodies served as controls (BD). After incubation, the cells were lysed, washed with PBS and fixed in 4% paraformaldehyde
  • Circulating apoptotic EC were defined as cells in which the endothelial mark ⁇ roteins CD 146 and vWF were positive and annexin V binding took place. In addition, only CD45 " cells were included in the analysis in order to exclude potentially contaminating leukocytes. A representative analysis is shown in FIG.
  • VEGF vascular endothelial growth factor
  • KDR VEGF receptor 1
  • vWF vascular endothelial growth factor receptor 1
  • CRP C-reactive protein
  • serum amyloid A serum amyloid A
  • FIG. 3 shows that treatment with atorvastatin significantly reduces both the number of circulating apoptotic ECs and their share in the total number of circulating ECs.
  • Statin therapy also significantly reduced the LDL cholesterol concentration (p ⁇ 0.02).

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Abstract

L'invention concerne un procédé d'analyse immunocytologique de cellules endothéliales apoptotiques et/ou de cellules précurseurs endothéliales dans des échantillons tels que de sang périphérique, p. ex. par cytométrie de flux ou par immuno-essai en phase solide, comme instrument de diagnostic chez des patients souffrant manifestement d'une maladie cardiovasculaire ou présentant un risque de développer une telle maladie et comme un instrument permettant d'améliorer la prévention et la gestion thérapeutique d'une maladie de ce type.
EP03779961A 2002-11-16 2003-11-17 Cellules endotheliales comme instrument de diagnostic de maladies cardiovasculaires Withdrawn EP1561104A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10253526 2002-11-16
DE10253526A DE10253526A1 (de) 2002-11-16 2002-11-16 Endothelzellen als Diagnoseinstrument bei kardiovaskulären Erkrankungen
PCT/EP2003/012859 WO2004046718A1 (fr) 2002-11-16 2003-11-17 Cellules endotheliales comme instrument de diagnostic de maladies cardiovasculaires

Publications (1)

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EP1561104A1 true EP1561104A1 (fr) 2005-08-10

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EP03779961A Withdrawn EP1561104A1 (fr) 2002-11-16 2003-11-17 Cellules endotheliales comme instrument de diagnostic de maladies cardiovasculaires

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US (1) US20050244897A1 (fr)
EP (1) EP1561104A1 (fr)
AU (1) AU2003288092A1 (fr)
CA (1) CA2506046A1 (fr)
DE (1) DE10253526A1 (fr)
WO (1) WO2004046718A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7645739B2 (en) * 2001-02-21 2010-01-12 Alavita Pharmaceuticals, Inc. Modified annexin compositions and methods of using same
US7635680B2 (en) * 2001-02-21 2009-12-22 Alavita Pharmaceuticals, Inc. Attenuation of reperfusion injury
US20050222030A1 (en) * 2001-02-21 2005-10-06 Anthony Allison Modified annexin proteins and methods for preventing thrombosis
US7635676B2 (en) * 2001-02-21 2009-12-22 Alavita Pharmaccuticals, Inc. Modified annexin proteins and methods for their use in organ transplantation
US20090291086A1 (en) * 2001-02-21 2009-11-26 Alavita Pharmaceuticals, Inc. Compositions and Methods for Treating Cerebral Thrombosis and Global Cerebral Ischemia
US7901950B2 (en) * 2005-08-12 2011-03-08 Veridex, Llc Method for assessing disease states by profile analysis of isolated circulating endothelial cells
MX2008013333A (es) * 2006-04-18 2008-11-10 Wellstat Biologics Corp Deteccion de celulas endoteliales circulantes.
ES2670991T3 (es) * 2013-12-18 2018-06-04 Siemens Healthcare Diagnostics Inc. Detección de enfermedad endotelial

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
None *
See also references of WO2004046718A1 *

Also Published As

Publication number Publication date
DE10253526A1 (de) 2004-06-09
WO2004046718A1 (fr) 2004-06-03
CA2506046A1 (fr) 2004-06-03
US20050244897A1 (en) 2005-11-03
AU2003288092A1 (en) 2004-06-15

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