KR101106015B1 - Pharmaceutical composition for preventing or treating diabetic wound - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating diabetic wounds, comprising a culture of endothelial progenitor cells or a fraction thereof as an active ingredient. By the present invention, it has been found that cultures (or fractions thereof) of EPCs cultured in vitro without the direct transplantation of endothelial progenitor cells can be usefully used for the treatment of chronic wounds such as diabetic wounds. Thus, cultures (or fractions thereof) of EPCs cultured in vitro can be usefully applied to the treatment of chronic wounds, such as diabetic wounds, without physical incorporation of the cells themselves through transplantation, which can cause carcinogenic problems. Furthermore, the culture (or fractions thereof) of the EPC can be usefully used as an alternative therapeutic agent without the immune side effects of allogeneic transplantation, which must be carefully considered during non-autologous cell injection.

Endothelial progenitor cells, diabetes, wounds

Description

Pharmaceutical composition for preventing or treating diabetic wound}

The present invention relates to a pharmaceutical composition for preventing or treating diabetic wounds, comprising a culture of endothelial progenitor cells or a fraction thereof as an active ingredient.

Endothelial progenitor cells (EPCs) are known as endothelial precursors that are derived from the bone marrow and collected into damaged tissue and finally contribute to tissue repair by promoting neovascularization. Various therapeutic activities of EPC, such as myocardial infarction, stroke, and peripheral arterial disease, have been reported for the treatment of ischemic diseases, and the present inventors also determined excisional full thickness wounds in normal mice. Topical injections of EPCs have been shown to significantly promote wound healing and promote revascularization in granulated tissue (Suh W, et al., Transplantation of endothelial progenitor cells accelerates dermal wound healing with increased). recruitment of monocytes / macrophages and neovascularization.Stem Cells 23: 1571-1578, 2005).

The wound healing process involves multiple stage biological and molecular processes, including inflammation, cell migration / proliferation, extracellular matrix deposition, and the like. This repair process proceeds through the action of various growth factors and cytokines, and also through the interaction of different types of cells such as inflammatory cells, fibroblasts, keratinocytes and endothelial cells. Acute wounds without pathophysiological defects can be easily healed through the sequential progression of the wound healing process, while chronic wounds in patients with pathophysiological abnormalities such as diabetes are present. Multilevel mechanisms affect the healing deficits of diabetic chronic wounds, of which reduced production of growth factors, decreased angiogenesis, and deterioration of the migration and proliferation of keratinocytes and fibroblasts are critical factors.

In addition, recent studies in diabetic mice have reported that a decrease in the number of EPCs in the bloodstream and wound tissue is another critical reason for exacerbating diabetic wound healing (Gallagher KA, et al., Diabetic impairments in NO). -mediated endothelial progenitor cell mobilization and homing are reversed by hyperoxia and SDF-1 alpha.J Clin Invest 117: 1249-1259, 2007), it has also been reported that the number of EPCs in peripheral blood is significantly lower in diabetic patients than in normal individuals. (Loomans CJ et al., Endothelial progenitor cell dysfunction: a novel concept in the pathogenesis of vascular complications of type 1 diabetes. Diabetes 53: 195-199, 2004 8; and Fadini GP et al., Number and function of endothelial progenitor cells as a marker of severity for diabetic vasculopathy.Arterioscler Thromb Vasc Biol 26: 2140-2146, 2006). In addition, EPCs obtained from normal mice significantly improved blood flow in injured tissues, but EPCs isolated from diabetic mice have been reported to exhibit impaired angiogenic function when injected into an ischemic disease animal model (Tamarat R et. al., Impairment in ischemia-induced neovascularization in diabetes: bone marrow mononuclear cell dysfunction and therapeutic potential of placenta growth factor treatment.Am J Pathol 164: 457-466, 2004).

Thus, such conventional reports suggest that for the treatment of chronic wounds, such as diabetic wounds, it may provide a therapeutic benefit when transplanting EPCs. In other words, when EPC is injected externally, growth factors are secreted from EPC and chronic wound injury is caused by EPC-mediated neovascularization mechanism through the physical incorporation of EPC into neovascularization. It is expected to facilitate treatment.

However, the use of non-autologus endothelial progenitor cells is inevitable when directly implanting EPCs for the treatment of chronic wounds, especially diabetic wounds. This is because patients with chronic wounds, such as diabetic patients with wounds, are significantly impaired in the revascularization function of autologus endothelial progenitor cells and are unable to achieve the desired therapeutic purpose. Therefore, direct transplantation of undifferentiated cells, such as non-autologus endothelial progenitor cells, can not rule out carcinogenesis problems that may arise from the transplanted cells, thus avoiding safety problems. Can not.

The inventors of the present invention, while studying the molecular biological mechanisms of endothelial progenitor cells in the treatment of chronic wounds, have surprisingly found that sufficient therapeutic effects of chronic wounds can be achieved with conditioned media of EPC. That is, it was found that the culture of endothelial progenitor cells exhibits therapeutically equivalent activity with the endothelial progenitor cells themselves through the paracrine effect in the treatment of chronic wounds such as diabetic wounds; Therefore, a culture of EPC cultured in vitro alone can heal chronic wounds, such as diabetic wounds, without directly transplanting the cells themselves, i.e., EPCs, and thus the problem of transplanting the cells themselves (e.g., carcinogenesis). It was found that sex) can be avoided.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of chronic wounds, such as diabetic wounds, comprising a culture of the endothelial progenitor cells as an active ingredient.

According to one aspect of the invention, there is provided a pharmaceutical composition for the prevention or treatment of diabetic wounds, comprising a culture of endothelial progenitor cells or a fraction thereof as an active ingredient.

The culture of the endothelial progenitor cells may comprise a supernatant obtained by centrifuging the culture solution of endothelial progenitor cells; Concentrate of the supernatant; Or lyophilisate of the supernatant. In addition, the fraction of the culture of the endothelial progenitor cells may include a fraction obtained by fractionating the supernatant obtained by centrifuging the culture solution of the endothelial progenitor cells to contain a protein of 10 kDa or more; Concentrate of said fraction; Or lyophilisate of the fraction. The culture of the endothelial progenitor cells can be obtained by culturing the endothelial progenitor cells in a serum-free medium such as M199 medium.

The endothelial progenitor cells are endothelial progenitor cells derived from umbilical cord blood, and the diabetic wounds are diabetic foot ulcers, diabetic angiopathy or diabetic foot infections. Preferably.

According to the present invention, it has been found that in the healing of chronic wounds such as diabetic wounds, the conditioned media of endothelial progenitor cells exhibit therapeutically equivalent activity with the EPC itself through the paracrine effect. lost. That is, it has been found that cultures (or fractions thereof) of EPCs cultured in vitro without direct transplantation of endothelial progenitor cells can be usefully used for the healing of chronic wounds such as diabetic wounds. Thus, cultures (or fractions thereof) of EPCs cultured in vitro can be usefully applied to the treatment of chronic wounds, such as diabetic wounds, without physical incorporation of the cells themselves through transplantation, which can cause carcinogenic problems. Furthermore, the culture (or fractions thereof) of the EPC can be usefully used as an alternative therapeutic agent without the immune side effects of allogeneic transplantation, which must be carefully considered during non-autologous cell injection.

In the present specification, "endothelial progenitor cells (EPCs)" are progenitor cells prior to differentiation into vascular endothelial cells, which are present in a small amount in bone marrow and blood but regressed into ischemic tissues due to vascular injury to renal blood vessels ( neovessel) refers to progenitor cells that promote production and include cells expressing CD31, KDR and Tie2 specific. The EPC is described, for example, in Suh W, et al., Transplantation of endothelial progenitor cells accelerates dermal wound healing with increased recruitment of monocytes / macrophages and neovascularization. Stem Cells 23: 1571-1578, 2005 may be isolated according to known separation methods, and may be isolated from various sources including bone marrow, umbilical cord blood, adipose tissue. Among them, EPC isolated from umbilical cord blood of a healthy person can be particularly preferably used because it can minimize the potential immune rejection response.

In addition, "diabetic wound" refers to a chronic wound (diabetic wound) that occurs in diabetic patients, the chronic refers to the damage of peripheral blood vessels due to diabetes is accompanied by blood circulation disorders delay wound healing Speaking of being wound. Wounds and wounds are used herein in the same sense. The diabetic wounds include diabetic foot ulcers, diaebtic angiopathy, diabetic foot infections, and the like.

The inventors have found that in the healing of chronic wounds, such as diabetic wounds, the conditioned media of the EPC exhibits therapeutically equivalent activity with the EPC itself through the paracrine effect. That is, it has been found that cultures (or fractions thereof) of EPCs cultured in vitro without transplantation of EPCs can be usefully used for the healing of chronic wounds such as diabetic wounds. This is very surprising given the reports that conventionally, angiogenesis-promoting activity of EPCs is achieved not only through the secretion of growth factors, but also through physical incorporation into vasculature (angiogenesis, vasculogenesis). Thus, according to the present invention, cultures (or fractions thereof) of EPCs cultured in vitro are useful for the treatment of chronic wounds, such as diabetic wounds, without physical incorporation of the cells themselves through transplantation, which can cause carcinogenic problems. It turns out that it can be applied. Furthermore, the culture (or fractions thereof) of the EPC can be usefully used as an alternative therapeutic agent without the immune side effects of allogeneic transplantation, which must be carefully considered during non-autologous cell injection.

The present invention provides a pharmaceutical composition for preventing or treating diabetic wounds, comprising a culture of endothelial progenitor cells or a fraction thereof as an active ingredient.

The endothelial progenitor cells can be cultured using conventional cell culture media. In particular, it has been found by the present invention that endothelial progenitor cells are preferably cultured in serum-free medium, such as M199 medium (Gibco, Cat. No. 12350039). That is, in order to promote cell proliferation and prevent cell death, endothelial progenitor cells, like other cells, are required to be cultured in a medium to which serum such as bovine serum is added. Surprisingly, however, endothelial progenitor cell death and autophagy were observed when short-term culture (eg, culture for about 3-4 days) using serum-free medium such as M199 without serum addition. Was not found. In particular, since it can be cultured without using animal serum such as bovine serum, it is possible to fundamentally avoid the problem of contamination due to the incorporation of heterologous serum. The M199 is a known cell culture medium, the specific composition of which is shown in Table 1a and Table 1b (http://www.invitrogen.com/site/us/en/home/support/Product-Technical-Resources/media_formulation .91.html).

ingredient Molecular Weight Concentration (mg / L) amino acid Glycine 75 50 L-alanine 89 25 L-arginine hydrochloride 211 70 L-aspartic acid 133 30 L-Cysteine Hydrochloride-H ₂ O 176 0.1 L-cystine 2HCl 240 26 L-glutamic acid 165 75 L-Glutamine 146 100 L-histidine hydrochloride-H ₂ O 210 21.88 L-hydroxyproline 131 10 L-isoleucine 131 40 L-leucine 131 60 L-lysine hydrochloride 183 70 L-methionine 149 15 L-phenylalanine 165 25 L-proline 115 40 L-serine 105 25 L-Threonine 119 30 L-Tryptophan 204 10 L-Tyrosine Disodium Salt Dihydrate 181 40 L-valine 117 25 vitamin Alpha-tocopherol phosphate 702 0.01 Ascorbic acid 176 0.05 Biotin 244 0.01 Choline chloride 140 0.5 D-Calcium Pantothenate 477 0.01 Folic Acid 441 0.01 Menadione (vitamin K3) 172 0.01 Niacinamide 122 0.025 Niacin (niacin) 123 0.025 Para-aminobenzoic acid 137 0.05 Pyridoxal hydrochloride 204 0.025 Pyridoxine hydrochloride 206 0.025 Riboflavin 376 0.01 Thiamine hydrochloride 337 0.01 Vitamin A (Acetate) 328 0.1 Vitamin D2 (Calciferol) 397 0.1 i-inositol 180 0.05

ingredient Molecular Weight Concentration (mg / L) Inorganic salts CaCl ₂ (anhydride) 111 140 Fe (NO ₃ ) -9H ₂ O 404 0.7 MgSO ₄ (anhydride) 120 97.67 KCl 75 400 KH ₂ PO ₄ 136 60 NaHCO ₃ 84 350 NaCl 58 7500 Na ₂ HPO ₄ -7H2O 268 90 Other ingredients Adenine sulfate 404 10 Adenosine 5'-triphosphate disodium salt
(Adenosine triphosphate disodium salt) 605 One Adenylic Acid 347 0.2 cholesterol 387 0.2 D-glucose (dextrose) 180 1000 Deoxyribose 134 0.5 Glutathione (reduced) 307 0.05 Guanine hydrochloride 188 0.3 HEPES 238 5960 Hypoxanthine Na 136 0.4 Phenolic Red 376.4 20 Riboose 150 0.5 Sodium acetate 82 50 Thymine 126 0.3 Tween 80® 20 Uracil 112 0.3 Xanthine-Na 152 0.34

The pharmaceutical composition of the present invention comprises a culture of endothelial progenitor cells or a fraction thereof obtained from the culture of endothelial progenitor cells as described above. The culture of the endothelial progenitor cells may comprise a supernatant obtained by centrifuging the culture solution of endothelial progenitor cells; Concentrate of the supernatant; Or lyophilisate of the supernatant. In addition, the fraction of the culture of the endothelial progenitor cells may include a fraction obtained by fractionating the supernatant obtained by centrifuging the culture solution of the endothelial progenitor cells to include a protein of 10 kDa or more, more preferably 10 to 200 kDa; Concentrate of said fraction; Or lyophilisate of the fraction.

The centrifugation may be performed for about 5 minutes at conditions that can be removed by sedimentation of endothelial progenitor cells, macromolecules, and media components, for example about 3,500 g. In addition, the concentrate may be obtained by concentrating the culture solution or fraction until a desired protein concentration is obtained by a conventional method such as vacuum concentration. The fraction can also be carried out using conventional membranes having a specific pore size (eg pore size of 10 kDa).

The pharmaceutical composition of the present invention contains the culture of the endothelial progenitor cells or fractions thereof as an active ingredient, and may include a pharmaceutically acceptable carrier, and may be a liquid, suspension, emulsion, lotion, ointment according to a conventional method. It may be formulated into a parenteral formulation, such as lyophilizer. Preferably, it may be formulated into a transdermal dosage form such as an external solution, an emulsion, an ointment. The pharmaceutically acceptable carrier includes an aqueous diluent or solvent such as phosphate buffered saline, purified water and sterile water, and a non-aqueous diluent or solvent such as propylene glycol, polyethylene glycol and olive oil. It includes. In addition, wetting agents, fragrances, preservatives, and the like may be included as necessary.

The dosage of the culture of the endothelial progenitor cells or fractions thereof contained in the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. Can be. For example, the culture of endothelial progenitor cells or fractions thereof may be administered at a dose of 1 μg / kg to 1000 μg / kg per day, and the administration may be administered once or several times a day. The pharmaceutical composition of the present invention may be administered alone or in combination with other wound therapeutics, and when administered in combination, may be administered sequentially or simultaneously with other therapeutic agents. When administered alone or in combination, the dosage of the pharmaceutical composition of the present invention is preferably administered in an amount that can achieve the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

1. Materials and Methods

(1) cell culture

CD34 obtained from human umbilical cord blood according to previously reported methods (Suh W, et al., Transplantation of endothelial progenitor cells accelerates dermal wound healing with increased recruitment of monocytes / macrophages and neovascularization.Stem Cells 23: 1571-1578, 2005). EPCs were isolated by incubating positive cells for 2-3 weeks in endothelial growth medium (EGM) -2MV Singlequot (Clonetics, Walkersville, MD, USA). The cells have been found to express CD31, KDR and Tie2 and also have established definitions of EPC phenotypes such as uptake of acetylated LDL and angiogenesis on matrigel (Kim KL, et al., Interaction between Tie receptors modulates angiogenic activity of angiopoietin2 in endothelial progenitor cells.Cardiovasc Res 72: 394-402, 2006). The Institutional Review Board of CHA approved all protocols and received informed consent from all providers.

EPC-derived medium (EPC-conditioned medium, EPC-CM) was obtained as follows: EPC was incubated for 24 hours at 37 ° C. in serum-free M199 media (Clonetics), followed by centrifugation at 3500 g. Fractions were obtained by centrifugation using a membrane of 10 Kda molecular weight cutoff (Amicon Division, Danvers, MA, USA).

(2) diabetic wound animal model

BALB / c nude mice (7-8 weeks old, male, 17-23 g body weight, Japan SLC Inc., Shizuoka, Japan), which are immunodeficient mice, induced diabetes. That is, the mice were fasted for 16 hours prior to injection of streptozotocin (STZ) (Sigma, St. Louis, MO, USA; 200 mg / kg). Every week after STZ administration, plasma glucose levels were measured using an Accu-Check Advantage glucometer (Roche, Indianopolis, IN, USA). Three weeks after infusion, mice with plasma glucose levels of> 300 mg / dL were considered to have STZ-induced diabetes.

STZ-induced diabetic mice were anesthetized by intraperitoneal injection of ketamine-silazine (79.5 mg / kg and 9.1 mg / kg, respectively), and two full-thickness excisional wounds [upper and lower wounds were Separation by 2.5 cm) was formed on the dorso-lateral site using a standard skin biopsy punch (0.5 cm in diameter, Acuderm Inc., Fort Lauderdale, FL, USA). Immediately after surgery, EPC (1 × 10 ⁶ cells suspended in 60 μl of phosphate buffered saline) and 60 μl of phosphate buffered saline were injected into three different epidermal sites near the upper or lower wound formed in the same mouse. To confirm the paracrine effect of EPC, 100 μl of M199 basal medium or EPC-CM (obtained from 1 × 10 ⁶ EPCs) was injected into the wound in the same manner as described above. All procedures were performed with the approval of the Institutional Animal Care and Use Committee (IACUC).

(3) wound analysis

Post-treatment 0, after treatment using previously reported methods (Suh W, et al., Transplantation of endothelial progenitor cells accelerates dermal wound healing with increased recruitment of monocytes / macrophages and neovascularization.Stem Cells 23: 1571-1578, 2005). Open wound immunity was measured at days 3, 5, 7, 9, and 12. That is, the wounds were taken with a digital camera and their images were analyzed by using Image-Pro Plus (Media Cybernetics, Inc., Silver Spring, MD, USA) to track the wound edges and calculate the pixel area. . Pixel counts were related to circular filter paper of the same diameter as the original wound, serving as a standard for all images. The measurement was performed twice, and the open wound area was expressed as a percentage divided by the wound area at each time by the initial wound area at day 0.

To determine granulation tissue area, wound tissue harvested on days 15 and 20 were cut halfway in lengthwise through the least healed portion, fixed with 4% paraformaldehyde, and then placed in paraffin, Sectioned perpendicular to the wound surface. Staining with wound sections hematoxylin and eosin (H & E) with the largest epithelial gap (maximum distance between epithelial edges; judged to be the center of the wound). Then, using Image-Pro Plus on the H & E stained digitized image, trace the wound boundary where there is a transition from the original hair follicle and normal to the hypertrophic epidermis, and calculating the pixel area, thereby resulting in ablated tissue area Was analyzed. The number of pieces observed for the following experiments ranged from 10 to 12 per wound.

(4) Immunohistochemical Analysis

The wound pieces were treated with 0.3% hydrogen peroxide to quench endogenous peroxidase and warmed in Target Retrieval Solution (DakoCytomation, Carpinteria, Calif., USA) to recover antigen epitopes. After blocking the pieces in 10% normal goat serum, anti-von Willebrand factor (vWF; Dako), anti-cytokeratin 14 (CK14; Abcam) , Cambridge, MA, USA), anti-vimentin, Dako, anti-Ki67 (anti-Ki67, BD Bioscienece, San Jose, CA, USA), anti-human CD31 (anti-human CD31, Abcam ), Anti-mouse CD31 (anti-mouse CD31, BD pharmingen) or irreversible nonspecific IgG. The pieces were incubated with biotinylated or fluorescence-conjugated secondary IgGs (Jackson ImmunoResearch Laboratories Inc., West Grove, Pa., USA), and the ABC kit (Elite kit, Vector Laboratories, Burlingame, CA) , USA) and Vector Blue (Vector Laboratories). For multicolor immunofluorescence analysis, the pieces were stained with 4,6-diamidino-2-phenylindole (DAPI) and fluorescence microscopy (Carl Zeiss, Oberkochen, Germany).

(5) statistical analysis

All data are expressed as mean ± SEM. Statistical significance pair t - was evaluated by way ANOVA, Bonferroni's post hoc multiple comparison test after using (ne-way analysis of variance) - test (paired t -test) or stage. P values less than 0.05 were considered statistically significant and the number of samples was expressed as n .

2. Results

(1) EPC transplantation promotes wound closure in STZ-induced diabetic mice

To evaluate the therapeutic effect of human cord blood-derived EPC on the treatment of diabetic wounds, diabetes was induced in athymic nude mice by STZ injection. As expected in previous studies, STZ-induced diabetic mice exhibited markedly delayed wound healing (Michaels J, et al., Db / db mice exhibit severe wound-healing impairments compared with other murine diabetic strains in a silicone-splinted excisional wound model.Wound Repair Regen 15: 665-670, 2007). The mice showed complete wound closure on day 15. In contrast, normal immunodeficient mice showed complete healing 7 days after wound induction.

When EPC was injected transdermally around the entire skin wounds formed on the back of the STZ-induced diabetic nude mice, wound healing was significantly promoted from 3 days post-wound compared to PBS-treated wounds (FIGS. 1A and B). . Significant decreases in the area of open wounds were observed until day 12 (EPC, 10.6 ± 5.3%; PBS, 45.7 ± 9.2%, day 12, p <0.05). The granulation tissue area at the middle site of the wound was measured from the tissue separated 20 days after the wound, and the EPC-treated wound had a substantially reduced cross-sectional area of granulation tissue compared to the PBS-treated wound (FIG. 1). C). These results indicate that EPC transplantation can promote wound healing in diabetic mice.

(2) EPC transplantation promotes proliferation of keratinocytes and fibroblasts during the initial wound healing process.

In order to confirm the cellular changes caused by EPC transplantation during the initial wound healing process, the wound fragments separated on the third and seventh days of the wound were analyzed immunohistochemically, and the results are shown in FIG. 2. From the beginning of the test (day 3), numerous proliferating keratinocytes (positive to both CK 14 and the proliferation marker Ki67) were found in EPC-treated wounds, especially the bottom layer and distal epidermis adjacent to the thickened wound edges. epidermis). In contrast, only a few proliferating keratinocytes were found in PBS-treated controls (FIG. 2A). In particular, in EPC-treated wounds, a large number of CK14-positive proliferating cells have been found in the outer root sheath of hair follicles in adjacent skin, a site known as the geratinite reservoir. The vesicular keratinocytes, which stopped growing during skin regeneration, mitosis and provide progeny to the epidermis after wound (Langton AK, et al., An extended epidermal response heals cutaneous wounds in the absence of a hair follicle stem cell contribution.J Invest Dermatol 128: 1311-1318, 2008), which has been reported to induce rapid re-epithelialization and is believed to play an important role in skin regeneration. CK14 / Ki67-positive cells inside hair follicles were observed more frequently in EPC-treated wounds than in PBS-treated controls. In addition, immunohistochemical analysis of granulated tissue 7 days after the wound showed that EPC-treated wounds showed more proliferating fibroblasts (positive to both nonmentin and Ki67) compared to PBS-treated controls. EPC transplantation has been shown to promote fibroblast proliferation in granulated tissue (FIG. 2B). The results indicate that EPC transplantation not only promotes the proliferation of fibroblasts in granulated tissue, but also activates keratinocytes in the epidermal bottom layer and hair follicles to promote epidermal regeneration.

(3) EPC-CM is therapeutically equivalent to EPC in its ability to promote wound healing and revascularization in STZ-induced diabetic mice.

EPC-CM (obtained from a culture cultured for 12 hours of 1 x 10 ⁶ EPCs) was transdermally injected into the same diabetic wound rodent model, and the effect on wound healing was analyzed and the effect of cell injection (1 x 10 ⁶ EPCs) It was compared with the case. Visual observation at the time points indicated in FIG. 3A showed that wounds treated with EPC-CM had an open wound area much smaller than the M199 basal medium control from the initial time point. In particular, a direct comparison between the EPC-CM-treated and EPC-infused groups showed that transdermal administration of EPC-CM promoted wound closure rates to the same extent as EPC infusion over the entire test period (FIG. 3B). The EPC-CM-treated wound had significantly smaller granulated tissue than the M199 control (FIG. 3C). In addition, the degree of reduction in granulation tissue area was similar to that seen in EPC-transplanted wounds. These results indicate that EPC-CM alone is therapeutically equivalent to EPC in promoting wound healing and reducing granulation tissue area in STZ-induced diabetic mice.

To examine whether infusion of EPC-CM can increase angiogenesis of wound tissue as observed in EPC-injected tissue, vWF-positive neovascularization in granulated tissue of wound pieces harvested on day 15 By counting the capillary density was measured. Representative images and quantitative analyzes shown in D-E of FIG. 3 indicate that capillary density in EPC-CM-treated wounds is significantly greater than M199-treated controls. Although slightly downsized, this increase is not significantly different from that found in EPC-treated wounds.

Immunohistologic analysis using species-specific CD31 antibodies revealed that very few neovascularizations in EPC-treated wounds were formed by mouse and human CD31-positive cells (FIG. 3F). These results indicate that partially injected human EPC contributes to revascularization of wound tissue through physical incorporation (EPC-mediated postnatal vasculogenesis) into newly formed mouse vasculature. However, no significant difference in capillary density between EPC-CM-treated and EPC-injected wounds was observed. These results show that EPC-mediated postnatal vasculogenesis is not a major mechanism for promoting neovascularization observed after EPC transplantation in diabetic wounds.

3. Consideration

EPC is known as an endothelial precursor that plays an important role in maintaining vascular homeostasis and in promoting vascular repair in damaged tissues. Thus, research on the therapeutic activity of EPCs derived from various sources, including bone marrow, adipose tissue, and peripheral blood, is actively progressing in various ischemic diseases such as myocardial infarction, stroke, and peripheral arterial disease. However, the vascular regeneration function of these cells has not yet been fully identified in terms of the treatment of chronic wounds found in many patients with symptoms such as diabetes. Chronic wounds are a major condition associated with diabetes, which is found in about 85% of all diabetes-related lower leg amputations, but it is difficult to achieve complete healing even in the best treatments (Brem H, Tomic-). Canic M: Cellular and molecular basis of wound healing in diabetes.J Clin Invest 117: 1219-1222, 2007). Treatment of such exacerbated diabetic wounds has recently been found to result from a fundamental lack of EPCs and their diabetes-associated dysfunction in diabetics. Therefore, in order to achieve satisfactory clinical results for wound healing, it is expected that EPCs from healthy donors in addition to autologous EPCs will be required (Gallagher KA, et al., Diabetic impairments in NO-mediated endothelial progenitor cell mobilization and homing are reversed by hyperoxia and SDF-1 alpha.J Clin Invest 117: 1249-1259, 2007). In this regard, we evaluated the therapeutic activity of EPC derived from human umbilical cord blood in the treatment of diabetic wounds.

In this trial, EPC transplantation significantly promoted wound closure from the beginning of the test, while promoting angiogenesis and keratinocyte / fibroblast proliferation. In particular, EPC transplantation has been shown to promote the proliferation of CK14-positive keratinocytes in the basal layer of the epidermis close to the wound and in the lateral hair roots of the hair follicles. This proliferation of keratinocytes may contribute to rapid epidermal re-epithelialization at the time of initial skin regeneration by facilitating the movement of keratinocytes to the center of the wound where little Ki67-positive proliferating cells are found. According to some recent studies, the activation of hair follicle keratinocytes is essential for initiating rapid epidermal re-epithelialization shortly after injury and also constitutes a significant portion of the epidermal layer where the progeny of these cells newly form at the site of injury. (Langton AK, et al., An extended epidermal response heals cutaneous wounds in the absence of a hair follicle stem cell contribution. J Invest Dermatol 128: 1311-1318, 2008). According to the previous reports, the abundant proliferation of vesicular keratinocytes observed in the EPC-treated group will help speed the onset of the wound healing process. The fact that EPC-treated wounds exhibit many Ki67-positive cells and rapid wound closure in the very early wound healing process suggests that EPC can directly activate the proliferation of keratinocytes and fibroblasts. However, subsequent blood supply for EPC-mediated angiogenesis and cell growth may be possible as a mechanism to support early wound healing. However, it is not clear which mechanism plays a major role in EPC-mediated tissue regeneration.

In this regard, the comparison of EPC-transplanted and EPC-CM-injected wounds has fully demonstrated what mechanisms are involved in the observed EPC-mediated wound healing effects. The results indicate that paracline factors secreted from EPC are sufficient for efficient wound healing in diabetic mice. The results also indicate that EPC-mediated vasculogenesis is not related to improved healing rate and enhanced neovascularization at the wound site observed in EPC. Although some injected human EPCs have been physically conjugated to mouse blood vessels, it has been shown that EPCs promote wound regeneration of wounds, primarily by stimulating mouse angiogenesis through the secretion of paracline factors.

Neovascularization of human EPCs in diabetic mice has recently been reported, and EPC transplantation has been reported to relieve diabetic neuropathy in rodents by promoting vascularization around peripheral nerves (Naruse K, et al., Therapeutic neovascularization using cord blood-derived endothelial progenitor cells for diabetic neuropathy Diabetes 54:... 1823-1828, 2005; and JO Jeong, et al, Dual angiogenic and neurotrophic effects of bone marrow-derived endothelial progenitor cells on diabetic neuropathy Circulation 119 : 699-708, 2009). However, this study does not directly disclose the effect on EPC transplantation on diabetic wound healing. This study was the first to identify the therapeutic effect of human umbilical cord blood-derived EPCs in diabetic wound healing. Importantly, the results demonstrate that EPC-CM is therapeutically equivalent to EPC, at least in terms of wound healing rate, granulation tissue formation, and neovascularization. EPC-CM can act as an alternative treatment without immune side effects for allogeneic transplantation, which must be carefully considered in non-autologous cell injection.

1 shows the results of evaluating the effect of EPC transplantation on incisional wound healing in STZ-induced diabetic mice. (A) shows images of EPC- and PBS-injected wounds of STZ-induced BALB / c nude mice at predetermined time points after wound induction. Scale bar is 2 mm. (BC) shows the open wound area (B) expressed as a percentage of the initial wound area and the relative granulated tissue area (C) on day 20 measured in wounds treated with EPC (white) or PBS (black). * p <0.05, n = 6. Data is mean ± SEM.

2 shows the results of evaluating the effect of EPC transplantation on in vivo proliferation of keratinocytes and fibroblasts during the initial wound healing process. (A) is an immunofluorescence image of epidermal tissue obtained from wounds treated with PBS- or EPC three days after wound induction. Proliferating keratinocytes were measured by coimmunofluorescence by staining with antibodies to Ki67 (red, nuclear) and cytokeratin (green, cytoplasmic). Nuclei were stained with DAPI (blue). Arrows indicate epidermal wound edges; e, epithelium; d, dermis; h, hair follicle; And es, eschar. The lower image shows a high magnification image of the wound edge, distal epidermis, and hyperthickened epidermis in the hair follicles shown in the upper image. (B) is an immunofluorescence image of granulated tissue obtained from wounds treated with PBS- or EPC on day 7. Proliferating fibroblasts were measured by coimmunofluorescence by staining with antibodies to Ki67 (red, nucleus) and non-mentin (green, cytoplasmic). Nuclei were stained with DAPI (blue). Scale bar is 100 μm.

Figure 3 shows the results of evaluating the therapeutic effect of EPC-CM and EPC on the enhancement of wound healing and neovascularization in STZ-induced diabetic mice. (A) shows images of wounds injected with EPC-CM- or M199 basal medium in STZ-induced diabetic BALB / c nude mice. Scale bar is 2 mm. (B) shows the result of quantifying the open wound area obtained from the four experimental groups at a predetermined time point (○, EPC; ●, PBS; □, EPC-CM; ■, M199). * p <0.05, n = 7. Data is mean ± SEM. (C) shows the granulated tissue area measured 15 days after wounding. * p <0.05, n = 7. NS, non-significant. Data is mean ± SEM. (D) is a vWF-stained image of granulated tissue obtained from M199-, EPC-CM-, PBS-, or EPC-treated mice. Incisional wound tissue was harvested 15 days after wound induction, and rodent vasculature in the granulated tissue was immunohistochemically stained with anti-vWF (arrow) or nonspecific IgG. Scale bar is 100 μm. (E) shows capillary density in granulated tissue measured as the number of vWF-positive vessels per high power field (hpf). * p <0.05, n = 5. Data is mean ± SEM. (F) represents infused human EPC directly incorporated into newly formed capillaries in granulated tissue. Immunohistochemical analysis was performed on EPC- or PBS-injected wounds using human (red) and mouse (green) specific CD31 antibodies. Scale bar is 50 μm.

Claims (7)

A pharmaceutical composition for preventing or treating diabetic wounds, comprising a culture of endothelial progenitor cells or a fraction thereof as an active ingredient. The method of claim 1, wherein the culture of the endothelial progenitor cells comprises a supernatant obtained by centrifuging the culture solution of endothelial progenitor cells; Concentrate of the supernatant; Or a lyophilisate of the supernatant. The method according to claim 1, wherein the fraction of the culture of the endothelial progenitor cells is a fraction obtained by fractionating the supernatant obtained by centrifuging the culture solution of the endothelial progenitor cells to contain at least 10 kDa protein; Concentrate of said fraction; Or a lyophilisate of the fraction. The pharmaceutical composition according to claim 2 or 3, wherein the culture of endothelial progenitor cells is obtained by culturing endothelial progenitor cells in serum-free medium. delete The pharmaceutical composition of claim 1, wherein the endothelial progenitor cells are endothelial progenitor cells derived from umbilical cord blood. The pharmaceutical composition of claim 1, wherein the diabetic wound is diabetic foot ulcers, diaebtic angiopathy, or diabetic foot infections.

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