Reviews Adv Clin Exp Med 2012, 21, 2, 249–254 ISSN 1899–5276
© Copyright by Wroclaw Medical University
Ewelina Drela, Katarzyna Stankowska, Arleta Kulwas, Danuta Rość
Endothelial Progenitor Cells in Diabetic Foot Syndrome Śródbłonkowe komórki progenitorowe w zespole stopy cukrzycowej Department of Pathophysiology, Nicolaus Copernicus University Collegium Medicum in Bydgoszcz, Poland
Abstract In the late 20th century endothelial progenitor cells (EPCs) were discovered and identified as cells capable of differentiating into endothelial cells. Antigens characteristic of endothelial cells and hematopoietic cells are located on their surface. EPCs can proliferate, adhere, migrate and have the specific ability to form vascular structure, and they have a wide range of roles: They participate in maintaining hemostasis, and play an important part in the processes of vasculogenesis and angiogenesis. They are sources of angiogenic factors, especially vascular endothelial growth factor (VEGF). EPCs exist in bone marrow, from which they are recruited into circulation in response to specific stimuli. Tissue ischemia is thought to be the strongest inductor of EPC mobilization. Local ischemia accompanies many pathological states, including diabetic foot syndrome (DFS). Impaired angiogenesis – in which EPCs participate – is typical of DFS. An analysis of the available literature indicates that in diabetic patients the number of EPCs declines and their functioning is impaired. Endothelial progenitor cells are crucial to vasculogenesis and angiogenesis during ischemic neovascularization. The pathomechanisms underlying impaired angiogenesis in patients with DFS is complicated, but the discovery of EPCs has shed new light on the pathogenesis of many diseases, including diabetes foot syndrome (Adv Clin Exp Med 2012, 21, 2, 249–254). Key words: endothelial progenitor cells, diabetic foot syndrome.
Streszczenie Śródbłonkowe komórki progenitorowe (EPCs – endothelial progenitor cells) zostały odkryte jako komórki wykazujące zdolność do różnicowania w komórki śródbłonka. Na swej powierzchni mają antygeny charakterystyczne zarówno dla komórek śródbłonka, jak i komórek hematopoetycznych. EPCs wykazują cechy proliferacji, adhezji, migracji oraz posiadają swoistą zdolność tworzenia struktur naczyniowych. Wachlarz ról pełnionych przez EPCs jest szeroki: biorą udział w utrzymaniu hemostazy, odgrywają istotną rolę w procesach waskulogenezy i angiogenezy. Są źródłem czynników angiogennych, zwłaszcza naczyniowo śródbłonkowego czynnika wzrostu (VEGF – vascular endothelial growth factor). EPCs egzystują w szpiku kostnym, skąd są rekrutowane do krążenia pod wpływem określonych bodźców. Za najsilniejszy czynnik mobilizujący EPCs do krwi krążącej jest uważane niedotlenienie tkanek. Miejscowa hipoksja towarzyszy wielu patologicznym stanom, w tym zespołowi stopy cukrzycowej (DFS – diabetic foot syndrome). DFS charakteryzuje się nieprawidłową angiogenezą, w której biorą udział EPCs. Z analizy dostępnej literatury wynika, iż liczba EPCs u pacjentów z cukrzycą jest zmniejszona oraz obserwuje się upośledzenie ich funkcji. Śródbłonkowe komórki progenitorowe są kluczowym elementem w budowaniu naczyń krwionośnych podczas neowaskularyzacji indukowanej niedotlenieniem. Patomechanizm leżący u podłoża nieprawidłowej angiogenezy u pacjentów z DFS jest skomplikowany, a odkrycie EPCs rzuciło nowe światło na patogenezę wielu schorzeń, w tym zespołu stopy cukrzycowej (Adv Clin Exp Med 2012, 21, 2, 249–254). Słowa kluczowe: śródbłonkowe komórki progenitorowe, zespół stopy cukrzycowej.
The existence of endothelial progenitor cells (EPCs) was suggested in the second half of the 20th century. Pearson noted that in 1963 Stump et al. published the results of a study in which they observed that dacron grafts implanted in a pig thoracic aorta were completely covered by a monolayer of non-thrombogenic cells that were histologically similar to cells of the endothelium
[1]. Pearson also pointed out that the origin of endothelial cells was described by Risau and Flamme in the 1990s, when they postulated that endothelial cells and hematopoietic cells derived from the same mesenchymal precursor: the hemangioblast, and described the differentiation of precursor cells into endothelial cells taking place during embryonic vasculogenesis [1].
250 As Pearson notes, a study conducted by Takayuki Asahara et al. in 1997 was a milestone in the discovery of EPCs: The authors isolated monocytic cells from human blood which, in appropriate culture conditions, showed the expression of markers characteristic of endothelial cells (CD31+, E-selectin+, eNOS+ and uptake of modified low-density lipoprotein [LDL]) [1]. Up until then, the widelyheld view was that the differentiation of mesenchymal cells occurs exclusively during embryonic development, but the findings reported by Asahara et al. demonstrated the existence of hematopoietic progenitor cells in adults. Urbich and Dimmeler noted that in 1998, Shi et al. described circulating endothelial progenitor cells (CEPCs) in adults [2]. EPCs and CEPCs have specific antigens for hematopoietic stem cells and for endothelial cells [2]. The discovery of endothelial progenitor cells has shed a new light on the pathogenesis of many diseases, including diabetes mellitus, and has opened a new avenue in their treatment.
Endothelial Progenitor Cells EPCs are cells with the ability to differentiate into endothelium cells. They demonstrate the expression of markers characteristic of hematopoietic cells (CD34+, CD133+) and endothelial cells (KDR, vWF, CD31+, VE-cadherin, eNOS+, uptake of acetylated LDL and binding lectins such as Ulex europaeus) [3–5]. EPCs are a heterogenic group of cells that show the expression of various surface antigens. Immature endothelial cells have a marker peculiar to stem cells: CD133 (also known as AC133). During the differentiation process this antigen gradually disappears; mature endothelial cells do not have CD133, but still show the expression of CD34, another stem cell marker [4, 6]. EPCs present in peripheral blood can be at different stages of endothelial differentiation, so researchers distinguish different subpopulations of EPCs. Silvestre demonstrated the presence of early and late EPCs derived from cultured human peripheral blood mononuclear cells [5]. Early EPCs express antigens characteristic of the monocytic lineage. The peak growth of this population of EPCs occurs during the 2nd and 3rd weeks of cultivation; they do not survive beyond the 4th week. Late EPCs, which demonstrate the expression of VE-cadherin, Flt-, KDR and CD45, appear between the 2nd and 3rd weeks of cultivation with expotential growth from the 4th to 8th weeks, and live for an average of 12 weeks [5]. It is known that circulating CD34+ cells have vasculogenic properties and are able to differentiate into cells with endothelial characteristics [4]. However, cells characterized as EPCs may derive from
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the CD14+myeloid/monocyte lineage [5]. Pearson wrote that in 2003 Rehman et al. conducted a study in which they observed that cells forming early EPC colonies were characterized by a weak expression of the CD34 antigen, but showed strong expression of CD45 (a panleukocyte marker) and CD14 (a monocyte marker); they also found that these cells were barely able to proliferate [1]. Rehman et al. suggested that these cells were not progenitors of the endothelium, but that they could participate in the angiogenesis process in vivo due to their secretion of angiogenic mediators such as vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor (G-CSF) [1]. In Urbich and Dimmeler’s opinion the same controversies concern the origin of EPCs isolated from mononuclear cells from human peripheral blood [2]. They wrote: “In peripheral blood mononuclear cells, several possible sources for endothelial cells may exist: (1) the rare number of hematopoietic stem cells, (2) myeloid cells, which may differentiate to endothelial cells under the cultivation selection pressure, (3) other circulating progenitor cells (eg, “side population” cells), and (4) circulating mature endothelial cells, which are shed off the vessel wall” [2]. Circulating blood is not only the source of EPCs. As Ranjan et al. pointed out [7], various researchers have isolated EPCs from umbilical cord blood [8], bone marrow [9], adipose tissue [10], skeletal muscles [11] and from such organs as the heart [12] and spleen [13]. EPCs exist in the bone marrow, from which they are recruited under the influence of specific stimulating signals and become circulating EPCs (CEPCs) [3, 14, 15]. In physiological conditions the number of circulating EPCs is low, but it increases in response to stimuli. Hypoxia is regarded as the strongest inductor for mobilizing EPCs from the bone marrow into circulation [3, 15, 16]. Hypoxia mobilizes EPCs via the hypoxia-sensing system, which involves hypoxia-inducible factor 1 (HIF-1). HIF-1 is a transcription factor composed of alpha and beta subunits. The HIF-1β subunit is expressed constitutively, but expression of the HIF-1α subunit is regulated by the concentration of oxygen [3]. At the correct oxygen concentration, the HIF-1α subunit is quickly degraded by ubiquitin proteolysis. The ischemia state slows down the degradation of the alpha subunit, which can dimerize with the beta subunit. The active HIF1 factor binds to the correct region of the DNA and activates the transcription of genes, including the vascular endothelial growth factor gene SDF1α and the erythropoietin gene [3]. The kinase insert domain receptor (KDR) is a receptor for VEGF located on the surface of bone marrow stem cells. The transduction of a signal by a EGF-KDR
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complex activates endothelial nitric oxide synthase (eNOS). Next, nitric oxide activates matrix metalloproteinase 9 (MMP-9), which releases the stem cell-active cytokine, soluble Kit ligand. This cytokine causes hematopoietic stem cells to leave their quiescent niche and pass to a proliferation niche, and stimulates them to enter circulation [15, 16]. Growth factors (mainly VEGF) regulate EPC migration, but chemokin SDF-1α plays a role in EPC homing to ischemic tissue [1, 2, 15]. Fadini et al (2007) put forward the hypothesis that an inability to mobilize EPCS from bone marrow is connected with the downregulation of HIF-1α and a reduced concentration of factors like VEGF and SDF-1α [3]. Endothelial progenitor cells can proliferate, adhere, migrate and have the specific ability to form vascular structures [3]. Their proliferation is manifested as the ability to expand and form colonies in culture, and it is induced by local ischemia or growth factors. Angiogenesis or reendothelialization require another property of EPCs: adhesion [3], which is possible because of the presence of an adhesion molecule and selectin on the surface of the cells (β2-integrins or L-selectin) [5]. Integrins play an important role in mediating cell-to-cell interactions. They enable various cells, including hematopoietic cells, to adhere to the extracellular matrix (ECM) and endothelial cells. β2-integrins and α4β1integrins are responsible for cell-to-cell interaction. β1-integrins are located on the surface of various cell types, including endothelial cells; β2-integrins are preferentially expressed by hematopoietic cells. β2-integrins can also be found on peripheral bloodderived EPCs and are needed for EPC adhesion to endothelial cells and transendothelial migration in vitro [2]. Thus, the presence of adhesion molecules is required for ischemic tissue to arrest EPCs. After adhesion, endothelial progenitor cells migrate through the ECM. The highest form of specialization for functional EPCs is the ability to form vascular tubes. This property is necessary for the creation of new vascular vessels [2, 5, 14]. Endothelial progenitor cells take part in hemostasis, constituting a circulating pool of cells with the ability to “patch” injuries to the endothelial layer by replacing cells which have succumbed to apoptosis or been lost in other circumstances [3, 17]. EPCs play an important role in vasculogenesis and angiogenesis. Vasculogenesis, which dominates in the embryonic period, causes vessels to be created de novo. It was previously thought that post-natal creation of blood vessels occurs via angiogenesis, through the migration and proliferation of mature endothelial cells, but the isolation of EPCs from peripheral blood indicates that these cells participate in vasculogenesis in adults [4, 18].
EPCs in Diabetic Foot Syndrome Bone-marrow-derived endothelial progenitor cells are the crucial elements in post-ischemic neovascularization [19]. Hypoxia is a pathological state that occurs in various diseases, especially in diabetes. Among the consequences of long-lasting unregulated type 2 diabetes mellitus (T2DM) are angiopathic complications: microangiopathy and macroangiopathy. Coexisting neuropathy and impaired wound healing contribute to the development of diabetic foot syndrome (DFS). DFS is the most frequent cause of amputation: According to the International Diabetes Federation (IDF), worldwide, every 30 seconds an amputation of a lower limb is performed because of diabetes [20]. The pathogenic model underlying abnormal angiogenesis in diabetic patients is complicated. The isolation of EPCs has contributed to understanding the processes involved. Studies conducted by independent groups of scientists show that abnormal EPC functioning and impaired EPC mobilization from the bone marrow in diabetic patients is caused by hyperglycemia [21]. The number of circulating EPCs is small under normal circumstances, but is elevated in response to hypoxia or injury [18]. Reduced numbers of EPCs and impairment of their functioning are observed in patients with diabetes and in animals with induced diabetes [17, 21]. A study conducted by Loomans et al. shows the influence of hyperglycemia on EPCs [17]. The study involved mice in which hyperglycemia had been induced by intraperitoneal injections of streptozotocin (80 mg/kg body weight) and a control group of healthy mice. The authors noted a 40% reduction in the number of EPCs and a 50% increase in the number of macrophages in the mice with hyperglycemia as compared to the control group. In the authors’ opinion, the reduction in the number of EPCs and their impaired functioning were the result of the toxic influence of hyperglycemia on myeloid bone marrow progenitors [17]. Similar findings were reported by Fadini et al. (2005), who showed that a high glucose concentration could initiate the process of apoptosis in cultured endothelial cells [21]. A common complication of diabetes mellitus is peripheral vascular disease (PVD). The group examined by Fadini et al. was made up of 51 patients: 24 diabetic patients with PVD, 16 diabetics without PVD and 11 nondiabetics with PVD. The control group consisted of 17 healthy volunteers. The researchers reported a decreased number of EPCs and circulating progenitor cells (CPCs) in diabetic patients as compared to
252 the healthy volunteers, stating that in patients with T2DM the number of EPCs in peripheral blood is reduced by about 40%. What is more, comparing only the subgroups with PVD, a reduced number of CPCs was noted in the patients with diabetes; and a decreased number of EPCs and CPCs was noted in diabetic patients with ischemic foot as compared to the control group and to diabetic patient with PVD but without ischemic foot [21]. The influence of hyperglycemia on circulating progenitor cells was also studied by Krankel et al., who observed that a high concentration of glucose was a crucial factor in inducing apoptosis and reducing the proliferation of CPCs. They also found that hyperglycemia reduced MMP-9 activity [22]. The decreased number of EPCs in diabetic patients is connected with impaired recruitment of these cells from the bone marrow. A study conducted by Gallagher et al. on an animal model demonstrated that reduced EPC mobilization is a result of impaired eNOS phosphorylation in the bone marrow [15]. The authors found a 50% reduction in circulating EPCs in diabetic mice as compared to mice without induced diabetes. They also observed decreased SDF-1α expression on myofibroblasts and endothelial cells isolated from diabetic peripheral cutaneous wounds [15]. Caballero et al. also noted reduced EPC mobilization from bone marrow and decreased EPC numbers in circulation in patients with diabetes [23]. CD34 cells isolated from diabetic patients demonstrated abnormal migration in response to SDF-1α EPCs isolated from diabetic individuals show disorders of proliferation, adhesion and deformability compared with nondiabetic EPCs. Caballero et al. hypothesized that EPC homing to wounds may be impaired by the influence of advanced end glycation products (AGEs) [23]. AGEs modify blood vessel walls, especially the basement membranes, through cross-linking; the altered vessel wall makes it difficult to recruit EPCs to the angiogenesis site and hindering their participation in the whole repair process [23]. Yan et al. suggested that abnormal EPC functioning results from coexisting oxidative stress [19]. Their results show that EPC concentration in peripheral blood was higher in the control group than in mice with type 1 or type 2 diabetes. Increased EPC migration induced via VEGF was observed in the control group as compared to the diabetic groups [19]. Some authors have observed an inverse correlation between glycated hemoglobin and the number of endothelial progenitor cells [17, 18, 24]. Churdchomjan et al. published their study “Comparison of endothelial progenitor cell function in type 2 diabetes with good and poor glycemic control compare EPC function in type 2 dia-
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betes with good and poor glycemic control” [14], in which they observed that the number of EPCs was higher in patients with good glycemic control than in patients with poor control. They suggested that the decreased number of EPCs was caused by decreased survival of these cells in circulation [14]. This study demonstrates the importance of diabetes management, but it is a problem that requires further research. In patients with diabetes, a paradox is observed: Depending on the kind of tissue, different concentrations of the same cytokines (VEGFs) and EPCs are found. Retinopathy shows increased VEGF level and EPCs in the retina, while diabetic patients with peripheral arterial disease demonstrate a decreased number of EPCs [25]. In diabetic foot syndrome, which is a late diabetes complication, local decreased VEGF synthesis and EPC reduction is observed. These changes contribute to abnormal angiogenic response. Impaired angiogenesis leads to tissue ischemia. A consequence of the long-lasting ischemia is the development of non-healing wounds and in severe cases necrosis, for which an amputation is the only effective therapy. Impairment of the blood vessel regeneration process seems to be an important reason for the amputation of lower limbs in patients with DFS. More and more attention is being paid to the genetic mechanism underlying impaired endothelial progenitor cell recruitment from bone marrow in patients with DFS. An example is the study investigating the relationship between the SDF-1 genotype and neovascularization in diabetes foot syndrome [26]. Humpert et al. noticed that in patients with DFS and accompanying macrovascular disease, heterozygous SDF-1 3. A genotype appeared more often [26]. This chemokine is responsible for EPC homing. On the other hand, it induces platelet aggregation and is expressed on atherosclerotic plaques [26]. It may be hypothesized that excessive expression of the specific SDF-1 genotype may increase the risk of macroangiopathy, thus limiting the role of homing cytokine. The authors concluded that the role of endothelial progenitor cells in post-natal neovasculariazation is indisputable. This process is very important for patients with diabetic foot syndrome. Abnormal biological activity and a decline in the mobilization of EPCs from bone marrow are results of the toxic influences of hyperglycemia. The decreased secretion of proangiogenic factors and reduced number of EPCs – elements that are crucial to the formation of blood vessels formation – contribute to an abnormal process of angiogenesis. Exploring the connection between EPCs, VEGF and DFS appears to be a worthwhile avenue for further research.
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Address for correspondence: Ewelina Drela Department of Pathophysiology Collegium Medicum in Bydgoszcz Skłodowskiej-Curie 9 85-094 Bydgoszcz Poland E-mail:
[email protected] Tel.: +48 52 585 35 91 Conflict of interest: None declared Received: 22.03.2011 Revised: 6.10.2011 Accepted: 29.03.2012
