Identification Of Endothelial Progenitor Cells Research Articles

Baicalin promotes migration and angiogenesis of endothelial progenitor cells but impedes thrombus formation via SIRT1/NF-κB signaling in a rat model of deep vein thrombosis.

Deep vein thrombosis (DVT) is the third most prevalent vascular disease worldwide, seriously threatening human health. Baicalin, a flavonoid isolated from the roots of Scutellaria baicalensis, has been identified to play a crucial role in various vascular diseases. The study aimed to explore the efficacy and underlying mechanisms of baicalin in DVT. Endothelial progenitor cells (EPCs) were differentiated from peripheral blood mononuclear cells isolated from rat bone marrow. Dil-ac-LDL/FITC-UEA-1 double staining and flow cytometry analysis were conducted for the identification of EPCs. The angiogenesis and migration of EPCs in vitro were tested by a tube formation assay and Transwell assay, respectively. DVT rat models were established by stenosis of the inferior vena cava (IVC). After the euthanasia of rats, thrombi in the IVC were collected and weighed, and histological alterations in IVC tissue were measured by H&E staining. The protein levels of SIRT1, p-P65, and p65 in rat IVC tissues were quantified via western blotting. EPCs used in this study displayed a spindle-like shape and were positive for endothelial cell-specific markers, suggesting the phenotypic characteristics of EPCs. Baicalin enhanced the migratory and angiogenetic abilities of EPCs in vitro. For in vivo experiments, baicalin reduced thrombus weight and mitigated DVT formation in model rats. Moreover, baicalin activated SIRT but repressed NF-κB signaling in IVC tissues of DVT rats. Baicalin facilitates migration and angiogenesis of EPCs but impedes thrombus formation via regulation of SIRT1/NF-κB signaling in DVT model rats.

Novel cell therapy with ex vivo cultured peripheral blood mononuclear cells significantly impacts angiogenesis in the murine ischemic limb model

IntroductionAutologous mononuclear cells (MNCs) have been used in vascular regenerative therapy since the identification of endothelial progenitor cells (EPCs). However, the efficacy of autologous EPC therapy for diseases such as diabetes and connective tissue disorders is limited due to deficiencies in the number and function of EPCs. To address this, we developed a novel RE-01 cells that enriches pro-angiogenic cells from peripheral blood MNCs (PBMNCs). MethodsPBMNCs were collected from healthy volunteers following ethical guidelines. RE-01 cells were cultured in the presence of specific growth factors for 5 days without media change. Flow cytometry was used to analyze cell surface markers. Tube formation assays, EPC culture assays, and mRNA analysis were performed to evaluate angiogenic potential. The efficacy of RE-01 cells upon transplantation into ischemic hind limbs of mice was evaluated. ResultsRE-01 cells exhibited a significant increase in pro-angiogenic cells such as M2 macrophages and angiogenic T cells, in contrast to PBMNCs, while the number of inflammatory cells reduced. In vitro assays demonstrated the enhanced angiogenic abilities of RE-01 cells, supported by increased mRNA expression of angiogenesis-related cytokines. In vivo studies using mouse ischemic hind limb models have shown that blood flow and angiogenesis improved following RE-01 cell transplantation. Transplantations for 3 consecutive days significantly improved the number of pericyte-recruited vessels in the severely ischemic hind limbs of mice. ConclusionsRE-01 cells showed promising results in enhancing angiogenesis and arteriogenesis, possibly owing to the presence of M2 macrophages and angiogenic T cells. These cells also demonstrated anti-fibrotic effects. The efficacy of RE-01 cells has been confirmed in mouse models, suggesting their potential for treating ischemic vascular diseases. Clinical trials are planned to validate the safety and efficacy of RE-01 cell therapy in patients with connective tissue disease and unhealed ulcers. We hope that this new RE-01 cell therapy will prevent many patients from undergoing amputation.

Effects of Garlic Extract (allicin) on Proliferation of Endothelial Progenitor Cells (EPC) in Patients with Stable Coronary Artery Disease

The reduced number and function of Endothelial Progenitor Cells (EPC) in stable coronary artery disease (SCAD) patients aggravate endothelial dysfunction and inhibit neovascularization, thus leading to atherosclerosis. Garlic is currently believed to increase the number and function of EPCs. Therefore, this in vitro study was conducted to analyse the effect of garlic extract (Allicin) on the proliferation of EPCs in patients with SCAD. Mononuclear cells were isolated from peripheral blood of eight SCAD patients and cultured on CFU-Hill media for three days. Samples were divided into 2 groups: a group treated with Allicin and a control group. The treatment group was then divided into 3 subgroups which received 10, 50, and 100 mg/ml of doses and incubated for 48 hours. EPC proliferation was assessed using MTT Cell Proliferation Assay. Immunohistochemical method of CD34+ were performed for EPC identification. Data was analysed using an independent T test and ANOVA. MTT Assay showed significant increase in EPC proliferation in Allicin group compared to control group (0.2811±0.008 vs 0.194±0.151, p<0.05) and significant improvements were observed in each dose increment. CFU-Hill quantification shows the addition of EPC colony in high-dose Allicin. Immunohistochemical method shows positive CD34+ expression. Allicin increases EPC proliferation dose-dependently from peripheral blood of SCAD patients.

Mechanisms of regeneration of the endothelium at diabetes mellitus

Vascular endothelium is the main organ, suffering from diabetes mellitus (DM) and cardiovascular diseases (CVD). In this review, the role of endothelial progenitor cells (EPCs) in the regeneration of the endothelium and in the formation of new blood vessels is considered. Mechanisms of migration and mobilization of EPCs from the bone marrow to the damage zone are described. The analyzed data show that CVD and DM cause the decrease in the number and the disturbance of the function of EPCs. The data on the heterogeneity of the population of EPCs are presented in article. The various combinations of surface markers for identification of these cells are assessed. At the same time, protocols for the identification of EPCs have not been developed, which confirms the relevance of the search for the phenotype of EPCs, which would be adopted as the standard.

Intrinsic Vascular Repair by Endothelial Progenitor Cells in Acute Coronary Syndromes: an Update Overview.

Bone marrow-derived endothelial progenitor cells (EPCs) play a key role in the maintenance of endothelial homeostasis and endothelial repair at areas of vascular damage. The quantification of EPCs in peripheral blood by flow cytometry is a strategy to assess this reparative capacity. The number of circulating EPCs is inversely correlated with the number of cardiovascular risk factors and to the occurrence of cardiovascular events. Therefore, monitoring EPCs levels may provide an accurate assessment of susceptibility to cardiovascular injury, greatly improving risk stratification of patients with high cardiovascular risk, such as those with an acute myocardial infarction. However, there are many issues in the field of EPC identification and quantification that remain unsolved. In fact, there have been conflicting protocols used to the phenotypic identification of EPCs and there is still no consensual immunophenotypical profile that corresponds exactly to EPCs. In this paper we aim to give an overview on EPCs-mediated vascular repair with special focus on acute coronary syndromes and to discuss the different phenotypic profiles that have been used to identify and quantify circulating EPCs in several clinical studies. Finally, we will synthesize evidence on the prognostic role of EPCs in patients with high cardiovascular risk.

Therapeutic Potential of Endothelial Progenitor Cells in the Field of Orthopaedics.

Inadequate blood supply frequently impedes the viability of tissue-engineered constructs in the initial phase after implantation, and can lead to improper cell integration or cell death. Vascularization using stem cells has continued to evolve as a potential solution to this problem. In this review, we summarize studies that utilize endothelial progenitor cells (EPCs) for musculoskeletal regeneration. This review will also highlight recent concepts for EPC identification in conjunction with the development of EPC biology research. EPCs promote bone regeneration in animal models through a variety of mechanisms. By differentiating toward endothelial cell lineages and osteoblasts, EPCs stimulate vasculogenesis, angiogenesis and osteogenesis. Moreover, EPCs influence supporting cells through the secretion of growth factors and cytokines. Phase I/II clinical trials have applied circulating CD34+ cells/EPCs to nonunion bone fractures and have exhibited promising results including accelerated bone healing. Similar mechanisms of angiogenesis and osteogenesis are proposed for anterior cruciate ligament (ACL) ruptured tissue derived CD34+ cells, and thus EPCs have implied a critical role at the site of tendon-bone integration. EPCs are an emerging strategy among other cell-based therapies in the field of orthopaedics for the promotion of musculoskeletal regeneration.

Concurrent hypermulticolor monitoring of CD31, CD34, CD45 and CD146 endothelial progenitor cell markers for acute myocardial infarction

The circulating endothelial progenitor cells (EPCs) in blood of acute myocardial infarction (AMI) patient have been monitored in many previous studies. The number of circulating EPC increases in the blood of patients at onset of the AMI. EPC is originated from bone marrow. It performs vessel regeneration. There are many markers used for detecting EPC. Four of these markers, CD31, CD34, CD45, and CD146, were concurrently detected at the single cell level for the identification of EPC in the present preliminary study. The CD45 negative cell sorting was performed to peripheral blood mononuclear cells (PBMCs) acquired from four AMI patients with a magnetic bead sorter, since, EPCs expressed CD45 negative or dim. The resultant PBMC eluents were treated with quantum-antibody conjugates for the probing four different markers of EPCs and then applied to a high-content single cell imaging cytometer using acousto-optical tunable filter (AOTF). The use of quantum dot, with narrow emission wavelength range and AOTF enabling cellular image at a particular single wavelength, is very advantageous for accurate high-content AMI diagnosis based on simultaneous monitoring of many markers. The number of EPC increased as compared with control in three of four AMI patients. In this approach, two EPC subtypes were found, CD31(+), CD34(+), CD45(−/dim), CD146(−) as early outgrowth EPCs and CD31(+), CD34(+), CD45(−/dim), CD146(+) as late outgrowth EPCs. Patient 1 had CD31(+), CD34(+), CD45(−/dim), CD146(+) cells whose percentage was 4.21% of cells. Patient 2 had 2.38% of CD31(+), CD34(+), CD45(−/dim), CD146(−) cells and patient 3 had 4.28% of CD31(+), CD34(+), CD45(−/dim), CD146(+) cells.

Identification of endothelial progenitor cells in the corpus cavernosum in rats.

The vascular wall resident progenitor cells seem to serve as a local reservoir of cells for vascular repair. It was hypothesized that the corpus cavernosum may contain vascular wall endothelial progenitor cells (EPCs). In this study, we investigated the identification and localization of EPCs in the corpus cavernosum in a rat model. Adult male Sprague-Dawley rats were used to isolate EPCs from corpora cavernosum. To verify the existence and localization of EPCs, EPC-specific markers (CD34, Flk-1, and VE-cadherin) were evaluated by flow cytometric analysis and confocal microscopy. The EPC markers were mainly expressed in the cavernosal sinusoidal endothelial space. EPC-marker-positive cells made up about 3.31% of the corpus cavernosum of normal rat by FACS analysis. As shown by confocal microscopy, CD34+/Flk-1+ and CD34+/VE-cadherin+ positive cells existed in the corpus cavernosum. Our findings imply that regulation of corpus cavernosal EPCs may be a new therapeutic strategy in the treatment of erectile dysfunction.

Authors′ Reply

Background Although in the general population circulating vascular progenitor cell levels have been implicated in the homeostasis of the vascular wall through differentiation into endothelium and/or smooth muscle cells, it has not yet been assessed in HIV-infected patients. We herein investigated the number of progenitor cell subpopulations in HIV-infected patients and its relationship to carotid intima-media thickness (c-IMT). Methods Blood samples were collected from 200 HIV-infected patients and CD34/KDR, CD34/VE-cadherin, and CD14/Endoglin progenitor cells were identified by flow cytometry. c-IMT was determined by ultrasonography. A group of 27 healthy subjects was used as control group. Results In our population (20 ART-naive patients and 180 treated patients), traditional cardiovascular risk factors were not found predictive of vascular progenitor cell levels. However, antiretroviral therapy (ART)-treatment was identified as the main predictive value for low CD34/KDR cells and high CD14/Endoglin cells after adjustment by cardiovascular risk factors (age, sex, hypertension, diabetes, and hyperlipidaemia) and HIV-related characteristics (HIV duration and ART treatment). Low levels of circulating CD34/KDR or CD34/VE-cadherin endothelial progenitor cells tended to be associated with increased c-IMT. However, a positive association was found between CD14/Endoglin cells and c-IMT. Low number of CD34/KDR cells was also associated with the longest exposure to nucleoside reverse transcriptase inhibitors and/or protease inhibitors. Conclusions ART exposure is the main predictor of circulating vascular progenitor cell levels. However, their levels are only partially associated with high c-IMT in HIV-infected patients. ART has already been found to have proatherogenic effect, but our data first describe its relationship with vascular progenitor cells and c-IMT.

Identification of CD133-, CD34- and KDR-positive cells in the bovine ovary: A new site of vascular wall resident endothelial progenitor cells

Intense angiogenesis, vascular remodelling as well as regression of its vasculature are prerequisites for ovarian function with its cyclically developing and regressing follicles and corpora lutea. So far neither a stringent explanation for the enormous angiogenic potential of the ovary nor its cellular origins have been suggested. In an earlier study of our work group, endothelial cells were isolated from the bovine corpus luteum and cultivated in vitro. They performed vasulogenesis in vitro and showed properties of progenitor cells. The present study aimed at in situ identification of endothelial progenitor cells (EPCs) in the bovine ovary. Immunohistochemical examinations, based on the detection of KDR and CD34 co-labelled cells - a marker combination that amongst others is commonly accepted as typical for EPC identification - were performed. Hormonal cycle dependent expression varieties were analysed by the measurement of mRNA amounts of CD34 and KDR as well as the stem cell marker CD133 (Prominin-1). Ovarian samples comprising corpora lutea of varying stages (developing and mature corpus luteum, corpus luteum in regression, corpus luteum of pregnancy) from 17 adult cows were examined. Results show that specific mRNA of CD133, CD34 and KDR was expressed in ovaries of all luteal stages. Expression data analysis revealed significant differences in CD133 and CD34 expression levels between the luteal stages but no significant differences in KDR expression. CD34/KDR co-immunoreactive cells were predominantly situated within the media of arterial vessel wall. The detection of ovarian EPCs represents an important step towards further understanding of the mechanisms involved in the reproductive biology and pathophysiology of the ovary.

CD45-CD34+ Endothelial Progenitor Cells (EPCs) from Human Adipose Tissue Promote Tumor Growth and Metastases

Abstract 2208A catalytic role has been proposed in neoplastic angiogenesis and cancer progression for bone marrow-derived endothelial progenitor cells (EPCs). However, in preclinical and clinical studies the quantitative role of marrow-derived EPCs in cancer vascularization was found to be extremely variable. Adipose tissue represents an attractive source of autologous adult stem cells due to its abundance and surgical accessibility.Lipotransfer aspirates (LAs) from patients undergoing breast reconstruction after breast cancer surgery were analyzed by six colors flow cytometry and tissue culture. After collagenase digestion, cells were stained with the nuclear binding antigen Syto16 and 7-AAD and with CD34, CD45, CD133, CD31, CD140b, CD105, CD90, CD44, CD13, CD144, CD10, CD29, CD109, CD117, CD146,CD16, CD11c, CD14, CD38, CXCR4, VEGFR-1, VEGFR-2, VEGFR-3, Tie-2. The absolute count of CD45-CD34+ cells was obtained using reference beads in Trucount tubes (BD, Mountain View, CA). LAs were found to contain a large amount of CD45-CD34+ cells fulfilling the most recent criteria for EPC identification. These CD45-CD34+ cells included two subpopulations: CD45-CD34++ CD13+ CD140b+ CD44+ CD90++ cells and CD45-CD34+ CD31+CD105+ cells. We found in the adipose tissue about 263 fold more CD45-CD34+ EPCs/mL when compared to the bone marrow. In particular, the median of CD45-CD34+CD31- cells/mL was 181,046 (range 35,970–465,357), and the median of CD45-CD34+CD31+ cells/mL was 76,946 (range13,982-191,287).When compared to marrow-derived CD34+ cells, purified CD45-CD34+ adipose cells expressed similar levels of stemness-related genes such as NANOG, SOX2, Lin28 and significantly increased levels of angiogenesis-related genes such as CD144, VEGFR2, ALK-1. In vitro, CD45-CD34+ cells generated mature endothelial cells and capillary tubes as well as mature mesenchymal cells.When coinjected with triple negative human breast cancer MDA-MB-436 and HCC1937 cells in the mammary fat of a murine model of human breast cancer, purified CD45-CD34+ cells significantly increased tumor growth, and immunohistochemistry studies demonstrated the presence of human CD31+, CD34+, CD105+ endothelial cells lining the vessels of orthotopic breast cancers growing in mice co-injected with human adipose tissue-derived CD45-CD34+ cells. Moreover, in a mouse model of breast cancer metastatization we found an increased number of lung and axillary lymph node metastases when purified CD34+ WAT cells were injected into the third mammary fat pad after the primary tumor resection.In conclusion our data demonstrate that the phenotype of adipose derived EPCs is consistent with that reported for both bone marrow and circulating EPCs, but their frequency in adipose tissue is more than 250 fold higher. Further studies are ongoing to clarify what cell populations residing in the adipose tissue can be used safely for breast reconstruction and what are at risk for supporting the growth of otherwise quiescent cancer cells still resident after surgery. Disclosures:No relevant conflicts of interest to declare.

Circulating endothelial progenitor cells, microparticles and vascular disease

Circulating endothelial progenitor cells, microparticles and vascular disease

Pro-angiogenic actions of Salvianolic acids on in vitro cultured endothelial progenitor cells and chick embryo chorioallantoic membrane model

Salvia miltiorrhiza (SM, also known as DanShen) is one of the well-known widely-used Chinese herbal medicines in clinical practice. In this study we aimed to demonstrate the pro-angiogenic effects of Salvianolic acids (SAs) to treat illnesses such as ischemic cardiovascular diseases, the main active components of aqueous extract of SM. To do this, new-born rat spleen mononuclear cells were isolated and endothelial progenitor cells (EPCs) were expanded (not more than 24h) SD. Then the pro-angiogenic activities of SAs were evaluated on in vitro cultured EPCs and chick embryo chorioallantoic membrane (CAM) model. And the adherent cells were stained with DiI complexed acetylated low-density lipoprotein (DiI-acLDL) and fluorescein Ulex Europaeus agglutinin-1 (FITC-UEA-1), and then viewed by laser scanning confocal microscope (LSCM) to confirm EPCs lineage. EPCs identification was also tested by ultrastructural analyses. EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, transwell chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. EPCs phenotype was confirmed by the presence of double positive cells for DiI-acLDL uptake and lectin binding and identification of Weibel-Palade body in cytoplasm by ultrastructural analyses. Incubation of EPCs with SAs increased the number of EPCs and promoted EPCs migratory, adhesive and in vitro vasculogenesis capacity. SAs also promoted angiogenesis as evidenced by CAM model. The results of the present study suggest that SAs may have utility for therapeutic postnatal vasculogenesis of ischemic tissue, contributing to the clinical benefit of SM therapy in patients with coronary artery disease.

Culture and identification of endothelial progenitor cells from human umbilical cord blood.

To elucidate a simple method for isolating endothelial progenitor cells (EPCs) from human umbilical cord blood mononuclear cells and observe the endothelial cell-specific expression profile during proliferation and differentiation in vitro. Human umbilical cord blood were isolated by Percoll density gradient centrifugation from human cord blood and cultured in vitro. The adherent cells were then identified by immunohistochemical staining and flow cytometric analysis. CD(34), vascular endothelial growth factor receptor-2 (VEGFR-2), EPCs specific antigen CD(133), as well as endothelial cell specific markers CD(31) and vWF were used. The cells were characterized by acetylated LDL (acLDL) up-taking and lectin binding by direct fluorescentstaining. During culture, the attached cells exhibited spindle-shape in early stage, and gradually display endothelium-like cobblestone morphology with outgrowth. On day 7, flow cytometric analysis showed that the positive staining rate of attached cells for CD(133), CD(34) and VEGFR-2 were 17.8%±3.7%, 22.1%±4.4% and 81.5%±5.0%, respectively. While, immunohistochemical staining showed that the adherent cells were positive to CD(31) and vWF at the rate of 92.7%±2.2% and 73.3%±4.2%, respectively. By direct fluorescentstaining, we observed that 83.0%±4.3% of the attached cells were double positive for DiI-acLDL and FITC-UEA-I. EPCs can be separated from human cord blood under certain conditions in vitro. This observation may provide a basis for study of relationship between EPCs and retinal neovascularization, as well as further clinical application of EPCs in ischemic retinal lesions.

Endothelial Nitric Oxide Synthase as A Marker for Human Endothelial Progenitor Cells

Endothelial progenitor cells (EPCs) have been proposed as a promising tool for therapeutic neovascularization, vascular repair, tumor pathology and tissue engineering, though their identification is still a subject of much discussion. EPCs consist of two different subpopulations, termed endothelial cell (EC)-like cells and endothelial outgrowth cells (EOCs). Both types of EPCs are derived from mononuclear cells, but they have different characteristics. Our aim was to characterize and compare the two types of EPCs to find reliable biological features of EPCs that can be used for identification of EPCs. In this study, human peripheral blood mononuclear cells were isolated by density gradient centrifugation and cultured on fibronectin-coated culture plates. While adherent cells were maintained, EC-like cells appeared within 4-7 days of culture, and EOCs developed after 2-3 weeks of culture. EOCs, which were characterized by high proliferation potential, were able to form capillary tubes on Matrigel, but not EC-like cells, despite the higher concentrations of three angiogenic cytokines, vascular endothelial growth factor, granulocyte colony-stimulating factor, and interleukin 8, in the conditioned medium of EC-like cells. In contrast, endothelial nitric oxide synthase (eNOS) was expressed in both types of EPCs, and both cell types could produce nitric oxide (NO), as judged by measuring the total amounts of nitrites and nitrates in culture media. In conclusion, the expression of eNOS and the production of NO could be used as common biological features to identify EPCs. These findings provide new insights into the identification of EPCs.

Endothelial progenitor cells dysfunction and senescence: contribution to oxidative stress.

The identification of endothelial progenitor cells (EPCs) has led to a significant paradigm in the field of vascular biology and opened a door to the development of new therapeutic approaches. Based on the current evidence, it appears that EPCs may make both direct contribution to neovascularization and indirectly promote the angiogenic function of local endothelial cells via secretion of angiogenic factors. This concept of arterial wall repair mediated by bone marrow (BM)-derived EPCs provided an alternative to the local “response to injury hypothesis” for development of atherosclerotic inflammation. Increased oxidant stress has been proposed as a molecular mechanism for endothelial dysfunction, in part by reducing nitric oxide (NO) bioavailability. EPCs function may also be highly dependent on a well-controlled oxidant stress because EPCs NO bioavailability (which is highly sensitive to oxidant stress) is critical for their in vivo function. The critical question is whether oxidant damage directly leads to an impairment in EPCs function. It was revealed that activation of angiotensin II (Ang II) type 1 receptor stimulates nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase in the vascular endothelium and leads to production of reactive oxygen species. We observed that Ang II accelerates both BM- and peripheral blood (PB)-derived EPCs senescence by a gp91phox-mediated increase of oxidative stress, resulting in EPCs dysfunction. Consistently, both Ang II receptor 1 blockers (ARBs) and angiotensin converting enzyme (ACE) inhibitors have been reported to increase the number of EPCs in patients with cardiovascular disease. In this review, we describe current understanding of the contributions of oxidative stress in cardiovascular disease, focusing on the potential mechanisms of EPCs senescence.

Endothelial Progenitors in Vascular Repair and Angiogenesis: How Many are Needed and What to do?

Defects in the regulation of neo blood vessel growth (angiogenesis) or in vessel repair are major complications in many diseases, such as cancer, diabetes, atherosclerosis and myocardial infarction. In these diseases it was shown that the number of circulating endothelial progenitor cells (EPC) was altered. This has been associated with the angiogenic status and patient prognosis. However, the regulation of angiogenesis depends not only on the number of circulating EPC but also on their functions. EPC are bone marrow derived cells that are recruited into the peripheral blood in situations of vascular repair/angiogenesis or vascular stress. EPC are believed to exert their function using mainly two strategies: activating locally the endothelial cells and/or differentiating into mature endothelial cells that integrate the damaged vessels. To do this, EPC must home to "angiogenic active" sites, adhere to the activated/damaged endothelial cells or to the extracellular matrix and participate in the endothelial activation/repair process. In vitro and in vivo experiments using animal models revealed the importance of various signalling pathways in these processes and, in patients, new therapeutic strategies are being developed based on the specific functions of EPC. Although the role of EPC in vessel repair in disease is not totally understood, it becomes clear that the activation state of these cells is critical for the vessel repair process. Our previous work generated a detailed gene expression profile of EPC during the endothelial differentiation process in vitro. With this information, it has been possible to identify numerous molecular targets crucial for EPC differentiation and function and to test their involvement in EPC function during wound healing or tumor angiogenesis. The importance of EPC identification, activation state and function in vascular repair and in angiogenesis in disease will be discussed in this review.

Vascular progenitor cells and diabetes: role in postischemic neovascularisation

Advances in the field of vascular biology lead to the identification of endothelial progenitor cells (EPC) and to the development of EPC-based cell therapy to induce new vessel formation in ischemic tissues and to accelerate re-endothelialisation of injured vessels in human and various animals models. However, recent studies have shown that age and other risk factors for cardiovascular diseases, such as diabetes, reduce the availability of EPC and impair their function to varying degrees, leading to reduction in postischemic vessel growth. This review focus on the cellular and molecular mechanisms governing EPC-related functions and analyzes the impact of diabetes in this setting.

Potencial terapéutico de las células progenitoras endoteliales en el ictus isquémico

To review the present knowledge about endothelial progenitor cells (EPCs), their relationship with stroke and their possible therapeutic potential. Activation of angiogenesis and vasculogenesis after cerebral ischemia is an attempt to recover damaged cerebral tissue. The role of EPCs in angiogenesis/vasculogenesis after brain ischemia remains unknown. Many studies have been published about the isolation, phenotyping and function of EPCs. However, there is not a unique definition for these cells; their origin and function are still an issue of controversy between different research groups. In this review, we summarize the currently used techniques and the most relevant publications about EPCs in experimental models of cerebral ischemia and their role in stroke. The identification of EPCs in peripheral blood as hematopoietic cells with the ability to differentiate into endothelial cells, broke the paradigm that vasculogenesis was only an embryogenic process. However, better knowledge about the origin and function of EPCs in cerebral ischemia is required. Stimulation of these cells opens a wide new field of cell-based angiogenic therapy that could improve the current stroke treatment.

Increased clonogenic potential of circulating endothelial progenitor cells in patients with type 1 diabetes and proliferative retinopathy

To the Editor: The identification of endothelial progenitor cells (EPCs) along with the description of their essential role in postnatal neovascularisation has been a major achievement for stem cell biology [1]. In particular the finding that the clonogenic potential of EPCs is reduced in patients with high cardiovascular risk [2] represents the first step towards the recognition of the therapeutic effect of EPC transplantation in cardiac and limbic ischaemia [3, 4]. However, two harmful side effects are expected to be associated with EPC transplantation as a consequence of their physiological function: malignant tumour neovascularisation and the induction of proliferative diabetic retinopathy [1]. The contribution of EPCs to tumour neovascularisation has been clearly demonstrated in animal models and confirmed, although to a lesser extent, also in humans [5]. Concerning diabetic retinopathy, recent reports have demonstrated that EPCs play a major role in the retinal neovascularisation of a mouse model of proliferative retinopathy [6], and that the concentration of stromalcell-derived factor 1, the most important chemokine mobilising EPCs, increases with the severity of diabetic retinopathy in vitreous samples of patients affected by type 2 diabetes [7]. These studies are in some way in contrast to two independent reports performed in non-complicated patients showing that type 1 diabetes per se is associated with a reduced number and function of EPCs [8], and that EPCs in type 2 diabetes are impaired in adhesion, proliferation and tubulisation [9]. Finally, a recent study has demonstrated an increased number of CD34+ mononuclear cells in patients affected by type 2 diabetes with both non-proliferative and proliferative retinopathy [10]. Although of interest, these results cannot be easily compared with the ones described above as EPCs represent only one of the different classes of circulating cells staining positively for CD34+ [1]. Taken together, and from a largely speculative point of view, these apparently discrepant results can be explained by a unifying hypothesis. Taking into account the fact that EPCs have a physiological role in the rescue and maintenance of the existing retinal capillary bed [11], it is possible to hypothesise that the reduced number and clonogenic potential of EPCs found in non-complicated diabetic patients [8, 9] might predispose these patients to the later development of diabetic retinopathy. Once the damage is widespread and specific chemokines are produced by the suffering retina, the bone marrow would respond by increasing the production of EPCs, which at this stage, possibly because of the high ambient glucose and/or the badly damaged retina, would give rise to an incompetent neoangiogenesis. To test this hypothesis and verify the possible association between the clonogenic potential of circulating EPCs and proliferative diabetic retinopathy, we isolated EPCs from whole blood obtained from 11 patients with type 1 diabetes and untreated proliferative retinopathy, from 12 patients without retinopathy despite similar age, gender distribution and duration of type 1 diabetes, and from 11 ageand sex-matched non-diabetic controls. Patients affected by type 1 diabetes took no medication other than insulin. Clinical characteristics of subjects included in the study are described in Table 1. Patients were classified with respect to the status of diabetic retinopathy by means of stereoscopic fundus examination and fluorescent angiography. Patients considered for this study either had no signs of diabetic V. Asnaghi . G. Mazzolari . M. R. Pastore . A. Maestroni . D. Ruggieri . L. Luzi . G. Zerbini (*) Renal Pathophysiology of Diabetes Unit, Division of Nutrition–Metabolism, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy e-mail: g.zerbini@hsr.it