Role of nox2-based NADPH oxidase in bone marrow and progenitor cell function involved in neovascularization induced by hindlimb ischemia

Bone marrow (BM) is the major reservoir for endothelial progenitor cells (EPCs). Postnatal neovascularization depends on not only angiogenesis but also vasculogenesis, which is mediated through mobilization of EPCs from BM and their recruitment to the ischemic sites. Reactive oxygen species (ROS) derived from Nox2-based NADPH oxidase play an important role in postnatal neovascularization; however, their role in BM and EPC function is unknown. Here we show that hindlimb ischemia of mice significantly increases Nox2 expression and ROS production in BM-mononuclear cells (BMCs), which is associated with an increase in circulating EPC-like cells. Mice lacking Nox2 show reduction of ischemia-induced flow recovery, ROS levels in BMCs, as well as EPC mobilization from BM. Transplantation of wild-type (WT)-BM into Nox2-deficient mice rescues the defective neovascularization, whereas WT mice transplanted with Nox2-deficient BM show reduced flow recovery and capillary density compared to WT-BM transplanted control. Intravenous infusion of WT- and Nox2-deficient BMCs into WT mice reveals that neovascularization and homing capacity are impaired in Nox2-deficient BMCs in vivo. In vitro, Nox2-deficient c-kit+Lin- BM stem/progenitor cells show impaired chemotaxis and invasion as well as polarization of actins in response to stromal derived factor (SDF), which is associated with blunted SDF-1-mediated phosphorylation of Akt. In conclusion, Nox2-derived ROS in BM play a critical role in mobilization, homing, and angiogenic capacity of EPCs and BM stem/progenitor cells, thereby promoting revascularization of ischemic tissue. Thus, NADPH oxidase in BM and EPCs is potential therapeutic targets for promoting neovascularization in ischemic cardiovascular diseases.     

ClC-3 Deficiency Impairs the Neovascularization Capacity of Early Endothelial Progenitor Cells by Decreasing CXCR4/JAK-2 Signalling

BackgroundEndothelial progenitor cell (EPC) therapy has been suggested as a major breakthrough in the treatment of ischemic diseases. However, the molecular mechanism that underlies EPC functional regulation is still unclear.MethodsWe examined the angiogenic capacity of EPCs in a hindlimb ischemia model of wild-type and ClC-3 knockout mice.ResultsMice lacking of ClC-3 exhibited reduced blood flow recovery and neovascularization in ischemic muscles 7 and 14 days after hind limb ischemia. Moreover, compared with wild-type EPCs, the hindlimb blood reperfusion in mice receiving ClC-3 knockout EPCs was significantly impaired, accompanied by reduced EPC homing and retention. In vitro, EPCs derived from ClC-3 knockout mice displayed impaired migratory, adhesive, and angiogenic activity. CXC chemokine receptor 4 (CXCR4) expression was significantly reduced in EPC from ClC-3 knockout mice compared with wild-type. Moreover, the expression and phosphorylation of Janus kinase 2 (JAK-2), a downstream signalling of CXCR4, was also reduced in ClC-3 knockout EPC, indicating that CXCR4/JAK-2 signalling is dysregulated by ClC-3 deficiency. Consistent with this assumption, the migratory capacity of wild-type EPCs was attenuated by either CXCR4 antagonist AMD3100 or JAK-2 inhibitor AG490. More importantly, the impaired migratory capacity of ClC-3 knockout EPCs was rescued by overexpression of CXCR4.ConclusionsClC-3 plays a critical role in the angiogenic capacity of EPCs and EPC-mediated neovascularization of ischemic tissues. Disturbance of CXCR4/JAK-2 signalling may contribute to the functional impairment of ClC-3 deficient EPCs. Thus, ClC-3 may be a potential therapeutic target for modulating neovascularization in ischemic diseases.     

Activation of Epac stimulates integrin-dependent homing of progenitor cells

Cell therapy is a novel promising option for treatment of ischemic diseases. Administered endothelial progenitor cells (EPCs) are recruited to ischemic regions and improve neovascularization. However, the number of cells that home to ischemic tissues is restricted. The GTPase Rap1 plays an important role in the regulation of adhesion and chemotaxis. We investigated whether pharmacologic activation of Epac1, a nucleotide exchange protein for Rap1, which is directly activated by cAMP, can improve the adhesive and migratory capacity of distinct progenitor cell populations. Stimulation of Epac by a cAMP-analog increased Rap1 activity and stimulated the adhesion of human EPCs, CD34(+) hematopoietic progenitor cells, and mesenchymal stem cells (MSCs). Specifically, short-term stimulation with a specific Epac activator increased the beta2-integrin-dependent adhesion of EPCs to endothelial cell monolayers, and of EPC and CD34(+) cells to ICAM-1. Furthermore, the Epac activator enhanced the beta1-integrin-dependent adhesion of EPCs and MSCs to the matrix protein fibronectin. In addition, Epac1 activation induced the beta1- and beta2-integrin-dependent migration of EPCs on fibronectin and fibrinogen. Interestingly, activation of Epac rapidly increased lateral mobility of beta1- and beta2-integrins, thereby inducing integrin polarization, and stimulated beta1-integrin affinity, whereas the beta2-integrin affinity was not increased. Furthermore, prestimulation of EPCs with the Epac activator increased homing to ischemic muscles and neovascularization-promoting capacity of intravenously injected EPCs in the model of hind limb ischemia. These data demonstrate that activation of Epac1 increases integrin activity and integrin-dependent homing functions of progenitor cells and enhances their in vivo therapeutic potential. These results may provide a platform for the development of novel therapeutic approaches to improve progenitor cell homing.     

Evidence for genetic regulation of endothelial progenitor cells and their role as biological markers of atherosclerotic susceptibility.

AIMS: Endothelial progenitor cells (EPCs) are found in the peripheral circulation and are capable of endothelial repair and neovascularization. EPC number and function are reduced in subjects with cardiovascular risk factors or proven coronary artery disease (CAD). We hypothesized that EPC number and/or function may be genetically regulated and may vary in healthy adult offspring depending on parental history of CAD. METHODS AND RESULTS: We studied 102 subjects comprising 24 healthy parent-healthy offspring pairs and 27 CAD parent-healthy offspring pairs. We measured the number of circulating CD34(+)VEGFR-2(+) and AC133(+)VEGFR-2(+) EPCs, the number of EPCs grown in culture, and the migration capacity of cultured EPCs towards vascular endothelial growth factor. There was significant correlation in the number of cultured EPCs between healthy parents and their offspring (R = 0.492, P = 0.015) and CAD parents and their offspring (R = 0.751, P < 0.001). Offspring of subjects with CAD had significantly higher numbers of circulating CD34(+)VEGFR-2(+) and AC133(+)VEGFR-2(+) cells (P = 0.018 and P < 0.001, respectively). There was no difference in migration capacity between groups. CONCLUSION: Our results suggest that EPC number is, at least in part, genetically regulated. Circulating EPCs may represent biological markers of occult vascular damage in offspring with hereditary risk of CAD.     

Glucose tolerance is negatively associated with circulating progenitor cell levels

AIMS/HYPOTHESIS: Circulating progenitor cells participate in cardiovascular homeostasis. Depletion of the pool of endothelial progenitor cells (EPCs) is associated with increased cardiovascular risk. Furthermore, EPCs are reduced in the presence of classical risk factors for atherosclerotic disease, including diabetes mellitus. This study was designed to evaluate progenitor cell levels in volunteers with different degrees of glucose tolerance. METHODS: Cardiovascular parameters and the levels of circulating CD34(+) and CD34(+) kinase insert domain receptor (KDR)(+) cells were determined in 219 middle-aged individuals with no pre-diagnosed alterations in carbohydrate metabolism. Glucose tolerance was determined by fasting and 2 h post-challenge glucose levels, with IFG and IGT considered as pre-diabetic states. RESULTS: CD34(+) and CD34(+)KDR(+) cells were significantly reduced in individuals who were found to have diabetes mellitus, and were negatively correlated with both fasting and post-challenge glucose in the whole population. While only CD34(+) cells, but not CD34(+)KDR(+) cells, were significantly reduced in pre-diabetic individuals, post-challenge glucose was an independent determinant of the levels of both CD34(+) and CD34(+)KDR(+) cells. CONCLUSIONS/INTERPRETATION: Glucose tolerance was negatively associated with progenitor cell levels in middle-aged healthy individuals. Depletion of endothelial progenitors with increasing fasting and post-meal glucose may be one cause of the high incidence of cardiovascular damage in individuals with pre-diabetes.     

Endothelial progenitor cells and peripheral neuropathy in subjects with type 2 diabetes mellitus

AimsTo examine for differences in circulating progenitor cells (CPCs) and endothelial progenitor cells (EPCs) in patients with and without diabetic peripheral neuropathy (DPN).MethodsA total of 105 participants were included: 50 patients with type 2 diabetes (T2DM) and DPN, 30 patients with T2DM without DPN and 25 healthy individuals. CPCs and 6 different EPCs phenotypes were assessed with flow cytometry. We also measured plasma levels of vascular endothelial growth factor (VEGF), stromal cell-derived factor 1 (SDF-1), vascular cell adhesion protein-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM) and tumor necrosis factor a (TNFa).ResultsNo difference was observed in the number of CPCs among the 3 groups. Patients with DPN had higher numbers of all 6 EPCs phenotypes when compared with patients without DPN and higher number of 5 EPCs phenotypes when compared with healthy individuals. Plasma VEFG, VCAM-1, ICAM-1 and TNFa levels did not differ among the 3 groups. Patients with DPN had lower SDF-1 levels in comparison with healthy individuals.ConclusionCirculating EPCs are increased while SDF-1 levels are decreased in the presence of DPN. Our findings suggest that DPN may be associated with impaired trafficking of EPCs and impaired EPCs homing to the injured endothelium.     

Endothelial progenitor cells: mobilization,differentiation, and homing

Postnatal bone marrow contains a subtype of progenitor cells that have the capacity to migrate to the peripheral circulation and to differentiate into mature endothelial cells. Therefore, these cells have been termed endothelial progenitor cells (EPCs). The isolation of EPCs by adherence culture or magnetic microbeads has been described. In general, EPCs are characterized by the expression of 3 markers, CD133, CD34, and the vascular endothelial growth factor receptor-2. During differentiation, EPCs obviously lose CD133 and start to express CD31, vascular endothelial cadherin, and von Willebrand factor. EPCs seem to participate in endothelial repair and neovascularization of ischemic organs. Clinical studies using EPCs for neovascularization have just been started; however, the mechanisms stimulating or inhibiting the differentiation of EPC in vivo and the signals causing their migration and homing to sites of injured endothelium or extravascular tissue are largely unknown at present. Thus, future studies will help to explore areas of potential basic research and clinical application of EPCs.     

Endothelial progenitor cells: isolation and characterization

Bone marrow of adults contains a subtype of progenitor cells that have the capacity to differentiate into mature endothelial cells and have therefore been termed endothelial progenitor cells (EPCs). Of the three cell markers (CD133, CD34, and the vascular endothelial growth factor receptor 2) that characterize the early functional EPCs, located predominantly in the bone marrow, EPCs obviously lose CD133/CD34 and start to express CD31, vascular endothelial cadherin, and von Willebrand factor when migrating to the circulation. Various isolation procedures of EPCs from different sources by using adherence culture or magnetic microbeads have been described, but published findings with regard to the number of EPCs in the peripheral circulation of healthy adults are scanty and no data regarding the lifetime of EPCs in vivo exist. Clinical studies employing EPCs for neovascularization of ischemic organs have just been started; however, the mechanisms stimulating or inhibiting the differentiation of bone marrow-derived EPCs in vivo and the signals causing their adhesion, migration, and homing to sites of injured tissue are largely unknown at present.     

Role of plasma kallikrein–kinin system activation in synovial recruitment of endothelial progenitor cells in experimental arthritis

Objective

To examine whether activation of the plasma kallikrein–kinin system (KKS) mediates synovial recruitment of endothelial progenitor cells (EPCs) in arthritis.

Methods

EPCs were isolated from Lewis rat bone marrow, and expression of progenitor cell–lineage markers and functional properties were characterized. EPCs were injected intravenously into Lewis rats with arthritis, and their recruitment and formation of de novo blood vessels in inflamed synovium were evaluated. The role of plasma KKS was examined using a plasma kallikrein inhibitor (EPI‐KAL2) and an antikallikrein antibody (13G11). A transendothelial migration assay was used to determine the role of bradykinin and its receptor in EPC mobilization.

Results

EPCs from Lewis rats exhibited a strong capacity to form tubes and vacuoles and expressed increased levels of bradykinin type 2 receptor (B2R) and progenitor cell markers CD34 and Sca‐1. In Lewis rats with arthritis, EPCs were recruited into inflamed synovium at the acute phase of disease and formed de novo blood vessels. Inhibition of plasma kallikrein by EPI‐KAL2 and 13G11 significantly suppressed synovial recruitment of EPCs and hyperproliferation of synovial cells. Bradykinin stimulated transendothelial migration of EPCs in a concentration‐dependent manner. This was mediated by B2R, as demonstrated by the finding that knockdown of B2R with silencing RNA completely blocked bradykinin‐stimulated transendothelial migration. Moreover, bradykinin selectively up‐regulated expression of the homing receptor CXCR4 in EPCs.

Conclusion

These observations demonstrate a novel role of plasma KKS activation in the synovial recruitment of EPCs in arthritis, acting via kallikrein activation and B2R‐dependent mechanisms. B2R might be involved in the mobilization of EPCs via up‐regulation of CXCR4.

     

Impaired development and dysfunction of endothelial progenitor cells in type 2 diabetic mice

AimDysfunction of circulating endothelial progenitor cells (EPCs) has been shown to affect the development of microvascular diseases in diabetes patients. The aim of this study was to elucidate the development and mechanical dysfunction of EPCs in type 2 diabetes (T2D).MethodsThe colony-forming capacity of EPCs and differentiation potential of bone marrow (BM) c-Kit(+)/Sca-I(+) lineage-negative mononuclear cells (KSL) were examined in T2D mice, db/db mice and KKA^y mice, using EPC colony-forming assay (EPC-CFA).ResultsT2D mice had fewer BM stem/progenitor cells, and proliferation of KSL was lowest in the BM of db/db mice. In T2D mice, the frequency of large colony-forming units (CFUs) derived from BM-KSL was highly reduced, indicating dysfunction of differentiation into mature EPCs. Only a small number of BM-derived progenitors [CD34(+) KSL cells], which contribute to the supply of EPCs for postnatal neovascularization, was also found. Furthermore, in terms of their plasticity to transdifferentiate into various cell types, BM-KSL exhibited a greater potential to differentiate into granulocyte macrophages (GMs) than into other cell types.ConclusionT2D affected EPC colony formation and differentiation of stem cells to mature EPCs or haematopoietic cells. These data suggest opposing regulatory mechanisms for differentiation into mature EPCs and GMs in T2D mice.     

Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization

Increasing evidence suggests that postnatal neovascularization involves the recruitment of circulating endothelial progenitor cells (EPCs). Hematopoietic and endothelial cell lineages share common progenitors. Cytokines formerly thought to be specific for the hematopoietic system have only recently been shown to affect several functions in endothelial cells. Accordingly, we investigated the stimulatory potential of erythropoietin (Epo) on EPC mobilization and neovascularization. The bone marrow of Epo-treated mice showed a significant increase in number and proliferation of stem and progenitor cells as well as in colony-forming units. The number of isolated EPCs and CD34+/flk-1+ precursor cells was significantly increased in spleen and peripheral blood of Epo-treated mice compared with phosphate-buffered saline-treated mice. In in vivo models of postnatal neovascularization, Epo significantly increased inflammation- and ischemia-induced neovascularization. The physiologic relevance of these findings was investigated in patients with coronary heart disease. In a multivariate regression model, serum levels of Epo and vascular endothelial growth factor were significantly associated with the number of stem and progenitor cells in the bone marrow as well as with the number and function of circulating EPCs. In conclusion, the present study suggests that Epo stimulates postnatal neovascularization at least in part by enhancing EPC mobilization from the bone marrow. Epo appears to physiologically regulate EPC mobilization in patients with ischemic heart disease. Thus, Epo serum levels may help in identifying patients with impaired EPC recruitment capacity.     

TGF-β1 signaling and Krüppel-like factor 10 regulate bone marrow-derived proangiogenic cell differentiation, function, and neovascularization

Emerging evidence demonstrates that proangiogenic cells (PACs) originate from the BM and are capable of being recruited to sites of ischemic injury where they contribute to neovascularization. We previously determined that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitor cells (GMPs) differentiate into PACs and possess robust angiogenic activity under ischemic conditions. Herein, we report that a TGF-β1-responsive Krüppel- like factor, KLF10, is strongly expressed in PACs derived from CMPs and GMPs, ～ 60-fold higher than in progenitors lacking PAC markers. KLF10(-/-) mice present with marked defects in PAC differentiation, function, TGF-β responsiveness, and impaired blood flow recovery after hindlimb ischemia, an effect rescued by wild-type PACs, but not KLF10(-/-) PACs. Overexpression studies revealed that KLF10 could rescue PAC formation from TGF-β1(+/-) CMPs and GMPs. Mechanistically, KLF10 targets the VEGFR2 promoter in PACs which may underlie the observed effects. These findings may be clinically relevant because KLF10 expression was also found to be significantly reduced in PACs from patients with peripheral artery disease. Collectively, these observations identify TGF-β1 signaling and KLF10 as key regulators of functional PACs derived from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states.     

Nonbone marrow-derived circulating progenitor cells contribute to postnatal neovascularization following tissue ischemia

Circulating progenitor cells home to sites of postnatal neovascularization and differentiate into endothelial cells but questions remain regarding the source of these cells. Indeed, a recent study suggests that nonbone marrow-derived cells may be even more important than bone marrow-derived cells in the setting of transplant arteriosclerosis. Thus, we aimed to thoroughly investigate the contribution of nonbone marrow-derived progenitor cells for neovascularization. We exclusively identified nonbone marrow-derived progenitor cells by combining a parabiosis model with reverse bone marrow transplantation followed by hindlimb ischemia. In this model, nonbone marrow-derived circulating progenitor cells attributed for 74+/-13% of the circulating progenitor cells that incorporated into the ischemic hindlimb. Increasing evidence suggests that organs such as small intestine and liver contain a considerable number of tissue resident progenitor cells and, thus, represent putative sources for nonbone marrow-derived progenitors. To track organ-derived progenitors, we transplanted sex-mismatched small intestine or liver, respectively, into rats followed by induction of hindlimb ischemia. These experiments show that organ-derived progenitor cells are contributing to postnatal vasculogenesis (intestine: 4.7+/-3.7%; liver: 6.3+/-2.2%). Based on the subsequent observation that liver-derived nonhematopoietic c-kit(+)CD45(-) progenitors are mobilized on induction of hindlimb ischemia, we prospectively isolated and intravenously infused these progenitors from murine livers. The isolated cells demonstrated a marked capacity for enhancing neovascularization and restoring blood flow to the ischemic hindlimb (no cells: 26.4+/-4.8% of normal blood flow; c-kit(+)CD45(-) cells: 67.0+/-8.0% of normal flow; P<0.01). In conclusion, we find that nonbone marrow-derived c-kit(+)CD45(-) progenitors contribute to postnatal neovascularization to an extent that is similar to that of bone marrow-derived progenitor cells. Intestine and liver represent a rich source for mobilized tissue-residing progenitor cells.     

Human CD34+AC133+VEGFR-2+ cells are not endothelial progenitor cells but distinct, primitive hematopoietic progenitors

OBJECTIVE: Endothelial progenitor cells (EPCs) are used for angiogenic therapies or as biomarkers to assess cardiovascular disease risk. However, there is no uniform definition of an EPC, which confounds EPC studies. EPCs are widely described as cells that coexpress the cell-surface antigens CD34, AC133, and vascular endothelial growth factor receptor-2 (VEGFR-2). These antigens are also expressed on primitive hematopoietic progenitor cells (HPCs). Remarkably, despite their original identification, CD34+AC133+VEGFR-2+ cells have never been isolated and simultaneously plated in hematopoietic and endothelial cell (EC) clonogenic assays to assess the identity of their clonal progeny, which are presumably the cellular participants in vascular regeneration. METHODS: CD34+AC133+VEGFR-2+ cells were isolated from human umbilical cord blood (CB) or granulocyte colony-stimulating factor-mobilized peripheral blood and assayed for either EPCs or HPCs. RESULTS: CD34+AC133+VEGFR-2+ cells did not form EPCs and were devoid of vessel forming activity. However, CD34+AC133+VEGFR-2+ cells formed HPCs and expressed the hematopoietic lineage-specific antigen, CD45. We next tested whether EPCs could be separated from HPCs by immunoselection for CD34 and CD45. CD34+CD45+ cells formed HPCs but not EPCs, while CD34+CD45- cells formed EPCs but not HPCs. CONCLUSIONS: Therefore, CD34+AC133+VEGFR-2+ cells are HPCs that do not yield EC progeny, and the biological mechanism for their correlation with cardiovascular disease needs to be reexamined.     

Phosphatidylinositol-3-kinase-gamma is integral to homing functions of progenitor cells

Endothelial progenitor cells (EPCs) and hematopoietic progenitor cells are recruited to ischemic regions, improving neovascularization. beta1 and beta2 integrins play a crucial role for progenitor cell homing to ischemic tissues. Integrin activity is regulated by chemokines and their respective G protein-coupled receptors. The phosphatidylinositol-3-kinase catalytic subunit gamma (PI3Kgamma) is the PI3K isoform that selectively transduces signals from G protein-coupled receptors. Here, we investigated the role of PI3Kgamma as a signaling intermediate in the chemokine-induced integrin-dependent homing functions of progenitor cells. A pharmacological PI3Kgamma inhibitor significantly reduced chemokine-induced chemotaxis and stromal cell-derived factor (SDF)1alpha-induced transmigration of human EPCs. Moreover, the PI3Kgamma inhibitor significantly reduced SDF1alpha-induced adhesion of EPCs to intercellular adhesion molecule-1 and human umbilical vein endothelial cell monolayers. These findings were corroborated with Lin(-) bone marrow-derived progenitor cells from PI3Kgamma-deficient mice that displayed reduced SDF1alpha-induced migration and intercellular adhesion molecule-1 adhesion as compared with wild-type cells. Pharmacological inhibition or genetic ablation of PI3Kgamma reduced SDF1alpha-induced integrin activation in human EPCs and in murine Lin(-) BM-derived progenitor cells, respectively. In vivo, the homing of PI3Kgamma-deficient Lin(-) progenitor cells to ischemic muscles after intravenous infusion in the model of hindlimb ischemia and their neovascularization-promoting capacity was reduced as compared with wild-type cells. In conclusion, PI3Kgamma is integral to the integrin-dependent homing of progenitor cells.     

CD45 regulates homing and engraftment of immature normal and leukemic human cells in transplanted immunodeficient mice

Bone marrow homing and engraftment by clinically transplanted hematopoietic stem and progenitor cells is a complex process that is not fully understood. We report that the pan-leukocyte CD45 phosphatase plays an essential role in trafficking and repopulation of the bone marrow by immature human CD34(+) cells and leukemic cells in transplanted nonobese diabetic severe combined immunodeficient mice. Inhibiting CD45 function by blocking antibodies or a CD45 inhibitor impaired the motility of both normal and leukemic human cells. Blocking CD45 inhibited homing and repopulation by immature human CD34(+) cells as well as homing of primary patient leukemic cells. In addition, CD45 inhibition negatively affected development of hematopoietic progenitors in?vitro and their recovery in transplanted recipients in?vivo, revealing the central role of CD45 in the regulation of hematopoiesis. Moreover, CD45 blockage induced a hyperadhesive phenotype in immature human progenitor cells as well as in murine leukocytes, leading to their defective adhesion interactions with endothelial cells. This phenotype was further manifested by the ability of CD45 blockage to prevent breakdown of adhesion interactions in the BM, which inhibited murine progenitor mobilization. The substantial effects of a direct CD45 inhibition point at its essential roles in cell trafficking, including murine progenitor cell mobilization and both normal immature and leukemic human hematopoietic cells as well as regulation of hematopoiesis and engraftment potential.     

The role of vascular cell adhesion molecule 1/ very late activation antigen 4 in endothelial progenitor cell recruitment to rheumatoid arthritis synovium

OBJECTIVE: Marrow-derived endothelial progenitor cells (EPCs) are important in the neovascularization that occurs in diverse conditions such as cardiovascular disorders, inflammatory diseases, and neoplasms. In rheumatoid arthritis (RA), synovial neovascularization propels disease by nourishing the inflamed and hyperproliferative synovium. This study was undertaken to investigate the hypothesis that EPCs selectively home to inflamed joint tissue and may perpetuate synovial neovascularization. METHODS: In a collagen-induced arthritis (CIA) model, neovascularization and EPC accumulation in mouse ankle synovium was measured. In an antibody-induced arthritis model, EPC recruitment to inflamed synovium was evaluated. In a chimeric SCID mouse/human synovial tissue (ST) model, mice were engrafted subcutaneously with human ST, and EPC homing to grafts was assessed 2 days later. EPC adhesion to RA fibroblasts and RA ST was evaluated in vitro. RESULTS: In mice with CIA, cells bearing EPC markers were significantly increased in peripheral blood and accumulated in inflamed synovial pannus. EPCs were 4-fold more numerous in inflamed synovium from mice with anti-type II collagen antibody-induced arthritis versus controls. In SCID mice, EPC homing to RA ST was 3-fold greater than to normal synovium. Antibody neutralization of vascular cell adhesion molecule 1 (VCAM-1) and its ligand component alpha4 integrin potently inhibited EPC adhesion to RA fibroblasts and RA ST cryosections. CONCLUSION: These data demonstrate the selective recruitment of EPCs to inflamed joint tissue. The VCAM-1/very late activation antigen 4 adhesive system critically mediates EPC adhesion to cultured RA fibroblasts and to RA ST cryosections. These findings provide evidence of a possible role of EPCs in the synovial neovascularization that is critical to RA pathogenesis.     

Synergistic effect of combined intramyocardial CD34+ cells and VEGF2 gene therapy after MI

Previous studies have shown that local angiogenic gene therapy acts, in part, by recruiting endothelial progenitor cells (EPCs) to ischemic tissue. Recent data indicate that patients with the most severe vascular disease may have insufficient or deficient EPCs and the poorest response to angiogenic therapy. Accordingly, we hypothesized that combining human CD34(+) cell implantation with local vascular endothelial growth factor 2 (phVEGF2) gene therapy might overcome these deficiencies. The addition of VEGF2 to EPC cultures resulted in significant and dose-dependent decreases in EPC apoptosis. Phosphorylated Akt (p-Akt) was increased in VEGF2-treated EPCs. In vivo, myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery in 34 immunodeficient rats. The animals were then randomized to one of four treatment groups: cell therapy alone with human CD34(+) cells; VEGF2 gene therapy alone; combination therapy with CD34(+) cells plus phVEGF2; or CD34(-) cells and 50 microg empty plasmid. Four weeks after MI, animals treated with combination therapy showed improved fractional shortening, increased capillary density, and reduced infarct size compared with the other three groups. Combination therapy was also associated with an increased number of circulating EPCs 1 week after MI. Combined subtherapeutic doses of cell and gene therapy result in a significant therapeutic effect compared to monotherapy. This approach may overcome therapeutic failures (e.g. inability of certain patients to mobilize sufficient EPCs) and may also offer safety advantages by allowing lower dosing strategies.     

Development of angiogenic cell and gene therapy by transplantation of umbilical cord blood with vascular endothelial growth factor gene.

Endothelial progenitor cells (EPCs) are present in the mononuclear cells (MNCs) of umbilical cord blood and peripheral blood. To establish the efficiency of angiogenic cell and gene therapies, we transfected the human vascular endothelial growth factor (hVEGF) gene into cord blood MNCs to enhance endothelialization. MNCs from cord blood and peripheral blood were isolated and transfected with pCR3 expressing hVEGF165 or GFP by the Hemagglutinating Virus of Japan (HVJ)-envelope and the cells were cultured in endothelium basal medium-2. The number of attached cells from cord blood was higher than that from peripheral blood. Attached cells expressed Flk-1, VE-cadherin, PECAM-1, CD34, and Tie-2. The increase in the number of attached cells was transient with the transfection of vascular endothelial growth factor (VEGF) gene early in the experimental period. Flt-1 mRNA was not expressed early in the culture period, but was expressed at 2 weeks after separation. VEGF gene transfer into MNCs at 12 days after separation, i.e., when Flt-1 mRNA was expressed continuously, increased the number of attached cells. We evaluated the effects of the transplantation of cord blood MNCs expressing the hVEGF gene on regional blood flow in an ischemic area in a rat model of chronic hindlimb ischemia. Blood flow was significantly improved in nude rats that received transplanted control MNCs. Transplantation of cord blood MNCs transfected with the hVEGF gene yielded greater improvements in blood flow. These results indicate that the hVEGF gene enhances endothelialization of EPCs, and that the transplantation of cord blood MNCs transfected with the VEGF gene may be feasible for the treatment of ischemic diseases as a type of angiogenic cell and gene therapy.