Outgrowth Endothelial Progenitor Cells Research Articles

Abstract 894: Leptin Potentiates the Angiogenic Properties of Endothelial Progenitor Cells In Vitro and In Vivo

Endothelial progenitor cells (EPC) have been reported to contribute to neovascularization. We have previously shown that the adipocytokine leptin may enhance the adhesive properties of EPC by upregulating specific integrins. To investigate whether the angiogenic effects of leptin may be mediated by modulation of EPC function, mononuclear cells were isolated from healthy human volunteers and cultivated under endothelial cell conditions for 7 days. In the matrigel assay, pretreatment of EPC with recombinant leptin for 24 hours dose-dependently enhanced their incorporation into tubular structures provided by mature endothelial cells. For example, 138.3 ± 7.6% (P = 0.001) and 145.3 ± 5.5% (P = 0.0001) CM-DiI-labeled EPC were detected after stimulation with 10 and 100 ng/mL leptin, respectively (control-treated EPC defined as 100%). Furthermore, in the spheroid angiogenesis assay, stimulation of EPC with 10 ng/mL leptin increased the number of sprouts (P < 0.0001) and tube length (P < 0.0001) of coincubated mature endothelial cells, and the outgrowth of EPC (P < 0.0001). Addition of 100-fold excess of leptin-neutralizing or leptin-receptor-binding antibodies completely reversed these effects. Moreover, EPC adhesion onto endothelial cell tubules could be reduced by addition of RGD peptides (from 159 ± 13.7% to 101.8 ± 14.6%; P = 0.02), or of neutralizing antibodies against αvβ3 (from 165.3 ± 11.8% to 103.8 ± 13.3%; P = 0.006) or αvβ5 (to 93.5 ± 15.8%; P = 0.005). Further experiments using specific signal transduction inhibitors (10 μM of LY294002, PD98059, or SB203580), as well as Western blot analysis, revealed that leptin signaling in EPC involves phosphoinositide-3 kinase and p42/44, but not by p38 MAP kinase. The effects of leptin could also be confirmed under in vivo conditions. Stimulation of EPC with 100 ng/mL leptin potentiated the insprout of newly formed avian vessels into collagen onplants placed on the chorion allantoic membrane of chicken embryos (angiogenic index, 0.58 ± 0.24) compared to control-treated EPC (0.44 ± 0.27; P = 0.07) and endothelial basal medium alone (0.31 ± 0.26; P = 0.0007). Thus, our in vitro and in vivo results suggest that the angiogenic effects of leptin may partly depend on its specific interaction with endothelial progenitor cells.

End-stage renal disease causes an imbalance between endothelial and smooth muscle progenitor cells

Patients with end-stage renal disease (ESRD) on hemodialysis have an increased risk of cardiovascular disease (CVD). Circulating endothelial progenitor cells (EPC) contribute to vascular regeneration and repair, thereby protecting against CVD. However, circulating smooth muscle progenitor cells (SPC) may contribute to adverse vascular remodeling. We hypothesized that an imbalance occurs between EPC and SPC in ESRD patients and sampled progenitor cells from 45 ESRD patients receiving regular treatment. Our study is the first to show reduced numbers of CD34+KDR+ hematopoietic stem cell (HSC)-derived EPC (type I EPC). Furthermore, monocyte-derived EPC cultured from mononuclear cells (type II EPC) were reduced in number and had a reduced capacity to stimulate endothelial cell angiogenesis. In contrast, SPC outgrowth was unaffected. In vitro incubation with uremic serum impaired type II EPC outgrowth from healthy donor mononuclear cells and did not influence SPC outgrowth. The hemodialysis procedure itself induced HSC apoptosis and caused an acute depletion of circulating EPC. Taken together, the decreased number and impaired function of EPC are compatible with impaired endogenous vascular repair in hemodialysis patients, whereas the unaffected SPC numbers suggest that the potential of progenitor cells to contribute to adverse remodeling is retained. This EPC-SPC imbalance may contribute to the acceleration of CVD in ESRD patients and could offer novel therapeutic targets.

IGF-1 receptor dependent effect of human insulin on the outgrowth of circulating endothelial progenitor cells in patients with diabetes type 2

Introduction: Endothelial progenitor cells (EPC) were shown to be involved in vascular regeneration and angiogenesis in experimental diabetes. EPC of patients with both diabetes type 1 and type 2 demonstrate impaired outgrowth and function. In previous studies it was shown, that insulin increases in vitro proliferation of EPC isolated from healthy volunteers. Since clinical studies indicate that insulin therapy enhances the number of circulating CD34+/CD133+ cells in type 2 diabetes patients, we studied effects of human insulin on in vitro outgrowth of EPC isolated from type 2 diabetes patients and receptors mediating these effects.

CD34+AC133+VEGFR-2+ Cells Are Primitive Hematopoietic Progenitors, Not Functional Endothelial Progenitor Cells.

Endothelial progenitor cells (EPCs) are currently used for angiogenic therapies or as biomarkers to assess cardiovascular disease risk and progression. However, there is no uniform definition of an EPC, which complicates interpretation of prior EPC studies. EPCs are primarily defined by expression of cell surface antigens. The most widely cited definition of an EPC is a cell which co-expresses CD34, AC133 and VEGFR-2. Importantly, these antigens are also expressed on the most primitive population of hematopoietic progenitor cells (HPCs), including high proliferative potential- (HPP-) and low proliferative potential-colony forming cells (LPP-CFCs). Remarkably, CD34+AC133+VEGFR-2+ cells have never been isolated and plated in endothelial cell (EC) or hematopoietic cell clonogenic assays to determine what cell progeny can be derived from a CD34+AC133+VEGFR-2+ cell. Utilizing human umbilical cord blood (CB), an enriched source of both EPCs and HPCs, we isolated and purified CD34+AC133+VEGFR-2+ cells by FACS and assayed for the presence of clonogenic endothelial CFCs (ECFCs) plus HPP- and LPP-CFCs. Surprisingly, CD34+AC133+VEGFR-2+ cells do not form ECFCs under any culture conditions previously described for outgrowth of EPCs. However, consistent with a HPC phenotype, CD34+AC133+VEGFR-2+ cells formed both HPP- and LPP-CFCs in multiple independent assays. In addition, all CD34+AC133+VEGFR-2+ cells were shown to co-express the specific hematopoietic cell surface antigen, CD45, which is not present on ECs. Based on this information, we plated CD34+CD45+ or CD34+CD45− cells to determine if EPCs could be separated from HPCs on the basis of CD45 expression. In multiple independent assays, CD34+CD45+ cells consistently formed both HPP- and LPP-CFCs but not EC colonies. In contrast, CD34+CD45− cells form EC colonies but not hematopoietic cell colonies. Taken together, these data demonstrate that circulating CD34+AC133+VEGFR-2+ cells are HPCs and the biologic mechanism for their correlation with cardiovascular disease needs to be re-examined. Furthermore, studies focused on determining the angiogenic potential of CD34+CD45− cells are needed given that this cell population harbors ECFCs.

Th-P15:248 Computational approaches towards prediction

Endothelial progenitor cells (EPCs) participate in the neovascularization processes in the development of hepatocellular carcinoma (HCC). We investigated whether interactions between EPCs and HCC cells affect chemotactic and pro-inflammatory activities of EPCs. Two distinct phenotypes of circulating EPCs, i.e., myeloid-derived EPCs (colony forming unit-endothelial cells, CFU-ECs) and outgrowth EPCs (endothelial-colony forming cells, ECFCs), were co-cultured with Huh7 and Hep3B cells by using transwell chamber and IBIDITM Culture-Inserts and μ-slide plates. Transwell and horizontal migration/invasion assays and time-lapse microscopy were used to monitor and analyze the migration and invasion of EPCs induced by these HCC cells. A human cytokine antibody array was used to compare protein expression profiles in EPCs and HCC cells. Flow cytometry and electromobility shift analysis were used to detect nuclear factor-κB (NF-κB)–DNA binding activity and pro-inflammatory adhesion molecule expression in EPCs. Ectopic full-length CC chemokine receptor 6 (CCR6) plasmid was used to transfect into ECFCs to investigate the role of CCR6 in HCC-induced EPC migration and invasion. The results show that co-culture with Huh7 and Hep3B cells induces the expression of endothelial cell (EC) markers KDR, Flt1, CD31 and VE-cadherin in CFU-ECs, but down-regulates the expressions of CD31 and VE-cadherin in ECFCs. These HCC cells induce migration and invasion of CFU-ECs, but not ECFCs, and do not affect the cell cycle distribution in these EPCs. Cytokine protein array identifies macrophage inflammatory protein-3α (MIP-3α) produced by HCC cells as a critical factor responsible for the HCC-induced chemotaxis of CFU-ECs, which highly express the specific MIP-3α counterreceptor CCR6. Overexpressing CCR6 in ECFCs significantly increases their chemotaxis in response to HCC cells. Co-culturing EPCs with HCC cells results in decreases in NF-κB binding activity and hence intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expressions in EPCs. Our results indicate that HCC cells exert differential effects on CFU-ECs and ECFCs, with increased chemotaxis for CFU-ECs, but not ECFCs. This HCC-induced chemotaxis of CFU-ECs is mediated by MIP-3α produced by HCC cells, which targets to CCR6 on CFU-ECs. Tumors may provide a humoral microenvironment to attenuate the pro-inflammatory activity of EPCs, which might be associated with the tumor escape mechanism.

Clonal Restriction in Endothelial Progenitor Cells in Patients with Multiple Myeloma.

In multiple myeloma (MM), neoplastic plasma cells and bone marrow microvascular cells have paracrine cross-regulatory interactions, and display abnormal features suggesting that these neoangiogenic cells are myeloma-related. Tumor neovascularization is closely linked to neoplastic growth. We have recently shown that circulating endothelial progenitor cells (EPCs) reflect disease severity as assessed by M protein and serum β2-microglobulin levels in MM. These observations linking circulating EPCs to disease activity in MM prompted us to study the genetic characteristics of EPCs using X-chromosome inactivation (XCI) analysis to determine their clonal restriction profile. In addition, we examined EPCs for the presence of genetic markers common to neoplastic MM cells by examining immunoglobulin heavy chain (IgH) rearrangement of germline DNA. Isolation and outgrowth of EPCs was done as previously described. Clonality within EPCs was evaluated by quantitation of XCI patterns in female patients by a human androgen receptor gene (AR) assay (HUMARA). DNA was prepared from EPCs, hair root, or PBMCs and digested by a methylase-sensitive restriction enzyme. The highly polymorphic X-linked human androgen receptor gene was amplified by PCR. In polyclonal populations, both alleles are seen as two distinct bands, while clonal populations yield a single band. Amplification of genomic DNA extracted from PBMCs showed that XCI status was informative in 7 of the 16 MM patients and in 7 of the 15 healthy controls. Next, EPCs and hair root cells from informative patients were studied for XCI patterns to determine clonality. DNA obtained from confluent EPCs outgrown from 7 of the 10 informative patients displayed extremely skewed XCI patterns (90% inactivation of one allele), while hair follicle cells in the same patients displayed a random pattern. These results suggest that EPCs share characteristics of clonal plasma cells in MM, and display clonal restriction that is lineage-specific. To further explore the genetic similarity between EPCs and MM cells, EPCs were assessed for clonotypic immunoglobulin heavy chain (IgH) VDJ sequences by PCR amplification of genomic DNA. In these experiments, DNA was extracted from bone marrow EPCs and used in a series of 7 PCR amplifications containing a specific VH forward primer and a consensus JH reverse primer. In EPCs from 2 patients, including one with skewed XCI, we found a single PCR product identical to the IgH VDJ rearrangement found in bone marrow containing 60% and 65 % plasma cells, respectively, suggesting that an identical IgH VDJ rearrangement was present in EPCs and plasma cells in the same patient. Controls for this experiment included HUVEC genomic DNA, which did not yield PCR products even when spiked with a positive control DNA (up to 50 %) extracted from the MM cell line U266, and PBMC genomic DNA from controls which yielded multiple bands produced from several of the VH primers. Our results show that EPCs in a population of MM patients display clonal restriction and genetic similarity to plasma cells. Thus, it is possible that neoplastic MM cells and EPCs are linked not only within a functional context by their paracrine cross-regulatory effects within bone marrow microenvironment, but also by their genetic ancestory. These conclusions await confirmation from ongoing molecular genetic comparisons of the two cell types.

Differentiation and Proliferation of Endothelial Progenitor Cells from Canine Peripheral Blood Mononuclear Cells 1,2

The isolation, differentiation, and expansion of endothelial progenitor cells (EPCs) from peripheral blood have potential applicability in areas of therapeutic neovascularization, vascular repair, and tissue engineering. The purpose of the current study was to elucidate a simple method of isolation and differentiation of EPCs by defining the endothelial morphology, surface marker expression, and proliferative capacity of EPC outgrowth from canine peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from fresh canine blood and cultured in fibronectin-coated plates in which EPCs were identified from cell morphology and outgrowth characteristics. Cell surface markers were determined with flow cytometry analysis to identify differentiation of cultured and subcultured colonies. A hematologic counter with phase contrast microscopy was used to study cell growth curves of EPCs as compared with mature human coronary artery endothelial cells. During the first week of canine PBMC culture, cells were morphologically round and varied in size, but in the course of the second and third week of culture, the cells, respectively, became spindle-shaped and displayed an endothelium-like cobblestone morphology with outgrowth. CD34 was significantly decreased at 21 days as compared with 7 days culture (36.04% to 21.37%), whereas vWF (from 77.26% to 96.37%) and eNOS (from 0% to 14.97%) were significantly increased. VEGFR-2 was slightly increased, and P1H12 (CD146) was unchanged. Subcultured canine EPCs displayed a higher proliferation rate as compared to mature human coronary artery endothelial cells in the same culture conditions. These data demonstrate that canine EPCs can be isolated and cultured from the canine PBMC fraction. These outgrowth cells displayed characteristics of endothelial morphology with endothelial cell-specific surface markers. Furthermore, it was revealed that canine EPCs have a greater growth potential as compared to mature endothelial cells. This study suggests that PBMCs could be used as a source of EPCs for potential applications in tissue engineering and vascular therapy.

A Comparison of the Ability of AMD3100 Versus G-CSF to Induce Angiogenesis and Mobilize Endothelial Progenitor Cells (EPCs).

There is emerging evidence that circulating EPCs are able to home to sites of vascular injury and facilitate revascularization. However, under normal conditions, the number of EPCs in the blood is small, potentially limiting revascularization following acute injury. Previous reports suggest that certain cytokines including G-CSF can mobilize EPCs into the blood, potentially circumventing this limitation. However, the kinetics and magnitude of EPC mobilization by G-CSF have not been carefully defined and the angiogenic capacity of mobilized EPCs unclear. Herein, we characterize the ability of G-CSF treatment to improve revascularization in a mouse model of acute hindlimb ischemia. Since it is possible that EPC mobilization by G-CSF is delayed (maximal mobilization of hematopoietic stem cells (HSC) occurs after 4-5 days), we also studied AMD3100, a CXCR4 antagonist, that has been shown to lead to rapid and robust mobilization of HSC. Four groups of mice (n=7–10, each) were treated with saline alone, G-CSF (250μg/kg per day for 7 days by miniosmotic pump), AMD3100 (5mg/kg SQ on days 1–3), or a combination of both G-CSF and AMD3100; treatment was initiated immediately after induction of unilateral hindlimb ischemia.Revascularization was monitored by laser Doppler imaging and the ratio of perfusion between ischemic and non-ischemic calculated. At 2 weeks post surgery, a significant improvement in ischemic limb perfusion was observed in G-CSF and G-CSF+AMD3100 treated mice compared with saline treated mice with a trend towards improved perfusion noted in the AMD3100 treated mice [ratio of ischemic vs. non-ischemic limb perfusion + SEM: 71.9 ± 5.2% (saline treated); 89.2 ± 4.7%, p<0.05 (G-CSF); 80.5 + 6.6%, p=NS (AMD3100); 94.5 ± 3.6%, p<0.01 (G-CSF+AMD3100)]. In all treatment groups, the kinetics of revascularization was more rapid compared with control mice. In particular, the combination of G-CSF and AMD3100 resulted in the most rapid increase in perfusion with significant differences observed as early as 2 days post surgery (28.2 ± 2.5% compared with 15.3 ± 1.4% for control mice, p<0.01).As described more completely in a separate abstract, we have initiated a phase II study to compare the safety and efficacy of AMD3100 versus G-CSF for mobilization and transplantation of HSC for HLA-matched sibling allografting. Donors are sequentially mobilized with AMD3100 and G-CSF. To compare the ability of AMD3100 and G-CSF to mobilize EPCs, we have begun to quantify the number of EPCs in the blood at baseline and after treatment with AMD3100 or G-CSF. A culture-based method was used to identify both early outgrowth EPCs (scored on day 7; predominantly CD14+, CD31+, monocytic cells) and late outgrowth EPCs (scored on day 14–21; predominantly CD45−, CD14−, CD31+ endothelial cells). To date only a single patient has been analyzed completely. Interestingly, treatment with G-CSF leads to a more robust mobilization of early EPCs (fold increase compared with baseline: 4.7-fold and 25-fold for AMD3100 and G-CSF, respectively). In contrast, treatment with AMD3100 leads to a more robust mobilization of late EPC. Surprisingly, no late outgrowth EPC were detected in 40 ml of baseline or G-CSF treated blood, whereas 26 were detected in 40 ml of AMD3100 treated blood. Studies are underway to confirm these findings with additional donors.Collectively, these data show that treatment with AMD3100 alone or in combination with G-CSF leads to enhanced revascularization following acute vascular injury and the rapid mobilization of EPCs.