Vascular endothelial growth factor-C promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels.

Neovascularization, angiogenesis, and collagen constriction are essential for wound healing. We tested whether vascular endothelial growth factor-C (VEGF-C) can promote collagen constriction, capillary sprouting (angiogenesis), and invasion/migration of bone marrow-derived endothelial progenitor cells into collagen (vasculogenesis). We used a recently characterized three-dimensional collagen matrix assay with either monolayers of human dermal microvascular endothelial cells (HMVECs) or bone marrow-derived endothelial progenitor cells (BMD EPCs), obtained from Tie-2 LacZ transgenic mice, overlaid with an acellular layer and then a cellular layer of collagen embedded with fibroblasts, that were nontransduced or transduced with either LacZ adenoviral vector (Ad5) or VEGF-C/Ad5. The ability of VEGF-C to enhance fibroblast-mediated collagen constriction was measured, and gels overlying HMVECs or BMD EPCs were co-cultured, harvested, and assayed for HMVEC migration, sprouting, and capillary-like formation; gels containing BMD EPCs were assayed for EPC invasion/migration into the collagen extracellular matrix. VEGF-C significantly increased collagen constriction and formation of capillary-like structures with true lumina (P < .05) assessed by von Willebrand factor and VEGF receptor-2 immunoassaying. VEGF-C induced a significant increase in HMVEC migration, tubular polarization, and branching sprouts associated with a significant up-regulation of membrane type 1 matrix metalloproteinase (MT1-MMP) ( P < .05). Fibroblasts were necessary to support BMD-EPC invasion/migration from the monolayer into the collagen. Moreover, fibroblasts overexpressing VEGF-C significantly enhanced EPC invasion/migration ( P < .05) into the extracellular matrix by two-fold, and this effect could not be achieved with equivalent levels of exogenous VEGF-C in the absence of fibroblasts. The addition of a soluble VEGF-C competitor protein only partially inhibited these responses, reducing the EPCs by three-fold, but significant numbers of EPCs still invaded/migrated into the extracellular matrix, suggesting that other fibroblast-specific signals also contribute to the vasculogenic response. Fibroblast-specific expression of VEGF-C promotes collagen constriction by fibroblasts and enhances microvascular endothelial cell migration, branching, and capillary sprouting in association with up-regulating MT1-MMP expression. Fibroblasts are necessary for BMD EPC invasion/migration into collagen, and their overexpression of VEGF-C enhances this fibroblast-mediated vasculogenic effect. Collectively, these findings suggest a role for VEGF-C in multiple biologic steps required for wound healing (angiogenesis, vasculogenesis, and collagen constriction). Ischemic wound healing remains an unsolved problem with no previously identified molecular target for therapeutic intervention. This study demonstrates that VEGF-C overexpression by fibroblasts stimulates multiple biologic processes known to impact wound healing, such as collagen constriction, capillary sprouting, and EPC invasion and migration through extracellular matrix. Most ischemic wounds fail to heal and frequently lead to major limb amputation. Available cytokine ointments are ineffective, and revascularization is often not technically feasible. Even when these procedures are accomplished, many ischemic wounds frequently still do not heal because of multifactorial tissue level impairments in the fibroblastic and neovascularization responses at the wound base. Our findings identify an important role for two novel tissue level targets, dermis-derived fibroblasts and VEGF-C, in collagen constriction, angiogenesis, and postnatal vasculogenesis from BMD EPCs. Thus the findings are particularly relevant to the unsolved clinical problem of ischemic wound healing.

Bauer SM ，Bauer RJ ，Liu ZJ ，Chen H ，Goldstein L ，Velazquez OC ... -  《JOURNAL OF VASCULAR SURGERY》

Combination of stromal-derived factor-1alpha and vascular endothelial growth factor gene-modified endothelial progenitor cells is more effective for ischemic neovascularization.

Recruitment and entrapment of bone marrow-derived endothelial progenitor cells (EPCs) is important in vascular endothelial growth factor (VEGF)-induced angiogenesis. EPC mobilization and differentiation are modulated by stromal-derived factor-1alpha (SDF-1alpha/CXCL12), another important chemokine. In this study, we investigated the hypothesis that SDF-1alpha and VEGF might act synergistically on EPC-mediated vasculogenesis. EPCs were isolated and cultured from human peripheral blood, then transduced with retroviral vectors pBabe containing human VEGF(165) complimentary DNA (Td/V-EPCs) and pBabe wild-type (Td/p-EPCs). EPC migration activity was investigated with a modified Boyden chamber assay. EPC apoptosis induced by serum starvation was studied by annexin V assays. The combined effect of local administration of SDF-1alpha and Td/V-EPC transplantation on neovascularization was investigated in a murine model of hind limb ischemia. Over-expression of hVEGF(165) increased SDF-1alpha-mediated EPC migration. SDF-1alpha-mediated migration was significantly increased when EPCs were modified with VEGF (Td/V-EPCs) vs when VEGF was not present (Td/p-EPCs) or when VEGF alone was present (Td/V-EPCs; 196.8 +/- 15.2, 81.2 +/- 9.8, and 67.4 +/- 7.4/mm(2), respectively P < .001). SDF-1alpha combined with VEGF reduced serum starvation-induced apoptosis of EPCs more than SDF-1alpha or VEGF alone (P < .001). To determine the effect of this combination in vivo, SDF-1alpha was locally injected alone into the ischemic hind limb muscle of nude mice or combined with systemically injected Td/V-EPCs. The SDF-1alpha plus VEGF group showed significantly increased local accumulation of EPCs, blood-flow recovery, and capillary density compared with the other groups. The ratio of ischemic/normal blood flow in Td/V-EPCs plus SDF-1alpha group was significantly higher (P < .01), as was capillary density (capillaries/mm(2)), an index of neovascularization (Td/V-EPCs plus SDF-1alpha group, 863 +/- 31; no treatment, 395 +/-13; SDF-1alpha, 520 +/- 29; Td/p-EPCs, 448 +/- 28; Td/p-EPCs plus SDF-1alpha, 620 +/- 29; Td/V-EPCs, 570 +/- 30; P < .01). To investigate a possible mechanistic basis, we showed that VEGF up-regulated the receptor for SDF-1alpha, CXCR4, on EPCs in vitro. The combination of SDF-1alpha and VEGF greatly increases EPC-mediated angiogenesis. The use VEGF and SDF-1alpha together, rather than alone, will be a novel and efficient angiogenesis strategy to provide therapeutic neovascularization.

Yu JX ，Huang XF ，Lv WM ，Ye CS ，Peng XZ ，Zhang H ，Xiao LB ，Wang SM ... -  《-》

The role of fibrin E on the modulation of endothelial progenitors adhesion, differentiation and angiogenic growth factor production and the promotion of wound healing.

Severe skin loss constitutes a major unsolved clinical problem worldwide. For this reason, in the last decades there has been a major push towards the development of novel therapeutic approaches to enhance skin wound healing. Neo-vessel formation through angiogenesis is a critical step during the wound healing process. Besides the contribution of pre-existing endothelial cells (EC), endothelial progenitor cells (EPCs) have also been implicated in wound healing acting either by differentiating into EC that incorporate the neo-vessels, or via the production of paracrine factors that improve angiogenesis. Here we tested the importance of different extracellular matrices (ECM) in regulating the angiogenic and wound healing potential of cord blood-derived EPC (CB-EPC). We compared the properties of several ECM and particularly of fibrin fragment E (FbnE) in regulating EPC adhesion, proliferation, differentiation and healing-promotion in vitro and in vivo. Our results show that CB-EPCs have increased adhesion and endothelial differentiation when plated on FbnE compared to collagens, fibronectin or fibrin. Using integrin neutralizing antibodies, we show that CB-EPC adhesion to FbnE is mediated by integrin α5β1. Gene expression analysis of CB-EPCs plated on different substrates revealed that CB-EPC grown on FbnE shows increased expression of paracrine factors such as VEGF-A, TGF-β1, SDF-1, IL-8 and MIP-1α. Accordingly, conditioned media from CB-EPC grown on FbnE induced EC tube formation and monocyte migration in vitro. To test the wound healing effects of FbnE in vivo we used an FbnE enriched scaffold in a cutaneous wound healing mouse model. In accordance with our in vitro data, co-administration of the FbnE enriched scaffold with CB-EPC significantly accelerated wound closure and wound vascularization, compared FbnE enriched scaffold alone or to using collagen-based scaffolds. Our results show that FbnE modulates several CB-EPC properties in vivo and in vitro, and as such promotes wound healing. We suggest the use of FbnE-based scaffolds represents a promising approach to resolve wound healing complications arising from different pathologies.

Caiado F ，Carvalho T ，Silva F ，Castro C ，Clode N ，Dye JF ，Dias S ... -  《-》

The bone marrow-derived endothelial progenitor cell response is impaired in delayed wound healing from ischemia.

Vasculogenesis relies on the recruitment of bone marrow-derived endothelial progenitor cells (BMD EPCs) and is stimulated by tissue-level ischemia. We hypothesized that the BMD EPC response is impaired in ischemic wounds and studied the relationship between BMD EPCs and wound healing. We used transgenic Tie-2/LacZ mice, which carry the beta-galactosidase (beta-gal) reporter gene under Tie-2 promoter control. Wild-type mice were lethally irradiated and reconstituted with Tie-2/LacZ bone marrow. Four weeks later, the mice underwent unilateral femoral artery ligation/excision and bilateral wounding of the hindlimbs. Ischemia was confirmed and monitored with laser Doppler imaging. A subset of mice received incisional vs excisional nonischemic bilateral hindlimb wounds, without femoral ligation. Excisional wound closure was measured by using daily digital imaging and software-assisted calculation of surface area. Ischemia resulted in significantly delayed wound healing and differentially affected the number of BMD EPCs recruited to wound granulation tissue and muscle underlying the wounds. At 3 days postwounding, the granulation tissue of the wound base contained significantly fewer numbers of BMD EPCs in ischemic wounds compared with the nonischemic wounds (P < .05). In contrast, significantly more BMD EPCs were present in the muscle underlying the ischemic wounds at this same time point compared with the muscle under the nonischemic wounds (P < .05). In ischemic wounds, eventual wound closure significantly correlated with a delayed rise in BMD EPCs within the wound granulation tissue (Kendall's correlation, -.811, P = .0005) and was significantly associated with a gradual recovery of hindlimb perfusion (P < .0001). By 7 days postwounding, BMD EPCs were incorporated into the neovessels in the granulation tissue. At 14 days and 75 days, BMD EPCs were rarely observed within the wounds. Granulation tissue of excisional ischemic wounds showed significantly less BMD EPCs 3 days postwounding, in association with significantly delayed wound closure. However, the number of BMD EPCs were increased in ischemic hindlimb skeletal muscle, consistent with the notion that ischemia is a powerful signal for vasculogenesis. To our knowledge, this is the first report identifying a deficit in BMD EPCs in the granulation tissue of ischemic skin wounds and reporting the key role for these cells in both ischemic and nonischemic wound healing.

Bauer SM ，Goldstein LJ ，Bauer RJ ，Chen H ，Putt M ，Velazquez OC ... -  《JOURNAL OF VASCULAR SURGERY》

VEGF and PlGF promote adult vasculogenesis by enhancing EPC recruitment and vessel formation at the site of tumor neovascularization.

There are growing data to suggest that tissue hypoxia represents a critical force that drives adult vasculogenesis. Vascular endothelial growth factor (VEGF) expression is dramatically up-regulated by hypoxia and results in enhanced neovascularization. Although the role of VEGF in angiogenesis has been well characterized, its role in adult vasculogenesis remains poorly understood. We used two distinct murine bone marrow transplantation (BMT) models to demonstrate that increased VEGF levels at the site of tumor growth promoted vasculogenesis in vivo. This effect of VEGF was downstream of its effect to enhance either mobilization or survival of circulating endothelial progenitor cells (EPCs). Both VEGFR1 (flt1) and VEGFR2 (flk1) are expressed on culture expanded human EPCs. Previous studies suggest that the effect of VEGF on endothelial cell migration is primarily mediated via VEGFR2; however, VEGF-induced EPC migration in vitro was mediated by both receptors, suggesting that VEGF-VEGFR1 interactions in EPCs are distinct from differentiated endothelial cells. We used specific blocking antibodies to these receptors to demonstrate that VEGFR1 plays an important role in human EPC recruitment to tumors. These findings were further supported by our finding that tumor-associated placental growth factor (PlGF), a VEGFR1-specific agonist, increased tumor vasculogenesis in a murine BMT model. We further showed that both VEGF receptors were necessary for the formation of functional vessels derived from exogenously administered human ex vivo expanded EPCs. Our data suggest local VEGF and/or PlGF expression promote vasculogenesis; VEGF plays a role in EPC recruitment and subsequent formation of functional vessels.

Li B ，Sharpe EE ，Maupin AB ，Teleron AA ，Pyle AL ，Carmeliet P ，Young PP ... -  《-》