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Objective— Endothelial progenitor cells (EPCs) comprise a heterogeneous population of cells, which improve therapeutic neovascularization after ischemia. The neovascularization-promoting potential of progenitor cells depends on survival and retention of the infused cells to the tissue. Caspases mediate apoptosis but are also involved in other critical biological processes. Therefore, we aimed to address the role of caspases in proangiogenic cells. Methods and Results— The caspase-8 inhibitor zIETD abrogated the ex vivo formation of EPCs, inhibited EPC adhesion and migration, and reduced their capacity to improve neovascularization in vivo. Consistently, cells isolated from caspase-8-deficient mice exhibited a reduced capacity for enhancing neovascularization when transplanted into mice after hindlimb ischemia. Because inhibition of Caspase-8 reduced the adhesion and homing functions of EPCs, we further determined the surface expression of integrins and receptors involved in cell recruitment to ischemic tissues. Pharmacological inhibition of caspase-8 and genetic depletion of caspase-8 reduced the expression of the fibronectin receptor subunits α5 and β1 and the SDF-1 receptor CXCR4. Moreover, we identified the E3 ubiquitin ligase Cbl-b, which negatively regulates integrin and receptor-mediated signaling, as a potential Caspase-8 substrate. Conclusion— In summary, our data demonstrate a novel apoptosis-unrelated role of caspase-8 in proangiogenic cells.

Circulating blood–derived vasculogenic cells improve neovascularization of ischemic tissue by a broad repertoire of potential therapeutic actions. Whereas initial studies documented that the cells incorporate and differentiate to cardiovascular cells, other studies suggested that short-time paracrine mechanisms mediate the beneficial effects. The question remains to what extent a physical incorporation is contributing to the beneficial effects of cell therapy. By using the inducible suicide gene thymidine kinase to deplete transplanted cells, we determined the contribution of physical incorporation in 3 animal models. After acute myocardial infarction, depletion of cells 14 days after infusion resulted in a reduction of capillary density and a substantial deterioration of heart function. Likewise, neovascularization of Matrigel plugs and ischemic limbs was significantly suppressed when infused cells were depleted 7 days after infusion. Induction of cell death in the previously transplanted cells reduced perfusion and led to vascular leakage as evidenced by Evans blue extravasation. These results indicate that physical incorporation and persistence of cells contribute to cell-mediated improvement of neovascularization and cardiac function. Long-term paracrine activities and/or cell intrinsic mechanisms may have contributed to the maintenance of functional improvement.

Endothelial progenitor cells (EPCs) are recruited to ischemic regions and improve neovascularization. Integrins contribute to EPC homing. High-mobility group box 1 (HMGB1) is a nuclear protein that is released extracellularly on cell necrosis and tissue damage, eliciting a proinflammatory response and stimulating tissue repair. In the present study, we investigated the effects of HMGB1 on EPC homing. EPCs express the HMGB1 receptors RAGE (receptor for advanced glycation end products) and TLR2 (Toll-like receptor 2). EPC migration was stimulated by HMGB1 in a RAGE-dependent manner. In addition, the HMGB1-induced migration of EPCs on fibronectin and fibrinogen was significantly inhibited by antibodies against β 1 and β 2 integrins, respectively. Short-term prestimulation of EPCs with HMGB1 also increased EPC adhesion to endothelial cell monolayers, and this effect was blocked by antibodies to β 2 integrins or RAGE. HMGB1 increased EPC adhesion to the immobilized integrin ligands intercellular adhesion molecule-1 and fibronectin in a RAGE-dependent manner. Strikingly, HMGB1 rapidly increased integrin affinity and induced integrin polarization. Using intravital microscopy in a tumor model of neovascularization, prestimulation of EPCs with HMGB1 enhanced the initial in vivo adhesion of EPCs to microvessels and the recruitment of EPCs in the tumor tissue. In addition, prestimulation of EPCs with HMGB1 increased the homing of EPCs to ischemic muscles. In conclusion, these data represent a link between HMGB1 and integrin functions of EPCs and demonstrate that HMGB1 stimulates EPC homing to ischemic tissues. These results may provide a platform for the development of novel therapeutic approaches to improve EPC homing.

β1-Integrins are essential for angiogenesis. The mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. Brag2 is a guanine nucleotide exchange factor for the small Arf-GTPases Arf5 and Arf6. The role of Brag2 in EC and angiogenesis and the underlying molecular mechanisms remain unclear. siRNA-mediated Brag2-silencing reduced EC angiogenic sprouting and migration. Brag2-siRNA transfection differentially affected α5β1- and αVβ3-integrin function: specifically, Brag2-silencing increased focal/fibrillar adhesions and adhesion on β1-integrin ligands (fibronectin and collagen), while reducing the adhesion on the αVβ3-integrin ligand, vitronectin. Consistent with these results, Brag2-silencing enhanced surface expression of α5β1-integrin, while reducing surface expression of αVβ3-integrin. Mechanistically, Brag2-mediated αVβ3-integrin-recycling and β1-integrin endocytosis and specifically of the active/matrix-bound α5β1-integrin present in fibrillar/focal adhesions (FA), suggesting that Brag2 contributes to the disassembly of FA via β1-integrin endocytosis. Arf5 and Arf6 are promoting downstream of Brag2 angiogenic sprouting, β1-integrin endocytosis and the regulation of FA. In vivo silencing of the Brag2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Furthermore, in vivo intravitreal injection of plasmids containing Brag2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveal that Brag2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating β1-integrin internalization and link for the first time the process of β1-integrin endocytosis with angiogenesis.

Progenitor cells (PC) are recruited to ischemic tissues and improve neovascularization. Beta2-integrins are essential for adhesion, transmigration and homing of PC to ischemic tissues. Developmental Endothelial Locus-1 (Del-1) is an extracellular matrix protein, that binds alphaVbeta3- and alphaVbeta5-integrins and is up-regulated during ischemia. Therefore, we investigated the role of endogenous Del-1 for angiogenesis and homing functions of PC. The Del-1-deficient mice (Del-1 −/− ) displayed a significantly increased angiogenic response in ischemic muscles in comparison to the wild type (WT) mice in the model of hind limb ischemia. However, when we assessed the role of Del-1 in HUVEC in vitro , silencing of Del-1 by siRNA did not affect angiogenic sprouting. Moreover, the ischemic muscles of Del-1 −/− mice displayed a higher infiltration with CD45 + hematopoietic cells than WT mice, suggesting that Del-1 may have an inhibitory effect on homing of PC and inflammatory cells to ischemic tissues. Interestingly, in adhesion assays human endothelial progenitor cells (EPC) and murine Lin − progenitor cells bound to Del-1 via beta2-integrins, but not via the alphaVbeta3- and alphaVbeta5- integrins. Furthermore, soluble Del-1 significantly inhibited the adhesion of EPC to HUVEC monolayers and to the major beta2-integrin-ligand, ICAM-1, raising the possibility that Del-1 is a beta2-integrin-inhibitor. Indeed, WT murine bone marrow mononuclear cells displayed higher adhesion rates on Del-1-deficient murine lung endothelial cells (LEC) than on WT LEC. In order to investigate the role of Del-1 for in vivo homing of PC, we intravenously injected murine fluorescence-labeled WT Lin − bone marrow PC in WT and Del-1 −/− mice 2 days after the induction of hind limb ischemia. Interestingly, the homing of injected Lin − cells to ischemic muscles was significantly increased in Del-1 −/− in comparison to WT mice (200± 30 % increase). Taken together, endogenous Del-1 is a new beta2-integrin ligand, which blocks beta2-integrin-dependent adhesion and homing of PC to ischemic tissues. It is conceivable, that endogenous Del-1 may reduce ischemia-induced neovascularization through an inhibitory effect on the beta2-integrins of progenitor and inflammatory cells.

Ras associated protein 1 (Rap1), a small GTPase of the Ras family, has attracted much attention because of its involvement in several aspects of cell adhesion, including integrin- and cadherin-mediated adhesion. Yet, the role of Rap1 genes for integrin signaling in endothelial cells (EC) and angiogenesis has not been investigated. Human umbilical vein endothelial cells (HUVEC) express Rap1a and Rap1b mRNA as assessed by RT-PCR. In order to determine the contribution of Rap1 activity for angiogenesis, we overexpressed Rap1GAP1, a GTPase activating protein, which specifically inhibits the activity of both Rap1a and Rap1b. Overexpression of Rap1GAP1 led to a significant inhibition of angiogenic sprouting of HUVEC under basal conditions and bFGF stimulation by 44 ± 5 % in a 3-dimensional spheroidal system and blocked tube formation in a matrigel assay, migration and adhesion. In order to separately investigate the role of Rap1a and Rap1b genes in angiogenesis, we performed gene silencing with siRNA. Silencing of either Rap1a or Rap1b significantly and additively blocked the sprouting of HUVEC under basal and bFGF-stimulated conditions (Rap1a-siRNA: 55 ± 5 %, Rap1b-siRNA: 61 ± 9 % and Rap1a+Rap1b siRNA: 73 ± 5% inhibition) and significantly reduced HUVEC migration and adhesion on fibronectin and collagen. Moreover, silencing of Rap1a and Rap1b reduced beta1-integrin affinity in HUVEC, suggesting the importance of Rap1a and Rap1b for inside-out integrin activation in EC. In addition, silencing of Rap1a and Rap1b prevented VEGF-induced PKB/Akt1 activation. These data prompted us to investigate the in vivo role of Rap1a using Rap1a-deficient mice. Interestingly, Rap1a −/− mice are born with a substantially reduced mendelian ratio. Rap1a +/− heterozygote mice displayed decreased microvessel density in comparison to wild-type mice (Rap1a +/+ ) in a matrigel plug assay. Moreover Rap1a +/− and Rap1a −/− displayed significantly reduced microvessel density in ischemic muscles in the model of hind limb ischemia in comparison to wild-type mice (Rap1a +/− : 32 ± 3 % ; Rap1a −/− : 43 ± 3 % inhibition). Thus, our data demonstrated a critical role of Rap1 in the regulation of β1-integrin signaling in endothelial cells and for postnatal neovascularization.

Endothelial progenitor cells (EPCs) and hematopoietic progenitor cells are recruited to ischemic regions, improving neovascularization. β1 and β2 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 γ (PI3Kγ) is the PI3K isoform that selectively transduces signals from G protein–coupled receptors. Here, we investigated the role of PI3Kγ as a signaling intermediate in the chemokine-induced integrin-dependent homing functions of progenitor cells. A pharmacological PI3Kγ inhibitor significantly reduced chemokine-induced chemotaxis and stromal cell–derived factor (SDF)1α-induced transmigration of human EPCs. Moreover, the PI3Kγ inhibitor significantly reduced SDF1α-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 PI3Kγ-deficient mice that displayed reduced SDF1α-induced migration and intercellular adhesion molecule-1 adhesion as compared with wild-type cells. Pharmacological inhibition or genetic ablation of PI3Kγ reduced SDF1α-induced integrin activation in human EPCs and in murine Lin−BM-derived progenitor cells, respectively. In vivo, the homing of PI3Kγ-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, PI3Kγ is integral to the integrin-dependent homing of progenitor cells.

Endothelial progenitor cells (EPC) are recruited to ischemic regions to improve neovascularization. β1- and β2-integrins play a crucial role for progenitor cell homing to ischemic tissues. Chemokines and their respective G-protein coupled receptors (GPCRs) are involved in the EPC homing to ischemic tissues. The phosphatidylinositol-3-kinase catalytic subunit gamma (PI3Kγ) is the PI3K isoform, which selectively transduces signals from GPCRs. Here, we investigated the role of PI3Kγ for integrin-dependent homing functions of progenitor cells. As assessed by western blot, EPC express the catalytic subunit PI3Kγ. We then studied the role of PI3Kγ for EPC migration. AS-605240 (100 nM), a selective PI3Kγ-inhibitor (Camps M, Nat. Med., 2005), significantly reduced the SDF1- and the IL-8-induced migration and the SDF1-induced transendothelial migration of human EPC. Adhesion is a further essential step during EPC homing to ischemic tissues. In this regard, the PI3Kγ-inhibitor significantly reduced the SDF1-induced adhesion of EPC on HUVEC monolayers by 69 ± 8 % and on ICAM-1, a β2-integrin ligand. However, the PI3Kγ-inhibitor did not affect the SDF1-induced adhesion of EPC on fibronectin, a β1-integrin ligand, suggesting that PI3Kγ in EPC is involved in the regulation of β2-, but not of β 1-integrin-dependent adhesion. In line with these results, inhibition PI3Kγ blocked the SDF1-induced increase of β2-, but not of β1-integrin-affinity in EPC. Beside EPC, the SDF1-induced migration and adhesion on ICAM-1 of murine bone marrow (BM)-derived Lin − progenitor cells from PI3Kγ-deficient mice (PI3Kγ − / − ) were reduced in comparison to wild type (WT) cells. In addition, PI3Kγ-deficiency led to a significant reduction of homing of murine BM-Lin − progenitor cells to ischemic muscles after intravenous infusion in the model of hind limb ischemia in comparison to WT cells (48 ± 8 % inhibition). In conclusion, these data demonstrate that PI3Kγ plays an essential role for the integrin-dependent homing of progenitor cells in vitro and in vivo. The understanding of the molecular mechanisms of progenitor cell homing is essential for the development of new therapeutic strategies in order to improve the efficacy of cell-based therapies in patients with ischemic disorders.