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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 (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.