Role of the small GTPase Rap1 for integrin activity regulation in endothelial cells and angiogenesis.

Ras-associated protein 1 (Rap1), a small GTPase, attracted attention because of its involvement in several aspects of cell adhesion, including integrin- and cadherin-mediated adhesion. Yet, the role of Rap1 genes and of Rap1 effectors for angiogenesis has not been investigated. Human umbilical vein endothelial cells (HUVECs) express Rap1a and Rap1b mRNA. To determine the contribution of Rap1 activity for angiogenesis, we overexpressed Rap1GAP1, a GTPase-activating protein that inhibits Rap1 activity. Overexpression of Rap1GAP1 significantly blocked angiogenic sprouting and tube-forming activity of HUVECs as well as migration and integrin-dependent adhesion. Silencing of Rap1a, Rap1b, or both significantly blocked HUVECs sprouting under basal and basic fibroblast growth factor-stimulated conditions and reduced HUVEC migration and integrin-dependent adhesion. We found that Rap1a and Rap1b are essential for the conformational activation of beta(1)-integrins in endothelial cells. Furthermore, silencing of Rap1a and Rap1b prevented phosphorylation of tyrosine 397 in focal adhesion kinase (FAK) and vascular endothelial growth factor-induced Akt1-activation. Rap1a(-/-)-deficient and Rap1a(+/-) heterozygote mice displayed reduced neovascularization after hind limb ischemia compared with wild-type mice. Silencing of RAPL significantly blocked the Rap1-induced sprouting of HUVECs, suggesting that the angiogenic activity of Rap1 is partly mediated by RAPL. Our data demonstrate a critical role of Rap1 in the regulation of beta(1)-integrin affinity, adhesion, and migration in endothelial cells and in postnatal neovascularization.

Long-term diabetes impairs repopulation of hematopoietic progenitor cells and dysregulates the cytokine expression in the bone marrow microenvironment in mice.

Diabetes is characterized by a chronic stage of hyperglycemia associated with endothelial progenitor cell dysfunction and reduced neovascularization in response to tissue ischemia. The underlying mechanisms are not entirely clear. The bone marrow niches provide the essential microenvironment for maintenance of stem cell function in the bone marrow. A disturbed stem cell niche might lead to stem cell dysfunction, thereby, impairing progenitor cell-dependent vascular repair. Therefore, we investigated the effects of streptozotocin-induced diabetes on the bone marrow stem cell niches and stem cell function in mice. Here, we show that long-term diabetes induced a reduction in Lin⁻Sca-1(+)c-kit(+) hematopoietic progenitor cells and reduced the repopulation capacity in a competitive engraftment experiment. Consistently, the expression of Bmi1, which prevents hematopoietic progenitor cell senescence, was significantly reduced in diabetic bone marrow cells. To address the mechanism underlying the progenitor cell dysfunction, we analyzed the composition of the stem cell niche and the cytokine environment. Although the morphology of the vascular and endosteal niche was not affected by diabetes, diabetic mice showed a significant deterioration of cytokine expression patterns in the bone marrow. In summary, these data indicate that diabetes imposes a long-term effect on the stem cell niche and affects important hematopoietic progenitor cell functions in mice.

The Wnt antagonist Dickkopf-1 mobilizes vasculogenic progenitor cells via activation of the bone marrow endosteal stem cell niche.

Therapeutic mobilization of vasculogenic progenitor cells is a novel strategy to enhance neovascularization for tissue repair. Prototypical mobilizing agents such as granulocyte colony-stimulating factor mobilize vasculogenic progenitor cells from the bone marrow concomitantly with inflammatory cells. In the bone marrow, mobilization is regulated in the stem cell niche, in which endosteal cells such as osteoblasts and osteoclasts play a key role. Because Wnt signaling regulates endosteal cells, we examined whether the Wnt signaling antagonist Dickkopf (Dkk)-1 is involved in the mobilization of vasculogenic progenitor cells. Using TOP-GAL transgenic mice to determine activation of beta-catenin, we demonstrate that Dkk-1 regulates endosteal cells in the bone marrow stem cell niche and subsequently mobilizes vasculogenic and hematopoietic progenitors cells without concomitant mobilization of inflammatory neutrophils. The mobilization of vasculogenic progenitors required the presence of functionally active osteoclasts, as demonstrated in PTPepsilon-deficient mice with defective osteoclast function. Mechanistically, Dkk-1 induced the osteoclast differentiation factor RANKL, which subsequently stimulated the release of the major bone-resorbing protease cathepsin K. Eventually, the Dkk-1-induced mobilization of bone marrow-derived vasculogenic progenitors enhanced neovascularization in Matrigel plugs. Thus, these data show that Dkk-1 is a mobilizer of vasculogenic progenitors but not of inflammatory cells, which could be of great clinical importance to enhance regenerative cell therapy.

Endogenous developmental endothelial locus-1 limits ischaemia-related angiogenesis by blocking inflammation.

We have recently identified endothelial cell-secreted developmental endothelial locus-1 (Del-1) as an endogenous inhibitor of ß2-integrin-dependent leukocyte infiltration. Del-1 was previously also implicated in angiogenesis. Here, we addressed the role of endogenously produced Del-1 in ischaemia-related angiogenesis. Intriguingly, Del-1-deficient mice displayed increased neovascularisation in two independent ischaemic models (retinopathy of prematurity and hind-limb ischaemia), as compared to Del-1-proficient mice. On the contrary, angiogenic sprouting in vitro or ex vivo (aortic ring assay) and physiological developmental retina angiogenesis were not affected by Del-1 deficiency. Mechanistically, the enhanced ischaemic neovascularisation in Del-1-deficiency was linked to higher infiltration of the ischaemic tissue by CD45+ haematopoietic and immune cells. Moreover, Del-1-deficiency promoted ß2-integrin-dependent adhesion of haematopoietic cells to endothelial cells in vitro, and the homing of hematopoietic progenitor cells and of immune cell populations to ischaemic muscles in vivo. Consistently, the increased hind limb ischaemia-related angiogenesis in Del-1 deficiency was completely reversed in mice lacking both Del-1 and the ß2-integrin LFA-1. Additionally, enhanced retinopathy-associated neovascularisation in Del-1-deficient mice was reversed by LFA-1 blockade. Our data reveal a hitherto unrecognised function of endogenous Del-1 as a local inhibitor of ischaemia-induced angiogenesis by restraining LFA-1-dependent homing of pro-angiogenic haematopoietic cells to ischaemic tissues. Our findings are relevant for the optimisation of therapeutic approaches in the context of ischaemic diseases.

Myoendothelial differentiation of human umbilical cord blood-derived stem cells in ischemic limb tissues.

Human umbilical cord blood (UCB) contains high numbers of endothelial progenitors cells (EPCs) characterized by coexpression of CD34 and CD133 markers. Prior studies have shown that CD34+/CD133+ EPCs from the cord or peripheral blood (PB) can give rise to endothelial cells and induce angiogenesis in ischemic tissues. In the present study, it is shown that freshly isolated human cord blood CD34+ cells injected into ischemic adductor muscles gave rise to endothelial and, unexpectedly, to skeletal muscle cells in mice. In fact, the treated limbs exhibited enhanced arteriole length density and regenerating muscle fiber density. Under similar experimental conditions, CD34- cells did not enhance the formation of new arterioles and regenerating muscle fibers. In nonischemic limbs CD34+ cells increased arteriole length density but did not promote formation of new muscle fibers. Endothelial and myogenic differentiation ability was maintained in CD34+ cells after ex vivo expansion. Myogenic conversion of human cord blood CD34+ cells was also observed in vitro by coculture onto mouse myoblasts. These results show that human cord blood CD34+ cells differentiate into endothelial and skeletal muscle cells, thus providing an indication of human EPCs plasticity. The full text of this article is available online at http://www.circresaha.org.

Electrophysiological properties of mouse bone marrow c-kit+ cells co-cultured onto neonatal cardiac myocytes.

OBJECTIVE: Controversy about hematopoietic stem cells reprogramming into cardiac myocytes is currently supported by positive and negative findings. In fact, some reports have shown the ability of stem cells from the bone marrow (BM) to differentiate into cardiac myocytes and to contribute to myocardium repair, while others have reported the opposite. METHODS: C-kit(+) cells from mouse bone marrow were co-cultured onto neonatal cardiac myocytes. Hematopoietic stem cell-derived cells were analyzed by investigating the expression of cardiac markers and ion channels and by single-cell electrophysiological recordings. RESULTS: Groups of undifferentiated c-kit(+) cells displayed only outward currents. Co-cultured c-kit(+) stem cells on neonatal cardiac myocytes expressed cardiac markers and Na(+) and Ca(2+) voltage-gated ion channels. However, Na(+) and Ca(2+) currents were not detected by electrophysiological patch-clamp recordings even if caffeine and cyclopiazonic acid treatment showed the presence of intracellular calcium stores. This suggests that these channels, although expressed, were not functional and thus do not allow the coupling between excitation and contraction that is typical of cardiac myocytes. Nevertheless, co-cultured cells had a more hyperpolarized resting membrane potential and, at least in a subset of cells, displayed voltage-gated inward rectifier currents and outward currents. Co-cultured c-kit(+)-derived cells were not connected to surrounding cardiac myocytes through gap junctions. To induce a more pronounced differentiation, co-cultured cells were treated with BMP-4 and TGF-beta, two factors that were shown to trigger a cardiac myocyte differentiation pathway in embryonic stem (ES) cells. Even under these conditions, c-kit(+) cells did not differentiate into functionally active cardiac myocytes. However, TGF-beta/BMP-4-treated cells were hyperpolarized and showed and increased inward rectifier current density. CONCLUSIONS: Our study shows that mouse BM hematopoietic stem cells exhibit a limited plasticity to transdifferentiate into cardiac myocytes in culture.

Functional properties of cells obtained from human cord blood CD34+ stem cells and mouse cardiac myocytes in coculture.

Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human CD34(+) cells from the human umbilical cord blood (hUCB), cocultured with neonatal mouse cardiomyocytes, acquire the functional properties of myocardial cells and express human cardiac genes. hUCB CD34(+) cells were cocultured onto cardiomyocytes following an infection with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After 7 days, mononucleated EGFP(+) cells were tested for their electrophysiological features by patch clamp and for cytosolic [Ca(2+)] ([Ca(2+)](i)) homeostasis by [Ca(2+)](i) imaging of X-rhod1-loaded cells. Human Nkx2.5 and GATA-4 expression was examined in cocultured cell populations by real-time RT-PCR. EGFP(+) cells were connected to surrounding cells by gap junctions, acquired electrophysiological properties similar to those of cardiomyocytes, and showed action potential-associated [Ca(2+)](i) transients. These cells also exhibited spontaneous sarcoplasmic reticulum [Ca(2+)](i) oscillations and the associated membrane potential depolarization. However, RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion, under our experimental conditions, hUCB CD34(+) cells cocultured with murine cardiomyocytes formed cells that exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However, the expression of human-specific cardiac genes was undetectable by RT-PCR.

Del-1, an endogenous leukocyte-endothelial adhesion inhibitor, limits inflammatory cell recruitment.

Leukocyte recruitment to sites of infection or inflammation requires multiple adhesive events. Although numerous players promoting leukocyte-endothelial interactions have been characterized, functionally important endogenous inhibitors of leukocyte adhesion have not been identified. Here we describe the endothelially derived secreted molecule Del-1 (developmental endothelial locus-1) as an anti-adhesive factor that interferes with the integrin LFA-1-dependent leukocyte-endothelial adhesion. Endothelial Del-1 deficiency increased LFA-1-dependent leukocyte adhesion in vitro and in vivo. Del-1-/- mice displayed significantly higher neutrophil accumulation in lipopolysaccharide-induced lung inflammation in vivo, which was reversed in Del-1/LFA-1 double-deficient mice. Thus, Del-1 is an endogenous inhibitor of inflammatory cell recruitment and could provide a basis for targeting leukocyte-endothelial interactions in disease.

SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells.

Chemokine stromal derived factor 1 (SDF-1) is involved in trafficking of hematopoietic stem cells (HSCs) from the bone marrow (BM) to peripheral blood (PB) and has been found to enhance postischemia angiogenesis. This study was aimed at investigating whether SDF-1 plays a role in differentiation of BM-derived c-kit(+) stem cells into endothelial progenitor cells (EPCs) and in ischemia-induced trafficking of stem cells from PB to ischemic tissues. We found that SDF-1 enhanced EPC number by promoting alpha(2), alpha(4), and alpha(5) integrin-mediated adhesion to fibronectin and collagen I. EPC differentiation was reduced in mitogen-stimulated c-kit(+) cells, while cytokine withdrawal or the overexpression of the cyclin-dependent kinase (CDK) inhibitor p16(INK4) restored such differentiation, suggesting a link between control of cell cycle and EPC differentiation. We also analyzed the time course of SDF-1 expression in a mouse model of hind-limb ischemia. Shortly after femoral artery dissection, plasma SDF-1 levels were up-regulated, while SDF-1 expression in the bone marrow was down-regulated in a timely fashion with the increase in the percentage of PB progenitor cells. An increase in ischemic tissue expression of SDF-1 at RNA and protein level was also observed. Finally, using an in vivo assay such as injection of matrigel plugs, we found that SDF-1 improves formation of tubulelike structures by coinjected c-kit(+) cells. Our findings unravel a function for SDF-1 in increase of EPC number and formation of vascular structures by bone marrow progenitor cells.