Embryonic Endothelial Progenitor Cells Research Articles

Adherent Platelets Recruit and Induce Differentiation of Murine Embryonic Endothelial Progenitor Cells to Mature Endothelial Cells In Vitro

The homing and differentiation mechanisms of endothelial progenitor cells (EPCs) at sites of vascular lesions are unclear. To investigate whether platelets play a role in the recruitment and differentiation of EPCs, we made use of a robust mouse embryonic EPC (eEPC) line that reliably differentiates to a mature endothelial phenotype. We found that platelets stimulate chemotaxis and migration of these murine eEPCs. Further, the substantial adhesion of murine eEPCs on immobilized platelets that occurs under dynamic flow conditions is inhibited by neutralizing anti-P-selectin glycoprotein ligand-1 and anti-VLA-4 (beta1-integrin) monoclonal antibodies but not by anti-CD11b (aM-integrin; macrophage antigen-1). Coincubation of murine eEPCs with platelets for 5 days induced differentiation of EPCs to mature endothelial cells as verified by positive von Willebrand factor immunofluorescence and detection of Weibel Palade bodies through electron microscopy. We conclude that platelets may play a critical part in the capture and subsequent differentiation of murine eEPCs at sites of vascular lesions, revealing a possible new role of platelets in neoendothelization after vascular injury.

Embryonic endothelial progenitor cells expressing a broad range of proangiogenic and remodeling factors enhance vascularization and tissue recovery in acute and chronic ischemia

Clonal embryonic endothelial progenitor cells (eEPCs) isolated from embryonic day 7.5 mice home specifically to hypoxic areas in mouse tumor metastases but spare normal organs and do not form carcinomas. Based on these results, we assessed the potential of eEPCs to enhance vascularization and limit organ dysfunction after ischemia in syngenic and xenotypic organisms. The angiogenic potential of eEPCs was evaluated in chronic ischemic rabbit hindlimbs after regional application by retroinfusion. eEPC treatment improved limb perfusion, paralleled by an increase in capillary density and collateral blood vessel number. Systemic eEPC infusion into mice after ischemic cardiac insult increased postischemic heart output measured by a marked improvement in left ventricle developed pressure and both systolic and diastolic functions. In vitro, eEPCs strongly induced vascular outgrowths from aortic rings. To address the molecular basis of this intrinsic angiogenic potential, we investigated the eEPC transcriptome. Genome-wide Affymetrix GeneChip analysis revealed that the eEPCs express a wealth of secreted factors known to induce angiogenesis, tissue remodeling, and organogenesis that may contribute to the eEPC-mediated beneficial effects. Our findings show that eEPCs induce blood vessel growth and cardioprotection in severe ischemic conditions providing a readily available source to study the mechanisms of neovascularization and tissue recovery.

Embryonic endothelial progenitor cells armed with a suicide gene target hypoxic lung metastases after intravenous delivery

We show that mouse embryonic endothelial progenitor cells (eEPCs) home preferentially to hypoxic lung metastases when administered intravenously. This specificity is inversely related to the degree of perfusion and vascular density in the metastasis and directly related to local levels of hypoxia and VEGF. Ex vivo expanded eEPCs that were genetically modified with a suicide gene specifically and efficiently eradicated lung metastases with scant patent blood vessels. eEPCs do not express MHC I proteins, are resistant to natural killer cell-mediated cytolysis, and can contribute to tumor vessel formation also in nonsyngeneic mice. These results indicate that eEPCs can be used in an allogeneic setting to treat hypoxic metastases that are known to be resistant to conventional therapeutic regimes.

Therapeutic angiogenesis. Devising new strategies based on past experiences.

Therapeutic angiogenesis improves tissue ischemia by supplementing and supporting the intrinsic process of angiogenesis. Besides direct administration of angiogenic growth factor proteins, injection of naked DNA, non-viral vectors and viral vector constructs carrying angiogenic genes have been used. A novel approach is to achieve therapeutic angiogenesis through cell mediated gene transfer. Genetically modulated cells carrying exogenous genes encoding for angiogenic factors and the cells with inherent ability to secrete angiogenic cytokines, such as bone marrow stem cells, embryonic stem cells and endothelial progenitor cells, have been used to achieve revascularization. This review discusses proof of the concept pre-clinical studies and phase-I/II human trials using VEGF, and cellular angiogenesis at length in the light of the literature and analyzes the problems and considerations of these approaches as a treatment strategy in the clinical perspective for the treatment of ischemic heart disease.

Multistep nature of microvascular recruitment of ex vivo-expanded embryonic endothelial progenitor cells during tumor angiogenesis.

Tissue neovascularization involves recruitment of circulating endothelial progenitor cells that originate in the bone marrow. Here, we show that a class of embryonic endothelial progenitor cells (Tie-2+, c-Kit+, Sca-1+, and Flk-1−/low), which were isolated at E7.5 of mouse development at the onset of vasculogenesis, retain their ability to contribute to tumor angiogenesis in the adult. Using intravital fluorescence videomicroscopy, we further defined the multistep process of embryonic endothelial progenitor cell (eEPC) homing and incorporation. Circulating eEPCs are specifically arrested in “hot spots” within the tumor microvasculature, extravasate into the interstitium, form multicellular clusters, and incorporate into functional vascular networks. Expression analysis and in vivo blocking experiments provide evidence that the initial cell arrest of eEPC homing is mediated by E- and P-selectin and P-selectin glycoprotein ligand 1. This paper provides the first in vivo insights into the mechanisms of endothelial progenitor cell recruitment and, thus, indicates novel ways to interfere with pathological neovascularization.

Endothelial progenitor cells for neovascularization.

Available evidence suggests that hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs)(Risau et al. 1988; Pardanaud et al. 1989) are derived from a common precursor, the hemangioblast (Flamme and Risau 1992; His 1900; Weiss and Orkin 1996). The identification of putative adult HSCs in peripheral blood and bone marrow (BM) and the demonstration of sustained hematopoietic reconstitution with these HSC transplants have constituted inferential evidence for the recognition of HSC (Brugger et al. 1995; Kessinger and Armitage 1991; Sheridan et al. 1992; Shpall et al. 1994). The related descendants of HSCs, EPCs, have been isolated along with HSCs in hematopoietic organs. Flk-1 and a second antigen, CD 34, shared by embryonic EPCs and HSCs were used to detect putative EPCs from a mononuclear cell fraction of peripheral blood (Asahara et al. 1997) (Fig. 1). In vitro, these cells differentiated into endothelial lineage cells, and in animal models of ischemia, heterologous, homologous, and autologous EPCs were shown to incorporate into sites of active neovascularization (Fig. 2).