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.

The origin and in vivo significance of murine and human culture-expanded endothelial progenitor cells.

In adults highly purified populations of early hematopoietic progenitors or cells derived from ex vivo expanded unmobilized human peripheral blood mononuclear cells contribute to new blood vessel formation. However, the source of these culture-expanded endothelial progenitor cells (CE-EPCs) remains controversial. We demonstrate that ex vivo expansion of unmobilized human peripheral blood generated CE-EPCs with similar numbers, kinetics, and antigen expression profile as compared to plating unfractionated CD34(+)/lin(-)-enriched bone marrow mononuclear cells. Both CE-EPC populations uniformly co-expressed myeloid and endothelial markers, suggesting that peripheral blood progenitor enumeration does not correlate with the numbers of early outgrowth CE-EPCs. Using purified myeloid subpopulations obtained from mice harboring the lacZ transgene driven by an endothelial-specific promoter, we showed that the immature myeloid lineage marker CD31(+) cells generated CE-EPCs with fourfold greater frequency than mature myeloid populations. Biphenotypic cells co-expressing myeloid/endothelial antigens were not detected in circulating human or murine peripheral blood or bone marrow but were associated with murine tumors. Unlike CE-EPCs, CD14(+) leukocytes admixed within tumors did not generate vWF-positive blood vessels during a similarly defined period of tumor growth, but some leukocytes up-regulated the endothelial marker VE-cadherin. Taken together, the data suggest that the local neovascular microenvironment may facilitate vasculogenesis by promoting endothelial differentiation and that CE-EPCs may accelerate such vasculo-genesis.

Enrichment of genes in the aortic intima that are associated with stratified epithelium: implications of underlying biomechanical and barrier properties of the arterial intima.

BACKGROUND: Arteries and veins are exposed to different pressures and are easily distinguished by morphology. Although several recent studies have focused on differential gene expression between the arterial and venous endothelium, the molecular distinctions that give rise to the dramatic structural distinctions between arteries and veins, such as in the organization of the intima, are not known. METHODS AND RESULTS: We used high-density oligonucleotide arrays to analyze the transcriptional profile of the mouse aorta and inferior vena cava (IVC), not restricting our analysis to the endothelium, to identify genes whose expression was enriched in aorta over other tissues and the IVC. By quantitative reverse transcription-polymerase chain reaction analysis, these genes have been shown to be highly expressed in the mouse aorta and were either expressed at low levels or were undetectable in the murine IVC. By immunofluorescence analysis of human tissue, we determined that a subset of these aorta-enriched proteins exhibited a primarily intima-restricted expression. Intimal expression of at least a subset of these genes, plakoglobin, galectin 7, sciellin, and SPRR3, was also detected in other types of arteries but not in veins. Furthermore, SPRR3 expression in the intima was primarily associated with atheromas. The proteins identified are functionally related in that they are known to also be enriched in stratified epithelia, where they play an important role in stress-bearing and barrier properties. CONCLUSIONS: Vascular expression of these genes has not been reported previously. Our observations suggest that they may play a significant role in the mechanisms by which large arteries may adapt to biomechanical stress.

Blood donor white blood cell reduction filters as a source of human peripheral blood-derived endothelial progenitor cells.

BACKGROUND: Neovascularization in tumors, wounds, and sites of ischemic injury occurs by both angiogenesis (proliferation from existing vessels) and by vasculogenesis (differentiation into endothelial cells from circulating endothelial progenitor cells [EPCs]). EPCs can be obtained from marrow, from cord blood, or by ex vivo expansion of human peripheral blood (PB). The ease of obtaining human PB EPCs has led many recent studies to utilize PB EPCs. The ability to obtain large numbers of PB EPCs would greatly facilitate characterization to further our understanding of EPC biology and their application in cellular gene therapy. STUDY DESIGN AND METHODS: Peripheral blood mononuclear cells (PBMNCs) from whole blood or from the material obtained from white blood cell (WBC) reduction filters were isolated. The cells from both sources were then cultured separately under defined conditions to quantify EPC yield from each source. RESULTS: The yield of EPCs per million PBMNCs plated was approximately 3.5-fold higher from fresh PB. Because greater numbers of PBMNCs were obtained from each filter, however, the mean yield of EPCs from one filter versus fresh blood was 5.4 million versus 0.4 million, respectively (approx. 14-fold increased yield). CONCLUSION: The use of WBC reduction filters provides a safe, inexpensive, and readily available source for large numbers of PBMNCs from which culture-expanded EPCs can be generated for further study.