An In Vitro Model for the Study of Human Bone Marrow Angiogenesis: Role of Hematopoietic Cytokines

Abstract

SUMMARY:This study describes a human bone marrow endothelial cell culture in which endothelial cells are organized into capillary tubes. These endothelial cells were positive for von Willebrand Factor, expressed CD34, CD31, and L-fucose residues, took up acetylated low-density lipoproteins, contained Weibel-Palade bodies, and were ensheathed in a basal lamina (which included laminin ␤1, EDaϩ and EDbϩ fibronectin, and collagen type iv). Pericytes expressing ␣-smooth muscle (␣-SM) actin were spatially associated with the capillary tubes and there was a highly significant correlation between the number of capillary tubes and pericytes. In this model, basal angiogenesis was found to be vascular endothelial growth factor (VEGF)-dependent, because neutralization of endogenous VEGF induced a dramatic regression in the number of tubes. However, the presence of ␣-SM actin-expressing pericytes in the linings of endothelial tubes partially prevented the VEGF-neutralized tube regression. We also observed that nitric oxide production contributed to basal angiogenesis and that upregulation of nitric oxide increased the number of tubes. Tube numbers also decreased when antibodies neutralizing the integrin ␣v␤5 were applied to the cultures. Moreover, addition of any of the hematopoietic cytokines, erythropoietin, stem cell factor, granulocytic colony stimulating factor, or granulomonocytic colony stimulating factor induced a highly significant increase in tube formation. When erythropoietin and granulocytic colony stimulating factor were added, this increase was larger than the maximum increase observed with VEGF. Thus, we have described an in vitro model for human bone marrow angiogenesis in which pericytes and basal lamina matrix were associated with endothelial cells and formed fully organized capillary tubes. In this model, cytokines known to regulate hematopoiesis also seemed to be mediators of angiogenesis. This culture system may therefore prove to be a valuable tool for the study of hematopoietic cytokines on angiogenesis. (Lab Invest 2000, 80:501-511).

Low Erythropoietin Plasma Levels during Exacerbations of COPD

“…Likewise, Epo appears to mobilize endothelial progenitor cells from the bone marrow (EPCs) and to increase neovascularization in coronary ischemia [6]. In fact, in an in vitro angiogenesis assay the stimulation of capillary outgrowth by Epo was comparable to that produced by the vascular endothelial growth factor (VEGF) [7,8]. On the other hand, it is known that the pro-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) suppress Epo gene expression and Epo protein secretion in vitro, and may be responsible for impaired Epo synthesis in inflammatory diseases in vivo [9].…”

Erythropoietin and hypoxia stimulate erythropoietin receptor and nitric oxide production by endothelial cells

“…1,5,11,12 In an in vitro angiogenesis assay, the stimulation of capillary outgrowth by EPO is comparable with that of vascular endothelial growth factor (VEGF). 13,14 EPO has been proposed to increase neovascularization induced by inflammation or ischemia associated with coronary heart disease, possibly via mobilizing endothelial progenitor cells from the bone marrow. 15 EPO is produced primarily in the fetal liver and adult kidney in a hypoxia-dependent manner.…”

Control of autocrine and paracrine myocardial signals: an emerging therapeutic strategy in heart failure

“…The G-CSF receptor (G-CSFR) is expressed by endothelial cells. In vitro, G-CSF has been shown to directly stimulate the migration and proliferation of endothelial cells [79], their repair of mechanically wounded endothelial monolayers [80], and the formation of capillary tube-like structures [81]. G-CSF also promotes vascular smooth muscle cell proliferation [82], which may be important for structural integrity in maturing vessels.…”