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).

Summary. We have previously described a culture system for human bone marrow endothelial cells that organize into capillary tubes associated to pericytes. In the present work, we used this model to assess the angiogenic properties of tachykinins, which have been demonstrated to be involved in neuro-immuno-haematopoietic interactions. The substance P (SP) and neurokinin A (NKA) were similarly potent at increasing in vitro angiogenesis, via NK1 and NK2 receptors respectively. These mediators were not produced by cells in culture, suggesting that in vivo they may be released by nerve fibres in the bone marrow. Therefore, we looked for in situ innervation of the human bone marrow, unknown to date, using immunohistochemistry techniques. As in rodents, arterioles were largely innervated, associated with between one and 10 nerve fibres. Capillary innervation was more restrictive as a unique thin nerve fibre was found in the vicinity of only 6% of these vessels. Finally, no nerve fibres were observed in the vicinity of sinus walls. In conclusion, both in vitro results and the anatomical display of nerve fibres suggest a role in human bone marrow for the vasoactive neuropeptides SP and NKA, which were secreted into a perivascular location. These neural mediators might modulate blood flow in the bone marrow both in the short term by adjusting vascular tone and in the long term by inducing angiogenesis.