In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse

Abstract

We have identified conditions for forming cultured human umbilical vein endothelial cells (HUVEC) into tubes within a threedimensional gel that on implantation into immunoincompetent mice undergo remodeling into complex microvessels lined by human endothelium. HUVEC suspended in mixed collagen͞ fibronectin gels organize into cords with early lumena by 24 h and then apoptose. Twenty-hour constructs, s.c. implanted in immunodeficient mice, display HUVEC-lined thin-walled microvessels within the gel 31 days after implantation. Retroviral-mediated overexpression of a caspase-resistant Bcl-2 protein delays HUVEC apoptosis in vitro for over 7 days. Bcl-2-transduced HUVEC produce an increased density of HUVEC-lined perfused microvessels in vivo compared with untransduced or control-transduced HUVEC. Remarkably, Bcl-2-but not control-transduced HUVEC recruit an ingrowth of perivascular smooth-muscle ␣-actin-expressing mouse cells at 31 days, which organize by 60 days into HUVEC-lined multilayered structures resembling true microvessels. This system provides an in vivo model for dissecting mechanisms of microvascular remodeling by using genetically modified endothelium. Incorporation of such human endothelial-lined microvessels into engineered synthetic skin may improve graft viability, especially in recipients with impaired angiogenesis.

Treatment of melanoma cells with a bcl-2/bcl-xL antisense oligonucleotide induces antiangiogenic activity

“…The involvement of bcl-2 in angiogenesis has been demonstrated in various human tumour histotypes (Biroccio et al, 2000;Fernandez et al, 2001;Iervolino et al, 2002) and in endothelial cells (Nor et al, 1999(Nor et al, , 2001Schechner et al, 2000). The expression of VEGF and the angiogenic potential of human breast carcinoma, prostate and melanoma cells exposed to hypoxia are increased after bcl-2 overexpression (Biroccio et al, 2000;Fernandez et al, 2001;Iervolino et al, 2002), and upregulation of bcl-2 in human endothelial cells enhances the number of capillary-like sprouts and the density of vascular structures (Nor et al, 1999(Nor et al, , 2001Schechner et al, 2000).…”

“…The expression of VEGF and the angiogenic potential of human breast carcinoma, prostate and melanoma cells exposed to hypoxia are increased after bcl-2 overexpression (Biroccio et al, 2000;Fernandez et al, 2001;Iervolino et al, 2002), and upregulation of bcl-2 in human endothelial cells enhances the number of capillary-like sprouts and the density of vascular structures (Nor et al, 1999(Nor et al, , 2001Schechner et al, 2000). Since bcl-2 is an inhibitor of apoptosis and confers varying degrees of resistance to chemotherapy (Biroccio et al, 1999;Del Bufalo et al, 2001) dependent on the drugs used (Del Bufalo et al, 2002a, b), a better knowledge of bcl-2 role in angiogenesis may help to design more effective combination therapies.…”

Working hypothesis to redefine endothelial progenitor cells

“…87,88 We hypothesized that the most definitive method to assay for postnatal vasculogenesis would be to suspend putative EPCs in a scaffold, implant the scaffold, and assay for de novo human vessel formation. Following the work of Schechner et al, 89,90 we established a de novo EPC differentiation model in NOD/SCID mice. 39,89,90 In this model, collagen I gels are mixed with EPC test cells and implanted into immunodeficient mice.…”

“…Following the work of Schechner et al, 89,90 we established a de novo EPC differentiation model in NOD/SCID mice. 39,89,90 In this model, collagen I gels are mixed with EPC test cells and implanted into immunodeficient mice. Upon scaffold excision 14 days later, the number and histological characteristics of human vessels are examined.…”

Synthesis of organs: In vitro or in vivo ?

“…1 and 2). A possible explanation for the eventual lack of physiological vascularization using this protocol is suggested by Schechner and coworkers (1). When these authors cultured endothelial cells in a collagenfibronectin gel they observed spontaneous reorganizations into multicellular cords, resembling incipient vessels; however, the cells soon thereafter showed morphologic evidence of apoptosis and died (1).…”