56: In vitro generation of human microvessel networks

Abstract

Introduction: Successful incorporation of bioengineered tissues requires vascularization. Attempts at de novo vascular generation or angiogenic induction have had little success in improving tissue perfusion. An alternate approach to neovascularization is to form a prevascularized construct that is capable of rapid inosculation and rapid cellular perfusion. Human parathyroid (PTH) tissue is uniquely capable of a rapid angiogenic induction, with restoration of calcium homeostasis within 6 weeks of PTH autografting. We hypothesized that human PTH cells could induce human microvessel fragments to form networks of capillary microvessels in vitro. Because PTH cells release VEGF and Angiopoietin-2 (Ang-2) after devascularization, we further hypothesized that PTH angiogenic induction is driven by PTH release of VEGF and Ang-2. Methods: Human subcutaneous fat was partially digested with collagenase to yield microvessel fragments. After sequential filtering, 30-350 micron fragments were embedded in DMEM + 10% FBS-containing collagen I matrix in 3 dimensional culture. PTH cells were cocultured on the matrix surface to induce an angiogenic response. Other matrix cultures were treated with VEGF (50 ng/ml) or VEGF + Ang-2 (50 ng/ml) and microvessel responses noted. Because Stat3 phosphorylation has been identified with endothelial cell (EC) angiogenic responses, we also tested for Stat3 activation in human dermal microvessel EC (HDMEC) after treatment with VEGF or VEGF + Ang-2. Results: Human microvessel fragments in culture are completely quiescent. Coculture with PTH cells induces the rapid development of neovascular networks. Treatment of the human microvessels in vitro with VEGF failed to induce an angiogenic event. VEGF + Ang-2 induced EC migration but no capillary formation. Both VEGF and Ang-2 induced Stat3 phosphorylation in HDMEC, but not in iliac vein EC. Conclusions: Human PTH cells release or induce release of all necessary angiogenic factors for capillary network angiogenic induction of human microvessels in vitro. The mechanism of angiogenic induction is more complex than VEGF stimulation alone. Stat 3 activity is upregulated in microvessel EC after VEGF or Ang-2 stimulation, but not in large vessel EC, suggesting a possible role in microvessel angiogenic processes. PTH cells have the angiogenic potential to induce capillary network development in vitro. This system may be capable of providing vascularization of bioengineered tissues for therapeutic intervention.