Introduction: Delays in vascularization of cultured skin substitutes (CSS) contribute to clinical failure. Our CSS were generated from the spontaneously immortalized NIKS human cell line, a consistent source of non-tumorigenic and pathogen-free progenitor keratinocytes. NIKS were genetically engineered to express transgenes encoding the pro-angiogenic growth factor VEGF-165 (CSS-VEGF) or a form of the transcription factor hypoxia inducible factor alpha lacking the oxygen sensitive domain (CSS-HIF1a). The purpose of this study was to determine whether our pro-angiogenic cell lines increase vascularity in grafted CSS. Methods: CSS-VEGF, CSS-HIF1a, and control CSS were grafted to the dorsum of nude mice (n=3 per CSS). After 9 days grafts were harvested with the wound bed and fresh frozen. Indirect immunofluorescence for CD31 was performed on sections and section images acquired with fluorescent microscopy. Blood vessel density (% area) was then quantified for each graft. Expression of pro-angiogenic and anti-angiogenic genes was also determined by microarray analysis for each cell line. Significant differences compared to control CSS were identified as either a >/= 2-fold increase or </= 0.5 decrease in gene expression. Results: Blood vessel density for CSS-VEGF was 4.5+/-0.7%, CSS-HIF1a=2.2+/-0.4%, and control CSS=1.9+/-0.3%. CSS-VEGF had more vascular in-growth than control CSS (p=0.03). Vessel growth induced by CSS-HIF1a was not statistically different from control. Microarray analysis revealed several differences in gene expression relative to controls. The NIKS-VEGF cell line showed increased expression of pro-angiogenic growth factors VEGF, IL-8 and midkine, and decreased expression of pro-angiogenic platelet derived growth factor alpha (PDGF-A).  The NIKS-HIF1a cell line showed increased expression of HIF-1a, cyclooxygenase-2 (COX-2), pleiotrophin, and tissue inhibitor of metallopeptidase 2 (TIMP-2). Conclusion: Non-viral genetic engineering of NIKS keratinocytes results in enhanced neo-vascularization in this preliminary study and no pathologic vessel formation was observed in any specimens. CSS-VEGF produced more functional vessels than both CSS-HIF1a and control CSS. Q-PCR analysis is underway to confirm microarray results, and additional clone selection in progress. Future studies will address whether enhanced vascularity results in improved wound healing.