Mediators of endothelial cell injury in low perfusion conditions

Abstract

Abstract Introduction: During free tissue transfer operations, endothelial cells (EC) are exposed to a period of ischemia that can affect outcome. We compared gene expression profiles of ECs in normal culture conditions to serum-deprived conditions as an in vitro model of tissue stress. Methods: ECs from multiple body sites were grown in EGM-2MV with 5% fetal bovine serum (FCS). For serum-deprived samples, ECs were switched to EGM-2MV with 0.25% FCS. After 24h, ECs were harvested and mRNA was extracted. Gene expression patterns were compared between the normal and serum-deprived samples using cDNA microarray. Specific gene expression was then confirmed by quantitative PCR. Results: There are 1083 genes that show more than a four fold difference in expression when comparing normal versus serum-deprived growth conditions. ECs exposed to serum deprivation up regulate inflammatory and coagulation factor genes while cell cycle genes are down regulated. Up regulated inflammatory mediators included MCP-1, interferon-alpha, TGF-alpha, colony stimulating factor 2, TNFC, and TNF receptor-associated factor 5. Coagulation factors included thrombin and the thrombin receptor. The down regulated cell cycle genes included cyclin B2 and c-myc binding protein. Conclusions: Our data suggest that serum deprivation induces gene expression of inflammatory mediators and coagulation factors that can lead to poor outcome during tissue transfer. Understanding the mechanism of EC injury may lead to the development of rationally designed therapies aimed at minimizing complications and improving outcomes.