Gene expression profile in laser dissected endothelial cells from vascularised heart transplant subjected to ischemia/reperfusion injury

Abstract

Introduction: Oligonucleotide microarray analysis can be powerful tool to determine gene expression changes after transplantation. However, the genes analyzed by this technique in transplanted organs do not provide information regarding specific cell types. Moreover, since transplanted organs are infiltrated with recipient lymphocytes, the changes in gene expression do not accurately reflect changes in either the donor organs or in the infiltrating lymphocytes. To obviate this difficulty we used the recently developed technique of laser microdissection (LMD) to obtain endothelial cells from heart allografts undergoing ischemia/ reperfusion injury (I/R). Methods: Endothelial cells were dissected from cryostat sections of coronary vessels of heart transplanted from C57 BL/6 (H-2b) donors and Balb/C (H-2d) recipients using the Leica® LS AMD laser dissector. RNA was subjected to three rounds of aRNA amplification and analyzed on an Affymetrix MG-U74A mouse Gene Chip®. Genes showing stastically significant changes from control endothelial cells dissected from normal C57 BL/6 mice were further analyzed for functional pathways effects using Simplified Gene Ontology and Kyoto Encyclopedia of Genes and Genomes public databases. Results: LMD yielded 550–600 endothelial cells from 20 sections of heart allografts and normal hearts. Adequate amount of RNA was obtained for microarray analysis. Ninety-eight genes were upregulated and 172 genes were downregulated (p < 0.05). Upregulated functional pathways included adhesion, cell communication, cell growth, cytoskeletal, integral membrane, kinase and signal transduction. Conclusion: A combination of laser microdissection and functionally based microarray analysis is a powerful method to analyze changes in gene expression in specific cell types after transplantation.