oa Characterization of Sex-Related Differences in Valvular Interstitial Cells

Although the pathogenesis of calcific aortic valve disease (CAVD) is not well understood, males have been identified as having a two-fold increased risk for the disease when compared to females. In this study, we examined gene expression profiles in healthy pigs and measured markers of disease in vitro to determine whether the differences in clinical risk between males and females translate into measurable intrinsic differences on the cellular scale. In addition, we also investigated potential sex-related differences in cellular response to TGF-β1, an inflammatory stimulus known to be elevated in calcified human aortic valve explants. mRNA was isolated from three male and three female porcine aortic valves (denuded of endothelial cells) and hybridized to Affymetrix® GeneChip Porcine Genome microarrays. Mean expression values of each probe set in the male samples were compared with those in the female samples and biological processes were analyzed from the dataset for overrepresentation using Gene Ontology term enrichment analysis. From the microarrays there were 183 genes identified as being significantly (fold change>2; P<0.05) different in healthy male versus female aortic valve leaflets. Within this significant gene list there were 298 overrepresented biological processes, several of which are relevant to pathways identified in CAVD pathogenesis. In particular, pathway analysis indicated that cellular proliferation, apoptosis, cell migration, ossification, and extracellular matrix reorganization were all significantly represented in the data set. In vitro culture of male and female porcine valve cells also revealed intrinsic differences between sexes, with male cells exhibiting higher proliferation, apoptosis, and expression of αSMA after five days of culture. When exposed to TGF-β1, male cells grown in serum-free culture were found to be more sensitive to the inflammatory cytokine, with dramatically decreased apoptosis and proliferation compared to a marginal decrease in apoptosis and no change in proliferation in female cells. These data suggest that some sex-related propensity for CAVD may be present on the cellular level in healthy subjects, possibly resulting in a differential response to systemic factors that promote disease onset and progression. These results also offer motivation for further sex-related studies with valve cells to better determine possible genetic contributors and to explore sex-related susceptibilities for valve calcification.