Multiomics Uncover Sexual Disparities in the Expression of Genes and Proteins in Rat Cerebral Microvessels

Abstract

Background The relationship between gene expression and corresponding protein levels are complex due to many factors. Therefore, we compared multiomics (RNA-Seq and proteomic) results to discover similarities and differences. Although RNA-Seq and proteomic approaches have been applied independently to the field of microvascular pathophysiology, to date there are no reported studies of sex-dependent differences in rat cerebral microvessels (MVs) by multiomics analyses. Methods Cerebral MVs were isolated from 8‒10 weeks old, male and female, Sprague-Dawley rats, and the presence of end-arterioles, capillaries, and venules in MVs was confirmed by light microscopy and by alkaline phosphate staining. RNA-Seq analysis was performed by Illumina NextSeq 550 sequencing system. Proteomics analysis was performed, using liquid chromatography/mass spectrometry (LC-MS/MS). The expression of top genes and proteins were validated by RT-qPCR and western blotting, respectively. Results In RNA-Seq analysis, among the 23,786 identified genes, a total of 298 (1.2%) significant (FDR < 0.05) differentially expressed (DE) genes were identified, of which 40% and 60% of DE genes were abundant in male and female MVs, respectively. Instead, among the 1,871 quantified proteins, a total of 198 (10.6%) significant DE proteins were quantified, of which 84% and 16% of DE proteins were abundant in male and female MVs, respectively. Nucleic acid binding, enzyme modulator, and transcription factor were the top three gene functions in male MVs. Whereas cytoskeletal protein, extracellular matrix protein, and transcriptional regulator were the top three gene functions in female MVs. Gene functions were supported by proteomic analysis. The gene-centric top three canonical pathways in male MVs involved glycosylphosphatidylinositol (GPI), biosynthesis, and post-translational modification (PTM) of GPI-anchored proteins. Similarly, mitochondrial respiration, ribosome, and 3´UTR mediated translational regulation pathways were the top gene-centric canonical pathways in female MVs. Surprisingly, despite large differences in the expression of some genes, the resultant proteins are not always consistent with these differences. There was a greater correlation between gene expression and protein synthesis in female MVs while, despite relatively large differences in gene expression favoring males, actual protein levels were either not different between the sexes or were opposite of expectations. Genes related with PTM and ubiquitin-mediated proteolysis were highly expressed in male MVs and probably play an important role on the formation, stability, and functions of various proteins. Conclusions We have provided the first comprehensive multiomics analysis of male and female MVs of rats. Our study indicates that male and female MVs have significant differences in gene expression and protein synthesis, which may explain the sex-dependent differences in the microcirculation during health and diseases.