Transcriptome analysis reveals sexual disparities in gene expression in rat brain microvessels

Abstract

Sex is an important determinant of brain microvessels (MVs) function and susceptibility to cerebrovascular and neurological diseases, but underlying mechanisms are unclear. Using high throughput RNA sequencing analysis, we examined differentially expressed (DE) genes in brain MVs from young, male, and female rats. Bioinformatics analysis of the 23,786 identified genes indicates that 298 (1.2%) genes were DE using False Discovery Rate criteria (FDR; p < 0.05), of which 119 (40%) and 179 (60%) genes were abundantly expressed in male and female MVs, respectively. Nucleic acid binding, enzyme modulator, and transcription factor were the top three DE genes, which were more highly expressed in male than female MVs. Synthesis of glycosylphosphatidylinositol (GPI), biosynthesis of GPI-anchored proteins, steroid and cholesterol synthesis, were the top three significantly enriched canonical pathways in male MVs. In contrast, respiratory chain, ribosome, and 3 ́-UTR-mediated translational regulation were the top three enriched canonical pathways in female MVs. Different gene functions of MVs were validated by proteomic analysis and western blotting. Our novel findings reveal major sex disparities in gene expression and canonical pathways of MVs and these differences provide a foundation to study the underlying mechanisms and consequences of sex-dependent differences in cerebrovascular and other neurological diseases.

Effects of aging on protein expression in mice brain microvessels: ROS scavengers, mRNA/protein stability, glycolytic enzymes, mitochondrial complexes, and basement membrane components

“…The MVs were isolated as previously described [ 12 , 13 ]. Large, superficial, blood-vessel-free, cortical tissue from mice brains was homogenized in ice cold Dulbecco’s phosphate-buffered saline (DPBS) (Life Technologies Corporation, NY, USA) and centrifuged at 3300 × g for 15 min.…”

“…An equal amount of each TMT-labeled sample was pooled in a single tube with SepPak purified (Waters, Ireland) using acidic reverse phase conditions. We used off-line fractionation to reduce the sample complexity, as previously described [ 12 , 13 ]. The fractionated, labeled peptide mixtures were run on a Dionex U3000 nano-flow system coupled to a Thermo Fisher Fusion Orbitrap mass spectrometer.…”

“…Last, the MV pellet was resuspended in DPBS and stored at − 80 °C until used. The MV preparation integrity has been validated as described in our studies [12][13][14][15].…”

“…8 Putative events leading to stress and dysfunction of the brain MVs in aging. Brown, alkaline phosphate staining [ref 12,13] in normal microscopy shows the isolated cortical MVs as a mixture of arterioles (a), capillaries, and venules (v). This study indicated that the reduction of ROS scavenging ability and subsequent increased oxidative damage, and decreased mRNA/ protein stability, represent early, precipitating events leading to energy failure and basement membrane (BM) disruptions in the cerebral microvasculature of the aging brain any studies which have considered the effects on glycolysis of aging brain cortical MVs.…”

“…Recently, we reported on our proteomics approaches to examine expression and interactions of large numbers of proteins in MVs in young male and female rodents, with an emphasis on mitochondrial and related proteins [ 12 , 13 ]. In the current study, we have expanded our investigation and have performed a more extensive examination of proteins involved in the structure and function of MVs using a discovery-based quantitative proteomics approach quantifying more than 4200 differentially expressed (DE) proteins/group in cortical MVs of young, middle-aged, and old male and female mice.…”