Search for age-dependent transcriptomic and epigenetic changes in murine cerebral endothelial cells

Abstract

Intracerebral hemorrhage (ICH) is associated with disruption of the blood-brain barrier (BBB) and rupture of blood vessels, leading to the leakage of blood into brain. In humans, increasing age is the biggest risk factor for ICH, with people above the age of 75 years being at the highest risk of developing the disease. Other risk factors for ICH include hypertension and cerebral amyloid angiopathy. Although hemorrhagic stroke is a leading global cause of death and disability, the mechanisms leading to blood-brain barrier dysfunction with age and development of ICH is poorly understood. The endothelial cells (ECs) constitute the core component of the BBB in the central nervous system vasculature and provide a physical barrier due to the presence of tight junctions, adherens junctions and basement membrane. This study in wild-type mice and mice having floxed, yet non-recombined Srf and Mrtf alleles, thereby having functional Srf and Mrtf genes (wild-type phenotype), demonstrates increased incidents of bleeding in brain with increasing age. Transgenic Cdh5-mT H2B-GFP mice were used to isolate pure population of ECs and RNA-seq was used to study the mRNA expression levels in ECs isolated from murine brains of increasing age – 2, 6, 12, 18 and 24 months. Linear regression analysis performed on the RNA-seq data reveal age-dependent dysregulation of 1388 genes, including many responsible for the maintenance of BBB and vascular integrity. However, no significant age-dependent changes in the expression levels of Srf and Mrtf, encoding two cooperating transcription factors whose endothelial knockout had previously been shown to cause hemorrhagic stroke-like symptoms, were observed. As epigenetic mechanisms are known to regulate gene expression, age-dependent changes at the CpG methylation and chromatin structure were investigated in this study. Reduced representation bisulfite sequencing (RRBS) was used to investigate change in CpG methylation with age. Interestingly, the older mice (18 months-old) show increased average CpG methylation. However, only two 1000 bp tiles annotated to Arid5b and Adgrg1 genes show a significant age-dependent change in methylation level, with both the tiles showing decrease in methylation with age. Assay for transposase-accessible chromatin sequencing (ATAC-seq) performed to investigate changes at the level of chromatin structure reveals approximately 2290 peaks that undergo age-dependent changes in the chromatin structure with a majority of the peaks (2013) closing with age. Comparing RNA-seq and ATAC-seq data reveals 27 peaks associated with 25 genes that show a correlation between age-dependent changes in chromatin structure and changes in gene expression. The study finds a strong age-dependent downregulation of the apelin receptor (Aplnr) gene, known to play a crucial role in positive regulation of vasodilation and implicated in vascular health. Also, the promoter of Aplnr gene shows an age-dependent reduction in chromatin accessibility as revealed by the ATAC-seq data. Interestingly, the study demonstrates an age-dependent reduction in the expression levels of apelin receptor protein in the brain, thereby suggesting a potential association of apelin receptor with increased risk of intracerebral hemorrhage.