Gene Expression and MicroRNA Expression Analysis in Small Arteries of Spontaneously Hypertensive Rats. Evidence for ER Stress

Abstract

Small arteries are known to develop functional and structural alterations in hypertension. However, the mechanisms of this remodeling are not fully understood. We hypothesized that altered gene expression is associated with the development of hypertension in mesenteric arteries of spontaneously hypertensive rats (SHR). Three sublines of SHR and normotensive Wistar Kyoto rats (WKY) were studied at 6 weeks and 5 months of age. MiRNA and mRNA microarray experiments were performed and analyzed with bioinformatical tools, including Ingenuity Pathway Analysis (IPA). Principal component analysis showed a clear separation in both miRNA and mRNA expression levels between both ages studied, demonstrating strong age-related changes in expression. At the miRNA level, IPA identified differences between SHR and WKY related to metabolic diseases, cellular growth, and proliferation. The mRNAs differentially expressed between SHR and WKY were related to metabolism, cellular movement and proliferation. The most strongly upregulated gene (9.2-fold) was thrombospondin 4 (Thbs4), a protein involved in the endoplasmic reticulum (ER) stress response that activates transcription factor 6α (ATF6α). ATF6α downstream targets were also differentially expressed in SHR vs. WKY. Differential expression of THBS4, the cleaved form of ATF6α, and two of its targets were further confirmed at the protein level by western blot. In summary, these data revealed a number of genes (n = 202) and miRNAs (n = 3) in mesenteric arteries of SHR that had not been related to hypertension previously. The most prominent of these, Thbs4, is related to vascular ER stress that is associated with hypertension.

Endoplasmic reticulum stress in the pathogenesis of hypertension

“…Likewise, vascular ER stress is present in spontaneously hypertensive rats and is accompanied by elevations in endothelium‐dependent contraction and myogenic constriction, as well as reductions in endothelium‐dependent vasodilatation (Palao et al . ; Carlisle et al . ; Choi et al .…”

Thrombospondin-4 knockout in hypertension protects small-artery endothelial function but induces aortic aneurysms

“…The ER stress response has indeed been linked to vascular dysfunction (8), and induction of ER stress increases the blood pressure and impairs the endothelial dependent relaxation in aorta of normotensive rats (18). In addition, in our previous study in mesenteric arteries of spontaneously hypertensive rats, we found that TSP-4, Atf6α and the ER stress response genes were significantly upregulated (16). Thus, we speculate that the impaired relaxation hypertension depends on the role of TSP-4 in ER stress.…”

“…For this purpose, wild-type and Thbs4 knockout mice were treated with Ang II to reach a hypertensive state. We analyzed several cardiovascular parameters, but focused on small arteries, where we previously identified a sizeable increase in Thbs4 expression (16).…”

“…This contributes to higher peripheral resistance, limited vascular reserve and aggravation of high blood pressure. In a recent study we identified differences at the gene expression level between small arteries of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) (16). In that study we identified thrombospondin-4 gene (Thbs4) as the most upregulated mRNA in the hypertensive arterial wall.…”

Insider trading: Extracellular matrix proteins and their non‐canonical intracellular roles

“…Therefore, the authors proposed that the conserved type 3 repeats of TSPs have a critical intracellular role in ER stress response by driving Atf6α‐dependent enhancement of protein secretion, to promote reconstruction of the ECM after injury. More recently, correlated upregulated expression of Thbs4 and Atf6‐target genes has been identified in vivo, in mesenteric arteries of the spontaneously hypertensive rat .…”