| Renin-angiotensin system (RAS) activation and phenotypic modulation of vascular smooth muscle cells (VSMCs) are common characteristics associated with human diseases, such as pulmonary hypertension, atherosclerosis and stroke. While elevation of the RAS hormone product, angiotensin II (AngII) is a well-established risk in these diseases; the mechanism of activation of RAS and modulation of VSMC phenotype by AngII is vague, suggesting that novel global regulators may mediate the risk by AngII. MicroRNAs (miRNAs) represent one such class of potential global regulators. MiRNAs are small (∼22 nt), endogenous, non-coding RNAs that act as post-transcriptional regulators of physiological processes. MiRNAs primarily function by binding to complementary target sites in the 3'-untranslated regions (3'-UTR) of mRNAs, causing translational repression and/or mRNA destabilization.;To elucidate the global miRNA expression profile following chronic Angiotensin II Type 1 Receptor (AT1R) activation by AngII, we performed microarray analysis in 23 biological and technical replicates derived from humans, rats and mice. We pharmacologically distinguished the AT1R-regulated miRNA profiles by comparing technical replicates treated with the specific AT1R-blocker, losartan and biological replicates following chronic AT2R activation by AngII. Thirty-two miRNAs are AngII-regulated universally. Most other miRNAs are regulated in a treatment- or species-specific manner. A few miRNAs are unique to specific cell types. s.;We have previously shown that a single miRNA can have multiple targets, potentially providing simultaneous regulation of the genes involved in a physiological pathway and accounting for a complex phenotype, such as human heart failure (JBC 284: 27487-27499). In the current study, we explored the extent to which AngII/AT1R-regulated miRNAs contribute to maintenance of RAS homeostasis and phenotypic modulation of VSMCs. A distinct AngII-regulated miRNA expression pattern emerged in the human and rat VSM cell lines in the profiling experiment, which was validated in our independent samples. Of the 17 miRNAs comprising the VSMC expression pattern, we selected miR-483 as a representative candidate for further study because of its location within the genome and its ability to potentially target multiple components of RAS. In addition, we show evidence that suggests muscle cell-specific expression of miR-483. We incorporated a miR-483 expression cassette into two distinct cell lines as a means to determine post-transcriptional inhibition of specific RAS component gene expression. The functionality of miR-483, in terms of modulating the cellular phenotype, was assessed in a wound-healing assay. Overall, miR-483 regulates both RAS and AngII-activated migration; specifically in VSMCs. Our results further suggest that following inhibition of several AT1R-activated kinases, the AngII-regulated MEK1 kinase signaling cascade most effectively mediates the steady state pool of miRNAs, which includes downregulation of mir-483. In this context, JAK2 inhibition is somewhat effective. This results in activation of RAS and switching of the VSMC phenotype associated with pathological states in vivo , such as hypertension. Further insight into these mechanisms will be valuable for a greater understanding of AngII biology. In the context of determining the full capacity of RAS as an intrinsic regulatory system, AngII-regulated miRNAs will likely have a strong influence on cardiovascular disease. |
