MicroRNAs that target Ca2+ transporters are involved in vascular smooth muscle cell calcification

Abstract

The role of microRNAs (miRNAs) in vascular calcification is currently unclear. To examine how miRNAs are involved in vascular smooth muscle cell (VSMC) calcification, we explored the alteration of miRNAs in VSMC calcification in vitro and in vivo. Klotho homozygous mutant mice (kl/kl) display vascular calcification and have perturbations of calcium handling. We therefore hypothesized that the calcium perturbations in VSMCs could be mediated by miRNAs. Using an miRNA array analysis, we demonstrated that miRNAs are aberrantly expressed in the aortic media of 3-week-old kl/kl mice compared with wild-type (WT) mice. The expression levels of miR-135a*, miR-762, miR-714, and miR-712* in the aortic media of kl/kl mice were significantly higher than in WT mice. We used quantitative real-time reverse transcriptase polymerase chain reaction to further confirm that these miRNAs were increased in the aortic media of kl/kl mice and in cultured VSMCs treated with high phosphate and calcium. A search of the miRNA database indicated that the Ca 2 þ efflux proteins NCX1, PMCA1, and NCKX4 frequently appeared as potential targets of these miRNAs. The transfection of miRNA mimics into cultured VSMCs reduced the protein levels of each potential target. Conversely, miRNA inhibitors reduced phosphate and calcium-induced VSMC calcification. Furthermore, these inhibitors decreased the intracellular Ca 2 þ concentration in cultured VSMCs after treatment with phosphate and calcium. Our results suggest that increased expression of miR-135a*, miR-762, miR-714, and miR-712* in VSMCs may be involved in VSMC calcification by disrupting Ca 2 þ efflux proteins.

MicroRNA-339 and microRNA-556 regulate Klotho expression in vitro

“…In the aorta, the absence of KL induces upregulation of miRs that regulate calcium ion transporters promoting an environment in which calcification can occur (Gui et al 2012). Likewise, expression profiling across multiple organs in KL knockout mice indicate an increase in a number of miRs, most prominently of the miR-29 family (Takahashi et al 2012).…”

“…Age downregulation of KL may be a result of miR expression changes with age. Although the absence of KL in mice alters miR expression (Gui et al 2012;Takahashi et al 2012), to date no information is available as to whether miRs regulate KL expression. Here, we report in vitro evidence of regulation of KL mRNA by miR-339 and miR-556.…”

Spironolactone ameliorates PIT1-dependent vascular osteoinduction in klotho-hypomorphic mice

“…The origin of the mice, breeding, and genotyping were described previously (8). As klotho-deficient mice show severe calcifications at 3 weeks of age (66,67), life-long treatment with spironolactone was maintained in this study. The klotho WT (WT) and kl/kl mice had access to vehicle drinking water without or with addition of 80 mg/l spironolactone (Sigma-Aldrich) and rodent chow ad libitum (68,69).…”

A current understanding of vascular calcification in CKD

“…The calcium efflux proteins NCX1, PMCA1, and NCKX 4 have been identified as potential targets of these miRNAs, and thus would be transcriptionally downregulated when these miRNAs were active, potentially enhancing intracellular calcium loading in the cells and increasing calcification potential. Indeed, when inhibitors of these identified miRNA were added to VSMC cultured in a high-phosphate, highcalcium medium the degree of intracellular calcium and calcification decreased (34). miR-133a is known to suppress Runx2 and impede the osteoblastic differentiation of mesenchymal cells and has now been shown to play a role in the regulation of the osteogenic differentiation of cultured VSMC.…”