Inorganic Phosphate Accelerates the Migration of Vascular Smooth Muscle Cells: Evidence for the Involvement of miR-223

et al. (2012) Inorganic Phosphate Accelerates the Migration of Vascular Smooth Muscle Cells: Evidence for the Involvement of miR-223. PLoS ONE 7(10): e47807. doi:10.1371/journal.pone.0047807 Inorganic Phosphate Accelerates the Migration of Vascular Smooth Muscle Cells: Evidence for the Involvement of miR-223 Ashraf Yusuf Rangrez 0 1 Ele onore M'Baya-Moutoula 0 1 Vale rie Metzinger-Le Meuth 0 1 Lucie He naut 0 1 Mohamed Seif el Islam Djelouat 0 1 Joyce Benchitrit 0 1 Ziad A. Massy 0 1 Laurent Metzinger 0 1 Fabio Martelli, IRCCS-Policlinico San Donato, Italy 0 Current address: Department of Internal Medicine III, Cardiology and Angiology, University of Kiel , Kiel , Germany 1 1 INSERM U1088, Amiens, France, 2 Faculty of Pharmacy and Medicine, Jules Verne University of Picardie, Amiens, France, 3 Division(s) of Pharmacology / Nephrology, Amiens University Hospital , Amiens, France, 4 UFR SMBH , University of Paris 13 , Bobigny , France Backgound: An elevated serum inorganic phosphate (Pi) level is a major risk factor for kidney disease and downstream vascular complications. We focused on the effect of Pi levels on human aortic vascular smooth muscle cells (VSMCs), with an emphasis on the role of microRNAs (miRNAs). Methodology/Principal Findings: Exposure of human primary VSMCs in vitro to pathological levels of Pi increased calcification, migration rate and concomitantly reduced cell proliferation and the amount of the actin cytoskeleton. These changes were evidenced by significant downregulation of miRNA-143 (miR-143) and miR-145 and concomitant upregulation of their targets and key markers in synthetic VSMCs, such as Kru ppel-like factors24 and 25 and versican. Interestingly, we also found that miR-223 (a marker of muscle damage and a key factor in osteoclast differentiation) is expressed in VSMCs and is significantly upregulated in Pi-treated cells. Over-expressing miR-223 in VSMCs increased proliferation and markedly enhanced VSMC migration. Additionally, we found that the expression of two of the known miR223 targets, Mef2c and RhoB, was highly reduced in Pi treated as well as miR-223 over-expressing VSMCs. To complement these in vitro findings, we also observed significant downregulation of miR-143 and miR-145 and upregulation of miR-223 in aorta samples collected from ApoE knock-out mice, which display vascular calcification. Conclusions/Significance: Our results suggest that (i) high levels of Pi increase VSMC migration and calcification, (ii) altered expression levels of miR-223 could play a part in this process and (iii) miR-223 is a potential new biomarker of VSMC damage. - Funding: This work was supported by grants from Region Picardie, France (MARNO-MPCC and Modulation des calcifications cardiovasculaires), including post-doctoral fellowships for AYR and EMM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. " These authors also contributed equally to this work. Vascular smooth muscle cells (VSMCs) are the predominant cells in the tunica media of arteries. They play a critical role in regulating the blood vessel tone, which in turn influences blood pressure. Vascular smooth muscle cells are not terminally differentiated and are able to switch from a differentiated (contractile) state to a dedifferentiated (synthetic) state in response to changing environmental signals. This phenotype modulation is critical in the pathogenesis of proliferative cardiovascular diseases (reviewed by House et al.[1]). The synthetic phenotype is concomitant with accelerated migration and ultimately results in the formation of vascular calcification - one of the main pathological lesions in chronic kidney disease[2]. Hyperphosphatemia is a major cause of this calcifying process[3,4] and the latter is correlated with increased cardiovascular mortality in dialysis patients[2]. In the present work, we sought to determine whether or not microRNAs (miRNAs) play a role in the VSMC calcification process induced by inorganic phosphate (Pi). MicroRNAs (miRNAs) are a novel class of small RNAs that negatively regulate gene expression via repression of the corresponding target mRNAs[5,6]. Several miRNAs have been found to have roles in the healthy and diseased vascular system[6,7]. MiR-143 and miR-145 (the most extensively studied species) have been correlated with human cardiovascular diseases, since VSMC maintenance and vascular homeostasis are altered in mir-143 and mir-145 knock-out (KO) mice[8]. The latter authors also determined that expression levels of miR-143 and miR-145 are low in the aortas of apolipoprotein E gene knockout (ApoE-KO) mouse. Boettger and colleagues also demonstrated that the mouse mir-143/145 cluster is necessary for acquisition of the contractile VSMC phenotype[9]. Additionally, a detailed study by Cordes et al.[10] showed that miR-145 can direct the fate of smooth muscle and regulate the synthetic phenotype of smooth muscle cells. In the present study, we investigated the molecular mechanisms by which Pi impacts on the phenotype of primary VSMC cultures derived from human aortas and correlated these effects with the expression of miRNAs. Evidence from our studies suggests that Pi alters cell proliferation and migration, reduces the amount of the actin cytoskeleton, downregulates miR-143 and miR-145 and upregulates miR-223. Indeed, increased levels of miR-223 have been reported in damaged skeletal muscle (in Duchenne muscular dystrophy[11]) and in cardiomyocytes[12]). Hence, we hypothesized that this miRNA might also be a marker of smooth muscle damage. Furthermore, miR-223 has been linked to osteogenesis and calcium-phosphate deposits(Ca*Pi) [13]. We found that upregulating miR-223 in VSMCs induces a significant increase in cell proliferation and migration and reduces the amount of the actin cytoskeleton. We also show that modulating the expression of miR-223 affects the expression of its reported targets Mef2c and RhoB in our in vitro model. Lastly, we detected the upregulation of miR-223 expression in vivo, in aorta collected from ApoE-KO mice (a well-established model of atherosclerosis and vascular calcification, displaying Ca*Pi [3]). Elevated Pi induces VSMC calcification and migration and reduces proliferation Hyperphosphatemia is associated with vascular calcification in various cardiovascular disorders[14]. In order to study the direct effect of Pi on VSMCs from the human aorta, we treated the cells for 10 days with 3.5 mM Pi (to reflect hyperphosphatemia [15]) in Dulbeccos modified Eagle Medium (DMEM) with 1% Fetal Bovine Serum (FBS; the concentration needed to obtain Ca*Pi deposits in vitro). We determined the level of calcification by alizarin staining and found that Pi indeed induced calcification in VSMCs. The calcification was 15- to (...truncated)