MicroRNAs are critical in regulating smooth muscle cell mineralization and apoptosis during vascular calcification

Abstract Vascular calcification refers to the pathological deposition of calcium and phosphate minerals into the vasculature. It is prevalent in atherosclerosis, ageing, type 2 diabetes mellitus and chronic kidney disease, thus, increasing morbidity and mortality from these conditions. Vascular calcification shares similar mechanisms with bone mineralization, with smooth muscle cells playing a critical role in both processes. In the last decade, a variety of microRNAs have been identified as key regulators for the differentiation, phenotypic switch, proliferation, apoptosis, cytokine production and matrix deposition in vascular smooth muscle cells during vascular calcification. Therefore, this review mainly discusses the roles of microRNAs in the pathophysiological mechanisms of vascular calcification in smooth muscle cells and describes several interventions against vascular calcification by regulating microRNAs. As the exact mechanisms of calcification remain not fully elucidated, having a better understanding of microRNA involvement in vascular calcification may give impetus to development of novel therapeutics for the control and treatment of vascular calcification.


| INTRODUC TI ON
Vascular calcification (VC) is the pathological deposition of calcium and phosphate minerals in the vasculature. It leads to vascular stiffness and fragility, impaired hemodynamics, and increased morbidity and mortality from cardiovascular diseases such as atherosclerosis, systolic hypertension and coronary artery disease . 1 Based on the location of hydroxyapatite precipitation, vascular calcification is classified into intimal and medial calcification. 2 Intimal calcification is usually associated with atherosclerosis, in the presence of risk factors such as hyperlipidemia. Lipid deposited in the intima induces complicated pathophysiological responses, including inflammatory cell infiltration, endothelial cells (ECs) apoptosis, smooth muscle cells (SMCs) proliferation and transdifferentiation, extracellular matrix (ECM) remodelling and oxidative stress. 3 Medial calcification is secondary to ageing, type 2 diabetes mellitus or chronic kidney disease (CKD), under the stimulation of hyperglycaemia and high circulating phosphate levels. 4,5 Epidemiological studies have highlighted that elevated inorganic phosphate (Pi) and calcium caused by disturbed mineral metabolism aggravates vascular calcification. 6 Additionally, hyperglycaemia accelerates the accumulation of free radicals (superoxide anion) that can activate several cellular pathways including advanced glycation end products (AGEs), protein kinase C (PKC) and nuclear factor-κB (NF-κB)-mediated vascular inflammation, which contribute to apatite formation in vasculature. 7 | 13565 WANG et Al.
Vascular smooth muscle cells (VSMCs) have been proven to play an essential role in both intimal and medial vascular calcification. This is characterized by VSMCs reprogramming and transdifferentiating into osteoblast-like cells, VSMCs apoptosis and VSMCs-derived calcifying matrix vesicle release. Besides VSMCs dysfunction, loss of calcification inhibitors, oxidative stress, endoplasmic reticulum stress and disturbed calcium-phosphate homeostasis contribute to the development of calcification. 8 MicroRNAs (miRs) are small non-coding RNAs with 18-25 nucleotides that bind to the 3'-untranslated region of target messenger RNA (mRNA) to silence gene expression by destabilizing the mRNA or compromising mRNA translation. MicroRNAs regulate the expression of many genes and a multitude of cellular functions. 8 In bone metabolism, miRs regulate the differentiation of bone precursor cells into mature bone cells. Likewise, a variety of miRs have been implicated in the development of vascular calcification. This review paper will introduce the role of miRs in the pathophysiological process of vascular calcification in VSMCs in order to identify potential therapeutics for vascular calcification associated diseases.

| MicroRNAs in VSMCs osteochondrogenic transdifferentiation
During bone formation, bone marrow-derived mesenchymal stem cells (MSCs) differentiate into chondrocytes or osteoblasts that are capable of synthesizing bone matrix and turning into osteocytes.
Osteoblastic differentiation is regulated by hormones and various transcription factors. Bone morphogenetic proteins (BMPs), which are members of the transforming growth factor beta (TGF-β) superfamily, mediate transdifferentiation of MSCs into osteoblasts through BMP/Smad signalling pathway. 9 The BMPs were identified to target Runx2 and Osterix in the process of bone formation. 9 As a member of the Runt-related transcription factors, Runx2 is the master upstream osteoblast transcription factor that regulates opulent bone matrix proteins expression. 10 In the transcriptional cascade of osteoblast differentiation, Msx2 and Osterix act as the upstream and downstream connectors of Runx2, respectively. 10,11 Moreover, BMPs can activate Wnt/β-catenin signalling pathway to promote alkaline phosphatase (ALP) expression and matrix mineralization. 12 Vascular calcification and bone mineralization share similar mechanisms. 2 At the molecular level, the signature of active osteogenic processes is found in virtually all calcified arterial segments. 13 The VSMCs normally express contractile molecules, including smooth muscle actin-α (α-SMA), transgelin (SM22a), smooth muscle myosin heavy chain (SM-MHC) and calponin 1 (CNN1). 14 However, when exposed to atherogenic or uraemic stimulus, they are capable of transdifferentiating into osteo/chondrocyte-like cells. This induces increased expression of bone-related transcription factors such as Msx2, Sox9, Runx2, Osterix, tissue non-specific alkaline phosphatase (TNAP), osteocalcin and osteopontin (OPN). 9,15,16 Studies have demonstrated that MiRs are essential regulators for osteoblast transdifferentiation of VSMCs. The majority of reported miRs are down-regulated during the process of SMCs transdifferentiation whereas some of them are up-regulated in this process. The regulation of osteogenic transdifferentiation of VSMCs by miRs is illustrated in Figure 1.
Upon BMPs binding to the receptor complex, Smad proteins translocate into the nucleus and modulate gene expression transcriptionally by directly interacting with the promoter region of target genes or post-transcriptionally through regulating miRs synthesis. The BMPs suppress miR-302 transcription via a Smad protein complex in a histone deacetylase (HDAC)-dependent manner. This results in elevation in the expression of BMP receptor II, which consequently improves BMP signalling in VSMCs. 17 Sun et al 18 found that overexpression of miR-302b in mice with chronic renal failure regulated calcium-phosphorus metabolism and inhibited VC through down-regulating BMP receptor II expression, which inhibits BMP-2/ Runx2/Osterix pathway.
In another study, miR-125b was reported to regulate cell proliferation during osteoblast differentiation in mouse MSCs. 19 Additionally, Knock-down of miR-125b aggravated osteogenic transdifferentiation and calcium deposition in VSMCs via increasing Osterix and Ets-1 protein expression. 20,22 The Ets1 is a potent transcription factor that promotes VSMCs remodelling through regulating genes encoding extracellular matrix proteins, such as osteopontin. 23 Rangrez and colleagues 24 found that miR-143 and miR-145 were down-regulated whereas miR-223 was markedly up-regulated in calcified VSMCs treated with high Pi and calcified aorta samples derived from ApoE deficient mice. However, circulating levels of miR-223 were significantly lower in CKD mice and in a cohort of patients with advanced CKD, while the expression of miR-223 increased in the calcified vascular SMCs, which indicated an accumulation of this microRNA in the vascular wall. 25,26 In VSMCs that were treated with high Pi, overexpression of miR-223 markedly inhibited α-SMA, improved VSMCs migration and induced calcification by targeting myocyte enhancer factor 2C (Mef2c) and ras homolog gene family member B (RhoB). 24 The Mef2c is involved in VSMCs differentiation and regulates myocardin(MYOCD) expression 27 whereas RhoB is a member of the Rho guanosine triphosphatases family of proteins and has been shown to increase VSMCs contractility and mediate adaptational changes to hypoxia. 28 Another microRNA, MiR-143, suppresses osteogenic differentiation via targeting transcription factor Osterix and is potentially related to VC 29 whereas MiR-145 was reported to target core-binding factor-beta (Cbfβ), which is the hetero-dimeric partner of Runx2, to transactivate molecular target of Runx2 in osteoblast differentiation. 30 There are several miRs that have been demonstrated to regu- Members of the miR-30 family were reported to suppress osteoblast differentiation in mouse bone marrow MSCs induced by BMP-2 through targeting Smad1 and Runx2. 39 For instance, MiR-30e is reciprocally associated with osteoblast and adipocyte differentiation. 40 Overexpression of miR-30e improved adipogenesis but repressed osteoblast differentiation in mouse bone marrow stromal cells by down-regulating Wnt/β-catenin signalling. 40 Additionally, significantly reduced expression of miR-30e and up-regulated Runx2, OPN and insulin-like growth factor 2 (IGF-2) were reported in aortas from aged ApoE deficient mice. As one of the targets of miR-30e, IGF-2 promotes OPN expression and calcium deposition. 41 It is probable that BMPs mediates phenotype switch of VSMCs during VC by regulating the expression of miRs. However, miRs are also capable of influencing BMPs-induced osteoblast differentiation by targeting osteoblastic transcription factors. Balderman et al 42 proved that BMP-2 markedly improved Runx2 protein expression by F I G U R E 1 Schematic representation of microRNAs in osteogenic transdifferentiation of VSMCs. Upon BMPs binding to the receptor complex, Smad proteins translocate into the nucleus and modulate gene expression transcriptionally by directly interacting with the promoter region of target genes (such as Runx2 and Osterix) or post-transcriptionally through regulating miRs synthesis. Then, these osteoblast transcription factors regulate opulent bone matrix proteins expression and promote transdifferentiation of VSMCs to osteoblastlike cells. MicroRNAs regulate osteogenic transdifferentiation of VSMCs through targeting key factors in the BMP/Smad/Runx2 signalling pathway and several inhibitors of osteoblastic differentiation. Besides, some miRs are able to regulate the expression of proteins involved in VSMCs differentiation, migration and contractility. ADAMTS-7, a disintegrin and metalloproteinase with thrombospondin motifs 7; ACVR2A, A receptor type IIA; ALP, alkaline phosphatase; BMPR, Bone morphogenetic proteins receptor; BMPs, Bone morphogenetic proteins; Cbfβ, core-binding factor beta; COMP, cartilage oligomeric matrix protein; CTNNBIP1, b-catenin interacting protein 1; DNMT3a, DNA methyltransferase 3A; Ets1, endothelial transcription factor 1; HDAC2, histone deacetylase 2; HDAC4, histone deacetylase 4; Hoxa2, Homeobox A2; IGF-2, Insulin-like growth factor 2; Mef2c, myocyte enhancer factor 2C; MMP2, matrix metallopeptidase 2; RhoB, ras homolog gene family member B; SATB2, Special AT-rich sequence-binding protein 2; Wnt, wingless-type MMTV integration site; α-SMA, smooth muscle actin-α.

| MicroRNAs in VSMCs apoptosis
Apoptosis of VSMCs is a key process in vascular calcification. The Topoisomerase II inhibitor (Topo II inhibitor) was reported to impair macrophage and SMCs invasion of the intima and inhibit the expression of inflammatory cytokines in the arterial wall. 86

| CON CLUS ION
In this review, we mainly discussed the involvement of miRs in osteogenic transdifferentiation and apoptosis of SMCs during the process of VC. It was found that numerous miRs regulate hydroxyapatite formation in the vasculature through targeting specific genes. In addition, the BMP/Smad/Runx2 signalling pathway is considered as the foremost pathway during VC. Therefore, the miRs regulating this pathway contribute to the process of VC. Some miRs are modulated through several interventions, which may be used in development of potential therapeutics in the future to control VC and prevent the development of cardiovascular diseases. Apart from playing a role in the reprogramming and transdifferentiating of SMCs, studies demonstrated that miRs are also associated with SMCs autophagy, increased SMCs oxidants and/or endoplasmic reticulum stress and loss of calcium-phosphate homeostasis. [94][95][96] To summarize, miRs involvement in vascular calcification is pretty complicated; hence, more studies are needed to fully explore the association between miRs and VC and discover more targets for VC therapeutic intervention.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.