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Vascular smooth muscle cells (VSMCs) exhibit remarkable plasticity during postnatal development. Vascular injury initiates and perpetuates VSMCs dedifferentiation to a synthetic phenotype, which has been increasingly recognized to play a central role in neointimal hyperplasia during the pathogenesis of vascular proliferative diseases. MicroRNAs (miRNAs) are a novel class of regulatory noncoding RNAs that regulate gene expression at the posttranscriptional level by binding to 3′ untranslated regions of target mRNAs, leading to either degrading mRNAs or inhibiting their translation. There is emerging evidence that miRNAs are critical regulators of widespread cellular functions such as differentiation, proliferation, and migration. Recent studies have indicated that a number of specific miRNAs play important roles in regulation of vascular cell functions and contribute to neointimal hyperplasia after vascular injury. Here, we review recent advance regarding functions of specific miRNAs in vasculature and discuss possible mechanisms by which miRNAs modulate proliferation and differentiation of VSMCs.
MicroRNA (miRNA) is a class of highly conserved, single-stranded, noncoding small RNAs that control cellular function by either degrading mRNAs or inhibiting their translation. MiRNAs are produced from longer primary RNA precursors (pri-miRNAs) containing stem-loop structures that are transcribed from genomes by RNA polymerase II and cleaved in the nucleus by the complex of the RNase III enzyme Drosha and its partner DGCR8/Pasha to form approximately 70-nucleotide pre-miRNAs [1-5]. Pre-miRNAs are transported into the cytoplasm by Exportin-5 and subsequently processed by the nuclease Dicer into the 20- to 24-nucleotide mature miRNA [6-9]. MiRNAs regulate gene expression at the posttranscriptional level by binding to 3′ untranslated regions (UTRs) of target mRNAs that are fully or partially complementary, leading to either translational repression or mRNA decay [10-13]. Bioinformatic and basic studies have revealed that a single miRNA can regulate hundreds of genes and that one gene can be modulated by a number of miRNAs . In recent years, a growing body of evidence suggests that miRNAs are critical regulators of widespread cellular functions, such as differentiation, proliferation, migration, and apoptosis . MiRNAs might play pivotal roles in the pathogenesis of a variety of human diseases, including cancer and vascular diseases.
Vascular smooth muscle cells (VSMCs), the predominant cells in tunica media of artery, are highly specialized cells that regulate blood pressure through the regulation of blood vessel tone. In contrast to terminally differentiated muscle cells, VSMCs in the postnatal organism retain remarkable plasticity and can switch between differentiated and dedifferentiated phenotypes in response to physiological and pathological cues, such as vascular injury, hypertension, and atherosclerosis . Differentiated VMSCs demonstrate a very low rate of proliferation, appropriate contractility to contractile cues, and express SMC-specific genes, such as smooth muscle α-actin, smooth muscle myosin heavy chain (SM-MHC), SM22α, and calponin. In response to vascular injury or growth factor signaling, VSMCs dedifferentiate and adopt a synthetic phenotype, which is characterized by increased proliferation, migration, enhanced production of collagens and matrix metalloproteinases, and diminished expression of SMC-specific contractile markers . Although VSMC dedifferentiation to the synthetic phenotype is believed to be critical for the response to vascular injury, this process has also been correlated with multiple vascular proliferative diseases, including restenosis after balloon angioplasty or stenting, atherosclerosis, and transplant vasculopathy [16, 18]. Recent studies have indicated that many miRNAs are highly expressed in vascular system and involved in the control of proliferation and differentiation of VSMCs.
Several recent reports demonstrate that miR-143 and miR-145 are enriched in VSMCs and play a significant role in regulating the phenotypic switching of VSMCs. In vitro overexpression of miR-145 or miR-143 was sufficient to promote differentiation and inhibit proliferation of cultured VSMCs [19, 20]. In contrast, miR-143- and miR-145-deficient VSMCs were absent of contractile abilities to vasopressive stimuli and maintained in the synthetic state [21, 22]. They also indicated a significant increase in the ability to migrate toward PDGF . To investigate the effect of miRNAs on VSMCs differentiation in vivo, miR-143/miR-145 mutant mice were generated and analyzed. The results revealed a significant decrease in the number of contractile VSMCs and a remarkable increase in the number of synthetic VSMCs in the aorta and the femoral artery of miR-143/miR-145 mutant mice, with a reduced media thickness [21-23]. VSMCs within miR-143/145 mutant artery showed a pro-synthetic morphological features and a significant downregulation in the expression of SMC-specific differentiated markers [21, 22]. Taken together, these results suggest that miR-143 and miR-145 have critical roles for maintaining the differentiated phenotype of VSMCs. Deficiency of miR-143 and miR-145 leads to VSMCs phenotypic switching from a contractile to synthetic state.
There is compelling evidence that transcriptional factors, such as serum response factor (SRF) and its coactivator myocardin (Myocd), mediate transcriptional response to physiological and pathological cues and orchestrate a SMC-differentiated phenotype [24, 25]. Myocd is selectively expressed in cardiomyocytes and VSMCs and activates SMC-specific gene expression to promote SMC differentiation by physically interacting with SRF [26, 27]. Cordes and colleagues have observed that potential binding sites for SRF were identified in the regulatory region of miR-143 and miR-145 . Expression of miR-143 and miR-145 can be directly activated by SRF–Myocd interaction. Deletion of miR-145 was sufficient to block the conversion from fibroblasts to VSMCs induced by Myocd and repress expression of several SMC markers. Interestingly, introduction of miR-145 could induce undifferentiated neural crest stem cells into the VSMC lineage, which characterized by expression of numerous markers of VSMC differentiation, including SMα-actin, SM22α, and SM-MHC . Bioinformatics assay also revealed highly conserved binding sites for miR-143 in the 3′ UTR of Elk-1 and for miR-145 in the 3′ UTR of Myocd, Klf4, and CamkIIδ. Luciferase assay confirmed that Elk-1 is direct transcriptional target of miR-143, and Myocd, Klf4, and CamkIIδ are direct targets of miR-145 . These findings suggest that miR-143 and miR-145 targeted a network of transcription factors to regulate differentiation and proliferation of VSMCs.
Recently, tropomyosin 4 (Tpm4) and angiotensin-converting enzyme (ACE) were identified as predicted targets of miR-145 . TPM-4 is a structural protein that is specifically upregulated in synthetic SMCs. ACE cleaves circulating angiotensin I to its active form angiotensin II, which bind to AT1 receptor on the surface of VSMCs, regulating both contractile functions and the contractile phenotype of VSMCs. The results suggest that miR-145 regulates expression of membrane-bound ACE in VSMCs to induce conversion of VSMC phenotype .
Recent reports have demonstrated that miR-221 and miR-222 are also implicated in modulation of VSMCs differentiation. In cultured VSMCs in vitro, miR-221 and miR-222 expression can be transcriptionally induced by PDGF signaling and mediate its action on the VSMCs phenotypic switching [28, 29]. Transfection of exogenous miR-221 into VSMCs reduced the expression of VSMCs differentiated markers and significantly elevated VSMCs migration and proliferation, which is similar to the effect of PDGF on VSMCs in vitro. Conversely, depletion of miR-221 increased the expression of VSMCs differentiated markers and remarkably abrogated the induction of PDGF on VSMCs migration and proliferation, which are fundamental characteristics of the dedifferentiated state of VSMCs . It suggests that miR-221 is essential for the PDGF-mediated stimulation of VSMCs phenotypic switching. Moreover, miR-221 was confirmed to target the 3′ UTR of c-Kit and p27Kip1 mRNAs in VSMCs . Overexpression of miR-221 diminished gene expression of c-Kit and Myocd, leading to repression of SMC marker genes. In contrast, deletion of miR-221 by anti-miR-221 transfection dramatically increases the levels of Myocd through its upstream modulator, c-Kit. Therefore, PDGF-mediated induction of miR-221 represses SMC-specific gene expression through downregulation of c-Kit, which subsequently inhibits Myocd . These findings suggest that miR-221-dependent downregulation of p27Kip1 promotes cell proliferation in VSMCs, and downregulation of c-Kit and Myocd induces VSMCs phenotypic switching from a differentiated, contractile state to a dedifferentiated, synthetic state.
Neointimal hyperplasia has generally been accepted as the main features of vascular repair response to various injuries. Importantly, multiple studies have demonstrated that neointimal progression is a much better-defined scenario of molecular and cellular events, including endothelial dysfunction and VSMC migration and dedifferentiation. The miRNAs, as powerful regulators of gene expression, are involved in the modulation of all these cellular events and have crucial roles in intimal thickening after vascular injury.
One important characteristic of miRNA expression is the tissue- and cell-specific expression manner [4, 30]. Microarray analysis indicated that miR-21 , miR-126 , miR-143, and miR-145 [19, 21, 22] are highly expressed in normal murine arteries. Remarkably, expression of miR-143 and miR-145 strongly parallels with the number of SMCs in different mouse tissues during embryonic development and is relatively stable in VSMCs of postnatal mouse . These findings suggest that miR-143 and miR-145 are selectively expressed in VSMCs. Moreover, miR-126 is the most abundant microRNA found in mouse endothelial cells [32, 33].
In response to vascular injury, expression of miRNAs exhibits a dynamic profile in injured vessel wall. At 7 days after balloon injury, 140 miRNAs detectable in arterial tissue, 60 miRNAs are highly upregulated, and 53 miRNAs are downregulated in the rat carotid arteries. At 28 days after the injury, 102 miRNAs are still expressed at significantly different levels . Remarkably, both miR-221 and miR-222 are increased in the balloon-injured carotid arteries and mainly localized in VSMCs of the vascular wall . In another vascular injury model, miR-143 and miR-145 expression was significantly decreased in ligated carotid arteries compared with contralateral control arteries . Furthermore, transcripts of miR-145 were also decreased to nearly undetectable levels in mouse arteriosclerotic lesions . Obviously, distinct expression profiles of specific miRNAs are involved in the pathological process after different stages of vascular injury.
MiRNAs contribute to the formation of neointimal lesions through regulating various cellular functions. In injured carotid artery, downregulation of miR-145 was coincident with decreased expression of the differentiation marker of VSMCs . In vitro miR-145 was also required for Myocd-induced conversion of cultured fibroblasts into VSMCs . miR-143/145-deficient VSMCs were maintained in the synthetic state . miR-143/145 knockout mice also indicated a significant reduction in systolic blood pressure due to reduced vascular tone [21, 23]. Old miR-143/145 mutant mice showed a significant increase in neointimal lesion formation with large amounts of VSMCs and macrophages and deposits of amorphous collagen I in the femoral arteries . Moreover, introduction of miR-145 in injured rat carotid arteries via adenovirus-mediated gene transfer remarkably inhibited neointimal lesion formation accompanied with upregulation of VSMC differentiation marker . Taken together, miR-143 and miR-145 play pivotal roles in the control of contractile phenotype of VSMCs and the response of the vascular wall to injury.
Recently, Xin and colleagues also reported the effect of miR-143 and miR-145 on neointimal hyperplasia after vascular injury in mouse . The carotid artery ligation model was established in miR-143−/−, miR-145−/−, or double knockout mice. Neointimal lesion development after ligation was significantly impaired in mice for either single miRNA deletion or the double miRNAs deletion. This result was contrary to other reports. It may account, at least in part, for different vascular injury models or genetic loss-of-function studies. But, it highlights the importance of genetic model systems for the complete understanding of the complex functions of miRNAs in disease settings .
MiR-221 and miR-222 are also critical modulators for VSMCs proliferation and neointimal lesion formation. Both of them are significantly increased in proliferative VSMCs . Knockdown of miR-221 and miR-222 can inhibit VSMC proliferation and intimal thickening in rat carotid artery after vascular injury . As target genes of miR-221 and miR-222, p27(Kip1), p57(Kip2), and c-Kit were involved in regulation of VSMC proliferation and neointimal formation [28, 29].
Reactive oxygen species contribute to the pathogenesis of atherosclerosis and restenosis after angioplasty . A recent study demonstrated that miR-21 was highly expressed in VSMCs treated by hydrogen peroxide . miR-21 has a positive effect on VSMC proliferation and a reverse effect on cell apoptosis through regulating expression of PTEN and Bcl-2 [31, 35]. In addition, in rat carotid artery balloon-injury model, neointimal hyperplasia of injured artery was significantly repressed by downregulation of miR-21 via antisense oligonucleotide-mediated miRNA depletion .
The important role of vascular endothelial cells in neointimal lesion formation has been convincingly demonstrated by a large number of studies. Recently, Fish and colleagues indicated that miR-126 is required for migration and proliferation of HUVECs in response to VEGF stimulation . Moreover, PIK3R2 and SPRED1 were identified as the direct target genes of miR-126 to modulate VEGF-dependent endothelial cell functions , whereas vascular cell adhesion molecule-1 as another target of miR-126 is involved in regulating endothelial cell interactions with leukocyte, which plays a critical role in leukocyte trafficking and vascular inflammation . Thus, it is critical for future studies to address the functions of specific miRNAs on vascular inflammation.
MiRNAs have become one of the most important gene regulators of multiple cellular functions. Since miRNA deficiencies or excesses have been implicated in the pathogenesis of several human diseases including cancer and cardiovascular diseases, abnormally expressed miRNAs seem to represent promising therapeutic targets for diagnostic and therapeutic approaches. Specific miRNA expression can be modulated by genetic approaches including overexpression or silencing of the prospective miRNA. The former can be achieved by introduction of corrective synthetic miRNA mimics, which is an effective approach for rescuing under-expressed miRNAs . Cordes et al. demonstrated that introduction of miR-145 in mouse ligated carotid arteries via lentiviral-mediated gene transfer is sufficient to suppress the proliferative response and promote the differentiated state of VSMCs . Consistent with this study, adenovirus-mediated restoration of miR-145 into rat balloon-injured carotid arteries in vivo significantly inhibited neointimal lesion formation . These findings suggest that delivery of miRNA mimics into the proper tissue can provide a therapeutic benefit by enhancing the levels of specific miRNAs whose expression is downregulated in the disease state. For specific miRNAs that are upregulated during disease, silencing of specific miRNAs would be beneficial. Currently, modified antisense oligonucleotides, also designated as antagomirs, represent a potential tool for miRNA silencing . Modified oligonucleotides can be designed to complement either the mature miRNA or its precursors leading to inhibition of specific miRNAs. Liu et al. have applied modified antisense oligonucleotides to successfully knock down miR-221 and miR-222 in cultured VSMCs and significantly inhibit cell proliferation and neointimal growth in rat balloon-injured carotid artery . In recent years, many studies have validated the efficacy of antisense oligonucleotides both in vitro and in vivo to reduce the levels of pathogenic or aberrantly expressed miRNAs. Although targeting miRNAs represents promising therapeutic strategies, substantial preclinical work still needs to be done to identify precise targets among the many predicted targets in vascular diseases.
Currently, more than 700 human miRNAs have been identified in the human genome; a few specific miRNAs that regulate vascular injury and remodeling have been validated [38-40]. Recent studies have shown that a number of specific miRNAs play critical roles in regulation of vascular cell functions and contribute to various pathological processes, such as atherosclerosis and neointimal lesion formation after vascular injury. However, little information is known about the cell type-specific signatures of miRNAs on target mRNA expression and the mechanisms by which miRNAs regulate target gene expression in the vasculature. As a single miRNA can bind to multiple targets whereas single gene may be regulated by multiple miRNAs, further studies need to analyze the complex interactions between vascular SMC-specific miRNAs and signaling pathways involved in vascular diseases. Identification of miRNAs and their targets and better understanding of in vivo mechanisms will lead us towards development of miRNA drugs designed against specific molecular targets for the treatment of vascular diseases.
The work was supported by the National Institutes of Health Grant HL087990 (Dr. Li) and by American Heart Association grant 0530166N (Dr. Li).
Zifang Song, Department of Neurosurgery, LSU Health Science Center, Shreveport, LA, USA.
Guohong Li, Department of Neurosurgery, LSU Health Science Center, Shreveport, LA, USA; Vascular Biology & Stroke Research Laboratory, Department of Neurosurgery, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130, USA ; Email: ude.cshusl@ilg.