SM22α deficiency: promoting vascular fibrosis via SRF-SMAD3-mediated activation of Col1a2 transcription following arterial injury.

Vascular fibrosis, characterized by increased Type I collagen expression, significantly contributes to vascular remodeling. Our previous studies show that disrupting the expression of SM22α (aka SM22, Tagln) induces extensive vascular remodeling following arterial injury, involving oxidative stress, inflammation, and chondrogenesis within the vessel wall. This study aims to investigate the molecular mechanisms underlying the transcription of Col1a2, a key fibrotic extracellular matrix marker. We observed upregulation of COL1A2 in the arterial wall of Sm22-/- mice following carotid injury. Bioinformatics and molecular analyses reveal that Col1a2 transcription depends on a CArG box in the promoter, activated synergistically by SRF and SMAD3. Notably, we detected enhanced nuclear translocation of both SRF and SMAD3 in the smooth muscle cells of the injured carotid artery in Sm22-/- mice. These findings demonstrate that SM22 deficiency regulates vascular fibrosis through the interaction of SRF and the SMAD3-mediated canonical TGF-ß1 signal pathway, suggesting SM22α as a potential therapeutic target for preventing vascular fibrosis.

Ubiquitylation-independent cotranslational degradation of dihydrofolate reductase and ubiquitin.

Nascent proteins are degraded during or immediately after synthesis, a process called cotranslational protein degradation (CTPD). Although CTPD was observed decades ago, it has never been fully explored mechanistically and functionally. We show here that dihydrofolate reductase (DHFR) and ubiquitin (Ub), two stable proteins widely used in protein degradation studies, are actually subject to CTPD. Unlike canonical posttranslational protein degradation, CTPD of DHFR and Ub does not require prior ubiquitylation. Our data also suggest that protein expression level and N-terminal folding pattern may be two critical determinants for CTPD. Thus, this study reveals that CTPD plays a role in regulating the homeostasis of long-lived proteins and provides insights into the mechanism of CTPD.

Insights into elastic fiber fragmentation: Mechanisms and treatment of aortic aneurysm in Marfan syndrome.

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder caused by mutations in fibrillin 1 (FBN1) gene. These mutations result in defects in the skeletal, ocular, and cardiovascular systems. Aortic aneurysm is the leading cause of premature mortality in untreated MFS patients. Elastic fiber fragmentation in the aortic vessel wall is a hallmark of MFS-associated aortic aneurysms. FBN1 mutations result in FBN1 fragments that also contribute to elastic fiber fragmentation. Although recent research has advanced our understanding of MFS, the contribution of elastic fiber fragmentation to the pathogenesis of aneurysm formation remains poorly understood. This review provides a comprehensive overview of the molecular mechanisms of elastic fiber fragmentation and its role in the pathogenesis of aortic aneurysm progression. Increased comprehension of elastic fragmentation has significant clinical implications for developing targeted interventions to block aneurysm progression, which would benefit not only individuals with Marfan syndrome but also other patients with aneurysms. Moreover, this review highlights an overlooked connection between inhibiting aneurysm and the restoration of elastic fibers in the vessel wall with various aneurysm inhibitors, including drugs and chemicals. Investigating the underlying molecular mechanisms could uncover innovative therapeutic strategies to inhibit elastin fragmentation and prevent the progression of aneurysms.

Homeostatic regulation of ribosomal proteins by ubiquitin-independent cotranslational degradation.

Ribosomes are the workplace for protein biosynthesis. Protein production required for normal cell function is tightly linked to ribosome abundance. It is well known that ribosomal genes are actively transcribed and ribosomal messenger RNAs (mRNAs) are rapidly translated, and yet ribosomal proteins have relatively long half-lives. These observations raise questions as to how homeostasis of ribosomal proteins is controlled. Here, we show that ribosomal proteins, while posttranslationally stable, are subject to high-level cotranslational protein degradation (CTPD) except for those synthesized as ubiquitin (Ub) fusion precursors. The N-terminal Ub moiety protects fused ribosomal proteins from CTPD. We further demonstrate that cotranslational folding efficiency and expression level are two critical factors determining CTPD of ribosomal proteins. Different from canonical posttranslational degradation, we found that CTPD of all the ribosomal proteins tested in this study does not require prior ubiquitylation. This work provides insights into the regulation of ribosomal protein homeostasis and furthers our understanding of the mechanism and biological significance of CTPD.

Estrogen distinctly regulates transcription and translation of lncRNAs and pseudogenes in breast cancer cells.

Estrogen drives key transcriptional changes in breast cancer and stimulates breast cancer cells' growth with multiple mechanisms to coordinate transcription and translation. In addition to protein-coding transcripts, estrogen can regulate long non-coding RNA (lncRNA) transcripts, plus diverse non-coding RNAs including antisense, enhancer, and intergenic. LncRNA genes comprise the majority of human genes. The accidental, or regulated, translation of their short open reading frames by ribosomes remains a controversial topic. Here we report for the first time an integrated analysis of RNA abundance and ribosome occupancy level, using Ribo-seq combined with RNA-Seq, in the estrogen-responsive, estrogen receptor α positive, human breast cancer cell model MCF7, before and after hormone treatment. Translational profiling can determine, in an unbiased manner, which fraction of the genome is actually translated into proteins, as well as resolving whether transcription and translation respond concurrently, or differentially, to estrogen treatment. Our data showed specific transcripts more robustly detected in RNA-Seq than in the ribosome-profiling data, and vice versa, suggesting distinct gene-specific estrogen responses at the transcriptional and the translational level, respectively. Here, we showed that estrogen stimulation affects the expression levels of numerous lncRNAs, but not their association with ribosomes, and that most lncRNAs are not ribosome-bound. For the first time, we also demonstrated the transcriptional and translational response of expressed pseudogenes to estrogen, pointing to new perspectives for drug-target development in breast cancer in the future.

Gestational Age Dependence of the Maternal Circulating Long Non-Coding RNA Transcriptome During Normal Pregnancy Highlights Antisense and Pseudogene Transcripts.

In the post-genomic era, our understanding of the molecular regulators of physiologic and pathologic processes in pregnancy is expanding at the whole-genome level. Longitudinal changes in the known protein-coding transcriptome during normal pregnancy, which we recently reported (Gomez-Lopez et al., 2019), have improved our definition of the major operant networks, yet pregnancy-related functions of the non-coding RNA transcriptome remain poorly understood. A key finding of the ENCODE (Encyclopedia of DNA Elements) Consortium, the successor of the Human Genome Project, was that the human genome contains approximately 60,000 genes, the majority of which do not encode proteins. The total transcriptional output of non-protein-coding RNA genes, collectively referred to as the non-coding transcriptome, is comprised mainly of long non-coding RNA (lncRNA) transcripts (Derrien et al., 2012). Although the ncRNA transcriptome eclipses its protein-coding counterpart in abundance, it has until recently lacked a comprehensive, unbiased, genome-scale characterization over the timecourse of normal human pregnancy. Here, we annotated, characterized, and selectively validated the longitudinal changes in the non-coding transcriptome of maternal whole blood during normal pregnancy to term. We identified nine long non-coding RNAs (lncRNAs), including long intergenic non-coding RNAs (lincRNAs) as well as lncRNAs antisense to or otherwise in the immediate vicinity of protein-coding genes, that were differentially expressed with advancing gestation in normal pregnancy: AL355711, BC039551 (expressed mainly in the placenta), JHDM1D-AS1, A2M-AS1, MANEA-AS1, NR_034004, LINC00649, LINC00861, and LINC01094. By cross-referencing our dataset against major public pseudogene catalogs, we also identified six transcribed pseudogenes that were differentially expressed over time during normal pregnancy in maternal blood: UBBP4, FOXO3B, two Makorin (MKRN) pseudogenes (MKRN9P and LOC441455), PSME2P2, and YBX3P1. We also identified three non-coding RNAs belonging to other classes that were modulated during gestation: the microRNA MIR4439, the small nucleolar RNA (snoRNA) SNORD41, and the small Cajal-body specific ncRNA SCARNA2. The expression profiles of most hits were broadly suggestive of functions in pregnancy. These time-dependent changes of the non-coding transcriptome during normal pregnancy, which may confer specific regulatory impacts on their protein-coding gene targets, will facilitate a deeper molecular understanding of pregnancy and lncRNA-mediated molecular pathways at the maternal-fetal interface and of how these pathways impact maternal and fetal health.

YY1 directly interacts with myocardin to repress the triad myocardin/SRF/CArG box-mediated smooth muscle gene transcription during smooth muscle phenotypic modulation.

Yin Yang 1 (YY1) regulates gene transcription in a variety of biological processes. In this study, we aim to determine the role of YY1 in vascular smooth muscle cell (VSMC) phenotypic modulation both in vivo and in vitro. Here we show that vascular injury in rodent carotid arteries induces YY1 expression along with reduced expression of smooth muscle differentiation markers in the carotids. Consistent with this finding, YY1 expression is induced in differentiated VSMCs in response to serum stimulation. To determine the underlying molecular mechanisms, we found that YY1 suppresses the transcription of CArG box-dependent SMC-specific genes including SM22α, SMα-actin and SMMHC. Interestingly, YY1 suppresses the transcriptional activity of the SM22α promoter by hindering the binding of serum response factor (SRF) to the proximal CArG box. YY1 also suppresses the transcription and the transactivation of myocardin (MYOCD), a master regulator for SMC-specific gene transcription by binding to SRF to form the MYOCD/SRF/CArG box triad (known as the ternary complex). Mechanistically, YY1 directly interacts with MYOCD to competitively displace MYOCD from SRF. This is the first evidence showing that YY1 inhibits SMC differentiation by directly targeting MYOCD. These findings provide new mechanistic insights into the regulatory mechanisms that govern SMC phenotypic modulation in the pathogenesis of vascular diseases.

A Long Non-coding RNA, LOC157273, Is an Effector Transcript at the Chromosome 8p23.1-PPP1R3B Metabolic Traits and Type 2 Diabetes Risk Locus.

AIMS: Causal transcripts at genomic loci associated with type 2 diabetes (T2D) are mostly unknown. The chr8p23.1 variant rs4841132, associated with an insulin-resistant diabetes risk phenotype, lies in the second exon of a long non-coding RNA (lncRNA) gene, LOC157273, located 175 kilobases from PPP1R3B, which encodes a key protein regulating insulin-mediated hepatic glycogen storage in humans. We hypothesized that LOC157273 regulates expression of PPP1R3B in human hepatocytes. METHODS: We tested our hypothesis using Stellaris fluorescent in situ hybridization to assess subcellular localization of LOC157273; small interfering RNA (siRNA) knockdown of LOC157273, followed by RT-PCR to quantify LOC157273 and PPP1R3B expression; RNA-seq to quantify the whole-transcriptome gene expression response to LOC157273 knockdown; and an insulin-stimulated assay to measure hepatocyte glycogen deposition before and after knockdown. RESULTS: We found that siRNA knockdown decreased LOC157273 transcript levels by approximately 80%, increased PPP1R3B mRNA levels by 1.7-fold, and increased glycogen deposition by >50% in primary human hepatocytes. An A/G heterozygous carrier (vs. three G/G carriers) had reduced LOC157273 abundance due to reduced transcription of the A allele and increased PPP1R3B expression and glycogen deposition. CONCLUSION: We show that the lncRNA LOC157273 is a negative regulator of PPP1R3B expression and glycogen deposition in human hepatocytes and a causal transcript at an insulin-resistant T2D risk locus.

The ubiquitin specific protease USP34 protects the ubiquitin ligase gp78 from proteasomal degradation.

The E3 ubiquitin (Ub) ligase gp78 plays an important role in endoplasmic reticulum (ER)-associated degradation (ERAD) and regulation of lipid biogenesis. Although a variety of substrates of gp78 have been described, the regulation of the degradation of gp78 itself remains poorly understood. To address this problem, we used co-immunoprecipitation-coupled liquid chromatography-tandem mass spectrometry (Co-IP/LC-MS/MS) to identify novel proteins interacting with gp78. One of the proteins identified in this study is the deubiquitylating (DUB) enzyme USP34 (Ub-specific protease 34). We demonstrate that knockdown of USP34 facilitates proteasomal degradation of gp78 and consequently impairs the function of gp78 in regulating lipid droplet formation. This study unveils a previously unknown function of USP34 in regulating the metabolic stability of gp78 and adds to our understanding of the relevance of partnering of DUBs and E3s in regulation of protein ubiquitylation.

SM22α suppresses cytokine-induced inflammation and the transcription of NF-κB inducing kinase (Nik) by modulating SRF transcriptional activity in vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) phenotypic modulation is characterized by the downregulation of SMC actin cytoskeleton proteins. Our published study shows that depletion of SM22α (aka SM22, Transgelin, an actin cytoskeleton binding protein) promotes inflammation in SMCs by activating NF-κB signal pathways both in cultured VSMCs and in response to vascular injury. The goal of this study is to investigate the underlying molecular mechanisms whereby SM22 suppresses NF-κB signaling pathways under inflammatory condition. NF-κB inducing kinase (Nik, aka MAP3K14, activated by the LTßR) is a key upstream regulator of NF-κB signal pathways. Here, we show that SM22 overexpression suppresses the expression of NIK and its downstream NF-κB canonical and noncanonical signal pathways in a VSMC line treated with a LTßR agonist. SM22 regulates NIK expression at both transcriptional and the proteasome-mediated post-translational levels in VSMCs depending on the culture condition. By qPCR, chromatin immunoprecipitation and luciferase assays, we found that Nik is a transcription target of serum response factor (SRF). Although SM22 is known to be expressed in the cytoplasm, we found that SM22 is also expressed in the nucleus where SM22 interacts with SRF to inhibit the transcription of Nik and prototypical SRF regulated genes including c-fos and Egr3. Moreover, carotid injury increases NIK expression in Sm22-/- mice, which is partially relieved by adenovirally transduced SM22. These findings reveal for the first time that SM22 is expressed in the nucleus in addition to the cytoplasm of VSMCs to regulate the transcription of Nik and its downstream proinflammatory NF-kB signal pathways as a modulator of SRF during vascular inflammation.

Primate-specific oestrogen-responsive long non-coding RNAs regulate proliferation and viability of human breast cancer cells.

Long non-coding RNAs (lncRNAs) are transcripts of a recently discovered class of genes which do not code for proteins. LncRNA genes are approximately as numerous as protein-coding genes in the human genome. However, comparatively little remains known about lncRNA functions. We globally interrogated changes in the lncRNA transcriptome of oestrogen receptor positive human breast cancer cells following treatment with oestrogen, and identified 127 oestrogen-responsive lncRNAs. Consistent with the emerging evidence that most human lncRNA genes lack homologues outside of primates, our evolutionary analysis revealed primate-specific lncRNAs downstream of oestrogen signalling. We demonstrate, using multiple functional assays to probe gain- and loss-of-function phenotypes in two oestrogen receptor positive human breast cancer cell lines, that two primate-specific oestrogen-responsive lncRNAs identified in this study (the oestrogen-repressed lncRNA BC041455, which reduces cell viability, and the oestrogen-induced lncRNA CR593775, which increases cell viability) exert previously unrecognized functions in cell proliferation and growth factor signalling pathways. The results suggest that oestrogen-responsive lncRNAs are capable of altering the proliferation and viability of human breast cancer cells. No effects on cellular phenotypes were associated with control transfections. As heretofore unappreciated components of key signalling pathways in cancers, including the MAP kinase pathway, lncRNAs hence represent a novel mechanism of action for oestrogen effects on cellular proliferation and viability phenotypes. This finding warrants further investigation in basic and translational studies of breast and potentially other types of cancers, has broad relevance to lncRNAs in other nuclear hormone receptor pathways, and should facilitate exploiting and targeting these cell viability modulating lncRNAs in post-genomic therapeutics.

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix.

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis(1). Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

Rapidly Translated Polypeptides Are Preferred Substrates for Cotranslational Protein Degradation.

Nascent polypeptides are degraded by the proteasome concurrently with their synthesis on the ribosome. This process, called cotranslational protein degradation (CTPD), has been observed for years, but the underlying mechanisms remain poorly understood. Equally unclear are the identities of cellular proteins genuinely subjected to CTPD. Here we report the identification of CTPD substrates in the yeast Saccharomyces cerevisiae via a quantitative proteomic analysis. We compared the abundance of individual ribosome-bound nascent chains between a wild type strain and a mutant defective in CTPD. Of 1,422 proteins acquired from the proteomic analysis, 289 species are efficient CTPD substrates, with >30% of their nascent chains degraded cotranslationally. We found that proteins involved in translation, ribosome biogenesis, nuclear transport, and amino acid metabolism are more likely to be targeted for CTPD. There is a strong correlation between CTPD and the translation efficiency. CTPD occurs preferentially to rapidly translated polypeptides. CTPD is also influenced by the protein sequence length; longer polypeptides are more susceptible to CTPD. In addition, proteins with N-terminal disorder have a higher probability of being degraded cotranslationally. Interestingly, the CTPD efficiency is not related to the half-lives of mature proteins. These results for the first time indicate an inverse correlation between CTPD and cotranslational folding on a proteome scale. The implications of this study with respect to the physiological significance of CTPD are discussed.

Dyclonine enhances the cytotoxic effect of proteasome inhibitor bortezomib in multiple myeloma cells.

The proteasome has become an important target for cancer therapy with the approval of bortezomib for the treatment of relapsed/refractory multiple myeloma (MM). However, numerous patients with MM do not respond to bortezomib and those responding initially often acquire resistance. Recent clinical studies have also demonstrated that bortezomib is also inefficacious in the treatment of other types of cancer. Therefore, it is imperative to develop novel approaches and agents for proteasome-targeting cancer therapy. In the present study, it was revealed that dyclonine, a major component of the cough droplets Sucrets, markedly enhances the cytotoxic effects of bortezomib and minimizes drug resistance in MM cells. It was demonstrated that a combination of bortezomib and dyclonine markedly induced apoptosis of MM cells. The present study suggests a novel therapeutic use of an over­the­counter medicine for the treatment of MM.

Nuclear import factor Srp1 and its associated protein Sts1 couple ribosome-bound nascent polypeptides to proteasomes for cotranslational degradation.

Cotranslational protein degradation plays an important role in protein quality control and proteostasis. Although ubiquitylation has been suggested to signal cotranslational degradation of nascent polypeptides, cotranslational ubiquitylation occurs at a low level, suggesting the existence of an alternative route for delivery of nascent polypeptides to the proteasome. Here we report that the nuclear import factor Srp1 (also known as importin α or karyopherin α) is required for ubiquitin-independent cotranslational degradation of the transcription factor Rpn4. We further demonstrate that cotranslational protein degradation is generally impaired in the srp1-49 mutant. Srp1 binds nascent polypeptides emerging from the ribosome. The association of proteasomes with polysomes is weakened in srp1-49. The interaction between Srp1 and the proteasome is mediated by Sts1, a multicopy suppressor of srp1-49. The srp1-49 and sts1-2 mutants are hypersensitive to stressors that promote protein misfolding, underscoring the physiological function of Srp1 and Sts1 in degradation of misfolded nascent polypeptides. This study unveils a previously unknown role for Srp1 and Sts1 in cotranslational protein degradation and suggests a novel model whereby Srp1 and Sts1 cooperate to couple proteasomes to ribosome-bound nascent polypeptides.

The N-terminal domain of Rpn4 serves as a portable ubiquitin-independent degron and is recognized by specific 19S RP subunits.

The number of proteasomal substrates that are degraded without prior ubiquitylation continues to grow. However, it remains poorly understood how the proteasome recognizes substrates lacking a ubiquitin (Ub) signal. Here we demonstrated that the Ub-independent degradation of Rpn4 requires the 19S regulatory particle (RP). The Ub-independent degron of Rpn4 was mapped to an N-terminal region including the first 80 residues. Inspection of its amino acid sequence revealed that the Ub-independent degron of Rpn4 consists of an intrinsically disordered domain followed by a folded segment. Using a photo-crosslinking-label transfer method, we captured three 19S RP subunits (Rpt1, Rpn2 and Rpn5) that bind the Ub-independent degron of Rpn4. This is the first time that specific 19S RP subunits have been identified interacting with a Ub-independent degron. This study provides insight into the mechanism by which Ub-independent substrates are recruited to the 26S proteasome.

The CCAAT box-binding transcription factor NF-Y regulates basal expression of human proteasome genes.

Protein degradation by the proteasome plays an important role in all major cellular pathways. Aberrant proteasome activity is associated with numerous human diseases including cancer and neurological disorders, but the underlying mechanism is virtually unclear. At least part of the reason for this is due to lack of understanding of the regulation of human proteasome genes. In this study, we found that a large set of human proteasome genes carry the CCAAT box in their promoters. We further demonstrated that the basal expression of these CCAAT box-containing proteasome genes is regulated by the transcription factor NF-Y. Knockdown of NF-YA, an essential subunit of NF-Y, reduced proteasome gene expression and compromised the cellular proteasome activity. In addition, we showed that knockdown of NF-YA sensitized breast cancer cells to the proteasome inhibitor MG132. This study unveils a new role for NF-Y in the regulation of human proteasome genes and suggests that NF-Y may be a potential target for cancer therapy.

Arterial injury promotes medial chondrogenesis in Sm22 knockout mice.

AIMS: Expression of SM22 (also known as SM22alpha and transgelin), a vascular smooth muscle cells (VSMCs) marker, is down-regulated in arterial diseases involving medial osteochondrogenesis. We investigated the effect of SM22 deficiency in a mouse artery injury model to determine the role of SM22 in arterial chondrogenesis. METHODS AND RESULTS: Sm22 knockout (Sm22(-/-)) mice developed prominent medial chondrogenesis 2 weeks after carotid denudation as evidenced by the enhanced expression of chondrogenic markers including type II collagen, aggrecan, osteopontin, bone morphogenetic protein 2, and SRY-box containing gene 9 (SOX9). This was concomitant with suppression of VSMC key transcription factor myocardin and of VSMC markers such as SM α-actin and myosin heavy chain. The conversion tendency from myogenesis to chondrogenesis was also observed in primary Sm22(-/-) VSMCs and in a VSMC line after Sm22 knockdown: SM22 deficiency altered VSMC morphology with compromised stress fibre formation and increased actin dynamics. Meanwhile, the expression level of Sox9 mRNA was up-regulated while the mRNA levels of myocardin and VSMC markers were down-regulated, indicating a pro-chondrogenic transcriptional switch in SM22-deficient VSMCs. Furthermore, the increased expression of SOX9 was mediated by enhanced reactive oxygen species production and nuclear factor-κB pathway activation. CONCLUSION: These findings suggest that disruption of SM22 alters the actin cytoskeleton and promotes chondrogenic conversion of VSMCs.

Resveratrol induces p53 and suppresses myocardin-mediated vascular smooth muscle cell differentiation.

Resveratrol (RSVL), a polyphenolic antioxidant present in red wine, has been shown to provide cardiovascular protection by improving endothelial function and reducing myocardial ischemia. However, little is known about how RSVL affects vascular smooth muscle cells (VSMCs) differentiation. RSVL blocks VSMC proliferation in vitro and neointimal formation following artery injury in vivo. Thus, one might expect that RSVL will promote VSMC differentiation. Unexpectedly, our results in this study show that RSVL induces VSMCs phenotypic modulation; this is characterized by suppressed transcription of SMC-specific marker genes Tagln, Acta2, Myh11, and Smtn in a dose-dependent and time-dependent manner in cultured VSMCs. Consistent with previous studies, RSVL induces the nuclear translocation of p53 and the expression of p53-responsive genes such as Cdkn1a, Gadd45a, Gadd45, and Fas. In an effort to identify the molecular mechanisms whereby RSVL represses VSMC differentiation, we found that RSVL inhibits the transcription of Myocardin (myocd) and Srf, the key VSMC transcriptional regulators. However, knockingdown and overexpressing p53 did not affect RSVL-induced VSMCs phenotypic modulation: this suggests that RSVL may induce VSMC dedifferentiation via p53-independent mechanisms. This study provides the first evidence showing that RSVL induces VSMC dedifferentiation by regulating Myocardin and SRF-mediated VSMC gene transcription.