Itga8-Cre-mediated deletion of YAP and TAZ impairs bladder contractility with minimal inflammation and chondrogenic differentiation.

A role of Yes1-associated transcriptional regulator (YAP) and WW domain-containing transcription regulator 1 (TAZ) in vascular and gastrointestinal contractility due to control of myocardin (Myocd) expression, which in turn activates contractile genes, has been demonstrated. Whether this transcriptional hierarchy applies to the urinary bladder is unclear. We found that YAP/TAZ are expressed in human detrusor myocytes and therefore exploited the Itga8-CreERT2 model for the deletion of YAP/TAZ. Recombination occurred in detrusor, and YAP/TAZ transcripts were reduced by >75%. Bladder weights were increased (by ≈22%), but histology demonstrated minimal changes in the detrusor, while arteries in the mucosa were inflamed. Real-time quantitative reverse transcription PCR (RT-qPCR) using the detrusor demonstrated reductions of Myocd (-79 ± 18%) and serum response factor (Srf) along with contractile genes. In addition, the cholinergic receptor muscarinic 2 (Chrm2) and Chrm3 were suppressed (-80 ± 23% and -80 ± 10%), whereas minute increases of Il1b and Il6 were seen. Unlike YAP/TAZ-deficient arteries, SRY (sex-determining region Y)-box 9 (Sox9) did not increase, and no chondrogenic differentiation was apparent. Reductions of smooth muscle myosin heavy chain 11 (Myh11), myosin light-chain kinase gene (Mylk), and Chrm3 were seen at the protein level. Beyond restraining the smooth muscle cell (SMC) program of gene expression, YAP/TAZ depletion silenced SMC-specific splicing, including exon 2a of Myocd. Reduced contractile differentiation was associated with weaker contraction in response to myosin phosphatase inhibition (-36%) and muscarinic activation (reduced by 53% at 0.3 µM carbachol). Finally, short-term overexpression of constitutively active YAP in human embryonic kidney 293 (HEK293) cells increased myocardin (greater than eightfold) along with archetypal target genes, but contractile genes were unaffected or reduced. YAP and TAZ thus regulate myocardin expression in the detrusor, and this is important for SMC differentiation and splicing as well as for contractility.NEW & NOTEWORTHY This study addresses the hypothesis that YAP and TAZ have an overarching role in the transcriptional hierarchy in the smooth muscle of the urinary bladder by controlling myocardin expression. Using smooth muscle-specific and inducible deletion of YAP and TAZ in adult mice, we find that YAP and TAZ control myocardin expression, contractile differentiation, smooth muscle-specific splicing, and bladder contractility. These effects are largely independent of inflammation and chondrogenic differentiation.

YAP and TAZ in Vascular Smooth Muscle Confer Protection Against Hypertensive Vasculopathy.

BACKGROUND: Hypertension remains a major risk factor for cardiovascular diseases, but the underlying mechanisms are not well understood. We hypothesize that appropriate mechanotransduction and contractile function in vascular smooth muscle cells are crucial to maintain vascular wall integrity. The Hippo pathway effectors YAP (yes-associated protein 1) and TAZ (WW domain containing transcription regulator 1) have been identified as mechanosensitive transcriptional coactivators. However, their role in vascular smooth muscle cell mechanotransduction has not been investigated in vivo. METHODS: We performed physiological and molecular analyses utilizing an inducible smooth muscle-specific YAP/TAZ knockout mouse model. RESULTS: Arteries lacking YAP/TAZ have reduced agonist-mediated contraction, decreased myogenic response, and attenuated stretch-induced transcriptional regulation of smooth muscle markers. Moreover, in established hypertension, YAP/TAZ knockout results in severe vascular lesions in small mesenteric arteries characterized by neointimal hyperplasia, elastin degradation, and adventitial thickening. CONCLUSIONS: This study demonstrates a protective role of YAP/TAZ against hypertensive vasculopathy.

Myocardin regulates exon usage in smooth muscle cells through induction of splicing regulatory factors.

Differentiation of smooth muscle cells (SMCs) depends on serum response factor (SRF) and its co-activator myocardin (MYOCD). The role of MYOCD for the SMC program of gene transcription is well established. In contrast, the role of MYOCD in control of SMC-specific alternative exon usage, including exon splicing, has not been explored. In the current work we identified four splicing factors (MBNL1, RBPMS, RBPMS2, and RBFOX2) that correlate with MYOCD across human SMC tissues. Forced expression of MYOCD family members in human coronary artery SMCs in vitro upregulated expression of these splicing factors. For global profiling of transcript diversity, we performed RNA-sequencing after MYOCD transduction. We analyzed alternative transcripts with three different methods. Exon-based analysis identified 1637 features with differential exon usage. For example, usage of 3´ exons in MYLK that encode telokin increased relative to 5´ exons, as did the 17 kDa telokin to 130 kDa MYLK protein ratio. Dedicated event-based analysis identified 239 MYOCD-driven splicing events. Events involving MBNL1, MCAM, and ACTN1 were among the most prominent, and this was confirmed using variant-specific PCR analyses. In support of a role for RBPMS and RBFOX2 in MYOCD-driven splicing we found enrichment of their binding motifs around differentially spliced exons. Moreover, knockdown of either RBPMS or RBFOX2 antagonized splicing events stimulated by MYOCD, including those involving ACTN1, VCL, and MBNL1. Supporting an in vivo role of MYOCD-SRF-driven splicing, we demonstrate altered Rbpms expression and splicing in inducible and SMC-specific Srf knockout mice. We conclude that MYOCD-SRF, in part via RBPMS and RBFOX2, induce a program of differential exon usage and alternative splicing as part of the broader program of SMC differentiation.

Antagonistic relationship between the unfolded protein response and myocardin-driven transcription in smooth muscle.

Smooth muscle cells (SMCs) are characterized by a high degree of phenotypic plasticity. Contractile differentiation is governed by myocardin-related transcription factors (MRTFs), in particular myocardin (MYOCD), and when their drive is lost, the cells become proliferative and synthetic with an expanded endoplasmic reticulum (ER). ER is responsible for assembly and folding of secreted proteins. When the load on the ER surpasses its capacity, three stress sensors (activating transcription factor 6 [ATF6], inositol-requiring enzyme 1α [IRE1α]/X-box binding protein 1 [XBP1], and PERK/ATF4) are activated to expand the ER and increase its folding capacity. This is referred to as the unfolded protein response (UPR). Here, we hypothesized that there is a reciprocal relationship between SMC differentiation and the UPR. Tight negative correlations between SMC markers (MYH11, MYOCD, KCNMB1, SYNPO2) and UPR markers (SDF2L1, CALR, MANF, PDIA4) were seen in microarray data sets from carotid arterial injury, partial bladder outlet obstruction, and bladder denervation, respectively. The UPR activators dithiothreitol (DTT) and tunicamycin (TN) activated the UPR and reduced MYOCD along with SMC markers in vitro. The IRE1α inhibitor 4µ8C counteracted the effect of DTT and TN on SMC markers and MYOCD expression. Transfection of active XBP1s was sufficient to reduce both MYOCD and the SMC markers. MRTFs also antagonized the UPR as indicated by reduced TN and DTT-mediated induction of CRELD2, MANF, PDIA4, and SDF2L1 following overexpression of MRTFs. The latter effect did not involve the newly identified MYOCD/SRF target MSRB3, or reduced production of either XBP1s or cleaved ATF6. The UPR thus counteracts SMC differentiation via the IRE1α/XBP1 arm of the UPR and MYOCD repression.

Human host defense peptide LL-37 facilitates double-stranded RNA pro-inflammatory signaling through up-regulation of TLR3 expression in vascular smooth muscle cells.

OBJECTIVE: The importance of human host defense peptide LL-37 in vascular innate immunity is not understood. Here, we assess the impact of LL-37 on double-stranded RNA (dsRNA) signaling in human vascular smooth muscle cells. MATERIALS AND METHODS: Cellular import of LL-37 and synthetic dsRNA (poly I:C) were investigated by immunocytochemistry and fluorescence imaging. Transcript and protein expression were determined by qPCR, ELISA and Western blot. Knockdown of TLR3 was performed by siRNA. RESULTS: LL-37 was rapidly internalized, suggesting that it has intracellular actions. Co-stimulation with poly I:C and LL-37 enhanced pro-inflammatory IL-6 and MCP-1 transcripts several fold compared to treatment with poly I:C or LL-37 alone. Poly I:C increased IL-6 and MCP-1 protein production, and this effect was potentiated by LL-37. LL-37-induced stimulation of poly I:C signaling was not associated with enhanced import of poly I:C. Treatment with poly I:C and LL-37 in combination increased expression of dsRNA receptor TLR3 compared to stimulation with poly I:C or LL-37 alone. In TLR3 knockdown cells, treatment with poly I:C and LL-37 in combination had no effect on IL-6 and MCP-1 expression, showing loss of function. CONCLUSIONS: LL-37 potentiates dsRNA-induced cytokine production through up-regulation of TLR3 expression representing a novel pro-inflammatory mechanism.

Identification of the intermediate filament protein synemin/SYNM as a target of myocardin family coactivators.

Myocardin (MYOCD) is a critical regulator of smooth muscle cell (SMC) differentiation, but its transcriptional targets remain to be exhaustively characterized, especially at the protein level. Here we leveraged human RNA and protein expression data to identify novel potential MYOCD targets. Using correlation analyses we found several targets that we could confirm at the protein level, including SORBS1, SLMAP, SYNM, and MCAM. We focused on SYNM, which encodes the intermediate filament protein synemin. SYNM rivalled smooth muscle myosin (MYH11) for SMC specificity and was controlled at the mRNA and protein levels by all myocardin-related transcription factors (MRTFs: MYOCD, MRTF-A/MKL1, and MRTF-B/MKL2). MRTF activity is regulated by the ratio of filamentous to globular actin, and SYNM was accordingly reduced by interventions that depolymerize actin, such as latrunculin treatment and overexpression of constitutively active cofilin. Many MRTF target genes depend on serum response factor (SRF), but SYNM lacked SRF-binding motifs in its proximal promoter, which was not directly regulated by MYOCD. Furthermore, SYNM resisted SRF silencing, yet the time course of induction closely paralleled that of the SRF-dependent target gene ACTA2. SYNM was repressed by the ternary complex factor (TCF) FLI1 and was increased in mouse embryonic fibroblasts lacking three classical TCFs (ELK1, ELK3, and ELK4). Imaging showed colocalization of SYNM with the intermediate filament proteins desmin and vimentin, and MRTF-A/MKL1 increased SYNM-containing intermediate filaments in SMCs. These studies identify SYNM as a novel SRF-independent target of myocardin that is abundantly expressed in all SMCs.

Loss of Vascular Myogenic Tone in miR-143/145 Knockout Mice Is Associated With Hypertension-Induced Vascular Lesions in Small Mesenteric Arteries.

OBJECTIVE: Pressure-induced myogenic tone is involved in autoregulation of local blood flow and confers protection against excessive pressure levels in small arteries and capillaries. Myogenic tone is dependent on smooth muscle microRNAs (miRNAs), but the identity of these miRNAs is unclear. Furthermore, the consequences of altered myogenic tone for hypertension-induced damage to small arteries are not well understood. APPROACH AND RESULTS: The importance of smooth muscle-enriched microRNAs, miR-143/145, for myogenic tone was evaluated in miR-143/145 knockout mice. Furthermore, hypertension-induced vascular injury was evaluated in mesenteric arteries in vivo after angiotensin II infusion. Myogenic tone was abolished in miR-143/145 knockout mesenteric arteries, whereas contraction in response to calyculin A and potassium chloride was reduced by ≈30%. Furthermore, myogenic responsiveness was potentiated by angiotensin II in wild-type but not in knockout mice. Angiotensin II administration in vivo elevated systemic blood pressure in both genotypes. Hypertensive knockout mice developed severe vascular lesions characterized by vascular inflammation, adventitial fibrosis, and neointimal hyperplasia in small mesenteric arteries. This was associated with depolymerization of actin filaments and fragmentation of the elastic laminae at the sites of vascular lesions. CONCLUSIONS: This study demonstrates that miR-143/145 expression is essential for myogenic responsiveness. During hypertension, loss of myogenic tone results in potentially damaging levels of mechanical stress and detrimental effects on small arteries. The results presented herein provide novel insights into the pathogenesis of vascular disease and emphasize the importance of controlling mechanical factors to maintain structural integrity of the vascular wall.

Expression of endothelin type B receptors (EDNRB) on smooth muscle cells is controlled by MKL2, ternary complex factors, and actin dynamics.

The endothelin type B receptor (ETB or EDNRB) is highly plastic and is upregulated in smooth muscle cells (SMCs) by arterial injury and following organ culture in vitro. We hypothesized that this transcriptional plasticity may arise, in part, because EDNRB is controlled by a balance of transcriptional inputs from myocardin-related transcription factors (MRTFs) and ternary complex factors (TCFs). We found significant positive correlations between the TCFs ELK3 and FLI1 versus EDNRB in human arteries. The MRTF MKL2 also correlated with EDNRB. Overexpression of ELK3, FLI1, and MKL2 in human coronary artery SMCs promoted expression of EDNRB, and the effect of MKL2 was antagonized by myocardin (MYOCD), which also correlated negatively with EDNRB at the tissue level. Silencing of MKL2 reduced basal EDNRB expression, but depolymerization of actin using latrunculin B (LatB) or overexpression of constitutively active cofilin, as well as treatment with the Rho-associated kinase (ROCK) inhibitor Y27632, increased EDNRB in a MEK/ERK-dependent fashion. Transcript-specific primers indicated that the second EDNRB transcript (EDNRB_2) was targeted, but this promoter was largely unresponsive to LatB and was inhibited rather than stimulated by MKL2 and FLI1, suggesting distant control elements or an indirect effect. LatB also reduced expression of endothelin-1, but supplementation experiments argued that this was not the cause of EDNRB induction. EDNRB finally changed in parallel with ELK3 and FLI1 in rat and human carotid artery lesions. These studies implicate the actin cytoskeleton and ELK3, FLI1, and MKL2 in the transcriptional control of EDNRB and increase our understanding of the plasticity of this receptor.

Cavin-3 (PRKCDBP) deficiency reduces the density of caveolae in smooth muscle.

Cavins belong to a family of proteins that contribute to the formation of caveolae, which are membrane organelles with functional roles in muscle and fat. Here, we investigate the effect of cavin-3 ablation on vascular and urinary bladder structure and function. Arteries and urinary bladders from mice lacking cavin-3 (knockout: KO) and from controls (wild type: WT) were examined. Our studies revealed that the loss of cavin-3 resulted in ∼40% reduction of the caveolae protein cavin-1 in vascular and bladder smooth muscle. Electron microscopy demonstrated that the loss of cavin-3 was accompanied by a reduction of caveolae abundance by 40-45% in smooth muscle, whereas the density of caveolae in endothelial cells was unchanged. Vascular contraction in response to an α1-adrenergic agonist was normal but nitric-oxide-dependent relaxation was enhanced, in parallel with an increased relaxation on direct activation of soluble guanylyl cyclase (sGC). This was associated with an elevated expression of sGC, although blood pressure was similar in WT and KO mice. Contraction of the urinary bladder was not affected by the loss of cavin-3. The proteomic response to outlet obstruction, including STAT3 phosphorylation, the induction of synthetic markers and the repression of contractile markers were identical in WT and KO mice, the only exception being a curtailed induction of the Golgi protein GM130. Loss of cavin-3 thus reduces the number of caveolae in smooth muscle and partly destabilizes cavin-1 but the functional consequences are modest and include an elevated vascular sensitivity to nitric oxide and slightly disturbed Golgi homeostasis in situations of severe cellular stress.

MicroRNA-Dependent Control of Serotonin-Induced Pulmonary Arterial Contraction.

BACKGROUND: Serotonin (5-HT) is considered to play a role in pulmonary arterial hypertension by regulating vascular remodeling and smooth muscle contractility. Here, arteries from mice with inducible and smooth muscle-specific deletion of Dicer were used to address mechanisms by which microRNAs control 5-HT-induced contraction. METHODS: Mice were used 5 weeks after Dicer deletion, and pulmonary artery contractility was analyzed by wire myography. RESULTS: No change was seen in right ventricular systolic pressure following dicer deletion, but systemic blood pressure was reduced. Enhanced 5-HT-induced contraction in Dicer KO pulmonary arteries was associated with increased 5-HT2A receptor mRNA expression whereas 5-HT1B and 5-HT2B receptor mRNAs were unchanged. Contraction by the 5-HT2A agonist TCB-2 was increased in Dicer KO as was the response to the 5-HT2B agonist BW723C86. Effects of Src and protein kinase C inhibition were similar in control and KO arteries, but the effect of inhibition of Rho kinase was reduced. We identified miR-30c as a potential candidate for 5-HT2A receptor regulation as it repressed 5-HT2A mRNA and protein. CONCLUSION: Our findings show that 5-HT receptor signaling in the arterial wall is subject to regulation by microRNAs and that this entails altered 5-HT2A receptor expression and signaling.

Emerging roles of the myocardin family of proteins in lipid and glucose metabolism.

Members of the myocardin family bind to the transcription factor serum response factor (SRF) and act as coactivators controlling genes of relevance for myogenic differentiation and motile function. Binding of SRF to DNA is mediated by genetic elements called CArG boxes, found often but not exclusively in muscle and growth controlling genes. Studies aimed at defining the full spectrum of these CArG elements in the genome (i.e. the CArGome) have in recent years, unveiled unexpected roles of the myocardin family proteins in lipid and glucose homeostasis. This coactivator family includes the protein myocardin (MYOCD), the myocardin-related transcription factors A and B (MRTF-A/MKL1 and MRTF-B/MKL2) and MASTR (MAMSTR). Here we discuss growing evidence that SRF-driven transcription is controlled by extracellular glucose through activation of the Rho-kinase pathway and actin polymerization. We also describe data showing that adipogenesis is influenced by MLK activity through actions upstream of peroxisome proliferator-activated receptor γ with consequences for whole body fat mass and insulin sensitivity. The recently demonstrated involvement of myocardin coactivators in the biogenesis of caveolae, Ω-shaped membrane invaginations of importance for lipid and glucose metabolism, is finally discussed. These novel roles of myocardin proteins may open the way for new unexplored strategies to combat metabolic diseases such as diabetes, which, at the current incidence, is expected to reach 333 million people worldwide by 2025. This review highlights newly discovered roles of myocardin-related transcription factors in lipid and glucose metabolism as well as novel insights into their well-established role as mediators of stretch-dependent effects in smooth muscle. As co-factors for serum response factor (SRF), MKLs regulates transcription of genes involved in the contractile function of smooth muscle cells. In addition to mechanical stimuli, this regulation has now been found to be promoted by extracellular glucose levels in smooth muscle. Recent reports also suggest that MKLs can regulate a subset of genes involved in the formation of lipid-rich invaginations in the cell membrane called caveolae. Finally, a potential role of MKLs in non-muscle cells has been discovered as they negatively influence adipocyte differentiation.

Inhibition of Polyamine Uptake Potentiates the Anti-Proliferative Effect of Polyamine Synthesis Inhibition and Preserves the Contractile Phenotype of Vascular Smooth Muscle Cells.

Increased vascular smooth muscle cell (VSMC) proliferation is a factor in atherosclerosis and injury-induced arterial (re) stenosis. Inhibition of polyamine synthesis by α-difluoro-methylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, attenuates VSMC proliferation with high sensitivity and specificity. However, cells can escape polyamine synthesis blockade by importing polyamines from the environment. To address this issue, polyamine transport inhibitors (PTIs) have been developed. We investigated the effects of the novel trimer44NMe (PTI-1) alone and in combination with DFMO on VSMC polyamine uptake, proliferation and phenotype regulation. PTI-1 efficiently inhibited polyamine uptake in primary mouse aortic and human coronary VSMCs in the absence as well as in the presence of DFMO. Interestingly, culture with DFMO for 2 days substantially (>95%) reduced putrescine (Put) and spermidine (Spd) contents without any effect on proliferation. Culture with PTI-1 alone had no effect on either polyamine levels or proliferation rate, but the combination of both treatments reduced Put and Spd levels below the detection limit and inhibited proliferation. Treatment with DFMO for a longer time period (4 days) reduced Put and Spd below their detection limits and reduced proliferation, showing that only a small pool of polyamines is needed to sustain VSMC proliferation. Inhibited proliferation by polyamine depletion was associated with maintained expression of contractile smooth marker genes. In cultured intact mouse aorta, PTI-1 potentiated the DFMO-induced inhibition of cell proliferation. The combination of endogenous polyamine synthesis inhibition with uptake blockade is thus a viable approach for targeting unwanted vascular cell proliferation in vivo, including vascular restenosis.

Thrombospondin-4 knockout in hypertension protects small-artery endothelial function but induces aortic aneurysms.

Thrombospondin-4 (TSP-4) is a multidomain calcium-binding protein that has both intracellular and extracellular functions. As an extracellular matrix protein, it is involved in remodeling processes. Previous work showed that, in the cardiovascular system, TSP-4 expression is induced in the heart in response to experimental pressure overload and infarction injury. Intracellularly, it mediates the endoplasmic reticulum stress response in the heart. In this study, we explored the role of TSP-4 in hypertension. For this purpose, wild-type and TSP-4 knockout (Thbs4(-/-)) mice were treated with angiotensin II (ANG II). Hearts from ANG II-treated Thbs4(-/-) mice showed an exaggerated hypertrophic response. Interestingly, aortas from Thbs4(-/-) mice treated with ANG II showed a high incidence of aneurysms. In resistance arteries, ANG II-treated wild-type mice showed impaired endothelial-dependent relaxation. This was not observed in ANG II-treated Thbs4(-/-) mice or in untreated controls. No differences were found in the passive pressure-diameter curves or stress-strain relationships, although ANG II-treated Thbs4(-/-) mice showed a tendency to be less stiff, associated with thicker diameters of the collagen fibers as revealed by electron microscopy. We conclude that TSP-4 plays a role in hypertension, affecting cardiac hypertrophy, aortic aneurysm formation, as well as endothelial-dependent relaxation in resistance arteries.

ApoA-I Milano stimulates lipolysis in adipose cells independently of cAMP/PKA activation.

ApoA-I, the main protein component of HDL, is suggested to be involved in metabolic homeostasis. We examined the effects of Milano, a naturally occurring ApoA-I variant, about which little mechanistic information is available. Remarkably, high-fat-fed mice treated with Milano displayed a rapid weight loss greater than ApoA-I WT treated mice, and a significantly reduced adipose tissue mass, without an inflammatory response. Further, lipolysis in adipose cells isolated from mice treated with either WT or Milano was increased. In primary rat adipose cells, Milano stimulated cholesterol efflux and increased glycerol release, independently of ß-adrenergic stimulation and phosphorylation of hormone sensitive lipase (Ser563) and perilipin (Ser522). Stimulation with Milano had a significantly greater effect on glycerol release compared with WT but similar effect on cholesterol efflux. Pharmacological inhibition or siRNA silencing of ABCA1 did not diminish Milano-stimulated lipolysis, although binding to the cell surface was decreased, as analyzed by fluorescence microscopy. Interestingly, methyl-ß-cyclodextrin, a well-described cholesterol acceptor, dose-dependently stimulated lipolysis. Together, these results suggest that decreased fat mass and increased lipolysis following Milano treatment in vivo is partly explained by a novel mechanism at the adipose cell level comprising stimulation of lipolysis independently of the canonical cAMP/protein kinase A signaling pathway.

Suppression of smooth muscle cell inflammation by myocardin-related transcription factors involves inactivation of TANK-binding kinase 1.

Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) suppress production of pro-inflammatory cytokines and chemokines in human smooth muscle cells (SMCs) through sequestration of RelA in the NF-κB complex, but additional mechanisms are likely involved. The cGAS-STING pathway is activated by double-stranded DNA in the cytosolic compartment and acts through TANK-binding kinase 1 (TBK1) to spark inflammation. The present study tested if MRTFs suppress inflammation also by targeting cGAS-STING signaling. Interrogation of a transcriptomic dataset where myocardin was overexpressed using a panel of 56 cGAS-STING cytokines showed the panel to be repressed. Moreover, MYOCD, MRTFA, and SRF associated negatively with the panel in human arteries. RT-qPCR in human bronchial SMCs showed that all MRTFs reduced pro-inflammatory cytokines on the panel. MRTFs diminished phosphorylation of TBK1, while STING phosphorylation was marginally affected. The TBK1 inhibitor amlexanox, but not the STING inhibitor H-151, reduced the anti-inflammatory effect of MRTF-A. Co-immunoprecipitation and proximity ligation assays supported binding between MRTF-A and TBK1 in SMCs. MRTFs thus appear to suppress cellular inflammation in part by acting on the kinase TBK1. This may defend SMCs against pro-inflammatory insults in disease.

Vascular smooth muscle-specific YAP/TAZ deletion triggers aneurysm development in mouse aorta.

Inadequate adaption to mechanical forces, including blood pressure, contributes to development of arterial aneurysms. Recent studies have pointed to a mechanoprotective role of YAP and TAZ in vascular smooth muscle cells (SMCs). Here, we identified reduced expression of YAP1 in human aortic aneurysms. Vascular SMC-specific knockouts (KOs) of YAP/TAZ were thus generated using the integrin α8-Cre (Itga8-Cre) mouse model (i8-YT-KO). i8-YT-KO mice spontaneously developed aneurysms in the abdominal aorta within 2 weeks of KO induction and in smaller arteries at later times. The vascular specificity of Itga8-Cre circumvented gastrointestinal effects. Aortic aneurysms were characterized by elastin disarray, SMC apoptosis, and accumulation of proteoglycans and immune cell populations. RNA sequencing, proteomics, and myography demonstrated decreased contractile differentiation of SMCs and impaired vascular contractility. This associated with partial loss of myocardin expression, reduced blood pressure, and edema. Mediators in the inflammatory cGAS/STING pathway were increased. A sizeable increase in SOX9, along with several direct target genes, including aggrecan (Acan), contributed to proteoglycan accumulation. This was the earliest detectable change, occurring 3 days after KO induction and before the proinflammatory transition. In conclusion, Itga8-Cre deletion of YAP and TAZ represents a rapid and spontaneous aneurysm model that recapitulates features of human abdominal aortic aneurysms.

Replicative aging induces endothelial to mesenchymal transition in human aortic endothelial cells: potential role of inflammation.

Thickening of the intimal layer of arteries characterized by expression of smooth muscle α-actin (SMαA), collagen deposition, and inflammation is an important pathophysiological change with aging assumed to be mediated by smooth muscle cells migrating from the medial layer. We tested the novel hypothesis that these characteristics could also reflect an endothelial-mesenchymal (smooth muscle-like) transition (EnMT). Late ('old') compared with early ('young') passage (45.0 ± 1.2 vs. 27.1 ± 0.5 population doublings) human aortic endothelial cells demonstrated greater smooth muscle (spindle) morphological changes, expression of SMαA and collagen I, nuclear factor-κB activation, and transforming growth factor-ß (TGF-ß) (all p < 0.05). Based on increases in SMαA, stimulation with the proinflammatory cytokine tumor necrosis factor-α, but not with TGF-ß, induced EnMT in early passage cells similar to that observed in late passage cells. Here, we present the first evidence for EnMT induced in a model of endothelial cell aging and provide support for proinflammatory signaling in mediating this phenotypic change.

Regulation of the Muscarinic M3 Receptor by Myocardin-Related Transcription Factors.

Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) are co-factors of serum response factor (SRF) that activate the smooth muscle cell (SMC) gene program and that play roles in cardiovascular development and mechanobiology. Gain and loss of function experiments have defined the SMC gene program under control of MRTFs, yet full understanding of their impact is lacking. In the present study, we tested the hypothesis that the muscarinic M3 receptor (CHRM3) is regulated by MRTFs together with SRF. Forced expression of MYOCD (8d) in human coronary artery (SMC) followed by RNA-sequencing showed increased levels of M2, M3, and M5 receptors (CHRM2: 2-fold, CHRM3: 16-fold, and CHRM5: 2-fold). The effect of MYOCD on M3 was confirmed by RT-qPCR using both coronary artery and urinary bladder SMCs, and correlation analyses using human transcriptomic datasets suggested that M3 may also be regulated by MRTF-B. Head-to-head comparisons of MYOCD, MRTF-A and MRTF-B, argued that while all MRTFs are effective, MRTF-B is the most powerful transactivator of CHRM3, causing a 600-fold increase at 120h. Accordingly, MRTF-B conferred responsiveness to the muscarinic agonist carbachol in Ca2+ imaging experiments. M3 was suppressed on treatment with the MRTF-SRF inhibitor CCG-1423 using SMCs transduced with either MRTF-A or MRTF-B and using intact mouse esophagus in culture (by 92±2%). Moreover, silencing of SRF with a short hairpin reduced CHRM3 (by >60%) in parallel with α-actin (ACTA2). Tamoxifen inducible knockout of Srf in smooth muscle reduced Srf (by 54±4%) and Chrm3 (by 41±6%) in the urinary bladder at 10days, but Srf was much less reduced or unchanged in aorta, ileum, colon, trachea, and esophagus. Longer induction (21d) further accentuated the reduction of Chrm3 in the bladder and ileum, but no change was seen in the aorta. Single cell RNA-sequencing revealed that Mrtfb dominates in ECs, while Myocd dominates in SMCs, raising the possibility that Chrm3 may be driven by Mrtfb-Srf in the endothelium and by Myocd-Srf in SMCs. These findings define a novel transcriptional control mechanism for muscarinic M3 receptors in human cells, and in mice, that could be targeted for therapy.

NG2/CSPG4, CD146/MCAM and VAP1/AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells.

The present work addressed the hypothesis that NG2/CSPG4, CD146/MCAM, and VAP1/AOC3 are target genes of myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MKL1, MRTF-B/MKL2) and serum response factor (SRF). Using a bioinformatics approach, we found that CSPG4, MCAM, and AOC3 correlate with MYOCD, MRTF-A/MKL1, and SRF across human tissues. No other transcription factor correlated as strongly with these transcripts as SRF. Overexpression of MRTFs increased both mRNA and protein levels of CSPG4, MCAM, and AOC3 in cultured human smooth muscle cells (SMCs). Imaging confirmed increased staining for CSPG4, MCAM, and AOC3 in MRTF-A/MKL1-transduced cells. MRTFs exert their effects through SRF, and the MCAM and AOC3 gene loci contained binding sites for SRF. SRF silencing reduced the transcript levels of these genes, and time-courses of induction paralleled the direct target ACTA2. MRTF-A/MKL1 increased the activity of promoter reporters for MCAM and AOC3, and transcriptional activation further depended on the chromatin remodeling enzyme KDM3A. CSPG4, MCAM, and AOC3 responded to the MRTF-SRF inhibitor CCG-1423, to actin dynamics, and to ternary complex factors. Coincidental detection of these proteins should reflect MRTF-SRF activity, and beyond SMCs, we observed co-expression of CD146/MCAM, NG2/CSPG4, and VAP1/AOC3 in pericytes and endothelial cells in the human brain. This work identifies highly responsive vascular target genes of MRTF-SRF signaling that are regulated via a mechanism involving KDM3A.

Inducible Deletion of YAP and TAZ in Adult Mouse Smooth Muscle Causes Rapid and Lethal Colonic Pseudo-Obstruction.

BACKGROUND & AIMS: YAP (Yap1) and TAZ (Wwtr1) are transcriptional co-activators and downstream effectors of the Hippo pathway, which play crucial roles in organ size control and cancer pathogenesis. Genetic deletion of YAP/TAZ has shown their critical importance for embryonic development of the heart, vasculature, and gastrointestinal mesenchyme. The aim of this study was to determine the functional role of YAP/TAZ in adult smooth muscle cells in vivo. METHODS: Because YAP and TAZ are mutually redundant, we used YAP/TAZ double-floxed mice crossed with mice that express tamoxifen-inducible CreERT2 recombinase driven by the smooth muscle-specific myosin heavy chain promoter. RESULTS: Double-knockout of YAP/TAZ in adult smooth muscle causes lethality within 2 weeks, mainly owing to colonic pseudo-obstruction, characterized by severe distension and fecal impaction. RNA sequencing in colon and urinary bladder showed that smooth muscle markers and muscarinic receptors were down-regulated in the YAP/TAZ knockout. The same transcripts also correlated with YAP/TAZ in the human colon. Myograph experiments showed reduced contractility to depolarization by potassium chloride and a nearly abolished muscarinic contraction and spontaneous activity in colon rings of YAP/TAZ knockout. CONCLUSIONS: YAP and TAZ in smooth muscle are guardians of colonic contractility and control expression of contractile proteins and muscarinic receptors. The knockout model has features of human chronic intestinal pseudo-obstruction and may be useful for studying this disease.