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.

Emerging role of YAP/TAZ in vascular mechanotransduction and disease.

Cells have an incredible ability to physically interact with neighboring cells and their environment. They can detect and respond to mechanical forces by converting mechanical stimuli into biochemical signals in a process known as mechanotransduction. This is a key process for the adaption of vascular smooth muscle and endothelial cells to altered flow and pressure conditions. Mechanical stimuli, referring to a physical force exerted on cells, are primarily sensed by transmembrane proteins and the actin cytoskeleton, which initiate a cascade of intracellular events, including the activation of signaling pathways, ion channels, and transcriptional regulators. Recent work has highlighted an important role of the transcriptional coactivators YAP/TAZ for mechanotransduction in vascular cells. Interestingly, the activity of YAP/TAZ decreases with age, providing a potential mechanism for the detrimental effects of aging in the vascular wall. In this review, we summarize the current knowledge on the functional role of YAP and TAZ in vascular endothelial and smooth muscle cells for mechanotransduction in homeostasis and disease. In particular, the review is focused on in vivo observations from conditional knockout (KO) models of YAP/TAZ and the potential implications these studies may have for our understanding of vascular disease development.

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.

Endothelial basement membrane laminin 511 is essential for shear stress response.

Shear detection and mechanotransduction by arterial endothelium requires junctional complexes containing PECAM-1 and VE-cadherin, as well as firm anchorage to the underlying basement membrane. While considerable information is available for junctional complexes in these processes, gained largely from in vitro studies, little is known about the contribution of the endothelial basement membrane. Using resistance artery explants, we show that the integral endothelial basement membrane component, laminin 511 (laminin α5), is central to shear detection and mechanotransduction and its elimination at this site results in ablation of dilation in response to increased shear stress. Loss of endothelial laminin 511 correlates with reduced cortical stiffness of arterial endothelium in vivo, smaller integrin ß1-positive/vinculin-positive focal adhesions, and reduced junctional association of actin-myosin II In vitro assays reveal that ß1 integrin-mediated interaction with laminin 511 results in high strengths of adhesion, which promotes p120 catenin association with VE-cadherin, stabilizing it at cell junctions and increasing cell-cell adhesion strength. This highlights the importance of endothelial laminin 511 in shear response in the physiologically relevant context of resistance arteries.

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.

Regulation of IRS-1, insulin signaling and glucose uptake by miR-143/145 in vascular smooth muscle cells.

Regulation of insulin signaling by microRNAs in smooth muscle cells may contribute to diabetic vascular disease. The two smooth muscle enriched miRNAs miR-143 and miR-145 have been reported to target mediators of insulin signaling in non-smooth muscle cells. In this study, we aimed to determine the importance of this regulation in vascular smooth muscle cells, where expression of miR-143/145 is much higher than in other cell types. Smooth muscle cells deficient of the miR-143/145 cluster were used, as well as smooth muscle cells transfected with mimics/inhibitors for either miR-143 or miR-145. We found that deletion of miR-143/145 in smooth muscle results in a dramatic upregulation IRS-1 expression and insulin signaling, and an increased insulin-induced glucose uptake. Furthermore, specific modulation of either miR-145 or miR-143 expression regulated specific targets (IRS-1, ORP8 and the IGF-1 receptor) in the insulin signaling pathway. Consequently, transient inhibition or overexpression of either miR-143 or miR-145 was sufficient to regulate insulin signaling in smooth muscle cells. In conclusion, the results of this study support an important role for both miR-143 and miR-145 in the regulation of insulin signaling and glucose uptake in vascular smooth muscle cells.

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.

Regulation of microRNA expression in vascular smooth muscle by MRTF-A and actin polymerization.

The dynamic properties of the actin cytoskeleton in smooth muscle cells play an important role in a number of cardiovascular disease states. The state of actin does not only mediate mechanical stability and contractile function but can also regulate gene expression via myocardin related transcription factors (MRTFs). These transcriptional co-activators regulate genes encoding contractile and cytoskeletal proteins in smooth muscle. Regulation of small non-coding microRNAs (miRNAs) by actin polymerization may mediate some of these effects. MiRNAs are short non-coding RNAs that modulate gene expression by post-transcriptional regulation of target messenger RNA. In this study we aimed to determine a profile of miRNAs that were 1) regulated by actin/MRTF-A, 2) associated with the contractile smooth muscle phenotype and 3) enriched in muscle cells. This analysis was performed using cardiovascular disease-focused miRNA arrays in both mouse and human cells. The potential clinical importance of actin polymerization in aortic aneurysm was evaluated using biopsies from mildly dilated human thoracic aorta in patients with stenotic tricuspid or bicuspid aortic valve. By integrating information from multiple qPCR based miRNA arrays we identified a group of five miRNAs (miR-1, miR-22, miR-143, miR-145 and miR-378a) that were sensitive to actin polymerization and MRTF-A overexpression in both mouse and human vascular smooth muscle. With the exception of miR-22, these miRNAs were also relatively enriched in striated and/or smooth muscle containing tissues. Actin polymerization was found to be dramatically reduced in the aorta from patients with mild aortic dilations. This was associated with a decrease in actin/MRTF-regulated miRNAs. In conclusion, the transcriptional co-activator MRTF-A and actin polymerization regulated a subset of miRNAs in vascular smooth muscle. Identification of novel miRNAs regulated by actin/MRTF-A may provide further insight into the mechanisms underlying vascular disease states, such as aortic aneurysm, as well as novel ideas regarding therapeutic strategies. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

MicroRNA-dependent regulation of KLF4 by glucose in vascular smooth muscle.

Diabetes is a major risk factor for cardiovascular disease and this is in part due to the effects of hyperglycemia on vascular smooth muscle cells. Small non-coding microRNAs are known to control smooth muscle phenotype and arterial contractility and are dysregulated in diabetes. The effect of microRNAs on smooth muscle differentiation is in part mediated by the transcription factor KLF4 but the role of this mechanism in diabetic vascular disease is not fully understood. Herein, we have investigated the importance of hyperglycemia and diabetes for the expression of KLF4 in vascular smooth muscle and the involvement of miRNAs in this regulation. Hyperglycemia down-regulated KLF4 in vascular smooth muscle cells and similar results were found in arteries of diabetic mice and patients. This correlated with a Foxa2-dependent up-regulation of miR-29c, which targeted KLF4 in vascular smooth muscle cells. Importantly, by preventing downregulation of KLF4, the induction of smooth muscle contractile protein markers by glucose was inhibited. In conclusion, miR-29 mediated inhibition of KLF4 in hyperglycemic conditions contributes to increased expression of contractile markers in vascular smooth muscle cells. Further studies are warranted to determine the therapeutic implications of miR-29 inhibition in diabetic vascular disease.

Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization.

Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells, but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hypercontractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, quantitative PCR, and Western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers were increased in isolated smooth muscle cells cultured under high compared with low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization, and myocardin-related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility.

Pyk2 inhibition promotes contractile differentiation in arterial smooth muscle.

Modulation from contractile to synthetic phenotype of vascular smooth muscle cells is a central process in disorders involving compromised integrity of the vascular wall. Phenotype modulation has been shown to include transition from voltage-dependent toward voltage-independent regulation of the intracellular calcium level, and inhibition of non-voltage dependent calcium influx contributes to maintenance of the contractile phenotype. One possible mediator of calcium-dependent signaling is the FAK-family non-receptor protein kinase Pyk2, which is activated by a number of stimuli in a calcium-dependent manner. We used the Pyk2 inhibitor PF-4594755 and Pyk2 siRNA to investigate the role of Pyk2 in phenotype modulation in rat carotid artery smooth muscle cells and in cultured intact arteries. Pyk2 inhibition promoted the expression of smooth muscle markers at the mRNA and protein levels under stimulation by FBS or PDGF-BB and counteracted phenotype shift in cultured intact carotid arteries and balloon injury ex vivo. During long-term (24-96 hr) treatment with PF-4594755, smooth muscle markers increased before cell proliferation was inhibited, correlating with decreased KLF4 expression and differing from effects of MEK inhibition. The Pyk2 inhibitor reduced Orai1 and preserved SERCA2a expression in carotid artery segments in organ culture, and eliminated the inhibitory effect of PDGF stimulation on L-type calcium channel and large-conductance calcium-activated potassium channel expression in carotid cells. Basal intracellular calcium level, calcium wave activity, and store-operated calcium influx were reduced after Pyk2 inhibition of growth-stimulated cells. Pyk2 inhibition may provide an interesting approach for preserving vascular smooth muscle differentiation under pathophysiological conditions.

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.

Patients with bicuspid and tricuspid aortic valve exhibit distinct regional microrna signatures in mildly dilated ascending aorta.

MicroRNAs are able to modulate gene expression in a range of diseases. We focused on microRNAs as potential contributors to the pathogenesis of ascending aorta (AA) dilatation in patients with stenotic tricuspid (TAV) or bicuspid aortic valve (BAV). Aortic specimens were collected from the 'concavity' and the 'convexity' of mildly dilated AAs and of normal AAs from heart transplant donors. Aortic RNA was analyzed through PCR arrays, profiling the expression of 84 microRNAs involved in cardiovascular disease. An in silico analysis identified the potential microRNA-mRNA interactions and the enriched KEGG pathways potentially affected by microRNA changes in dilated AAs. Distinct signatures of differentially expressed microRNAs are evident in TAV and BAV patients vs. donors, as well as differences between aortic concavity and convexity in patients only. MicroRNA changes suggest a switch of SMC phenotype, with particular reference to TAV concavity. MicroRNA changes potentially affecting mechanotransduction pathways exhibit a higher prevalence in BAV convexity and in TAV concavity, with particular reference to TGF-ß1, Hippo, and PI3K/Akt/FoxO pathways. Actin cytoskeleton emerges as potentially affected by microRNA changes in BAV convexity only. MicroRNAs could play distinct roles in BAV and TAV aortopathy, with possible implications in diagnosis and therapy.

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.

Regulation of smooth muscle dystrophin and synaptopodin 2 expression by actin polymerization and vascular injury.

OBJECTIVE: Actin dynamics in vascular smooth muscle is known to regulate contractile differentiation and may play a role in the pathogenesis of vascular disease. However, the list of genes regulated by actin polymerization in smooth muscle remains incomprehensive. Thus, the objective of this study was to identify actin-regulated genes in smooth muscle and to demonstrate the role of these genes in the regulation of vascular smooth muscle phenotype. APPROACH AND RESULTS: Mouse aortic smooth muscle cells were treated with an actin-stabilizing agent, jasplakinolide, and analyzed by microarrays. Several transcripts were upregulated including both known and previously unknown actin-regulated genes. Dystrophin and synaptopodin 2 were selected for further analysis in models of phenotypic modulation and vascular disease. These genes were highly expressed in differentiated versus synthetic smooth muscle and their expression was promoted by the transcription factors myocardin and myocardin-related transcription factor A. Furthermore, the expression of both synaptopodin 2 and dystrophin was significantly reduced in balloon-injured human arteries. Finally, using a dystrophin mutant mdx mouse and synaptopodin 2 knockdown, we demonstrate that these genes are involved in the regulation of smooth muscle differentiation and function. CONCLUSIONS: This study demonstrates novel genes that are promoted by actin polymerization, that regulate smooth muscle function, and that are deregulated in models of vascular disease. Thus, targeting actin polymerization or the genes controlled in this manner can lead to novel therapeutic options against vascular pathologies that involve phenotypic modulation of smooth muscle cells.

Reticulon 4 is necessary for endoplasmic reticulum tubulation, STIM1-Orai1 coupling, and store-operated calcium entry.

Despite recent advances in understanding store-operated calcium entry (SOCE) regulation, the fundamental question of how ER morphology affects this process remains unanswered. Here we show that the loss of RTN4, is sufficient to alter ER morphology and severely compromise SOCE. Mechanistically, we show this to be the result of defective STIM1-Orai1 coupling because of loss of ER tubulation and redistribution of STIM1 to ER sheets. As a functional consequence, RTN4-depleted cells fail to sustain elevated cytoplasmic Ca(2+) levels via SOCE and therefor are less susceptible to Ca(2+) overload induced apoptosis. Thus, for the first time, our results show a direct correlation between ER morphology and SOCE and highlight the importance of RTN4 in cellular Ca(2+) homeostasis.

Induction of angiotensin-converting enzyme after miR-143/145 deletion is critical for impaired smooth muscle contractility.

MicroRNAs have emerged as regulators of smooth muscle cell phenotype with a role in smooth muscle-related disease. Studies have shown that miR-143 and miR-145 are the most highly expressed microRNAs in smooth muscle cells, controlling differentiation and function. The effect of miR-143/145 knockout has been established in the vasculature but not in smooth muscle from other organs. Using knockout mice we found that maximal contraction induced by either depolarization or phosphatase inhibition was reduced in vascular and airway smooth muscle but maintained in the urinary bladder. Furthermore, a reduction of media thickness and reduced expression of differentiation markers was seen in the aorta but not in the bladder. Supporting the view that phenotype switching depends on a tissue-specific target of miR-143/145, we found induction of angiotensin-converting enzyme in the aorta but not in the bladder where angiotensin-converting enzyme was expressed at a low level. Chronic treatment with angiotensin type-1 receptor antagonist restored contractility in miR-143/145-deficient aorta while leaving bladder contractility unaffected. This shows that tissue-specific targets are critical for the effects of miR-143/145 on smooth muscle differentiation and that angiotensin converting enzyme is one such target.

HIF-mediated metabolic switching in bladder outlet obstruction mitigates the relaxing effect of mitochondrial inhibition.

Prior work demonstrated increased levels of hypoxia-inducible factor-1α (HIF-1α) in the bladder following outlet obstruction, associated with bladder growth and fibrosis. Here we hypothesized that HIF induction in outlet obstruction also switches energetic support of contraction from mitochondrial respiration to glycolysis. To address this hypothesis, we created infravesical outlet obstruction in female Sprague-Dawley rats and examined HIF induction and transcriptional activation. HIF-1α increased after 6 weeks of outlet obstruction as assessed by western blotting and yet transcription factor-binding site analysis indicated HIF activation already at 10 days of obstruction. Accumulation HIF-2α and of Arnt2 proteins were found at 10 days, providing an explanation for the lack of correlation between HIF-1α protein and transcriptional activation. HIF signature targets, including Slc2a1, Tpi1, Eno1 and Ldha increased in obstructed compared with sham-operated bladders. The autophagy markers Bnip3 and LC3B-II were also increased at 6 week of obstruction, but electron microscopy did not support mitophagy. Mitochondria were, however, remodeled with increased expression of Cox4 compared with other markers. In keeping with a switch toward glycolytic support of contraction, we found that relaxation by the mitochondrial inhibitor cyanide was reduced in obstructed bladders. This was mimicked by organ culture with the HIF-inducer dimethyloxalylglycine, which also upregulated expression of Ldha. On the basis of these findings, we conclude that HIF activation in outlet obstruction involves mechanisms beyond the accumulation of HIF-1α protein and that it results in a switch of the energetic support of contraction to anaerobic glycolysis. This metabolic adaptation encompasses increased expression of glucose transporters and glycolytic enzymes combined with mitochondrial remodeling. Together, these changes uphold contractility when mitochondrial respiration is limited.