Recent work has uncovered a role of the microRNA (miRNA) miR-29 in remodeling of the extracellular matrix. Partial bladder outlet obstruction is a prevalent condition in older men with prostate enlargement that leads to matrix synthesis in the lower urinary tract and increases bladder stiffness. Here we tested the hypothesis that miR-29 is repressed in the bladder in outlet obstruction and that this has an impact on protein synthesis and matrix remodeling leading to increased bladder stiffness. c-Myc, NF-κB and SMAD3, all of which repress miR-29, were activated in the rat detrusor following partial bladder outlet obstruction but at different times. c-Myc and NF-κB activation occurred early after obstruction, and SMAD3 phosphorylation increased later, with a significant elevation at 6 weeks. c-Myc, NF-κB and SMAD3 activation, respectively, correlated with repression of miR-29b and miR-29c at 10 days of obstruction and with repression of miR-29c at 6 weeks. An mRNA microarray analysis showed that the reduction of miR-29 following outlet obstruction was associated with increased levels of miR-29 target mRNAs, including mRNAs for tropoelastin, the matricellular protein Sparc and collagen IV. Outlet obstruction increased protein levels of eight out of eight examined miR-29 targets, including tropoelastin and Sparc. Transfection of human bladder smooth muscle cells with antimiR-29c and miR-29c mimic caused reciprocal changes in target protein levels in vitro. Tamoxifen inducible and smooth muscle-specific deletion of Dicer in mice reduced miR-29 expression and increased tropoelastin and the thickness of the basal lamina surrounding smooth muscle cells in the bladder. It also increased detrusor stiffness independent of outlet obstruction. Taken together, our study supports a model where the combined repressive influences of c-Myc, NF-κB and SMAD3 reduce miR-29 in bladder outlet obstruction, and where the resulting drop in miR-29 contributes to matrix remodeling and altered passive mechanical properties of the detrusor.

Caveolae are membrane organelles that play roles in glucose and lipid metabolism and in vascular function. Formation of caveolae requires caveolins and cavins. The make-up of caveolae and their density is considered to reflect cell-specific transcriptional control mechanisms for caveolins and cavins, but knowledge regarding regulation of caveolae genes is incomplete. Myocardin (MYOCD) and its relative MRTF-A (MKL1) are transcriptional coactivators that control genes which promote smooth muscle differentiation. MRTF-A communicates changes in actin polymerization to nuclear gene transcription. Here we tested if myocardin family proteins control biogenesis of caveolae via activation of caveolin and cavin transcription. Using human coronary artery smooth muscle cells we found that jasplakinolide and latrunculin B (LatB), substances that promote and inhibit actin polymerization, increased and decreased protein levels of caveolins and cavins, respectively. The effect of LatB was associated with reduced mRNA levels for these genes and this was replicated by the MRTF inhibitor CCG-1423 which was non-additive with LatB. Overexpression of myocardin and MRTF-A caused 5-10-fold induction of caveolins whereas cavin-1 and cavin-2 were induced 2-3-fold. PACSIN2 also increased, establishing positive regulation of caveolae genes from three families. Full regulation of CAV1 was retained in its proximal promoter. Knock down of the serum response factor (SRF), which mediates many of the effects of myocardin, decreased cavin-1 but increased caveolin-1 and -2 mRNAs. Viral transduction of myocardin increased the density of caveolae 5-fold in vitro. A decrease of CAV1 was observed concomitant with a decrease of the smooth muscle marker calponin in aortic aneurysms from mice (C57Bl/6) infused with angiotensin II. Human expression data disclosed correlations of MYOCD with CAV1 in a majority of human tissues and in the heart, correlation with MKL2 (MRTF-B) was observed. The myocardin family of transcriptional coactivators therefore drives formation of caveolae and this effect is largely independent of SRF.

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.Diabetes confers a 2-4-fold excess risk for a wide range of cardiovascular diseases, including macrovascular complications leading to coronary heart disease and ischemic stroke, as well as microvascular diseases, such as nephropathy and retinopathy (1, 2). Based on current trends, the rising incidence of diabetes (expected to reach 333 million people worldwide by 2025) will undoubtedly equate to increased cardiovascular mortality. Chronic hyperglycemia has long been recognized as an independent risk factor for cardiovascular disease (3, 4). Importantly, the progressive relationship between glucose levels and cardiovascular risk extends below the threshold for diabetes diagnosis (fasting plasma glucose Ն7.0 mmol/liter or 2-h plasma glucose Ն11.1 mmol/liter (2, 3)), and more recently, even transient hyper-and hypoglycemia have emerged as important determinants of cardiovascular disease (5). Despite the vast clinical and epidemiological experience linking blood glucose and poor glucose control to the development and progression of cardiovascular disease, the underlying molecular mechanisms leading to vascular dysfunction and disease are poorly understood (6).It has been well established that hyperglycemia results in vascular hyperreactivity in diabetic patients (7) and animal models (8 -10). Part of this effect may be attributed to a decrease in nitric oxide (NO) bioavailability as well as a reduced respon...

Developmental changes in the regulation of smooth muscle contraction were examined in urinary bladder smooth muscle from mice. Maximal active stress was lower in newborn tissue compared with adult, and it was correlated with a lower content of actin and myosin. Sensitivity to extracellular Ca2+ during high-K+ contraction, was higher in newborn compared with 3-wk-old and adult bladder strips. Concentrations at half maximal tension (EC50) were 0.57 ± 0.01, 1.14 ± 0.12, and 1.31 ± 0.08 mM. Force of the newborn tissue was inhibited by ∼45% by the nonmuscle myosin inhibitor Blebbistatin, whereas adult tissue was not affected. The calcium sensitivity in newborn tissue was not affected by Blebbistatin, suggesting that nonmuscle myosin is not a primary cause for increased calcium sensitivity. The relation between intracellular [Ca2+] and force was shifted toward lower [Ca2+] in the newborn bladders. This increased Ca2+ sensitivity was also found in permeabilized muscles (EC50: 6.10 ± 0.07, 5.77 ± 0.08, and 5.55 ± 0.02 pCa units, in newborn, 3-wk-old, and adult tissues). It was associated with an increased myosin light chain phosphorylation and a decreased rate of dephosphorylation. No difference was observed in the myosin light chain phosphorylation rate, whereas the rate of myosin light chain phosphatase–induced relaxation was about twofold slower in the newborn tissue. The decreased rate was associated with a lower expression of the phosphatase regulatory subunit MYPT-1 in newborn tissue. The results show that myosin light chain phosphatase activity can be developmentally regulated in mammalian urinary bladders. The resultant alterations in Ca2+ sensitivity may be of importance for the nervous and myogenic control of the newborn bladders.

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

BACKGROUND AND PURPOSECaveolin-1-deficiency is associated with substantial urogenital alterations. Here, a mechanical, histological and biochemical characterization of female detrusors from wild-type and caveolin-1-deficient (KO) mice was made to increase the understanding of detrusor changes caused by lack of caveolae. EXPERIMENTAL APPROACHLength-tension relationships were generated, and we recorded responses to electrical field stimulation, the muscarinic receptor agonist carbachol and the purinoceptor agonist ATP. Tyrosine nitration and the contents of caveolin-1, cavin-1, muscarinic M3 receptors, phospholipase Cb1, muscle-specific kinase (MuSK) and L-type Ca 2+ channels were determined by immunoblotting. Innervation was assessed by immunohistochemistry. KEY RESULTSBladder to body weight ratio was not changed, nor was there any change in the optimum circumference for force development. Depolarization-and ATP-induced stress was reduced, as was carbachol-induced stress between 0.1 and 3 mM, but the supramaximal relative (% K + ) response to carbachol was increased, as was M3 expression. The scopolamine-sensitive component of the electrical field stimulation response was impaired, and yet bladder nerves contained little caveolin-1. The density of cholinergic nerves was unchanged, whereas CART-and CGRP-positive nerves were reduced. Immunoblotting revealed loss of MuSK. CONCLUSIONS AND IMPLICATIONSAblation of caveolae in the female detrusor leads to generalized impairment of contractility, ruling out prostate hypertrophy as a contributing factor. Cholinergic neuroeffector transmission is impaired without conspicuous changes in the density of cholinergic nerves or morphology of their terminals, but correlating with reduced expression of MuSK. AbbreviationsCART, cocaine-and amphetamine-regulated transcript; Cav-1 or Cav1, caveolin-1; CaV1.2, pore-forming subunit of L-type Ca 2+ channel; CGRP, calcitonin gene-related peptide; EFS, electrical field stimulation; HK, K + -high solution (60 mM); HTX, Mayer's haematoxylin; KO, caveolin-1 knockout; L0, optimum circumference for force development; M3, type 3 muscarinic acetylcholine receptor; MuSK, muscle-specific kinase; NPK, neuropeptide K; PLCb1, phospholipase Cb1; VAChT, vesicular acetylcholine transporter; WT, wild type

Diet-induced weight loss leads to mainly reduced levels of metabolites that are elevated in obese insulin resistant individuals. We identified multiple new associations with metabolic risk factors and validated several previous findings related to weight loss-mediated metabolite changes. Levels of specific metabolites, such as xylitol, may be predictive of the response to non-surgical weight loss already at baseline.

MicroRNAs have emerged as important regulators of smooth muscle phenotype and may play important roles in pathogenesis of various smooth muscle related disease states. The aim of this study was to investigate the role of miRNAs for urinary bladder function. We used an inducible and smooth muscle specific Dicer knockout (KO) mouse which resulted in significantly reduced levels of miRNAs, including miR-145, miR-143, miR-22, miR125b-5p and miR-27a, from detrusor preparations without mucosa. Deletion of Dicer resulted in a disturbed micturition pattern in vivo and reduced depolarization-induced pressure development in the isolated detrusor. Furthermore, electrical field stimulation revealed a decreased cholinergic but maintained purinergic component of neurogenic activation in Dicer KO bladder strips. The ultrastructure of detrusor smooth muscle cells was well maintained, and the density of nerve terminals was similar. Western blotting demonstrated reduced contents of calponin and desmin. Smooth muscle α-actin, SM22α and myocardin were unchanged. Activation of strips with exogenous agonists showed that depolarization-induced contraction was preferentially reduced; ATP- and calyculin A-induced contractions were unchanged. Quantitative real time PCR and western blotting demonstrated reduced expression of Cav1.2 (Cacna1c). It is concluded that smooth muscle miRNAs play an important role for detrusor contractility and voiding pattern of unrestrained mice. This is mediated in part via effects on expression of smooth muscle differentiation markers and L-type Ca2+ channels in the detrusor.