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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., Graphical abstract

Caveolae are 50-100nm large invaginations in the cell membrane that are considered to play roles in receptor signaling. Here we aimed to investigate the expression and distribution of the arginine-vasopressin (AVP) V1a receptor and its functional dependence on caveolin-1 (Cav1) in the mouse urethra. Female Cav1 knockout (KO) and wild type (WT) mice were used, and urethral preparations were micro-dissected for mechanical experiments. Methyl-β-cyclodextrin (mβcd) was used to deplete cholesterol and to disrupt caveolae. Protein expression and localization was determined using immunofluorescence and western blotting and transcript expression was determined by qRT-PCR. We found that Cav1 and AVP V1a receptors were expressed in urethral smooth muscle cells with apparent co-localization at the cell membrane. AVP caused urethral contraction that was inhibited by the V1a receptor antagonist SR49059. Concentration-response curves for AVP were right-shifted and maximal contraction was reduced in Cav1 KO mice and after mβcd treatment. In addition to caveolin-1 we also detected caveolin-2, cavin-1 and cavin-3 in the mouse urethra by western blotting. Caveolin-2, cavin-1 and cavin-3 as well as V1a receptor expression was reduced in KO urethra. We conclude that AVP regulates urethral contractility via the V1a receptor through a Cav1-dependent mechanism involving, in part, altered V1a receptor expression.

Purpose: Restenosis is a complex and heterogeneous pathophysiological phenomenon occurring in patients submitted to revascularization procedures. Previous studies proved the antirestenotic properties of injected allogenic mesenchymal stromal cells (MSCs) in an experimental model of rat carotid (re)stenosis induced through arteriotomy. In this study we describe some of the effects subsequent to MSC treatment of rats submitted to carotid arteriotomy and possibly responsible for their antirestenotic effect. Methods: Rat MSCs were isolated from bone marrow, expanded in vitro and characterized. Subsequently, we evaluated the effects of MSC administration via tail vein at 3 and 7 days after carotid arteriotomy both in rat serum and in injured carotids, focusing on DNA oxidative damage (8-oxo-dG detection), cell proliferation index (BrdU incorporation assay), apoptotic index (TUNEL assay), the expression of inflammation- and proliferation-related genes (RT-PCR), the release of growth factors and of inflammation-related cytokines (antibody arrays and ELISA). Results: MSC administration induced a greater cell proliferation in carotids after arteriotomy, together with an increased level of VEGF in the serum and with the higher expression of VEGF mRNA in injured carotids. Serum analysis also revealed a decreased level of the pro-inflammatory cytokines CXCL1, CXCL5, L-Selectin, ICAM-1 and LIX, and of TIMP1 and SDF-1alpha in MSC-treated rats. The MSC immunomodulatory activity was confirmed by the decreased expression of TLR2 and TLR4 in injured carotids. Conclusions: MSCs play an immunomodulatory paracrine role when injected in rats submitted to carotid arteriotomy, accompanied by the release of VEGF, possibly contributing to the accelerated repair of the injured vascular wall.

We examined the spatial expression of the relaxin-3 gene in the developing zebrafish brain, one of the vertebrate model systems in which this gene has been identified. Until the pharyngula stage, the gene is expressed diffusely in the brain, where, starting at about 40 hpf, the transcripts appear restricted in a midbrain cell cluster of the periaqueductal gray. Later, at 72 hpf, the transcripts are still evident in that cluster and distributed in a larger cell number; at this stage, the gene is also expressed posteriorly, in a smaller cell group that, as we report for the first time, could be homologous to mammalian nucleus incertus. The gene expression persists in both cell clusters at 96 hpf. This pattern indicates both conserved and divergent expression features of the relaxin-3 gene among developing zebrafish and rat brains, where only scattered cells express the gene in the periaqueductal gray. Developmental Dynamics 237:3864–3869, 2008. © 2008 Wiley-Liss, Inc.

Much evidence highlights the importance of polyamines for VSMC (vascular smooth muscle cell) proliferation and migration. Cav-1 (caveolin-1) was recently reported to regulate polyamine uptake in intestinal epithelial cells. The aim of the present study was to assess the importance of Cav-1 for VSMC polyamine uptake and its impact on cell proliferation and migration. Cav-1 KO (knockout) mouse aortic cells showed increased polyamine uptake and elevated proliferation and migration compared with WT (wild-type) cells. Both Cav-1 KO and WT cells expressed the smooth muscle differentiation markers SM22 and calponin. Cell-cycle phase distribution analysis revealed a higher proportion of Cav-1 KO than WT cells in the S phase. Cav-1 KO cells were hyper-proliferative in the presence but not in the absence of extracellular polyamines, and, moreover, supplementation with exogenous polyamines promoted proliferation in Cav-1 KO but not in WT cells. Expression of the solute carrier transporters Slc7a1 and Slc43a1 was higher in Cav-1 KO than in WT cells. ODC (ornithine decarboxylase) protein and mRNA expression as well as ODC activity were similar in Cav-1 KO and WT cells showing unaltered synthesis of polyamines in Cav-1 KO cells. Cav-1 was reduced in migrating cells in vitro and in carotid lesions in vivo. Our data show that Cav-1 negatively regulates VSMC polyamine uptake and that the proliferative advantage of Cav-1 KO cells is critically dependent on polyamine uptake. We provide proof-of-principle for targeting Cav-1-regulated polyamine uptake as a strategy to fight unwanted VSMC proliferation as observed in restenosis., We demonstrate that caveolin-1 negatively regulates vascular smooth muscle cell polyamine uptake and show that caveolin-1-regulated polyamine uptake is critical for proliferative advantage of caveolin-1 deficient cells, providing proof-of-principle for targeting this mechanism as a strategy to fight unwanted proliferation.

Objectives: Polyamines are organic polycations playing an essential role in cell proliferation and differentiation, as well as in cell contractility, migration and apoptosis. These processes are known to contribute to restenosis, a pathophysiological process often occurring in patients submitted to revascularization procedures. We aimed to test the effect of alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, on vascular cell pathophysiology in vitro and in a rat model of carotid arteriotomy-induced (re) stenosis. Methods: The effect of DFMO on primary rat smooth muscle cells (SMCs) and mouse microvascular bEnd. 3 endothelial cells (ECs) was evaluated through the analysis of DNA synthesis, polyamine concentration, cell viability, cell cycle phase distribution and by RT-PCR targeting cyclins and genes belonging to the polyamine pathway. The effect of DFMO was then evaluated in arteriotomy-injured rat carotids through the analysis of cell proliferation and apoptosis, RT-PCR and immunohistochemical analysis of differential gene expression. Results: DFMO showed a differential effect on SMCs and on ECs, with a marked, sustained anti-proliferative effect of DFMO at 3 and 8 days of treatment on SMCs and a less pronounced, late effect on bEnd. 3 ECs at 8 days of DFMO treatment. DFMO applied perivascularly in pluronic gel at arteriotomy site reduced subsequent cell proliferation and preserved smooth muscle differentiation without affecting the endothelial coverage. Lumen area in DFMO-treated carotids was 49% greater than in control arteries 4 weeks after injury. Conclusions: Our data support the key role of polyamines in restenosis and suggest a novel therapeutic approach for this pathophysiological process. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

Previous studies on BAV (bicuspid aortic valve)-related aortopathy, whose aetiology is still debated, have focused mainly on severe dilatations. In the present study, we aimed to detect earlier signs of aortopathy. Specimens were collected from the ‘concavity’ (lesser curvature) and the ‘convexity’ (greater curvature) of mildly dilated AAs (ascending aortas; diameter ≤4 cm) with stenotic TAV (tricuspid aortic valve) or BAV and from donor normal aortas. Specimens were submitted to morphometry, immunohistochemistry and differential gene-expression analysis, focusing on SMC (smooth muscle cell) phenotype, remodelling, MF (myofibroblast) differentiation and TGFβ (transforming growth factor β) pathway. Smoothelin and myocardin mRNAs decreased in all the samples from patients, with the exception of those from BAV convexity, where a change in orientation of smoothelin-positive SMCs and an increase of α-SMA (α-smooth muscle actin) mRNA occurred. Dilated aortas from BAV and TAV patients showed both shared and distinct alterations concerning the TGFβ pathway, including an increased TGFβ and TGFβR2 (TGFβ receptor 2) expression in both groups and a decreased TGFβR1 expression in BAV samples only. Despite a decrease of the mRNA coding for the ED-A (extra domain-A) isoform of FN (fibronectin) in the BAV convexity, the onset of the expression of the corresponding protein in the media was observed in dilated aortas, whereas the normal media from donors was negative for this isoform. This discrepancy could be related to modifications in the intima, normally expressing ED-A FN and showing an altered structure in mild aortic dilatations in comparison with donor aorta. Our results suggest that changes in SMC phenotype and, likely, MF differentiation, occur early in the aortopathy associated with valve stenosis. The defective expression of TGFβR1 in BAV might be a constitutive feature, while other changes we reported could be influenced by haemodynamics.

Polyamines are organic polycations expressed by all living organisms, which are known to play an essential role in cell proliferation and differentiation. Recent studies revealed their involvement also in cell contractility and migration and in programmed cell death. These processes are known to contribute to restenosis, a pathophysiological process occurring in 10-20% of patients submitted to revascularization procedures. The advent of bare metal stents and of drug-eluting stents has significantly reduced but not eliminated the incidence of restenosis, which thus remains a clinically relevant problem. Despite the potential role of the polyamine pathway as a therapeutic target due to its involvement in proliferation, apoptosis and migration of vascular cells, experimental inhibition of polyamine synthesis and/or uptake has been poorly investigated in animal models of vascular disease. Here we review the current knowledge about molecular mechanisms related to polyamine functions, with particular reference to the role played by polyamines in vascular cell pathophysiology, together with experimental evidence obtained so far in animal models of (re) stenosis. We also evaluate the advantages of different routes of administration of polyamine synthesis/transport inhibitors and polyamine analogue molecules. Increasing knowledge about the molecular mechanisms and functions of polyamines is expected to shed new light on their potential role as a therapeutic target for restenosis reduction.

Restenosis rates following vascular interventions still limit their long-term success. Oxidative stress plays a relevant role in this pathophysiological phenomenon, but less attention has been devoted to its effects on DNA damage and to the subsequent mechanisms of repair. In the present study, we analysed in a model of arteriotomy-induced stenosis in rat carotid arteries the time-dependent expression of DNA damage markers and of DNA repair genes, together with the assessment of proliferation and apoptosis indexes. The expression of the oxidative DNA damage marker 7,8-dihydro-8-oxo-2′-deoxyguanosine was increased at 3 and 7 days after arteriotomy, with immunostaining distributed in the injured vascular wall and perivascular tissue. Expression of the DNA damage marker phospho-H2A.X was less relevant, but increased from 4 h to 7 days after arteriotomy, with immunostaining prevalently present in the adventitia and, to a lesser extent, in medial smooth muscle cells at the injury site. RT (reverse transcription)–PCR indicated a decrease in eight out of 12 genes involved in the DNA repair machinery we selected from 4 h to 7 days after arteriotomy, with the exception of an increase in the Mutyh and Slk genes (P