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.

Voltage-dependent conformational changes of Kv1.3 channels activate cell proliferation.

The voltage-dependent potassium channel Kv1.3 has been implicated in proliferation in many cell types, based on the observation that Kv1.3 blockers inhibited proliferation. By modulating membrane potential, cell volume, and/or Ca2+ influx, K+  channels can influence cell cycle progression. Also, noncanonical channel functions could contribute to modulate cell proliferation independent of K+ efflux. The specificity of the requirement of Kv1.3 channels for proliferation suggests the involvement of molecule-specific interactions, but the underlying mechanisms are poorly identified. Heterologous expression of Kv1.3 channels in HEK cells has been shown to increase proliferation independently of K+ fluxes. Likewise, some of the molecular determinants of Kv1.3-induced proliferation have been located in the C-terminus region, where individual point mutations of putative phosphorylation sites (Y447A and S459A) abolished Kv1.3-induced proliferation. Here, we investigated the mechanisms linking Kv1.3 channels to proliferation exploring the correlation between Kv1.3 voltage-dependent molecular dynamics and cell cycle progression. Using transfected HEK cells, we analyzed both the effect of changes in resting membrane potential on Kv1.3-induced proliferation and the effect of mutated Kv1.3 channels with altered voltage dependence of gating. We conclude that voltage-dependent transitions of Kv1.3 channels enable the activation of proliferative pathways. We also found that Kv1.3 associated with IQGAP3, a scaffold protein involved in proliferation, and that membrane depolarization facilitates their interaction. The functional contribution of Kv1.3-IQGAP3 interplay to cell proliferation was demonstrated both in HEK cells and in vascular smooth muscle cells. Our data indicate that voltage-dependent conformational changes of Kv1.3 are an essential element in Kv1.3-induced proliferation.

Myocardin-Dependent Kv1.5 Channel Expression Prevents Phenotypic Modulation of Human Vessels in Organ Culture.

OBJECTIVE: We have previously described that changes in the expression of Kv channels associate to phenotypic modulation (PM), so that Kv1.3/Kv1.5 ratio is a landmark of vascular smooth muscle cells phenotype. Moreover, we demonstrated that the Kv1.3 functional expression is relevant for PM in several types of vascular lesions. Here, we explore the efficacy of Kv1.3 inhibition for the prevention of remodeling in human vessels, and the mechanisms linking the switch in Kv1.3 /Kv1.5 ratio to PM. Approach and Results: Vascular remodeling was explored using organ culture and primary cultures of vascular smooth muscle cells obtained from human vessels. We studied the effects of Kv1.3 inhibition on serum-induced remodeling, as well as the impact of viral vector-mediated overexpression of Kv channels or myocardin knock-down. Kv1.3 blockade prevented remodeling by inhibiting proliferation, migration, and extracellular matrix secretion. PM activated Kv1.3 via downregulation of Kv1.5. Hence, both Kv1.3 blockers and Kv1.5 overexpression inhibited remodeling in a nonadditive fashion. Finally, myocardin knock-down induced vessel remodeling and Kv1.5 downregulation and myocardin overexpression increased Kv1.5, while Kv1.5 overexpression inhibited PM without changing myocardin expression. CONCLUSIONS: We demonstrate that Kv1.5 channel gene is a myocardin-regulated, vascular smooth muscle cells contractile marker. Kv1.5 downregulation upon PM leaves Kv1.3 as the dominant Kv1 channel expressed in dedifferentiated cells. We demonstrated that the inhibition of Kv1.3 channel function with selective blockers or by preventing Kv1.5 downregulation can represent an effective, novel strategy for the prevention of intimal hyperplasia and restenosis of the human vessels used for coronary angioplasty procedures.

Myocardin related transcription factor and galectin-3 drive lipid accumulation in human blood vessels.

OBJECTIVE: Diabetes and hypertension are important risk factors for vascular disease, including atherosclerosis. A driving factor in this process is lipid accumulation in smooth muscle cells of the vascular wall. The glucose- and mechano-sensitive transcriptional coactivator, myocardin-related transcription factor A (MRTF-A/MKL1) can promote lipid accumulation in cultured human smooth muscle cells and contribute to the formation of smooth muscle-derived foam cells. The purpose of this study was to determine if intact human blood vessels ex vivo can be used to evaluate lipid accumulation in the vascular wall, and if this process is dependent on MRTF and/or galectin-3/LGALS3. Galectin-3 is an early marker of smooth muscle transdifferentiation and a potential mediator for foam cell formation and atherosclerosis. APPROACH AND RESULTS: Human mammary arteries and saphenous veins were exposed to altered cholesterol and glucose levels in an organ culture model. Accumulation of lipids, quantified by Oil Red O, was increased by cholesterol loading and elevated glucose concentrations. Pharmacological inhibition of MRTF with CCG-203971 decreased lipid accumulation, whereas adenoviral-mediated overexpression of MRTF-A had the opposite effect. Cholesterol-induced expression of galectin-3 was decreased after inhibition of MRTF. Importantly, pharmacological inhibition of galectin-3 with GB1107 reduced lipid accumulation in the vascular wall after cholesterol loading. CONCLUSION: Ex vivo organ culture of human arteries and veins can be used to evaluate lipid accumulation in the intact vascular wall, as well as adenoviral transduction and pharmacological inhibition. Although MRTF and galectin-3 may have beneficial, anti-inflammatory effects under certain circumstances, our results, which demonstrate a significant decrease in lipid accumulation, support further evaluation of MRTF- and galectin-3-inhibitors for therapeutic intervention against atherosclerotic vascular 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.

Role of smooth muscle YAP and TAZ in protection against phenotypic modulation, inflammation, and aneurysm development.

A ruptured arterial aneurysm, especially in the aorta, represents one of the most acute and mortal conditions encountered in clinical medicine. Population-based screening in elderly men, treatment of risk factors, such as hypertension, and endovascular or open repair of rupture-prone lesions, represent cornerstones in management. Surgical repair has a sizeable effect on life-expectancy, but medical therapy that retards aneurysm growth still represents a considerable and unmet clinical need. In the current review we survey recent findings implicating the mechano-responsive transcriptional co-activators YAP and TAZ in protection from aneurysm development. Arteries from mouse mutants that lack YAP and TAZ in vascular smooth muscle respond inadequately to mechanical stimulation, and they develop aneurysms characterized by elastin fragmentation, proteoglycan infiltration, and severe inflammation at breathtaking speed. This seems to be due, at least in part, to unscheduled activation of STING (stimulator of interferon genes), an arm of innate immunity that responds to double-stranded DNA in the cytoplasm. YAP and TAZ protect from STING activation by securing nuclear integrity. These novel insights suggest unanticipated medical therapies for sporadic and genetic aneurysms alike, involving inhibition of kinases in the Hippo pathway using small molecules, or inhibition of STING signaling itself. Translation of these novel findings into clinical therapies now represents an important priority.

miR-126 contributes to the epigenetic signature of diabetic vascular smooth muscle and enhances antirestenosis effects of Kv1.3 blockers.

OBJECTIVES: Restenosis after vessel angioplasty due to dedifferentiation of the vascular smooth muscle cells (VSMCs) limits the success of surgical treatment of vascular occlusions. Type 2 diabetes (T2DM) has a major impact on restenosis, with patients exhibiting more aggressive forms of vascular disease and poorer outcomes after surgery. Kv1.3 channels are critical players in VSMC proliferation. Kv1.3 blockers inhibit VSMCs MEK/ERK signalling and prevent vessel restenosis. We hypothesize that dysregulation of microRNAs (miR) play critical roles in adverse remodelling, contributing to Kv1.3 blockers efficacy in T2DM VSMCs. METHODS AND RESULTS: We used clinically relevant in vivo models of vascular risk factors (VRF) and vessels and VSMCs from T2DM patients. RESUKTS: Human T2DM vessels showed increased remodelling, and changes persisted in culture, with augmented VSMCs migration and proliferation. Moreover, there were downregulation of PI3K/AKT/mTOR and upregulation of MEK/ERK pathways, with increased miR-126 expression. The inhibitory effects of Kv1.3 blockers on remodelling were significantly enhanced in T2DM VSMCs and in VRF model. Finally, miR-126 overexpression confered "diabetic" phenotype to non-T2DM VSMCs by downregulating PI3K/AKT axis. CONCLUSIONS: miR-126 plays crucial roles in T2DM VSMC metabolic memory through activation of MEK/ERK pathway, enhancing the efficacy of Kv1.3 blockers in the prevention of restenosis in T2DM patients.

Elastin-like recombinamer-based devices releasing Kv1.3 blockers for the prevention of intimal hyperplasia: An in vitro and in vivo study.

Coronary artery disease (CAD) is the most common cardiovascular disorder. Vascular surgery strategies for coronary revascularization (either percutaneous or open) show a high rate of failure because of restenosis of the vessel, due to phenotypic switch of vascular smooth muscle cells (VSMCs) leading to proliferation and migration. We have previously reported that the inhibition of Kv1.3 channel function with selective blockers represents an effective strategy for the prevention of restenosis in human vessels used for coronary angioplasty procedures. However, delivery systems for controlled release of these drugs have not been investigated. Here we tested the efficacy of several formulations of elastin like recombinamers (ELRs) hydrogels to deliver the Kv1.3 blocker PAP-1 in various restenosis models. The dose and time course of PAP-1 release from ELRs click hydrogels was able to inhibit human VSMC proliferation in vitro as well as remodeling of human vessels in organ culture and restenosis in in vivo models. We conclude that this combination of active compound and advanced delivery method could improve the outcomes of vascular surgery in patients. STATEMENT OF SIGNIFICANCE: Vascular surgery strategies for coronary revascularization show a high rate of failure, because of occlusion (restenosis) of the vessel, due to vascular smooth muscle cells proliferation and migration. We have previously reported that blockers of Kv1.3 channels represent an effective anti-restenosis therapy, but delivery systems for their controlled release have not being explored. Here we tested the efficacy of several formulations of elastin like recombinamers (ELRs) hydrogels to deliver the Kv1.3 blocker PAP-1 in various restenosis models, both in vivo and in vitro, and also in human vessels. We demonstrated that combination of active compound and advanced delivery method could improve the outcomes of vascular surgery in patients.