Steatotic Liver Disease: Pathophysiology and Emerging Pharmacotherapies.

Steatotic liver disease (SLD) displays a dynamic and complex disease phenotype. Consequently, the metabolic dysfunction-associated steatotic liver disease (MASLD)/metabolic dysfunction-associated steatohepatitis (MASH) therapeutic pipeline is expanding rapidly and in multiple directions. In parallel, noninvasive tools for diagnosing and monitoring responses to therapeutic interventions are being studied, and clinically feasible findings are being explored as primary outcomes in interventional trials. The realization that distinct subgroups exist under the umbrella of SLD should guide more precise and personalized treatment recommendations and facilitate advancements in pharmacotherapeutics. This review summarizes recent updates of pathophysiology-based nomenclature and outlines both effective pharmacotherapeutics and those in the pipeline for MASLD/MASH, detailing their mode of action and the current status of phase 2 and 3 clinical trials. Of the extensive arsenal of pharmacotherapeutics in the MASLD/MASH pipeline, several have been rejected, whereas other, mainly monotherapy options, have shown only marginal benefits and are now being tested as part of combination therapies, yet others are still in development as monotherapies. Although the Food and Drug Administration (FDA) has recently approved resmetirom, additional therapeutic approaches in development will ideally target MASH and fibrosis while improving cardiometabolic risk factors. Due to the urgent need for the development of novel therapeutic strategies and the potential availability of safety and tolerability data, repurposing existing and approved drugs is an appealing option. Finally, it is essential to highlight that SLD and, by extension, MASLD should be recognized and approached as a systemic disease affecting multiple organs, with the vigorous implementation of interdisciplinary and coordinated action plans. SIGNIFICANCE STATEMENT: Steatotic liver disease (SLD), including metabolic dysfunction-associated steatotic liver disease and metabolic dysfunction-associated steatohepatitis, is the most prevalent chronic liver condition, affecting more than one-fourth of the global population. This review aims to provide the most recent information regarding SLD pathophysiology, diagnosis, and management according to the latest advancements in the guidelines and clinical trials. Collectively, it is hoped that the information provided furthers the understanding of the current state of SLD with direct clinical implications and stimulates research initiatives.

PAI-1 Regulates the Cytoskeleton and Intrinsic Stiffness of Vascular Smooth Muscle Cells.

BACKGROUND: Plasma concentration of PAI-1 (plasminogen activator inhibitor-1) correlates with arterial stiffness. Vascular smooth muscle cells (SMCs) express PAI-1, and the intrinsic stiffness of SMCs is a major determinant of total arterial stiffness. We hypothesized that PAI-1 promotes SMC stiffness by regulating the cytoskeleton and that pharmacological inhibition of PAI-1 decreases SMC and aortic stiffness. METHODS: PAI-039, a specific inhibitor of PAI-1, and small interfering RNA were used to inhibit PAI-1 expression in cultured human SMCs. Effects of PAI-1 inhibition on SMC stiffness, F-actin (filamentous actin) content, and cytoskeleton-modulating enzymes were assessed. WT (wild-type) and PAI-1-deficient murine SMCs were used to determine PAI-039 specificity. RNA sequencing was performed to determine the effects of PAI-039 on SMC gene expression. In vivo effects of PAI-039 were assessed by aortic pulse wave velocity. RESULTS: PAI-039 significantly reduced intrinsic stiffness of human SMCs, which was accompanied by a significant decrease in cytoplasmic F-actin content. PAI-1 gene knockdown also decreased cytoplasmic F-actin. PAI-1 inhibition significantly increased the activity of cofilin, an F-actin depolymerase, in WT murine SMCs, but not in PAI-1-deficient SMCs. RNA-sequencing analysis suggested that PAI-039 upregulates AMPK (AMP-activated protein kinase) signaling in SMCs, which was confirmed by Western blotting. Inhibition of AMPK prevented activation of cofilin by PAI-039. In mice, PAI-039 significantly decreased aortic stiffness and tunica media F-actin content without altering the elastin or collagen content. CONCLUSIONS: PAI-039 decreases intrinsic SMC stiffness and cytoplasmic stress fiber content. These effects are mediated by AMPK-dependent activation of cofilin. PAI-039 also decreases aortic stiffness in vivo. These findings suggest that PAI-1 is an important regulator of the SMC cytoskeleton and that pharmacological inhibition of PAI-1 has the potential to prevent and treat cardiovascular diseases involving arterial stiffening.

Inhibition of the histone methyltransferase EZH2 induces vascular stiffness.

Vascular stiffness increases with aging, obesity and hypertension and predicts cardiovascular risk. The levels of histone H3-lysine-27 methylation (H3K27me) and the histone methyltransferase EZH2 both decrease in aging vessels, driving vascular stiffness. The impact of EZH2 inhibitors on vascular stiffness is unknown. We tested the hypothesis that the EZH2 inhibitor GSK126, currently in development for cancer treatment, increases vascular stiffness and explored underlying molecular mechanisms. Young (3 month) and middle-aged (12 month) male mice were treated with GSK126 for 1-2 months and primary human aortic smooth muscle cells (HASMCs) from young male and female donors were treated with GSK126 for 24-48 h. Stiffness was measured in vivo by pulse wave velocity and in vitro by atomic force microscopy (AFM) and vascular structure was quantified histologically. Extracellular matrix proteins were studied by qRT-PCR, immunoblotting, zymography and chromatin immunoprecipitation. GSK126 treatment decreased H3K27 methylation (H3K27me) and increased acetylation (H3K27ac) in mouse vessels and in HASMCs. In GSK126-treated mice, aortic stiffness increased without changes in vascular fibrosis. EZH2 inhibition enhanced elastin fiber degradation and matrix metalloprotease-2 (MMP2) expression. In HASMCs, GSK126 treatment increased synthetic phenotype markers and intrinsic HASMCs stiffness by AFM with altered cytoskeletal structure and increased nuclear actin staining. GSK126 also increased MMP2 protein expression, activity and enrichment of H3K27ac at the MMP2 promoter in HASMCs. GSK126 causes vascular stiffening, inducing MMP2 activity, elastin degradation, and modulation of SMC phenotype and cytoskeletal stiffness. These findings suggest that EZH2 inhibitors used to treat cancer could negatively impact the vasculature by enhancing stiffness and merits examination in human trials.

Diabetic Vasculopathy: Molecular Mechanisms and Clinical Insights.

Clinical and basic studies have documented that both hyperglycemia and insulin-resistance/hyperinsulinemia not only constitute metabolic disorders contributing to cardiometabolic syndrome, but also predispose to diabetic vasculopathy, which refers to diabetes-mellitus-induced microvascular and macrovascular complications, including retinopathy, neuropathy, atherosclerosis, coronary artery disease, hypertension, and peripheral artery disease. The underlying molecular and cellular mechanisms include inappropriate activation of the renin angiotensin-aldosterone system, mitochondrial dysfunction, excessive oxidative stress, inflammation, dyslipidemia, and thrombosis. These abnormalities collectively promote metabolic disorders and further promote diabetic vasculopathy. Recent evidence has revealed that endothelial progenitor cell dysfunction, gut dysbiosis, and the abnormal release of extracellular vesicles and their carried microRNAs also contribute to the development and progression of diabetic vasculopathy. Therefore, clinical control and treatment of diabetes mellitus, as well as the development of novel therapeutic strategies are crucial in preventing cardiometabolic syndrome and related diabetic vasculopathy. The present review focuses on the relationship between insulin resistance and diabetes mellitus in diabetic vasculopathy and related cardiovascular disease, highlighting epidemiology and clinical characteristics, pathophysiology, and molecular mechanisms, as well as management strategies.

Endothelial cell-specific mineralocorticoid receptor activation promotes diastolic dysfunction in diet-induced obese male mice.

Widespread consumption of diets high in fat and fructose (Western diet, WD) has led to increased prevalence of obesity and diastolic dysfunction (DD). DD is a prominent feature of heart failure with preserved ejection fraction (HFpEF). However, the underlying mechanisms of DD are poorly understood, and treatment options are still limited. We have previously shown that deletion of the cell-specific mineralocorticoid receptor in endothelial cells (ECMR) abrogates DD induced by WD feeding in female mice. However, the specific role of ECMR activation in the pathogenesis of DD in male mice has not been clarified. Therefore, we fed 4-wk-old ECMR knockout (ECMRKO) male mice and littermates (LM) with either a WD or chow diet (CD) for 16 wk. WD feeding resulted in DD characterized by increased left ventricle (LV) filling pressure (E/e') and diastolic stiffness [E/e'/LV inner diameter at end diastole (LVIDd)]. Compared with CD, WD in LM resulted in increased myocardial macrophage infiltration, oxidative stress, and increased myocardial phosphorylation of Akt, in concert with decreased phospholamban phosphorylation. WD also resulted in focal cardiomyocyte remodeling, characterized by areas of sarcomeric disorganization, loss of mitochondrial electron density, and mitochondrial fragmentation. Conversely, WD-induced DD and associated biochemical and structural abnormalities were prevented by ECMR deletion. In contrast with our previously reported observations in females, WD-fed male mice exhibited enhanced Akt signaling and a lower magnitude of cardiac injury. Collectively, our data support a critical role for ECMR in obesity-induced DD and suggest critical mechanistic differences in the genesis of DD between males and females.

Obesity, Adipose Tissue and Vascular Dysfunction.

Cardiovascular diseases are the leading cause of death worldwide. Overweight and obesity are strongly associated with comorbidities such as hypertension and insulin resistance, which collectively contribute to the development of cardiovascular diseases and resultant morbidity and mortality. Forty-two percent of adults in the United States are obese, and a total of 1.9 billion adults worldwide are overweight or obese. These alarming numbers, which continue to climb, represent a major health and economic burden. Adipose tissue is a highly dynamic organ that can be classified based on the cellular composition of different depots and their distinct anatomical localization. Massive expansion and remodeling of adipose tissue during obesity differentially affects specific adipose tissue depots and significantly contributes to vascular dysfunction and cardiovascular diseases. Visceral adipose tissue accumulation results in increased immune cell infiltration and secretion of vasoconstrictor mediators, whereas expansion of subcutaneous adipose tissue is less harmful. Therefore, fat distribution more than overall body weight is a key determinant of the risk for cardiovascular diseases. Thermogenic brown and beige adipose tissue, in contrast to white adipose tissue, is associated with beneficial effects on the vasculature. The relationship between the type of adipose tissue and its influence on vascular function becomes particularly evident in the context of the heterogenous phenotype of perivascular adipose tissue that is strongly location dependent. In this review, we address the abnormal remodeling of specific adipose tissue depots during obesity and how this critically contributes to the development of hypertension, endothelial dysfunction, and vascular stiffness. We also discuss the local and systemic roles of adipose tissue derived secreted factors and increased systemic inflammation during obesity and highlight their detrimental impact on cardiovascular health.

Inhibition of sphingomyelinase attenuates diet - Induced increases in aortic stiffness.

Sphingomyelinases ensure ceramide production and play an integral role in cell turnover, inward budding of vesicles and outward release of exosomes. Recent data indicate a unique role for neutral sphingomyelinase (nSMase) in the control of ceramide-dependent exosome release and inflammatory pathways. Further, while inhibition of nSMase in vascular tissue attenuates the progression of atherosclerosis, little is known regarding its role on metabolic signaling and arterial vasomotor function. Accordingly, we hypothesized that nSMase inhibition with GW4869, would attenuate Western diet (WD) - induced increases in aortic stiffness through alterations in pathways which lead to oxidative stress, inflammation and vascular remodeling. Six week-old female C57BL/6L mice were fed either a WD containing excess fat (46%) and fructose (17.5%) for 16 weeks or a standard chow diet (CD). Mice were variably treated with GW4869 (2.0 µg/g body weight, intraperitoneal injection every 48 h for 12 weeks). WD feeding increased nSMase2 expression and activation while causing aortic stiffening and impaired vasorelaxation as determined by pulse wave velocity (PWV) and wire myography, respectively. Moreover, these functional abnormalities were associated with aortic remodeling and attenuated AMP-activated protein kinase, Sirtuin 1, and endothelial nitric oxide synthase activation. GW4869 treatment prevented the WD-induced increases in nSMase activation, PWV, and impaired endothelium dependent/independent vascular relaxation. GW4869 also inhibited WD-induced aortic CD36 expression, lipid accumulation, oxidative stress, inflammatory responses, as well as aortic remodeling. These findings indicate that targeting nSMase prevents diet - induced aortic stiffening and impaired vascular relaxation by attenuating oxidative stress, inflammation and adverse vascular remodeling.

Mineralocorticoid Receptor Activation in Vascular Insulin Resistance and Dysfunction.

Systemic insulin resistance is characterized by reduced insulin metabolic signaling and glucose intolerance. Mineralocorticoid receptors (MRs), the principal receptors for the hormone aldosterone, play an important role in regulating renal sodium handling and blood pressure. Recent studies suggest that MRs also exist in tissues outside the kidney, including vascular endothelial cells, smooth muscle cells, fibroblasts, perivascular adipose tissue, and immune cells. Risk factors, including excessive salt intake/salt sensitivity, hypertension, and obesity, can lead to the activation of vascular MRs to promote inflammation, oxidative stress, remodeling, and fibrosis, as well as cardiovascular stiffening and microcirculatory impairment. These pathophysiological changes are associated with a diminished ability of insulin to initiate appropriate intracellular signaling events, resulting in a reduced glucose uptake within the microcirculation and related vascular insulin resistance. Therefore, the pharmacological inhibition of MR activation provides a potential therapeutic option for improving vascular function, glucose uptake, and vascular insulin sensitivity. This review highlights recent experimental and clinical data that support the contribution of abnormal MR activation to the development of vascular insulin resistance and dysfunction.

Western diet induces renal artery endothelial stiffening that is dependent on the epithelial Na+ channel.

Consumption of a Western diet (WD) induces central aortic stiffening that contributes to the transmittance of pulsatile blood flow to end organs, including the kidney. Our recent work supports that endothelial epithelial Na+ channel (EnNaC) expression and activation enhances aortic endothelial cell stiffening through reductions in endothelial nitric oxide (NO) synthase (eNOS) and bioavailable NO that result in inflammatory and oxidant responses and perivascular fibrosis. However, the role that EnNaC activation has on endothelial responses in the renal circulation remains unknown. We hypothesized that cell-specific deletion of the α-subunit of EnNaC would prevent WD-induced central aortic stiffness and protect the kidney from endothelial dysfunction and vascular stiffening. Twenty-eight-week-old female αEnNaC knockout and wild-type mice were fed either mouse chow or WD containing excess fat (46%), sucrose, and fructose (17.5% each). WD feeding increased fat mass, indexes of vascular stiffening in the aorta and renal artery (in vivo pulse wave velocity and ultrasound), and renal endothelial cell stiffening (ex vivo atomic force microscopy). WD further impaired aortic endothelium-dependent relaxation and renal artery compliance (pressure myography) without changes in blood pressure. WD-induced renal arterial stiffening occurred in parallel to attenuated eNOS activation, increased oxidative stress, and aortic and renal perivascular fibrosis. αEnNaC deletion prevented these abnormalities and support a novel mechanism by which WD contributes to renal arterial stiffening that is endothelium and Na+ channel dependent. These results demonstrate that cell-specific EnNaC is important in propagating pulsatility into the renal circulation, generating oxidant stress, reduced bioavailable NO, and renal vessel wall fibrosis and stiffening.

Discovery of novel L-type voltage-gated calcium channel blockers and application for the prevention of inflammation and angiogenesis.

BACKGROUND: The ways in which microglia activate and promote neovascularization (NV) are not fully understood. Recent in vivo evidence supports the theory that calcium is required for the transition of microglia from a surveillance state to an active one. The objectives of this study were to discover novel L-type voltage-gated channel (L-VGCC) blockers and investigate their application for the prevention of inflammation and angiogenesis. METHODS: Pharmacophore-based computational modeling methods were used to screen for novel calcium channel blockers (CCBs) from the ZINC compound library. The effects of CCBs on calcium blockade, microglial pro-inflammatory activation, and cell toxicity were validated in BV-2 microglial cell and freshly isolated smooth muscle cell (SMC) cultures. Laser-induced choroidal neovascularization (NV) and the suture-induced inflammatory corneal NV models of angiogenesis were used for in vivo validation of the novel CCBs. CX3CR1gfp/+ mice were used to examine the infiltration of GFP-labeled microglial cells. RESULTS: We identified three compounds from the ZINC database (Zinc20267861, Zinc18204217, and Zinc33254827) as new blockers of L-type voltage-gated calcium channels (L-VGCC) using a structure-based pharmacophore approach. The effects of the three CCBs on Ca2+ influx into cells were verified in BV-2 microglial cells using Fura-2 fluorescent dye and in freshly isolated SMCs using the voltage-patch clamp. All three CCBs reduced microglial cell migration, activation stimulated by lipopolysaccharide (LPS), and reduced the expression of the inflammatory markers NF-κB (phospho-IκBα) and cyclooxygenase-2 (COX-2) as well as reactive oxygen species. Of the three compounds, we further examined the in vivo activity of Zinc20267861. Topical treatment with Zinc20267861 in a rat model of suture-induced inflammatory cornea neovascularization demonstrated efficacy of the compound in reducing monocyte infiltration and overall corneal NV response. Subconjunctival administration of the compound in the choroidal NV mouse model effectively prevented CNV and microglial infiltration. CONCLUSIONS: Our findings suggest that the novel CCBs identified here are effective anti-inflammatory agents that can be further evaluated for treating NV disorders and can be potentially applied in the treatment of ocular inflammatory and pathological angiogenetic disorders.

Diabetic Cardiomyopathy: An Update of Mechanisms Contributing to This Clinical Entity.

Heart failure and related morbidity and mortality are increasing at an alarming rate, in large part, because of increases in aging, obesity, and diabetes mellitus. The clinical outcomes associated with heart failure are considerably worse for patients with diabetes mellitus than for those without diabetes mellitus. In people with diabetes mellitus, the presence of myocardial dysfunction in the absence of overt clinical coronary artery disease, valvular disease, and other conventional cardiovascular risk factors, such as hypertension and dyslipidemia, has led to the descriptive terminology, diabetic cardiomyopathy. The prevalence of diabetic cardiomyopathy is increasing in parallel with the increase in diabetes mellitus. Diabetic cardiomyopathy is initially characterized by myocardial fibrosis, dysfunctional remodeling, and associated diastolic dysfunction, later by systolic dysfunction, and eventually by clinical heart failure. Impaired cardiac insulin metabolic signaling, mitochondrial dysfunction, increases in oxidative stress, reduced nitric oxide bioavailability, elevations in advanced glycation end products and collagen-based cardiomyocyte and extracellular matrix stiffness, impaired mitochondrial and cardiomyocyte calcium handling, inflammation, renin-angiotensin-aldosterone system activation, cardiac autonomic neuropathy, endoplasmic reticulum stress, microvascular dysfunction, and a myriad of cardiac metabolic abnormalities have all been implicated in the development and progression of diabetic cardiomyopathy. Molecular mechanisms linked to the underlying pathophysiological changes include abnormalities in AMP-activated protein kinase, peroxisome proliferator-activated receptors, O-linked N-acetylglucosamine, protein kinase C, microRNA, and exosome pathways. The aim of this review is to provide a contemporary view of these instigators of diabetic cardiomyopathy, as well as mechanistically based strategies for the prevention and treatment of diabetic cardiomyopathy.

Arteriolar vasodilation involves actin depolymerization.

It is generally assumed that relaxation of arteriolar vascular smooth muscle occurs through hyperpolarization of the cell membrane, reduction in intracellular Ca2+ concentration, and activation of myosin light chain phosphatase/inactivation of myosin light chain kinase. We hypothesized that vasodilation is related to depolymerization of F-actin. Cremaster muscles were dissected in rats under pentobarbital sodium anesthesia (50 mg/kg). First-order arterioles were dissected, cannulated on glass micropipettes, pressurized, and warmed to 34°C. Internal diameter was monitored with an electronic video caliper. The concentration of G-actin was determined in flash-frozen intact segments of arterioles by ultracentrifugation and Western blot analyses. Arterioles dilated by ~40% of initial diameter in response to pinacidil (1 × 10-6 mM) and sodium nitroprusside (5 × 10-5 mM). The G-actin-to-smooth muscle 22α ratio was 0.67 ± 0.09 in arterioles with myogenic tone and increased significantly to 1.32 ± 0.34 ( P < 0.01) when arterioles were dilated with pinacidil and 1.14 ± 0.18 ( P < 0.01) with sodium nitroprusside, indicating actin depolymerization. Compared with control vessels (49 ± 5%), the percentage of phosphorylated myosin light chain was significantly reduced by pinacidil (24 ± 2%, P < 0.01) but not sodium nitroprusside (42 ± 4%). These findings suggest that actin depolymerization is an important mechanism for vasodilation of resistance arterioles to external agonists. Furthermore, pinacidil produces smooth muscle relaxation via both decreases in myosin light chain phosphorylation and actin depolymerization, whereas sodium nitroprusside produces smooth muscle relaxation primarily via actin depolymerization. NEW & NOTEWORTHY This article adds to the accumulating evidence on the contribution of the actin cytoskeleton to the regulation of vascular smooth muscle tone in resistance arterioles. Actin depolymerization appears to be an important mechanism for vasodilation of resistance arterioles to pharmacological agonists. Dilation to the K+ channel opener pinacidil is produced by decreases in myosin light chain phosphorylation and actin depolymerization, whereas dilation to the nitric oxide donor sodium nitroprusside occurs primarily via actin depolymerization.

N-Cadherin, a novel and rapidly remodelling site involved in vasoregulation of small cerebral arteries.

KEY POINTS: N-cadherin formed punctate adherens junctions (AJ) along the borders between vascular smooth muscle cells (VSMCs) in the pressurized rat superior cerebellar artery. The formation of N-cadherin AJs in the vessel wall depends on the intraluminal pressure and was responsive to treatment with phenylephrine (PE) (10-5  m) and ACh (10-5  m). N-cadherin-coated beads were able to induce clustering of N-cadherin-enhanced green fluorescent protein (EGFP) on the plasma membrane of isolated VSMCs, whereas treatment with PE (10-5  m) or sodium nitroprusside (10-5  m) induced a significant increase or decrease in the N-cadherin-EGFP clustering, respectively. Application of pulling force (∼1 nN) to the N-cadherin-coated beads via an atomic force microscope induced a localized mechanical response from the VSMCs that opposed the pulling. ABSTRACT: N-cadherin is the major cell-cell adhesion molecule in vascular smooth muscle cells (VSMCs). We tested the hypothesis that N-cadherin is part of a novel mechanosensory mechanism in VSMCs and plays an active role in both the arteriolar myogenic response and during changes in vascular tone induced by vasomotor agonists. Intact and pressurized rat superior cerebellar arteries were labelled for confocal immunofluorescence imaging. N-cadherin formed punctate adherens junctions (AJ) along the borders between VSMCs. When the lumen pressure was raised from 50 to 90 mmHg, both the density and the average size of N-cadherin AJs increased significantly. Similarly, arteriolar constriction with phenylephrine (PE) (10-5  m) induced a significant increase of N-cadherin AJ density at 50 mmHg, whereas vasodilatation induced by ACh (10-5  m) was accompanied by a significant decrease in density and size of N-cadherin AJs. An atomic force microscope (AFM) was employed to further examine the mechano-responsive properties of N-cadherin adhesion sites in isolated VSMCs. AFM probes with an attached N-cadherin-coated microbead (5 µm) induced a progressive clustering of N-cadherin-enhanced green fluorescent protein (EGFP) on the VSMC surface. Application of pulling force (∼1 nN) to the N-cadherin-coated-beads with the AFM induced a localized mechanical response from the VSMCs that opposed the pulling. Treatment with PE (10-5  m) or sodium nitroprusside (10-5  m) induced a significant increase or decrease of the N-cadherin-EGFP clustering, respectively. These observations provide compelling evidence that N-cadherin AJs are sensitive to pressure and vasomotor agonists in VSMCs and support a functional role of N-cadherin AJs in vasomotor regulation.

Nonenzymatic glycation interferes with fibronectin-integrin interactions in vascular smooth muscle cells.

OBJECTIVE: We aimed to investigate whether advanced nonenzymatic glycation of the ECM protein, fibronectin, impacts its normal integrin-mediated interaction with arteriolar VSMC. METHODS: AFM was performed on cultured VSMC from rat cremaster arterioles to study native and glycated fibronectin (FN and gFN) interactions with cellular integrins. AFM probes were functionalized with FN or gFN or with native or glycated albumin (gAlb) as controls. RESULTS: VSMC showed increased adhesion probability to gFN (72.9±3.5%) compared with native FN (63.0±1.6%). VSMC similarly showed increased probability of adhesion (63.8±1.7%) to gAlb compared with native Alb (40.1±4.7%). Adhesion of native FN to VSMC was α5 and ß1 integrin dependent whereas adhesion of gFN to VSMC was integrin independent. The RAGE-selective inhibitor, FPS-ZM1, blocked gFN (and gAlb) adhesion, suggesting that adhesion of glycated proteins was RAGE dependent. Interaction of FN with VSMC was not altered by soluble gFN while soluble native FN did not inhibit adhesion of gFN to VSMC. In contrast, gAlb inhibited adhesion of gFN to VSMC in a concentration-dependent manner. CONCLUSIONS: Glycation of FN shifts the nature of cellular adhesion from integrin- to RAGE-dependent mechanisms.

Mechanical activation of angiotensin II type 1 receptors causes actin remodelling and myogenic responsiveness in skeletal muscle arterioles.

KEY POINTS: Candesartan, an inverse agonist of the type 1 angiotensin II receptor (AT1 R), causes a concentration-dependent inhibition of pressure-dependent myogenic tone consistent with previous reports of mechanosensitivity of this G protein-coupled receptor. Mechanoactivation of the AT1 R occurs independently of local angiotensin II production and the type 2 angiotensin receptor. Mechanoactivation of the AT1 R stimulates actin polymerization by a protein kinase C-dependent mechanism, but independently of a change in intracellular Ca2+ . Using atomic force microscopy, changes in single vascular smooth muscle cell cortical actin are observed to remodel following mechanoactivation of the AT1 R. ABSTRACT: The Gq/11 protein-coupled angiotensin II type 1 receptor (AT1 R) has been shown to be activated by mechanical stimuli. In the vascular system, evidence supports the AT1 R being a mechanosensor that contributes to arteriolar myogenic constriction. The aim of this study was to determine if AT1 R mechanoactivation affects myogenic constriction in skeletal muscle arterioles and to determine underlying cellular mechanisms. Using pressure myography to study rat isolated first-order cremaster muscle arterioles the AT1 R inhibitor candesartan (10-7 -10-5  m) showed partial but concentration-dependent inhibition of myogenic reactivity. Inhibition was demonstrated by a rightward shift in the pressure-diameter relationship over the intraluminal pressure range, 30-110 mmHg. Pressure-induced changes in global vascular smooth muscle intracellular Ca2+ (using Fura-2) were similar in the absence or presence of candesartan, indicating that AT1 R-mediated myogenic constriction relies on Ca2+ -independent downstream signalling. The diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) reversed the inhibitory effect of candesartan, while this rescue effect was prevented by the protein kinase C (PKC) inhibitor GF 109203X. Both candesartan and PKC inhibition caused increased G-actin levels, as determined by Western blotting of vessel lysates, supporting involvement of cytoskeletal remodelling. At the single vascular smooth muscle cell level, atomic force microscopy showed that cell swelling (stretch) with hypotonic buffer also caused thickening of cortical actin fibres and this was blocked by candesartan. Collectively, the present studies support growing evidence for novel modes of activation of the AT1 R in arterioles and suggest that mechanically activated AT1 R generates diacylglycerol, which in turn activates PKC which induces the actin cytoskeleton reorganization that is required for pressure-induced vasoconstriction.

Brief serotonin exposure initiates arteriolar inward remodeling processes in vivo that involve transglutaminase activation and actin cytoskeleton reorganization.

Inward remodeling of the resistance vasculature is strongly associated with life-threatening cardiovascular events. Previous studies have demonstrated that both actin polymerization and the activation of transglutaminases mediate early stages of the transition from a structurally normal vessel to an inwardly remodeled one. Ex vivo studies further suggest that a few hours of exposure to vasoconstrictor agonists induces inward remodeling in the absence of changes in intraluminal pressure. Here we report that a short, 10-min, topical exposure to serotonin (5-HT) + N(ω)-nitro-l-arginine methyl ester hydrochloride (l-NAME) was sufficient to initiate inward remodeling processes in rat cremasteric feed arterioles (100-200 µm lumen diameter), in vivo. Addition of the transglutaminase inhibitor, cystamine, blocked the in vivo remodeling. We further demonstrate that, in isolated arterioles, 5-HT + l-NAME activates transglutaminases and modulates the phosphorylation state of cofilin, a regulator of actin depolymerization. The 5-HT + l-NAME-induced remodeling process in isolated arterioles was also inhibited by an inhibitor of Lim Kinase, the kinase that phosphorylates and inactivates cofilin. Therefore, our results indicate that a brief vasoconstriction induced by 5-HT + l-NAME is able to reduce the passive structural diameter of arterioles through processes that are dependent on the activation of transglutaminases and Lim kinase, and the subsequent phosphorylation of cofilin.

Small artery mechanobiology: Roles of cellular and non-cellular elements.

Small arteries and their component cellular and non-cellular elements are continually subjected to, and interact with, mechanical forces. Such interactions are key in both short- and long-term adaptation of the structure and function of the microcirculation to its local environment and metabolic requirements. Following this brief introduction is a series of papers presented as a symposium (Small Artery Mechanobiology: Roles of Cellular and Non-Cellular Elements) at the World Congress for Microcirculation, Kyoto 2015.

Small Artery Elastin Distribution and Architecture-Focus on Three Dimensional Organization.

The distribution of ECM proteins within the walls of resistance vessels is complex both in variety of proteins and structural arrangement. In particular, elastin exists as discrete fibers varying in orientation across the adventitia and media as well as often resembling a sheet-like structure in the case of the IEL. Adding to the complexity is the tissue heterogeneity that exists in these structural arrangements. For example, small intracranial cerebral arteries lack adventitial elastin while similar sized arteries from skeletal muscle and intestinal mesentery exhibit a complex adventitial network of elastin fibers. With regard to the IEL, several vascular beds exhibit an elastin sheet with punctate holes/fenestrae while in others the IEL is discontinuous and fibrous in appearance. Importantly, these structural patterns likely sub-serve specific functional properties, including mechanosensing, control of external forces, mechanical properties of the vascular wall, cellular positioning, and communication between cells. Of further significance, these processes are altered in vascular disorders such as hypertension and diabetes mellitus where there is modification of ECM. This brief report focuses on the three-dimensional wall structure of small arteries and considers possible implications with regard to mechanosensing under physiological and pathophysiological conditions.

Adiponectin Receptor Agonist, AdipoRon, Causes Vasorelaxation Predominantly Via a Direct Smooth Muscle Action.

OBJECTIVE: AdipoRon, an adiponectin receptor agonist, was recently proposed for treating insulin resistance and hyperglycemia. As adiponectin is vasoprotective via NO-mediated signaling, it was hypothesized that adipoRon similarly exerts potentially beneficial vasodilator effects. We therefore examined if adipoRon induces vasorelaxation and via what contributing mechanisms. METHODS: Vascular function was assessed in skeletal muscle arteries from rats and cerebral/coronary arteries from mice using pressure and wire myography. RESULTS: Using qPCR, mRNA for adiponectin receptors was demonstrated in skeletal muscle, cerebral and coronary arteries. AdipoRon-caused vasorelaxation was not abolished by compound C (10 µM; AMPK inhibitor). Inhibition of endothelium-dependent relaxation with combinations of l-NAME/indomethacin/apamin/TRAM-34 only slightly reduced adipoRon-mediated vasorelaxation in cerebral and coronary arteries. EC-denuded cremaster arteries showed similar relaxant responses to adipoRon as in intact vessels, suggesting adipoRon directly impacts VSMCs. K(+) currents measured in VSMCs isolated from mouse basilar and LAD arteries were not altered by adiopRon. In cremaster arteries, adipoRon induced vasorelaxation without a marked decrease in VSMC [Ca(2+)]i . Adiponectin, itself, caused vasodilation in intact cremaster arteries while failing to cause significant dilation in EC-denuded arteries, consistent with endothelium dependency of adiponectin. CONCLUSIONS: AdipoRon exerts vasodilation by mechanisms distinct to adiponectin. The dominant mechanism for adipoRon-induced vasorelaxation occurs independently of endothelium-dependent relaxing factors, AMPK activation, K(+) efflux-mediated hyperpolarization and reductions in cytosolic [Ca(2+)]i .