Response Of Smooth Muscle Cells Research Articles

Irradiation abolishes smooth muscle investment into vascular lesions in specific vascular beds.

The long-term adverse effects of radiotherapy on cardiovascular disease are well documented. However, the underlying mechanisms responsible for this increased risk are poorly understood. Previous studies using rigorous smooth muscle cell (SMC) lineage tracing have shown abundant SMC investment into atherosclerotic lesions, where SMCs contribute to the formation of a protective fibrous cap. Studies herein tested whether radiation impairs protective adaptive SMC responses during vascular disease. To do this, we exposed SMC lineage tracing (Myh11-ERT2Cre YFP+) mice to lethal radiation (1,200 cGy) followed by bone marrow transplantation prior to atherosclerosis development or vessel injury. Surprisingly, following irradiation, we observed a complete loss of SMC investment in 100% of brachiocephalic artery (BCA), carotid artery, and aortic arch lesions. Importantly, this was associated with a decrease in multiple indices of atherosclerotic lesion stability within the BCA. Interestingly, we observed anatomic heterogeneity, as SMCs accumulated normally into lesions of the aortic root and abdominal aorta, suggesting that SMC sensitivity to lethal irradiation occurs in blood vessels of neural crest origin. Taken together, these results reveal an undefined and unintended variable in previous studies using lethal irradiation and may help explain why patients exposed to radiation have increased risk for cardiovascular disease.

Role of Bisphosphonates in Vascular calcification and Bone Metabolism: A Clinical Summary.

Background:Vascular calcification is known to be a strong risk factor for cardiovascularadverse events and mortality. Atherosclerosis, diabetes, aging, abnormal bone mineral homeostasisand high uremic milieu such as chronic kidney disease are major factors that contribute to theprogression of vascular calcification. Several mechanisms such as the osteoblastic transition of vascularsmooth muscle cells in response to oxidative stress have shed light on the active nature ofvascular calcification, which was once thought to be a passive process. The fine interplay of regulatoryfactors such as PTH, vitamin D3, FGF 23 and klotho reflect the delicate balance between vascularcalcification and bone mineralization. Any disturbance affecting this equilibrium of the bonemineral-vascular axis results in accelerated vascular calcification. Bisphosphonates share similar mechanism of action as statins, and hence several studies were undertakenin humans to verify if the benefits proven to be obtained in animal models extended tohuman models too. This yielded conflicting outcomes which are outlined in this review. This wasattributed mainly to inadequate sample size and flaws in the study design. Therefore, this benefitcan only be ascertained if studies addressing this are undertaken.Conclusion:This review seeks to highlight the pathophysiologic phenomena implicated in vascular and valvular calcification and summarize the literature available regarding the use of bisphosphonates in animal and human models. We also discuss novel treatment approaches for vascular calcification,with emphasis on chronic kidney disease and calciphylaxis.

Contractility of Airway Smooth Muscle Cell in Response to Zinc Oxide Nanoparticles by Traction Force Microscopy.

Zinc oxide nanoparticles (ZnO-NPs) have been widely used in engineering and biomedicine. However, their adverse pathological effects and mechanisms, especially the biomechanical effects on respiratory system where airway smooth muscle cell (ASMC) contractility regulates the airway response and lung function, are not fully understood. Herein, we used traction force microscopy (TFM)method to investigate whether ZnO-NPs of different concentrations (0.1-10μg/mL) can alter ASMC contractility (basal and agonist-stimulated) after a short-term exposure and the potential mechanisms. We found that ZnO-NPs exposure led to a decrease of ASMC viability in a dose-dependent manner. Notably, basal contractility was enhanced when the concentration of ZnO-NPs was less than0.1μg/mL and decreased afterwards, while KCl-stimulated contractility was reduced in all cases of ZnO-NPs treated groups. Cytoskeleton structure was also found to be significantly altered in ASMC with the stimulation of ZnO-NPs. More importantly, it seems that ZnO-NPs with low concentration (< 0.1μg/mL) would change ASMC contractility without any apparent cytotoxicity through disruption of the microtubule assembly. Moreover, our results also emerged that ASMC contractility responses were regulated by clathrin-mediated endocytosis and cytoskeleton remodeling. Together, these findings indicate the susceptibility of cell mechanics to NPs exposure, suggesting that cell mechanical testing will contribute to uncover the pathological mechanisms of NPs in respiratory diseases.

The homeostatic ensemble for cells

Cells are quintessential examples of out-of-equilibrium systems, but they maintain a homeostatic state over a timescale of hours to days. As a consequence, the statistics of all observables is remarkably consistent. Here, we develop a statistical mechanics framework for living cells by including the homeostatic constraint that exists over the interphase period of the cell cycle. The consequence is the introduction of the concept of a homeostatic ensemble and an associated homeostatic temperature, along with a formalism for the (dynamic) homeostatic equilibrium that intervenes to allow living cells to evade thermodynamic decay. As a first application, the framework is shown to accurately predict the observed effect of the mechanical environment on the in vitro response of smooth muscle cells. This includes predictions that both the mean values and diversity/variability in the measured values of observables such as cell area, shape and tractions decrease with decreasing stiffness of the environment. Thus, we argue that the observed variabilities are inherent to the entropic nature of the homeostatic equilibrium of cells and not a result of in vitro experimental errors.

Low Cell-Matrix Adhesion Reveals Two Subtypes of Human Pluripotent Stem Cells

SummaryWe show that a human pluripotent stem cell (hPSC) population cultured on a low-adhesion substrate developed two hPSC subtypes with different colony morphologies: flat and domed. Notably, the dome-like cells showed higher active proliferation capacity and increased several pluripotent genes’ expression compared with the flat monolayer cells. We further demonstrated that cell-matrix adhesion mediates the interaction between cell morphology and expression of KLF4 and KLF5 through a serum response factor (SRF)-based regulatory double loop. Our results provide a mechanistic view on the coupling among adhesion, stem cell morphology, and pluripotency, shedding light on the critical role of cell-matrix adhesion in the induction and maintenance of hPSC.

Anti-Atherogenic Effects of Vaspin on Human Aortic Smooth Muscle Cell/Macrophage Responses and Hyperlipidemic Mouse Plaque Phenotype.

Vaspin (visceral adipose tissue-derived serine protease inhibitor) was recently identified as a novel adipocytokine with insulin-sensitizing effects. Serum vaspin levels are reported either increased or decreased in patients with coronary artery disease. Our translational research was performed to evaluate the expression of vaspin in human coronary atherosclerotic lesions, and its effects on atherogenic responses in human macrophages and human aortic smooth muscle cells (HASMC), as well as aortic atherosclerotic lesion development in spontaneously hyperlipidemic Apoe−/− mice, an animal model of atherosclerosis. Vaspin was expressed at high levels in macrophages/vascular smooth muscle cells (VSMCs) within human coronary atheromatous plaques. Vaspin significantly suppressed inflammatory phenotypes with nuclear factor κB down-regulation in human macrophages. Vaspin significantly suppressed oxidized low-density lipoprotein-induced foam cell formation with CD36 and acyl-coenzyme A: cholesterol acyltransferase-1 down-regulation and ATP-binding cassette transporters A1 and G1, and scavenger receptor class B type 1 up-regulation in human macrophages. Vaspin significantly suppressed angiotensin II-induced migration and proliferation with ERK1/2 and JNK down-regulation, and increased collagen production with phosphoinositide 3-kinase and Akt up-regulation in HASMCs. Chronic infusion of vaspin into Apoe−/− mice significantly suppressed the development of aortic atherosclerotic lesions, with significant reductions of intraplaque inflammation and the macrophage/VSMC ratio, a marker of plaque instability. Our study indicates that vaspin prevents atherosclerotic plaque formation and instability, and may serve as a novel therapeutic target in atherosclerotic cardiovascular diseases.

Gene expression profile analysis of aortic vascular smooth muscle cells reveals upregulation of cadherin genes in myocardial infarction patients.

Myocardial infarction (MI) induced by acute coronary arterial occlusion is usually secondary to atherosclerotic plaque rupture. Dysregulated response of vascular smooth muscle cells (VSMCs) in atherosclerotic plaques may promote plaque rupture. Cadherins (CDHs) form adherens junctions and are known stabilizers of atherosclerotic plaques. To date, the expression patterns of cadherin have not been well investigated in MI aortic VSMCs. We aimed to investigate the expression of cadherin genes in the aortic wall of patients with and without MI. Laser capture microdissected VSMCs were obtained from aortic tissue samples of patients undergoing coronary artery bypass graft surgery. Integrative bioinformatic analysis of the microarray profiles of the VSMCs revealed that MI is discriminated at the whole transcriptome level by hundreds of differentially expressed genes, including genes involved in cell adhesion, of which the cadherin superfamily genes were among the top structural category. Eleven significantly deregulated candidates of the cadherin superfamily were chosen and formed a new classifier that collectively discriminated MI vs. non-MI with ~95% accuracy. Significance validation was performed with an independent cohort by quantitative RT-quantitative PCR, confirming overexpression of CDH2, CDH12, PCDH17, and PCDH18 in MI VSMCs. The dysregulation of these cadherin superfamily genes might be related to an MI-induced remote effect on aortic wall VSMCs and to imbalances in signaling pathways and myocardial repair mechanisms. Although pathophysiological significance of our findings requires functional studies, mRNA upregulation of the identified cadherin superfamily members in VSMCs might be associated with the progression of atherosclerosis and angiogenesis activation in MI.

Short-Term Angiotensin II Treatment Affects Large Artery Biomechanics and Function in the Absence of Small Artery Alterations in Mice.

Induction of hypertension by angiotensin II (AngII) is a widely used experimental stimulus to study vascular aging in mice. It is associated with large artery stiffness, a hallmark of arterial aging and a root cause of increased cardiovascular risk. We reported earlier that long term (4 week) AngII treatment in mice altered the active, contractile properties of the arteries in a vascular bed-specific manner and that, in healthy mice aorta, active contractile properties of the aortic wall determine isobaric aortic stiffness. Given the huge physiological relevance of large artery stiffening, we aimed to characterize the early (1 week) changes in the active properties of the aorta of AngII-treated mice. We were not able to detect a significant effect of AngII treatment on anesthetized blood pressure or abdominal aorta pulse wave velocity. Ex vivo biomechanical and functional studies of the aorta revealed increased arterial stiffness and altered vascular smooth muscle cell (VSMC) and endothelial cell reactivity. Interestingly, the AngII-associated changes in the aorta could be largely attributed to alterations in basal VSMC tone and basal nitric oxide efficacy, indicating that, besides structural remodeling of the arterial wall, dysfunctional active components of the aorta play a crucial role in the pathophysiological mechanisms by which AngII treatment induces arterial stiffness.

Novel Polymer-Free Everolimus-Eluting Stent Fabricated using Femtosecond Laser Improves Re-endothelialization and Anti-inflammation

The aim of this study was to fabricate a novel polymer-free everolimus-eluting stent with nanostructure using a femtosecond laser (FSL). The stent were coated with everolimus (EVL) using FSL and electrospinning processes. The surface was rendered hydrophobic, which negatively affected both platelet adhesion (82.1%) and smooth muscle cell response. Animal study was performed using a porcine coronary restenosis model. The study groups were divided into 1) bare metal stent (BMS), 2) poly(L-lactide) (PLA)-based EVL drug eluting stent (DES), 3) commercial EVL-eluting DES, and 4) FSL-EVL-DES. After four weeks of stent implantation, various analyses were performed. Quantitative analysis showed that the amount of in-stent restenosis was higher in the BMS group (BMS; 27.8 ± 2.68%, PLA-based DES; 12.2 ± 0.57%, commercial DES; 9.8 ± 0.28%, and FSL-DES; 9.3 ± 0.25%, n = 10, p < 0.05). Specifically, the inflammation score was reduced in the FSL-DES group (1.9 ± 0.39, n = 10, p < 0.05). The increment in re-endothelialization in the FSL-DES group was confirmed by immunofluorescence analysis. Taken together, the novel polymer-free EVL-eluting stent fabricated using FSL can be an innovative DES with reduced risk of ISR, thrombosis, and inflammation.

RETRACTED ARTICLE: The anti-proliferative and anti-inflammatory response of COPD airway smooth muscle cells to hydrogen sulfide

BackbroundCOPD is a common, highly debilitating disease of the airways, primarily caused by smoking. Chronic inflammation and structural remodelling are key pathological features of this disease caused, in part, by the aberrant function of airway smooth muscle (ASM). We have previously demonstrated that hydrogen sulfide (H2S) can inhibit ASM cell proliferation and CXCL8 release, from cells isolated from non-smokers.MethodsWe examined the effect of H2S upon ASM cells from COPD patients. ASM cells were isolated from non-smokers, smokers and patients with COPD (n = 9). Proliferation and cytokine release (IL-6 and CXCL8) of ASM was induced by FCS, and measured by bromodeoxyuridine incorporation and ELISA, respectively.ResultsExposure of ASM to H2S donors inhibited FCS-induced proliferation and cytokine release, but was less effective upon COPD ASM cells compared to the non-smokers and smokers. The mRNA and protein expression of the enzymes responsible for endogenous H2S production (cystathionine-β-synthase [CBS] and 3-mercaptopyruvate sulphur transferase [MPST]) were inhibited by H2S donors. Finally, we report that exogenous H2S inhibited FCS-stimulated phosphorylation of ERK–1/2 and p38 mitogen activated protein kinases (MAPKs), in the non-smoker and smoker ASM cells, with little effect in COPD cells.ConclusionsH2S production provides a novel mechanism for the repression of ASM proliferation and cytokine release. The ability of COPD ASM cells to respond to H2S is attenuated in COPD ASM cells despite the presence of the enzymes responsible for H2S production.

Preparation of recombinant human-like collagen/fibroin scaffold and its promoting effect on vascular cells biocompatibility

A novel recombinant human-like collagen/fibroin scaffold has been prepared previously, which has high porosity, controllable pore size, and much better mechanical properties than the reported fibroin-based scaffold. In this research, the cell responses of vascular smooth muscle cells to this blend scaffold were examined in vitro. Cell morphology, adherence, and growth in scaffolds were observed by scanning electron microscopy, laser scanning confocal microscopy after staining of the cells with propidium iodide at 1, 3, 5, and 7 days, respectively. A wide range of measurements, including 3-[4,5–dimethylthiazol-2-yl]-2, 5-diphenyl tetrasodium bromide assay, and total intracellular protein content at the end of 7 days culture, were conducted. An increase of viability and protein content of vascular smooth muscle cells cultured in recombinant human-like collagen/fibroin scaffold was found. The laser scanning confocal microscopy and scanning electron microscopy results confirm that the cells readily adhered and proliferation in the blend than in fibroin scaffold, and indicate a better adhesion process. The positive effects were especially significant for vascular smooth muscle cells. The recombinant human-like collagen/fibroin scaffold could be a promising biomaterial for vascular tissue engineering.

Traction Force Screening Enabled by Compliant PDMS Elastomers

Actomyosin contractility is an essential element of many aspects of cellular biology and manifests as traction forces that cells exert on their surroundings. The central role of these forces makes them a novel principal therapeutic target in diverse diseases. This requires accurate and higher-capacity measurements of traction forces; however, existing methods are largely low throughput, limiting their utility in broader applications. To address this need, we employ Fourier-transform traction force microscopy in a parallelized 96-well format, which we refer to as contractile force screening. Critically, rather than the frequently employed hydrogel polyacrylamide, we fabricate these plates using polydimethylsiloxane rubber. Key to this approach is that the polydimethylsiloxane used is very compliant, with a lower-bound Young’s modulus of ∼0.4 kPa. We subdivide these monolithic substrates spatially into biochemically independent wells, creating a uniform multiwell platform for traction force screening. We demonstrate the utility and versatility of this platform by quantifying the compound and dose-dependent contractility responses of human airway smooth muscle cells and retinal pigment epithelial cells. By directly quantifying the endpoint of therapeutic intent, airway-smooth-muscle contractile force, this approach fills an important methodological void in current screening approaches for bronchodilator drug discovery, and, more generally, in measuring contractile response for a broad range of cell types and pathologies.

10-Hydroxy-trans-2-decenoic acid attenuates angiotensin II-induced inflammatory responses in rat vascular smooth muscle cells

10-Hydroxy-trans-2-decenoic acid (10-HDA) is a major medium chain fatty acid in royal jelly. In this study, the rat vascular smooth muscle cells (VSMCs) were pretreated with 10-HDA (250 μM, 500 μM, 1000 μM), followed by incubation with Angiotensin II (Ang II, 100 nM). 10-HDA significantly reduced the productions of Ang II induced inflammatory cytokines interleukin 6 and C-reactive protein, through downregulating the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. 10-HDA also attenuated the Ang II-stimulated reactive oxygen species production and the NADPH oxidase 4 (NOX4) expression. Further mechanistic studies showed that 10-HDA reduced the elevated abundances of the angiotensin II type 1 receptor, and inhibited cAMP-response element-binding protein binding to the cAMP-response element site in the IL-6 promoter region. The results suggested that 10-HDA effectively attenuated Ang II-induced inflammatory responses and ROS production via regulating multiple steps in NF-κB and MAPK signaling pathways in VSMCs.

Inflammatory Response of Pulmonary Artery Smooth Muscle Cells Exposed to Oxidative and Biophysical Stress

Pulmonary hypertension in the neonate requires treatment with oxygen and positive pressure ventilation, both known to induce lung injury. The direct response of pulmonary artery smooth muscle cells, the most abundant cells in the artery wall, to the stress of positive pressure and hyperoxia has not been previously studied. Pulmonary artery smooth muscle cells were cultured in temperature- and pressure-controlled air-tight chambers under conditions of positive pressure or hyperoxia for 24h. Control cells were cultured in room air under atmospheric pressure. After the exposure period, culture medium was collected and samples were analyzed by ELISA, Human Cytokine 25-Plex Panel using a Luminex 200 analyzer and Western blot. Secretion of various inflammatory mediators, specifically IL-6, IL-8, IL-2R, MIP-1β, MCP-1, IP-10, IL-7, IL-1RA, and IFN-α, was higher in the positive pressure and hyperoxia groups compared with control. The level of cyclin D1 was decreased in the hyperoxia and positive pressure group compared with control. Levels of fibronectin and α-smooth muscle actin were not different among the groups. Pulmonary artery smooth muscle cells directly produce multiple inflammatory mediators in response to oxidative and biophysical stress in vitro, which may be part of a cascade that leads to the vascular and perivascular changes in pulmonary hypertension.

β1-Subunit of the calcium-sensitive potassium channel modulates the pulmonary vascular smooth muscle cell response to hypoxia.

Accessory subunits associated with the calcium-sensitive potassium channel (BKCa), a major determinant of vascular tone, confer functional and anatomical diversity. The β1 subunit increases Ca2+ and voltagesensitivity of the BKCa channel and is expressed exclusively in smooth muscle cells. Evidence supporting the physiological significance of the β1 subunit includes the observations that murine models with deletion of the β1 subunit are hypertensive and that humans with a gain-of-function β1 mutation are at a decreased risk of diastolic hypertension. However, whether the β1 subunit of the BKCa channel contributes to the low tone that characterizes the normal pulmonary circulation or modulates the pulmonary vascular response to hypoxia remains unknown. To determine the role of the BKCa channel β1 subunit in the regulation of pulmonary vascular tone and the response to acute and chronic hypoxia, mice with deletion of the Kcnmb1 gene that encodes for the β1 subunit ( Kcnmb1-/-) were placed in chronic hypoxia (10% O2) for 21-24 days. In normoxia, right ventricular systolic pressure (RVSP) did not differ between Kcnmb1+/+ (controls) and Kcnmb1-/- mice. After exposure to either acute or chronic hypoxia, RVSP was higher in Kcnmb1-/- mice compared with Kcnmb1+/+ mice, without increased vascular remodeling. β1 subunit expression was predominantly confined to pulmonary artery smooth muscle cells (PASMCs) from vessels ≤ 150 µm. Peripheral PASMCs contracted collagen gels irrespective of β1 expression. Focal adhesion expression and Rho kinase activity were greater in Kcnmb1-/- compared with Kcnmb1+/+ PASMCs. Compromised PASMC β1 function may contribute to the heightened microvascular vasoconstriction that characterizes pulmonary hypertension.

In Hypertension, Global Endothelial Calcium Underlies a Protective Mechanism that Limits Arterial Vasoconstriction.

In small arteries, increased vascular reactivity contributes to the raised blood pressure that characterizes established hypertension. Endothelial function is altered in hypertension and may confer protection against the increased vascular reactivity but the precise mechanisms involved are unresolved. Here, in isolated small mesenteric arteries from hypertensive SHR (arterial BP, 190 ± 4 mmHg) and age‐matched normotensive WKY (arterial BP, 120 ± 4 mmHg) rats, the mechanisms of altered endothelial function were examined using simultaneous measurement of arterial contraction and endothelial Ca2+ signalling in large fields (~2000) of cells.At rest, average Ca2+ concentration was higher in endothelium from SHR rats, compared to that in normotensive WKY controls (n = 8 in each group). In both groups, phenylephrine (1 μM) evoked an arterial contraction that had no accompanying change in endothelial Ca2+ levels during the contraction. However, phenylephrine‐induced (1 μM) contraction was reduced in arteries from SHR, compared to WKY rats (p &lt; 0.05; n = 5 in each group). In both normotensive and hypertensive arteries, selective disruption of endothelial Ca2+ signalling, using BAPTA‐AM (30 μM), inhibited ACh‐mediated endothelial Ca2+ signalling and artery relaxation and increased the contractions evoked by phenylephrine (n = 5 per group). After BAPTA treatment, the response to phenylephrine was normalised among groups – i.e. 1 μM phenylephrine evoked a maximal contraction in both groups. The nitric oxide synthase inhibitor L‐NAME also normalised phenylephrine‐induced contractions in pressurised arteries. These results suggest that basal, Ca2+‐dependent, nitric oxide production may be upregulated in the endothelium of SHR rats to limit arterial contractions. To explore this possibility further, local spontaneous endothelial Ca2+ signals were examined using high spatial (100X, NA = 1.4) and temporal (20 Hz) resolution imaging. In the absence of stimulation with pharmacological agents, spontaneous local Ca2+ events occurred frequently in the endothelium of blood vessels from WKY rats. These events had a wide spectrum of amplitudes and durations. A spatial map of the location and distribution of the local Ca2+ sites showed the majority of local events occurred close to junctions with smooth muscle cells to a greater extent than would be expected if the Ca2+ events initiated randomly throughout the cytoplasm (p &lt; 0.05; n = 8 in each group). These findings suggest that the local endothelial Ca2+ rises may account for an endothelial‐dependent opposition to phenylephrine‐induced tone. However, the propensity of local Ca2+ signals was significantly decreased in arteries from SHR compared to those from WKY rats (p &lt; 0.05; n = 8 in each group). This result suggests that alterations in local endothelial Ca2+ signal dynamics are unlikely to explain the increased endothelium‐dependent negative feedback that opposes phenylephrine‐evoked contraction in hypertension.We suggest the elevated global Ca2+ concentration in endothelial cells at rest accounts for increased NO production and suppressed phenylephrine‐evoked contraction. The increased endothelial global Ca2+ concentration may be a compensatory mechanism to offset the increased contractile responsiveness of smooth muscle cells in the hypertensive rat arteries.Support or Funding InformationThis work was funded by the Wellcome Trust (202924/Z/16/Z) and the British Heart Foundation (PG/16/54/32230; PG16/82/32439), whose support is gratefully acknowledged. C.W. is supported by a Sir Henry Wellcome Postdoctoral Research Fellowship (204682/Z/16/Z).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

O‐GlcNAc Transferase Regulates Phenotypic Transformation of Vascular Smooth Muscle Cells in Response to Hyperglycemia

Risks of vascular complications are enhanced two‐to‐four fold in diabetes, accounting for increased morbidity and mortality in these patients. Hyperglycemia is an important risk factor for macrovascular complications associated with diabetes. Diabetic patients exhibit an increased incidence of vascular smooth muscle cell (VSMC) migration and proliferation, a hallmark of VSMC phenotypic switching from the quiescent contractile state to a synthetic proliferative phenotype. Hyperglycemia increases glucose flux through the hexosamine biosynthetic pathway triggering an enhanced signaling via O‐linked N‐acetylglucosamine (O‐GlcNAc) transferase (OGT), the key enzyme catalyzing addition of O‐GlcNAc moieties to proteins. Protein O‐GlcNAcylation is a unique post‐translational protein modification implicated in diabetes and related cardiovascular complications. Previous studies have suggested both cardio‐protective and cardio‐detrimental effects of protein O‐GlcNAcylation in diabetes. However, the precise role of OGT in the etiology of diabetic atherogenesis remains incompletely understood. The goal of the current study was to investigate whether OGT modulates VSMC transition to a de‐differentiated phenotype under hyperglycemic conditions. Immunoblotting experiments revealed that in primary cultures of human aortic smooth muscle cells (HASMC) in vitro, benzyl 2‐deoxy α‐D‐galactopyranoside (BG), a pharmacological inhibitor of OGT, decreased high glucose‐induced OGT and O‐GlcNAc protein expression, and this was accompanied with attenuated PCNA (proliferation marker), reduced vimentin (SM synthetic marker) and increased SM‐MHC (SM contractile marker) expression. Consistent with these findings, deletion of OGT using siRNA gene silencing significantly lowered protein O‐GlcNAc and PCNA expression in glucose‐stimulated HASMC compared with glucose‐treated cells transfected with control siRNA. Notably, under glucose‐stimulated conditions, OGT knockdown enhanced calponin (SM contractile marker) expression in HASMC transfected with OGT siRNA vs cells transfected with control siRNA. Finally, electrophoretic mobility shift assay (EMSA) revealed that specific OGT inhibitors (BG and ST045849) attenuated high glucose‐induced DNA‐binding activity of YY1 and Elk1 (transcriptional repressors of SM contractile phenotype) in HASMC compared to cells treated with glucose alone. Taken together, these data clearly demonstrate that loss of OGT inhibits VSMC phenotypic de‐differentiation in response to hyperglycemia, suggesting OGT as a potential therapeutic target in diabetic atherosclerosis.Support or Funding InformationAmerican Heart Association ‐ Grant‐in‐Aid 16GRNT31200034This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Estrogen Receptor Signaling and Intracellular Calcium Regulation in Human Airway Smooth Muscle

PurposeBronchial asthma is a chronic inflammation of the airways that is characterized by airway remodeling, hyperactive responsiveness and acute bronchoconstriction of airway smooth muscle (ASM) cells. Clinical findings suggest that there is a higher incidence of asthma in pre‐menopausal women and aging men, strongly indicating the role of sex steroids in modulating the airway tone. A major sex steroid, estrogen mediates its role through estrogen receptors (ER) ‐ ERα and ERβ. Our recent study demonstrated that these receptors are expressed in both male and female ASM cells and their expression is upregulated in the presence of inflammation/asthma. However, the role of ER signaling in asthma or genomic mechanisms in intracellular Ca2+ ([Ca2+]i ) regulation in human ASM are unknown. In this study, we evaluated the genomic effect of specific ERα and ERβ modulators on the [Ca2+]i in human ASM cells.MethodsHuman ASM cells were enzymatically dissociated from tissue obtained incidental to lung surgery from Mayo Clinic (IRB approved). Cells were plated onto 8‐well Lab‐Tek plates and grown to 70% confluence in a 5% CO2 humidified incubator. Cells were exposed to pro‐inflammatory cytokines TNFα (20ng/ml) or IL‐13 (50ng/ml) in the presence and absence of 1nM 17β‐estradiol (E2), 10nM PPT (ERα agonist) or WAY (ERβ agonist) for 24h. Cells were washed with HBSS and incubated with 3 μm Fluo‐3 for 1h for calcium imaging. The [Ca2+]i levels are measured from the intensity change observed following exposure to 10μM histamine under both physiological (2 mM) and zero extracellular calcium using Biotek LFX Fluorescence Imager.ResultsIn fluo‐3 loaded ASM cells, 24h exposure to WAY (and to a lesser extent PPT and E2) was effective in reducing [Ca2+]i levels. TNFα and IL‐13 significantly increased [Ca2+]i compared to vehicle. In the presence of TNFα or IL‐13, WAY significantly reduced [Ca2+]i in ASM cells, while PPT did not draw out any notable changes compared to TNFα or IL‐13 treatment alone. Addition of nifedipine (1μM, L‐type calcium channel blocker) resulted in a ~40% decrease in [Ca2+]i compared to control. This nifedipine blunting effect was similar in PPT treated group and to a higher extend to the WAY on [Ca2+]i responses to histamine, suggesting that ER effects were mediated via L‐type calcium channels. To verify the ER effect on sarcoplasmic reticulum (SR) Ca2+ reuptake, extracellular Ca2+ was removed by perfusion with zero Ca2+ HBSS, and Ca2+ influx was non‐specifically blocked with 1 mM LaCl3. Under these conditions, the [Ca2+]i responses were significantly reduced in the presence of WAY. Further, we examined effects of ER signaling on [Ca2+]i sequestration to SR (SERCA activity). In both control ASM cells as well as in those exposed to cytokines, WAY increased the rate of fall of [Ca2+]i responses to histamine, suggesting greater sequestration of [Ca2+]i.ConclusionsOverall, our data highlights that both ERα and ERβ specific agonist were able to reduce [Ca2+]i in the inflamed ASM via receptor‐mediated genomic action. Specifically, ERβ activation is more effective in the presence of inflammation.Support or Funding InformationSupported by NIH grants R01 HL123494 and R01 HL123494‐02S1 (Venkatachalem)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Apelin impairs myogenic response to induce diabetic nephropathy in mice.

The cause of the invalid reaction of smooth muscle cells to mechanical stimulation that results in a dysfunctional myogenic response that mediates the disruption of renal blood flow (RBF) in patients with diabetes is debatable. The present study revealed that increased apelin concentration in serum of diabetic mice neutralized the myogenic response mediated by apelin receptor (APJ) and resulted in increased RBF, which promoted the progression of diabetic nephropathy. The results showed that apelin concentration, RBF, and albuminuria:creatinine ratio were all increased in kkAy mice, and increased RBF correlated positively with serum apelin both in C57 and diabetic mice. The increased RBF was accompanied by decreased phosphorylation of myosin light chain (MLC), β-arrestin, and increased endothelial NOS in glomeruli. Meanwhile, calcium, phosphorylation of MLC, and β-arrestin were decreased by high glucose and apelin treatment in cultured smooth muscle cells, as well. eNOS was increased by high glucose and increased by apelin in cultured endothelial cells (ECs). Knockdown of β-arrestin expression in smooth muscle cells cancelled phosphorylation of MLC induced by apelin. Therefore, apelin may induce the progression of diabetic nephropathy by counteracting the myogenic response in smooth muscle cells.-Zhang, J., Yin, J., Wang, Y., Li, B., Zeng, X. Apelin impairs myogenic response to induce diabetic nephropathy in mice.

Transcriptomic sequencing reveals diverse adaptive gene expression responses of human vascular smooth muscle cells to nitro-conjugated linoleic acid.

Nitro-conjugated linoleic acid (NO2-CLA) is formed by metabolic and inflammatory reactions of nitric oxide and nitrite, and represents the most abundant nitro-fatty acid species in humans. These electrophilic fatty acid nitroalkene derivatives mediate pleiotropic cell signaling responses. Here, we report a systematic approach to investigate the effect of NO2-CLA on human coronary artery smooth muscle cells (hCASMC), based on the RNA-Seq and bioinformatics analysis. There were extensive differentially expressed genes in NO2-CLA vs. control (510) and NO2-CLA vs. CLA (272) treatment groups, respectively. Notably, only minimal alterations were observed in CLA vs. control conditions, indicating that the electrophilic character of NO2-CLA is requited to induce differential gene expression responses independently from native CLA. Functional enrichment analysis of differentially expressed genes reveals multiple cellular processes to be affected under NO2-CLA treatment, including cell proliferation, lipid metabolism, antioxidant and inflammatory-related gene expression responses. These findings reveal that nitro-fatty acid derivatives such as NO2-CLA regulate a broad array of adaptive gene expression responses by hCASMC.