Response Of Vascular Smooth Muscle Cells Research Articles

A microtubule stability switch alters isolated vascular smooth muscle Ca2+ flux in response to matrix rigidity

ABSTRACT During ageing, the extracellular matrix of the aortic wall becomes more rigid. In response, vascular smooth muscle cells (VSMCs) generate enhanced contractile forces. Our previous findings demonstrate that VSMC volume is enhanced in response to increased matrix rigidity, but our understanding of the mechanisms regulating this process remain incomplete. In this study, we show that microtubule stability in VSMCs is reduced in response to enhanced matrix rigidity via Piezo1-mediated Ca2+ influx. Moreover, VSMC volume and Ca2+ flux is regulated by microtubule dynamics; microtubule-stabilising agents reduced both VSMC volume and Ca2+ flux on rigid hydrogels, whereas microtubule-destabilising agents increased VSMC volume and Ca2+ flux on pliable hydrogels. Finally, we show that disruption of the microtubule deacetylase HDAC6 uncoupled these processes and increased α-tubulin acetylation on K40, VSMC volume and Ca2+ flux on pliable hydrogels, but did not alter VSMC microtubule stability. These findings uncover a microtubule stability switch that controls VSMC volume by regulating Ca2+ flux. Taken together, these data demonstrate that manipulation of microtubule stability can modify VSMC response to matrix stiffness.

Sex differences and role of lysyl oxidase-like 2 in angiotensin II-induced hypertension in mice.

Hypertension, a disease with known sexual dimorphism, accelerates aging-associated arterial stiffening, partly because of the activation of matrix remodeling caused by increased biomechanical load. In this study, we tested the effect of biological sex and the role of the matrix remodeling enzyme lysyl oxidase-like 2 (LOXL2) in hypertension-induced arterial stiffening. Hypertension was induced by angiotensin II (ANG II) infusion via osmotic minipumps in 12- to 14-wk-old male and female mice. Blood pressure and pulse wave velocity (PWV) were measured noninvasively. Wire myography and uniaxial tensile testing were used to test aortic vasoreactivity and mechanical properties. Aortic wall composition was examined by histology and Western blotting. Uniaxial stretch of cultured cells was used to evaluate the effect of biomechanical strain. LOXL2's catalytic function was examined using knockout and inhibition. ANG II infusion-induced hypertension in both genotypes and sexes. Wild-type (WT) males exhibited arterial stiffening in vivo and ex vivo. Aortic remodeling with increased wall thickness, intralamellar distance, higher LOXL2, and collagen I and IV content was noted in WT males. Female mice did not exhibit increased PWV despite the onset of hypertension. LOXL2 depletion improved vascular reactivity and mechanics in hypertensive males. LOXL2 depletion improved aortic mechanics but worsened hypercontractility in females. Hypertensive cyclic strain contributed to LOXL2 upregulation in the cell-derived matrix in vascular smooth muscle cells (VSMCs) but not endothelial cells. LOXL2's catalytic function facilitated VSMC alignment in response to biomechanical strain. In conclusion, in males, arterial stiffening in hypertension is driven both by VSMC response and matrix remodeling. Females are protected from PWV elevation in hypertension. LOXL2 depletion is protective in males with improved mechanical and functional aortic properties. VSMCs are the primary source of LOXL2 in the aorta, and hypertension increases LOXL2 processing and shifts to collagen I accumulation. Overall, LOXL2 depletion offers protection in young hypertensive males and females.NEW & NOTEWORTHY We examined the effect of sex on the evolution of angiotensin II (ANG II)-induced hypertension and the role of lysyl oxidase-like 2 (LOXL2), an enzyme that catalyzes matrix cross linking. While ANG II led to hypertension and worsening vascular reactivity in both sexes, aortic remodeling and stiffening occurred only in males. LOXL2 depletion improved outcomes in males but not females. Thus males and females exhibit a distinct etiology of hypertension and LOXL2 is an effective target in males.

Instability in Computational Models of Vascular Smooth Muscle Cell Contraction

PurposeThrough their contractile and synthetic capacity, vascular smooth muscle cells (VSMCs) can regulate the stiffness and resistance of the circulation. To model the contraction of blood vessels, an active stress component can be added to the (passive) Cauchy stress tensor. Different constitutive formulations have been proposed to describe this active stress component. Notably, however, measuring biomechanical behaviour of contracted blood vessels ex vivo presents several experimental challenges, which complicate the acquisition of comprehensive datasets to inform complex active stress models. In this work, we examine formulations for use with limited experimental contraction data as well as those developed to capture more comprehensive datasets.MethodsFirst, we prove analytically that a subset of constitutive active stress formulations exhibits unstable behaviours (i.e., a non-unique diameter solution for a given pressure) in certain parameter ranges, particularly for large contractile deformations. Second, using experimental literature data, we present two case studies where these formulations are used to capture the contractile response of VSMCs in the presence of (1) limited and (2) extensive contraction data.ResultsWe show how limited contraction data complicates selecting an appropriate active stress model for vascular applications, potentially resulting in unrealistic modelled behaviours.ConclusionOur data provide a useful reference for selecting an active stress model which balances the trade-off between accuracy and available biomechanical information. Whilst complex physiologically motivated models’ superior accuracy is recommended whenever active biomechanics can be extensively characterised experimentally, a constant 2nd Piola-Kirchhoff active stress model balances well accuracy and applicability with sparse contractile data.

Network-based prioritization and validation of regulators of vascular smooth muscle cell proliferation in disease

Aberrant vascular smooth muscle cell (VSMC) homeostasis and proliferation characterize vascular diseases causing heart attack and stroke. Here we elucidate molecular determinants governing VSMC proliferation by reconstructing gene regulatory networks from single-cell transcriptomics and epigenetic profiling. We detect widespread activation of enhancers at disease-relevant loci in proliferation-predisposed VSMCs. We compared gene regulatory network rewiring between injury-responsive and nonresponsive VSMCs, which suggested shared transcription factors but differing target loci between VSMC states. Through in silico perturbation analysis, we identified and prioritized previously unrecognized regulators of proliferation, including RUNX1 and TIMP1. Moreover, we showed that the pioneer transcription factor RUNX1 increased VSMC responsiveness and that TIMP1 feeds back to promote VSMC proliferation through CD74-mediated STAT3 signaling. Both RUNX1 and the TIMP1–CD74 axis were expressed in human VSMCs, showing low levels in normal arteries and increased expression in disease, suggesting clinical relevance and potential as vascular disease targets.

Enhanced expression of miR-20a driven by nanog exacerbated the degradation of extracellular matrix in thoracic aortic dissection

Thoracic aortic dissection (TAD) is a life-threatening vascular disease manifested as intramural bleeding in the medial layers of the thoracic aorta. The key histopathologic feature of TAD is medial degeneration, characterized by depletion of vascular smooth muscle cells (VSMCs) and degradation of extracellular matrix (ECM). MicroRNA, as essential epigenetic regulators, can inhibit the protein expression of target genes without modifying the sequences. This study aimed to elucidate the role and underlying mechanism of miR-20a, a member of the miR-17-92 cluster, in regulating ECM degradation during the pathogenesis of TAD. The expression of the miR-17-92 cluster was significantly increased in synthetic VSMCs derived from TAD lesions compared to contractile VSMCs isolated from normal thoracic aortas. Notably, the expression of miR-20a was increased in VSMCs in response to serum exposure and various stimuli. In TAD lesions, the expression of miR-20a was significantly negatively correlated with that of elastin. Elevated expression of miR-20a was also observed in thoracic aortas of TAD mice induced by β-aminopropionitrile fumarate and angiotensin II. Overexpression of miR-20a via mimic transfection enhanced the growth and invasive capabilities of VSMCs, with no significant impact on their migratory activity or the expression of phenotypic markers (α-SMA, SM22, and OPN). Silencing of miR-20a with inhibitor transfection mitigated the hyperactivation of MMP2 in VSMCs stimulated by PDGF-bb, as evidenced by reduced levels of active-MMP2 and increased levels of pro-MMP2. Subsequently, TIMP2 was identified as a novel target gene of miR-20a. The role of miR-20a in promoting the activation of MMP2 was mediated by the suppression of TIMP2 expression in VSMCs. In addition, the elevated expression of miR-20a was found to be directly driven by Nanog in VSMCs. Collectively, these findings indicate that miR-20a plays a crucial role in maintaining the homeostasis of the thoracic aortic wall during TAD pathogenesis and may represent a potential therapeutic target for TAD.

Abstract 1006: Pathological PERK/ATF4 Activation In Vascular Smooth Muscle Cells Drives Abdominal Aortic Aneurysm Development

Introduction: Abdominal aortic aneurysms (AAA) are characterized by vascular smooth muscle cell (VSMC) apoptosis leading to pathological aortic remodeling. Activation of the endoplasmic reticulum (ER) stress response has been linked to VSMC dysfunction but the driving factors behind ER stress activation and its role on AAA development remains poorly defined. The epigenetic enzyme, MLL1, plays a critical role in establishing cellular phenotype and regulating gene expression. We examine the MLL-mediated epigenetic regulation of the ER stress response in VSMCs during AAA development and establish the ER stress pathways as a novel target in the treatment AAAs. Methods: Single-cell sequencing was conducted on human AAA and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and then fed a saturated fat-enriched diet and infused with AngII (1 μg/kg/min) with or without 4-PBA (20mg/kg/day). Mice with vascular smooth muscle cell-specific knockout of PERK (PERK SMKO ) underwent tamoxifen injection. AAA maximum diameters were quantified and VSMCs were isolated. Eif2a, Atf4 , and Chop gene expression was examined. Apoptosis was quantified by Annexin V staining. Results: Using single-cell RNA sequencing of human aortic tissue, we identified EIF2A, ATF4 , and CHOP were profoundly upregulated in aneurysmal VSMCs as well as two murine AAA models. Mechanistically, TNFα signaling in VSMCs leads to activation of MLL1 which trimethylates histone 3 lysine 4 (H3K4me3) at NF-κB binding sites on the EIF2A and ATF4 gene promoters thereby upregulating the ER stress response and driving VSMC apoptosis. Both In vivo pharmacological inhibition of the PERK/eIF2A/ATF4 ER stress response via 4-PBA injection and vascular smooth muscle depletion of PERK (PERK SMKO ) inhibited AAA formation resulting with in significant reduction in VSMC apoptosis, elastin fragmentation, and inflammatory cytokine infiltration. Conclusions: Taken together, our results identify MLL1-mediated activation of the PERK/eIF2A/ATF4 ER stress response as a critical regulator of VSMC apoptosis in AAA formation and suggest inhibition of ER stress response as a mechanistic target for the management of AAAs.

Abstract 131: Local Pcsk6 Inhibition Attenuates VSMC Response In Vascular Injury And Restenosis

Intro: Vascular smooth muscle cell (VSMC) proliferation following endovascular interventions leads to restenosis, treated with drug-eluting stents. However, current drugs completely abolish vascular healing, cause thrombosis and clinical events. PCSK6 is a key protease in vascular remodeling and VSMCs activation upon vessel injury, via regulation of MMP2/14 activity. Here, we investigate whether local PCSK6 inhibition reduces VSMC proliferation and intimal hyperplasia. Methods: PCSK6 peptide inhibitor was tested in vitro on VSMCs migration, proliferation, apoptosis in comparison to rapamycin and paclitaxel. Internalization of a FITC labelled inhibitor was studied by flow cytometry and immunofluorescence. In vivo , PCSK6 inhibitor was administered at 3mg/kg IP using cationic microbubbles, followed by ultrasound-mediated local delivery in mice developing intimal hyperplasia after carotid ligation (n=8 vs 11). Carotid ligation in Tagln Cre+ /Pcsk6 fl/fl mice investigated the effect of PCSK6 specific ablation in VSMCs on intimal hyperplasia (n=7 vs 9). Carotids were collected from these and constitutive Pcsk6 -/- mice vs. controls for transcriptomic and histological analyses. Results: In vitro , PCSK6 inhibitor was internalized and inhibited both VSMC migration and proliferation (p<0.0001), reducing the expression of PCSK6, MMP2 and GDF15 . In comparison, paclitaxel showed severe VSMC toxicity, whereas rapamycin inhibited more strongly VSMC migration and proliferation than the PCSK6 inhibitor. In vivo, the inhibitor reduced intimal hyperplasia (p=0.026), again, by repressing PCSK6 and MMP2 expression in treated vs. control mice, with a favorable safety profile. Likewise, conditional VSMC Pcsk6 knockouts showed less intimal hyperplasia (p=0.053). In carotids from Pcsk6 -/- mice, cytoskeletal (Synm) and peptidase (Pgpep1l, Klk1) genes were strongly downregulated, showing its importance for vascular matrix composition. Conclusion: Our proof-of-concept translational study shows that local application of a PCSK6 inhibitor is a viable method for specifically modulating VSMC activation in intimal hyperplasia following vascular injury

Salvianolic Acid B Alleviates High Glucose-Induced Vascular Smooth Muscle Cell Inflammation by Upregulating the miR-486a-5p Expression.

The macrovascular complications of diabetes cause high mortality and disability in patients with type 2 diabetes mellitus (T2DM). The inflammatory response of vascular smooth muscle cell (VSMC) runs through its pathophysiological process. Salvianolic acid B (Sal B) exhibits beneficial effects on the cardiovascular system. However, its role and mechanism in diabetic vascular inflammatory response remain unclear. In this study, we found that Sal B reduced vascular inflammation in diabetic mice and high glucose- (HG-) induced VSMC inflammation. Subsequently, we found that Sal B reduced HG-induced VSMC inflammation by downregulating FOXO1. Furthermore, miR-486a-5p expression was obviously reduced in HG-treated VSMC. Sal B attenuated HG-induced VSMC inflammation by upregulating miR-486a-5p. Loss- and gain-of-function experiments had proven that the transfection of the miR-486a-5p mimic inhibited HG-induced VSMC inflammation whereas that of the miR-486a-5p inhibitor promoted HG-induced VSMC inflammation, thereby leading to the amelioration of vascular inflammation in the diabetic mice. Furthermore, studies had shown that miR-486a-5p inhibited FOXO1 expression by directly targeting its 3'-UTR. In conclusion, Sal B alleviates the inflammatory response of VSMC by upregulating miR-486a-5p and aggravating its inhibition of FOXO1 expression. Sal B exerts a significant anti-inflammatory effect in HG-induced VSMC inflammation by modulating the miR-486a-5p/FOXO1 axis.

Piezo1-mediated regulation of smooth muscle cell volume in response to enhanced extracellular matrix rigidity.

Decreased aortic compliance is a precursor to numerous cardiovascular diseases. Compliance is regulated by the rigidity of the aortic wall and the vascular smooth muscle cells (VSMCs). Extracellular matrix stiffening, observed during ageing, reduces compliance. In response to increased rigidity, VSMCs generate enhanced contractile forces that result in VSMC stiffening and a further reduction in compliance. Mechanisms driving VSMC response to matrix rigidity remain poorly defined. Human aortic-VSMCs were seeded onto polyacrylamide hydrogels whose rigidity mimicked either healthy (12 kPa) or aged/diseased (72 kPa) aortae. VSMCs were treated with pharmacological agents prior to agonist stimulation to identify regulators of VSMC volume regulation. On pliable matrices, VSMCs contracted and decreased in cell area. Meanwhile, on rigid matrices VSMCs displayed a hypertrophic-like response, increasing in area and volume. Piezo1 activation stimulated increased VSMC volume by promoting calcium ion influx and subsequent activation of PKC and aquaporin-1. Pharmacological blockade of this pathway prevented the enhanced VSMC volume response on rigid matrices whilst maintaining contractility on pliable matrices. Importantly, both piezo1 and aquaporin-1 gene expression were up-regulated during VSMC phenotypic modulation in atherosclerosis and after carotid ligation. In response to extracellular matrix rigidity, VSMC volume is increased by a piezo1/PKC/aquaporin-1 mediated pathway. Pharmacological targeting of this pathway specifically blocks the matrix rigidity enhanced VSMC volume response, leaving VSMC contractility on healthy mimicking matrices intact. Importantly, upregulation of both piezo1 and aquaporin-1 gene expression is observed in disease relevant VSMC phenotypes.

Abstract 12885: Pathological Perk/Atf4 Activation in Abdominal Aortic Aneurysm Formation

Introduction: Abdominal aortic aneurysms (AAA) are characterized by vascular smooth muscle cell (VSMC) apoptosis leading to pathological aortic remodeling. Activation of the endoplasmic reticulum (ER) stress response has been linked to VSMC dysfunction but the driving factors behind ER stress activation and its role on AAA development remains poorly defined. The epigenetic enzyme, MLL1, plays a critical role in establishing cellular phenotype and regulating gene expression. We examine the MLL-mediated epigenetic regulation of the ER stress response in VSMCs during AAA development and establish the ER stress pathways as a novel target in the treatment AAAs. Methods: Single-cell sequencing was conducted on human AAA and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and then fed a saturated fat-enriched diet and infused with AngII (1 μg/kg/min) with or without 4-PBA (20mg/kg/day). The elastase induced AAA model was used as a secondary murine model. AAA maximum diameters were quantified and VSCMs were isolated. Eif2a, Atf4 , and Chop gene expression was examined. Apoptosis was quantified by Annexin V staining. Results: Using single-cell RNA sequencing of human aortic tissue, we identified EIF2A, ATF4 , and CHOP were profoundly upregulated in aneurysmal VSMCs as well as two murine AAA models. Mechanistically, TNFα signaling in VSMCs leads to activation of MLL1 which trimethylates histone 3 lysine 4 (H3K4me3) at NF-κB binding sites on the EIF2A and ATF4 gene promoters thereby upregulating the ER stress response and driving VSMC apoptosis. In vivo pharmacological inhibition of the eIF2A/ATF4 ER stress response via 4-PBA injection inhibited AAA formation in both the AngII-induced and elastase-induced AAA model resulting with in significant reduction in VSMC apoptosis, elastin fragmentation, and inflammatory cytokine infiltration. Conclusions: Taken together, our results identify MLL1-mediated activation of the eIF2A/ATF4 ER stress response as a critical regulator of VSMC apoptosis in AAA formation and suggest inhibition of ER stress response as a mechanistic target for the management of AAAs.

Alkaloids from <i>Peganum harmala</i> attenuated contractile responses of vascular smooth muscle cells

Purpose: To investigate the contractile responses of vascular smooth muscle cells (VSMCs) to spasmogens after incubation with harmaline, harmine, and harmalol, which are alkaloids obtained from Peganum harmala L., a member of the Zygophyllaceae family.
 Methods: Contractile responses of VSMCs to norepinephrine (NE; 1 µmol/L) and potassium chloride (KCl; 60 mmol/L) were recorded in rat aortic ring preparations pre-incubated with 0.5, 1, 5 and 10 µmol/L of each alkaloid for 15 min. Responses were expressed as mean values of contractions in incubated preparations, relative to the recorded tension prior to treatment with alkaloids.
 Results: Pre- incubation with harmaline at concentration of 10 µmol/L significantly reduced contractile responses to NE by 69.0 ± 3.0 % (p < 0.00002), and decreased KCl-induced contraction by 34.0 ± 9.0 % (p < 0.05). Harmalol was the most effective in inhibiting contractions to KCl (48.0 ± 9.0 %, p < 0.01). However, harmalol produced relatively moderate inhibitory effects on NE-induced contractions (46.0 ± 4.0 %, p < 0.005), followed by harmine (52.0 ± 8.0 %, p < 0.02), but it did not significantly affect contractile responses to KCl.
 Conclusion: These results highlight the differential effects of pre-incubation with alkaloids from P. harmala and their potential effects on the prevention of VSMC spasms induced by either chemicals or stimuli that change the membrane potential.

Calcineurin Is a Universal Regulator of Vessel Function-Focus on Vascular Smooth Muscle Cells.

Calcineurin, a serine/threonine phosphatase regulating transcription factors like NFaT and CREB, is well known for its immune modulatory effects and role in cardiac hypertrophy. Results from experiments with calcineurin knockout animals and calcineurin inhibitors indicate that calcineurin also plays a crucial role in vascular function, especially in vascular smooth muscle cells (VSMCs). In the aorta, calcineurin stimulates the proliferation and migration of VSMCs in response to vascular injury or angiotensin II administration, leading to pathological vessel wall thickening. In the heart, calcineurin mediates coronary artery formation and VSMC differentiation, which are crucial for proper heart development. In pulmonary VSMCs, calcineurin/NFaT signaling regulates the release of Ca2+, resulting in increased vascular tone followed by pulmonary arterial hypertension. In renal VSMCs, calcineurin regulates extracellular matrix secretion promoting fibrosis development. In the mesenteric and cerebral arteries, calcineurin mediates a phenotypic switch of VSMCs leading to altered cell function. Gaining deeper insights into the underlying mechanisms of calcineurin signaling will help researchers to understand developmental and pathogenetical aspects of the vasculature. In this review, we provide an overview of the physiological function and pathophysiology of calcineurin in the vascular system with a focus on vascular smooth muscle cells in different organs. Overall, there are indications that under certain pathological settings reduced calcineurin activity seems to be beneficial for cardiovascular health.

Atherogenic Activation of Human Vascular Smooth Muscle Cells by Monosodium Urate Crystals

Gout is strongly associated with atherosclerosis and other cardiovascular comorbidities. Furthermore, sites of extra-articular monosodium urate (MSU) crystal deposits in gout can include heart valves and atherosclerotic artery plaques, but with unclear effects therein. Hence, we seminally explored cultured vascular smooth muscle cell (VSMC) responsiveness to MSU crystals. To limit confounding effects, we cultured human aortic VSMCs under serum-free conditions to assess MSU crystal effects on VSMC differentiation and function, differentially expressed genes (DEGs) via RNA sequencing, and selected atherogenic changes in cytokines and the lipidome. MSU crystals induced p38 phosphorylation, IL-6, and VSMC vacuolization with dysregulated autophagy. MSU-crystal-induced DEGs included decreased late-stage autophagosome maturation mediator GABARAPL1, decreased physiologic VSMC differentiation regulators (LMOD1 and SYNPO2), increased ATF4, CHOP, and the intrinsic apoptosis signaling pathway in response to ER stress, and neointimal atherogenic nuclear receptors (NR4A1 and NR4A3). MSU crystals alone increased the levels of cholesterol biosynthetic intermediates 14-demethyl-lanosterol (14-DML), desmosterol, and zymosterol. Adding MSU crystals increased oxidized LDL’s capacity to increase intracellular 27-OH cholesterol, and MSU crystals and oxidized LDL synergistically induced a marked release of arachidonate. In conclusion, MSU crystals deposited in arterial media and neointima have the potential to dysregulate VSMC differentiation and proteostasis, and to induce further atherogenic effects, which include enhanced VSMC loading of oxidized cholesterol intermediates and release of IL-6 and arachidonic acid (AA).

Inhibition of lncRNA NFIA-AS1 Alleviates Abnormal Proliferation and Inflammation of Vascular Smooth Muscle Cells in Atherosclerosis by Regulating miR-125a-3p/AKT1 Axis.

Vascular smooth muscle cells (VSMCs) are critical elements of the vascular wall and play a crucial role in the genesis and development of atherosclerosis (AS). Increasingly, studies have indicated that long noncoding RNAs (lncRNAs) regulate VSMC proliferation, apoptosis, and other biological processes. Nevertheless, the role of lncRNA NFIA-AS1 (hereinafter referred to as NFIA-AS1) in VSMCs and AS remains unclear. Quantitative real-time PCR (qRT-PCR) was performed to analyze the messenger RNA (mRNA) levels of NFIA-AS1 and miR-125a-3p. CCK-8 and EdU staining were performed to detect VSMC proliferation. VSMC apoptosis was evaluated by flow cytometry. The expression of various proteins was detected using western blotting. The levels of inflammatory cytokines secreted by VSMCs were measured by enzyme linked immunosorbent assay (ELISA). The binding sites of NFIA-AS1 and miR-125a-3p, as well as miR-125a-3p and AKT1, were analyzed using bioinformatics methods and validated using a luciferase reporter assay. The function of NFIA-AS1/miR-125a-3p/AKT1 in VSMCs was clarified through loss- and gain-of-functional experiments. We confirmed that NFIA-AS1 was highly expressed in AS tissues and VSMCs induced by oxidized low-density lipoprotein (Ox-LDL). Knockdown of NFIA-AS1 restrained the exceptional growth of Ox-LDL-induced VSMCs, promoted their apoptosis, and decreased the secretion of inflammatory factors and expression of adhesion factors. In addition, NFIA-AS1 regulated the proliferation, apoptosis, and inflammatory response of VSMCs through the miR-125a-3p/AKT1 axis, suggesting that NFIA-AS1 may be a potential therapeutic target for AS.

Pericytes protect rats and mice from sepsis-induced injuries by maintaining vascular reactivity and barrier function: implication of miRNAs and microvesicles

BackgroundVascular hyporeactivity and leakage are key pathophysiologic features that produce multi-organ damage upon sepsis. We hypothesized that pericytes, a group of pluripotent cells that maintain vascular integrity and tension, are protective against sepsis via regulating vascular reactivity and permeability.MethodsWe conducted a series of in vivo experiments using wild-type (WT), platelet-derived growth factor receptor beta (PDGFR-β)-Cre + mT/mG transgenic mice and Tie2-Cre + Cx43flox/flox mice to examine the relative contribution of pericytes in sepsis, either induced by cecal ligation and puncture (CLP) or lipopolysaccharide (LPS) challenge. In a separate set of experiments with Sprague–Dawley (SD) rats, pericytes were depleted using CP-673451, a selective PDGFR-β inhibitor, at a dosage of 40 mg/(kg·d) for 7 consecutive days. Cultured pericytes, vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) were used for mechanistic investigations. The effects of pericytes and pericyte-derived microvesicles (PCMVs) and candidate miRNAs on vascular reactivity and barrier function were also examined.ResultsCLP and LPS induced severe injury/loss of pericytes, vascular hyporeactivity and leakage (P < 0.05). Transplantation with exogenous pericytes protected vascular reactivity and barrier function via microvessel colonization (P < 0.05). Cx43 knockout in either pericytes or VECs reduced pericyte colonization in microvessels (P < 0.05). Additionally, PCMVs transferred miR-145 and miR-132 to VSMCs and VECs, respectively, exerting a protective effect on vascular reactivity and barrier function after sepsis (P < 0.05). miR-145 primarily improved the contractile response of VSMCs by activating the sphingosine kinase 2 (Sphk2)/sphingosine-1-phosphate receptor (S1PR)1/phosphorylation of myosin light chain 20 pathway, whereas miR-132 effectively improved the barrier function of VECs by activating the Sphk2/S1PR2/zonula occludens-1 and vascular endothelial-cadherin pathways.ConclusionsPericytes are protective against sepsis through regulating vascular reactivity and barrier function. Possible mechanisms include both direct colonization of microvasculature and secretion of PCMVs.

Regulation by Nrf2 of IL-1β-induced inflammatory and oxidative response in VSMC and its relationship with TLR4.

Introduction: Vascular oxidative stress and inflammation play an important role in the pathogenesis of cardiovascular diseases (CVDs). The proinflammatory cytokine Interleukin-1β (IL-1β) participates in the vascular inflammatory and oxidative responses and influences vascular smooth muscle cells (VSMC) phenotype and function, as well as vascular remodelling in cardiovascular diseases. The Toll-like receptor 4 (TLR4) is also involved in the inflammatory response in cardiovascular diseases. A relationship between Interleukin-1β and Toll-like receptor 4 pathway has been described, although the exact mechanism of this interaction remains still unknown. Moreover, the oxidative stress sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) promotes the transcription of several antioxidant and anti-inflammatory genes. Nuclear factor-erythroid 2-related factor 2 activators have shown to possess beneficial effects in cardiovascular diseases in which oxidative stress and inflammation are involved, such as hypertension and atherosclerosis; however, the molecular mechanisms are not fully understood. Here, we analysed the role of Toll-like receptor 4 in the oxidative and inflammatory effects of Interleukin-1β as well as whether nuclear factor-erythroid 2-related factor 2 activation contributes to vascular alterations by modulating these effects. Materials: For this purpose, vascular smooth muscle cells and mice aortic segments stimulated with Interleukin-1β were used. Results: Interleukin-1β induces MyD88 expression while the Toll-like receptor 4 inhibitor CLI-095 reduces the Interleukin-1β-elicited COX-2 protein expression, reactive oxygen species (ROS) production, vascular smooth muscle cells migration and endothelial dysfunction. Additionally, Interleukin-1β increases nuclear factor-erythroid 2-related factor 2 nuclear translocation and expression of its downstream proteins heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and superoxide dismutase-2, by an oxidative stress-dependent mechanism; moreover, Interleukin-1β reduces the expression of the nuclear factor-erythroid 2-related factor 2 inhibitor Keap1. The nuclear factor-erythroid 2-related factor 2 activator tert-butylhydroquinone (tBHQ) reduces the effects of Interleukin-1β on the increased reactive oxygen species production and the expression of the proinflammatory markers (p-p38, p-JNK, p-c-Jun, COX-2), the increased cell proliferation and migration and prevents the Interleukin-1β-induced endothelial dysfunction in mice aortas. Additionally, tert-butylhydroquinone also reduces the increased MyD88 expression, NADPHoxidase activity and cell migration induced by lipopolysaccharide. Conclusions: In summary, this study reveals that Toll-like receptor 4 pathway contributes to the prooxidant and proinflammatory Interleukin-1β-induced effects. Moreover, activation of nuclear factor-erythroid 2-related factor 2 prevents the deleterious effects of Interleukin-1β, likely by reducing Toll-like receptor 4-dependent pathway. Although further research is needed, the results are promising as they suggest that nuclear factor-erythroid 2-related factor 2 activators might protect against the oxidative stress and inflammation characteristic of cardiovascular diseases.

Gene expression of proinflammatory cytokines in human coronary artery smooth muscle cells exposed to alkylating mutagen

Background. It is known that DNA damage in smooth muscle cells can trigger their clonal expansion and transformation into foam cells. Thus, the study of the molecular genetic mechanisms of the vascular smooth muscle cells response to genotoxic exposure is important and relevant in the context of an in-depth understanding of atherogenesis.Aim. To study mRNA level and concentration of proinflammatory cytokines IL6 and IL8 in the human coronary artery smooth muscle cells exposed to alkylating mutagen.Methods. Gene expression signature of studied cytokines in the human coronary artery smooth muscle cells was accessed by quantitative polymerase chain reaction in the two timepoints – immediately after six-hour exposure to mitomycin C (point 1) and after six-hour exposure to mitomycin C followed by 24 hours of cells being cultivated on mitomycin C-free cell growth medium (point 2). Smooth muscle cells cultured according to the above scheme without genotoxin were used as controls. HPRT1, GAPDH and B2M were used as the reference genes. Gene expression level was calculated by ΔCt method. IL6 and IL8 concentration was evaluated in the culture media in points 1 and 2 by enzyme-linked immunosorbent assay. Statistical analysis was performed in GraphPad Prism 9 software.Results. Immediately after mutagenic exposure (point 1) we discovered no significant changes in the expression level of IL6 and IL8 in the mitomycin C exposed smooth muscle cells compared to controls. Removal of mutagen increased expression of IL6 and IL8 in the experimental group (0,36- and 0,67-fold, respectively). At the same time, we discovered no significant differences in the studied cytokines concentration in the culture medium of mutagen-exposed cells compared to the nonexposed controls.Conclusion. Genotoxic stress in human coronary artery smooth muscle cells exposed to alkylating mutagen (mitomycin C) leads to differential expression but not secretion of proinflammatory cytokines IL6 and IL8. Thus, exposure of smooth muscle cells to mitomycin C do not trigger their proinflammatory phenotype.

Bisphenol A exposure inhibits vascular smooth muscle cell responses: Involvement of proliferation, migration, and invasion

Previous studies have associated bisphenol A (BPA) with malignant tumor formation, infertility, and atherosclerosis in vitro and in vivo. However, the precise mechanisms through which BPA affects the cardiovascular system under normal conditions remain unclear. Therefore, this study investigated the biological mechanisms through which BPA affects the responses of aortic vascular smooth muscle cells (VSMCs). BPA treatment inhibited the proliferative activity of VSMCs and induced G2/M-phase cell cycle arrest via stimulation of the ATM-CHK2-Cdc25C-p21WAF1-Cdc2 cascade in VSMCs. Furthermore, BPA treatment upregulated the phosphorylation of mitogen-activated protein kinase (MAPK) pathways such as ERK, JNK, and p38 MAPK in VSMCs. However, the phosphorylation level of AKT was down-regulated by BPA treatment. Additionally, the phosphorylation of ERK, JNK, and p38 MAPK was suppressed when the cells were treated with their respective inhibitors (U0126, SP600125, and SB203580). BPA suppressed MMP-9 activity by reducing the binding activity of AP-1, Sp-1, and NF-κB, thus inhibiting the invasive and migratory ability of VSMCs. These data demonstrate that BPA interferes with the proliferation, migration, and invasion capacities of VSMCs. Therefore, our findings suggest that overexposure to BPA can lead to cardiovascular damage due to dysregulated VSMC responses.

Lin28b delays vasculature aging by reducing platelet-derived growth factor-beta resistance in senescent vascular smooth muscle cells

Background and aimsPlatelet-derived growth factor-β (PDGFB) is an important mediator of vascular smooth muscle cell (VSMC) proliferation, and PDGFB resistance is observed in senescent VSMCs. Lin28b is a stemness regulator in the embryo; however, its role in vasculature aging and VSMC senescence is unknown. We aimed to investigate whether Lin28b could restore the VSMC response to PDGFB and delay vasculature aging. MethodsApoE−/− mice were fed a high-fat diet for different weeks to establish an aging model. PDGFB resistance was observed using EdU staining in vessel culture in vitro. Quantitative polymerase chain reaction and in situ hybridization were used to detect let-7 expression. Senescence was identified by Western blotting, senescence-associated beta-galactosidase activity or Sudan Black B staining, and VSMC function was determined using CCK-8, migration, and enzyme-linked immunosorbent assays. ResultsVessels from aged mice showed poor responses to PDGFB stimulation compared with those from young mice; similar results were found in senescent VSMCs. The expression levels of Lin28b and PDGF receptor-β were downregulated in aging vasculature and senescent VSMCs, whereas let-7 family levels increased with aging and VSMC passage growth. Transfection of VSMCs with let-7c induced PDGFB resistance and accelerated VSMC senescence, whereas blocking let-7c restored PDGFB reactions in VSMCs. Overexpression of Lin28b protein by lentivirus resulted in the restoration of PDGFB reactions and delayed VSMC senescence, which was blocked by a let-7c mimic. ConclusionsThis study reveals the role of Lin28b in delaying vasculature aging by decreasing senescent VSMC PDGFB resistance mediated by let-7.

Abstract P054: Loss Of The Smooth-muscle-cell-angiotensin Ii-sensitive Lncrna Leads To Smc Hypertrophic And Hypertensive Remodeling Due To Cell Cycle Dysregulation.

Vascular smooth muscle cells (SMC) responses to increased levels of Ang II during hypertensive remodeling included enhanced vasoconstriction and hypertrophy due to the enlargement of the SMC on the vessel wall. Hypertrophic SMC usually present defects in cell division and cell polyploidy. However, the molecular mechanisms controlling SMC hypertrophy and polyploidy vs. hyperplasia are still not fully understood. Long-non-coding-RNAs (LncRNAs) are epigenetic regulators of gene expression influencing biological processes, including cell division fidelity. We discovered a novel lncRNA decreased in dedifferentiated SMC, the SMC-Ang II-Sensitive (SAS) lncRNA, which expression is reduced in response to Ang-II in cultured SMC and the aorta of hypertensive mice, suggesting a role in mediating hypertension-induced SMC hypertrophy. Publicly available transcriptional datasets revealed that SAS is preferentially expressed in SMC-rich tissues, including the aorta and renal artery, in humans and mice. Yet, the functional relevance of SAS in SMC has never been investigated. Knockdown of SAS reduces proliferation and migration in SMC treated with Platelet-Derived Growth Factor (PDGF-BB). SAS knockdown was also associated with distinct SMC hypertrophic morphological changes, including enlargement in cell size and polynucleation in vitro. We have generated a SAS KO mouse, and aortas from these mice present a higher number of polynucleated medial SMC than their WT littermates. These data correlate with a cell cycle arrest in G1 and senescence phenotype that SAS deficient SMC present due to dysregulation in cyclins expression. Furthermore, SAS defective cells show mitochondria hyperfusion and increased oxygen consumption that correlates with the observed senescence and arrest on the G1/S checkpoint, and it is exacerbated by treatment with Ang-II. Interestingly, treatment with Losartan, an Ang-II receptor inhibitor, rescues SAS expression on Ang-II treated SMC and diminishes SMC hypertrophy. Together, these observations suggest that a decrease in SAS causes SMC hypertrophy due to defects in cell cycle completion. SAS is a potent regulator of SMC morphology and is required for proper cell division and mitochondria organization.