Coronary Vascular Smooth Muscle Research Articles

Smooth Muscle Cofilin Confers Elasticity to Coronary Resistance Microvessels in Diabetes

Coronary resistance microvessels (CRMs) from type 2 diabetic (T2D) mice and pigs are less stiff compared to normal, a finding that is dictated by less stiff coronary vascular smooth muscle cells (VSMCs) Cofilin is an endogenous actin regulatory protein that depolymerizes filamentous F-actin, and recent studies have shown that portions of F-actin bound to cofilin are less stiff compared to their unbound F-actin counterparts. In this study, we tested the hypothesis that augmented cofilin confers elasticity to diabetic CRMs. CRMs were isolated and pooled (n=6 pooled CRMs per biological replicate) for proteomic data collection from both normal (n=3) and diabetic (n=5) mice. In separate experiments, we utilized deidentified normal and T2D human primary coronary VSMCs obtained from commercial vendors (Lonza and ATCC; n=4 per group) and experiments were performed on passage 5-8 VSMCs. Using these cells, we determined cofilin expression by Western blot, and cofilin siRNA (Cell Signaling Technology) was used 48 hours prior to experimentation to knock down cofilin expression. Elastic modulus, a measure of cell stiffness, was assessed by atomic force microscopy (AFM). Globular G-actin was labeled with fluorescein-deoxyribonuclease I (DNAse I) and F-actin was labeled with phalloidin. Images were taken in blinded manner and Image J was used to assess expression. In the proteomics screen, we observed zero cofilin spectral hits in the normal CRMs and 3-4 spectral hits in db/db CRMs (0.00 ± 0.0 vs. 3.40 ± 0.25, p<0.0001). Cofilin protein was increased in the coronary VSMCs of diabetic patients compared to normal coronary VSMCs (1.00 ± 0.16 vs. 1.837± 0.35, p=0.052). In human coronary VSMCs, silencing cofilin caused a significant increase in the F/G actin ratio when normal and diabetic data were pooled (scramble 3.029 ± 0.49 vs. siRNA 4.029 ± 0.68, p= 0.018, n=8). This increased F/G ratio was significant in the diabetic coronary VSMCs (scramble 2.636 ± 0.46 vs. siRNA 3.685 ± 0.41, p=0.049, n=4), and trended in the normal cells but did not reach statistical significance (scramble 3.423 ± 0.89 vs. cofilin siRNA 4.373 ± 1.37, p=0.224, n=4). Cofilin knockdown also caused a significant increase in elastic modulus by AFM when normal and diabetic data were pooled (scramble siRNA 1.814 ± 0.25 vs. cofilin siRNA 3.074 ± 0.35, p=0.0057, n=8). This trend persisted in the normal VMSCs (scramble siRNA 2.038 ± 0.31 vs. cofilin siRNA 3.445± 0.30, p=0.075, n=4) and in the diabetic VSMCs (scramble siRNA 1.589 ± 0.40 vs. cofilin siRNA 2.702 ± 0.62, p=0.085, n=4) but did not reach significance due to low available sample number. Overall, we demonstrate increased cofilin in human diabetic coronary VSMCs and in CRMs from diabetic mice, and silencing cofilin increased F/G actin ratio and increased cellular stiffness. These data support the notion that increased cofilin in T2D VSMCs may be the cause of decreased diabetic CRM stiffness. Support: NIH R00 HL116769, R01 HL165124, and S10 OD023438 and Nationwide Children’s Hospital to AJT. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Coronary Spasm Testing with Acetylcholine: A Powerful Tool for a Personalized Therapy of Coronary Vasomotor Disorders.

Coronary vasomotor disorders (CVD) are characterized by transient hypercontraction of coronary vascular smooth muscle cells, leading to hypercontraction of epicardial and/or microvascular coronary circulation. CVDs play a relevant role in the pathogenesis of ischemia, angina and myocardial infarction with non-obstructive coronary arteries. Invasive provocative testing with intracoronary Acetylcholine (ACh) administration is the gold standard tool for addressing CVD, providing relevant therapeutic and prognostic implications. However, safety concerns preclude the widespread incorporation of the ACh test into clinical practice. The purpose of this review is to shed light on the pathophysiology underlying CVD and on the clinical role of the ACh test, focusing on safety profile and prognostic implications. We will also discuss contemporary evidence on the management of CVD and the role of the ACh test in driving a personalized approach of patients with CVD.

2 - Lack of SIRT1 increases risk of imflammaging in coronary artery vascular smooth muscle cells

2 - Lack of SIRT1 increases risk of imflammaging in coronary artery vascular smooth muscle cells

Phosphoglycerate Dehydrogenase Overexpression Inhibits Ferroptosis to Repress Calcification of Human Coronary Artery Vascular Smooth Muscle Cells via the P53/SLC7A11 Pathway.

Coronary artery calcification (CAC) is in almost all patients with coronary artery disease and requires more effective therapies. We aim to explore the effects of phosphoglycerate dehydrogenase (PHGDH) on CAC. We identified the differentially expressed genes through bioinformatic analysis and selected PHGDH for further verification. Human coronary artery smooth muscle cells (HCASMCs) cultured with calcifying medium were used as models of CAC in vitro. Erastin was administered to induce ferroptosis. We determined the cell viability by the cell count kit-8 assay. The alkaline phosphatase activity, calcium content, and the expression of glutathione were evaluated by the corresponding detection kits. The calcification level was detected by alizarin red staining. Then we performed Western blot to examine the expression of runt-related transcription factor 2, bone morphogenetic protein 2, cyclooxygenase 2, glutathione peroxidase 4, P53, and solute carrier family 7a member 11 (SLC7A11). We acquired 201 differentially expressed genes and selected PHGDH to verify. In calcifying medium-induced HCASMCs, PHGDH overexpression increased the cell viability and decreased the alkaline phosphatase activity, calcium content, calcification level, and the expression of bone morphogenetic protein 2 and runt-related transcription factor 2. Additionally, we found higher levels of glutathione, glutathione peroxidase 4, and SLC7A11 and lower levels of cyclooxygenase 2 and P53 after up-regulating PHGDH. Erastin reversed the effects of PHGDH on calcification of HCASMCs. PHGDH overexpression suppresses the calcification level of HCASMCs by inhibiting ferroptosis through the P53/SLC7A11 signaling pathway, suggesting PHGDH as a promising therapeutic target of CAC.

The selective Rho Kinase 2 inhibitor Chroman-1, is a potent vasodilator but does not compromise left ventricular function in-vivo

Abstract Background In the field of cardioprotection, focus is turning to limiting microvascular obstruction (MVO) post myocardial infarction. The development of MVO carries a poor prognosis, which is independent of infarct burden [1]. Commonly used vasodilators in the treatment of MVO e.g., Ca2+ blockers, beta-blockers can induce negatively inotropic effects. This is a serious problem as hypotension and shock during acute MI are harmful. Rho kinase (ROCK) inhibitors induce vasodilation by inhibiting the phosphorylation of myosin light chain kinase (MLC2) in vascular smooth muscle (VSMC) [2]. ROCK1/2 inhibitors are known to cause transient negative inotropic effects [3]. However, the potential side-effects of the selective ROCK2i, Chroman-1 (Ch1) are unknown. Purpose We aimed to i) Confirm the expression of ROCK2 mRNA in aorta and coronary vasculature, ii) To investigate the vasodilatory properties of Ch1 iii) To confirm that Ch1 does not acutely impair left ventricular ejection fraction (LVEF%) and blood pressure in-vivo. Methods RNA scope in-situ hybridisation was performed with a fluorescent, multiplex assay for ROCK1/2 & VSMC mRNA in whole hearts, coronary vasculature and aorta from male SD rats. Thoracic aorta was dissected and 3mm segments suspended into a tissue bath. Vascular rings were constricted with 1µM PE and treated with [10-12]-[10-5]mM Ch1. In a separate set of in-vivo experiments, male SD rats 250-300g were anaesthetised with isoflurane, intubated and ventilated. LVEF% was measured in a closed chest model, following the addition of Ch1 (30-300µg/kg) or DMSO vehicle, administered i.p at 15-minute intervals. Mean arterial pressure (MAP, mmHg) and heart rate (BPM) were measured throughout. Results ROCK2 mRNA was localised to myocardium, aorta and coronary circulation VSMC. Figure 1 demonstrates that Ch1 induced dose dependent vasodilation of PE constricted aortic rings vs control (EC50 Ch1 95% CI [5.1e-008, 2.2e-007] n=6). In-vivo, Ch1 at all doses (30-300µg/kg) did not significantly affect LVEF% (p>0.05, n=5) nor average MAP at doses 30-100µg/kg (p>0.05, n=5). At the highest dose, i.e 300µg/kg, Ch1 decreased average MAP (mmHg) compared to control +/-SEM (76+/-7.1 vs 92+/-2.8, p=0.02, n=5), but not below 70mmHg threshold. Ch1 did not significantly affect mean heart rate at any dose (p>0.05, n=5). Conclusion The selective ROCK2 inhibitor Ch1, induces potent arterial vasodilation, without compromising LVEF% or haemodynamics. It is therefore, a suitable drug for further preclinical studies of MVO in ischaemia/reperfusion (I/R).Figure 1:Ch1 induces dose dependent vasodilation of aortic rings, **p<0.005, ***p<0.001

Plasma polymerized nanoparticles are a safe platform for direct delivery of growth factor therapy to the injured heart.

Introduction: Heart failure due to myocardial infarction is a progressive and debilitating condition, affecting millions worldwide. Novel treatment strategies are desperately needed to minimise cardiomyocyte damage after myocardial infarction and to promote repair and regeneration of the injured heart muscle. Plasma polymerized nanoparticles (PPN) are a new class of nanocarriers which allow for a facile, one-step functionalization with molecular cargo. Methods: Here, we conjugated platelet-derived growth factor AB (PDGF-AB) to PPN, engineering a stable nano-formulation, as demonstrated by optimal hydrodynamic parameters, including hydrodynamic size distribution, polydisperse index (PDI) and zeta potential, and further demonstrated safety and bioactivity in vitro and in vivo. We delivered PPN-PDGF-AB to human cardiac cells and directly to the injured rodent heart. Results: We found no evidence of cytotoxicity after delivery of PPN or PPN-PDGFAB to cardiomyocytes in vitro, as determined through viability and mitochondrial membrane potential assays. We then measured contractile amplitude of human stem cell derived cardiomyocytes and found no detrimental effect of PPN on cardiomyocyte contractility. We also confirmed that PDGF-AB remains functional when bound to PPN, with PDGF receptor alpha positive human coronary artery vascular smooth muscle cells and cardiac fibroblasts demonstrating migratory and phenotypic responses to PPN-PDGF-AB in the same manner as to unbound PDGF-AB. In our rodent model of PPN-PDGF-AB treatment after myocardial infarction, we found a modest improvement in cardiac function in PPN-PDGF-AB treated hearts compared to those treated with PPN, although this was not accompanied by changes in infarct scar size, scar composition, or border zone vessel density. Discussion: These results demonstrate safety and feasibility of the PPN platform for delivery of therapeutics directly to the myocardium. Future work will optimize PPN-PDGF-AB formulations for systemic delivery, including effective dosage and timing to enhance efficacy and bioavailability, and ultimately improve the therapeutic benefits of PDGF-AB in the treatment of heart failure cause by myocardial infarction.

4 - Increased pro-inflammatory transcriptome signature in coronary artery vascular smooth muscle cells

4 - Increased pro-inflammatory transcriptome signature in coronary artery vascular smooth muscle cells

Abstract 11380: Exercise Induced ST Segment Elevation Caused by Coronary Vasospasm

Case Presentation: A 52-year-old male with no known medical history was seen in the cardiology clinic for occasional pressure-like chest pain on exertion. Due to an intermediate pretest probability of CAD, ischemic workup with a nuclear perfusion exercise stress test was planned. Patient achieved maximum predicted heart rate after 12 minutes of exercising, with no anginal symptoms, ST segment changes or arrhythmias during the entire period. However, 3 minutes into the recovery phase, he developed chest pain and diaphoresis, with EKG now showing 7mm ST elevations in the inferior leads and BP falling to 77/56 mmHg. STEMI code was called, and patient was emergently taken to the cardiac cath lab for coronary angiogram and possible PCI. Angiogram found the coronary arteries to be non-obstructive but revealed vessels possibly having vasospasm. Intra coronary nitroglycerin was given with no further PCI. The patient’s symptoms subsided after the angiogram, and he remained asymptomatic thereupon and was eventually discharged on Verapamil and nitrate therapy. Discussion: Typically, vasospastic angina occurs at rest. Exercise induced and post-exercise recovery phase ST segment elevation on EKG is associated with severe atherosclerotic disease with significant coronary obstruction. However, exercise induced ST elevations in the post exercise recovery phase caused by non-obstructive, vasospastic coronary arteries, as seen in this patient, is a rare occurrence. The vasospasm is hypothesized to be due to coronary vascular smooth muscle hyperactivity and endothelial cell dysfunction. Treatment is usually with calcium channel blockers, nitrates and lifestyle modification such as smoking cessation and alcohol restriction. This case illustrates the importance of identifying the etiology of the angina, noting that post exercise ST segment elevations are not always associated with coronary vessel obstruction and vasospasm should be considered as a possible etiology.

Determining the Association Between Density of Muscarinic Acetylcholine Receptor M3 in Myocardium and Tunica Media of Coronary Vasculature and Self‐Reported Disease States

Myocardial infarction causes parasympathetic dysfunction in cardiovascular tissue, where central parasympathetic drive decreases but local acetylcholine levels are unchanged. However, muscarinic acetylcholine receptor M3 (AChM3R) is shown to have mediating effects in cardiac tissue and vasculature, such as regulation of heart rate and vasodilation of coronary arteries. The objective of this study is to determine the association between AChM3R levels in the heart wall and in the coronary vasculature with self‐reported disease states from cadaver donor records.Biopsies of the left anterior descending (LAD) artery and the underlying anterior interventricular septum (AIVS) were taken from 14 cadavers as opportunity samples. A 5 mm biopsy of the LAD and its underlying AIVS were harvested 2.54‐3.81cm from the bifurcation point of the left coronary artery, depending on the tissue integrity of the cadaver specimen. The tissues were immunostained for AChM3R (CHRM3 NB100‐58975 at 1:2000) and visualized using a DAB chromogen. Slides were digitally scanned into a virtual image at 20x using the Motic Easyscan (Motic, Inc.). Images were segmented for tunica media and myocardium using Adobe Photoshop CS (Adobe, Inc.). We developed code in Python to calculate the strong positive and positive staining of the tissues. The number of pixels stained was normalized to the tissue area. The donors from which tissue was biopsied were classified into cardiovascular, cerebrovascular, Alzheimer’s disease dementia (AD), or both cerebrovascular and AD disease categories based on self‐reported donor declarations. We used SPSS (v27, IBM, Inc.) to run a Pearson correlation to determine the association of staining positivity in the anatomical regions and a MANOVA to determine significant differences in the amount of pixel positivity in the tunica media and myocardium as a function of disease classification.Strong positive and positive staining of the myocardium were not correlated with strong positive in the tunica media. There was a significant, strong, and positive relationship between strong myocardium staining and positive tunica media staining (r =.727, n=14, p=.003) as well as positive myocardium and positive tunica media staining (r=.674, n=14, p=.008). There is no significant difference between the amount of AChM3R staining and disease classification.The lack of correlation between strong positive in the tunica media and positive and strong positive in the myocardium suggests that changes in the AChM3R of coronary vascular smooth muscle are independent of the AChM3R state in the heart wall. However, significant correlation between strong positive in the myocardium and positive in the tunica media indicates that changes in AChM3R in the myocardium could be dependent on the presence of a baseline amount of AChM3R in the coronary vascular smooth muscle. Categorization of donor disease states may be too broad to provide statistical significance. Many donors reported co‐morbid diseases, which could have affected the influence of cardiovascular disease and dementia on AChM3R modulation. These findings highlight that interventions targeting parasympathetic dysfunction of coronary vasculature may leave the underlying heart wall unaffected. Future studies should consider cardiovascular disease diagnoses to assess AChM3R distribution in the heart wall and coronary vasculature.

Substrate Stiffness Modulates Normal and Diabetic Coronary Smooth Muscle Cell Stiffness

We have previously reported that coronary resistance microvessels (CRMs) from 16‐week‐old type 2 diabetic (db/db) mice were less stiff compared to normal heterozygous Db/db controls, a finding that was driven by less stiff coronary vascular smooth muscle cells (VSMCs). Because the surrounding extracellular matrix (ECM) substrates in vivo may impact cellular properties such as elastic modulus (a measure of stiffness), the aim of this study was to test the hypothesis that modulating substrate stiffness correspondingly modulates the elastic modulus of normal and diabetic coronary VSMCs in vitro. In this study, we plated low‐passage coronary VSMCs isolated from Db/db and db/db mice onto 60 mm polyacrylamide dishes (Matrigen) that were of varying stiffnesses – 4 kPa, 25 kPa, and 50 kPa (n=4 per group). Cells were serum starved for 24 hours, and then cell stiffness was measured by atomic force microscopy (AFM). We observed a stepwise increase in cellular elastic modulus with each increase in polyacrylamide substrate stiffness in both the normal and diabetic coronary VSMCs (Db/db 4 kPa = 3.39±0.48 kPa, Db/db 25 kPa = 5.46±0.78 kPa, Db/db 50 kPa = 7.13±0.29 kPa; db/db 4 kPa = 2.49±0.23, db/db 25 kPa = 5.00±0.52 kPa, db/db 50 kPa = 5.58±0.16 kPa, p<0.0001). Similar to what we have previously reported on cells grown on plastic, diabetic coronary VSMCs were consistently less stiff compared to normal at each substrate stiffness. We did not observe statistically significant differences in cellular filamentous (F) actin, globular (G) actin, or the F/G actin ratio on the varying polyacrylamide substrate stiffness for neither Db/db nor db/db coronary VSMCs. However, there was a preliminary trend for lower F actin (Db/db: 1141.00 ± 472.20 AU vs. db/db: 422.50 ± 240.30 AU at 4 kPa, p=0.24, n=2 per group) and F/G actin ratio (Db/db: 4.01 ± 0.92 AU vs. db/db: 2.55 ± 0.35 AU at 4 kPa, p=0.21, n=2 per group) in the db/db VSMCs, corroborating our past data demonstrating that db/db coronary VSMCs are less stiff compared to normal Db/db coronary VSMCs. Overall, these data show that substrate stiffness correspondingly modulates normal and diabetic coronary VSMC stiffness.

Regulation of Epicardial Cell Fate during Cardiac Development and Disease: An Overview.

The epicardium is the outermost cell layer in the vertebrate heart that originates during development from mesothelial precursors located in the proepicardium and septum transversum. The epicardial layer plays a key role during cardiogenesis since a subset of epicardial-derived cells (EPDCs) undergo an epithelial–mesenchymal transition (EMT); migrate into the myocardium; and differentiate into distinct cell types, such as coronary vascular smooth muscle cells, cardiac fibroblasts, endothelial cells, and presumably a subpopulation of cardiomyocytes, thus contributing to complete heart formation. Furthermore, the epicardium is a source of paracrine factors that support cardiac growth at the last stages of cardiogenesis. Although several lineage trace studies have provided some evidence about epicardial cell fate determination, the molecular mechanisms underlying epicardial cell heterogeneity remain not fully understood. Interestingly, seminal works during the last decade have pointed out that the adult epicardium is reactivated after heart damage, re-expressing some embryonic genes and contributing to cardiac remodeling. Therefore, the epicardium has been proposed as a potential target in the treatment of cardiovascular disease. In this review, we summarize the previous knowledge regarding the regulation of epicardial cell contribution during development and the control of epicardial reactivation in cardiac repair after damage.

Loss of GATA4 C-Terminus by p.S335X Mutation Modulates Coronary Artery Vascular Smooth Muscle Cell Phenotype.

Coronary artery disease (CAD) has been the leading cause of morbidity and mortality worldwide, and its pathogenesis is closely related with the proliferation and migration of vascular smooth muscle cell (VSMC). We previously reported a truncated GATA4 protein lacking C-terminus induced by p.S335X mutation in cardiomyocyte from ventricular septal defect (VSD) patients. However, it is still unclear whether GATA4 p.S335X mutation could influence the development of CAD. GATA4 wild-type (WT) and p.S335X mutant (MU) overexpression plasmids were constructed and transfected transiently into rat coronary artery smooth muscle cell (RCSMC) to observe the proliferative and migratory abilities by MTS and wound healing assay, respectively. PCR array was used to preliminarily detect the expression of phenotypic modulation-related genes, and QRT-PCR was then carried out to verify the screened differentially expressed genes (DEGs). The results showed that, when stimulated by fetal bovine serum (10%) for 24 h or tumor necrosis factor-α (10 or 30 ng/ml) for 10 or 24 h, deletion of GATA4 C-terminus by p.S335X mutation in GATA4 enhanced the proliferation of RCSMC, without alteration of the migration capability. Twelve DEGs, including Fas, Hbegf, Itga5, Aimp1, Cxcl1, Il15, Il2rg, Il7, Tnfsf10, Il1r1, Irak1, and Tlr3, were screened and identified as phenotypic modulation-related genes. Our data might be beneficial for further exploration regarding the mechanisms of GATA4 p.S335X mutation on the phenotypic modulation of coronary VSMC.

Characterization of cholesteryl ester-loaded human coronary vascular smooth muscle cell secretome. A source of potential biomarkers of coronary artery disease

Background and Aims: Human coronary vascular smooth muscle cells (hcVSMC) become foam cells by the selective uptake of cholesteryl ester (CE) from aggregated LDL (agLDL). We developed peptides that specifically inhibited agLDL formation and intracellular CE accumulation. Despite the relevance of CE-loaded hcVSMC for atherothrombosis, knowledge about their clinical implications for Coronary Artery Disease (CAD) diagnosis is limited. The objective of this work was to identify the proteins released by CE-loaded hcVSMC to identify potential new circulating CAD biomarkers.

Metabolic syndrome inhibits store-operated Ca2+ entry and calcium-induced calcium-release mechanism in coronary artery smooth muscle

Background and purposeMetabolic syndrome causes adverse effects on the coronary circulation including altered vascular responsiveness and the progression of coronary artery disease (CAD). However the underlying mechanisms linking obesity with CAD are intricated. Augmented vasoconstriction, mainly due to impaired Ca2+ homeostasis in coronary vascular smooth muscle (VSM), is a critical factor for CAD. Increased calcium–induced calcium release (CICR) mechanism has been associated to pathophysiological conditions presenting persistent vasoconstriction while increased store operated calcium (SOC) entry appears to activate proliferation and migration in coronary vascular smooth muscle (VSM). We analyze here whether metabolic syndrome might alter SOC entry as well as CICR mechanism in coronary arteries, contributing thus to a defective Ca2+ handling and therefore accelerating the progression of CAD. Experimental approachMeasurements of intracellular Ca2+ ([Ca2+]i) and tension and of Ca2+ channels protein expression were performed in coronary arteries (CA) from lean Zucker rats (LZR) and obese Zucker rats (OZR). Key resultsSOC entry stimulated by emptying sarcoplasmic reticulum (SR) Ca2+ store with cyclopiazonic acid (CPA) was decreased and associated to decreased STIM-1 and Orai1 protein expression in OZR CA. Further, CICR mechanism was blunted in these arteries but Ca2+ entry through voltage-dependent L-type channels was preserved contributing to maintain depolarization-induced increases in [Ca2+]i and vasoconstriction in OZR CA. These results were associated to increased expression of voltage-operated L-type Ca2+ channel alpha 1C subunit (CaV1.2) but unaltered ryanodine receptor (RyR) and sarcoendoplasmic reticulum Ca2+‐ATPase (SERCA) pump protein content in OZR CA. Conclusion and implicationsThe present manuscript provides evidence of impaired Ca2+ handling mechanisms in coronary arteries in metabolic syndrome where a decrease in both SOC entry and CICR mechanism but preserved vasoconstriction are reported in coronary arteries from obese Zucker rats. Remarkably, OZR CA VSM at this state of metabolic syndrome seemed to have developed a compensation mechanism for impaired CICR by overexpressing CaV1.2 channels.

Abstract 466: Vascular Claudin-11 Plays a Protective Role for Human Atherosclerosis Progression

Background and Aims: The expression and the role of tight junction protein, Claudin-11 (CLDN11) in vasculat smooth muscle cell (vSMC) is unknown. Methods: To understand the role of CLDN11 in the vSMC, we transfected siRNA-CLDN11 into human coronary vascular smooth muscle cell line (hCSMC). cDNA microarray using Agilent Human mRNA arrays, immunocytochemistry, tube formation assay, FACS, and cell cycle analysis were performed 7 days after CLDN11 knock-down. To know the role of CLDN11 in the human cardiovascular system, we obtained vascular smooth muscle layer from autopsied left anterior descending artery and CLDN11 mRNA expression was evaluated following modified AHA Consensus Classification Based on Morphologic Descriptions (three groups, total n=45). Results: CLDN11 was well expressed in vascular smooth mucscle layer in immunohistochemisty and western blot analysis. We observed the angiogenesis (CXCL8, SOX17, HEY1), cell proliferation (EGR3, ITGB2), and extracellular matrix (BMPER, WNT1) associated gene expression. Following CLDN11-siRNA transfection, the tube formation assay and proliferating cellular phase was markedly increased in siRNA treatment group (p<0.01, respectively). In the human sample, CLDN11 expression was inversely correlated with the progression of coronary atherosclerosis (p=0.0026), and the sudden cardiac death with complicated coronary atherosclerosis (p<0.05). Conclusions: These results indicate that CLDN11 could tightly regulate the vascular smooth muscle physiology. And CLDN11 might play a certain role for atherosclerosis propagation to regulate the vSMCs plasticity and possible new etiology for cardiovascular events.

Molecular mechanisms of coronary disease revealed using quantitative trait loci for TCF21 binding, chromatin accessibility, and chromosomal looping

BackgroundTo investigate the epigenetic and transcriptional mechanisms of coronary artery disease (CAD) risk, as well as the functional regulation of chromatin structure and function, we create a catalog of genetic variants associated with three stages of transcriptional cis-regulation in primary human coronary artery vascular smooth muscle cells (HCASMCs).ResultsWe use a pooling approach with HCASMC lines to map regulatory variants that mediate binding of the CAD-associated transcription factor TCF21 with ChIPseq studies (bQTLs), variants that regulate chromatin accessibility with ATACseq studies (caQTLs), and chromosomal looping with Hi-C methods (clQTLs). We examine the overlap of these QTLs and their relationship to smooth muscle-specific genes and transcription factors. Further, we use multiple analyses to show that these QTLs are highly associated with CAD GWAS loci and correlate to lead SNPs where they show allelic effects. By utilizing genome editing, we verify that identified functional variants can regulate both chromatin accessibility and chromosomal looping, providing new insights into functional mechanisms regulating chromatin state and chromosomal structure. Finally, we directly link the disease-associated TGFB1-SMAD3 pathway to the CAD-associated FN1 gene through a response QTL that modulates both chromatin accessibility and chromosomal looping.ConclusionsTogether, these studies represent the most thorough mapping of multiple QTL types in a highly disease-relevant primary cultured cell type and provide novel insights into their functional overlap and mechanisms that underlie these genomic features and their relationship to disease risk.

Inflammatory Drivers of Cardiovascular Disease: Molecular Characterization of Senescent Coronary Vascular Smooth Muscle Cells.

The senescence of vascular smooth muscle cells (VSMCs) has been implicated as a causal pro-inflammatory mechanism for cardiovascular disease development and progression of atherosclerosis, the instigator of ischemic heart disease. Contemporary limitations related to studying this cellular population and senescence-related therapeutics are caused by a lack of specific markers enabling their detection. Therefore, we aimed to profile a phenotypical and molecular signature of senescent VSMCs to allow reliable identification. To achieve this goal, we have compared non-senescent and senescent VSMCs from two in vitro models of senescence, replicative senescence (RS) and DNA-damage induced senescence (DS), by analyzing the expressions of established senescence markers: cell cycle inhibitors- p16 INK4a, p14 ARF, p21 and p53; pro-inflammatory factors-Interleukin 1β (IL-1β), IL-6 and high mobility group box-1 (HMGB-1); contractile proteins-smooth muscle heavy chain- (MYH11), smoothelin and transgelin (TAGLN), as well as structural features (nuclear morphology and LMNB1 (Lamin B1) expression). The different senescence-inducing modalities resulted in a lack of the proliferative activity. Nucleomegaly was seen in senescent VSMC as compared to freshly isolated VSMC Phenotypically, senescent VSMC appeared with a significantly larger cell size and polygonal, non-spindle-shaped cell morphology. In line with the supposed switch to a pro-inflammatory phenotype known as the senescence associated secretory phenotype (SASP), we found that both RS and DS upregulated IL-1β and released HMGB-1 from the nucleus, while RS also showed IL-6 upregulation. In regard to cell cycle-regulating molecules, we detected modestly increased p16 levels in both RS and DS, but largely inconsistent p21, p14ARF, and p53 expressions in senescent VSMCs. Since these classical markers of senescence showed insufficient deregulation to warrant senescent VSMC detection, we have conducted a non-biased proteomics and in silico analysis of RS VSMC demonstrating altered RNA biology as the central molecular feature of senescence in this cell type. Therefore, key proteins involved with RNA functionality, HMGB-1 release, LMNB-1 downregulation, in junction with nuclear enlargement, can be used as markers of VSMC senescence, enabling the detection of these pathogenic pro-inflammatory cells in future therapeutic studies in ischemic heart disease and atherosclerosis.

Development of Innovative Antiatherosclerotic Peptides through the Combination of Molecular Modeling and a Dual (Biochemical‐Cellular) Screening System

AbstractCardiovascular disease (CVD) is a leading cause of death worldwide. Approximately 60% of patients treated with low‐density lipoprotein (LDL)‐lowering drug treatments, with on‐target plasma cholesterol levels, are still suffering clinical acute ischemic events. Mechanisms, such as LDL aggregation, underlie extracellular and intracellular cholesterol accumulation in the vasculature. A peptide sequence (P3) of the low‐density lipoprotein receptor‐related protein 1 (LRP1) efficiently protects LDL from sphingomyelinase (SMase‐) and phospholipase A2 (PLA2)‐induced LDL aggregation. The aim is to design families of peptide derivatives from P3 with enhanced potency and proteolytic stability. New peptides are designed through in silico conformational sampling and ApoB‐100 molecular docking, and are tested in dual (biochemical‐cellular) screening assays. A total of 46 new peptides including linear, fragment, cyclic, and alanine scanning derivatives are generated through two consecutive optimization rounds. Structurally and functionally optimized peptides contain hotspot residues that are replaced by alanine. This strategy confers an increased capacity to form prone alpha‐helix conformations crucial for the electrostatic interaction with ApoB‐100. These new compounds are highly efficient at inhibiting LDL aggregation and human coronary vascular smooth muscle cell‐cholesteryl ester loading and should be studied in preclinical models of atherosclerosis.

Reduced stiffness and augmented traction force in type 2 diabetic coronary microvascular smooth muscle.

Type 2 diabetic (T2DM) coronary resistance microvessels (CRMs) undergo inward hypertrophic remodeling associated with reduced stiffness and reduced coronary blood flow in both mice and pig models. Since reduced stiffness does not appear to be due to functional changes in the extracellular matrix, this study tested the hypothesis that decreased CRM stiffness in T2DM is due to reduced vascular smooth muscle cell (VSMC) stiffness, which impacts the traction force generated by VSMCs. Atomic force microscopy (AFM) and traction force microscopy (TFM) were conducted on primary low-passage CRM VSMCs from normal Db/db and T2DM db/db mice in addition to low-passage normal and T2DM deidentified human coronary VSMCs. Elastic modulus was reduced in T2DM mouse and human coronary VSMCs compared with normal (mouse: Db/db 6.84 ± 0.34 kPa vs. db/db 4.70 ± 0.19 kPa, P < 0.0001; human: normal 3.59 ± 0.38 kPa vs. T2DM 2.61 ± 0.35 kPa, P = 0.05). Both mouse and human T2DM coronary microvascular VSMCs were less adhesive to fibronectin compared with normal. T2DM db/db coronary VSMCs generated enhanced traction force by TFM (control 692 ± 67 Pa vs. db/db 1,507 ± 207 Pa; P < 0.01). Immunoblot analysis showed that T2DM human coronary VSMCs expressed reduced β1-integrin and elevated β3-integrin (control 1.00 ± 0.06 vs. T2DM 0.62 ± 0.14, P < 0.05 and control 1.00 ± 0.49 vs. T2DM 3.39 ± 1.05, P = 0.06, respectively). These data show that T2DM coronary VSMCs are less stiff and less adhesive to fibronectin but are able to generate enhanced force, corroborating previously published computational findings that decreasing cellular stiffness increases the cells' ability to generate higher traction force.NEW & NOTEWORTHY We show here that a potential causative factor for reduced diabetic coronary microvascular stiffness is the direct reduction in coronary vascular smooth muscle cell stiffness. These cells were also able to generate enhanced traction force, validating previously published computational models. Collectively, these data show that smooth muscle cell stiffness can be a contributor to overall tissue stiffness in the coronary microcirculation, and this may be a novel area of interest for therapeutic targets.

Coronary Vascular Cell Stiffness Influences Coronary Blood Flow

Our laboratory has previously shown that type 2 diabetic (T2DM) coronary resistance microvessels (CRMs) undergo inward hypertrophic remodeling associated with reduced stiffness. Reduced T2DM CRM tissue stiffness is associated with decreased elastic modulus of coronary vascular smooth muscle cells (VSMCs). The goal of this study was to test the hypothesis that reducing or augmenting coronary vascular cell stiffness will increase or decrease coronary blood flow, respectively. Coronary blood flow was measured from hearts isolated from 16‐week‐old control Db/db and diabetic db/db mice and perfused on a Langendorff system at a constant pressure of 80 mmHg. Latrunculin B (1 μM final concentration; n=2 per group) was infused to depolymerize actin and reduce vascular cell stiffness and jasplakinolide (0.2 μM final concentration; n=4 per group) was infused to increase actin polymerization and increase vascular cell stiffness. After drug infusions, hearts were immediately transferred to an adjacent Langendorff apparatus and were perfusion fixed with 4% paraformaldehyde for 2 mins at 1 mL/min. Hearts were then placed in 4% PFA for 24–48 hours and were stored in 70% EtOH until embedding in paraffin and sectioning. Sections were evaluated for F‐ and G‐actin by immunofluorescence. In pooled Db/db and db/db analyses, Latrunculin B caused an increase in coronary blood flow over baseline (34.1 ± 15.0% above baseline, n=7), and jasplakinolide caused a 26.5 ± 4.1% reduction in coronary blood flow from baseline (n=8). Immunohistochemical analysis confirmed a decreased trend in F/G actin in the coronary microvessels of latrunculin B‐perfused hearts (1.00 vs. 0.82 ± 0.08, n=1–4 per group), while F/G actin tended to be increased in the CRMs of jasplakinolide‐perfused hearts (1.00 ± 0.00 vs. 1.50 ± 0.16, p=0.12, n=2–6 per group). Importantly, the F/G actin ratio in jasplakinolide‐treated hearts was not different measured in the myocardium 50–100 μm away from a CRM (1.00 ± 0.06 in vehicle‐perfused hearts vs. 1.01 ± 0.10 in jasplakinolide‐perfused hearts, p=0.96, n=2–6 per group), demonstrating that the drug was efficacious only in the peri‐vascular region. These data support the hypothesis that coronary blood flow increases as vascular cell stiffness is decreased and coronary blood flow decreases as vascular cell stiffness is increased. Collectively, these data suggest that coronary vascular cell stiffness can modulate coronary blood flow.Support or Funding InformationKRM was supported by an American Heart Association Summer Undergraduate Research Fellowship (17UFEL33420025). NIH R00HL116769, R21EB026518, and S10OD023438 to AJT.