Human Coronary Vascular Smooth Muscle Research Articles

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.

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.

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.

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.

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.

Myoendothelial Junctions of Mature Coronary Vessels Express Notch Signaling Proteins

The Notch signaling pathway, via cell‐cell communication, is critical during normal vascular development and homeostasis, and it is known to regulate vascular smooth muscle (VSMC) phenotype, including proliferation; however, few studies have addressed the role of Notch signaling in the mature vasculature. The current study tested the hypothesis that Notch signaling is present and active at mature myoendothelial junctions (MEJs), signaling hubs that occur at breaks in the internal elastic lamina (IEL). In mature, intact normal adult mouse coronary resistance microvessels (CRMs) embedded within heart sections, we observed Notch1, Notch2, Notch3, and Jagged1 expression within the fenestrae of the CRM IEL where MEJs are known to occur. Immunofluorescence clearly shows bridging from the VSMCs to endothelial cells (ECs). Immunofluorescence in larger mouse left anterior descending (LAD) coronary arteries demonstrate the expression Notch2 and Notch3 protein immunofluorescence within fenestrae of the IEL. We found that, on average, 39% and 33% of IEL fenestrae were positive for Notch2 and Notch3 expression, respectively. We also confirmed the presence of Notch3 expression in the MEJ by immunogold electron microscopy. In normal mouse femoral arteries, we found that, on average, 46%, 51%, and 58% of IEL fenestrae were positive for Notch1, Notch3, and Jagged1 expression, respectively. To determine whether Notch signaling components were localized to the MEJ, we co‐cultured primary human coronary ECs (hcECs) and primary human coronary VSMCs (hcVSMCs) across transwell inserts, and we utilized fluorescent microscopy to immunostain for Pai‐1, Jagged1, Notch1, and Notch2. PAI‐1 immunofluorescence was observed on the EC side of the transwells and within the transwell pores, indicating the formation of in vitro MEJs. We also identified distinct staining of Notch1, Notch2, and Jagged1 in the pores of the co‐culture transwells, which co‐localized with PAI‐1 within the pores. To assess whether Notch signaling was active at the MEJ, we co‐cultured hcECs and hcVSMCs on transwells to create in vitro MEJs. Smooth muscle cells were first transfected with a Notch‐sensor luciferase plasmid followed by transwell co‐culture of the transfected cells with endothelial cells. Notch activity was stimulated by 3‐fold across the MEJ in hcVSMC (1.00 ± 0.15 vs. 3.02 ± 0.43, p&lt;0.001), which was completely abrogated by the non‐selective Notch γ‐secretase inhibitor, DAPT (1.15 ± 0.16, p&lt;0.01). In this study, we demonstrate for the first time that Notch receptors and ligands are expressed within and are active at mature coronary MEJs, demonstrating a previously unrecognized mode of Notch signaling regulation between the endothelium and smooth muscle.Support or Funding InformationFunding: EW was supported by NIH R25HD086885. NIH R00HL116769, R21EB026518, and S10OD023438 to AJT.

Immunization with the Gly1127-Cys1140 amino acid sequence of the LRP1 receptor reduces atherosclerosis in rabbits. Molecular, immunohistochemical and nuclear imaging studies.

Background: The LRP1 (CR9) domain and, in particular, the sequence Gly1127-Cys1140 (P3) plays a critical role in the binding and internalization of aggregated LDL (agLDL). We aimed to evaluate whether immunization with P3 reduces high-fat diet (HFD)-induced atherosclerosis.Methods: Female New Zealand White (NZW) rabbits were immunized with a primary injection and four reminder doses (R1-R4) of IrP (irrelevant peptide) or P3 conjugated to the carrier. IrP and P3-immunized rabbits were randomly divided into a normal diet group and a HFD-fed group. Anti-P3 antibody levels were determined by ELISA. Lipoprotein profile, circulating and tissue lipids, and vascular pro-inflammatory mediators were determined using standardized methods while atherosclerosis was determined by confocal microscopy studies and non-invasive imaging (PET/CT and Doppler ultrasonography). Studies treating human macrophages (hMΦ) and coronary vascular smooth muscle cells (hcVSMC) with rabbit serums were performed to ascertain the potential impact of anti-P3 Abs on the functionality of these crucial cells.Results: P3 immunization specifically induced the production of anti-P3 antibodies (Abs) and did not alter the lipoprotein profile. HFD strongly induced cholesteryl ester (CE) accumulation in the aorta of both the control and IrP groups, and their serum dose-dependently raised the intracellular CE of hMΦ and hcVSMC, promoting TNFR1 and phospho-NF-kB (p65) overexpression. These HFD pro-inflammatory effects were dramatically decreased in the aorta of P3-immunized rabbits and in hMΦ and hcVSMC exposed to the P3 rabbit serums. Microscopy studies revealed that P3 immunization reduced the percentage of lipids, macrophages, and SMCs in the arterial intima, as well as the atherosclerotic extent and lesion area in the aorta. PET/CT and Doppler ultrasonography studies showed that the average standardized uptake value (SUVmean) of the aorta and the arterial resistance index (ARI) of the carotids were more upregulated by HFD in the control and IrP groups than the P3 group.Conclusions: P3 immunization counteracts HFD-induced fatty streak formation in rabbits. The specific blockade of the LRP1 (CR9) domain with Anti-P3 Abs dramatically reduces HFD-induced intracellular CE loading and harmful coupling to pro-inflammatory signaling in the vasculature.

Type 2 Diabetic Human Coronary Vascular Smooth Muscle Cells Are Less stiff and Less Adhesive to Fibronectin

Previous data from our laboratory has demonstrated that 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. Further analysis revealed that the reduced stiffness was in part due to increased tissue elastin expression and decreased vascular smooth muscle cell (VSMC) stiffness in mice. The goal of the present study was to test the hypothesis that T2DM coronary VSMCs from humans are less stiff than normal. De‐identified, low‐passage normal and T2DM human coronary VSMCs were purchased from Lonza and ATCC for all experiments (n=4–5 per group). Using an atomic force microscope with probes coated with fibronectin (FN), a nano‐indentation protocol was used to measure cellular stiffness and adhesion to FN. Elastic modulus, a measurement of cellular stiffness, was reduced in T2DM human coronary VSMCs compared to normal (Normal: 3.60 ± 0.32 kPa vs. T2DM: 2.75 ± 0.22 kPa, p&lt;0.05). Additionally, T2DM human coronary VSMCs were less adhesive to fibronectin compared to normal human coronary VSMCs (Normal: 58.3 ± 7.7 vs. T2DM: 43.5 ± 4.9 pN, p=0.17). Immunoblot analysis showed that T2DM human coronary VSMC expressed reduced vinculin (Normal: 1.0 ± 0.17 vs. T2DM: 0.31 ± 0.11, p&lt;0.05) and increased cofilin (Normal: 1.0 ± 0.16 vs. T2DM: 1.8 ± 0.35, p=0.052) compared to normal human coronary VSMCs; this expression profile favors a less stiff cell phenotype given that vinculin deficient mice exhibit reduced cytoskeletal stiffness and that increased cofilin binding to F‐actin reduces stiffness. These data show that reductions in stiffness and adhesion to the ECM of diabetic CRM VSMCs may be due to underlying alterations in the cytoskeletal arrangement. Collectively, reduced stiffness in T2DM human coronary VSMCs may drive whole tissue microvascular mechanical properties to account for reduced CRM stiffness. These findings will inform future studies on the mechanisms of stiffness in the diabetic coronary circulation.Support or Funding InformationNIH R00 HL116769, NIH R21 EB026518, and Nationwide Children's Hospital to AJTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cell Type‐Specific Ephrin expression in the Diabetic Coronary Circulation

Ephrin signaling between vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) is crucial during normal vascular development and homeostasis, primarily by influencing cell behavior, resulting in alterations of the cell cytoskeleton and cell adhesion. Ephrins are re‐expressed in vascular injury and disease states such as cancer and chronic inflammation; therefore the goal of this study was to determine the impact of type 2 diabetes on ephrin expression in coronary VSMCs and ECs. Low‐passage primary coronary VMSCs were isolated from normal (n=6) and type 2 diabetic db/db mice (n=4) and were either cultured alone or co‐cultured with normal or type 2 diabetic human coronary microvascular endothelial cells (hCMECs), respectively, and RNA was collected for PCR analysis. Normal (n=5) and type 2 diabetic (T2DM) (n=4) human coronary vascular smooth muscle cells (CVSMCs) were also cultured and protein lysates collected for Western Blot analysis. Ephrin B2 ligand gene expression was significantly increased in diabetic CRM VSMCs in both mono‐ and co‐culture conditions (1.00 ± 0.13 vs. 1.57 ± 0.24, p=0.049 and 1.00 ± 0.14 vs. 1.60 ± 0.22, p=0.044, respectively). There were no significant differences in ephrin B4 receptor levels in normal vs. diabetic coronary VMSCs. In human coronary VSMCs, ephrin B2 ligand protein expression was also significantly increased in diabetic coronary VSMCs over control VSMCs (normal 1.00±0.05 vs. T2DM 2.91±0.29, p=0.019). Interestingly, there was a different trend in the diabetic hCMECs. Ephrin B2 gene expression was significantly reduced in diabetic hCMECs in both mono‐ and co‐culture conditions (1.00 ± 0.05 vs. 0.75 ± 0.07, p=0.02 and 1.00 ± 0.10 vs. 0.50 ± 0.07, p=0.007, respectively). Ephrin B4 receptor gene expression was significantly reduced in diabetic hCMECs in both mono‐ and co‐culture conditions (1.00 ± 0.07 vs. 0.55 ± 0.05, p=0.002 and 1.00 ± 0.03 vs. 0.77 ± 0.03, p=0.0008, respectively). Collectively, these data demonstrate that, in both mice and humans, diabetes alters ephrin expression in VSMCs and ECs in a cell‐specific manner. Further studies will address how alterations in ephrin expression influence coronary cell behavior in the setting of diabetes.Support or Funding InformationNIH R00 HL116769, NIH R21 EB026518, and Nationwide Children's Hospital to AJTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Apabetalone downregulates factors and pathways associated with vascular calcification

Background and aimsApabetalone is an inhibitor of bromodomain and extraterminal (BET) proteins. In clinical trials, apabetalone reduced the incidence of major adverse cardiac events (MACE) in patients with cardiovascular disease and reduced circulating factors that promote vascular calcification (VC). Because VC contributes to MACE, effects of apabetalone on pro-calcific processes were examined. Methods and resultsApabetalone inhibited extracellular calcium deposition and opposed induction of transdifferentiation markers in human coronary artery vascular smooth muscle cells (VSMCs) under osteogenic culture conditions. Tissue-nonspecific alkaline phosphatase (TNAP) is a key contributor to VC, and apabetalone suppressed osteogenic induction of the mRNA, protein and enzyme activity. The liver is a major source of circulating TNAP, and apabetalone also downregulated TNAP expression in primary human hepatocytes. BRD4, a transcriptional regulator and target of apabetalone, has been linked to calcification. Osteogenic transdifferentiation of VSMCs resulted in disassembly of 100 BRD4-rich enhancers, with concomitant enlargement of remaining enhancers. Apabetalone reduced the size of BRD4-rich enhancers, consistent with disrupting BRD4 association with chromatin. 38 genes were uniquely associated with BRD4-rich enhancers in osteogenic conditions; 11 were previously associated with calcification. Apabetalone reduced levels of BRD4 on many of these enhancers, which correlated with decreased expression of the associated gene. Bioinformatics revealed BRD4 may cooperate with 7 specific transcription factors to promote transdifferentiation and calcification. ConclusionsApabetalone counters transdifferentiation and calcification of VSMCs via an epigenetic mechanism involving specific transcription factors. The mechanistic findings, combined with evidence from clinical trials, support further development of apabetalone as a therapeutic for VC.

Cell-Specific Protective Signaling Induced by the Novel AT2R-Agonist NP-6A4 on Human Endothelial and Smooth Muscle Cells.

Cardiovascular disease incidence continues to rise and new treatment paradigms are warranted. We reported previously that activation of Angiotensin II receptor (encoded by the X-linked Agtr2 gene) by a new peptide agonist, NP-6A4, was more effective in protecting mouse cardiomyocyte HL-1 cells and human coronary artery vascular smooth muscle cells (hCAVSMCs) from acute nutrient deficiency than other drugs tested. To elucidate further the protective effects of NP-6A4 in human cells, we studied the effects of NP-6A4 treatment on functions of human coronary artery endothelial cells (hCAECs), and hCAVSMCs. In hCAVSMCs, NP-6A4 (1 μM) increased Agtr2 mRNA (sixfold, p < 0.05) after 12-h exposure, whereas in hCAECs, significant increase in Agtr2 mRNA (hCAECs: eightfold) was observed after prolonged exposure. Interestingly, NP-6A4 treatment (1 μM, 12 h) increased AT2R protein levels in all human cells tested. Pre-treatment with AT2R-antagonist PD123319 (20 μM) and anti-AT2R siRNA (1 μM) suppressed this effect. Thus, NP-6A4 activates a positive feedback loop for AT2R expression and signaling in hCAVSMCs and hCAECs. NP-6A4 (1–20 μM) increased cell index (CI) of hCAVSMCs as determined by real time cell analyzer (RTCA), indicating that high concentrations of NP-6A4 were not cytotoxic for hCAVSMCs, rather promoting better cell attachment and growth. Seahorse Extracellular Flux Assay revealed that NP-6A4 (1 μM) treatment for 7 days increased whole cell-based mitochondrial parameters of hCAVSMCs, specifically maximal respiration (p < 0.05), spare respiratory capacity (p < 0.05) and ATP production (p < 0.05). NP-6A4 (1 μM; 7 days) also suppressed Reactive Oxygen Species (ROS) in hCAVSMCs. Exposure to Doxorubicin (DOXO) (1 μM) increased ROS in hCAVSMCs and this effect was suppressed by NP-6A4 (1 μM). In hCAECs grown in complete medium, NP-6A4 (1 μM) and Ang II (1 μM) exerted similar changes in CI. Additionally, NP-6A4 (5 μM: 12 h) increased expression of eNOS (sixfold, p < 0.05) and generation of nitric oxide (1.3-fold, p < 0.05) in hCAECs and pre-treatment with PD123319 (20 μM) suppressed this effect partially (65%). Finally, NP-6A4 decreased phosphorylation of Jun-N-terminal kinase, implicated in apoptosis of ECs in atherosclerotic sites. Taken together, NP-6A4, through its ability to increase AT2R expression and signaling, exerts different cell-specific protective effects in human VSMCs and ECs.

Mining the Stiffness-Sensitive Transcriptome in Human Vascular Smooth Muscle Cells Identifies Long Noncoding RNA Stiffness Regulators

Vascular extracellular matrix stiffening is a risk factor for aortic and coronary artery disease. How matrix stiffening regulates the transcriptome profile of human aortic and coronary vascular smooth muscle cells (VSMCs) is not well understood. Furthermore, the role of long noncoding RNAs (lncRNAs) in the cellular response to stiffening has never been explored. This study characterizes the stiffness-sensitive (SS) transcriptome of human aortic and coronary VSMCs and identifies potential key lncRNA regulators of stiffness-dependent VSMC functions. Aortic and coronary VSMCs were cultured on hydrogel substrates mimicking physiological and pathological extracellular matrix stiffness. Total RNAseq was performed to compare the SS transcriptome profiles of aortic and coronary VSMCs. We identified 3098 genes (2842 protein coding and 157 lncRNA) that were stiffness sensitive in both aortic and coronary VSMCs (false discovery rate <1%). Hierarchical clustering revealed that aortic and coronary VSMCs grouped by stiffness rather than cell origin. Conservation analyses also revealed that SS genes were more conserved than stiffness-insensitive genes. These VSMC SS genes were less tissue-type specific and expressed in more tissues than stiffness-insensitive genes. Using unbiased systems analyses, we identified MALAT1 as an SS lncRNA that regulates stiffness-dependent VSMC proliferation and migration in vitro and in vivo. This study provides the transcriptomic landscape of human aortic and coronary VSMCs in response to extracellular matrix stiffness and identifies novel SS human lncRNAs. Our data suggest that the SS transcriptome is evolutionarily important to VSMCs function and that SS lncRNAs can act as regulators of stiffness-dependent phenotypes.

Abstract P353: Attenuation of Cardiac Fibrosis, Hypertrophy and Myopathy by AT2R Agonist NP-6A4

Adverse cardiac remodeling (hypertrophy, fibrosis and myopathy) underlies cardiac dysfunction and heart failure. Male Zucker obese (ZO:fa/fa) rat that suffers from cardiac dysfunction, adverse remodeling, and heart failure with preserved ejection fraction is a useful model to study the effects of cardioprotective drugs. We reported that NP-6A4 (Novopyxis Inc. Cambridge, MA), a peptide agonist of the Angiotensin II Type 2 Receptor (AT2R), protected mouse cardiomyoblast HL-1 cells and human coronary artery vascular smooth muscle cells (hCAVSMCs) from acute nutrient serum deficiency stress better than β-AR-Blockers, ARBs, and AT2R agonist CGP42112A. This effect was inhibited by AT2R-specific antagonist PD123319, confirming that NP-6A4 acts through AT2R. AT2R is a cardiac and vascular reparative molecule that protects the heart and vasculature from structural damage and fibrosis. No current drugs increase AT2R expression. We report that NP-6A4 treatment (1μM) increased AT2R mRNA (up to 4 fold; p≤0.05) in hCAVSMCs and human coronary artery endothelial cells (up to 8 fold; p≤0.05). This effect was inhibited by PD123319 (10 μM). Treatment of 11-week old male ZO rats with heart disease with NP-6A4 (1.8mg/kg/day in saline, delivered once daily subcutaneously; N=7) increased cardiac AT2R expression and mitigated cardiac dysfunction and adverse remodeling. Quantitative RT-PCR and immunohistochemistry analysis showed that 2 weeks of NP-6A4 treatment increased cardiac AT2R mRNA (up to 9 fold) and protein (1.5-3 fold) compared to controls (N=6) receiving saline (p&lt;0.01). NP-6A4 treatment improved cardiac parameters; endocardial circumferential strain (p≤0.05), myocardial performance index (MPI) (p≤0.005), and E/E’ ratio (p≤0.002). NP-6A4 treatment also increased cardiac capillary density (118% compared to saline treatedl; p≤0.002), and reduced interstitial fibrosis (77% compared to saline treated; p≤0.039.) At 19-weeks (after 8 weeks of treatment), cardiac tissues from 5 out of 6 control rats exhibited regions of myopathy. Only 2 out 7 NP-6A4 treated rats exhibited similar levels of myopathy in the heart. We propose that NP-6A4 attenuates adverse remodeling and improves cardiac function by increasing AT2R expression in cardiac cells.

Doxorubicin Dose Dependently Attenuates Cyclooxygenase‐2 in Female Human Coronary Vascular Smooth Muscle Cells

Doxorubicin (Dox), an anthracycline‐based chemotherapeutic agent, is highly effective at reducing recurrence and mortality in breast cancer patients. However, this anti‐cancer drug elicits dose‐dependent cardiotoxicity. Multiple mechanisms have been implicated in its pathogenesis, one of which involves the development and progression of inflammation mediated by activation of the TLR4/NFkappaB pathway. However, we recently demonstrated that chronic in vivo Dox treatment reduced expression of the NFkappaB downstream proinflammatory mediator, cyclooxygenase‐2 (COX‐2), in cerebral and peripheral vasculatures isolated from ovariectomized female rats. Therefore, in the current study we tested whether Dox similarly alters vascular proinflammatory enzyme expression in the human vasculature and whether this response is dose dependent. We further investigated whether Dox alters upstream mediators of COX‐2 expression by assessing levels of expression and activation of NFkappaB and an additional downstream gene product, TLR4. Primary human coronary vascular smooth muscle cells of female origin were studied at passage 6 and treated with vehicle or Dox (0.3, 1.0, and 5 μM). Following drug treatment, cells were isolated, homogenized, and lysates (whole cell; nuclear and cytosolic) analyzed for NFkappaBp65, COX‐2, and TLR4 protein levels via immunoblotting. Our results demonstrate that while total NFkappaB levels were unaffected following Dox, enhanced translocation of NFkappaB to the nucleus was observed. However, even in the presence of Dox‐induced NFkappa translocation, basal expression of its downstream mediator, COX‐2, was dose dependently attenuated. Similar to COX‐2, a trend for a decrease in TLR4 levels was observed. Dox had no effect on cell morphology or density, as visualized via light microscopy. In conclusion, although others have suggested the involvement of NFkappaB activation during the development and progression of Dox‐induced inflammation in cardiac muscle, our studies demonstrate a possible novel action for the anticancer agent targeting important downstream mediators of inflammation in the female vasculature. Future studies will evaluate the impact of Dox on COX‐2 at the transcriptional or translational level to determine the mode of molecular regulation.Support or Funding InformationUniversity of Arizona Sarver Heart Center (RG and MS); Mayo Clinic Dept. of Medicine Extramural Chair Award in collaboration with Arizona State University (SA)

A novel inhibitory effect of oxazol-5-one compounds on ROCKII signaling in human coronary artery vascular smooth muscle cells.

The selectivity of (4Z)-2-(4-chloro-3-nitrophenyl)-4-(pyridin-3-ylmethylidene)-1,3-oxazol-5-one (DI) for zipper-interacting protein kinase (ZIPK) was previously described by in silico computational modeling, screening a large panel of kinases, and determining the inhibition efficacy. Our assessment of DI revealed another target, the Rho-associated coiled-coil-containing protein kinase 2 (ROCKII). In vitro studies showed DI to be a competitive inhibitor of ROCKII (Ki, 132 nM with respect to ATP). This finding was supported by in silico molecular surface docking of DI with the ROCKII ATP-binding pocket. Time course analysis of myosin regulatory light chain (LC20) phosphorylation catalyzed by ROCKII in vitro revealed a significant decrease upon treatment with DI. ROCKII signaling was investigated in situ in human coronary artery vascular smooth muscle cells (CASMCs). ROCKII down-regulation using siRNA revealed several potential substrates involved in smooth muscle contraction (e.g., LC20, Par-4, MYPT1) and actin cytoskeletal dynamics (cofilin). The application of DI to CASMCs attenuated LC20, Par-4, LIMK, and cofilin phosphorylations. Notably, cofilin phosphorylation was not significantly decreased with a novel ZIPK selective inhibitor (HS-38). In addition, CASMCs treated with DI underwent cytoskeletal changes that were associated with diminution of cofilin phosphorylation. We conclude that DI is not selective for ZIPK and is a potent inhibitor of ROCKII.

High cut-off dialysis in chronic haemodialysis patients reduces serum procalcific activity

Vascular calcification is enhanced in chronic dialysis patients, possibly due to the insufficient removal of various intermediate molecular weight uraemic toxins such as interleukins with conventional membranes. In this study, we assessed the modulation of in vitro vascular calcification with the use of high cut-off (HCO) membranes in chronic dialysis patients. In a PERCI trial, 43 chronic dialysis patients were treated with conventional high-flux and HCO filters for 3 weeks in a randomized order following a 2-period crossover design. After each phase, serum predialysis samples were drawn. Calcifying human coronary vascular smooth muscle cells (VSMCs) were incubated with the patient's serum samples. Calcification was assessed with alkaline phosphatase (ALP) and alizarin red staining. In the clinical trial, HCO dialysis reduced the serum levels of the soluble tumour necrosis factor receptor (sTNFR) 1 and 2, vascular cell adhesion molecule 1 (VCAM-1) and soluble interleukin-2 receptor (sIL2R). We therefore investigated the in vitro effects of these mediators on vascular calcification. VSMCs incubated with HCO dialysis serum showed a 26% reduction of ALP with HCO serum compared with high-flux serum. Alizarin was 43% lower after incubation with the HCO serum compared with the high-flux serum. While sIL2R and sTNFR 1 and 2 showed no effects on VSMC calcification, VCAM-1 caused a dose-dependent enhancement of calcification. The use of HCO dialysis membranes in chronic dialysis patients reduces the procalcific effects of serum on VSMC in vitro. The mechanisms of the strong effect of HCO on in vitro calcification are not completely understood. One factor may be lower levels of VCAM-1 in HCO serum samples, since VCAM-1 was able to induce vascular calcification in our experiments. Neither sTNFR 1, sTNFR 2 nor sIL2R enhance vascular calcification in vitro. Regardless of the mechanisms, our results encourage further studies of highly permeable filters in chronic dialysis patients.

Abstract P623: Nutrient Stress Response of Cardiovascular Cells to β-Blockers, ARB and AT2R Agonists

To determine how cardioprotective drugs modulate cell response to nutrient starvation, we investigated the effects of β-blockers (Nebivolol [Neb], Carvedilol [Car], Metoprolol [Met] and Atenolol [Aten] (3μM each), Angiotensin II (Ang II) (300nM), AT1R blocker (ARB-Losartan (1μM)) and AT2R agonists (CGP42112A [CGP] and a novel peptide agonist NP-6A4 (300nM each)) on serum-starved HL-1 cardiomyocytes. The Xcelligence Real-Time Cell Analyzer (RTCA), which measures area covered by cells in microtiter plates and displays it as a unitless quantity called Cell Index (CI), was used to assess cellular changes in response to drug treatment. To find whether changes in CI are due to altered proliferation or cell size, we utilized the MTS Proliferation Assay, a colorimetric assay that measures formazan dye produced by viable cells, and fluorophore-conjugated Wheat Germ Agglutinin (WGA) labeling to measure cell size respectively. Difference in CI units is reported as % of CI units of cells treated with vehicle. CI units were suppressed by β-blockers (Aten≤15%; Met≤15%; Neb≤17%; Car≤8%), increased by Ang II (≥9.6%), CGP (≥14%) and NP-6A4 (≥25%), but not by losartan (n≥4 and p≤0.05 for all treatments). MTS assay revealed that only NP-6A4 increased (19%) formazan dye as measured at 490nm (n=3, p&lt;0.05). Differences in cell size of WGA stained cells was calculated as % of size of cells treated with vehicle. Neb and Car decreased cell size (Neb≤14%, Car≤10%, p&lt;0.05 and p&lt;0.1 respectively), suggesting their mechanism for decrease in CI. Other treatments showed no significant change. Next, we determined changes in Myeloid cell leukemia 1 (MCL-1) levels, an essential protein for cardiomyocyte survival, in response to drug treatments by immunoblotting and immunofluorescence. β-Blockers suppressed MCL-1 expression in HL-1 cardiomyocytes (Neb≤26%, Car≤24%, Met≤24%, Aten≤16%) while AT2R agonists increased (CGP≥17%, NP-6A4≥28%, n=3; p&lt;0.05 for all). Treatment of Female Human Coronary Artery Vascular Smooth Muscle Cells by these drugs also showed a similar expression pattern of expression for MCL-1 (CGP≥23%, NP-6A4≥43%). Therefore, AT2R agonist NP-6A4 is more effective in protecting serum starved cardiovascular cells.

Circulating soluble low-density lipoprotein receptor-related protein 1 (sLRP1) concentration is associated with hypercholesterolemia: A new potential biomarker for atherosclerosis

BackgroundThere is clinical interest in identifying novel lipid biomarkers for evaluating cardiovascular risk and targeting lipid-lowering treatment. Low-density lipoprotein receptor-related protein 1 (LRP1) plays a crucial role in the dysregulated cholesterol transfer from modified lipoproteins to human coronary vascular smooth muscle cells (hVSMCs), promoting hVSMC-derived foam cell formation. LRP1 has a soluble and circulating form (sLRP1) generated from LRP1. Cholesterol modulates the release of the soluble form of LRP1. Using in vitro, ex vivo and patient-based approaches, we tested the association between circulating sLRP1 concentrations and hypercholesterolemia and the potential of sLRP1 as a biomarker of atherosclerosis. Methods and resultsCirculating sLRP1 concentrations were higher in severe hypercholesterolemia compared to moderate hypercholesterolemia or normocholesterolemia (Study 1). Circulating sLRP1 was significantly associated with established pro-atherogenic lipid parameters in two different hypercholesterolemic populations (Studies 2 and 3). sLRP1 concentrations decreased after statin treatment and increased after statin withdrawal (Study 3). In vitro experiments showed that native LDL, aggregated LDL and VLDL+IDL lipoproteins induced the release of sLRP1 from hVSMC. sLRP1 levels were increased in the conditioned medium of coronary atherosclerotic plaque areas extracted from patients compared to non-atherosclerotic areas of the same coronary artery and patient. Circulating sLRP1 concentrations were independently associated with the occurrence of carotid atherosclerosis in a hypercholesterolemic population (Study 2). The later association was higher than that observed for other classical or novel lipid parameters. ConclusionsCirculating sLRP1 is a new lipid-related parameter potentially useful as a biomarker for atherosclerosis.

Abstract 391: Enhanced Coronary Vasoconstriction in Western Diet-Induced Obesity is Associated with Alterations in NHE1, SERCA2a and 3

Cardiovascular disease (CVD) remains a major risk factor and cause of mortality in type 2 diabetes but, the effect of western diet (WD) on coronary artery vasoconstriction has not been well characterized. Therefore, the purpose of this study was to identify potential calcium (Ca 2+ ) molecular mechanism(s) underlying alterations in coronary constriction in WD-induced obesity. C57Bl/6J mice were fed a WD or control diet for 16wks. Coronary vascular function was assessed with wire myography and human coronary vascular smooth muscle cells (VSMC) were cultured. WD enhanced coronary constriction to the thromboxane analog U46619. This was associated with increased coronary VSMC expression of sodium-hydrogen exchanger 1 (NHE1) and SERCA3 with decreased expression of SERCA2a. Oxidation of SERCA2a cysteine-674, a signal for SERCA2a degradation, was increased in WD coronary VSMC. Exposure of cultured human coronary VSMC to hyperinsulinemic conditions elicited similar changes in SERCA2a, 3 and NHE1. This study suggests that enhanced thromboxane-mediated coronary constriction in WD and insulin resistance/hyperinsulinemia may result from NHE1-dependent changes in intracellular pH and/or impaired Ca 2+ handling at the level of the sarcoplasmic reticulum.