Coronary Artery Smooth Muscle Cells Research Articles

Inhibitory effects of the atypical antipsychotic, clozapine, on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

To investigate the adverse effects of clozapine on cardiovascular ion channels, we examined the inhibitory effect of clozapine on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Clozapine-induced inhibition of Kv channels occurred in a concentration-dependent manner with an half-inhibitory concentration value of 7.84 ± 4.86 µM and a Hill coefficient of 0.47 ± 0.06. Clozapine did not shift the steady-state activation or inactivation curves, suggesting that it inhibited Kv channels regardless of gating properties. Application of train pulses (1 and 2 Hz) progressively augmented the clozapine-induced inhibition of Kv channels in the presence of the drug. Furthermore, the recovery time constant from inactivation was increased in the presence of clozapine, suggesting that clozapine-induced inhibition of Kv channels is use (state)-dependent. Pretreatment of a Kv1.5 subtype inhibitor decreased the Kv current amplitudes, but additional application of clozapine did not further inhibit the Kv current. Pretreatment with Kv2.1 or Kv7 subtype inhibitors partially blocked the inhibitory effect of clozapine. Based on these results, we conclude that clozapine inhibits arterial Kv channels in a concentration- and use (state)-dependent manner. Kv1.5 is the major subtype involved in clozapine-induced inhibition of Kv channels, and Kv2.1 and Kv7 subtypes are partially involved.

The epoxy fatty acid pathway enhances cAMP in mammalian cells through multiple mechanisms

The cellular mechanism by which epoxy fatty acids (EpFA) improves disease status is not well characterized. Previous studies suggest the involvement of cellular receptors and cyclic AMP (cAMP). Herein, the action of EpFAs derived from linoleic acid (LA), arachidonic acid (ARA), and docosahexaenoic acid on cAMP levels was studied in multiple cell types to elucidate relationships between EpFAs, receptors and cells’ origin. cAMP levels were enhanced in HEK293 and LLC-PK1 cells by EpFAs from LA and ARA. Using selective antagonists, the EpFA effects on cAMP levels appear dependent on the prostaglandin E2 receptor 2 (EP2) but not 4 (EP4). Human coronary artery smooth muscle cells responded similarly to the EpFAs. However, we were not able to show the involvement of any of the receptors tested in this cell type. The results pinpointed distinct cell lines and receptor subtypes that natively respond to EpFA.

Retraction Statement.

Retraction Statement.

Mechano-biological phenotype in post-infarction cardiac remodeling

Ischemic heart disease is one of the leading cause of death in developed countries. In 30% of patients with heart infarction, inadequate cardiac fibrosis leads to terminal failure. We believe that local mechanical forces (inherent to the pump function of the heart) after cardiac infarction play a key role in the outcome of cardiac remodeling, in particular by modifying the response of microvascular endothelial cells (MECs), main players accompanying myocardial healing processes. Dynamic data, accessible by ultrasound, are however not widely used in the clinic, and could help to understand why patients remain refractory to treatments for heart failure. We aimed at studying the mechano-biological coupling that exists between mechanical stress applied to ischemic lesions, and the local biological response of cardiac cells. Using a mouse model of myocardial infarction (coronary artery ligation), we analyzed heart deformation using dynamic ultrasound imaging 1 month post-infarction (speckle tracking, VevoLabs). Mice were then sacrificed, and the hearts were harvested and processed for immunohistochemical analysis. Multiparametric mechanical maps (strain, displacement and velocity) were obtained using a morphometry program developed in the laboratory, and compared to a spatial transcriptomic analysis in the corresponding section planes (Visium, 10X genomics), in 4 infarcted heart of various size and localization. The transcriptomic data were validated in situ by immunofluorescence, in contiguous mouse heart sections ( Fig. 1 ). Generated big data highlight a panel of transcripts which expression highly correlates with radial strain and displacement of the heart wall. Among these factors, neuregulin-1 (involved in cardiac remodeling), and TRPC6 or PECAM-1 (sensors of mechanical forces) were found highly expressed in the mouse heart sections (immunofluorescence). Neuregulin-1 and TRPC6 strongly co-localized with MECs and coronary artery SMCs bordering the infarcted area, while PECAM-1 activation (phosphorylation) in MECs was found highly correlated with radial strain. These results support a mechano-biological coupling in infarcted hearts, modulating the expression of factors involved in its repair. Understanding the biology associated with mechanical stress could improve the management of patients after a heart attack, on the sole basis of dynamic imaging.

ONOO- -mediated oxidative modification of extracellular matrix aggravates atherosclerosis by affecting biological behaviors of vascular smooth muscle cells.

Atherosclerosis (AS) is a principal contributor to stroke and coronary heart disease in humans characterized by chronic low-grade inflammation. The extracellular matrix (ECM) plays critical roles in regulating the function of arteries. However, the effect of changes in ECM on AS development is rarely studied. In this context, we intend to study the effect of oxidizing agent peroxynitrite (ONOO- )-mediated oxidization of ECM proteins on the biological behaviors of vascular smooth muscle cells(SMCs) and the development of AS. AS mouse models were established, and mouse coronary artery smooth muscle cells (MCASMCs) were cultured in vitro to derive ECM (SMC-ECM), which was obtained by deoxycholate (DOC)-based decellularization. Further, MCASMCs were subjected to the determination of ECM oxidative damage and ECM protein structure. Finally, roles of ONOO- -mediated oxidization of ECM in SMC adhesion and migration and in AS development were explored through Transwell assay, transcriptome sequencing, and gene enrichment analysis. High concentration of ONOO- was found in the serum of AS mice, and ONOO- could stimulate the development of AS. SMC-ECM with intact structure can be obtained in vitro by DOC treatment. Functionally, ONOO- -mediated oxidization destroyed the three-dimensional structure of SMC-ECM proteins, affected SMC adhesion and migration and promoted the absorption efficiency of lipids while reducing the efflux of cholesterol. In addition, the expression of inflammation- and oxidative stress-related genes was significantly increased in ECM subjected to ONOO- -mediated oxidization, thereby contributing to AS progression. ONOO- -mediated oxidative modification of ECM aggravates AS by affecting the biological behavior of SMCs.

Inhibitory mechanism of tangeretin, a citrus flavone on the sphingosylphosphorylcholine (SPC)-induced vascular smooth muscle contraction

We previously discovered that the SPC/Fyn/Rho-kinase (ROK) pathway mediates the Ca2+-sensitization of coronary arterial smooth muscle (CASM) contraction leading to vasospasm, a major cause of sudden death. Lately, we have been trying to find and develop more natural edible compounds which can treat and/or prevent the SPC-induced abnormal CASM contraction, and finally the first to discover that tangeretin (5,6,7,8,4′-pentamethoxyflavone), a natural compound extracted from citrus plants, can inhibit the SPC-induced CASM contraction both in the pretreatment and posttreatment. In porcine CASM tissues, tangeretin showed remarkable inhibitory effects on the SPC-induced contraction with modest inhibitory effects on the high K+-depolarization-induced Ca2+-dependent contraction, both in pretreatment and posttreatment at the optimal concentrations; Regarding the mechanisms, tangeretin markedly abolished the SPC-induced cell contraction through inhibiting the SPC-induced activation and translocation of Fyn and ROK from the cytoplasm to the cell membrane in cultured CASM cells, resulting in the reduction of phosphorylation of myosin light chain. Taken together, these findings indicate that tangeretin, upon pre- or post-treatment, inhibits the SPC-induced CASM contraction through suppressing the Fyn/ROK signaling pathway, thereby suggesting that tangeretin can be a potential candidate for the treatment and/or prevention of vasospasm.

Peroxynitrous acid-modified extracellular matrix alters gene and protein expression in human coronary artery smooth muscle cells and induces a pro-inflammatory phenotype

Leukocytes produce oxidants at inflammatory sites, including within the artery wall during the development of atherosclerosis. Developing lesions contain high numbers of activated leukocytes that generate reactive nitrogen species, including peroxynitrite/peroxynitrous acid (ONOO−/ONOOH), as evidenced by the presence of oxidized/nitrated molecules including extracellular matrix (ECM) proteins. ECM materials are critical for arterial wall integrity, function, and determine cell phenotype, with smooth muscle cells undergoing a phenotypic switch from quiescent/contractile to proliferative/synthetic during disease development. We hypothesized that ECM modification by ONOO−/ONOOH might drive this switch, and thereby potentially contribute to atherogenesis. ECM generated by primary human coronary artery smooth muscle cells (HCASMCs) was treated with increasing ONOO−/ONOOH concentrations (1–1000 μM). This generated significant damage on laminin, fibronectin and versican, and lower levels on collagens and glycosaminoglycans, together with the increasing concentrations of the damage biomarker 3-nitrotyrosine. Adhesion of naïve HCASMC to ECM modified by 1 μM ONOO−/ONOOH was enhanced, but significantly diminished by higher ONOO−/ONOOH treatment. Cell proliferation and metabolic activity were significantly enhanced by 100 μM ONOO−/ONOOH pre-treatment. These changes were accompanied by increased expression of genes involved in mitosis (PCNA, CCNA1, CCNB1), ECM (LAMA4, LAMB1, VCAN, FN1) and inflammation (IL-1B, IL-6, VCAM-1), and corresponding protein secretion (except VCAM-1) into the medium. These changes induced by modified ECM are consistent with HCASMC switching to a synthetic/proliferative/pro-inflammatory phenotype, together with ECM remodelling. These changes model those in atherosclerosis, suggesting a link between oxidant-modified ECM and disease progression, and highlight the potential of targeting oxidant generation as a therapeutic strategy.

MO185: Post-Translational Carbamylation of Sortilin is Associated with Cardiovascular Calcification in Chronic Kidney Disease

Abstract BACKGROUND AND AIMS Sortilin, an intracellular sorting receptor, has been identified as a cardiovascular (CV) risk factor in the general population. Patients with chronic kidney disease (CKD) are highly susceptible to developing CV complications such as CV calcification. However, specific CKD-induced post-translational protein modifications of sortilin and their link to CV calcification remain unknown. METHODS Circulating sortilin isolated from two independent CKD cohorts was analysed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF mass spectrometry). The binding partner affinity was analysed by surface plasmon resonance spectroscopy. The effect of carbamylated sortilin on vascular calcification was analysed in vitro using human coronary artery smooth muscle cells. RESULTS In CKD patients, targeted mass spectrometric analyses of circulating sortilin revealed an increase in carbamylated lysine residues with kidney function decline. Carbamylation did not affect dimer formation of sortilin assessed by mass spectrometry. We observed an increased affinity of interleukin 6 to in vitro carbamylated sortilin. Carbamylated sortilin increased SMC calcification in vitro that was accelerated by interleukin 6, while carbamylated albumin and collagen type I did not affect SMC calcification. Mass-spectrometry imaging of human calcified arteries revealed in situ carbamylated sortilin. In CKD patients, sortilin carbamylation was associated with coronary artery calcification volume, independent of age, kidney function and other risk factors. Moreover, patients with carbamylated sortilin displayed significantly faster coronary artery calcification progression than patients without sortilin carbamylation. CONCLUSION Carbamylated sortilin is a risk factor for CV calcification and may contribute to elevated CV complications in patients with CKD.

Abstract 195: Transcriptional Regulation Of Transcriptional Regulation Of Triggering Receptor Expressed On Myeloid Cells-1 By Low Shear Stress In A Three-dimensional Co-culture Of Coronary Artery Smooth Muscle Cells, Endothelial Cells, And Monocytes

Objectives: To identify transcriptional factors involved in the expression of Triggering Receptor Expressed on Myeloid Cells-1 (TREM-1) in response to low shear stress in an in-vitro model of coronary artery atherosclerosis. Methods: Human coronary artery smooth muscle cells were mixed with neutralized type I collagen solution and plated on glass coverslips. The human coronary artery endothelial cells (HCAECs) were then plated on top of the collagen gels followed by plating monocytes (THP-1 cells) on top of the HCAECs. After 48 h, the co-culture was treated with or without low shear stress (LSS, 5±3 dyne/cm 2 ) using the Flexcell Streamer for 30 min. Protein and mRNA of TREM-1 expression was assessed by immunofluorescence staining and real time RT-PCR. The expression of transcriptional factors involved in the expression of TREM-1 was assessed by ChIP assay. Results: LSS significantly stimulated TREM-1 expression as evidenced by IF staining and mRNA expression (fold increase vs control: 43.2 ± 9.7, Fig 1A and 1B ). ChIP assay demonstrated that, under control condition (no flow), NF-κB (p65), PU.1 and activation transcription factor 2 (ATF2) bound to TREM-1 promoter region 2, but not to region 1 ( Fig 2A ). In response to LSS for 30 min, however, binding of the NF-κB (p65), PU.1 and ATF2 to TREM-1 promoter region 2 was significantly enhanced; furthermore, LSS treatment induced binding of NF-κB (p65), PU.1 and ATF2 to TREM-1 promoter region 1 ( Fig 2B) . Conclusions: Transcriptional factors NF-κB (p65), PU.1 and ATF2 are critically involved in the TREM-1 expression in response to LSS stimulation in an in-vitro model of human coronary artery atherosclerosis.

Abstract 505: Cross Talk Between T Cells And Vascular Smooth Muscle Cells May Contribute To Phenotypic Changes In Human Coronary Plaque

Background: The atherosclerotic plaque is a complex niche composed of immune cells including T cells and macrophages as well as vascular smooth muscle cells. How T cells interact with the plaque microenvironment is not well understood. Objective: To map the crosstalk between T cells, macrophages, and smooth muscle cells. Methods/Results: We performed single cell RNAseq using the 10x transcriptomics platform in coronary plaque samples from 12 patients, who underwent heart transplantation. We found a high proportion of macrophages as well as T cells, many of which display a memory phenotype that was confirmed by immunohistochemistry. Ligand-receptor computational analysis demonstrated significant cross talk between T cells, macrophages, and smooth muscle cells. One cytokine of interest is amphiregulin (AREG), which has been shown to mediate tissue healing and fibrosis. Of note, AREG expression appears to increase as coronary plaques progress from early lipid-rich stages to more advanced stages. To elucidate the effects of AREG on smooth muscle cells, we treated human coronary artery smooth muscle cells (hCASMCs) in vitro with recombinant AREG and found increased cell proliferation compared to control (P<0.001). Bulk RNAseq profiling of hCASMCs showed activation of pathways that promote inflammation, proliferation, and fibrosis (Figure 1). Conclusion: Taken together, our findings suggest that cross talk between T cells and smooth muscle cells, partially through AREG production by T cells, may contribute to plaque progression.

C1q/gC1qR‐Mediated Activation of Extrinsic Coagulation

Vascular diseases can be characterized by and are highly associated with inflammation and thrombosis. Understanding the relationship between these two processes would allow us to treat vascular diseases more effectively. Activation of complement component 1 (C1) is an important contributor to innate immunity and drives the lysis of pathogens. Upon C1 activation, significant concentrations of circulating C1q are observed. C1q has an affinity for gC1qR, a cell surface receptor molecule that is commonly found on cell types associated with cardiovascular disease development, including vascular smooth muscle cells and adventitial fibroblasts. When C1q interacts with gC1qR, many pathological pathways initiate including the inflammation observed during atherosclerosis and the initiation of intrinsic coagulation. The intrinsic cascade plays a minor role in physiological and pathological thrombosis as compared with the extrinsic cascade; however, the effects of C1 and the role of C1q/gC1qR on extrinsic coagulation have not been studied. We hypothesized that C1q association with gC1qR would increase the amount of sub‐endothelial tissue factor expressed by both aortic adventitial fibroblasts (HAAFs) and coronary artery smooth muscle cells (HCASMCs). We investigated these cells as they are the primary TF bearing cells. Further, we hypothesized that the newly expressed TF would bind Factor VII (FVII), which is a crucial step in the initiation of the extrinsic coagulation cascade. The objective of this study was to elucidate the role of C1q/gC1qR‐mediated TF expression and its ability to bind FVII. Using a solid‐phase ELISA, TF expression was observed on both HAAFs and HCASMCs. Cells were conditioned for one hour with either platelet poor plasma (PPP), purified human C1q, LPS, or a combination of anti‐gC1qR antibodies (60.11 or 74.5.2 clones) and purified human C1q, PPP, or LPS. A capture ELISA approach was used to evaluate the binding of FVII to TF following exposure after the same conditions. Further, a capture ELISA was used to assess released TF. Finally, a live/dead assay to assess cell viability and cell density, as well as an MTT assay to assess cell metabolic activity were performed to monitor cell culture conditions. Our results indicate that HAAF and HCASMC expression of TF increased significantly by approximately 15% after incubation with C1q as compared with unconditioned cells. Cells conditioned with gC1qR blocking antibodies prior to conditioning with C1q exhibited no change in TF expression when compared to unconditioned cells. Additionally, a 20‐30% increase in bound FVII was observed in both cell types when conditioned with C1q as compared to unconditioned cells. Our results show no significant differences in metabolic activity or cell viability under these conditions; however, cell density decreased by approximately 25% when conditioned with C1q. Overall, this data illustrates a role for C1q in activation of extrinsic coagulation. Furthermore, our data suggests that gC1qR acts a mediator for both inflammation and thrombosis. This work serves as a meaningful step in identifying a convergence of inflammatory and thrombotic pathways that can be therapeutically targeted to mitigate vascular diseases. Future work will determine if the expressed TF and the binding of FVII can support downstream extrinsic coagulation activity. Thanks to NIH for supporting this research.

Abstract 222: The Molecular Mechanisms Of Coronary Artery Disease Risk At The Pdgfd Locus

Background: Atherosclerotic cardiovascular diseases, including coronary artery disease (CAD), are the worldwide leading cause of death. Recent genome wide association studies (GWAS) have now identified over 250 genetic loci that are associated with CAD, and efforts in this lab and others are investigating the mechanisms of association at these loci. The CAD GWAS locus at 11q22.3 is of potential interest because it encodes the PDGFD gene, a member of the platelet-derived growth factor family whose expression is up-regulated in atherosclerosis. But whether disease allelic variation regulates PDGFD expression and how this growth factor might influence the risk of CAD has not been studied. Methods: The causal variant and gene were investigated with colocalization and in vitro transcription studies in cultured human coronary artery smooth muscle cells (SMC). Single-cell transcriptomic profiling (scRNAseq) and histological analyses were conducted in SMC-specific lineage tracing in an in vivo Pdgfd knockout mouse model of atherosclerosis. Results: The causal variant rs2019090 was linked to PDGFD expression in SMC. In silico analysis of transcription factor binding site prediction and in vitro haplotype-specific chromatin immunoprecipitation combined with qPCR analysis revealed another causal CAD factor forkhead box c1 (FOXC1) preferentially binds to the A-allele of variant rs2019090 and controls the expression level of PDGFD . scRNAseq data identified that loss of Pdgfd resulted in a marked increase in differentiated SMC but a decrease in fibromyocyte (FMC) and chondromyocyte (CMC) transition populations, as well as a significant decrease in plaque macrophages. Differentially expressed gene analysis in SMC lineage cells was highly enriched for CMC genes such as Col2a1 and Ibsp. Lesion histology analysis identified an increase in medial SMC, but a decrease in fibrous cap SMC, with decreased Cd68 and Col2a1 stained area. Conclusion: PDGFD is a causal CAD gene that increases disease risk by promoting SMC transition primarily to the CMC phenotype, and recruitment of monocyte-macrophage lineage cells to disease lesions. These findings support suppression of PDGFD as a possible therapy for human atherosclerotic disease.

Abstract 532: Dioxin Modifies The Transcriptomic And Epigenetic Landscape Of Smooth Muscle Cells Leading To Adverse Remodeling Of Atherosclerosis

Background: Environmental exposure to dioxin, an active component of cigarette smoke and air pollution has been linked to increased myocardial infarction. Smooth muscle cells (SMC) in the coronary vasculature play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity. We have previously found strong dioxin response element activity in modulated SMC of atherosclerotic lesion. In this study, we aimed to understand the effect of dioxin on vascular SMC and atherosclerosis phenotype. Methods: Primary human coronary artery SMC (HCASMC) treated in culture with 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) or vehicle control were used to perform RNA-Seq and ATAC-Seq. ChIP-Seq was performed with antibodies against Aryl-hydrocarbon receptor (AHR) and TCF21. Tamoxifen inducible SMC-lineage tracing reporter mice were used to assess the in-vivo effect of TCDD on aortic SMC. After 8 weeks of high fat diet (HFD), followed by 8 weeks of TCDD injection and HFD, the aortic sinus was collected for histology and single-cell sequencing (10X Genomics). Results: Analysis of the RNA-Seq from TCDD treated HCASMC showed differential enrichment of pathways related to cell migration and vascular development. Further, ATAC-Seq data showed a significant enrichment for pathways regulating vascular development, cell migration, and apoptosis. There was an overall increase in chromatin accessibility suggesting transcriptional activation with TCDD. We observed enrichment for AHR ChIP-Seq peaks in the open chromatin regions along with a significant reciprocal reduction in TCF21 occupancy. The scRNA-Seq of mice treated with TCDD exposure showed differential gene expression of SMC-lineage cells enriching for extracellular matrix (ECM) organization, inflammation, unfolded protein response, and apoptotic process, and histological analysis of the aortic sinus showed increased lesion area with increased presence of SMC-lineage cells and Cd68 + cells compared to control. Conclusion: Dioxin adversely remodels atherosclerotic plaque affecting pathways of ECM organization, inflammation and unfolded protein response in SMC, partly by modifying chromatin accessibility and occupancy of key transcription factors of phenotypic modulation.

Electrospun Biodegradable α-Amino Acid-Substituted Poly(organophosphazene) Fiber Mats for Stem Cell Differentiation towards Vascular Smooth Muscle Cells.

Mesenchymal stem cells, derived from human-induced pluripotent stem cells (iPSC), are valuable for generating smooth muscle cells (SMCs) for vascular tissue engineering applications. In this study, we synthesized biodegradable α-amino acid-substituted poly(organophosphazene) polymers and electrospun nano-fibrous scaffolds (~200 nm diameter) to evaluate their suitability as a matrix for differentiation of iPSC-derived mesenchymal stem cells (iMSC) into mature contractile SMCs. Both the polymer synthesis approach and the electrospinning parameters were optimized. Three types of cells, namely iMSC, bone marrow derived mesenchymal stem cells (BM-MSC), and primary human coronary artery SMC, attached and spread on the materials. Although L-ascorbic acid (AA) and transforming growth factor-beta 1 (TGF-β1) were able to differentiate iMSC along the smooth muscle lineage, we showed that the electrospun fibrous mats provided material cues for the enhanced differentiation of iMSCs. Differentiation of iMSC to SMC was characterized by increased transcriptional levels of early to late-stage smooth muscle marker proteins on electrospun fibrous mats. Our findings provide a feasible strategy for engineering functional vascular tissues.

Synthesis and cellular evaluation of click-chemistry probes to study the biological effects of alpha, beta-unsaturated carbonyls

Humans are commonly exposed to α,β-unsaturated carbonyls as both environmental toxins (e.g. acrolein) and therapeutic drugs (e.g. dimethylfumarate, DMFU, a front-line drug for the treatment of multiple sclerosis and psoriasis). These compounds undergo rapid Michael addition reactions with amine, imidazole and thiol groups on biological targets, with reaction at protein Cys residues being a major reaction pathway. However, the cellular targets of these species (the ‘adductome’) are poorly understood due to the absence of readily identifiable tags or reporter groups (chromophores/fluorophores or antigens) on many α,β-unsaturated carbonyls. Here we report a ‘proof of concept’ study in which we synthesize novel α,β-unsaturated carbonyls containing an alkyne function introduced at remote sites on the α,β-unsaturated carbonyl compounds (e.g. one of the methyl groups of dimethylfumarate). The presence of this tag allows ‘click-chemistry’ to be used to visualize, isolate, enrich and characterize the cellular targets of such compounds. The probes show similar selectivity and reactivity to the parent compounds, and compete for cellular targets, yielding long-lived (stable) adducts that can be visualized in intact cells (such as primary human coronary artery smooth muscle cells), and extracted and enriched for subsequent target analysis. It is shown using this approach that dimethylfumarate forms adducts with multiple intracellular targets including cytoskeletal, organelle and nuclear species, with these including the rate-limiting glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This approach should be amenable to use with multiple α,β-unsaturated carbonyls and a wide variety of targets containing nucleophilic sites.

Dual surface modification of poly(L-lactide) scaffold achieved by thermal incorporation of aligned nanofiber and click immobilization of VEGF to enhance endothelialization and blood compatibility

Rapid endothelialization is very important in the development of blood-contact biomaterials because of the inhibitory effects of endothelium in preventing thrombosis and restenosis. In this study, topographical and biochemical cues were generated to accelerate endothelialization on the poly(L-lactide) (PLLA) scaffold via a simple, bio-orthogonal, and biocompatible process. Topographical guidance was incorporated into a PLLA scaffold via thermal attachment of aligned PLLA nanofibers using electrospinning. Vascular endothelial growth factor (VEGF) was covalently immobilized onto the PLLA scaffold by aminolysis and copper-catalyzed azide/alkyne cycloaddition-mediated click chemistry. Various surface analyses confirmed the presence of aligned topography and immobilized VEGF without delamination or leaching. In vitro experiments with human coronary artery endothelial cells revealed that proliferation, CD31 expression, and migration was significantly enhanced on the nanofiber-attached VEGF-immobilized PLLA scaffold. The aligned nanofiber also accommodated the sustained release of heparin, which suppressed the proliferation of human coronary artery smooth muscle cells and platelet activation without affecting surface endothelialization. The heparin-loaded, aligned, nanofiber-attached VEGF-immobilized PLLA scaffold was evidenced to be biocompatible and a promising candidate for blood-contact materials where fast endothelialization and suppressed platelet activation is required.

Enhancer promoter interactome and Mendelian randomization identify network of druggable vascular genes in coronary artery disease

Coronary artery disease (CAD) is a multifactorial disorder, which is partly heritable. Herein, we implemented a mapping of CAD-associated candidate genes by using genome-wide enhancer-promoter conformation (H3K27ac-HiChIP) and expression quantitative trait loci (eQTL). Enhancer-promoter anchor loops from human coronary artery smooth muscle cells (HCASMC) explained 22% of the heritability for CAD. 3D enhancer-promoter genome mapping of CAD-genes in HCASMC was enriched in vascular eQTL genes. By using colocalization and Mendelian randomization analyses, we identified 58 causal candidate vascular genes including some druggable targets (MAP3K11, CAMK1D, PDGFD, IPO9 and CETP). A network analysis of causal candidate genes was enriched in TGF beta and MAPK pathways. The pharmacologic inhibition of causal candidate gene MAP3K11 in vascular SMC reduced the expression of athero-relevant genes and lowered cell migration, a cardinal process in CAD. Genes connected to enhancers are enriched in vascular eQTL and druggable genes causally associated with CAD.

Involvement of ischemia-driven 5-lipoxygenase-resolvin-E1-chemokine like receptor-1 axis in the resolution of post-coronary artery bypass graft inflammation in coronary arteries.

Expression status of pro-resolvin lipid mediators (PLM) and receptors in the post-Coronary artery bypass grafting (CABG) coronary arteries are largely unknown. Here, we aim to investigate the expression of the enzymes involved in PLM synthesis and their receptors in the atherosclerotic post-CABG swine (AS) left anterior descending (LAD) compared to without CABG (LAD-AS), and in isolated coronary artery smooth muscle cells (CASMCs) cultured under ischemia. The arteries of interest were harvested from post-CABG atherosclerotic swine and the histomorphology and the expression status of key PLM mediators were quantified using immunostaining. Smooth muscle cells (SMCs) were cultured under ischemia and confirmed the expression on PLM mediators at transcript and protein level. The histomorphometric analysis revealed considerable alterations in the tissue architecture in LAD-CABG and LAD-AS arteries compared to control. PLM synthetic enzyme 5-lipoxygenases (5LO) was significantly upregulated in LAD-CABG and LAD-AS whereas the other enzymes including 12LO, 15LO, and cyclooxygenase-2, and the receptors including Chemokine like receptor 1 (ChemR23), 7-transmembrane G-protein coupled receptor-18 (GPCR18), GPCR120 were decreased in LAD-CABG than control. LO enzymes and PLM receptors were upregulated in ischemic CASMCs with respect to control. Western blot showed the upregulation of 5LO, and ChemR23. Additionally, higher level of resolvin-E1 (RvE1) was observed in ischemic control CASMCs which was decreased following reperfusion. These findings suggest that the CASMCs withstand the ischemia-triggered proinflammatory episodes by increasing the secretion of RvE1 mediated through 5LO and ChemR23 signaling.

The inhibitory effects of pimozide, an antipsychotic drug, on voltage-gated K+ channels in rabbit coronary arterial smooth muscle cells

Pimozide is an antipsychotic drug used to treat chronic psychosis, such as Tourette’s syndrome. Despite its widespread clinical use, pimozide can cause unexpected adverse effects, including arrhythmias. However, the adverse effects of pimozide on vascular K+ channels have not yet been determined. Therefore, we investigated the effects of pimozide on voltage-gated K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Pimozide concentration-dependently inhibited the Kv currents with an IC50 value of 1.78 ± 0.17 μM and a Hill coefficient of 0.90 ± 0.05. The inhibitory effect on the Kv current by pimozide was highly voltage-dependent in the voltage range of Kv channel activation, and additive inhibition of the Kv current by pimozide was observed in the full activation voltage range. The decay rate of inactivation was significantly accelerated by pimozide. Pimozide shifted the inactivation curve to a more negative potential. The recovery time constant from inactivation increased in the presence of pimozide. Furthermore, pimozide-induced inhibition of the Kv current was augmented by applying train pulses. Although pretreatment with the Kv2.1 subtype inhibitor guangxitoxin and the Kv7 subtype inhibitor linopirdine did not alter the degree of pimozide-induced inhibition of the Kv currents, pretreatment with the Kv1.5 channel inhibitor DPO-1 reduced the inhibitory effects of pimozide on Kv currents. Pimozide induced membrane depolarization. We conclude that pimozide inhibits Kv currents in voltage-, time-, and use (state)-dependent manners. Furthermore, the major Kv channel target of pimozide is the Kv1.5 channel.

Apolipoprotein (a)/Lipoprotein(a)-Induced Oxidative-Inflammatory α7-nAChR/p38 MAPK/IL-6/RhoA-GTP Signaling Axis and M1 Macrophage Polarization Modulate Inflammation-Associated Development of Coronary Artery Spasm.

Objective Apolipoprotein (a)/lipoprotein(a) (Lp(a)), a major carrier of oxidized phospholipids, and α7-nicotinic acetylcholine receptor (α7-nAChR) may play an important role in the development of coronary artery spasm (CAS). In CAS, the association between Lp(a) and the α7-nAChR-modulated inflammatory macrophage polarization and activation and smooth muscle cell dysfunction remains unknown. Methods We investigated the relevance of Lp(a)/α7-nAChR signaling in patient monocyte-derived macrophages and human coronary artery smooth muscle cells (HCASMCs) using expression profile correlation analyses, fluorescence-assisted cell sorting flow cytometry, immunoblotting, quantitative real-time polymerase chain reaction, and clinicopathological analyses. Results There are increased serum Lp(a) levels (3.98-fold, p = 0.011) and macrophage population (3.30-fold, p = 0.013) in patients with CAS compared with patients without CAS. Serum Lp(a) level was positively correlated with high-sensitivity C-reactive protein (r2 = 0.48, p < 0.01), IL-6 (r2 = 0.38, p = 0.03), and α7-nAChR (r2 = 0.45, p < 0.01) in patients with CAS, but not in patients without CAS. Compared with untreated or low-density lipoprotein- (LDL-) treated macrophages, Lp(a)-treated macrophages exhibited markedly enhanced α7-nAChR mRNA expression (p < 0.01) and activity (p < 0.01), in vitro and ex vivo. Lp(a) but not LDL preferentially induced CD80+ macrophage (M1) polarization and reduced the inducible nitric oxide synthase expression and the subsequent NO production. While shRNA-mediated loss of α7-nAChR function reduced the Lp(a)-induced CD80+ macrophage pool, both shRNA and anti-IL-6 receptor tocilizumab suppressed Lp(a)-upregulated α7-nAChR, p-p38 MAPK, IL-6, and RhoA-GTP protein expression levels in cultures of patient monocyte-derived macrophages and HCASMCs. Conclusions Elevated Lp(a) levels upregulate α7-nAChR/IL-6/p38 MAPK signaling in macrophages of CAS patients and HCASMC, suggesting that Lp(a)-triggered inflammation mediates CAS through α7-nAChR/p38 MAPK/IL-6/RhoA-GTP signaling induction, macrophage M1 polarization, and HCASMC activation.