Gene Expression In Smooth Muscle Cells Research Articles

PAI-1 Regulates the Cytoskeleton and Intrinsic Stiffness of Vascular Smooth Muscle Cells.

Plasma concentration of PAI-1 (plasminogen activator inhibitor-1) correlates with arterial stiffness. Vascular smooth muscle cells (SMCs) express PAI-1, and the intrinsic stiffness of SMCs is a major determinant of total arterial stiffness. We hypothesized that PAI-1 promotes SMC stiffness by regulating the cytoskeleton and that pharmacological inhibition of PAI-1 decreases SMC and aortic stiffness. PAI-039, a specific inhibitor of PAI-1, and small interfering RNA were used to inhibit PAI-1 expression in cultured human SMCs. Effects of PAI-1 inhibition on SMC stiffness, F-actin (filamentous actin) content, and cytoskeleton-modulating enzymes were assessed. WT (wild-type) and PAI-1-deficient murine SMCs were used to determine PAI-039 specificity. RNA sequencing was performed to determine the effects of PAI-039 on SMC gene expression. In vivo effects of PAI-039 were assessed by aortic pulse wave velocity. PAI-039 significantly reduced intrinsic stiffness of human SMCs, which was accompanied by a significant decrease in cytoplasmic F-actin content. PAI-1 gene knockdown also decreased cytoplasmic F-actin. PAI-1 inhibition significantly increased the activity of cofilin, an F-actin depolymerase, in WT murine SMCs, but not in PAI-1-deficient SMCs. RNA-sequencing analysis suggested that PAI-039 upregulates AMPK (AMP-activated protein kinase) signaling in SMCs, which was confirmed by Western blotting. Inhibition of AMPK prevented activation of cofilin by PAI-039. In mice, PAI-039 significantly decreased aortic stiffness and tunica media F-actin content without altering the elastin or collagen content. PAI-039 decreases intrinsic SMC stiffness and cytoplasmic stress fiber content. These effects are mediated by AMPK-dependent activation of cofilin. PAI-039 also decreases aortic stiffness in vivo. These findings suggest that PAI-1 is an important regulator of the SMC cytoskeleton and that pharmacological inhibition of PAI-1 has the potential to prevent and treat cardiovascular diseases involving arterial stiffening.

Effects of high-frequency mechanical stimuli on flow related vascular cell biology.

Mechanical forces related to blood pressure and flow patterns play a crucial role in vascular homeostasis. Perturbations in vascular stresses and strain resulting from changes in hemodynamic may occur in pathological conditions, leading to vascular dysfunction as well as in vascular prosthesis, arteriovenous shunt for hemodialysis and in mechanical circulation support. Turbulent-like blood flows can induce high-frequency vibrations of the vessel wall, and this stimulus has recently gained attention as potential contributors to vascular pathologies, such as development of intimal hyperplasia in arteriovenous fistula for hemodialysis. However, the biological response of vascular cells to this stimulus remains incompletely understood. This review provides an analysis of the existing literature concerning the impact of high-frequency stimuli on vascular cell morphology, function, and gene expression. Morphological and functional investigations reveal that vascular cells stimulated at frequencies higher than the normal heart rate exhibit alterations in cell shape, alignment, and proliferation, potentially leading to vessel remodeling. Furthermore, vibrations modulate endothelial and smooth muscle cells gene expression, affecting pathways related to inflammation, oxidative stress, and muscle hypertrophy. Understanding the effects of high-frequency vibrations on vascular cells is essential for unraveling the mechanisms underlying vascular diseases and identifying potential therapeutic targets. Nevertheless, there are still gaps in our understanding of the molecular pathways governing these cellular responses. Further research is necessary to elucidate these mechanisms and their therapeutic implications for vascular diseases.

Abstract 2141: An IL12/IL23 Positive Feedback Loop Modulates Smooth Muscle Differentiation Via Setd4 During Macrophage-mediated Inflammation

Diabetic patients have increased risk of vascular disease (e.g., atherosclerosis), which is driven by macrophage-mediated inflammation. In diseases such as atherosclerosis, smooth muscle cells (SMC) undergo phenotypic modulation from a mature, contractile to a synthetic phenotype, thus leading to increased neointima formation. We hypothesize that pro-inflammatory macrophages as in the setting of diabetes epigenetically pattern SMC during vascular disease. In this study, we use single cell-RNA sequencing (scRNA-seq) of human arteries, loss of function experiments, and mouse models of disease to identify epigenetic mechanisms that regulate SMC phenotype. We identified the H3K4 and H3K9 histone lysine methyltransferase Setd4, which was increased in aortic SMC from diabetic mice and in our scRNA-seq dataset from human atherosclerotic arteries. siRNA mediated knockdown of Setd4 in mouse aortic SMC increased expression of the early SMC differentiation markers, Acta2 and Tagln (p<0.05). To model the pro-inflammatory environment in vitro, supernatant from LPS-stimulated macrophages increased Setd4 expression and inhibited smooth muscle gene expression ( Acta2, Tagln, Cnn1, Myh11 ), which was reversed by siRNA-mediated knockdown of Setd4 in SMC (p<0.05). IL23 increased Setd4 expression in SMC and enrichment at smooth muscle gene promoters, while deceasing SMC-specific gene expression (p<0.05). Treatment of SMC with an IL23 receptor neutralizing antibody prevented downregulation of SMC gene expression by macrophage supernatant (p<0.05). Intriguingly, ELISA of mouse aortic SMC supernatant revealed that IL23 increased SMC secretion of IL12, which also inhibited SMC gene expression via Setd4 (p<0.05). In conclusion, we demonstrate that pro-inflammatory macrophage IL23 represses smooth muscle gene expression via SETD4, thereby shifting the SMC to a synthetic, diseased phenotype. IL23 also stimulates vascular SMC secretion of IL12, which further increases production of inflammatory cytokines from macrophages and leads to additional repression of smooth muscle gene expression. Thus, targeting the IL23-SETD4 axis in SMC may be a potential therapy for the treatment of atherosclerosis, especially in patients who also have diabetes.

Translatome profiling reveals Itih4 as a novel smooth muscle cell-specific gene in atherosclerosis.

Vascular smooth muscle cells (SMCs) and their derivatives are key contributors to the development of atherosclerosis. However, studying changes in SMC gene expression in heterogeneous vascular tissues is challenging due to the technical limitations and high cost associated with current approaches. In this paper, we apply translating ribosome affinity purification sequencing to profile SMC-specific gene expression directly from tissue. To facilitate SMC-specific translatome analysis, we generated SMCTRAP mice, a transgenic mouse line expressing enhanced green fluorescent protein (EGFP)-tagged ribosomal protein L10a (EGFP-L10a) under the control of the SMC-specific αSMA promoter. These mice were further crossed with the atherosclerosis model Ldlr-/-, ApoB100/100 to generate SMCTRAP-AS mice and used to profile atherosclerosis-associated SMCs in thoracic aorta samples of 15-month-old SMCTRAP and SMCTRAP-AS mice. Our analysis of SMCTRAP-AS mice showed that EGFP-L10a expression was localized to SMCs in various tissues, including the aortic wall and plaque. The TRAP fraction demonstrated high enrichment of known SMC-specific genes, confirming the specificity of our approach. We identified several genes, including Cemip, Lum, Mfge8, Spp1, and Serpina3, which are known to be involved in atherosclerosis-induced gene expression. Moreover, we identified several novel genes not previously linked to SMCs in atherosclerosis, such as Anxa4, Cd276, inter-alpha-trypsin inhibitor-4 (Itih4), Myof, Pcdh11x, Rab31, Serpinb6b, Slc35e4, Slc8a3, and Spink5. Among them, we confirmed the SMC-specific expression of Itih4 in atherosclerotic lesions using immunofluorescence staining of mouse aortic roots and spatial transcriptomics of human carotid arteries. Furthermore, our more detailed analysis of Itih4 showed its link to coronary artery disease through the colocalization of genome-wide association studies, splice quantitative trait loci (QTL), and protein QTL signals. We generated a SMC-specific TRAP mouse line to study atherosclerosis and identified Itih4 as a novel SMC-expressed gene in atherosclerotic plaques, warranting further investigation of its putative function in extracellular matrix stability and genetic evidence of causality.

Identification of immune-related signatures and pathogenesis differences between thoracic aortic aneurysm patients with bicuspid versus tricuspid valves via weighted gene co-expression network analysis.

Thoracic aortic aneurysm (TAA) occurs due to pathological aortal dilation, and both individuals with normal tricuspid aortic valves (TAV) or abnormal bicuspid aortic valves (BAV), the latter being a congenital condition, are at risk. However, some differences are present between TAA/BAV and TAA/TAV with respect to their pathophysiological processes and molecular mechanisms, but their exact nature is still mostly unknown. Therefore, it is necessary to elucidate TAA developmental differences among BAV vs. TAV patients. Publically-available gene expression datasets, aortic tissue derived from TAA/BAV and TAA/TAV individuals, were analyzed by weighted gene co-expression network analysis (WGCNA) to identify gene modules associated with those conditions. Gene Ontology (GO) enrichment analysis was performed on those modules to identify the enriched genes within those modules, which were verified by Gene Set Variation Analysis (GSVA) on a dataset derived from aortic smooth muscle cell gene expression between TAA/TAV and TAV/BAV patients. Immune cell infiltration patterns were then analyzed by CIBERSORT, and a protein-protein interaction (PPI) network was constructed based on WGCNA and enrichment analysis results to identify hub genes, followed by validation via stepwise regression analysis. Three signatures most strongly associated with TAA/TAV were confirmed by receiver operating characteristic (ROC) and decision curve analyses (DCA) between prior-established training and testing gene sets. WGCNA delineated 2 gene modules being associated with TAA/TAV vs. TAA/BAV; both were enriched for immune-associated genes, such as those relating to immune responses, etc., under enrichment analysis. TAA/TAV and TAA/BAV tissues also had differing infiltrating immune cell proportions, particularly with respect to dendritic, mast and CD4 memory T cells. Identified three signatures, CD86, integrin beta 2 (ITGB2) and alpha M (ITGAM), as yielding the strongest associations with TAA/TAV onset, which was verified by areas under the curve (AUC) at levels approximating 0.8 or above under ROC analysis, indicating their predictive value for TAA/TAV onset. However, we did not examine possible confounding variables, so there are many alternative explanations for this association. TAA/TAV pathogenesis was found to be more associated with immune-related gene expression compared to TAA/BAV, and the identification of three strongly-associated genes could facilitate their usage as future biomarkers for diagnosing the likelihood of TAA/TAV onset vs. TAA/BAV, as well as for developing future treatments.

Epigenetic Induction of Smooth Muscle Cell Phenotypic Alterations in Aortic Aneurysms and Dissections.

Smooth muscle cell (SMC) phenotypic switching has been increasingly detected in aortic aneurysm and dissection (AAD) tissues. However, the diverse SMC phenotypes in AAD tissues and the mechanisms driving SMC phenotypic alterations remain to be identified. We examined the transcriptomic and epigenomic dynamics of aortic SMC phenotypic changes in mice with angiotensin II-induced AAD by using single-cell RNA sequencing and single-cell sequencing assay for transposase-accessible chromatin. SMC phenotypic alteration in aortas from patients with ascending thoracic AAD was examined by using single-cell RNA sequencing analysis. Single-cell RNA sequencing analysis revealed that aortic stress induced the transition of SMCs from a primary contractile phenotype to proliferative, extracellular matrix-producing, and inflammatory phenotypes. Lineage tracing showed the complete transformation of SMCs to fibroblasts and macrophages. Single-cell sequencing assay for transposase-accessible chromatin analysis indicated that these phenotypic alterations were controlled by chromatin remodeling marked by the reduced chromatin accessibility of contractile genes and the induced chromatin accessibility of genes involved in proliferation, extracellular matrix, and inflammation. IRF3 (interferon regulatory factor 3), a proinflammatory transcription factor activated by cytosolic DNA, was identified as a key driver of the transition of aortic SMCs from a contractile phenotype to an inflammatory phenotype. In cultured SMCs, cytosolic DNA signaled through its sensor STING (stimulator of interferon genes)-TBK1 (tank-binding kinase 1) to activate IRF3, which bound and recruited EZH2 (enhancer of zeste homolog 2) to contractile genes to induce repressive H3K27me3 modification and gene suppression. In contrast, double-stranded DNA-STING-IRF3 signaling induced inflammatory gene expression in SMCs. In Sting-/- mice, the aortic stress-induced transition of SMCs into an inflammatory phenotype was prevented, and SMC populations were preserved. Finally, profound SMC phenotypic alterations toward diverse directions were detected in human ascending thoracic AAD tissues. Our study reveals the dynamic epigenetic induction of SMC phenotypic alterations in AAD. DNA damage and cytosolic leakage drive SMCs from a contractile phenotype to an inflammatory phenotype.

Abstract P3024: Endothelial To Mesenchymal Transition Frequency Increases In Williams Syndrome Pulmonary Artery Endothelial Cells In Novel 3D Bioprinted In Vitro Arterial Model

Introduction: Pulmonary artery stenosis (PAS) affects 1 in 2000 live births worldwide, with several causes, most commonly by genetic mutations that lead to or are associated with other congenital heart diseases (CHD). Despite its prevalence, long-term treatment strategies for patients with PAS remains ineffective due to its heterogenous etiology. In the last few decades, researchers have found hemodynamic forces to be a great contributor to stenosis development because it induces endothelial cell (EC) dysfunction through endothelial-to-mesenchymal transition (EndMT). Current pulmonary artery models, in vivo and in vitro , are ineffective in uncovering this mechanism in PAS because they either are unable to recapitulate the phenotype or the micromechanical environment. Methods: To address these limitations, we developed and optimized a high-throughput 3D bioprinted in vitro vascular model that recapitulates patient-specific disease states to determine how EndMT plays a role in stenosis development. This bioprinted model will be a cellularized bilayer comprised of EC and smooth muscle cells (SMC) sourced from either human primary cells or our established Williams Syndrome (WS) induced-pluripotent stem cell (iPSC) lines. WS was chosen due to its high predisposition in developing PAS, around 50%. Our hypothesis is that utilizing our 3D bioprinted model, we will demonstrate how alterations in hemodynamic forces, specifically flow regiments, will further promote EndMT in the WS group. Results: Encapsulated SMC functionality and differentiated phenotype was confirmed by calcium measurement and immunohistochemistry, respectively. Single-cell RNA sequencing (scRNA-seq) was used to examine EC, SMC, EC-SMC crosstalk, and EndMT gene expression profiles. Conclusion: This work represents a new and substantive departure from conventional in vitro vascular modeling, for it will allow us to identify initial vascular cellular responses in acute non-homeostatic conditions and uncover unique signaling pathways found in multiple CHD related PAS. This fundamental knowledge from our vascular mimics can then be applied to facilitate translational advancements in treatment of PAS via surgical and transcatheter intervention methods.

Final 36-Month Outcomes from the Multicenter DynamX Study Evaluating a Novel Thin-Strut Novolimus-Eluting Coronary Bioadaptor System and Supporting Preclinical Data.

The DynamX Novolimus-Eluting Coronary Bioadaptor System ( Bioadaptor) has uncaging elements that disengage after the resorption of the polymer coating, aiming to restore vessel function in the treated segment and to avoid long-term adverse outcomes associated with the permanent caging of the coronary artery seen with conventional stenting. This prospective, multicenter, single-arm first-in-human study enrolled 50 patients in Belgium and Italy who were treated with the DynamX Bioadaptor. Eligible patients had de novo lesions in coronary arteries measuring between 2.5 and 3.5 mm in diameter and 24 mm in length. Clinical follow-up was performed up to 36 months. This analysis includes the intention-to-treat population and is based on data available. The preclinical studies include optical coherence tomography (OCT) analyses of 5 DynamX Bioadaptors implanted in 3 mini Yucatan pigs (at 3, 12 and 24 months), and assessment of smooth muscle cell gene expression profile in 8 pigs of which each was implanted with the DynamX Bioadaptor and the Xience drug-eluting stent. To assess the gene expression profile by quantitative real-time polymerase chain reaction, animals were sacrificed at 3, 6, 9 and 12 months. Target lesion failure at 36 months was 8.7% (4/46), consisting of one clinically-driven target lesion revascularization and 3 cardiac deaths (all site-reported to be unrelated to the device or procedure). There were no additional target vessel revascularization and no definite or probable scaffold thrombosis. Preclinical data confirmed late lumen enlargement (from 7.02 1.31 at baseline to 8.46 1.31 at 24 months) and identified an increased expression of contractile genes around 9 months compared to a conventional drug-eluting stent. The DynamX Bioadaptor demonstrated very good 36-month clinical outcomes, highlighted by the absence of target-vessel myocardial infarction and definite or probable device thrombosis, and only one target lesion revascularization up to 36 months. These data are supported by preclinical studies that showed late lumen enlargement by OCT and an increased expression of contractile genes around 9 months compared to conventional drug-eluting stents, indicating faster vessel healing. Larger clinical studies are necessary to compare outcomes against contemporary drug-eluting stents. https://clinicaltrials.gov/: NCT03429894.

Modulation of Smooth Muscle Cell Phenotype for Translation of Tissue-Engineered Vascular Grafts.

Translation of small-diameter tissue-engineered vascular grafts (TEVGs) for the treatment of coronary artery disease (CAD) remains an unfulfilled promise. This is largely due to the limited integration of TEVGs into the native vascular wall-a process hampered by the insufficient smooth muscle cell (SMC) infiltration and extracellular matrix deposition, and low vasoactivity. These processes can be promoted through the judicious modulation of the SMC toward a synthetic phenotype to promote remodeling and vascular integration; however, the expression of synthetic markers is often accompanied by a decrease in the expression of contractile proteins. Therefore, techniques that can precisely modulate the SMC phenotypical behavior could have the potential to advance the translation of TEVGs. In this review, we describe the phenotypic diversity of SMCs and the different environmental cues that allow the modulation of SMC gene expression. Furthermore, we describe the emerging biomaterial approaches to modulate the SMC phenotype in TEVG design and discuss the limitations of current techniques. In addition, we found that current studies in tissue engineering limit the analysis of the SMC phenotype to a few markers, which are often the characteristic of early differentiation only. This limited scope has reduced the potential of tissue engineering to modulate the SMC toward specific behaviors and applications. Therefore, we recommend using the techniques presented in this review, in addition to modern single-cell proteomics analysis techniques to comprehensively characterize the phenotypic modulation of SMCs. Expanding the holistic potential of SMC modulation presents a great opportunity to advance the translation of living conduits for CAD therapeutics.

Exploring the expression and potential function of follicle stimulating hormone receptor in extragonadal cells related to abdominal aortic aneurysm.

Follicle stimulating hormone (FSH) is identified to play a role in postmenopausal disease and hypothesized to affect abdominal aortic aneurysm (AAA) onset/progression in postmenopausal women. We aimed to detect FSHR gene expression in AAA tissue and cell types involved in AAA formation. FSH stimulation of human umbilical cord endothelial cells (HUVECs), smooth muscle cells (HUCs) and PMA-differentiated macrophages to assess gene expression of FSHR and various markers. Human macrophages activated with various stimuli were assessed for FSHR gene expression. AAA dataset, AAA tissue samples and AAA-derived smooth muscle cells (SMC) obtained from elderly female donors were assessed for FSHR gene expression. AAA-SMCs were stimulated with FSH to assess its effect on gene expression. Lastly, oxidized low-density-lipoprotein (ox-LDL) uptake and abundance of cell surface protein markers were assessed by flow cytometry after FSH stimulation of human monocytes. FSH stimulation showed similar levels of gene expression in HUVECs and HUCs. Only ACTA2 was downregulated in HUCs. In PMA-differentiated macrophages, gene expression of inflammation markers was unchanged after FSH stimulation. FSHR gene expression was found to be low in the AAA datasets. Female AAA-SMCs show occasional FSHR gene expression at a very low level, yet stimulation with FSH did not affect gene expression of SMC- or inflammation markers. FSH stimulation did not impact ox-LDL uptake or alter cell surface protein expression in monocytes. While FSHR gene expression was detected in human testis tissue, it was below quantification level in all other investigated cell types, even upon activation of macrophages with various stimuli. Despite previous reports, we did not detect FSHR gene expression in various extragonadal cell types, except in occasional female AAA-SMCs. No clear effect on cell activation was observed upon FSH stimulation in any cell type. Our data suggest that a direct effect of FSH in AAA-related extragonadal cells is unlikely to influence AAA.

Inflammatory Expression Profiles in Bladder Exstrophy Smooth Muscle: Normalization Over Time

ObjectiveTo test the hypothesis that phenotypes in bladder exstrophy result from alterations in detrusor smooth muscle cell (SMC) gene expression. MethodsWe generated primary human bladder smooth muscle cell lines from patients with classic bladder exstrophy (CBE) undergoing newborn closure (n = 6), delayed primary closure (n = 5), augmentation cystoplasty (n = 6), and non-CBE controls (n = 3). Gene expression profiles were then created using RNA sequencing and characterized using gene set enrichment analysis (GSEA). ResultsWe identified 308 differentially expressed genes in bladder exstrophy SMC when compared to controls, including 223 upregulated and 85 downregulated genes. Bladder exstrophy muscle cell lines from newborn closure and primary delayed closures shared expression changes in 159 genes. GSEA analysis revealed increased expression in the inflammatory response and alteration of genes for genitourinary development in newborn and delayed closure SMC. However, these changes were absent in SMC from older exstrophy patients after closure. ConclusionBladder exstrophy SMC demonstrate gene expression changes in the inflammatory response and genitourinary development. However, gene expression profiles normalized in exstrophy SMC from older patients after closure, suggesting a normalization of exstrophy SMC over time. Our in vitro findings regarding the normalization of exstrophy SMC gene expression following bladder closure suggest that the development of poor detrusor compliance in bladder exstrophy has a complex multifactorial etiology. Taken together, our findings suggest that alterations in SMC gene expression may explain abnormalities in the exstrophy bladder seen prior to and immediately after closure and suggest that surgical closure may allow exstrophy SMC to normalize over time.

Meet the First Authors

Meet the First Authors

Genetic Regulation of SMC Gene Expression and Splicing Predict Causal CAD Genes.

Coronary artery disease (CAD) is the leading cause of death worldwide. Recent meta-analyses of genome-wide association studies have identified over 175 loci associated with CAD. The majority of these loci are in noncoding regions and are predicted to regulate gene expression. Given that vascular smooth muscle cells (SMCs) play critical roles in the development and progression of CAD, we aimed to identify the subset of the CAD loci associated with the regulation of transcription in distinct SMC phenotypes. We measured gene expression in SMCs isolated from the ascending aortas of 151 heart transplant donors of various genetic ancestries in quiescent or proliferative conditions and calculated the association of their expression and splicing with ~6.3 million imputed single-nucleotide polymorphism markers across the genome. We identified 4910 expression and 4412 splicing quantitative trait loci (sQTLs) representing regions of the genome associated with transcript abundance and splicing. A total of 3660 expression quantitative trait loci (eQTLs) had not been observed in the publicly available Genotype-Tissue Expression dataset. Further, 29 and 880 eQTLs were SMC-specific and sex-biased, respectively. We made these results available for public query on a user-friendly website. To identify the effector transcript(s) regulated by CAD loci, we used 4 distinct colocalization approaches. We identified 84 eQTL and 164 sQTL that colocalized with CAD loci, highlighting the importance of genetic regulation of mRNA splicing as a molecular mechanism for CAD genetic risk. Notably, 20% and 35% of the eQTLs were unique to quiescent or proliferative SMCs, respectively. One CAD locus colocalized with a sex-specific eQTL (TERF2IP), and another locus colocalized with SMC-specific eQTL (ALKBH8). The most significantly associated CAD locus, 9p21, was an sQTL for the long noncoding RNA CDKN2B-AS1, also known as ANRIL, in proliferative SMCs. Collectively, our results provide evidence for the molecular mechanisms of genetic susceptibility to CAD in distinct SMC phenotypes.

Significance of the p38MAPK-CRP2 axis in myofibroblastic phenotypic transition.

We have recently demonstrated that a LIM domain protein, cysteine and glycine-rich protein 2 (CSRP2 [CRP2]), plays a vital role in the functional expression of myofibroblasts and cancer-associated fibroblasts. CRP2 binds directly to myocardin-related transcription factors (MRTF [MRTF-A or MRTF-B]) and serum response factor (SRF) to stabilize the MRTF/SRF/CArG-box complex, leading to the expression of smooth muscle cell (SMC) genes such as α-smooth muscle actin (α-SMA) and collagens. These are the marker genes for myofibroblasts. Here, we show that the adhesion of cultured human skin fibroblasts (HSFs) to collagen reduces the myofibroblastic features. HSF adhesion to collagen suppresses the expression of CRP2 and CSRP2-binding protein (CSRP2BP [CRP2BP]) and reduces the expression of SMC genes. Although CRP2BP is known as an epigenetic factor, we find that CRP2BP also acts as an adaptor protein to enhance the function of CRP2 mentioned above. This CRP2BP function does not depend on its histone acetyltransferase activity. We also addressed the molecular mechanism of the reduced myofibroblastic features of HSFs on collagen. HSF adhesion to collagen inhibits the p38MAPK-mediated pathway, and reducing the p38MAPK activity decreases the expression of CRP2 and SMC genes. Thus, the activation of p38MAPK is critical for the myofibroblastic features. We also show evidence that CRP2 plays a role in the myofibroblastic transition of retinal pigment epithelial cells (RPEs). Like HSFs, such phenotypic modulation of RPEs depends on the p38MAPK pathway.Key words: CRP2, p38MAPK, MRTF, myofibroblasts, retinal pigment epithelial cells.

Role of CRP2-MRTF interaction in functions of myofibroblasts

Inflammatory response induces phenotypic modulation of fibroblasts into myofibroblasts. Although transforming growth factor-βs (TGF-βs) evoke such transition, the details of the mechanism are still unknown. Here, we report that a LIM domain protein, cysteine-and glycine-rich protein 2 (CSRP2 [CRP2]) plays a vital role in the functional expression profile in myofibroblasts and cancer-associated fibroblasts (CAFs). Knock-down of CRP2 severely inhibits the expression of smooth muscle cell (SMC) genes, cell motility, and CAF-mediated collective invasion of epidermoid carcinoma. We elucidate the following molecular bases: CRP2 directly binds to myocardin-related transcription factors (MRTF-A/B [MRTFs]) and serum response factor (SRF) and stabilizes the MRTF/SRF/CArG-box complex to activate SMC gene expression. Furthermore, a three-dimensional structural analysis of CRP2 identifies the amino acids required for the CRP2-MRTF-A interaction. Polar amino acids in the C-terminal half (serine-152, glutamate-154, serine-155, threonine-156, threonine-157, and threonine-159 in human CRP2) are responsible for direct binding to MRTF-A. On the other hand, hydrophobic amino acids outside the consensus sequence of the LIM domain (tryptophan-139, phenylalanine-144, leucine-153, and leucine-158 in human CRP2) play a role in stabilizing the unique structure of the LIM domain.Key words: CRP2, 3D structure, myocardin-related transcription factor, myofibroblast, cancer-associated fibroblasts.

A Human atlas of smooth muscle cell gene expression; insights from the FANTOM consortium CAGE dataset

Abstract Background Under normal physiological conditions, smooth muscle cells (SMC's) have a role in both vessel contraction and generation of the extra-cellular matrix. It has now become apparent that SMCs throughout the vasculature play a key role in several cardiovascular conditions including hypertension, atherosclerosis, vasculopathy and myocardial infarction. However, how SMC gene expression compares in different vascular and non-vascular anatomical regions remains unknown. Purpose To create a human SMC gene expression map using data from the FANTOM consortium Cap-analysis gene expression (CAGE) dataset, a primary database of almost 600 human primary cell samples. Methods The gene level expression data was obtained by aggregating expression levels of all the promoters belonging to same gene as defined by the FANTOM CAGE dataset. The anatomical regions (n: 13) included the following vascular sites: aortic, brachiocephalic, carotid, coronary, internal thoracic & pulmonary arteries. Non-vascular regions included colon, oesophagus, prostate, uterine, trachea and bronchial. Weighted correlation network analysis (WGCNA) was used as an unsupervised approach to identify clustering of gene sets. Results Hierarchical clustering (Figure 1) demonstrates a distinct clustering of vascular SMC gene sets (aortic, carotid, coronary, internal thoracic, pulmonary & subclavian arteries). The same pattern of vascular SMC clustering was also seen in WGCNA (Figure 2). Gene ontology enrichment of vascular SMCs that clustered in WGCNA identified common gene sets relating to cytokine and chemokine activity. KEGG pathway analysis highlighted several pathways linked to inflammation (TNF, IL17 & NF-κB signalling) as well as pathways linked to atherosclerosis and lipid metabolism. Conclusion Vascular SMCs distinctly cluster away from non-vascular SMC types in humans and the separation is primarily driven by gene sets relating to inflammation, lipid metabolism & atherosclerosis. Mapping SMC gene expression at various anatomical regions in both healthy and diseased states may give us better insight into the role of SMCs in disease progression and could identify novel mechanistic targets for future therapy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

The role and mechanism of epidermal growth factor receptor in hemodynamic induction of abdominal aortic aneurysm formation.

BackgroundAbdominal aortic aneurysm (AAA) is a serious threat to human health, and in the event of aneurysm rupture, the rates of disability and mortality are high. At present, the treatment of AAA mainly includes craniotomy and endovascular therapy. With advances in technology, although the safety of the treatment is improving, there is still a risk associated with surgery. Therefore, the exploration of non-invasive treatment options for aneurysms is worthwhile. The etiology of aneurysms must be investigated to identify the target of non-invasive treatment.MethodsIn the experimental group, bilateral common carotid arteries were ligated, while in the control group, bilateral common carotid arteries were exposed. After 5 days, the rabbits were sacrificed, and the hearts were perfused with normal saline. Smooth muscle cells (SMCs) of abdominal aorta were isolated by enzyme digestion. Total RNA was extracted from SMC samples and detected using an Agilent Rabbit 4×44K Gene Expression Microarray. Then, some genes were selected and real-time polymerase chain reaction (RT-PCR) was conducted to verify the credibility of the chip results. Finally, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed. The transcriptome contained in the identified differentially expressed gene (DEG) was analyzed.ResultsMicroarray revealed 947 DEGs, of which 617 genes were increased and 330 genes were decreased in the experimental group compared with the control group. The microchips of PTHLH, ENPP1, IGF1, and others were selected to verify the altered genes by PCR.ConclusionsThe gene expression of rabbit abdominal aorta SMCs was significantly changed under high blood flow load. Through data analysis, it was found that some specific genes and transcription factors may play an important role in hemodynamically-induced vascular remodeling.

Medial Collagen Type and Quantity Influence Mechanical Properties of Aneurysm Wall in Bicuspid Aortic Valve Patients

Collagen has an essential role in aortic biomechanics, and collagen remodeling has been associated with the development and progression of aortic aneurysm. However, the exact mechanisms behind collagen remodeling and the biomechanical implications are not well understood. This study presents an investigation of the relationship between collagen remodeling in the aortic wall and biomechanics, by means of collagen assays, smooth muscle cell gene expression, and mechanical testing on human aortic specimens collected from patients with bicuspid aortic valve. Collagen assay analysis was employed to determine collagen-I and total collagen content; quantitative real-time PCR was used to determine amountsCOL1A1andCOL3A1expression in the tissue. These parameters were compared with the local biomechanical properties determined from biaxial and uniaxial tensile testing. Collagen-I content was found to relate to improved mechanical properties, while total collagen content did not exhibit a relationship with biomechanics.COL1A1andCOL3A1expression were found to relate to the collagen-I content of the tissue, but not the total collagen content or biomechanical performance. Relationships between variables appeared to be dependent on the collagen content in specific layers of the aortic wall. The effect of age is also noted, as total collagen content and biomechanics were found to have significant associations with increasing age, while collagen-I content and collagen gene expression did not exhibit any correlation. Varying relationships were observed when looking at younger versus older patients. Findings highlight the importance of type and location in determining the influence of collagen on aortic biomechanics, as well as the role of gene expression in the onset and progression of collagen remodeling in aortic aneurysm.

Abstract 466: Fhl5 Enhances Vascular Calcification By Regulating Smooth Muscle Cell Transcriptional Networks

Coronary artery calcium (CAC) is a hallmark of coronary artery disease (CAD) with increased CAC levels associated with increased risk for CAD related morbidity and mortality. The development of CAC is a pathological process driven by the modulation of smooth muscle cells (SMCs) and maladaptive extracellular matrix remodeling. In the most recent multi-ancestry meta-analysis of more than 35,000 individuals, FHL5 (Four-and-a-half LIM domains 5) was prioritized as a novel CAC gene through Bayesian colocalization and epigenomic analyses. scRNA-seq studies of human coronary and carotid arteries revealed high enrichment of FHL5 gene expression in SMC and pericyte populations. These preliminary studies motivate our hypothesis that FHL5 functions as a transcriptional regulator of vascular remodeling pathways associated with increased CAC risk. Following treatment of SMCs with a calcifying cocktail, stable FHL5 overexpression promoted the osteogenic SMC phenotypic transition, characterized by increased levels of osteogenic activators and reduced expression of SMC contractile markers. This osteogenic gene signature correlated with increased SMC calcification, consistent with the direction of effect of the CAC risk allele. To provide insights into the transcriptional program regulated by FHL5 that contributes to the vascular calcification phenotype, we mapped FHL5 binding sites in SMCs using the CUT&RUN method. We identified 17,201 FHL5 binding sites that overlapped the active enhancer mark H3K27ac. FHL5 target genes were enriched in pathways linked to vascular remodeling, such as cell adhesion and extracellular matrix organization. Moreover, risk variants for pulse pressure, a metric of arterial stiffness that is highly correlated with CAC, was enriched in FHL5 binding sites. Lastly, integration with FHL5 trans-eQTL target genes in STARNET arterial tissues revealed a network of CAD-associated candidate genes regulated by FHL5 in vitro and in vivo that impact SMC proliferation, migration, and calcification. Taken together, we provide evidence that FHL5 regulates a network of genes linked to arterial stiffness, which may explain the underlying mechanism of its genetic association with CAC in individuals from the general population.

Glucose-6-phosphate dehydrogenase and MEG3 controls hypoxia-induced expression of serum response factor (SRF) and SRF-dependent genes in pulmonary smooth muscle cell.

Although hypoxia induces aberrant gene expression and dedifferentiation of smooth muscle cells (SMCs), mechanisms that alter dedifferentiation gene expression by hypoxia remain unclear. Therefore, we aimed to gain insight into the hypoxia-controlled gene expression in SMCs. We conducted studies using SMCs cultured in 3% oxygen (hypoxia) and the lungs of mice exposed to 10% oxygen (hypoxia). Our results suggest hypoxia upregulated expression of transcription factor CP2-like protein1, krüppel-like factor 4, and E2f transcription factor 1 enriched genes including basonuclin 2 (Bcn2), serum response factor (Srf), polycomb 3 (Cbx8), homeobox D9 (Hoxd9), lysine demethylase 1A (Kdm1a), etc. Additionally, we found that silencing glucose-6-phosphate dehydrogenase (G6PD) expression and inhibiting G6PD activity downregulated Srf transcript and hypomethylation of SMC genes (Myocd, Myh11, and Cnn1) and concomitantly increased their expression in the lungs of hypoxic mice. Furthermore, G6PD inhibition hypomethylated MEG3, a long non-coding RNA, gene and upregulated MEG3 expression in the lungs of hypoxic mice and in hypoxic SMCs. Silencing MEG3 expression in SMC mitigated the hypoxia-induced transcription of SRF. These findings collectively demonstrate that MEG3 and G6PD codependently regulate Srf expression in hypoxic SMCs. Moreover, G6PD inhibition upregulated SRF-MYOCD-driven gene expression, determinant of a differentiated SMC phenotype.