Population Of Smooth Muscle Cells Research Articles

Tobacco smoking is associated with sex- and plaque-type specific upregulation of CRLF1 in atherosclerotic lesions

Background and aimsTobacco smoking is a known risk factor for atherosclerotic disease, with more elevated risks in women compared to men. We hypothesized that atherosclerotic plaques from smokers show different gene expression patterns compared to non-smokers, in a sex-specific manner. MethodsGene expression data of 625 carotid plaques (151 females and 474 males) were analyzed for differential gene expression between current smokers (n = 226) and non-smokers (n = 399). All analyses were stratified by sex and by molecular plaque characteristics. Finally, we projected the activity of gene regulatory networks and utilized single-cell transcriptomics from 38 plaques (26 males and 12 females) to interpret the sex- and plaque-type specific signals. ResultsWe observed higher expression levels of CRLF1 gene in atherosclerotic plaques from smokers compared to non-smokers (log2FC = 0.48, FDR = 0.012). CRLF1 upregulation was interacting with sex (p = 0.01) and was more pronounced in females (log2FC = 0.93, p = 1.53E-05) compared to males (log2FC = 0.35, p = 0.0018). Through single-cell RNA-seq analysis, we identified the highest CRLF1 expression within the transitioning and synthetic smooth muscle cell populations. CRLF1 expression was increased in fibro-inflammatory and fibro-cellular plaque types. Gene annotations pointed to increased expression of CRLF1 in networks with extracellular matrix related genes. ConclusionsAtherosclerotic plaques from current smokers show sex-dependent upregulation of smooth muscle cell gene CRLF1. This may explain the different contributions of smoking to cardiovascular risk in females.

Abstract 2075: Vascular Smooth Muscle Cell Derived Pdgfd Promotes Vascular Remodeling And Plaque Progression In Atherosclerosis

We have recently identified rs2019090 and PDGFD as the functional variant and gene mediating CAD risk at the 11q22.3 locus, with our initial analysis using a global knockout (KO) model showing that this gene may promote phenotypic changes in smooth muscle cells (SMCs) in the plaque and contribute to neointimal vascular calcification. Nonetheless, the specific cell-type and phenotypic states through which PDGFD may confer disease risk remains unexplored.To delineate the impact of SMC-derived PDGFD signalling on cell state transitions and plaque progression in atherosclerosis, we have developed a novel SMC-specific lineage tracing and Pdgfd KO mouse ( Pdgfd ΔSMC/ΔSMC , Myh11 CreERT2 , ROSA tdT/+ , ApoE -/- ) allowing us to confidently define the impact of SMC-derived Pdgfd in the vascular SMC lineage as well as in neighbouring populations. Leveraging our lineage tracing KO mutants on a hypercholesterolemic diet, we have employed single cell RNA sequencing (scRNAseq) and histological analyses to characterize the cellular and molecular effect of Pdgfd in vascular disease. SMC-specific Pdgfd deletion resulted in alterations in the distribution of transitioning SMC populations, as well as previously unexplored gene expression differences within these cell types. This was accompanied by a significant reduction in atherosclerotic burden and plaque size across the aorta, including aortic root as well as descending abdominal aorta. Histological analysis revealed decreased monocyte recruitment, show by quantifying CD68+ cells within the plaque. To explore this further we interrogated the scRNAseq dataset to identify major pathways of cell-cell communication and the impact of altered Pdgfd signalling across different cell types. Overall, these data reveal that SMC-derived PDGFD substantially alters lesional SMC cell phenotype transitions, as well as inflammatory cell recruitment, implicating it as a major regulator of atherosclerotic disease progression.

Tobacco smoking upregulates CRLF1 expression in the atherosclerotic lesions in sex- and plaque-type specific manner

Abstract Background Tobacco smoking is known to impact the development of atherosclerotic disease. Interestingly, females who smoke have a higher risk of cardiovascular disease( CVD) than males. It is established that smoking can lead to changes in DNA methylation and gene expression. We, therefore, hypothesised that smoking influences gene expression in a sex- and plaque-type-specific manner. Method We executed bulk RNA sequencing of 654 carotid plaques and single sequencing in 46 plaques. We analysed the transcriptomes of 625 carotid plaques from smokers (n= 226) and non-smokers (n = 399) and performed the differential gene expression in plaques between smokers and non-smokers. The analysis was repeated separately in males and females. The observed gene expression differences were also associated with pathological and molecular plaque characteristics. Results After correction for multiple testing, CRLF1 was found to be significantly higher expressed in smokers than non-smokers (Log2FC=0.48, FDR =0.012) (Figure 1a). Single-cell RNA-seq analyses revealed specific expression of CRLF1 in the synthetic smooth muscle cell population. The smoking-associated CRLF1 overexpression was larger in females (log2FC=0.93) compared to males (log2FC=0.35) (Figure1b). Furthermore, the effect was restricted to plaque types with a higher content of smooth muscle cells. Conclusion Our results show the sex and plaque type-specific effect of smoking on gene expression in carotid plaques. The higher upregulation of the CRLF1 gene in female smokers suggests mechanistic clues to a higher risk of smoking-related CVD in women.Figure 1

Abstract 15566: Dissecting the Genetic Architecture of Intracranial Aneurysms

Introduction: The genetic risk of intracranial aneurysm (IA) development has been ascribed largely to smoking and hypertension. The contribution of IA risk loci to aberrant gene programs within cerebrovascular cell types remains unclear. Hypothesis: We hypothesized that candidate causal genes at IA susceptibility loci are enriched within extracellular matrix producing cell types. Methods: We identified participants enrolled in the Million Veterans Program (MVP) with and without IA. Thirty-two million genotyped and imputed variants were tested for association with IA using mixed effect regression modeling, controlling for age, sex, and population structure prior to external replication and meta-analysis. Candidate causal genes were prioritized through expression quantitative trait loci colocalization and transcriptome wide association studies. We constructed a cerebrovascular single nucleus RNA sequencing (snRNA-seq) dataset and integrated IA summary statistics to compute a ‘disease relevance score’ per nucleus using the scDRS pipeline. Cell type level analysis was used to assess association of cerebrovascular cell types with IA. Results: We identified 3165 participants (2280 European, 627 African, and 258 Hispanic ancestry) with and 592927 without IA in the MVP. After replication and meta-analysis with data from an additional 12273 cases and 591046 controls, we identified 22 loci (5 novel) associated with IA. We prioritized 16 candidate causal genes. Gene expression analysis of snRNA-seq genes revealed enrichment of several candidate genes, including EDNRA and PLCE1 within smooth muscle cells (SMCs) and pericytes. Integration of summary statistics and snRNA-seq data revealed significant association of matrix-producing pericytes and smooth muscle cell populations with IA. Conclusion: Here, we identify novel loci associated with IA and found enrichment of candidate causal genes in pericytes and SMCs. Integration of summary statistics with cerebrovascular snRNA-seq reveals association of cell-types involved in matrix production, suggesting an intrinsic deficit in matrix production and vascular integrity that may drive IA pathogenesis independent of systemic hypertension.

Abstract 16902: Molecular Basis of the Vascular Cell Phenotypic Switch and Wall Remodeling in Abdominal Aortic Aneurysm: NOX4 NADPH Oxidase-Derived Mitochondrial Oxidative Stress and DNA Damage

Vascular cells phenotypic changes and wall remodeling are the hallmarks of abdominal aortic aneurysm (AAA) pathology. Mitochondrial oxidative stress (mtOS) is associated with the vascular smooth muscle cells (SMC) and macrophage phenotypic switch. We previously reported that NOX4 NADPH oxidase expression is increased in cardiovascular cells in response to pathological insult inducing mtOS and dysfunction and vascular disease progression. We tested the hypothesis that NOX4-dependent mtOS and DNA damage induce vascular phenotypic changes and remodeling resulting in AAA progression. In angiotensin II (Ang II)-induced AAA model Apoe -/- / Nox4 Tg mice with mitochondria-targeted overexpression of NOX4 had the highest, whereas Apoe -/- / Nox4 -/- displayed the lowest incidence of AAA, abdominal aorta area and diameter as compared with Apoe -/- mice. The aortic wall ROS levels, oxidative DNA damage and DNA damage response markers, elastin degradation index, MMP2 expression, and macrophage infiltration were significantly higher in AngII-treated Apoe -/- / Nox4 TG mice compared with Apoe -/- or Apoe -/- / Nox4 -/- mice. Spectral flow cytometry analysis of AAA cell suspension from Ang II-infused mice revealed a decrease in contractile SMCs but an increase in macrophage-like SMC populations, as well as increased number of infiltrating pro-inflammatory macrophages in Apoe -/- / Nox4 TG mice. As compared with wild-type or Nox4 -/- mice, Ang II-treated SMC from Nox4 TG mice showed significantly elevated mtOS, DNA damage, dysfunction, as well as activation of cGAS-STING pathway and NLRP3/AIM2 inflammasome expression. Flow cytometry analysis of Ang II-treated Nox4 TG mice SMC contained a lower proportion of cells with contractile markers (ACTA2 + , MYH11 + , TAGLN + ) and a higher proportion of cells with macrophage-like markers (CD68 + , CD11b + , LGALS3 + ). These data suggest that NOX4-dependent mitochondrial DNA damage and activation of DNA-sensing pathways have a causal role in AAA development, leading to macrophage-like SMCs, increased inflammation, and vessel wall remodeling. NOX4 inhibition and mitochondrial function augmentation may be beneficial in preserving aortic wall integrity in AAA-susceptible patients.

Distribution of smooth muscle actin and collagen in superficial peritoneal endometriotic lesions varies from the surrounding microenvironment

Research questionDo different subtypes of superficial peritoneal endometriotic lesions exist, based on the presence and morphology of smooth muscle, collagen fibres and immune cell populations? DesignA retrospective cohort study of 24 patients, from across the menstrual cycle, with surgically and histologically confirmed endometriosis. Immunofluorescence was used to delineate the CD10 stromal area of lesions (n = 271 lesions from 67 endometriotic biopsies), and then smooth muscle actin (SMA) positive tissue and immune cell populations (CD45+ and CD68+) were quantified within and adjacent to these lesions. Second harmonic generation microscopy was used to evaluate the presence and morphology of type-1 collagen fibres within and surrounding lesions. ResultsOverall, immune cell numbers and the area of SMA and collagen within endometriotic lesions tended to be low, but a spectrum of presentations significantly varied, particularly in the adjacent tissue microenvironment, based on lesion locations, the morphology of endometriotic gland profiles, or both. Lesions in which collagen fibres formed well aligned capsules around the CD10+ stromal border were identified compared with lesions in which collagen fibre distribution was random. Considerable inter- and intra-patient variability in the morphology of SMA and collagen was observed within and surrounding lesions. ConclusionThese data demonstrate considerable diversity in the presence of immune cells and morphology of SMA and collagen within, but even more so, surrounding endometriotic lesions, even within individual patients. This heterogeneity, especially within individual patients, presents a challenge to incorporating these cell and tissue types into any new endometriosis classification systems or prognostic approaches.

Sinus venosus adaptation models prolonged cardiovascular disease and reveals insights into evolutionary transitions of the vertebrate heart

How two-chambered hearts in basal vertebrates have evolved from single-chamber hearts found in ancestral chordates remains unclear. Here, we show that the teleost sinus venosus (SV) is a chamber-like vessel comprised of an outer layer of smooth muscle cells. We find that in adult zebrafish nr2f1a mutants, which lack atria, the SV comes to physically resemble the thicker bulbus arteriosus (BA) at the arterial pole of the heart through an adaptive, hypertensive response involving smooth muscle proliferation due to aberrant hemodynamic flow. Single cell transcriptomics show that smooth muscle and endothelial cell populations within the adapting SV also take on arterial signatures. Bulk transcriptomics of the blood sinuses flanking the tunicate heart reinforce a model of greater equivalency in ancestral chordate BA and SV precursors. Our data simultaneously reveal that secondary complications from congenital heart defects can develop in adult zebrafish similar to those in humans and that the foundation of equivalency between flanking auxiliary vessels may remain latent within basal vertebrate hearts.

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.

Stiffness sensing by smooth muscle cells: Continuum mechanics modeling of the acto-myosin role

Aortic smooth muscle cells (SMCs) play a vital role in maintaining homeostasis in the aorta by sensing and responding to mechanical stimuli. However, the mechanisms that underlie the ability of SMCs to sense and respond to stiffness change in their environment are still partially unclear. In this study, we focus on the role of acto-myosin contractility in stiffness sensing and introduce a novel continuum mechanics approach based on the principles of thermal strains. Each stress fiber satisfies a universal stress-strain relationship driven by a Young's modulus, a contraction coefficient scaling the fictitious thermal strain, a maximum contraction stress and a softening parameter describing the sliding effects between actin and myosin filaments. To account for the inherent variability of cellular responses, large populations of SMCs are modeled with the finite-element method, each cell having a random number and a random arrangement of stress fibers. Moreover, the level of myosin activation in each stress fiber satisfies a Weibull probability density function. Model predictions are compared to traction force measurements on different SMC lineages. It is demonstrated that the model not only predicts well the effects of substrate stiffness on cellular traction, but it can also successfully approximate the statistical variations of cellular tractions induced by intercellular variability. Finally, stresses in the nuclear envelope and in the nucleus are computed with the model, showing that the variations of cytoskeletal forces induced by substrate stiffness directly induce deformations of the nucleus which can potentially alter gene expression. The predictability of the model combined to its relative simplicity are promising assets for further investigation of stiffness sensing in 3D environments. Eventually, this could contribute to decipher the effects of mechanosensitivity impairment, which are known to be at the root of aortic aneurysms.

Is carotid web an arterial wall dysplasia? A histological series

IntroductionDescribed for 60 years under various names, the carotid web is a suspected cause of cryptogenic stroke, especially in young patients. The web creates an intraluminal protrusion that may contribute to turbulent flow and thrombus embolization into cerebral arteries. Although the carotid web has frequently been related to arterial fibrodysplasia, its natural history and pathological description remain unclear. PatientsAmong all consecutive patients admitted to the stroke unit of Sainte-Anne Hospital and referred to the vascular surgery department from January 2015 to December 2022, we retrospectively identified 9 patients with a carotid web. The surgical specimens of the 9 patients were submitted to systematic pathological analysis. ResultsThe patients with a histologically confirmed carotid web were young (median age was 42 years), prominently women (7/9), and presenting with low cardiovascular risk. Eight patients had a stroke proven by a magnetic resonance imaging, and 1 had transient monocular amaurosis. The typical pathological lesion supporting the imaging pattern of the carotid web was a focal eccentric intimal hyperplasia forming a protruding lesion characterized by a population of vascular smooth muscle cells intermingled in an abundant, most often loose extracellular matrix. Pathologically proven thrombus was observed in 4 cases. Importantly atherosclerosis was absent. ConclusionHistological features in our 9 cases strengthen carotid web characterization as a homogeneous pattern of localized intimal hyperplasia. It is a unique entity consistent with intimal fibroplasia, distinct from medial fibromuscular dysplasia and early atherosclerosis.

Abstract 490: High-dimensional Proteogenomics Of Human Carotid Atherosclerosis Reveals Significant Diversity Of Smooth Muscle Cells Within Plaques

Atherosclerotic lesions are cellularly heterogeneous tissue comprised of diverse cell types including smooth muscle cells (SMCs), endothelial cells, and immune cells. The development of single cell technologies has provided the granular resolution required to identify new cell types present in plaques, aiding in our understanding of the pathophysiology of the disease. Transdifferentiation of SMCs during atherosclerosis progression to a synthetic phenotype has a major influence on disease severity and development. Therefore, to deeply characterize and immunophenotype SMCs in atherosclerotic plaques we performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) on six patients and scRNA-sequencing on fifteen patients undergoing carotid endarterectomy. Our initial analysis identified 25 unique cell populations, which consisted of 4 SMC populations which comprised about 14% of all cells in the dataset. Three of the SMC populations appeared to have a contractile phenotype, with high expression of SMC-specific genes like ACTA2, MYH11, and CNN1. The fourth SMC cluster had a modulated phenotype with downregulation of SMC genes and an up regulation of fibroblast genes such as COL1A2 and LUM. We also identified novel and uncharacterized SMC-specific cell surface proteins such as CD29, CD142, and EGR. The modulated SMC cluster specifically expressed CD90, giving us the unique ability to differentiate these similar yet distinct cell types. We then performed a subclustering analysis on SMC and fibroblast populations, identifying 8 SMC subpopulations. This was followed by Canonical Pathway analysis with Ingenuity Pathway Analysis (IPA) using the most highly expressed genes in each cluster to identify enriched pathways. This revealed a diverse array of functional differences between SMC populations. Our data support the idea that SMCs have a vast array of phenotypic states and functions. Future work will uncover the relevance of these newly identified cell types in the development of atherosclerosis and clinical events.

Abstract 396: Plasticity Of The Tumor Vasculature Is Induced By Tumor Cells Via Cell Surface Receptor Tetherin

It is increasingly clear that cardiovascular disease and cancer have shared pathophysiological mechanisms beyond traditional risk factors. Murine models have demonstrated tumors grow more quickly in the presence of cardiovascular abnormalities, and targeting of shared signaling pathways has proven efficacious in both diseases. It is unknown, however, if smooth muscle cell (SMC) plasticity as seen in atherogenesis represents yet another biologic parallel. Our aim was to understand if SMC plasticity occurs in tumor vasculature, and how those transitions are induced. Syngeneic tumor cell lines were implanted on the flanks of SMC lineage-traced mice and allowed to grow for 11 days. Fluorescent microscopy of the tumors revealed a population of SMCs that moved away from angiogenic vessels and lost canonical SMC markers. We used single cell RNA sequencing to characterize the in vivo transcriptional phenotype of SMCs within the tumor. Principal component analysis demonstrated distinct SMC populations, with pseudotime analysis suggesting transitions from a contractile SMC towards a more macrophage-like transcriptional state. CellChat ligand-receptor analysis was leveraged to examine differential interactions between tumor cells and classic macrophages versus macrophage-like SMCs. The only unique interaction was between cell surface receptors Tetherin on tumor cells and paired immunoglobulin-like receptor A2 (PIRA2) on macrophage-like SMCs. In vitro murine SMCs treated with recombinant Tetherin decreased contractile SMC markers and increased macrophage markers by qPCR. Exposure to recombinant Tetherin also increased overall proliferative rate, migratory capacity, and phagocytic ability compared to untreated SMCs. The knockdown of PIRA2 in murine SMCs abrogates these apparent effects of Tetherin. Our findings demonstrate that SMCs in tumor vasculature, like those in atherogenesis, demonstrate significant degrees of plasticity. One of the phenotypic changes is towards a macrophage-like state. In vitro studies suggest interaction between Tetherin and PIRA2 receptors is sufficient to induce this switching. Ongoing murine studies will establish whether in vivo inhibition of the Tetherin-PIRA2 axis decreases vascular plasticity.

MP27-04 IDENTIFICATION AND CHARACTERIZATION OF NOVEL ATYPICAL SMOOTH MUSCLE CELLS IN THE MOUSE CORPUS CAVERNOSUM

You have accessJournal of UrologyCME1 Apr 2023MP27-04 IDENTIFICATION AND CHARACTERIZATION OF NOVEL ATYPICAL SMOOTH MUSCLE CELLS IN THE MOUSE CORPUS CAVERNOSUM Karen I. Hannigan, Kenton M. Sanders, Sang Don Koh, and Caroline A. Cobine Karen I. HanniganKaren I. Hannigan More articles by this author , Kenton M. SandersKenton M. Sanders More articles by this author , Sang Don KohSang Don Koh More articles by this author , and Caroline A. CobineCaroline A. Cobine More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003255.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Highly coordinated relaxation of smooth muscle cells (SMCs) is a requirement for penile erection. Previous studies in the corpus cavernosum (CC) have shown that blocking the Ca2+ activated Cl- channel Ano1 inhibited contractile, electrical and intracellular Ca2+ activity. Penile activity is influenced by neural inputs with SMCs thought to be directly targeted by neurotransmitters due to their expression of soluble guanylate cyclase (sGC), the receptor for nitric oxide (NO). However, as the CC is sparsely innervated, a cellular mediator could be present between nerves and CCSMCs. Despite this, the specific cell type that expresses Ano1 has not been identified, with previous studies relying on morphological identification of SMCs alone. Platelet derived growth factor receptor-α (PDGFRα)+ cells have been identified in other visceral smooth muscles including the gastrointestinal and urinary tracts where they mediate inhibitory neurotransmission. In the renal pelvis, PDGFRα+ cells express Ano1. The aim of this study is to identify and characterize the cell type that expresses Ano1 and to determine whether these cells mediate neurotransmission in the CC. METHODS: Cells were isolated from the CC of mice expressing eGFP in PDGFRα+ cells (PDGFRα+-eGFP mice) and SMCs (SmMHC-eGFP mice). Isolated cells were sorted with FACS. Enriched PDGFRα and SMC populations were examined using qPCR for expression of cell specific markers (Pdfgra and smooth muscle myosin heavy chain (Myh11)) to ensure purity. Purified PDGFRα+ cells and SMCs were evaluated with ddPCR to examine the expression of Ano1 and genes encoding signaling proteins from the NO pathway. RESULTS: PDGFRα+ cells isolated with FACS belonged to bright and dim populations. While qPCR revealed that the bright and dim populations expressed similar levels of Pdgfra, the dim population also expressed Myh11. In contrast, SMCs purified using FACS expressed Myh11 and Pdgfra, similar to the dim population of PDGFRα+ cells. ddPCR revealed that both the dim population of PDGFRα+ cells and SMCs expressed Ano1 and genes encoding signaling proteins from the NO pathway. CONCLUSIONS: These data suggest that Ano1 is expressed by atypical SMCs that also express PDGFRα. Therefore, these cells are a potential novel target for the treatment of erectile dysfunction. Future directions of this project include investigating the interactions between PDGFRa+ cells, SMCs and neurons. Source of Funding: Urology Care Foundation Research Scholar Award, sponsored by the Sexual Medicine Society of North America © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e363 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Karen I. Hannigan More articles by this author Kenton M. Sanders More articles by this author Sang Don Koh More articles by this author Caroline A. Cobine More articles by this author Expand All Advertisement PDF downloadLoading ...

Long non-coding RNAs: The growth controller of vascular smooth muscle cells in cardiovascular diseases.

The active proliferation and migration of vascular smooth muscle cells supports the healing of vessel damage while their abnormal aggression or destitution contribute to the aberrant intima-medial structure and function in various cardiovascular diseases, so the understanding of the proliferation disorders of vascular smooth muscle cell and the related mechanism is the basis of effective intervention and control for cardiovascular diseases. Recently, long non-coding RNAs have stood out as upstream switchers for multiple proliferative signaling pathways and molecules, and many of them have been shown to conduce to the dysregulated proliferation and apoptosis of vascular smooth muscle cells under various pathogenic stimuli. This article discusses the long non-coding RNAs disclosed and linked to atherosclerosis, pulmonary hypertension, and aneurysms, and focuses upon their modulation of vascular smooth muscle cell population affecting three deadly cardiovascular diseases.

Single-Cell Analysis Reveals Distinct Immune and Smooth Muscle Cell Populations that Contribute to Chronic Thromboembolic Pulmonary Hypertension.

Rationale: Chronic thromboembolic pulmonary hypertension (CTEPH) is a sequela of acute pulmonary embolism (PE) in which the PE remodels into a chronic scar in the pulmonary arteries. This results in vascular obstruction, pulmonary microvasculopathy, and pulmonary hypertension. Objectives: Our current understanding of CTEPH pathobiology is primarily derived from cell-based studies limited by the use of specific cell markers or phenotypic modulation in cell culture. Therefore, our main objective was to identify the multiple cell types that constitute CTEPH thrombusy and to study their dysfunction. Methods: Here we used single-cell RNA sequencing of tissue removed at the time of pulmonary endarterectomy surgery from five patients to identify the multiple cell types. Using invitro assays, we analyzed differences in phenotype between CTEPH thrombus and healthy pulmonary vascular cells. We studied potential therapeutic targets in cells isolated from CTEPH thrombus. Measurements and Main Results: Single-cell RNA sequencing identified multiple cell types, including macrophages, T cells, and smooth muscle cells (SMCs), that constitute CTEPH thrombus. Notably, multiple macrophage subclusters were identified but broadly split into two categories, with the larger group characterized by an upregulation of inflammatory signaling predicted to promote pulmonary vascular remodeling. CD4+ and CD8+ T cells were identified and likely contribute to chronic inflammation in CTEPH. SMCs were a heterogeneous population, with a cluster of myofibroblasts that express markers of fibrosis and are predicted to arise from other SMC clusters based on pseudotime analysis. Additionally, cultured endothelial, smooth muscle, and myofibroblast cells isolated from CTEPH fibrothrombotic material have distinct phenotypes from control cells with regard to angiogenic potential and rates of proliferation and apoptosis. Last, our analysis identified PAR1 (protease-activated receptor 1) as a potential therapeutic target that links thrombosis to chronic PE in CTEPH, with PAR1 inhibition decreasing SMC and myofibroblast proliferation and migration. Conclusions: These findings suggest a model for CTEPH similar to atherosclerosis, with chronic inflammation promoted by macrophages and T cells driving vascular remodeling through SMC modulation, and suggest new approaches for pharmacologically targeting this disease.

Acute injury to the mouse carotid artery provokes a distinct healing response.

Treatment of vascular stenosis with angioplasty results in acute vascular damage, which may lead to restenosis. Owing to the highly complex cellularity of blood vessels, the healing response following this damage is incompletely understood. To gain further insight into this process, scRNA-seq of mouse carotid tissue after wire injury was performed. Stages of acute inflammation, resolution and remodeling were recapitulated in these data. To identify cell types which give rise to neointima, analyses focused on smooth muscle cell and fibroblast populations, and included data integration with scRNA-seq data from myocardial infarction and atherosclerosis datasets. Following carotid injury, a subpopulation of smooth muscle cells which also arises during atherosclerosis and myocardial infarction was identified. So-called stem cell/endothelial cell/monocyte (SEM) cells are candidates for repopulating injured vessels, and were amongst the most proliferative cell clusters following wire-injury of the carotid artery. Importantly, SEM cells exhibit specific transcriptional profiles which could be therapeutically targeted. SEM cell gene expression patterns could also be detected in bulk RNA-sequencing of neointimal tissue isolated from injured carotid vessels by laser capture microdissection. These data indicate that phenotypic plasticity of smooth muscle cells is highly important to the progression of lumen loss following acute carotid injury. Interference with SEM cell formation could be an innovative approach to combat development of restenosis.

A vein wall cell atlas of murine venous thrombosis determined by single-cell RNA sequencing

Deep vein thrombosis (DVT) is a common clinical problem, but its cellular and molecular mechanisms remain incompletely understood. In this study, we performed single-cell RNA sequencing on mouse inferior vena cava (IVC) 24 h after thrombus-inducing IVC ligation or sham operation. 9 cell types composed of multiple subpopulations were identified. Notable transcriptomic changes induced by DVT included a marked inflammatory response, elevated hypoxia, and globally reduced myogenesis. Analysis of individual cell populations revealed increased inflammation and reduced extracellular matrix production across smooth muscle cells and fibroblasts, juxtaposed against an early phenotypic shift in smooth muscle cell populations away from a contractile state. By characterizing the transcriptomic changes in the vein wall during acute venous thrombosis at the single-cell level, this work provides novel insights into early pathological events in the vein wall that may potentiate thrombus formation and result in long term adverse venous remodeling.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

Single-cell transcriptomics reveals skewed cellular communication and phenotypic shift in pulmonary artery remodeling.

A central feature of progressive vascular remodeling is altered smooth muscle cell (SMC) homeostasis; however, the understanding of how different cell populations contribute to this process is limited. Here, we utilized single-cell RNA sequencing to provide insight into cellular composition changes within isolated pulmonary arteries (PAs) from pulmonary arterial hypertension and donor lungs. Our results revealed that remodeling skewed the balanced communication network between immune and structural cells, in particular SMCs. Comparative analysis with murine PAs showed that human PAs harbored heterogeneous SMC populations with an abundant intermediary cluster displaying a gradient transition between SMCs and adventitial fibroblasts. Transcriptionally distinct SMC populations were enriched in specific biological processes and could be differentiated into 4 major clusters: oxygen sensing (enriched in pericytes), contractile, synthetic, and fibroblast-like. End-stage remodeling was associated with phenotypic shift of preexisting SMC populations and accumulation of synthetic SMCs in neointima. Distinctly regulated genes in clusters built nonredundant regulatory hubs encompassing stress response and differentiation regulators. The current study provides a blueprint of cellular and molecular changes on a single-cell level that are defining the pathological vascular remodeling process.

Role of smooth muscle-derived S100A4 in an atherosclerotic mouse model

Abstract Background S100A4, a small calcium binding protein, plays an important role in vascular smooth muscle cell (SMC) phenotypic switch but its implication in atherosclerotic plaque development and particularly in SMC phenotypic plasticity is not clear yet. By neutralizing S100A4 using a monoclonal antibody, we have previously shown a reduction in overall atherosclerotic burden. However, this strategy did not distinguish the different contribution between SMCs or other S100A4-expressing cells (e.g. macrophages). Methods Herein, we used a lineage tracing mouse model in which we induced a SMC specific deletion of S100A4 (SMC-S100A4Δ/Δ) in an ApoE−/− background. High cholesterol diet was maintained for 12 weeks, after which, SMC-S100A4Δ/Δ and control mice (SMC-S100A4wt/wt) were sacrificed and aortas processed for staining and single cell RNA sequencing (scRNA-seq). Results We showed that S100A4 deletion modulated plaque composition rather than plaque size. ScRNA-seq analysis from SMC-S100A4Δ/Δ and SMC-S100A4wt/wt showed a reduction in macrophages and dendritic cells and an increase in total SMCs, likely participating in fibrous cap stabilization. SMC populations showed a decrease in macrophage-like inflammatory phenotype and an increase in fibroblast-like repair phenotype, as well as retention of specific SMC markers, namely Acta2 and Myh11. Gene expression analysis revealed a reduction in the expression of inflammation markers, fatty acid metabolism, and extracellular matrix-related genes, and an increase in classical SMC markers and maintenance of the contractile phenotype. Conclusion We report an intrinsic function of S100A4 in the establishment of SMC fate within the plaque and, surprisingly, a global impact on the atherosclerotic plaque microenvironnement and inflammation status. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation