Smooth Muscle Cell Function Research Articles

Sorbs2 Deficiency and Vascular BK Channelopathy in Diabetes.

Vascular large conductance Ca2+-activated K+ (BK) channel, composed of the α-subunit (BK-α) and the β1-subunit (BK-β1), is a key determinant of coronary vasorelaxation and its function is impaired in diabetic vessels. However, our knowledge of diabetic BK channel dysregulation is incomplete. The Sorbs2 (Sorbin homology [SoHo] and Src homology 3 [SH3] domains-containing protein 2), is ubiquitously expressed in arteries, but its role in vascular pathophysiology is unknown. The role of Sorbs2 in regulating vascular BK channel activity was determined using patch-clamp recordings, molecular biological techniques, and in silico analysis. Sorbs2 is not only a cytoskeletal protein but also an RNA-binding protein that binds to BK channel proteins and BK-α mRNA, regulating BK channel expression and function in coronary smooth muscle cells. Molecular biological studies reveal that the SH3 domain of Sorbs2 is necessary for Sorbs2 interaction with BK-α subunits, while both the SH3 and SoHo domains of Sorbs2 interact with BK-β1 subunits. Deletion of the SH3 or SoHo domains abolishes the Sorbs2 effect on the BK-α/BK-β1 channel current density. Additionally, Sorbs2 is a target gene of the Nrf2 (nuclear factor erythroid-2-related factor 2), which binds to the promoter of Sorbs2 and regulates Sorbs2 expression in coronary smooth muscle cells. In vivo studies demonstrate that Sorbs2 knockout mice at 4 months of age display a significant decrease in BK channel expression and function, accompanied by impaired BK channel Ca2+-sensitivity and BK channel-mediated vasodilation in coronary arteries, without altering their body weights and blood glucose levels. Importantly, Sorbs2 expression is significantly downregulated in the coronary arteries of db/db type 2 diabetic mice. Sorbs2, a downstream target of Nrf2, plays an important role in regulating BK channel expression and function in vascular smooth muscle cells. Vascular Sorbs2 is downregulated in diabetes. Genetic knockout of Sorbs2 manifests coronary BK channelopathy and vasculopathy observed in diabetic mice, independent of obesity and glucotoxicity.

LPL rs264, PROCR rs867186 and PDGF rs974819 Gene Polymorphisms in Patients with Unstable Angina.

Coronary artery disease is caused by changes in the coronary arteries due to the atherosclerotic process and thrombotic changes. A very important role in the development of the atherosclerotic process in the coronary vessels is played by the inflammatory process and the immune response. Due to the important role of lipids and the coagulation process in the atherosclerotic process, research has also focused on genes affecting lipid metabolism and the coagulation system. Lipoprotein lipase (LPL) is an enzyme that metabolises lipids, hydrolysing triglycerides to produce free fatty acids and glycerol. Protein C (PC) is an essential component of coagulation and fibrinolysis. It is activated on the endothelial surface by the membrane-bound thrombin-thrombomodulin complex. Platelet-derived growth factor (PDGF) has a number of important functions in processes related to fibroblast and smooth muscle cell function. Due to their influence on lipid metabolism and coagulation processes, LPL, PROCR (endothelial cell protein C receptor) and PDGF may affect the atherosclerotic process and, thus, the risk of coronary heart disease. The aim of the study was to examine the associations between the LPL rs264, PROCR rs867186 and PDGF rs974819 gene polymorphisms and the risk of unstable angina and selected clinical parameters. The study included 232 patients with unstable angina and 144 healthy subjects as the control group. Genotyping was performed using real-time PCR. There were no statistically significant differences in the distribution of the polymorphisms tested between the patients with unstable angina and the control subjects. The results showed associations between the PROCR rs867186 and PDGF rs974819 polymorphisms and some clinical parameters in patients with unstable angina. In patients with the PDGF rs974819 CC genotype, there were increased values for cholesterol and LDL serum levels in comparison with patients with the PDGF rs974819 CT and TT genotypes. In patients with the PROCR rs867186 AA genotype, HDL serum levels were lower than in patients with the GA genotype. The results of our study did not show that the LPL rs264, PROCR rs867186 and PDGF rs974819 gene polymorphisms were significant risk factors for unstable angina in our population. The results of the study suggest that PDGF rs974819 and PROCR rs867186 may be associated with some parameters of lipid metabolism.

The Thirty-Fifth Anniversary of K+ Channels in O2 Sensing: What We Know and What We Don’t Know

On the thirty-fifth anniversary of the first description of O2-sensitive K+ channels in the carotid body chemoreceptors O2 sensing remains a salient issue in the literature. Whereas much has been learned about this subject, important questions such as the identity of the specific K+ channel subtype(s) responsible for O2 sensing by chemoreceptors and the mechanism(s) by which their activities are altered by hypoxia have not yet been definitively answered. O2 sensing is a fundamental biological process necessary for the acute and chronic responses to varying environmental O2 levels which allow organisms to adapt to hypoxia. Whereas chronic responses depend on the modulation of hypoxia-inducible transcription factors which determine the expression of numerous genes encoding enzymes, transporters and growth factors, acute responses rely mainly on the dynamic modulation of ion channels by hypoxia, causing adaptive changes in cell excitability, contractility and secretory activity in specialized tissues. The most widely studied oxygen-sensitive ion channels are potassium channels, but oxygen sensing by members of both the calcium and sodium channel families has also been demonstrated. Given the explosion of information on this topic, in this review, we will focus on the mechanisms of physiological oxygen chemotransduction by PO2-dependent K+ channels, with particular emphasis on their function in carotid body chemoreceptor cells (CBCC) and pulmonary artery smooth muscle cells (PASMC), highlighting areas of consensus and controversy within the field. We will first describe the most well-established concepts, those reproduced in multiple laboratories, and then discuss selected observations or questions that remain unresolved, and that limit our progress in this field.

Mzb1 Attenuates Atherosclerotic Plaque Vulnerability in ApoE-/- Mice by Alleviating Apoptosis and Modulating Mitochondrial Function.

In this study, we investigated the protective role of Mzb1 in atherosclerotic plaque vulnerability. To explore the impact of Mzb1, we analyzed Mzb1 expression, assessed apoptosis, and evaluated mitochondrial function in atherosclerosis (AS) mouse models and human vascular smooth muscle cells (HVSMCs). We observed a significant decrease in Mzb1 expression in AS mouse models and ox-LDL-treated HVSMCs. Downregulation of Mzb1 increased ox-LDL-induced apoptosis and cholesterol levels of HVSMCs, while Mzb1 overexpression alleviated these effect. Mzb1 was found to enhance mitochondrial function, as evidenced by restored ATP synthesis, mitochondrial membrane potential, and reduced mtROS production. Moreover, Mzb1 overexpression attenuated atherosclerotic plaque vulnerabilityin ApoE-/- mice. Our findings suggest that Mzb1 overexpression regulates the AMPK/SIRT1 signaling pathway, leading to the attenuation of atherosclerotic plaque vulnerability. This study provides compelling evidence for the protective effect of Mzb1 on atherosclerotic plaques by alleviating apoptosis and modulating mitochondrial function in ApoE-/- mice.

SOCS5, targeted by miR-155-5p, plays a negative regulatory role in pulmonary hypertension through inhibiting JAK2/STAT3 signaling pathway

Pulmonary hypertension (PH) is a chronic pulmonary vascular disease and causes massive deaths. Here, we intended to investigate the function and mechanism of SOCS5 in PH. We engineered a hypoxia-induced PH model in mice. HE staining were implemented to evaluate pathological alterations in the lung tissues. The potential mechanism of SOCS5 in regulating hypoxia-induced pulmonary artery smooth muscle cell (PASMC) function was explored in vitro. RT-qPCR and western blot revealed that the level of SOCS5 was decreased both in PH mice and hypoxia-induced HPASMCs. Functional assays were performed for confirming the role of SOCS5 in modulating the cell phenotype and JAK2/STAT3 pathway in HPASMCs. Results revealed that overexpression of SOCS5 suppressed proliferation, migration and contraction of HPASMCs and negatively regulated the JAK2/STAT3 signaling pathway in HPASMCs under hypoxia in vitro, while knockdown of SOCS5 accelerated it. As evidenced by mechanism studies, SOCS5 was targeted and regulated by miR-155-5p, hence affecting on HPASMC proliferation, migration and contraction. These outcomes indicated that the decreased level of SOCS5 in hypoxia-induced HPASMCs promoted the cell proliferation, cell migration, and cell contraction through activating JAK2/STAT3 signaling pathway. Moreover, SOCS5 was targeted by miR-155-5p. All in all, our work hinted that miR-155-5p/SOCS5/JAK2/STAT3 axis played a crucial part in PH.

Calcineurin inhibition, cardiovascular consequences, vascular resistance, and potential responses.

To place the consequences of calcineurin inhibition in a cardiovascular context. Literature review coupled with personal encounters. Calcineurin is a calcium-binding and calmodulin-binding protein that is conserved across evolution from yeast to mammals. The enzyme functions as a calcium-dependent, calmodulin-stimulated protein phosphatase. Its role in regulating physiology has largely been elucidated by observing calcineurin inhibition. Calcineurin inhibition transformed organ transplantation from an experiment into a therapy and made much of general immunotherapy possible. The function of this phosphatase and how its inhibition leads to toxicity concern us to this date. Initial research from patients and animal models implicated a panoply of factors contributing to hypertension and vasculopathy. Subsequently, the role of calcineurin in regulating the effective fluid volume, sodium reabsorption, and potassium and hydrogen ion excretion was elucidated by investigating calcineurin inhibition. Understanding the regulatory effects of calcineurin on endothelial and vascular smooth muscle cell function has also made substantial progress. However, precisely how the increase in systemic vascular resistance arises requires further mechanistic research. Calcineurin inhibition continues to save lives; however, options to counteract the negative effects of calcineurin inhibition should be vigorously pursued.

Pharmacological inhibition of MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) induces ferroptosis in vascular smooth muscle cells

MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a human paracaspase protein with proteolytic activity via its caspase-like domain. The pharmacological inhibition of MALT1 by MI-2, a specific chemical inhibitor, diminishes the response of endothelial cells to inflammatory stimuli. However, it is largely unknown how MALT1 regulates the functions of vascular smooth muscle cells (SMCs). This study aims to investigate the impact of MALT1 inhibition by MI-2 on the functions of vascular SMCs, both in vitro and in vivo. MI-2 treatment led to concentration- and time-dependent cell death of cultured aortic SMCs, which was rescued by the iron chelator deferoxamine (DFO) or ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, but not by inhibitors of apoptosis (Z-VAD-fmk), pyroptosis (Z-YVAD-fmk), or necrosis (Necrostatin-1, Nec-1). MI-2 treatment downregulated the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy polypeptide 1 (FTH1), which was prevented by pre-treatment with DFO or Fer-1. MI-2 treatment also activated autophagy, which was inhibited by Atg7 deficiency or bafilomycin A1 preventing MI-2-induced ferroptosis. MI-2 treatment reduced the cleavage of cylindromatosis (CYLD), a specific substrate of MALT1. Notably, MI-2 treatment led to a rapid loss of contractility in mouse aortas, which was prevented by co-incubation with Fer-1. Moreover, local application of MI-2 significantly reduced carotid neointima lesions and atherosclerosis in C57BL/6J mice and apolipoprotein-E knockout (ApoE−/−) mice, respectively, which were both ameliorated by co-treatment with Fer-1. In conclusion, the present study demonstrated that MALT1 inhibition induces ferroptosis of vascular SMCs, likely contributing to its amelioration of proliferative vascular diseases.

Cholesteryl Hemiazelate Present in Cardiovascular Disease Patients Causes Lysosome Dysfunction in Murine Fibroblasts.

There is growing evidence supporting the role of fibroblasts in all stages of atherosclerosis, from the initial phase to fibrous cap and plaque formation. In the arterial wall, as with macrophages and vascular smooth muscle cells, fibroblasts are exposed to a myriad of LDL lipids, including the lipid species formed during the oxidation of their polyunsaturated fatty acids of cholesteryl esters (PUFA-CEs). Recently, our group identified the final oxidation products of the PUFA-CEs, cholesteryl hemiesters (ChE), in tissues from cardiovascular disease patients. Cholesteryl hemiazelate (ChA), the most prevalent lipid of this family, is sufficient to impact lysosome function in macrophages and vascular smooth muscle cells, with consequences for their homeostasis. Here, we show that the lysosomal compartment of ChA-treated fibroblasts also becomes dysfunctional. Indeed, fibroblasts exposed to ChA exhibited a perinuclear accumulation of enlarged lysosomes full of neutral lipids. However, this outcome did not trigger de novo lysosome biogenesis, and only the lysosomal transcription factor E3 (TFE3) was slightly transcriptionally upregulated. As a consequence, autophagy was inhibited, probably via mTORC1 activation, culminating in fibroblasts' apoptosis. Our findings suggest that the impairment of lysosome function and autophagy and the induction of apoptosis in fibroblasts may represent an additional mechanism by which ChA can contribute to the progression of atherosclerosis.

The Role of Reactive Oxygen Species in the Regulation of Blood Vessel Tone in Perinatal and Early Postnatal Ontogenesis

Reactive oxygen species (ROS) have been considered for many years as negative regulators in the cardiovascular system. Indeed, excessive production of ROS characterizes many cardiovascular diseases. The damaging effect of ROS can be especially pronounced in a newborn organism, since during this period their contractile effect in pulmonary arteries remains as high as in the pre-term fetus arteries and the antioxidant systems have not yet formed. Therefore, in the first hours and days of independent life, pulmonary arteries tend to contract, primarily due to the low bioavailability of endothelial NO, which increases the risk of developing pulmonary hypertension in newborns. At the same time, during the perinatal period, ROS play an important role in the adaptive reactions of the circulatory system. ROS provide occlusion of the ductus arteriosus and separation of the pulmonary and systemic circulations soon after birth, and also contribute to the contraction of peripheral vessels during hypoxia, which often accompanies the delivery, and therefore provide priority blood supply to the brain in these conditions. The vasomotor effect of ROS is also pronounced in early postnatal ontogenesis, but it has a different character. In the first weeks of life, the action of ROS serves as one of the mechanisms for increasing endothelium-dependent relaxation of pulmonary vessels. In addition, during early postnatal ontogeny, ROS may play an important role in the regulation of systemic vascular tone. This review outlines the current understanding of the vasomotor role of ROS in the vessels of the pulmonary and systemic circulation and considers the mechanisms of ROS effects on the functioning of vascular endothelial and smooth muscle cells in the perinatal and early postnatal periods.

Cardiovascular phenotype of the duchenne muscular dystrophy carrier female rat

Abstract Background Duchenne muscular dystrophy (DMD) is a lethal, severely progressive muscle-wasting disease. It is an X-linked recessive disease caused by mutations in the DMD gene encoding dystrophin protein and consequently the absence of dystrophin protein synthesis. Besides the skeletal muscle abnormalities, DMD patients are often suffering cardiovascular symptoms include cardiac fibrosis, dilated cardiomyopathy, arrhythmia, and congestive heart failure. Heterozygous female DMD carriers rarely acquire skeletal muscle symptoms, however the cardiac abnormalities are often underrepresented. There are few studies to date characterizing cardiovascular abnormalities in female DMD carriers and the mechanisms underlying the cardiac manifestations are still not fully known. Aims and Methods: Therefore, the aim of this study was to investigate the cardiovascular phenotype of 9 months old female DMD carrier rats and compare with age-matched wildtype rats. Objectives included assessment of cardiac function via transthoracic echocardiography, vascular function using wire myography, cardiac fibrosis and dystrophin via histology, mRNA expression of inflammatory markers via RT-qPCR, measurements of ACE1 and ACE2 activity, as well as expression of dystrophin, SERCA, sarcolipin, BAG3, and Connexin43 (Cx43) using western blotting. The cardiac function, vascular endothelial function, ACE1 and ACE2 activity, and collagen deposition in cardiac tissue were similar between the female DMD carrier and wildtype rats. The echocardiography data revealed thinning of the left-ventricular posterior wall indicating trend towards cardiac atrophy and dysfunction (p<0.05). Wire myography data showed trends of impaired endothelium-independent relaxation and hypercontractility, suggesting the potential vascular abnormalities. Histological analysis showed thickening of intima-media layer in coronary arteries, addition to reduced dystrophin staining, further affirming dystrophin loss impairs smooth muscle cell function (p<0.05, respectively). Protein expression of dystrophin, SERCA2, and BAG3 was decreased in female DMD carrier rats in the myocardium. We also found decreased levels of Cx43 at the intercalated discs, with a concomitant increase in the phosphorylation of Serine368 on Cx43 that very often precedes ubiquitination and degradation of Cx43 channels. In agreement, higher levels of the ubiquitin ligase Nedd4 were observed in hearts of female DMD carrier rats, implicating Cx43 degradation in cardiac dysfunction observed in these animals. Conclusions These findings indicate that female DMD carrier rats do not develop severe cardiac systolic dysfunction nor dilated cardiomyopathy at the age of 9 months unlike male DMD rats as previously described(1). However, the female DMD carrier rats begin to manifest tendencies towards cardiac and vascular dysfunction in association with a changes of calcium handling protein and Cx 43 expression in the heart.

Importance of smooth muscle p110alpha/AKT/FOXO1 signaling for the development of abdominal aortic aneurysm

Abstract Background Abdominal aortic aneurysms (AAA) are characterized by loss of vascular smooth muscle cells (SMCs) and extracellular matrix (ECM) degradation. The catalytic PI 3-kinase isoform p110α signals downstream of growth factor receptors. Mice harboring an SMC-specific p110α deficiency (SM-p110α-/-) are characterized by impaired SMC proliferation and survival. Aim We hypothesized that p110α deficiency in SMCs exacerbates the development and progression of AAA as it promotes SMC loss and thus weakens the aortic wall in healthy and diseased animals. Therefore, we investigated how p110α deficiency in SMCs affects AAA formation, vascular integrity as well as downstream signaling in SMCs. Methods and results Medial thickness of the abdominal aorta in SM-p110α-/- mice was significantly decreased compared to wild-type (WT) littermate controls (29.0±3.1µm vs. 42.5±4.1µm). Morphological characterization of the aortic wall by transmission electron microscopy revealed impaired elastic fibers, detached SMCs and apoptotic cells in SM-p110α-/- mice. SMC differentiation markers including calponin and myosin heavy chain were downregulated in the aortic wall of SM-p110α-/- mice. Western blots demonstrated that p110α deficiency impaired the expression of elastin. Consequently, elastin fibers were characterized by reduced fiber width and length. In addition, growth factor stimulated proliferation of p110α-/- SMCs was significantly impaired compared to WT controls. AAA development in SM-p110α-/- mice and WT littermates were studied using the porcine pancreatic elastase (PPE) model. PPE was infused into the infrarenal aorta. Ultrasound examination of the aorta revealed an increased aortic diameter in all PPE-treated mice. However, the increase in aortic diameter was significantly elevated (p<0.05) in SM-p110α-/- mice (70.2±10.8%, n=9) compared to WT animals (42.4±6.0%, n=10). Furthermore, PPE treated SM-p110-/- mice were characterized by increased ECM degradation, decreased vascular remodelling, and less proliferating cells. Mechanistically, growth factor-dependent phosphorylation and inactivation of crucial phenotypic modulators of SMCs - FOXO-1, -3 and -4 - were shown to be significantly decreased in p110α-/- SMCs compared to WT cells. Specific inhibition of FOXO1 rescued the impaired proliferation of p110α-/- SMCs and enhanced the expression of elastin and calponin. Analysis of FOXO expression in humans revealed that FOXO1 was significantly upregulated in AAA patients compared to healthy individuals. Conclusion p110α deficiency in SMCs promotes the development and progression of AAA, as p110α controls crucial SMC functions which are important for structural processes in the aortic wall. Thereby, SMC proliferation is controlled by p110α dependent phosphorylation and inactivation of FOXO1. Since FOXO1 is upregulated in AAA patients, the p110α/AKT/FOXO1 signaling cascade may provide a key target for modulating aortic wall stability in AAA.

Nicotine affects mitochondrial structure and function in human airway smooth muscle cells.

Exposure to cigarette smoke and e-cigarettes, with nicotine as the active constituent, contributes to increased health risks associated with asthma. Nicotine exerts its functional activity via nicotinic acetylcholine receptors (nAChRs), and the alpha7 subtype (α7nAChR) has recently been shown to adversely affect airway dynamics. The mechanisms of α7nAChR action in airways, particularly in the context of airway smooth muscle (ASM), a key cell type in asthma, are still under investigation. Mitochondria have garnered increasing interest for their role in regulating airway tone and adaptations to cellular stress. Here mitochondrial dynamics such as fusion versus fission, and mitochondrial Ca2+ ([Ca2+]m), play an important role in mitochondrial homeostasis. There is currently no information on effects and mechanisms by which nicotine regulates mitochondrial structure and function in ASM in the context of asthma. We hypothesized that nicotine disrupts mitochondrial morphology, fission-fusion balance, and [Ca2+]m regulation, with altered mitochondrial respiration and bioenergetics in the context of asthmatic ASM. Using human ASM (hASM) cells from nonasthmatics, asthmatics, and smokers, we examined the effects of nicotine on mitochondrial dynamics and [Ca2+]m. Fluorescence [Ca2+]m imaging of hASM cells with rhod-2 showed robust responses to 10 μM nicotine, particularly in asthmatics and smokers. In both asthmatics and smokers, nicotine increased the expression of fission proteins while decreasing fusion proteins. Seahorse analysis showed blunted oxidative phosphorylation parameters in response to nicotine in these groups. α7nAChR siRNA blunted nicotine effects, rescuing [Ca2+]m, changes in mitochondrial structural proteins, and mitochondrial dysfunction. These data highlight mitochondria as a target of nicotine effects on ASM, where mitochondrial disruption and impaired buffering could permit downstream effects of nicotine in the context of asthma.NEW & NOTEWORTHY Asthma is a major healthcare burden, which is further exacerbated by smoking. Recognizing the smoking risk of asthma, understanding the effects of nicotine on asthmatic airways becomes critical. Surprisingly, the mechanisms of nicotine action, even in normal and especially asthmatic airways, are understudied. Accordingly, the goal of this research is to investigate how nicotine influences asthmatic airways in terms of mitochondrial structure and function, via the a7nAChR.

Phenotype and function of smooth muscle cells derived from the human normal great saphenous vein in response to hypoxia.

The contribution of hypoxia to the pathophysiology of vascular smooth muscle cells (VSMCs) has not yet been fully elucidated. This study evaluated the effect of hypoxia on the phenotype and function of SMCs derived from the human normal great saphenous veins (NGSVs). Fifteen NGSV tissue samples were collected. SMCs were isolated and cultured. Proliferation, migration, adhesion, senescence, and the structure of cytoskeletal filaments in SMCs were observed. mRNA and protein expression of Bax, Bcl-2, caspase-3, matrix metalloproteinases (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 was detected by fluorescent quantitative polymerase chain reaction and immunoblotting in the cobalt chloride (CoCl2) and the control groups. A decrease in the number of cytoskeletal filaments was observed. mRNA and protein expression of Bas and caspase-3 was significantly decreased, while the quantity of proliferation, migration, adhesion, senescence, and mRNA and protein expression of Bcl-2, MMP-2, MMP-9, TIMP-1, and TIMP-2 in SMCs in the CoCl2 group were significantly increased compared with the control group. Under hypoxic conditions, the phenotype and function of SMCs derived from the human NGSVs were dysregulated, suggesting that VSMCs switch from the contractile phenotype to the secretory or synthetic phenotype, and more dedifferentiate, resulting in extracellular matrix deposition and apoptotic decrease through the intrinsic pathway.

Direct investigation of cell contraction signal networks by light-based perturbation methods.

Cell contraction plays an important role in many physiological and pathophysiological processes. This includes functions in skeletal, heart, and smooth muscle cells, which lead to highly coordinated contractions of multicellular assemblies, and functions in non-muscle cells, which are often highly localized in subcellular regions and transient in time. While the regulatory processes that control cell contraction in muscle cells are well understood, much less is known about cell contraction in non-muscle cells. In this review, we focus on the mechanisms that control cell contraction in space and time in non-muscle cells, and how they can be investigated by light-based methods. The review particularly focusses on signal networks and cytoskeletal components that together control subcellular contraction patterns to perform functions on the level of cells and tissues, such as directional migration and multicellular rearrangements during development. Key features of light-based methods that enable highly local and fast perturbations are highlighted, and how experimental strategies can capitalize on these features to uncover causal relationships in the complex signal networks that control cell contraction.

Vav2 promotes ductus arteriosus anatomic closure via the remodeling of smooth muscle cells by Rac1 activation.

The ductus arteriosus (DA), bridging the aorta and pulmonary artery, immediately starts closing after birth. Remodeling of DA leads to anatomic obstruction to prevent repatency. Several histological changes, especially extracellular matrices (ECMs) deposition and smooth muscle cells (SMCs) migration bring to anatomic closure. The genetic etiology and mechanism of DA closure remain elusive. We have previously reported a novel copy number variant containing Vav2 in patent ductus arteriosus (PDA) patients, but its specific role in DA closure remains unknown. The present study revealed that the expression of Vav2 was reduced in human patent DA, and it was less enrichment in the adjacent aorta. Matrigel experiments demonstrated that Vav2 could promote SMC migration from PDA patient explants. Smooth muscle cells with Vav2 overexpression also presented an increased capacity in migration and downregulated contractile-related proteins. Meanwhile, SMCs with Vav2 overexpression exhibited higher expression of collagen III and lessened protein abundance of lysyl oxidase, and both changes are beneficial to DA remodeling. Overexpression of Vav2 resulted in increased activity of Rac1, Cdc42, and RhoA in SMCs. Further investigation noteworthily found that the above alterations caused by Vav2 overexpression were particularly reversed by Rac1 inhibitor. A heterozygous, rare Vav2 variant was identified in PDA patients. Compared with the wild type, this variant attenuated Vav2 protein expression and weakened the activation of downstream Rac1, further impairing its functions in SMCs. In conclusion, Vav2 functions as an activator for Rac1 in SMCs to promote SMCs migration, dedifferentiation, and ECMs production. Deleterious variant potentially induces Vav2 loss of function, further providing possible molecular mechanisms about Vav2 in PDA pathogenesis. These findings enriched the current genetic etiology of PDA, which may provide a novel target for prenatal diagnosis and treatment. KEY MESSAGES: Although we have proposed the potential association between Vav2 and PDA incidence through whole exome sequencing, the molecular mechanisms underlying Vav2 in PDA have never been reported.This work, for the first time, demonstrated that Vav2 was exclusively expressed in closed DAs. Moreover, we found that Vav2 participated in the process of anatomic closure by mediating SMCs migration, dedifferentiation, and ECMs deposition through Rac1 activation. Our findings first identified a deleterious Vav2 c.701C>T variant that affected its function in SMCs by impairing Rac1 activation, which may lead to PDA defect. Vav2 may become an early diagnosis and an effective intervention target for PDA clinical therapy.

Fat and inflammation: adipocyte-myeloid cell crosstalk in atherosclerosis.

Adipose tissue inflammation has been implicated in various chronic inflammatory diseases and cancer. Perivascular adipose tissue (PVAT) surrounds the aorta as an extra layer and was suggested to contribute to atherosclerosis development. PVAT regulates the function of endothelial and vascular smooth muscle cells in the aorta and represent a reservoir for various immune cells which may participate in aortic inflammation. Recent studies demonstrate that adipocytes also express various cytokine receptors and, therefore, may directly respond to inflammatory stimuli. Here we will summarize current knowledge on immune mechanisms regulating adipocyte activation and the crosstalk between myeloid cells and adipocytes in pathogenesis of atherosclerosis.

Long noncoding RNA TUG1 regulates smooth muscle cell differentiation via KLF4-myocardin axis.

Abdominal aortic aneurysms (AAAs) are asymptomatic vascular diseases that have life-threatening outcomes. Smooth muscle cell (SMC) dysfunction plays an important role in AAA development. The contribution of non-coding genome, specifically the role of long non-coding RNAs (lncRNAs) in SMC dysfunction, is relatively unexplored. We investigated the role of lncRNA TUG1 in SMC dysfunction. To identify potential lncRNAs relevant to SMC functionality, lncRNA profiling was performed in angiotensin-II-treated SMCs. AAA was induced by angiotensin-II treatment in mice. Transcriptional regulation of TUG1 was studied using promoter luciferase and chromatin-immuno-precipitation experiments. Gain-or-loss-of-function experiments were performed in vitro to investigate TUG1-mediated regulation of SMC function. Immunoprecipitation experiments were conducted to elucidate the mechanism underlying TUG1-mediated SMC dysfunction. TUG1 was upregulated in SMCs following angiotensin-II treatment. Similarly, TUG1 levels were elevated in abdominal aorta in a mouse model of angiotensin-II-induced AAA. Further investigations showed that angiotensin-II-induced TUG1 expression could be suppressed by inhibiting Notch-signaling pathway, both in vitro and in mouse AAA model and that TUG1 is a direct transcriptional target of the Notch pathway. In aneurysmal tissues, TUG1 expression was inversely correlated with the expression of SMC contractile genes. Overexpression of TUG1 repressed SMC differentiation in vitro, whereas siRNA/shRNA-mediated TUG1 knockdown showed an opposite effect. Mechanistically, TUG1 interacts with transcriptional repressor KLF4 and facilitates its recruitment to myocardin promoter ultimately leading to the repression of SMC differentiation. In summary, our study uncovers a novel role for the lncRNA TUG1 wherein it modulates SMC differentiation via the KLF4-myocardin axis, which may have potential implications in AAA development.NEW & NOTEWORTHY TUG1 is an angiotensin-II-induced long noncoding RNA that mediates smooth muscle cell (SMC) dysfunction through interaction with transcriptional repressor KLF4.

Genetic Overlap of Spontaneous Dissection of Either the Thoracic Aorta or the Coronary Arteries

Ascending thoracic aortic dissection (ATAD) is a well-known vascular cause of sudden death. Spontaneous coronary artery dissections (SCAD) are emerging as an important cause of early-onset myocardial infarction and sudden death. Genetic variants in multiple connective tissue genes have been recognized to underlie ATAD; other genetic variants have similarly been recognized to underlie SCAD. Little data are available regarding any genetic commonality between ATAD and SCAD. Our objective is to determine and characterize any genetic overlap between genes coding for ATAD and SCAD. We identified and reviewed 17 retrospective and prospective genetic studies of thoracic aortic dissection and SCAD published between 2016 and 2022 identified through PubMed and Orbis. Articles highlighting the significant plausible triggers for ATAD or SCAD individually were analyzed. No previous study reviewed both ATAD and SCAD genetics together. Separate lists of causative genes were constructed for ATAD and SCAD-and then commonalities were sought. A Venn diagram was constructed to display the genetic overlap and common physiologic pathways involved. We identified a definite, meaningful overlap of 15 independent genes based on a genome-wide association study or other genetic methods. The associated genetic pathways involved various biologic processes including elastin degradation, smooth muscle cell function, and the TGFβ-pathway. The overlapping genes included the following: COL3A1, TGFB2, SMAD3, MYLK, TGFBR2, TGFBR1, LOX, FBN1, NOTCH1, ELN, COL5A1, COL5A2, COL1A2, MYH11, and TLN1. The corresponding molecular pathways were investigated and correlated for both diseases. We are not aware of other studies searching for genetic commonalities between ATAD and SCAD. We have successfully identified overlapping genes-and their corresponding molecular pathways-for ATAD and SCAD. We hope that these insights will lead to further clinical and scientific understanding of each disease through study of their fundamental commonalities.

Regulation of DNA damage and transcriptional output in the vasculature through a cytoglobin-HMGB2 axis

Identifying novel regulators of vascular smooth muscle cell function is necessary to further understand cardiovascular diseases. We previously identified cytoglobin, a hemoglobin homolog, with myogenic and cytoprotective roles in the vasculature. The specific mechanism of action of cytoglobin is unclear but does not seem to be related to oxygen transport or storage like hemoglobin. Herein, transcriptomic profiling of injured carotid arteries in cytoglobin global knockout mice revealed that cytoglobin deletion accelerated the loss of contractile genes and increased DNA damage. Overall, we show that cytoglobin is actively translocated into the nucleus of vascular smooth muscle cells through a redox signal driven by NOX4. We demonstrate that nuclear cytoglobin heterodimerizes with the non-histone chromatin structural protein HMGB2. Our results are consistent with a previously unknown function by which a non-erythrocytic hemoglobin inhibits DNA damage and regulates gene programs in the vasculature by modulating the genome-wide binding of HMGB2.

Abstract P3044: MicroRNA-127-3p Regulates Inflammatory Response In Injury-induced Vascular Remodeling And Cellular Senescence In Vascular Smooth Muscle Cells

The accumulation of senescent cells in the vasculature over the lifespan contributes to vascular remodeling due to the severely impaired functional properties of the cells. MicroRNAs (miRs) are promising therapeutical targets as they exhibit diverse and cell-specific functions and can be effectively targeted in vivo . Here, we propose that targeting miR-127-3p might be an attractive approach to enhance vascular healing and regeneration in a cell-specific manner, thereby limiting angioplasty-related complications. The initial screenings revealed miR-127-3p as a highly regulated miR in the process of restenosis 7 and 21 days (p<0.001) after wire induced-injury and in aged (20 months) C57BL/6 mice (p<0.05) in vivo . Additionally, replicative senescent vascular smooth muscle cells (SMC) and human coronary artery endothelial cells (EC) showed an enhanced expression of miR-127-3p (p<0.05) compared to non-senescent cells. Functional effects of miR-127-3p on SMCs and ECs were assessed via transfection with either pre-miR or antagomir. Overexpression of miR-127-3p in vitro reduced SMC proliferation (p<0.05) and altered migrational capacity. Downregulation of miR-127-3p resulted in a reverse effect. In contrast, cellular functions were not altered in ECs. We further identified potential targets with effects on inflammation and cellular aging by in silico analysis and verified them on mRNA and protein levels by qRT-PCR and western blot analysis. As a direct target of miR-127-3p, the senescence-associated gene CD44 was identified by TargetScan. Expression of CD44 was altered on mRNA and protein levels after overexpression respectively downregulation of miR-127-3p in SMCs. Further, p14arf and laminB1 were downregulated after overexpression of miR-127-3p in SMCs, contrary to the effect after downregulation of miR-127-3p with increased mRNA levels of p14arf and laminB1. In this study, we identified miR-127-3p as a contributing factor in SMC function during the development of cellular senescence and show that it might therewith also be a modulating factor in the process of restenosis. However, further studies need to be carried out to validate the potential of miR-127-3p as a therapeutic target in vascular aging and remodeling in vitro and in vivo .