Vascular Smooth Muscle Cell Dysfunction Research Articles

Glucagon-like peptide-1 receptor agonists: new strategies and therapeutic targets to treat atherosclerotic cardiovascular disease.

Atherosclerotic cardiovascular disease (ASCVD) is a leading cause of cardiovascular mortality and is increasingly prevalent in our population. Glucagon-like peptide-1 receptor agonists (GLP-1RAs) can safely and effectively lower glucose levels while concurrently managing the full spectrum of ASCVD risk factors and improving patients' long-term prognosis. Several cardiovascular outcome trials (CVOTs) have been carried out to further investigate the cardiovascular benefits of GLP-1RAs. Analyzing data from CVOTs can provide insights into the pathophysiologic mechanisms by which GLP-1RAs are linked to ASCVD and define the use of GLP-1RAs in clinical practice. Here, we discussed various mechanisms hypothesized in previous animal and preclinical human studies, including blockade of the production of adhesion molecules and inflammatory factors, induction of endothelial cells' synthesis of nitric oxide, protection of mitochondrial function and restriction of oxidative stress, suppression of NOD-like receptor thermal protein domain associated protein three inflammasome, reduction of foam cell formation and macrophage inflammation, and amelioration of vascular smooth muscle cell dysfunction, to help explain the cardiovascular benefits of GLP-1RAs in CVOTs. This paper provides an overview of the clinical research, molecular processes, and possible therapeutic applications of GLP-1RAs in ASCVD, while also addressing current limitations in the literature and suggesting future research directions.

Vascular smooth muscle cell-specific Igf1r deficiency exacerbates the development of hypertension-induced cerebral microhemorrhages and gait defects.

Cerebrovascular fragility and cerebral microhemorrhages (CMH) contribute to age-related cognitive impairment, mobility defects, and vascular cognitive impairment and dementia, impairing healthspan and reducing quality of life in the elderly. Insulin-like growth factor 1 (IGF-1) is a key vasoprotective growth factor that is reduced during aging. Circulating IGF-1 deficiency leads to the development of CMH and other signs of cerebrovascular dysfunction. Here our goal was to understand the contribution of IGF-1 signaling on vascular smooth muscle cells (VSMCs) to the development of CMH and associated gait defects. We used an inducible VSMC-specific promoter and an IGF-1 receptor (Igf1r) floxed mouse line (Myh11-CreERT2 Igf1rf/f) to knockdown Igf1r. Angiotensin II in combination with L-NAME-induced hypertension was used to elicit CMH. We observed that VSMC-specific Igf1r knockdown mice had accelerated development of CMH, and subsequent associated gait irregularities. These phenotypes were accompanied by upregulation of a cluster of pro-inflammatory genes associated with VSMC maladaptation. Collectively our findings support an essential role for VSMCs as a target for the vasoprotective effects of IGF-1, and suggest that VSMC dysfunction in aging may contribute to the development of CMH.

Circ_0004872 deficiency attenuates ox-LDL-induced vascular smooth muscle cell dysfunction by miR-424-5p-dependent regulation of FRS2.

Atherosclerosis (AS) is a pivotal pathological basis of cardiovascular and cerebrovascular diseases, and circular RNAs (circRNAs) has been disclosed to exert a vital part in the progression of AS. However, the functions of circ_0004872 in the progression of AS is indistinct. In this context, we aimed to elucidate the role of circ_0004872 and the potential mechanism in AS. The level of circ_0004872, miR-424-5p and fibroblast growth factor receptor substrate 2 (FRS2) was detected using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was monitored by Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine (EDU) assays. The invasion and migration capabilities of VSMCs were tested by transwell assays and wound-healing assay, respectively. Western blot was adopted to check the protein levels of CyclinD1, Vimentin and FRS2. Dual-luciferase reporter and RNA immunoprecipitation assay were executed to manifest the interaction between miR-424-5p and circ_0004872 or FRS2. The level of circ_0004872 was increased in the serum samples of AS patients and ox-LDL-exposed VSMCs. Ox-LDL exposure triggered cell proliferation, invasion and migration ability of VSMCs. depletion of circ_0004872 partly weakened ox-LDL-mediated effects in VSMCs. Mechanistically, circ_0004872 functioned as a sponge of miR-424-5p, and miR-424-5p inhibition partly alleviated circ_0004872 deficiency-mediated influences in VSMCs. Additionally, miR-424-5p interacted with FRS2, and miR-424-5p constrained dysfunction in ox-LDL-stimulated VSMCs via reducing FRS2 level. Notably, circ_0004872 functioned as a sponge of miR-424-5p to elevate FRS2 expression. Circ_0004872 accelerated ox-LDL-induced damage via mediating miR-424-5p/FRS2 axis.

IGF1R deficiency in vascular smooth muscle cells impairs myogenic autoregulation and cognition in mice.

Cerebrovascular pathologies contribute to cognitive decline during aging, leading to vascular cognitive impairment and dementia (VCID). Levels of circulating insulin-like growth factor 1 (IGF-1), a vasoprotective hormone, decrease during aging. Decreased circulating IGF-1 in animal models leads to the development of VCID-like symptoms, but the cellular mechanisms underlying IGF-1-deficiency associated pathologies in the aged cerebrovasculature remain poorly understood. Here, we test the hypothesis that vascular smooth muscle cells (VSMCs) play an integral part in mediating the vasoprotective effects of IGF-1. We used a hypertension-based model of cerebrovascular dysfunction in mice with VSMC-specific IGF-1 receptor (Igf1r) deficiency and evaluated the development of cerebrovascular pathologies and cognitive dysfunction. VSMC-specific Igf1r deficiency led to impaired cerebral myogenic autoregulation, independent of blood pressure changes, which was also associated with impaired spatial learning and memory function as measured by radial arm water maze and impaired motor learning measured by rotarod. In contrast, VSMC-specific IGF-1 receptor knockdown did not lead to cerebral microvascular rarefaction. These studies suggest that VSMCs are key targets for IGF-1 in the context of cerebrovascular health, playing a role in vessel stability alongside other cells in the neurovascular unit, and that VSMC dysfunction in aging likely contributes to VCID.

PCSK9 increases vulnerability of carotid plaque by promoting mitochondrial dysfunction and apoptosis of vascular smooth muscle cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been recognized as a novel lipid-lowing target. Recent clinical studies suggested the value of inhibiting PCSK9 in decreasing the vulnerability of coronary plaques. However, the evidence of PCSK9-regulated evolution of unstable carotid plaques is unclear, which has limited the use of PCSK9 inhibitor in carotid plaques. This study aimed to determine the effect and molecular mechanisms of PCSK9 on vulnerability of carotid plaques, to provide potential therapeutic targets for stabilizing carotid plaques. The expression of PCSK9 in stable and unstable carotid plaques were examined in tissue and plasma. Human aortic vascular smooth muscle cells (VSMCs) and carotid VSMCs were employed to transfect lentivirus for overexpression and knockdown of PCSK9, respectively. Morphological and functional changes of mitochondria were observed by live-cell imaging. Cell apoptosis was evaluated by propidium iodide staining. RNA-sequencing and biological examinations were performed to explore and validate the underlying mechanisms. Truncated plasmids were employed to identify the functional domain of PCSK9 in regulation of VSMCs' mitochondrial morphology, function and apoptosis. Clinically, PCSK9 was closely related with vulnerability of human carotid plaques. Increased expression of PCSK9 in human VSMCs was accompanied by higher level of apoptosis. At subcellular level of VSMCs, the morphology of mitochondria was shifted toward the fission state, followed by mitochondrial dysfunction. Inhibition of p38 MAPK activation partially rescued the above morphological and behavioral changes caused by PCSK9. Furthermore, inhibiting of dynamin-related protein 1 (DRP1) attenuated PCSK9-related mitochondrial dysfunction and cell apoptosis. The 1-149aa domain of PCSK9 protein was essential to achieve functional regulation to VSMCs. Our findings demonstrated that PCSK9 induced morphology-related mitochondrial dysfunction and apoptosis of VSMCs, which may be related to increased vulnerability of carotid plaque.

Paeonol Attenuates Atherosclerosis by Inhibiting Vascular Smooth Muscle Cells Senescence via SIRT1/P53/TRF2 Signaling Pathway.

Atherosclerosis is a chronic inflammatory disease leading to various vascular diseases. Vascular smooth muscle cell (VSMC) senescence promotes atherosclerotic inflammation and the formation of plaque necrosis core, in part through telomere damage mediated by a high-fat diet. Our previous research found that paeonol, a potential anti-inflammatory agent extracted from Cortex Moutan, could significantly improve VSMCs dysfunction. However, the impact of paeonol on the senescence of VSMCs remains unexplored. This study presents the protective effects of paeonol on VSMCs senescence, and its potential activity in inhibiting the progression of atherosclerosis in vivo and in vitro. Sirtuin 1 (SIRT1) is a nuclear deacetylase involved in cell proliferation, senescence, telomere damage, and inflammation. Here, SIRT1 was identified as a potential target of paeonol having anti-senescence and anti-atherosclerosis activity. Mechanistic studies revealed that paeonol binds directly to SIRT1 and then activates the SIRT1/P53/TRF2 pathway to inhibit VSMCs senescence. Our results suggested that SIRT1-mediated VSMCs senescence is a promising druggable target for atherosclerosis, and that pharmacological modulation of the SIRT1/P53/TRF2 signaling pathway by paeonol is of potential benefit for patients with atherosclerosis.

Higher matrix stiffness promotes VSMC senescence by affecting mitochondria-ER contact sites and mitochondria/ER dysfunction.

Abdominal aortic aneurysm (AAA) is a prevalent condition characterized by the weakening and bulging of the abdominal aorta. This study aimed to investigate the impact of a stiff matrix on vascular smooth muscle cells (VSMCs) in AAA development. Bioinformatics analysis revealed that differentially expressed genes (DEGs) in VSMCs of an AAA mouse model were enriched in cellular senescence and related pathways. To simulate aging-related changes, VSMCs were cultured on stiff matrices, and compared to those on soft matrices, the VSMCs cultured on stiff matrices exhibited cellular senescence. Furthermore, the mutual distance between mitochondria and endoplasmic reticulum (ER) in VSMCs was increased, indicating altered mitochondria-endoplasmic reticulum contacts (MERCs). The observed upregulation of reactive oxygen species (ROS) levels, antioxidant gene expression, and decreased mitochondrial membrane potential suggested the presence of mitochondrial dysfunction in VSMCs cultured on a stiff matrix. Additionally, the induction of ER stress-related genes indicated ER dysfunction. These findings collectively indicated impaired functionality of both mitochondria and ER in VSMCs cultured on a stiff matrix. Moreover, our data revealed that high lipid levels exacerbated the effects of high matrix stiffness on VSMCs senescence, MERC sites, and mitochondria/ER dysfunction. Importantly, treatment with the antilipemic agent CI-981 effectively reversed these detrimental effects. These findings provide insights into the role of matrix stiffness, mitochondrial dysfunction, ER stress, and lipid metabolism in AAA development, suggesting potential therapeutic targets for intervention.

Hypothyroidism increases angiotensinogen gene expression associated with vascular smooth muscle cells cholesterol metabolism dysfunction and aorta remodeling in Psammomys obesus

It has been previously shown that clinical cardiovascular manifestations can be caused by mild changes in thyroid function. However, the implication of angiotensinogen (Agt) and vascular smooth muscle cells (VSMCs) dysfunction in the pathophysiology of cardiovascular manifestations in hypothyroidism have not yet been investigated. We induced experimental hypothyroidism in Psammomys obesus by administering carbimazole for five months. At the end of the experiment, the animals were sacrificed and histopathological analysis was performed using Masson's trichrome staining of the aorta and thyroid gland. The expression of the Agt gene and the genes implicated in cholesterol metabolism regulation in the liver and VSMCs was determined by qRT-PCR. Histological observations revealed profound remodeling of the aorta structure in animals with hypothyroidism. In addition, Agt gene expression in the liver was significantly increased. In vitro study, showed that VSMCs from hypothyroid animals overexpressed 3-hydroxy-3-methylglutaryl coenzyme A reductase (Hmgcr) and Acyl CoA:cholesterol acyltransferase (Acat) 1, with failure to increase the efflux pathway genes (ATP-binding cassette subfamily G member (Abcg) 1 and 4). These results suggest that hypothyroidism leads to vascular alterations, including structural remodeling, VSMCs cholesterol metabolism dysfunction, and their switch to a synthetic phenotype, together with hepatic Agt gene overexpression.

Abstract 12885: Pathological Perk/Atf4 Activation in Abdominal Aortic Aneurysm Formation

Introduction: Abdominal aortic aneurysms (AAA) are characterized by vascular smooth muscle cell (VSMC) apoptosis leading to pathological aortic remodeling. Activation of the endoplasmic reticulum (ER) stress response has been linked to VSMC dysfunction but the driving factors behind ER stress activation and its role on AAA development remains poorly defined. The epigenetic enzyme, MLL1, plays a critical role in establishing cellular phenotype and regulating gene expression. We examine the MLL-mediated epigenetic regulation of the ER stress response in VSMCs during AAA development and establish the ER stress pathways as a novel target in the treatment AAAs. Methods: Single-cell sequencing was conducted on human AAA and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and then fed a saturated fat-enriched diet and infused with AngII (1 μg/kg/min) with or without 4-PBA (20mg/kg/day). The elastase induced AAA model was used as a secondary murine model. AAA maximum diameters were quantified and VSCMs were isolated. Eif2a, Atf4 , and Chop gene expression was examined. Apoptosis was quantified by Annexin V staining. Results: Using single-cell RNA sequencing of human aortic tissue, we identified EIF2A, ATF4 , and CHOP were profoundly upregulated in aneurysmal VSMCs as well as two murine AAA models. Mechanistically, TNFα signaling in VSMCs leads to activation of MLL1 which trimethylates histone 3 lysine 4 (H3K4me3) at NF-κB binding sites on the EIF2A and ATF4 gene promoters thereby upregulating the ER stress response and driving VSMC apoptosis. In vivo pharmacological inhibition of the eIF2A/ATF4 ER stress response via 4-PBA injection inhibited AAA formation in both the AngII-induced and elastase-induced AAA model resulting with in significant reduction in VSMC apoptosis, elastin fragmentation, and inflammatory cytokine infiltration. Conclusions: Taken together, our results identify MLL1-mediated activation of the eIF2A/ATF4 ER stress response as a critical regulator of VSMC apoptosis in AAA formation and suggest inhibition of ER stress response as a mechanistic target for the management of AAAs.

TGF-β/Smads signaling pathway, Hippo-YAP/TAZ signaling pathway, and VEGF: Their mechanisms and roles in vascular remodeling related diseases.

Vascular remodeling is a basic pathological process in various diseases characterized by abnormal changes in the morphology, structure, and function of vascular cells, such as migration, proliferation, hypertrophy, and apoptosis. Various growth factors and pathways are involved in the process of vascular remodeling. The transforming growth factor-β (TGF-β) signaling pathway, which is mainly mediated by TGF-β1, is an important factor in vascular wall enhancement during vascular development and regulates the vascular response to injury by promoting the accumulation of intimal tissue. Vascular endothelial growth factor (VEGF) has an important effect on initiating the formation of blood vessels. The Hippo-YAP/TAZ signaling pathway also plays an important role in angiogenesis. In addition, studies have shown that there is a certain interaction between the TGF-β/Smads signaling pathway, Hippo-YAP/TAZ signaling pathway, and VEGF. Many studies have shown that in the development of atherosclerosis, hypertension, aneurysm, vertebrobasilar dolichoectasia, pulmonary hypertension, restenosis after percutaneous transluminal angioplasty, and other diseases, various inflammatory reactions lead to changes in vascular structure and vascular microenvironment, which leads to vascular remodeling. The occurrence of vascular remodeling changes the morphology of blood vessels and thus changes the hemodynamics, which is the cause of further development of the disease process. Vascular remodeling can cause vascular smooth muscle cell dysfunction and vascular homeostasis regulation. This review aims to explore the mechanisms of the TGF-β/Smads signaling pathway, Hippo-YAP/TAZ signaling pathway, and vascular endothelial growth factor in vascular remodeling and related diseases. This paper is expected to provide new ideas for research on the occurrence and development of related diseases and provide a new direction for research on the treatment of related diseases.

Resveratrol Inhibits Abdominal Aortic Aneurysm Progression by Reducing Extracellular Matrix Degradation, Apoptosis, Autophagy, and Inflammation of Vascular Smooth Muscle Cells via Upregulation of HMOX1.

This study aimed to explore the therapeutic effect of resveratrol (RES) against abdominal aortic aneurysm (AAA) and the role of HMOX1 underlying this effect. Vascular smooth muscle cells (VSMCs) were induced by angiotensin II (Ang II) to construct the microenvironment of AAA. HMOX1 expression was downregulated by the short hairpin ribonucleic acid (RNA) specific to HMOX1 in RES-pretreated VSMCs. The levels of matrix metalloproteinase (MMP)-2, MMP-9, and elastin were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. Apoptosis rate was detected. The levels of apoptosis-related proteins (caspase-3 and Bax/Bcl-2), inflammatory cytokines (interleukin [IL]-6, tumor necrosis factor [TNF]-α, and IL-1β), and autophagy-related proteins (Beclin 1, light chain 3 [LC3] II/I, and p62) were detected by western blot. The secretion of inflammatory factors in cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The number of autophagic vesicles in VSMCs was observed and analyzed by transmission electron microscopy. A rat model of pancreatic elastase-induced AAA was established to verify the effect and action mechanism of RES. Stimulation of Ang II increased the messenger RNA (mRNA) and protein levels of MMP-2 and MMP-9, decreased elastin expression, and enhanced apoptosis, secretion of inflammatory factors, and autophagy in VSMCs, whereas RES pretreatment ameliorated Ang II-induced VSMC dysfunction. In addition, HMOX1 mRNA and heme oxygenase-1 (HO-1) protein levels were significantly increased in VSMCs pretreated with RES compared with Ang II treatment alone. Silencing of HMOX1 abolished the effects of RES on VSMC dysfunction. Consistently, RES suppressed the development of AAA in rats by increasing the expression of HMOX1. Resveratrol protects against AAA by inhibiting extracellular matrix degradation, apoptosis, autophagy, and inflammation of VSMCs via HMOX1 upregulation. Our study found that angiotensin II (Ang II) stimulated increased the levels of MMP-2 and MMP-9 in vascular smooth muscle cells (VSMCs), decreased elastin expression, and promoted apoptosis, autophagy occurrence, and secretion of inflammatory factors, while resveratrol (RES) pretreatment improved this effect. In addition, downregulation of HMOX1 expression eliminated the effect of RES on the function of VSMCs. Our study elucidates that RES improves AAA progression through HMOX1 at both cellular and animal levels. This work can help doctors better understand the pathological mechanism of the occurrence and development of AAA, and provide a theoretical basis for clinicians to find better treatment options.

Roles of endothelial and smooth muscle cell dysfunction and vasa vasorum in vasomotor disorders in ischemia with no obstructive coronary artery disease

Recently, the importance has emerged of ischemia with no obstructive coronary artery disease (INOCA), for which endothelial and vascular smooth muscle cell (VSMC) dysfunctions and alterations in coronary vasa vasorum are involved. Regarding endothelial vasodilator functions, both endothelium-derived nitric oxide and endothelium-derived hyperpolarizing factor play important roles in modulating vascular tone, especially in the microcirculation. Recent studies have suggested systemic endothelial dysfunction in INOCA. Regarding VSMC dysfunction, Rho-kinase has been identified as a key molecular mechanism of VSMC hyperconstriction in INOCA. Finally, recent advances of coronary imaging have demonstrated the important role of altered adventitial vasa vasorum functions in INOCA.

T cells in abdominal aortic aneurysm: immunomodulation and clinical application.

Abdominal aortic aneurysm (AAA) is characterized by inflammatory cell infiltration, extracellular matrix (ECM) degradation, and vascular smooth muscle cell (SMC) dysfunction. The inflammatory cells involved in AAA mainly include immune cells including macrophages, neutrophils, T-lymphocytes and B lymphocytes and endothelial cells. As the blood vessel wall expands, more and more lymphocytes infiltrate into the outer membrane. It was found that more than 50% of lymphocytes in AAA tissues were CD3+ T cells, including CD4+, CD8+T cells, γδ T cells and regulatory T cells (Tregs). Due to the important role of T cells in inflammatory response, an increasing number of researchers have paid attention to the role of T cells in AAA and dug into the relevant mechanism. Therefore, this paper focuses on reviewing the immunoregulatory role of T cells in AAA and their role in immunotherapy, seeking potential targets for immunotherapy and putting forward future research directions.

Based on network pharmacology, gastrodin attenuates hypertension-induced vascular smooth muscle cell proliferation and PI3K/AKT pathway activation

The effects and underlying mechanisms of gastrodin treatment on hypertensive vascular dysfunction and proliferation of vascular smooth muscle cells (VSMCs) were determined in vitro and in vivo. Using a pharmacological target network interaction analysis, 151 common targets and a PPI network were identified containing the top 10 hub genes. Kyoto encyclopedia of genes and genomes (KEGG) analysis identified the PI3K/AKT pathway as a significantly enriched pathway. Both spontaneous hypertensive rats (SHRs) and Wistar Kyoto rats were used to assess the therapeutic effects of gastrodin on hypertension. Gastrodin treatment of the SHRs resulted in a marked attenuation of elevated blood pressure, pulse wave velocity, and pathological changes in the abdominal aorta. Moreover, gastrodin treatment significantly inhibited cell growth and downregulated the expression of PCNA as well as the p-PI3K/PI3K and p-AKT/AKT levels in angiotensin II-stimulated VSMCs. Taken together, gastrodin treatment attenuates blood pressure elevation, vascular dysfunction, and proliferation of VSMCs and inhibits the activation of the PI3K/AKT pathway.

Ginsenoside Rb1 protects human vascular smooth muscle cells against resistin-induced oxidative stress and dysfunction.

Resistin has been shown to play a key role in inducing vascular smooth muscle cells (VSMCs) malfunction in the atherosclerosis progression. Ginsenoside Rb1 is the main component of ginseng, which has been used for thousands of years and has been reported to have a powerful vascular protective effect. The aim of this study was to explore the protective effect of Rb1 on VSMCs dysfunction induced by resistin. In the presence or absence of Rb1, human coronary artery smooth muscle cells (HCASMC) were treated at different time points with or without 40 ng/ml resistin and acetylated low-density lipoprotein (acetylated LDL). Cell migration and proliferation were analyzed using wound healing test and CellTiter Aqueous Cell Proliferation Assay (MTS) test, respectively. Intracellular reactive oxygen species (ROS) (H2DCFDA as a dye probe) and superoxide dismutase (SOD) activities were measured by a microplate reader and the differences between groups were compared. Rb1 significantly reduced resistin-induced HCASMC proliferation. Resistin increased HCASMC migration time-dependently. At 20 µM, Rb1 could significantly reduce HCASMC migration. Resistin and Act-LDL increased ROS production to a similar level in HCASMCs, while Rb1 pretreated group reversed the effects of resistin and acetyl-LDL. Besides, the mitochondrial SOD activity was significantly reduced by resistin but was restored when pretreated with Rb1. We confirmed the protection of Rb1 on HCASMC and suggested that the mechanisms involved might be related to the reduction of ROS generation and increased activity of SOD. Our study clarified the potential clinical applications of Rb1 in the control of resistin-related vascular injury and in the treatment of cardiovascular disease.

Exosomal miR-663b from "M1" macrophages promotes pulmonary artery vascular smooth muscle cell dysfunction through inhibiting the AMPK/Sirt1 axis.

Inflammatory mediators from macrophages are proven to be involved in pulmonary vascular remodeling in pulmonary hypertension (PH). Here, this study intends to explore the mechanism of "M1" macrophage-derived exosomal miR-663b in pulmonary artery smooth muscle cells (PASMCs) dysfunctions and pulmonary hypertension. Hypoxia-treated PASMCs were utilized for constructing an in-vitro pulmonary hypertension model. THP-1 cells were treated with PMA (320 nM) and LPS (10 μg/mL) + IFN-γ (20 ng/ml) for eliciting macrophage "M1" polarization. Exosomes derived from "M1" macrophages were isolated and added into PASMCs. The proliferation, inflammation, oxidative stress, and migration of PASMCs were evaluated. RT-PCR or Western blot examined the levels of miR-663b and the AMPK/Sirt1 pathway. Dual luciferase activity assay and RNA pull-down assay were carried out for confirming the targeted association between miR-663b and AMPK. An in-vivo PH model was built. Macrophage-derived exosomes with miR-663b inhibition were used for treating the rats, and alterations of pulmonary histopathology were monitored. miR-663b was obviously up-regulated in hypoxia-elicited PASMCs and M1 macrophages. miR-663b overexpression boosted hypoxia-induced proliferation, inflammation, oxidative stress, and migration in PASMCs, whereas miR-663b low expression resulted in the opposite situation. AMPK was identified as a target of miR-663b, and miR-663b overexpression curbed the AMPK/Sirt1 pathway. AMPK activation ameliorated the damaging impact of miR-663b overexpression and "M1" macrophage exosomes on PASMCs. In vivo, "M1" macrophage exosomes with miR-663b low expression alleviated pulmonary vascular remodeling in pulmonary hypertension rats. Exosomal miR-663b from "M1" macrophage facilitates PASMC dysfunctions and PH development by dampening the AMPK/Sirt1 axis.

IPSCs-based generation of vascular smooth muscle cells to study genotype-phenotype correlation in patients with ACTA2-related hereditary thoracic aortic aneurysm and dissection (HTAAD): A multicentric pilot study

Hereditary thoracic aortic aneurysm and dissection (HTAAD) is an autosomal dominant disorder characterized by dilatation or dissection of the thoracic aorta in families without Marfan- or related syndromes. ACTA2 gene, encoding alpha-smooth muscle actin, is the most frequent cause of non-syndromic HTAAD (12 to 21%). The penetrance is incomplete, with a dominant negative effect, and probably affected by the mutation location. Thus, the cumulative risk to aortic event ranges from 76% at the age of 85 in the classical form, to 100% by the age of 25 years in the highly penetrant ACTA2 variant at p.R179 which is associated with multisystem smooth muscle cell dysfunction syndrome (SMDS). SMDS is characterized by patent ductus arteriosus or aorto-pulmonary fenestration, early aortic dissection without dilatation, and cerebrovascular or coronary disease (moya-moya, stenosis). In clinical practice, this clinical heterogeneity doesn’t allow a relevant and personalized health care prevention. We proposed to develop and validate a new cellular model to explore in vitro the functional effects of ACTA2 variants (Class V) and to identify key mediator of vascular smooth muscle cells (VSMCs) dysfunction. Three patients with different ACTA2 pathogenic variants (p.G.148R, p.Thr212Lys, p.P355G) were included. Their phenotype was aortic dissection before 30-years old, and a more severe phenotype with ischemic stroke at 13-years old mimicking the SMDS for the patient with the pG.148R variant. Peripheric blood mononuclear cells (PBMCs) were collected from patient blood samples and induced pluripotent stem cells (iPSCs) were reprogrammed. Human iPSCs were differentiated toward the neural crest lineage, and subsequently to VSMCs. VSMCs were analyzed for their proliferation rate and migration capacity. Their actin polymerization was examined. To identify phenotypic difference between these three ACTA2 variants we performed RNAseq analysis. Patients VSMCs from healthy controls were used as controls. We observed difference of proliferation using FACS analysis, and of migration between these three iPSC-derived VSMCs compared to controls. Transcriptomic analysis is currently analysed and results will be presented at the congress. Our study identifies a robust protocol to obtain iPSC-derived VSMCs and provides novel insight on the genotype-phenotype correlation in patients with ACTA2 mutations.

[Latest Findings on the Pathogenic Mechanisms of Thoracic Aortic Dissection].

Thoracic aortic dissection (TAD) is a cardiovascular disease entailing a high lethality between 65% and 85%. Surgery-assissed implant/interventional stenting is the prevailing treatment of TAD. However, surgical treatment can cause severe postoperative complications and patients incur a relatively higher risk of postoperative mortality. Since the pathogenic mechanism underlying TAD is not clear, effective medication therapies are still not available. In recent years, along with advances in single-cell sequencing and other molecular biological technologies, there have been prelimiary findings suggesting the special role of dysfunctional vascular smooth muscle cells (VSMCs) in the pathogenesis and development of TAD. Furthermore, the molecular mechanisms regulating the dysfunction of VSMCs have been initially explored. It is expected that these new findings will contribute to the development of new strategies to prevent TAD and lead to new ideas for the identifiction of potential drug therapeutic targets. Herein, we summarized the critical role of dysfunctional VSMCs in the pathogenesis and development of TAD and presented in detail the biological factors and the related molecular mechanisms that regulate the dysfunction of VSMCs. We hope this review will provide a reference for further investigation into the central role of dysfunctional VSMCs in the pathogenesis and development of TAD and exploration for effective molecular drug targets for TAD.

Ox-LDL-Induced Vascular Smooth Muscle Cell Dysfunction Partly Depends on the Circ_0044073/miR-377-3p/AURKA Axis in Atherosclerosis.

Atherosclerosis (AS) is the main reason for most cardiovascular diseases. Circular RNA hsa_circ_0044073 (circ_0044073) has been found to promote AS progression. However, the specific regulatory mechanism of circ_0044073 in AS progression remains unclear.In this study, oxidized low-density lipoprotein (Ox-LDL) -stimulated human vascular smooth muscle cells (VSMCs) were used as AS cell models. The expression changes of circ_0044073 in serum samples and Ox-LDL-stimulated human VSMCs were assessed via real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability, proliferation, colony formation, migration, and invasion were assessed using 3- (4,5-Dimethylthiazol-2-yl) -2,5-Diphenyltetrazolium Bromide (MTT), 5-ethynyl-2'-deoxyuridine (EDU), colony formation, and transwell assays. Some protein levels were detected via Western blotting. The regulatory mechanism of circ_0044073 was predicted using bioinformatics analysis and validated by dual-luciferase reporter and RNA pull-down assays.We observed an overt increase in circ_0044073 expression in serum samples derived from AS patients and Ox-LDL-stimulated human VSMCs. Circ_0044073 was identified as a miR-377-3p sponge. Either circ_0044073 knockdown or miR-377-3p overexpression could impair Ox-LDL-induced human VSMC proliferation, migration, invasion, and inflammation. AURKA served as a miR-377-3p target, and circ_0044073 regulated AURKA expression by adsorbing miR-377-3p. Furthermore, AURKA overexpression partly reversed the effects of circ_0044073 inhibition on Ox-LDL-induced human VSMC proliferation, migration, invasion, and inflammation.Circ_0044073 promoted AS progression by elevating AURKA expression by functioning as a miR-377-3p sponge. Providing a proof-of-concept demonstration to support circ_0044073 might be a target for AS treatment.

MiR-216b-5p regulates proliferation and apoptosis of ox-LDL-stimulated VSMCs and HUVECs via IGF2.

Atherosclerosis (AS) is one of the principal causes of cardiovascular disorder. Reportedly, vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) play key roles in AS development, and microRNAs (miRNAs) regulate their functions. The function of miR-216b-5p in AS remains unknown. Human VSMCs and human HUVECs were treated with ox-LDL to establish the in vitro model of AS. MiR-216b-5p and IGF2 expressions in VSMCs and HUVECs were probed by qRT-PCR and western blot. The viability, cell cycle progression, and apoptosis of VSMCs and HUVECs were evaluated by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine, and flow cytometry assays, respectively. The binding sites between IGF2 3'UTR and miR-216b-5p were validated by dual-luciferase reporter assay. miR-216b-5p expression was declined in ox-LDL-induced VSMCs and HUVECs. In VSMCs, miR-216b-5p overexpression inhibited excessive proliferation and induced apoptosis. MiR-216b-5p could markedly restrain the viabiblity of VSMCs induced by ox-LDL and enhanced the viability of HUVECs. Additionally, IGF2 was confirmed as the direct target of miR-216b-5p and transfection of IGF2 overexpression plasmids rescued the effects of miR-216b-5p on VSMCs and HUVECs. miR-216b-5p alleviates the dysfunction of VSMCs and HUVECs caused by ox-LDL via repressing IGF2, and exerts protective functions to block the development of AS.