Smooth Muscle Cell Migration Research Articles

Chitosan inhibits vascular intimal hyperplasia via LINC01615/MIR-185-5p/PIK3R2 signaling pathway

The abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are the main pathological processes which are involved in the formation of new intima. In our previous study, we found that chitosan can inhibit the formation of new intima in the arteriovenous fistulas of uremic patients, and the expression of LINC01615 was significantly increased in patients after treatment with chitosan. Therefore, this study aims to further explore the effect of chitosan on the intimal hyperplasia and elucidate the potential molecular mechanism. In vitro, we found that in chitosan-treated VSMC, the levels of Il-1β, IL-6 and TNF-α decreased, and the intimal hyperplasia was inhibited along with significantly downregulated PIK3R2 and upregualted PI3K, AKT and p-AKT. Meanwhile, we observed the phenotypic transformation of hVSMCs after LINC01615 was upregulated. In addition, inflammatory factors showed the same changes in the process of up-regulating LINC01615. Moreover, only in the LINC01615 overexpression and miR-185-5p mimic experimental group, the inhibition of intimal hyperplasia was the most obvious. The interaction between LINC01615 and miR-185-5p, miR-185-5p and PIK3R2 was further confirmed by the dual luciferase assay. These results suggest that chitosan has a potential preventive effect on neointimal hyperplasia and related vascular remodeling.

The Mechanism of Triacetyl Andrographolide in Inhibiting Proliferation of Pulmonary Artery Smooth Muscle Cells

Article The Mechanism of Triacetyl Andrographolide in Inhibiting Proliferation of Pulmonary Artery Smooth Muscle Cells Zhe Wang 1,#, Yi-Xuan Zhang 2,#, Jun-Zhuo Shi 1,#, Chen-Chen Wang 1, Meng-Qi Zhang 1, Yi Yan 3, Yan-Ran Wang 1, Lu-Ling Zhao 1, Jie-Jian Kou 4, Qing-Hui Zhao 5, Xin-Mei Xie 1, Yang-Yang He 1,2, Jun-Ke Song 6,*, Guang Han 1,7,*, and Xiao-Bin Pang 1,2,* 1 School of Pharmacy, Henan University, Kaifeng 475004, China 2 Department of Anesthesiology, Huaihe Hospital of Henan University, Kaifeng 475004, China 3 Heart Center and Shanghai Institute of Pediatric Congenital Heart Disease, Shanghai Children's Medical Center, National Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai 200217, China 4 Department of Pharmacy, Huaihe Hospital of Henan University, Kaifeng 475004, China 5 Institute of Physical Culture, Huanghuai University, Zhumadian 463000, China 6 Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China 7 Henan Province Engineering Research Center of High Value Utilization to Natural Medical Resource in Yellow River Basin, Kaifeng 475004, China. * Correspondence: smilejunke@imm.ac.cn (Jun-Ke Song); hang@henu.edu.cn ( Guang Han); pxb@vip.henu.edu.cn ( Xiao-Bin Pang) Received: 17 April 2023 Accepted: 27 July 2023 Abstract: This study examines the impact of triacetyl-diacyllactone (ADA) on the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and elucidates its underlying mechanism. PASMCs derived from SD rats were cultured in vitro and randomly divided into four groups: control group, administration group, model group, and model administration group. The appropriate concentration of ADA for intervention was determined using the MTT assay. The proliferation ability of PASMCs in each group was assessed using the EdU assay. The migration ability of PASMCs in each group was evaluated using the Scratch wound healing assay and Transwell assay. Western blot analysis was performed to determine the protein expression levels of BMPR2, PCNA, and TGF-β1, as well as the phosphorylation levels of SMAD1 and SMAD2/3 in PASMCs from each group. Results show that at a concentration of 5 µmol/L, ADA did not impact the cell activity of PASMCs and instead exerted inhibitory effects on both the proliferation and migration of PASMCs induced by PDGF-BB. PDGF-BB was found to upregulate the expression levels of PCNA and TGF-β1, while downregulating the expression of BMPR2. Furthermore, PDGF-BB led to enhanced protein phosphorylation of SMAD1 and SMAD2/3. However, following ADA intervention, the expression levels of PCNA and TGF-β1 decreased, while the expression of BMPR2 increased. Additionally, protein phosphorylation of SMAD1 and SMAD2/3 decreased. Therefore, ADA can hinder the proliferation and migration of PASMCs induced by PDGF-BB, as well as suppress the upregulation of PCNA and TGF-β1 caused by PDGF-BB. Furthermore, the downregulation of BMPR2 may be associated with the inhibition of SMAD1 and SMAD2/3 signaling pathways.

Retracted: Role of miR-181b/Notch1 Axis in circ_TNPO1 Promotion of Proliferation and Migration of Atherosclerotic Vascular Smooth Muscle Cells.

[This retracts the article DOI: 10.1155/2022/4086935.].

Contents of matrix metalloproteinase type 2 (MMP-2) and complexes MMP-9/TIMP-1, MMP-2/TIMP-2, and total cholesterol in practically healthy people and patients with hypertension

Arterial hypertension (AH) is the most common age-associated disease among the working population. Polyetiology of AH requires a more thorough definition and study of its predictors aiming for understanding the role of external influences, as well as detection of genetic polymorphisms at early stages, in order to limit their implementation. The scientific reviews discuss the important role of abnormal vascular remodeling in the pathogenesis of hypertension. The main factor of such alterations is the rearrangement of extracellular matrix due to matrix metalloproteinases (MMP), the zinc-dependent enzymes with a wide substrate specificity. In addition, in more than 70% of cases, AH is combined with lipid metabolism disorders, including an increase in cholesterol levels. These mechanisms should be taken into account when assessing risk factors for development of cardiovascular accidents. The role of MMP-2 and the complexes MMP- 9/ TIMP- 1, MMP-2/TIMP-2 in pathogenesis of vascular restructuring is insufficiently described in the review articles. The article presents our results of studying the system of proteolytic enzymes and the level of total cholesterol in 110 practically healthy people and 90 persons with AH aged 18 to 74 years old. The levels of MMP-2, MMP-9/ TIMP- 1, MMP-2/TIMP-2 complexes in blood serum were studied by the sandwich variant of ELISA technique. Statistical processing of the obtained data was carried out using the analytical software IBM SPSS Statistics, v. 22.0.1. It was found that in the groups of women, regardless of the presence, or absence of pathology, the level of total cholesterol is higher if compared with male subjects. MMP-2 values were low both in the general group of practically healthy people, regardless of gender, and in the group of men with hypertension. Proteolytic activity of MMP-2 shows a broader substrate activity in women with hypertension. In the group of practically healthy men and women, we have registered lower levels of the studied MMP complexes and their tissue inhibitors. An imbalance in the proteolysis/antiproteolysis system is observed both in the group of hypertensive women with more pronounced effects upon migration, proliferation and apoptosis of smooth muscle cells, endothelial and inflammatory cells, thus determining formation of intima and arterial remodeling, as well as in the group of men with hypertension with predominance of remodeling factors that affects the rigidity of vascular wall.

CircDiaph3 influences PASMC apoptosis by regulating PI3K/AKT/mTOR pathway through IGF1R

The pathogenesis of pulmonary hypertension has not been elucidated. We investigated the role of a circular ribonucleic acid, circDiaph3, in the proliferation and migration of pulmonary artery smooth muscle cells during pulmonary hypertension. CircDiaph3 overexpression in blood samples of patients with pulmonary hypertension was analyzed by real-time quantitative polymerase chain reaction. Subsequently, a rat model of pulmonary arterial hypertension was established under hypoxic conditions. Pulmonary artery smooth muscle cells were harvested from the rat model for subsequent experiments with small interfering ribonucleic acid-mediated knockdown of circDiaph3. In cell model, we found that PI3K, AKT, mTOR and insulin-like growth factor 1 signaling pathway (IGF1R) and smooth muscle cell marker genes (α-SMA, Vcam1) were significantly downregulated. The overexpression of Igf1r in pulmonary artery smooth muscle cells rescued the downregulated smooth muscle cell genes, IGF1R signaling pathway proteins, increased smooth muscle cell proliferation, and reduced apoptosis. CircDiaph3 regulates the PI3K/AKT/mTOR signaling pathway via IGF1R to inhibit apoptosis and promote proliferation of smooth muscle cells. Additionally, adenovirus-mediated in vivo inhibition of circDiaph3 was carried out in rats with pulmonary arterial hypertension, followed by harvesting of their pulmonary artery smooth muscle cells for subsequent experiments. Excessive proliferation of smooth muscle cells in the pulmonary artery has narrowed the pulmonary artery lumen, thereby causing pulmonary hypertension, and our results suggest that circDiaph3 has important value in the treatment of pulmonary hypertension.

Dipyridamole and vascular healing following stent implantation.

Patients undergoing coronary stent implantation incur a 2% annual rate of adverse events, largely driven by in-stent restenosis (ISR) due to neointimal (NI) tissue proliferation, a process in which smooth muscle cell (SMC) biology may play a central role. Dipyridamole (DP) is an approved therapeutic agent with data supporting improved vascular patency rates. Pre-clinical data supports that DP may enact its vasculoprotective effects via adenosine receptor-A2B (ADOR-A2B). We sought to evaluate the efficacy of DP to mitigate ISR in a pre-clinical rabbit stent model. 24 New Zealand White Rabbits were divided into two cohorts-non-atherosclerosis and atherosclerosis (n = 12/cohort, 6 male and 6 female). Following stent implantation, rabbits were randomized 1:1 to control or oral dipyridamole therapy for 6 weeks followed by optical coherence tomography (OCT) and histology assessment of NI burden and stent strut healing. Compared to control, DP demonstrated a 16.6% relative reduction in NI volume (14.7 ± 0.8% vs. 12.5 ± 0.4%, p = 0.03) and a 36.2% relative increase in optimally healed stent struts (37.8 ± 2.8% vs. 54.6 ± 2.5%, p < 0.0001). Atherosclerosis demonstrated attenuated effect with no difference in NI burden (15.2 ± 1.0% vs. 16.9 ± 0.8%, p = 0.22) and only a 14.2% relative increase in strut healing (68.3 ± 4.1% vs. 78.7 ± 2.5%, p = 0.02). DP treated rabbits had a 44.6% (p = 0.045) relative reduction in NI SMC content. In vitro assessment of DP and coronary artery SMCs yielded dose-dependent reduction in SMC migration and proliferation. Selective small molecule antagonism of ADOR-A2B abrogated the effects of DP on SMC proliferation. DP modulated SMC phenotypic switching with ADOR-A2B siRNA knockdown supporting its role in the observed effects. Dipyridamole reduces NI proliferation and improves stent healing in a preclinical model of stent implantation with conventional antiplatelets. Atherosclerosis attenuates the observed effect. Clinical trials of DP as an adjunctive agent may be warranted to evaluate for clinical efficacy in stent outcomes.

Glycosylation-Engineered Platelet Membrane-Coated Interleukin 10 Nanoparticles for Targeted Inhibition of Vascular Restenosis.

The purpose of this study was to improve the immune compatibility and targeting abilities of IL10 nanoparticles coated with platelet membrane (IL10-PNPs) by glycosylation engineering in order to effectively reduce restenosis after vascular injury. In this study, we removed sialic acids and added α (1,2)-fucose and α (1,3)-fucose to platelet membrane glycoprotein, thus engineering the glycosylation of IL10-PNPs (IL10-GE-PNPs). In vitro and in vivo experiments were conducted to evaluate the targeting and regulatory effects of IL10-GE-PNPs on macrophage polarization, as well as the influence of IL10-GE-PNPs on the phenotypic transformation, proliferation, and migration of smooth muscle cells, and its potential in promoting the repair function of endothelial cells within an inflammatory environment. In order to assess the distribution of IL10-GE-PNP in different organs, in vivo imaging experiments were conducted. IL10-GE-PNPs were successfully constructed and demonstrated to effectively target and regulate macrophage polarization in both in vitro and in vivo settings. This regulation resulted in reduced proliferation and migration of smooth muscle cells and promoted the repair of endothelial cells in an inflammatory environment. Consequently, restenosis after vascular injury was reduced. Furthermore, the deposition of IL10-GE-PNPs in the liver and spleen was significantly reduced compared to IL10-PNPs. IL10-GE-PNPs emerged as a promising candidate for targeting vascular injury and exhibited potential as an innovative drug delivery system for suppressing vascular restenosis. The engineered glycosylation of IL10-PNPs improved their immune compatibility and targeting abilities, making them an excellent therapeutic option.

Matrix-Binding, N-Cadherin-Targeting Chimeric Peptide Inhibits Intimal Thickening but Not Endothelial Repair in Balloon-Injured Carotid Arteries.

Treatment of occluded vessels can involve angioplasty, stenting, and bypass grafting, which can be limited by restenosis and thrombosis. Drug-eluting stents attenuate restenosis, but the current drugs used are cytotoxic, causing smooth muscle cell (SMC) and endothelial cell (EC) death that may lead to late thrombosis. N-cadherin is a junctional protein expressed by SMCs, which promotes directional SMC migration contributing to restenosis. We propose that engaging N-cadherin with mimetic peptides can act as a cell type-selective therapeutic strategy to inhibit polarization and directional migration of SMCs without negatively impacting ECs. We designed a novel N-cadherin-targeting chimeric peptide with a histidine-alanine-valine cadherin-binding motif, combined with a fibronectin-binding motif from Staphylococcus aureus. This peptide was tested in SMC and EC culture assays of migration, viability, and apoptosis. Rat carotid arteries were balloon injured and treated with the N-cadherin peptide. Treating scratch-wounded SMCs with the N-cadherin-targeting peptide inhibited migration and reduced polarization of wound-edge cells. The peptide colocalized with fibronectin. Importantly, EC junction, permeability, or migration was not impacted by peptide treatment in vitro. We also demonstrated that the chimeric peptide persisted for 24 hours after transient delivery in the balloon-injured rat carotid artery. Treatment with the N-cadherin-targeting chimeric peptide reduced intimal thickening in balloon-injured rat carotid arteries at 1 and 2 weeks after injury. Reendothelialization of injured vessels after 2 weeks was unimpaired by peptide treatment. These studies show that an N-cadherin-binding and fibronectin-binding chimeric peptide is effective in inhibiting SMC migration in vitro and in vivo and limiting neointimal hyperplasia after balloon angioplasty without affecting EC repair. These results establish the potential of an advantageous SMC-selective strategy for antirestenosis therapy.

Gas station in blood vessels: An endothelium mimicking, self-sustainable nitric oxide fueling stent coating for prevention of thrombosis and restenosis

Stenting is the primary treatment for vascular obstruction-related cardiovascular diseases, but it inevitably causes endothelial injury which may lead to severe thrombosis and restenosis. Maintaining nitric oxide (NO, a vasoactive mediator) production and grafting endothelial glycocalyx such as heparin (Hep) onto the surface of cardiovascular stents could effectively reconstruct the damaged endothelium. However, insufficient endogenous NO donors may impede NO catalytic generation and fail to sustain cardiovascular homeostasis. Here, a dopamine-copper (DA-Cu) network-based coating armed with NO precursor L-arginine (Arg) and Hep (DA-Cu-Arg-Hep) is prepared using an organic solvent-free dipping technique to form a nanometer-thin coating onto the cardiovascular stents. The DA-Cu network adheres tightly to the surface of stents and confers excellent NO catalytic activity in the presence of endogenous NO donors. The immobilized Arg functions as a NO fuel to generate NO via endothelial nitric oxide synthase (eNOS), while Hep works as eNOS booster to increase the level of eNOS to decompose Arg into NO, ensuring a sufficient supply of NO even when endogenous donors are insufficient. The synergistic interaction between Cu and Arg is analogous to a gas station to fuel NO production to compensate for the insufficient endogenous NO donor in vivo. Consequently, it promotes the reconstruction of natural endothelium, inhibits smooth muscle cell (SMC) migration, and suppresses cascading platelet adhesion, preventing stent thrombosis and restenosis. We anticipate that our DA-Cu-Arg-Hep coating will improve the quality of life of cardiovascular patients through improved surgical follow-up, increased safety, and decreased medication, as well as revitalize the stenting industry through durable designs.

Nemopilema nomurai jellyfish venom attenuates phenotypic modulation of PDGF BB-induced vascular smooth muscle cells and κ-carrageenan-induced rat tail thrombosis

Jellyfish venoms have long been recognized as a potentially rich source of natural bioactive compounds with pharmacological potential for the creation of innovative drugs. Our previous study demonstrated that Nemopilema nomurai jellyfish venom (NnV) has a chymotrypsin-like serine protease with fibrinolytic activity in vitro. Therefore, the present study aims to investigate the potential effect of NnV on cell migration, proliferation, and differentiation of vascular smooth muscle cells (VSMC; A7r5 cells) involved in the probable mechanism pathways. We also determined its anti-thrombotic effect through κ-carrageenan-induced Sprague–Dawley (SD) rat tail thrombus model. NnV inhibits on Platelet-derived growth factor (PDGF)-BB-stimulated A7r5 cells migration and proliferation by decreasing matrix metalloproteinase 2 (MMP-2) level and phosphorylation of ERK and Akt in a dose-dependent manner, but not p38. Furthermore, NnV regulates the phenotype transition of differentiation in PDGF-BB-stimulated A7r5 cells via ɑ-SMA and calponin in a dose-dependent manner. In an in vivo study, NnV treatment demonstrated clear anti-thrombotic activity in a dose-dependent manner, which was associated with decreased thrombus formation and length in κ-carrageenan-induced SD rat tail. These findings suggested that NnV has a novel fibrinolytic enzyme that can be used to prevent and/or treat thrombosis-related cardiovascular disorders.

Hsa_circ_0031891 targets miR-579-3p to enhance HMGB1 expression and regulate PDGF-BB-induced human aortic vascular smooth muscle cell proliferation, migration, and dedifferentiation.

Atherosclerosis (AS) is an underlying cause of the majority of coronary artery disease (CAD), in which proliferation, migration, and dedifferentiation of vascular smooth muscle cells (VSMCs) exert vital roles. It has been reported that circular RNAs (circRNAs) are associated with the VSMCs function. Here, we undertook to explore the biological function and mechanism of hsa_circ_0031891 in a platelet-derived growth factor-BB (PDGF-BB)-induced AS cell model. Hsa_circ_0031891 and microRNA-579-3p (miR-579-3p) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation and migration were detected using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), wound healing, and transwell assay. Protein levels of alpha-smooth muscle actin (α-SMA), smooth muscle protein 22-α (SM22-α), Osteopontin, and High mobility group box-1 (HMGB1) were determined using western blot assay. After predicting via a variety of bioinformatics software, the binding between miR-579-3p and hsa_circ_0031891 or HMGB1 was validated using dual-luciferase reporter and RNA pull-down assays. Increased hsa_circ_0031891 and HMGB1 and reduced miR-579-3p were found in CAD patients and PDGF-BB-induced human aortic vascular smooth muscle cells (HA-VSMCs). Moreover, hsa_circ_0031891 deficiency relieved PDGF-BB-mediated HA-VSMC proliferation, migration, and dedifferentiation. Mechanically, hsa_circ_0031891 modulated HMGB1 expression via sponging miR-579-3p. Hsa_circ_0031891 boosted PDGF-BB-induced proliferation, migration, and dedifferentiation partly by regulating the miR-579-3p/HMGB1 axis, hinting at a feasible therapeutic strategy for AS.

MiR-21-3p in extracellular vesicles from vascular fibroblasts of spontaneously hypertensive rat promotes proliferation and migration of vascular smooth muscle cells

Enhanced proliferation and migration of vascular smooth muscle cells (VSMCs) contributes to vascular remodeling in hypertension. Adventitial fibroblasts (AFs)-derived extracellular vesicles (EVs) modulate vascular remodeling in spontaneously hypertensive rat (SHR). This study shows the important roles of EVs-mediated miR-21-3p transfer in VSMC proliferation and migration and underlying mechanisms in SHR. AFs and VSMCs were obtained from aorta of Wistar-Kyoto rat (WKY) and SHR. EVs were separated from AFs culture with ultracentrifugation method. MiR-21-3p content in the EVs of SHR was increased compared with those of WKY. MiR-21-3p mimic promoted VSMC proliferation and migration of WKY and SHR, while miR-21-3p inhibitor attenuated proliferation and migration only in the VSMCs of SHR. EVs of SHR stimulated VSMC proliferation and migration, which were attenuated by miR-21-3p inhibitor. Sorbin and SH3 domain containing 2 (SORBS2) mRNA and protein levels were reduced in the VSMCs of SHR. MiR-21-3p mimic inhibited, while miR-21-3p inhibitor promoted SORBS2 expressions in the VSMCs of both WKY and SHR. EVs of SHR reduced SORBS2 expression, which was prevented by miR-21-3p inhibitor. EVs of WKY had no significant effect on SORBS2 expressions. SORBS2 overexpression attenuated the roles of miR-21-3p mimic and EVs of SHR in promoting VSMC proliferation and migration of SHR. Overexpression of miR-21-3p in vivo promotes vascular remodeling and hypertension. These results indicate that miR-21-3p in the EVs of SHR promotes VSMC proliferation and migration via negatively regulating SORBS2 expression.

Bilayer vascular grafts with on-demand NO and H2S release capabilities

Nitric oxide (NO) and hydrogen sulfide (H2S) gasotransmitters exhibit potential therapeutic effects in the cardiovascular system. Herein, biomimicking multilayer structures of biological blood vessels, bilayer small-diameter vascular grafts (SDVGs) with on-demand NO and H2S release capabilities, were designed and fabricated. The keratin-based H2S donor (KTC) with good biocompatibility and high stability was first synthesized and then electrospun with poly (l-lactide-co-caprolactone) (PLCL) to be used as the outer layer of grafts. The electrospun poly (ε-caprolactone) (PCL) mats were aminolyzed and further chelated with copper (II) ions to construct glutathione peroxidase (GPx)-like structural surfaces for the catalytic generation of NO, which acted as the inner layer of grafts. The on-demand release of NO and H2S selectively and synergistically promoted the proliferation and migration of human umbilical vein endothelial cells (HUVECs) while inhibiting the proliferation and migration of human umbilical artery smooth muscle cells (HUASMCs). Dual releases of NO and H2S gasotransmitters could enhance their respective production, resulting in enhanced promotion of HUVECs and inhibition of HUASMCs owing to their combined actions. In addition, the bilayer grafts were conducive to forming endothelial cell layers under flow shear stress. In rat abdominal aorta replacement models, the grafts remained patency for 6 months. These grafts were capable of facilitating rapid endothelialization and alleviating neointimal hyperplasia without obvious injury, inflammation, or thrombosis. More importantly, the grafts were expected to avoid calcification with the degradation of the grafts. Taken together, these bilayer grafts will be greatly promising candidates for SDVGs with rapid endothelialization and anti-calcification properties.

Current Biomarkers for Carotid Artery Stenosis: A Comprehensive Review of the Literature.

Carotid artery stenosis (CAS), an atherosclerotic disease of the carotid artery, is one of the leading causes of transient ischemic attacks (TIA) and cerebrovascular attacks (CVA). The atherogenic process of CAS affects a wide range of physiological processes, such as inflammation, endothelial cell function, smooth muscle cell migration and many more. The current gold-standard test for CAS is Doppler ultrasound; however, there is yet to be determined a strong, clinically validated biomarker in the blood that can diagnose patients with CAS and/or predict adverse outcomes in such patients. In this comprehensive literature review, we evaluated all of the current research on plasma and serum proteins that are current contenders for biomarkers for CAS. In this literature review, 36 proteins found as potential biomarkers for CAS were categorized in to the following nine categories based on protein function: (1) Inflammation and Immunity, (2) Lipid Metabolism, (3) Haemostasis, (4) Cardiovascular Markers, (5) Markers of Kidney Function, (6) Bone Health, (7) Cellular Structure, (8) Growth Factors, and (9) Hormones. This literature review is the most up-to-date and current comprehensive review of research on biomarkers of CAS, and the only review that demonstrated the several pathways that contribute to the initiation and progression of the disease. With this review, future studies can determine if any new markers, or a panel of the proteins explored in this study, may be contenders as diagnostic or prognostic markers for CAS.

Downregulation of PDCD4 through STAT3/ATF6/autophagy mediates MIF-induced PASMCs proliferation/migration and vascular remodeling

To address the molecular mechanisms underlying macrophage migration inhibitory factor (MIF) induced pulmonary artery smooth muscle cells (PASMCs) proliferation, migration and vascular remodeling in pulmonary hypertension (PH), primary cultured rat PASMCs and monocrotaline (MCT)-induced rats with PH were applied in the present study. The results showed that MIF increased signal transducer and activator of transcription 3 (STAT3) phosphorylation, and then stimulated activating transcription factor 6 (ATF6) activation, subsequently triggered autophagy activation, which further led to programmed cell death factor 4 (PDCD4) lysosomal degradation, and eventually promoted PASMCs proliferation/migration. In lung tissues of MCT rats, MIF protein expression was elevated, phosphorylation of STAT3 and activation of ATF6 were increased, activation of autophagy was evident, and reduction of PDCD4 was observed. Intervention with MIF inhibitor 4-Iodo-6-phenylpyrimidine (4-IPP), ATF6 blocker melatonin or autophagy inhibitor chloroquine, confirmed the in vitro interaction among MIF, STAT3, ATF6, autophagy and PDCD4 in MCT induced rats with PH. Targeting MIF/STAT3/ATF6/autophagy/PDCD4 axis effectively prevented the development of PH by suppressing PASMCs proliferation and vascular remodeling. In conclusions, we demonstrate that MIF activates the STAT3/ATF6/autophagy cascade and then degrades PDCD4 leading to PASMCs proliferation/migration and pulmonary vascular remodeling, suggesting that intervention this axis might have potential value in management of PH.

Abstract P3043: Mir92b-3p: A Key Regulator In Vascular Homeostasis And Remodelling

In vascular disease, cells undergo important alterations in cellular function, influencing vascular homeostasis. Apoptosis is one of these functions, affecting vascular tone and plaque stability. Influencing this process for example, by targeting microRNAs is key for therapeutic strategies tackling vascular diseases in a cell-specific manner. We identified the critical role of miR92b-3p on the functional properties of vascular cells. Studying human primary coronary artery smooth muscle cells (SMCs) and human umbilical vein endothelial cells (ECs), methods included expression analysis in mice tissue after restenosis by wire-induced injury and in atherosclerosis. The role of miR-92b on cellular function and target regulation was determined by pre- and antagomir transfection. Initial analysis in vivo showed miR92b-3p to be significantly upregulated in atherosclerosis (p&lt;0,01) and regulated in restenosis (p&lt;0,05). Under growth conditions in both ECs and SMCs, miR-92b-3p was significantly higher expressed (p&lt;0,05). In SMCs, upregulation of miR92b-3p resulted in increased proliferation (p&lt;0,01) and significantly enhanced cell migration in SMCs (p&lt;0,001) but hampered migration in ECs (p&lt;0,01). Downregulation of miR-92b-3p in SMC resulted in decreased migrational (p&lt;0.001) and proliferative capacity (p&lt;0.001), accompanied by a reduction in metabolic activity (p&lt;0.05) and, exclusively in SMCs, increased apoptosis (p&lt;0.05). In ECs, proliferation was enhanced following downregulation (p&lt;0,01). Multiple targets were identified, using Western blot and qPCR including KLF4 (p&lt;0,01), DKK3 (p&lt;0,05), Notch1 (p&lt;0,01), PTEN (p&lt;0,05), and especially in SMC, Bim (p&lt;0,001). Regulation of Bim by miR92b-3p and subsequent apoptosis was then evaluated by siRNA-knockdown experiments, confirming their strong interaction. We further verified the crucial role of miR-92b-3p by reversing cell death in apoptotic SMCs through miR-92b-3p overexpression experiments (p&lt;0.05). This study reports miR 92b-3p to be a crucial regulator of vascular function. Therapeutic regulation might present a promising strategy for stabilizing critical plaques, restoring the function of vascular cells, and thereby preventing vascular remodeling.

Abstract P3045: MicroRNA-31-5p Is Upregulated During Neointima Formation And Promotes Smooth Muscle Cell Proliferation And Migration By Regulating Krüppel-like Factor 4 (KLF4)

In the context of CVDs, microRNAs (miRs) have emerged as potential therapeutic targets for preventing adverse vascular remodeling and restenosis, given their ability to modulate multiple signaling pathways and cellular processes. Here, we propose that targeting miR-31-5p might be an effective approach to enhance vascular healing and regeneration in a cell-specific manner, thereby limiting angioplasty-related complications. A screening of murine neointimal tissue for differentially regulated miRs revealed robustly increased expression levels of miR-31-5p over time. Subsequently, expression levels of miR-31-5p were confirmed by qRT-PCR in human coronary artery endothelial cells (EC) and -smooth muscle cells (SMC). Further, the effect of miR-31-5p on cellular function was investigated and possible targets were identified and confirmed on mRNA and protein level. Expression analysis revealed a significant upregulation of miR-31-5p (p&lt;0.0001) in C57BL/6J mice following wire-induced injury of the femoral artery at 7 and 21 days. In vitro, miR-31-5p was significantly upregulated in SMC (p&lt;0,05 ) following serum stimulation, whereas only a slightly enhanced expression was observed in EC. On a functional level, miR-31-5p revealed dissenting effects on EC and SMC: Migration and proliferation were not altered in EC (p&gt;0.05). In contrast, knockdown of miR-31-5p significantly reduced the migration (p&lt;0,05) and proliferation in SMC (p&lt;0,01) without detectable apoptotic effects. Systematic in silico target screens suggested KLF4 as potential target. Knock down of miR-31-5p resulted in a significant upregulation of KLF4 in SMC (p&lt;0,01), whereas overexpression resulted in a significant downregulation of KLF4 in SMC (p&lt;0,05) on mRNA- and protein level. Here, we report that miR-31-5p is robustly upregulated during neointima formation and seems to exert a functional role in SMC rather than EC since knockdown of miR-31-5p was able to prevent SMC proliferation and migration. Mechanistically, KLF4 identified as a direct target of miR-31-5p can mediate the observed effects. Thus, miR-31-5p might represent an attractive target to selectively modulate SMC dysfunction following vascular intervention, limiting neointima formation in treated vessels.

Abstract P3020: O-GlcNAc Transferase Regulates Vascular Smooth Muscle Cell Cycle Progression In Diabetes

Diabetic patients show increased proclivity for vascular smooth muscle cell (VSMC) migration and proliferation. Hyperglycemia, hallmark of diabetes, increases intracellular glucose signaling via O-linked N-acetylglucosamine transferase (OGT), a key regulator of protein O-GlcNAcylation. We previously showed that smooth muscle OGT (smOGT) deletion prevents hyperglycemia-induced atherosclerosis. Goal of this work was to interrogate the role of smOGT in VSMC cell cycle progression in diabetes. For this, we used conditional SMC-specific OGT knockout (smOGT KO ) and wild-type (smOGT WT ) mice generated via tamoxifen (tmx)-inducible VSMC-restricted Cre driver mice ( Itga8-CreER T2 ). Briefly, following tmx injection, 8-wks-old smOGT KO and smOGT WT mice were placed on Western diet feeding regimen for study duration. At 14-wks-age, mice were injected with 50mg/Kg/day streptozotocin i.p. once daily for 5 consecutive days. Mice were harvested at 16-18-wks-age and plasma and aortic tissues were collected for biochemical and molecular studies. Immunoblotting of aortic lysates revealed a significant decrease (59%) in OGT and O-GlcNAc expression, validating the smOGT KO mice. Loss of smOGT reduced (40%) CDK6 expression (regulator of G1-S phase cell cycle progression) concomitant to enhanced (62%) p21 Cip1 expression (cyclin-dependent kinase inhibitor) in aortic vessels from diabetic smOGT KO vs. smOGT WT mice. This was further accompanied with 69% attenuation in SRF expression (transcriptional regulator of SM proliferation) in diabetic smOGT KO vs. smOGT WT aortic vessels. Similar results were obtained with OGT loss-of-function studies in human coronary artery SMC (HCASMC) primary cultures under diabetic conditions in vitro . Briefly, control (siCtrl) and OGT siRNA (siOGT)-transfected HCASMC were treated with or without 25mM glucose+100μM PUGNAc for 48 hours. Immunoblotting revealed that reduced OGT-mediated O-GlcNAcylation in siOGT cells in a diabetic milieu significantly attenuated SRF expression coupled with augmented p21 Cip1 expression compared to diabetic siCtrl cells. Together, our results suggest a direct regulatory role of smooth muscle OGT in VSMC cell cycle progression in diabetes.

Abstract P3033: Thrombospondin-1 Promotes Vascular Smooth Muscle Cell De-differentiation In Diabetes

Diabetes is a major risk factor for atherosclerosis. Diabetic patients are prone to increased vascular smooth muscle cell (VSMC) migration and proliferation, hallmarks of VSMC de-differentiation, critical for the evolution of atherosclerosis. Multiple studies, including our earlier work, support the role of thrombospondin-1 (TSP1), a proatherogenic matricellular protein, as a putative link between diabetes and vascular disease. However, molecular mechanism(s) by which TSP1 promotes diabetic vasculopathy are incompletely understood. The goal of this work was to explore whether smooth muscle TSP1 (smTSP1) regulates VSMC phenotypic switch in diabetes. We generated conditional SMC-specific TSP1 knockout (smTSP1 KO ) mice by crossing female TSP1 fl/fl mice with tamoxifen (tmx)-inducible Myh11-CreER T2 male mice. For Cre recombination, 5-wks-old male TSP1 fl/fl Cre tg and age-matched TSP1 +/+ Cre tg littermate mice were treated with 60 mg/Kg/day tmx i.p. once daily for 5 consecutive days. To induce type 2 diabetes, mice cohorts were placed on western diet at 7-wks-age followed by streptozotocin (50 mg/Kg/day, i.p. x 5 days) at 16-wks-age. Mice were harvested at 20-wks-age for plasma and tissue collection. Immunoblotting of aortic lysates revealed significant decrease in TSP1 expression in tmx-treated TSP1 fl/fl Cre tg , validating smTSP1 KO mice. Lack of smTSP1 increased ACTA2 (SM contractile marker) expression by 2-fold accompanied with 50% reduction in CDK4 (cell cycle regulator) expression in diabetic smTSP1 KO vs. smTSP1 WT aortic vessels. Consistently, in murine aortic SMC (MASMC) primary cultures derived from smTSP1 KO mice, loss of smTSP1 augmented (&gt;65%) LMOD1 (SM contractile marker) and SRF (transcriptional activator of SM contractile genes) expression coupled with diminished (45%) vimentin (SM synthetic marker) expression in 25mM glucose-treated smTSP1 KO vs. smTSP1 WT cells. Further, propidium iodide staining via flow cytometry revealed reduced S-phase cell counts concomitant to increased G 0 -G 1 cell distribution in glucose-treated smTSP1 KO vs. smTSP1 WT MASMC. Together, our data demonstrates a direct role of TSP1 in VSMC phenotypic transition and opens novel avenues for cell-specific TSP1-based therapies for treatment of diabetes.

Synthesis of 2′,4′,6′-trimethoxy-3′,5′-dimethylchalcone derivatives and their anti-diabetic and anti-atherosclerosis effects

AMP-activated protein kinase (AMPK) is an important regulator of energy metabolism and is considered a promising target for the treatment of metabolic diseases including diabetes. However, no AMPK agonist is used clinically at present. Here, we synthesized various derivatives of 2′,4′,6′-trimethoxy-3′,5′-dimethylchalcone and tested whether they activated AMPK and improved glucose tolerance in an obese mouse model. The derivatives activated AMPK at a much lower concentration than 5-aminoimidazole-4-carboxamide ribonucleotide, a well-known AMPK agonist, in C2C12 myotubes. Among them, compounds 2c and 2i showed predominant AMPK activation effects and increased the fatty acid oxidation (FAO) rate more than 2-fold in C2C12 myotubes. When compound 2i was administered to high fat diet-induced obese mice for two weeks, the glucose tolerance was improved and skeletal muscle FAO rate increased (1.6-fold). In addition, several derivatives, including compound 2i, suppressed the migration of vascular smooth muscle cells treated with platelet-derived growth factor, and inhibited tumor necrosis factor-α-induced adhesion between monocytes and endothelial cells. These results suggest these derivatives have anti-atherosclerosis effects. Taken together, the derivatives, especially compound 2i, might be therapeutic agents against diabetes and atherosclerosis.