MiR-411-5p Promotes Vascular Smooth Muscle Cell Phenotype Switch by Inhibiting Expression of Calmodulin Regulated Spectrin-Associated Protein-1.

When stimulated, vascular smooth muscle cells (VSMCs) change from a differentiated to a dedifferentiated phenotype. Dedifferentiated VSMCs have a key activity in cardiovascular diseases such as in-stent restenosis. MicroRNAs (miRNAs) have crucial functions in conversion of differentiated VSMCs to a dedifferentiated phenotype. We investigated the activity of miR-411-5p in the proliferation, migration, and phenotype switch of rat VSMCs.Based on a microRNA array assay, miR-411-5p expression was found to be significantly increased in cultured VSMCs stimulated by platelet-derived growth factor-BB (PDGF-BB). A CCK-8 assay, transwell assay, and scratch test were performed to measure the effect of miR-411-5p on the proliferation and migration of PDGF-BB-treated VSMCs. MiR-411-5p promoted expression of dedifferentiated phenotype markers such as osteopontin and tropomyosin 4 in PDGF-BB-treated VSMCs. Using mimics and inhibitors, we identified the target of miR-411-5p in PDGF-BB-treated VSMCs and found that calmodulin-regulated spectrin-associated protein-1 (CAMSAP1) was involved in the phenotypic switch mediated by PDGF-BB.By inhibiting expression of CAMSAP1, miR-411-5p enhanced the proliferation, migration, and phenotype switch of VSMCs.Blockade of miR-411-5p interaction with CAMSAP1 is a promising approach to treat in-stent restenosis.

IL1ß-NFκß-Myocardin signaling axis governs trophoblast-directed plasticity of vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) plasticity is fundamental in uterine spiral artery remodeling during placentation in Eutherian mammals. Our previous work showed that the invasion of trophoblast cells into uterine myometrium coincides with a phenotypic change of VSMCs. Here, we elucidate the mechanism by which trophoblast cells confer VSMC plasticity. Analysis of genetic markers on E13.5, E16.5, and E19.5 in the rat metrial gland, the entry point of uterine arteries, revealed that trophoblast invasion is associated with downregulation of MYOCARDIN, α-smooth muscle actin, and calponin1, and concomitant upregulation of Smemb in VSMCs. Myocardin overexpression or knockdown in VSMCs led to upregulation or downregulation of contractile markers, respectively. Co-culture of trophoblast cells with VSMCs decreased MYOCARDIN expression along with compromised expression of contractile markers in VSMCs. However, co-culture of trophoblast cells with VSMCs overexpressing MYOCARDIN inhibited their change in phenotype, whereas, overexpression of transactivation domain deleted MYOCARDIN failed to elicit this response. Furthermore, the co-culture of trophoblast cells with VSMCs led to the activation of NFκß signaling. Interestingly, despite producing IL-1ß, trophoblast cells possess only the decoy receptor, whereas, VSMCs possess the IL-1ß signaling receptor. Treatment of VSMCs with exogenous IL-1ß led to a decrease in MYOCARDIN and an increase in phosphorylation of NFκß. The effect of trophoblast cells in the downregulation of MYOCARDIN in VSMCs was reversed by blocking NFκß translocation to the nucleus. Together, these data highlight that trophoblast cells direct VSMC plasticity, and trophoblast-derived IL-1ß is a key player in downregulating MYOCARDIN via the NFκß signaling pathway.

Single-Cell Transcriptome Analysis Reveals Dynamic Populations of Vascular Cells in Neointimal Hyperplasia.

BACKGROUND: Neointimal hyperplasia (NIH) is the pathological basis of vascular injury disease. Vascular cells are the dominant cells in the process of NIH, but the extent of heterogeneity amongst them is still unclear. METHODS: A mouse model of NIH was constructed by inducing carotid artery ligation. Single-cell sequencing was then used to analyze the transcriptional profile of vascular cells. Cluster features were determined by functional enrichment analysis, gene set scoring, pseudo-time analysis, and cell-cell communication analysis. Additionally, immunofluorescence staining was conducted on vascular tissues from fibroblast lineage-traced (PdgfraDreER-tdTomato) mice to validate the presence of Pecam1+Pdgfra+tdTomato+ cells. RESULTS: The left carotid arteries (ligation) were compared to right carotid arteries (sham) from ligation-induced NIH C57BL/6 mice. Integrative analyses revealed a high level of heterogeneity amongst vascular cells, including fourteen clusters and seven cell types. We focused on three dominant cell types: endothelial cells (ECs), vascular smooth muscle cells (vSMCs), and fibroblasts. The major findings were: (1) four subpopulations of ECs, including ECs4, mesenchymal-like ECs (ECs1 and ECs2), and fibro-like ECs (ECs3); (2) four subpopulations of fibroblasts, including pro-inflammatory Fibs-1, Sca1+ Fibs-2, collagen-producing Fibs-3, and mesenchymal-like Fibs-4; (3) four subpopulations of vSMCs, including vSMCs-1, vSMCs-2, vSMCs-3, and vSMCs-3-derived vSMCs; (4) ECs3 express genes related to extracellular matrix (ECM) remodeling and cell migration, and fibro-like vSMCs showed strong chemokine secretion and relatively high levels of proteases; (5) fibro-like vSMCs that secrete Vegfa interact with ECs mainly through vascular endothelial growth factor receptor 2 (Vegfr2). CONCLUSIONS: This study presents the dynamic cellular landscape within NIH arteries and reveals potential relationships between several clusters, with a specific focus on ECs3 and fibro-like vSMCs. These two subpopulations may represent potential target cells for the treatment of NIH.

Nanoparticles targeting OPN loaded with BY1 inhibits vascular restenosis by inducing FTH1-dependent ferroptosis in vascular smooth muscle cells.

Vascular restenosis following angioplasty continues to pose a significant challenge. The heterocyclic trioxirane compound [1, 3, 5-tris((oxiran-2-yl)methyl)-1, 3, 5-triazinane-2, 4, 6-trione (TGIC)], known for its anticancer activity, was utilized as the parent ring to conjugate with a non-steroidal anti-inflammatory drug, resulting in the creation of the spliced conjugated compound BY1. We found that BY1 induced ferroptosis in VSMCs as well as in neointima hyperplasia. Furthermore, ferroptosis inducers amplified BY1-induced cell death, while inhibitors mitigated it, indicating the contribution of ferroptosis to BY1-induced cell death. Additionally, we established that ferritin heavy chain1 (FTH1) played a pivotal role in BY1-induced ferroptosis, as evidenced by the fact that FTH1 overexpression abrogated BY1-induced ferroptosis, while FTH1 knockdown exacerbated it. Further study found that BY1 induced ferroptosis by enhancing the NCOA4-FTH1 interaction and increasing the amount of intracellular ferrous. We compared the effectiveness of various administration routes for BY1, including BY1-coated balloons, hydrogel-based BY1 delivery, and nanoparticles targeting OPN loaded with BY1 (TOP@MPDA@BY1) for targeting proliferated VSMCs, for prevention and treatment of the restenosis. Our results indicated that TOP@MPDA@BY1 was the most effective among the three administration routes, positioning BY1 as a highly promising candidate for the development of drug-eluting stents or treatments for restenosis.

IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells.

Introduction: IL-2Rα knock out (KO) mice have been instrumental to discovering the immunoregulatory properties of IL-2Rα. While initially thought of only as a stimulatory cytokine, IL-2 and IL-2Rα KO mice revealed that this cytokine-receptor system controls immune responses through restimulation-induced cell death and by promoting the survival of T regulatory cells. Although described mostly in the context of lymphocytes, recent studies by our laboratory showed that IL-2R is expressed in smooth muscle cells. Given this finding, we sought to use IL-2Rα KO to determine the function of this receptor in vascular smooth muscle cells. Surprisingly, we found that IL-2Rα KO vascular smooth muscle cells had detectable IL-2Rα. Methods: We used multiple gene and protein-based methods to determine why IL-2Rα KO vascular smooth muscle cells exhibited IL-2Rα protein. These methods included: genomic sequencing, assessing cells and tissues for evidence of maternal microchimerism, and determining the half-life of IL-2Rα protein. Results: Our studies demonstrated the following: (1) in addition to the cell surface, IL-2Rα is localized to the nucleus; (2) the genetic deletion of IL-2Rα is intact in IL-2Rα KO mice; (3) both IL-2Rα KO and WT tissues show evidence of maternal microchimerism, the likely source of IL-2Rα (4) IL-2Rα is transmitted between cells; (5) IL-2Rα has a long half-life; and (6) nuclear IL-2Rα contributes to the regulation of cell proliferation and size. Conclusion: Our findings suggest that the phenotype of complete IL-2Rα loss is more severe than demonstrated by IL-2Rα KO mice, and that IL-2Rα plays a here-to-fore unrecognized role in regulating cell proliferation in non-lymphoid cells.

Deciphering smooth muscle cell heterogeneity in atherosclerotic plaques and constructing model: a multi-omics approach with focus on KLF15/IGFBP4 axis.

BACKGROUND: Ruptured atherosclerotic plaques often precipitate severe ischemic events, such as stroke and myocardial infarction. Unraveling the intricate molecular mechanisms governing vascular smooth muscle cell (VSMC) behavior in plaque stabilization remains a formidable challenge. METHODS: In this study, we leveraged single-cell and transcriptomic datasets from atherosclerotic plaques retrieved from the gene expression omnibus (GEO) database. Employing a combination of single-cell population differential analysis, weighted gene co-expression network analysis (WGCNA), and transcriptome differential analysis techniques, we identified specific genes steering the transformation of VSMCs in atherosclerotic plaques. Diagnostic models were developed and validated through gene intersection, utilizing the least absolute shrinkage and selection operator (LASSO) and random forest (RF) methods. Nomograms for plaque assessment were constructed. Tissue localization and expression validation were performed on specimens from animal models, utilizing immunofluorescence co-localization, western blot, and reverse-transcription quantitative-polymerase chain reaction (RT-qPCR). Various online databases were harnessed to predict transcription factors (TFs) and their interacting compounds, with determination of the cell-specific localization of TF expression using single-cell data. RESULTS: Following rigorous quality control procedures, we obtained a total of 40,953 cells, with 6,261 representing VSMCs. The VSMC population was subsequently clustered into 5 distinct subpopulations. Analyzing inter-subpopulation cellular communication, we focused on the SMC2 and SMC5 subpopulations. Single-cell subpopulation and WGCNA analyses revealed significant module enrichments, notably in collagen-containing extracellular matrix and cell-substrate junctions. Insulin-like growth factor binding protein 4 (IGFBP4), apolipoprotein E (APOE), and cathepsin C (CTSC) were identified as potential diagnostic markers for early and advanced plaques. Notably, gene expression pattern analysis suggested that IGFBP4 might serve as a protective gene, a hypothesis validated through tissue localization and expression analysis. Finally, we predicted TFs capable of binding to IGFBP4, with Krüppel-like family 15 (KLF15) emerging as a prominent candidate showing relative specificity within smooth muscle cells. Predictions about compounds associated with affecting KLF15 expression were also made. CONCLUSION: Our study established a plaque diagnostic and assessment model and analyzed the molecular interaction mechanisms of smooth muscle cells within plaques. Further analysis revealed that the transcription factor KLF15 may regulate the biological behaviors of smooth muscle cells through the KLF15/IGFBP4 axis, thereby influencing the stability of advanced plaques via modulation of the PI3K-AKT signaling pathway. This could potentially serve as a target for plaque stability assessment and therapy, thus driving advancements in the management and treatment of atherosclerotic plaques.

In Vitro Experimental Approach for Studying Human Pulmonary Artery Smooth Muscle Cells and Endothelial Cells Proliferation and Migration.

Pulmonary arterial hypertension (PAH) is a severe vascular disease characterized by persistent precapillary pulmonary hypertension, leading to right heart failure and death. Despite intense research in the last decades, PAH remains an incurable disease with high morbidity and mortality. New directions and therapies to improve understanding and treatment of PAH are desperately needed. The pathological mechanisms leading to this fatal disorder remain mostly undetermined, although structural remodeling of the pulmonary vessels is known to be an early feature of PAH. Pulmonary vascular remodeling includes proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs). The use of in vitro approaches is useful to delineate the mechanisms involved in the pathogenesis of PAH and to identify new therapeutic strategies for PAH. In this chapter, we describe protocols for culturing and assessing proliferation and migration of human PASMCs and PAECs.

Mir-195-5p targets Smad7 regulation of the Wnt/ß-catenin pathway to promote osteogenic differentiation of vascular smooth muscle cells.

In this study, we sought to investigate the mechanisms of action of miR-195-5p in the osteogenic differentiation of vascular smooth muscle cells (VSMCs), and thereby provide novel insights and a reference for the targeted therapy of arterial media calcification. VSMC differentiation was induced using sodium ß-glycerophosphate, and we investigated the effects of transfecting cells with miR-195-5p mimics, vectors overexpressing Smad7, and the Wnt/ß-catenin pathway inhibitor (KYA1797K) on VSMC differentiation by determining cell viability and apoptosis, and the mRNA and protein expression of factors associated with osteogenic differentiation and the Wnt/ß-catenin pathway. The results revealed that miR-195-5p mimics enhanced the osteogenic differentiation of VSMCs induced by ß-glycerophosphate, whereas the overexpression of Smad7 reversed this phenomenon. In addition, KYA1797K was found to promote the effects of Smad7 overexpression. In conclusion, by targeting, Smad7, miR-195-5p promotes the Wnt/ß-catenin pathway. and thus the osteogenic differentiation of VSMCs. These findings will provide a reference for elucidating the mechanisms whereby miR-195-5p regulates osteogenic differentiation.

Activation of heme oxygenase-1 by laminar shear stress ameliorates high glucose-induced endothelial cell and smooth muscle cell dysfunction.

High glucose (HG)-induced endothelial cell (EC) and smooth muscle cell (SMC) dysfunction is critical in diabetes-associated atherosclerosis. However, the roles of heme oxygenase-1 (HO-1), a stress-response protein, in hemodynamic force-generated shear stress and HG-induced metabolic stress remain unclear. This investigation examined the cellular effects and mechanisms of HO-1 under physiologically high shear stress (HSS) in HG-treated ECs and adjacent SMCs. We found that exposure of human aortic ECs to HSS significantly increased HO-1 expression; however, this upregulation appeared to be independent of adenosine monophosphate-activated protein kinase, a regulator of HO-1. Furthermore, HSS inhibited the expression of HG-induced intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and reactive oxygen species (ROS) production in ECs. In an EC/SMC co-culture, compared with static conditions, subjecting ECs close to SMCs to HSS and HG significantly suppressed SMC proliferation while increasing the expression of physiological contractile phenotype markers, such as α-smooth muscle actin and serum response factor. Moreover, HSS and HG decreased the expression of vimentin, an atherogenic synthetic phenotypic marker, in SMCs. Transfecting ECs with HO-1-specific small interfering (si)RNA reversed HSS inhibition on HG-induced inflammation and ROS production in ECs. Similarly, reversed HSS inhibition on HG-induced proliferation and synthetic phenotype formation were observed in co-cultured SMCs. Our findings provide insights into the mechanisms underlying EC-SMC interplay during HG-induced metabolic stress. Strategies to promote HSS in the vessel wall, such as continuous exercise, or the development of HO-1 analogs and mimics of the HSS effect, could provide an effective approach for preventing and treating diabetes-related atherosclerotic vascular complications.

An integrated multi-omics analysis reveals osteokines involved in global regulation.

Bone secretory proteins, termed osteokines, regulate bone metabolism and whole-body homeostasis. However, fundamental questions as to what the bona fide osteokines and their cellular sources are and how they are regulated remain unclear. In this study, we analyzed bone and extraskeletal tissues, osteoblast (OB) conditioned media, bone marrow supernatant (BMS), and serum, for basal osteokines and those responsive to aging and mechanical loading/unloading. We identified 375 candidate osteokines and their changes in response to aging and mechanical dynamics by integrating data from RNA-seq, scRNA-seq, and proteomic approaches. Furthermore, we analyzed their cellular sources in the bone and inter-organ communication facilitated by them (bone-brain, liver, and aorta). Notably, we discovered that senescent OBs secrete fatty-acid-binding protein 3 to propagate senescence toward vascular smooth muscle cells (VSMCs). Taken together, we identified previously unknown candidate osteokines and established a dynamic regulatory network among them, thus providing valuable resources to further investigate their systemic roles.

Nardilysin in vascular smooth muscle cells controls blood pressure via the regulation of calcium dynamics.

Blood pressure is a crucial physiological parameter and its abnormalities can cause a variety of health problems. We have previously reported that mice with systemic deletion of nardilysin (NRDC), an M16 family metalloprotease, exhibit hypotension. In this study, we aimed to clarify the role of NRDC in vascular smooth muscle cell (VSMC) by generating VSMC-specific Nrdc knockout (VSMC-KO) mice. Our findings reveal that VSMC-KO mice also exhibit hypotension. Aortas isolated from VSMC-KO mice exhibited a weakened contractile response to phenylephrine, accompanied by reduced phosphorylation of myosin light chain 2 and decreased rhoA expression. VSMC isolated from VSMC-KO aortas showed a reduced increase in intracellular Ca2+ concentration induced by α-stimulants. These findings suggest that NRDC in VSMC regulates vascular contraction and blood pressure by modulating Ca2+ dynamics.

Vascular smooth muscle cell-derived nerve growth factor regulates sympathetic collateral branching to innervate blood vessels in embryonic skin.

Blood vessels serve as intermediate conduits for the extension of sympathetic axons towards target tissues, while also acting as crucial targets for their homeostatic processes encompassing the regulation of temperature, blood pressure, and oxygen availability. How sympathetic axons innervate not only blood vessels but also a wide array of target tissues is not clear. Here we show that in embryonic skin, after the establishment of co-branching between sensory nerves and blood vessels, sympathetic axons invade the skin alongside these sensory nerves and extend their branches towards these blood vessels covered by vascular smooth muscle cells (VSMCs). Our mosaic labeling technique for sympathetic axons shows that collateral branching predominantly mediates the innervation of VSMC-covered blood vessels by sympathetic axons. The expression of nerve growth factor (NGF), previously known to induce collateral axon branching in culture, can be detected in the vascular smooth muscle cell (VSMC)-covered blood vessels, as well as sensory nerves. Indeed, VSMC-specific Ngf knockout leads to a significant decrease of collateral branching of sympathetic axons innervating VSMC-covered blood vessels. These data suggest that VSMC-derived NGF serves as an inductive signal for collateral branching of sympathetic axons innervating blood vessels in the embryonic skin.

The Role of Fatty Acid Synthase in the Vascular Smooth Muscle Cell to Foam Cell Transition.

Vascular smooth muscle cells (VSMCs), in their contractile and differentiated state, are fundamental for maintaining vascular function. Upon exposure to cholesterol (CHO), VSMCs undergo dedifferentiation, adopting characteristics of foam cells-lipid-laden, macrophage-like cells pivotal in atherosclerotic plaque formation. CHO uptake by VSMCs leads to two primary pathways: ABCA1-mediated efflux or storage in lipid droplets as cholesterol esters (CEs). CE formation, involving the condensation of free CHO and fatty acids, is catalyzed by sterol O-acyltransferase 1 (SOAT1). The necessary fatty acids are synthesized by the lipogenic enzyme fatty acid synthase (FASN), which we found to be upregulated in atherosclerotic human coronary arteries. This observation led us to hypothesize that FASN-mediated fatty acid biosynthesis is crucial in the transformation of VSMCs into foam cells. Our study reveals that CHO treatment upregulates FASN in human aortic SMCs, concurrent with increased expression of CD68 and upregulation of KLF4, markers associated with the foam cell transition. Crucially, downregulation of FASN inhibits the CHO-induced upregulation of CD68 and KLF4 in VSMCs. Additionally, FASN-deficient VSMCs exhibit hindered lipid accumulation and an impaired transition to the foam cell phenotype following CHO exposure, while the addition of the fatty acid palmitate, the main FASN product, exacerbates this transition. FASN-deficient cells also show decreased SOAT1 expression and elevated ABCA1. Notably, similar effects are observed in KLF4-deficient cells. Our findings demonstrate that FASN plays an essential role in the CHO-induced upregulation of KLF4 and the VSMC to foam cell transition and suggest that targeting FASN could be a novel therapeutic strategy to regulate VSMC phenotypic modulation.

Proteasome inhibitor bortezomib prevents proliferation and migration of pulmonary arterial smooth muscle cells.

Pulmonary vascular remodeling is a key pathological process of pulmonary arterial hypertension (PAH), characterized by uncontrolled proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). Bortezomib (BTZ) is the first Food and Drug Administration (FDA)-approved proteasome inhibitor for multiple myeloma treatment. Recently, there is emerging evidence showing its effect on reversing PAH, although its mechanisms are not well understood. In this study, anti-proliferative and anti-migratory effects of BTZ on PASMCs were first examined by different inducers such as fetal bovine serum (FBS), angiotensin II (Ang II) and platelet-derived growth factor (PDGF)-BB, while potential mechanisms including cellular reactive oxygen species (ROS) and mitochondrial ROS were then investigated; finally, signal transduction of ERK and Akt was examined. Our results showed that BTZ attenuated FBS-, Ang II- and PDGF-BB-induced proliferation and migration, with associated decreased cellular ROS production and mitochondrial ROS production. In addition, the phosphorylation of ERK and Akt induced by Ang II and PDGF-BB was also inhibited by BTZ treatment. This study indicates that BTZ can prevent proliferation and migration of PASMCs, which are possibly mediated by decreased ROS production and down-regulation of ERK and Akt. Thus, proteasome inhibition can be a novel pharmacological target in the management of PAH.

KLF15 maintains contractile phenotype of vascular smooth muscle cells and prevents thoracic aortic dissection by interacting with MRTFB.

Thoracic aortic dissection (TAD) is a highly dangerous cardiovascular disorder caused by weakening of the aortic wall, resulting in a sudden tear of the internal face. Progressive loss of the contractile apparatus in vascular smooth muscle cells (VSMCs) is a major event in TAD. Exploring the endogenous regulators essential for the contractile phenotype of VSMCs may aid the development of strategies to prevent TAD. Krüppel-like factor 15 (KLF15) overexpression was reported to inhibit TAD formation; however, the mechanisms by which KLF15 prevents TAD formation and whether KLF15 regulates the contractile phenotype of VSMCs in TAD are not well understood. Therefore, we investigated these unknown aspects of KLF15 function. We found that KLF15 expression was reduced in human TAD samples and ß-aminopropionitrile monofumarate-induced TAD mouse model. Klf15KO mice are susceptible to both ß-aminopropionitrile monofumarate- and angiotensin II-induced TAD. KLF15 deficiency results in reduced VSMC contractility and exacerbated vascular inflammation and extracellular matrix degradation. Mechanistically, KLF15 interacts with myocardin-related transcription factor B (MRTFB), a potent serum response factor coactivator that drives contractile gene expression. KLF15 silencing represses the MRTFB-induced activation of contractile genes in VSMCs. Thus, KLF15 cooperates with MRTFB to promote the expression of contractile genes in VSMCs, and its dysfunction may exacerbate TAD. These findings indicate that KLF15 may be a novel therapeutic target for the treatment of TAD.

Curcumol reduces lower limb arteriosclerosis in rats by inhibiting human arterial smooth muscle cell activity.

Cardiovascular diseases, particularly those involving arterial stenosis and smooth muscle cell proliferation, pose significant health risks. This study aimed to investigate the therapeutic potential of curcumol in inhibiting platelet-derived growth factor-BB (PDGF-BB)-induced human aortic smooth muscle cell (HASMC) proliferation, migration and autophagy. Using cell viability assays, 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays and Western Blot analyses, we observed that curcumol effectively attenuated PDGF-BB-induced HASMC proliferation and migration in a concentration-dependent manner. Furthermore, curcumol mitigated PDGF-BB-induced autophagy, as evidenced by the downregulation of LC3-II/LC3-I ratio and upregulation of P62. In vivo experiments using an arteriosclerosis obliterans model demonstrated that curcumol treatment significantly ameliorated arterial morphology and reduced stenosis. Additionally, curcumol inhibited the activity of the KLF5/COX2 axis, a key pathway in vascular diseases. These findings suggest that curcumol has the potential to serve as a multi-target therapeutic agent for vascular diseases.

Exploring the anti-atherosclerosis mechanism of ginsenoside Rb1 by integrating network pharmacology and experimental verification.

Ginsenoside Rb1 is the major active constituent of ginseng, which is widely used in traditional Chinese medicine for the atherosclerosis treatment by anti-inflammatory, anti-oxidant and reducing lipid accumulation. We explored cellular target and molecular mechanisms of ginsenoside Rb1 based on network pharmacology and in vitro experimental validation. In this study, we predicted 17 potential therapeutic targets for ginsenoside Rb1 with atherosclerosis from public databases. We then used protein-protein interaction network to screen the hub targets. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment showed that the effects of ginsenoside Rb1 were meditated through multiple targets and pathways. Next, molecular docking results revealed that in the 10 core targets, CCND1 has the highest binding energy with ginsenoside Rb1. Vascular cell proliferation plays a critical role in atherosclerosis development. However, the effect and direct target of ginsenoside Rb1 in regulating vascular cell proliferation in atherosclerosis remains unclear. Edu straining results indicated that ginsenoside Rb1 inhibited the cell proliferation of endothelial cells, macrophages, and vascular smooth muscle cells. The protein immunoprecipitation (IP) analysis showed that ginsenoside Rb1 inhibited the vascular cell proliferation by suppressing the interaction of CCDN1 and CDK4. These findings systematically reveal that the anti-atherosclerosis mechanism of ginsenoside Rb1 by integrating network pharmacology and experimental validation, which provide evidence to treat atherosclerosis by using ginsenoside Rb1 and targeting CCND1.

Smooth muscle cell-specific matrix metalloproteinase 3 deletion reduces osteogenic transformation and medial artery calcification.

AIMS: Vascular calcification is highly prevalent in atherosclerosis, diabetes, and chronic kidney disease. It is associated with increased morbidity and mortality in patients with cardiovascular disease. Matrix metalloproteinase 3 (MMP-3), also known as stromelysin-1, is part of the large matrix metalloproteinase family. It can degrade extracellular matrix components of the arterial wall including elastin, which plays a central role in medial calcification. In this study, we sought to determine the role of MMP-3 in medial calcification. METHODS AND RESULTS: We found that MMP-3 was increased in rodent models of medial calcification as well as in vascular smooth muscle cells (SMCs) cultured in a phosphate calcification medium. It was also highly expressed in calcified tibial arteries in patients with peripheral arterial disease (PAD). Knockdown and inhibition of MMP-3 suppressed phosphate-induced SMC osteogenic transformation and calcification, whereas the addition of a recombinant MMP-3 protein facilitated SMC calcification. In an ex vivo organ culture model and a rodent model of medial calcification induced by vitamin D3, we found that MMP-3 deficiency significantly suppressed medial calcification in the aorta. We further found that medial calcification and osteogenic transformation were significantly reduced in SMC-specific MMP-3-deficient mice, suggesting that MMP-3 in SMCs is an important factor in this process. CONCLUSION: These findings suggest that MMP-3 expression in vascular SMCs is an important regulator of medial calcification and that targeting MMP-3 could provide a therapeutic strategy to reduce it and address its consequences in patients with PAD.

High glucose promotes atherosclerosis by regulating miRNA let7d-5p level.

BACKGROUND: MiRNA let7d-5p has been recently reported to be abnormally expressed in diabetes-associated atherosclerosis (AS). However, it still remains unknown how let7d-5p contributes to the process of atherosclerosis. METHODS: Twenty fresh tissues and a total of 28 wax block specimens from carotid endarterectomy procedures were obtained from the Luoyang Central Hospital affiliated to Zhengzhou University. The expression of let7d-5p was assessed using quantitative RT-PCR (qRT-PCR). A series of in vitro experiments was used to determine the roles of let7d-5p knockdown and overexpression in vascular smooth muscle cells (VSMCs). RESULTS: We discovered that the carotid plaques from diabetic patients had lower expression levels of miR let7d-5p. In VSMCs, the expression of miRNA let7d-5p was significantly lower in high glucose conditions compared with low glucose situations. The proliferation and migration of VSMCs were also inhibited by the overexpression of let7d-5p, whereas the opposite was true when let7d-5p was inhibited, according to gain and loss of function studies. Mechanically, let7d-5p might activate the GSK3ß/ß-catenin signaling pathway via binding to the high mobility group AT-Hook 2 (HMGA2) mRNA in VSMCs. Additionally, GLP-1RA liraglutide may prevent the migration and proliferation of VSMCs by raising let7d-5p levels. CONCLUSIONS: High glucose stimulated the proliferation and migration of VSMCs by regulating the let7d-5p/HMGA2/GSK3ß/ß-catenin pathway, and liraglutide may slow atherosclerosis by increasing the levels of miR let7d-5p.

In vitro Evaluation of the Calcification Inhibitory Properties of Policosanol, Genistein, and Vitamin D (Reduplaxin®) either Alone or in Combination.

INTRODUCTION: The process of vascular calcification has severe clinical consequences in a number of diseases, including diabetes, atherosclerosis, and end-stage renal disease. In the present study, we investigated the effect of policosanol (Poli), genistein (Gen), and vitamin D (VitD) separately and in association to evaluate the possible synergistic action on inorganic phosphate (Pi)-induced calcification of vascular smooth muscle cells (VSMCs). METHODS: Primary human VSMCs were cultured with either growth medium or growth medium supplemented with calcium and phosphorus (calcification medium) in combination with Poli, Gen, and VitD. Alizarin Red staining, mineralization, and the protein expression of RUNX2 and superoxide dismutase-2 (SOD2) were investigated. RESULTS: All three substances tested were effective at reducing osteogenic differentiation of VSMCs in a dose-dependent manner. Poli+Gen, Poli+VitD, Gen+VitD treatment induced a greater inhibition of calcification and RUNX2 expression compared to single compounds treatments. Moreover, the association of Poli+Gen+VitD (Reduplaxin®) was more effective at inhibiting VSMCs mineralization and preventing the increase in RUNX2 expression induced by calcification medium but not modified SOD2 expression. CONCLUSIONS: The association of Pol, Gen, and VitD (Reduplaxin®) has an additive inhibitory effect on the calcification process of VSMCs induced in vitro by a pro-calcifying medium.