Proliferation Of VSMCs Research Articles

A study of lovastatin and L-arginine co-loaded PLGA nanomedicine for enhancing nitric oxide production and eNOS expression.

Nitric oxide (NO) is an exceptional endogenous biological gas that mediates and regulates physiological and pathological processes in the human body. However, its synthesis process is impaired during athero-progression and formation. Hence, a strategy to boost NO production and target endothelial nitric oxide synthase (eNOS) is crucial and intriguing in atherosclerosis (AS) management. Herein, we prepare L-arginine (LA) and lovastatin (LV) co-loaded PLGA nanomedicine to achieve sustainable release for enhancing NO production. The utilization of LA reveals that LA has dual contributions, acting as a NO donor and enhancing the solubility of LV by stabilizing PLGA NPs. PLGA-LA/LV demonstrated its potential to boost NO in vitro and in vivo confirmed using DAF-FM DA, augment eNOS and p-eNOS mRNA and protein levels, and suppress the ki67 proliferation marker in VSMCs; in addition, it lowers the total cholesterol level of blood plasma in C57BL/6 mice. Moreover, PLGA can protect the compound delivered and enhance the bioavailability to reach and get released in the blood circulation after oral administration. Collectively, our results endow a safe and efficient nanomedicine outcome, specifically with potential for AS management.

Metformin suppresses vascular smooth muscle cell senescence by promoting autophagic flux

IntroductionVascular smooth muscle cell (VSMC) senescence in the vasculature results in vascular aging as well as age-related diseases, while metformin improves the inflamm-aging profile by enhancing autophagy. However, metformin’s impact on VSMC senescence is largely undefined. ObjectivesTo test the hypothesis that metformin exerts an anti-senescence role by restoring autophagic activity in VSMCs and vascular tissues. MethodsAnimal models established by angiotensin II (Ang II) induction and physiological aging and senescent primary VSMCs from the aortas of elderly patients were treated with metformin. Cellular and vascular senescence were assessed by measuring the amounts of senescence-associated β-galactosidase and senescence markers, including p21 and p53. Autophagy levels were assessed by autophagy-related protein expression, transmission electron microscope, and autolysosome staining. In order to explore the underlying mechanism of the anti-senescence effects of metformin, 4D label-free quantitative proteomics and bioinformatic analyses were conducted, with subsequent experiments validating these findings. ResultsAng II-dependent senescence was suppressed by metformin in VSMCs and vascular tissues. Metformin also significantly improved arterial stiffness and alleviated structural changes in aged arteries, reduced senescence-associated secretory phenotype (SASP), and improved proliferation and migration of senescent VSMCs. Mechanistically, the proteomic analysis indicated that autophagy might contribute to metformin’s anti-senescence effects. Reduced autophagic flux was observed in Ang II-induced cellular and vascular senescence; this reduction was reversed by metformin. Specifically, metformin enhanced the autophagic flux at the autophagosome-lysosome fusion level, whereas blockade of autophagosome-lysosome fusion inhibited the anti-senescence effects of metformin. ConclusionsMetformin prevents VSMC and vascular senescence by promoting autolysosome formation.

The Association and Pathogenesis of SERPINA3 in Coronary Artery Disease.

Background: Serine proteinase inhibitor A3 (SERPINA3) has been discovered in the pathogenesis of many human diseases, but little is known about the role of SERPINA3 in coronary artery disease (CAD). Therefore, we aim to determine its relationship with CAD and its function in the pathogenesis of atherosclerosis.Methods: In total 86 patients with CAD and 64 patients with non-CAD were compared. The plasma SERPINA3 levels were measured using ELISA. Logistic regression analysis and receiver-operating characteristic (ROC) analysis were performed to illustrate the association between plasma SERPINA3 levels and CAD. In vitro, real-time PCR (RT-PCR) and immunofluorescence staining were used to determine the expression of SERPINA3 in atherosclerotic plaques and their component cells. Then rat aortic smooth muscle cells (RASMCs) were transfected with siRNA to knock down the expression of SERPINA3 and human umbilical vein endothelial cells (HUVECs) were stimulated by SERPINA3 protein. EdU assay and scratch assay were used for assessing the capability of proliferation and migration. The cell signaling pathway was evaluated by western blot and RT-PCR.Results: Patients with CAD [104.4(54.5–259.2) μg/mL] had higher levels of plasma SERPINA3 than non-CAD [65.3(47.5–137.3) μg/mL] (P = 0.004). After being fully adjusted, both log-transformed and tertiles of plasma SERPINA3 levels were significantly associated with CAD. While its diagnostic value was relatively low since the area under the ROC curve was 0.64 (95% CI: 0.55–0.73). Secreted SERPINA3 might increase the expression of inflammatory factors in HUVECs. Vascular smooth muscle cells had the highest SERPINA3 expression among the aorta compared to endothelial cells and inflammatory cells. The knockdown of SERPINA3 in RASMCs attenuated its proliferation and migration. The phosphorylated IκBα and its downstream pathway were inhibited when SERPINA3 was knocked down.Conclusions: Elevated plasma SERPINA3 levels were associated with CAD. SERPINA3 can increase inflammatory factors expression in HUVECs. It can regulate VSMCs proliferation, migration, and releasing of inflammatory factors through the NF-κB signaling pathway. Thus, SERPINA3 played a significant role in the pathogenesis of atherosclerosis.

Abstract 11489: Oral Rhamnan Sulfate Reduces Vascular Inflammation and Atherosclerotic Plaque Formation

Introduction: The glycocalyx, an interior lining of blood vessels, forms a barrier against inflammation and atherosclerotic risk factors. It is degraded during plaque development, allowing fat deposition in the intima. Rhamnan sulfate (RS), a polysaccharide from green seaweed, resembles GAGs such as heparan sulfate in the glycocalyx. Hypothesis: We hypothesize that RS can replace and rejuvenate the glycocalyx to enhance barrier function and prevent plaque formation. Methods: In vitro, we tested the effects of RS on proliferation, migration and permeability to LDL. We used western blots to measure the efficacy of RS in inhibiting the NF-κb pathway. We also performed atomic force microscopy on endothelial cells to test effects of RS on glycocalyx stiffness and thickness post-inflammation. In vivo, we conducted a study in female and male ApoE -/- mice on a high fat diet for 13 weeks where RS treatment was provided orally (n = 10 per group). Results: Rhamnan sulfate decreased growth factor induced proliferation and migration of endothelial and vascular smooth muscle cells. It increased heparan sulfate coverage and decreased the permeability of endothelial cells to LDL. It also decreased the activation of NF-κb in response to TNF-α treatment while also binding to NF-κb subunits. In ApoE -/- mice, oral consumption of RS decreased cholesterol levels in the plasma by 22.5% in female mice. En face analysis of aorta showed that plaque area decreased by 45.2% in female and 36.4% in male mice. In female mice, lipid deposition and stenosis decreased in the aortic arches and thoracic aortas while only lipid deposition was reduced in the aortic arches of male mice. Inflammatory factors were also decreased in the aortic arches of female mice. In livers of these mice, RS treatment reduced NAFLD activity scores and lipid deposition in females and unsaturated lipid content in males. RNA sequencing showed significant differences in genes related to metabolism, circadian rhythm and lipid regulation between high fat diet and RS groups and male and female mice. Conclusions: Rhamnan sulfate reduces inflammation in endothelial cells and proliferation of vSMCs. It also reduces plaque formation in aorta of ApoE -/- mice.

LncRNA SNHG16 accelerates atherosclerosis and promotes ox-LDL-induced VSMC growth via the miRNA-22–3p/HMGB2 axis

Long non-coding RNAs (LncRNAs) are essential regulators in the occurrence and development of AS. Here we aim to explore the underlying molecular mechanism of LncRNA SNHG16 in regulating ox-LDL-induced VSMC proliferation, migration and invasion. After constructing AS in vivo and in vitro models, the expressions of SNHG16, miR-22–3p, HMBG2, proliferation- and metastasis-related proteins were determined by qRT-PCR and Western blot assays. Detection of serological lipids, H&E and Masson staining analysis were conducted to evaluate the AS injury in mice. The effects of ox-LDL treatment on VSMCs were examined by CCK-8, wound scratch and Transwell Chamber assays. The targeted relationship was measured by luciferase reporter and RIP assays. The results showed that SNHG16 and high-mobility group box 2 (HMGB2) expressions were increased while miRNA-22–3p expression was decreased in AS mice and ox-LDL-stimulated VSMCs. Functionally, sh-SNHG16 restrained ox-LDL-induced VSMC growth and migration. SNHG16 suppressed miRNA-22–3p expression by direct binding. Furthermore, in ox-LDL-treated VSMCs, miRNA-22–3p mimic prevented proliferation, migration, and invasion. Further explorations showed that HMGB2 was a target of miRNA-22–3p, SNHG16 upregulated HMGB2 levels by acting as a competing endogenous RNA (ceRNA) of miRNA-22–3p. More importantly, sh-HMGB2 partially reversed the effects of sh-SNHG16 together with miR-22–2p inhibitor on ox-LDL-induced VSMC proliferation, migration and invasion. Collectively, SNHG16 accelerated atherosclerotic plaque (AP) formation and enhanced ox-LDL-activated VSMCs proliferation and migration by miRNA-22–3p/HMGB2 axis.

The promoting role of Cx43 on the proliferation and migration of arterial smooth muscle cells for angiotensin II-dependent hypertension

BackgroundRecent studies have shown that endothelin-1 and angiotensin II (AngII) can increase gap junctional intercellular communication (GJIC) by activating Mitogen-activated protein kinases (MAPKs) pathway. However, not only the precise interaction of AngII with Connexin43(Cx43) and the associated functions remain unclear, but also the regulatory role of Cx43 on the AngII-mediated promotion proliferation and migration of VSMCs is poorly understood. Material and methodsOur research applicated pressure myography measurements, immunofluorescence and Western blot analyses to investigate the changes in physiological indicators in spontaneously hypertensive rats (SHRs) and AngII-stimulated proliferation and migration of A7r5 SMCs(Rat vascular smooth muscle cells). The aim was to elucidate the role of CX43 in hypertension induced by AngII. ResultsChronic ramipril (angiotensin converting enzyme inhibitor) management for SHRs significantly attenuated blood pressure and blood vessel wall thickness, also reduced contraction rate in the cerebral artery. The cerebral artery contraction rates, mRNA and protein expression of Cx43, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) protein expression in the SHR + ramipril and SHR + ramipril + carbenoxolone (CBX, Cx43 specific blocker) groups were significantly lower than those in the SHR group. Cx43 protein expression and Ser368 phosphorylated Cx43 protein levels increased significantly in AngII-stimulated A7r5 cells. However, the levels of phosphorylated Cx43 decreased after pre-treatment with candesartan (AT1 receptor blocker), GF109203X (protein kinase C (PKC) blocker) and U0126 (mitogen-activated protein kinases/extracellular signal-regulated kinase1/2(MEK/ERK1/2)-specific blocker) in AngII-stimulated A7r5 cells. Cx43 was widely distributed in the cell membrane, nucleus, and cytoplasm of the SMCs. Furthermore, pre-treatment of the AngII- stimulated A7r5 cells with Gap26 (Cx43 blocker) significantly inhibited cell migration and decreased the expression levels of MEK1/2, ERK1/2, P-MEK1/2, and P-ERK1/2. ConclusionOur research confirms that Cx43 plays an important role in the regulation of proliferation and migration of VSMCs via MEK/ERK and PKC signal pathway in AngII-dependent hypertension.

Deletion of Fam172a accelerates advanced atherosclerosis and induces plaque instability

Background and aimsVascular smooth muscle cells (VSMCs) play a critical role in atherosclerosis. The family with sequence similarity 172, member A (FAM172A) is a novel protein and its role in atherosclerosis has not been explored so far. Therefore, our aim is to investigate whether FAM172A affects atheroprogression through VSMCs and its possible mechanism. MethodsFam172a−/− mice were generated using CRISPR/Cas9 technology. Fam172a−/− and Apoe−/− double knockout (Fam172a−/−/Apoe−/−) mice and their littermates (Fam172a+/+/Apoe−/−) were fed with a Western diet for 18 weeks to induce advanced atherosclerotic lesions. The role and mechanism of Fam172a in phenotypic switching, proliferation and migration of VSMCs were investigated through in vivo and in vitro experiments. ResultsCompared with Fam172a+/+/Apoe−/− mice, Fam172a−/−/Apoe−/− mice showed increased atherosclerotic lesion size and plaque instability such as increased necrotic core area and decreased fiber deposition. Additionally, knockout of Fam172a promoted expression of CD68 and KLF4 and decreased expression of α-SMA and SM22α in atherosclerotic lesions. Furthermore, overexpression of Fam172a promoted Movas cells proliferation and migration, increased expression of α-SMA and SM22α and decreased expression of KLF4. Meanwhile, knockdown of Fam172a in Movas cells and deletion of Fam172a in VSMCs from Fam172a−/−/Apoe−/− mice showed opposite phenotypes. Similar phenotypes were also observed in human aortic smooth muscle cells. ConclusionsOur results provide the first direct evidence that Fam172a has a protective role in advanced atherosclerosis by increasing atherosclerotic plaque stability and inhibiting transition of VSMCs from contractile to synthetic phenotype, which may be through KLF4-dependent pathway.

Angiotensin II-induced overexpression of sirtuin 1 contributes to enhanced expression of Giα proteins and hyperproliferation of vascular smooth muscle cells.

Angiotensin II (ANG II) plays an important role in the regulation of various physiological functions including proliferation, hypertrophy of vascular smooth muscle cells (VSMCs) through the overexpression of Giα proteins. Sirtuin 1 (Sirt1), a class III histone deacetylase and epigenetic regulator is implicated in a wide range of cellular functions, including migration and growth of VSMCs and in ANG II-induced hypertension. The present study was undertaken to examine the role of Sirt1 in ANG II-induced overexpression of Giα proteins and hyperproliferation of aortic VSMCs. We show that ANG II treatment of VSMCs increased the expression of Sirt1, which was attenuated by AT1 and AT2 receptor antagonists, losartan, and PD123319, respectively. In addition, the knockdown of Sirt1 by siRNA attenuated ANG II-induced overexpression of Giα-2 and Giα-3 proteins, hyperproliferation of VSMCs and the overexpression of cell cycle proteins, cyclin D1, Cdk4, and phosphorylated retinoblastoma proteins. Furthermore, ANG II-induced increased levels of superoxide anion (O2-) and NADPH oxidase activity and increased phosphorylation of ERK1/2 and Akt that are implicated in enhanced expression of Giα proteins and hyperproliferation of VSMCs were also attenuated to control levels by silencing of Sirt1. In addition, depletion of Sirt1 by siRNA also attenuated ANG II-induced enhanced phosphorylation of platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), and insulin-like growth factor receptor (IGFR) in VSMCs. In summary, our results demonstrate that ANG II increased the expression of Sirt1, which through oxidative stress, growth factor receptor-mediated mitogen-activated protein (MAP) kinase/Akt signaling pathway enhances the expression of Giα proteins and cell cycle proteins and results in the hyperproliferation of VSMCs.NEW & NOTEWORTHY ANG II regulates various physiological functions including proliferation of VSMCs through the overexpression of Giα proteins. Sirt1, a class III histone deacetylase, is implicated in several cellular functions, including VSMC growth and ANG II-induced hypertension. We showed for the first time that ANG II increased the expression of Sirt1, which through oxidative stress, growth factor receptor-mediated MAP kinase/Akt signaling pathway enhances the levels of Giα and cell cycle proteins resulting in the hyperproliferation of VSMCs.

Jujuboside B Inhibits Neointimal Hyperplasia and Prevents Vascular Smooth Muscle Cell Dedifferentiation, Proliferation, and Migration via Activation of AMPK/PPAR-γ Signaling.

The uncontrolled proliferation and migration of vascular smooth muscle cells is a critical step in the pathological process of restenosis caused by vascular intimal hyperplasia. Jujuboside B (JB) is one of the main biologically active ingredients extracted from the seeds of Zizyphus jujuba (SZJ), which has the properties of anti-platelet aggregation and reducing vascular tension. However, its effects on restenosis after vascular intervention caused by VSMCs proliferation and migration remain still unknown. Herein, we present novel data showing that JB treatment could significantly reduce the neointimal hyperplasia of balloon-damaged blood vessels in Sprague-Dawley (SD) rats. In cultured VSMCs, JB pretreatment significantly reduced cell dedifferentiation, proliferation, and migration induced by platelet-derived growth factor-BB (PDGF-BB). JB attenuated autophagy and reactive oxygen species (ROS) production stimulated by PDGF-BB. Besides, JB promoted the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ). Notably, inhibition of AMPK and PPAR-γ partially reversed the ability of JB to resist the proliferation and migration of VSMCs. Taken as a whole, our findings reveal for the first time the anti-restenosis properties of JB in vivo and in vitro after the endovascular intervention. JB antagonizes PDGF-BB-induced phenotypic switch, proliferation, and migration of vascular smooth muscle cells partly through AMPK/PPAR-γ pathway. These results indicate that JB might be a promising clinical candidate drug against in-stent restenosis, which provides a reference for further research on the prevention and treatment of vascular-related diseases.

Protective role of the mitochondrial fusion protein OPA1 in hypertension.

Hypertension is associated with excessive reactive oxygen species (ROS) production in vascular cells. Mitochondria undergo fusion and fission, a process playing a role in mitochondrial function. OPA1 is essential for mitochondrial fusion. Loss of OPA1 is associated with ROS production and cell dysfunction. We hypothesized that mitochondria fusion could reduce oxidative stress that defect in fusion would exacerbate hypertension. Using (a) Opa1 haploinsufficiency in isolated resistance arteries from Opa1+/- mice, (b) primary vascular cells from Opa1+/- mice, and (c) RNA interference experiments with siRNA against Opa1 in vascular cells, we investigated the role of mitochondria fusion in hypertension. In hypertension, Opa1 haploinsufficiency induced altered mitochondrial cristae structure both in vascular smooth muscle and endothelial cells but did not modify protein level of long and short forms of OPA1. In addition, we demonstrated an increase of mitochondrial ROS production, associated with a decrease of superoxide dismutase 1 protein expression. We also observed an increase of apoptosis in vascular cells and a decreased VSMCs proliferation. Blood pressure, vascular contractility, as well as endothelium-dependent and -independent relaxation were similar in Opa1+/- , WT, L-NAME-treated Opa1+/- and WT mice. Nevertheless, chronic NO-synthase inhibition with L-NAME induced a greater hypertension in Opa1+/- than in WT mice without compensatory arterial wall hypertrophy. This was associated with a stronger reduction in endothelium-dependent relaxation due to excessive ROS production. Our results highlight the protective role of mitochondria fusion in the vasculature during hypertension by limiting mitochondria ROS production.

Down Regulation of Let-7 Triggers a Critical Double-Negative Feedback Loop to Determinate the Malignant VSMCs Fate During Restenosis

Background: The excessive proliferation and migration of VSMCs is the main cause of restenosis (RS). Our previous results suggested that overexpression of Lin28a is responsible for this “malignant” behaver of VSMCs. However, the mechanisms driving the specific VSMCs behavior in RS are far less understood. Methods and Findings: Using an atherosclerosis followed by PTA treatment rats as RS model, we discovered a decreased expression of let-7c, let-7g, let-7i, miR98 and miR202, that associated with an increased expression of Lin28a in RS plaques compared to those of atherosclerosis. Combined with in vitro results, we demonstrated that let-7c, let-7g, and miR98 were Lin28a downstream targets in RS. More importantly, the decreased expression of let-7c, let-7g, in turn, will enhance the expression of Lin28a, indicating a double-negative feedback loop between Lin28a and let-7c/let-7g, which may be responsible for the vicious behavior of the VSMCs in RS. Transfection of let-7c and let-7g in vivo by AAV9 attenuated the aggregation of VSMCs and decreased the RS plaque size. In addition, unlike the other member of let-7, let-7d downregulates Lin28a and plays a restrictive regulator on the Lin28a/let-7 loop, that may bias VSMCs fate decision. Conclusion: In this study, by using a “two-step lesion” protocol to create RS models, we discovered that the double negative feedback system of Lin28a/let-7 axis act as a regulation center in vicious behavior of VSMCs in RS. These discoveries give us a better understanding of RS in lower extremity arterial disease and expected to present opportunities for discovering new therapeutic approaches. Funding: This work was supported by the National Natural Science Foundation of China Grants (No. 81670757, 81770822, 81800732, 81900685), Shandong Provincial Natural Science Foundation of China Grants (No. ZR2017LH025, ZR2020MH101), Shandong Provincial Medicine and Health Science and Technology Development Program (No. 2017WS461), China Postdoctoral Science Foundation (No. 2020M682193). Declaration of Interest: The authors confirm that there are no conflicts of interest. Ethical Approval: The protocol was authorized by the Ethics Committee of Qilu Hospital, Shandong University.

55-OR: Targeted Deletion of Insulin Receptor in Vascular Smooth Muscle Cells Promoted Atherosclerosis and Unstable Plaque

Risks of unstable arterial plaques are increased in diabetes or insulin resistance. We hypothesized that insulin resistance in VSMCs contributes to reduced plaque stability since we have reported that insulin receptors (IR) on VSMCs are potent regulators of its proliferation and apoptosis. Further, insulin induced Akt phosphorylation was decreased in the aorta of diabetic mice. Lipid deposition were increased in the aortas of ApoE-/- and VSMCs IR double knockout (SMIRKO/ApoE-/-) mice vs. control mice. Pathological analysis of the atherosclerotic plaques of SMIRKO/ApoE-/- mice demonstrated features of unstable plaques, including reduced VSMCs proliferation and reduced cap thickness and collagen, but increased VSMCs apoptosis and more macrophages and necrosis area. VSMCs from SMIRKO/ApoE-/- mice exhibited less insulin induced proliferation and more apoptosis which were not inhibited by insulin compared to VSMCs from ApoE-/- mice. Artery relaxation induced by sodium nitroprusside (SNP) (NO donor) was impaired in artery from SMIRKO/ApoE-/- mice vs. control mice. Insulin and SNP induced VASP phosphorylation, a downstream signaling protein of NO in VSMCs, were reduced in SMIRKO/ApoE-/- mice vs. control mice. Expressions of MMP2, THBS1, sFRP3, and COMP were increased in the aorta of SMIRKO/ApoE-/- mice vs. ApoE-/- mice, which were also observed in the aorta of diabetic mice fed with high fat diet or diabetes induced by STZ. Insulin induced Akt and Foxo1 phosphorylation were impaired in cultured VSMCs from SMIRKO/ApoE-/- mice vs. control mice. Overexpression of constitutively activated Akt increased VASP phosphorylation and downregulated THBS1 expression in cultured VSMCs, whereas overexpression of FoxO1 increased THBS1 expression. This is the first report that selective inhibition of IR activation of pAkt in VSMCs is partially causing the elevated risk of unstable atherosclerotic plaques found in insulin resistance or diabetes. Disclosure Q. Li: None. J. Fu: None. K. Park: None. I. Wu: None. G. L. King: Consultant; Self; Agios, Inc., Medtronic, Other Relationship; Self; Janssen Pharmaceuticals, Inc. Funding National Institutes of Health (R01DK053105)

MiR-181b and miR-204 suppress the VSMC proliferation and migration by downregulation of HCK

BackgroundVSMC proliferation and migration pathways play important roles in plaque formation in the vessel stenosis and re-stenosis processes. The microRNAs affect the expression of many genes that regulate these cellular processes. The aim of this study was to investigate the effects of miR-181b, miR-204, and miR-599 on the gene and protein expression levels of hematopoietic cell kinase (HCK) in VSMCs. MethodsmiR-181b, miR-204 were predicted for the suppression of HCK in the chemokine signaling pathway using bioinformatics tools. Then, the VSMCs were transfected by PEI-containing microRNAs. The HCK gene and protein expression levels were evaluated using RT-qPCR and Western blotting techniques, respectively. Moreover, the cellular proliferation and migration were evaluated by MTT and scratch assay methods. ResultsThe miR-181b and miR-204 decreased significantly the HCK gene and (total and phosphorylated) protein expression levels. Also, the miR-599 did not show any significant effects on the HCK gene and protein levels. The data also showed that miR-181b, miR-204, and miR-599 prevent significantly the proliferation and migration of VSMCs. ConclusionThe downregulation of HCK by miR-181b and miR-204 suppressed the VSMC proliferation and migration.

THE ROLE OF MATRIX METALLOPROTEINASE (MMP)-2 TO TYPE I COLLAGEN TURNOVER CONTRIBUTING TO HYPERTROPHIC ARTERIAL REMODELING IN RENOVASCULAR HYPERTENSION

Objective: Type I collagen proteolytic products contribute to VSMC proliferation via integrin receptor and focal adhesion kinase (FAK) activation. We then evaluated whether the early activation of MMP-2 in hypertension induces VSMC proliferation, due to type I collagen proteolysis, which may activate integrin and FAK to develop hypertrophic arterial remodeling. Design and method: Sprague Dawley rats were submitted to 2K1C surgery to induce hypertension and they were orally treated with doxycycline or vehicle (five days at first week (acute) and eight weeks at ten weeks of hypertension (chronic)); (CEUA-USP 165/2019). In the aorta, we evaluated the expression of MMP-2 and type I collagen by Western blotting; MMP-2 activity by gelatin and in situ zymography, arterial morphology by HE, and collagen deposition by picrosirius red. All statistical analyzes used two-way ANOVA followed by Tukey post hoc test with p < 0.05. Results: Our results showed that 2K1C rats developed high systolic blood pressure at the first week (156 ± 13 mmHg vs. Sham (p < 0,001)), which was progressively higher at ten weeks (219 ± 6 mmHg, p < 0,0001). Moreover, there was a significant increase in the MMP-2 expression and activity in the 2K1C rats at the first and tenth weeks of hypertension (p < 0.05). Treatment with doxycycline reduced MMP-2 activity only in the acute situation (p < 0.05). The 2K1C showed increased levels of type I collagen as well as its potential degradation products (p < 0.05) in the aorta during the first week of hypertension. However, the deposition of collagen was notably resynthesized in the aortas of chronic 2K1C rats (p < 0.05). Conclusions: Treatment with doxycycline reduced such effects in hypertensive rats. At this moment, we concluded that increased activity of MMP-2 may contribute to type I collagen proteolysis in aortas at early stages of hypertension, with its potential re-synthesis at late stages. This proteolytic effect of MMP-2 on collagen may contribute to stimulating VSMCs proliferation through integrins and FAK, mechanisms that we are currently investigating.

Glutamine switches vascular smooth muscle cells to synthetic phenotype through inhibiting miR-143 expression and upregulating THY1 expression

AimsVascular smooth muscle cells (VSMCs) are involved in the pathogenesis of many human cardiovascular diseases. They modulate their phenotype from “contractile” to “synthetic” in response to changes in local environmental cues. How glutamine regulates the differentiation of VSMCs and the underlying mechanisms remain largely unknown. Main methodsHere, we explored the effects of various doses of glutamine (0 mM, 1 mM, 2 mM, and 4 mM) on the proliferation, migration, and phenotypic switch of human VSMCs in vitro. Glutamine dose-dependently enhanced VSMC proliferation, and markedly increased VSMC migration. Key findingsNotably, glutamine promoted the phenotypic switch of VSMCs towards a synthetic phenotype, as evidenced by significantly decreased expression of contractile markers myosin heavy chain 11 (MYH11) and calponin while increased expression of synthetic markers collagen I and vimentin. Importantly, these changes upon glutamine treatments were attenuated after additional treatments with glutamine metabolism inhibitor BPTES. Additionally, glutamine downregulated miR-143 expression, and miR-143 inactivation alone resulted in enhanced proliferation, migration, and promoted the synthetic phenotype of VSMCs. Moreover, Thy-1 cell surface antigen (THY1) was validated as a downstream target of miR-143, and THY1 expression was upregulated by glutamine in VSMCs. Furthermore, either miR-143 overexpression or THY1 silencing abolished the effect of glutamine on proliferation, migration, and phenotypic switch of VSMCs, supporting a novel glutamine-miR-143-THY1 pathway in modulating VSMC functions. SignificanceThis study demonstrated a novel mechanism of glutamine in modulation of VSMC phenotypic switch by targeting miR-143 and THY1, and provides significant insight on targeted therapy of patients with cardiovascular diseases.

Regulation of autophagy by controlling Erk1/2 and mTOR for platelet-derived growth factor-BB-mediated vascular smooth muscle cell phenotype shift

AimsVascular smooth muscle cell (VSMC) phenotype shift is involved in the pathophysiology of vascular injury or platelet-derived growth factor (PDGF)-induced abnormal proliferation and migration of VSMCs. We aimed to investigate the underlying mechanism involved in PDGF-mediated signaling pathways and autophagy regulation followed by VSMC phenotype shift. Main methodsThe proliferation, migration and apoptosis of cultured rat aortic VSMCs were measured, and cells undergoing phenotype shift and autophagy were examined. Specific inhibitors for target proteins in signaling pathways were applied to clarify their roles in regulating cell functions. Key findingsPDGF-BB stimulation initiated autophagy activation and synthetic phenotype transition by decreasing α-smooth muscle-actin (SMA), calponin and myosin heavy chain (MHC) and increasing osteopontin (OPN) expression. However, U0126, a potent extracellular signal-regulated kinase 1/2 (Erk1/2) inhibitor, decreased PDGF-BB-induced LC3 expression, while rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), increased it. Furthermore, U0126 decreased the expresseion of autophagy-related genes (Atgs) such as beclin-1, Atg7, Atg5, and Atg12-Atg5 complex, indicating that Erk1/2 is a regulator of PDGF-BB-induced VSMC autophagy. Regardless of autophagy inhibition by U0126 or activation by rapamycin, the PDGF-BB-induced decrease in SMA, calponin and MHC and increase in OPN expression were inhibited. Furthermore, PDGF-BB-stimulated VSMC proliferation, migration and proliferating cell nuclear antigen (PCNA) expression were inhibited by U0126 and rapamycin. SignificanceThese findings suggest that PDGF-BB-induced autophagy is strongly regulated by Erk1/2, an mTOR-independent pathway, and any approach for targeting autophagy modulation is a potential therapeutic strategy for addressing abnormal VSMC proliferation and migration.

Vascular Smooth Muscle FTO Promotes Aortic Dissecting Aneurysms via m6A Modification of Klf5.

Background: Aortic dissecting aneurysm (ADA) represents an aortic remodeling disease with a high mortality rate. Fat mass and obesity-associated protein (FTO) exerts RNA demethylation function to regulate gene expression related to stem cell differentiation, DNA damage repair, and tumorigenesis, but the role of FTO in ADA is still unclear.Methods: The expression and location of FTO in 43 ADA tissues and 11 normal tissues were determined by RT-qPCR, WB, immunohistochemistry, and immunofluorescence staining. Detecting proliferation and migration of VSMCs. M6A methylated RNA immuno-precipitation qRT-PCR and dual luciferase reporter assay were performed for determining m6A level and interaction between m6A modulation and Klf5 mRNA, respectively.Results: FTO are highly expressed in VSMCs. FTO was positively correlated with BMI, triglyceride, and D-dimer (all P < 0.05). Functionally, both AngII-induced FTO expression and over expression of FTO promote cell proliferation and migration, whereas knockdown of FTO inhibits these functions. Mechanically, we identified Krüppel-like factor 5 (Klf5) as a target of FTO mediating m6A modification. Overexpression of FTO reduced m6A modification on Klf5 mRNA and promoted Klf5 mRNA expression. Furthermore, the p-GSK3β and Klf5 levels increased after FTO overexpression. Finally, knockdown of FTO suppresses the p-GSK3β levels and Klf5 expression regardless of AngII treatment.Conclusions: Our study revealed that FTO expression significantly contributes to the phenotype conversion of VSMCs and the ADA by the demethylation function (m6A), thereby providing a novel therapeutic target.

Abstract 15501: Repeated Social Defeat Exaggerates Cacl2-induced Abdominal Aortic Aneurysm Expansion by Suppression of Periaortic Fibrosis in Tissue Repair Phase

Background and Objective: Depression is an independent risk factor of cardiovascular disease and significantly associated with the prevalence of abdominal aortic aneurysm (AAA). We investigated the impact of repeated social defeat (RSD) on AAA formation. Methods and Results: Eight-week-old male WT mice were exposed to RSD by housing with a larger CD-1 mouse in a shared cage. They were subjected to vigorous physical contact daily for 10 consecutive days. Control mice were housed in the same cage without physical contact. After social interaction test to confirm depressive-like behaviors, mice underwent application of 0.5M calcium chloride (CaCl 2 ) on the infrarenal aorta. At one-week, maximum diameter and circumference of external elastic membrane were comparable between the two groups. The number of F-4/80 and MMP-9-positive cells in immunofluorescent images were also comparable. Consistently, fluorescent image taken by IVIS revealed no difference in MMP activity, suggesting that acute inflammatory response was comparable between the two groups. In contrast, at two weeks, maximum diameter and circumference of external elastic membrane were significantly increased in defeated mice ( p &lt; 0.01). Intriguingly, periaortic fibrotic area in aneurysmal portion was markedly decreased in defeated mice (12.5х10 3 μm 2 vs. 3.7х10 3 μm 2 , Control vs. Defeat, p &lt; 0.01). Consistently, accumulation of α-SMA-positive cells in adventitia of aneurysmal portion was much lesser in defeated mice (876 cells/mm 2 vs. 319 cells/mm 2 , Control vs. Defeat, p &lt; 0.05), whereas those in tunica media of non-aneurysmal portion did not show any difference between the two groups. LPS-induced iNOS gene and protein expressions in bone marrow-derived macrophages (BMDMs) were significantly decreased in defeated mice, whereas the TGF-β-induced phosphorylation of Smad2 and ERK1/2 in primary cultured aortic VSMCs were comparable between the two groups. Conclusions: Our findings demonstrate that RSD enhances AAA expansion by suppressing the periaortic fibrosis following inflammatory response, and suggest that decreased NO production from accumulated macrophages contributes to the reduced VSMCs proliferation and collagen deposition.

IRF-1 contributes to the pathological phenotype of VSMCs during atherogenesis by increasing CCL19 transcription.

Atherosclerosis (AS) is a chronic inflammatory disease that mainly involves the large and middle arteries, but the specific mechanism is not precise. Chemokine ligand 19 (CCL19) has been reported highly expressed in peripheral blood of patients with atherosclerosis, but its role lacks explicit data. By ELISA assay and immunohistochemical (IHC) analysis, we found that the CCL19 was significantly up-regulated in AS. Therefore, we tried to clarify whether CCL19 expression was related to the progression of AS. QRT-PCR and western blot demonstrated that overexpression of CCL19 promoted the secretion of inflammatory factors and the deposition of the extracellular matrix, and facilitated the proliferation and migration of VSMCS. Besides, knockdown of CCL19 reduced the inflammation, collagen secretion, proliferation and migration of VSMCS induced by PGDF-BB. The results of database analysis, chromatin immunoprecipitation (ChIP) and luciferase assay showed that interferon regulatory factor 1 (IRF-1) activated the expression of CCL19 at the transcriptional level. Importantly, silencing IRF-1 inhibited atherosclerosis in high-fat-fed mice, inhibited the proliferation and migration of VSMCS, and down-regulated the expression of CCL19. Summing up, the results demonstrated that IRF-1 contributed to the pathological phenotype of VSMCs during atherogenesis by increasing CCL19 transcription.

SIRT1-mediated deacetylation of NF-κB inhibits the MLCK/MLC2 pathway and the expression of ET-1, thus alleviating the development of coronary artery spasm.

Coronary artery spasm (CAS) is an intense vasoconstriction of coronary arteries that causes total or subtotal vessel occlusion. The cardioprotective effect of sirtuin-1 (SIRT1) has been extensively highlighted in coronary artery diseases. The aims within this study include the investigation of the molecular mechanism by which SIRT1 alleviates CAS. SIRT1 expression was first determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis in an endothelin-1 (ET-1)-induced rat CAS model. Interaction among SIRT1, nuclear factor-kappaB (NF-κB), myosin light chain kinase/myosin light chain-2 (MLCK/MLC2), and ET-1 was analyzed using luciferase reporter assay, RT-qPCR, and Western blot analysis. After ectopic expression and depletion experiments in vascular smooth muscle cells (VSMCs), contraction and proliferation of VSMCs and expression of contraction-related proteins (α-SMA, calponin, and SM22α) were measured by collagen gel contraction, 5-ethynyl-2'-deoxyuridine (EdU) assay, RT-qPCR, and Western blot analysis. The obtained results showed that SIRT1 expression was reduced in rat CAS models. However, overexpression of SIRT1 inhibited the contraction and proliferation of VSMCs in vitro. Mechanistic investigation indicated that SIRT1 inhibited NF-κB expression through deacetylation. Moreover, NF-κB could activate the MLCK/MLC2 pathway and upregulate ET-1 expression by binding to their promoter regions, thus inducing VSMC contraction and proliferation in vitro. In vivo experimental results also revealed that SIRT1 alleviated CAS through regulation of the NF-κB/MLCK/MLC2/ET-1 signaling axis. Collectively, our data suggested that SIRT1 could mediate the deacetylation of NF-κB, disrupt the MLCK/MLC2 pathway, and inhibit the expression of ET-1 to relieve CAS, providing a theoretical basis for the prospect of CAS treatment and prevention.NEW & NOTEWORTHY Rat coronary artery spasm models exhibit reduced expression of SIRT1. Overexpression of SIRT1 inhibits contraction and proliferation of VSMCs. SIRT1 inhibits NF-κB through deacetylation to modulate VSMC contraction and proliferation. NF-κB activates the MLCK/MLC2 pathway. NF-κB upregulates ET-1 to modulate VSMC contraction and proliferation.