Tryptanthrin Regulates Vascular Smooth Muscle Cell Phenotypic Switching in Atherosclerosis by AMP-Activated Protein Kinase/Acetyl-CoA Carboxylase Signaling Pathway.

Atherosclerosis (AS) is one of the most severe cardiovascular diseases involved in the phenotypic switching of vascular smooth muscle cells (VSMCs). Tryptanthrin is a natural product with broad biological activities. However, the effect of tryptanthrin on atherosclerotic progression is unclear. The aim of this study was to determine the role of tryptanthrin in AS and explore the potential mechanism. In vitro, primary VSMCs were stimulated with platelet-derived growth factor-BB (PDGF) to induce cell dedifferentiation. Treatment with tryptanthrin (5 μM or 10 μM) suppressed the proliferation and recovered the contractility of VSMCs in the presence of PDGF. The contractile proteins (α-smooth muscle actin, calponin, and SM22α) were increased, and the synthetic protein vimentin was decreased by tryptanthrin in PDGF-induced VSMCs. ApoE-/- mice fed with high-fat diet were used as an in vivo model of AS. Similarly, gavage administration of tryptanthrin (50 mg/kg or 100 mg/kg) attenuated VSMC phenotypic changes from a contractile to a synthetic state in aortic tissues of AS mice. The serum lipid level, atherosclerotic plaque formation, and arterial intimal hyperplasia were attenuated by tryptanthrin. Furthermore, tryptanthrin increased the expression levels of phosphorylated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) both in vitro and in vivo. Administration of compound C, an AMPK inhibitor, reversed the inhibitory effect of tryptanthrin on VSMC dedifferentiation in vitro. Thus, we demonstrate that tryptanthrin protects against AS progression through the inhibition of VSMC switching from a contractile to a pathological synthetic phenotype by the activation of AMPK/ACC pathway. It provides novel insights into AS prevention and treatment.

Shi X ，Zhang Y ，Han J ，Peng W ，Fang Z ，Qin Y ，Xu X ，Lin J ，Xiao F ，Zhao L ，Lin Y ... -  《-》

Liraglutide, a GLP-1 receptor agonist, inhibits vascular smooth muscle cell proliferation by enhancing AMP-activated protein kinase and cell cycle regulation, and delays atherosclerosis in ApoE deficient mice.

Several studies have demonstrated that both native glucagon-like peptide-1 (GLP-1) and GLP-1 receptor agonists suppress the progression of atherosclerosis in animal models. We investigated whether liraglutide, a GLP-1 analogue, could prevent the development of atherosclerosis in apolipoprotein E knockout mice (ApoE-/-) on a high-fat diet. We also examined the influence of liraglutide on angiotensin II-induced proliferation of rat vascular smooth muscle cells (VSMCs) via enhancement of AMP-activated protein kinase (AMPK) signaling and regulation of cell cycle progression. Treatment of ApoE-/- mice with liraglutide (400 μg/day for 4 weeks) suppressed atherosclerotic lesions and increased AMPK phosphorylation in the aortic wall. Liraglutide also improved the endothelial function of thoracic aortas harvested from ApoE-/- mice in an ex vivo study. Furthermore, liraglutide increased AMPK phosphorylation in rat VSMCs, while liraglutide-induced activation of AMPK was abolished by exendin 9-39, a GLP-1 antagonist. Moreover, angiotensin (Ang) II-induced proliferation of VSMCs was suppressed by liraglutide in a dose-dependent manner, and flow cytometry of Ang II-stimulated VSMCs showed that liraglutide reduced the percentage of cells in G2/M phase (by arrest in G0/G1 phase). These findings suggest that liraglutide may inhibit Ang II-induced VSMC proliferation by activating AMPK signaling and inducing cell cycle arrest, thus delaying the progression of atherosclerosis independently of its glucose-lowering effect.

Jojima T ，Uchida K ，Akimoto K ，Tomotsune T ，Yanagi K ，Iijima T ，Suzuki K ，Kasai K ，Aso Y ... -  《-》

Soluble epoxide hydrolase is involved in the development of atherosclerosis and arterial neointima formation by regulating smooth muscle cell migration.

Epoxyeicosatrienoic acids (EETs) have beneficial effects on cardiovascular disease. Soluble epoxide hydrolase (sEH) metabolizes EETs to less active diols, thus diminishing their biological activity. sEH inhibitors can suppress the progression of atherosclerotic lesions in animal models. However, the regulation of sEH in vascular smooth muscle cells (VSMCs) and role of sEH in patients with atherosclerosis have not been evaluated. We hypothesize that sEH in VSMCs plays a pivotal role in atherosclerosis and injury-induced neointima formation. In this study, sEH expression in human autopsy atherosclerotic plaque was determined by immunohistochemistry. In cultured rat and human VSMCs, the phenotypic switching marker and sEH expression induced by platelet-derived growth factor-BB (PDGF-BB) were examined by Western blot analysis. Carotid-artery balloon injury was performed after adenovirus-mediated overexpression of sEH or oral administration of a potent sEH inhibitor in Sprague-Dawley rats. sEH was highly expressed in VSMCs of the intima and media within human atherosclerotic plaque. In vitro, PDGF-BB upregulated the expression in VSMCs after transcription and promoted cell proliferation and migration; the latter effect could be largely attenuated by an sEH inhibitor. Adenovirus-mediated overexpression of sEH could mimic the effect of PDGF-BB and induce VSMC proliferation and migration. In vivo, the sEH inhibitor led to a significant decrease in injury-induced neointima formation in a rat carotid-artery injury model. These data establish the effect of sEH expression on atherosclerotic progression and vascular remodeling after injury, thus identifying a novel integrative role for sEH in VSMC phenotypic modulation and migration. Blocking sEH activity may be a potential therapeutic approach for ameliorating vascular occlusive disease.

Wang Q ，Huo L ，He J ，Ding W ，Su H ，Tian D ，Welch C ，Hammock BD ，Ai D ，Zhu Y ... -  《-》

Intermedin reduces neointima formation by regulating vascular smooth muscle cell phenotype via cAMP/PKA pathway.

Vascular smooth muscle cell (VSMC) dedifferentiation contributes to neointima formation, which results in various vascular disorders. Intermedin (IMD), a cardiovascular paracrine/autocrine polypeptide, is involved in maintaining circulatory homeostasis. However, whether IMD protects against neointima formation remains largely unknown. The purpose of this study is to investigate the role of IMD in neointima formation and the possible mechanism. IMD1-53 (100ng/kg/h) or saline water was used on rat carotid-artery balloon-injury model. The mouse left common carotid-artery ligation-injury model was established using IMD-transgenic and C57BL/6J mice. Immunohistochemistry and immunofluorescence staining was used to detect the protein expression in rat carotid arteries. Radioimmunoassay was used to determine the serum IMD level. The hematoxylin andeosin staining was used for carotid arteries morphological testing. In vitro, for rat primary cultured VSMC phenotype transition, proliferation and migration assays, platelet-derived growth factor-BB (PDGF-BB) reagent and IMD1-53 peptide were added to the culture media at the final concentration of 20 ng/mL and 10-7mol/L respectively. Quantification of VSMC proliferation involved MTT and BrdU assay and migration was detected by wound-healing assay. Western blot and realtime PCR were used to detect the protein and mRNA levels of tissues or cells. With the rat carotid-artery balloon-injury model, IMD was significantly downregulated in injured arteries and plasma. Exogenous IMD1-53 greatly inhibited neointima formation and prevented VSMC from switching to a synthetic phenotype. With the left common carotid-artery ligation-injury model, IMD-transgenic mice showed less neointima formation than C57BL/6J mice. PDGF-BB reduced IMD mRNA expression in rat primary cultured VSMCs but increased that of its receptors, calcitonin receptor-like receptor or receptor activity-modifying proteins. Furthermore, PDGF-BB promoted VSMC proliferation and migration and transformed VSMCs to the synthetic phenotype, which was reversed with IMD1-53 treatment. Mechanistically, IMD1-53 maintained the contractile VSMC phenotype via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway. IMD attenuated neointima formation both in the rat model of carotid-artery balloon injury and mouse model of common carotid-artery ligation injury. IMD protection may be mediated by maintaining a VSMC contractile phenotype via the cAMP/PKA pathway.

Zhu Q ，Ni XQ ，Lu WW ，Zhang JS ，Ren JL ，Wu D ，Chen Y ，Zhang LS ，Yu YR ，Tang CS ，Qi YF ... -  《-》

Ruxolitinib attenuates intimal hyperplasia via inhibiting JAK2/STAT3 signaling pathway activation induced by PDGF-BB in vascular smooth muscle cells.

Cardiovascular diseases are associated with proliferation and phenotypic switch. Platelet-derived growth factor-BB (PDGF-BB) is a major initiating factor for proliferative vascular diseases, such as neointimal lesion formation, restenosis after angioplasty, and atherosclerosis. Ruxolitinib, a potent Janus kinase (JAK) 1 and 2 inhibitor, has been reported to significantly block the proliferation-related signaling pathway of JAK2/signal transducers and activators of transcription 3 (STAT3) and harbor a broad spectrum of anti-cancer activities, including proliferation inhibition, apoptosis induction, and anti-inflammation. However, the role of ruxolitinib in regulating PDGF-BB-induced VSMC proliferation remains to be elucidated. Thus, this study investigates the role of ruxolitinib in regulating PDGF-BB-induced VSMC proliferation and its underlying mechanisms. In vivo, the medial thickness of the carotid artery was evaluated using a mouse carotid ligation model, ruxolitinib was administered orally to the mice every other day, and the mice were euthanized on day 28 to evaluate the therapeutic effects of ruxolitinib. Cell proliferation markers were measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. In vitro, VSMCs were treated with ruxolitinib with or without PDGF-BB at an indicated time and concentration. Cell proliferation and apoptosis were measured using Cell Counting Kit-8 assay, MTS assays and flow cytometry. The JAK2/STAT3 signaling pathway involved in the effects of ruxolitinib on VSMCs was detected by western blotting with the specific pathway inhibitor AG490. In vivo, ruxolitinib significantly decreased the ratio-of-intima ratio (I/M ratio) by inhibiting the expression of PCNA and cyclinD1 (p <0.05). In vitro, ruxolitinib inhibited PDGF-BB-induced VSMC proliferation compared with the PDGF-BB treatment group (p <0.05). In addition, ruxolitinib inhibited the PDGF-BB-induced activation of the JAK2/STAT3 signaling pathway and decreased the expression of proliferation related-proteins cyclinD1 and PCNA in VSMCs (p <0.05). Our findings suggest that ruxolitinib inhibits VSMC proliferation in vivo and in vitro by suppressing the activation of the JAK2/STAT3 signaling pathway. Therefore, ruxolitinib has a therapeutic potential for proliferative vascular diseases.

Song HT ，Cui Y ，Zhang LL ，Cao G ，Li L ，Li G ，Jia XJ ... -  《-》