Kanglexin counters vascular smooth muscle cell dedifferentiation and associated arteriosclerosis through inhibiting PDGFR.

BACKGROUND: Dysregulation of vascular smooth muscle cell (VSMC) function leads to a variety of diseases such as atherosclerosis and hyperplasia after injury. However, antiproliferative drug targeting VSMC exhibits poor specificity. Therefore, there is an urgent to develop highly specific antiproliferative drugs to prevention and treatment VSMC dedifferentiation associated arteriosclerosis. Kanglexin (KLX), a new anthraquinone compound designed by our team, has potential to regulate VSMC phenotype according to the physicochemical properties. PURPOSE: This project aims to evaluate the therapeutic role of KLX in VSMC dedifferentiation and atherosclerosis, neointimal formation and illustrates the underlying molecular mechanism. METHODS: In vivo, the ApoE-/- mice were fed with high-fat diet (HFD) for a duration of 13 weeks to establish the atherosclerotic model. And rat carotid artery injury model was performed to establish the neointimal formation model. In vitro, PDGF-BB was used to induce VSMC dedifferentiation. RESULTS: We found that KLX ameliorated the atherosclerotic progression including atherosclerotic lesion formation, lipid deposition and collagen deposition in aorta and aortic sinus in atherosclerotic mouse model. In addition, The administration of KLX effectively ameliorated neointimal formation in the carotid artery following balloon injury in SD rats. The findings derived from molecular docking and surface plasmon resonance (SPR) experiments unequivocally demonstrate that KLX had potential to bind PDGFR-ß. Mechanism research work proved that KLX prevented VSMC proliferation, migration and dedifferentiation via activating the PDGFR-ß-MEK -ERK-ELK-1/KLF4 signaling pathway. CONCLUSION: Collectively, we demonstrated that KLX effectively attenuated the progression of atherosclerosis in ApoE-/- mice and carotid arterial neointimal formation in SD rats by inhibiting VSMC phenotypic conversion via PDGFR-ß-MEK-ERK-ELK-1/KLF4 signaling. KLX exhibits promising potential as a viable therapeutic agent for the treatment of VSMC phenotype conversion associated arteriosclerosis.

Asprosin aggravates atherosclerosis via regulating the phenotype transformation of vascular smooth muscle cells.

Phenotype transformation of vascular smooth muscle cells (VSMCs) plays an important role in the development of atherosclerosis. Asprosin is a newly discovered adipokine, which is critical in regulating metabolism. However, the relationship between asprosin and phenotype transformation of VSMCs in atherosclerosis remains unclear. The aim of this study is to investigate whether asprosin affects the progression of atherosclerosis by inducing phenotype transformation of VSMCs. We established an atherosclerosis model in ApoE-/- mice and administered asprosin recombinant protein and asprosin antibody to mice. Knocking down asprosin was also as an intervention. Interestingly, we found a correlation between asprosin levels and atherosclerosis. Asprosin promoted plaque formation and phenotype transformation of VSMCs. While, AspKD or asprosin antibody reduced the plaque lesion and suppressed vascular stiffness in ApoE-/- mice. Mechanistically, asprosin induced phenotype transformation of MOVAs by binding to GPR54, leading to Gαq/11 recruitment and activation of the PLC-PKC-ERK1/2-STAT3 signaling pathway. Si GPR54 or GPR54 antagonist partially inhibited the action of asprosin in MOVAs. Mutant GPR54-(267, 307) residue cancelled the binding of asprosin and GPR54. In summary, this study confirmed asprosin activated GPR54/Gαq/11-dependent ERK1/2-STAT3 signaling pathway, thereby promoting VSMCs phenotype transformation and aggravating atherosclerosis, thus providing a new target for the treatment of atherosclerosis.

Myeloid-derived growth factor suppresses VSMC dedifferentiation and attenuates postinjury neointimal formation in rats by activating S1PR2 and its downstream signaling.

Restenosis after angioplasty is caused usually by neointima formation characterized by aberrant vascular smooth muscle cell (VSMC) dedifferentiation. Myeloid-derived growth factor (MYDGF), secreted from bone marrow-derived monocytes and macrophages, has been found to have cardioprotective effects. In this study we investigated the effect of MYDGF to postinjury neointimal formation and the underlying mechanisms. Rat carotid arteries balloon-injured model was established. We found that plasma MYDGF content and the level of MYDGF in injured arteries were significantly decreased after balloon injury. Local application of exogenous MYDGF (50 µg/mL) around the injured vessel during balloon injury markedly ameliorated the development of neointimal formation evidenced by relieving the narrow endovascular diameter, improving hemodynamics, and reducing collagen deposition. In addition, local application of MYDGF inhibited VSMC dedifferentiation, which was proved by reversing the elevated levels of osteopontin (OPN) protein and decreased levels of α-smooth muscle actin (α-SMA) in the left carotid arteries. We showed that PDGF-BB (30 ng/mL) stimulated VSMC proliferation, migration and dedifferentiation in vitro; pretreatment with MYDGF (50-200 ng/mL) concentration-dependently eliminated PDGF-BB-induced cell proliferation, migration and dedifferentiation. Molecular docking revealed that MYDGF had the potential to bind with sphingosine-1-phosphate receptor 2 (S1PR2), which was confirmed by SPR assay and Co-IP analysis. Pretreatment with CCG-1423 (Rho signaling inhibitor), JTE-013 (S1PR2 antagonist) or Ripasudil (ROCK inhibitor) circumvented the inhibitory effects of MYDGF on VSMC phenotypic switching through inhibiting S1PR2 or its downstream RhoA-actin monomers (G-actin) /actin filaments (F-actin)-MRTF-A signaling. In summary, this study proves that MYDGF relieves neointimal formation of carotid arteries in response to balloon injury in rats, and suppresses VSMC dedifferentiation induced by PDGF-BB via S1PR2-RhoA-G/F-actin-MRTF-A signaling pathway. In addition, our results provide evidence for cross talk between bone marrow and vasculature.

A novel small molecule AdipoR2 agonist ameliorates experimental hepatic steatosis in hamsters and mice.

Adiponectin receptor 2 (AdipoR2) can be activated by its endogenous ligand adiponectin to reduce hepatic steatosis, and is regarded as a therapeutic target for metabolic associated fatty liver disease (MAFLD). This study proposes a novel anthraquinone compound, emodin succinate monoethyl ester (ESME), which activates AdipoR2, inhibits hepatic lipogenesis, promotes fatty acid oxidation, and alleviates experimental hepatic steatosis in hamsters and mice. Molecular docking shows that ESME has strong binding potential with AdipoR2 by forming a arene-arene interaction. AdipoR2 on the cytomembrane of HepG2 cells can be labeled by fluorescent ESME (Cy5-ESME). ESME activates AdipoR2, AMPK and PPARα, and reduces lipid deposition in palmitic acid or oleic acid-induced HepG2 and L02 cells. Suppression of AdipoR2 expression or AMPK activation completely eliminates the effect of ESME on reducing lipid accumulation in hepatocytes. Oral administration of ESME reduces liver lipid production and accumulation, and alleviates hepatic steatosis in hamsters and Apoe-/- mice induced by high-fat diet. Compared with statins and emodin, ESME showed prepotent efficacy and safety in reducing hepatic steatosis and protecting hepatocytes. Furthermore, ESME activates CaMKK2 and LKB1 in liver to activate AMPK and reduce lipogenesis through stimulating AdipoR2. Taken together, ESME reduces hepatic lipid accumulation and alleviates hepatic steatosis by agonizing AdipoR2. ESME is a promising new agent for clinical treatment of MAFLD.

Differentially Expressed Circular Non-coding RNAs in Atherosclerotic Aortic Vessels and Their Potential Functions in Endothelial Injury.

Background: Circular non-coding RNA (circRNA) has a variety of biological functions. However, the expression profile and potential effects of circRNA on atherosclerosis (AS) and vascular endothelial injury have not been fully elucidated. This study aims to identify the differentially expressed circRNAs in atherosclerotic aortic vessels and predict their potential functions in endothelial injury. Method: ApoE-/- mice were fed with high-fat diet for 12 weeks to induce AS. Atherosclerotic plaques were evaluated by H&E and Masson staining and immunohistochemistry; differentially expressed circRNAs were detected by Arraystar Circular RNA Microarray and verified by RT-PCR; the potential target mircoRNAs of circRNAs were predicted by miRanda, Tarbase, Targetscan and their expression changes were verified by RT-PCR; the potential target genes of mircoRNAs were predicted by Targetscan and verified by Western blot; the signaling pathways that they might annotate or regulate and their potential functions in vascular endothelial injury were predicted by gene enrichment analysis. Results: Fifty two circRNAs were up-regulated more than twice and 47 circRNAs were down-regulated more than 1.5 times in AS aortic vessels. Mmmu_circRNA_36781 and 37699 were up-regulated both in AS aortic vessels and H2O2-treated mouse aortic endothelial cells (MAECs). The expression of miR-30d-3p and miR-140-3p, the target microRNA of circRNA_37699 and circRNA_36781, were downregulated both in AS vessels and H2O2-treated MAECs. On the contrary, MKK6 and TP53RK, the potential target gene of miR-140-3p and miR-30d-3p, were upregulated both in AS aortic roots and H2O2-treated MAECs. Besides, gene enrichment analysis showed that MAPK and PI3K-AKT signaling pathway were the most potential signaling pathways regulated by the differentially expressed circRNAs in atherosclerosis. Conclusions: Mmu_circRNA_36781 (circRNA ABCA1) and 37699 (circRNA KHDRBS1) were significantly up-regulated in AS aortic vessels and H2O2-treated MAECs. They have potential regulatory effects on atherosclerosis and vascular endothelial injury by targeting miR-30d-3p-TP53RK and miR-140-3p-MKK6 axis and their downstream signaling pathways.

Kanglexin accelerates diabetic wound healing by promoting angiogenesis via FGFR1/ERK signaling.

Diabetic foot is one of the main causes of non-traumatic amputation. However, there is still lack of effective drugs to treat diabetic foot in clinical practice. Kanglexin (KLX) is a new anthraquinone compound with cardiovascular protective effects. Here we report that KLX accelerates diabetic wound healing by promoting angiogenesis via FGFR1/ERK signaling. Firstly, KM mice were injected (ip) with streptozocin to establish type 1 diabetic model. The full thickness wound with the diameter of 5 mm was prepared on the back of each mice. The wounds were treated with KLX once a day for 14 consecutive days. Results showed that KLX significantly accelerated the closure of diabetic wounds. Pathological studies of skin tissues around the wounds showed that KLX promoted the formation of granulation tissue and new blood vessels, increased collagen deposition and reduced inflammatory cell infiltration. Besides, KLX significantly alleviated advanced glycation end products (AGEs) - induced abnormal proliferation, migration and tubule formation of human umbilical vein endothelial cells (HUVECs), and up-regulated phospho-ERK1/2 both in the diabetic wound tissue and AGEs - treated HUVECs. Moreover, molecular docking results indicated that KLX had the potential to bind with FGF receptor 1 (FGFR1), and subsequent experiments confirmed that FGFR1 inhibitor PD173074 reversed the effect of KLX on promoting the phosphorylation of ERK1/2 and angiogenesis, suggesting that KLX promoted angiogenesis through FGFR1/ERK signaling. In conclusion, our study provides a new effective compound for treating diabetic wounds. More importantly, KLX has the potential to be developed as a topical drug to promote diabetic wound healing.

Diosmin Regulates Oxidative Stress and Inflammatory Marker Levels in N-Methyl-N-Nitrosourea-Induced Gastric Carcinogenesis in Rats.

Gastric cancer is the most typical oncological illness globally. Though the incidence of gastric cancer has dropped dramatically over the last few years, the survival rate is yet concerning due to poor diagnostic strategy. The etiology of gastric cancer is majorly associated with dietary factors. For this reason, application of nontoxic natural agents with anticancer effects for patients is needed. Diosmin has been commonly utilized to treat various diseases both traditionally and now clinically. However, its effect on gastric carcinogenesis remains unclear. The effects of diosmin were used to evaluate how N-methyl-N-nitrosourea (MNU) induces gastric carcinogenesis in rats. The general and gross assessment of MNU in experimental animals was tabulated. Biological tumor markers (gastrin, ALP, LDH, and γ-GT) were examined. Oxidative markers (LPO) and antioxidative markers (GSH, vitamin E, and vitamin C) were determined. In addition, inflammatory cytokines (thioredoxin, glutaredoxin, NF-κB, TNF-α, IL-6, PGE2) were explored and justified with histopathological studies. Overall, the results showed positive anticancer activity demonstrated by improved body weight, reduced tumor rate, decreased oxidative marker value by increased antioxidative rate, and suppressed tumor biomarkers and inflammatory cytokines. The histopathological analysis was congruent with the data observed. Our conclusion that diosmin exerts its anticancer activity by up-regulation of antioxidants which might be responsible for amelioration of oxidative stress and inflammatory cytokines in carcinogenesis to prevent gastric cancer.

Kang Le Xin Reduces Blood Pressure Through Inducing Endothelial-Dependent Vasodilation by Activating the AMPK-eNOS Pathway.

Hypertension is a major risk factor for stroke and cardiovascular events in clinic, which is accompanied by the abnormality of vascular tone and endothelial dysfunction of small artery. Here we report that Kang Le Xin (KLX), a novel anthraquinones compound, could reduce blood pressure and the underlying mechanisms involves that KLX induces endothelium-dependent vasodilation. KLX significantly decreases the arterial blood pressure of spontaneous hypertensive rats (SHR), decreases the contractile reactivity of superior mesenteric artery to phenylephrine and increases the vasodilatory reactivity of superior mesenteric artery to carbachol in a dose-dependent manner. Besides, KLX reduces vascular tension of endothelium-intact mesenteric artery pre-constricted with phenylephrine in a dose-dependent manner, while this effect is inhibited by depriving vascular endothelium or pretreating vascular rings with L-NAME (endothelial nitric oxide synthase inhibitor) or compound C (AMP-activated protein kinase inhibitor). Moreover, KLX increases nitric oxide (NO) generation, endothelial nitric oxide synthase (eNOS), AKT and AMP-activated protein kinase (AMPK) phosphorylation in cultured human umbilical vein endothelial cells (HUVECs), while these effects are inhibited by pretreating cells with compound C. In conclusion, KLX is a new compound with the pharmacological action of reducing arterial blood pressure. The underlying mechanism involves KLX induces endothelium-dependent vasodilation through activating AMPK-AKT-eNOS signaling pathway.

Shenxian-Shengmai Oral Liquid Reduces Myocardial Oxidative Stress and Protects Myocardium from Ischemia-Reperfusion Injury.

BACKGROUND/AIMS: Shenxian-shengmai (SXSM) oral liquid, a Chinese patent compound medicine, has been used to treat sinus bradyarrhythmias induced by mild sick sinus syndrome in clinical practice. Myocardial ischemia, in particular in serious or right coronary-related heart diseases, can cause bradyarrhythmias and cardiac dysfunction. Moreover, reperfusion of ischemic myocardium is associated with additional myocardial damage known as myocardial ischemia-reperfusion (I/R) injury. This study was designed to evaluate the effects of SXSM on bradyarrhythmias and cardiac dysfunction induced by myocardial I/R injury, and to explore the underlying mechanisms. METHODS: Administration of SXSM to adult male Sprague Dawley (SD) rats was achieved orally by gavage and control rats were given equivalent deionized water every day for 14 days. After the last administration, the heart was connected with the Langendorff perfusion apparatus and both groups were subjected to ischemia for 20 min followed by reperfusion for 40 min to induce myocardial I/R injury. Heart rate (HR), left ventricular developed pressure (LVDP), the maximal increase rate of left ventricular pressure (+dp/dtmax) and the maximal decrease rate of left ventricular pressure (-dp/dtmax) were recorded by a physiological signal acquisition system. The heart treated with ischemic preconditioning (IPC) for 3 times at a range of 5 min/time before ischemia served as a positive control group. The hearts without I/R injury served as control group. After reperfusion, superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GSH-Px) activities in the myocardium were determined by appropriate assay kits. Myocardial SOD1 and glutamate cysteine ligase catalytic subunit (GCLC) expression were assessed by western blot analysis. For the in vitro study, SXSM serum was prepared according to the serum pharmacological method and neonatal rat cardiomyocytes were isolated from the heart of new born SD rats. Neonatal rat cardiomyocytes were pretreated with SXSM serum and subjected to H2O2 or anoxia/ reoxygenation (A/R) treatment to induce oxidative damage. Cell viability was evaluated using a Cell Counting Kit-8 (CCK8) assay. Levels of reactive oxygen species (ROS), SOD, GSH and GSH-Px in cardiomyocytes were determined by appropriate assay kits. SOD1 and GCLC expression were assessed by western blot analysis. Buthionine-[S, R]-sulfoximine (BSO), a GCLC inhibitor, and SOD1 siRNA were also used for identifying the cardiac protective targets of SXSM. RESULTS: SXSM and ischemic preconditioning (IPC) significantly increased heart rate during myocardial reperfusion and protected cardiac function against myocardial I/R injury, including an increase in left ventricular diastolic pressure (LVDP), the maximal increase rate of left ventricular pressure (+dp/dtmax) and the maximal decrease rate of left ventricular pressure (-dp/dtmax). We also found that SXSM and IPC improved the expansion of myocardial interstitium, the structural abnormality and morphological changes of cardiomyocytes induced by I/R injury. Meanwhile, SXSM protected cardiomyocytes against the oxidative damage induced by H2O2 and A/R injury through reducing intracellular ROS levels. Moreover, SXSM increased SOD activity through enhancing SOD1 expression and increased GSH content through promoting GCLC expression as well as GSH-Px activity. BSO and SOD1 siRNA counteracted anti-arrhythmic and cardiac protective effect of SXSM, suggesting that the therapeutic targets of SXSM might be SOD1 and GCLC. CONCLUSION: SXSM is effective in protecting the myocardium from I/R injury, with myocardial SOD1 and GCLC being the potential therapeutic targets.

An integration of fast alignment and maximum-likelihood methods for electron subtomogram averaging and classification.

Motivation: Cellular Electron CryoTomography (CECT) is an emerging 3D imaging technique that visualizes subcellular organization of single cells at sub-molecular resolution and in near-native state. CECT captures large numbers of macromolecular complexes of highly diverse structures and abundances. However, the structural complexity and imaging limits complicate the systematic de novo structural recovery and recognition of these macromolecular complexes. Efficient and accurate reference-free subtomogram averaging and classification represent the most critical tasks for such analysis. Existing subtomogram alignment based methods are prone to the missing wedge effects and low signal-to-noise ratio (SNR). Moreover, existing maximum-likelihood based methods rely on integration operations, which are in principle computationally infeasible for accurate calculation. Results: Built on existing works, we propose an integrated method, Fast Alignment Maximum Likelihood method (FAML), which uses fast subtomogram alignment to sample sub-optimal rigid transformations. The transformations are then used to approximate integrals for maximum-likelihood update of subtomogram averages through expectation-maximization algorithm. Our tests on simulated and experimental subtomograms showed that, compared to our previously developed fast alignment method (FA), FAML is significantly more robust to noise and missing wedge effects with moderate increases of computation cost. Besides, FAML performs well with significantly fewer input subtomograms when the FA method fails. Therefore, FAML can serve as a key component for improved construction of initial structural models from macromolecules captured by CECT. Availability and implementation: http://www.cs.cmu.edu/mxu1.

Shensong Yangxin capsules prevent ischemic arrhythmias by prolonging action potentials and alleviating Ca2+ overload.

Shensong Yangxin capsules (SSYX) are an effective traditional Chinese medicine that has been used to treat coronary heart disease clinically. The present study aimed to establish whether SSYX prevent ischemic arrhythmias in rats, and to explore the underlying mechanisms. Male rats were pretreated with distilled water, SSYX and amiodarone for one week. Acute myocardial ischemia (AMI) was performed to induce ischemic arrhythmias. The incidence and severity of ischemic arrhythmias were evaluated. The action potential, transient outward K+ current (Ito) and inward rectifier K+ current (IK1) of rat cardiomyocytes were measured using the patch­clamp technique. The intracellular Ca2+ concentration of the cardiomyocytes was measured using a laser scanning confocal microscope. The results revealed that SSYX lowered the incidence of arrhythmia markedly during AMI. Furthermore, SSYX delayed the appearance, and reduced the severity, of ischemic arrhythmias compared with the control. In addition, SSYX markedly decreased the ratio of the myocardial infarction region to the whole heart. In an in vitro study, SSYX prolonged the action potential duration of rat cardiomyocytes, and inhibited Ito and IK1 markedly. Additionally, SSYX inhibited Ca2+ elevation induced by KCl in cardiomyocytes. These results suggested that SSYX prevents ischemic arrhythmia, and the underlying mechanism responsible for this process may include prolonging the action potential and alleviating Ca2+ overload.

Jujuboside B Reduces Vascular Tension by Increasing Ca2+ Influx and Activating Endothelial Nitric Oxide Synthase.

Jujuboside B has been reported to have protective effect on many cardiovascular diseases. However, the effects of Jujuboside B on vascular tension and endothelial function are unknown. The present study investigated the effects of Jujuboside B on reducing vascular tension, protecting endothelial function and the potential mechanisms. The tension of isolated rat thoracic aorta ring was measured by Wire myograph system. The concentration of nitric oxide (NO) and the activity of endothelial nitric oxide synthase (eNOS) in human aortic endothelial cells (HAECs) were determined by Griess reagent method and enzyme-linked immune sorbent assay. The protein levels of eNOS and p-eNOS at Serine-1177 were determined by western blot analysis. Intracellular Ca2+ concentration in HAECs was measured by laser confocal imaging microscopy. Results showed that Jujuboside B reduced the tension of rat thoracic aorta rings with intact endothelium in a dose-dependent manner. L-NAME, KN93, EGTA, SKF96365, iberiotoxin and glibenclamide significantly attenuated Jujuboside B-induced vasodilation in endothelium-intact tissues. In contrast, indometacin and 4-DAMP had no such effects. Jujuboside B also promoted NO generation and increased eNOS activity, which were attenuated by L-NAME, EGTA and SKF96365. Moreover, Jujuboside B increased intracellular Ca2+ concentration dose-dependently, which was inhibited by EGTA and SKF96365. Besides, Jujuboside B induced a rapid Ca2+ influx instantaneously after depleting intracellular Ca2+ store, which was significantly inhibited by SKF96365. In conclusion, this study preliminarily confirmed that Jujuboside B reduced vascular tension endothelium-dependently. The underlying mechanisms involved that Jujuboside B increased extracellular Ca2+ influx through endothelial transient receptor potential cation (TRPC) channels, phosphorylated eNOS and promoted NO generation in vascular endothelial cells. In addition, Jujuboside B-induced vasodilation involved endothelium-dependent hyperpolarizaiton through endothelial potassium channels. Jujuboside B is a natural compound with new pharmacological effects on improving endothelial dysfunction and treating vascular diseases.

Construction of redox-active multilayer film for electrochemically controlled release.

An electrochemically controlled drug release from a redox-active multilayer film is reported. The multilayer film is fabricated by alternate assembly of the electrochemical redox-active micelles and DNA. The buildup of multilayer films is monitored by spectroscopic ellipsometry, UV-vis spectroscopy, and fluorescence spectroscopy. A ferrocene-modified poly (ethyleneimine) (PEI-Fc) is used to form a hydrophobic ferrocene core and hydrophilic PEI shell micelle, showing the electrochemical redox-active properties. Hydrophobic pyrene (Py) molecules are then incorporated into the micelles. The PEI-Fc@Py micelles are assembled into the (PEI-Fc@Py/DNA) multilayer film by layer-by-layer assembly. Thanks to ferrocene groups with the properties of the hydrophilic-to-hydrophobic switch based on the electrical potential trigger, pyrene molecules can be control released from the multilayer film. The electrochemically controlled release of pyrene is investigated and confirmed by electrochemical quartz crystal microbalance and electrochemistry workstation. The (PEI-Fc@drug/DNA) multilayer film may have potential applications in the field of biomedical and nanoscale devices.