Liping Meng

Polyphenols and Polypeptides in Chinese Rice Wine Inhibit Homocysteine-induced Proliferation and Migration of Vascular Smooth Muscle Cells.

Abstract: The beneficial effect of Chinese rice wine on atherosclerosis has been proved, but the exact components that have the cardiovascular protective effect are still unknown. This study aimed to explore the exact ingredients in Chinese rice wine that could inhibit homocysteine (Hcy)-induced vascular smooth muscle cell (VSMC) proliferation and migration. VSMCs were divided into 7 groups: control, Hcy (1 mmol/L), Hcy + oligosaccharide, Hcy + polypeptides, Hcy + polyphenols, Hcy + alcohol, and Hcy + Chinese rice wine. methyl thiazolyl tetrazolium (MTT) assay, Transwell chambers, and wound-healing assay were used to test the proliferation and migratory ability of the VSMCs. Western blot and gelatin zymography were used to investigate the expressions and activities of metal matrix proteinase 2/9 (MMP-2/9) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in VSMCs. Polypeptides and polyphenols in the Chinese rice wine reduced the proliferation and migration ability of the VSMCs. Furthermore, they also decreased the expression and activity of MMP-2/9 but had no obvious impact on the expression of TIMP-2 in each group. This study further confirms that polypeptides and polyphenols in the Chinese rice wine could inhibit Hcy-induced proliferation and migration of VSMCs and maintain the balance between matrix metalloproteinases (MMPs) and TIMPs.

Chinese Yellow Wine Could Inhibit Production of Matrixmetalloproteinase-2 Induced by Homocysteine in Cultured Rat Vascular Endothelial Cells

Background: The effects of Chinese yellow wine on the production of homocysteine (Hcy) induced intracellular MMP-2 in cultured rats vascular endothelial cells (VECs) has not been investigated. Methods: Isolation, cultivation, purification and identification of vascular endothelial cells of rat thoracic aorta in vitro were conducted. The VECs in passages 3 to 4 were used in all studies. HCY was used to induce VECs to over expressing MMP-2. Cells were divided into 5 groups: Control, Hcy, Hcy+yellow wine, Hcy+red wine, Hcy+ethanol and the cells were given different treatment for 48 h. The mRNA expression of MMP-2 was detected by FQ-PCR. The western blotting and gelatin zymography were applied to test the protein levels and the enzymatic activity of MMP-2. Results: Hcy could significantly increase the expression and activity of MMP-2 compared with the control group, and could reach the maximum at 500 μ mol/L, cultured for 48 h. Compared with those in Hcy group, the expression and activity of MMP-2 in yellow wine and red wine groups was significantly decreased. No significant difference was shown as between the ethanol group and the Hcy group and no significant discrepancy between the yellow wine and red wine group was found. Conclusions: The result suggest that Hcy promotes the expression and activity of MMP-2, which may play an important role in pathogenesis of atherosclerosis (AS). Treatment with yellow wine or red wine decreases Hcy-induced MMP-2 production in VECs. The attenuation of MMP-2 activation by yellow wine and red wine might contribute to their beneficial effects on the cardiovascular system.

The differential diagnostic value of serum homocysteine for white coat hypertension

Objective To assess the value of serum homocysteine (Hcy) in differential diagnosis of white coat hypertension (WCH). Results In this retrospective study, serum Hcy levels were elevated in hypertensive patients (P < 0.001) compared to WCH patients. Serum Hcy levels were positively correlated with 24-h mean systolic blood pressure, r = 0.1378, P < 0.001. The results of the receiving operating characteristic (ROC) curve showed that the AUC value of Hcy was 0.80 (95% CI, 0.77–0.83), the cut-off value was 13.8 μmol/L, the sensitivity was 68.58% and the specificity 87.21%. In the prospective study, the AUC value of Hcy was 0.73 (95% CI: 0.67–0.78), higher than N - terminal pro - brain natriuretic peptide(NT-pro-BNP) (0.64, 95% CI:0.58–0.70) and cystatin C (Cys-C) (0.62, 95% CI:0.55–0.68). Hcy, NT-proBNP and Cys-C combined, provided a better indication of a differential diagnosis of WCH, than Hcy alone. Materials and Methods This investigation involved both a retrospective and a prospective study. Clinical data including blood pressure, age, sex, height, weight, BMI, smoking status, past history, and behavioral electrocardiogram of patients who had undergone 24-hour ambulatory blood pressure monitoring (ABPM) with elevated clinical blood pressure (BP) were recorded. Pearson correlation analysis was used to test the correlation between Hcy and BP. The ROC curve was used to analyze the value of measuring Hcy levels in differential diagnosis of WCH. Conclusions Serum Hcy was decreased in WCH patients and therefore could be a biomarker for differential diagnosis of WCH.

Primary Culture of Rat Aortic Vascular Smooth Muscle Cells: A New Method

Background Developing a simple and efficient method of obtaining primary cultured VSMCs is necessary for basic cardiovascular research. Material/Methods The procedure of our new method mainly includes 6 steps: isolation of the aortic artery, removal of the fat tissue around the artery, separation of the media, cutting the media into small tissue blocks, transferring the tissue blocks to cell culture plates, and incubation until the cells reach confluence. The cells were identified as VSMCs by morphology and immunofluorescence. Then, VSMCs obtained by this new tissue explants method, the traditional tissue explants method, the enzyme digestion method, and A7r5 cell line were divided into 4 groups. The purity of cells was test by multiple fluorescent staining. Western blotting was used to investigate the phenotype of VSMCs obtained by different methods. Results Cells began to grow out at about 8 days and became relatively confluent within 16 days. Compared with VSMCs from the traditional tissue explants method and enzyme digestion method or A7r5 cell line, VSMCs obtained by our method showed higher purity and manifested a more “contractile” phenotype characteristic. Conclusions We have conquered the disadvantages in the previous primary culture methods and established a simple and reliable way to isolate and culture rat aortic VSMCs with high purity and stability.

Effects of Chinese yellow wine on nitric oxide synthase and intercellular adhesion molecule-1 expressions in rat vascular endothelial cells.

Objective The objective of this study was to determine similarities in the effect of yellow wine as compared to statin and the possibility that yellow wine inhibits tumour necrosis factor- α(TNF-α)-induced nitric oxide (NO) synthesis, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1) in cultured rat vascular endothelial cells (VECs). Methods We isolated VECs, and cultivated and purified Sprague Dawley (SD) rat thoracic aortas in vitro. We selected the optimal wine concentration using clonogenic and MTT assays to measure cell survival. Next, we divided the cells into 9 groups: (1) control, (2) TNF-α, (3) TNF- α+ rosuvastatin (10 μmol/L), (4) TNF- α+ ethanol 0.5%, (5) TNF- α+ yellow wine 0.5%, (6) TNF- α+ ethanol 1.0%, (7) TNF- α+ yellow wine 1.0%, (8) TNF- α+ ethanol 1.5%, and (9) TNF- α+ yellow wine 1.5% and they were given the corresponding treatment for 24 h. We determined NO production with nitrate reductase. We then measured eNOS activity, and detected eNOS, iNOS, and ICAM-1 protein levels by Western blotting. Results Compared with the TNF-á group, NO production, eNOS activity, and eNOS protein expression in the rosuvastatin, and yellow wine 1.0%, and 1.5% groups were significantly increased. Protein expression of iNOS and ICAM-1 in the rosuvastatin, yellow wine 1.0%, and 1.5% groups were significantly decreased. Compared with the rosuvastatin group, eNOS, iNOS, and ICAM-1 protein expression in the yellow wine (0.5% -1.5%) groups were significantly different. Conclusion Treatment with yellow wine increased NO production, eNOS activity, and eNOS protein expression, which decreases iNOS and ICAM-1 protein expression. We conclude that yellow wine may have similar beneficial effects as rosuvastatin on the cardiovascular system. These effects may be attributed to their anti-atherosclerotic actions.

Folic acid delays development of atherosclerosis in low‐density lipoprotein receptor‐deficient mice

Many studies support the cardioprotective effects of folic acid (FA). We aimed to evaluate the utility of FA supplementation in preventing the development of atherosclerotic in low‐density lipoprotein receptor‐deficient (LDLR−/−) mice and to elucidate the molecular processes underlying this effect. LDLR−/− mice were randomly distributed into four groups: control group, HF group, HF + FA group and the HF + RAPA group. vascular smooth muscle cells (VSMCs) were divided into the following four groups: control group, PDGF group, PDGF + FA group and PDGF + FA + RAPA group. Blood lipid levels, oxidative stress and inflammatory cytokines were measured. Atherosclerosis severity was evaluated with oil red O staining. Haematoxylin and eosin (H&E) staining was used to assess atherosclerosis progression. Immunohistochemical staining was performed with antismooth muscle α‐actin (α‐SMA) antibodies and anti‐osteopontin (OPN) antibodies that demonstrate VSMC dedifferentiation. The protein expression of α‐SMA, OPN and mechanistic target of rapamycin (mTOR)/p70S6K signalling was detected by Western blot analysis. FA and rapamycin reduced serum levels of total cholesterol, triacylglycerol, LDL, inhibiting oxidative stress and the inflammatory response. Oil red O and H&E staining demonstrated that FA and rapamycin inhibited atherosclerosis. FA and rapamycin treatment inhibited VSMC dedifferentiation in vitro and in vivo, and FA and rapamycin attenuated the mTOR/p70S6K signalling pathway. Our findings suggest that FA attenuates atherosclerosis development and inhibits VSMC dedifferentiation in high‐fat‐fed LDLR−/− mice by reduced lipid levels and inhibiting oxidative stress and the inflammatory response through mTOR/p70S6K signalling pathway.

Knockdown of Herp alleviates hyperhomocysteinemia mediated atherosclerosis through the inhibition of vascular smooth muscle cell phenotype switching

BACKGROUND Phenotypic switching of vascular smooth muscle cells (VSMCs) plays a key role in atherosclerosis. We aimed to investigate whether Homocysteine-responsive endoplasmic reticulum protein (Herp) was involved in VSMC phenotypic switching and affected atheroprogression. METHODS To assess the role of Herp in homocysteine (Hcy)-associated atherosclerosis, Herp-/- and LDLR-/- double knockout mice were generated and fed with a high methionine diet (HMD) to induce Hyperhomocysteinemia (HHcy). Atherosclerotic lesions, cholesterol homeostasis, endoplasmic reticulum (ER) stress activation, and the phenotype of VSMCs were assessed in vivo. We used siRNAs to knockdown Herp in cultured VSMCs to further validate our findings in vitro. RESULTS HMD significantly activated the activating transcription factor 6 (ATF6)/Herp arm of ER stress in LDLR-/- mice, and induced the phenotypic switch of VSMCs, with the loss of contractile proteins (SMA and calponin) and an increase of OPN protein. Herp-/-/LDLR-/- mice developed reduced atherosclerotic lesions in the aortic sinus and the whole aorta when compared with LDLR-/- mice. However, Herp deficiency had no effect on diet-induced HHcy and hyperlipidemia. Inhibition of VSMC phenotypic switching, decreased proliferation and collagen accumulation were observed in Herp-/-/LDLR-/- mice when compared with LDLR-/- mice. In vitro experiments demonstrated that Hcy caused VSMC phenotypic switching, promoted cell proliferation and migration; this was reversed by Herp depletion. We achieved similar results via inhibition of ER stress using 4-phenylbutyric-acid (4-PBA) in Hcy-treated VSMCs. CONCLUSION Herp deficiency inhibits the phenotypic switch of VSMCs and the development of atherosclerosis, thus providing novel insights into the role of Herp in atherogenesis.