Xinping Luo

Salvianolic acid B inhibits platelets as a P2Y12 antagonist and PDE inhibitor: Evidence from clinic to laboratory

Salviae miltiorrhiza (Danshen) has been used for thousands of years in China and some other Asian countries to treat atherothrombotic diseases. Salvianolate which consists of three water-soluble ingredients purified from Salviae miltiorrhiza, has been approved by Chinese SFDA to treat coronary artery disease. So far, there is no evidence clearly showing the clinical efficiency of salvianolate and the underlying mechanism. This study is to evaluate the effects of salvianolate on platelets in patients with acute coronary syndrome and explore the underlying mechanism. We evaluated the effects of salvianolate on platelets in patients with acute coronary syndrome by measuring ADP-induced PAC-1 binding and P-selectin expression on platelets. Salvianolate significantly potentiated the antiplatelet effects of standard dual antiplatelet therapy. We also investigated the antiplatelet effects of salvianolatic acid B (Sal-B), the major component which composes 85% of salvianolate. Sal-B inhibits human platelet activation induced by multiple agonists in vitro by inhibiting phosphodiesterase (PDE) and antagonizing P2Y12 receptor. For the first time, we show the antiplatelet efficiency of salvianolate in ACS patients undergoing treatment with clopidogrel plus aspirin, and demonstrate that Sal-B, the major component of salvianolate inhibits human platelet activation via PDE inhibition and P2Y12 antagonism which may account for the clinical antiplatelet effects of salvianolate. Our results suggest that Sal-B may substitute salvianolate for clinical use.

Plasma Cathepsin L and Its Related Pro/Antiangiogenic Factors Play Useful Roles in Predicting Rich Coronary Collaterals in Patients with Coronary Heart Disease

Cathepsin L enhances angiogenesis by increasing extracellular matrix degradation and remodelling. This study investigated whether plasma cathepsin L could be used as a biomarker to predict collateral formation in patients with coronary heart disease (CHD). Patients with CHD (n = 218; aged 67 ± 11 years) underwent coronary angiography and were categorized as having either ‘poor’ or ‘rich’ collaterals. Plasma cathepsin L, the proangiogenic placenta growth factor (PLGF) and the antiangiogenic factors, cystatin C and endostatin, were measured. Elevated cathepsin L and PLGF levels were independently and significantly associated with enhanced collateral formation in patients with CHD; subgroup analyses also showed a significant correlation in patients with diabetes and acute coronary syndrome. Plasma endostatin and cystatin C levels were not significantly correlated with coronary collateral formation. Plasma cathepsin L and PLGF, acting as important modulators of angiogenesis, could be used as biomarkers to predict coronary collateral formation in patients with CHD.

Tissue factor pathway inhibitor-2 is downregulated by ox-LDL and inhibits ox-LDL induced vascular smooth muscle cells proliferation and migration

INTRODUCTION Tissue factor pathway inhibitor-2 (TFPI-2) is a member of the Kunitz-type family of serine protease inhibitors, which inhibits several matrix metalloproteinases activity involved in extracellular matrix degradation. Studies have shown low TFPI-2 expression in the shoulder regions of atherosclerotic plaques. But studies evaluating its role in the progression of atherosclerotic plaque are scarce. Vascular smooth muscle cells (VSMCs) are important components of atherosclerotic plaques and oxidized low density lipoprotein (ox-LDL) is an important detrimental factor of atherosclerosis. The aim of this study is to elucidate the effect of TFPI-2 on smooth muscle cell proliferation and migration induced by ox-LDL. METHODS Retroviruses expressing human TFPI-2 were constructed. Cell proliferation was determined by CCK-8 assay. Cell apoptosis was analyzed by double staining of FITC-Annexin V and propidium iodide. Cell migration was studied through a Transwell chamber and with a scratch-wound assay. The matrix metalloproteinase-2 and -9 activities were analyzed by gelatin zymography. Phosphorylation of FAK was analyzed by western blot. RESULTS TFPI-2 over-expression of mRNA and protein was confirmed in infected cells. CCK-8 assay showed that TFPI-2 inhibit VSMCs proliferation induced by ox-LDL while without cytotoxicity to VSMCs. Transwell and scratch wound assay confirmed TFPI-2 over-expression can inhibit VSMC migration. Zymography assay showed that TFPI-2 can inhibit MMP-2, 9 activity induced by ox-LDL. Western blot assay showed TFPI-2 can inhibit cyclinD1 expression and FAK phosphorylation. CONCLUSION TFPI-2 over-expression may strongly inhibit the proliferation and migration of VSMCs and suppresses MMP-2, 9 activity induced by ox-LDL, making it a promising candidate for treatment of atherosclerotic process.

The effects of simvastatin on angiogenesis: studied by an original model of atherosclerosis and acute myocardial infarction in rabbit

Statins have shown pleiotropic effects, many of them independent of their impact on lipids. Angiogenesis can be beneficial in the acute myocardial infarction to improve circulation. However, it also can be harmful due to worsening of atherosclerosis. Here, we established a new minimal invasive rabbit model to study ischemic myocardium and atherosclerosis together to mimic clinical scenario. We demonstrated that simvastatin has the effect of pro-angiogenesis and further improve cardiac function in ischemic myocardium, as well as the effect of anti-angiogenesis to improve atherosclerosis in aorta vessels.

Cardiac matrix remodeling following intracoronary cell transplantation in dilated cardiomyopathic rabbits

Cellular cardiomyoplasty has been proposed as a promising therapeutic strategy for chronic heart failure. Previous studies focused on structural changes in cardiomyocytes to explain the potential benefits for contractile function. However, limited information is available about the cardiac matrix remodeling following cell transplantation in dilated cardiomyopathy (DCM). Here, we established a new animal model of intracoronary bone marrow mononuclear cells (BMMNCs) transplantation to explore extracellular matrix remodeling in adriamycin-induced cardiomyopathic rabbits. In vivo studies demonstrated that BMMNCs transplantation can dramatically delay the progress of collagen metabolism and decrease myocardial collagen volume fraction. The beneficial effects were mediated by attenuating stress-generated over-expression of matrix metalloproteinases (MMPs) in ventricular remodeling. Improved cardiac function may be contributed in part by stem-associated inhibition of extracellular matrix remodeling.

Recombinant TFPI-2 enhances macrophage apoptosis through upregulation of Fas/FasL.

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor with inhibitory activity toward activated factor XI, plasma kallikrein, plasmin, certain matrix metalloproteinases, and the tissue factor-activated factor VII complex. In addition, TFPI-2 has other functions such as promoting cell migration and inducing apoptosis. In the present study, we investigated if TFPI-2 induced apoptosis in cultured U937-derived macrophages and the possible signal pathways that involved in the apoptotic process. Apoptotic DNA fragment detection and caspase-3,9 activity measurements indicated that rTFPI-2 promoted U937-derived macrophage apoptosis. Hoechst 33342 assay and flow cytometry further showed that rTFPI-2 induced apoptosis in cultured macrophages in a dose-dependent manner. Because death receptors of the TNF family such as Fas are the best-understood death pathways that recruit Fas-associated death domain (FADD) and procaspase-8 to the receptor in macrophages, we investigated the expression of Fas and its ligand (FasL) and downstream signal caspase-8 by Western blot analysis. The results indicated that the process of apoptosis triggered by rTFPI-2 was, at least in part, actively conducted by U937-derived macrophages possibly through Fas/FasL signal pathway. In brief, rTFPI-2 may have the potential usefulness in inducing macrophages apoptosis, which suggest TFPI-2 might have antiatherogenic effects.

Platelets Express Activated P2Y12 Receptor in Patients With Diabetes Mellitus.

Background: Platelets from patients with diabetes mellitus are hyperactive. Hyperactivated platelets may contribute to cardiovascular complications and inadequate responses to antiplatelet agents in the setting of diabetes mellitus. However, the underlying mechanism of hyperactivated platelets is not completely understood. Methods: We measured P2Y12 expression on platelets from patients with type 2 diabetes mellitus and on platelets from rats with diabetes mellitus. We also assayed platelet P2Y12 activation by measuring cAMP and VASP phosphorylation. The antiplatelet and antithrombotic effects of AR-C78511 and cangrelor were compared in rats. Finally, we explored the role of the nuclear factor-&kgr;B pathway in regulating P2Y12 receptor expression in megakaryocytes. Results Platelet P2Y12 levels are 4-fold higher in patients with type 2 diabetes mellitus compared with healthy subjects. P2Y12 expression correlates with ADP-induced platelet aggregation (r=0.89, P<0.01). P2Y12 in platelets from patients with diabetes mellitus is constitutively activated. Although both AR-C78511, a potent P2Y12 inverse agonist, and cangrelor have similar antiplatelet efficacy on platelets from healthy subjects, AR-C78511 exhibits more powerful antiplatelet effects on diabetic platelets than cangrelor (aggregation ratio 36±3% versus 49±5%, respectively, P<0.05). Using a FeCl3-injury mesenteric arteriole thrombosis model in rats and an arteriovenous shunt thrombosis model in rats, we found that the inverse agonist AR-C78511 has greater antithrombotic effects on GK rats with diabetes mellitus than cangrelor (thrombus weight 4.9±0.3 mg versus 8.3±0.4 mg, respectively, P<0.01). We also found that a pathway involving high glucose-reactive oxygen species-nuclear factor-&kgr;B increases platelet P2Y12 receptor expression in diabetes mellitus. Conclusions Platelet P2Y12 receptor expression is significantly increased and the receptor is constitutively activated in patients with type 2 diabetes mellitus, which contributes to platelet hyperactivity and limits antiplatelet drug efficacy in type 2 diabetes mellitus.

Over-expression of TFPI-2 promotes atherosclerotic plaque stability by inhibiting MMPs in apoE−/− mice

MMPs in apoE−/− mice Junjie Pan , Duan Ma , Feng Sun , Wang Liang , Rui Liu , Wei Shen , Huijun Wang , Yong Ji , Rui Hu , Rongle Liu , Xinping Luo ⁎, Haiming Shi a,⁎ a Department of Cardiovascular Medicine, Huashan Hospital Affiliated to Fudan University, 12 Middle Urumqi Road, Building 3, Room 1602, Shanghai, 200040, China b Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai Medical College, Fudan University, Shanghai 200032, China c ICU, Nanjing Government Hospital, Nanjing, China d Key Laboratory of Cardiovascular Disease and Molecular Intervention, Key Laboratory of Human Functional Genomics, Atherosclerosis Research Centre, Nanjing Medical University, Nanjing, China e Department of Biostatistics and Computational Biology, University of Rochester, Rochester, NY, USA

Modulating Autophagy Improves Cardiac Function in a Rat Model of Early-Stage Dilated Cardiomyopathy

Objectives: Previous studies reported that autophagy is activated in human dilated cardiomyopathy (DCM). It is still unknown whether modulating autophagy can improve cardiac function of the failing heart. Methods: We immunized rats with porcine cardiac myosin to set up a model of DCM. Rapamycin, a kind of mTOR inhibitor upregulating autophagy, was given to rats weeks after the immunization at low (1 mg/kg · day i.p.), intermediate (2 mg/kg · day i.p.) and high dose (4 mg/kg · day i.p.) for 2 weeks. Results: Compared to the control group (ejection fraction, EF = 81.3 ± 3.8%), the average EF decreased in both the DCM group (EF = 56.1 ± 3.3%) and the high-dose rapamycin group (EF = 55.9 ± 3.6%), but recovered in the low-/intermediate-dose rapamycin groups (EF = 64.9 ± 4.6/69.4 ± 4.4%). Phosphorylation of p70s6k and 4E-BP1 decreased and the expression of LC3BI/II increased in all rapamycin groups. Autophagic vacuoles were easily found in these groups. However, body weight was significantly reduced in the rapamycin groups. Furthermore, mortality was increased in the high-dose rapamycin group. Conclusions: Rapamycin could improve cardiac function of early-stage DCM, but the effect of rapamycin turned out to be biphasic and the effective range appeared narrow.

Growth differentiation factor 15 and coronary collateral formation.

The coronary collateral circulation can reduce sudden cardiac death,myocardial cell loss,and infarct size.Growth differentiation factor 15(GDF‐15),a member of the transforming growth factor‐β (TGF‐β) superfamily,has been reported to have a prognostic predicting value in coronary artery disease.