Effects of Rosuvastatin on Fibrinolytic System of Human Umbilical Vein Endothelial Cells In Vitro Fei He, MD, Jing Zhao, MD, Rong Guo, MD and Ying Liang, MD
Abstract: Background: Besides its lipid-lowering effect, rosuvastatin has an antithrombotic effect, the exact underlying mechanism of which is still unclear. The aim of this study was to investigate the effects of rosuvastatin on the ﬁbrinolytic system, including tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) with its receptor (u-PAR) and plasminogen activator inhibitor-1 (PAI1), in vascular endothelial cells exposed to oxidized low-density lipoprotein (oxLDL). Methods: Human umbilical vein endothelial cells (HUVEC) were cultured and divided into 7 groups: control, rosuvastatin alone (10 nM), oxLDL alone (100 mg/L), oxLDL plus rosuvastatin (0.1, 1.0 and 10 nM, respectively) and oxLDL plus rosuvastatin (10 nM) with mevalonate (100 mM). The lactate dehydrogenase activity and concentrations of t-PA, u-PA, u-PAR and PAI-1 protein in culture medium were measured, whereas the expressions of t-PA, u-PA, u-PAR and PAI-1 mRNA in endothelial cells were detected by real-time polymerase chain reaction at 24 hours after treatment. Results: Compared with the control group, oxLDL caused a signiﬁcant increase in lactate dehydrogenase activity. It could reduce the expression of t-PA mRNA and protein (P , 0.05) and increase the expression of PAI-1 mRNA and protein (P , 0.05). Rosuvastatin could protect the endothelial cells, improve t-PA production and reduce PAI-1 production (P , 0.05), whether in unstimulated HUVEC or in HUVEC exposed to oxLDL. The effects of rosuvastatin on the ﬁbrinolytic system could be reversed by mevalonate. No signiﬁcant differences in u-PA and u-PAR production were seen among different groups. Conclusions: Rosuvastatin has protective effects on oxLDL-induced damaged human vascular endothelial cells; its antithrombotic effects may be mediated by the regulation of the ﬁbrinolytic system. Key Indexing Terms: Rosuvastatin; Fibrinolysis; Vascular endothelium. [Am J Med Sci 2014;348(4):319–323.]
high level of low-density lipoprotein cholesterol (LDL-C) in plasma is known as a very important risk factor for atherosclerosis.1 Statins are widely used to lower cholesterol levels by inhibiting 3-hydroxy-3-methyl-glutaryl-CoA (HMGCoA) reductase, which plays a central role in the production of From the Department of Cardiology (FH, JZ, YL), The First Afﬁliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China; and Department of Hematology (RG), The First Afﬁliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China. Submitted July 20, 2013; accepted in revised form January 28, 2014. This study was supported by China Scholarship Council (No. 2011841094), Henan Provincial Medical Scientiﬁc Research Fund (Nos. 2011020012 and 201203041) and Henan Provincial Health Technology Innovation Talent Project: Young and Middle-aged Technology Innovation Talent Project, National Natural Science Foundation of China (Nos. 81070445 and 30972790), Guangdong Natural Science Funds (No. 06020896), Guangdong Provincial Medical Science Research Funds (No. A2010021), the Key Scientiﬁc Research Project of Henan Education Department (No. 13A320443) and the Youth Fund of the First Afﬁliated Hospital of Zhengzhou University (No. 20110901). The ﬁrst 2 authors contributed equally to this work as co-ﬁrst authors. The authors have no ﬁnancial or other conﬂicts of interest to disclose. Correspondence: Fei He, MD, Department of Cardiology, The First Afﬁliated Hospital of Zhengzhou University, No.1 Jianshe East Road, Zhengzhou, Henan Province 450052, China (E-mail: [email protected]
The American Journal of the Medical Sciences
cholesterol in the liver.2 Numerous large clinical trials3,4 have shown that statins can reduce the risk of major cardiovascular events and improve the survival of patients with cardiovascular disease. Many studies in recent years5,6 have also suggested that statins have pleiotropic effects independent of reductions in LDL-C, such as improvements in endothelial function, decreases in smooth muscle cell proliferation and vascular inﬂammation. The pleiotropic effects of statins may play an important role in reducing cardiovascular morbidity and mortality.5,6 Rosuvastatin is a novel statin that was nicknamed “superstatin” because its ability to reduce LDL-C is greater than that of previously available statins.7 A few studies8,9 have also suggested that rosuvastatin has antithrombotic effects besides its lipid-lowering effect. Patterson et al9 believed that rosuvastatin might prevent thrombosis through its effects on the ﬁbrinolytic system. Normal vascular endothelial cells play a very important role in the prevention of thrombosis by regulating the ﬁbrinolytic system,10,11 which is mainly composed of tissuetype plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) with its receptor (u-PAR) and plasminogen activator inhibitor (PAI-1).12 Vascular endothelial cells are the major sites of t-PA, u-PA and PAI-1 production. t-PA and u-PA can convert the inactive zymogen plasminogen into the active protease plasmin to promote blood clot lysis, whereas the protein PAI-1 can form the inactive complexes by binding with t-PA and u-PA. These inactive complexes can prevent plasminogen activation and thus blood clot lysis.12 However, reports about the effects of rosuvastatin on t-PA, u-PA, u-PAR and PAI-1 in vascular endothelial cells are few. In this study, our aim was to investigate the effects of rosuvastatin on t-PA, u-PA, u-PAR and PAI-1 in vascular endothelial cells exposed to oxidized LDL (oxLDL).
METHODS All experimental protocols adhered to rules for the protection of human subjects, and the collection of informed consent was reviewed and approved by the ethics committee of the First Afﬁliated Hospital of Zhengzhou University. Isolation and Oxidation of LDL According to previous experiments,13 human blood sample was obtained from healthy subjects in the First Afﬁliated Hospital of Zhengzhou University, and native LDL was isolated by the ultracentrifugation method. Oxidized LDL was obtained from native LDL (1 mg protein/mL) and CuSO4 (1 mmol/L) in PBS coincubated for 24 hours at 23°C. The degree of oxidation was determined by the relative mobility between oxLDL and native LDL on agarose gel electrophoresis. The mobility of oxLDL on agarose gel electrophoresis was 3.5-fold to 4.0-fold compared with that of native LDL. Human Umbilical Vein Endothelial Cell Culture The protocol for harvesting and culturing human umbilical vein endothelial cell (HUVECs) was according to a previous study.14 HUVECs were cultured in RPMI 1640 medium
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(Gibco, New York, NY) with 10% fetal bovine serum (Gibco, New York, NY), 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C in a fully humidiﬁed atmosphere of 5% CO2. Treatment Groups Twenty-four hours before the experiment, HUVECs from 2 to 4 passages were inoculated in 6-well plates (density: 2 3 105 cells/well). These were then divided into 7 groups (n 5 6): control, rosuvastatin alone (10 nM), oxLDL alone (100 mg/L), oxLDL plus low-dose rosuvastatin (0.1 nM rosuvastatin), oxLDL plus medium-dose rosuvastatin (1.0 nM rosuvastatin), oxLDL plus high-dose rosuvastatin (10 nM rosuvastatin) and oxLDL plus rosuvastatin (10 nM) with mevalonate (100 mM). Rosuvastatin was obtained from AstraZeneca Pharmaceutical Company (London, United Kingdom). Mevalonate was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). The culture medium was collected, and endothelial cells were harvested at 24 hours after treatment. Measurement of Lactate Dehydrogenase Activity HUVEC injury was evaluated through the lactate dehydrogenase (LDH) activity leakage from cells. The LDH activity in the culture medium was detected by the enzymatic dynamics method. Real-Time Polymerase Chain Reaction The total RNA was collected from HUVECs and then converted to cDNA according to a previous report.15 Quantitative real-time polymerase chain reaction (PCR) was established for quantiﬁcation of mRNA expressions of t-PA, u-PA, u-PAR and PAI-1. Real-time PCR reactions were performed with the use of the SYBR Green Master Mix (Bio-Rad, Hercules, CA) in an iCycler PCR system (Bio-Rad). All samples were run in triplicate, and beta-actin was used as internal control. The following primers were used: (1) t-PA (encoding gene: PLAT) forward: 59-CTCTGCTGTGTGCTGCTGCT-39, reverse: 59-GCGCTGCT GTTCCAGTTGGT-39; (2) u-PA (encoding gene: PLAU) forward: 59-GGAGATGAAGTTTGAGGTGG-39, reverse: 59-GGTCTGTA TAGTCCGGGATG-39; (3) u-PAR (encoding gene: PLAUR) forward: 59-CACAAAACTGCCTCCTTCCT-39, reverse: 59-AAT CCCCGTTGGTCTTACAC-39; (4) PAI-1 (encoding gene SERPINE1) forward: 59-GGCCATGCTCCAGCTGACAA-39, reverse: 59-CATTCACCAGCACCAGCCGT-39 and (5) betaactin (encoding gene: ACTB) forward: 59-GCCATGTACGTTG CTATCCA-39, reverse: 59-CATCTCTTGCTCGAAG-39. The target mRNA expression was normalized to the beta-actin expression and expressed as a relative value through the comparative threshold cycle (Ct) method (22DDCt) according to the manufacturer’s instructions. Measurement of t-PA, u-PA, u-PAR and PAI-1 Protein Culture Medium The concentrations of t-PA, u-PA, u-PAR and PAI-1 protein were measured by enzyme-linked immunosorbent assay. t-PA and PAI-1 assay kits were purchased from Shanghai Sun Biological Products Co Ltd (Shanghai, China). Assay kits for u-PA and u-PAR were obtained from R&D Systems (Minneapolis, MN). Statistical Analysis The results were expressed as mean 6 SD. Multiple comparisons were analyzed by analysis of variance, and comparisons between 2 groups were evaluated by the StudentNewman-Keuls post hoc test. P values less than 0.05 were considered statistically signiﬁcant.
RESULTS Lactate Dehydrogenase Activity Rosuvastatin alone did not affect LDH activity. When compared with the control group, the LDH activity in the oxLDL alone group increased signiﬁcantly (P , 0.05). After rosuvastatin treatment, LDH activities decreased dramatically (P , 0.05; Figure 1). This phenomenon could be observed with a low or high dose of rosuvastatin. Mevalonate (100 mM) could reverse the effect of rosuvastatin on LDH activity (P , 0.05). Real-Time PCR Rosuvastatin could increase the expression of t-PA mRNA and decrease the expression of PAI-1 mRNA (P , 0.05, respectively) in unstimulated HUVECs. Compared with the control group, oxLDL could cause a dramatic decrease in t-PA mRNA expression and a signiﬁcant increase in PAI-1 mRNA expression (P , 0.05, respectively). Rosuvastatin could signiﬁcantly inhibit the effects of oxLDL on expressions of t-PA and PAI-1 mRNA (P , 0.05, respectively); even lowdose rosuvastatin had this effect. Mevalonate (100 mM) could reverse the effects of rosuvastatin on t-PA and PAI-1 mRNA (P , 0.05, respectively) (Figure 2). For u-PA and u-PAR mRNA, no signiﬁcant differences among the different groups were observed. Concentrations of t-PA, u-PA, u-PAR and PAI-1 Protein In accordance with the results of real-time PCR, rosuvastatin alone could increase the concentration of t-PA and decrease the concentration of PAI-1 (P , 0.05, respectively) in unstimulated HUVECs. Compared with the control group, oxLDL could not only signiﬁcantly decrease the concentration of t-PA but also dramatically increase PAI-1 concentration (P , 0.05, respectively). Similarly, rosuvastatin doses ranging from low to high could signiﬁcantly reverse the effects of oxLDL on t-PA and PAI-1 concentrations (P , 0.05, respectively) (Figures 3A and 3B). Mevalonate (100 mM) could reverse the effects of rosuvastatin on t-PA and PAI-1 concentrations (P , 0.05, respectively). There were no signiﬁcant differences in u-PA and u-PAR protein among different groups (Figures 3C and 3D).
DISCUSSION HMG-CoA reductase inhibitors or statins are the most effective agents currently available for hyperlipidemia.2 Large clinical trials3,4 have shown that statins reduce not only the incidence of cardiovascular events but also the mortality in patients with high or “average” cholesterol levels. Beyond their lipid-lowering capacity, statins have been shown to have multiple “vasoprotective” effects, such as improvements in endothelial function and prevention of thrombus formation.5,6 Rosuvastatin is a novel synthetic statin that has a greater number of binding interactions with HMG-CoA reductase and a higher afﬁnity for the active site of the enzyme when compared with other statins.16 Therefore, its lipid-lowering ability is the best among currently available statins. Clinical trials17,18 have shown the advantages of rosuvastatin in therapy for atherosclerosis. A few studies8,9 have also shown that rosuvastatin can prevent arterial and venous thrombosis through its non–lipid-lowering effects. Yanagi et al19 and Qu et al20 found that rosuvastatin could lower the PAI-1 level in patients with diabetes or hypercholesterolemia. Patterson et al9 showed that rosuvastatin could reduce deep vein thrombosis in ApoE gene– deleted mice hyperlipidemia by decreasing the PAI-1 level. Volume 348, Number 4, October 2014
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FIGURE 1. Comparison of LDH activity expressions in different groups. Values are mean 6 SD (n 5 6); Ros, rosuvastatin; M, mevalonate; *P , 0.05 versus control, #P , 0.05 versus oxLDL alone, DP , 0.05 versus oxLDL plus high-dose rosuvastatin.
These studies9,19,20 suggested that rosuvastatin may prevent thrombosis through its effects on the ﬁbrinolytic system. Vascular endothelial cells are the major sites for t-PA, u-PA and PAI-1 production.21 However, the exact effect of rosuvastatin on the ﬁbrinolytic system in vascular endothelial cells is still unclear. Therefore, in this study, we investigated the effects of rosuvastatin on t-PA, u-PA, u-PAR and PAI-1 in vascular endothelial cells exposed to oxLDL. At present, it is widely accepted that endothelial dysfunction plays a critical role in the development of atherosclerosis and thrombosis disease1 because vascular endothelial cells can not only keep the blood vessel intact but also secrete a large amount of active substances, such as nitric oxide, t-PA, u-PA and PAI-1.1,22 A high level of LDL-C is an important risk factor for atherosclerosis because LDL-C can be converted to oxLDL, which can damage vascular endothelial cells.1,23 Previous research has shown that oxLDL could injure
vascular endothelial cells ex vivo and in vivo.24,25 Following past studies,26 including our previous experiment,27 we used 100 mg/L oxLDL to cause injury to HUVECs in this study. LDH activity is a marker of cell damage.28 The results showed that oxLDL could cause an increase in LDH released from endothelial cells, whereas rosuvastatin could protect vascular endothelial cells, whether in low- or high-dose rosuvastatin groups. The ﬁbrinolytic system participates not only in thrombus formation but also in atherosclerosis progression. t-PA and u-PA can cause thrombus ﬁbrinolysis, whereas PAI-1 can lead to thrombus formation by inhibiting t-PA and u-PA.10–12 PAI-1 is the major physiologic inhibitor of t-PA and u-PA and therefore plays a key role in the regulation of ﬁbrinolysis.29 A high level of plasma PAI-1 and a decreased level of t-PA are associated with atherosclerosis disease. PAI-1 mRNA has been found in human atherosclerotic lesions, underlining its role in
FIGURE 2. Comparison of t-PA, u-PA, u-PAR and PAI-1 mRNA expressions in different groups. Values are mean 6 SD (n 5 6); Ros, rosuvastatin; M, mevalonate; *P , 0.05 versus control, #P , 0.05 versus oxLDL alone, DP , 0.05 versus oxLDL plus high-dose rosuvastatin.
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He et al
FIGURE 3. (A) Comparison of t-PA concentrations in different groups. Values are mean 6 SD (n 5 6); Ros, rosuvastatin; M, mevalonate; *P , 0.05 versus control, #P , 0.05 versus oxLDL alone, DP , 0.05 versus oxLDL plus high-dose rosuvastatin. (B) Comparison of PAI-1 concentrations in different groups. Values are mean 6 SD (n 5 6); Ros, rosuvastatin; M, mevalonate; *P , 0.05 versus control, #P , 0.05 versus oxLDL alone, DP , 0.05 versus oxLDL plus high-dose rosuvastatin. (C) Comparison of u-PA concentrations in different groups. Values are mean 6 SD (n 5 6); Ros, rosuvastatin; M, mevalonate; *P , 0.05 versus control, #P , 0.05 versus oxLDL alone, DP , 0.05 versus oxLDL plus high-dose rosuvastatin. (D) Comparison of u-PAR concentrations in different groups. Values are mean 6 SD (n 5 6); Ros, rosuvastatin; M, mevalonate; *P , 0.05 versus control, #P , 0.05 versus oxLDL alone, DP , 0.05 versus oxLDL plus high-dose rosuvastatin.
the development of these disorders.30,31 Our experiments showed that oxLDL could decrease t-PA production and increase PAI-1 production in vascular endothelial cells. These results suggested that vascular endothelial cells can be damaged, after which ﬁbrinolytic system disorder can occur in hyperlipidemia. To be precise, it is easier for thrombus to form in blood vessels. In this study, we found that rosuvastatin, even in low doses, could improve t-PA production and reduce PAI-1 production, whether in unstimulated HUVSCs or in HUVSCs exposed to oxLDL. We also found that rosuvastatin could increase the expression of t-PA mRNA and decrease the expression of PAI-1 mRNA. Previous studies32–35 have also shown that simvastatin,32,33 lovastatin33 and ﬂuvastatin34,35 have similar effects on the ﬁbrinolytic system in vascular endothelial cells. Past reports9,19,20 have suggested that rosuvastatin may prevent thrombosis through its effects on the ﬁbrinolytic system. However, the exact effect of rosuvastatin on the ﬁbrinolytic system in vascular endothelial cells is still unclear. From our experiments, we can conclude that rosuvastatin has proﬁbrinolytic effects in vitro. We did not ﬁnd any effects of rosuvastatin or oxLDL on u-PA and u-PAR. Wiesbauer et al33 found that 6 other statins had no effects on u-PA and u-PAR. The reason for this is not very clear. Perhaps, u-PA and u-PAR are more frequently expressed in migrating cells, and their major function is pericellular proteolysis. In our experiment, we also found that all the effects of rosuvastatin on the ﬁbrinolytic
system were reversed by mevalonate, which is the primary product of the HMG-CoA reductase enzyme.36 The results suggested that rosuvastatin inﬂuenced t-PA and PAI-1 through inhibition of the isoprenoid pathway. It should be noted, however, that our experiment in vitro only provides a proposition, and further investigation is still required. In conclusion, our study conﬁrmed that rosuvastatin has protective effects on oxLDL-injuring human vascular endothelial cells and that its antithrombotic effects may be involved in the regulation of the ﬁbrinolytic system in vascular endothelial cells. ACKNOWLEDGMENTS The authors thank Dr. Huizhi Wang for his careful reading and editing of this article. REFERENCES 1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801–9. 2. Witztum JL. Current approaches to drug therapy for the hypercholesterolemic patient. Circulation 1989;80:1101–14. 3. Scandinavian Simvastatin Survival Study Group. Randomized trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 1994;344:1383–9. 4. The Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) Study Group. Prevention of cardiovascular events and death
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33. Wiesbauer F, Kaun C, Zorn G, et al. HMG CoA reductase inhibitors affect the ﬁbrinolytic system of human vascular cells in vitro: a comparative study using different statins. Br J Pharmacol 2002;135:284–92. 34. Dunoyer-Geindre S, Fish RJ, Kruithof EK. Regulation of the endothelial plasminogen activator system by ﬂuvastatin. Role of Rho family proteins, actin polymerisation and p38 MAP kinase. Thromb Haemost 2011;105:461–72. 35. Mussoni L, Banﬁ C, Sironi L, et al. Fluvastatin inhibits basal and stimulated plasminogen activator inhibitor 1, but induces tissue type plasminogen activator in cultured human endothelial cells. Thromb Haemost 2000;84:59–64. 36. Thurnher M, Nussbaumer O, Gruenbacher G. Novel aspects of mevalonate pathway inhibitors as antitumor agents. Clin Cancer Res 2012;18:3524–31.