Beneficial Effects Of Intensive Statin Treatment:Is The Endothelial Progenitor Cells Mobilization The Missing Link?

Background and objects:The beneficial efficacy of an intensive statin pretreatment, in the setting of percutaneous coronary intervention (PCI), has been first demonstrated by randomized protocols in the ARMYDA trial, then in the MRACL, PROVE-IT TIMI22trail. Endothelial progenitor cells (EPCs) circulate in the peripheral blood and are thought to participate in vasculogenesis and ongoing repair of the vascular endothelium. Enumeration and functional assessment of EPCs has been considered as a novel technique for the assessment of the vascular damage and reparative capacity. Whether the beneficial effects of intensive statin treatment associated with a higher EPCs count, compared to standard treatment, remain poorly understood. And PI-3K/Akt signal pathway is well established to play an important role in cell apoptosis, survival and proliferation. Some data reveal that statin induced the activation of PI-3K/Akt signal pathway. The underlying mechanism, however, of how intensive statin exert effects on EPCs remain to be determined. The aim of the study, first, to evaluate, in a randomized clinical trial, the effect of an intensive versus standard treatment with statins on EPCs mobilization in patients of non-ST elevated ACS (NSTACS) scheduled for PCI; secondary, we investigate the possible mechanism underlying the effects of intensive statins on mobilization of EPCs in mouse myocardial infarction (MI) model, and thereby intensive statin exert beneficial effects on myocardial neovascularization, left ventricular function, remodeling.Part One:Effects of Intensive Atorvastatin on the Mobilization of Endothelial Progenitor Cells in ACS PatientsMethods and results:From December2010,48NSTACS patients scheduled for PCI were randomly into two groups at admission. The intensive dose statin treatment group received a two days of pretreatment with atorvastatin80mg/QN, then after PCI with atorvastatin40mg/QN up to one month follow up. Frequent dose statin treatment group received atorvastatin20mg/QN from enrollment to one month follow up. The blood sample to measure EPCs were collected on the enrollment (baseline), then on the dayl, day2, day3, day4after statin administration. Flow cytometric analysis was performed to quantify the early EPCs, which were defined as CD45low/-/CD34+/CD133+/KDR+or VEGFR2+cells. There was no difference in the levels of early EPCs between two groups at baseline (P=0.893). The early EPCs increased on day1and day2(P=0.000) in intensive group before PCI, while the level of early EPCs kept still in frequent group. After the PCI, the early EPCs in both two groups went up (P<0.05), but the level of early EPCs in intensive group was still higher than the frequent group (P<0.01). And there were no different in the levels of hsCRP between these two groups. All above results suggested that intensive atorvastatin could promote the mobilization of EPCs.Part Two:Mechanisms Underlying the Effects of Intensive Atorvastatin on the Mobilization of Endothelial Progenitor Cells in ACS patientsMethods and results:The plasma levels of VEGF and SDF-1alpha were respectively determined in duplicate from frozen plasma samples using commercially available ELISA kits according to the manufacturer’s guidelines. There was no difference in the levels of VEGF and SDF-1alfa between two groups at baseline (P>0.05). The plasma VEGF levels increased on day1and day2(P<0.01) in intensive group before PCI, while the levels of VEGF kept still in frequent group. After the PCI, VEGF levels in both two groups goes up (P<0.05), but the level of VEGF in intensive group was still higher than the frequent group (P<0.01). And there were no different among the levels of SDF-1alfa in these two groups at each time point (P>0.05). And there was a significant positive relationship between the level of EPCs and VEGF at day2(r=0.77, P<0.01), indicating that intensive atorvastatin might promote the mobilization of EPCs via up regulation of plasma VEGF. Part Three:Effects of atorvastatin on the mobilization of Endothelial Progenitor Cells in mouse myocardial infarctionMethods and results:Coronary artery ligation was performed to establish the mouse myocardial infarction (MI) model. The mice were divided into three groups after operation:the intensive dose atorvastatin treatment group was administered15mg/kg*day, and frequent dose atorvastatin treatment group with5mg/kg*day, then the control group was gavaged with normal saline. The blood samples were taken before (0d) and at days1,3,5,7,10,14,28post-operatively. Flow cytometric analysis was performed to quantify the early EPCs, which were defined as expression of three surface antigens, c-Kit, Sca-1, and VEGFR2or Flk-1in mice. There was no difference in the level of early EPCs among these three groups at the0d (pre-MI), and early EPCs increased at dayl in all groups, and peaked at day7, then gradually decreased, suggesting that the MI could induce the mobilization of EPCs. Compared with control group and frequent statin treatment group, the intensive group maintained a significant higher level of early EPCs from day1to day14after MI (all P<0.01), suggesting that intensive atorvastatin could promote the mobilization of EPCs from bone marrow. And echocardiography revealed that intensive statin significantly improved heart function. We further observed significant enhanced angiogenesis and reduced fibrosis in the intensive statin group. Part Four:Mechanisms Underlying the Effects of Intensive Atorvastatin on the Mobilization of Endothelial Progenitor Cells in Mouse Myocardial InfarctionMethods and results:In this part, we investigated the possible mechanisms underlying the effects of intensive atorvastatin on mobilization of EPCs in mouse myocardial infarction (MI) model. The plasma VEGF were determined in duplicate using commercially available ELISA kits according to the manufacturer’s guidelines. The expression of Akt, eNOS, and their corresponding Phospho-Akt, Phospho-eNOS in bone marrow were detected by Western Blot. Acute MI led to a significant time-dependent increase of plasma VEGF. The plasma VEGF peaked on the day7, and then gradually decreased. The VEGF levels in the intensive group were significant higher than that of control group and frequent group (P<0.01). And the VEGF levels in the frequent group were higher than that of control group (P<0.05). And the expression of Phospho-Akt and Phospho-eNOS were up regulated in the bone marrow (P<0.01). Moreover, more EPCs were found in border zone. All above results suggested that the intensive statin might promote the mobilization of EPCs via improvement the plasma VEGF level, and activation of PI-3K/Akt signal pathway, then up regulation of Phospho-Akt and Phospho-eNOS. Conclusion:1Intensive atorvastatin could promote the mobilization of EPCs. And acute myocardial infarction and PCI could induce the mobilization of EPCs.2Intensive atorvastatin can improve heart function, enhance angiogenesis, and reduce fibrosis in myocardial infarction, and these beneficial effects might be the result of intensive statin-induced improvement of EPCs mobilization.3The effects of intensive atorvastatin promote the mobilization of EPCs might via improvement the plasma VEGF level, and activation of PI-3K/Akt signal pathway, then up regulation of Phospho-Akt and Phospho-eNOS.