Effects of Statins on Endothelial Progenitor Cell Proliferation from Peripheral Blood of Stable Coronary Artery Disease Patient

Background: Atherosclerotic lesions develop as the result of an inflammatory process initiated by endothelial damage. Endothelial progrenitor cells (EPCs), which is derived from bone marrow, participate in endothelial repair and new vessel growth. Cardiovascular pharmacotherapies have been shown to improve overall numbers and function of EPCs in patients with cardiovascular risk and cardiovascular disease. Studies have reported that statins exert beneficial effects on EPCs by enhancing EPC proliferation and differentiation. Thus, we require a research to analyze the effects of three different statins on EPC proliferation. The objective of this study is to analyze the effect of statins on EPC proliferation from peripheral blood of stable coronary artery disease patient. Methods: This is an in vitro true experimental post-test only control group design. The mononuclear cells (MNCs) were isolated from peripheral blood of stable coronary artery disease patient and were cultured in CFU-Hill medium for three days. Then samples were put into four groups, simvastatin experiment group, atorvastatin experiment group, rosuvastatin experiment group and control group. Each experiment group was divided into three subgroups with different doses, 0.1 μmol/L, 0.5 μmol/L, and 2.5 μmol/L then incubated for 48 hours. EPC proliferation was evaluated afterwards with MTT cell proliferation assay. Immunocytochemistry method was performed for EPC identification to evaluate expression of CD34+. CFU-Hills were observed to confirm functional characteristics of EPC. Data were analyzed by independent T-test and ANOVA. Results: MTT cell proliferation assay showed a significant increase of EPC proliferation in simvastatin, atorvastatin, and rosuvastatin groups compared with control group (0.237±0.007, 0.248±0.01, 0.231±0.008 vs 0.17±0.008, p<0.05). It also revealed significant difference in EPC proliferation between each experiment groups, which atorvastatin showed the highest effect. EPC proliferation in atorvastatin is higher than simvastatin group (0.248±0.01 vs. 0.237±0.007, p<0.05), and simvastatin is also higher than rosuvastatin group (0.237±0.007 vs. 0.231±0.008, p<0.05). CFU-Hill counts demonstrated highest number in rosuvastatin group, followed by atorvastatin, and simvastatin. Immunocytochemistry showed positive expression of CD34. Conclusion: Statins increase EPC proliferation from peripheral blood of stable coronary artery disease patient. Atorvastatin showed the highest EPC proliferation, followed by simvastatin, and rosuvastatin. Each statins increased EPC proliferation dose-dependently.


Introduction
C oronary heart disease is a major cause of morbidity and mortality worldwide. Improvements in the quality of therapy for this disease were not accompanied by a significant reduction in recurrence rates. The discovery that cells originating from the bone marrow known as endothelial progenitor cells (EPC) play a role in doi: 10.30701/ijc.v40i1.840 Indonesian Journal of Cardiology endothelial cell repair and new vascular growth has changed the model of the pathogenesis of cardiovascular disease1. EPC is part of a single nucleated cell or mononuclear cell (MNC) that has several hematopoetic stem cell markers, namely CD34 and CD133. Some physiologica l stimuli, ischaemic response, and some drugs have been known affecting the number and function of EPC 2 . Statins or HMG-CoA reductase inhibitors reduce cholesterol levels through inhibition of HMG-CoA reductase. Some studies report statins to have beneficial effects on EPC by increasing EPC proliferation and migration through activation of the endothelial nitric oxide synthase (eNOS) pathway 3 . In a previous study, it was found that simvastatin, atorvastatin, and rosuvastatin provided an increase of EPC proliferation in peripheral blood of SCAD patients when compared with controls, where atorvastatin showed the most significant proliferation result 4 .

Methods
Blood samples were taken from patients with SCAD which were men, 40-59 years old, had narrowing of ≥50% in the left main coronary artery and ≥70% in one or several of the major coronary arteries from angiography.
Patients with history of acute myocardial infarction, acute limb ischemia, stent placement, coronary artery bypass grafting, diabetes mellitus, smoking, and anemia were excluded.
Written informed consent was obtained from patients before peripheral blood drawing. The protocol was approved by local ethics committee (0379/KEPK/ VII/2018).

Isolation and culture of EPCs
Forty ml of peripheral blood from SCAD patient was diluted with phosphate buffered saline (PBS) containing 2% of fetal bovine serum (FBS). It was then centrifuged on Ficoll-Histopaque density gradients and interface mononuclear cells (MNCs) were collected. After two washes in PBS containing 2% of FBS, pellet were diluted with stemline II hematopoietic stemcell expansion medium, and these cells were counted using hemocytometry. 5x106cells/ml mononuclear cells were plated on 6-fibronectin coated well plates. After two days, nonadherent cells were transferred into new fibronectin coated well plates and incubated again for 24 hours.

Statin
After 3 days, EPC culture was divided into 4 groups. Each group was given simvastatin 0.5μmol/L, atorvastatin 0.5μmol/L, rosuvastatin 0.5μmol/L, and control, then cell culture was incubated for next 48 hours

EPC migration assay
After EPC culture was incubated for 72 hours, 5 x 10 5 cells / ml were transferred to the transwell upper chamber. The bottom chamber transwell was given 2 ml of media. After 24 hours of incubation, the nonmigratory cells on the upper side of upper transwell insert were manually removed using sterile cotton swabs. The upper and lower side of insert system were washed using PBS. Cells on the bottom side of transwell insert system were detached by trypsination method (Figure 1). The new medium was placed on the receiver plate and added 500μl of trypsin + EDTA solution 0.5%, then the insert was returned to the receiver plate and incubated for 10 minutes. The detached cells on the receiver plate were read using a cell counter with tryphan blue coloring to determine the number of migrating cells.

Immunofluorescence assay
Cells were washed with PBS and fixed with 3% formaldehyde for 15 minutes. Cells then added with 1% serum and washed again with PBS. Cells were stained with a FITC anti-CD34 antibody. Expression of the cells was documented with fluorescence microscope

Statistical analysis
Data were expressed as mean±SD. To analyse differences, one-way ANOVA was used. Differences were considered significant if p-values were <0.05. All statistical analyses were performed with SPSS for Windows (IBM Corp., Armonk, NY).

Immunofluorescence assay
CD34 is one of the positive marker for EPC. Before migration assay, one of the well was washed with PBS and stained with a FITC anti-CD34 antibody. Expression of CD34 was green under fluorescence microscope (Figure3).

Discussion
The results of the this study demonstrated that statin significantly increased EPC migration in patients with SCAD. There were different level in EPC migration induced by each statins. Atorvastatin had the highest effect of EPC migration, and simvastatin could promote EPC migration more than rosuvastatin.
Many studies have reported the beneficial effects of statin on EPCs. Similar to this result, Llevadot, et al. reported that simvastatin increased EPC proliferative activity and function of EPC using EPC culture assay of peripheral blood from simvastatin-treated animals in vivo 5 . Spiel, et al. explored the effect of statin using simvastatin and rosuvastatin. The results showed that five days pre-treatment with 80 mg of simvastatin increased CFU 3.5-fold, whereas pre-treatment with 40 mg of rosuvastatin led to a 2.6 fold increase as compared to baseline values 6 . Dimmeler, et al. also used three different types of statins, which were simvastatin, mevastatin, and atorvastatin. The results demonstrated that incubation of isolated human MNCs with statin increased the number of EPCs. Atorvastatin augmented the number and function of EPCs more than simvastatin 7 .
Dimmeler, et al. demonstrated that statins enhanced the mobilization of EPCs from bone marrow to newly forming blood vessels through the PI3K/Akt pathway, which lead to an increase in endothelial nitric oxide Indonesian Journal of Cardiology synthase (eNOS) and nitric oxide (NO) production, as NO is essential for EPC proliferation 7,8 . The mechanism underlying different effect of statins on EPC migration still needs to be determined. Although all statins share a common mechanism of action, they differ in terms of their chemical structures, pharmacokinetic profiles, and lipid modifying efficacy. These differences might influence the effect on EPC migration induced by different types of statin.

Conclusion
The results of this study define that statin increases EPC migration from peripheral blood of SCAD patient. Our data suggests that atorvastatin has the highest EPC migration effect, followed by simvastatin, and rosuvastatin.