The acute impact of high-dose lipid-lowering treatment on endothelial progenitor cells in patients with coronary artery disease-The REMEDY-EPC early substudy

Abstract

Rationale and objective: Endothelial progenitor cells (EPCs) play a role in vascular repair, while circulating endothelial cells (CECs) are biomarkers of vascular damage and regeneration. Statins may promote EPC/CEC mobilization in the peripheral blood. We evaluated whether pre-procedural exposure to different lipid-lowering drugs (statins±ezetimibe) can acutely increase levels/activity of EPCs/CECs in patients with stable coronary artery disease (CAD).

Methods: In a planned sub-analysis of the Rosuvastatin For REduction Of Myocardial DamagE During Coronary AngioplastY (REMEDY) trial, 38 patients with stable CAD on chronic low-dose statin therapy were randomized, in a double-blind, placebo-controlled design, into 4 groups before PCI: i. placebo (n = 11); ii. atorvastatin (80 mg+40 mg, n = 9); iii. rosuvastatin (40 mg twice, n = 9); and iv. rosuvastatin (5 mg) and ezetimibe (10 mg) twice, (n = 9). At baseline and 24 h after treatment-before PCI-, patients underwent blinded analyses of EPCs [colony forming units-endothelial cells (CFU-ECs), endothelial colony-forming cells (ECFCs) and tubulization activity] and CECs in peripheral blood.

Results: We found no significant treatment effects on parameters investigated such as number of CECs [Median (IQR): i. 0(0), ii. 4.5(27), iii. 1.9(2.3), iv. 1.9(2.3)], CFU-ECs [Median (IQR): i. 27(11), ii. 19(31), iii. 47(36), iv. 30(98)], and ECFCs [Median (IQR): i. 86(84), ii. 7(84), iii. 8/(42.5), iv. 5(2)], as well as tubulization activity [total tubuli (well), Median (IQR): i. 19(7), ii. 5(4), iii. 25(13), iv. 15(24)].

Conclusions: In this study, we found no evidence of acute changes in levels or activity of EPCs and CECs after high-dose lipid-lowering therapy in stable CAD patients.

Fig 1. Study design of the REMEDY-EPC early substudy.

Coronary artery disease (CAD) patients candidate for an elective percutaneous coronary interventions (n = 60) were screened, Forty-eight of them, providing written informed consent, were enrolled and randomly allocated to 4 treatment strategy groups comprising placebo or 3 lipid-lowering treatments, as illustrated. The final analysis consisted of n = 38 patients due to some missing sampling (indicated by *), as illustrated. Sampling for the identification and quantification of endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs) were performed at the time of randomization (sample R), and 1 hour before the diagnostic angiography and PCI, at time of treatment reload (placebo or lipid-lowering treatments, sample T0).

Fig 2. Colony forming units-endothelial cells and late endothelial colony forming cells.

Representative images of colony forming units-endothelial cells or CFU-Hill (A) and late endothelial colony forming cells or ECFCs (B) in patients at time of treatment reload.

Fig 3. Flow cytometry specificities and reagents.

Reagents composing the lyophilized panel are evidenced in bold face. Other reagents added to the basic panel are listed in plain fonts. *Catalog number of the lyophilized combination. Abbreviations: PE = R-phycoerythrin; 7-AAD = 7-AminoActinomycin D; Cy7 = PE-Cyanine; APC-H7 = Allophycocyanin-Hilite^®7; BD = Becton Dickinson Biosciences (San Jose, CA, USA).

Fig 4. Composition of the flow cytometry panel and control tubes.

Reagents composing the lyophilized panel are evidenced in bold face. Other reagents added as liquid drop in to the basic panel are listed in plain fonts. Abbreviations: PE = R-phycoerythrin; 7-AAD = 7-AminoActinomycin D; Cy7 = PE-Cyanine 7; APC = Allophycocyanin = APC; APC-H7 = Allophycocyanin-Hilite^®7.

Fig 5. Baseline characteristics of the population studied, divided in the various study groups.

Abbreviations: SD = standard deviation; CAD = coronary artery disease; PCI = percutaneous coronary intervention; CABG = coronary artery bypass graft surgery; CAD = coronary artery disease.

Fig 6. Selected markers of myocardial damage, parameters of angiogenesis and clonogenic activities, and surface antigens before and after treatment.

For each parameter, at each time point, numbers represent the median and the interquartile range (this latter in parenthesis). Abbreviations; Plac = placebo; At80+40 = atorvastatin 80 mg + 40 mg; Ro40+40 = rosuvastatin 40 mg + 40 mg; RoEz = rosuvastatin + ezetimibe (these are the treatment groups as defined in Methods); T0, T1 = time 0, time 1, as defined in Methods; Δ T1-T0 = Difference T1-T0; CK-MB = creatine kinase-MB isoform; Early and Late Colonies = as defined in Methods; CD = cluster of differentiation—see Methods for details.

Fig 7. Correlation analysis (Spearman rho) of the differences (Δ = after minus before treatments) in angiogenesis and clonogenic activities (independent variable), and the differences (Δ = after minus before treatments) in surface antigens.

Abbreviations; AU = Arbitrary Units; Early (A) and early (B) = Hill colony analysis performed by two independent investigators. Only the underlined correlation coefficients were significant. Treaments = atorvastatin 80 mg + 80 mg, rosuvastatin 40 mg + 40 mg, rosuvastatin 10 mg + ezetimibe 10 mg.