HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway

Abstract

HMG-CoA reductase inhibitors (statins) have been developed as lipid-lowering drugs and are well established to reduce morbidity and mortality from coronary artery disease. Here we demonstrate that statins potently augment endothelial progenitor cell differentiation in mononuclear cells and CD34-positive hematopoietic stem cells isolated from peripheral blood. Moreover, treatment of mice with statins increased c-kit(+)/Sca-1(+)--positive hematopoietic stem cells in the bone marrow and further elevated the number of differentiated endothelial progenitor cells (EPCs). Statins induce EPC differentiation via the PI 3-kinase/Akt (PI3K/Akt) pathway as demonstrated by the inhibitory effect of pharmacological PI3K blockers or overexpression of a dominant negative Akt construct. Similarly, the potent angiogenic growth factor VEGF requires Akt to augment EPC numbers, suggesting an essential role for Akt in regulating hematopoietic progenitor cell differentiation. Given that statins are at least as potent as VEGF in increasing EPC differentiation, augmentation of circulating EPC might importantly contribute to the well-established beneficial effects of statins in patients with coronary artery disease.

Figures

Figure 1

Statins increase the number of adherent EPCs. (a and b) MNCs were incubated with atorvastatin as indicated, and adherent DiLDL/lectin-positive cells were counted (13). Basal control values represent 388 ± 146 cells/mm2. The solvent DMSO had no effect on EPC differentiation. Data are mean ± SEM, n = 4–6. (c) MNCs were incubated with atorvastatin (1 μM), simvastatin (1 μM), or mevastatin (1 μM) for 24 hours, and adherent cells’ DiLDL uptake (red) and lectin binding (green) were assessed. Double positive cells appear yellow in the overlay. Representative images are shown from at least three experiments. (d) MNCs were incubated for 4 days. Adherent cells were analysed for expression of KDR, CD31, VE-cadherin, and vWF by FACS. Dotted lines represent isotype controls. Similar expression patterns were observed after stimulation of MNCs with atorvastatin (1 μM, 24 hours) (data not shown). Representative images from n = 4 experiments are shown. (e) MNCs were incubated with VEGF for 24 hours and adherent DiLDL/lectin-positive cells were counted. Data are mean ± SEM, n = 3–6.

Figure 2

Statins and VEGF induce differentiation of CD34+ cells. (a) CD34+ cells were isolated and purity of CD34+ cell fraction was analysed by FACS. Isotype control is represented by a dotted line and CD34 by a solid line. (b) CD34+ cells were incubated with atorvastatin (AT; 1 μM) or VEGF (100 ng/ml) for 24 hours, and adherent DiLDL/lectin-positive cells were counted. Data are mean ± SEM, n = 4–7, *P < 0.05 versus control.

Figure 3

Statins increase EPCs and HSCs in mice. (a) Mice were fed with simvastatin for 3 weeks. Splenocytes were isolated and incubated for 4 days. Adherent DiLDL/lectin-positive cells were counted. Data are mean ± SEM, *P < 0.05 versus control. (b) Bone marrow cells were isolated and the number of c-kithigh cells was measured by FACS. Data are mean ± SEM, *P = 0.006 versus control (Mann-Whitney test).

Figure 4

Statin- and VEGF-induced EPC differentiation is mediated by the PI3K/Akt pathway. (a and b) MNCs were incubated with atorvastatin (1 μM) or VEGF (100 ng/ml) and the respective inhibitors (Ly294002, 10 μM; wortmannin, 10 nM; PD98059, 10 μM) for 24 hours. Adherent DiLDL/lectin-positive cells were counted. Neither the substances alone nor the solvents had any toxic effect (data not shown). Data are mean ± SEM, n = 3–6, *P < 0.05 versus VEGF or AT. (c) Western blot against phosphorylated Akt (Ser 473) of MNCs incubated with atorvastatin for the respective time points. Equal loading was confirmed by reprobe of the membranes with total Akt. Protein isolation and Western blot analysis was performed as outlined previously (25). A representative experiment is shown (n = 3). (d and e) MNCs were transfected with pcDNA3.1.-GFP as control vector or pcDNA3.1. dominant negative (dom. neg.) Akt and were incubated with atorvastatin (1 μM) or recombinant hVEGF (100 ng/ml) for 24 hours. Adherent DiLDL/lectin-positive cells were counted. Atorvastatin was similarly effective in empty vector–transfected cells compared with GFP. Data are expressed as mean ± SEM, n = 4–6, *P < 0.05 versus control vector plus VEGF, **P < 0.05 versus control vector plus atorvastatin.

Figure 5

Characterization of the statin effect. (a) Schematic illustration of the statin effects on intracellular pathways. (b) MNCs were incubated with atorvastatin (AT; 1 μM) for 24 hours with or without mevalonate (100 μM), GGPP (5 μM), the Rho-kinase inhibitor HA 1077 (10 μM), or NG-mono-methyl-L-arginin (LNMA, 1mM). DiLDL/lectin-positive cells were counted. Data are expressed as mean ± SEM, P < 0.05 versus AT; n = 3–7.