HMG-CoA reductase inhibitor mobilizes bone marrow--derived endothelial progenitor cells

Abstract

Endothelial progenitor cells (EPCs) have been isolated from circulating mononuclear cells in peripheral blood and shown to incorporate into foci of neovascularization, consistent with postnatal vasculogenesis. These circulating EPCs are derived from bone marrow and are mobilized endogenously in response to tissue ischemia or exogenously by cytokine stimulation. We show here, using a chemotaxis assay of bone marrow mononuclear cells in vitro and EPC culture assay of peripheral blood from simvastatin-treated animals in vivo, that the HMG-CoA reductase inhibitor, simvastatin, augments the circulating population of EPCs. Direct evidence that this increased pool of circulating EPCs originates from bone marrow and may enhance neovascularization was demonstrated in simvastatin-treated mice transplanted with bone marrow from transgenic donors expressing beta-galactosidase transcriptionally regulated by the endothelial cell-specific Tie-2 promoter. The role of Akt signaling in mediating effects of statin on EPCs is suggested by the observation that simvastatin rapidly activates Akt protein kinase in EPCs, enhancing proliferative and migratory activities and cell survival. Furthermore, dominant negative Akt overexpression leads to functional blocking of EPC bioactivity. These findings establish that augmented mobilization of bone marrow-derived EPCs through stimulation of the Akt signaling pathway constitutes a novel function for HMG-CoA reductase inhibitors.

Figures

Figure 1

Augmented angiogenic properties of EPCs in response to simvastatin via Akt signaling. (a) Representative Western immunoblots of Akt phosphorylation are shown as time-dependent changes in Akt phosphorylation at serine 473 after treatment with simvastatin (1 μM). Akt protein level was normalized by whole Akt1. Increased Akt phosphorylation was detected as early as 5 minutes. (b) Dose-dependent increase in cell number was observed in day 7 cultured EPCs. (d) MTS assay of human EPCs in response to simvastatin. A moderate dose-dependent mitogenic response to simvastatin was observed in day 7 cultured EPCs. (f) Migration assay of human EPCs in response to simvastatin. Migratory effect was augmented by simvastatin treatment with a peak at 1 μM concentration (c, e, and g). Cell number, mitogenic response, and migratory effect to simvastatin were precluded by dnAkt overexpression. –, simvastatin-negative; +, simvastatin-positive. *P < 0.01.

Figure 2

Increase in EPC survival induced by simvastatin via Akt signaling. (a) Apoptosis was evaluated after serum starvation of cultured EPCs. Annexin-V staining disclosed that simvastatin reduced the number of EPCs positive for Annexin-V or propidium odide, a marker for cell death. (b) Quantification of Annexin-V–positive cells: Tf/β-gal + no simvastatin versus Tf/β-gal + simvastatin, 29% ± 3% vs. 6% ± 1% Annexin-positive cells (*P < 0.01); Tf/dnAkt + no simvastatin versus Tf/dnAkt + simvastatin, 31% ± 3% vs. 27% ± 1% Annexin-positive cells (NS). (c) Hoechst 33342 staining was also performed to detect the frequency of apoptosis by counting pyknotic nuclei. Simvastatin attenuated apoptosis (control versus simvastatin, 24% ± 5% vs. 6% ± 1% pyknotic nuclei [**P < 0.02]). (d) Dominant negative Akt overexpression abolished positive cell survival effect of simvastatin (Tf/β-gal + no simvastatin versus Tf/β-gal + simvastatin, 21% ± 6% vs. 5% ± 1% pyknotic nuclei [*P < 0.01]; Tf/dnAkt + no simvastatin versus Tf/dnAkt + simvastatin, 26% ± 2% vs. 21% ± 2% pyknotic nuclei [NS]). –, simvastatin-negative; +, simvastatin-positive.

Figure 3

EPC mobilization induced by simvastatin in vitro and in vivo. (a) Representative fluorescence photomicrographs of EPCs cultured 4 days after isolation from peripheral blood. EPCs are identified as double positive cells owing to uptake of acLDL-DiI (red) and BS-1 lectin reactivity (green). (b) Quantification of cultured EPC population isolated from mouse peripheral blood. EPC population increased after simvastatin administration. †P < 0.05. (c) The effect of simvastatin on EPC chemotactic activity in vitro was analyzed in a transwell assay. Chemotactic effect was augmented by simvastatin with a peak at 1 μM concentration. The potency of this effect reached 70% of chemotactic response to recombinant murine GM-CSF. *P < 0.01. (d) FACS analysis of mouse peripheral blood. Ratio of Flk-1–positive cells increased after simvastatin treatment. *P < 0.01.

Figure 4

Enhanced contribution of BM-derived EPCs to corneal neovascularization. (a) Representative photos show corneal neovascularization (left, vehicle; right, simvastatin). (b) Whole-mounted corneal X-gal staining. Blue dots show X-gal–positive cells (left, vehicle; right, simvastatin). (c) Representative photomicrograph after fluorescent histochemistry examination of paraffin-embedded corneas in neovascularization assay of Tie2/LacZ/BMT mice. Double positive cells indicate that Tie2-expressing BM-derived EPCs incorporated into foci of neovascularization. Red shows β-gal, and green shows isolectin B4 binding. Double positive cell (yellow) indicates BM-derived EPCs incorporated into neovasculature. (d) Representative photomicrograph after fluorescent histochemistry examination of whole-mounted corneas in neovascularization assay of Tie2/LacZ/BMT mice. Red shows β-gal–positive cells, and green shows BS-1–lectin–stained corneal neovasculature. (e) Quantification of BM-derived EPCs incorporated into neovasculature. Ratio indicates percentage of BM-derived EPCs among total endothelial cells comprising corneal neovasculature. †P < 0.05.