Effect of eplerenone on endothelial progenitor cells and oxidative stress in ischemic hindlimb.
BACKGROUND
We have demonstrated that angiotensin II receptor blocker (ARB) improved endothelial progenitor cells (EPCs) dysfunction through the antioxidative mechanism. Therefore, we investigate whether the selective mineralocorticoid receptor (MR) antagonist eplerenone improves EPCs function in rat hindlimb ischemia.


METHODS
Unilateral hindlimb ischemia was surgically induced in Wistar rats. After induced ischemia, rats received eplerenone (30 mg/kg/day), valsartan (3 mg/kg/day), or vehicle for 3 weeks. Peripheral blood mononuclear cells were isolated, subjected to flow cytometric analysis to determine the number of circulating EPCs, cultured to assay EPC colony formation, and subjected to a migration chamber assay to evaluate EPCs migration.


RESULTS
Blood perfusion by laser Doppler image was significantly higher in eplerenone than in vehicle. Capillary density by isolectin B4 stained of ischemic muscle was significantly increased in eplerenone compared with vehicle. Eplerenone significantly increased the number, colony formation, and migration of EPCs. Levels of endothelial nitric oxide synthase (eNOS) and angiogenic factor such as vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), and angiopoietin-2 (Ang-2) protein expression by western blot were significantly higher in eplerenone than in vehicle. Eplerenone significantly decreased the NAD(P)H oxidase p22(phox), p47(phox), gp91(phox) and MR expression and expression of aldosterone effector kinase serum and glucocorticoid-induced protein kinase 1 (Sgk1). These effects of eplerenone are similar extent as valsartan.


CONCLUSIONS
This study showed that eplerenone improves the proliferation and function of EPCs in rat hindlimb ischemia, suggesting that eplerenone may provide a novel and effective therapeutic strategy for the repair of cardiovascular diseases.
Kobayashi
Naohiko
N
Department of Hypertension and Cardiorenal Medicine, Dokkyo Medical University School of Medicine, Tochigi, Japan. nao-koba@dokkyomed.ac.jp
Fukushima
Hiromichi
H
Takeshima
Hiroshi
H
Koguchi
Wataru
W
Mamada
Yasuko
Y
Hirata
Hisato
H
Machida
Yoshifumi
Y
Suzuki
Noriko
N
Yokotsuka
Fumie
F
Tabei
Kyoko
K
Kobayashi
Eri
E
Fukuda
Noboru
N
Ishimitsu
Toshihiko
T
D042683:Angiopoietin-1 / Q000032:analysis; D042702:Angiopoietin-2 / Q000032:analysis; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology; D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects*; D000077545:Eplerenone; D006614:Hindlimb / Q000098:blood supply*; D017874:Immediate-Early Proteins; D007511:Ischemia / Q000503:physiopathology*; D008297:Male; D000451:Mineralocorticoid Receptor Antagonists / Q000494:pharmacology*; D009000:Monocytes / Q000187:drug effects; D019255:NADPH Oxidases / Q000032:analysis; D052250:Nitric Oxide Synthase Type III / Q000032:analysis; D018384:Oxidative Stress / Q000187:drug effects*; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D017208:Rats, Wistar; D013148:Spironolactone / Q000031:analogs & derivatives* / Q000494:pharmacology; D013234:Stem Cells / Q000187:drug effects*; D013777:Tetrazoles / Q000494:pharmacology; D014633:Valine / Q000031:analogs & derivatives / Q000494:pharmacology; D000068756:Valsartan; D042442:Vascular Endothelial Growth Factors / Q000032:analysis; D000099184:Serum-Glucocorticoid Regulated Kinases
D042683:Angiopoietin-1; D042702:Angiopoietin-2; D047228:Angiotensin II Type 1 Receptor Blockers; D000959:Antihypertensive Agents; D000077545:Eplerenone; D017874:Immediate-Early Proteins; D000451:Mineralocorticoid Receptor Antagonists; D019255:NADPH Oxidases; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; D013148:Spironolactone; D013777:Tetrazoles; D014633:Valine; D000068756:Valsartan; D042442:Vascular Endothelial Growth Factors; D000099184:Serum-Glucocorticoid Regulated Kinases