To test these hypotheses, we first studied the pro-fibrotic activity of MBG in salt-loaded rats with diabetes type 2 (DM), in which stimulation of MBG and cardiac remodeling were observed in the absence of BP elevation. DM was induced by early postnatal administration of streptozotocin, and 8-week old DM rats were salt loaded (1.8% NaCl instead of water) for 4 weeks, following which BP did not change, MBG levels increased 2-fold and erythrocyte NKA was inhibited vs. control group. During the last week of salt loading, rats were administered anti-MBG mAb or vehicle. Isolated aortic rings from NaCl-loaded DM rats exhibited reduced sensitivity to sodium nitroprusside (SNP; a donor of NO). Collagen-1 abundance in rat aortae increased in parallel to decline in Fli1 level. Increase in collagen-5 levels indicated that another pro-fibrotic pathway is involved in DM. We detected an increase of TGFβ and other proteins, implicated in TGFβ pro-fibrotic pathway (fibronectin, SMAD-5) in aortae from DM rats. Anti-MBG mAb administration did not affect BP, and restored erythrocyte NKA, which was inhibited following high salt intake. Importantly, aortic rings from DM rats, treated with 3E9 mAb, demonstrated restored responsiveness to SNP to control parameters, and decreased levels of aortic proteins implicated in pro-fibrotic TGFβ-signaling protein levels vs. vehicle-treated DM rats. Next, we studied effects of MR antagonist canrenone (10 μM), an active metabolite of spironolactone, on MBG-induced synthesis of collagen-1 in vitro in the explants of rat thoracic aortae. Incubation of aortic rings with 100 nM MBG for 24 h resulted in a reduction of Fli1 level and a concomitant increase in collagen-1 levels, accompanied by reduced sensitivity of aortae to the vasorexation effect of SNP. Addition of canrenone to the incubation media reversed the pro-fibrotic effect of MBG, and restored the vasorelaxation of aortae. In a pilot study in patients with RH (7 men and 9 women, 562 years) we assessed BP, vascular stiffness as a pulse wave velocity (PWV), plasma levels of MBG and activity of erythrocyte NKA before and after a six-month of addition of placebo or spironolactone (50 mg/day) to the conventional triple therapy (lisinopril/amlodipine/hydrochlorothiazide). RH patients demonstrated greater BP and PWV, higher serum creatinine, reduced GFR, elevated plasma MBG, and decreased erythrocyte NKA activity vs. normotensive control. Administration of spironolactone to RH patients for 6 months was associated with restoration of NKA activity even in the presence of unchanged MBG levels. Spironolactone treatment, unlike placebo, decreased systolic and diastolic BP, and significantly reduced PWV. Thus, MR antagonists reversed MBG-induced vascular collagen deposition that may compromise relaxation of rat aortae. These data together with the clinical finding that spironolactone can reduce arterial stiffness in RH patients and restore NKA in the presence of increased MBG levels, indicate the deep relation between MBG and arterial stiffness. Thus MBG and MBG-induced arterial remodeling and arterial stiffness are novel targets for MR antagonists. Conclusion: Thus, both immunoneutralization of heightened MBG levels and modulation of NKA/MBG interaction by aldosterone antagonists demonstrated anti-fibroric effects in various pathological conditions, when increased MBG levels participate in development of vascular stiffness via activation of Fli-1 and/or TGFβ signaling.
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