Novel anti-inflammatory mechanisms of N-Acetyl-Ser-Asp-Lys-Pro in hypertension-induced target organ damage

Abstract

High blood pressure (HBP) is an important risk factor for cardiac, renal, and vascular dysfunction. Excess inflammation is the major pathogenic mechanism for HBP-induced target organ damage (TOD). N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a tetrapeptide specifically degraded by angiotensin converting enzyme (ACE), reduces inflammation, fibrosis, and TOD induced by HBP. Our hypothesis is that Ac-SDKP exerts its anti-inflammatory effects by inhibiting: 1) differentiation of bone marrow stem cells (BMSC) to macrophages, 2) activation and migration of macrophages, and 3) release of the proinflammatory cytokine TNF-alpha by activated macrophages. BMSC were freshly isolated and cultured in macrophage growth medium. Differentiation of murine BMSC to macrophages was analyzed by flow cytometry using F4/80 as a marker of macrophage maturation. Macrophage migration was measured in a modified Boyden chamber. TNF-alpha release by activated macrophages in culture was measured by ELISA. Myocardial macrophage activation in mice with ANG II-induced hypertension was studied by Western blotting of Mac-2 (galectin-3) protein. Interstitial collagen deposition was measured by picrosirius red staining. We found that Ac-SDKP (10 nM) reduced differentiation of cultured BMSC to mature macrophages by 24.5% [F4/80 positivity: 14.09 +/- 1.06 mean fluorescent intensity for vehicle and 10.63 +/- 0.35 for Ac-SDKP; P < 0.05]. Ac-SDKP also decreased galectin-3 and macrophage colony-stimulating factor-dependent macrophage migration. In addition, Ac-SDKP decreased secretion of TNF-alpha by macrophages stimulated with bacterial LPS. In mice with ANG II-induced hypertension, Ac-SDKP reduced expression of galectin-3, a protein produced by infiltrating macrophages in the myocardium, and interstitial collagen deposition. In conclusion, this study demonstrates that part of the anti-inflammatory effect of Ac-SDKP is due to its direct effect on BMSC and macrophage, inhibiting their differentiation, activation, and cytokine release. These effects explain some of the anti-inflammatory and antifibrotic properties of Ac-SDKP in hypertension.

Fig. 1.

FACS analysis to compare expression of F4/80 by macrophages. A: surface expression of F4/80 by macrophages obtained by treating bone marrow stem cells (BMSC) with macrophage growth medium (MGM). B: isotype control for A. C: surface F4/80 expression in cells treated with MGM + N-acetyl-serylaspartyl-lysyl-proline (Ac-SDKP; 10 nM). D: isotype control for C. FL1-H, fluorescence intensity on the FL1, (FITC) channel; M_1, marker used to define positive events.

Fig. 2.

Quantitative analysis of the effect of Ac-SDKP on decreased F4/80 surface expression. BMSC incubated in MGM for 7 days demonstrated surface expression of F4/80. F4/80 expression was significantly reduced by concomitant Ac-SDKP treatment for 7 days; n = 3. *P < 0.05, vehicle versus Ac-SDKP.

Fig. 3.

Measurement of macrophage migration in the Boyden chamber. Cells were plated on the upper surface of the Boyden chamber and a chemoattractant [galectin-3 or macrophage colony-stimulating factor (M-CSF)] with and without Ac-SDKP was added to the lower chamber. A: effects of Ac-SDKP on macrophage migration stimulated with galectin-3. B: effects of Ac-SDKP on macrophage migration stimulated with M-CSF. Ac-SDKP inhibited both galectin-3- and M-CSF-dependent macrophage migration in vitro. A.U., arbitrary units; n = 4.

Fig. 4.

Effect of Ac-SDKP on TNF-α release by cultured macrophages stimulated with LPS. Macrophages were grown in the 96-well culture plate; TNF-α levels were measured in the culture supernatant and were normalized to the viable cells present on the corresponding culture wells. Bacterial LPS increased the release of TNF-α by mature macrophages, which was significantly reduced by Ac-SDKP; n = 6.

Fig. 5.

Effect of Ac-SDKP on ANG II-induced interstitial collagen deposition in the mice heart. A: representative images of the picrosirious-stained sections of the heart. B: quantification of the interstitial collagen contents in the picrosirious-stained sections. ANG II significantly increased interstitial collagen volume fraction in mice heart, which was inhibited by Ac-SDKP; n = 5–7.

Fig. 6.

Measurement of left ventricular (LV) galectin-3 levels in mice treated with vehicle (n = 5), ANG II (n = 7), or ANG II + Ac-SDKP (n = 7) for 8 wk. A: representative galectin-3 immunoblot on the protein extracted from mice heart treated with vehicle, ANG II, or ANG II + Ac-SDKP. Actin immunoblot was performed on the same sample concurrently to normalize the galectin-3 levels. B: quantification of galectin-3 levels in the immunoblots. ANG II treatment increased LV galectin-3 expression levels, which was significantly reduced by Ac-SDKP; n = 5–7.