Prevention of aortic fibrosis by N-acetyl-seryl-aspartyl-lysyl-proline in angiotensin II-induced hypertension

Abstract

Fibrosis is an important component of large conduit artery disease in hypertension. The endogenous tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) has anti-inflammatory and antifibrotic effects in the heart and kidney. However, it is not known whether Ac-SDKP has an anti-inflammatory and antifibrotic effect on conduit arteries such as the aorta. We hypothesize that in ANG II-induced hypertension Ac-SDKP prevents aortic fibrosis and that this effect is associated with decreased protein kinase C (PKC) activation, leading to reduced oxidative stress and inflammation and a decrease in the profibrotic cytokine transforming growth factor-beta1 (TGF-beta1) and phosphorylation of its second messenger Smad2. To test this hypothesis we used rats with ANG II-induced hypertension and treated them with either vehicle or Ac-SDKP. In this hypertensive model we found an increased collagen deposition and collagen type I and III mRNA expression in the aorta. These changes were associated with increased PKC activation, oxidative stress, intercellular adhesion molecule (ICAM)-1 mRNA expression, and macrophage infiltration. TGF-beta1 expression and Smad2 phosphorylation also increased. Ac-SDKP prevented these effects without decreasing blood pressure or aortic hypertrophy. Ac-SDKP also enhanced expression of inhibitory Smad7. These data indicate that in ANG II-induced hypertension Ac-SDKP has an aortic antifibrotic effect. This effect may be due in part to inhibition of PKC activation, which in turn could reduce oxidative stress, ICAM-1 expression, and macrophage infiltration. Part of the effect of Ac-SDKP could also be due to reduced expression of the profibrotic cytokine TGF-beta1 and inhibition of Smad2 phosphorylation.

Fig. 1.

Effect of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on angiotensin II (ANG II)-induced hypertension and aortic hypertrophy. Rats were treated with vehicle, Ac-SDKP, ANG II, or ANG II + Ac-SDKP. Top: systolic blood pressure (n = 9 or 10/group). Bottom: aortic medial cross-sectional area (n = 5/group). Data are means ± SE. *P < 0.0001, vehicle vs. ANG II.

Fig. 2.

Effect of Ac-SDKP on ANG II-induced collagen deposition in the aortic media. Top: representative images showing collagen deposition (red) stained with picrosirius red in aortas from rats treated with vehicle, Ac-SDKP, ANG II, or ANG II + Ac-SDKP. Magnification ×200. Bottom: collagen deposition in the aortic media was quantified with Olympus Microsuite Biological imaging software; positively stained area is expressed as % of the medial area. n = 5–8/group.

Fig. 3.

mRNA expression of collagen type I and III in rat aorta by semiquantitative PCR. Rats were treated with vehicle, Ac-SDKP, ANG II, or ANG II + Ac-SDKP. A: collagen type I mRNA expression. B: collagen type III mRNA expression. Bar graphs show mRNA expression of collagen type I or III indicated as the density ratio of collagen I or III to 18S. Data are means ± SE; n = 6 rats/group.

Fig. 4.

Effect of Ac-SDKP on ANG II-induced elastin deposition in the aortic media. Top: representative images showing elastic fibers (purple) stained with Verhoeff van Giesson in aortas from rats treated with vehicle, Ac-SDKP, ANG II, or ANG II + Ac-SDKP. Magnification ×200. Bottom: quantitative evaluation of elastin density in the aortic media. The stained area is indicated as % of the medial area. n = 8 or 9/group.

Fig. 5.

Western blot for protein kinase C (PKC) phosphorylation in aortas. Rats were treated with vehicle, Ac-SDKP, ANG II, or ANG II + Ac-SDKP. The bands of phosphorylated (p-)PKC and β-actin were measured by densitometric analysis, and the quantitative data are indicated as the ratio of p-PKC to β-actin. Data are means ± SE; n = 5 or 6 rats/group.

Fig. 6.

Effect of Ac-SDKP on ANG II-induced oxidative stress in the aorta, as indicated by staining for 4-hydroxy-2-nonenal (4-HNE; a marker for lipid peroxidation) and nitrotyrosine (a marker for peroxynitrite). Top: representative images showing 4-HNE staining (brown) in aortas from rats treated with vehicle, Ac-SDKP, ANG II, or ANG II + Ac-SDKP. Magnification ×400. Bottom: quantification graphs for 4-HNE (left) and nitrotyrosine (right) staining. The positively stained area is expressed as % of the medial + intimal area. Values are means ± SE; n = 7 or 8/group (4-HNE) or 8 or 9/group (nitrotyrosine).

Fig. 7.

Effect of Ac-SDKP on ANG II-induced macrophage infiltration in the aorta. Top: representative cross sections of aortas from rats treated with vehicle, Ac-SDKP, ANG II, or ANG II + Ac-SDKP. ED-1-positive cells (stained reddish-brown) are indicated by arrows. Magnification ×400. Bottom: quantitative analysis of macrophage infiltration, represented by the number of ED-1-positive cells per mm² of intimal + medial area. n = 5/group.

Fig. 8.

Effect of Ac-SDKP on ANG II-induced aortic intercellular adhesion molecule (ICAM)-1 mRNA expression. Rats were treated with vehicle, Ac-SDKP, ANG II, or ANG II + Ac-SDKP. Top: representative bands for ICAM-1 and 18S by semiquantitative PCR. Bottom: expression of ICAM-1 in the aorta, expressed as the density ratio of ICAM-1 to 18S. Data are means ± SE; n = 6/group.

Fig. 9.

TGF-β1 and Smad2 and -7 expression in the aortas of rats treated with vehicle, Ac-SDKP, ANG II, or ANG II + Ac-SDKP. Top: representative immunoblots. Bottom: quantification graphs. A: TGF-β1 expression. B: Smad2 phosphorylation. T-Smad2, total Smad2. C: Smad7 expression. Values are means ± SE; n = 5 or 6/group.

Fig. 10.

Hypothesized mechanism by which Ac-SDKP inhibits aortic fibrosis in ANG II-induced hypertension. As illustrated, ANG II activates PKC and also induces ICAM-1 expression and macrophage activation. PKC activates macrophages and also NADPH, both of which can increase reactive oxygen species (ROS) production. Macrophages and ROS induce expression of TGF-β1, which, via receptor-associated Smads (Smad2 and -3), induces fibrosis. Ac-SDKP acts by decreasing PKC activation, ROS generation, macrophage infiltration, TGF-β1 expression, and inhibition of Smad2 phosphorylation. Ac-SDKP increases inhibitory Smad7, which blocks the effects of TGF-β1. Ac-SDKP may also directly inhibit fibrosis by blocking collagen synthesis and fibroblast proliferation (46).