Effects of salsalate therapy on recovery from vascular injury in female Zucker fatty rats

Abstract

Objective: Salsalate is a dimeric form of salicylic acid that has been shown to have anti-inflammatory activity and to reduce glucose levels, insulin resistance, and cytokine expression. However, the effect of salsalate on vascular injury has not been determined. The objective of this study is to investigate the effect of salsalate on vascular injury and repair in a rat model of carotid artery balloon catheter injury.

Research design and methods: Salsalate treatment was started in female Zucker fatty rats (insulin resistant) 1 week before carotid artery balloon catheter injury and continued for 21 days, at which time the animals were killed and studied.

Results: Treatment with salsalate significantly decreased the intima-to-media ratio and upregulated the expression of aortic endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS) (ser 1177), and manganese superoxide dismutase (MnSOD) and reduced serum interleukin (IL)-6 with concomitant downregulation of nuclear factor (NF) κB subunit p65 and vascular endothelial growth factor (VEGF) expression in the balloon-injured carotid artery of female Zucker fatty rats.

Conclusions: The present study shows that salsalate treatment decreases vascular damage caused by balloon catheter injury in female Zucker fatty rats. The beneficial effect of salsalate on vascular injury was associated with upregulation of eNOS, p-eNOS, and MnSOD, which reduce oxidative stress and have anti-inflammatory properties, as evidenced by reduction in serum IL-6 and the downregulation of VEGF and NFκB, which promote inflammation without changing glucose levels. These results suggest that salsalate may be useful in reducing vascular injury and restenosis following interventional revascularization procedures.

FIG. 1.

Representative sections of H&E-stained carotid arteries from uninjured (A) and injured and untreated/control (B) Zucker fatty rats. The hyperplastic response that follows injury was significantly reduced when the rats were treated with salsalate (C), and quantitation of the reduction in intimal hyperplasia is depicted in D. Balloon catheter injury results in a hyperplastic response in the injured segment of the artery and salsalate produces a significant attenuation of the response. The reduction in intimal hyperplasia could be attributed, at least in part, to treatment with salsalate. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

VEGF protein immunostained carotid artery sections of injured, untreated/control (A) and injured and treated with salsalate (B) Zucker fatty rats. The intimal staining intensity was significantly decreased by 22% in salsalate-treated rats (62 ± 7) versus untreated rats (78 ± 6). P < 0.05.

FIG. 3.

The RDUs obtained from the densitometric scan of the Western blots of eNOS (after normalizing expression levels to corresponding β-actin) showed a marked increase in eNOS expression in aorta samples from salsalate-treated rats when compared with the control group. The mean relative density of scans from control rats that were balloon catheter injured and not treated was 0.027 ± 0.009; the mean relative density of the salsalate-treated rats was 0.148 ± 0.066 and was significantly different (P < 0.05). Compared with the control rats, the expression of eNOS in the salsalate-treated rats was increased fivefold (0.15/0.03).

FIG. 4.

The RDUs obtained from the densitometric scan of the Western blots for the expression of phosphorylated eNOS (ser 1177) (after normalizing expression levels to corresponding β-actin) in aortic tissue showed an upregulation of p-eNOS by salsalate therapy. There was a significant difference in the mean RDU between the control and salsalate-treated rats (0.2 ± 0.06 vs. 2.0 ± 0.5; P < 0.005). Compared with the control rats, the expression of p-eNOS was increased 10-fold (2.0/0.2). The ratio of the expression of p-eNOS to eNOS in the control animals was 6:1 (0.2/0.03) and in the salsalate-treated rats was 13:1 (2.0/0.15).

FIG. 5.

The Western blot analysis of aortic arch NF-κB subunit p65 indicated a significant decrease expression in the salsalate-treated group (mean relative densities of the salsalate-treated and control groups: 0.1599 ± 0.1092 vs. 0.6003 ± 0.11; P = 0.01). The data indicates that salsalate treatment reduced the inflammatory response following the balloon catheter injury of the carotid artery.

FIG. 6.

The MnSOD expression of aortic arch in balloon catheter injured and untreated/control Zucker fatty rats and balloon catheter injured and treated with salsalate. There was a significant increase in the mean relative density in salsalate-treated rats when compared with control rats (control was 0.673 ± 0.07 and salsalate 1.2 ± 0.2; P < 0.05). The increase in the expression of this antioxidant defense enzyme suggests that salsalate therapy enhances ROS quenching thereby reducing tissue injury.