Hypertensive vascular remodeling was inhibited by Xuezhikang through the regulation of Fibulin-3 and MMPs in spontaneously hypertensive rats

Abstract

Fibulin-3, an extracellular glycoprotein, has been suggested as having functions in vessels. In hypertension, extracellular matrix, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play important roles in cardiovascular remodeling. However, the role of Fibulin-3 as an extracellular glycoprotein in hypertensive vascular remodeling remains unclear. Our study was to determine whether Fibulin-3 and TIMPs/MMPs would affect vascular structure during hypertension and the treatment of Xuezhikang. Thirty spontaneously hypertensive rats (SHRs) aged 8 weeks were randomized to three groups: SHRs control group (SHRs group, n=10), group treated with low dose Xuezhikang (XZK-L, 20 mg/kg/d, n=10) and group treated with high dose Xuezhikang (XZK-H, 200 mg/kg/d, n=10), the normal group was comprised of ten Wistar-Kyoto (WKY) rats of the same age. We showed that serum nitric oxide (NO) in control group was significantly lower than WKY group (P<0.05). Concomitantly, serum oxidized low-density lipoprotein (ox-LDL) was higher than WKY group (P<0.05). The treatment of high dose Xuezhikang significantly dicreased ox-LDL, left ventricular mass index (LVMI) and Wall-to-lumen area ratio (W/L) of thoracic aorta (P<0.05), while serum NO was significantly increasing (P<0.05). Moreover, the expressions of Fibulin-3 and MMP-2, 9 at both protein and mRNA levels were significantly higher in thoracic aorta of SHRs group compared to WKY group by immunohistochemistry and western blotting (P<0.05). However, the levels of Fibulin-3 and MMP-2, 9 were significantly decreased in XZK-H group compared to control group (P<0.05). The level of TIMP-3 had no significance difference between SHRs and WKY groups (P>0.05). So the levels of Fibulin-3 and MMP-2, 9 in SHRs could be inhibited by Xuezhikang. Furthermore, a strong correlation in transcript expression was established between Fibulin-3, and MMP-2 (r=0.81, P<0.05) and MMP-9 (r=0.92, P<0.05) through immunohistochemistry. In summary, the overexpression of Fibulin-3 and MMP-2, 9 levels were associated with hypertension and vascular remodeling and inhibited by Xuezhikang. Fibulin-3 is a candidate in the pathogenesis of cardiovascular remodeling in hypertension.

Keywords: Fibulin-3; hypertension; matrix metalloproteinase; spontaneously hypertensive rat; vascular remodeling.

Figure 1

Histological Analysis of thoracic aorta. A. Representative images of thoracic aorta (H&E stain). B. The quantitative analysis on the thickness of thoracic aortic wall. The thickness of vascular wall of SHRs was significantly higher than that of WKY group. C. The quantitative analysis on wall-to-lumen ratio of thoracic aorta. Wall-to-lumen area ratio of aorta in SHRs group was significantly higher than that of WKY group. At the same time, wall-to-lumen area ratio of aorta was significantly decreased in the XZK-H group compared to SHRs group. Compare to WKY group, *P<0.05; Compare to SHRs group, #P<0.05.

Figure 2

The protein expression level of Fibulin-3, MMP-2, MMP-9 and TIMP-3 were detected in all groups by immunohistochemistry. A. Fibulin-3, MMP-2 and MMP-9 In SHRs group were significantly increased in the thoracic aorta compared to WKY group. Fibulin-3 and MMP-2, 9 were significantly decreased in XZK-H group compared to SHRs group. TIMP-3 had no significance in the four groups. Positive staining is shown as brown in cells cytoplasm (×400). B. Quantitative analysis of Fibulin-3, MMP-2, MMP-9 and TIMP-3 expression in different experimental groups was performed. The data are expressed in terms of the fold of the WKY control group as mean ± SD. Compare to WKY group, *P<0.05; Compare to SHRs group, #P<0.05.

Figure 3

The protein expression level of Fibulin-3, MMP-2, MMP-9 and TIMP-3 of thoracic aorta were detected in all groups by western blotting. The levels of Fibulin-3, MMP-2 and MMP-9 in SHRs group were significantly higher than those in WKY group. Protein expression levels were normalized to GADPH.

Figure 4

The transcription levels of Fibulin-3, MMP-2 and MMP-9 mRNA of thoracic aorta were analyzed by Real-time PCR. A. Real-time PCR examination of Fibulin-3, MMP-2 and MMP-9 in SHRs, XZK-H, XZK-L and WKY groups. The mRNA expression levels are presented as the increasing fold compared with WKY group or SHRs group and were normalized to GAPDH. Bars represent the SEM. Compare to WKY group, *P<0.05; Compare to SHRs group, #P<0.05. B. The scatter diagram showed the strong correlation between Fibulin-3 and MMP-9 on mRNA level. C. The scatter diagram showed the strong correlation between Fibulin-3 and MMP-2 on mRNA level.