| Objective: Stiffening of the large arteries is the characteristic of arterial aging.Elastin and collagen are two main components of the arterial wall and their normalmetabolism are regulated by a number of enzymes, the most important of which ismatrix metalloproteinases (MMPs) system. It is reported that aortic MMPs activity isincreased in the development of hypertension; but its relationship with hypertensiveaortic remodeling has not been clarified so far. Aneurysm is a kind of outward arterialremodeling. Recent researches show that the increased activity of MMPs plays animportant role in the development and rupture of aneurysm. Accordingly,non-specificity MMPs inhibitor (doxycycline) can effectively delay the dilation ofaneurysm. Therefore, the purpose of present study is to investigate the effect ofdoxycycline on hypertensive aortic remodeling by its property of broad-spectrumMMPs inhibition.Methods: 80 male SHR-SPs of 7 weeks old were randomly divided into 2 groups(n=40 for vehicle-treated group and doxycycline-treated group). 5 mate Wistar-Kyotorats (WKY) at the same age were served as normal controls. We treated SHR-SPschronically from the end of 7 weeks after birth with doxycycline (30 mg·Kg-1·day-1) indrinking water. The amount of drug was adjusted weekly by body weight. Throughoutthe intervention, body weight and indirect tail-cuff systolic blood pressure werecontinuously measured. The intervention was terminated when mortality ofvehicle-treated group reached 50％. After perfusion with phosphate buffered solution, aorta was dissected and quickly frozen by liquid nitrogen and stored in -80℃forfurther use. Some arteries were embedded in paraffin, sectioned into 5μm, stainedwith hematoxylin and eosin, Victoria blue (elastic fiber staining) and von Kossa silver(calcium staining) pathological staining. Others were prepared for cross-linkedcollagen assay, gelatin zymography, western blot analysis. The sections were scannedby digital CCD. The intima perimeter, adventitia perimeter, cross sectional area ofmedia, elastic fiber/media area percentage, calcium/media area percentage wereanalyzed by image analysis software.Results:1. Analysis of gelatin zymography and western blot suggested that doxycycline couldinhibit the activity of aortic MMP-2 and MMP-9, and had no effect on the proteinexpression level of MMP-2. The result of gelatin zymography analysis: comparedwith CON group, the activity of MMP-2 and -9 were greatly decreased in DOX group(MMP-2:3.15±0.23 vs. 2.43±0.14, P＜0.05; MMP-9: 14.1±1.50 vs. 9.71±1.31,P＜0.05). Western blot: there is no statistical difference in the protein expression levelof MMP-2 between CON and DOX group (0.625±0.194 vs. 0.576±0.165, P＞0.05).2. Analysis of Victoria blue staining: compared with WKY group, aortic elastic fiber/media area was elevated in CON group [(2.19±0.997)％vs. (5.72±3.87)％, P＜0.05].Compared with CON group, the ratio was dramatically decreased in DOX group[(5.72±3.87)％vs. (3.46±1.62)％, P＜0.05].3. Sircol soluble collagen assay: the aortic cross-linked collagen in CON and DOXgroup was lower than that of WKY (28.0±8.71μg/mg, 26.4±6.98μg/mg vs.55.2±13.3μg/mg, all P＜0.05). There was no statistical difference between CON andDOX group (28.0±8.71μg/mg vs. 26.4±6.98μg/mg, P＞0.05).4. von Kossa silver staining: aortic calcium/media area in CON and DOX group waslarger than that of WKY[(0.960±0.999)％, (0.688±0.718)％vs. (0.232±0.237)％,P＜0.05]. There was no statistical difference between CON and DOX group, but the latter had a decreased tendency [(0.960±0.999)％vs. (0.688±0.718)％, P=0.177].5. There was no difference in intima perimeter, adventitia perimeter and media areabetween three groups: there was no statistical difference in aortic intima perimeteramong CON, WKY and DOX group, respectively (6322.4±640.2μm vs.6127.4±331.1μm vs. 6294.4±271.6μm, all P＞0.05). Compared with WKY group, theaortic adventitia perimeter in CON and DOX group had elevated tendency(6952.3±493.1μm vs. 7418.9±786.2μm, 7434.0±392.2μm, P=0.132, P=0.121,respectively). There was no statistical difference between CON and DOX group(7418.9±786.2μm vs. 7434.0±392.2μm, P＞0.05). Compared with WKY group, thecross sectional area of media in CON and DOX group also had elevated tendency(757559.2±291711.6μm2 vs. 905547.6±344201.9μm2, 990607.8±175468.7μm2,P=0.268, P=0.09, respectively). There was no statistical difference between CON andDOX group (905547.6±344201.9μm2 vs. 990607.8±175468.7μm2, P＞0.05).Conclusions: The present work shows that doxycycline, a broad-spectrum matrixmetalloproteinases inhibitor, can inhibit aortic MMP-2 and MMP-9 activity inSHR-SPs, but have no effects on protein expression level of MMP-2 and cross-linkedcollagen, and can decrease the content of elastic fiber. Therefore, non-selective MMPsinhibition in the setting of uncontrolled hypertension, may aggravate the process ofaortic stiffening. |
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