| BackgroundFibulins is a family of seven extracellular matrix proteins characterized by tandem arrays of epidermal growth factor (EGF)-like domains and a C-terminal fibulin-type module. They are widely distributed and often associated with vasculature and elastic tissues. Fibulin-3 is widely distributed in the extracellular matrix (ECM) in vertebrates. It is essential for normal development and maintenance in the microenvironment of embryonic and adult tissues. Matrix metalloproteinases (MMPs) are believed to play a significant role in the degradation of ECM components. The tissue inhibitor of metalloproteinase (TIMP), which binds to MMP with a 1:1 molar stoichiometry, is an endogenous inhibitor of MMPs and regulates the matrix remodeling by the MMP. Fibulin-3 could target endothelial cell expression of MMPs and TIMPs, thereby potentially reducing ECM proteolysis and remodeling. Remodeling of ECM is an important regulator of the physiological and pathological processes in the vessel wall. Tumors and cultured cells overexpressing Fibulin-3 also showed elevated expression and activity of MMPs, such as MMP-2 and MMP-9.While, there was a different report that Fibulin-3 negatively modulated the invasiveness of lung cancer cells via regulation of MMP-7 and MMP-2. Moreover, Fibulin-3 could decrease MB 114 endothelial cell expression of MMP-2 and MMP-3 while simultaneously increasing that of the MMP inhibitor, TIMP-1 and TIMP-3.However, the role of Fibulin-3 in the hypertensive vascular remodeling remains unclear. We hoped to find the relation between Fibulin-3, MMPs and hypertensive vascular remodeling in hypertensive animal model. In this study, we have analyzed the presence of Fibulin-3, MMP-2, MMP-9 and TIMP-3 in aorta of spontaneously hypertensive rats (SHR) and serum levels in the patients with hypertension.Part I The expressions of Fibulin-3 in thoracic aorta of spontaneously hypertensive ratsObjective:To study the expressions of Fibulin-3, MMP-2 and MMP-9 in the vascular remodeling in SHRs.Methods:Ten SHRs aged 8 weeks and ten SHRs aged 16 weeks were used as experimental groups. Ten Wistar-Kyoto(WKY) rats aged 8 weeks and ten WKY rats aged 16 weeks were used as control groups. The thoracic aorta were harvested, sectioned and stained with hematoxylin and eosin. Histological analysis was used to analyse the vascular remodeling. The expressions of Fibulin-3, MMP-2, MMP-9 and TIMP-3 proteins were detected by immunohistochemistry and western blotting. The levels of mRNA of Fibulin-3, MMP-2 and MMP-9 were detected by RT-PCR. Immunohistochemical expression was evaluated using Image-Pro Plus 6.0 software.Results:The average systolic blood pressure was significantly higher in SHRs aged 16 weeks[(222±14.66)mmHg] than in the other three groups[(142±8.89) mmHg in SHRs aged 8 weeks, (118±14.3)mmHg in WKY rats aged 8 weeks and (120±8.5)mmHg in WKY rats aged 16 weeks,P<0.001]. The thickness of thoracic aortic wall and wall-to-lumen ratio of SHRs aged 16 weeks were significantly higher than that of the other three groups(P<0.05).Moreover, the expressions of Fibulin-3 and MMP-2,9 proteins and mRNA levels were significantly higher in thoracic aorta of SHRs aged 16 weeks than in the other three groups. There were no significant differences in the levels of TIMP-3 protein between the four groups. Furthermore, immunohistochemistry revealed there was a positive correlation in the expressions between Fibulin-3, and MMP-2 (r=0.518, P=0.001) as well as beween Fibulin-3 and MMP-9 (r=0.672, P=0.000).Conclusions:The upregulating expression of Fibulin-3 and MMP-2,9 were associated with hypertensive vascular remodeling in SHRs’ thoracic aorta aged 16 weeks. The expressions of Fibulin-3 and MMP-2,9 may be take roles in the process of hypertensive vascular remodeling.Part II The effect of Fibulin-3 on blood pressure and vascular remodeling in spontaneously hypertensive ratsObjective:To determine the relation between Fibulin-3, MMP-2, MMP-9 and TIMP-3 and changes of vascular fibrous tissues and oxidative stress in vascular remodeling in spontaneously hypertensive rats.Methods:Ten Wistar-Kyoto(WKY) rats and thirty spontaneously hypertensive rats(SHRs) aged 8 weeks were used. SHRs were randomized to three groups:SHRs treated with physiological saline (I.V.) (placebo group,n=10); SHRs treated with low-dose Fibulin-3 protein (I.V.,120 ng/kg, FBLN1 group,n=10); SHRs treated with high-dose Fibulin-3 protein (I.V.,240ng/kg, FBLN2 group,n=10). Fibulin-3 protein was injected through tail vein of rats every week for 8 weeks, whereas the placebo group received normal saline. The control group (WKY rats, n=10) received no addition.Histological analysis was used to analyse the vascular remodeling. The aortic collagen was showed by Masson trichrome stain. The aortic fibre tissue was stained by aldehyde fuchsin staining. The aortic net fibre was stained by ammonium hydroxide liquid silver dip method. After incubation by dihydroethidium (DHE), frozen sections were stimulated by green light to show the levels of oxidative stress. The expressions of Fibulin-3, MMP-2, MMP-9 and TIMP-3 proteins were detected by immunohistochemistry and western blotting. The levels of mRNA of Fibulin-3, MMP-2 and MMP-9 were detected by RT-PCR.Results:The average systolic blood pressure (SBP) in SHRs[(224±15)mmHg in placebo group, (207±14.5) mmHg in FBLN1 group and (180±13.7)mmHg in FBLN2 group) was significantly higher than that in WKY rats[(122±10.0) mmHg, P<0.01]. SBP in FBLN2 group was significantly decreased compared to placebo group (P<0.05). The thickness of thoracic aorta in placebo group was higher than that in control group(97.78±5.39um vs.82.23±6.02um, P<0.05).The thickness of thoracic aorta in FBLN1 group (105.99±5.92um) and FBLN2 group (121.7±10.81um) were higher than that in placebo group(P<0.05). The thickness of aorta in FBLN2 group was increased significantly compared to FBLN1 group(P<0.05).The wall-to-lumen ratio in placebo, FBLN1 and FBLN2 groups was significantly higher than that in control group(7.91±0.6%,8.15±0.38% and 7.96±0.41% vs 6.35±0.55%, P<0.05). However, there was no significance between placebo group, FBLN1 group and FBLN2 group(P>0.05).In control group, collagen tissue was scattered distribution lined in the membrane elastic fiber, elastic fiber membrane integrity, and neatly arranged in the thoracic aortic wall.In the placebo group, the collagen fiber content increased significantly, elastic fibers arranged in loose, disorder, or even broken.The number of muscle fiber was hypertrophy and hyperplasia obviously;Compared to the placebo group, collagen fibers were reduced in the FBLN1 group and FBLN2 group, even FBLN2 group was similar to the control group.The number of elastic fibers was increased significantly in the FBLN1 group and FBLN2 group compared to the placebo group, showed more complete, neat, crude in fiber bundle diameter, and elastic fiber of FBLN2 group thickening obviously.The diameter of elastic fiber was calculated by using Image-Pro Plus6.0.The diameter of elastic fiber in all the SHRs[(4.56±0.70)um in placebo group, (5.03±0.86)um in FBLNl group and (7.61±0.89)um in FBLN2 group] was higher than that in the control group(3.52±0.63 um)(P<0.05). The diameter of elastic fiber in the FBLN2 group was higher than those in placebo group and FBLNl group(P<0.01).The net fibers were increased in the media of aorta in SHRs (placebo group, FBLNl group and FBLN2 group), surrounding around elastic fibers.But there were no significant difference between the three groups.In experiments of vascular oxidative stress, ROS-positive cells in the whole nucleus were stained red. It showed little or no positive cells in aorta of control group.We had seen more positive cells stained nuclei red in the placebo group, indicating that aorta was oxidative highly. The number of positive cells decreased in the FBLN1 group and FBLN2 group compared to the placebo group.Moreover, the expressions of Fibulin-3 and MMP-2,9 at both protein and mRNA levels were significantly higher in thoracic aorta of placebo group compared to control group. The levels of MMP-2 and MMP-9 had significantly decreased in FBLN2 group compared to placebo group. But the levels of TIMP-3 had no significance in the four groups.Conclusions:The upregulating expressions of Fibulin-3 and MMP-2,9 were associated with hypertensive vascular remodeling in SHRs’thoracic aorta compared to WKY rats. The collagen fibers appear significantly increased, elastic fibers arranged in loose, disorder, or even broken, levels of vascular oxidative stress elevated in the hypertensive vascular remodeling. Moreover, Fibulin-3 could decreased the level of SBP in SHR and reduce MMP-2,9 in hypertensive vascular remodelin. Fibulin-3 might have the roles of repairing the damage of elastic fibers, inhibiting the proliferation of collagen fibers and the level of vascular oxidative stress. Fibulin-3 might be a growth factor in the arteries.Thus we thought that increasing Fibulin-3 might be beneficial for improving vascular health and offsetting some cardiovascular risk factors of hypertension.Part III The expressions of serum Fibulin-3, MMP-9 and TIMP-3 as well as the correlation between Fibulin-3 and Pulse Wave Velocity in patients with hypertensionObjective:To investigate the changes and significances of the levels of serum Fibulin-3, MMP-9 and TIMP-3 as well as correlation between these indexes and branchial-ankle Pulse Wave Velocity (baPWV) in patients with hypertension.Methods:A total of 50 patients with hypertension and 38 healthy people were enrolled finally, whose fasting blood were collected. The levels of Fibulin-3, MMP-9 and TIMP-3 were detected by enzyme-linked immunosorbent assay. The baPWV was detected by pulse wave velocity automatic analyzer.Results:The level of serum Fibulin-3 in hypertension group was significantly lower than that in control group (52.64±24.30 ng/ml vs 78.44±23.37 ng/ml, F<0.001).There was no significance of serum MMP-9 and TIMP-3 in the two groups (40.82±13.73 ng/ml vs 40.57±11.29 ng/ml,P>0.05; 5.41±2.29ng/ml vs 6.32±2.67 ng/ml, P>0.05). The baPWV in hypertension group was higher than that in control group (1725.28±366.86 cm/s vs 1365.50±235.33 cm/s,P<0.001). There was a negative correlation between the level of Fibulin-3 and baPWV (r=-0.328, P=0.002).While correlations between the level of MMP-9 and baPWV had no significance(r=-0.030, P=0.784). The correlations between the level of TIMP-3 and baPWV had no significance (r=0.002, P=0.984).Conclusions:The level of serum Fibulin-3 and baPWV can be looked as index of functional changes in the early stage of hypertensive vascular remodeling, and may be used for forecasting the organ damage in hypertension. |
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