The Effects Of All-trans Retinoic Acid On VEGF, MMP-9and Vascular Remodeling In Asthmatic Rats

Objective: We have established a OVA-challenged asthmatic rat model.To study the effects of all-trans retinoic acid on the expression of MMP-9,VEGF and vascular remodeling in asthmatic rats. The expression level ofMMP-9, VEGF in lung tissue and serum, the airway wall thickness andvascular density were measured in the lung tissues of rats. Throughcomparing the differences of MMP-9, VEGF, airway wall thickness andvascular density between all-trans retinoic acid intervention group and asthmagroup, we investigate the effects of all-trans retinoic acid on asthma vascularremodeling.Method: Thirty cleaning male SD rats, which were six weeks old, andweight from100to120grams, were randomly divided into three groups (10ineach group): control group, asthma group, all-trans retinoic acid interventiongroup. The asthma group rats were sensitized on the first day and the eighthday with ovalbumin (100mg) and Al(OH)3(100mg) which mixed in2mlnormal saline by intraperitoneal injection. And then these rats were inhaledaerosolized OVA in an on completed closed box from the fifteenth day to theend. Rats were inhaled30minutes each time, every other day, there are20times in total. The all-trans retinoic acid intervention group was given all-transretinoic acid (20mg/Kg) for30minutes before OVA challenged, the otherswere the same as the asthma group. The control group rats were sensitized andchallenged with normal saline, the others were the same as the asthma group.After24hours of the last challenge, rats were anesthetized by1%pentobarbital sodium. Then we collected bronchoalveolar lavage fluid (BALF),the lung tissue and serum in each group. The total and different cell’s numbersin BALF were counted. Lung tissues were sliced and stained with HE. Theexpressions of MMP-9and VEGF in lung tissues were observed by immunohistochemistry combined with the micro-image analysis. The contentsof VEGF in serum were measured by ELISA. And mean optical densities ofVEGF and MMP-9were measured by image analysis software. The vasculardensity and airway wall thickness were measured by image analysis software.Results：1.Results of total cells and eosinophils in the BALF(×106): The numbersof total cells and eosinophils in the BALF in the three groups were as follows:Control group were (9.68±2.71,1.17±0.48).Asthma group were (33.91±3.67,5.38±0.63). All-trans retinoic acid intervention group were (12.88±2.72,2.19±0.53). The all-trans retinoic acid intervention group were significantlyless than the asthma group(P<0.01),and all the cells were significantly morethan that in the control group (F=64.860P<0.05, F=90.599P<0.05).2.Pathological findings: The obvious inflammatory cells infiltration,goblet cells and vascular hyperplasia, airway wall thickness were observed inthe asthma group. The pathological changes were not observed in the controlgroup. The pathological changes in the lung tissue of all-trans retinoic acidintervention group were all better than the asthma group.3.Results of image analysis: The airway wall thickness (μm2/μm) andvascular density (a/mm2) in the all-trans retinoic acid intervention group(86.20±2.36,40.75±4.70) were significantly less than the asthma group(98.78±2.86,56.84±8.37)(P<0.01).The airway wall thickness and vasculardensity in the two groups were significantly more than the controlgroup(70.87±1.80,20.11±6.08)(F=58.736P<0.01, F=98.886P<0.05).4.Result of ELISA: The all-trans retinoic acid intervention group(168.89±16.66) was significantly lower than The content of VEGF in serum ofthe asthma group (203.86±17.00)(P<0.01).The contents of VEGF in serum ofthe two groups were significantly higher than the control group (137.70±17.02)(F=60.452, P<0.05).5.Results of immunohisto chemistry: The expression levels of VEGF andMMP-9in lung of the asthma group and all-trans retinoic acid weresignificantly higher than the control group(0.22±0.04,0.22±0.01)(F=57.966 P<0.01, F=75.261P<0.01).The expression levels of VEGF and MMP-9inlung tissue of the asthma group (0.46±0.05,0.63±0.02) were significantlyhigher than the all-trans retinoic acid intervention group (0.28±0.06,0.41±0.02)(F=73.38P<0.05, F=56.921P<0.05).6.Results of correlation analysis: There was a significant positivecorrelation between the content of VEGF in serum and vascular density(r=0.856P<0.01). There was a significant positive correlation between theexpression levels of VEGF and MMP-9in lung (r=0.797P<0.01).There wasalso a significant positive correlation between the airway wall thickness andvascular density (r=0.893P<0.01). There were significant positive correlationsbetween the vascular density and the expression levels of VEGF andMMP-9in lung (r=0.795P<0.01, r=0.890P<0.01)Conclusions:1.We observed obviously inflammatory cells infiltration in lung tissue ofthe asthma group. It demonstrated that bronchial asthma is a chronic airwayinflammatory disease which usually has various inflammatory cellsparticipated. And all-trans retinoic acid can significantly relieve the airwayinflammation.2.All-trans retinoic acid reduced the extent of glandular hyperplasia,vascular hyperplasia and airway wall thickening in lung tissue of the asthmagroup. All-trans retinoic acid also reduced the vascular density and the airwaywall thickness. There was a obviously positive correlation between vasculardensity and the airway wall thickness. Vascular remodeling plays an importantrole on airway remodeling. All-trans retinoic acid reduced vascularremodeling and airway remodeling.3.The expression levels of VEGF and MMP-9in asthmatic group weresignificantly increased. The expression levels of VEGF and MMP-9inall-trans retinoic acid group can be significantly reduced. And there was asignificant positive correlation between VEGF and MMP-9. It comes toconclusion that VEGF and MMP-9play an important role on the pathogenesisof bronchial asthma. It maybe has a cooperation effect between VEGF and MMP-9. All-trans retinoic acid can significantly inhibit the expression ofVEGF and MMP-9.4.The results of correlation analysis displayed that there was a significantpositive correlation between the expression level of VEGF, MMP-9, and thevascular density. There was also a significant positive correlation between thevascular density and airway wall thickness. It showed that VEGF MMP-9mayplay an important role on vascular remodeling of bronchial asthma. MMP-9and VEGF can increase vascular remodeling. Our data also showed thatall-trans retinoic acid could effectively relieve the vascular remodeling, airwayinflammation, and airway remodeling.