Pulmonary Interstitial Changes And Its Mechanism Studies In Rats With Increased Blood Flow-induced Pulmonary Artery Hypertension

Establishment of Rat Models of Increased Blood Flow-induced Pulmonary Artery HypertensionAim To establish two rat models of increased blood flow-induced pulmonary artery hypertension using the right lung pneumonectomy and abdominal aorta-inferior vena cava shunting (A-V) procedures, and to compare the dynamic changes of hemodynamics and pulmonary vascular morphology in the two models so as to determine the optimal model for further studies.Methods160Spargue-Dawley rats were randomly divided into three groups:Control (n=40), PE (n=60) and A-V (n=60). Each group was further divided into four subgroups based on the time points:Week1, Week2, Week4and Week6.10rats were assigned in each subgroup of Control and15were in each subgroup of PE and A-V. At each time point, mean pulmonary artery pressure (mPAP) was detected; the right ventricle and left ventricle plus septum were separated and weighted for calculation of the weight ratio of the two parts [RV/(LV+SP)]; Lung tissues were collected for HE staining, Masson staining and a-SMA immunohistochemistry analysis, respectively. The pathomorphological changes of the small and medium-sized pulmonary arteries were observed, and the percentage of pulmonary vascular wall thickness (WT%) and the percentage of muscularized vessel (MA%) were evaluated.Results①The numbers that rats died during the process of modeling establishment was significantly greater in the PE group than that in the A-V group. Bleeding and heart failure were the major reasons of death for the PE group.②The trend of variations of mPAP in the A-V and PE groups were similar:quickly increased at Week1, decreased to the lowest at Week2, and gradually started to go up from Week4.Compared to the Control group, mPAP in the PE group was significantly increased at each time point (Week1, Week2, Week4and Week6)(P<0.01); mPAP in the A-V group was significantly increased at Week4and Week6(P<0.01). Compared to the A-V group, mPAP in the PE group was significantly increased at each time point (P<0.05).③RVHI in both the A-V and PE groups were gradually increased over time. Compared to the Control group, RVHI (right ventricular hypertrophy index) in both the A-V and PE groups were significantly increased at each time point (P<0.05). Compared to the A-V group, RVHI in the PE group was significantly increased at Week4, Week2and Week1(P<0.05).④WT%in both the A-V and PE groups were gradually increased over time. Compared to the Control group, WT%in both the A-V and PE groups were significantly increased from Week2(P<0.01or P<0.05) Compared to the A-V group, WT%in the PE groups was significantly increased at each time point (P<0.01or P<0.05).⑤)MA%in both the A-V and PE groups were gradually increased over time. Compared to the Control group, MA%in both the A-V and PE groups were significantly increased from Week2(P<0.01or P<0.05). Compared to the A-V group, MA%in the PE groups was significantly increased from Week4(P<0.01)Conclusions①Both PE and A-V rat models demonstrated the pathophysiologic characteristics of congenital heart disease-related pulmonary hypertension, showing clinical manifestations of pulmonary hypertension and right ventricular hypertrophy, and histopathological changes of increased media membrane wall thickness and increased numbers of muscularized pulmonary small arteries. Both of the two models could be used as effective and reliable animal models for studies of congenital heart disease-related pulmonary hypertension.②Compared to the A-V model, the PE model showed several advantages: more significant changes of hemodynamics, more obvious manifestations of pulmonary vessel remodeling, and less time required for establishment. Part II The Pulmonary Interstitial Changes in the Rat Model of Increased Blood Flow-induced Pulmonary Artery HypertensionAim To observe the changes in the pulmonary interstitium in the rat model of increased blood flow-induced pulmonary artery hypertension.Methods40Spargue-Dawley rats were randomly divided into four groups:Sham,2weeks after the PE procedure (PE-2W),4weeks after the PE procedure (PE-4W) and6weeks after the PE procedure (PE-6W).10rats were assigned in each group. The rat model with increased pulmonary blood flow-induced pulmonary artery hypertension was established using the right lung pneumonectomy (PE) procedure. The rats were killed at respective time point, and their lung tissues were collected for further studies. After HE staining and Masson staining, the pathological changes in the lung tissue was observed by microscope, and the cell microstructures was observed by transmission electron microscope. The amount of hydroxyproline in the lung tissue was detected for evaluating the collagen metabolism. The fibroblast proliferation was examined by the PCNA immunohistochemistry analysis. The protein expression of the Col-Ⅰ和Col-Ⅲ were detected by immunohistochemistry analysis and Western blot analysis. The mRNA amount of the Col-Ⅰ和Col-Ⅲ were detected by real-time fluorescence quantitative PCR (Q-PCR).Results①Under the microscope, the lung tissue in the sham group showed clear structures, no edema or inflammation, no thickening of alveolar septa, and distribution of a small amount of slim collagen. However, all the three PE groups showed inflammation reactions. The early phase of inflammation was obvious, mainly infiltrated with neutrophils. Inflammation of the late phase was alleviated, mainly manifested with lymphoid follicles. With the extension of time after the PE procedure, the alveolar septa gradually became thicker, a large number of fibroblasts proliferated, collagen fibers diffused from the perivascular tissues to the interstitial tissue, and parts of the alveolar cavity structures were damage.②The amount of hydroxyproline was gradually increased with the extension of time after the PE procedure. Compared to the Sham group, there was significant difference in the amount of hydroxyproline in the PE-4W and PE-6W groups (P<0.01) The amount of hydroxyproline was positively in proportion to mPAP, WT%and MA%.③In the Sham group, there was a small amount of expression of Col-Ⅰ and Col-Ⅲ in the interstitial tissue. In the PE groups, the expression of Col-I and Col-III was significantly increased from Week4. Col-Ⅰ and Col-Ⅲ interweaved into nets, which were mainly present around the blood vessels. The protein expression of Col-I was significantly increased at Week4and was slightly decreased at Week6, as detected by Western blot analysis (P<0.01). The protein expression of Col-Ⅲ was gradually and significantly increased from Week4to Week6(P<0.01).④The mRNA amount of Col-I was increased from Week2and was slightly decreased at Week6. Compared to the Sham group, there was significant increase in the mRNA amount of Col-I in three PE groups (P<0.01or P<0.05). The mRNA amount of Col-III was gradually increased over time in three PE groups, showing significant difference compared to the Sham group (P<0.01or P<0.05)⑤The mRNA amount of Col-I and Col-III were positively in proportion to mPAP, WT%and MA%(P<0.05)Conclusions①The rat PE model showed the pulmonary interstitial changes, and the histopathologic features were similar to that of the hybrid non-specific interstitial pneumonia.②The expression of Col-Ⅰ and Col-Ⅲ gradually increased as the increased blood flow-induced pulmonary artery hypertension progressed over time. Part ⅢExpression and Effects of TGF-β1and CTGF on the Pulmonary Interstitial Changes in the Rat Model of Increased Blood Flow-induced Pulmonary Artery HypertensionAim To observe the dynamic expression of TGF-β1and CTGF, and to explore the effects of TGF-β1and CTGF on the changes of pulmonary interstitium in the rat model of increased blood flow-induced pulmonary artery hypertensionMethods40Spargue-Dawley rats were randomly divided into four groups:Sham,2weeks after the PE procedure (PE-2W),4weeks after the PE procedure (PE-4W) and6weeks after the PE procedure (PE-6W). Each group contained10rats. The rat model with increased pulmonary blood flow-induced pulmonary artery hypertension was established using the right lung pneumonectomy (PE) procedure. The rats were killed at respective time point, and their lung tissues were collected for further studies. The expression of TGF-β1 and CTGF were detected by immunohistochemistry analysis. The protein expression of TGF-β1and CTGF were detected by Western blot analysis. The mRNA amount of TGF-β1and CTGF were detected by real-time fluorescence quantitative PCR(Q-PCR).Results①The protein expression of TGF-β1in the PE-2W and PE-4W groups were significantly increased, and were decreased in the PE-6W group. The protein expression of TGF-β1in the four groups were significantly different by pair comparison (all P<0.01or P<0.05). The mRNA amount of TGF-β1was highest in the PE-2W group, and was gradually decreased from Week4to Week6. The mRNA amount of TGF-β1in the four groups were significantly different by pair comparison (all P<0.01or P<0.05).②The protein expression of CTGF in the PE-4W and PE-6W groups were significantly increased compared to the PE-2W group and the Sham group (P<0.01). There was no significant difference between the Sham and the PE-2W group, or between the PE-4W and PE-6W group. The mRNA amount of CTGF was gradually increased in all three PE groups, showing significance compared to the Sham group (P<0.01). There was significant increase in mRNA amount of CTGF in the PE-4W and PE-6W groups, compared to the PE-2W group (P<0.01). However, no significant difference between the PE-4W and PE-6W groups was observed.③The protein expression of TGF-β1was positively in proportion to the protein expression and mRNA amount of Col-I (P<0.05). The protein expression and mRNA amount of CTGF were positively in proportion to the protein expression and mRNA amount of Col-Ⅲ (P<0.05). The mRNA amount of TGF-β1was negatively in proportion to the protein expression and mRNA amount of CTGF (P<0.05) Conclusions①During the process of increased blood flow-induced pulmonary artery hypertension, the expression of TGF-β1and CTGF were increased not only in the pulmonary vascular wall but also in the interstitium with slight fibrosis.②As crucial factors resulting in fibrosis, TGF-β1and CTGF may be involved in the fibrosis process of the pulmonary interstitium in the rat model of increased blood flow-induced pulmonary artery hypertension.