Vascular Intima and Angiogenic Factors Alterations in a Rat Model of Venous Thromboembolism
|School||Peking Union Medical College , China|
|Keywords||animal model of venous thromboembolism deep vein thrombosis pulmonary thromboembolism vessel intima vascular endothelial growth factor basic fibroblast growth factor natural history|
[Backgroud]Deep vein thrombosis （DVT） and pulmonary thromboembolism （PTE） are distinct but related aspects of the same dynamic disease process knowed as venous thromboembolism （VTE）. PTE results from sudden occlusion of pulmonary arteries by thromboemboli originating in the deep veins in the majority of cases. Additionally, both DVT and PTE present with the same risk factors and management strategies. Therefore, it is necessary to study them as a whole.Development of VTE appears to be the result of multiple simultaneously operating factors identified by one or more predisposing genetic and/or acquired （environmental） risk factors for thrombosis. The pathogenesis of VTE comprises the triad of interdependent factors described by Virchow, consisting of blood stasis, hypercoagulable states and vascular endothelium injury. Modern studies show that endothelial cell microdamage and/or dysfunction play an important role in thrombosis, which in turn may result in the occurrence of recurrent thromboembolic events, incomplete resolution of embolic residuals and even progression to chronic thromboembolic pulmonary hypertension. Thus, in order to further understand the underlying pathogenesis, pathophysiology, and the natural history of VTE, it is important to explore its vascular intimal alterations and angiogenic factors such as vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF）.[Objective]1. To establish a rat model of VTE and measure its cardiovascular and respiratory pathophysiology.2. To observe the vascular intimal alterations of femoral veins and pulmonary arteries associated with VTE in rats.3. To determine changes and the location of VEGF and bFGF expression in the lung and serum and their roles in the pathology of VTE.[Methods]1. 120 male or female Sprague-Dawley rats were randomly divided into DVT group （n=18）, DVT-PTE group （n=54） and sham group （n=48）. Furthermore, the DVT group is divided into three subgroups according to the different duration of thrombosis （1 day, 4days and 7 days） knowed as DVT1 subgroup （D1） （n=6）, DVT4 subgroup （D4） （n=6） and DVT7 subgroup （D7） （n=6）. Similarly, DVT-PTE group is also divided into nine subgroups according to the different duration of thrombosis （1 day, 4days and 7 days） and the length of time since embolization （1 day, 4days and 7 days） knowed as DVT1-PTE1 subgroup （D1P1） （n=6）, DVT1-PTE4 subgroup （D1P4） （n=6）, DVT1-PTE7 subgroup （D1P7） （n=6）, DVT4-PTE1 subgroup （D4P1） （n=6）, DVT4-PTE4 subgroup （D4P4） （n=6）, DVT4-PTE7 subgroup （D4P7） （n=6）, DVT7-PTE1 subgroup （D7P1） （n=6）, DVT7-PTE4 subgroup （D7P4） （n=6）, and DVT7-PTE7 subgroup （D7P7） （n=6）. An additional 48 rats underwent sham operations （twelve subgroups） corresponding to DVT subgroups and DVT-PTE subgroups as described above （n=4）. Animals were sacrificed at 1, 4, 7 days after DVT and 1, 4, 7 days after DVT-PTE, respectively.2. A rat model of femoral vein thrombosis was established by the indwelling blood vessel clip to reduce blood flow. Emboli were occluded in pulmonary arteries by injecting the laminated thrombi induced in vivo from another lateral femoral vein.3. Mean pulmonary arterial pressure （mPAP）, mean fight ventricular pressure （mRVP）, arterial partial pressure of oxygen （PaO2） and mixed venous oxygen pressure （PvO2） were detected.4. Vascular intima of pulmonary arteries and femoral veins and emboli lodged in vessels were observed on the light microscope and electric microscope.4. Immunohistochemical analysis was used to investigate the expression and location of VEGF and bFGF in the lung. Both VEGF and bFGF mRNA expression and protein concentration in lung tissue were determined by RT-PCR and Western blot, respectively. The serum level of VEGF and bFGF was measured with ELISA. 5. Data were analyzed statistically by the analysis of variance and nonparametric tests. A value of p＜0.05 was accepted as significant.[Results]1. Establishment of a new model of VTE in rats.It is successful to establish a model of DVT in femoral veins by the indwelling blood vessel clip and produce DVT-PTE by injecting the laminated thrombi. Of the 54 animals with DVT-PTE, one died within 12 hours in D1P1 subgroup. Autopsy disclosed fresh emboli occluding nearly all lobar vessels.2. Changes of cardiovascular and respiratory pathophysiology in VTEThe animals in D1P1 group and D7P4 group demonstrated that a significant elevation of mPAP and mRVP （p＜0.05）. The level of PaO2 and PvO2 in D4P4, D4P7, D7P1, D7P4 and D7P7 subgroups decreased significantly （p＜0.05）. The duration of DVT or PTE made an independent inflence on mPAP, mRVP and PaO2 （p＜0.05）, their combined effects were also obvious （p＜0.01）. Although the duration of DVT or PTE did not make an independent influence on PvO2, their combined effects were significant （p＜0.01）.3. Changes in the shape of non-embolized and embolized pulmonary arteries:There was an increase in the ratio of the non-embolized arterial wall area and vessel area, especially that in D1P7, D4P4, DTP4 and D7P7 subgroups increased significantly compared to that in sham group （p＜0.05）. Although the ratio of the embolized arterial wall area and vessel area did not vary compared to that in sham group, there was a trend of a mild decrease, then a mild increase at 1 day and 7 days after DVT-PTE, respectively.4. Pathology of VTE in rats:On gross and/or microscopic examination, there were reddish thrombi, mixed （fibrin-platelet） thrombi and whitish （organized） thrombi at 1, 4, 7 days after DVT, respectively. When the two latter thrombi were obstructed in the pulmonary arteries, the extent of emboli resolution became slow and the process of organization became early, simultaneously, the changes of vessel wall were severe and early, the inflammation of parenchyma and intra-alveolar hemorrage were extensive and located more proximally. The juncture of endothelial cells was ruptured and the intra-elastic layer was exposed in the first day afte DVT, then they became disappear in the seventh day after DVT. Additionally, pulmonary arterial intimal hyperplasia was observed at 4 days or 7 days after reddish and/or fibrin-platelet emboli occluded in pulmonary arteries. Furthermore, pulmonary arterial intima may be disappear in the first day after organized emboti lodged in pulmonary arteries.5. Immunohistochemical analysis of the lungImmunohistochemical analysis indicated that VEGF antigen was located to the pulmonary vascular smooth muscle cells and alveolar endothelial cells in the first day after DVT-PTE, then, it was present in the pulmonary vascular endothelial cells in the forth day after DVT-PTE. In addition to, VEGF antigen expression was also present in the fibroblasts witin the organized emboli. Contrarily, VEGF antigen expressed in bronchia endothelial cells in sham group.Immunohistochemistry showed bFGF antigen overexpression was present in the pulmonary vascular smooth muscle cells at 1 day after DVT-PTE, and then bFGF antigen was located to the extracellular matrix around the pulmonary arteries at 4 days or 7 days after DVT-PTE. Meanwhile, bFGF antigen was also present in the fibroblasts witin the organized emboli. Contrarily, bFGF antigen expressed in bronchia endothelial cells and plmonary vascular smooth muscle cells in sham group.6. Changes of VEGF and bFGF in the lung and serumThe lung VEGF mRNA and protein expression rose to maximum at 1 day after DVT-PTE （p＜0.05）. At 7 days after DVT-PTE, the elevation of the mRNA expression was persisted （p＜0.05）, and the protein levels remained elevated above baseline, but was not significantly. Meanwhile, Both VEGF mRNA and protein expression in the lung in DVT group did not change. Furthermore, the serum VEGF levels in DVT and DVT-PTE group were not significantly altered compared to those in sham group.In DVT-PTE group, the lung bFGF mRNA and protein expression began to increase in the first day and in the seventh day after DVT-PTE, respectively （p＜0.05）, but in the DVT group the lung bFGF mRNA and protein expression did not change compared to those in sham group. Interestingly, the serum bFGF was significantly raised at 1 day after DVT, then decreased at 7 days after DVT. Additionally, the serum bFGF was lower in D7P4 and D7P7 subgroups than those in sham group（p＜0.05）.[Conclusions]1. A new rat model of venous thromboembolism can be successfully established owing to either laminated thrombi produced in the femoral vein by indwelling blood vessel clip or pulmonary emboli developed by injecting these different age of thrombi from another lateral femoral vein.2. The age and nature of thrombi before embolization are related directively to the extent of embolic residuals lodged in the pulmonary arteries, and together with the length of time since embolization, which can condition pathophysiological dysfunctions and pathological changes of VTE （DVT-PTE）, including pulmonary arterial intimal alterations.3. VEGF and bFGF have a role in the process of thrombus organization and reparation of pulmonary arterial intima associated with VTE.