Effect Of Doxycycline On The Development Of Pulmonary Hypertension Induced By Four Methods In Rats

Objective Based on establishment of four rat models of experimental pulmonary hypertention(PH), namely induced by hypoxia (H), monocrotaline injection(M), high pulmonary blood flow(P) and pneumonectomy plus monocrotaline(PM) , we examined the inhibition of matrix metallproteinases(MMPs) by Doxycycline and its effect on the development of PH and associated pulmonary vascular remodeling.We explored whether MMPs played an important role in the process of pulmonary vascular remodeling and consequently if the role represented a common pathologic passway in the development of PH of various etiology.Methods Healthy male Spraque-Dawley rats(weight 350g to 400g)were randomly divided into nine groups: Normal control group(N), four model groups(H, M, P, PM) and their corresponding drug intervention groups(HD, MD, PD, PMD) in which Doxycycline was given by gavage on 20mg/kg daily dosage. High blood flow model(P) and neointima formation model(PM)were established by left lung pneumonectomy in normal rats and in monocrotalin-injected rats, respectively.On day 28(day 35, PM and PMD models), animals were catheterized to record mean pulmonary arterial pressure(mPAP) and then sacrifised. Fulton Inedx[RV/(LV+S)]were measured immediately. Samples taken from right lower lobe of lung underwent histological and morphometric analyses. Morphometric parameters, including percent vascular wall thickness, muscularizition of non-musclarised peripheral pulmonary arterioles, collagen distribution in pulmonary arteria and neointima formation , were determined microscopically.Immunohistochemical stains of PCNA and Caspases-3 were used to investigate cellular proliferation and apoptosis , respectively. Expression of MMP-2 , MMP-9 and tissue inhibitor of Metallproteinases-1(TIMP-1) were assessed by both immunohistochemical stain and real-time PCR(RT-PCR) of relevant mRNAs.Meanwhile,the activity of MMPs was measured by gelatin zymography in the lung tissue.Results①Rats in all model groups(H, M, P, PM) developed significant pulmonary arterial hypertention and right ventricular hypertrophy in comparison with their corresponding drug intervention groups(HD, MD, PD, PMD) and normal control group(N).(p<0.01)②The percent vascular wall thickness , percentage of muscularization of non-muscular pulmonary arterioles, extent of cellular proliferation and collagen deposition in pulmonary arterioles were significantly increased in all model groups compared with drug intervention groups and normal group.(P<0.01)③Expression of Caspases-3, which is related to celluar apoptosis, was decreased in model groups compared to the drug intervention groups significantly.(P<0.01)④Neointima was observed only in group PM and group PMD.Rats in group PM demonstrated more severe neointima hyperplasia than the group PMD.(P<0.01)⑤Expression and activity of MMPs were inhibited by doxycycline effectively as assessed by the immunohistochemical stain and gelatin zymography(P<0.01), which was consistant with the results of RT-PCR which examined the expression of MMP mRNAs.(P<0.01)⑥Model groups had higher TIMP-1 expression than their corresponding drug intervention groups both suggested by immunohistochemical stain and RT-PCR.(P<0.01).Conclusion Doxycycline attenuated PH and associated pulmonary vascular remodeling in all rat PH models. Inhibition of MMPs by Doxycycline was confirmed by the immunohistochemical stain and gelatin zymography.The study suggests that high expression and enhanced activity of MMPs may play a crutial role in the development of PH. Such phenomenon seems to be common in a variety of PH models of different etiology. The underlying mechanism of the drug is possiblly ascrible to the reduced smooth muscle cells(SMCs) proliferation , suppressed migration and promoted cell apoptosis, which are thought to be associated to the MMPs inhibition.These results are strongly in favor of the novel MMPs inhibition approach to the prevention and therapy of PH.