The Role And Mechanism Of Cyclophilin A/CD147 Signal Pathway In The Development Of Right Ventricular Dysfunction After Acute Pulmonary Embolism In Rats

Acute pulmonary embolism(APE)is a serious and life-threatening disease with a high and increasing incidence.Although there is a progress on the diagnosis and treatment of the disease,the 30-day mortality rate within hospitalized patients is still as high as 10%.In the clinical practice for intermediate-and high-risk patients,the existing treatment is focusing on improving mechanical obstruction of pulmonary vascular bed by thrombolysis or removing thrombus.This strategy,though significantly improves the survival rate for patients with hemodynamic instability,contributes little to the patients whose hemodynamics is stable but present right ventricular dysfunction(RVD).With the advent of population aging society in our country and the rising incidence of malignant tumor,APE will be more common in clinical work in the future.Thus,further clarifying the pathogenesis and looking for ways to reduce the incidence of RVD and improve the prognosis of patients with APE is meaningful and urgent.After the presence of APE,right ventricular dysfunction due to increased pulmonary vascular resistance is the prime cause of death.Previous studies suggested that the tension of right ventricular wall caused by the high right ventricular pressure and the ischemia hypoxia caused by the compression of coronary artery are the main causes of myocardial tissue injury,which contributes to RVD.However,with the deepening of research,researchers gradually realized that inflammation response following APE also played an important role in the development of RVD.In this regard,APE associated inflammatory cells inflow to right ventricular tissue,followed by a rapid release and activation of large amounts of matrix metalloproteinases(MMPs).Activated MMPs(especially MMP-2 and MMP-9)could degrade cardiac troponin-I(c Tn I),myosin light chain-1,and other sarcomeric and cytoskeletal proteins,thus causing myocardial contractile dysfunction following APE.Numerous studies have shown that anti-inflammatory compounds or the inhibition of MMPs attenuates cardiomyocyte injury and improves survival rate in animal models of APE.More importantly,several studies have also proved that the blocking of MMPs can also weaken the contraction of pulmonary vascular following APE,thus attenuating APEinduced increases in pulmonary arterial pressure.These results suggested that inflammatory response following APE may participate both in pulmonary vasoconstriction and myocardial tissue structure damage process.Inhibition of this response may alleviate pulmonary artery pressure and right ventricular myocardial injury simultaneously,which may reduce the incidence or severity of RVD and improving the prognosis of patients with APE.However,the exact mechanism by which inflammatory cells are recruited is still unclear.Within the last decade,studies have demonstrated that extracellular cyclophilin A(Cy PA)is highly expressed in a variety of inflammatory diseases and is involved in the inflammatory response process of these diseases as a key factor.In the stimulation of hypoxia,infection or oxidative stress,Cy PA could be secreted from a variety of inflammatory cells,smooth muscle cells and activated platelets cell.By interacting with its cell-surface signaling receptor cluster of differentiation 147(CD147),extracellular Cy PA could activate ERK1/2-nuclear factor(NF)-κB pathways,stimulate cytokine release,accelerate leukocyte recruitment,and boost MMPs activation in stimulus sites.The Cy PA/CD147 interaction has been implicated in the pathological inflammatory process of many diseases,such as rheumatoid arthritis,atherosclerosis,pulmonary hypertension caused by hypoxia,myocardial infarction,early brain injury caused by subarachnoid hemorrhage and cerebral infarction.Given this evidence,we hypothesized that Cy PA also participated in the inflammatory response following APE and contributed to the development of RVD.Therefore,the purpose of this study was to investigate the role of extracellular Cy PA and CD147 in pulmonary hypertension and right ventricular injury following APE and to examine whether inhibition of Cy PA with cyclosporine A(Cs A)or inhibition of CD147 with CD147 m Ab(anti-CD147)attenuates the increase of mean pulmonary arterial pressure(m PAP)and right ventricular injury and dysfunction associated with APE.Part Ⅰ The establishment of APE model and the expression of Cy PA and CD147 in the lung and right ventricular tissue of embolized ratsObjective: To establish the rat model of APE and to investigate the expression of Cy PA and CD147 in the lung and right ventricular tissue of the embolized animals.Methods: The APE model was induced by intravenous injections of Sephadex G-50 microspheres in Sprague-Dawley(SD)rats,with a dose of 12 mg/kg.Thirty-six rats were randomly divided into six groups,including six rats each,a sham group,and five experimental groups arranged by time: 6,12,24,48,and 72 hours after APE.Sham group animals underwent the same procedure as described,but an equivalent volume of normal saline was injected into the vein instead of microspheres.All the animals were euthanized at the above-mentioned post-APE time points.Plasma samples were collected and Cy PA levels were ascertained by Western blotting.The lung and right ventricular tissues were excised and the expression of Cy PA or CD147 was accessed by Western blotting or immunofluorescence analysis.Results: Compared with the sham group,the protein levels of Cy PA and CD147 in the lung and the right ventricular tissues from the APE groups increased and peaked at 24 hours and then decreased.However,the protein levels of Cy PA and CD147 were still higher than that in the sham group,even 72 hours after APE.The Cy PA levels in plasma also increased following APE and showed similar changing process.Conclusions: The expressions of Cy PA and CD147 increased synchronously in the lung and right ventricular tissues following APE and peaked at 24 hours.Cy PA and CD147 might participate in the pathological progression following APE,and 24 hours after APE may be a suitable time point for further research.Part Ⅱ The effects of Cy PA/CD147 interaction inhibition on the pulmonary arterial pressure after APEObjective: To observe the changes of mean pulmonary arterial pressure(m PAP)following inhibition of Cy PA/CD147 interaction in APE models and to investigate its possible mechanism.Methods: The establishment of rat APE model was as same as the first part of the study.Eighty-four rats were randomly assigned to seven groups,which included 12 rats each: a sham group,a sham + Cs A group,a sham + anti-CD147 group,an APE control group,an APE + Cs A group,an APE + anti-CD147 group,and an APE + Cs A + anti-CD147 group.Cs A was injected through right femoral vein 20 minutes before microspheres injection,with a dose of 10 mg/kg;and anti-CD147 was injected immediately after microspheres injection,with a dose of 3 mg/kg.m PAP was measured at 5,10,20,30,40,50,and 60 minutes after APE induction in 6 animals in each group.For the other 6 rats in each group,m PAP was measured 24 hours after APE,which was chosen based on the first part of the experiment.Plasma samples of the animals that underwent manometry at 24 hours were collected to detect the level of tumor necrosis factor(TNF-α)and interleukin-6(IL-6);and the animals were sacrificed.The quantification of neutrophil accumulation was evaluated by myeloperoxidase(MPO)activity.Western-blot analysis was used to analyze the expressions of Cy PA,CD147,extracellular signal-regulated protein kinase(ERK)1/2,phosphorylated ERK1/2,NF-κB p65 and phosphorylated NF-κB p65 in lung tissue.Gelatin zymography was used to access the activation of MMP-2 and MMP-9.Results: Compared with the Sham group,the m PAP in the APE group increased significantly within one hour and at 24 hours after the injection of microspheres(p < 0.01);the TNF-α and IL-6 levels in plasma increased significantly 24 hours after APE(p < 0.01);the MPO activity in the lung tissue increased significantly(p < 0.01);the expressions of Cy PA,CD147,phosphorylation of ERK1/2 and phosphorylation of NF-κB p65 protein in the lung tissue increased significantly(p < 0.01)and the activity of MMP-9 in the lung tissue also increased significantly(p < 0.01).Compared with the APE group,the anti-CD147 intervention attenuated APE-induced increases in m PAP at 50 minutes,60 minutes and 24 hours;Cs A intervention attenuated APE-induced increases in m PAP at 60 minutes and 24 hours;and the combined effect of Cs A and anti-CD147 is more obvious.Their effect of lowering m PAP was associated with reduced right ventricular neutrophil infiltration,decreased levels of TNF-α and IL-6 in plasma,prevention of MMP-9 activation and decreased expression of phosphorylated ERK1/2 and NF-κB p65 protein in the lung tissue.The Cy PA inhibitor Cs A attenuated both the increases of Cy PA and CD147,and the antibody of CD147 abrogated the elevation of CD147 in the lung tissue but not the increase of Cy PA.Conclusions 1.There is a significant neutrophil aggregation in lung tissue after APE,and the MMP-9 expression and activation in lung tissue are increased.2.Inhibition of Cy PA/CD147 interaction attenuates increases in m PAP and this effect is associated with reduced neutrophil infiltration and prevention of MMP-9 activation.3.CD147 may participate in the APE-induced pulmonary hypertension by accelerating the inflammatory response,at least partly through the ERK1/2-nuclear factor-κB pathway.Part Ⅲ The effects of Cy PA/CD147 interaction inhibition on the right ventricular morphology and function after APEObjective: To observe the changes of right ventricular morphology and function following inhibition of Cy PA/CD147 interaction in APE models and to investigate its possible mechanism.Methods: The establishment of rat APE model was as same as the first part of the study.Eighty-four rats were randomly assigned to seven groups,which included 12 rats each: a sham group,a sham + Cs A group,a sham + anti-CD147 group,an APE control group,an APE + Cs A group,an APE + anti-CD147 group,and an APE + Cs A + anti-CD147 group.Cs A was injected through right femoral vein 20 minutes before microspheres injection,with a dose of 10 mg/kg;and anti-CD147 was injected immediately after microspheres injection,with a dose of 3 mg/kg.At 24 hours postAPE,which was chosen based on the first part of the experiment,the right ventricular to left ventricular end-diastolic inner diameter ratio(RVID/LVID),the Tei index of right ventricle and the diameter of pulmonary artery were assessed via echocardiography in 6 animals in each group;and the right ventricular systolic pressure(RVSP)was measured via catheterization in the other 6 rats in each group.Plasma samples of the animals were collected to detect the level of c-Tn I;and the animals were sacrificed.The quantification of neutrophil accumulation in right ventricular tissue was evaluated by MPO activity.Western-blot analysis was used to analyze the expressions of Cy PA,CD147,ERK1/2,phosphorylated ERK1/2,NF-κB p65 and phosphorylated NF-κB p65 in right ventricular tissue andimmunofluorescence analysis was performed for Cy PA to detect its expression in the myocardium.Gelatin zymography was used to access the activation of MMP-2 and MMP-9.Results: Compared with the Sham group,the RVSP,RVID/LVID,Tei index of right ventricle tissue and the diameter of pulmonary artery in the APE group increased significantly at 24 hours after the injection of microspheres(p < 0.01);the c-Tn I level in plasma,the myeloperoxidase activity,the expressions of Cy PA,CD147,phosphorylation of ERK1/2 and phosphorylation of NF-κB p65 protein and the activity of MMP-2 and MMP-9 in the right ventricular tissue also increased significantly.Treatment with Cs A,anti-CD147,or both attenuated APE-induced increases in RVSP,plasma c-Tn I concentration,the RVID/LVID,and the Tei index,measured by echocardiography 24 hours after APE induction.These beneficial effects were associated with reduced right ventricular tissue neutrophil infiltration and prevention of MMP-9 and MMP-2 activation.In addition,the expression of phosphorylated ERK1/2 and phosphorylated NF-κB p65 protein also decreased significantly in the three intervention groups.The Cy PA inhibitor Cs A attenuated both the increases of Cy PA and CD147,and the antibody of CD147 abrogated the elevation of CD147 in the right ventricular tissue but not the increase of Cy PA.Conclusions 1.There is a significant neutrophil aggregation in the right ventricular tissue after APE,and the expression and activation of MMP-2 and MMP-9 in the right ventricular tissue increased.2.Inhibition of Cy PA/CD147 interaction attenuates APE-associated right ventricular cardiomyocyte injury and dysfunction and these effects are associated with reduced neutrophil infiltration and prevention of MMP-2 and MMP-9 activation.3.Cy PA/CD147 may participate in the APE-induced right ventricular cardiomyocyte injury by accelerating inflammatory response,at least partly through the ERK1/2-nuclear factor-κB pathway.4.Cy PA/CD147 interaction may represent a potential therapeutic target for managing APE with RVD.