Activation Of Circulating Platelet And Leukocyte In Chronic Pulmonary Artery Hypertensive Rats And Effects Of HMG-CoA Reductase Inhibitors

Background:Primary Pulmonary Hypertension (PPH) is an idiopathic malignant pulmonary vascular disease. It is characterized by progressive elevation of pulmonary artery pressure and vascular resistance that leads to right ventricular failure and death. Currently, there is no effective treatment on PPH. The basic pathological features contributing to PPH include injury of endothelium, blood vessel wall remodeling, thrombosis in situ and inflammation. Thrombosis and inflammation are closely related to patho-physiological processes, in which platelet and leukocyte activation play pivotal roles. Evidence is accumulating that there are complex interactions, or "cross talk," between platelet and leukocyte. The interactions may enhance each other's functions and contribute to thrombosis and inflammation. Platelet leukocyte aggregates(PLAs) are one of the important manifestation of these interactions. They are also sensitive markers for platelet activation. Presently, however, there are no systemic evaluation of platelet and leukocyte activation and their interactions in PPH. Whether there exists enhanced platelet leukocyte activation and interactions in PPH, and how they may contribute to the pathogenesis and pathophysiology of PPH, remains a mystery. Mitogen activated protein kinase (MAPK) has been implicated in the pathophysiology of PPH. Hyperphosphorylation of the MAPKs, e.g., ERK and p38, have been demonstrated in PPH vasculature. ERK and p38 have been shown as important signaling molecules in mediating platelet cytoskeleton reorganization and thus platelet activation. It is not known whether the MAPK signaling pathways areactivated in PPH platelets. 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors, also known as statins, have pleiotropic effects which are independent to its lipid-lowering properties. Those beneficial effects of statins include the improvement of endothelial functionality, anti-inflammatory, anticoagulant, antioxidative, and antiplatelet effects. Recent studies have indicated that statins are beneficial in treating PPH, although the exact mechanism remains to be elucidated. By employing a rat model of monocrotaline (MCT) induced pulmonary artery hypertension (MCT-PAH), the aims of the present study are 1) to investigate the contribution of platelet and leukocyte in the pathogenesis and pathophysiology of PPH. 2) to elucidate the possible signaling events underlying the activation of PPH platelet. 3) to evaluate the therapeutic effects of different statins on PPH.Methods:Animals and monocrotaline treatmentTo induce pulmonary hypertension, 108 male Sprague-Dawley (SD) rats were injected subcutaneously with 60 mg/kg of MCT. Control animals were injected subcutaneously with 1 mL of saline. The degree of pulmonary hypertension was assessed 3 weeks after saline or MCT injection by measuring right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP).Statins treatmentThree weeks after MCT injection, rats were treated for 4 weeks with atrovastatin 50mg/kg/d (Group MCT_+A50, n=20), atrovastatin 10mg/kg/d (Group MCT_+A10, n=20), provastatin 10mg/kg/d (Group MCT_P10,n=20). The control groups were not given MCT nor statins (group Control, n= 16) or were given MCT injection only and observed for 3 weeks (group MCT_3W, n=16) or 7 weeks (group MCT_7W, n=32).Laboratory measurementsEchocardiography, RVSP and mPAP were measured 3 weeks and 7 weeks after MCT injection. Right ventricular hypertrophy was assessed by comparing the ratio ofright cardiac ventricular weight to the combined weights of the septum and left cardiac ventricular wall. Morphometric analysis of medial wall thickness of pulmonary artery was determined on hematoxylin-eosin and Masson stained sections. Flow cytometric analysis was used to determine the platelet (fibrinogen binding), leukocyte (CD11b expression) activation markers, and platelet-leukocyte aggregation. SDS page and western blot analysis were employed to determine platelet p38 phosphorylation.Results:MortalityBecause of the severity of the animal model, mortality was significantly higher in the untreated MCT rats compared with the control rats. During the 7-week experimental period, mortality was 56%, 20%, 25%, 25%, and 0% in groups MCT_7W MCT_+A50, MCT_+A10 MCT_+P10, and Control, respectively (p<0.05, when statins treated goups compared with group of 7 weeks after MCT injection.)HemodynamicsThree weeks after MCT injection, untreated rats exhibited higher RVSP and mPAP as compared with controls(mPAP: 21.03±2.74 vs 11.25±0.92 mmHg, p<0.01). Seven weeks after MCT injection, mPAP was further increased to 26.83±3.29 mmHg . mSAP, however, decreased when compared with controls. Administration of 10 mg/kg/day or 50 mg/kg/day atrovastatin or 10 mg/kg/day provastatin had similar effects and significantly decreased mPAP and RVSP (P < 0.01, respectively). At the same time, mSAP were increased to normal in all three statin treated groups.Right ventricular hypertrophyThree weeks after MCT injection, rats developed right ventricular hypertrophy, ratio of right cardiac ventricular weight to the combined weights of the septum and left cardiac ventricular wall increased significantly, as compared to the controls (p<0.01), seven weeks after MCT injection, ratio further increased. Statin administration significantly decreased the right ventricular hypertrophy (p<0.01,p<0.05, and p<0.01, respectively, when groups of MCT_+A50, MCT_+A10, MCT_+P10 compared with MCT_7W).Echocardiographic measurementsConsistent with the increased RV weight in MCT-injected rats, echocardiography showed an increased RVWT 3 weeks after MCT injection, although PAAT/CL did not alter significantly at this time point. RVWT increased more obviously 7weeks after MCT injection, compared with that of 3 weeks. PAAT/CL was much smaller in MCT_7W- A functionally altered morphology of the RV in MCT-induced RV failure was demonstrated by increases in RVEDD 7 weeks after MCT injection. Statin administration significantly improved RVWT, RVEDD, and PAAT/CL. Among control, MCT, and statin administration groups, LVIDd, LVIDs and EF did not differ.MorphometricsMCT induced vascular remodeling characterized by vascular medial wall thickening in both small and moderately sized pulmonary arteries, resulting in lumen narrowing or near obstruction. Administration of statins significantly attenuated MCT-induced vascular remodeling in both small and moderately sized arteries.Whole blood flow cytometryThe basal level of platelet fibrinogen binding was significantly enhanced in animals received MCT. A similar increase was observed 3 weeks and 7 weeks after MCT injection. Atrovastatin 10mg/kg/d and provastatin 10mg/kg/d decreased fibrinogen binding significantly, while atrovastatin 50mg/kg/d further decreased fibrinogen binding to the control level. ADP 10^-6 M stimulation drastically increased platelet fibrinogen binding. Platelet sensitivity towards ADP was increased in MCT_7W group. This hyperreactivity was corrected by all 3 regimes of statin administration.CD11b expression of monocyte and neutrophil, but not lymphocyte was increased significantly 3 weeks after MCT injection, more obvious increase was observed 7 weeks after MCT injection. Leukocyte specific agonist fMLP 10^-7 Mstimulated more marked CD11b increase in MCT treated animals, compared with healthy controls. Increased leukocyte CD11b basal level and sensitivity to fMLP by MCT was decreased after statin administration, with atrovastatin 50mg/kg/d showed most obvious inhibition. Both dosage of atrovastatin showed inhibition to neutrophils at basal level and by stimulation, while provastatin only showed inhibition to neutrophils at basal level.Basal- and ADP stimulated- platelet leukocyte aggregation were increased in MCT injected rats. Statin administration significantly decreased both basal and ADP stimulated platelet and leukocyte aggregation. Both atrovastatin and provastatin had similar inhibition in platelet-neutrophil aggregation.Western blotSeven weeks after animals receiving MCT injection, platelet P38 phosphorylation was significantly increased, when compared to that of controls. Admininstration of atrovastatin 50 mg/kg/d for 4 weeks decreased p38 phosphorylation to the control level. Atrovastatin at 10 mg/kg/d or 10 mg/kg/d provastatin administration also demonstrated significant decrease of p38 phosphorylation.Conclusions:1. SD rat developed pulmonary hypertension, right ventricular hypertrophy, and thickening of small pulmonary artery medial wall three weeks after MCT injection, confirmed the establishment of chronic pulmonary hypertensive rat model.2. MCT - PAH rats demonstrated enhanced circulating platelets and leukocytes activation and interaction, as well as enhanced sensitivity towards in vitro stimulations, indicating activated platelet and leukocyte and their interaction may contribute to the pathogenesis and pathophysiology of PAH.3. Platelet from MCT-PAH rats was associated with hyperphosphorylation of p38 MAPK.4. Both atrovastatin at different dosage and provastatin showed similar effects in decreasing RVSP, mPAP, motality and reducing hypertrophy of right ventricular indecreasing RVSP, mPAP, motality and reducing hypertrophy of right ventricular in MCT-PAH rats.5. Both atrovastatin at different dosage and provastatin decreased circulating platelet and leukocyte activation and their interaction in MCT-PAH rats. Atrovastatin and provastatin decreased platelet p38 hyperphosphorylation in MCT-PAH rats, with large dosage of atrovastatin showing more prominent effects.