Activation Of Circulating Platelet And Leukocyte And Platelet Signal Transduction Pathway In Pulmonary Artery Hypertensive Rats

AIM:Idiopathic Pulmonary Arterial Hypertention(IPAH)is a kind of rare pulmonary vascular disease.It is characterized by sustained elevations of pulmonary artery pressure and pulmonary vascular resistance which ultimately leads to right ventricular failure and death. The pathological findings include intimal proliferation and fibrosis, medial hypertrophy,in situ thrombosis,and inflammation in the small muscular pulmonary arterial.According to the clinical statistics,about 50%patients with IPAH have the complication of pulmonary vascular thromboembolism.Platelets are the most important cells in the progress of forming thromboembolism.Platelets activation is a prerequisite for leading to thrombosis.But it still remains obscure whether platelets are activated in IPAH.It is known that there are complex interactions between platelets and leukocytes.The interactions may enhance each other's functions and contribute to thrombosis and inflammation.It is reported that platelet-leukocyte aggregations(PLAs)is one form of interaction,and PLAs play important role in accelerating process of some diseases,such as artherosclerosis,acute lung injury.Thrombosis and inflammation are common complications in IPAH,however,there is no systemic evaluation of platelet and leukocyte activation and their interactions in IPAH. Whether there exists enhanced platelets and leukocytes activation and interactions in IPAH,and how they may contribute to the pathogenesis and pathophysiology of IPAH,all the questions are very interesting and remain mysteries.Mitogen activated protein kinase(MAPK)is a key enzyme in the signaling pathway leading to cellular growth and proliferation and has been implicated in the pathophysiology of IPAH.Hyperphosphorylation of the MAPKs,e.g.,ERK and p38,have been demonstrated in IPAH vasculature,p38 MAP kinase play essential role in hypoxia-mediated human pulmonary artery fibroblast proliferation.Inhibition of p38 MAPK attenuates inflammation and the development of pulmonary hypertension in monocrotaline-treated rats.Thus,p38 MAPK might play a central role in the molecular events that underlie the development and progression of pulmonary hypertension.It has been reported that activation of JNK and ERK and p38 kinase appears to be associated with hypoxia-induced pulmonary arterial remodeling.It has been known ERK and p38 are important signaling molecules in mediating platelet cytoskeleton reorganization and thus leading platelet activation and inducing platelets aggregation and adhesion.Also,ERK2 and p38,involved in PAR4-induced platelet spreading,contribute to the stabilization of platelet thrombi at sites of high thrombin production.However,it is not known whether MAPK signaling pathways are activated in IPAH platelets.Whether MAPK signaling pathways activation induces platelets activation,and then promotes thrombosis, remains unclear.METHODS:1 Induce pulmonary hypertension in rats:Prague-Dawley(SD)rats were injected subcutaneously with 60 mg/kg of 2%monocrotaline(MCT). Control animals were injected subcutaneously with 1 mL of saline.2 Hematoxylin-eosin stained sections:Morphometric analysis of medial wall thickness of pulmonary artery was determined 3 weeks after MCT injection.3 Hemodynamics:RVSP and mPAP were measured 3 weeks afler MCT injection.4 Whole blood flow cytometric analysis:Flow cytometric analysis was used to determine the platelet(fibrinogen binding),leukocyte(CD11b expression)activation markers,and platelet-leukocyte aggregation. 5 Western blot analysis:western blot was used to determine platelet p38 MAPK,ERK1/2 and JNK phosphorylation.RESULTS:1 Animal model:3 weeks after MCT injection,rats have a syndrome of tachypnea,dyspnea,less foodintake,losing body weight,cyanosis.2 Morphometrics:MCT induced vascular remodeling characterized by vascular medial wall thickening in both small and moderately sized pulmonary arteries,resulting in lumen narrowing or near obstruction.3 Hemodynamics:Three weeks after MCT injection,PAH rats exhibited higher mPAP and RVSP as compared with controls(mPAP:21.03±2.74 vs 11.25±0.92 mmHg,p＜0.01;RVSP:24.50±2.00 vs 44.71±4.13, p＜0.01).4 Whole blood flow cytometry:The ratio of platelets fibrinogen binding was enhanced in rats 3 weeks after MCT injection than that of the control group[(4.08±1.59)%vs(1.45±0.61)%,P＜0.01].CD11b expression of monocyte and Neutrophil,but not lymphocyte was increased significantly 3 weeks after MCT injection(p＜0.01).Platelet-Neutrophil aggregations increased in MCT injected rats as compared with controls[(8.28±6.68)% vs(23.84±7.93)%,p＜0.01].5 Western blot analysis:platelet phosphorylation of p38 and JNK was significantly increased,when compared to that of controls.However, ERK1/2 phosphorylation did not change. CONCLUSION:1 MCT induced rats PAH developed increased mPAH and RVSP and thickening of small pulmonary artery medial wall.2 MCT induced PAH rats demonstrated enhanced circulating platelets and leukocytes activation and interaction,as well as enhanced sensitivity towards in vitro stimulations.3 Platelet from MCT induced PAH rats was associated with hyperphosphorylation of p38 MAPK,and JNK.