PART ?:the Effects Of Nicorandil On Pulmonary Arterioles Remodeling In Monocrotaline-induced Pulmonary Artery Hypertension Of Mice PART ?:Spatial And Temporal Distribution Of Pulmonary Artery Mast Cells In Pulmonary Artery Hypertension Of Rats

Objective:Pulmonary artery hypertension is a vascular disease of different etiologies.The progressive increase in pulmonary vascular resistance causes severe elevation of pulmonary artery pressure,leading to increased right ventricular systolic pressure and right ventricular hypertrophy,ultimately death from right heart failure.Pulmonary artery hypertension is a great threat to human beings with difficult diagnosis,limited treatment and poor prognosis.Nicorandil is a KATP channel opener that has been shown to inhibit the proliferation of pulmonary artery smooth muscle cells.In this study,monocrotaline-induced pulmonary artery hypertension in mice were used to investigate the effect of nicorandil on the pulmonary vascular remolding.Methods:18 CD1 male mice were randomly divided into the control group,the monocrotaline group,and the monocrotaline+nicorandil group(n=6 for each group).The latter 2 groups were subcutaneously injected with 400mg/kg monocrotaline weekly for 8 weeks to induce pulmonary artery hypertension.The mice in the monocrotaline+nicorandil group were treated with 10.5mg/kg nicorandil daily by intragastric administration.After 8 weeks,the right ventricular systolic pressure(RVSP)were evaluated by right ventricular puncture through the diaphragm;the index of right ventricular hypertrophy were calculated;the morphologic changes of right ventricle cardialmyocytes were detected by HE staining;the thickness of pulmonary arterial media wall were measured by elastin staining;a-smooth muscle actin(a-SMA)immunohistochemitry was used to assay pulmonary arterioles muscularization.Results:The RVSP of the monocrotaline group was significantly increased than the control group(33.45±1.37mmHg vs 23.43±2.32mmHg,P<0.01).The index of right ventricular hypertrophy of the monocrotaline group was significantly higher than the control group(0.27±0.03 vs 0.24±0.02,P<0.05).Daily treated with nicorandil significantly decreased RVSP(27.12±2.06mmHg vs 33.45±1.37mmHg,P<0.01)and attenuated the index of right ventricular hypertrophy(0.24±0.01 vs 0.27±0.03,P<0.05).The thickness of pulmonary arterial media was significantly increased in the monocrotaline group than the control group(11.06±0.80%vs 3.89±0.61%,P<0.01).Nicorandil alleviated the thickness of pulmonary arterial media wall(5.31±0.51%vs 11.06± 0.80%,P<0.01).A significant higher ratio of partial-muscularized pulmonary arterioles was found in the monocrotaline group(36.01±18.68%)than that of the control group(25.64±6.63%,P<0.05)and the ratio of fully-muscularized pulmonary arterioles was increased in the monocrotaline group(57.79±9.51%)than that of the control group(15.94±5.63%,P<0.01).Morever the ratio of non-muscularized pulmonary arterioles in the monocrotaline group(6.11±1.70%)was significantly decreased than the control group(58.41±8.01%,P<0.01).Nicorandil declined the ratio of fully-muscularized pulmonary arterioles(42.76±4.11%)compared with the monocrotaline group(57.79±9.51%,P<0.01)and increased the ratio of partial-muscularized pulmonary arterioles(47.31±4.61%vs 36.01±8.68%,P<0.05).Nicorandil could prevent the injury of right ventricle cardialmyocytes induced by monocrotaline.Conclusion:Nicorandil could relieve monocrotaline-induced pulmonary artery hypertension by inhibiting pulmonary vascular remodeling.It could be a potential drug for preventing and treating pulmonary artery hypertension.Objective:Pulmonary artery hypertension is a clinical syndrome with a progressive increase in pulmonary vascular resistance and pulmonary artery pressure.Pulmonary vascular remodeling is a typically pathological change of pulmonary artery hypertension,including damage and repair of intima,thickness of medial wall and infiltration of inflammation cells in adventitial wall.The inflammation of adventitial wall and pulmonary vascular remodeling are interdependent.The infiltration of inflammation cells promoted the development of pulmonary artery hypertension.Mast cells(MCs)are important immune cells which secrete lots of biologically active substances such as histamine,leukotriene,interleukin-3(IL-3),proteolytic enzymes,transforming growth factor-?(transforming growth factor-?,TGF-?)and other cytokines.Through the cytokines secreted by mast cells inflammatory cells were infiltrated and promoted the vessels fibrosis.Mast cells participated and regulated the pulmonary vascular remodeling.In this study,monocrotaline-induced pulmonary artery hypertension of rats were used to investigate the spatial and temporal distribution of MCs in the pulmonary vascular remolding.Methods:40 SD male rats were randomly divided into the control group and the monocrotaline group(4 time groups,n=8 for each group).The control group were intraperitoneally injected with saline 0.8ml and the monocrotaline groups were intraperitoneally injected with monocrotaline 60mg/kg.The rats were sacrificed on the 3rd,7th,14th and 28th day respectively.The index of right ventricular hypertrophy(RVHI)were calculated.The thickness of pulmonary medial wall and the cross-sectional area of the myocardial cells were calculated by HE staining.The distribution of MCs and the thickness of pulmonary medial wall were observed and calculated by gomorji aldehyde fuchsin(GAF)staining.The spatial and temporal distribution of MCs were calculated by toluidine blue(TB)staining.Picrosirius red(PSR)staining was used to calculated the cardiac fibrosis.Immunohistochemical staining was performed with primary antibodies against ?-smooth muscle actin(?-SMA),S100A4,tryptase and CD68 and were used to assay pulmonary arterioles muscularization and the infiltration of MCs and macrophages.Results:The index concerning RV hypertrophy and myocardial hypertrophy demonstrated statistical significance on day 14,28 after MCT injection in RVHI(0.322±0.04,0.57±0.06 vs 0.25±0.02,P<0.05)and on day 28 in CSA(334.1±38.5 um2 vs 203.3±27.4um2,P<0.05).The analysis of RV fibrosis using PSR staining identified the remolding of RV was remarkable on day 28 after MCT injection(12.14±5.94%vs 1.44±0.57%,P<0.05).The thickness of pulmonary media wall were significantly increased on day 28 compared with the control group(0.58±0.10 vs 0.24±0.02,P<0.05)by GAF staining.The density of muscularized distal pulmonary vessels increased compared with that in the control group(3.25±0.86 vs 0.97±0.18,P<0.05)by ?-SMA immunohistoehemitry.By immunohistochemistry with S100A4,the expression of S100A4 in pulmonary arteries was increased with time,and peaked on 28th day.On day 28,a number of MCs accumulated around mtra-acinar arteries(5.86±1.86cells/vessel vs 0.04±0.09cells/vessel,P<0.05)and infiltrated into alveolar septa(34.00±11.27celLs/MM2vs 0.06±0.14cells/mm2,P<0.05)by TB staining.The number of MCs without degranulation was significantly lower than those in normal rats remarkably on day 28(31.24±2.87%vs 98.76±3.04%,P<0.05).The density of CD68-positive macrophages increased on day 14 in small arteries(5.82±1.46cells/vessel vs 0.98±0.14cells/vessel,P<0.05)and alveolar septa(7.56± 1.37cells/mm2 vs 0.96±0.12cells/mm2,P<0.05)and increased remarkably on day 28 in small arteries(7.68±1.63cells/vessel vs 0.98±0.14cells/vessel,P<0.05)and alveolar septa(23.2715.35cells/mm2 vs 0.±0.12cells/mm2,P<0.05).On day 28 the number of tryptase-positive MCs in small arteries and alveolar septa were 5.87± 1.67cells/vessel and 24.17±4.78cells/mm2,there were significantly differences between the control group(0.25±0.04cells/vessel and 0.18±0.06cells/mm2,P<0.05).No increase of MCs was found in the inflammatory infiltration area and the fibrosis area by TB staining(P>0.05).The data showed that neither CD68-positive macrophages nor tryptase-positive MCs redistributed or accumulated in RV tissues after MCT injection in rats(P>0.05).Conclusion:In the late stage of PAH rat model,large numbers of MCs infiltrated in the pulmonary arteries and alveolar septa,and the number of degranulated MCs was significantly increased.CD68+macrophages were upregulated prior to mast cells,suggesting that the infiltration of mast cells may be regulated by CD68+macrophages,both of which were involved in pulmonary vascular remodeling.