The Role Of Platelet Derived Growth Factor In Cigarette Smoke-induced Pulmonary Vascular Remodeling In Rats And The Related Mechanisms

Part one Relationship between platelet derived growth factor expression in pulmonary arteries and pulmonary artery remodeling in rats exposed to smokeObjectiveTo observe pathological, ultrastructure changes and right ventricular systolic pressure, platelet derived growth factor (PDGF) expression in pulmonary artery in rats by smoke exposure. To explore the relationship between smoke-induced pulmonary artery remodeling and PDGFB/PDGFR-β expression in pulmonary arteries.MethodsForty male Sprague-Dawley rats were randomly assigned into control group and smoke exposure group, and the latter was further divided into three time points of1,2,3months. Arterial oxygen partial pressure (PaO2), right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) were observed. Pulmonary arteries pathological changes were observed by Hematoxylin-Eosin staining, and collagen contents were investigated by Masson staining. Ultrastructure of small pulmonary arteries was observed by transmission electron microscope. Immunohistochemistry methods were used to determine a-SMA expression. The expressions of PDGFB/PDGFRβ mRNA and protein in pulmonary arteries were evaluated by RT-PCR and Western-blotting.Results1.Arterial oxygen partial pressure of four groups (C, S1m, S2m, S3m) were respectively(96.2±4.3),(92.1±5.2),(90.5±4.1),(89.6±4.8)mmHg, there were no statistically differences between each group(P>0.05, respectively).2. The mRVSP and RVHI in S3m group (65.63±5.6,54.79±7.13) were significantly higher than those in C group (14.07±4.18,32.41±0.26)(P<0.01), There were no statistically differences in mRVSP and RVHI between S1m group (16.85±1.26,36.62±1.32), S2m group (23.57±4.51,40.23±2.12)and C group (P>0.05, respectively).3.The pulmonary artery WA%of four groups (C, S1m, S2m, S3m) were respectively (31.29±1.95)%,(42.68±2.24)%,(51.84±2.38)%,(62.11±1.39)%.Compared to control group,there were significant increases in smoke exposure groups(P<0.01respectively). There were markedly differences among three smoke groups (P<0.01respectively).4.The collagen thickness of pulmonary artery wall of four groups (C, Sim, S2m, S3m) were respectively(4.67±0.36)μm,(8.96±1.80)μm,(11.8±2.12)μm,(15.3±1.65)μm. Collagen was abnormally deposited in pulmonary artery walls of smoke exposure groups. Compared to control group, there were significant increases in smoke exposure groups (P<0.01respectively). There were markedly differences among three smoke groups (P<0.01respectively).5. Ultrastructure changes of intra-acinar pulmonary arteries: intimal layer in control group showed no increased so as endothelial cells and collagen fiber. Observation showed degeneration and distortion of endothelial cells and proliferation of smooth muscle cells in gmup in one-month smoke group, Mitochondria in the endothelial cells could also be observed swelled. The internal elastic laminar was irregular, smooth muscle was observed irregular and fracture, collagen fibers increased in two-month smoke group. The organizational structure showed disordered in three-month smoke group, endothelial cells severely deformed, The muscularized arteries were observed, The hyperplasia of intermediate cells was present. Collagen fibers increased dramatically.6.The α-SMA expressions of pulmonary artery walls of four groups (C, S1m, S2m, S3m) were respectively (37.5±7.5),(52.6±14.3),(72.2±13.6),(98.1±15.6).Compared to control group, there were significant increases in smoke exposure groups(.P<0.01respectively). There were markedly differences among three smoke groups (P<0.01respectively).7. The PDGFB/PDGFRβ mRNA and protein in pulmonary artery of four groups.(1)The PDGFB mRNA expressions of pulmonary artery of four groups (C, S1m, S2m, S3m) were respectively (0.31±0.09),(0.35±0.02),(0.42±0.01),(0.55±0.03),Compared to control group,there were significant increases in smoke exposure groups(P<0.01respectively).(2)The PDGFB protein expressions of pulmonary artery of four groups (C, S1m, S2m, S3m) were respectively (1.38±0.04),(1.50±0.02),(1.58±0.06),(1.44±0.02),Compared to control group,there were significant increases in smoke exposure groups(P<0.01respectively).(3)The PDGFR-(3mRNA expressions of pulmonary artery of four groups (C, S1m,S2m, S3m)were respectively (0.21±0.09),(0.41±0.02),(0.61±0.03),(0.83±0.08),Compared to control group, there were significant increases in smoke exposure groups(P<0.01respectively).(4)The PDGFR-P protein expressions of pulmonary artery of four groups (C, S1m, S2m, S3m) were respectively (1.23±0.01),(1.50±0.01),(1.38±0.02),(1.57±0.04),Compared to control group, there were significant increases in smoke exposure groups(respectively P<0.01).8. The correlation analysis:There was a positive correlation between the mRVSP and RVHI (P<0.05). Both PDGFB mRNA and protein expressions of pulmonary artery were positively correlated with WA%(r=0.941,r=0.605, respectively P<0.05).Both PDGFR-β mRNA and protein expressions of pulmonary artery were positively correlated with WA%(r=0.936, r=0.820respectively, P<0.05).PDGFB mRNA expressions of pulmonary artery were positively correlated with PDGFR-β mRNA expression (r=0.921,P<0.05).PDGFB protein expressions of pulmonary artery were not correlated with PDGFR-P protein expressions (r=0.379, P>0.05).Conclusions1. Smoke exposure could induce obvious pathological changes and ultrastructural changes and causing arteries remodeling, leading to an increase in right ventricular systolic pressure.2. PDGFB/PDGFR-β expression in pulmonary arteries in smoke exposure groups obviously increased and may play an important role in pulmonary arteries remodeling. Part two Effect of platelet derived growth factor on proliferation of rat pulmonary artery smooth muscle cells induced by cigarette smoke extractObjectiveTo investigate the effects of cigarette smoke extract on proliferation of rPASMCs and to evaluate the relevant roles of platelet derived growth factor.MethodsCultured rPASMCs were studied in the following conditions:1. rPASMCs were exposed to different concentrations of CSE (0,2.5%,5%,10%,20%) for24h, then cell proliferation was examined by MTT colorimetric assay and cell cycle analysis.2. rPASMCs were exposed to different concentrations of CSE (0,2.5%,5%,10%,20%) for24h, PDGFB/PDGFR-β mRNA expression was detected by Reverse transcription-polymerase chain reaction (RT-PCR) and protein expression by Western-blotting.3. rPASMCs were pre-incubated with different concentrations of the PDGF receptor antagonist Imatinib (1μM/L,2μM/L,4μM/L,8μM/L) for1h before exposure10%CSE for24h,then MTT colorimetric assay was used for detection of cell proliferation.4. rPASMCs were pre-incubated with8μM/L Imatinib for1h before exposure10%CSE for24h, PDGFB/PDGFR-p protein expression was detected by Western-blotting.Results1. The absorbance (A) value in rPASMCs from different concentrations of CSE (2.5%,5%,10%,20%) were (1.839±0.128,2.241±0.182,2.951±0.072,4.329±0.176respectively) and were significantly increased compared to those of control group (0.977±0.156,P<0.01). S phase cell fraction in rPASMCs from CSE exposure group (17.08±0.31,18.47±0.21,23.19±0.16,23.71±0.29respectively) were significantly increased compared to that of control group (13.04±2.39, P<0.01).G2phase cell fraction in rPASMCs from CSE exposure group (45.21±5.20,47.05±14.14,42.05±8.97,41.15±6.80respectively) were significantly increased compared to that of control group (22.77±3.04, P<0.01).2. PDGFB mRNA levels in rPASMCs from CSE exposure group (0.647±0.035,0.819±0.065,0.776±0.046,0.623±0.048respectively) were significantly increased compared to those of control group (0.411±0.026,P<0.05). PDGFR-β mRNA expression in rPASMCs from CSE exposure group (0.393±0.021,0.739±0.034,0.745±0.032,0.620±0.019respectively) were significantly increased compared to those of control group (0.411±0.011, P<0.05). PDGFB protein expression in PASMCs from CSE exposure group (0.367±0.015,0.716±0.033,0.897±0.041,1.202±0.039respectively) were significantly increased compared to those of control group (0.544±0.025, P<0.05). PDGFR-β protein expression in rPASMCs from CSE exposure group (0.718±0.019,0.920±0.039,0.998±0.081,1.061±0.057respectively) were significantly increased compared to those of control group (0.544±0.025, P<0.05).3.The absorbance (A) value in rPASMCs from10%CSE exposure group (2.643±0.099) were significantly increased compared to those of control group (0.856±0.017,P<0.01). PDGF receptor antagonist Imatinib (1μM/L,2μM/L,4μM/L,8μM/L) pre-incubation reduced10%CSE induced rPASMCs proliferation (2.012±0.91,1.997±0.379,1.458±0.495,1.022±0.102respectively).4.After exposure to10%CSE for24h, PDGFB protein expression in rPASMCs (1.662±0.271)were much higher than that of control group (0.996±0.181,.P<0.05), PDGFR receptor antagonist Imatinib pre-incubation reduced10%CSE induced expression of PDGFB protein(1.231±0.192).Moreover, PDGFR-β protein expression in rPASMCs (1.581±0.153)were much higher than that of control group (0.986±0.131, P<0.05), PDGF receptor antagonist Imatinib pre-incubation reduced10%CSE induced expression of PDGFB protein (1.211±0.192).Conclusions1. CSE promotes cell proliferation of rPASMCs.2. CSE leads to cell cycle progression of rPASMCs and promotes G1phase converting to S phase and G2phase.3. PDGFB/PDGFR-β signaling pathway is activated after the CSE treatment. Both mRNA and protein levels of PDGFB/PDGFR-P were upregulated after the CSE treatment.4. PDGFB expressions of pulmonary artery were positively correlated with PDGFR-β expression. Their expression variations may be associated with proliferation of rPASMCs induced by CSE.5. PDGF receptor antagonist Imatinib inhibits10%CSE induced cell proliferation of rPASMCs and inhibits the upregulation of the protein level of PDGFB/PDGFR-β signaling pathway. The underlying molecular mechanisms need to be further investigated. Part Three Effect of protein kinase C-δ on proliferation of rat pulmonary artery smooth muscle cells induced by cigarette smoke extract and the related mechanismsObjectiveTo investigate the effect of CSE on PDGFB/PDGFR-P expression in rPASMCs and the possible role of PKC-δ in this process.MethodCultured rPASMCs were studied in the following conditions:1. rPASMCs were pre-incubated with different concentrations of the PKC-δ specific inhibitor Rottlerin (lnM/L,2nM/L,4nM/L,8nM/L) for1h before exposure10%CSE for24h,then MTT colorimetric assay was used for detection of cell proliferation.2. rPASMCs were pre-incubated with Rottlerin (8nM/L) for1h before exposure10%CSE for24h, cell cycle distribution was measured using flow cytometry.3. rPASMCs were pre-incubated with different concentrations of Rottlerin (lnM/L,2nM/L,4nM/L,8nM/L) for1h before exposure10%CSE for24h, PKC-δ mRNA expression was detected by Reverse transcription polymerase chain reaction (RT-PCR) and protein expression by Western-blotting.4. rPASMCs were pre-incubated with different concentrations of Rottlerin (lnM/L,2nM/L,4nM/L,8nM/L) for1h before exposure10%CSE for24h, PDGFB/PDGFR-β mRNA expression were detected by Reverse transcription polymerase chain reaction(RT-PCR) and protein expression by Western-blotting.Results1. The absorbance (A) value in rPASMCs from10%CSE exposure group (3.535±0.353) were significantly increased compared to those of control group (0.873±0.184,P<0.01). The PKC-δ specific inhibitor Rottlerin (lnM/L,2nM/L,4nM/L,8nM/L) pre-incubation reduced10%CSE induced rPASMCs proliferation (2.322±0.358,2.227±0.291,1.903±0.212,0.944±0.109respectively)(P<0.01).2. S Phase cell fraction in rPASMCs from10%CSE exposure group (22.39±1.09) was significantly increased compared to those of control group (6.51±1.23, P<0.01). S Phase cell fraction from10%CSE+Rottlerin (12.21±1.96) reduced compared to that of10%CSE (P<0.01); G2Phase cell fraction in rPASMCs from10%CSE exposure group (17.12±0.45) was significantly increased compared to those of control group (4.03±2.96, P<0.05).G2Phase cell fraction from10%CSE+Rottlerin (8.59±2.05) reduced compared to that of10%CSE (P<0.01).3. PKC-δ mRNA and protein expression in rPASMCs.(1) PKC-δ mRNA and protein expression in rPASMCs were respectively control (0.955±0.042,0.758±0.131),10%CSE(1.068±0.079,0.873±0.011),10%CSE+Rottlerin(lnM/L,2n M/L,4nM/L,8nM/L)(1.027±0.113,0.813±0.174),(0.971±0.118,0.723±0.117),(0.851±0.065,0.904±0.011),(0.887±0.053,0.881±0.025).There were no statistically difference between each group (P>0.05respectively).(2)Phosphorylated PKC-δ protein expression in rPASMCs were respectively: control(0.985±0.009),10%CSE(1.485±0.058),10%CSE±Rottlerin(lnM/L,2nM/L,4nM/L,8nM/L)(1.257±0.05),(1.146±0.174),(1.051±0.019),(1.006±0.018),After exposure to10%CSE, phosphorylated PKC-δ protein expression were much higher than that in control group (P<0.05), Rottlerin(lnM,2nM,4nM,8nM) pre-incubation reduced10%CSE induced expression of phosphorylated PKC-8protein (P<0.05respectively).4. PDGFB mRNA and protein expression(1) PDGFB mRNA expression from10%CSE exposure group (1.245±0.020) were higher than that in control group (0.649±0.046, P<0.05).Rottlerin (lnM/L,2nM/L,4nM/L,8nM/L) preincubation reduced10%CSE induced expression of PDGFB mRNA (1.235±0.086),(0.722±0.035),(0.679±0.023),(0.676±0.035).There were significantly difference compared to those of10%CSE group (P<0.05respectively).(2) PDGFB protein expression from10%CSE exposure group (1.746±0.132) were higher than that in control group (0.654±0.009, P<0.05). Rottlerin (lnM/L,2nM/L,4nM/L,8nM/L) preincubation reduced10%CSE induced expression of PDGFB protein (1.322±0.066),(0.913±0.020),(0.932±0.219),(0.918±0.013).There were significantly difference compared to those of10%CSE group (P<0.05respectively).(3) PDGFR-β mRNA expression from10%CSE exposure group (1.728±0.029) were higher than that in control group (0.973±0.069, P<0.05), Rottlerin (InM/L,2nM/L,4nM/L,8nM/L) pre-incubation reduced10%CSE induced expression of PDGFR-β mRNA (1.225±0.056),(1.021±0.074),(0.935±0.051),(0.745±0.074).There were significantly difference compared to those of10%CSE group (P<0.05respectively).(4) PDGFR-β protein expression from10%CSE exposure group (1.207±0.058) were higher than that in control group (0.657±0.009, P<0.05), Rottlerin (InM/L,2nM/L,4nM/L,8nM/L) pre-incubation reduced10%CSE induced expression of PDGFR-β mRNA and protein (1.176±0.050),(0.848±0.174),(0.843±0.019),(0.838±0.018). There were significantly differences compared to those of10%CSE group (P<0.05respectively).Conclusions1.10%CSE had no obvious effect on the total mRNA and protein levels of PKC-δ.2.10%CSE upregulated the phosphorylation protein levels of PKC-δ of rPASMCs; PKC-δ specific inhibitor Rottlerin inhibits the upregulation of the levels of the phosphorylation of PKC-δ protein expression.3. PKC-δ specific inhibitor Rottlerin inhibits10%CSE induced cell proliferation of rPASMCs.4. PKC-δ specific inhibitor Rottlerin inhibits10%CSE induced cell cycle progression.5. PKC-δ specific inhibitor Rottlerin can decrease the CSE induced upregulation of the expression of PDGFB/PDGFR-β; It suggests that CSE induced cell proliferation of rPASMCs occurred by upregulating the PKC-δ-PDGFB signaling pathway.