The Study On Mechanism Of Inhibitory Effect Of Sildenafil On Proliferation Of Pulmonary Artery Smooth Muscle Cells

Pulmonary hypertension (PH) is one of rare and fatal diseases, which is characterized by an increase in pulmonary vascular resistance, thrombsis formation in situ, pulmonary vascular remodeling. Accumulating evidence shows that the proliferation, hyperplesia and migration of pulmonary artery SMCs cells play a key role in the pathogensis and progression of PH. PDEs inhibitors which once were regarded as effective pulmonary vasodiltors can suppress pulmonary vascular remodeling including narrowing of vascular lumen, thicken of pulmonary arteriole wall, fibrosis of adventitial in the monocrotaline PH model in rats. Moreover recent researches vertify that sildenafil can significantly inhibit the proliferation of human pulmonary artery SMCs. However the molecular mechanism of inhibitory effect of sildenafil on proliferation of smooth muscle cells is not clearly elucidated now. Here we report our work on this field. It is made up of three parts:Partâ… : The effect of sildenafil on phosphorylation of MAPK44/42 (ERK1/2) in the proliferation of pulmonary artery smooth muscle cellsObjective: To study the antimitogenic effect of sildenafil on the proliferation of pulmonary artery smooth muscle cells and the expression of phosphorylation of ERK1/2．Methods: Porcine pulmonary artery SMCs were cultured. The SMCs at 3～5 passage were divided into 4 groups: control group(C), PDGF group (P): cells were incubated with PDGF at 20ng/ml. Sildenafil groups (PS₁, PS₂) : 20min prior to PDGF administration, 24,96μmol/l sildenafil were incubated with cells respectively. MTT assay was used to assess cell proliferation after 3 days treatment. 5-Brdu incorporation and western blot were used to evaluate DNA synthesis and expression of proliferative cell nuclear antigen (PCNA) after 24h incubation. The phosphorylation of ERK1/2 after 1h treatment was also analyzed with western blot.Results: Compared with P group, pulmonary artery SMCs proliferation index was inhibited to 145% and 117% from 179% in PS₁ and PS₂ groups respectively. The ratio of positive cell number in group P remarkably elevated compared with C group while the ratio markedly reduced in PS₁ and PS₂ groups (P <0．05) . PCNA was highly expressed after 24 hours PDGF stimulation and significantly suppressed in PS₁ and PS₂ group (P <0．05) . Phosphorylation of ERK1/2 was markedly increased 10min after PDGF stimulation and subsided 4 hours later. The phosphorylation of ERK1/2 in PS₁ and PS₂ group was lower than P group, while the difference of the total ERK1/2 among groups was not so evident.Conclusion: Sildenafil inhibited proliferation of porcine pulmonary artery SMCs by suppressing ERK1/2 phosphorylation and down-regulating PCNA expression.Partâ…¡: the Effect of Sildenafil on mRNA and Protein Expression of MKP-1 in pulmonary artery Smooth Muscle CellsObjective: To investigate the effect of sildenafil on mRNA and protein expression of MKP-1 in pulmonary artery SMCs.Methods: Porcine pulmonary artery SMCs were primary cultured with explant method.1．Pulmonary artery SMCs at 3～5 passage were incubated with sildenafil at different concentration (0～192μmol/l). RT-PCR and western blot were used to measure mRNA and protein expression of MKP-1 respectively.2．Pulmonary artery SMCs were divided into 4 groups. Control group, PDGF group: cells were incubated with 20ng/ml PDGF, sildenafil group: cells were treated with sildenafil at concentration of 96．mol/l, SP group: 20min prior to stimulation of PDGF, cells were incubated with 96．mol/l sildenfil. RT-PCR and western blot methods were used to evaluate the mRNA and protein expression of MKP-1 respectively.3．Pulmonary artery SMCs were divided into 7 groups. Control group, DMSO group: cells were incubated with 0．3% DMSO, PKG inhibitor group: cells were incubated with Rp-8BrcGMP at 25μmol/l. sildenafil group: cells were incubated with sildenafil at 96μmol/l, G group: cells were incubated with 8-BrcGMP at 100μmol/l. RS group: 30min prior to incubation with sildenafil, cells were treated with 25μmol/l Rp-8BrcGMP. GS group: 30min prior to incubation with sildenafil, cells were treated with 8-BrcGMP. MKP-1 expression was measured with western blot.Results: (1) In contrast to RT-PCR result that the difference of mRNAlevel was not so evident among groups, protein expression of MKP-1 induced by sildenafil was in concentration-dependent manner at certain range (0～96μmol/l). (2) The mRNA level was markedly elevated in PDGF group and SP group, compared with C and S group (P <0．05) , and the protein expression of MKP-1 was significantly elevated in P group, S group and SP group compared with group C (P <0．05) . (3) Pre-exposure to Rp-8BrcGMP at 25μmol/l signicifantly repressed the up-regulation of MKP-1 induced by sildenafil (96μmol/l), however Rp-8BrcGMP alone had no impact on MKP-1 expression per se. In addition to dramatical upregulation of MKP-1, 8-BrcGMP at 100μmol/l also enhanced MKP-1 expression induced by sildenafil.Conclusion: Protein expression of MKP-1 induced by sildenafil at certain range (0～96μmol/l) was in concentration-dependant manner, albeit was independentof MKP-1 mRNA level. Upregulation of MKP-1 induced by sildenafil is partiallydue to involvement activation of cGMP/PKG signal pathway.Partâ…¢: Effect of MKP-1 on the Proliferation of pulmonary artery SMCs induced by PDGFObjective: To observe the effect of MKP-1 on the proliferation of pulmonary artery SMCs induced by PDGF.Methods: Porcine pulmonary artery smooth muscle cells at 3～5 passage were divided into 8 groups.Control group, PKG inhibitor group: cells were incubated with Rp-8BrcGMP at 25μmol/l. Vanadate group: cells treated with vanadate (a MKP-1 inhibitor) at concentration of 12．5．mol/L PDGF group: cells were incubated with 20ng/ml PDGF. SP group: 20min prior to stimulation of PDGF, 96 . mol/l sildenfil were admininstered, VSP group: with preexposure to vanadate for 30min followed by 20min treatment of sildenafil, PDGF was administered to cells. RSP group: a 30min preincubation with Rp-8BrcGMP followed by exposure to sildenafil for 20min, thereafter PDGF was administered. GP group: cells were incubated with 8-BrcGMP for 20min, followed by PDGF. Control group, treated like the other groups except that the same volume of sterile PBS was substituted for the different agents. MTT, FACS analysis were used to measure the proliferation and cell cycle progression of pulmonary artery SMCs. Western blot was used to analyze the expression of MKP-1 and the phosphorylation of ERK1/2．Results: (1) After stimulation of PDGF, the phosphorylation of ERK1/2 dramatically increased, the percentage of cell in S phase increased from 4．5% to 20．8%, accompanied with the increase in viable cell number, namely, the increase of OD value from 0．204 in control group to 0．434 in PDGF group (P<0．01) . (2) Pretreatment with sildenafil markedly suppressed the phosphorylation of ERK1/2, meanwhile the percentage of cells in S phase reduced about 50%, accompanied with 25% reduction in OD value compared with PDGF group (P<0．05) . (3) Vanadate and Rp-8BrcGMP reversed the inhibitory effect of sildenafil on the phosphorylation of ERK1/2, cell cycle progression and cell proliferation. The percentage of cells in VSP and RSP group signicantly elevated from 10．8% in PS group to 19．1% and 14．6% respectively, accompanied with increase in OD value from 0．32 in PS group to 0．41 and 0．40 respectively (P<0．01) . (4) 8-BrcGMP at 100．mol/l has similar inhibitory effect of 96．mol/l sildenafil (26% versus 23% reduction in OD value respectively) on the proliferation of pulmonary artery SMCs stimulated by PDGF. The magnitude of suppression by sildenafil and 8-BrcGMP of cell percentage in S phase relative to PDGF group (from 21% toll% versus from 21% to 9．3% respectively) is also comparable.Conclusion: Sildenafil probably upregulated the expression of MKP-1 and promoted degradation of the phosphorylation of ERK1/2, which suppressed the proliferation of pulmonary artery SMCs induced by PDGF.