Study Of Role And Mechanism Of Akt/PTEN In Pulmonary Vascular Remodeling Induced By Hypoxia

A great deal of pulmonary artery smooth muscle cells (PASMCs) migrating from tunica media to endomemebrane and abnomal proliferating is an improtant contributor to the vascular remodeling that occurs in chronic hypoxic pulmonary hypertension (HPH). Different growth factors, cytokine and proinflammatory mediators convey extracelluar signal to the nucleus by interacting with receptors on the cell surface, then regulate expression of target genes and contribute to cell differentiation, growth and proliferation.Now the main studies about signal transmitting passway of vascular remodeling in HPH focused on STATs passway, MLCK passway and PKC passway. Serine/threonine kinase (Akt) participates in the signal transduction actived by phosphoinositide 3 kinase (PI3K), which is closely correlated to cell differentiation,cell growth,apoptosis,migration and other biological actions. Phosohatase and tensin homolog deleted on chromosome 10(PTEN) is the first discovered anticoncogene with lipid-phosphatase activity so far. PTEN can carry out 3-location dephosphorylation, that can lower the level of PIP3 in cell and down regulate Akt signal condutive passway. When hypoxia actived intracellular signal transdutive passway and inducted a series of pathological actions of (PASMCs phenotype switching, proliferation and migration ), what role did the PTEN/Akt play in hypoxia lung vascular remodeling? Along the nature course of disease, whether the expression of PTEN/Akt descended even depleted?Methods1. After PASMCs were primarily cultured, expression of PTEN/Akt1,2,3 mRNA was detected by RT-PCR under normoxic or hypoxia conditions (3% O2); expressions of SOCS3 proteins were performed by immunocytochemistry and Western blot under normoxic or hypoxia conditions.2. Amplification, appraisement, transfective effciency,MOI and titre measuration of Ad-PTEN.3. The PASMCs transfected with Ad-PTEN and the control cells were cultured under normoxic and hypoxia conditions respectively. The expression of PTEN mRNA and protein activity were performed by RT-PCR and Western blot. The changes of cell proliferation were observed by MTT,flow cytometric DNA analysis and 3H-TdR incorporation.4. After PASMCs transfected with Ad-PTEN, level of migratory distance was detected under normoxia or hypoxia conditions.5. Expression of Akt1,2,3 mRNA in PASMCs before and after transfection were detected by RT-PCR under normoxic and hypoxia conditions.Results1. Rat PASMCs primarily cultured were confirmed by immunocytochemistry usingα-SM-actin antibody. The fourth generation was freezed and reserved. There existed a certain extent of degree diversity in growth curve and protein content of PASMCs between cultured by tissue clump or enzymic method, but it was not remarkable(p>0.05). Both reached its peak after 7~8d, and then declined due to contact inhibition. The content of intracellular Ca2+ of PASMC cultured by tissue clump method was (195.32±14.62) nmol/L, and (150.18±11.87) nmol/l by the enzymic method. The difference between both was obvious (P<0.05).2. Expression of PTEN/Akt1,2,3 mRNA and proteins in PASMCs was detected by RT-PCR and western blot under normoxic and hypoxia conditions. When PASMCs were cultured under hypoxia conditions, the level of expression of PTEN mRNA and protein gradually heightened. The experession of Akt1,2,3 mRNA was detected highter at 2 h of hypoxia and reached its maximal at 6 h or 12 h, declined to previous normoxic level at 24 h. The change of protein expression induced by hypoxia was similar to that of mRNA. Immunocytochemistry was used to confirm Akt1,2,3 protein only related to cytoplasmic distribution of rat PASMCs,but PTEN protein dealt with to be both in cytoplasmic and mucelus distribution both.3. About 2 ml Ad-PTEN was freezed after amplification. The titer was about 1.03×109 pfu/ml. It was identificated that the exogenous objective gene PTEN fragment existed in recombinative adenovirus. Ad-PTEN has high efficiency of infection for PASMCs, and the best MOI was 60. The hyperexpression of PTEN gene was found in PASMCs after Ad-PTEN transfection, And that was the same under hypoxia condition.4. In MTT study, proliferation of PASMCs was enhanced and was hypoxia time lengthen related. The proliferation of PASMCs in transfected by Ad-PTEN decresed obviouly. 3H-TdR incorporation increased obviously in hypoxia in contrast to normoxia condition. And 3H-TdR incorporation n transfected by Ad-PTEN began to decrease obviously. In contrast to the same hour in normal PASMCs exposed to hypoxia, cells transfected with Ad-PTEN at G0/G1 increased, cells in S+G2/M decreased significantly.5. In normoxia culture, the migratory distance of PASMCs gradually increased along with cultivate time prolongation. The migratory distance in 48h and 72h increased obviously in contrast to 36h(p<0.05). The hypoxia could stimulate PASMCs migration. The migratory distance in infected by Ad-PTEN cells decreased obviously no matter whether it was under normoxic or hypoxia condition.6. The expression of Akt1,2,3 mRNA increased after exposure of cells to hypoxia and reached its maximum at 6 h, then declined at 12 h, and decreased to level of normoxia at 24h. The magnitude of expression enhancement of Akt1,2,3 mRNA in PTEN gene-transfected cells were significantly lower than that in normal PASMCs whether under normoxic or hypoxia condition.Conclusions1. Akt1,2,3 and PTEN mRNA and proteins are detected in rat PASMCs exposed to normoxia. Hypoxia can stimulate the expression intensity of Akt1,2,3 and PTEN.2. Ad-PTEN amplificated can transfect PASMCs and caused hyperexpress PTEN in cells.3. Maybe hypoxia promotes proliferation of PASMCs through inducting the expression and activation of PTEN/Akt. And the expression of activation of Akt may plays a more important role.4. Hypoxia may promotes migration of PASMCs through inducting the expression and activation of PTEN/Akt. And the expression of activation of Akt plays a more important role.5. Hyperexpressive PTEN in Ad-PTEN transfected PASMCs can obviously inhibit the transcription of Akt1,2,3. that maybe the molecular basis of Ad-PTEN inhibit the proliferation and imgration of PASMCs under hypoxia condition.