Effects Of Classical Protein Kinase C Isoform On Proliferation Of Hypoxia Induced Pulmonary Artery Smooth Muscle Cells In Mice And Its Mechanisms

Objective:Proliferation of pulmonary artery smooth muscle cells?PASMCs?is an important pathologic change of the pulmonary vascular remodeling that occurs in hypoxic pulmonary hypertension?HPH?.Structural changes of the distal pulmonary arterials,in particular,play a vital role in the occurrence and development of HPH.Since hypoxia condition,several growth factors and other factors are involved in the initiation and regulation of abnormal cell proliferation through intracellular signaling pathways,it is of great significance to find out the pathologic mechanism of it and identify possible therapeutic targets that may reverse the vascular remodeling induced by HPH.In recent years,the regulation of protein kinase transduction pathway has attracted wide attention.It is found in these studies of protein kinase that classical protein kinase C?cPKCs?may be associated with hypoxia-induced proliferation of PASMCs and HPH.In addition,with the application of murine gene animal model in pulmonary artery disease research,primary culture technology of PASMCs from mouse distal pulmonary artery becomes one basis tool for cellular and molecular researches.However,it is not clear whether the cPKCs isoforms are involved in and further regulate the abnormal proliferation of PASMCs induced by hypoxia.Therefore,this experiment is designed to explore the following questions:1.To develop the stable primary culture technique of PASMCs in the distal pulmonary artery of mice by improving PASMCs magnetic separation culture scheme used abroad.2.To establish the hypoxic model by the primary cultured distal PASMCs of mice,and to investigate the effects of hypoxia on the proliferation of PASMCs and the expression of cPKCs isoforms.3.To find out the effects of specific isoforms of cPKCs on hypoxia-induced proliferation and the expression of ERK1/2 and Akt by drug intervention or virus transfection in vitro.Methods:1.Using Dynal MPC-1 magnetic particle concentrator,iron-containing pulmonary arterioles fragments were separated,and cells were primary cultured;cell morphology was observed under microscope,and immunofluorescence double staining was carried out by using smoothelin polyclonal antibody and myosin heavy chain polyclonal antibody to identify cultured cells.2.The expression of cPKCs isoforms in PASMCs of normal mice was identified by immunoblotting and immunofluorescence using specific antibodies of cPKCs isoforms.3.The normal mice PASMCs was cultured in hypoxia condition?3%O₂?,and the hypoxia model was established.4.CCK and Brdu were employed to detect the proliferation of cells under the condition of normal oxygen and hypoxia.5.Immunofluorescence,Western blot and indirect immunofluorescence flow cytometry were used to detect the expressions of cPKCs protein at different time points under the condition of normal oxygen and hypoxia.6.When the cells were intervened by PKC agonists?PMA?,PKC?inhibitors?safingol?,PKC?I inhibitors?Go6976?,and PKC?II inhibitors?LY333531?respectively,the changes in protein expressions of cPKCs,and the phosphorylation levels of ERK1/2,Akt were observed by immunoblotting under the condition of normal oxygen and hypoxia.7.The lentiviral vector of PKC?and PKC?was used to specifically knock down the activity of target gene by virus transfection techniques,and Western blotting was used to observe the protein expressions of cPKCs,and the phosphorylation levels of ERK1/2,Akt in hypoxia-induced PASMCs in mice.Results:1.The primary cultured cells from the distal pulmonary artery of the mice were identified as smooth muscle cells by morphology and the smooth muscle cell markers?smoothelin and myosin heavy chain?immunofluorescence.2.Stable and high-purity mice PASMCs could be cultured with the improved magnetic separation and primary culture techniques.Besides,the improved techniques also aided to make the experimental process easier to operate and more precise to repeat.3.The expressions of cPKC??75 kDa?,cPKC?I?77 kDa?and cPKC?II?77 kDa?in mice PASMCs were detected by immunoblotting and immunofluorescence,while no expression of cPKC??78 kDa?was detected.4.After being cultured in the condition of hypoxia and normal oxygen with a period of 24h,48h,and 72h,CCK and Brdu were applied to test the proliferation of cells.It was found that the absorbance value of CCK in PASMCs and Brdu positive cells were significantly higher than those in the control group;and the highest proliferation was found in cells being cultured for 72h.5.Immunofluorescence qualitative analysis showed that under hypoxia culture,the protein expression of cPKC?at 24h,48h and 72h were significantly higher than that in the normoxic group.Compared with the control group,the protein expressions of cPKC?I and?II at 48h and 72h were higher than that in normoxic group.6.Western blotting and flow cytometry analysis showed that the protein expression of cPKC?,?I,?II under hypoxia culture at 24h,48h,and 72h were significantly higher than in normoxic group.7.With Brdu method,the proliferation of PASMCs induced by hypoxia was detected to be significantly inhibited when safingol,Go6976,LY333531 were used to inhibit cPKC?,?I and?II respectively.We also observed that PMA could significantly promote the proliferation of PASMCs under normoxic condition.In addition,after transfection with PKC??PKC?lentiviral vector,it was also found that PASMCs proliferation in hypoxia transfection group was significantly lower than that in the control group.8.Using Western blotting,we also observed that after being inhibited by safingol,Go6976,LY333531 respectively,the phosphorylation levels of ERK1/2 and Akt in PASMCs induced by hypoxia was significantly lower than the control group,while PMA could promote the phosphorylation levels of ERK1/2 and Akt under normoxic condition.Using transfection technique to specifically knock down the expression of cPKC?and?,we found that under hypoxic conditions,transfection of cells could significantly lower the phosphorylation levels of ERK1/2,while no evident change was shown on the phosphorylation levels of Akt.Conclusion:1.In this experiment,we have successfully carried out the primary culture of distal PASMCs in mice by improved magnetic separation method.The purity and survival rate of cells obtained were high,which could be used in the following procedures.Being highly operable and reproducible,the improved method made it much easier to obtain experimental tissues from the distal pulmonary arterioles.2.This experiment confirmed that hypoxia could induce excessive proliferation of PASMCs from the distal pulmonary artery of the mice.3.This experiment also showed that there existed expression of cPKC?,cPKC?I and cPKC?II on mice PASMCs,but no expression of cPKC?.4.Hypoxia could increase the protein expression of cPKC?,cPKC?I and cPKC?II in the distal PASMCs of mice,and the abnormal proliferation of the distal PASMCs induced by hypoxia might be regulated by up-regulation of the signaling pathway of cPKC?,cPKC?I and cPKC?II.5.Drug intervention or virus transfection could not only reduce the expression of cPKC?,cPKC?I?cPKC?II in PASMCs,but inhibit the hypoxia-induced proliferation of PASMCs significantly,which may be related to the up regulation of cPKC?,cPKC?I and cPKC?II protein expression.6.Hypoxia may lead to phosphorylation of ERK1/2 by promoting cPKC?,cPKC?I and cPKC?II protein expression respectively,which eventually induces abnormal proliferation of PASMCs from the distal pulmonary artery of the mice and further participates in the developing of HPH.7.Regulation of the expression of cPKC?,cPKC?I and cPKC?II may help to improve the formation of pulmonary vascular remodeling.Target therapy based on cPKCs is expected to be a new direction for HPH therapy in the future.