AKT/mTOR/NF-?B Signaling Pathway Promotes Repair Of Pulmonary Arterial Smooth Muscle Cells Damage By Increasing CXCR4 On Hypoxic Pulmonary Hypertension In Rats

Pulmonary hypertension(PH) is a chronic and progressive disease leading to right heart failure and ultimately death if untreated. The main feature is the increased pulmonary artery pressure and pulmonary vascular resistance. Hypoxia plays a very important role in the development of PH, therefore, the pathogenesis of hypoxic pulmonary hypertension(HPH) is a study hotspot in recent years. HPH is characterized by pulmonary vascular smooth muscle cell proliferation and pulmonary vascular remodeling.Once the pulmonary vascular remodeling occurs, it will be difficult to reverse, and there is no effective methods of prevention and treatment. Pathogenesis of pulmonary vascular remodeling include the following three aspects: pulmonary vascular endothelial cells into mesenchymal cells; mesenchymal cells migrate into the bottom of endothelial; the proliferation and anti-apoptosis of smooth muscle cells, fibroblasts. Therefore, to studypathogenesis of pulmonary vascular remodeling and find out the key molecular pathways,thereby inhibiting the occurrence and development of HPH is the key issues we need to focus.It has been reported PI3K/AKT/mTOR signaling pathway promotes hypoxia-induced pulmonary vascular cell proliferation and anti-apoptosis; mammalian transcription factor NF-?B is activated in lung macrophages, lymphocytes, endothelial cells and pulmonary artery smooth muscle cells(PASMCs) of patients with end-stage PH; CXC chemokine receptor-4(CXCR4) is highly expressed in lung tissue of HPH. Many articles reported that NF- ? B is activated by PI3K/AKT/mTOR signaling pathway in tumor cells; the expression of CXCR4 is regulated by NF-?B, and then played an important role in the promotion of cell migration and invasion; CXCR4 inhibitors could inhibit the formation of HPH in rats, but the exact mechanism is unclear. This study will clarify whether these molecules related in PH, and whether there is a correlation between them.Aims(1) To clarify the relationship between PI3K/AKT/mTOR signaling pathway, NF-?B and CXCR4; and clear the specific molecular mechanism in the hypoxia-induced PASMCs.(2) To clear the role between PI3K/AKT/mTOR signaling pathway, NF-?B and CXCR4 in migration of hypoxia-induced PASMCs.(3) To clear the treatment of CXCR4 inhibitors in PH, and in order to establish whether CXCR4 is a novel therapeutic target for PH.Methods and Results(1) First, we established damage model with PASMCs, and found that hypoxia can promote cell migration compared with the normoxic group. PI3K/mTOR dual inhibitor BEZ-235, mTORC1/C2 dual inhibitor OSI-027, mTORC1 inhibitor Rapamycin were added to hypoxia-induced damage model in PASMCs at the same time, it was found that cell migration were inhibited. These results suggest that PI3K/AKT/mTOR signaling pathway can promote the migration of hypoxia-induced PASMCs.(2) We established hypobaric hypoxia model of PH in SD rats, right ventricular pressure and right ventricular hypertrophy index were increased compared with normoxic groupafter 4 weeks; small arteries of lung tissue by HE staining showed pulmonary vascular wall thickening in hypoxia group. Western-blot detected that AKT, mTOR and NF-?B is activated on lung tissue of HPH in rats.(3) The phosphorylation levels of AKT, mTOR, and NF- ? B protein were increased compared with normoxic group in hypoxia-induced PASMCs. PI3K/AKT inhibitor LY294002 was added to hypoxia-induced PASMCs, it was found that phosphorylation levels of mTOR and NF-?B protein was inhibited. Inhibiting the expression of mTORC1,phosphorylation levels of NF-?B was inhibited, but phosphorylation levels of AKT was not significantly changed. These results suggest that AKT/mTOR signaling pathway promotes the activation of NF-?B.(4) mTOR and IKK? could be combined by co-immunoprecipitation in hypoxia-induced PASMCs. Targeted inhibition of Raptor, we found that IKK? is specific binding mTORC1.(5) The phosphorylation levels of IKK?/? and IKB? protein were increased compared with normoxic group in hypoxia-induced PASMCs. Inhibiting the expression of mTORC1,phosphorylation levels of IKK?/? was inhibited. Inhibiting the expression of IKK?/?,phosphorylation levels of IKB? and NF- ? B were inhibited. Activation of mTORC1 activated IKK?/?, activation of IKK?/? also made the downstream molecules IKB? and NF-?B activation.(6) Whether we increased or decreased the protein expression of NF-?B in hypoxia-induced PASMCs, CXCR4 protein expression levels along with change synchronously. We examined the influence of NF- ? B on the transcription level of CXCR4 by RT-PCR, it was found that the transcription level of CXCR4 is also regulated by NF-?B. Inhibiting the expression of CXCR4 could inhibit the cell migration in hypoxia-induced PASMCs. These results suggest that activation of NF-?B promotes cell migration by regulating the expression of CXCR4 in hypoxia-induced PASMCs.(7) We proved CXCR4 is highly expressed on lung tissue of HPH in rats by Western blot and Immunohistochemistry. CXCR4 inhibitor AMD3100 were gave by intraperitoneal injection to hypobaric hypoxia model of PH in rats. The results showed that right ventricle pressure and right ventricular hypertrophy index of AMD3100 group was significantlyreduced compared with the hypoxia group; small arteries of lung tissue by HE staining showed pulmonary vascular wall thickening was significantly reduced. The results showed CXCR4 inhibitor AMD3100 inhibit HPH in rats.ConclusionIn summary, our data showed that AKT/mTOR signaling pathway promotes HPH in rats; mTORC1 activate IKB? and NF- ? B by regulating IKK?/? in hypoxia-induced PASMCs, activation of NF-?B promotes cell migration by regulating the expression of CXCR4 in hypoxia-induced PASMCs. This signaling pathway plays an important role on HPH in rats, and CXCR4 inhibitor AMD3100 can inhibit the formation. Inhibiting the expression of CXCR4 for the treatment of HPH may be a new way.