The Mechanisms Of RAS Mediated The Proliferation Of Bone Marrow Mesenchymal Stem Cells Under Hypoxia Via PI3K/AKT Pathway

Background: Traditionally, mesenchymal stem cells, the major cells in the bonemarrow hematopoietic stem cell microenvironment, exist only in the bone marrow.They perform a crucial role in the generation of bone and cartilage. Bone marrowmesenchymal stem cells are undifferentiated cells with the functions of self-replicationand differentiation. They can differentiate into osteoblasts, chondrocytes, adipocytesand embryonic layer-derived cells like stem cells and nerve cells. The growth anddevelopment of the bone marrow mesenchymal stem cells are influenced by a variety ofintrinsic mechanisms and micro-environment. Bone marrow mesenchymal stem cellscan adapt to lower oxygen concentrations. There has been limited data on thephysiological effect and mechanism of hypoxia on bone marrow mesenchymal stemcells. Renin-angiotensin system (RAS) is an important endocrine system related to thedevelopment. Recent studies have demonstrated that local RAS exists in a variety ofcells and plays an important role in the differentiation and proliferation via autocrine oradjacent secretion routes. In this project, we investigate the biological functions andmechanisms of RAS mediating the development of bone marrow mesenchymal stemcells in the cellular and molecular levels under the condition of hypoxia. Part Ⅰ Theexpression of Local RAS in the primary culture ofbone marrow mesenchymal stem cellsObjective: To observe the distribution and expression of local RAS in primarilycultured bone marrow mesenchymal stem cells. Methods: The single mononuclear cellswere obtained by density gradient centrifugation to digestive subculture,usingimmunocytochemistry and flow cytometry to identify the trait and purity of the cells.Identify the multi-differentiation capacity of the cells in vitro by inducing them intomyocardial cells and adipocytes. Additionally, The cells were determined byimmunocytochemistry. The growth curve of primary cultured bone marrowmesenchymal stem cells was selected and the best experimental time was choosed.Distribution and abundance of AT1, AT2in the cells was assessed byimmunofluorescence staining and the content of AngII and ACE in the culture mediumof the bone marrow mesenchymal stem cells was evaluated by radioimmunoassay.Results: The purity of bone marrow-derived mesenchymal stem cells in the culture ismore than95%. Thereafter, we successfully induced cellular differentiate intomyocardial cells and fat cells. AngII receptors, AT1Rand AT2R, were widely distributedon the membrane of the bone marrow mesenchymal stem cells. Conclusions: The bonemarrow-derived mesenchymal stem cells from the primary culture maintained highpurity and potentials of differentiation characteristics. AngII receptors，AT1R and AT2R,were shown on the bone marrow mesenchymal stem cells. Part Ⅱ The influences of the local RAS on the growth ofhypoxia-induced bone marrow mesenchymal stem cellsObjective: To evaluate the effect of AngII on the growth and proliferation ofhypoxia-induced bone marrow mesenchymal stem cells usig exogenous and endogenous AngII. Methods: Cell counting and flow cytometry were used to assess the impact ofhypoxia on bone marrow mesenchymal stem cell growth, cell cycle, and apoptosis.RT-PCR method was employed to detect the effect of hypoxia on the Oct-4, Sox-2, andRex-1mRNA expression in bone marrow mesenchymal stem cells. Concentrations ofAngII and ACE in the culture medium of hypoxia-treated bone marrow mesenchymalstem cells were determined by radioimmunoassay. Influences of hypoxia on AngI,AngII and ACE in bone marrow mesenchymal stem cells was evalued by Western-blotarray. AngII was added into the normoxia group, as the exogenous AngII group.Different concentrations of Losartan (AT1R blocker) and PD123319(AT2R blocker)were added into bone marrow mesenchymal stem cells in normoxia and hypoxia group.The number of bone marrow mesenchymal stem cells was counted by the cytometry.Results: The ratio and proliferation index of bone marrow mesenchymal stem cells inS-phase both increased after hypoxia72hours. The number of cells increasedsignificantly in the hypoxia group compared to the normoxia. Multi-directional markersof bone marrow mesenchymal stem cells in the hypoxia group and normoxia groupwere highly expressed. Sox-2expression in the hypoxia group was higher. The levels ofAngII and ACE in the cell culture medium increased significantly in the hypoxia groupcompared to the normal group. AT1-blocker de the cells proliferation stimulated by thehypoxia. Conclusions: Hypoxia stimulated the proliferation of bone marrowmesenchymal decreased stem cells and increased the levels of the components of thelocal RAS, suggesting RAS may mediate the proliferation of bone marrowmesenchymal stem cells under the hypoxic conditions via AT1receptor. Part Ⅲ AngII-mediated on hypoxia-induced growth and migrationof bone marrow mesenchymal stem cellsObjective: To investigate the proliferation signaling pathway and migrationmechanism of bone marrow mesenchymal stem cells. The cells were cultured usingexogenous AngII under hypoxia. Methods: PI3K/AKT pathway protein expressions ofbone marrow mesenchymal stem cells in different periods of hypoxia within24h (0h,0.5h,1h,4h,12h,24h) were examined by western-blot array. The two time points atwhich the protein expression varied obviously were selected. Captopril (ACE inhibitor),Losartan (AT1receptor blocking agent), LY294002(PI3K specific blocker), andPD123319(AT2receptor blocker) were added to the normoxia+AngII group and thehypoxia group. Then the protein expression of PI3K/AKT pathway was determined. Theprotein expression of HIF-1α and VEGF in bone marrow mesenchymal stem cells atdifferent time periods (1h,4h,12h,24h) was assessed by western-blot array. VEGFexpression of the bone marrow mesenchymal stem cells incubated with differentblocking agents in the normoxia+AngII group and the hypoxia group respectively after24h under hypoxia was evaluated. Results:The expressions of AKT and p-AKT proteinin cells at different time periods was tested. The results showed that AKT expressed inboth the endogenous AngII group and hypoxia group, while there was no significantdifference at all time points between the two groups. p-AKT expression reached thepeak at4h under the hypoxia or AngII stimulation, and down to basal levels at24h.Block of the signaling pathways at different phases of bone marrow mesenchymal stemcells in normoxia+AngII group and hypoxia group showed similar results at4h and12h.The PI3K/AKT pathway protein expressions decreased significantly after blocking ofAT1, while no changes after blocking of AT2. Blockage of PI3K, PI3K/AKT signalingpathway protein expression decreased significantly. PI3K/AKT signaling pathwayprotein expression increased in the AngII group compared to the hypoxia group afterblocking of ACE. Following24h hypoxia, HIF-1α and VEGF protein expression inbone marrow mesenchymal stem cells increased in a time-dependent manner. VEGF protein expression reduced significantly in the AngII group and hypoxia group afterblocking of AT1. Conclusions: The influences of the two models, AngII and hypoxia,were through the same signaling pathway in the bone marrow mesenchymal stem cells.HIF-1α, the central molecule of the PI3K/AKT pathway, translated into the nucleus afterthe pathway was activated and promotes the up-regulation of VEGF expression.Ultimately, the series of courses promoted the migration of the cells.