The Studies Of Biologically Functional Effect Of Shear Stress On Human Bone Mesenchymal Stem Cells Molecular Mechanism Involved

Blood vessel grafts are urgently needed for serious wound repair and surgical therapy for some severe diseases. Lacking of autograft (autogenous vessel graft), the transplantation of vessel graft often fails for the thrombosis, especially in vessels application with minor diameter (diameter < 6 mm) or some huge vessels that the speed of internal blood flow is very low, such as vein. Tissue engineered blood vessels (TEBVs) is one of the most hot spot in life science study and human bone mesenchymal stem cells is one of the most important seed cells. But the adhesion and vasoreactivity of seeded cells are not good under shear stress, consequently leading to cell apoptosis and the failure of graft. It is the key problem limited tissue engineered blood vessels study. But there is no research on functional effect of shear stress on human bone mesenchymal stem cells and the mechanisms involved. Therefore, our studies focus on biological functional effect of shear stress on hBMSCs and the mechanisms involved, which is critical to establish of TEBVs.Aims1. To explore the effect of various duration/intensity of shear stress on apoptosis, proliferation and biological functions of human bone mesenchymal stem cells.2. To screen related genes of human bone mesenchymal stem cells and the key proteins that may be critical targets for enhancing function of cells under shear stress.3. To demonstrate the molecular mechanisms involved in effect of shear stress on hBMSCs, and to introduce the feasible strategy to improve the function of seed cells.Material and Methods1. We utilized density gradient centrifugation (DGC) to isolate and culture human bone mesenchymal stem cells, and then utilized flow cytometry to identify the specific surface CD molecules. Human bone mesenchymal stem cells were induced to differentiate to adipocytes and osteoplasts, which were determined by oil red-O dyeing and osteocalcin immunohistochemistry.2. The third generation of hBMSCs were exposed to different intensity of shear stress (3, 10, or 30 dyne/cm~2) for 2h, 6h, or 24h differently, and LDH release ,cell counts, flow cytometry, and Caspase3 activity were applied to assess the apoptosis, proliferation and cell death of human bone mesenchymal stem cells.3. The third-generation of hBMSCs was exposed to no shear stress or shear stress of 3 dyne/cm~2 for 6 hours. To identify its effect at protein level, we utilized two-dimensional gel electrophoresis to separate the proteins and used MALDI-TOF-Ms, immunofluorescence and western blotting to prove the key proteins.4. hBMSCs were exposed to shear stress (3 dyne/cm~2 for 6 hours),and the extracted protein was determined by Oligo GEARRAY adhesion molecular and apoptosis microarray. After that, rt-PCR was applicated to double check the accuracy of microarray and to confirm the key genes of hBMSCs under shear stress. And the cIAP1 protein level of hBMSCs was determined by western blotting.5. hBMSCs were exposed to different levels of shear stress, and cell count for cell proliferation, caspase3 activity for apoptosis and LDH release for cell death were detected. As the possible key factor, human recombinant cIAP1 was administrated to hBMSCs. And its antagonist, recombinant DIABLO was also dosed to double-confirm the role of cIAP1 to hBMSCs during shear stress exposure.Results1. Flow cytometry of isolated cells showed that CD105 and CD29 positive, CD14, CD34 and CD45 negative, which is supposed to be specific characteristics of hBMSCs. Cultured cells could also be induced into adipocytes (oil-red O positive), and osteoplasts (osteocalcin positive).2. A high raise of multiplication (cells at S+G2 period) was observed under shear stress of 3 dyne/cm~2 for 2h or 6h, compared to their counterparts at 10 and 30 dyne/cm~2. And a visible increase of apoptosis and necrosis was also assessed, after the duration of 6h under different intensity of shear stress, and this tendency is even higher as the time elongation. However, neither LDH nor Caspase3 activity of Human bone mesenchymal stem cells under shear stress (at 3 dyne/cm~2 for 6h) changed, compared to control counterparts.3. 2-DE map of Human bone mesenchymal stem cells were subjected to shear stress (at 3 dyne/cm~2 for 6h). There were 32 specific proteins which were identified. Among them, 3 proteins decreased, 10 proteins were up-regulated, and 19 proteins kept unchanged. sustained increases in the expression of GAPDH and AnnexinA2 had been specifically induced by shear stress ,which has also been proved by western blotting.4. The results of gene microarray exposed that there were 11 genes significantly varied after shear stress exposure (3 dyne/cm~2 for 6h). Four adheresion related genes, ECM-1,ICAM-1,cSPG7,TIMP2 were up-regulated, and 2 (LAMA4 and VCAM1) were down-regulated at the same time. And among them, two apoptosis genes increased dramatically (APAF1 and BOK). And all of these results were double confirmed by rt-PCR, which verified the accuracy of gene microarray. cIAP1 protein level increased under shear stress at 3 dyne/cm~2 for 2h, but decreased when exposed to high level of shear stress at 30 dyne/cm~2 for 2h and even lower for 24h.5. Human recombinant cIAP1 and human recombinant DIABLO were administrated to hBMSCs to double confirm whether cIAP1 is the key molecule of apoptosis pathway activation during shear stress exposure to hBMSCs. After cIAP1 was medicated, both Caspase3 activity and LDH release level declined, and cell proliferation increased. And if DIABLO was administrated, both Caspase3 activity and LDH release level increased, and cell proliferation reduced.ConclutionsWe have made several novel and important observations in the current study. 1. We demonstrated for the first time that low shear stress exposure (3dyne/cm~2, less than 6h) did not affect apoptosis, but significantly increased proliferation of hBMSCs, as well as cIAP1 expression. And higher and longer exposure of shear stress inhibited proliferation, deteriorated apoptosis and cell death, and impaired biologically function of hBMSCs. Higher exposure of shear stress, more serious injury on exposed hBMSCs, which suggested that pre-condition of shear stress, is critical to improve biological function of hBMSCs as seed cells.2. It is an initial found that the low level of shear stress (3 dyne/cm~2) for 6h could up-regulate the expression of AnnexinA2 and GAPDH (evidenced by proteomics and western blotting), and 6 adhesion-related genes and 5 apoptosis-related genes were significantly changed (determined by gene chips). Most interestingly, APAF1 and BOK, key genes in apoptosis, were obviously increased in shear stress exposure, but caspase 3 activity was not elevated, which demonstrated that such exposure to shear stress already activated apoptotic proceeding, and APAF1 and BOK were involved. The presented studies suggested the molecular targets of treatment against shear stress caused seed cell dysfunction.3. Originally studied and verified that hBMSCs apoptosis caused by shear stress was through mitochondrial apoptosis pathway and finally resulted in hBMSCs dysfunction. Administrated by Caspase9 inhibitor, human recombinant cIAP1 could reduce Caspase3 activity and LDH release, further inhibit apoptosis and cell death of hBMSCs, promote cell proliferation and improve biological function. Vice versa when we treated hBMSCs with human recombinant DIABLO, one cIAP1 antagonist. All these results demonstrated that apoptosis of hBMSCs enhanced by shear stress via internal apoptosis pathway.4. Moreover, we demonstrated the first time that administrated by Caspase9 inhibitor, human recombinant cIAP1 could reduce Caspase3 activity and LDH release, further inhibit apoptosis and cell death, promote cell proliferation and improve biological function of hBMSCs under shear stress. Vice versa when we treated hBMSCs with human recombinant DIABLO, one cIAP1 antagonist. This strongly suggested that cIAP1 could be a key point of intervene to protect seed cells from apoptosis and cell death during shear stress exposure, and improve the biological function of TEBVs.Through gene microarray screening, we firstly reported that there were 6 adhesion related genes and 5 apoptosis related genes changed. Among these genes, APAF1 and BOK are major points of cellular apoptosis pathway, and responsible for activation of hBMSCs apoptosis under shear stress. This supports our previous study and provides evidence for next step studies.