Therapeutic ultrasound protects HUVECs from ischemia/hypoxia-induced apoptosis via the PI3K-Akt pathway.

Background: Previous studies have demonstrated that therapeutic ultrasound (TUS) ameliorates angiogenesis on ischemic hind limb animals and also promotes human umbilical vein endothelial cells (HUVECs) tube formation. Apoptosis plays a key role in post-ischemic angiogenesis pathogenesis. However, the mechanisms underlying the anti-apoptotic effects of TUS are not clear. Therefore we put forward the hypothesis that TUS might promote angiogenesis during ischemia/hypoxia (I/H) by decreasing apoptosis. Methods: We investigated the cytoprotective role of TUS and the underlying mechanisms in I/H-induced HUVEC apoptosis. HUVECs were treated under hypoxic serum-starved conditions for 36 h and then treated with or without TUS (9 minutes, 1 MHz, 0.3 W/cm2). The cell viability was examined by the CCK-8 assay, apoptosis cell rate was determined by TUNEL staining and flow cytometry assay. In addition, the mitochondrial-dependent apoptosis pathway was evaluated by the protein activity of Bax, Bcl-2 and Caspase-3. Results: 1) apoptosis could be induced by I/H in HUVECs. 2) TUS attenuates HUVECs cell apoptosis induced by I/H. 3) TUS inhibits the protein expression of apoptosis modulators and effectors that regulate the mitochondrial pathway of apoptosis in HUVECs. 4) TUS increases the phosphorylation of Akt, which demonstrates the activation of the phosphoinositide 3-kinase (PI3K)- serine/threonine kinase (Akt) signal pathway. Conclusions: The present study indicates that exposure to TUS exerts a protective effect against I/H-induced apoptosis among HUVECs and that this process is mediated through the mitochondrial-dependent intrinsic apoptotic pathway. We also confirm that the PI3K-Akt signal cascade may be taken part in the TUS effects on apoptosis.

Significance of low mTORC1 activity in defining the characteristics of brain tumor stem cells.

Background: The significance of mammalian target of rapamycin complex 1 (mTORC1) activity in the maintenance of cancer stem cells (CSCs) remains controversial. Previous findings showed that mTORC1 activation depleted the population of leukemia stem cells in leukemia, while maintaining the stemness in pancreatic CSCs. The purpose of this study was to examine the currently unknown role and significance of mTORC1 activity in brain tumor stem cells (BTSCs). Methods: Basal mTORC1 activity and its kinetics were investigated in BTSC clones isolated from patients with glioblastoma and their differentiated progenies (DIFFs). The effects of nutrient deprivation and the mTORC1 inhibitors on cell proliferation were compared between the BTSCs and DIFFs. Tissue sections from patients with brain gliomas were examined for expression of BTSC markers and mTORC1 activity by immunohistochemistry. Results: BTSCs presented lower basal mTORC1 activity under each culture condition tested and a more rapid decline of mTORC1 activity after nutrient deprivation than observed in DIFFs. The self-renewal capacity of BTSCs was unaffected by mTORC1 inhibition, whereas it effectively suppressed DIFF proliferation. In agreement, immunohistochemical staining of glioma tissues revealed low mTORC1 activity in tumor cells positive for BTSC markers. In in vitro culture, BTSCs exhibited resistance to the antitumor agent temozolomide. Conclusions: Our findings indicated the importance of low mTORC1 activity in maintaining the undifferentiated state of BTSCs, implicating the relevance of manipulating mTORC1 activity when developing future strategies that target BTSCs.

Angiogenesis effect of therapeutic ultrasound on HUVECs through activation of the PI3K-Akt-eNOS signal pathway.

Therapeutic angiogenic effects of low-intensity ultrasound have been reported in endothelial cells and animal models of hind limb ischemia. It has been shown that the proliferation, migration, and tube formation of endothelial cells play critical roles in angiogenesis. The purpose of this study was to determine the underlying mechanism of low-intensity continuous therapeutic ultrasound on angiogenesis in endothelial cells. In the present study, human umbilical vein endothelial cells (HUVECs) were simulated of low-intensity therapeutic ultrasound (TUS, 1 MHz, 0.3 W/cm(2), 9 minute per day) for 3 days, and we observed migration, tube formation, and expression of endothelial nitric oxide synthase (eNOS) and serine/threonine kinase (Akt) in HUVECs. Specific inhibitors of eNOS and phosphoinositide 3-kinase (PI3K) were added to the culture medium and TUS-induced changes in the pathways that mediate angiogenesis were investigated. After exposure to TUS, HUVECs tube formation and migration were significantly promoted, which was blocked by the eNOS inhibitor Immunofluorescence assay and Western blotting analysis demonstrated that eNOS expression in the HUVECs was significantly increased after TUS exhibition. Proteins of phosphorylated eNOS and Akt were both up-regulated after TUS stimulation. However, the specific inhibitor of PI3K not only significantly decreased the expression of p-Akt, but also down-regulated the p-eNOS. This suggested that the PI3K/Akt signal pathway might participate in modulating the activity of eNOS. In short, TUS therapy promotes angiogenesis through activation of the PI3K-Akt-eNOS signal cascade in HUVECs.

Pulsed electromagnetic field improves postnatal neovascularization in response to hindlimb ischemia.

Pulsed electromagnetic fields (PEMF) have been shown to promote proliferation and regeneration in the damaged tissue. Here, we examined whether PEMF therapy improved postnatal neovascularization using murine model of hindlimb ischemia, and the underlying cellular/molecular mechanisms were further investigated. Hindlimb ischemia was induced by unilateral femoral artery resection using 6-8 week-old male C57BL6 mice. Then, mice were exposed to extracorporeal PEMF therapy (4 cycles, 8min/cycle, 30 ± 3 Hz, 5 mT) every day until day 14. Our data demonstrated that PEMF therapy significantly accelerated wound healing, decreased prevalence of gangrene and increased postnatal neovascularization. Moreover, the levels of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS) and Akt phosphorylation in ischemic muscles were markedly enhanced following PEMF therapy. In vitro, PEMF inhibited the process of hypoxia-induced apoptosis and augmented tube formation, migration and proliferative capacities of human umbilical vein endothelial cells (HUVECs). Additionally, PEMF exposure increased VEGF secretion, as well as the eNOS and Akt phosphorylation, and these benefits could be blocked by either phosphoinositide 3-kinase (PI3K) or eNOS inhibitor. In conclusion, our data indicated that PEMF therapy enhanced ischemia-mediated angiogenesis, through up-regulating VEGF expression and activating the PI3K-Akt-eNOS pathway. Therefore, PEMF should be a valuable treatment for the patients with critical limb ischemia.

Evaluation of osteopontin as a potential biomarker for central nervous system embryonal tumors.

Osteopontin (OPN) is a protein linked to tumor growth, progression and metastasis of cancers. However, its role in the progression of central nervous system (CNS) embryonal tumors such as atypical teratoid/rhabdoid tumor (AT/RT), medulloblastoma (MB) and primitive neuroepithelial tumors (PNET) remains elusive. In this study, we investigated the value of OPN staining in differential diagnosis of AT/RT from MB and PNET, and assessed the correlation between OPN expression and patients' prognosis. This retrospective study was conducted on tissue sections obtained from children cases with CNS embryonal tumors treated in Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine from 2006 to 2012 by immunohistochemistry (IHC). 49 cases were collected (11 AT/RTs, 25 MBs, and 13 PNETs), with a median follow-up time of 28.9 months. OPN expression in AT/RT was significantly higher than MB and PNET with the positive rates of 100, 32, and 23 %, respectively (P < 0.01). The specificity and sensitivity of OPN staining in diagnosing AT/RT are 97.4 and 90.9 %, respectively, as judged by strong OPN IHC staining level (+++). Patients who had positive OPN staining have increased risks of poorer median overall survival (hazard risk 5.54, 95 % CI 1.87-16.38) and tumor progression (hazard risk 14.47, 95 % CI 4.47-46.85). OPN is a valuable biomarker to aid in the differential diagnosis between AT/RT and MB/PNET. Moreover, OPN is a potential novel prognostic marker for CNS embryonal tumors.

Nanoparticle-based bio-barcode assay for the detection of bluetongue virus.

The ultrasensitive bio-barcode amplification assay (BCA) technique was developed for the specific detection of the outer-core protein VP7 of bluetongue virus (BTV). The target antigen VP7 was first captured by gold nanoparticles (GNPs) coated with polyclonal antibodies. Magnetic microparticles (MMP) coated with VP7 monoclonal antibody were then added to form a sandwich immuno-complex. After the immuno-complex was formed, signal DNA annealed to DNA strands covalently bound to the GNPs were released by heating and characterized by PCR and real-time fluorescence PCR. A detection limit of 0.1fg/ml was measured for purified VP7, seven orders of magnitude more sensitive than that of conventional antigen capture ELISA. The BCA demonstrated the same enhanced sensitivity for detecting BTV in serum samples from sheep. In the following work it is demonstrated that this assay is a highly sensitive method for the detection of BTV proteins that could be adapted to measure other proteins.

Extracorporeal whole blood immuno-adsorption of passively transferred myasthenia gravis rabbits by cellulose-tryptophan column.

The whole blood immuno-adsorption (WBIA) system, using an adsorbent to remove pathogenic antibodies of Myasthenia Gravis (MG), was studied. Cellulose-tryptophan adsorbent was synthesized and purified in our lab. Experimental autoimmune myasthenia gravis (EAMG) rabbits were passively transferred with immunoglobulin from patients with myasthenia gravis. The rabbits underwent extracorporeal whole blood adsorption for 2 hours. Results showed no significant damage to blood cells and no changes in the concentrations of electrolytes. Total protein decreased by 12.6% (P<0.05) and globulin protein decreased 21.9% (P<0.05). The overall removal of antibodies against nicotinic acetylcholine receptor (nAChR) was 49.85%. The percentage of decrement of compound muscle action potential in 3, 5, 10 Hz of EAMG rabbits all dropped down after the treatment. The quantity of neuromuscular junctions per unit area (25 mm(2)) increased significantly after treatment (P<0.05). In conclusion, the adsorbent was biocompatible, safe for whole blood immuno-adsorption. Whole blood immuno-adsorption improved clinical manifestation and neuromuscular function of the passively transferred EAMG rabbits.