Blockade of insulin receptor substrate-1 inhibits biological behavior of choroidal endothelial cells.

AIM: To investigate the effects of blockade of insulin receptor substrate-1 (IRS-1) on the bio-function of tube formation of human choroidal endothelial cells (HCECs). METHODS: Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to determine the expression level of IRS-1 and phospho-IRS-1 in HCECs. Tube formation of HCECs was analyzed using three dimensional in vitro Matrigel assay with or without IRS-1 blockage via IRS-1 inhibitor (GS-101) and vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor. In addition, cell counting kit (CCK)-8 and Transwell migration assay were exerted to analyze the effects of blockade of IRS-1 on the bio-function of proliferation and migration of HCECs, respectively. The apoptosis of HCECs was examined using flow cytometry (FCM). RESULTS: RT-PCR and Western blot revealed that IRS-1 phospho-IRS-1 were expressed in HCECs and the expression level was enhanced by stimulation of VEGF-A. The number of tube formation was decreased significantly in GS-101 treated groups compared to phosphate buffered saline (PBS) treated control groups. Furthermore, both cell proliferation and migration of HCECs were decreased in the presence of GS-101. FCM analysis showed that the apoptosis of HCECs was enhanced when the cells were treated with GS-101. Western blot also showed that the expression level of cleaved-caspase 3 in GS-101 treated group was higher than that in control group. CONCLUSION: Blockade of IRS-1 can inhibit tube formation of HCECs through reducing cell proliferation and migration and promoting cell apoptosis.

Monocyte chemoattractant protein 1 and fractalkine play opposite roles in angiogenesis via recruitment of different macrophage subtypes.

AIM: To explore the interaction between macrophages and chemokines [monocyte chemoattractant protein 1 (MCP-1/CCL2) and fractalkine/CX3CL1] and the effects of their interaction on neovascularization. METHODS: Human peripheral blood mononuclear cells, donated by healthy volunteers, were separated and cultured in RPMI-1640 medium containing 10% fetal bovine serum, then induced into macrophages by stimulation with 30 µg/L granulocyte macrophage-colony stimulating factor (GM-CSF). The expression of CCR2 and/or CX3CR1 in the macrophages was examined using flow cytometry. Macrophages were then stimulated with recombinant human CCL2 (rh-CCL2) or recombinant human CX3CL1 (rh-CX3CL1). The expression of angiogenesis-related genes, including VEGF-A, THBS-1 and ADAMTS-1 were examined using real-time quantitative polymerase chain reaction (PCR). Supernatants from stimulated macrophages were used in an assay of human retinal endothelial cell (HREC) proliferation. Finally, stimulated macrophages were co-cultured with HREC in a migration assay. RESULTS: The expression rate of CCR2 in macrophages stimulated by GM-CSF was 42%±1.9%. The expression rate of CX3CR1 was 71%±3.3%. Compared with vehicle-treated groups, gene expression of VEGF-A in the macrophages was greater in 150 mg/L CCL2-treated groups (P<0.05), while expression of THBS-1 and ADAMTS-1 was significantly lower (P<0.05). By contrast, compared with vehicle-treated groups, expression of VEGF-A in 150 mg/L CX3CL1-treated groups was significantly lower (P<0.05), while expression of THBS-1 and ADAMTS-1 was greater (P<0.05). Supernatants from CCL2 treated macrophages promoted proliferation of HREC (P<0.05), while supernatants from CX3CL1-treated macrophages inhibited the proliferation of HREC (P<0.05). HREC migration increased when co-cultured with CCL2-treated macrophages, but decreased with CX3CL1-treated macrophages (P<0.05). CONCLUSION: CCL2 and CX3CL1 exert different effects in regulation of macrophage in expression of angiogenesis-related factors, including VEGF-A, THBS-1 and ADAMTS-1. Our findings suggest that CCL2 and CX3CL1 may be candidate proteins for further exploration of novel targets for treatment of ocular neovascularization.

Anti-apoptosis effects of vascular endothelial cadherin in experimental corneal neovascularization.

AIM: To explore the effects and mechanism of vascular endothelial cadherin (VE-cadherin) on experimental corneal neovascularization (CRNV). METHODS: Mouse corneas were burned with sodium hydroxide to build a CRNV model. The burned corneas were locally administrated with anti-mouse VE-cadherin neutralizing antibody. Annexin V and cluster of differentiation 31 (CD31) double staining was used to measure vascular endothelial cell apoptosis with the use of flow cytometry (FCM). The protein expression of NADPH oxidase 2 (Nox2), caspase-3, and protein kinase C (PKC) in the burned corneas were examined by Western blot. Human retinal endothelial cell (HREC) proliferation was detected using a Cell Counting Kit 8 (CCK-8) assay in vitro. RESULTS: The amount of CRNV peaked two weeks after the alkali burn. FCM confirmed that VE-cadherin neutralizing antibody treatment increased CD31 positive cell apoptosis. Western blot revealed that the intracorneal protein expression of Nox2 and caspase-3 were up-regulated, while PKC was down-regulated in the VE-cadherin neutralizing antibody administrated group. CCK-8 assay showed that VE-cadherin neutralizing antibody markedly inhibited HREC proliferation. CONCLUSION: VE-cadherin exhibited an anti-apoptosis effect through enhanced PKC signaling and an enhanced cell proliferation pathway.

B7-H4 expression associates with cancer progression and predicts patient's survival in human esophageal squamous cell carcinoma.

A retrospective cohort study including 112 patients suffering from esophageal squamous cell carcinoma (ESCC) was performed to investigate the expression of B7-H4 in ESCC and determine its association with patient's clinicopathological parameters and survival. Expression levels of B7-H4 on tumor cells and densities of tumor infiltrating lymphocytes (TILs) in the surgical specimens of ESCC tissues were characterized using immunohistochemical assays. Uni- and multivariate analyses were performed to evaluate the prognostic value of B7-H4 expression levels and densities of TILs in tumor sections. Positive B7-H4 immunostaining was observed in 107 of 112 (95.5%) of ESCC tissue sections. We further divided all patients into two major subgroups, a lower B7-H4 expression group with 46 patients and a higher B7-H4 expression group with 66 patients. We found that expression levels of B7-H4 on tumor cells were significantly correlated with patient's gender (P = 0.0288), distant metastasis (P = 0.0500), and TNM stage (P = 0.0258). Moreover, tumor cell B7-H4 expression was inversely correlated with densities of CD3(+) T cells in tumor nest (P = 0.0424) and CD8(+) T cells in tumor stroma (P = 0.0229). The overall survival rate of the patients with higher B7-H4 expression was significantly worse than that of the patients with lower B7-H4 expression (P = 0.0105, Hazard Ratio: 1.854, 95%CI:1.152-2.902). Markers of cell-mediated immune responses such as CD3, CD8, and T-bet were associated with better patient survival. The present study demonstrated that B7-H4 expression in human ESCC is associated with cancer progression, reduced tumor immunosurveillance and worse patient outcomes. B7-H4 can serve as a novel prognostic predictor for human ESCC and a potential target for the immune therapy against this malignancy.

[Prokaryotic expression and biological activity of recombinant human IL-17F/His protein].

AIM: To Prepare recombinant human IL-17F/His protein and investigate its biological activity in vitro. METHODS: The gene region of human IL-17F was cloned by RT-PCR. After identification by sequencing, the hIL-17F gene encoding function domain was cloned into expression plasmid PQE3.0 and transfected into E.coli M15. By the induction of Isopropyl-ß-D-Thiogalacto-Pyranoside(IPTG), recombinant IL-17F/His protein was effectively expressed in E.coli M15. The recombinant protein was identified by Western blot. RESULTS: After renaturation and purification by HiTrap(TM); affinity column, the recombinant protein can up-regulate macrophages to secret TNF-α, IL-6 and other relative cytokines. It also promoted proliferation of HeLa cells in vitro. CONCLUSION: hIL-17F/His recombinant protein is of high biological activity, which can be used to make further study of its special characteristic.

The negative co-signaling molecule b7-h4 is expressed by human bone marrow-derived mesenchymal stem cells and mediates its T-cell modulatory activity.

Though experimental evidence shows that human bone marrow-derived mesenchymal stem cells (hBMSCs) are able to suppress T-cell activation and proliferation, the precise mechanisms are still not completely understood. Here, we investigated the role of the negative costimulatory molecule B7-H4 in the immunosuppressive effect of hBMSCs on T-cell activation. We showed that B7-H4 expresses abundantly on hBMSCs assessed by reverse transcription, immunofluorescence staining, and flow cytometric analysis. Further studies demonstrated that B7-H4 expressed on hBMSCs inhibits T-cell activation and proliferation via induction of cell cycle arrest and inhibition of NF-kappaB nuclear translocation. Blocking B7-H4 would decrease the secretion of transforming growth factor-beta1 (TGF-beta1) in the supernatant of activated T cells co-cultured with hBMSCs. Addition of neutralizing antibodies against B7-H4 significantly attenuated the inhibitory effects of hBMSCs on T-cells. Thus, our study established the novel role of B7-H4 molecule in the suppressive effect of hBMSCs on T-cell activation and proliferation. Taken together, these results highlight the complex role of hBMSCs in regulating the immune response, asserting the possibility of their therapeutic application in transplantation, the treatment of graft-versus-host disease (GVHD), and autoimmune diseases.

[Prokaryotic expression, purification and biological activity of recombinant human IL-17/His protein].

AIM: To investigate the biological activity of recombinant human IL-17 protein in vitro. METHODS: The gene region of human IL-17 was cloned by RT-PCR. After identification by sequencing, the hIL-17 gene encoding function domain was cloned into expression plasmid PQE3.0 and transfect into E.coli M15. By the induction of Isopropyl-beta-D-Thiogalacto-Pyranoside (IPTG), recombinant IL-17/His protein was effectively expressed in E.coli M15. The recombinant protein was identified by Western blot. RESULTS: After denaturation, renaturation and purification by HiTrap affinity column, the recombinant protein stimulated HeLa, a human uterine cervix cancer cell line, to excrete IL-6 and GM-CSF in vitro. CONCLUSION: IL-17/His recombinant protein is of high biological activity, which can be used to make further study of auto-immune diseases.

[Preparation and characterization of two novel functional monoclonal antibodies against human OX40L].

AIM: To prepare novel anti-OX40L functional monoclonal antibodies and characterize their distinct biological functions. METHODS: Routine immunization of BALB/c mice by a tansfected cell line L929/OX40L expressing high level of OX40L as antigens. Then fuse the immunized spleen with Sp2/0, a kind of myloma, and screen the positive clones by FCS with L929/OX40L as a positive control.After acquisition of the hybidomas secreting anti-OX40L mAb, investigation of their biological activities by Western blot, rapid isotyping analysis, karyotype analysis, competitive inhibition test, indirect immunofluorescence and MTT incorporation assay were followed. RESULTS: Obtain two stable hybridomas, 4D6 and 5C2, which could continuously secret specific anti-OX40L monoclonal antibodies. The following biological activity studies showed that these monoclonal antibodies could both recognize the natural OX40L expressed on the mature DC, especially the OX40L on the several leukemia cell lines, such as Jurkat, SHI-1, U937 etc. Furthermore, they could also suppress the proliferation of SHI-1 in vitro. CONCLUSION: Two hybridomas secreting anti-OX40L monoclonal antibodies continuously and steadily have been established. These monoclonal antibodies could specifically recognize human OX40L and effect the proliferation of leukemia cell line SHI-1 through OX40/OX40L costimulatory signals.

Recombinant human B7-H4 expressed in Escherichia coli inhibits T lymphocyte proliferation and IL-2 secretion in vitro.

AIM: To explore the biofunctions of human B7-H4 generated from prokaryotic system. METHODS: The gene of human B7-H4 extracellular region (IgV-like and IgC-like domains) was obtained by PCR from human cDNA FLJ22418 and then inserted into the prokaryotic expression vector pGEX-5X-3 expressing glutathione s-transferase (GST) fusion protein. After being identified by restriction enzyme digestion and sequencing, the recombinant vector was transferred into host strain E coli BL21-RIL(DE3). A 47 kDa fusion protein (GST/hB7-H4) was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by standard methods reported in the prokaryotic system. The inhibitory effect of GST/hB7-H4 on proliferation of T cells was observed in vitro by CD3mAb activated T-cell culturing system and [(3)H]-thymidine incorporation assay. The concentrations of interleukin-2 and iterferon-g in the supernatants of T cells were determined by ELISA. RESULTS: We successfully constructed the method for high-level expression and purification of the hB7-H4 extracellular domain as GST fusion protein from E coli. The GST/hB7-H4 fusion protein produced in bacteria had obvious biological activity to inhibit T-lymphocyte proliferation and IL-2 secretion. CONCLUSION: The prokaryote expression system could be used to generate hB7-H4 protein with natural spatial conformations and biological functions, which provided an efficient and economical way for the preparation of this target protein.