Effect Of Recombinant Adenovirus Vector-mediated Granulysin Gene Therapy On Mice Infected With Mycobacterium Tuberculosis Endothelial Cell-specific Molecule 2 (Ecsm2) Localization And Function In Endothelial Cells

【Objective】Recombinant adenovirus-vectored (Ad) tuberculosis (TB) vaccine platform has demonstrated great potential to be used either as a stand-alone or a boost vaccine in murine models. Granulysin is a cytolytic and proinflammatory molecule expressed by activated human cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Recombinant 9 kDa granulysin is broadly antimicrobial. In this study, we constructed the recombinant adenovirus rAdhGA expressing human granulysin, amplified and purified rAdhGA, tested its titer and distribution; then we examined its treatment efficacy in U937 cells infected with M.smegnatis and BALB/c mice challenge with virulent Mycobacterium tuberculosis H37Rv.【Methods】HEK293 cells were transfected with pBHG and pDChGA expressing human granulysin to package the recombinant adenovirus rAdhGA, then confirmed it respectively through RT-PCR and Western blot from mRNA and protein level.We tested expression of rAdhGA in U973 and its distribution in BALB/c mice by immunofluorescence,examined its treatment efficacy in U937 cells infected with M.smegnatis using acid fast staining,evaluated its treatment efficacy in BALB/c mice challenge with virulent Mycobacterium tuberculosis H37Rv according to bacterial loading in lung and pathology analysis. 【Results】The titer of recombinant rAdhGA was 3.95x1010ifu/ml, and it mainly expressed in lung rather than other organs. Treatment of BALB/c mice with rAdhGA resulted in significant treatment efficacy against challenge with virulent Mycobacterium tuberculosis H37Rv when compared with the control group. rAdhGA could directly kill Mycobacterium tuberculosis. The most significant reduction in bacterial load of lung was obtained in mice treated with high dosage of rAdhGA.【Conclusion】Our study indicates that rAdhGA may be used as an efficient non-invasive treatment against TB with the strong ability to directly kill the Mycobacterium tuberculosis. 【Background and objective】Despite its first discovery by in silico cloning of novel endothelial cell-specific genes a decade ago, the biological functions of endothelial cell-specific molecule 2 (ECSM2) have only recently begun to be understood. Limited data suggest its involvement in cell migration and apoptosis. However, the underlying signaling mechanisms and novel functions of ECSM2 remain to be explored.【Methods】A rabbit anti-ECSM2 monoclonal antibody (RabMAb) was generated through hybridoma techniques. Immunoblotting, immunoprecipitation, deglycosylation, immunostaining and confocal microscopy were used to find the that localization and characteristics of ECSM2 in vascular endothelial cells (ECs). Cell aggregation and transwell assays were performed to test the ECSM2 effections on basic fibroblast growth factor (bFGF)-driven EC migration. Gain or loss of function assays by overrexpression or knockdown of ECSM2 in ECs were performed to demonstrate the mechanism underlying ECSM2 modulated the bFGF-directed EC motility.【Results】A rabbit anti-ECSM2 monoclonal antibody (RabMAb) was generated and used to characterize the endogenous ECSM2 protein. Immunoblotting, immunoprecipitation, deglycosylation, immuno staining and confocal microscopy validated that endogenous ECSM2 is a plasma membrane glycoprotein and is preferentially expressed in vascular endothelial cells (ECs). Expression patterns of heterologously expressed and endogenous ECSM2 identified that ECSM2 was particularly concentrated at cell-cell contacts. Cell aggregation and transwell assays showed that ECSM2 promoted cell-cell adhesion and attenuated basic fibroblast growth factor (bFGF)-driven EC migration. Gain or loss of function assays by overrexpression or knockdown of ECSM2 in ECs demonstrated that ECSM2 modulated the bFGF-directed EC motility via the FGF receptor (FGFR)-extracellular regulated kinase (ERK)-focal adhesion kinase (FAK) pathway. The counterbalance between FAK tyrosine phosphorylation (activation) and ERK-dependent serine phosphorylation of FAK was critically involved. A model of how ECSM2 signals to impact bFGF/FGFR-driven EC migration was proposed.【Conclusions】ECSM2 is likely a novel EC junctional protein, promotes cell-cell adhesion, and inhibits bFGF-mediated cell migration. Mechanistically, ECSM2 attenuates EC motility through the FGFR-ERK-FAK pathway. The findings suggest that ECSM2 could be a key player in coordinating receptor tyrosine kinase (RTK)-, integrin-, and EC junctional component-mediated signaling and may have important implications in disorders related to endothelial dysfunction and impaired EC junction signaling.