| 研究実績の概要 |
The goal of this project is to clarify the mechanism of VEGF-TFEB mediated antimicrobial effects for endothelial cell defense against GAS infection. There are three specific aims were proposed, and I had completed specific aim 1 and specific aim 2. The first specific aim found that VEGF-enhanced endosomal degradation. I used VEGF-A165, a most ubiquitously exists in host and strongly binds on receptor for signals, to treat endothelial cells. I confirmed that VEGF suppressed GAS growth inside endothelial cells. Next, the localization between bacteria and endosomal markers, including Rab7, LAMP-1 and acidification indicator (lysotracker), with VEGF treatment were identified with a higher patten under confocal microscopy observation. Since GAS hemolysins were suppressed by acidity and VEGF promoted endosomal acidification, GAS-vesicle was detected with lysotracker shown a higher patten and a lower Galectin3 recruitment, a vesicle damage marker. I found VEGF promotes GAS trapped inside an efficient maturated endosome/lysosome prior to causing membrane damage.
Second, I had demonstrated VEGF-mediate antimicrobial activity through TFEB activation. I found the protein level of TFEB and its downstream turn-on gene expression were higher under VEGF treatment. TFEB nuclear translocation in endothelial cells was increased under GAS infection but low efficiency. VEGF treatment increased TFEB efficiency in GAS-infected endothelial cells. My results linked the relationship between VEGF and TFEB in this stage.
|
| 今後の研究の推進方策 |
I had almost completed the specific aim 1 and 2. I will continue specific aim 3. I will try to demonstrate the detail of VEGF-mediated antimicrobial activity through TFEB activation. I will checked whether VEGF affect Ca2+mediated calcineurin activations. mTORC1 activity will be detected by observe S6K phosphorylation. Ca2+ level in cell will be confirmed by Fluo8 stain. Calcineurin phosphatase activation will be investigated by the observing the de-phosphorylation of TFEB and TFEB nuclear trans-localization by fluorescence microscope observation. Bacterial number under TFEB over-expression or siRNA knockdown will be performed by colony forming assay to confirm bacteria clearance is contribution of TFEB.
In the half of this year. I will try to link into specific aim 3 and to clarify whether VEGF-mediated TFEB activation rescue xenophagy in endothelial cells. Since TFEB activation also increase autophagy, we will detect whether autophagy related genes are upregulated by VEGF. The recruitments of autophagic related proteins, such as FIP200 and LC3, on GAS-containing vesicles w/o VEGF will be compared under confocal microscope observation. My previous findings showed acidity was lost in LC3-positive GAS vesicle in endothelial cells. We will detect whether VEGF reverse such acidification. Most importantly, whether VEGF mediate a successful autophagosome, isolation membrane (IM), target on GAS will need to be confirmed by electron microscope.
I expect that VEGF-mediated TFEB activation will even more rescue xenophagy against GAS in endothelial cells.
|
