（ 様 式 ３ ）論文

要旨

Low grade inflammation inhibits VEGF induced HUVECs migrationin p53 dependent manner

小規模の炎症反応は、VEGF で誘導されるヒト血管内皮細胞の遊走能を p53 分子依存性に抑制する。

Sushil Panta【序論及び目的】

The signaling pathway for endothelial cell migration involve inflammatory factor like TNF andangiogenic factor like VEGF. Once TNF is secreted in response to toxins, its first target is theendothelium. VEGF is a major factor for endothelial cell migration and its understanding will be useful tocontrol the different pathological conditions. P53 is a hub for the cell signaling pathway and relays therespective signals to their targets. Cells must acquire lethal level of P53 faster to undergo apoptosis. Since
P53 has been reported to attenuate cancer cell migration, we hypothesize that TNF induced mild
upregulation of P53 must be involved in controlling cell migration induced by VEGF. Our aim in this study
is to explore the role of low grade inflammation in growth factor induced angiogenesis.

【材料及び方法】

HUVECs were incubated in EBM-2 basal medium containing 0.2% FBS for experimental purposes.
Wound healing assay and transwell migration assay were performed to observe and quantify cell migration.
Western blotting, ELISA and quantitative real-time RT-PCR was used to quantify different proteins andtheir mRNA levels respectively. The importance of the proteins in cell migration were established bysilencing the proteins of interests also. Localization of the proteins were observed by immunofluorescenceimaging. Data were presented as means ± S.D. of several determinations. Differences between groups weretested for significance using Student’s t-test and P ≤ 0.05 was considered as statistically significant. In
these assays, similar results were obtained in at least three independent experiments.

【結果】Pretreatment of HUVECs with low dose TNF and co-incubation with VEGF resulted into reduced
migration compared to VEGF treatment only. MTT assay and VEGFR ELISA revealed that the decrease inthe HUVECs migration by TNF pretreatment was not due to the decrease in cell number or VEGF
receptors in TNF treated groups. TNF and VEGF co-incubated cells showed mild elevation and nuclear
localization of P53 compared to TNF and/or VEGF only treated groups. RT-PCR analysis showed that
pretreatment with TNF did not make much difference in p53 mRNA level. TNF pretreatment could notinhibit the VEGF induced migration in P53 silenced group. TNF pretreatment inhibited the shuttling ofId1 from the nucleus to the cytoplasm while VEGF treatment released it to the cytoplasm. In P53 silenced
groups, distribution of Id1 in cytoplasm was evident. The upregulation of P53 by the concerted effort ofTNF and VEGF was followed by downregulation of Id-1. Compared to scramble, Id1 level was
drastically upregulated in P53 silenced group. TNF pretreatment and VEGF co-incubation made no
significant difference between 1-integrin levels. 5 3–integrin is an important one regarding its ability tobind to wide variety of plasma proteins and matrix and expression on angiogenic vessels. Id-1 have beenreported to control 53-integrins. We silenced Id1 in HUVECs and checked the level of 3-integrin.
Compared to scramble, Id1 silenced cells had significantly decreased level of 3-integrin.

【結論及び考察】Low grade acute inflammation have been used to treat bladder cancer in the past. We used TNF in ourexperiment because classical features of inflammation in endothelial cells can be achieved by treating thecells with TNF. TNF can be pro-tumorogenic or tumoricidal agent depending on its local tissue
concentration. We found that in HUVECs low dose TNF exerts the anti-migratory activity against VEGF.
We focused on inhibition of cell migration by a nonlethal and mild upregulation of p53 for which we usedmuch lower dose of TNF. TNF pretreated and VEGF co-incubated group always had mild increase ofP53 levels. Comparison of control versus P53 silenced group showed that P53 is an important conductor oflow grade inflammation triggered antiangiogenic response. We choose Id1 as a potential candidate to beinvestigated in P53 controlled cell migration. TNF pretreatment downregulated the VEGF induced Id1. In
P53 silenced group, TNF pretreatment was unable to downregulate the Id1 induced by VEGF treatment.
Recruitment of the downstream target of Id1 i.e. αv3-integrin to lamellipodia is important in endothelial
cell migration. 3-integrin was significantly downregulated in Id1 silenced HUVECs. In summary, theanti-angiogenic signal of TNF is relayed by P53 to suppress Id1 activity. Suppression of Id1 activity
caused downregulation of 3-integrin that decreased the HUVECs ability to migrate.

（Biochemical and Biophysical Research Communications

2017 年IN PRESS）