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Phospholipase D1 mediate flow dependent vascular inflammation

Yujin Jang, Sanguk Yun*
Department of Biotechnology, Inje University, 197 Inje-ro, Gimhae-si, Gyengsangnam-do, Korea

Abstract Result

PLD1 has been implicated in a variety of inflammatory diseases,
including arthritis, asthma, and cancer. However, little is known about
the role of PLD1 in vascular inflammation. As a result of checking the
mRNA level using qPCR, it was confirmed that PLD1 was knocked down.
In EC, siRNA-mediated PLD-1 gene knockdown led to the blockade of
disturbed flow-induced increased activity of NF-kB. In addition, it was
confirmed that siPLD knockdown in EC increased the perturbation-
induced NF-kB activity and decreased the Yap activity. These results
suggest that PLD1 may mediate vascular inflammation and Figure 1. qPCR quantification of Bovine PLD1. It was confirmed whether
PLD1 was KD using qPCR.
atherosclerosis by regulating EC mechanization.

Introduction

PLD1 is responsible for the degradation of phospholipids in the plasma
membrane, which hydrolyzes phosphatidylcholine to produce
phosphatidic acid and choline. In addition, various membrane-induced
phospholipid changes themselves play important roles in vesicle
migration and other membrane-related events. Phospholipase D 1(PLD1)
has been implicated in various inflammatory diseases including arthritis,
Figure 2. The change of NF-kB was confirmed using disturbed flow. The
asthma and cancer. However, little is known about the role of PLD1 in
disturbed flow was performed for 12 hours. (A) Western blot was performed to
vascular inflammation. So, to find out how disturbed flow affects PLD, confirm the NF-kB(p65) protein level in control and PLD KD cells. (B) p-p65 was
NF-kB and yap phosphorylation were investigated in control and PLD confirmed by quantification with t-p65. N=3, (**p=0.00454)
knockdown cell.

Method

- PLD1 expression was investigated using qPCR analysis.
- We investigated NF-kB activation by inducing disturbed flow in the
siRNA-mediated PLD1 gene knockdown cell. Fibronectin was mixed in 1X
PBS coat in the glass, and kept in a 37℃ incubator for 1 hour, UV
treatment for 20 minutes after suction, and coated with 0.2% dBSA in a Figure 3. The change in yap was confirmed using disturbed flow. The
37 ℃ incubator for 20 minutes, and used for cell culture. disturbed flow was performed for 12 hours. (A) Western blot was performed to
confirm the Yap protein level in control and PLD KD cells. (B) Yap was confirmed by
quantification with actin.
- All experiments were carried out by culturing BAEC in 10% FBS and p/s
DMEM using GenDPOT’s FBS and Hyclone’s Penicillin Streptomycin. In
the disturb flow experiment, 1% FBS, p/s DMEM was used.
Conclusion
- Knockdown of PLD was confirmed using qPCR.
- In addition, changes in NF-kB and Yap were observed using in
disturbed flow using control and PLD Knockdown cell. NF-kB is
increasing and Yap is decreasing in PLD knockdown cells.
- These results suggest that PLD1 may mediate vascular inflammation
and atherosclerosis by regulating EC mechanization.

Reference

Huang, Chunfa, et al. "Shear stress induces cell apoptosis via a c-Src-
phospholipase D-mTOR signaling pathway in cultured
podocytes." Experimental cell research 318.10 (2012): 1075-1085.

Kang, Dong Woo. "Platelet derived growth factor increases
phospholipase D1 but not phospholipase D2 expression via NFκB
signaling pathway and enhances invasion of breast cancer cells." Cancer
Created with BioRender.com letters 294.1 (2010): 125-133.