P1628Angiopoietin-like protein (Angptl) 2 secreted from epicardial adipose tissue induces atrial myocardial fibrosis
Authors: Kira S , Abe I , Teshima Y , Ishii Y , Miyoshi M , Oniki T , Fukui A , Shinohara T , Shimada T , Yufu K , Nakagawa M , Takahashi N 
Published: October 2019

Abstract

Background
Using excised human left atrial appendage samples, we previously demonstrated that epicardial adipose tissue (EAT) are highly associated with atrial myocardial fibrosis as a substrate of atrial fibrillation (AF). We also reported the relationship between Angptl2 in EAT and atrial fibrosis. However, the mechanism is not clear. The purpose is to clarify the mechanisms underlying the effect of EAT on the atrial myocardium.

Methods
Human peri-left atrial EAT and abdominal subcutaneous adipose tissue (SAT) samples were obtained from 6 cases (2 females, 70.2±13.2 years). 50 mg of EAT and SAT were quickly washed with PBS and centrifuged 1min at 1200rpm. After 3 times this procedures, adipose tissues were cultured in DMEM F12 medium with Fetal bovine Serum (FBS) overnight.
After pre-incubation, EAT and SAT tissues were washed and centrifuge d three times and cultured in medium without FBS for 24hours. Finally, we collected oozed medium (conditioned medium) and used for experiments.
Concentrations of Angptl2 in conditioned medium were measured by ELISA.
To study the effects of conditioned medium, we used “organo-culture” system. Isolated atrium from 8week old male Sprague-Dawley rats were placed on the porous membrane with the endothelial face toward the membrane. After that, loading medium (conditioned medium:culture medium = 1:4), culture medium (control), or recombinant Angptl2 were dropped onto the epicardial face of the atrium once a day and incubated for 7 days (37°C, 5% CO2).
Then, histological and immunohistochemical analysis were performed. We also performed quantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis.
Next, we isolated and cultured neonatal rat fibroblast and loaded Angptl2 for 24 hours.After collected these cells, we performed western blotting analysis.

Results
Atria organo-culture incubated for 7 days with conditioned medium showed global fibrosis. At epicardial side, fibrotic area of EAT group was significantly greater compared to that of SAT and control group (P<0.05).
mRNA of Col1a1, col3a1 and TGFβ1 were significantly increased in EAT group compared with the SAT and control group.
And, the concentration of conditioned medium created from EAT was significant higher than that from SAT (P<0.05).
Then, we dropped 500 ng/ml of recombinant Angptl2 onto the rat atria. Fibrotic area of Angptl22 group significantly greater than that of control with increasing number of α-SMA positive cells, and mRNA of col3a1 and TGFβ1 were significantly increased in Angptl2 group compared with control group.
In cultured fibroblasts, α-SMA and p-ERK expression were increased in Angptl2 group measured by western blotting analysis.

Conclusions
Our results demonstrated that EAT rather than SAT induces atrial myocardial fibrosis. There is a possibility that Angptl2 effused from EAT plays a part in atrial fibrosis thought EAT paracrine effect.

Acknowledgement/Funding
ONO PHARMACEUTICAL CO