Thrombin and factor Xa promote premature senescence and remodeling in areas at risk of the left appendage and in atrial endothelial cells: potential modulation by SGLT1 and/or SGLT2 inhibition
Authors: Fakih W , Hasan H , Gong D , Ohlmann P , Morel O , Schini Kerth V , Jesel L ,   INSERM, UMR 1260
Published: October 2021

Abstract

Introduction
Atrial fibrillation (AF), the most common cardiac arrhythmia, promotes prothrombotic responses particularly in the distal part of the left atrial appendage (LAA) characterized by reduced flow and low shear stress. AF occurrence and duration are closely associated with endothelial dysfunction and left atrium remodeling.
The contribution of coagulation factors thrombin and factor Xa (FXa) to LA remodeling remains poorly studied. This study investigated whether thrombin and FXa promote endothelial dysfunction and profibrotic and prothrombotic responses in the distal and proximal parts of the LAA. Since SGLT2 inhibitors have shown pronounced cardiac protection, the contribution of SGLT2 was examined.

Methods
LAAs were harvested from porcine hearts. Proximal (low stasis, high shear) and distal (high stasis, low shear) parts were cut and incubated with a coagulation factor (thrombin or FXa). Left atrial endothelial cells (AECs) were collected enzymatically, cultured and used at passage 1. The level of oxidative stress was assessed using the redox-sensitive probe dihydroethidium (DHE), fibrosis by Sirius red histological staining, senescence-associated β-galactosidase (SA-β-gal) activity using X-gal staining in tissues and the fluorogenic substrate C12FDG and flow cytometry in cells. The expression of target proteins was detected by immunofluorescence staining and Western blot analysis.

Results
The distal part of LAA displayed higher levels of oxidative stress in the endothelium and the atrial wall than the proximal part. SA-β-gal activity, ICAM-1, VCAM-1, and MMP-9 staining was more pronounced in the endothelium of the distal part compared to the proximal part whereas eNOS staining was decreased. The distal LAA wall showed higher levels of fibrosis. Western blot analysis showed higher expression levels of tissue factor (TF), VCAM-1, MCP-1, MMP-9, TGF-β and SGLT-2 in the distal part compared to the proximal one. Both thrombin and FXa increased these signals to a greater extent in the distal part of the LAA whereas the eNOS expression level was decreased. Exposure of AECs to either thrombin or FXa induced a pro-oxidant response and increased the expression level of ICAM-1, TGF-β, MMP-2, MMP-9, SGLT1 and SGLT-2. Thrombin promoted SA-β-gal activity in AECs. The selective SGLT-2 inhibitor empagliflozin and the dual SGLT1/2 inhibitor sotagliflozin prevented the pro-oxidant response and SA-β-gal activity in response to thrombin.

Conclusion
Thus, the distal part of the LAA is prematurely affected by endothelial dysfunction and senescence associated with a pro-oxidant, pro-remodeling and pro-inflammatory response that is further increased by thrombin and FXa. Hence, the coagulation cascade appears to prematurely promote atrial endothelium and wall dysfunction predominantly in areas at risk that may pave the way to thrombus formation. The findings also suggest a potential protective effect of SGLT1 and/or SGLT2 inhibition on atrial dysfunction.

Funding Acknowledgement
Type of funding sources: Private company. Main funding source(s): Unrestricted research grant from Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany