Introduction

Vascular inflammation is a crucial contributor to the progression of atherosclerosis, in which oxidative stress, endoplasmic reticulum (ER) stress and transition of endothelial to mesenchymal cells (EndMT) are of critical importance. 

Vitamin-K-antagonists (VKA) promote vascular dysfunction, while supplementation of vitamin K was proposed to reduce incidence of coronary artery disease. Key enzyme for regeneration of vitamin K is the Vitamin K epoxide reductase complex subunit 1 (VKORC1), which also represents the pharmacological target of VKA. VKOR-like1 (VKORC1L1) is an isoenzyme of VKORC1, which resides at the ER-membrane, exerts antioxidative properties and is involved in vitamin K maintenance. 

Aim of this study was to investigate in vitro the role of VKORC1 and VKORC1L1 in inflammation of coronary endothelial cells.

Methods and Results

In silico analyses of the proteome of human coronary atherosclerosis revealed increased expression of VKORC1L1, but not of its isoenzyme VKORC1. In vitro induction of oxidative stress (H2O2) and ER-Stress (tunicamycin) in human coronary artery endothelial cells (HCAEC) promoted time- and dose-dependent enhanced expression of VKORC1L1, without altering VKORC1 expression (H2O2: 2.3-fold vs. 0.8-fold after 40 minutes, p=0.04; tunicamycin: 1 μg/ml: 1.43-fold vs. 0.8-fold, p<0.01). Moreover, inducing EndMT by treatment with a differentiation medium (containing TGF-ß2, IL-1ß, hydrocortisone and ascorbic acid) resulted in a reduction of VKORC1L1, but not of VKORC1 (Fig.1).

To analyze the relevance of the VKOR-enzymes on endothelial cell function, HCAEC were transfected with siRNA against VKORC1 or VKORC1L1, resulting in downregulation of the enzymes.

VKORC1L1 downregulation increased formation of reactive oxygen species (ROS) (DCFDA signal: 0.98-fold vs. 1.31-fold, p = 0.03) and reduced proliferation (EdU signal: 0.56 ± 0.02 vs. control, p < 0.01), whereas viability of HCAEC remained unchanged. ELISA and qPCR experiments revealed enhanced expression of a variety of markers of vascular inflammation after VKORC1L1 (e.g., IL-6: 3.42-fold ± 0.53, NF-κB: 1.95-fold ± 0.13, VCAM-1: 1.59-fold ± 0.12, vs. control). Additionally, expression of ER-stress markers was increased (e.g., GRP78; 1.32-fold ± 0.04 vs. control, p<0.01) (Fig.1). 

In contrast, VKORC1 downregulation significantly promoted proliferation (EdU signal: 3.9 ± 1.7 vs. control, p<0.01) as well as viability (relative alamarBlue® absorbance: 1.07 ± 0.08 vs. control, p=0.04), and decreased formation of ROS (0.88 ± 0.17 vs. control, p=0.03). Expression of pro-inflammatory and ER-stress proteins were either unaltered or reduced after VKORC1 knockdown (e.g., GRP78: 0.89-fold, VCAM-1: 0.68-fold) (Fig.2).

Interestingly, VKORC1 downregulation promotes an increase in VKORC1L1 expression (1.66-fold, Fig.2), suggesting that protective effects of VKORC1 downregulation on endothelial cells may be mediated by an upregulation of VKORC1L1. To further elucidate these findings, we will perform overexpression experiments and will downregulate VKORC1 in cells with stable VKORC1L1-knockdown. 

Conclusion

VKORC1 and its isoenzyme VKORC1L1 exhibit divergent effects on endothelial cell inflammation. Further studies are warranted to shed light on the regulation of the enzymes. These results will improve our understanding regarding vascular side effects of VKA treatment and beneficial effects of vitamin K supplementation.