Introduction

Vascular inflammation is a crucial contributor to the progression of atherosclerosis, in which oxidative stress, ER stress and transition of endothelial to mesenchymal cells (EndMT) are of critical importance. Vitamin K supplementation is discussed to possess protective properties in cardiovascular diseases. The recently described VKOR-isoenzyme Vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1), located at the endoplasmic reticulum (ER) membrane, is involved in Vitamin K maintenance and has antioxidative functions in the vasculature.

Purpose

The aim of this study was to investigate the role of VKORC1L1 and Vitamin K2 (Menaquinone-7; MK7) in oxidative stress, ER stress and EndMT during vascular inflammation and calcification.

Methods and Results

In vitro, human coronary artery endothelial (HCAEC) were transfected with siRNA against VKORC1L1 or incubated with MK7. 

Treatment with H2O2 promoted time-dependent enhanced expression of VKORC1L1, but not of VKORC1 (2.3-fold vs. 0.8-fold after 40 minutes, p=0.04). VKORC1L1- downregulation by siRNA resulted in an increased intracellular formation of reactive oxygen species (ROS) (125% ± 12% vs. scrambled siRNA, p = 0.095) and a significantly reduced proliferation rate (EdU fluorescence signal: 54661 ± 3299 vs. 31092 ± 1473, p =0.002, Fig.1C), whereas viability and migration of HCAEC remained unchanged. qPCR and ELISA experiments revealed enhanced expression of markers of vascular inflammation after VKORC1L1-Knockdown (IL-6; 3.42-fold ± 0.53 vs. control, p = 0.045, NF-κB; 1.95-fold ± 0.13 vs. control, p = 0.001, and VCAM-1; 1.59-fold ± 0.12 vs. control, p = 0.01, Fig.1A,D). VKORC1L1 knockdown further increased expression of the main ER Stress moderator, glucose-regulated protein 78 kDa (GRP78; 1.32-fold ± 0.04 vs. control, p=0.006, Fig.1A). Moreover, treatment with the ER Stress inductor Tunicamycin promoted a dose-dependent increase in VKORC1L1, but not VKORC1 expression (e.g., 1 μg/ml: 1.43-fold vs. 0.8-fold, p=0.009, Fig.1B). Induction of EndMT by a differentiation medium (containing TGF-ß2, IL-1ß, FBS, Hydrocortisone and Ascorbic acid) resulted in a reduction of VKORC1L1, but not VKORC1 expression.

Next, the effect of MK7 on endothelial cell biology was investigated. MK7 treatment led to a reduction of ROS formation (0.54-fold ± 0.04 vs. control, p = 0.011, Fig.2A). After co-incubation with the atherogenic stimuli oxLDL and Tunicamycin, dose-dependent protective effects of MK7 on inflammation (IL-6; MK7 10 μM + oxLDL 0.57-fold ± 0.16 vs. only oxLDL 1.28-fold ± 0.10, p=0.0131, VCAM-1; MK7 10μM + oxLDL 0.25-fold ± 0.13 vs. only oxLDL 1.42-fold  ± 0.19, p=0.0089, Fig.2B) and ER stress (GRP78; MK7 10μM + Tunicamycin 4.37 fold ± 1.33 vs. only Tunicamycin 9.92-fold ± 2.31, p=0.0359, Fig.2C) could be demonstrated, whereas the viability was not altered. Intriguingly, MK7 supplementation was able to inhibit in vitro induction of EndMT by maintaining expression of endothelial markers and suppressing expression of mesenchymal markers (eNOS3; 2.32-fold vs. 0.01-fold positive control, and SM22-alpha; 0.28-fold vs. 12.03-fold positive control, Fig.2D,E).

Conclusion

VKORC1L1 and MK 7 play a pivotal role in vascular inflammation, ER Stress and EndMT. Further in vivo studies are warranted to investigate their effects on atherogenesisand calcification.