Introduction: Vitamin K antagonism is known to promote vascular calcification and oxidative stress whereas Vitamin K supplementation is discussed to be protective. Vitamin K epoxide reductase complex subunit 1 like 1 (VKORC1L1) is an isoenzyme of VKORC1-Protein family with anti-oxidative properties, located at the endoplasmic reticulum (ER) membrane. ER stress is known to be a critical contributor to cardiovascular disease. 

Purpose: Aim of this study is to investigate the role of VKORC1L1 and Vitamin K₂ (Menaquinone-7; MK7) supplementation on vascular inflammation and ER stress during atherogenesis.

Methods: In vitro, human coronary artery endothelial (HCAEC) and smooth muscle cells (HCASMC) were transfected with siRNA against VKORC1L1 or incubated with MK7. Cell viability, cell proliferation, reactive oxygen species (ROS) formation, and cell migration were analysed. Gene Expression was analysed by qPCR and PCR Profiler arrays. ELISA was utilized to determine protein expression.

In vivo, carotid wire injury was performed in 6-8 weeks old C57/Bl6 mice followed by randomization into three food regimes. They received either vehicle, vitamin K1 (1.5 mg/g food) or Warfarin (2mg/g food) + vitamin K1 (1.5 mg/g food). Fourteen days after the procedure, vessels were subjected to histological analysis, gene expression and haemostatic analysis.

Results: 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 down-regulation resulted in enhanced viability of HCASMC, but impaired viability of HCAEC. Intracellular ROS formation was increased in HCASMC, but not in HCAEC. Intriguingly, down-regulation of VKORC1L1 led to increased migration in HCASMC (60% vs. 49% re-migration, p=0.02), while migration in HCAEC was not altered (63% vs. 64%, p=0.94). qPCR and ELISA experiments revealed enhanced expression of markers of vascular inflammation after VKORC1L1-Knockdown (IL6, NF-κB) in both HCASMC and HCAEC. PCR Profiler arrays revealed broad dysregulation of pro-atherosclerotic and ER Stress markers in aortic tissue of VKORC1L1^-/--Mice. qPCR confirmed higher levels of GRP78 and CHOP after VKORC1L1-Knockdown. Treatment with ER Stress inductors Tunicamycin and Thapsigargin promoted dose-dependent upregulation of VKORC1L1, but not of VKORC1 (1.3 fold vs. 0.7 fold, p=0.03).

Next, the effect of the most potent Vitamin K (MK7) was investigated in vitro. Treatment of HCASMC with MK7 showed dose-dependent significantly reduced level of inflammatory and ER Stress markers and lower rate of both PDGF-induced adverse migration and proliferation.

In vivo, treatment with Warfarin enhanced neointima formation significantly, while sole treatment with Vitamin K tended to reduce neointima formation. In Warfarin treated mice, these effects were accompanied by a lower expression of VKORC1L1 mRNA in aortic tissue. Coagulation activity was not altered.

Conclusion: Vitamin K Antagonism promotes adverse vascular remodelling and reduces vascular VKORC1L1 expression in vivo. Direct inhibition of VKORC1L1 promotes ER stress, inflammation, oxidative stress and remodelling in a cell-type specific manner, while Vitamin K alleviates ER stress, inflammation, oxidative stress and remodelling.