Vitamin K Epoxide Reductase Complex Subunit 1-Like 1 (VKORC1L1) Inhibition Induces a Proliferative and Pro-inflammatory Vascular Smooth Muscle Cell Phenotype

Abstract

Background: Vitamin K antagonists (VKA) are known to promote adverse cardiovascular remodeling. Contrarily, vitamin K supplementation has been discussed to decelerate cardiovascular disease. The recently described VKOR-isoenzyme Vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1) is involved in vitamin K maintenance and exerts antioxidant properties. In this study, we sought to investigate the role of VKORC1L1 in neointima formation and on vascular smooth muscle cell (VSMC) function. Methods and Results: Treatment of wild-type mice with Warfarin, a well-known VKA, increased maladaptive neointima formation after carotid artery injury. This was accompanied by reduced vascular mRNA expression of VKORC1L1. In vitro, Warfarin was found to reduce VKORC1L1 mRNA expression in VSMC. VKORC1L1-downregulation by siRNA promoted viability, migration and formation of reactive oxygen species. VKORC1L1 knockdown further increased expression of key markers of vascular inflammation (NFκB, IL-6). Additionally, downregulation of the endoplasmic reticulum (ER) membrane resident VKORC1L1 increased expression of the main ER Stress moderator, glucose-regulated protein 78 kDa (GRP78). Moreover, treatment with the ER Stress inducer tunicamycin promoted VKORC1L1, but not VKORC1 expression. Finally, we sought to investigate, if treatment with vitamin K can exert protective properties on VSMC. Thus, we examined effects of menaquinone-7 (MK7) on VSMC phenotype switch. MK7 treatment dose-dependently alleviated PDGF-induced proliferation and migration. In addition, we detected a reduction in expression of inflammatory and ER Stress markers. Conclusion: VKA treatment promotes neointima formation after carotid wire injury. In addition, VKA treatment reduces aortal VKORC1L1 mRNA expression. VKORC1L1 inhibition contributes to an adverse VSMC phenotype, while MK7 restores VSMC function. Thus, MK7 supplementation might be a feasible therapeutic option to modulate vitamin K- and VKORC1L1-mediated vasculoprotection.

Keywords: ER stress; oxidative stress; vascular inflammation; vascular remodeling; vitamin K.

Figure 1

Vitamin K antagonism promotes vascular remodeling. (A) Schematic diagram of the design of the carotid artery injury experiment. After the procedure on 6–8 week-old C57Bl/6J wild-type mice, they were randomized in three groups that received either vehicle (group A), vitamin K1 (1.5 mg/g food)-enriched diet (group B), or vitamin K1 (1.5 mg/g food) and warfarin (2 mg/g food)-enriched diet (group C), n = 6 per group. (B) Images of neointima formation 14 days after carotid artery injury. Vessels were subjected to histological analysis by H.E. staining. (C) Quantitative assessment of neointima formation expressed as the media/intima ratio. (D) VKORC1L1 mRNA expression in the abdominal aorta expressed as 2^−ddCT relative to control. (E) Factor X activity measured in citrate plasma samples after 14 days of treatment. Data are presented as the mean ± SEM; *p < 0.05; one-way ANOVA + Bonferroni's multiple comparison test for (C-E).

Figure 2

VKORC1L1-downregulation enhances HCASMC viability and migration. (A) Viability of HCASMC measured by alamarblue® assay after treatment with 1 μmol warfarin for 24 h. (B) VKORC1L1 protein expression in untreated HCASMC visualized by western blot. ß-Actin was used as housekeeping protein. (C) mRNA expression of VKORC1L1 in HCASMC after treatment with 1 μmol warfarin for 24 or 48 h. mRNA expression was measured by qPCR and quantified by the 2^−ddCT method. n = 5. (D) Viability of HCASMC measured by alamarblue® after transfection of siRNAs directed against VKORC1L1 or a scrambled control. (E) Migration of siRNA-transfected HCASMC following a wound–scratch assay, measured by microscopic visualization after 8 and 12 hours. n = 7. (F) Representative pictures of migration. *p < 0.05. Data are presented as the mean ± SEM; *p < 0.05; one-way ANOVA + Bonferroni's multiple comparison test for (A, C, D).

Figure 3

VKORC1L1-inhibition promotes reactive oxygen species formation. (A, B) ROS formation in HCASMC after transfection of siRNAs directed against VKORC1L1 or a scrambled control. Measured by using L-012 (A) and DCFDA assays (B). Angiotensin II (Ang II) was used as a positive control. n = 6–8. *p < 0.05 (C) H₂O₂ Generation in HCASMC was measured by an Amplex^TM Red assay after transfection with siRNA against VKORC1L1 or a scrambled control. Treatment with H₂O₂ was used as a positive control. n = 6. *p < 0.05. Data are presented as the mean ± SEM; *p < 0.05; one-way ANOVA + Bonferroni's multiple comparison test for (A-C).

Figure 4

VKORC1L1 regulates vascular inflammation. (A) mRNA expression of the pro-inflammatory markers NF-κB and IL-6 in HCASMC after transfection with siRNAs directed against VKORC1L1 or a scrambled control. Control mRNA expression (=1) is shown as a dotted line. mRNA expression was measured by qPCR and quantified by the 2^−ddCT method. n = 3–8. (B) mRNA expression of ER stress markers GRP78 and CHOP in HCASMC after transfection with siRNAs directed against VKORC1L1 or a scrambled control. Control expression (=1) is shown as a dotted line. mRNA expression was measured by qPCR and quantified by the 2^−ddCT method. n = 4–8. (C) mRNA expression of VKORC1L1 and VKORC1 in HCASMC after treatment with the ER stress inductor, tunicamycin (6 h, 5 μg/ml). Control expression (DMSO treatment, =1) is shown as a dotted line. n = 4. Data are presented as the mean ± SEM; *p < 0.05. Unpaired t-test for (A-C).

Figure 5

Menaquinone-7 (MK7) alleviates HCASMC remodeling, inflammation and ER stress. (A) Proliferation of HCASMC untreated, treated with MK7 (1 μM, 24 h), PDGF (30 ng/ml, 24 h) or PDGF + MK7 (1 μM or 10 μM, 24 h). Measured by an EdU fluorescence assay. n = 5. (B) Migration of HCASMC after a wound–scratch assay, measured by microscopic visualization after 12 h. Cells treated with MK7 (1 μM, 24 h), PDGF (30 ng/ml, 24 h) or PDGF + MK7 (1 μM or 10 μM, 24 h). n = 4. (C) Representative pictures of cell migration 0 and 8 h after scratch assay. (D, E) mRNA expression of the pro-inflammatory markers NF-κB and IL-6 in HCASMC. Cells treated with MK7 (1 μM, 24 h), oxLDL (25 ng/ml, 24 h) or oxLDL + MK7 (1 μM or 10 μM, 24 h). RNA expression was measured by using qPCR and quantified by the 2^−ddCT method. n = 3–4. (F) mRNA expression of the ER stress marker GRP78 in HCASMC. Cells were treated with MK7 (1 μM, 24 h), tunicamycin (TNM, 5 μg/ml, 6 h) or TNM + MK7 (1 μM or 10 μM, 24 h). RNA expression was measured by using qPCR and quantified by the 2^−ddCT method. n = 3. Data are presented as the mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA + Bonferroni's multiple comparison test for (A-E).