Epoxide Reductase Complex Subunit Research Articles

Is a Vitamin K Epoxide Reductase Complex Subunit 1 (VKORC1) Polymorphism a Risk Factor for Nephrolithiasis in Sarcoidosis?

Sarcoidosis is a systemic inflammatory disorder characterized by granuloma formation in various organs. It has been associated with nephrolithiasis. The vitamin K epoxide reductase complex subunit 1 (VKORC1) gene, which plays a crucial role in vitamin K metabolism, has been implicated in the activation of proteins associated with calcification, including in the forming of nephrolithiasis. This study aimed to investigate the VKORC1 C1173T polymorphism (rs9934438) in a Dutch sarcoidosis cohort, comparing individuals with and without a history of nephrolithiasis. Retrospectively, 424 patients with sarcoidosis were divided into three groups: those with a history of nephrolithiasis (Group I: n = 23), those with hypercalcemia without nephrolithiasis (Group II: n = 38), and those without nephrolithiasis or hypercalcemia (Group III: n = 363). Of the 424 sarcoidosis patients studied, 5.4% had a history of nephrolithiasis (Group I), only two of whom possessed no VKORC1 polymorphisms (OR = 7.73; 95% CI 1.79-33.4; p = 0.001). The presence of a VKORC1 C1173T variant allele was found to be a substantial risk factor for the development of nephrolithiasis in sarcoidosis patients. This study provides novel insights into the genetic basis of nephrolithiasis in sarcoidosis patients, identifying VKORC1 C1173T as a potential contributor. Further research is warranted to elucidate the precise mechanisms and explore potential therapeutic interventions based on these genetic findings.

A physiologically-based pharmacokinetic/pharmacodynamic modeling approach for drug-drug-gene interaction evaluation of S-warfarin with fluconazole.

Warfarin is a widely used anticoagulant, and its S-enantiomer has higher potency compared to the R-enantiomer. S-warfarin is mainly metabolized by cytochrome P450 (CYP) 2C9, and its pharmacological target is vitamin K epoxide reductase complex subunit 1 (VKORC1). Both CYP2C9 and VKORC1 have genetic polymorphisms, leading to large variations in the pharmacokinetics (PKs) and pharmacodynamics (PDs) of warfarin in the population. This makes dosage management of warfarin difficult, especially in the case of drug-drug interactions (DDIs). This study provides a whole-body physiologically-based pharmacokinetic/PD (PBPK/PD) model of S-warfarin for predicting the effects of drug-drug-gene interactions on S-warfarin PKs and PDs. The PBPK/PD model of S-warfarin was developed in PK-Sim and MoBi. Drug-dependent parameters were obtained from the literature or optimized. Of the 34 S-warfarin plasma concentration-time profiles used, 96% predicted plasma concentrations within twofold range compared to observed data. For S-warfarin plasma concentration-time profiles with CYP2C9 genotype, 364 of 386 predicted plasma concentration values (~94%) fell within the twofold of the observed values. This model was tested in DDI predictions with fluconazole as CYP2C9 perpetrators, with all predicted DDI area under the plasma concentration-time curve to the last measurable timepoint (AUClast ) ratio within twofold of the observed values. The anticoagulant effect of S-warfarin was described using an indirect response model, with all predicted international normalized ratio (INR) within twofold of the observed values. This model also incorporates a dose-adjustment method that can be used for dose adjustment and predict INR when warfarin is used in combination with CYP2C9 perpetrators.

Kinetic-pharmacodynamic model of warfarin for prothrombin time-international normalized ratio in Japanese patients.

Genotype-guided dosing algorithms can explain about half of the interindividual variability in prothrombin time-international normalized ratio (PT-INR) under warfarin treatment. This study aimed to refine a published kinetic-pharmacodynamic model and guide warfarin dosage for an optimal PT-INR based on renal function. Using a retrospective cohort of adult patients (>20 years) who were administered warfarin and underwent PT-INR measurements, we refined the kinetic-pharmacodynamic model with age and the genotypes of cytochrome P450 2C9 and vitamin K epoxide reductase complex subunit 1 using the PRIOR subroutine in the nonlinear-mixed-effect modelling programme. We searched the significant covariates for parameters, such as the dose rate for 50% inhibition of coagulation (EDR50 ), using a stepwise forward and backward method. Monte Carlo simulation determined a required daily dose of warfarin with a target range of PT-INR (2.0-3.0 or 1.6-2.6) based on the significant covariates. A total of 350 patients with 2762 PT-INR measurements were enrolled (estimated glomerular filtration rate [eGFR]: 47.5 [range: 2.6-199.0] mL/min/1.73 m2 ). The final kinetic-pharmacodynamic model showed that the EDR50 changed power functionally with body surface area, serum albumin level and eGFR. Monte Carlo simulation revealed that a lower daily dose of warfarin was required to attain the target PT-INR range as eGFR decreased. Model-informed precision dosing of warfarin is a valuable approach for estimating its dosage in patients with renal impairment.

Frequencies of VKORC1-1639G>A and rs397509427 in Patients on Warfarin and Healthy Syrian Subjects.

Vitamin K epoxide reductase complex subunit 1 (VKORC1) gene encodes a key enzyme with multiple cellular activities, namely, the reduction of vitamin K to its active form. VKORC1-1639G>A (rs9923231) is a common single nucleotide polymorphism with a crucial impact on warfarin dosing and possibly other physiological functions. This study aimed at investigating the frequencies of VKORC1-1639G>A alleles and genotypes in Syrian healthy subjects and patients on warfarin for different indications. A total of 138 individuals were enrolled in this cross-sectional study. Genomic DNA was extracted from both patients on warfarin and healthy subjects, and polymerase chain reaction (PCR) specific amplicons were genotyped via standard sequencing which also allowed the detection of rs397509427. Comparisons of -1639G>A frequency with other populations were drawn. Of 94 patients on warfarin, 53 (56.38%) were with idiopathic venous thromboembolism (VTE). Despite comparable frequencies of the -1639A allele (47% and 50%), the AA and GA genotypes were at disparate frequencies of 93.2% versus 79.8% in the healthy subjects (n = 44) versus patients on warfarin, respectively. Carriers of the GG genotype were at a four-fold increased risk of VTE in comparison with those of the AA and GA genotypes (odds ratio (OR) = 4, 95% CI = 1.105 - 13.6, P = 0.0469). All study subjects were wild-type for the rs397509427 variant. Our results prove a high -1639A prevalence in Syrian healthy subjects and patients on warfarin at frequencies comparable to other Mediterranean and Middle Eastern populations. The A allele carriers are at a lower VTE risk, whereas a global prevalence gradient of the G allele is suggested to be associated with VTE risk.

The vitamin-k-cycle-enzyme VKORC1L1, but not its isoenzyme VKORC1, reduces inflammation in coronary endothelial cells

Abstract Introduction Vascular inflammation is a crucial contributor to 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 vitamin K intake 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 the role of VKORC1 and VKORC1L1 in inflammation of human coronary artery endothelial cells (HCAEC). Methods and Results In silico analyses of the proteome of human coronary atherosclerosis revealed increased expression of VKORC1L1, but not of VKORC1. In vitro induction of oxidative stress (H2O2) and ER-Stress (tunicamycin) in 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) resulted in a reduction of VKORC1L1, but not of VKORC1 (Fig.1). To analyze the relevance of the VKOR-enzymes on endothelial cell function, VKORC1 and VKORC1L1 were downregulated by siRNA. 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 (alamarBlue® absorbance: 1.07 ± 0.08 vs. control, p=0.04), and decreased ROS (0.88 ± 0.17 vs. control, p=0.03). Pro-inflammatory and ER-stress proteins were either unaltered or decreased after VKORC1 knockdown (e.g., GRP78: 0.89-fold, VCAM-1: 0.68-fold) (Fig.2). To further elucidate these findings, we will perform overexpression experiments and stable knockdown of the VKOR-enzymes. 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 to improve our understanding regarding vascular side effects of VKA treatment and beneficial effects of vitamin K supplementation.

Effect of asciminib and vitamin K2 on Abelson tyrosine-kinase-inhibitor-resistant chronic myelogenous leukemia cells

BackgroundAbelson (ABL) tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML); however, many patients develop resistance during ABL TKI therapy. Vitamin K2 (VK2) is a crucial fat-soluble vitamin used to activate hepatic coagulation factors and treat osteoporosis. Although VK2 has demonstrated impressive anticancer activity in various cancer cell lines, it is not known whether VK2 enhances the effects of asciminib, which specifically targets the ABL myristoyl pocket (STAMP) inhibitor.MethodIn this work, we investigated whether VK2 contributed to the development of CML cell lines. We also investigated the efficacy of asciminib and VK2 by using K562, ponatinib-resistant K562 (K562 PR), Ba/F3 BCR–ABL, and T315I point mutant Ba/F3 (Ba/F3 T315I) cells.ResultsBased on data from the Gene Expression Omnibus (GEO) database, gamma-glutamyl carboxylase (GGCX) and vitamin K epoxide reductase complex subunit 1 (VKORC1) were elevated in imatinib-resistant patients (GSE130404). UBIA Prenyltransferase Domain Containing 1 (UBIAD1) was decreased, and K562 PR cells were resistant to ponatinib. In contrast, asciminib inhibited CML cells and ponatinib resistance in a dose-dependent manner. CML cells were suppressed by VK2. Caspase 3/7 activity was also elevated, as was cellular cytotoxicity. Asciminib plus VK2 therapy induced a significantly higher level of cytotoxicity than use of each drug alone. Asciminib and VK2 therapy altered the mitochondrial membrane potential.ConclusionsAsciminib and VK2 are suggested as a novel treatment for ABL-TKI-resistant cells since they increase treatment efficacy. Additionally, this treatment option has intriguing clinical relevance for patients who are resistant to ABL TKIs.

Regulation of VKORC1L1 is critical for p53-mediated tumor suppression through vitamin K metabolism

Here, we identified vitamin K epoxide reductase complex subunit 1 like 1 (VKORC1L1) as a potent ferroptosis repressor. VKORC1L1 protects cells from ferroptosis by generating the reduced form of vitamin K, a potent radical-trapping antioxidant, to counteract phospholipid peroxides independent of the canonical GSH/GPX4 mechanism. Notably, we found that VKORC1L1 is also a direct transcriptional target of p53. Activation of p53 induces downregulation of VKORC1L1 expression, thus sensitizing cells to ferroptosis for tumor suppression. Interestingly, a small molecular inhibitor of VKORC1L1, warfarin, is widely prescribed as an FDA-approved anticoagulant drug. Moreover, warfarin represses tumor growth by promoting ferroptosis in both immunodeficient and immunocompetent mouse models. Thus, by downregulating VKORC1L1, p53 executes the tumor suppression function by activating an important ferroptosis pathway involved in vitamin K metabolism. Our study also reveals that warfarin is a potential repurposing drug in cancer therapy, particularly for tumors with high levels of VKORC1L1 expression.

The Association Between Bone Remodeling Markers, genes polymorphisms with Incidence of Osteopenia Among Young Saudi Female.

Osteopenia and osteoporosis, are prevalent skeletal systemic conditions, cause weaker bones and an increased risk of fragility fractures. This work is aimed to evaluate the relation between bone-remolding markers and genotypes of four single nucleotide polymorphisms in young Saudi females (rs2297480 of farnesyl diphosphate synthase (FDPS), rs3736228 of Low-density lipoprotein receptor-related protein 5(LRP5), rs1234612 of sclerostin (SOST), and rs9934438 of Vitamin K epoxide reductase complex subunit 1 (VKORC1) ). For this purpose, 750 premenopausal females aged 18 to 40 years old, either university students, postgraduates, or university employees were recruited and divided into three groups according to bone mineral density BMD (g/cm2) divided by T score into osteoporosis (n = 12), osteopenia (n = 147), and normal (n = 591). Serum SOST, BALP, calcium, phosphate, ALP, albumin, beta-CTXs and human VDR levels were determined. TaqMan SNP Genotyping assays were used to genotype four polymorphisms using real-time PCR (applied biosystem). Results showed that BALP, CTX-1 and SOST were significantly higher in the osteoporosis and osteopenia groups than in the normal group. Bone mineral density readings were considerably lower in females with the GG genotype in FDPS rs2297480 and TT genotype in LRP5 rs3736228, which increase the risk for osteopenia by 3. 6-fold and 3. 06-fold than control respectively. Also, females with the TT genotype in LRP5 rs3736228 have decreased average values for Bone Mineral Density. In conclusion, the GG genotype of FDPS rs2297480 and the TT genotype of LRP5 rs3736228 was shown to be strongly associated with osteopenia in young Saudi females with low bone mineral density and SOST levels.

Vkorc1 gene polymorphisms confer resistance to anticoagulant rodenticide in Turkish rats.

Mutations in Exon 1, 2 and 3 of the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene are known to lead to anticoagulant rodenticide resistance. In order to investigate their putative resistance in rodenticides, we studied the genetic profile of the Vkorc1 gene in Turkish black rats (Rattus rattus) and brown rats (Rattus norvegicus). In this context, previously recorded Ala21Thr mutation (R. rattus) in Exon 1 region, Ile90Leu mutation (R. rattus, R. norvegicus) in Exon 2 region and Leu120Gln mutation (R. norvegicus) in Exon 3 region were identified as "missense mutations" causing amino acid changes. Ala21Thr mutation was first detected in one specimen of Turkish black rat despite the uncertainty of its relevance to resistance. Ile90Leu mutation accepted as neutral variant was detected in most of black rat specimens. Leu120Gln mutation related to anticoagulant rodenticide resistance was found in only one brown rat specimen. Furthermore, Ser74Asn, Gln77Pro (black rat) and Ser79Pro (brown rat) mutations that cause amino acid changes in the Exon 2 region but unclear whether they cause resistance were identified. In addition, "silent mutations" which do not cause amino acid changes were also defined; these mutations were Arg12Arg mutation in Exon 1 region, His68His, Ser81Ser, Ile82Ile and Leu94Leu mutations in Exon 2 region and Ile107Ile, Thr137Thr, Ala143Ala and Gln152Gln mutations in Exon 3 region. These silent mutations were found in both species except for Ser81Ser which was determined in only brown rats.

Prediction of a Stable Complex of Compounds in the Ethanol Extract of Celery Leaves (Apium graveolens L.) Function as a VKORC1 Antagonist

The vitamin K cycle, specifically the protein VKORC1 (Vitamin K epoxide reductase complex subunit 1), is closely linked to coagulation mechanisms in the body. Disruptions in this cycle will be associated with vascular diseases such as myocardial infarction and stroke. Warfarin is commonly used as an anticoagulant, but it still has side effects in interactions with numerous drug compounds; thus, it is necessary to search for safer candidate compounds. The ethanol extract of celery leaves (Apium graveolens L.) has potential anticoagulant activity, but the compound responsible for this activity has not been identified yet. The CADD method has been developed to predict in silico a compound's potential to interact with binding sites. This study aimed to predict interactions and obtain a stable complex of the compounds in the ethanol extract of celery leaves, which function as a VKORC1 antagonist. A total of 23 compounds were simulated using Autodock 4.2, and AMBER 18 was then used to simulate the stability of the five compounds with the best interactions. The docking simulation results of 17 test compounds (ligands) yielded five selected compounds, namely 6-isopentenyloxy-isobergapten (S1), Heratomin (S2), Apigenin (S3), Lanatin (S4), and Isoimperatorin (S5), with G values of −9.27, −9.26, −9.22, −9.13, and −8.94 kcal/mol, respectively. The MD simulation continued to produce 6-isopentenyloxy-isobergapten as the most effective ligand for stabilizing the complex for 100 ns from these five ligands. In conclusion, the 6-isopentenyloxy-isobergapten from celery leaf has the potential as a candidate anticoagulant, VKORC1.

The prevalence of pharmacogenetic variants of vitamin K epoxide reductase complex subunit 1 gene (rs9923231), cytochrome P450 family 2 subfamily C member 9 gene (rs1799853) and cytochrome P450 family 3 subfamily-A member-5 gene (rs776746) among 13 ethnic groups of Pakistan.

Pharmacogenomics (PGx) plays a central role in the selection of targeted therapies that underpins precision-medicine. We investigated the prevalence of three important pharmacogenetic variants of VKORC1, CYP2C9, and CYP3A5 genes among Pakistani populations. A total of 1104 individuals were included representing thirteen major ethnicities. Samples were genotyped by using PCR-RFLP analysis. The allelic and genotypic frequencies of the three SNV's were calculated and were compared with the world's population data (ALFA, gnomAD, and 1000Genome, 1K databases), using the chi-square test. We found overall frequencies of functional-alleles of VKORC1 0.43, CYP2C9 0.94, and CYP3A5 0.14 in our population. Data showed a low prevalence of homozygous functional genotypes of VKORC1 (0.18; 0.0-0.45) and CYP3A5 (0.04; 0.0-0.22), and a high frequency of CYP2C9 (0.885; 0.80-1.0) across ethnicities. Genotyping distribution of VKCOR1 functional genotype was varied across ethnic groups such as 0.0-0.10 in Brahuis and Mohanas, Sindhis, Rajputs, and Gujjars populations, 0.11-0.20 in Makranis, Parsis, and Burusho populations, and 0.20-0.30 in Kalash, Kashmiris and Baloch populations. The highest VKORC1 (CC) was found in Pathans (0.45) and Hazaras (0.39) populations. Interestingly, we found a high prevalence of functional genotype CYP2C9 (rs1799853; C) and non-functional genotype of CYP3A5 (rs776746; T) across various ethnic groups of Pakistan. Data regarding prevalence of clinically important pharmacogenomics SNVs could be useful in drug adjustment and avoiding adverse drug reactions in a specific ethnic population. This could help in moving current medical practices toward precision medicine in our part of the world.

Mechanism of VKORC1 and VKORC1L1 signaling in the effects of sodium dehydroacetate on coagulation factors in rat hepatocytes

Sodium dehydroacetate (Na-DHA) is widely used as an antibacterial and preservative additive in food and cosmetics. Previously, we reported that repeated oral administration of Na-DHA induces coagulation disorders, and inhibited liver vitamin K epoxide reductase complex subunit 1 (VKORC1) and VKORC1-like protein 1 (VKORC1L1) in rats. However, the effects of Na-DHA on coagulation factors in rat hepatocytes and the mechanism of VKORC1 and VKORC1L1 signaling in that process are unclear. Here, we constructed stable Vkorc1 and Vkorc1l1 overexpressing cell lines using lentiviruses and transfected small interfering RNAs into buffalo rat liver BRL3A cells for Vkorc1 and Vkorc1l1 overexpression and silencing, respectively. After treatment with 5 mmol/L Na-DHA for 24 h, VKORC1 and VKORC1L1 expression levels were detected by real-time PCR and western blotting. Vitamin K (VK) and factor IX (FIX) contents were detected using enzyme linked immunosorbent assays. We observed that Na-DHA inhibited VKORC1 and VKORC1L1 expression levels and reduced VK and FIX levels in rat hepatocytes. Overexpression or silencing of Vkorc1 and Vkorc1l1 increased or decreased, respectively, the production and secretion of VK and FIX in rat hepatocytes, and alleviated or aggravated the inhibitory effects of Na-DHA on VKORC1 and VKORC1L1 expression levels. Taken together, the results indicated that both VKORC1 and VKORC1L1 signaling play regulatory roles in the effects of Na-DHA on coagulation factors in rat hepatocytes.

Novel Gamma-Glutamyl Carboxylase Mutation Causing Hemorrhagic and Non-Hemorrhagic VKCFD1 Phenotypes

Background: Vitamin K clotting factor deficiency (VKCFD) types 1 and 2 are very rare autosomal recessive disorders associated with reduced clotting factors activities for all vitamin K dependent clotting factors. Less than 30 cases are reported with defects in GGCX (gamma-glutamyl carboxylase) leading to VKCFD Type 1 (VKCFD1) or VKORC1 (vitamin K epoxide reductase complex subunit 1) leading to VKCFD Type 2 (VKCFD2). In addition to clotting factor deficiency, GGCX mutations can be associated with skeletal abnormalities such as bone hypoplasia, conductive hearing loss, mental retardation, and skin hyperlaxity. Here, we report an Arabic family with VKCFD1 harboring an unreported, new pathogenic variant in the GGCX gene. The proband presented at 17 months of age with severe bruising following trauma and work up demonstrated deficiencies of all Vitamin K dependent (VKD) coagulation proteins. Another sibling born 8 years later was also diagnosed with the same condition with abnormal coagulation parameters at birth. Additionally, the non-hemorrhagic manifestations diagnosed in both siblings were mid-facial hypoplasia with flat nasal bridge, dental abnormalities, and shortened distal phalanges representing a keutel syndrome-like phenotype. Both patients are currently managed with oral vitamin K supplementation with resolution of bleeding symptoms. Here we present the in-vitro assessment of the mutations effects to vitamin K supplementation and potential clinical implications. Methods: The proband, affected brother and mother, all underwent genetic evaluation at the University Clinic Bonn in Germany. DNA was isolated from the patient's blood and Sanger Sequencing was performed for the genes VKORC1 and GGCX. The identified new pathogenic variant in GGCX was further characterized by the in-vitro assay published by Ghosh et al. in 2021. Results: The brothers were found to be homozygous for c.[416G>A];[416G>A] p.(Cys139Tyr), a genetic variant in exon 4 of the GGCX gene. Mutations in VKORC1 gene leading to reduced activity for the VKD clotting factors were excluded confirming the diagnosis of VKCFD Type 1 (VKCFD1). The mother was found to be heterozygous for the same GGCX variant. The affected siblings and the mother were further genotyped for the promoter single nucleotide polymorphism in VKORC1 gene NG_011564.1:g.3588G>A (VKORC1:c.-1639 G>A) since this might correlate with the severity of the phenotypes in patients with skeletal manifestations. The brothers and mother were found to harbor VKORC1:c.-1639 GA, which has been associated with a 33% reduced VKORC1 expression. Treatment with 5mg of vitamin K resulted in increased activities of the VKD clotting factors (Table 1). However, clotting factor activities did not normalized after vitamin K supplementation. The in vitro characterization of the new identified pathogenic variant GGCX:p.C139Y (Figure 1) showed reduced activities for all VKD clotting factors, which did not reach wild type levels despite increasing concentrations of vitamin K. The levels of Matrix Gla protein (MGP), Bone Gla protein (BGLAP), and Gla-rich protein (GRP) showed almost no response to increasing amount of vitamin K. Discussion: Here, we report a new pathogenic variant in exon 4 of GGCX c.[416G>A];[416G>A] p.(Cys139Tyr) leading to VKCFD1 with reduced clotting factor activities and non-hemorrhagic manifestations. The pre and post vitamin K supplementation factor levels in the patients correlate with the in-vitro data and demonstrate failure of the clotting factor levels to normalize despite vitamin K supplementation. The in vitro data for the non-hematological VKD proteins indicate that the patients might develop a pseudoxanthoma elasticum (PXE)-like phenotype resulting into hyperlaxity of skin due to the almost zero levels of carboxylated GRP. The skeletal/bone phenotypes can be explained by the almost zero values for carboxylated MGP and GRP. To assess this in-vivo, levels of human MGP and BGLAP are currently being analyzed in the patients and mothers blood samples. These patients highlight the nuances associated with the diagnosis and management of patients with VKCFD. The antenatal management and timing of Vitamin K supplementation to prevent intrauterine bleeding is also unknown, and there may be benefit to starting early to prevent the bone and skin manifestations. Further research is warranted in rare bleeding disorders such as VKCFD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The screening for anticoagulant rodenticide gene VKORC1 polymorphism in the rat Rattus norvegicus, Rattus tanezumi and Rattus losea in Hong Kong

Anticoagulants are a major component of rodenticides used worldwide, which function by effectively blocking the vitamin K cycle in rodents. The rat Vitamin K epoxide Reductase Complex (VKORC) subunit 1 is the enzyme responsible for recycling vitamin K, and five substitution mutations (Tyr139Cys, Tyr139Ser, Tyr139Phe and Leu128Gln and Leu120Gln) located in the VKORC1 could result in resistance to anticoagulant rodenticides. This study carried out a VKORC1-based survey to estimate the anticoagulant rodenticide resistance in three Rattus species (R. losea, R. norvegicus, and R. tanezumi) collected in Hong Kong. A total of 202 rats captured in Hong Kong between 2017 and 2021 were analysed. Sequencing of molecular marker cytochrome c oxidase subunit 1 (COX1) was carried out to assist the species identification, and the identities of 52 lesser ricefield rats (R. losea), 81 common rats (R. norvegicus) and 69 house rats (R. tanezumi) were confirmed. Three VKORC1 exons were amplified from individuals by PCR followed by Sanger sequencing. A total of 47 R. tanezumi (68.1%) contained Tyr139Cys mutation in VKORC1 gene, and half of them were homozygous. None of the collected R. losea and R. norvegicus were detected with the five known substitutions leading to anticoagulant rodenticides resistance, and previously undescribed missense mutations were revealed in each species. Whole genome sequencing was further carried out on some individuals, and single nucleotide polymorphisms (SNPs) were also identified in the introns. This is the first study investigating the situation of anticoagulant rodenticide resistance in the rats collected in Hong Kong. Given that the efficacy of rodenticides is crucial for effective rodent management, regular genetic testing as well as population genomic analyses will be required to both monitor the situation and understand the adaption of different rat haplotypes for integrated pest management. Susceptibility tests for individual rodenticides should also be conducted regularly to assess their effectiveness on local species.

Proteomic Profiling and Pathway Analysis of Acid Stress-Induced Vasorelaxation of Mesenteric Arteries In Vitro.

Although metabolic acidosis is associated with numerous pathophysiological conditions and its vasorelaxation effects have been well described in different animal and culture models, the molecular mechanisms of acidosis-induced vasorelaxation are not fully understood. Mesenteric artery models have been used extensively to examine the vascular response to various pathophysiological conditions. Our previous studies and several other reports have suggested the vascular responses of goat mesenteric arteries and human arteries to various stimuli, including acidic stress, are highly similar. In this study, to further identify the signaling molecules responsible for altered vasoreactivity in response to acidic pH, we examined the proteomic profile of acid stress-induced vasorelaxation using a goat mesenteric artery model. The vascular proteomes under acidic pH were compared using 2D-GE with 7 cm IPG strips and mini gels, LC-MS/MS, and MALDI TOF MS. The unique proteins identified by mass spectroscopy were actin, transgelin, WD repeat-containing protein 1, desmin, tropomyosin, ATP synthase β, Hsp27, aldehyde dehydrogenase, pyruvate kinase, and vitamin K epoxide reductase complex subunit 1-like protein. Out of five protein spots identified as actin, three were upregulated > 2-fold. ATP synthase β was also upregulated (2.14-fold) under acid stress. Other actin-associated proteins upregulated were transgelin, desmin, and WD repeat-containing protein 1. Isometric contraction studies revealed that both receptor-mediated (histamine) and non-receptor-mediated (KCl) vasocontraction were attenuated, whereas acetylcholine-induced vasorelaxation was augmented under acidosis. Overall, the altered vasoreactivity under acidosis observed in the functional studies could possibly be attributed to the increase in expression of actin and ATP synthase β.

Pharmacogenetics of clopidogrel and warfarin in the treatment of cardiovascular diseases: an overview of reviews.

Pharmacogenetics (PGx) is the relationship between an individual's genetic variations and the response to pharmacological treatment. Wechose to perform an overview of reviews on PGx testing-guided treatment for cardiovascular diseases, based on clinically relevant gene-drug pairs. We conducted a search on Medline, Embase and Cochrane Library, from their inception to 18 June 2020. The most studied gene-drug pairs were clopidogrel and warfarin associated with cytochrome p450 and vitamin K epoxide reductase complex subunit 1 genes (CYP2C19, CYP2C9 and VKORC1), classified as critically low quality. There is a need for more quality primary studies and systematic reviews that assess the risk of bias, with consistent definitions of clinical outcomes to consider the benefits of PGx testing for cardiovascular diseases.

VKORC1 gene polymorphism (-1639G>A) in warfarin therapy patients of Pakistani population.

To observe vitamin K epoxide reductase complex subunit 1-1639 G>A polymorphism in patients resistant to warfarin therapy, and to calculate the allele frequency of the polymorphism in local patients. The cross-sectional case-control study was conducted at the Punjab Institute of Cardiology, Lahore, from 2013 to 2014 and comprised patients with heart valve replacement. Thy were divided into warfarin-resistant group 1 taking 10mg/day, 70mg/week and control group 2 taking a standard dose of 5mg/day, 35mg/week. The vitamin K epoxide reductase complex subunit 1-1639 G>A polymorphism analysis was done by polymerase chain reaction, followed by restriction fragment length polymorphism technique. Data was analysed using SPSS 20. Of the 146 patients, there were 73(50%) in each of the two groups. In group 1, there were 37(50.68%) males and 36(49.32%) were females with an overall mean age of 33±12 years, while group 2 had 36(49.32%) males and 37(50.68%) females with an overall mean age of 37±13 years. There were no significant differences in mean values of age, serum cholesterol, triglycerides and albumin levels between the groups (p>0.05). The G allele was the most frequently found in both groups, with 140(96%) in group-1 and 137(94%) in group-2. Overall, the homozygous GG genotype was significantly higher in the sample 132(90.4%) (p<0.05). There was evidence found that vitamin K epoxide reductase complex subunit 1-1639 G>A polymorphism alone may not be the dominant genetic factor associated with warfarin response variability.

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

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.

Response to anticoagulation in chronic thromboembolic pulmonary hypertension

Abstract Introduction Chronic thromboembolic pulmonary hypertension (CTEPH) is caused by fibrotic thrombus in the pulmonary arteries, likely originating from pulmonary embolism. Inadequate anticoagulation is one of the suspected mechanisms of disease in CTEPH. The aim of our study was to assess phenprocoumon dosing and genetic polymorphisms of vitamin K epoxide reductase complex subunit 1 (VKORC 1) that are known to affect the dose required to reach therapeutic range. Methods The ratio of mean weekly phenprocoumon dose in relation to mean INR levels was assessed in CTEPH patients on phenoprocoumon oral anticoagulation for at least 6 months, compared with PAH patients. VKORC 1 (–1639, –3730) single nucleotide polymorphisms (SNPs) were determined by polymerase chain reaction (PCR). Results In 225 patients (167 CTEPH, 58 PAH) mean treatment duration was 51.7±44.7 months, and mean age was 62.9±13.87 years (49.7% female). Median weekly dose of phenprocoumon was 10.5 mg (IQR 9.0–16.5) for PAH vs. 14.25 mg (IQR 10.5–18.0) in the CTEPH cohort (p=0.016). Although median INR was not significantly different among the two groups (2.28 vs. 2.40 in the PAH vs. CTEPH, respectively, p=0.084), achieving the mentioned INR require significantly higher weekly doses in the CTEPH group. While distribution of VKORC1 variants was according to the Hardy Weinberg equilibrium, patients with CTEPH and VKORC1 –1639 GG homozygous wild type required significantly higher phenprocoumon doses compared with VKORC1 –1639 AA homozygous mutants (P&amp;lt;0.001). Conclusion CTEPH patients require more vitamin K antagonists in relation to INR levels than PAH patients. Unmet phenprocoumon dosing requirements may be one mechanism of disease in CTEPH. Funding Acknowledgement Type of funding sources: None.

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) like Warfarin are known to promote adverse cardiovascular remodelling. 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 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 mediate the protective properties of VKORC1L1. 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-induced neointima formation is associated with reduced vascular VKORC1L1 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. Funding Acknowledgement Type of funding sources: None.