Development of the BioHybrid Assay: Combining Primary Human Vascular Smooth Muscle Cells and Blood to Measure Vascular Calcification Propensity.

BACKGROUND: Vascular calcification is an active process that increases cardiovascular disease (CVD) risk. There is still no consensus on an appropriate biomarker for vascular calcification. We reasoned that the biomarker for vascular calcification is the collection of all blood components that can be sensed and integrated into a calcification response by human vascular smooth muscle cells (hVSMCs). METHODS: We developed a new cell-based high-content assay, the BioHybrid assay, to measure in vitro calcification. The BioHybrid assay was compared with the o-Cresolphthalein assay and the T50 assay. Serum and plasma were derived from different cohort studies including chronic kidney disease (CKD) stages III, IV, V and VD (on dialysis), pseudoxanthoma elasticum (PXE) and other cardiovascular diseases including serum from participants with mild and extensive coronary artery calcification (CAC). hVSMCs were exposed to serum and plasma samples, and in vitro calcification was measured using AlexaFluor®-546 tagged fetuin-A as calcification sensor. RESULTS: The BioHybrid assay measured the kinetics of calcification in contrast to the endpoint o-Cresolphthalein assay. The BioHybrid assay was more sensitive to pick up differences in calcification propensity than the T50 assay as determined by measuring control as well as pre- and post-dialysis serum samples of CKD patients. The BioHybrid response increased with CKD severity. Further, the BioHybrid assay discriminated between calcification propensity of individuals with a high CAC index and individuals with a low CAC index. Patients with PXE had an increased calcification response in the BioHybrid assay as compared to both spouse and control plasma samples. Finally, vitamin K1 supplementation showed lower in vitro calcification, reflecting changes in delta Agatston scores. Lower progression within the BioHybrid and on Agatston scores was accompanied by lower dephosphorylated-uncarboxylated matrix Gla protein levels. CONCLUSION: The BioHybrid assay is a novel approach to determine the vascular calcification propensity of an individual and thus may add to personalised risk assessment for CVD.

Off-target effects of oral anticoagulants - vascular effects of vitamin K antagonist and non-vitamin K antagonist oral anticoagulant dabigatran etexilate.

INTRODUCTION: Vitamin K antagonists (VKA) and non-vitamin K oral antagonist anticoagulants (NOAC) are used in the clinic to reduce risk of thrombosis. However, they also exhibit vascular off-target effects. The aim of this study is to compare VKA and NOAC on atherosclerosis progression and calcification in an experimental setup. MATERIAL AND METHODS: Female Apoe-/- mice (age 12 weeks) were fed Western-type diet as control or supplemented with dabigatran etexilate or warfarin for 6 or 18 weeks. Vascular calcification was measured in whole aortic arches using µCT and [18 F]-NaF. Atherosclerotic burden was assessed by (immuno)histochemistry. Additionally, in vitro effects of warfarin, thrombin, and dabigatran on primary vascular smooth muscle cells (VSMC) were assessed. RESULTS: Short-term treatment with warfarin promoted formation of atherosclerotic lesions with a pro-inflammatory phenotype, and more rapid plaque progression compared with control and dabigatran. In contrast, dabigatran significantly reduced plaque progression compared with control. Long-term warfarin treatment significantly increased both presence and activity of plaque calcification compared with control and dabigatran. Calcification induced by warfarin treatment was accompanied by increased presence of uncarboxylated matrix Gla protein. In vitro, both warfarin and thrombin significantly increased VSMC oxidative stress and extracellular vesicle release, which was prevented by dabigatran. CONCLUSION: Warfarin aggravates atherosclerotic disease activity, increasing plaque inflammation, active calcification, and plaque progression. Dabigatran lacks undesired vascular side effects and reveals beneficial effects on atherosclerosis progression and calcification. The choice of anticoagulation impacts atherosclerotic disease by differential off target effect. Future clinical studies should test whether this beneficial effect also applies to patients.

A Novel Biomimetic Tool for Assessing Vitamin K Status Based on Molecularly Imprinted Polymers.

Vitamin K was originally discovered as a cofactor required to activate clotting factors and has recently been shown to play a key role in the regulation of soft tissue calcification. This property of vitamin K has led to an increased interest in novel methods for accurate vitamin K detection. Molecularly Imprinted Polymers (MIPs) could offer a solution, as they have been used as synthetic receptors in a large variety of biomimetic sensors for the detection of similar molecules over the past few decades, because of their robust nature and remarkable selectivity. In this article, the authors introduce a novel imprinting approach to create a MIP that is able to selectively rebind vitamin K1. As the native structure of the vitamin does not allow for imprinting, an alternative imprinting strategy was developed, using the synthetic compound menadione (vitamin K3) as a template. Target rebinding was analyzed by means of UV-visible (UV-VIS) spectroscopy and two custom-made thermal readout techniques. This analysis reveals that the MIP-based sensor reacts to an increasing concentration of both menadione and vitamin K1. The Limit of Detection (LoD) for both compounds was established at 700 nM for the Heat Transfer Method (HTM), while the optimized readout approach, Thermal Wave Transport Analysis (TWTA), displayed an increased sensitivity with a LoD of 200 nM. The sensor seems to react to a lesser extent to Vitamin E, the analogue under study. To further demonstrate its potential application in biochemical research, the sensor was used to measure the absorption of vitamin K in blood serum after taking vitamin K supplements. By employing a gradual enrichment strategy, the sensor was able to detect the difference between baseline and peak absorption samples and was able to quantify the vitamin K concentration in good agreement with a validation experiment using High-Performance Liquid Chromatography (HPLC). In this way, the authors provide a first proof of principle for a low-cost, straightforward, and label-free vitamin K sensor.

Vitamin K-antagonists accelerate atherosclerotic calcification and induce a vulnerable plaque phenotype.

BACKGROUND: Vitamin K-antagonists (VKA) are treatment of choice and standard care for patients with venous thrombosis and thromboembolic risk. In experimental animal models as well as humans, VKA have been shown to promote medial elastocalcinosis. As vascular calcification is considered an independent risk factor for plaque instability, we here investigated the effect of VKA on coronary calcification in patients and on calcification of atherosclerotic plaques in the ApoE(-/-) model of atherosclerosis. METHODOLOGY/PRINCIPAL FINDINGS: A total of 266 patients (133 VKA users and 133 gender and Framingham Risk Score matched non-VKA users) underwent 64-slice MDCT to assess the degree of coronary artery disease (CAD). VKA-users developed significantly more calcified coronary plaques as compared to non-VKA users. ApoE(-/-) mice (10 weeks) received a Western type diet (WTD) for 12 weeks, after which mice were fed a WTD supplemented with vitamin K(1) (VK(1), 1.5 mg/g) or vitamin K(1) and warfarin (VK(1)&W; 1.5 mg/g & 3.0 mg/g) for 1 or 4 weeks, after which mice were sacrificed. Warfarin significantly increased frequency and extent of vascular calcification. Also, plaque calcification comprised microcalcification of the intimal layer. Furthermore, warfarin treatment decreased plaque expression of calcification regulatory protein carboxylated matrix Gla-protein, increased apoptosis and, surprisingly outward plaque remodeling, without affecting overall plaque burden. CONCLUSIONS/SIGNIFICANCE: VKA use is associated with coronary artery plaque calcification in patients with suspected CAD and causes changes in plaque morphology with features of plaque vulnerability in ApoE(-/-) mice. Our findings underscore the need for alternative anticoagulants that do not interfere with the vitamin K cycle.