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

Abstract

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.

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition <em>In Vitro</em>

“…Potential further applications of the described method include patients' serum screening for in vitro calcification development as a surrogate marker for personalized development of vascular calcification. The platform has demonstrated its sensitivity toward dialysis and vitamin K treatment, in addition to both metabolic and non-metabolic diseases that are linked to patients with poor cardiovascular status and prognosis 20 .…”

“…In this paper, we detail the method for the application of a novel assay that utilizes hVSMCs with a fluorescent imaging probe to determine in vitro VC progression as well as function as a singular end-stage calcification assay. We previously demonstrated that this assay is directly comparable to the o-cresolphthalein and Alizarin Red methods and can be used to distinguish between varying culture conditions 20 . In addition to real-time measurements, this assay may be used to determine the propensity of serum or plasma samples as a surrogate marker for clinical VC development 20 .…”

“…We previously demonstrated that this assay is directly comparable to the o-cresolphthalein and Alizarin Red methods and can be used to distinguish between varying culture conditions 20 . In addition to real-time measurements, this assay may be used to determine the propensity of serum or plasma samples as a surrogate marker for clinical VC development 20 . This will aid in the application of biological strategies of cardiovascular sciences and disease modeling.…”

Application of the mineral-binding protein fetuin-A for the detection of calcified lesions

“…Accordingly, mice lacking the functional fetuin-A gene develop severe soft tissue mineralization starting in the lumen of microvasculature and have impaired bone formation [10][11][12][13]. Previously we showed that fluorescent derivatives of the mineral-binding protein fetuin-A/α2-Heremans-Schmid glycoprotein (Ahsg) serve as sensitive and specific reagents to detect early-stage calcifications ex vivo and in vivo [13][14][15][16]. We hypothesized that the mineral binding properties of fetuin-A might be improved by optimizing the protein-mineral binding interface.…”

What is the real predictor of coronary artery calcification in patients with chronic kidney disease?

“…did not consider the impact of renal replacement therapy on IL-6, did not test the level of vitamin K, did not mention whether CKD patients were taking vitamin K antagonists, such as warfarin, and did not conduct subgroup analysis of peritoneal dialysis patients and HD patients. Maybe the BioHybrid assay [ 7 ], a new cell-based high-content assay, to measure the vascular calcification propensity of an individual should be widely used.…”