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

Cells. 2021 Aug 16;10(8):2097. doi: 10.3390/cells10082097.


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.

Keywords: BioHybrid; cardiovascular disease; fetuin-A; matrix Gla protein; vascular calcification; vascular smooth muscle cells; vitamin K; vitamin K antagonist.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers / blood
  • Calcium-Binding Proteins / blood
  • Cardiovascular Diseases / blood
  • Cardiovascular Diseases / diagnosis
  • Cardiovascular Diseases / drug therapy
  • Cells, Cultured
  • Extracellular Matrix Proteins / blood
  • Fluorescent Dyes / chemistry
  • Hematologic Tests
  • Humans
  • Kinetics
  • Matrix Gla Protein
  • Muscle, Smooth, Vascular / metabolism*
  • Renal Dialysis
  • Renal Insufficiency, Chronic / blood
  • Renal Insufficiency, Chronic / diagnosis
  • Renal Insufficiency, Chronic / therapy
  • Vascular Calcification / blood*
  • Vascular Calcification / diagnosis
  • Vitamin K 1 / therapeutic use
  • alpha-2-HS-Glycoprotein / chemistry
  • alpha-2-HS-Glycoprotein / metabolism


  • Biomarkers
  • Calcium-Binding Proteins
  • Extracellular Matrix Proteins
  • Fluorescent Dyes
  • alpha-2-HS-Glycoprotein
  • Vitamin K 1