PB1335 - Tissue factor pathway inhibitor attenuates vascular smooth muscle cell calcification by supporting a contractile phenotype

Tuesday, June 25, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Vanessa Bröker

PhD student
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Limburg, Netherlands

Co-Author(s)

AA

Asim C. Akbulut

Post Doc
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Limburg, Netherlands
DW

David Weber

Scientist
TRON – Translational Oncology University Medical Center of the Johannes-Gutenberg-University Mainz, Mainz, Germany
Mainz, Rheinland-Pfalz, Germany
FF

Francesco Forin

PhD student
Institute for Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany
Bonn, Nordrhein-Westfalen, Germany
Phillip Wenzel, Professor, MD

Deputy Director
University Medical Center Mainz
Mainz, Rheinland-Pfalz, Germany
Tilman M. Hackeng, PhD

Professor
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Netherlands
Leon J. Schurgers, PhD

Professor
Department of BIochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Limburg, Netherlands

Background: Tissue factor pathway inhibitor (TFPI) acts as a constitutive inhibitor of the extrinsic coagulation pathway by inhibiting the tissue factor/FVIIa/FXa complex. TFPI also exhibits a protective role in thrombo-inflammatory processes. In the vasculature, vascular smooth muscle cells (VSMC) play a dual role in coagulation and calcification. The role of VSMC-derived TFPI in vascular calcification has not been studied so far.

Aims: The aim of this project was to identify the role of TFPI in vascular remodeling and calcification.

Methods: TFPI-deficient iPSC-derived vascular smooth muscle cells (TFPI-KD2-KO iVSMC; NP_006278.1:p.(Tyr137_Arg140del)) were generated using CRISPR/Cas9 and transcriptomic differences in wild-type and mutant iVSMC were elucidated using bulk RNAseq. In addition, contractile and synthetic phenotypes of iVSMC were analyzed for expression of VSMC-, inflammatory-, and calcification-related markers. Ultimately, cells were treated with calcification-inducing media to assess the calcifying potential of TFPI-KD2-KO cells.

Results: CRISPR/Cas9 modification resulted in iVSMC synthesizing partially functional TFPI. RNAseq in wild-type vs. mutated cells (TFPI-K2-KO) resulted in 815 differentially expressed genes including modulators of iVSMC phenotype and calcium metabolism. A significant decrease in contractile markers αSMA and CNN1 was observed in TFPI-K2-KO cells. While αSMA expression was reduced in both contractile and synthetic iVSMC, a decline of CNN1 was seen in contractile iVSMC only. Expression of the inflammatory chemokine MCP-1 was considerably reduced in contractile TFPI-K2-KO iVSMC. No differential gene expression was observed for synthetic marker S100A4, the inflammatory cytokine IL-6, and the osteogenic marker BMP4. Ultimately, calcifying medium significantly increased calcification of TFPI-K2-KO iVSMC compared to wild-type iVSMC. Extracellular addition of recombinant TFPI and reverse CRISPR/Cas9-engineered TFPI repair reversed this effect.

Conclusion(s): We show that functional TFPI plays a protective role in preventing development of vascular remodeling and calcification. Enhanced calcification in TFPI-K2-KO iVSMC might derive from a FXa-dependent phenotypic synthetic switch.