Development of a calcific aortic valve disease-mimicking microtissue platform for disease modeling applications

Thesis

Calcific aortic valve disease (CAVD) is a progressive, inflammation-linked disorder in which valvular interstitial cells (VICs) transition from quiescent fibroblasts to myofibroblasts and osteoblast-like cells, culminating in leaflet stiffening and obstruction. Despite its growing global burden, there are no approved drugs that halt or reverse progression; clinical care remains largely limited to surgical or transcatheter valve replacement, underscoring the need for tractable, physiologically relevant disease models to elucidate early mechanisms and enable therapeutic discovery.

This thesis develops a CAVD-mimicking microtissue platform focused on inflammation as a driver of fibro-osteogenic remodeling. First, a three-dimensional porcine VIC (pVIC) monoculture system embedded in collagen was established and challenged with pro-calcifying phosphate and tumor necrosis factor-α (TNF-α). The model reproducibly undergoes myofibroblastic activation (α-SMA), NF-κB/IL-6 pathway engagement, and dose-dependent osteogenic commitment (RUNX2), with peak calcification at ~day 16. Pharmacological perturbations validate mechanistic control points. MEK1/2 inhibition (selumetinib) and dual-SMAD blockade (SB431542 + noggin) reduce osteogenic readouts and calcium burden, demonstrating the platform’s utility for drug screening. To capture endothelial regulation, a bilayer pVIC–pVEC co-culture microtissue was engineered with a surface CD31⁺ valvular endothelial cell (pVEC) layer over a VIC-rich collagen core. Endothelialization exerted a potent anti-calcific effect (≈5–8-fold reduction in calcium vs monoculture) while permitting early osteogenic transcription (BMP2/RUNX2), yet imposing a maturation bottleneck by suppressing effector markers (SPP1/BGLAP) as well as apoptosis and inflammation (CASP3/IL6/NFKB1). Under inflammatory and pro-fibrotic cues, the model enabled interrogation of endothelial-to-mesenchymal transition (EndMT). TNF-α and TGF-β downregulated endothelial markers and markedly upregulated MMP9, revealing that endothelial protection can be locally overcome by EndMT-linked ECM remodeling.

Collectively, these studies position inflammation via NF-κB/IL-6 crosstalk with osteogenic programs as a central orchestrator of VIC fate, and identify endothelial integrity as a brake on mineral competence. The resulting monoculture and co-culture platforms provide complementary, physiologically relevant avenues for mechanism-guided pharmacology in CAVD.

Authors: Lau, QY

• DOI: 10.5287/ora-b7nzyjwba
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English