(E-167) 2D and 3D valve interstitial cell models to examine the effects of diabetes on calcific aortic valve disease

Introduction:: Calcific aortic valve disease (CAVD) is the most prevalent form of aortic stenosis, and diabetes is a prominent risk factor. CAVD causes fibro-calcific remodeling and thickening of the valve leaflets which impairs valve function and leads to heart failure if left untreated. There is no effective pharmacological treatment to prevent or delay its progression¹. Valve interstitial cells (VICs) have been identified as key mediators of valve calcification via osteochondrogenic transdifferentiation. We have developed 2D VIC culture models and examined osteochondrogenic differentiation and calcification in response to diabetogenic conditions. However, 2D cultures fail to mimic cell-ECM and mechanical interactions known to occur in CAVD and that may control responses to diabetic conditions². To better mimic the in vivo setting, we are developing and characterizing 3D collagen I hydrogel-based VIC organoids for future studies.


Materials and Methods:: 2D primate VIC (pVIC) culture: For gene expression studies, pVICs were grown in 6-well plates for 2 days in DMEM with 5% FBS and 1% Penicillin-Streptomycin containing normal glucose (NG, 5.5 mM) or high glucose (HG, 25 mM) and then treated with osteochondrogenic media (OCM) containing 2.6 mM Pi for 5 days. For calcification studies, pVICs were grown in 6-well plates for 2 days in high glucose (HG, 25mM) and then treated with OCM for 7 days.

mRNA Quantitation: RNA extraction and RT-qPCR were performed for selected genes and normalized to 18S ribosomal RNA.

Calcium Assay: Calcium levels were determined using the o-cresolphthalein complexone kit (Teco Diagnostics, C503-480) as previously described³ and normalized to the total protein using the Micro BCA Protein Assay Kit (Thermo Fisher Scientific, 23235).

3D pVIC organoid cultures: pVICs were encapsulated into organoids by mixing with 2 mg/mL type I collagen dissolved in HG DMEM containing 5% FBS and adding adequate 0.1 mol/L NaOH to neutralize and initiate gelation. 3D pVIC organoids were maintained in HG DMEM with 5% FBS and 1% Penicillin-Streptomycin. Cell viability was analyzed using the Invitrogen LIVE/DEAD Cell Imaging Kit (488/570) and visualized using a Leica SP8 confocal.

Results, Conclusions, and Discussions:: 2D pVIC cultured in OCM for 5 days in either NG or HG conditions showed elevated expression of osteochondrogenic markers, SOX9 and RUNX2, and loss of smooth muscle marker, SM22, compared to controls, suggesting osteochondrogenic transdifferentiation. HG + OCM treatment significantly increased SOX9, but not RUNX2, compared to NG + OCM treatment, suggesting that HG preferentially drives pVIC differentiation toward the chondrocytic lineage rather than the osteoblastic lineage. On the other hand, no significant differences in gene expression were observed between pVIC cultured in NG or HG in the absence of OCM. In addition, HG + OCM significantly promoted mineralization of pVIC compared to HG alone.

3D pVIC organoids were created and characterized. pVIC organoids seeded at 2.5 x 10⁵ cells/mL and 5 x 10⁵ cells/mL showed a viability of 64.8% live cells and 52.4% live cells, respectively. Higher seeding densities promoted cell death, with the pVIC organoid seeded at 7.5 x 10⁵ cells/mL showing a viability of only 15.9% live cells. Thus, lower seeding densities of pVICs form organoids and maintain cell viability closer to 2D culture (84% at 6 days). Studies to examine gene expression and mineralization of the 3D pVIC organoids under diabetic conditions will be presented.


Acknowledgements (Optional): : This project is supported by R35 HL 139602 to Cecilia Giachelli. Confocal imaging is supported by NIH S10 OD016240 and the UW Student Technology Fee.

References (Optional): :

1. Donato M, et al. Int J Mol Sci. 2020 Nov 4;21(21):8263.


2. Hutson H.N. PLoS One. 2016;11;e0163858.


3. Scatena, M. Cardiovasc Pathol. 2018;34; 28–37.