A Co-culture Model to Study Cellular Crosstalk in Aortic Valve Stenosis Sexual Dimorphism

Introduction: The aortic valve is located on the left side of the heart and undergoes a larger pressure gradient than its right-side counterparts, causing it to become diseased at a higher rate. Approximately 13% of the elderly population have aortic valve stenosis (AVS) [1], which is the stiffening and narrowing of the valve that occludes proper flow of oxygenated blood. In addition to age, biological sex impacts the presentation of AVS with men typically developing AVS due to calcification of the valve leaflet tissues whereas women typically develop AVS due to fibrosis or excessive scarring [2]. Despite these differences, no sex-specific treatments exist and AVS is only treated at severe stages by surgical replacement with a valve prosthesis, which are prone to calcification within 10-15 years and/or require indefinite anti-coagulation therapy. Currently, no non-surgical therapies exist to slow down or stop the initiation of AVS.

AVS is a cell-driven disease with resident fibroblast-like valve interstitial cells (VICs), exterior-lining valve endothelial cells (VECs) (Fig.1A), and infiltrating monocytes implicated in disease progression [3]. However, the crosstalk between these cell types remains largely unstudied, yet could reveal sex-specific and druggable targets. Herein, we engineer a three-dimensional co-culture model comprised of VICs encapsulated within a synthetic hydrogel and VECs seeded on the outer surface with the goal of studying monocyte infiltration and interaction with valve cells. Looking forward, the co-culture model will allow discovery of cellular mechanisms of sex-specific AVS initiation and serve as an in vitro test bed for drug screening of candidate therapies.

Materials and

Methods: Previously published methods [4,5] were modified to generate dome-shaped co-culture models of the aortic valve leaflet tissue. First, aortic valve cells were isolated from porcine hearts using previously published methods [6] and grown using EGM-2 Endothelial Cell Growth Medium-2 BulletKit (Lonza) for 3 days to encourage the VEC phenotype (Fig.1B). Afterwards, Dynabeads (ThermoFisher) were used to magnetically separate CD31+ VECs from VICs, at which point VICs are seeded within M199 media (ThermoFisher) supplemented with 10% Fetal Bovine Serum (FBS) (ThermoFisher), 1 𝜇g/ml amphotericin B, 50 𝜇g/ml streptomycin, and 50 U/ml of penicillin for 24 hours. Then, the VICs are encapsulated at a density of 1 million cells/ml within a hydrogel comprised of 40 kDa, 8-arm Poly (ethylene glycol) molecules functionalized with norbornene end groups, degradable peptide cross linkers, and CRGDS adhesive peptides (stiffness ~300 Pa). The encapsulated VICs are grown in M199 media for three days before VECs are seeded on top of the constructs at a density of 2 million cells/ml and the media changed to EGM-2. After three additional days of culture, the co-culture model was fixed and stained for markers indicative of VECs (CD31) and VICs (alpha smooth muscle actin, ⍺SMA) as well as their nuclei (DAPI) before imaging was conducted using a Nikon AXR laser scanning confocal microscope.

Results, Conclusions, and Discussions: Magnetic separation of heterogenous aortic valve cells using CD31+ Dynabeads resulted in virtually pure populations of VECs and VICs (Fig.1C). Specifically, the cells subjected to positive isolation stained positive for CD31 (Fig.1C, center panel), which is an endothelial marker that is highly expressed at intercellular junctions. The cells remaining in the supernatant largely stained negative for CD31 and positive for ⍺SMA, suggesting that they were mainly comprised of VICs (Fig.1C, right panel). However, this population was heterogenous with some cells staining positive for CD31 and negative for ⍺SMA. It is likely that these cells are VECs undergoing endothelial-to-mesenchymal transition to VICs [7] that do not express CD31 to an appreciable extent and therefore were not positively isolated.

Immunostaining revealed successful engineering of a 3D valve co-culture model. Specifically, CD31 positive VECs were spatially restricted to the top surface of the construct (Fig.1D), which mimics how native VECs line the exterior surface of the valve leaflet tissue (Fig.1A). In addition, ⍺SMA positive VICs spread and formed protrusions within the mid-substance of the co-culture, recapitulating the dispersion of VICs throughout the interstitium of the valve (Fig.1A). The center of the construct displayed a characteristic dome-shape (Fig.1D, left panel), which mimics the curved nature of the belly region of aortic valve leaflet tissues (Fig.1A, left panel). Furthermore, the periphery of the constructs approaches a taper on one end (Fig.1D, center and right panel), much like the commissure region of aortic valves (Fig.1A, left panel).

Future work will involve isolating monocytes from commercial porcine blood using published methods [8] and seeding them on the valve co-culture model to examine their infiltration trajectories, interaction with valve cells, and to monitor for development of a fibrotic or calcific niche. In addition, we will examine the effects of valve cells on monocyte polarization within the pro- (M1) to anti-inflammatory (M2) macrophage spectrum.

Acknowledgements (Optional): We acknowledge funding from National Institutes of Health T32HL007822 awarded to AK and National Institutes of Health R01 HL142935 and R01 HL132353 awarded to KSA. The imaging work was performed at the BioFrontiers Institute Advanced Light Microscopy Core (RRID: SCR_018302). The Nikon AXR Laser Scanning Confocal is supported by NIH grant 1S10OD034320.

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