Reproducible in vitro model for dystrophic calcification of cardiac valvular interstitial cells: insights into the mechanisms of calcific aortic valvular disease

Abstract

Calcific aortic valvular disease (CAVD) is the most prevalent valvular pathology in the United States. Development of a pharmacologic agent to slow, halt, or reverse calcification has proven to be unsuccessful as still much remains unknown about the mechanisms of disease initiation. Although in vitro models of some features of CAVD exist, their utility is limited by the inconsistency of the size and time course of the calcified cell aggregates. In this study, we introduce and verify a highly reproducible in vitro method for studying dystrophic calcification of cardiac valvular interstitial cells, considered to be a key mechanism of clinical CAVD. By utilizing micro-contact printing, we were able to consistently reproduce cell aggregation, myofibroblastic markers, programmed cell death, and calcium accumulation within aggregates of 50-400 μm in diameter on substrates with moduli from 9.6 to 76.8 kPa. This method is highly repeatable, with 70% of aggregates staining positive for Alizarin Red S after one week in culture. Dense mineralized calcium-positive nanoparticles were found within the valvular interstitial cell aggregates as shown by scanning electron microscopy (SEM) and energy dispersive spectrometry (EDS). The area of micro-contact printed aggregates staining positive for caspase 3/7 activity increased from 5.9 ± 0.9% to 12.6 ± 4.5% over one week in culture. Z-VAD-FMK reduced aggregates staining positive for Alizarin Red S by 60%. The state of cell stress is hypothesized to play a role in the disease progression; traction force microscopy indicates high substrate stresses along the aggregate periphery which can be modulated by altering the size of the aggregates and the modulus of the substrate. Micro-contact printing is advantageous over the currently used in vitro model as it allows the independent study of how cytokines, substrate modulus, and pharmacologic agents affect calcification. This controlled method for aggregate creation has the potential to be used as an in vitro assay for the screening of promising therapeutics to mitigate CAVD.

Heterogeneity Profoundly Alters Emergent Stress Fields in Constrained Multicellular Systems

“…These directly measured traction forces were averaged from six different aggregates to create a representative aggregate with a radially symmetric prototypical traction force distribution. On average, traction forces were highest at the edge and lowest in the center, as seen in previous studies (6,16). For the homogeneous model, the calculated average normal stresses are highest in the center and lowest along the edge (Fig.…”

“…Individual cell studies used polyacrylamide gels coated with monomeric collagen by functionalization of the surface using Sulfo-SANPAH (Thermo Fischer Scientific, Waltham, MA). Multicellular aggregate studies used polyacrylamide gels that were microcontact printed with monomeric collagen using polydimethylsiloxane stamps of 200-400 mm diameter circular posts, as previously described (16).…”

“…Assumptions of material homogeneity may be acceptable for cell monolayers in which unconstrained migration and spreading results in regional uniformity in cell density and orientation (15); however, in constrained systems (e.g., micropatterned protein islands in vitro and tissues with confined growth in vivo), regional differences in cell behavior markers indicative of variations in cell properties are commonly reported. Higher rates of proliferation (2), increased circumferential alignment (6), enhanced tumorigenicity (5), and heightened contractility markers (2,16) are reported near multicellular system edges compared to central regions (1)(2)(3).…”

Reproducible In Vitro Tissue Culture Model to Study Basic Mechanisms of Calcific Aortic Valve Disease: Comparative Analysis to Valvular Interstitials Cells

“…Even though our understanding of cellular and molecular mechanisms in the progression of CAVD has increased enormously in the last years, the lack of reproducible tissue models mimicking natural conditions and accurately replicating pathological mechanisms has proven to be challenging for researchers in this field [ 23 , 24 , 31 , 34 , 35 ]. The maladaptations of the highly organized valvular ECM, which is constantly remodeled, either enzymatically or non-enzymatically are not simply a consequence of impaired valve cells but rather contribute to the progression of CAVD by altering various fundamental biological processes [ 11 , 36 , 37 , 38 , 39 ].…”

“…The role of VICs and VECs in the development and progression of CAVD is difficult to study, and models that can accurately replicate the pathological mechanism in CAVD are lacking [ 23 ]. Explanted calcified AVs from patients undergoing SAVR are of great value, but disease mechanisms cannot be extrapolated from the end-stage pathology [ 24 ]. In general, most research regarding CAVD is based on experiments using two-dimensional (2D) cell culture or artificially created three-dimensional (3D) environments of VICs, most commonly neglecting VECs.…”