(775d) Heterogeneity in Valvular Interstitial Cell Phenotype Is a Predictor of Cell Activation and Acquisition of Degenerative Properties

Valvular interstitial cells (VICs), the resident cells in heart valves, play major roles in valvular pathophysiology. VICs are quiescent (qVIC) in healthy heart valves and maintain tissue homeostasis. They, however, become contractile and activated (aVIC) under chemically- or mechanically-induced degenerative conditions. It is well-known that qVIC transform to aVIC on stiffer substrates, at higher passage numbers and in the presence of paracrine signals. Regardless of quiescence or activation states, investigations of culture heterogeneity can provide insights into culture likelihood for degenerative transformation. In this study, we quantify six different VIC morphologies â?? rhomboid, spindled, tailed, round, triangle and multi-extension. We then correlate the distributions of these six morphologies with cell activation state. The main goal of this study is to demonstrate that the distribution of different VIC morphologies changes significantly with culture conditions and those changes, in turn, correlate with the amounts of degenerative cells (aVIC).

Porcine aortic VICs from passage P2 to P4 were cultured on soft polyacrylamide gels and on plastic culture dishes. In a separate experiment, the same cells were grown in the presence or absence of high numbers of progenitor VICs (pVICs). pVICs have the ability to differentiate in culture, and potentially become either qVIC or aVIC. Cell monolayers were imaged under phase and fluorescence microscopy of actin fibers. Three cell morphology parameters were analyzed: maximum cell span, span ratio and cell area. All six distinct morphologies of VICs were represented and their relative proportions in culture were calculated. Immunoblotting of protein lysates and immunofluorescence staining of monolayers were also employed in the determination of qVIC and aVIC content in all experiments.

An increase in α-smooth muscle actin (α-SMA) expression, the main marker for aVICs, was detectable with increasing passage number, increasing substrate stiffness and upon contact with pVICs. Round, rhomboid and triangle VICs were prominent in conditions favoring aVIC transformation (later passages, stiffer substrates and pVICs signals) whereas spindled and tailed morphologies were prominent in conditions favoring VIC quiescence. Span ratio, maximum span and cell area were found to be significantly different (p<0.5) between each pair of conditions tested.

The substantial increase of α-SMA expression, or aVIC content, indicates that VICs can sense their environment and become activated in response to increasing matrix stiffness. The substantial increase in α-SMA due to passage number indicates that VICs become activated over time as they multiply in vitro. The increase in α-SMA due to pVIC contact indicates that pVIC will differentiate to aVIC phenotype due to paracrine communication. Since earlier passages and soft substrates contained more spindled and tailed morphologies, these can be associated with physiological tissue conditions. Later passages and stiffer substrates expressed more aVICs, characterized by high numbers of round, rhomboid and triangle morphologies.

Our conclusion is that characterization of these six morphologies is arguably more important for engineering of valvular tissues than the traditional qVIC and aVIC classification. Follow-up experiments in progress include 3D characterization studies of heterogeneous populations as well as isolations of these six morphological cases. These studies will lead the way to proper maintenance of spindled and tailed morphologies, guide future differentiation studies of pVICs and potentially provide unique ways to target and inhibit degenerative proteins in aVIC.