Mechano-osmotic Stress Regulates Compaction and Elongation of the Fetal AV Valve in Collaboration with Developed Matrix Scaffolding
David M. Bassen, Duc Pham, Rashmi Rao, Jonathan T. Butcher, Dr.
Cornell University, Ithaca, NY, USA.
OBJECTIVE: It has been shown that interstitial osmotic stress can be induced by mechanical stretching that may further result in significant cellular tension or compression. Developing valve cushions experience such stretch in vivo and the mechanical effects of osmotic stress have not been evaluated. Here, we examine the influence of osmotic stress on compacting and elongating valve cushions, interactions with changing matrix components and potential signaling activation.
METHODS: Avian atrioventricular valve cushions were cultured in hanging drop culture for 24 hours to evaluate stage dependent compaction, measured by area ratio % (final to initial area), and elongation behaviors in hypertonic and hypotonic medias. IF and IHC were used to assess cell morphology, alignment, polarity, apoptosis, proliferation and potential signaling activity. Micropipette aspiration was used to measure relative stiffness.
RESULTS: Cushions in control conditions spontaneously compact to at HH25-36 by 50%, even given an increasing collagen matrix density, until after HH36 where compaction stops at 80%. HH40 matrix maintains a thin, saddle-like morphology that is lost via collagenase II treatment, despite having a stiffness corresponding to HH25 cushions. Cushions in hypotonic conditions compact 10% more than controls, an effect that diminishes at later stages indicating a greater resistance by collagen matrix. Cushions in hypertonic conditions compact only to 80% through HH36, indicating a decrease in cell contractility regardless of matrix content. Rho/Rac inhibitors did not show differences in compaction. AV endocardial disruption occurred with ROCK inhibition. At HH25, active RhoA is seen predominantly in the endothelium, while distinct RhoA-GTP patterning is seen in the endothelium of the hypertonic case that is disrupted by ROCK inhibition. Treatment with blebbistatin arrests compaction and causes tissue with little collagen to relax and expand by 20%, supporting that compaction is myosin II dependent.
CONCLUSIONS: This study demonstrates that osmotic stress has developmentally relevant effects on valve shape fidelity and growth and reveals hypo and hypertonic stresses as potential means for both mechanical perturbation and mechanotransduction. Further, we identify that ECM extent and composition helps provide a substrate for directional compaction and elongation crucial for forming valve leaflets.
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