Proteoglycan Metabolism During Cardiac Valve Development and Disease

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Summary: Approximately 1.5% of individuals have a Bicuspid Aortic Valve (BAV) that can result in insufficient blood flow and organ damage (1, 2). A BAV is also an independent risk factor for ascending aortic wall complications that can lead to rupture and sudden death. However, the cell and molecular basis for the BAV is unknown. Even basic aspects of BAV formation are undefined due to contributions from multiple cell lineages and a lack of highly penetrant viable BAV mouse models. Dysfunctional aortic valves exhibit massive accumulation of the extracellular matrix (ECM) proteoglycans versican (Vcan) and aggrecan (Acan) (3-7) but the origin and consequence of excess proteoglycans is largely unknown. Since proteoglycans are highly stable, abundance is regulated primarily by proteolytic degradation which led us to investigate a role for proteoglycan cleavage. We discovered that loss of a single ECM proteoglycanase, Adamts5, results in enlarged aortic valves with ascending aortopathies (100%). These defects co-localize with substantial increases in Vcan and Acan in the affected tissues (8-10) that mirror the human condition. Mechanistically, in Adamts5-/- aortic valve primordia with excess Vcan there is a reduction of pSmad2, and when Smad2 is reduced further by generating Adamts5-/- ;Smad2+/- mice, there is a high penetrance of BAV (75%), much higher than seen in other mouse BAV models. The objective of this proposal is to utilize the viable Adamts5-/-;Smad2+/- mice that exhibit a high percentage of BAV, to define morphological events, cell behaviors and factors that when disrupted contribute to BAV formation. Since excess Vcan is a hallmark of dysfunctional and diseased valves, use of a model with mutations that impact the control of Vcan content, may shed light on how proteoglycan metabolism is regulated in development and may also give insight into disease. Experiments test the hypothesis that ECM Vcan cleavage coordinates mesenchymal cell behaviors and myocardial cell contributions that are required for the tricuspid morphology of aortic valves. The hypothesis is tested in two aims: Aim 1 tests the impact of altered Vcan cleavage on mesenchymal cell lineage behaviors that are required for aortic valve formation. Our preliminary data show that loss of Vcan cleaved fragments and excess intact Vcan, disrupted lineage-specific patterning in the developing aortic valves of the BAV Adamts5-/-;Smad2+/-mice. A combination of ex vivo, and in vivo approaches will decipher the lineage-specific cell behaviors, and guidance factors that are dependent on Vcan cleavage and to prevent BAV. Although dogma states that valve cusps arise from mesenchymal cells, Aim 2 investigates the consequence of excess Vcan on the myocardial cell lineage contributions to the developing aortic valve in Adamts5-/-;Smad2+/- mice and other murine models of BAV. Our preliminary data show myocardial lineage expression of Adamts5 is required to clear Vcan-rich ECM and to form the non-coronary cusp of the aortic valve. Due to the emerging prevalence of excess Vcan in BAV, the investigation into ECM turnover will allow insight into the molecular and cellular origins of aortic valve diseases, which may lead to therapeutic advances.