(H-292) An in vitro alveolar-capillary model to investigate the therapeutic potential of cdc42 binding kinase MRCKα for treatment of Acute Respiratory Distress Syndrome

Acute Respiratory Distress Syndrome (ARDS) is a life-threatening clinical condition that causes pulmonary inflammation, edema and hypoxemia with high mortality rates of up to 50% in the U.S. Our lab previously demonstrated that gene delivery of the myotonic dystrophy kinase-associated CDC42 binding protein kinase alpha (MRCKα) lead to upregulation of tight junction and adherens junction expression as well as barrier function in LPS-injured alveolar epithelial type I and microvascular endothelial cells in vitro, and increases alveolar fluid clearance and barrier restoration in vivo in both small (mouse) and large (pig) animal models of ARDS. My project aims to deepen our understanding of MRCKα signaling mechanisms in order to develop it as a therapeutic target for ARDS. Alveoli consist of two important cell types that contribute to barrier function and fluid clearance: alveolar epithelial cells and capillary endothelial cells. Our lab’s data point to MRCKα effects in both of them, therefore, it is crucial to investigate MRCKα signaling mechanisms in both epithelial and endothelial cells of the alveolar-capillary barrier and whether possible crosstalk between them plays a role in MRCKα signaling pathways. Our lab optimized an in vitro alveolar-capillary barrier model that allows co-culturing of epithelial and endothelial cells in close proximity, with robust capabilities for assessment of barrier function and signaling pathways. Using this model, the roles of cellular crosstalk during gene transfer of MRCKα to either one or both of the cell types at the alveolar-capillary barrier are investigated.

In our alveolar-capillary model, primary rat epithelial type I cells and microvascular endothelial cells are seeded on two sides of a 100nm thin and porous silicon nitride membrane coated with extracellular matrix proteins to promote adhesion. To evaluate successful culture of both cell types on the model, morphology, barrier function, and junctional complex protein expression are evaluated using immunofluorescence imaging and macromolecular permeability assays. The macromolecular permeability assay is developed specifically for our model based on conventional permeability assay that measures transport of fluorescent tracers across the cell monolayer via paracellular pathways. To investigate the effects of gene transfer of MRCKα to the alveolar-capillary barrier, the MRCKα plasmid is transfected (via lipofection) to either epithelial or endothelial cells or both cell types on the co-culture model. The experimental groups include naïve; MRCKα transfection to epithelial or endothelial cells only; MRCKα transfection to both cell types; and GFP transfection (control). Successful transfection of plasmids into cells is determined by evaluation of expression of a transfected GFP plasmid in the control group. Barrier function at 48 hours post-transfection is assessed using our macromolecular permeability assay with 10kDa Dextran-Cascade Blue tracer. At 48 hours post-transfection, cells are also lysed for evaluation of expressions of exogenous MRCKα (distinguished from endogenous MRCKα with a Flag tag), tight junction protein Zo-1 (epithelial cells), and adherens junction protein VE-Cadherin (endothelial cells) by RT-qPCR assay. Cells are also immunofluorescent stained for Zo-1 and VE-Cadherin for qualitative evaluation of changes in junctional expressions.

Our alveolar-capillary model successfully supports adhesion and growth of alveolar epithelial type I and microvascular endothelial cells in both mono- and coculture settings. Both cell types show expected morphology, high junctional complex protein  expression, and mature barrier function by day 5 in culture. Gene transfer of MRCKα upregulates junctional protein expression and barrier function in both epithelial and endothelial cells. Transfection of MRCKα to either epithelial or endothelial cells alone significantly increases barrier function in the co-culture model, however, the effect is most significant when MRCKα is transfected to both cell types.  Ongoing experiments will evaluate potential crosstalk between epithelial and endothelial cells that may promote junctional complex protein expression, changes in junctional protein expression in both cell types will be evaluated when MRCKa is transfected to only one cell type and compared to the expression in naïve cells. These results indicate that our in vitro air-blood barrier model successfully supports the coculture of both epithelial and endothelial cells of the alveolar-capillary barrier and promotes their barrier maturation and secretome communication. Gene transfer of MRCKα to either cell type successfully increases barrier function in the entire co-culture system, suggesting robust therapeutic capability of MRCKα.