Molecular mechanisms relating Poldip2 to endothelial permeability

SUMMARY Endothelial cells line the lumen of all blood vessels and play a critical role in maintaining the barrier function of the vasculature. Diminished barrier function and the consequent increase in vascular permeability to plasma proteins and leukocytes has been described in pathological conditions such as acute respiratory distress syndrome and acute lung injury. The resulting tissue edema is associated with loss of aerated lung tissue and high mortality and morbidity. We have discovered that Polymerase delta interacting protein-2 (Poldip2), a protein with multiple binding partners, is an important modulator of lung endothelial barrier function. Remarkably, using a lipopolysaccharide (LPS)-induced lung injury model or P. aeruginosa infection, we observed that heterozygous deletion of Poldip2 nearly abolishes barrier dysfunction and extravasation of leukocytes into the lung, markedly improving survival. The effect of Poldip2 depletion on permeability is phenocopied in mice with endothelial- specific deletion of Poldip2. The phenotype of these animals is striking, suggesting that we have identified a novel and previously unappreciated major target for diseases related to endothelial barrier dysfunction. However, very little is known about the molecular mechanisms by which Poldip2 regulates endothelial permeability, and almost nothing has been published about how Poldip2 itself is regulated. To ultimately test the therapeutic potential of Poldip2 inhibitors, it is critical to first understand how Poldip2 depletion maintains the endothelial barrier. In this project, we will address these gaps in our knowledge, as well as develop small molecules with which to explore Poldip2’s function and therapeutic potential. In the first aim, we plan to define the mechanism by which endothelial Poldip2 regulates barrier function, focusing on the role of RhoA oxidation. In the second Aim, we will focus on post-translational modifications of Poldip2 as a novel mechanism of regulation, using mass spectrometry to identify modifications of interest and site-directed mutagenesis to test their effect on endothelial permeability. Finally, in Aim 3, we plan to investigate whether a putative pharmacological inhibitor of Poldip2 can be used experimentally to develop new, more specific inhibitors that will enable us to probe function and test whether pharmacological inhibition of Poldip2 can mitigate lung edema and inflammatory cell incursion in a mouse model of acute lung injury. Together, these three aims will allow us to gain new insight into the regulation and downstream signaling pathways of Poldip2, a novel therapeutic target for treatment of lung edema.