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.