Inhibition of the Arp2/3 complex protects the endothelial barrier during inflammation in vivo

The endothelium, a monolayer of endothelial cells, forms a semi‐permeable barrier in the vasculature that separates blood from underlying tissues. During inflammation, this barrier is altered to increase endothelial permeability and allow for leukocyte recruitment. Such alterations critically depend on endothelial actin dynamics and actin‐binding proteins (ABP) to modify junction architecture and thus permeability. An ABP critical for actin remodeling is the Arp2/3 complex, which forms branched actin networks. The Arp2/3 complex needs to be tightly regulated which is achieved by activation via nuclear promoting factors (NPFs) and inhibition via proteins that locally antagonize the activity of NPFs such as the newly discovered arpin. However, effects of Arp2/3 complex regulation on endothelial barrier during inflammation are not well understood. We hypothesize that Arp2/3 regulation interferes with actin and junction remodeling which in turn may alter barrier functions. We use two different approaches to address this hypothesis. First, we perform permeability assays in vivo with the small molecule inhibitor CK‐666, which maintains Arp2/3 in an inactive conformation and blocks binding of NPFs; and second, by depleting the newly discovered endogenous Arp2/3 inhibitor arpin in endothelial cells. Treatment with CK‐666 in modified Miles permeability assays in Balb/c mice showed a protective effect against histamine‐induced hyperpermeability, suggesting that the Arp2/3 complex indeed is involved in maintaining the integrity of the endothelial barrier in vivo during inflammation. By contrast, CK‐666 did not affect permeability under basal conditions in vivo . Mechanistic studies to unravel the reason for this effect are currently under way. Next, we analyzed expression of arpin in different endothelial cell types and found that arpin is expressed at high levels in bend3, bend5, murine lung endothelial cells, and human umbilical vein endothelial cells (HUVEC). To analyze arpin functions in endothelial permeability regulation, HUVEC were transfected with lentiviruses encoding specific short hairpin RNAs (shRNAs) against arpin and scrambled control. Arpin‐depleted HUVEC were then selected using puromycin to generate stable clones. Western blot analysis revealed that arpin expression in HUVEC was consistently reduced by more than 85%. Of note, expression of the junctional proteins VE‐Cadherin, Claudin‐5, Occludin and ZO‐1 did not change in arpin‐depleted HUVEC with and without treatment with 15 ng/mL TNF‐α for 18 hours. Mechanistic studies are currently being performed to analyze localization of junctional proteins, the Arp2/3 complex, and the actin cytoskeleton. Our data suggest an important role of Arp2/3 complex regulation for endothelial barrier integrity during inflammation. Future studies will reveal the underlying molecular mechanisms controlling Arp2/3‐dependent control of the endothelial actin cytoskeleton and junction architecture. Support or Funding Information CONACyT This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .