Vascular leakage is a hallmark of the inflammatory response. Acute changes in endothelial permeability are due to posttranslational changes in intercellular adhesion and cytoskeleton proteins. However, little is known about the mechanisms leading to long-term changes in vascular permeability. Here, we show that interleukin-6 (IL-6) promotes an increase in endothelial monolayer permeability that lasts over 24 h and demonstrate that activation of Src and MEK/ERK pathways is required only for short-term increases in permeability, being dispensable after 2 h. In contrast, Janus kinase (JAK)-mediated STAT3 phosphorylation at Y705 (but not S727) and de novo synthesis of RNA and proteins are required for the sustained permeability increases. Loss of junctional localization of VE-cadherin and ZO-1 is evident several hours after the maximal IL-6 response, thus suggesting that these events are a consequence of IL-6 signaling, but not a cause of the increased permeability. Understanding the mechanisms involved in sustaining vascular permeability may prove crucial to allow us to directly target vascular leakage and minimize tissue damage, thus reducing the rates of mortality and chronic sequelae of excessive edema. Targeting endothelial-specific mechanisms regulating barrier function could provide a new therapeutic strategy to prevent vascular leakage while maintaining the immune response and other beneficial aspects of the inflammatory response that are required for bacterial clearance and tissue repair.

Airway smooth muscle cell (ASMC) remodeling contributes to the structural changes in the airways that are central to the clinical manifestations of asthma. Ca2+ signals play an important role in ASMC remodeling through control of ASMC migration and hypertrophy/proliferation. Upregulation of STIM1 and Orai1 proteins, the molecular components of the store-operated Ca2+ entry (SOCE) pathway, has recently emerged as an important mediator of vascular remodeling. However, the potential upregulation of STIM1 and Orai1 in asthmatic airways remains unknown. An important smooth muscle migratory agonist with major contributions to ASMC remodeling is the platelet-derived growth factor (PDGF). Nevertheless, the Ca2+ entry route activated by PDGF in ASMC remains elusive. Here, we show that STIM1 and Orai1 protein levels are greatly upregulated in ASMC isolated from ovalbumin-challenged asthmatic mice, compared to control mice. Furthermore, we show that PDGF activates a Ca2+ entry pathway in rat primary ASMC that is pharmacologically reminiscent of SOCE. Molecular knockdown of STIM1 and Orai1 proteins inhibited PDGF-activated Ca2+ entry in these cells. Whole-cell patch clamp recordings revealed the activation of Ca2+ release-activated Ca2+ (CRAC) current by PDGF in ASMC. These CRAC currents were abrogated upon either STIM1 or Orai1 knockdown. We show that either STIM1 or Orai1 knockdown significantly inhibited ASMC proliferation and chemotactic migration in response to PDGF. These results implicate STIM1 and Orai1 in PDGF-induced ASMC proliferation and migration and suggest the potential use of STIM1 and Orai1 as targets for ASMC remodeling during asthma.

Maintaining VE-cadherin levels by inhibiting its endocytosis through p120-catenin binding is not sufficient for forming a restrictive barrier. Instead, p120-catenin binding to VE-cadherin is required to allow tyrosine-phosphorylated VE-cadherin to contribute to barrier formation.

Vascular endothelial cadherin (VE‐cad) is an Adherens Junction (AJ) transmembrane protein found at the cell‐cell junction in endothelial cells (EC) which participates in cell‐cell adhesion. The juxtamembrane domain of VE‐cad binds to p120 to regulate the levels of VE‐cad. Previously, we found that depletion of p120 using shRNA results in a decrease in the level of VE‐cad and a decrease in monolayer integrity as assessed by trans‐endothelial electrical resistance (TEER). Re‐expression of p120 can restore both the decrease in TEER and VE‐cad levels. In contrast, re‐expression of VE‐cad to normal levels in the absence of p120 will not rescue TEER even though VE‐cad is found at cell‐cell junctions. We hypothesize that re‐expression of VE‐cad does not rescue TEER in the absence of p120 due to an increase in VE‐cad endocytosis. To test this hypothesis, we expressed a VE‐cad mutant (DEE‐VE‐cad) that has limited endocytosis even in absence of p120 binding. Expression of DEE‐VE‐cad, following p120 depletion, was unable to rescue TEER even though the levels of DEE‐VE‐cad were equal to or above levels of endogenous VE‐cad and was localized at the cell‐cell junction. These results indicate that prevention of VE‐cad endocytosis is not the only mechanism by which p120 regulates the formation of a mature AJ in EC monolayers.

New growth of blood vessels, angiogenesis, is essential in cancer development since the proliferation, as well as metastatic spread, of cancer cells depends on an adequate supply of oxygen and nutrients and the removal of waste products. Endothelial cells, which line the inside of the entire vasculature, play an important role in angiogenesis. Specifically, endothelial cells detach from an existing blood vessel by down‐regulating their junctional cell‐cell adhesion protein complexes, they migrate away from the existing blood vessel, and they proliferate, all in an attempt to form a new blood vessel. Resolvin D1 (RvD1) is an autocoid molecule which possesses the ability to strengthen endothelial cell‐cell adhesion leading us to hypothesize that RvD1 may inhibit some of necessary steps for angiogenesis to occur. Here we show that treatment of endothelial monolayers with RvD1 increases the amount of junctional vascular endothelial‐cadherin (VE‐Cad) and slows endothelial cell migration. We also found that RvD1 slightly decreased endothelial cell proliferation. Taken together, we found that treatment of the endothelium with RvD1 inhibits several of the essential steps required for angiogenesis to occur. This suggests that RvD1 may be a useful therapeutic in the treatment cancer as it may decrease angiogenesis, preventing cancer cell proliferation and metastasis.Support or Funding InformationThis work was supported in part through the Hartwick College Faculty research grants program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.