The overall objective of this proposal is to understand how lung microvasculature acquires ?proinflammatory and leaky? phenotype during sepsis with a view to identifying viable therapeutic target to control sepsis-induced acute lung injury (ALI). Specifically, we will determine the role of Spleen Tyrosine Kinase (Syk) in the mechanism of lung vascular endothelial cell (EC) barrier disruption and inflammation and assess the therapeutic benefit of targeting Syk against ALI in mice with sepsis. The rationale for the study is based on our novel findings that Syk acts as a critical regulator of EC permeability and inflammation and that EC-restricted Syk knockout mice are markedly protected against LPS-induced lung vascular injury. We also have evidence that support the notion that Syk may exert its barrier disruptive effect via loss of cell surface vascular endothelial cadherin (VE-cadherin) to cause disassembly of adherens junctions, and its proinflammatory effect via recruitment of histone acetyltransferase (HAT) p300 to increase NF-?B signaling. Additionally our data show that Syk inhibitor R788 (fostamatinib), which has shown positive clinical benefits in rheumatoid arthritis, ameliorates lung tissue edema and improves survival in mouse models of sepsis. These new exciting findings have led us to hypothesize that Syk/VE-cadherin and Syk/p300 axes in the endothelium are critical components of lung vascular inflammation and injury, and that inhibiting Syk may be an effective therapeutic approach to control ALI in mice with sepsis.
Aim 1 will test the possibility that Syk causes disruption of endothelial adherens junctions (AJs) by mediating tyrosine phosphorylation and proteolysis/endocytosis of VE-cadherin.
Aim 2 will test the possibility that Syk regulates EC proinflammatory phenotype, leading to EC-neutrophil (PMN) interactions and transendothelial migration of PMN via recruitment of p300 which catalyzes acetylation of RelA to increase NF-?B signaling.
Aim 3 (i) will evaluate the causal role of endothelial Syk in inflammatory lung injury using EC-ablated Syk mice and (ii) evaluate the therapeutic potential of Syk inhibition to control ALI. We will use a combination of cellular, molecular, and biochemical approaches, and take advantage of EC-ablated Syk and mouse models of sepsis (cecal ligation puncture [CLP] or i.p. LPS challenge) to pursue these studies. The creative integration of in vitro and in vivo approaches will provide valuable mechanistic information concerning the role of Syk in ALI and may lead to novel therapeutic interventions involving inhibition of Syk to control ALI/ARDS associated with sepsis.
/ RELEVANCE TO PUBLIC HEALTH Acute lung Injury (ALI) is a common cause of respiratory failure in critically ill patients such as those with sepsis. Despite the use of state-of-the-art treatment, ALI is associated with an unacceptably high mortality rate and accounts for ~75000 deaths in the United States every year. All current therapies for ALI and its more severe form Acute Respiratory Distress Syndrome (ARDS) rely on supportive care to improve clinical outcome. No effective drugs have been developed. The proposed studies are designed to understand the role of Spleen Tyrosine Kinase (Syk) in the mechanism of lung vascular inflammation and injury with a view to identifying it as a critical target for modulation of ALI. Knowledge from this work may lead to development of Syk inhibition as a therapeutic strategy to control ALI/ARDS in sepsis.
