Doctor of Philosophy
Physiology and Pharmacology
Gill, Sean E.
Sepsis is a life-threatening disease characterized by a dysregulated immune response to infection. Septic organ dysfunction is due to endothelial cell (EC) injury, including pulmonary microvascular ECs (PMVEC), resulting in loss of barrier function. Metalloproteinases, including matrix metalloproteinases (MMP), a disintegrin and metalloproteinases (ADAMs), and ADAMs with thrombospondin motifs (ADAMTS), may contribute to EC barrier dysfunction through cleavage of cell-cell junctions. The activity of metalloproteinases is regulated by tissue inhibitors of metalloproteinases (TIMPs). Although, previous studies suggest that metalloproteinases can cleave junctional proteins and thus disrupt the PMVEC barrier, the specific role of individual metalloproteinases in septic PMVEC barrier disruption is not known. I hypothesize that the inhibition of metalloproteinase activity by TIMPs supports normal EC barrier function. Moreover, loss of metalloproteinase inhibition leads to septic EC barrier dysfunction through inter-EC junctional protein degradation.
Isolated mouse and human PMVEC were examined under basal (PBS) and septic (mouse: cytomix [equimolar tumour necrosis factor α, interferon γ, and interleukin 1β] + lipopolysaccharide [LPS]; human: cytomix) conditions. Gene expression was assessed using qRT-PCR (mouse) and RNA-Seq (human), and metalloproteinase activity was assessed using substrate-based assays. PMVEC permeability was quantified using Evans blue-labelled albumin, and surface localization of vascular endothelial (VE)-cadherin (adherens junction) and claudin 5 (tight junction) was assessed by immunofluorescence.
Septic stimulation as well as the loss of TIMP3 led to disruption of inter-PMVEC junctional proteins and barrier dysfunction. ADAM17 activity was increased in the absence of TIMP3 and under septic conditions. Mmp13 expression and activity was increased in septic mouse PMVEC and the expression of ADAMTSs was increased in septic human PMVEC. Generally, treatment of septic mouse and human PMVEC as well as mouse PMVEC lacking TIMP3 with broad and selective metalloproteinase inhibitors reduced septic permeability and disruption of inter-PMVEC junctional proteins.
Collectively, my findings indicate that the expression and activity of specific metalloproteinases mediates PMVEC barrier dysfunction and that TIMP3 supports barrier function through inhibition of metalloproteinases.
Summary for Lay Audience
Sepsis is a serious disease associated with an infection that currently has no effective treatment. After an infection, out-of-control inflammation occurs that leads to damage to cells lining small blood vessels, called microvascular endothelial cells. Damage to these cells and the blood vessels results in increased blood vessel leak and tissue swelling leading to the failure of major organs in our body. The family of enzymes called metalloproteinases are responsible for cutting proteins that attach endothelial cells to each other, causing blood vessels to leak protein and fluid, which can be especially problematic if metalloproteinases are overactive. My work demonstrated that the activity of certain metalloproteinases increased in sepsis leading to blood vessel leak and that the loss of one of the inhibitors of metalloproteinases also led to increased leak. By using synthetic inhibitors to block the activity of metalloproteinases, I was able to protect endothelial cells and reduce protein leak during sepsis.
Jayawardena, Devika P., "Regulation of Metalloproteinase Activity is Critical for Pulmonary Microvascular Endothelial Cell Barrier Function" (2023). Electronic Thesis and Dissertation Repository. 9265.
Available for download on Wednesday, April 30, 2025