Date of Award

5-2016

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Edmund Miller

Abstract

Sepsis remains a significant source of morbidity and mortality in critically ill patients.  Acute  respiratory distress  syndrome  (ARDS)  is  a  common  clinical presentation of sepsis, a sign of organ failure and an important prognostic factor. Treatment strategies are largely supportive. Cytokine storm and vascular instability are two characteristic features of sepsis and their role in the pathophysiology is still poorly understood. Evidence suggests that there is a link between exocytosis, a driving force of cytokine storm, and cytoskeletal stability, a determinant of vascular function. N-ethylmaleimide sensitive factor (NSF) is a critical component of endothelial trafficking machinery and a regulator of cytoskeleton. Angiopoietin-2 (Ang-2) is an inflammatory mediator and permeability inducing factor, known to be secreted by machineries involving NSF, and the plasma levels closely correlate with sepsis and ARDS morbidity and mortality. We therefore tested the hypothesis that NSF is a common regulator of 1) cytokine storm and 2) vascular instability that are important in sepsis pathophysiology. To test this hypothesis we developed a Gram positive, acute lung injury mouse model that also demonstrates key chemical and physiologic features of sepsis. In this model, lipoteichoic acid (LTA)-peptidoglycan (PGN), Gram positive bacteria cell wall components, are instilled intracheally to induce alveolar inflammation which subsequently induces a cytokine storm and vascular instability.

Transactivating  regulatory  protein  (TAT)-NSF700,  a  synthetic  peptide inhibitor of NSF, had no significant effect on the local, alveolar inflammation. However, it had a significant effect on systemic inflammation, evidenced by cytokine storm. We also investigated secretory mechanisms of Ang-2 in vitro with a clinically relevant human pulmonary endothelial cell line (HPMEC-ST1.6R). We identified two distinctive secretory pathways of Ang-2, constitutive and LTA-PGN regulated. Constitutive secretion resulted in a slow but continuous increase in Ang-2 in culture medium over time. It was regulated by cAMP-protein kinase A (PKA)-Ca2+ and nitric oxide (NO)-cGMP-protein kinase G (PKG)-Ca2+ pathways and partially regulated by NSF-Ca2+ pathways. Notably, nonspecific inhibition of phosphodiesterase (PDE) resulted in augmentation of cAMP-PKA-Ca2+ and NO-cGMP-PKG-Ca2+, as a result, significant elevation in Ang-2 extracellular concentration. It suggests that constitutive pathway may play an active role in certain pathologic conditions. LTA-PGN stimulation caused a rapid (< 2 min) and potent increase followed by gradual decrease of Ang-2. It was partially regulated by both Ral A- phospholipase D (PLD) and NSF-Ca2+ pathways.

In vivo, TAT-NSF700 pre-treatment significantly improved LTA-PGN induced oxygen desaturation. This was without any improvement in respiratory rate or heart rate. TAT-NSF700 also prevented LTA-PGN induced pulse distention (a measurement of carotid artery vessel diameter) change. Together, it suggests that TAT-NSF700 effect of oxygen saturation improvement is most likely secondary to favorable vascular stability and local tissue perfusion. In vitro, TAT-NSF700 pre-treatment reduced LTA-PGN induced myosin light chain (MLC) phosphorylation in HPMEC-ST1.6R supporting the in vivo data interpretation that TAT-NSF700 has a stabilizing effect on endothelial cell cytoskeleton.

These findings indicate that TAT-NSF700 has an inhibitory effect on development of cytokine storm and vascular instability, potentially important mechanisms involved in pathophysiology of Gram positive sepsis. Future studies should address the potential therapeutic use of NSF inhibition to reduce the morbidity and mortality associated with sepsis.

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