Amelioration of Sepsis by TIE2 Activation- Induced Vascular Protection Jenn Hou Burke Group Literature Seminar June 18 th 2016
Sepsis Globally, more than 19 million people are affected each year Causes mortality in half of sepsis patients Unregulated immune response to bacterial and viral infections Hallmarks: massive inflammation, blood vessel dilation, vascular leakage, multipleorgan failure (heart, kidney, lungs and/or liver) Risk Factors: Immuno-compromised (age, chemotherapy, HIV) ICU hospitalization Bodily injury and wounds Catheterization Image from: https://my.vanderbilt.edu/sepsismonitor/files/2013/10/sirs.jpg Image from: http://www.westvalleytrauma.com/2015/03/sepsis-in-the-trauma-patient/ https://www.nlm.nih.gov/medlineplus/sepsis.html http://global-sepsis-alliance.org/global-burden/ http://www.mayoclinic.org/diseases-conditions/sepsis/symptoms-causes/dxc-20169787
The Pathophysiology of Sepsis Parikh SM (2016) Targeting Tie2 and the host vascular response in sepsis. Science Translational Medicine. 8(335): 1-3. Brindle NPJ, Saharinen P, Alitalo K (2006) Signaling and functions of angiopoitein-1 in vascular protection. Circulation Research. 98: 1014-1023.
ANG2-Binding and TIE2-Activating Antibody (ABTAA) Associates with ANG2 to Promote TIE2 Activation Can ABTAA bind to ANG2 to trigger TIE2 phosphorylation in cells? K D =0.2nM K D =78pM Confluent, serum starved HUVEC ABTAA binding to ANG2 promotes TIE2 activation and cell-cell localization in vitro.
ABTAA + ANG2 Promote Downstream TIE2 Signaling How does ABTAA + ANG2 binding affect cell signaling? Confluent, serum starved HUVEC
TIE2 Knockdown Abrogates ABTAA Mediated AKT and ERK Phosphorylation How does the loss of function of TIE2 affect AKT and ERK signaling? TIE2 function is critical for downstream signaling.
ABTAA Complexes with ANG2 and TIE2 Is ANG2 required for ABTAA and TIE2 interactions? Size Exclusion Chromatography A2D: ANG2 s fibrinogen-like domain T2E: TIE2 s extracellular domain A2D and T2E associate 1:1 Non-Reducing SDS-PAGE Multi-Angle Light Scattering Enzyme Linked Immunosorbent Assay
ABTAA Promotes ANG2 Oligomerization Hypothesis: ABTAA can interact with multiple ANG2 to enhance its oligomerization. 1330 kd 998 kd 677 kd
How Does ABTAA Affect Survival in a Murine Sepsis Model? High-Grade Cecal Ligation and Puncture (CLP) Hypothesis: ABTAA will improve the survival of septic mice compared to ABA. Treat mice with Fc, ABA, or ABTAA 6 to 18 hours post-clp. Track progress of mice in blind study. Image from: http://www.nature.com/nrd/journal/v4/n10/images/nrd1854-f3.jpg Image from: http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit5/innate/u1fig26l1.html Image from: https://en.wikipedia.org/wiki/large_intestine#/media/file:blausen_0604_largeintestine2.png
ABTAA Improves the Survival of Septic Mice Post-CLP ABTAA: 40% survival ABA: 13% survival How does co-administration of ABTAA and broad spectrum antibiotics impact survival outcomes? Imipenem: 20% survival ABTAA + Imipenem: 70% survival
Pre-Administration of ABTAA Prior to CLP Provides Protective Effects in Mice Does pretreatment with ABTAA improve survival in septic mice? ABTAA pre-treat: 50% survival ABA pre-treat: 17% survival Higher doses of ABA does not improve septic mouse survival.
ABTAA Rescues Mice from Endotoxemia and Bacteremia How general is the protective effect of ABTAA against two alternative mouse models of sepsis? Structure of Lipopolysaccharide ABTAA: 63% survival ABA: 33% survival Fc: 28% survival ABTAA: 55% survival ABA: 9% survival Fc: 8% survival Image from: https://www.researchgate.net/figure/233962820_fig1_figure-1-lipopolysaccharide-lps-a-schematic-diagram-of-the-structure-of-lps-b
How Does Loss of Function of ABTAA s Binding Partners Affect Its Efficacy Against Sepsis? Loss of ANG1 accelerates death. ANG2 antagonizes TIE2. ABTAA association with TIE2 is required for survival.
ABTAA Protects Against Acute Lung Injury by Decreasing Leakage Experimental Set-Up
ABTAA Protects Against Acute Lung Injury by Maintaining Pericyte Coverage CLP was performed, and mice were treated 6 and 18 hours afterwards. Pericyte coverage = NG2 area /CD31 area ABTAA activates TIE2 to prevent EC-pericyte dissociation. Bergers G, Song S. (2005) The role of pericytes in blood-vessel formation and maintenance. Neuro. Oncol. 7(4): 452-464. Huang H et al. (2010) Targeting the ANGPT-TIE2 pathway in malignancy. Nature Reviews Cancer. 10: 575-585.
ABTAA Protects Against Sepsis by Retaining Endothelial Glycocalyx Integrity Thickness of Subpleural Microvasculature Endothelial surface layer
ABTAA Protects Against Sepsis by Retaining Endothelial Glycocalyx Integrity ABTAA reduces the expression of heparanase to maintain blood vessel wall integrity.
ABTAA Protects Against TNF-α Mediated Vascular Leakage
ABTAA Reduces Proinflammatory Cytokine and ANG2 Levels Damas P et al. (1992) Cytokine serum level during severe sepsis in human IL-6 as a marker of severity. Ann. Surg. 215(4): 356-362.
Proposed Model of ABTAA Mediated Amelioration of Sepsis
Conclusions ABTAA binds to ANG2 to activate TIE2 receptors and trigger downstream phosphorylation. ABTAA rescues mice in three separate sepsis models. Co-administration of ABTAA with antibiotics further improves septic mouse survival. ABTAA alleviates septic severity by protecting against vascular leakage, reducing lung edema, maintaining glycocalyx integrity, and decreasing proinflammatory signals. ABTAA could act as a viable new therapeutic candidate for the prophylaxis of and treatment of sepsis.