The pulmonary vasculature is a target for inflammation and thrombosis in conditions including acute lung injury (ALI), ischemia/reperfusions (I/R) and sepsis. The endothelial cell (EC) transmembrane glycoprotein, thrombomodulin (TM), controls these processes directly and via binding and "redirecting" the activities of thrombin, from fibrin formation and its pro-inflammatory ability to activate vascular cells, to activate protein C (APC) that inhibits coagulation and inflammation. Loss of TM in EC aggravates lung diseases. Infusions of recombinant APC and TM are helpful in treatment of some forms of ALI, but these drugs are rapidly eliminated, do not anchor onto the EC lumen, lack interaction with EC partners such as Endothelial Protein C Receptor (EPCR), and predispose to bleeding. We posit that anchoring TM on EC will provide more specific, comprehensive, efficient and safe effects and may be used to design advanced targeted therapeutic interventions. We fused a single-chain variable fragment (scFv) of Cell Adhesion Molecule PECAM-1 antibody with active recombinant TM or its domains (scFv/TM). In pilot studies, scFv/TM, but not TM, bound to the pulmonary EC and protected from thrombosis, inflammation and edema in mouse models of lung I/R and LPS/hyperoxia, without bleeding. In this grant application we will define mechanism(s) of effects of scFv/TM and its proposed mutated iterations that specifically target selected vascular areas and pathological pathways, in order to initiate translation of this promising novel therapeutic strategy into the clinical domain. We will pursue the following specific aims:
Aim 1 : Assess "benefit/risk" ratio of scFv/TM therapy of LPS/hyperoxia injury;
Aim 2 : Refine scFv/TM variants with precisely controlled selective activities;
Aim 3 : Define scFv/TM-EPCR cooperativity. This study will uncover molecular mechanisms of scFv/TM functions and guide design of targeted TM therapeutics tailored for selective treatment of inflammation or/and thrombosis in acute pulmonary and, likely, other diseases.

Public Health Relevance

Current treatment of the pathological conditions involving thrombosis and inflammation in lung diseases, including acute lung injury, hyperoxia, sepsis and ischemia/reperfusion are insufficient. We have designed a new agent comprising the endothelial anti-inflammatory and anti-thrombotic protein thrombomodulin (TM) genetically fused with an antibody fragment that anchors TM onto the endothelium. In this grant application we will study this novel agent and its proposed iterations in cell cultures and lab animals, in order to: i) dissect the mechanisms of its protective activities in lung diseases;and, ii) develop a novel specific and safe treatment for these diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL091950-04
Application #
8304940
Study Section
Lung Injury, Repair, and Remodeling Study Section (LIRR)
Program Officer
Moore, Timothy M
Project Start
2009-08-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
4
Fiscal Year
2012
Total Cost
$389,813
Indirect Cost
$142,313
Name
University of Pennsylvania
Department
Pharmacology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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