Reactive oxygen species (ROS) are broadly implicated in the pathogenesis of cardiovascular disease (CVD). ROS-mediated vascular dysfunction occurs, in part, via inactivation of the vasodilator nitric oxide (NO) by ROS superoxide anion and/or direct downstream signaling pathways promoting vasoconstriction. A major source of vascular ROS is the NADPH oxidases or Nox proteins. The matricellular protein thrombospondin-1 (TSP1) is significantly elevated in the vasculature in CVD and is associated with vascular dysfunction. We reported that TSP1, via its cognate receptor CD47, inhibits vasodilatation, however the exact mechanism remain unclear. In addition to CD47, vascular smooth muscle cells (VSMCs) also express signal regulatory protein alpha (SIRP- ?), a membrane receptor protein that has been linked to ROS production in inflammatory cells, but SIRP-?'s role in VSMC ROS is entirely unknown. Ischemia reperfusion (I/R) is a disease in which increased ROS leads to impairment in vascular flow. The mouse hind-limb preparation is a widely-accepted I/R model and previous data from our laboratory show that CD47 blockade protects vessels from I/R-associated flow impairment. SIRP-?'s role in I/R is not known. Preliminary data show that TSP1 potently stimulates (1) Nox-derived superoxide anion production in VSMCs via CD47;(2) VSMC hydrogen peroxide via SIRP-?-dependent signaling;and (3) ROS-mediated vascular tone dysfunction. These findings inform our overarching hypothesis that TSP1 promotes ROS production in VSMCs via CD47- and SIRP-?-dependent signaling, leading to marked impairment in vascular relaxation and/or enhanced constriction in I/R. This wholly innovative proposal investigates via multi-faceted novel actions of TSP1 on distinct synergizing receptor/signaling pathways, leading to pathological ROS formation and I/R-induced vascular dysfunction. This will be tested via the following aims: (1) examining for the first time whether TSP1 binding to VSMC CD47 increases superoxide anion levels via G-protein activation and mitogen-activated kinase pathways and, in turn, Nox activation;(2) interrogating for the first time whether SIRP-?, and SHP-1/2 signaling, plays a role in TSP1-induced hydrogen peroxide production in VSMCs and via Nox;(3) exploring in vivo whether CD47 and SIRP-? activation lead to decreased blood flow in the mouse hind limb I/R model. Based on a strong foundation of preliminary findings, the current proposal employs multiple molecular and genetic tools to explore (a) a novel role for matricellular protein TSP1, in vascular ROS production via CD47 and SIRP-?;(b) novel downstream mediators and oxidase sources involved;and, in turn, identify novel therapeutic targets in I/R-induced vascular injury. This research plan is novel at al levels and has potential implications for the role of myriad other matricellular proteins in CVD.

Public Health Relevance

In patients suffering from heart attack, stroke, and tissue trauma as well as complications from organ transplantation, ischemia-reperfusion (I/R) injury of blood vessels is a serious sequela. A major contributor to this injury is reactive oxygen species generated upon the restoration of oxygenated blood to vessels. Despite this knowledge, existing treatments for I/R injury are limited. This project aims to open an entirely new field of inquiry b (a) unraveling completely novel agonists and signaling pathways leading to I/R-associated vascular oxidant production;and (b) identifying previously unknown therapeutic targets to mitigate vascular I/R dysfunction.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL112914-02
Application #
8720053
Study Section
Special Emphasis Panel (ZRG1-VH-J (03))
Program Officer
Charette, Marc F
Project Start
2013-08-13
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
2
Fiscal Year
2014
Total Cost
$376,993
Indirect Cost
$131,993
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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