Nitric oxide (NO) is an important signaling molecule involved in the regulation of many physiological and pathological functions of the vascular endothelium. The objectives of this proposal are to investigate the mechanisms regulating endothelial NO synthase (eNOS) activity in intact microvessels under inflammatory conditions and their direct relationship with microvessel permeability. The hypothesis to be tested is that Ca2+/calmodulin (CaM) and heat shock protein 90 (hsp90) play antagonistic roles to caveolin-1 in the regulation of eNOS activity and microvessel permeability through competing protein-protein interactions in intact microvessels, and agonist-stimulated NO production is critical for increases in permeability under inflammatory conditions. Individually perfused intact venular microvessels in rat mesentery will be used to perform the proposed studies. The unique advantage of using single vessel perfusion technique over other whole animal approaches for this specific study is that it allows the NO effect on blood cell/endothelium interactions and on the changes in hemodynamics in the vasculature to be distinguished from its role in the regulation of microvessel permeability. Agonist-stimulated NO production will be quantitatively measured at levels of individual endothelial cells in intact microvessels with temporal and spatial resolution using fluorescence imaging. The role of Ca2+/CaM in the regulation of eNOS activity will be investigated by measuring corresponding changes in NO production when agonist-induced Ca2+ influx is modified following changes in electrochemical driving force for Ca2+ entry in endothelial cells. Our newly developed methods also allow a targeted regulatory protein for eNOS such as caveolin-1 or hsp90 to be internalized or genetically expressed in endothelial cel|s that form intact microvessels. In this proposal we combined our newly developed molecular approaches with our previously established techniques to investigate the mechanisms regulating eNOS activity in intact microvessels. Because of the nonuniformity nature of leaky site formation along the microvessel walls during acute inflammation, the spatial heterogeneity correlation between endothelial [Ca2+]i and NO production will be quantitatively evaluated at cellular levels in intact microvessels. The proposed research will provide new information that bridges the studies using whole animal, organ, or vascular beds and studies with purified proteins or endothelial cells in culture.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL084338-05
Application #
7996615
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Charette, Marc F
Project Start
2007-01-19
Project End
2013-12-31
Budget Start
2011-01-01
Budget End
2013-12-31
Support Year
5
Fiscal Year
2011
Total Cost
$329,625
Indirect Cost
Name
West Virginia University
Department
Physiology
Type
Schools of Medicine
DUNS #
191510239
City
Morgantown
State
WV
Country
United States
Zip Code
26506
Li, Xiang; Xu, Sulei; He, Pingnian et al. (2015) In vitro recapitulation of functional microvessels for the study of endothelial shear response, nitric oxide and [Ca2+]i. PLoS One 10:e0126797
Yuan, Dong; Xu, Sulei; He, Pingnian (2014) Enhanced permeability responses to inflammation in streptozotocin-induced diabetic rat venules: Rho-mediated alterations of actin cytoskeleton and VE-cadherin. Am J Physiol Heart Circ Physiol 307:H44-53
Xu, Sulei; Zhou, Xueping; Yuan, Dong et al. (2013) Caveolin-1 scaffolding domain promotes leukocyte adhesion by reduced basal endothelial nitric oxide-mediated ICAM-1 phosphorylation in rat mesenteric venules. Am J Physiol Heart Circ Physiol 305:H1484-93
Zhou, Xueping; Yuan, Dong; Wang, Mingxia et al. (2013) H2O2-induced endothelial NO production contributes to vascular cell apoptosis and increased permeability in rat venules. Am J Physiol Heart Circ Physiol 304:H82-93
Yuan, Dong; He, Pingnian (2012) Vascular remodeling alters adhesion protein and cytoskeleton reactions to inflammatory stimuli resulting in enhanced permeability increases in rat venules. J Appl Physiol 113:1110-20
Zhou, Xueping; He, Pingnian (2011) Improved measurements of intracellular nitric oxide in intact microvessels using 4,5-diaminofluorescein diacetate. Am J Physiol Heart Circ Physiol 301:H108-14
Zhou, Xueping; He, Pingnian (2011) Temporal and spatial correlation of platelet-activating factor-induced increases in endothelial [Ca²⁺]i, nitric oxide, and gap formation in intact venules. Am J Physiol Heart Circ Physiol 301:H1788-97
Zhou, Xueping; He, Pingnian (2010) Endothelial [Ca2+]i and caveolin-1 antagonistically regulate eNOS activity and microvessel permeability in rat venules. Cardiovasc Res 87:340-7
Zhang, Gengqian; Xu, Sulei; Qian, Yan et al. (2010) Sphingosine-1-phosphate prevents permeability increases via activation of endothelial sphingosine-1-phosphate receptor 1 in rat venules. Am J Physiol Heart Circ Physiol 299:H1494-504
He, Pingnian (2010) Leucocyte/endothelium interactions and microvessel permeability: coupled or uncoupled? Cardiovasc Res 87:281-90

Showing the most recent 10 out of 13 publications