The long-term goals of this research are: 1) to understand the molecular mechanisms that govern the permeability and growth suppressive properties of vascular cell gap junctions and 2) to develop from that knowledge the rationale for gene therapies that target connexin expression in endothelial cells with the goal of limiting susceptibility to and facilitating recovery from ischemic injury. Three gap junction proteins are commonly expressed in vascular cells, connexin (Cx) 37, Cx40 and Cx43;in the endothelium Cx37 and Cx40 normally predominate, but during vasculogenesis and with stress, injury and disease, Cx37 is down-regulated and Cx43 up-regulated. The consequences of this change in expression on the vessel's ability to form new vessels and to maintain vessel functions during vascular remodeling remain uncertain. In our previous studies, we demonstrated that these connexins form gap junction channels with vastly different permselective and growth suppressive properties that are regulated by growth factor activated signaling cascades in a connexin- specific manner. In the current proposal we hypothesize that connexin-specific, phosphorylation-dependent regulation of junctional permselectivity provides vascular cells a strategy for maintaining coordinated contraction/relaxation functions of vessels while simultaneously supporting the proliferative response of cells therein. We address this hypothesis in Aim 1 by examining the mechanistic basis for how phosphorylation events in the carboxyl terminal domain (CT) lead to altered permselective properties of the associated pore domain.
In aim 2 we extend the observations of Aim 1 and determine whether the growth suppressive properties of these connexins rely on their permselective properties and/or their direct interactions with proteins involved in cell cycle control and progression. These growth studies make use of Cx-deficient cell lines, endothelial cells isolated from wild type or Cx37 deficient mice, and an in vivo hindlimb ischemic injury model, asking whether Cx37 works in conjunction with or in opposition to Cx43 to regulate the angiogenic response induced by injury while preserving junctional permselective properties. A combination of electrophysiology and fluorescence microscopy will be used to quantify the permselective properties of junctions and molecular approaches will be used to identify essential regions/sites of interaction between connexin domains and between connexins and elements of the cell cycle machinery. Isolated endothelial cells and an in vivo ischemia model will be used to determine the benefit of connexin expression or silencing to the extent and speed of vascular remodeling following injury. Our studies can be expected to lend new insights on the mechanistic basis for phosphorylation-dependent regulation of the permeability and growth suppressive functions of the vascular connexins and to the possible use of gene therapy to manipulate connexin expression in the endothelium to maximize/minimize angiogenesis, as appropriate, in settings of vascular injury and disease.

Public Health Relevance

Successful recovery of tissues from ischemic and traumatic injury requires restoration of blood flow through a process that involves regulated proliferation of vascular cells. Gap junctions and their comprising proteins, the connexins, serve as regulators of cell proliferation. Three connexins are expressed in vascular cells;we are studying how the function of these connexins and the intercellular communication channels they comprise is regulated in normal and pathological settings. Insights gained from our studies may help in the development of clinical strategies that will facilitate more rapid and complete recovery of tissue function following ischemic or traumatic injuries.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL058732-14
Application #
8209153
Study Section
Electrical Signaling, Ion Transport, and Arrhythmias Study Section (ESTA)
Program Officer
Srinivas, Pothur R
Project Start
1997-07-15
Project End
2014-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
14
Fiscal Year
2012
Total Cost
$378,750
Indirect Cost
$128,750
Name
University of Arizona
Department
Physiology
Type
Schools of Medicine
DUNS #
806345617
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Ek Vitorín, José F; Pontifex, Tasha K; Burt, Janis M (2016) Determinants of Cx43 Channel Gating and Permeation: The Amino Terminus. Biophys J 110:127-40
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Koval, Michael; Molina, Samuel A; Burt, Janis M (2014) Mix and match: investigating heteromeric and heterotypic gap junction channels in model systems and native tissues. FEBS Lett 588:1193-204
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Good, Miranda E; Ek-Vitorín, José F; Burt, Janis M (2014) Structural determinants and proliferative consequences of connexin 37 hemichannel function in insulinoma cells. J Biol Chem 289:30379-86
Nelson, Tasha K; Sorgen, Paul L; Burt, Janis M (2013) Carboxy terminus and pore-forming domain properties specific to Cx37 are necessary for Cx37-mediated suppression of insulinoma cell proliferation. Am J Physiol Cell Physiol 305:C1246-56
Fang, Jennifer S; Angelov, Stoyan N; Simon, Alexander M et al. (2013) Compromised regulation of tissue perfusion and arteriogenesis limit, in an AT1R-independent fashion, recovery of ischemic tissue in Cx40(-/-) mice. Am J Physiol Heart Circ Physiol 304:H816-27
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Ek-Vitorin, Jose F; Burt, Janis M (2013) Structural basis for the selective permeability of channels made of communicating junction proteins. Biochim Biophys Acta 1828:51-68

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