Vascular smooth muscle cells (VSMC) and endothelial cells (EC) in the resistance vessels are functionally linked, and the point of contact between the two cells, the myoendothelial junction (MEJ), plays a key role in many elements of vascular function. However, the location and size of the MEJ have made it extremely difficult to study in vivo. We have developed a model of the MEJ by co-culturing VSMC and EC that show connexin- dependent dye transfer and a coupled pool of Ca2+. Moreover, the mode of intercellular calcium signaling depends on which of the two cell types is stimulated, i.e. intercellular second messenger signaling is polarized, and this appears to mimic signaling patterns seen in vivo. We propose to determine the structural and molecular basis for the intercellular coupling that occurs at the MEJ, which we believe has important implications for the operation of the MEJ in vivo. We will determine which connexins and second messengers are involved in intercellular signaling, and will test whether polarization of calcium communication is determined by selective permeability of the gap junctions at the MEJ or differential expression of second messenger receptors at the MEJ in the two cell types. We propose 3 specific experimental aims:
Specific Aim 1 - Use light and electron microscopy-based immunocytochemistry to assess and compare the in vivo and in vitro placement of: a.) connexins, b.) ryanodine receptors, and c.) inositol 1,4,5 triphosphate-receptors at the MEJ;
Specific Aim 2 - Measure the effect of modification of the gap junctional connexin composition on: a.) Ca2+ and b.) inositol 1,4,5 triphosphate -mediated intercellular signaling;and lastly, Specific Aim 3 - Assess the effects of cell specific deletion of a) ryanodine receptors and b) inositol 1,4,5 triphosphate-receptors on polarized calcium signaling. Our experiments should enhance understanding of the coordination of VSMC and EC and will provide insights into basic questions of vasomotor control and a variety of pathophysiological responses. The methods by which vascular cells communicate are key for understanding vascular processes such as hypertension and control of blood flow. Our model and the experiments proposed offer the first opportunity to investigate the capabilities of the myoendothelial junction and to understand its role in the vessel wall. We propose to study the ways in which vascular cells utilize this structure to maintain vascular function.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL088554-05
Application #
8208057
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Gao, Yunling
Project Start
2008-01-01
Project End
2014-06-30
Budget Start
2012-01-01
Budget End
2014-06-30
Support Year
5
Fiscal Year
2012
Total Cost
$374,963
Indirect Cost
$127,463
Name
University of Virginia
Department
Physiology
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Billaud, Marie; Lohman, Alexander W; Johnstone, Scott R et al. (2014) Regulation of cellular communication by signaling microdomains in the blood vessel wall. Pharmacol Rev 66:513-69
Butcher, Joshua T; Johnson, Tyler; Beers, Jody et al. (2014) Hemoglobin ? in the blood vessel wall. Free Radic Biol Med 73:136-42
Lohman, Alexander W; Isakson, Brant E (2014) Differentiating connexin hemichannels and pannexin channels in cellular ATP release. FEBS Lett 588:1379-88
Straub, Adam C; Butcher, Joshua T; Billaud, Marie et al. (2014) Hemoglobin ?/eNOS coupling at myoendothelial junctions is required for nitric oxide scavenging during vasoconstriction. Arterioscler Thromb Vasc Biol 34:2594-600
Isakson, Brant E; Thompson, Roger J (2014) Pannexin-1 as a potentiator of ligand-gated receptor signaling. Channels (Austin) 8:118-23
Straub, Adam C; Zeigler, Angela C; Isakson, Brant E (2014) The myoendothelial junction: connections that deliver the message. Physiology (Bethesda) 29:242-9
Billaud, Marie; Johnstone, Scott R; Isakson, Brant E (2012) Loss of compliance in small arteries, but not in conduit arteries, after 6 weeks exposure to high fat diet. J Cardiovasc Transl Res 5:256-63
Billaud, Marie; Lohman, Alexander W; Straub, Adam C et al. (2012) Characterization of the thoracodorsal artery: morphology and reactivity. Microcirculation 19:360-72
Heberlein, Katherine R; Han, Jenny; Straub, Adam C et al. (2012) A novel mRNA binding protein complex promotes localized plasminogen activator inhibitor-1 accumulation at the myoendothelial junction. Arterioscler Thromb Vasc Biol 32:1271-9
Straub, Adam C; Billaud, Marie; Johnstone, Scott R et al. (2011) Compartmentalized connexin 43 s-nitrosylation/denitrosylation regulates heterocellular communication in the vessel wall. Arterioscler Thromb Vasc Biol 31:399-407

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