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 #
5R01HL088554-03
Application #
7744651
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Tolunay, Eser
Project Start
2008-01-01
Project End
2012-12-31
Budget Start
2010-01-01
Budget End
2010-12-31
Support Year
3
Fiscal Year
2010
Total Cost
$378,750
Indirect Cost
Name
University of Virginia
Department
Physiology
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Brown, Isola A M; Diederich, Lukas; Good, Miranda E et al. (2018) Vascular Smooth Muscle Remodeling in Conductive and Resistance Arteries in Hypertension. Arterioscler Thromb Vasc Biol 38:1969-1985
Biwer, Lauren A; Good, Miranda E; Hong, Kwangseok et al. (2018) Non-Endoplasmic Reticulum-Based Calr (Calreticulin) Can Coordinate Heterocellular Calcium Signaling and Vascular Function. Arterioscler Thromb Vasc Biol 38:120-130
Hong, Kwangseok; Cope, Eric L; DeLalio, Leon J et al. (2018) TRPV4 (Transient Receptor Potential Vanilloid 4) Channel-Dependent Negative Feedback Mechanism Regulates Gq Protein-Coupled Receptor-Induced Vasoconstriction. Arterioscler Thromb Vasc Biol 38:542-554
Kuhn, Viktoria; Diederich, Lukas; Keller 4th, T C Stevenson et al. (2017) Red Blood Cell Function and Dysfunction: Redox Regulation, Nitric Oxide Metabolism, Anemia. Antioxid Redox Signal 26:718-742
Begandt, Daniela; Good, Miranda E; Keller, Alex S et al. (2017) Pannexin channel and connexin hemichannel expression in vascular function and inflammation. BMC Cell Biol 18:2
Biwer, L A; Isakson, B E (2017) Endoplasmic reticulum-mediated signalling in cellular microdomains. Acta Physiol (Oxf) 219:162-175
Keller 4th, T C Stevenson; Butcher, Joshua T; Broseghini-Filho, Gilson BrĂ¡s et al. (2016) Modulating Vascular Hemodynamics With an Alpha Globin Mimetic Peptide (Hb?X). Hypertension 68:1494-1503
Ivanov, Stoyan; Scallan, Joshua P; Kim, Ki-Wook et al. (2016) CCR7 and IRF4-dependent dendritic cells regulate lymphatic collecting vessel permeability. J Clin Invest 126:1581-91
Good, Miranda E; Begandt, Daniela; DeLalio, Leon J et al. (2016) Small Interfering RNA-Mediated Connexin Gene Knockdown in Vascular Endothelial and Smooth Muscle Cells. Methods Mol Biol 1437:71-82
Liu, Shufeng; DeLalio, Leon J; Isakson, Brant E et al. (2016) AXL-Mediated Productive Infection of Human Endothelial Cells by Zika Virus. Circ Res 119:1183-1189

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