Approaches to visualising myoendothelial connections using laser scanning confocal microscopy

Hamill, C., Gonzalez, J.M., Methven, L., Daly, C.J. and McGrath, J.C. (2008) Approaches to visualising myoendothelial connections using laser scanning confocal microscopy. Journal of Vascular Research, 45(Supple), p. 8. (doi: 10.1159/000113929)

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Introduction: Endothelial cells connect to smooth muscle cells through fenestrations in the internal elastic lamina, an elastic membrane that separates the two vascular layers. These cellular projections traversing the IEL are the only points of contact between endothelial and smooth muscle cells and bring the cell membranes into close apposition allowing for the formation of myoendothelial gap junctions (MEGJs). In rat mesenteric vessels it has been proposed that MEGJs are involved in the EDHF response, be it electrical or chemical.1,2 Previously, cellular projections that traverse the IEL and their associated gap junction plaques have been shown using electron microscopy.3 This is the first study to develop a technique to allow the visualisation of myoendothelial connections passing through fenestrae in the IEL of pressurised arteries. Methods and Results: Third order mesenteric resistance arteries from 6 month old WKY rats were pressure fixed (70 mmHg) and stained using the membrane dye FM4-64. Rings of 10 μm thickness were cut from these stained arteries and visualised using laser scanning confocal microscopy (LSCM), exploiting the auto fluorescent properties of elastin in combination with the membrane stain. This allowed simultaneous imaging of fenestrae, defined as dark gaps in the autofluorescent lamina, and the membrane stained cellular projections passing through. Further techniques are in development combining CLSM and pressure myography to study the transfer of luminally loaded fluorescent compounds in a live vessel. Calcein AM is a cell-permeant cytoplasmic dye that is converted to its fluorescent cell-impermeant form by esterases. Calcein AM was loaded luminally and taken up by endothelial cells and smooth muscle cells, with its presence in smooth muscle cells being suggestive of gap-junctional transfer. Preliminary work has been performed using a combination of the gap junction inhibitors 37,43Gap27 and 40Gap27 with luminally loaded Calcein AM and shows that these inhibitors prevent the transfer of fluorescent Calcein AM from endothelial cells to smooth muscle cells. Conclusion: Fluorescent dye transfer experiments with gap junction inhibitors is a novel approach to studying GJ function, utilizing CLSM, and has the advantage over electron microscopy of being in near physiological conditions in the live pressurized vessel. Previous work has shown that fenestrae area is significantly smaller in SHR compared with WKY at 6 months,4 suggesting that myoendothelial connectivity would be impaired in hypertension due to the smaller space through which the cells can connect. This preliminary work of establishing methods will form the basis for subsequent studies comparing myoendothelial connections in WKY and SHR at different age points to determine if reduced myoendothelial connectivity could account for the reduced EDHF response in the SHR strain.

Item Type:Articles
Additional Information:Suppl. 1. Resistance Arteries
Glasgow Author(s) Enlighten ID:McGrath, Professor John and Methven, Dr Laura and Daly, Professor Craig
Authors: Hamill, C., Gonzalez, J.M., Methven, L., Daly, C.J., and McGrath, J.C.
College/School:College of Medical Veterinary and Life Sciences > School of Life Sciences
Journal Name:Journal of Vascular Research
Journal Abbr.:J. Vasc. Res.
ISSN (Online):1423-0135

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