Submitted on February 6, 2003
Accepted on July 30, 2003
Localization of the permeability barrier to solutes in isolated arteries by confocal microscopy
Paul M. A. van Haaren1, Ed VanBavel1, Hans Vink1, and Jos A. E. Spaan1*
1 Department of Medical Physics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
* To whom correspondence should be addressed. E-mail: j.a.spaan{at}amc.uva.nl.
Endothelial cells are covered by a surface layer of membrane associated proteoglycans, glycosaminoglycans, glycoproteins, glycolipids, and associated plasma proteins. This layer may limit transendothelial solute transport. We determined dimension and transport properties of this endothelial surface layer in isolated arteries. Rat mesenteric small arteries (d~150 µm) were isolated and cannulated with a double-barreled 
-pipet on the inlet side and a regular pipet on the outlet side. Dynamics and localization of intra-arterial fluorescence by fluorescein-isothiocyanate (FITC)-labeled dextrans (FITC-
s) and the endothelial membrane dye DiI were determined with confocal microscopy. Large FITC- (148 kD) filled a core volume inside the arteries within 1 minute, but was excluded from a 2.6 ± 0.5 µm wide region on the luminal side of the endothelium during 30 minutes of dye perfusion. Medium-sized FITC- (50.7 kD) slowly penetrated this endothelial surface layer (ESL) within 30 minutes but did not permeate into the arterial wall. Small FITC- (4.4 kD) quickly passed the ESL and accumulated in the arterial wall. Prolonged luminal fluorochrome illumination with a bright mercury lamp destroyed the ~3 µm exclusion zone for FITC-148 within a few minutes. This study demonstrates the presence of a thick endothelial surface layer that contributes to the permeability barrier to solutes. The layer is sensitive to phototoxic stress and its damage could form an early event in atherosclerosis.
