Quantitative Laser Scanning Confocal Microscopy on Single Capillaries: Permeability Measurement

Adamson, Roger H.; Lenz, J. F.; Curry, F. E.

doi:10.3109/10739689409146752

Cited by 56 publications

(53 citation statements)

References 21 publications

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“…Model interpretation of these enzyme data resulted in an estimate of ESL thickness up to 7 m (21), roughly a factor of 3 larger than found presently from more direct observations of dye distributions. However, permeability studies are usually limited to a time period of ϳ2 min (2,(19)(20)(21)25), whereas fluorescence profiles are still significantly changing after this time, as is clear from our experiments.…”

Section: Discussionsupporting

confidence: 57%

Localization of the permeability barrier to solutes in isolated arteries by confocal microscopy

Haaren

vanBavel

Vink

et al. 2003

American Journal of Physiology-Heart and Circulatory Physiology

131

104

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. Localization of the permeability barrier to solutes in isolated arteries by confocal microscopy. Am J Physiol Heart Circ Physiol 285: H2848-H2856, 2003. First published August 7, 2003 10.1152/ajpheart.00117.2003.-Endothelial cells are covered by a surface layer of membraneassociated 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 (ESL) in isolated arteries. Rat mesenteric small arteries (diameter ϳ150 m) were isolated and cannulated with a double-barreled -pipette on the inlet side and a regular pipette on the outlet side. Dynamics and localization of intraarterial fluorescence by FITC-labeled dextrans (FITC-⌬s) and the endothelial membrane dye DiI were determined with confocal microscopy. Large FITC-⌬ (148 kDa) filled a core volume inside the arteries within 1 min but was excluded from a 2.6 Ϯ 0.5-m-wide region on the luminal side of the endothelium during 30 min of dye perfusion. Medium FITC-⌬ (50.7 kDa) slowly penetrated this ESL within 30 min but did not permeate into the arterial wall. Small FITC-⌬ (4.4 kDa) quickly passed the ESL and accumulated in the arterial wall. Prolonged luminal fluorochrome illumination with a bright mercury lamp destroyed the ϳ3-m exclusion zone for FITC-⌬148 within a few minutes. This study demonstrates the presence of a thick ESL 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. vascular permeability; isolated artery; endothelial surface layer; confocal microscopy; glycocalyx ENDOTHELIAL CELLS (ECs) are covered by a surface layer of membrane-associated proteoglycans, glycosaminoglycans, glycoproteins, glycolipids, and associated plasma proteins, known as the endothelial surface layer (ESL) (30). The functional properties of the ESL have been extensively described only recently. Biochemical research elucidates receptor functions of glycosaminoglycans within the ESL and the binding patterns of proteins to heparan sulfates (8,31,34). Biodegradation of sialic acid, an important constituent of the ESL, by neuraminidase inhibits shear-induced nitric oxide production (12, 29). The role of the ESL in the control of vascular wall permeability has been addressed in experimental studies (1, 21, 38) on microvessels and in new theoretical transport models (18). Vascular permeability forms an important parameter in the regulation of water and solute exchange between the circulation and tissues (10, 26). It is important that the intrusion of certain macromolecules into the arterial wall is limited. Inclusion of albumin and lowdensity lipoproteins into the subendothelial space forms part of the process of atherogenesis. Thus ESL dysfunction may contribute to the microvascular disease phenotype of atherosclerosis (4, 23, 24). An altered vascular permeability is one of the earliest detectable symptoms...

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Section: Discussionsupporting

confidence: 57%

Localization of the permeability barrier to solutes in isolated arteries by confocal microscopy

Haaren

vanBavel

Vink

et al. 2003

American Journal of Physiology-Heart and Circulatory Physiology

131

104

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“…Time-lapse microscopy was used to measure the flux of dextran into the collagen gel, and the resulting diffusion profile was fitted to a dynamic mass conservation equation as previously described 31 , with the diffusive permeability coefficient (P D ) defined by the following relation:

J = P_{D} false(C_{vessel} - C_{ECM} false)

where J is the mass flux of dextran, c vessel is the concentration of dextran in the vessel, and c ECM is the concentration of dextran in the perivascular ECM.…”

Section: Methodsmentioning

confidence: 99%

A non-canonical Notch complex regulates adherens junctions and vascular barrier function

Polacheck

Kutys

Yang

et al. 2017

Nature

283

315

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The vascular barrier that separates blood from tissues is actively regulated by the endothelium and is essential for transport, inflammation, and hemostasis1. Hemodynamic shear stress plays a critical role in maintaining endothelial barrier function2, but how this occurs remains unknown. Here, using an engineered organotypic model of perfused microvessels and confirming in mouse models, we identify that activation of the Notch1 transmembrane receptor directly regulates vascular barrier function through a non-canonical, transcription independent signaling mechanism that drives adherens junction assembly. Shear stress triggers Dll4-dependent proteolytic activation of Notch1 to reveal the Notch1 transmembrane domain – the key domain that mediates barrier establishment. Expression of the Notch1 transmembrane domain is sufficient to rescue Notch1 knockout-induced defects in barrier function, and does so by catalyzing the formation of a novel receptor complex in the plasma membrane consisting of VE-cadherin, the transmembrane protein tyrosine phosphatase LAR, and the Rac1 GEF Trio. This complex activates Rac1 to drive adherens junction assembly and establish barrier function. Canonical Notch transcriptional signaling is highly conserved throughout metazoans and is required for many processes in vascular development, including arterial-venous differentiation3, angiogenesis4, and remodeling5; here, we establish the existence of a previously unappreciated non-canonical cortical signaling pathway for Notch1 that regulates vascular barrier function, and thus provide a mechanism by which a single receptor might link transcriptional programs with adhesive and cytoskeletal remodeling.

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“…To evaluate whether our vascular chip recapitulates in vivo barrier-like features of vessels and whether hBMSCs contribute to that barrier, we seeded different ratios of hBMSCs to HUVECs (0:1, 1:1, 1:5, 1:10, and 1:100) (SI Appendix, Fig. S3) and quantified permeability by measuring the extravasation of fluorescently labeled 10 kDa or 70 kDa dextran from the vessel lumen (34) (Fig. 1C).…”

Section: Bicellular Perfusable 3d Platform Recapitulates Pericyte Rolmentioning

confidence: 99%

Three-dimensional biomimetic vascular model reveals a RhoA, Rac1, and N -cadherin balance in mural cell–endothelial cell-regulated barrier function

Alimperti

Mirabella

Bajaj

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The integrity of the endothelial barrier between circulating blood and tissue is important for blood vessel function and, ultimately, for organ homeostasis. Here, we developed a vessel-on-a-chip with perfused endothelialized channels lined with human bone marrow stromal cells, which adopt a mural cell-like phenotype that recapitulates barrier function of the vasculature. In this model, barrier function is compromised upon exposure to inflammatory factors such as LPS, thrombin, and TNFα, as has been observed in vivo. Interestingly, we observed a rapid physical withdrawal of mural cells from the endothelium that was accompanied by an inhibition of endogenous Rac1 activity and increase in RhoA activity in the mural cells themselves upon inflammation. Using a system to chemically induce activity in exogenously expressed Rac1 or RhoA within minutes of stimulation, we demonstrated RhoA activation induced loss of mural cell coverage on the endothelium and reduced endothelial barrier function, and this effect was abrogated when Rac1 was simultaneously activated. We further showed that N-cadherin expression in mural cells plays a key role in barrier function, as CRISPRmediated knockout of N-cadherin in the mural cells led to loss of barrier function, and overexpression of N-cadherin in CHO cells promoted barrier function. In summary, this bicellular model demonstrates the continuous and rapid modulation of adhesive interactions between endothelial and mural cells and its impact on vascular barrier function and highlights an in vitro platform to study the biology of perivascular-endothelial interactions.

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Quantitative Laser Scanning Confocal Microscopy on Single Capillaries: Permeability Measurement

Abstract: Therefore, the loss of sodium fluorescein through the upper mesothelium does not significantly alter permeability under these conditions. These techniques enable detailed analysis of solute concentration gradients surrounding microvessels.

Cited by 56 publications

References 21 publications

Localization of the permeability barrier to solutes in isolated arteries by confocal microscopy

Localization of the permeability barrier to solutes in isolated arteries by confocal microscopy

A non-canonical Notch complex regulates adherens junctions and vascular barrier function

Three-dimensional biomimetic vascular model reveals a RhoA, Rac1, and N -cadherin balance in mural cell–endothelial cell-regulated barrier function

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