Endothelial stomatal and fenestral diaphragms in normal vessels and angiogenesis

Abstract

Vascular endothelium lines the entire cardiovascular system where performs a series of vital functions including the control of microvascular permeability, coagulation inflammation, vascular tone as well as the formation of new vessels via vasculogenesis and angiogenesis in normal and disease states. Normal endothelium consists of heterogeneous populations of cells differentiated according to the vascular bed and segment of the vascular tree where they occur. One of the cardinal features is the expression of specific subcellular structures such as plas-malemmal vesicles or caveolae, transendothelial channels, vesiculo-vacuolar organelles, endothelial pockets and fenestrae, whose presence define several endothelial morphological types. A less explored observation is the differential expression of such structures in diverse settings of angiogenesis. This review will focus on the latest developments on the components, structure and function of these specific endothelial structures in normal endothelium as well as in diverse settings of angiogenesis.

1

Endothelium of continuous (A) and fenestrated (B) types as seen by freeze fracture. (A) The P face of the abluminal membrane of a heart endothelial cell shows the numerous caveolae. These are organized in linear arrays, better seen in the higher magnification images (insets). (B) Two endothelial cells of a jejunal capillary show the disposition of fenestrae in sieve plates. Higher magnification insets compare the linear disposition of caveolae (left) and fenestrae (right), suggesting attachment to cytoskeletal elements. Reproduced from reference [12], with permission.

2

Caveolae with (B) and without (A) stomatal diaphragms, TEC (C) and Fenestrae (D) as seen in rapidly frozen deeply etched specimens. The stomatal diaphragms of caveolae and TEC look alike. Fenestrae have an octagonal symmetry, their fenestral diaphragms being constituted of radial fibrils starting at the rim and interweaving in a central mesh. Reproduced from [17] with permission.

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Proposed model for PV1 integration in the structure of the diaphragms.(A) Perpendicular sections of a caveola provided with stomatal diaphragm (left), TEC (middle) and fenestra (right).(B) En face views of stomatal diaphragms (left) and fenestral diaphragms (right), as shown by deep-etch rapid–freeze techniques, demonstrating the fibrils in the fenestral diaphragms and the hints of fibrils in their stomatal diaphragms counterparts. Reprinted from Bearer and Orci, JCB, 1985, with permission. (C) Schematic of the membrane insertion and features of the PV1 monomer. (D) Model of PV1 integration in the endothelial diaphragms:PV1 dimers participate in the formation of the fibrils inserted in the rim (via PV1 N-terminus) and interweaving in the central mesh (via PV1 C terminus). The glycan antennae (accounting for ∼15% of PV1 mass) are situated near the membrane, which would keep the protein ‘afloat’ by preventing collapse on the plasma membrane.

4

PMA induces TEC (B, I), fenestrae (A, B, G, I) as well as caveolar stomatal diaphragms (C, D, I) only in endothelial cells and not in non-endothelial cell types such as fibroblasts (F). Non-treated control endothelial cells (H) as well as fibroblasts (E) contain only caveolae devoid of stomatal diaphragms. PMA also upregulates PV1, which is found in the newly formed stomatal diaphragms and fenestral diaphragms as demonstrated by immunogold labeling (G, I).(J) PV1 mRNA silencing with siRNA prevents the formation of stomatal diaphragms of caveolae as well as the TEC and fenestrae altogether in HUVEC (lower panels) while the scrambled siRNA counterpart does not (scr, upper panels). Bars 100 nm.

5

Transfection of PV1 in HUVEC leads to de novo formation of stomatal diaphragms of caveolae. No TEC or fenestrae are formed.(A) control HUVEC (B–C) HUVEC transfected with PV1-HA and stained with anti-HA gold (B).

3

Immunolocalization of PV1 by immunodifussion to stomatal diaphragms of caveolae in lung (A–B, E), stomatal diaphragms of TEC (D–E) and fenestral diaphragms (C–D, F), as detected with anti-PV1 antibodies directed against its C-terminus.(A–B) lung, C–E) kidney, (F) intestine. The label was found specifically associated with the stomatals and fenestral diaphragms at both fronts of the cell. No label was found on the plasmalemma proper, clathrin-coated pits and vesicles, intercellular junctions as well as non-endothelial cellular types. Bars 100 nm.

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PV1 (red) is expressed in tumor vessels where it colocalizes with CD31 (green)