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Correspondence to: Ann M. Dvorak, Dept. of Pathology, East Campus, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. Fax: 617 667 2943
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Summary |
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A newly defined endothelial cell permeability structure, termed the vesiculovacuolar organelle (VVO), has been identified in the microvasculature that accompanies tumors, in venules associated with allergic inflammation, and in the endothelia of normal venules. This organelle provides the major route of extravasation of macromolecules at sites of increased vascular permeability induced by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), serotonin, and histamine in animal models. Continuity of these large sessile structures between the vascular lumen and the extracellular space has been demonstrated in kinetic studies with ultrastructural electron-dense tracers, by direct observation of tilted electron micrographs, and by ultrathin serial sections with three-dimensional computer reconstructions. Ultrastructural enzyme-affinity cytochemical and immunocytochemical studies have identified histamine and VPF/VEGF bound to VVOs in vivo in animal models in which these mediators of permeability are released from mast cells and tumor cells, respectively. The high-affinity receptor for VPF/VEGF, VEGFR-2, was localized to VVOs and their substructural components by pre-embedding ultrastructural immunonanogold and immunoperoxidase techniques. Similar methods were used to localize caveolin and vesicle-associated membrane protein (VAMP) to VVOs and caveolae, indicating a possible commonality of formation and function of VVOs to caveolae.
(J Histochem Cytochem 49:419431, 2001)
Key Words: vesiculo-vacuolar organelle, endothelial cell, permeability, vascular permeability factor/, vascular endothelial growth, factor, venules, tumor biology, allergic inflammation, mast cell, histamine, electron microscopy
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Introduction |
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The microcirculation functions to provide rapid exchange of nutrients and waste products between blood and tissues. In normal tissues, this exchange occurs primarily across capillaries, which are the most abundant of the microvessels and have the greatest surface area. Whereas small molecules freely traverse most capillary endothelia, the passage of circulating macromolecules, such as plasma proteins, is severely restricted. It is likely that very small hydrophilic molecules (diameter ,3 nm) pass through intact interendothelial cell junctions, or, if lipid-soluble, exit vessels by diffusion in endothelial cell plasma membranes. Other small molecules cross the endothelial cell barrier by specific transport mechanisms. Large molecules, such as plasma proteins, exit most capillaries at low rates and, reportedly (
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VVOs Are the Major Site of Hyperpermeability of Tumor Blood Vessels |
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The microvessels that supply tumors, as well as those found in other settings characterized by angiogenesis, are hyperpermeable to plasma proteins and other circulating macromolecules (
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VVOs were found to provide a transcytotic pathway by which soluble macromolecular tracers extravasated from leaky tumor blood vessels (11 nm) was not found to exit tumor vessels through interendothelial cell junctions at any time interval up to 1 hr. The small protein tracer HRP (d.
5 nm) did exit tumor vessels through normally apposed interendothelial junctions, but only after a delay of at least 5 min, at a time when extensive HRP extravasation had already taken place through VVOs; such intercellular extravasation is similar to that reported in normal non-tumor microvessels (
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Subsequent study revealed that morphologically similar VVOs were present, and with equal frequency, in the venular endothelium supplying skin and many other tissues in normal animals as well as remote from tumor sites in tumor-bearing animals (
Some of the vessels supplying MOT tumors were fenestrated and prominent collections of ferritin were also found in and beneath individual fenestrae, suggesting that these structures had provided a second pathway for tracer extravasation (
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VVO Structure |
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The bunches of grape-like clusters of VVOs are deployed at intervals in the cytoplasm of endothelial cells (
VVO structure cannot be fully appreciated by electron microscopy of standard 70- to 100-nm sections. Therefore, we prepared serial 12- to 14-nm ultrathin sections for electron microscopy (Fig 4). Computer-assisted three-dimensional reconstructions of these serial sections revealed a network of interconnecting vesicles and vacuoles and established that VVOs provide a continuous, often serpentine pathway across venular endothelium, extending to both the lumen and ablumen at multiple sites (
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The large size and complexity of cutaneous VVOs suggest that they are sessile structures and that their component vesicles and vacuoles do not shuttle back and forth across endothelial cell cytoplasm, as has been proposed for capillary caveolae (
Morphometric measurements (
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VVO Function |
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The venules of normal tissues permit only minimal extravasation of circulating macromolecules. Consistent with this finding, the VVOs of normal tissues, although structurally similar to those of tumor vessels, differ functionally from tumor endothelial cell VVOs in that they permit only minimal entry and passage of macromolecular tracers (Table 1) (
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In addition, another such mediator, histamine, could act similarly in opening stomata in hyperpermeable vessels in allergic inflammation. In fact, using a new enzyme affinitygold postembedding method to detect histamine (
If VVO function is regulated by VPF/VEGF or other vasoactive mediators, then these mediators would be expected to increase the microvascular permeability of normal venules by opening VVO stomata to the passage of macromolecules. In fact, this proved to be the case. Injection of small amounts of VPF/VEGF, histamine, or serotonin into the normal flank or scrotal skin of guinea pigs, rats, and mice greatly increased the permeability of local venules (Table 1) (
VPF/VEGF, a potent multifunctional cytokine, permeabilizes vascular endothelia to plasma proteins, and reprograms endothelial cell gene expression so as to induce angiogenesis (
We used ultrastructural pre-embedding immunoperoxidase and immunonanogold methods to localize VEGFR-2 (flk-1/KDR) in vascular endothelium in three model systems in which VPF/VEGF is highly expressed: (a) glomerular and peritubular capillaries of normal mouse kidney; (b) microvessels supplying a well-characterized mouse mammary carcinoma; and (c) new vessels induced by an adenoviral vector engineered to overexpress VPF/VEGF (adeno-vpf/vegf) (
Localization of VPF/VEGFR-2 to VVO membranes and to the luminal and abluminal plasma membranes of vascular endothelium, but not to the lateral plasma membranes at interendothelial cell junctions, is consistent with the mechanisms that we have proposed for the increased microvascular permeability induced by VPF/VEGF and other vasoactive mediators (
The biochemical mechanisms by which VPF/VEGF and other mediators regulate VVO permeability have not yet been elucidated but must ultimately involve actions that take place at the level of the stomata and the diaphragms that close stomata and restrict the passage of macromolecules. When stomata are functionally closed, as in normal venules, very little macromolecular tracer enters or passes through VVOs. On the other hand, when stomata open, as in response to vasoactive mediators, tracers enter freely and pass across the endothelial barrier by way of the VVO network. We have searched for morphological changes in stomata that might correlate with the passage of macromolecular tracers. Thus far, we have found only one, i.e., that the mean stomatal diameters of VVO vesicles-vacuoles increased significantly (by 79%) in response to local injections of VPF/VEGF or serotonin (Table 2). In contrast, the much smaller stomatal diameters of capillary caveolae did not increase detectably after exposure to VPF/VEGF or serotonin, probably reflecting the selectivity of these mediators for venular as compared with capillary endothelium. We suggest that enlargement of stomatal diameter may be an important reflection of stomata opened to the passage of macromolecules.
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Stomatal diaphragms are likely the structures that regulate VVO permeability (
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VVO Formation |
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Given the similarities between caveolae and VVO vesicles and vacuoles, it is possible that VVOs might form from the linking together of individual caveolae. It is also possible that the larger vesicles and vacuoles of VVOs might form from the fusion of two or more caveola-sized vesicles. We did ultrastructural pre-embedding immunoperoxidase to localize caveolin in VVOs in vitro and in vivo (0.00015 µm3; this value corresponds to a spheroid of diameter
60 nm, i.e., the size of typical capillary caveolae. Vacuoles corresponding to the fusion of as many as 10 unit vesicles were detected. The data are therefore consistent with the hypothesis that larger VVO vesicles and vacuoles arise from the fusion of different numbers of caveola-sized unit vesicles.
The mechanism(s) responsible for vesicle fusion and organization into VVOs in venular endothelial cells is currently being investigated. N-ethylmaleimide (NEM) alkylates NEM-sensitive factor (NSF), an ATPase that is involved with fusion events in exocytosis and endocytosis (
We also have begun to investigate the substructural localization of other proteins of importance to vesicular trafficking and fusion, generally. One such protein, vesicle-associated membrane protein-2 (VAMP-2), also known as synaptobrevin (
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VVO Fate |
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Once formed, VVOs are sessile, multichambered structures occupying vast expanses of venular cytoplasm. They restrict the passage of macromolecules from the blood vascular spaces at their narrow points, or stomata, which are closed by thin diaphragms. These diaphragms are opened, making VVOs porousa process that is rapidly induced by exposure to well-known permeabilizing molecules, e.g., VPF/VEGF, histamine, and serotonin.
We envision at least three possible fates for VVOs in venular endothelium: (a) opened, leaky VVOs could close, thereby returning vessels to their prior non-leaky state; (b) opened, enlarged stomata in permeabilized VVOs could open further, allowing retention of opened vesicles and vacuoles in their fused state to form large transcellular pores through which circulating endogenous and exogenous particulates could freely enter the extravascular space (
At present, there are no data to confirm the first possibility, but long-term studies of VVO architecture after transient permeability events are in progress. Considerable data have accrued in support of the second proposed fate of VVOs (50 nm) as a tracer, a particulate that is, for the most part, too large to enter or pass through the stomata of VVOs, we observed a substantial (three- to 30-fold) increase in endothelial cell openings (
Using carbon as tracer, we did serial sections and computer three-dimensional reconstructions of endothelial cell openings (
The third possible fate of VVOs as a membrane store for rapidly expanding endothelial plasma membrane has gained some support from studies of transendothelial cell neutrophil migration in acute inflammation (
The fate of VVOs therefore includes at least three possibilities: to recover in place by reforming diaphragms between their components or to persist by remaining in place without reclosure of stomata (e.g., in acute and chronic inflammation), to expand further to form large transcellular pores (to facilitate transcellular trafficking in inflammation), or to provide rapid expansion of endothelial plasma membranes by externalizing their membrane in acute inflammatory and angiogenesis models.
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Acknowledgments |
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Supported by NIH grants AI-33372 and AI-44066.
We thank Peter K. Gardner, for editorial assistance in the preparation of the manuscript, and Ellen Morgan, Patricia Fox, Kathryn Pyne, and Rita MonahanEarley for technical and photographic assistance.
Received for publication August 4, 2000; accepted November 27, 2000.
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