Journal of Histochemistry and Cytochemistry, Vol. 49, 419-432, April 2001, Copyright © 2001, The Histochemical Society, Inc.


REVIEW

The Vesiculo–Vacuolar Organelle (VVO): A New Endothelial Cell Permeability Organelle

Ann M. Dvoraka and Dian Fenga
a The Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts

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


  Summary
Top
Summary
Introduction
VVOs Are the Major...
VVO Structure
VVO Function
VVO Formation
VVO Fate
Literature Cited

A newly defined endothelial cell permeability structure, termed the vesiculo–vacuolar 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 49:419–431, 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


  Introduction
Top
Summary
Introduction
VVOs Are the Major...
VVO Structure
VVO Function
VVO Formation
VVO Fate
Literature Cited

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 (Simionescu et al. 1975 ; Granger and Perry 1983 ; Simionescu 1983 ; Renkin 1985 ; Schnitzer 1993 ), by two routes: (a) vesicular transport via the shuttling of 50- to 70-nm cytoplasmic vesicles present in endothelial cells (or by interconnected vesicles that form transendothelial cell channels) and (b) interendothelial cell gaps.We identified a new endothelial cell organelle, which we termed the vesiculo–vacuolar organelle (VVO). This organelle provides the major route of extravasation of macromolecules at sites of augmented vascular permeability, induced by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), a tumor-derived cytokine (Dvorak et al. 1995 , Dvorak et al. 1999 ; Brown et al. 1997 ), in venules associated with experimental tumors (Kohn et al. 1992 ; Dvorak et al. 1996 ). We also reported extensive VVOs in venular endothelium in an animal model of allergic inflammatory eye disease, characterized by histamine secretion from augmented mast cell expression (Dvorak et al. 1994 ) (Fig 1). Our ultrastructural studies of human biopsy specimens in the performance of diagnostic ultrastructural pathology have verified the presence of VVOs in human endothelial cells of the microcirculation in health and disease, including primary and metastatic tumors in multiple tissue sites, acute and chronic inflammatory disorders, and in allergic inflammation (Dvorak and Monahan-Earley 1992 , Dvorak and Monahan-Earley 1995a , Dvorak and Monahan-Earley 1995b ). These observations were the stimulus for further studies using multiple ultrastructural methods designed to determine (a) the substructure and contents of VVOs, (b) the formation of VVOs, (c) the mechanism of upregulated function of tumor vessel VVOs, and (d) whether tumor cytokines and mediators of inflammation with permeabilizing properties could recapitulate the upregulated VVO function of tumor vessels in normal vessels (Kohn et al. 1992 ; Dvorak et al. 1994 , Dvorak et al. 1996 ; Qu-Hong et al. 1995 ; Feng et al. 1996 , Feng et al. 1997 , Feng et al. 2000a , Feng et al. 2000b ; Vasile et al. 1999 ).



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Figure 1. Cross-section of a venule from an inflamed eyelid of an interleukin-4 transgenic mouse. Two pericytes (P) embrace this vessel; two endothelial cell nuclei (N) are evident. Portions of five endothelial cells line the venule; individual endothelial cells are connected by interendothelial cell junctions (solid arrowheads), none of which show gaps. Focal clusters of vesicles and vacuoles, termed VVOs (open arrowheads), span the cytoplasm of individual endothelial cells that comprise this vessel. L, lumen. Bar = 3 µm. From Dvorak et al. 1994 , with permission.


  VVOs Are the Major Site of Hyperpermeability of Tumor Blood Vessels
Top
Summary
Introduction
VVOs Are the Major...
VVO Structure
VVO Function
VVO Formation
VVO Fate
Literature Cited

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 (Dvorak et al. 1995 ; Brown et al. 1997 ). Tumor microvessels are typically four- to 10-fold more permeable to circulating macromolecules than comparable normal vessels. To elucidate the structural basis for this hyperpermeability, we injected several different macromolecular tracers IV into mice or guinea pigs bearing syngenic solid or ascites tumors and followed the extravasation of these tracers from tumor and normal vessels over time by light and electron microscopy (Kohn et al. 1992 ; Dvorak et al. 1996 ). These studies led to the identification of VVOs in the endothelium of tumor microvessels (Kohn et al. 1992 ; Dvorak et al. 1996 ; Feng et al. 1996 ) (Fig 2). The vesicles and vacuoles of individual VVOs extend across endothelial cells, interconnecting with each other and with the luminal, abluminal, and often the lateral plasma membranes by stomata that may be open or that are guarded by thin diaphragms. These stomata and their closing diaphragms likely provide the structural basis for regulation of tracer passage across the microvascular endothelium.



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Figure 2. A VVO in the endothelium of a mouse tumor vessel shows a bunch of grape-like cluster of interconnected vesicles and vacuoles in the cytoplasm of an endothelial cell. Individual vesicles exhibit multiple pale, membrane-bound stomata (large arrow). Interconnecting vesicles are constricted focally at stomatal attachment points of diaphragms that close stomata between vesicles (small arrow). Several stomata exhibit central, dense knobs (Bruns and Palade 1968 ). Bar = 270 nm. From Dvorak et al. 1996 , with permission.

VVOs were found to provide a transcytotic pathway by which soluble macromolecular tracers extravasated from leaky tumor blood vessels (Kohn et al. 1992 ; Dvorak et al. 1996 ) (Fig 3). Within seconds of IV injection into mice bearing either the MOT (mouse ovarian tumor) or TA3/St mammary carcinoma, macromolecular tracers such as anionic ferritin and horseradish peroxidase (HRP) were found in VVO vesicles that opened to the vascular lumen. Initially, both tracers were found in VVO vesicles that opened to the vascular lumen, but VVO vesicles and vacuoles were rapidly labeled at all levels of endothelial cell cytoplasm. Vesicles that opened to the endothelial cell ablumen spilled both tracers into the underlying basal lamina. Ferritin (d. ~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 (Hilal 1973 ).



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Figure 3. Electron microscopic tracer studies of transendothelial cell leakage through VVOs of mouse tumor microvascular endothelial cells. (A) At 10 sec after IV injection of HRP, the electron-dense tracer fills the vessel lumen and a cytoplasmic VVO, which has released a small amount to the basal lamina focally (open arrowhead). The interendothelial cell junction (closed arrowhead) does not contain HRP. Bar = 260 nm. (B) A tumor endothelial cell VVO connected to the abluminal surface shows an open, HRP-containing stoma. The abluminal surface of the endothelial cell plasma membrane is focally stained with HRP beneath this cell; the basal lamina (arrow) remains unstained. Bar = 80 nm. (C) At 10 sec after IV HRP, several vesicles in this VVO contain electron-dense HRP, similar in intensity to the overlying HRP-filled vessel lumen. One HRP-loaded vesicle (arrowhead) is fused with the abluminal plasma membrane and has released a cloud of HRP focally into the underlying basal lamina and surrounding connective tissue. Bar = 154 nm. (D) At 30 min after IV injection of anionic ferritin, a VVO vesicle stoma at the basilar endothelial cell front is filled with ferritin particles. Individual ferritin particles (arrows) have dispersed widely in the extracellular matrix beneath this endothelial cell. Bar = 63 nm. From Dvorak et al. 1996 , with permission.

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 (Kohn et al. 1992 ; Dvorak et al. 1996 ; Feng et al. 1996 ). However, normal venules extravasated only very small amounts of macromolecules and their VVOs were minimally labeled with ferritin and HRP. Earlier studies (Bundgaard et al. 1979 ; Frokjaer-Jensen 1980 ) of normal capillary endothelia described closely aligned plasmalemmal vesicles that were interpreted to be connected either to luminal or to abluminal surfaces but not to both and, therefore, not to be transendothelial channels based on serial sections. Tracer studies were not done.

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 (Kohn et al. 1992 ). In contrast, the vessels supplying the TA3/St tumor were not fenestrated, and tracer extravasated exclusively by the transcellular VVO route. Others have described gaps in tumor vessels and have suggested that these provide a pathway for macromolecule extravasation (Roberts and Palade 1995 , Roberts and Palade 1997 ; Roberts et al. 1998 ). Gaps were exceedingly rare in the vessels supplying the experimental and human tumors we have studied (Dvorak and Monahan-Earley 1992 , Dvorak and Monahan-Earley 1995a , Dvorak and Monahan-Earley 1995b ; Kohn et al. 1992 ; Dvorak et al. 1996 ).


  VVO Structure
Top
Summary
Introduction
VVOs Are the Major...
VVO Structure
VVO Function
VVO Formation
VVO Fate
Literature Cited

The bunches of grape-like clusters of VVOs are deployed at intervals in the cytoplasm of endothelial cells (Kohn et al. 1992 ; Dvorak et al. 1994 , Dvorak et al. 1996 ; Feng et al. 1996 ) (Fig 1). They are often concentrated near lateral borders of endothelial cells, i.e., parajunctionally, and provide a direct link between the vascular lumen and ablumen. Approximately 12% of VVOs extend from the luminal to the abluminal plasma membranes when viewed in single, random 80-nm electron microscopic sections; in serial sections this value approaches 100% (Feng et al. 1996 ). The individual vesicles and vacuoles comprising VVOs are bounded by membranes and interconnect with each other and with the endothelial cell plasma membranes by means of stomata that are closed by thin diaphragms (Kohn et al. 1992 ; Dvorak et al. 1996 ; Feng et al. 1996 ) (Fig 2). The stomatal diaphragms measure less in diameter than the individual VVO vesicles and vacuoles (Feng et al. 1999 ). The presence of tracers (Fig 3) (i.e., HRP, ferritin) within adjoined vesicles–vacuoles, as well as within the stomata connecting them, provides prima facie evidence that such vesicles and vacuoles are in open communication with each other (Kohn et al. 1992 ; Dvorak et al. 1996 ; Feng et al. 1996 ). [Specimen tilting in the electron microscope also demonstrated continuities between joined vesicles and vacuoles (Dvorak et al. 1996 ).] However, in some cases, the stomata joining individual vesicles and vacuoles were closed by a diaphragm, and passage of macromolecular tracers was restricted such that one vesicle or vacuole contained tracer whereas the other did not. It therefore appears that the diaphragms which separate individual stomata serve to restrict the passage of cargo and may be opened and closed individually.

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 (Feng et al. 1996 ) (Fig 5). Subsequent studies have revealed that the VVOs of adjacent endothelial cells also open to the interendothelial cleft and, in some instances, VVOs from adjacent endothelial cells connect across the intercellular cleft between adjacent endothelial cells, raising the possibility that plasma may extravasate by a VVO pathway that extends across overlapping endothelial cells.



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Figure 4. Serial sections (14 nm thick) of a single VVO in normal mouse skin. Three sequences of interconnecting vesicles–vacuoles are illustrated, the most luminal of which is designated "a, b, c." Each sequence nearly forms a transendothelial cell channel in just two consecutive sections. Note the closed interendothelial cell junction at the left of panels. R, red blood cell; L, lumen. Bar = 200 nm. From Feng et al. 1996 , with permission.



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Figure 5. Computer-generated 3-D reconstruction of a portion of a VVO (15 consecutive serial ultrathin EM sections illustrating 25 interconnected vesicles–vacuoles) from a venule in normal mouse skin. (A–F) The reconstruction in successive rotations is around a horizontal axis at intervals of 30° (except 15°, C–D). There are two openings (E,F) to the vascular lumen and four to the abluminal surface (A). From Feng et al. 1996 , with permission.

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 (Palade 1988 ). Indeed, in evaluation of many sets of ultrathin (12- to 14-nm-thick) serial sections, only 9 of 1395 (0.65%) uncoated vesicles were found to be single entities, free and unattached in the cytoplasm, i.e., >99% were attached to other vesicles and vacuoles as parts of VVOs (Feng et al. 1996 ).

Morphometric measurements (Dvorak et al. 1996 ; Feng et al. 1996 , Feng et al. 1999 ) revealed that VVOs occupied a substantial portion (16–18%) of venular endothelial cytoplasm (Feng et al. 1996 ). Individual VVOs commonly extended through endothelial cytoplasm for distances of 1–2 µm. On the basis of serial sections and reconstructions, typical VVOs were found to be composed of 124 (median) vesicles and vacuoles (range 79 to >400). The smallest VVO vesicles closely resembled capillary caveolae. However, on average, the vesicles and vacuoles comprising VVOs measured 108 ± 32 nm (mean ± SD; range 50–415 nm) in internal diameter and were therefore significantly larger and more heterogeneous in size than capillary caveolae whose internal diameters measured, on average, 58 ± 9 nm (range 38–141 nm). Therefore, the vesicles and vacuoles comprising VVOs differ from the caveolae of capillary endothelium in several important respects: significantly larger average size, greater size heterogeneity, and organization into a cohesive organelle, the VVO, that occupies nearly one fifth of the venular endothelial cell cytoplasm and that links the vascular lumen with the ablumen.


  VVO Function
Top
Summary
Introduction
VVOs Are the Major...
VVO Structure
VVO Function
VVO Formation
VVO Fate
Literature Cited

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) (Kohn et al. 1992 ; Dvorak et al. 1996 ; Feng et al. 1996 ). To account for the functional differences between the VVOs of tumor vessels and those of normal venules, we postulated that VVO function was regulated by vasoactive mediators that in some way opened the stomata that connected individual VVO vesicles and vacuoles with each other and with the venular lumen and ablumen. We also postulated that one such mediator, VPF/VEGF (Dvorak et al. 1979 ; Senger et al. 1983 ; Ferrara and Henzel 1989 ; Gospodarowicz et al. 1989 ; Leung et al. 1989 ; Ferrara et al. 1992 ), was likely responsible for opening these stomata in hyperpermeable tumor vessels, thereby accounting for the relatively free passage of macromolecular tracers through tumor vessel VVOs. Consistent with this hypothesis, the tumors we studied synthesize and secrete large amounts of VPF/VEGF (Dvorak et al. 1995 , Dvorak et al. 1999 ; Brown et al. 1997 ; Feng et al. 2000b ). Moreover, tumor cell-secreted VPF/VEGF was found to localize on the surface of tumor microvascular endothelial cells as well as in association with the VVO vesicles and vacuoles in their cytoplasm (Qu-Hong et al. 1995 ) (Fig 6B).



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Figure 6. Specifically labeled VVO components in IL-4 transgenic mouse inflammatory eye disease (A), tumor-induced mouse peritoneal wall microvasculature (B), and tumor-induced mouse subcutaneous microvasculature (C). In A, a diamine oxidase-gold method was used to detect histamine. Two gold particles indicating histamine (arrow) are bound to a single stoma in a single vesicle within the VVO. Bar = 160 nm. From Dvorak et al. 1994 , with permission. (B) A pre-embedding immunoperoxidase technique, using specific antibody to VPF/VEGF, shows a single stoma (arrow) to contain this tumor cytokine in the VVO. Note the central, electron-lucent spot within the VPF/VEGF-positive stoma. This spot corresponds to a stomatal structure that has been termed a knob (Bruns and Palade 1968 ), as described for plasmalemmal caveolae. It does not stain for VPF/VEGF. Bar = 104 nm. From Qu-hong et al. (1995), with permission. (C) A pre-embedding immunoperoxidase method was used with a specific antibody to the VPFR-2. Note that a single caveolar diaphragm, which closes a vesicle at the luminal front of the endothelium, is positive for VPFR-2 (arrow). Several other vesicles in the VVO have positive vesicle membranes, indicating the presence of VPFR-2. L, lumen. Bar = 95 nm.


 
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Table 1. Percent of plasma concentration of ferritin in VVOs and immediately subjacent subendothelial space in normal mouse skin vs skin injected with VPF, serotonin or Hanks' balanced salt solution (HBSS)

In addition, another such mediator, histamine, could act similarly in opening stomata in hyperpermeable vessels in allergic inflammation. In fact, using a new enzyme affinity–gold postembedding method to detect histamine (Dvorak et al. 1993 ), we have localized histamine, which is secreted from mast cells in the allergic eye disease mouse model, to VVOs (Fig 6A) in involved vessels (Dvorak et al. 1994 ).

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) (Feng et al. 1996 ). Electron microscopy demonstrated that the circulating tracer (anionic ferritin) exited such venules primarily by way of VVOs, just as in tumor microvessels (Feng et al. 1996 ). Within seconds of intradermal injection of these mediators, and continuing for some minutes thereafter, increasing amounts of circulating ferritin entered VVO vesicles contiguous with the venular lumen and proceeded across the endothelium through a succession of interconnecting VVO vesicles and vacuoles to reach the vascular ablumen and underlying basal lamina, i.e., tracers followed the same pathway across normal dermal venule endothelial cells as in leaky tumor microvessels. Most stomata connecting adjacent VVO vesicles and vacuoles to one another and to the luminal and abluminal plasma membranes were functionally open because the passage of ferritin was not restricted. However, diaphragms closed some stomata to the passage of ferritin because ferritin molecules accumulated in immediately proximal vesicles or vacuoles. Thus, stomatal diaphragms were able to serve as barriers that limited the further transcellular passage of macromolecular tracers. Endothelial cell junctions remained intact and ferritin was never observed in them (Feng et al. 1996 ). Together, these data establish VVOs as the major pathway by which soluble plasma proteins exit non-tumor venules in response to several mediators that increase venular hyperpermeability.

VPF/VEGF, a potent multifunctional cytokine, permeabilizes vascular endothelia to plasma proteins, and reprograms endothelial cell gene expression so as to induce angiogenesis (Brown et al. 1997 ; Ferrara and Davis-Smyth 1997 ; Dvorak et al. 1999 ; Ferrara 1999 ). VPF/VEGF induces its biological effects by binding to two tyrosine kinase receptors, VEGFR-1 (fms-like tyrosine kinase receptor, or Flt) and VEGFR-2 (fetal liver kinase 1, or Flk-1 in rodents; kinase insert domain-containing receptor or KDR in human), which are selectively expressed in vascular endothelium (Matthews et al. 1991 ; Terman et al. 1991 , Terman et al. 1992 ; de Vries et al. 1992 ; Quinn et al. 1993 ; Mustonen and Alitalo 1995 ). Both receptors are strikingly upregulated in tumors, wounds, and in certain types of inflammation (e.g., rheumatoid arthritis, psoriasis) in which VPF/VEGF is overexpressed (Brown et al. 1997 ). Recently, a third non-tyrosine kinase receptor for VPF/VEGF, neuropilin, has also been described (Soker et al. 1998 ). Expression of both VEGFR-1 and VEGFR-2 has been localized to microvascular endothelium of normal kidneys, tumors, healing wounds, and inflammatory sites by in situ hybridization (Brown et al. 1997 ). VEGFR-2 has also been identified in the blood vessels of human placentas, breast cancers, and gastric carcinomas by light microscopic immunohistochemistry (Vuckovic et al. 1996 ; Tanigawa et al. 1997 ; de Jong et al. 1998a , de Jong et al. 1998b ).

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) (Feng et al. 2000a ). Microvascular endothelial cells were positive for VEGFR-2 in all three models and, in the latter two, could be localized to the luminal and abluminal surfaces and to the membranes of cytoplasmic VVOs. The stomatal diaphragms of some VVOs and caveolae were VEGFR-2-positive, best seen with the peroxidase reporter when the entire vesicle membrane was not stained (Feng et al. 2000a ) (Fig 6C).

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 (Feng et al. 1996 , Feng et al. 1997 , Feng et al. 1999 ). For example, VVOs, structures that are often strategically concentrated in parajunctional zones of normal venule and tumor endothelium, provide a primary transcellular pathway for extravasation of macromolecules across continuous vascular endothelium. In response to vasoactive mediators, the diaphragms separating individual VVO vesicles from each other and from the vascular lumen and ablumen open, allowing the transendothelial passage of tracers from one vesicle or vacuole to the next until they finally cross the endothelial barrier. This stands in contrast to the view (Majno et al. 1969 ; Baluk et al. 1997 ) that vasoactive mediators promote endothelial cell contractions that pull apart adjacent endothelial cells to generate interendothelial cell gaps through which plasma can leak. A more detailed review of the controversy surrounding the pathways taken by circulating macromolecules as they cross vascular endothelium in response to vasoactive mediators is presented in several recent reviews (Dvorak et al. 1999 ; Feng et al. 1999 ; McDonald et al. 1999 ; Michel and Neal 1999 ).

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 7–9%) 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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Table 2. Comparison of VVO stomatal diameter in venular endothelium with caveolae of capillary endothelium of mouse skin; tissue was harvested 5 min after intradermal injection of VPF/VEGF, serotonin, or HBSS

Stomatal diaphragms are likely the structures that regulate VVO permeability (Feng et al. 1999 , Feng et al. 2000a ). Support for this concept is based on the strategic localization of VEGFR-2 on this diaphragm, which closes stomata in VVOs (Fig 6C) (Feng et al. 2000a ) and of VPF/VEGF bound to VVOs (Fig 6B) in endothelia present in animal tumor models (Qu-Hong et al. 1995 ).


  VVO Formation
Top
Summary
Introduction
VVOs Are the Major...
VVO Structure
VVO Function
VVO Formation
VVO Fate
Literature Cited

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 (Vasile et al. 1999 ) to investigate the first possibility. These studies showed, both in vivo and in vitro, that endothelial cell VVOs were caveolin-positive (Vasile et al. 1999 ), like capillary caveolae (Rothberg et al. 1992 ; Vasile et al. 1999 ), lending support for formation of caveolin-positive VVO clusters from the joining of individual caveolin-positive caveolae. We used an ultrastructural morphometric approach (Feng et al. 1999 ) to investigate the second possibility. For example, a fusion mechanism has been proposed in the generation of several types of cytoplasmic secretory granules in which small progranules of unit size fuse with each other in various combinations to form larger mature granules whose volumes represent multiples of the volume of the unit progranule (Hammel et al. 1983 , Hammel et al. 1985 , Hammel et al. 1993 , Hammel et al. 1998 ; Dvorak et al. 1984 ). Both mast cell and pancreatic acinar cell granules have been shown to have such a periodic distribution of their granules (Hammel et al. 1983 , Hammel et al. 1985 , Hammel et al. 1993 , Hammel et al. 1998 ; Dvorak et al. 1984 ). By analogy, the volume of the various VVO vesicles and vacuoles would not be expected to fall on a continuum but instead would represent multiples of the volume of the smaller unit vesicle. Measurements of the volumes of vesicles and vacuoles comprising VVOs revealed a heterogeneous distribution in which the volumes of individual vesicles and vacuoles were not continuous but exhibited a periodic modal distribution (Feng et al. 1999 ). The most frequently occurring vesicle-vacuole had a unit volume of ~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 (Predescu et al. 1994 ; Schnitzer et al. 1995a ). In addition, transport of caveolae across capillary endothelium is believed to employ mechanisms and proteins similar to those involved in the intracellular movements of other cytoplasmic vesicles, such as endoplasmic reticular-Golgi transport vesicles and synaptic vesicles (Schnitzer et al. 1995b ; Predescu et al. 1997 ). Such caveolar trafficking is inhibited by NEM (Predescu et al. 1994 ; Schnitzer et al. 1995a ). By analogy, exposure of cultured endothelial cells to NEM greatly reduced caveolin-positive clusters, as determined by light microscopy (Vasile et al. 1999 ). By electron microscopy, NEM-treated endothelial cells exhibited a reduction in their VVOs but retained individual caveolae (Vasile et al. 1999 ). Thus, the inhibition of VVO formation by NEM lends support to fusion of individual caveolae as a possible mechanism for VVO formation (Vasile et al. 1999 ).

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 (Calakos and Scheller 1996 ), a vesicle SNARE (soluble NEM-sensitive factor attachment protein receptor) is localized to the cytoplasmic side of caveolar and VVO membranes (Feng et al. in press ). The presence of this vesicle SNARE protein in these locations lends support to the proposed construction of VVOs from fused caveolae.


  VVO Fate
Top
Summary
Introduction
VVOs Are the Major...
VVO Structure
VVO Function
VVO Formation
VVO Fate
Literature Cited

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 porous—a 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 (Feng et al. 1997 , Feng et al. 1998b ); and (c) VVOs could serve as an extensive intracellular store of membranes that, in certain circumstances, could rapidly and greatly expand the endothelial plasma membrane. This expansion could be instrumental in the rapid formation of large vessels ("mother" vessels) in VPF/VEGF angiogenesis models (Feng et al. 2000a , Feng et al. 2000b ; Pettersson et al. 2000 ; Sundberg et al. in press ) and in the enhanced endothelial surface process formation that accompanies endothelial thinning and loss of VVOs at points of neutrophil transmigration in an animal model of acute inflammation (Feng et al. 1998a ).

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 (Feng et al. 1997 , Feng et al. 1998b , Feng et al. 1999 ). In studies of VPF/VEGF and other vasoactive mediators in combination with soluble macromolecular tracers, we encountered relatively few openings across venular endothelium of the type previously described (Majno et al. 1961 , Majno et al. 1967 , Majno et al. 1969 ; Joris et al. 1987 ; Baluk et al. 1997 ). In such experiments, ferritin and HRP extravasated across venular endothelium primarily by way of VVOs (Kohn et al. 1992 ; Dvorak et al. 1996 ; Feng et al. 1996 ). However, when we used colloidal carbon (d. ~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 (Feng et al. 1997 ). Similar types of openings have been described in the vascular endothelium lining some tumor microvessels (Roberts and Palade 1995 , Roberts and Palade 1997 ; Roberts et al. 1998 ), although we have not found them in the tumors we have studied (Dvorak and Monahan-Earley 1992 , Dvorak and Monahan-Earley 1995a , Dvorak and Monahan-Earley 1995b ; Kohn et al. 1992 ; Qu-Hong et al. 1995 ; Dvorak et al. 1996 ; Vasile et al. 1999 ; Feng et al. 1999 , Feng et al. 2000a , Feng et al. 2000b ).

Using carbon as tracer, we did serial sections and computer three-dimensional reconstructions of endothelial cell openings (Feng et al. 1997 ). They were found to be separate from endothelial cell junctions and to pass through endothelial cell cytoplasm. It is possible that aggregates of carbon delayed or prevented pore closure, accounting for the pore increases in our experiments. It is also possible that VVOs and endothelial pores are related structures. First, they share a common anatomic distribution in the microvasculature. Both are most numerous in venular endothelium, both concentrate in parajunctional zones of individual endothelial cells, and both may open to the intercellular clefts between lateral surfaces of adjacent endothelial cells as well as to the luminal and abluminal surfaces. In addition, both VVOs and pores form pathways for extravasation of macromolecules that can extend across adjacent endothelial cells (Feng et al. 1997 ). Taken together, these findings suggest that, in response to vasoactive mediators, pores may develop from a rearrangement of VVO vesicles and vacuoles to form larger membrane-lined vacuolar structures and, eventually, channels of sufficient size to allow the passage of particulate tracers as large as erythrocytes. Channel-like openings could arise from VVOs by a progressive opening or dissolution of the diaphragms that interconnect individual VVO vesicles and vacuoles with each other and with the endothelial cell luminal and abluminal plasma membrane. Transendothelial cell pores have also been described in response to supraphysiological increases in intraluminal pressure; such pressure might be expected to force open VVO diaphragms, providing transendothelial cell pores (Neal and Michel 1996 ).

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 (Feng et al. 1998a ). Here, we noted that VVOs became less numerous in thinned endothelium through which neutrophils were traveling, and that a markedly expanded endothelial cell luminal membrane covered many extended endothelial cell cytoplasmic processes. By analogy, the markedly and rapidly enlarged "mother" vessels in tumor and VPF/VEGF-induced angiogenesis models lend support to the third possibility, because VVOs were not a conspicuous component of the cytoplasm of the enlarged "mother" vessels and the formation of mother vessels occurred rapidly in an angiogenesis model (Feng et al. 2000a ; Pettersson et al. 2000 ; Sundberg et al. in press ). VVOs may provide membrane for the rapid formation of these vessels in angiogenesis.

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.


  Acknowledgments

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 Monahan–Earley for technical and photographic assistance.

Received for publication August 4, 2000; accepted November 27, 2000.


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Summary
Introduction
VVOs Are the Major...
VVO Structure
VVO Function
VVO Formation
VVO Fate
Literature Cited

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