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. 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, 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. 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 .
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, we localized histamine, which is secreted from mast cells in the allergic eye disease mouse model, to VVOs in involved vessels.
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 macro-molecules. 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 . Electron microscopy demonstrated that the circulating tracer
(anionic ferritin) exited such venules primarily by way of VVOs, just as in tumor microvessels. Rapidly after 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, that is, 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 in that the passage of ferritin was not restricted. However, diaphragms closed some stomata to the passage of ferritin since 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. Together, these data establish VVOs as the major pathway by which soluble plasma proteins exit nontumor venules in response to several mediators that increase venu-lar hyperpermeability.
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 man), which are selectively expressed in vascular endothelium and are both strikingly upregulated in tumors, wounds, and inflammation in which VPF/VEGF is overexpressed. We used ultrastructural preembedding immunoperoxidase and immunonanogold methods to localize VEGFR-2 (flk-1/KDR) in vascular endothelium in model systems in which VPF/VEGF is highly expressed: (1) glomerular and peritubular capillaries of normal mouse kidney; (2) microvessels supplying a well-characterized mouse mammary carcinoma; and (3) new vessels induced by an adenoviral vector, engineered to overexpress VPF/VEGF (adeno-vpf/vegf) . Microvascular endothe-lial cells were positive for VEGFR-2 in all three models and, in the latter two, could be localized to the luminal and ablu-minal 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. Localization of VPF/VEGFR-2 to VVO membranes, 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 microvascu-lar permeability that is induced by VPF/VEGF and other vasoactive mediators .
Stomatal diaphragms are likely the structures that regulate VVO permeability. Support for this concept is based on the strategic localization of VEGFR-2 on this diaphragm which closes stomata in VVOs and of VPF/VEGF bound to VVOs in endothelia present in animal tumor models .
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