Vascular hyperpermeability, angiogenesis, and stroma generation

Abstract

It has been known for more than half a century that the tumor microvasculature is hyperpermeable to plasma proteins. However, the identity of the leaky vessels and the consequences of vascular hyperpermeability have received little attention. This article places tumor vascular hyperpermeability in a broader context, relating it to (1) the low-level "basal" permeability of the normal vasculature; (2) the "acute," short-term hyperpermeability induced by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A) and other vascular permeabilizing agents; and (3) the "chronic" hyperpermeability associated with longer-term exposure to agents such as VPF/VEGF-A that accompanies many types of pathological angiogenesis. Leakage of plasma protein-rich fluids is important because it activates the clotting system, depositing an extravascular fibrin gel provisional matrix that serves as the first step in stroma generation.

Figure 1.

Basal vascular permeability (BVP). (Upper) Electron micrograph illustrating a typical capillary endothelial cell with numerous caveolae. Many of these are connected to the luminal or abluminal plasma membranes (arrows). Scale bar, 100 nm. (Lower) Schematic diagram illustrating pathways by which molecules can cross the capillary barrier. (1) Interendothelial cell cleft, (2) caveolae that shuttle across the capillary endothelium or form a chain of vesicles connecting the lumen and albumen. (BL) Basal lamina, (EC) endothelial cell, (BVP) basal vascular permeability. (Figure adapted from Nagy et al. 2008a; reproduced, with permission, from Springer © 2008.)

Figure 2.

Transmission electron micrographs of normal venules (A,B) and hyperpermeable microvessels (C–G). (A) Typical normal venules lined by cuboidal endothelium. The cytoplasm contains prominent VVOs and is enveloped by a complete coating of pericytes (P). (R) Red blood cell. (B) Ultrathin section from a set of 36 consecutive serial sections demonstrates three sets of VVO vesicles (x,y,z) that could be traced from the vascular lumen to albumen. (C) Mother vessels (MVs) are characterized by greatly enlarged size, extensive endothelial cell thinning, striking reduction in VVOs and other cytoplasmic vesicles, prominent nuclei that project into the vascular lumen, mitotic figures (arrows), and pericyte (P) detachment. MV lumens are packed with red blood cells, indicating extensive plasma extravasation. (Inset) The normal venule depicted in A is reproduced in C at the same magnification as the MVs to illustrate relative size differences. (D–G) Leakage of ferritin tracer (black dots, some encircled) through MV VVOs (D,E) and fenestrae (F, arrows), and through VVOs of a normal venule in response to VPF/VEGF-A (G). Scale bars, 1 µm (A); 50 nm (B,D,G); 5 µm (C); 200 nm (E,F). (A, C, and E adapted from Nagy et al. 2006; reproduced, with permission, from Nature Publishing © 2006. D adapted from Kohn et al. 1992; reproduced, with permission, from Nature Publishing © 1992. B and G adapted from Feng et al. 1996; reproduced, with permission, from The Rockefeller University Press © 1996. F adapted from Feng et al. 1999a; reproduced, with permission, from John Wiley and Sons © 1999.)

Figure 3.

Pathways of acute and chronic vascular hyperpermeability. (A) Schematic diagram of a normal venule comprised of cuboidal endothelium with prominent VVOs and closed interendothelial cell junctions. Note that some VVO vesicles attach to the intercellular cleft below the tight and adherens junctions. 1 and 2 indicate potential pathways for transcellular (VVO) and intercellular (paracellular) plasma extravasation, respectively. Basal lamina (BL) is intact and the endothelium is covered by pericytes. (B) AVH. Acute exposure to VEGF-A causes VVOs to open, allowing transcellular passage of plasma contents, possibly by mechanical pulling apart of stomatal diaphragms. Others have suggested that fluid extravasation takes place through an opening of intercellular junctions. (C) CVH. Prolonged VEGF-A stimulation causes venules to transform into MVs. Plasma may extravasate either through residual VVO vesicles (4) or through fenestrae (5). (Figure adapted from Nagy et al. 2008a; reproduced, with permission, from Springer © 2008.)

Figure 4.

Computer-generated three-dimensional reconstruction of serial electron microscope sections of a colloidal carbon-filled pore induced in rat cremaster venule by VPF/VEGF-A. Panels represent successive rotations toward the viewer around a horizontal axis at the angles indicated. 0° (not shown) corresponds to a vascular cross section taken at right angles to the direction of blood flow and 90° corresponds to a view looking down on the luminal surface; views ≥240° illustrate the abluminal surface. Endothelial cells are labeled 1 (yellow) and 2 (orange-brown). Tracer colloidal carbon (black) fills the pores in the left panels. In the middle panels, carbon was deleted from the reconstruction to facilitate visualization into the pore interior, which is seen to pass transcellularly through the cytoplasm of both endothelial cells, revealing the underlying turquoise background. A small portion of the large pore, which passes through cell 1 (yellow), does not traverse cell 2, as evidenced by the underlying orange-brown background (middle panel, 30° and 90°). Also, a second smaller pore, as well as small portions of the larger pore, penetrate cell 2 without passing through cell 1 (recognized by yellow instead of turquoise background in panels ≥240°). Right panels depict endothelial cells 1 and 2 separately, again with carbon deleted. (L) Lumen. (Figure adapted from Feng et al. 1997; reproduced, with permission, from Wiley-Blackwell © 1997.)

Figure 5.

Chronic vascular hyperpermeability and tumor stroma generation. (A–C) Blood vessels (arrows) supplying line 10 guinea pig tumors are hyperpermeable to circulating macromolecular fluoresceinated dextran, outlining tumor nodules. (D,E) Line 10 tumor cells 48 h after transplant into the subcutaneous space of syngeneic strain 2 guinea pigs. Fibrin forms a water-trapping gel (F) that serves as a provisional stroma that separates tumor cells into discrete islands and that provides a favorable matrix for fibroblast (white arrows) and endothelial cell migration. (F,G) Immunohistochemical demonstration of fibrin (brown staining) in guinea pig line 1 and human colorectal adenocarcinoma, respectively. (H) Fibroblasts and blood vessels (black arrows) invade line 1 tumor fibrin gel, replacing it with fibrous connective tissue. (I) Fibroblasts (arrows) migrate through fibrin gel (F) in culture. (J) Implanted fibrin gel (F) in subcutaneous space is replaced by ingrowing fibroblasts and new blood vessels, creating granulation-like vascular connective tissue. Scale bars, 25 µm (B,I), 50 µm (A,C,D,H), and 100 µm (E,F,J). (A–F and H–J adapted from Dvorak 2003; reproduced, with permission, from the author. G adapted from Dvorak et al. 1988; reproduced, with permission, from the American Journal of Pathology © 1988.)