Regulated angiogenesis and vascular regression in mice overexpressing vascular endothelial growth factor in airways

Abstract

Angiogenesis and vascular remodeling occurs in many inflammatory diseases, including asthma. In this study, we determined the time course and reversibility of the angiogenesis and vascular remodeling produced by vascular endothelial growth factor (VEGF) in a tet-on inducible transgenic system driven by the CC10 promoter in airway epithelium. One day after switching on VEGF expression, endothelial sprouts arose from venules, grew toward the epithelium, and were abundant by 3 to 5 days. Vessel density reached twice baseline by 7 days. Many new vessels were significantly larger than normal, were fenestrated, and penetrated the epithelium. Despite their mature appearance at 7 days suggested by their pericyte coat and basement membrane, the new vessels started to regress within 3 days when VEGF was switched off, showing stasis and luminal occlusion, influx of inflammatory cells, and retraction and apoptosis of endothelial cells and pericytes. Vessel density returned to normal within 28 days after VEGF withdrawal. Our study showed the dynamic nature of airway angiogenesis and regression. Blood vessels can respond to VEGF by sprouting angiogenesis within a few days, but regress more slowly after VEGF withdrawal, and leave a historical record of their previous extent in the form of empty basement membrane sleeves.

Figure 1-4207

VEGF overexpression induces angiogenesis of airway blood vessels. A–F: Blood vessels stained by perfusion of biotinylated L. esculentum lectin in flattened whole mounts of mouse tracheas. A: WT control mouse drinking normal water. Blood vessels form regular arcades of arterioles (A) and venules (V) in regions between the cartilages and capillaries (arrows) overlying the cartilages. B: VEGF-Tg mouse, 5 days of drinking doxycycline (dox)-water. Endothelial sprouts arise from postcapillary venules (arrows). C: Enlargement of part of B showing endothelial sprouts (arrows). D: VEGF-Tg mouse, 7 days dox-water. Numerous endothelial loops and sprouts increase the vessel area density. E and F: Nomarski images of enlarged regions of D. E: Endothelial sprouts have various shapes, including pointed (arrow) and blunt (arrowheads). Some are field with stagnant erythrocytes. F: Numerous endothelial loops form bulbous protrusions (arrows) toward the epithelium. G and H: Toluidine blue-stained sections of tracheal mucosa. G: WT control, normal water. Blood vessels (arrows) are located far from the epithelium in the connective tissue overlying the cartilage (C). H: VEGF-Tg mouse, 7 days dox-water. Blood vessels (arrows) are more abundant and are located much closer to, or even within, the epithelium. Scale bar: 200 μm (A, B, D); 50 μm (C, E, F); 25 μm (G, H).

Figure 2-4207

Time course of onset of angiogenesis and VEGF expression after doxycycline. A: Percentage area density occupied by blood vessels perfused and stained with L. esculentum lectin in whole mount preparations of the mouse trachea (group size at least four mice per point). B: Concentration of VEGF in bronchoalveolar lavage fluid as measured by enzyme-linked immunosorbent assay. *, Significant difference from baseline (P < 0.05).

Figure 3-4207

Ultrastructural features of newly formed airway blood vessels. A: WT control, normal water. Capillary (*) distant from epithelium. B: VEGF-Tg mouse, 7 days dox-water. Newly formed blood vessels (*) are close to and within the distorted epithelium containing vacuoles (arrowhead). Endothelial cells are accompanied by pericytes and periendothelial cells (arrow) and are separated by empty-looking space from epithelium. C: WT control, normal water. Endothelial cells of normal capillary are accompanied by basement membrane (arrowheads) and processes of a pericyte (arrow) and periendothelial cell beyond (double arrow). D: VEGF-Tg mouse, 7 days dox-water. D: Capillary has many fenestrations and delicate basement membrane (arrowheads). Thin processes of a pericyte (arrow) and periendothelial cells (double arrow) accompany the new blood vessel. A platelet (*), erythrocyte, and coagulated material (**) appear trapped inside lumen. E and F: Enlarged regions of C and D. G: Growing blood vessel has a narrow slit-like lumen (arrowheads). H: Another growing blood vessel has plump endothelial cells with many ribosomes. An erythrocyte is trapped between the endothelial cell junctions (*). Pericyte processes (arrows) accompany blood vessel. Scale bar: 5 μm (A, B); 1 μm (C, D); 0.5 μm (E, F); 2 μm (G, H).

Figure 4-4207

Immunohistochemical features of normal endothelial cells and pericytes. A: Overview of a whole mount preparation of tracheal microvascular arcades stained for endothelial cells (hamster anti-mouse CD31, green) and pericytes (desmin, red). B: Higher magnification view of desmin-positive smooth muscle cells (red) on arteriole (A) and pericytes on capillaries (C) and venules (V) stained with rat anti-mouse CD31 antibody (green). C and D: Periendothelial cells of tracheal microvascular arcades stained for α-smooth muscle actin (red) and NG2 (green). Arterioles (arrow) and venules are stained for α-smooth muscle actin, but NG2 immunoreactivity is weak. Pericytes on normal capillaries (arrowheads) are strongly positive for NG2, but negative for α-smooth muscle actin immunoreactivity. E: Staining for endothelial cells (hamster CD31, green) and VEGFR-2 (red). VEGFR-2 is more strongly expressed on capillaries (arrowheads) than on arterioles (A) or venules (V). VEGFR-2 is also expressed on lymphatic vessels (L). F: Type IV collagen immunoreactivity (red) on normal blood vessels that have relatively smooth outlines. Bubble-like bulges (arrowheads) indicate location of pericyte nuclei. Weak staining for type IV collagen is also present on nerve bundles (N) and in patches on lymphatic vessels (L). Scale bar: 65 μm (A); 50 μm (B, D); 200 μm (C); 100 μm (E); 36 μm (F).

Figure 5-4207

Immunohistochemical features of growing endothelial cells and pericytes. A: Low magnification of blood vessels from VEGF-Tg mouse treated with dox-water for 3 days and stained for endothelial cells (CD31, green) and type IV collagen immunoreactivity (red). Profiles of blood vessels are rough and jagged. Regions indicated by arrows and arrowheads are enlarged in B and C, respectively. Endothelial sprouts (green, arrowheads) are covered by a delicate irregular coat of type IV collagen immunoreactivity (red). D: Section of tracheal wall from VEGF-Tg mouse treated for 7 days with dox-water stained for endothelial cells (green) and type IV collagen immunoreactivity (red). Newly formed blood vessels are located close to the epithelial basement membrane (red line, arrowheads) or penetrate (arrows) into the epithelium (E). E–K: Pericytes (red) on growing blood vessels stained for CD31-immunoreactivity (green). E and F: VEGF-Tg mouse, 3 days dox-water. Pericytes (red) stained for desmin in E, and NG2 in F, are absent from the extreme tips of growing endothelial sprouts (arrows). G: VEGF-Tg mouse, 7 days dox-water. Pericytes stained for desmin are present on most endothelial loops and sprouts (arrows). Some pericytes cross between adjacent endothelial loops. H–M: Up-regulation of PDGFR-β and VEGFR-2 receptor fluorescence intensity (red) in newly formed blood vessels. Endothelial cells labeled with CD31 (green). Pairs of photos are printed with the same contrast and intensity settings, so intensity is a measure of receptor expression. H and I: Expression of PDGFR-β immunoreactivity (red) in WT mice (H) and VEGF-Tg mice treated with dox-water for 7 days (I). PDGFR-β immunoreactivity is weak in blood vessels of WT mice, but is stronger in newly formed blood vessels. J and K: VEGFR-2 expression (red) is present on endothelial cells of WT mouse (J) and is greatly increased in VEGF-Tg mouse (K) treated with dox-water for 7 days. L and M: Surface plots of fluorescence intensities of PDGFR-β and VEGFR-2 immunoreactivity shown in H to K. Warmer, redder colors indicate greater pixel intensities. N: Leakage of RCA1 lectin (red, arrows) from blood vessels in VEGF-Tg mouse treated with dox-water for 7 days. Scale bar: 50 μm (A, E, F, H–K); 20 μm (B, C); 40 μm (D); 25 μm (G, N).

Figure 6-4207

Time course of regression of blood vessels and VEGF after withdrawal of doxycycline. VEGF-Tg mice were given dox-water for 7 days, and then normal water. A: Percentage area density occupied by blood vessels stained by biotinylated L. esculentum lectin in whole mount preparations of the trachea. B: Concentration of VEGF in bronchoalveolar lavage fluid as measured by enzyme-linked immunosorbent assay. *, Significant difference from baseline; †, significant difference from initial peak value at 7 days of doxycycline treatment (P < 0.05).

Figure 7-4207

Features of regressing endothelial cells and pericytes. A–D: Low (A, C)- and high (B, D)-magnification views and of blood vessels in tracheal whole mount preparations stained by perfusion of L. esculentum lectin in VEGF-Tg mouse treated with dox-water for 7 days, and then with normal water for 3 days (A, B) or 28 days (C, D). Three days after withdrawal of doxycycline (A, B), fewer endothelial loops and many blood vessels are blocked with erythrocytes (arrows) and are weakly stained by lectin. At 28 days after doxycycline withdrawal (C, D), the vasculature has almost returned to the baseline appearance of Figure 2A. A few vessels are blocked by erythrocytes and are weakly stained by lectin (arrows). E–I: VEGF-Tg mouse treated with dox-water for 7 days (E, F, H, I) or 28 days (G) and then normal water for 7 days. Endothelial cells are stained for CD31 (green) and (E–G) basement membrane for type IV collagen (red), and (H, I) pericytes for desmin (red). Many type IV collagen-immunoreactive sleeves devoid of CD31 staining (arrows in E–G) and CD31-positive cells are present (arrowheads). Desmin-positive pericytes (arrows in H and I) are confined to blood vessels. J–M: Two pairs of images from trachea of VEGF-Tg mouse on dox-water for 7 days, then normal water for 7 days. Triple staining for CD31 (green in all panels) activated caspase-3, an apoptosis marker (red in J and L), and nidogen/entactin immunoreactivity, a marker for basement membrane (red in K and M). Endothelial cell (arrow in J) and pericyte (arrow in L) are labeled for activated caspase-3. Double arrows indicate segments of empty basement membrane sleeves. Nerve bundle (N) in M also has basement membrane. Scale bar: 200 μm (A, C); 50 μm (B, D); 160 μm (E, H); 40 μm (F, G, I, J–M).