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First published online 5 August 2003
doi: 10.1242/jcs.00681

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Endothelial expression of the 6ß4 integrin is negatively regulated during angiogenesis

¹ Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208, USA
² The Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, NY 12208, USA
³ The Institute for Vascular Health and Disease, Albany Medical College, Albany, NY 12208, USA
⁴ The Center for Cardiovascular Sciences, Albany Medical College, Albany, NY 12208, USA

View larger version (42K): [in a new window]   Fig. 1. The 6ß4 integrin is expressed by human dermal microvascular endothelial cells. (A-D) Sections of human neonatal foreskin were double immunostained with antibodies to the endothelial marker von Willebrand Factor (vWF) (A), and with 3E1 antibodies to the ß4 subunit (B). The white dotted line indicates the epidermal-dermal interface. (C) The colocalization of ß4 and vWF is seen in the merged image as yellow, with a representative ß4-positive vessel indicated by the arrow. (D) A microvessel in cross section examined at higher magnification using confocal laser scanning microscopy, showing vWF (green) and ß4 (red). (E-J) Cells isolated from human neonatal foreskin were double immunostained to detect ß4 and endothelial cell-, smooth muscle cell-, and keratinocyte-specific markers: (E) cells stained with pAb to PECAM-1 (red) and 3E1 to ß4 (green); (G) cells stained with 1A4 to smooth muscle actin (red) and 3E1 to ß4 (green); and (I) cells stained with AE1/AE3 to human epidermal keratins (red) to detect keratinocytes and 439-9B to ß4 (green). (E,G,I) The corresponding phase contrast images are shown in F, H and J, respectively. The cells that stained brightly with only ß4 in E and G are probably keratinocytes. Scale bars: (A-C) 50 µm; (D-J) 10 µm.

View larger version (90K): [in a new window]   Fig. 2. ß4 integrin is expressed by endothelial cells of the vasa vasorum but not by endothelial cells lining the lumen of human saphenous vein. (A) Frozen section of human saphenous vein stained with Hematoxylin and Eosin; the asterisk indicates the lumen of the saphenous vein. The dashed box shows an example of the region chosen for higher magnification immunohistochemical imaging in BE. Cross sections of saphenous vein were stained with antibodies to vWF (B), marking endothelial cells lining the main lumen (arrow) and the vasa vasorum (arrowheads) and 3E1 to ß4 (D), selectively staining endothelial cells of the vasa vasorum (arrowheads). (C,E) Control immunostaining in the absence of primary antibody, for B and D, respectively. Scale bars: (A) 500 µm; (B-E) 200 µm.

View larger version (79K): [in a new window]   Fig. 3. Angiogenic endothelial cells do not express the 6ß4 integrin in explant cultures. Segments of human saphenous vein were grown as explant cultures in fibrin gels for 14 days. The plus sign indicates the explant tissue, with the outer surface of the original explant tissue outlined with a dotted blue line in A,C,E. (A,B) Cross sections of explant and outgrowth stained with antibodies to vWF, which stained outgrowing endothelial cells as well as endothelial cells remaining in the vasa vasorum and lining the saphenous vein lumen. A higher magnification of a region of outgrowth is shown in B. (C,E) Cross sections stained with 3E1 to ß4 (C) showing no ß4 expression on either the explant saphenous vein or the outgrowing endothelial cells, and 1A4 to smooth muscle actin (E) which stained the original explant, but not outgrowing cells. (D,F) Phase contrast images of C and E, respectively. Arrows indicate the direction of outgrowth. Controls with secondary antibody alone showed insignificant staining similar to the controls for the immunostainings in Fig. 2 (data not shown). Scale bars: (A,C-F) 200 µm; (B) 20 µm.

View larger version (67K): [in a new window]   Fig. 4. 6ß4-integrin-positive endothelial cells of the vasa vasorum contribute to angiogenesis in explant cultures of human saphenous vein. Human saphenous vein was treated with collagenase to strip the endothelial lining from the main lumen. (A,B) Cross sections of collagenase-treated explants were stained with pAb to PECAM-1 (A) and 3E1 to ß4 (B). Endothelial cells of the vasa vasorum maintained PECAM-1 and ß4 expression (black arrowheads); however, expression of PECAM-1 was not observed on the lumenal surface (open arrowhead), indicating the endothelial cells had been successfully removed. (C,D) Phase contrast images of endothelial outgrowth from collagenase-treated (D) or untreated (C) segments of saphenous vein after 14 days in culture in fibrinogen gels. Little angiogenic outgrowth occurred in the collagenase-treated lumen (D); however, robust endothelial outgrowth was observed from the vasa vasorum (E). (F-I) Cross sections of collagenase-treated saphenous vein explants were immunostained after 14 days in culture with either pAb to PECAM-1 (F,G), or with 3E1 to ß4 (H), indicating the outgrowing endothelial cells (arrow) do not express ß4. A region of outgrowth stained for PECAM-1 is shown at higher magnification in G. (I) A phase contrast image of H. (F,H) The perimeter of the explant is outlined by the dotted blue line. Controls with secondary antibody alone showed insignificant staining similar to the controls for immunostainings in Fig. 2 (data not shown). (J) Day-12 explant of human neonatal foreskin double stained with AE1/AE3 to human epidermal keratins (red) and 439-9B to ß4 (green), demonstrating that keratinocytes do not downregulate ß4 in explant cultures. Asterisks indicate vessel lumen. Plus signs indicate original explanted tissue. Arrows indicate outgrowing endothelial cells or keratinocytes. Scale bars: (A,B,F,H,I) 100 µm; (G) 20 µm; (J) 50 µm.

View larger version (114K): [in a new window]   Fig. 5. 6ß4 is expressed by endothelial cells in the adult mystacial pad. (A) Schematic representation of the vasculature associated with a whisker follicle (wf) in the adult mouse mystacial whisker pad. Small fur hairs (h) and a large whisker (w) are shown in dark gray. The epidermis (e), small hair follicles, and large whisker follicle are shown in light gray. The whisker follicle is surrounded by a vascular sinus (pink) which is enclosed by a dense collagen capsule (medium gray). Arteries, arterioles and capillary beds are red; veins and venules are blue; nerves are green; and piloerector muscles are brown (Fundin et al., 1997a; Fundin et al., 1997b; Rice et al., 1997). Dashed rectangles indicate areas comparable to sites in B,C, and in higher magnification in D-I. (B-I) Immunofluorescence staining of longitudinal sections of an adult whisker follicle. Adjacent sections were immunostained with anti-s100 (B,C: green) for Schwann cells on nerve axons. Double immunostaining with MEC 13.3 (B: red) labels PECAM-1 on the endothelium of all blood vessels. Double immunostaining with 346-11A (C: red) labels ß4 on the perineurium of nerves and endothelium of many blood vessels. Regions of B and C (dotted boxes) are shown at higher magnification (D,F,H) and (E,G,I) respectively. Arrows indicate nerves. Arrowheads indicate examples of ß4-positive vessels; dotted ovals indicate ß4-negative vessels. ß4 labeling is also present on basal keratinocytes (E, arrows) of the epidermis and hair follicles. Scale bars: 100 µm.

View larger version (83K): [in a new window]   Fig. 8. Confocal microscope images showing double immunostaining of microvascular endothelial cells for ß4 (red) and PECAM-1 (green). Each image is a maximum intensity projection image of a sub-stack of six optical sections taken from a larger stack of images collected at 0.3 µm intervals. The images were collected in the region just below the epidermal-dermal junction from sections of skin from adult (A-C) and P3 (D-F) mice. In the adult, almost all the vessels in this region showed immunoreactivity for ß4 and PECAM-1 (A-C, arrowheads). At P3, vessels expressing ß4 were rare (D-E, solid arrowhead), most lacked ß4 (open arrowheads). No vessels in this location showed ß4 immunoreactivity at P0 (not shown). Arrows indicate ß4 immunoreactivity on basal keratinocytes in the epidermis (e) and hair follicles (f). Scale bar: 25 µm.

View larger version (86K): [in a new window]   Fig. 6. Following the caudal to rostral and deep to superficial development of the whisker pad vasculature (E), embryonic microvascular endothelial cells of the whisker pad only express the ß4 integrin subunit in the caudal and deep vasculature in the whisker pad from an E19.5 p.c. animal. Sections from the caudal region (A,B) are shown immunostained with s100 (green) and either MEC 13.3 to PECAM-1 (A: red) or with 346-11A to ß4 (B: red). Neighboring sections from a more rostral region are shown immunostained with MEC 13.3 to PECAM-1 (C) or 346-11A to ß4 (D). The majority of the vasculature was ß4 negative, however, some larger caliber vessels deeper in the tissue were ß4 positive (arrowheads). (E) Schematic representation of the whisker pad showing six whisker follicles along the caudal to rostral axis, where the filled arrowhead indicates a whisker and the open arrowhead indicates a hair follicle. Intense ß4 labeling is present on basal keratinocytes of the epidermis (e), hair follicles (f) and whisker follicles (wf). Scale bars: 100 µm.

View larger version (109K): [in a new window]   Fig. 7. Microvascular endothelial cells turn on ß4 expression during postnatal development. The progression of endothelial ß4 expression was examined during postnatal development of the whisker pad. Neighboring sections were immunostained with s100 (A-F: green) and either MEC 13.3 to PECAM-1 (A,C,E: red) or with 346-11A to ß4 (B,D,F: red). Caudal regions from P0 embryos (A,B) and P3 embryos (C,D), and rostral region from P7 embryos (E,F) are shown. Arrowheads indicate examples of vessels that express ß4 (A-F). At P0, only vessels in the most caudal and deep regions of the whisker pad showed expression of ß4. By P3, the same regions show more vascular ß4 expression. By P7, ß4 expression is observed throughout the vasculature of the whisker pad, as shown on the rostral-most whisker follicles. Dotted ovals indicate examples of regions where vessels do not express ß4. Scale bars: 100 µm.