Figure 5

Effect of syndecan-1 expression on in vitro and in vivo MMEC angiogenesis. (a) Representative micrographs of capillary-like structure formation on Matrigel formed by BMECs (top left), MMECs (top right), MMEC syndecan-1 siRNA (bottom left) and MMEC scr-siRNA (bottom right) after 24 h of incubation. Original magnification, × 100. (b) Morphometric evaluation of capillary-like structures formed onto Matrigel after 5 h (grey bars) and 24 h (black bars). Data are expressed as the mean±s.d. of the length of capillary-like structures evaluated, in arbitrary units by the computer analysis system, in six different experiments performed in triplicate. Analysis of variance with Newman–Keuls multi-comparison test was performed (^*P<0.001 MMECs and MMEC scr-siRNA versus HUVECs; ^†P<0.001 MMECs and MMEC scr-siRNA versus BMECs; ^‡P<0.001 MMEC syndecan-1 siRNA versus MMECs; ^§P<0.05 MMEC syndecan-1 siRNA versus BMECs). (c, d) HUVECs, BMECs, MMECs, MMEC syndecan-1 shRNA and, as control, MMEC scr-shRNA within Matrigel were injected subcutaneously into SCID mice. Mice were killed after 7 days and Matrigel plugs were submitted for histological analysis. (c) Representative micrographs (haematoxylin/eosin staining) of Matrigel-containing BMECs (top left), MMECs (top right), MMEC syndecan-1 shRNA (bottom left) and MMEC scr-shRNA (bottom right). BMECs, MMEC syndecan-1 shRNA and MMEC scr-shRNA showed formation of vessels containing blood erythrocytes. Original magnification, × 200. (d) Representative confocal micrograph showing expression of human CD31 (left) and human leukocyte antigen class-I (right) on neo formed vessels in Matrigel by MMECs. Original magnification, × 400. (e) Quantitative evaluation of angiogenesis in a section of Matrigel plugs stained by haematoxylin/eosin staining. Angiogenesis was evaluated as the percentage of vessels area in five different fields at × 100 magnification. Only vascular structures containing red blood cells were counted as vessels. The data are the mean±s.d. of five individual experiments. Analysis of variance with Newman–Keuls multi-comparison test was performed (^*P<0.001 MMECs and MMEC scr-shRNA versus HUVECs; ^†P<0.001 MMECs and MMEC scr-siRNA versus BMECs; ^‡P<0.001 MMEC syndecan-1 siRNA versus MMECs).