Fig 2: TSP1 (thrombospondin-1)-induced CD47 (cluster of differentiation) activation blocks VEGF (vascular endothelial growth factor)-C–stimulated lymphangiogenic signaling. A through D, Lymphatic endothelial cells (LECs) were pretreated with TSP1 (22 nM, 16 h) in 0.5% fetal bovine serum (FBS) containing basal media MV2, stimulated with VEGF-C (15 min), and cell lysates subjected to Western blot analysis. A, Representative Western blot images are shown. B through D, Bar diagrams represent mean protein levels expressed as a ratio of phospho-to-total proteins, AKT (B), eNOS (C), and ERK1/2 (D; n=3). E, LECs were treated with control or CD47-siRNA (48 h) and immunoblotting done to determine CD47 expression (ab175388). F through I, Control or CD47-siRNA–treated cells were treated as in Figure 4A and Western blot experiments executed. F, Representative Western blot images are shown. G through I, Bar diagrams represent pAKT/total AKT (G), peNOS/total eNOS (H), and pERK1/2/total ERK1/2 (I; n=3). J, Control or CD47-silenced LECs were pretreated with TSP1 (22 nM, 4 h), stimulated with VEGF-C (100 ng/mL, 1 h) and analyzed for NO (nitric oxide) production using DAF-FM diacetate. K, Control or CD47-silenced LECs were pretreated with vehicle or TSP1 (1 h), incubated with H2DCFDA solution, and fluorescence analyzed using flow cytometry. Statistical analyses were performed using 1-way ANOVA (B–D), 2-way ANOVA with Tukey test for multiple comparisons (G–I and K), and 2-tailed unpaired Student t test (J). Data represent mean±SEM.

Fig 3: CD47 (cluster of differentiation) mediates TSP1 (thrombospondin-1)-induced inhibition of lymphangiogenesis. A, Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the relative mRNA levels of CD47 and CD36 in human lymphatic endothelial cells (HLECs) and human aortic endothelial cells (HAoEC). Bar graph represents mRNA levels in comparison to CD36 (n=9). B, Control and CD47 siRNA-treated lymphatic endothelial cells (LECs; 48 h) were utilized to quantify CD47 transcript expression using qRT-PCR (n=6). C, WST (water-soluble tetrazolium)-1 assay was conducted to investigate the effects of CD47 silencing on LEC proliferation in response to TSP1 treatment as described in Figure 2A. Data are representative of 3 independent experiments performed at least in quadruplicate. D, Control and CD47-silenced LECs were used to evaluate cell migration. Scale bar, 200 µm. xBar graph represents the percentage of migrated cells (n=3–4). E through G, Control and CD47-silenced cells were pretreated as in Figure 2D and seeded in wells of a Matrigel-coated plate in basal medium containing VEGF (vascular endothelial growth factor)-C±TSP1 and tube formation determined. Representative images of tube formation are shown (E). Scale bar, 1000 µm. Tube length (F) and number of branching points (G) quantified (n=7). H, Wild-type male mice were injected SC with Matrigel solutions premixed with either VEGF-C, VEGF-C+TSP1+IgG, or VEGF-C+TSP1+CD47-blocking antibody. Plugs were isolated after 10 days, sectioned, and immunostained for LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1). Representative images of LYVE-1 staining of the cross-sections of the Matrigel plugs and quantification of LYVE-1–positive area are shown (n=5–7). Scale bar, 20 µm. Statistical analyses were performed using 2-way ANOVA (A, C, D, F, and G) with Bonferroni (A), Tukey (C, F, and G), and Sidak (D) multiple comparisons test, 2-tailed unpaired Student t test (B), and Kruskal-Wallis test for multiple comparisons (H). Data represent mean±SEM.

Fig 4: Lymphatic endothelial cell (LEC)–specific deletion of Cd47 (cluster of differentiation) in mice increases arterial lymphatic vessel (LV) density. A through D, Aortic root cross-sections from male AAV (adeno-associated virus) 8-PCSK9–injected Cd47WT and Cd47?LEC mice (16-wk Western diet) were immunostained for CD68, iNOS (inducible NO [nitric oxide] synthase), Arg1 (arginase 1), and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1). Nuclei were counterstained with DAPI (4’,6-diamidino-2-phenylindole; blue). Representative confocal images of iNOS (green; A) and CD68 (red); Arg1 (green; B) and CD68 (red; scale bar, 50 µm); and LYVE-1 staining (red; C; scale bar, 20 µm) are shown (n=5–6). Statistical analyses were performed using a 2-tailed unpaired Student t test (A and B) and a Mann-Whitney U test (C). Data represent mean±SEM. A indicates adventitia; M, media; and P, plaque.