Fig 2: Effect of Lacidipine on the endothelial cell functions. Effect of Lacidipine on cell proliferation in HUVEC cultured as a monolayer (a) or in the angiogenesis assay (b); n = 6 per concentration, cell count was assessed after 48 h of assay initiation. Data expressed as mean ± SEM, one-way ANOVA followed by Bonferroni’s post hoc test, *** p < 0.001 compared with vehicle. (c) Representative fluorescence images following OrisTM migration assay of HUVEC treated with vehicle or Lacidipine (1, 2, and 5 µM) for 22 h. The yellow dotted line represents the cell edge at 0 h after removing the stoppers. Scale bar = 0.5 mm. (Right panel): quantification of cell migration. The graph represents the average per cent of gap closure ± S.E.M. from six independent experiments. Unpaired student’s t-test was used. No significant differences between groups were detected. (d) The effect of Lacipidine (1, 2, and 5 µM) on tube formation was assessed in a coculture angiogenesis assay where HUVECs were plated on a confluent human dermal fibroblast layer. The medium containing the compound was refreshed on days 3 and 5 following the plating of endothelial cells. Cocultures were stained with an antibody against CD31 and imaged 7 days after endothelial cell plating. The representative fluorescence images of the cocultures treated with Lacidipine (5 µM) and DMSO (vehicle). Inset, “White arrows” indicate the newly formed lumens. (Left panel), total tube length was quantified in nine fields for each well (n = 6 wells per group). Error bars, mean ± SEM, one-way ANOVA followed by Bonferroni’s post hoc test, **** p < 0.001 as compared with vehicle. Scale bar = 100 µm.

Fig 3: Identification of PNA binding ligands on endothelial cell surface. (A) PNA-agarose affinity purification followed by mass spectrometry analysis reveals MCAM and PECAM as two major cell surface glycan to be extracted by PNA affinity purification from HUVECs (insert shows silver staining of the eluted proteins eluted from PNA-agarose and separated by SDS-PAGE). (B) Representative confocal microscopy images show co-localization of PNA with MCAM on HUVEC cell surface. (C) MCAM immunoblots of PNA-agarose precipitates, without or with pre-treatment of the proteins with N- (PNGase-F) or O-glycanase. Treatment of the PNA precipitates with N-, but not O-glycanase, substantially reduces MCAM in the precipitates.

Fig 4: GPNMB/OA-expressing human mammary tumors display enhanced vascular density.(A) Human breast tumors were stained with CD31 and classified into two groups, those with low or high microvascular density (MVD). (B) Laser capture microdissection was used to extract RNA specifically from the tumor epithelium of low or high MVD breast tumors. Analysis of GPNMB/OA mRNA expression values for each tumor revealed a significant correlation between high levels of GPNMB/OA expression in the tumor epithelium and high microvascular density. *, P = 0.008, Student's t-test.

Fig 5: The expression of proangiogenic molecules is distinctly changed in recurrent GBM tumors. Representative images of FFPE tissue sections of primary and recurrent tumors stained for (a) VEGF (red), (c) CXCL2 (green), (e) IL8 (green), (g) blood vessels (CD31; red), (i) CXCR2 (green), and cell nuclei (DAPI; blue); scale bars: 100 µm. (b,d) Graphs show calculation of the stained area of VEGF (b) and CXCL2 (d) as a percentage in relation to the total analyzed tumor area (n = 38). (f) The percentage of patients expressing IL8 in primary and recurrent tumors was assessed (n = 38). (h,j) Graphs depict vessels/mm2 and the percentage of CXCR2+ vessels (n = 28–30). All primary tumors are depicted in black and recurrent tumors in grey. Box plots depicting mean (shown as “+”), median, 1st and 3rd quartile (shown as the box), and min to max (shown as the whiskers). * p < 0.05. Paired Student’s t-test; box plots depicting mean (shown as “+”) and median ± standard deviation.