Figure 1.
COSMC is overexpressed in proliferating hemangiomas.
(A) Immunohistochemistry of human hemangioma tissues. Paraffin-embedded hemangiomas in proliferating (N = 13), involuting (N = 21), and involuted (N = 9) phases were immunostained with anti-COSMC antibody or biotin-conjugated peanut agglutinin (PNA). Representative images are shown. Scale bars: 50 µm. Negative controls did not show specific staining (data not shown). (B) COSMC is overexpressed in proliferating and involuting hemangiomas compared with involuted hemangiomas. Intensity of immunostaining was quantified. N = patient numbers. Data are presented as means ± SEM. **P<0.01; ***P<0.001.
Figure 2.
Expression of COMSC in HUVECs and EA.hy926 cells.
(A) Proliferating infantile hemangiomas (IHs) (n = 3 patients) express higher levels of COSMC than HUVECs (n = 3 batches). COSMC mRNA levels were analyzed by real-time RT-PCR. *P<0.05. In lower panel, COSMC is overexpressed in HUVECs. (B) Western blots showing COSMC overexpression (left panel) and knockdown (right panel) in HUVECs. β-actin is a loading control. Relative intensity of signals on Western blots was quantified by ImageQuant5.1. (C) COSMC overexpression and knockdown in human endothelial cell line EA.hy926. Relative intensity of signals on Western blots was quantified by ImageQuant5.1. (D) COSMC overexpression and knockdown modulate cell surface carbohydrates on HUVECs. Left panel, COSMC overexpression enhances T synthase and T antigen expression in HUVECs. HUVECs were cell surface biotinylated, lysed, pulled down (PD) with PNA, and then blotted with streptavidin-HRP. The arrow indicated that a protein band with molecular mass of 220-kDa has increased PNA binding in COSMC-transfected HUVECs. Cell lysates were Western blotted for detecting T synthase expression and β-actin was used as a loading control. Right panel, COSMC knockdown increased glycoproteins pulled down by VVA, which recognizes Tn antigen.
Figure 3.
COSMC overexpression enhances cell growth in HUVECs.
(A) Cell growth of HUVECs transfected with pcDNA3.1 control plasmid (open bars) or COSMC/pcDNA3.1 (closed bars) analyzed by trypan blue exclusion assays. (B) Cell growth of HUVECs transfected with control siRNA (open bars) or COSMC siRNA (closed bars) analyzed by trypan blue exclusion assays. (C) Cell growth of EA.hy926 cells overexpressing COSMC. (D) Cell growth of EA.hy926 cells with COSMC knockdown. Results are presented as means ± SD from three independent experiments. *P<0.05 and **P<0.01, compared with mock.
Figure 4.
Roles of AKT and ERK signaling pathways in COSMC-enhanced cell proliferation.
(A) COSMC overexpression enhances phosphorylation of AKT and ERK. Western blotting was performed to analyze protein expression. Representative images are presented. Relative intensity of signals was quantified by ImageQuant5.1 and shown. (B) COSMC knockdown inhibits phosphorylation of AKT and ERK. (C) Effects of AKT and ERK inhibitors on cell proliferation. HUVECs transfected with mock or COSMC plasmids were treated with DMSO control, 5 µM of LY294002, or 10 µM of PD98059. Cell viability was analyzed by MTT assays at different time points. Results are presented as means ± SD from three independent experiments. *P<0.05; **P<0.01.
Figure 5.
COSMC overexpression modulates O-glycans on VEGFR2.
(A) Changes in O-glycans on VEGFR2 in COSMC overexpressing HUVECs. Cell lysates of HUVECs transfected with control or COSMC plasmids were treated with or without neuraminidase, pulled down with VVA or PNA lectins, then immunoblotted with anti-VEGFR2 antibody. β-actin is an internal control. (B) COSMC knockdown in HUVECs decreases binding of PNA to VEGFR2. (C) O-glycans are present on VEGFR2 in human primary hemangiomas. Tissue lysates of proliferating and involuted hemangiomas with (+) or without (−) neuraminidase treatment were pulled down (PD) by PNA and then immunoblotted with anti-VEGFR2 antibody. β-actin is an internal control. (D) COSMC overexpression enhances phosphorylation of VEGFR2 in HUVECs. HUVECs were serum starved for 4 h and then treated with 20 ng/ml of VEGF for different time periods. Phosphorylation of VEGFR2, AKT, and ERK were analyzed by Western blotting. β-actin is a loading control. Representative images from two independent experiments were shown. Signals on Western blots were quantified by ImageQuant5.1. (E) COSMC knockdown suppresses phosphorylation of VEGFR2 in HUVECs. Signals on Western blots were quantified by ImageQuant5.1.
Figure 6.
COSMC modulates protein degradation of VEGFR2.
(A) COSMC overexpression delays degradation of VEGFR2. HUVECs were treated with cycloheximide (10 µg/ml) to block protein synthesis and 20 ng/ml of VEGF to trigger internalization and degradation of VEGFR2 for indicated time points. Upper panel shows representative Western blots. Lower panel shows signals on Western blots quantified by ImageQuant5.1 for HUVECs transfected with pcDNA3.1 control plasmid (dashed line) and UVECs transfected with COSMC/pcDNA3.1 (solid line). (B) COSMC knockdown facilitates degradation of VEGFR2. VEGFR2 degradation in HUVECs transfected with control siRNA (dashed line) or COSMC siRNA (solid line) was shown. Representative data from two independent experiments are presented.