NG2-stimulated EC migration. (A) MAEC were tested for migration through Transwell membranes coated on the underside with type I collagen (30 μg/ml), Matrigel (30 μg/ml), or various concentrations of NG2/EC⁺ (0.1–100 μg/ml). Three high-power microscope fields were counted in each replicate well, and results were expressed as cells per field. Each bar represents the mean ± SD (n = 3). Statistically significant differences compared with the control are indicated (^*p < 0.001; ^**p < 0.01). Insets show representative micrographs of stained cells on the underside of control (left) and NG2-coated (25 μg/ml) wells (right). Scale bar, 50 μm. (B) Transwells were coated with type I collagen (30 μg/ml), Matrigel (30 μg/ml), or NG2/EC+ (10 μg/ml), and preincubated with NG2 antibody or control IgG (20 μg/ml) for 1 h before MAEC were added. Cell migration was quantified as described above. Each bar represents the mean ± SD (n = 3). A statistically significant difference was obtained for anti-NG2 compared with the value obtained in the presence of control IgG (^*p < 0.001). (C) HMVEC migration was quantified in Transwells coated with Matrigel (30 μg/ml) or various concentrations of NG2/EC+ (5–100 μg/ml). Each bar represents the mean ± SD (n = 3). Statistically significant differences compared with the control are indicated (^*p < 0.0001; ^**p < 0.01).

Corneal angiogenesis. (A) Hydron pellets containing PBS (control) or NG2/EC⁺ (500 ng) were implanted into Sprague-Dawley rat corneas. Five days later, vessels growing toward the pellet implant from the limbus were photographed. (B) Rat and mouse corneal angiogenesis in summary. Pellets containing 200 ng of NG2/EC⁺ or 50 ng of bFGF were implanted in the case of BALB/c and C57Bl/6 mice, and 500 or 100 ng, respectively, were used for Sprague-Dawley rats. For antibody inhibition, anti-NG2 mAb or isotype-matched control IgG (2 μg/pellet) was directly added to the pellet before implantation into corneas of C57Bl/6 mice. “Mock purified” material was also tested in C57Bl/6. n.d., not determined.

Inhibition of EC migration by antigalectin-3 antibody. (A) Detection of galectin-3 on the surface of ECs. Immunofluorescence (left panels) and flow cytometry (right panels) were performed on unpermeabilized MAEC (top panels) and HMVEC (bottom panels) using antigalectin-3 antibody. Scale bar, 20 μm. (B) MAEC were preincubated with either nonimmune rat IgG, anti-CD44, or antigalectin-3 antibody (15 μg/ml) for 15 min and assayed for cell motility in Transwells coated with Matrigel (30 μg/ml), type I collagen (30 μg/ml), fibronectin (10 μg/ml), or NG2/EC⁺ (10 μg/ml). Each bar represents the mean ± SD (n = 3). A statistically significant difference was obtained for antigalectin-3 on NG2/EC-coated Transwells compared with the value obtained in the presence of control IgG (^*p < 0.02).

EC response to NG2 involves both galectin-3 and α3β1 integrin. (A) HMVEC preincubated for 15 min with either nonimmune IgG, anti-α3, or antigalectin-3 antibody (15 μg/ml) were assayed for motility in Transwells coated with Matrigel (30 μg/ml) or NG2/EC⁺ (50 μg/ml). Each bar represents the mean ± SD (n = 3). ^*p < 0.02 indicates statistically significant differences from the value obtained in the presence of control IgG, and ^**p < 0.05 from the value obtained in the presence of either α3 or galectin-3 antibody alone. (B) Detergent extracts of MAEC were immunoprecipitated with anti-β1 integrin antibody or control IgG. Immunoprecipitates were fractionated by SDS-PAGE and immunoblotted with antibodies against α3 integrin, β1 integrin, and galectin-3. Crude MAEC lysate was also loaded as a positive control. (C) Detergent extracts of A375 cells were immunoprecipitated with anti-NG2 antibody, anti-β1 integrin antibody or control IgG. Immunoprecipitates were fractionated by SDS-PAGE, and immunoblotted with antibodies against NG2, α3 integrin, β1 integrin, and galectin-3. NCAM was immunoblotted as a negative control to show the specificity of the immunoprecipitations.

Attachment and spreading of ECs on NG2. (A) NG2 binding to the EC surface. NG2 immunoreactivity is seen on the surfaces of MAEC incubated with soluble NG2/EC⁺, but not on untreated cells (control). DAPI was used as a nuclear counterstain. Scale bar, 20 μm. (B) EC attachment to NG2-coated surfaces. MAEC were tested for their ability to adhere to wells coated with Matrigel (30 μg/ml), type I collagen (30 μg/ml), and various concentrations of NG2/EC⁺ (1–100 μg/ml). Three high-power microscope fields were counted in each replicate well, and results were expressed as cells per field. Each bar represents the mean ± SD (n = 3). Statistically significant differences compared with the control are indicated (^*p < 0.005; ^**p < 0.05). Insets show the stained cells in control (left) and NG2-coated (100 μg/ml) wells (right). Adherent cells in the right-hand panel represent roughly 90% of the input cells. Scale bar, 50 μm. (C) Spreading of ECs on NG2-coated surfaces. Staining with rhodamine-phalloidin was used to visualize F-actin organization in MAEC after adherence for 1 h to wells coated with BSA (control), type I collagen (30 μg/ml), NG2/EC⁺ (10 μg/ml), or NG2/EC^- (10 μg/ml). Scale bar, 20 μm.

EC network formation in three-dimensional collagen gels. (A) Formation of cord-like endothelial networks. MAEC were plated onto type I collagen gels in the absence of NG2 (control) or in the presence of either NG2/EC⁺ or NG2/EC^- (50 μg/ml). Morphogenesis was analyzed after a 48-h incubation. Cell nuclei are indicated by arrowheads. Scale bar, 100 μm. (B) Quantitative analysis of morphogenesis. The total length of cord-like networks in each gel was measured in four random fields, and the total length per field was calculated. Each bar represents the mean ± SD (n = 3). Statistically significant differences compared with the control are indicated (^*p < 0.01; ^**p < 0.05).

Identification of galectin-3 as an NG2-binding protein. (A) Identification of NG2-interacting proteins. Extracts from MAEC were incubated with either NG2 beads (lane 3) or CL-4B Sepharose beads as a control (lane 2). Bound proteins were visualized on 4–12% Tris-glycine gels by silver staining. NG2 beads alone were also loaded as another control (lane 1). MAEC proteins bound specifically to NG2 are labeled with arrowheads. The 30-kDa protein band (arrow) was analyzed by MALDI-TOF mass spectrometry. (B) Amino acid sequence of galectin-3. Shaded sequences indicate tryptic peptides derived from the 30-kDa band, as determined by mass spectrometry. (C) MAEC proteins bound to NG2 beads (lane 2) or CL-4B Sepharose beads (lane 1) were analyzed by immunoblotting with antigalectin-3 antibody. A crude lysate of MAEC was also loaded (lane 3). (D) NG2/EC⁺ or NG2/EC^- were incubated with glutathione-agarose beads carrying either GST-galectin-3 fusion protein (lane 3 and 4) or GST (lane 2) as a control. Bound proteins were eluted from GST-galectin-3 fusion protein beads with 100 mM lactose (lane 3), and the stripped beads were boiled in SDS-PAGE loading buffer (lane 4). Samples were separated by SDS-PAGE, and immunoblotted using anti-NG2 antibody. (lane 1) NG2/EC was loaded as a positive control. NG2/EC⁺ migrates as a broad smear because of the presence of the chondroitin sulfate chain. NG2/EC^- appears as three distinct bands because of proteolytic degradation during its purification. (E) GST-galectin-3 fusion protein was incubated with either NG2 beads (lane 3 and 4) or control CL-4B beads (lane 2). After washing, bound proteins were eluted from NG2 beads with lactose (lane 4). Stripped beads were also analyzed (lane 3). Samples were treated as described above and immunoblotted using antigalectin-3 antibody. (lane 1) GST-galectin-3 fusion protein was loaded as a positive control.

Identification of α3β1 integrin as an NG2 receptor. (A and B) Serum-starved MAEC (A) or HMVEC (B) were incubated with or without NG2/EC⁺ (25 μg/ml) for 30 min and then immunostained with mAb 9EG7 (A) or HUTS-21 (B) that recognizes activated β1 integrins. DAPI was used as a nuclear counterstain. Scale bar, 20 μm. (C) Detergent extracts of the indicated cells were fractionated by SDS-PAGE and immunoblotted with antibodies against various integrin subunits. (D) Identification of NG2-binding integrin. Extracts from MAEC were incubated with NG2/EC⁺ and immunoprecipitated with anti-NG2 antibody or control IgG. Immunoprecipitates were fractionated by SDS-PAGE and immunoblotted with anti-NG2, anti-α3, anti-α6, anti-β1, and antigalectin-3 antibodies. Crude MAEC lysate was also loaded as a positive control.

Coexpression of NG2 and α3β1 integrin at the pericyte-EC interface. Sections from healing wound tissue at 7 days after surgery were immunostained for NG2, α3 integrin subunit, and CD31. Cross section of a newly formed capillary reveals the investment of CD31-positive EC by NG2-positive pericytes (bottom panels). α3 integrin is also expressed in EC surrounded by NG2-expressing pericytes (top panels). Galectin-3 staining is also found in these blood vessels, but is not restricted to the vasculature (unpublished data). Scale bar, 20 μm.