Mitsugumin 53 Inhibits Angiogenesis Through Regulating Focal Adhesion Turnover and Tip Cell Formation

Abstract

Our previous studies have identified mitsugumin 53 (MG53) as a novel regulator for angiogenesis by directly entering endothelial cells and modulating focal adhesion kinase (FAK) activation, but little is known about how rhMG53 is taken up by cells and how rhMG53 mediates cell movement. In the present study, we demonstrated that the knockdown of caveolin-1 and the clathrin inhibitor, pitstop-2, both significantly reduced the entry of rhMG53 into endothelial cells, indicating caveolae-dependent and clathrin-dependent endocytosis during this process. The internalised rhMG53 remarkably inhibited the phosphorylation of FAK and the downstream signalling molecule paxillin, consequently resulting in a significant decrease in focal adhesion turnover during endothelial cell spreading and migration. Using a 3D collagen culture model, we further found that rhMG53 significantly inhibited tip cell formation and tubulogenesis. Furthermore, rhMG53 also remarkably prevented alkaline injury-induced corneal neovascularization in vivo. Taken together, these results indicate that rhMG53 inhibits angiogenesis through regulating focal adhesion turnover and tip cell formation. This may elucidate novel molecular mechanisms involved in rhMG53 uptake and rhMG53-modulated endothelial cell function and provide evidence for the potential utility of rhMG53 in treating diseases with excessive angiogenesis.

Keywords: angiogenesis; endocytosis; focal adhesion; mitsugumin 53; tip cell.

FIGURE 1

Cav‐1 is required for the uptake of rhMG53 into endothelial cells. HUVECs were transfected with scrambled or Cav‐1 siRNA, followed by stimulation with rhMG53 (20 μg/mL) for 60 min. (A) Total proteins were collected and western blotting was carried out to evaluate Cav‐1 expression and intracellular MG53. Representative images are shown on the left. The densitometric analysis of Cav‐1 or MG53 normalised to GAPDH was performed (n = 5 biological replicates). (B) HUVECs were fixed and then immunohistochemical staining was performed for MG53 (red) and Cav‐1 (green). DAPI‐stained nuclei are shown in blue. Representative images from four independent experiments are shown. The scale bar is 10 μm. The average fluorescent intensity of MG53 per cell was determined (n = 20 fields of view per group, from 4 independent experiments). For the above, data are presented as mean ± SD. ***p < 0.001 (one‐way ANOVA with Tukey's multiple comparisons in A, two‐tailed unpaired Student's t test in B).

FIGURE 2

Pitstop‐2 decreases rhMG53 uptake into endothelial cells. HUVECs were pretreated with pitstop‐2 (5 μM) or the negative control (NC, 5 μM) for pitstop‐2 for 30 min, followed by stimulation with rhMG53 (20 μg/mL) for 60 min. (A) Cell lysates were harvested and intracellular MG53 was detected by western blotting. Representative images are shown on the left. The densitometric analysis of MG53 normalised to β‐Actin was performed (n = 5 biological replicates). (B) HUVECs were fixed and incubated with a primary anti‐MG53 antibody, followed by immunostaining using a FITC‐conjugated secondary antibody (green). DAPI‐stained nuclei are shown in blue. Representative images from four independent experiments are shown. The scale bar is 10 μm. The average fluorescence intensity of MG53 per cell was determined (n = 20 fields of view per group, from four independent experiments). For the above, data are presented as mean ± SD. ***p < 0.001 (one‐way ANOVA with Tukey's multiple comparisons).

FIGURE 3

Internalised rhMG53 is targeted to the endosome and lysosome. HUVECs were stimulated with rhMG53 (20 μg/mL) for 1 h or 4 h. The co‐localization of MG53 (red) with the early endosomal marker EEA1 (green), with the late endosomal marker Rab7 (green) and with the lysosomal marker LAMP1 was identified by immunostaining. DAPI‐stained nuclei are shown in blue. Representative images from four independent experiments are shown. Arrowheads indicate examples of co‐localization. The scale bar is 10 μm.

FIGURE 4

rhMG53 inhibits FAK‐paxillin signalling pathway activation. HUVECs were incubated for 24 h with either vehicle control or rhMG53 (20 μg/mL), and then the cell lysates were prepared. Phosphorylation of FAK^Y397, FAK^Y925 and paxillin^Y118, and total FAK and paxillin were analysed by western blotting. Representative images from four independent experiments are shown. The densitometric analysis of phosphorylation of FAK and paxillin normalised to total FAK and paxillin was carried out. All data shown is presented as mean ± SD. ***p < 0.001 (two‐tailed unpaired Student's t test).

FIGURE 5

rhMG53 Inhibits FA turnover during cell migration. (A) HUVECs were allowed to grow to 90% confluence and incubated for 24 h with either vehicle control or rhMG53 (20 μg/mL). Afterward, the cells were scratched with a 200 μL pipette tip and allowed to migrate for an additional 0, 2 and 4 h, followed by immunostaining for F‐Actin (red) and vinculin (green). Representative images from four independent experiments are shown. The scale bar is 20 μm. (B) The number of FAs at the edge of the scratch was counted and the length of the scratch edge was measured. Then the ratio of FA number normalised to the edge length was quantitatively analysed (n = 20 fields of view per group, from four independent experiments). Data are presented as mean ± SD. ***p < 0.001 (two‐tailed unpaired Student's t test).

FIGURE 6

rhMG53 Inhibits endothelial cell spreading and FA turnover during cell spreading. HUVECs were incubated for 24 h with either vehicle control or rhMG53 (20 μg/mL). (A) The cells were then detached and seeded onto FN (10 μg/mL)‐coated 24‐well plates for 15, 30, 60 and 120 min, respectively. F‐Actin was stained with rhodamine‐conjugated phalloidin and representative images from 4 independent experiments are shown. The scale bar is 20 μm. The cell areas were measured, and quantitative assessment of four independent experiments was performed (n > 200 cells per group, from four independent experiments). (B) HUVECs were seeded onto FN (10 μg/mL) for 2 h, followed by staining for F‐Actin with rhodamine‐conjugated phalloidin and an antibody recognising vinculin. Representative images from 4 independent experiments are shown and the scale bar is 10 μm. The number of FAs (arrowheads) was calculated and quantitatively analysed. For the above, data are presented as mean ± SD. ***p < 0.001 (two‐tailed unpaired Student's t test).

FIGURE 7

rhMG53 Inhibits tip cell formation and tubulogenesis in 3D collagen matrices. HUVECs were incubated for 24 h with either vehicle control or rhMG53 (20 μg/mL). Cells are then collected and seeded into 3D collagen gel cultures, followed by feeding with media containing reduced serum supplement II (RSII), ascorbic acid and FGF‐2 at 40 ng/mL. (A) After 24 h, the cultures were fixed with 4% paraformaldehyde and stained with rhodamine‐conjugated phalloidin to visualise F‐Actin (red). DAPI‐stained nuclei are shown in blue. Representative images from four independent experiments are shown. The scale bar is 50 μm. The number of tip cells was calculated and quantitatively analysed (n = 80 fields of view per group, from four independent experiments). (B) Cultures were allowed to assemble into capillary networks for 72 h, and then the cultures were fixed in 4% paraformaldehyde and stained with rhodamine‐conjugated phalloidin. Representative images from four independent experiments are shown. The scale bar is 50 μm. The length of the tube was measured and quantitatively analysed (n > 50 fields of view per group, from four independent experiments). For the above, data are presented as mean ± SD. ***p < 0.001 (two‐tailed unpaired Student's t test).

FIGURE 8

rhMG53 inhibits alkaline injury‐induced corneal angiogenesis. The corneas were exposed to a 2 mm disk of filter paper soaked in NaOH (1 mol/L) for 40 s to induce injury, followed by topically incubation with PBS or rhMG53 (2 μg/mL) for 30 mL/eye twice daily for a total of 7 days. (A) Images were taken to show the lateral views of each eye and the ratio of corneal vessel area relative to the whole corneal area was calculated. (B) Mouse corneal neovascularization was observed by whole‐cornea immunofluorescence staining for IB4 (green). Intensity of IB4 signal was used as index of vascularization of the cornea at 7 days post–alkaline injury. For the above, data are presented as mean ± SD. ***p < 0.001 (two‐tailed unpaired Student's t test).

FIGURE 9

Schematic diagrams representing the molecular mechanisms underlying rhMG53‐regulated angiogenesis. rhMG53 enters endothelial cells via caveolae‐dependent and clathrin‐dependent endocytosis and then reduces FAK‐paxillin signalling activation, leading to decreased FA turnover and tip cell formation, ultimately inhibiting angiogenesis.