Figure 3
Figure 3. Ang-1–induced IL-8 plays a significant role in EC migration proliferation. (A) Representative examples of MSCV and MSCV-Ang-1 cell wounding experiments. MSCV (left panels) and MSCV-Ang-1 cells (right panel) were wounded (time 0) and maintained for 12 hours in conditioned media derived from MSCV and MSCV-Ang-1 cells, respectively, and were neutralized with IgG control antibody. Arrows point to the edges of the wounds. Note that wound healing (measured after 12 hours) was faster in MSCV-Ang-1 cells compared with MSCV cells. (B top panel) Mean (± SEM; n = 6) of wound healing in MSCV and MSCV-Ang-1 cells maintained for 12 hours in conditioned media of donor MSCV and MSCV-Ang-1 cells, respectively. MSCV or MSCV-Ang-1 cells were grown for 24 hours in basal medium. The media was collected and incubated with anti–IL-8 neutralizing antibody or IgG control antibody. Fresh MSCV and MSCV-Ang-1 cells were seeded into 24-well tissue culture plates and cultured in complete medium containing 20% FBS to nearly confluent cell monolayers. The cells were then carefully wounded using a pipette tip. After making the wounds, culture media were replaced with conditioned media of MSCV and MSCV-Ang-1 cells neutralized with anti–IL-8 or control IgG antibody. Wounds were then photographed (time = 0, and 12 hours later). Migration was evaluated by measuring the reduction in the diameter of the wound after migration of the cells into the cell-free zone. *P < .05 compared with MSCV cells maintained in IgG-neutralized conditioned media. #P < .05 compared with MSCV-Ang-1 maintained in IgG-neutralized conditioned medium. (B bottom panel) Means (± SEM; n = 6) of the number of HUVECs migrating toward conditioned media derived from MSCV or MSCV-Ang-1 cells. EC migration was performed in 24-well trans-well fibronectin-coated polycarbonate inserts. HUVECs were suspended in basal medium and seeded in the upper compartment. MSCV or MSCV-Ang-1 cells were grown in basal media for 24 hours, and conditioned media were then collected and incubated with anti–IL-8 and control IgG antibodies. Conditioned media were then placed into the lower compartment of the migration apparatus. Migration was quantified 5 hours later by counting cells in 10 fields per well. Symbols are the same as in panel B. (C) BrdU incorporation and cell number of MSCV and MSCV-Ang-1 cells maintained for 2 days in basal medium containing 2% FBS and IgG or anti–IL-8 antibodies. *P < .05 compared with MSCV cells maintained in the presence of IgG antibody. #P < .05 compared with MSCV-Ang-1 cells maintained in the presence of IgG antibody. (D,E) A representative immunoblot of cleaved caspase-3 and means (± SEM; n = 4) of cleaved caspase-3 intensity measured in MSCV and MSCV-Ang-1 cells after 2 days of culture in basal medium containing 2% FBS and IgG or anti–IL-8 antibodies. Symbols are the same as in panel B.

Ang-1–induced IL-8 plays a significant role in EC migration proliferation. (A) Representative examples of MSCV and MSCV-Ang-1 cell wounding experiments. MSCV (left panels) and MSCV-Ang-1 cells (right panel) were wounded (time 0) and maintained for 12 hours in conditioned media derived from MSCV and MSCV-Ang-1 cells, respectively, and were neutralized with IgG control antibody. Arrows point to the edges of the wounds. Note that wound healing (measured after 12 hours) was faster in MSCV-Ang-1 cells compared with MSCV cells. (B top panel) Mean (± SEM; n = 6) of wound healing in MSCV and MSCV-Ang-1 cells maintained for 12 hours in conditioned media of donor MSCV and MSCV-Ang-1 cells, respectively. MSCV or MSCV-Ang-1 cells were grown for 24 hours in basal medium. The media was collected and incubated with anti–IL-8 neutralizing antibody or IgG control antibody. Fresh MSCV and MSCV-Ang-1 cells were seeded into 24-well tissue culture plates and cultured in complete medium containing 20% FBS to nearly confluent cell monolayers. The cells were then carefully wounded using a pipette tip. After making the wounds, culture media were replaced with conditioned media of MSCV and MSCV-Ang-1 cells neutralized with anti–IL-8 or control IgG antibody. Wounds were then photographed (time = 0, and 12 hours later). Migration was evaluated by measuring the reduction in the diameter of the wound after migration of the cells into the cell-free zone. *P < .05 compared with MSCV cells maintained in IgG-neutralized conditioned media. #P < .05 compared with MSCV-Ang-1 maintained in IgG-neutralized conditioned medium. (B bottom panel) Means (± SEM; n = 6) of the number of HUVECs migrating toward conditioned media derived from MSCV or MSCV-Ang-1 cells. EC migration was performed in 24-well trans-well fibronectin-coated polycarbonate inserts. HUVECs were suspended in basal medium and seeded in the upper compartment. MSCV or MSCV-Ang-1 cells were grown in basal media for 24 hours, and conditioned media were then collected and incubated with anti–IL-8 and control IgG antibodies. Conditioned media were then placed into the lower compartment of the migration apparatus. Migration was quantified 5 hours later by counting cells in 10 fields per well. Symbols are the same as in panel B. (C) BrdU incorporation and cell number of MSCV and MSCV-Ang-1 cells maintained for 2 days in basal medium containing 2% FBS and IgG or anti–IL-8 antibodies. *P < .05 compared with MSCV cells maintained in the presence of IgG antibody. #P < .05 compared with MSCV-Ang-1 cells maintained in the presence of IgG antibody. (D,E) A representative immunoblot of cleaved caspase-3 and means (± SEM; n = 4) of cleaved caspase-3 intensity measured in MSCV and MSCV-Ang-1 cells after 2 days of culture in basal medium containing 2% FBS and IgG or anti–IL-8 antibodies. Symbols are the same as in panel B.

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