Resveratrol stimulates brain endothelial cell proliferation, migration, and tube formation. (A) Serum-starved brain endothelial cells were treated with resveratrol (0.1 to 10 μM) for 24 hours and cell growth was assessed for proliferation assay by WST. Bar graph shows that the proliferation of brain endothelial cells incubated with indicated concentration of resveratrol increased. Results were expressed as percentage of cells in relation to basal (untreated) and represent the mean of triplicates. ^*P<0.05 versus control, n=4. (B) Effect of resveratrol on cell-cycle distribution. Brain endothelial cells were treated with resveratrol for 24 hours. Flow cytometry analysis showed that resveratrol significantly increased the proportion of cells in S phase (gray bar) and decreased the proportion of cells in the G₀/G₁ phase (black bar). Data are presented as the mean±s.d. ^*P<0.05, ^**P<0.01 versus control. (C) Migration of serum-starved brain endothelial cells in response to resveratrol (0.1 to 10 μM) were examined using transwell chamber assay for 10 hours. Cells undergoing migration in response do resveratrol were captured using a light microscope at × 200 magnification. (D) Increased migration of brain endothelial cells treated with resveratrol. The values represent the average number of cells migrated (mean±s.d.) of three independent experiments in duplicate. ^*P<0.01 versus control. (E) Resveratrol promoted matrigel angiogenesis. Brain endothelial cells (8 × 10⁴) were plated on Matrigel-coated, 24-well plates and were incubated for 20 hours in medium alone or with resveratrol. Photomicrographs represent the matrigel tube formation after 20-hour incubation detected by phase-contrast microscopy (magnification, × 200). (F) Bar graph shows that resveratrol promoted endothelial tube formation. ^*P<0.05, ^**P<0.01 versus control. Each experiment was performed four times in duplicate. The results are expressed as mean±s.d. (G) Cytoskeleton rearrangement in brain endothelial cells following treatment with resveratrol. F-actin rearrangement in brain endothelial cells in response to resveratrol (5 μM) was examined using immunofluorescence analysis by phalloidin staining. Endothelial cells with media alone showed stress fibers aligned across the cell axis. Stress fibers disappeared forming membrane ruffles in brain endothelial cells treated with 5 μM of resveratrol in media for 20 minutes. Immunocytochemistry to show localization of β-catenin and VE-cadherin in endothelial cells treated with resveratrol was performed. β-Catenin was localized on the endothelial membrane of cells treated with media alone, whereas it was found more in the cytoplasm and nucleus of cells treated with 5 μM of resveratrol. VE-cadherin was localized at the intercellular junctions of cells treated with media alone, whereas it was found more in the cytoplasm of cells treated with 5 μM of resveratrol.

Endothelial nitric oxide synthase (eNOS) is regulated by resveratrol and downstream of phosphoinositide 3-Kinase (PI3-K) and Mitogen-Activated Protein Kinase (MAPK) signaling pathway. (A) Serum-starved brain endothelial cells were treated with 5 μM resveratrol for indicated time period. Representative images from four individual experiments are shown. (B) Bar graph represent p-eNOS/total eNOS levels expressed as the average percentage increase (mean±s.d.) over basal levels (n=4). ^*P<0.05 versus control. (C) Serum-starved brain endothelial cells were pretreated with a selective PI3-K (LY294002, 10 μM) and MEK (U0126, 10 μM) inhibitor for 1 hour before incubation with 5 μM resveratrol for 20 minutes. Representative images from four individual experiments are shown. (D) Bar graph represent p-eNOS/total eNOS levels expressed as the average percentage increase (mean±s.d.) over basal levels (n=4). ^*P<0.05, ^**P<0.01 versus control. eNOS phosphorylation on Ser1177 was measured by in-cell Western blot analysis using a specific antibody as described in the Materials and methods section. The results are represented as a ratio of phosphorylated to total protein and expressed as percentage of control. (E) Serum-starved brain endothelial cells were treated with 5 μM resveratrol for up to 30 minutes. The stable end product of nitric oxide, nitrite, was measured in the medium using the Griess reaction. Medium total nitrite content corrected for protein content of the cell layer was estimated according with the company product. Composite data from four experiments are shown relative to control (mean±s.d.). ^*P<0.05 versus control. (F) Serum-starved cells were pretreated with a selective PI3-K (LY294002, 10 μM), MEK (U0126, 10 μM), and NOS (L-NAME, 3 mM) inhibitor for 1 hour before incubation with 5 μM resveratrol for 20 minutes. Composite data from four experiments are shown relative to control (mean±s.d.). ^*P<0.05 versus control, ^#P<0.05 versus resveratrol group.

Vascular endothelial growth factor (VEGF) induced by resveratrol is nitric oxide synthase (NOS) dependent. (A) Serum-starved brain endothelial cells were treated with 5 μM resveratrol for 24 hours. Immunocytochemistry for VEGF demonstrated a strong stained for resveratrol–brain endothelial cells. (B) Serum-starved cells were treated with 5 μM resveratrol for indicated time periods and performed Western blotting. Representative blots from four individual experiments are shown. Deferoxamine (DFX) was used as a positive control for VEGF generation. (C) Bar graph represent VEGF/β-actin levels expressed as the average percentage increase (mean±s.d.) over basal levels (n=4). ^*P<0.05, ^**P<0.01 versus control group. The cell lysate were collected and Western blotting was performed. (D) VEGF induced by resveratrol is NOS dependent. Serum-starved cells were pretreated with a selective NOS inhibitor (L-NAME, 3 mM) for 1 hour before incubation with 5 μM resveratrol for 24 hours. Representative blots from four individual experiments are shown. (E) Bar graph represent VEGF/β-actin levels expressed as the average percentage increase (mean±s.d.) over basal levels (n=4). ^*P<0.05 versus control group; ^#P<0.05 versus resveratrol group. (F) VEGF levels in the conditioned media, as confirmed by ELISA, were also higher in resveratrol and DFX-treated cells. The VEGF concentrations were calculated from the standard curve. ^*P<0.05, ^**P<0.01 versus control. (G) VEGF levels in the conditioned media, as confirmed by ELISA, were higher in resveratrol and decreased when cells were pretreated with a selective NOS inhibitor (L-NAME, 3 mM). ^*P<0.05 versus control; ^#P<0.05 versus resveratrol group. (H) Serum-starved cells were pretreated with a selective VEGF antagonist (sFLT-1, 10 μM) for 1 hour before incubation with 5 μM resveratrol for 24 hours. Representative blots for p-VEGFR2/total VEGFR2 from four individual experiments are shown. (I) Bar graph represent p-VEGFR2/total VEGFR2 levels expressed as the average percentage increase (mean±s.d.) over basal levels (n=4). ^*P<0.05 versus control group; ^#P<0.05 versus resveratrol group.

Involvement of vascular endothelial growth factor (VEGF), VEGFR2, and matrix metalloproteinases (MMPs) signaling pathway in resveratrol-induced capillary tube formation. (A) Serum-starved brain endothelial cells were pretreated with a selective VEGF (sFLT-1, 10 μM), VEGFR2 (SU1498, 5 μM), and MMPs (GM6001, 10 μM) inhibitors for 1 hour before incubation with 5 μM resveratrol. Photomicrographs represent the matrigel tube formation after 20-hour incubation detected by phase-contrast microscopy (magnification, × 200). (B) Resveratrol-induced brain endothelial cell capillary tube formation was significantly inhibited when cells were preincubated with inhibitors. Results are mean values±s.d. of four experiments in duplicate and expressed as percentage of control. ^*P<0.05 versus control; ^#P<0.05 versus resveratrol group.

Regulation of phosphoinositide 3-Kinase (PI3-K) and Mitogen-Activated Protein Kinase (MAPK) signaling pathway by resveratrol. (A) Exposure to 5 μM resveratrol triggered PI3-K/Akt signaling as indicated by elevated levels of phospho-Akt for indicated time periods. Representative images from four individual experiments are shown. (B) Bar graph represent p-Akt/total Akt levels expressed as the average percentage increase (mean±s.d.) over basal levels (n=4). ^*P<0.05, ^**P<0.01 versus control. (C) Serum-starved cells were pretreated with a selective PI3-K inhibitor (LY294002, 10 μM) for one hour before incubation with 5 μM resveratrol for 10 minutes. Representative blot from four individual experiments are shown. (D) Bar graph represent p-Akt/total Akt levels expressed as the average percentage increase (mean±s.d.) over basal levels (n=4). ^*P<0.05, ^**P<0.01 versus control. (E) Serum-starved brain endothelial cells were treated with 5 μM resveratrol for up 30 minutes and Erk phosphorylation was measured by Western blot analysis. Representative images from four individual experiments are shown. (F) Bar graph represent p-Erk/total Erk levels expressed as the average percentage increase (mean±s.d.) over basal levels (n=4). ^*P<0.05, ^**P<0.01 versus control. (G) Serum-starved brain endothelial cells were pretreated with a selective MEK inhibitor (U0126, 10 μM) for 1 hour before incubation with 5 μM resveratrol for 10 minutes. Representative images from four individual experiments are shown. (H) Bar graph represent p-Erk/total Erk levels expressed as the average percentage increase (mean±s.d.) over basal levels (n=4). ^*P<0.05, ^**P<0.01 versus control. The cell lysates were subjected to Western blotting with the indicated antibodies. The results are represented as a ratio of phosphorylated to total protein and expressed as percentage of control.

Involvement of phosphoinositide 3-Kinase (PI3-K), Mitogen-Activated Protein Kinase (MAPK), and nitric oxide synthase (NOS) signaling pathway in resveratrol-induced capillary tube formation. (A) Serum-starved brain endothelial cells were pretreated with a selective PI3-K (LY294002, 10 μM), MEK (U0126, 10 μM), and NOS (L-NAME, 3 mM) inhibitors for 1 hour before incubation with 5 μM resveratrol. Photomicrographs represent the matrigel tube formation after 20-hour incubation detected by phase-contrast microscopy (magnification, × 200). (B) Resveratrol-induced brain endothelial cell capillary tube formation was significantly inhibited when cells were preincubated with inhibitors. Results are mean values±s.d. of four experiments in duplicate. ^*P<0.05 versus control; ^#P<0.05 versus resveratrol group.

Effect of resveratrol on matrix metalloproteinase (MMP)-2 and MMP-9 secretion. Gelatin zymograph analysis (A) shows MMP-2 and MMP-9 activities in the conditioned medium for 24 hours harvested from control and different concentrations of resveratrol (0.1 to 10 μM). (B) Serum-starved brain endothelial cells were pretreated with a selective vascular endothelial growth factor (VEGF) (sFLT-1, 10 μM), VEGFR2 (SU1498, 5 μM), and nitric oxide synthase (NOS) (L-NAME, 3 mM) inhibitors for 1 hour before incubation with 5 μM resveratrol for 24 hours. VEGF was used as a positive control. Representative zymograms from four individual experiments are shown. (C) Bar graph represent MMP-2 and MMP-9 levels expressed as the average percentage increase over basal levels (n=4). ^*P<0.05 versus control group; ^#P<0.05 versus resveratrol group. Data shown are mean values±s.d. The values represented are relative to basal.

A schematic diagram showing the proposed mechanism by which intracellular signaling pathway involved in resveratrol-induced brain endothelial cell angiogenesis.