Fig 1.
RSVT is a potent stimulator of Notch signaling.
(A) RSVT does not induce HMEC apoptosis at 1–10 μM. HMEC cells were treated with increasing concentrations of RSVT +/- DAPT. Cellular apoptosis was indirectly examined by monitoring cleavage of cleaved caspase 3 from pro-caspase 3 by western blot. HMEC cells were treated with UV light as a positive apoptosis control and protein loading was monitored by western blotting for α-actin. Data shows that RSVT induces apoptosis at 100 μM. (B) Notch activation measured by three luciferase reporter constructs in HAVSMC cells. Student’s t-test was performed to determine statistical significance compared to 0 μM RSVT control. P-values are reported as * < .05, ** < .01, *** < .001. Data represents n≥5. Data shows that RSVT induces Notch target gene transcription in HAVSMC cells. (C) Notch activation measured by three luciferase reporter constructs in HMEC-1 cells. Student’s t-test was performed to determine statistical significance compared to 0 μM RSVT control. P-values are reported as * < .05, ** < .01, *** < .001. Data represents n≥5. Data shows that RSVT induces Notch target gene transcription in HMEC-1 cells. (D) Cell viability of HMEC-1 cells measured using WST-1 cell viability assay. Cell viability was quantified 24 hours after treatment with nine different polyphenolic compounds. Cell viability as a percentage of DMSO control (0 μM) is graphed. Data represents the average of three replicate experiments. Structures of each polyphenol are shown above respective graph. Bolded line depicts cell viability measured for each polyphenol. Student’s t-test was performed to determine statistical significance compared to DMSO control. P-values are reported as * < .05, ** < .01, *** < .001. Data demonstrates that most polyphenols did not drastically compromise cell viability when used at 1 μM and 10 μM concentrations. However, 100 μM polyphenol concentrations significantly reduced cell viability for all nine polyphenols tested.
Fig 2.
Other polyphenolic compounds are Notch activators.
Notch activation measured by 4xCSL luciferase assays in 293T cells, by nine different polyphenols compared to DMSO (-) control, in the presence (+) or absence of N1ICD (-) overexpression. Student’s t-test was performed to determine statistical significance. P-values are reported as * < .05, ** < .01, *** < .001. Data represents n≥5. Data shows that resveratrol (RSVT), apigenin (APIG), chrysin (CHRY), genistein (GENI), and piceatannol (PICE) activate Notch target gene transcription, but only in the presence of NICD. Luteolin (LUTE), myricetin (MYRI), pterostilbene (PTER), and quercetin (QUER) do not activate Notch target gene transcription.
Fig 3.
Polyphenolic regulation of endothelial cell proliferation.
Proliferation of HMEC-1 cells measured by WST-1 proliferation assays. HMEC-1 cells which had been transduced with lentiviral particles encoding N1ICD under the control of a doxycycline inducible promoter were used. One-way ANOVA followed by Bonferroni’s post-hoc tests was performed to determine statistical significance. Differing letters represent statistical significant differences. Data represents n = 6. Polyphenols which inhibit HMEC-1 proliferation in a Notch dependent manner include RSVT, APIG, CHRY, MYRI and GENI. LUTE and PICE inhibit HMEC-1 proliferation during both basal and high Notch activity. PTER and QUER do not alter HMEC-1 proliferation.
Fig 4.
Polyphenolic regulation of endothelial cell migration.
Migration of endothelial cells measured through scratch assay analysis. (A) HMEC-1 cells were grown in 10 μM luteolin or DMSO control for 24 hours prior to wounding. Micrograph images where taken at 0hrs and 18hrs after treatment. Area of wound is outlined. (B) HMEC-1 cells were grown to confluency and treated with 10 μM polyphenols or DMSO control for 24 hours prior to wounding. Data depicts % wound closure after 18 hours. Student’s t-test was performed to determine statistical significance. RSVT, APIG, CHRY, GENI, LUTE, MYRI, and PICE inhibit HMEC-1 cell migration, whereas PTER and QUER do not alter migration. P-values are reported as * < .05, ** < .01, *** < .001. Data represents n = 3.
Table 1.
Summary.