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Global Translational Medicine Resveratrol’s EC barrier protection is dependent on KLF2

normoxia, fluorescein isothiocyanate (FITC)-dextran was the upregulation of several key endothelial tight junction
added to the transwell insert. Aliquots of medium collected factors, including occludin, claudin 12, junctional adhesion
from the bottom chamber and the fluorescence density of molecule 1 (JAM-1), and AF-6/afadin . To determine
[11]
samples were analyzed on a microplate fluorometer. if resveratrol can induce some or all of these factors and
whether the induction is KLF2 dependent, the ability of
2.7. In vivo permeability assay resveratrol to induce a panel of tight junction factors was
Following stereotactic injection of TNF-α, tail vein injection assessed in the presence and absence of KLF2. Human
of 2% Evans blue dye (EBD; 4 mL/kg) was performed. 2 h primary brain microvascular ECs were transfected with
later, the mice were euthanized and subjected to saline KLF2 siRNA (siKLF2) or control siRNA (non-specific,
perfusion, followed by brain tissue removal, weighing, and NS), followed by resveratrol treatment. Resveratrol
homogenizing in 50% trichloroacetic acid (TCA). Following specifically induced the expression of occludin (Figure 2B),
EBD extraction, the concentration (ng EBD/mg brain) was AF-6 (Figure 2C), and JAM-1 (Figure 2D). No significant
determined by fluorescence intensity (excitation 620 nm changes in the mRNA levels of claudin 3, claudin 5, and
and emission 680 nm) based on an EBD standard curve. zona occludens protein 1 (ZO-1) were observed (data not
shown). Following siRNA-mediated KLF2 knockdown,
2.8. Statistical analysis resveratrol’s induction of KLF2 expression was significantly
Data were expressed as mean ± standard error of the inhibited (Figure 2A), and the induction of occludin, AF-6,
mean. One-way analysis of variance (ANOVA) was used and JAM-1 was abrogated (Figure 2B–D). These findings
to compare the differences across 3 or more levels within show that KLF2 is necessary for resveratrol’s induction
1 variable. When comparing a single variable in multiple of several key tight junction factors and suggests that an
groups, one-way ANOVA (followed by Dunnett’s multiple additional mechanism by which resveratrol exerts its
comparisons test) was performed. Two-way ANOVA vasculoprotective effects may be through the regulation of
(followed by Dunnett’s post hoc test) was used for two- endothelial barrier function.
factor analysis. Statistical analyses were performed using 3.3. Resveratrol’s protection of endothelial barrier
Prism 9.0 software. P < 0.05 was considered statistically function is KLF2 dependent
significant.
To examine the functional impact of resveratrol on
3. Results endothelial barrier in vitro, transwell assays were performed
3.1. Resveratrol induces KLF2 in microvascular in human primary brain microvascular ECs. The cells were
endothelial cells plated onto a transwell, treated with 100 μM of resveratrol
for 16 h, and subjected to OGD. The permeability of FITC-
Previous data have established that resveratrol is able dextran was, then, measured to assess barrier integrity.
to induce KLF2 expression in human umbilical vein Under these conditions, resveratrol protects against OGD-
ECs in both time- and dose-dependent manners . mediated endothelial barrier disruption with a significant
[12]
We demonstrate similar findings in human primary decrease observed in fluorescence intensity when compared
brain microvascular ECs. The peak induction of KLF2 to control (Figure 3A). To determine if resveratrol’s
mRNA was observed at 8 h after resveratrol treatment endothelial barrier protective effects are KLF2 dependent,
(Figure 1A), while all times tested showed significant transwell experiments with OGD were conducted in ECs
increase over baseline conditions (0 h). Peak KLF2 following siRNA-mediated KLF2 knockdown. Indeed, the
mRNA expression was observed at a concentration of protective effects of resveratrol were significantly abrogated
100 μM, with lower doses also showing upregulation in the absence of KLF2 (Figure 3B). When the control
(Figure 1B). Complementary to our mRNA data, siRNA was transfected into microvascular ECs, resveratrol
resveratrol also induced KLF protein expression in a treatment showed decreased FITC-dextran fluorescence
dose-dependent manner as confirmed by western blot under OGD conditions (comparing siControl + vehicle
analysis (Figure 1C). Taken together, these data show [OGD] versus siControl + resveratrol [OGD]) (Figure 3B).
that resveratrol is a potent activator of KLF2 expression When siRNA specific for KLF was introduced, this effect
within microvascular ECs. was eliminated (comparing siKLF2 + vehicle [OGD] versus
siKLF2 + resveratrol [OGD]). To determine the physiologic
3.2. Resveratrol’s induction of key endothelial tight relevance of our findings in vivo, EBD incorporation assays
junction factors is KLF2 dependent were performed on both control and KLF2-deficient mice.
We have recently identified KLF2 to be an important Inflammatory cytokine TNFα was stereotactically injected
regulator of vascular endothelial barrier function through into the striatum of both control and KLF2 knockout mice,

Volume 2 Issue 1 (2023) 3 https://doi.org/10.36922/gtm.v2i1.218