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

resveratrol exerts its barrier protective effects remain Products Company) through daily gastric gavage for
largely unknown. 10 days.
Krϋppel-like factors (KLFs) are a family of zinc finger- 2.3. Stereotactic intracerebral injection of tumor
containing transcription factors that have been shown necrosis factor (TNF)-α
to have diverse regulatory roles in biological processes,
such as cell proliferation, differentiation, and survival, Male (15 – 20-week-old) control (tamoxifen-treated CAG-
2
organ development, and metabolism . Several published CreERT mice) and postnatal KLF2 knockout mice were
[7]
reports by us and others have demonstrated that KLF2 stereotactically injected with TNF-α (1 μg/kg in 4 μL 1%
serves as a nodal regulator of endothelial biology. It bovine serum albumin/phosphate-buffered saline (PBS)
promotes a healthy vascular phenotype by affecting key over 20 – 30 min) into the striatum using the following
aspects of vascular function and disease [8,9] . Importantly, coordinates from bregma: anteroposterior, 0.0 mm; lateral,
in vivo studies from our laboratory have identified 1.5 mm; and ventral, 2.1 mm.
KLF2’s ability to induce endothelial tight junction 2.4. In vitro cell culture studies and western blotting
factors (e.g., occludin) [10,11] and protect the integrity of
the vascular barrier of the brain, thereby also serving as Primary human brain microvascular ECs were purchased
a stroke protective factor . These studies have firmly from cell systems and cultured in endothelial basal
[11]
established the role of KLF2 as a critical positive regulator medium-2 (EBM-2) that was supplemented with growth
of vascular endothelial barrier function. factors. Resveratrol was obtained from Sigma. Polyclonal
rabbit anti-KLF2 antibody was provided as a gift by
Interestingly, in vitro studies have demonstrated that Huck-Hui Ng (Singapore). Mouse anti-β-actin antibody
resveratrol induces KLF2 expression in endothelial cells was obtained from Santa Cruz Biotechnology. All small
(ECs), implicating KLF2 as a potential regulator of its interfering RNA (siRNA) oligonucleotides were obtained
beneficial vascular effects . However, the physiologic from Dharmacon. Transfection of siRNA into endothelial
[12]
importance of this regulation in vivo and its specific role cells was carried out as described [10,11] . Protein isolation
in mediating vascular endothelial barrier function has and western blot analysis using the indicated antibodies
not been investigated thus far. In this study, we examine were performed as previously described .
[10]
resveratrol’s dependence on KLF2 for its protective effects
on vascular endothelial barrier function. 2.5. Quantitative real-time polymerase chain
reaction (RT-PCR)
2. Materials and methods
TRIzol reagent (Invitrogen) was used to extract the
2.1. Animals total RNA from primary human brain microvascular
Postnatal KLF2 knockout mice were generated as endothelial cells following the manufacturer’s instructions.
previously described . KLF2-floxed mice were crossed Two micrograms of total RNA were used for reverse
[11]
with CAG-CreERT strain (Jackson Laboratory) to transcription to generate complementary deoxyribonucleic
2
generate KLF2 -CAG-CreERT mice. Postnatal deletion acid (cDNA) using iScript Reverse Transcription kit (Bio-
fl/fl
2
of KLF2 was induced through intraperitoneal (IP) Rad). RT-PCR was performed with Universal SYBR Green
injection of tamoxifen (0.1 mL at 20 mg/mL, dissolved PCR Master Mix on Applied Biosystems Step One Real-
in sunflower seed oil; T5648, Sigma-Aldrich) to 8 week- Time PCR system. Gene expression was normalized to
old male KLF2 -CAG-CreERT mice. Age-matched glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
2
fl/fl
male CAG-CreERT mice that were subjected to the same using the ∆∆Ct method.
2
tamoxifen regimen were used as controls. All animals were
maintained in a clean animal facility. All mouse studies 2.6. In vitro endothelial permeability assay
were approved by an Institutional Animal Care and Use In vitro endothelial permeability analysis was performed
Committee (IACUC) at Case Western Reserve University using a transwell assay as described in our prior
and were conducted in accordance with the National publication . To achieve oxygen-glucose deprivation
[11]
Institutes of Health (NIH) Guide for the Care and Use of (OGD), cultured endothelial cells were exposed to
Laboratory Animals. deoxygenated PBS in a modular incubator chamber
flushed with 1% oxygen (O ), 5% carbon dioxide (CO ),
2.2. Resveratrol pretreatment and 94% nitrogen (N ), sealed, and placed at 37°C for
2
2
2
Male 17–20-week-old KLF2 and control mice were treated 30 min, followed by a 2-h exposure to normal culture
-/-
with 75 mg/kg of resveratrol (Sigma) in a saline solution media under normoxic conditions (21% O , 5% CO ) at
2
2
of 20% hydroxypropyl-β-cyclodextrin (American Maize- 37°C. Immediately on re-exposure to normal media and
Volume 2 Issue 1 (2023) 2 https://doi.org/10.36922/gtm.v2i1.218