Epigallocatechin-3-Gallate and PEDF 335 Peptide, 67LR Activators, Attenuate Vasogenic Edema, and Astroglial Degeneration Following Status Epilepticus

Non-integrin 67-kDa laminin receptor (67LR) is involved in cell adherence to the basement membrane, and it regulates the interactions between laminin and other receptors. The dysfunction of 67LR leads to serum extravasation via blood-brain barrier (BBB) disruption. Polyphenol (–)-epigallocatechin-3-O-gallate (EGCG) and pigment epithelium-derived factor (PEDF) bind to 67LR and inhibit neovascularization. Therefore, in the present study, we investigated the effects of EGCG and NU335, a PEDF-derive peptide, on BBB integrity and their possible underlying mechanisms against vasogenic edema formation induced by status epilepticus (SE, a prolonged seizure activity). Following SE, both EGCG and NU335 attenuated serum extravasation and astroglial degeneration in the rat piriform cortex (PC). Both EGCG and NU335 reversely regulated phosphatidylinositol 3 kinase (PI3K)/AKT–eNOS (endothelial nitric oxide synthase) mediated BBB permeability and aquaporin 4 (AQP4) expression in endothelial cells and astrocytes through the p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways, respectively. Furthermore, EGCG and NU335 decreased p47Phox (a nicotinamide adenine dinucleotide phosphate oxidase subunit) expression in astrocytes under physiological and post-SE conditions. Therefore, we suggest that EGCG and PEDF derivatives may activate 67LR and its downstream effectors, and they may be considerable anti-vasogenic edema agents.


Introduction
Status epilepticus (SE), defined as a prolonged seizure activity, is a common neurological emergency with considerable morbidity and mortality. In addition, SE is one of the high risk factors of developing acquired epilepsy [1]. As well as neuronal death, SE results in serum extravasation into the brain parenchyma (vasogenic edema) due to the blood-brain barrier (BBB) breakdown [2,3]. Vasogenic edema is widely considered to be detrimental for outcome following SE and leads to the paroxysmal neuronal discharge, neuroinflammation, and astroglial dysfunction, which is involved in epileptogenesis [4,5]. In addition, vasogenic edema results in undesirable secondary complications in various neurological diseases including an abrupt increase in intracranial pressure, abnormal neuronal excitability, and gray and white matter injuries [6][7][8][9]. SE-induced BBB breakdown is relevant to the generation of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) in endothelial cells and reactive oxygen species (ROS) by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in astrocytes, respectively [10]. Thus, it is likely that SE may result in BBB dysfunctions via endothelial-astroglial interactions through the nitrosactive and oxidative stresses. However, the underlying mechanisms and the related signaling pathways for eNOS and NADPH oxidase activations during SE-induced vasogenic edema formation are still unknown.
Here, we demonstrate for the first time that EGCG and PEDF peptide (NU335) attenuated SE-induced vasogenic edema formation by regulating phosphatidylinositol 3 kinase (PI3K)/AKT-mediated BBB permeability and AQP4 expression in endothelial cells and astrocytes through p38 MAPK and ERK1/2 pathways, respectively. Furthermore, these anti-vasogenic edema effects were closely relevant to the inhibitions of eNOS and NADPH oxidase expression in endothelial cells and astrocytes, respectively. Therefore, our findings suggest that 67LR may play a protective role against oxidative and nitrosactive stresses following SE, and its downstream effectors may be considerable therapeutic targets, which have important implications for the development and use of EGCG and PEDF derivatives as anti-vasogenic edema agents.

SE Induction and EEG Recording
Rats were injected intraperitoneally with LiCl (127 mg/kg) 24 h prior to the administration of pilocarpine (30 mg/kg). Twenty minutes before pilocarpine injection, animals were given atropine methylbromide (5 mg/kg) to block the peripheral effect of pilocarpine. SE induction was stopped 2 h after pilocarpine injection by the administration of diazepam (10 mg/kg, i.p.). Diazepam was repeatedly administered as needed. To validate the effect of EGCG or NU335 on seizure susceptibility induced by pilocarpine, electroencephalogram (EEG) signals of electrode-implanted animals were measured with a DAM 80 differential amplifier (0.1-3000 Hz bandpass; World Precision Instruments, Sarasota, FL, USA). EEG activity was measured (2 h recording session), digitized (400 Hz), and analyzed using LabChart Pro v7 (AD Instruments, New South Wales, Australia). The time of seizure onset was defined as the time point showing paroxysmal discharges (4-10 Hz with 2 times higher amplitude than the basal level) that lasted more than 3 seconds. Spectrograms were also automatically calibrated using a Hanning sliding window with 50% overlap [21][22][23].

Tissue Processing
Three days after SE induction, animals were perfused transcardially with phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) under urethane anesthesia (1.5 g/kg intraperitoneally (i.p.)). The brains were removed and stored in the same fixative for 4 h. Subsequently, brains were shifted to PB) containing 30% sucrose at 4 • C for 2 days. Coronal sections (30 µm) were prepared with a cryostat, and consecutive sections were collected in six-well plates containing PBS. For Western blots, animals were rats were sacrificed by decapitation under urethane anesthesia. The PC was rapidly dissected out and homogenized in lysis buffer containing protease inhibitor cocktail (Roche Applied Sciences, Branford, CT, USA) and phosphatase inhibitor cocktail (PhosSTOP ® , Roche Applied Science, Branford, CT, USA). The protein concentration in the supernatant was determined using a Micro BCA Protein Assay Kit (Pierce Chemical, Dallas, TX, USA).

Western Blot
Western blot was performed by the standard protocol. Briefly, sample proteins (10 µg) were separated on a Bis-Tris sodium dodecyl sulfate-poly-acrylamide electrophoresis gel (SDS-PAGE). Then, separated proteins were transferred to polyvinylidene fluoride membranes that were blocked overnight at 4 • C with 2% bovine serum albumin (BSA) in Tris-buffered saline (TBS; in mM 10 Tris, 150 NaCl, pH 7.5, and 0.05% Tween 20) and then incubated in primary antibodies (Table 1). Thereafter, the membranes were incubated for 1 h at room temperature in a solution containing horseradish peroxidase (HRP)-conjugated secondary antibodies. Immunoblots were quantified by membrane scanning in an ImageQuant LAS4000 system (GE Healthcare Korea, Seoul, Korea).
Optical densities of proteins were calculated by the protein/β-actin ratio. The ratio of phosphoprotein to total protein was described as the phosphorylation level.

Immunohistochemistry
Free-floating sections were washed 3 times in PBS (0.1 M, pH 7.3). After blocking the endogenous peroxidase with 3% H 2 O 2 and 10% methanol in PBS (0.1 M) for 20 min at room temperature, sections were incubated in 10% normal goat serum (Vector, Burlingame, CA, USA). Later, sections were incubated in biotinylated rat immunoglobulin G (IgG) and avidin-biotin complex (ABC) kit (Vector, #PK-6100, USA, diluted 1:200) and visualized by 3,3 -diaminobenzidine in 0.1 M Tris buffer. Some sections were incubated in a mixture of primary antibodies shown in Table 1 (in PBS containing 0.3% Triton X-100) at room temperature, overnight. For negative control, tissues were incubated in pre-immune serum instead of primary antibody. After washing 3 times, tissues were incubated with a fluorescein isothiocyanate (FITC)-or Cy3-conjugated secondary antibodies (Vector, Burlingame, CA, USA) for 1 h at room temperature.

Statistical Analysis
The values on normality of results were evaluated using Shapiro-Wilk W-test. Data were analyzed by one-way ANOVA or repeated measure ANOVA to determine statistical significance. Newman-Keuls test was used for post hoc comparisons. A p < 0.05 was considered to be statistically different.

Effects of EGCG and NU335 Peptide on Seizure Susceptibility in Response to Pilocarpine
First, we explored whether EGCG or NU335 influences susceptibility to SE induction. Both EGCG and NU335 did not result in behavioral seizures. As compared to vehicle, both EGCG and NU335 did not affect baseline EEG, latency of seizure onset, and seizure intensity in response to pilocarpine ( Figure 1A-C). Therefore, these findings indicate that EGCG and NU335 may not influence the seizure susceptibility in response to pilocarpine.

Effects of EGCG and NU335 Peptide on Seizure Susceptibility in Response to Pilocarpine
First, we explored whether EGCG or NU335 influences susceptibility to SE induction. Both EGCG and NU335 did not result in behavioral seizures. As compared to vehicle, both EGCG and NU335 did not affect baseline EEG, latency of seizure onset, and seizure intensity in response to pilocarpine ( Figure 1A-C). Therefore, these findings indicate that EGCG and NU335 may not influence the seizure susceptibility in response to pilocarpine.

Effects of EGCG and NU335 Peptide on Serum Extravasation Following SE
Next, we investigated whether EGCG or NU335 affects 67LR expression and BBB permeability

Effects of EGCG and NU335 Peptide on Serum Extravasation Following SE
Next, we investigated whether EGCG or NU335 affects 67LR expression and BBB permeability under post-SE conditions. Since vasogenic edema peaks in the PC at 3 days after SE [36], we explore the effects of EGCG and NU335 on vasogenic edema formation using 3-day post-SE animals. Following SE, 67LR expression was significantly reduced in the PC (p < 0.05 vs. control animals, one-way ANOVA, n = 7; Figure 2A,B). This SE-induced 67LR reduction was unaffected by EGCG or NU335 (Figure 2A,B). SE led to severe serum extravasation in the PC (p < 0.05 vs. control animals, one-way ANOVA, n = 7; Figure 2A,C). As compared to vehicle, EGCG and NU335 decreased SE-induced serum extravasation to 0.58-and 0.77-fold of vehicle level, respectively (p < 0.05 vs. vehicle, one-way ANOVA, n = 7; Figure 2A,C). EGCG and NU335 showed approximately 66% and 33% reduction in the volume of vasogenic edema in the PC, respectively (p < 0.05 vs. vehicle, one-way ANOVA, n = 7; Figure 2D,E). These findings indicate that EGCG and NU335 may ameliorate SE-induced vasogenic edema formation without altering 67LR expression and seizure susceptibility in response to pilocarpine.
Antioxidants 2020, 9, x FOR PEER REVIEW 6 of 21 induced vasogenic edema formation without altering 67LR expression and seizure susceptibility in response to pilocarpine.

Effects of EGCG and NU335 Peptide on Astroglial Damage under Physiological and Post-SE Conditions
Since astroglial loss is closely relevant to vasogenic edema formation [21,36,38], we explored the effects of EGCG and NU335 on astroglial viability under physiological and post-SE conditions. As compared to vehicle, EGCG and NU335 did not lead to reactive astrogliosis and astroglial loss in the PC under physiological conditions. Thus, there was no difference in the GFAP-positive area in the PC among the three groups ( Figure 3A,B). Following SE, the GFAP-positive area was reduced to approximately 0.37-fold of control level in the PC due to massive astroglial loss (p < 0.05, one-way ANOVA, n = 7, respectively; Figure 3A,B). EGCG and NU335 attenuated the GFAP-deleted area in the PC; thus, the GFAP-positive area was 0.74-and 0.54-fold of the control level (p < 0.05, one-way ANOVA, n = 7, respectively; Figure 3A,B). These findings indicate that EGCG and NU335 may reduce astroglial vulnerability to SE in the PC.

The Effects of EGCG and NU335 on Phosphorylations of p38 MAPK and ERK1/2
The blockade of 67LR functionality increases p38 MAPK and ERK1/2 activities [20][21][22][23]. Thus, we explored the effects of EGCG and NU335 on their phosphorylations (activities). Under physiological condition, neither EGCG nor NU335 affected p38 MAPK and ERK1/2 expressions in the PC. However, EGCG reduced p38 MAPK and ERK1/2 phosphorylations to approximately 0.65-and 0.77-fold of the vehicle level, respectively (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 4A,C). In addition, NU335 decreased them to approximately 0.85-and 0.83-fold of the vehicle level (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 4A,C). Immunohistochemical studies revealed that both EGCG and NU335 did not affect the SMI-71 (an endothelial BBB marker) expression level in the PC (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 5A,B). However, EGCG reduced p-p38 MAPK and pERK1/2 signals to 0.68-and 0.59-fold of the vehicle level in the PC. In addition, NU335 diminished them to 0.74-and 0.71-fold of the vehicle level, respectively (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 5A,C-E). These alterations in p-p38 MAPK and pERK1/2 signals were preferentially observed in endothelial cells and astrocytes, respectively ( Figure 5A). Immunohistochemical studies revealed that both EGCG and NU335 did not affect the SMI-71 (an endothelial BBB marker) expression level in the PC (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 5A,B). However, EGCG reduced p-p38 MAPK and pERK1/2 signals to 0.68-and 0.59-fold of the vehicle level in the PC. In addition, NU335 diminished them to 0.74-and 0.71-fold of the vehicle level, respectively (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 5A,C-E). These alterations in p-p38 MAPK and pERK1/2 signals were preferentially observed in endothelial cells and astrocytes, respectively ( Figure 5A). Following SE, the p38 MAPK phosphorylation ratio was increased to 1.2-fold of the control level, but its expression was not increased in the PC (p < 0.05 vs. control animals, one-way ANOVA; n = 7, respectively; Figure 4A,B). In contrast, SE reduced ERK1/2 expression and its phosphorylation level. The ERK1/2 phosphorylation ratio was 0.7-fold the control level (p < 0.05 vs. control animals, oneway ANOVA; n = 7, respectively; Figure 4A,C). EGCG attenuated SE-induced p38 MAPK phosphorylation to approximately 0.86-fold the control level (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 4A,B). EGCG reinforced the reduction in ERK1/2 phosphorylation ratio to 0.54-fold the control level following SE (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 4A,C). Similar to EGCG, NU335 ameliorated the SE-induced p38 MAPK phosphorylation ratio 0.99-fold of the control level (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 4A,B). NU335 also further reduced the ERK1/2 phosphorylation ratio to 0.56-fold the control level following SE (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 4A,C). Therefore, these findings indicate that EGCG and NU335 may inhibit p38 MAPK and ERK1/2 phosphorylations in the PC Following SE, the p38 MAPK phosphorylation ratio was increased to 1.2-fold of the control level, but its expression was not increased in the PC (p < 0.05 vs. control animals, one-way ANOVA; n = 7, respectively; Figure 4A,B). In contrast, SE reduced ERK1/2 expression and its phosphorylation level. The ERK1/2 phosphorylation ratio was 0.7-fold the control level (p < 0.05 vs. control animals, one-way ANOVA; n = 7, respectively; Figure 4A,C). EGCG attenuated SE-induced p38 MAPK phosphorylation to approximately 0.86-fold the control level (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 4A,B). EGCG reinforced the reduction in ERK1/2 phosphorylation ratio to 0.54-fold the control level following SE (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 4A,C). Similar to EGCG, NU335 ameliorated the SE-induced p38 MAPK phosphorylation ratio 0.99-fold of the control level (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 4A,B). NU335 also further reduced the ERK1/2 phosphorylation ratio to 0.56-fold the control level following SE (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 4A,C). Therefore, these findings indicate that EGCG and NU335 may inhibit p38 MAPK and ERK1/2 phosphorylations in the PC under physiological and post-SE conditions.
Antioxidants 2020, 9, x FOR PEER REVIEW 10 of 21 explored if EGCG and NU335 affect PI3K/AKT activities and AQP4 expression in the PC. Under physiological conditions, EGCG did not influence PI3K and AKT expression levels in the PC ( Figure  6A-C). However, EGCG reduced pPI3K-Y458 and pAKT-T308 phosphorylation levels to 0.74-and 0.76-fold the vehicle level, respectively (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 6A-C). NU335 also decreased PI3K and AKT phosphorylation levels to 0.85-and 0.88-fold the vehicle level, respectively (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 6A-C). Furthermore, EGCG and NU335 increased AQP4 expression to 1.26-and 1.13-fold the vehicle level (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 6A,D). Immunofluorescent study demonstrated that pAKT signals were observed in endothelial cells and astrocytes ( Figure 7A). EGCG and NU335 reduced pAKT fluorescent intensity in astrocytes and endothelial cells (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 7A,B), while they Immunofluorescent study demonstrated that pAKT signals were observed in endothelial cells and astrocytes ( Figure 7A). EGCG and NU335 reduced pAKT fluorescent intensity in astrocytes and endothelial cells (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 7A,B), while they enhanced AQP4 expression level in astrocytes (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 7C,D). Following SE, PI3K and AKT were increased to 1.25-and 1.35-fold the control level (p < 0.05 vs. control animals, one-way ANOVA; n = 7, respectively; Figure 6A-C). However, AQP4 expression was reduced to 0.72-fold the control level in the PC (p < 0.05 vs. control animals, one-way ANOVA; n = 7, respectively; Figure 6A,D). EGCG and NU335 abrogated the SE-induced increases in PI3K and AKT phosphorylations to vehicle-treated control animal level (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 6A-C). They also attenuated the SE-induced decrease in AQP4 expression (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 6A,D). These findings indicate that EGCG and NU335 may ameliorate vasogenic edema by inhibiting the alterations in PI3K/AKT-mediated BBB permeability and AQP4 expression induced by SE. These findings indicate that EGCG and NU335 may ameliorate vasogenic edema by inhibiting the alterations in PI3K/AKT-mediated BBB permeability and AQP4 expression induced by SE.

The Effects of EGCG and NU335 on eNOS and NADPH Oxidase Expression
SE leads to nitrosactive and oxidative stresses in endothelial cell and astrocytes, respectively [10]. SE triggers the production of NO derived from endothelial nitric oxide synthase (eNOS) in

The Effects of EGCG and NU335 on eNOS and NADPH Oxidase Expression
SE leads to nitrosactive and oxidative stresses in endothelial cell and astrocytes, respectively [10]. SE triggers the production of NO derived from endothelial nitric oxide synthase (eNOS) in endothelial cells, which increases BBB permeability [45,46]. SE also activates astroglial NADPH oxidase, which is the major source of ROS. p47phox plays an important role in the assembly of the NADPH complex that is composed of p40, p47, p67, gp91, and P22phox [47,48]. Indeed, apocynin, an NADPH oxidase inhibitor, attenuates SE-induced vasogenic edema [10]. Thus, we investigated whether EGCG and NU355 affect eNOS and p47Phox expression. Under physiological conditions, EGCG decreased eNOS and p47Phox expression levels to 0.78-and 0.81-fold the vehicle level, respectively (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 8A-C). NU335 also reduced eNOS and p47Phox expression levels to 0.77-and 0.83-fold the vehicle level, respectively (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 8A-C). Immunofluorescent study demonstrated that EGCG and NU335 reduced eNOS expression in endothelial cells (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 9A,B). In addition, EGCG and NU335 declined p47Phox expression in astrocytes (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 9C,D). Consistent with our previous study [10], eNOS and p47Phox expression were increased to 2.06-and 1.77-fold the control level following SE (p < 0.05 vs. control animals, one-way ANOVA; n = 7, respectively; Figure 8A-C). EGCG abrogated the SE-induced increases in eNOS and p47Phox levels to 1.54-and 1.35-fold the vehicle-treated control animal level (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 8A-C). NU335 also ameliorated SEinduced increases in eNOS and p47Phox levels to 1.63-and 1.42-fold the vehicle-treated control animal level (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 8A-C). These findings indicate that EGCG and NU335 may mitigate SE-induced vasogenic edema by abolishing endothelial NO synthesis and astroglial ROS generation. Immunofluorescent study demonstrated that EGCG and NU335 reduced eNOS expression in endothelial cells (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 9A,B). In addition, EGCG and NU335 declined p47Phox expression in astrocytes (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 9C,D). Consistent with our previous study [10], eNOS and p47Phox expression were increased to 2.06-and 1.77-fold the control level following SE (p < 0.05 vs. control animals, one-way ANOVA; n = 7, respectively; Figure 8A-C). EGCG abrogated the SE-induced increases in eNOS and p47Phox levels to 1.54-and 1.35-fold the vehicle-treated control animal level (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 8A-C). NU335 also ameliorated SE-induced increases in eNOS and p47Phox levels to 1.63-and 1.42-fold the vehicle-treated control animal level (p < 0.05 vs. vehicle, one-way ANOVA; n = 7, respectively; Figure 8A-C). These findings indicate that EGCG and NU335 may mitigate SE-induced vasogenic edema by abolishing endothelial NO synthesis and astroglial ROS generation.

Discussion
The major findings in the present study are that EGCG and NU335 attenuated SE-induced vasogenic edema formation. In addition, EGCG and NU335 reversely regulated PI3K/AKT-mediated BBB permeability and AQP4 expression in endothelial cells and astrocytes, which were modulated by p38 MAPK-ERK1/2 and NADPH oxidase-ERK1/2 signaling pathways, respectively ( Figure 10).

Discussion
The major findings in the present study are that EGCG and NU335 attenuated SE-induced vasogenic edema formation. In addition, EGCG and NU335 reversely regulated PI3K/AKT-mediated BBB permeability and AQP4 expression in endothelial cells and astrocytes, which were modulated by p38 MAPK-ERK1/2 and NADPH oxidase-ERK1/2 signaling pathways, respectively ( Figure 10). Figure 10. Scheme of the underlying mechanisms of EGCG and NU335 against vasogenic edema formation. The reduced 67LR functionality induced by SE activates p38 MAPK-PI3K/AKT-eNOS and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-ERK1/2-PI3K/AKT signaling pathways in endothelial cells and astrocytes, respectively. Subsequently, the increased nitric oxide (NO) generation enhances BBB permeability. In addition, astroglial AKT activation reduces AQP4 expression, leading to the impaired water efflux from the brain parenchyma, which worsens vasogenic edema. EGCG and NU335 attenuate vasogenic edema formation via 67LR activation that plays inhibitory roles in these p38 MAPK and NADPH oxidase-mediated signaling pathways in endothelial cells and astrocytes.
The BBB plays an important role in the maintenance of the microenvironment for brain functions by separating the brain from the systemic circulatory systems and regulating the transports of intravascular substances into the brain [49]. Under pathophysiological conditions, BBB is disrupted, and the subsequent serum-derived molecules are leaked in the brain parenchyma. This serum extravasation leads to neuronal excitability and neuroinflammation [50][51][52][53]. Thus, the preservation of BBB integrity is one of the therapeutic strategies to inhibit undesirable consequences from brain insults.
PEDF is a 50-kDa protein secreted by the retinal pigment epithelium in the eye as well as other tissues [56]. PEDF has anti-angiogenic, anti-tumorigenic, anti-inflammatory, anti-oxidative, Figure 10. Scheme of the underlying mechanisms of EGCG and NU335 against vasogenic edema formation. The reduced 67LR functionality induced by SE activates p38 MAPK-PI3K/AKT-eNOS and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-ERK1/2-PI3K/AKT signaling pathways in endothelial cells and astrocytes, respectively. Subsequently, the increased nitric oxide (NO) generation enhances BBB permeability. In addition, astroglial AKT activation reduces AQP4 expression, leading to the impaired water efflux from the brain parenchyma, which worsens vasogenic edema. EGCG and NU335 attenuate vasogenic edema formation via 67LR activation that plays inhibitory roles in these p38 MAPK and NADPH oxidase-mediated signaling pathways in endothelial cells and astrocytes.
The BBB plays an important role in the maintenance of the microenvironment for brain functions by separating the brain from the systemic circulatory systems and regulating the transports of intravascular substances into the brain [49]. Under pathophysiological conditions, BBB is disrupted, and the subsequent serum-derived molecules are leaked in the brain parenchyma. This serum extravasation leads to neuronal excitability and neuroinflammation [50][51][52][53]. Thus, the preservation of BBB integrity is one of the therapeutic strategies to inhibit undesirable consequences from brain insults.
In previous studies, we have reported that astroglial degeneration/dysfunction is not a primary cause of vasogenic edema formation [21][22][23]. In addition, AQP4 deletion cannot evoke serum extravasation [6,79]. However, AQP4 deletion or its inhibition in astrocytes deteriorates vasogenic edema due to the impaired vasogenic water elimination from parenchyma to vessels, which subsequently worsens astroglial degeneration [6,36,79]. Recently, we have also reported that U0126 aggravated SE-induced vasogenic edema and astroglial loss in the PC, while 3CAI alleviated these post-SE events [23]. On the contrary, the present study demonstrates that EGCG and NU335 attenuated the SE-induced AQP4 reduction in the PC, which is accompanied by decreasing ERK1/2 and AKT phosphorylations. In addition, EGCG and NU335 ameliorated SE-induced astroglial loss in the PC. These findings suggest that the EGCG-and NU335-induced inhibition of the ERK1/2-AKT signaling pathway may increase astroglial viability, which would be involved in the prevention of SE-induced AQP4 downregulation. On the other hand, astroglial NADPH oxidase activation reduces AQP4 expression and astroglial viability [10]. Interestingly, ROS generation from NADPH oxidase triggers downstream pathways for the ERK1/2 and AKT, but not p38 MAPK phosphorylations in astrocytes [80,81]. NADPH oxidase-mediated ROS activate ERK1/2 and AKT, but not p38 MAPK [82]. In the present study, ECGC and NU335 reduced astroglial p47Phox expression under physiological and post-SE conditions. Thus, it is likely that 67LR-mediated NADPH oxidase inhibition may decrease ERK1/2 and AKT phosphorylations, which would upregulate AQP4 expression. Indeed, EGCG inhibits the activities of NADPH oxidase [83] and ERK1/2 via 67LR [75,84]. PEDF also abrogates ERK1/2 and NADPH oxidase activities [85][86][87]. Thus, our findings suggest that EGCG and NU335 may lead to astroglial AQP4 upregulation by 67LR-medaited NADPH oxidase inhibition, which would decrease ERK1/2-AKT activities.
As aforementioned, most SE models show serum extravasation in the brain parenchyma, which is involved in ictogenesis and epileptogenesis [2][3][4][5]. Pilocarpine-induced SE generally leads to more intense damage in more areas of the brain than kainic acid (KA)-induced SE of similar duration [88]. In addition, KA results in cytotoxic edema in the hippocampus and piriform cortex where pilocarpine induces severe serum extravasation, although it develops vasogenic edema in the frontal cortex, thalamus, and the striatum [10,22,36,89,90]. Based on these features, we believe that a pilocarpine model may be better to evaluate the efficacy of EGCG and NU335 against SE-induced serum extravasation than the KA model. Using the same rationale, our findings suggest that EGCG and NU335 may be effective and possibly strong agents against vasogenic edema.
On the other hand, various insults such as stroke and head trauma also evoke vasogenic edema that increases intracranial pressure, leading to fatal conditions [91]. Furthermore, retinal vascular permeability is increased in diabetic retinopathy and exudative macular degeneration that cause blindness [28,92,93]. Currently, corticosteroids and agents blocking VEGF are the predominant treatment options for brain edema and retina macular edema, respectively. Unfortunately, both corticosteroids and anti-VEGF therapy induce adverse effects [94]. Thus, it is likely that EGCG and/or NU335 may be attractive and novel therapeutic agents for vasogenic cerebral edema and retinal macular edema. However, EGCG reaches the brain parenchyma even at a very low concentration [95]. In addition, the permeability of NU335 into the BBB is unknown. Therefore, the development of novel BBB-permeable derivatives of EGCG and NU335 would be needed for clinical trials concerning prevention treatment for vasogenic edema.

Conclusions
To the best of our knowledge, the present data reveal for the first time that EGCG and NU335, as 67LR activators, attenuated SE-induced vasogenic edema formation by inhibiting p38 MAPK-PI3K/AKT-eNOS axis in endothelial cells. In addition, they increased AQP4 expression by abrogating the NADPH oxidase-ERK1/2-PI3K/AKT-AQP4 signaling pathway in astrocytes ( Figure 10). Therefore, our findings suggest that the 67LR activation may be one of the therapeutic strategies for the prevention of vasogenic edema formation and its complications induced by nitrosactive and oxidative stresses.