PB101, a VEGF- and PlGF-targeting decoy protein, enhances antitumor immunity and suppresses tumor progression and metastasis.

Antiangiogenic therapy is a recognized method for countering the immunosuppressive tumor microenvironment (TME) and improving anti-tumor immunity. PB101 is a glycosylated decoy receptor that binds to VEGF-A and PlGF with high affinity, based on the VEGFR1 backbone. Here, we elucidated PB101-induced remodeling of tumor angiogenesis and immunity, which enhances anti-PD-L1 immune checkpoint blockade. PB101 inhibited tumor growth by suppressing angiogenesis and enhancing CD8+ T cell infiltration into the tumors. PB101 induced robust reprogramming of antitumor immunity and activates intratumoral CD8+ T cells. Anti-tumor efficacy of PB101 is mostly dependent on CD8+ T cells and IFN-γ. PB101 reprograms tumor immunity in a manner distinct from that of the conventional VEGF decoy receptor, VEGF-trap. With its potent immune-modulating capability, PB101 synergizes with an anti-PD-L1, triggering strengthened antitumor immunity. Combining PB101 and anti-PD-L1 could establish durable protective immunity against tumor recurrence and metastasis. The findings of this study offer scientific rationales for further clinical development of PB101, particularly when used in combination with immune checkpoint inhibitors, as a potential treatment for advanced cancers.

Noggin-mediated effects on metabolite profiles of microglia and oligodendrocytes after ischemic insult.

Recent studies show that shifts in energy metabolism in activated microglia are linked to their functions and immune responses in the ischemic brain. We previously reported that an antagonist of the bone morphogenetic protein, noggin, enhanced myelination in the ischemic brain during the chronic phase, and conditioned media (CM) from activated BV2 microglia treated with noggin after ischemia/reperfusion (I/R) increased the expression of myelin basic protein (MBP) in oligodendrocytes (MO3.13). To determine whether noggin induced changes in cell metabolism, metabolite profiles in BV2 and MO3.13 cells were analyzed by untargeted metabolomics using 1H nuclear magnetic resonance spectroscopy. Compared to vehicle-treated BV2 cells, noggin treatment (100 ng/mL for 3 h after I/R) suppressed the I/R-induced increase in intracellular glucose and lactate levels but increased extracellular levels of glucose and several amino acids. When MO3.13 cells were exposed to noggin CM from BV2 cells, most of the vehicle CM-induced changes in the levels of metabolites such as choline, formate, and intermediates of oxidative phosphorylation were reversed, while the glycerol level was markedly increased. An increase in glycerol level was also observed in the noggin-treated ischemic brain and was further supported by the expression of glycerol-3-phosphate dehydrogenase 1 (required for glycerol synthesis) in the cytoplasm of MBP-positive oligodendrocytes in the ischemic brains treated with noggin. These results suggest that noggin-induced changes in the metabolism of microglia provide a favorable environment for myelin synthesis in oligodendrocytes during the recovery phase after ischemic stroke.

Neuroprotective Effect of Statins in a Rat Model of Chronic Ocular Hypertension.

Glaucoma is an optic neuropathy in which the degeneration of retinal ganglion cells (RGCs) results in irreversible vison loss. Therefore, neuroprotection of RGCs from glaucomatous afflictions is crucial for glaucoma treatment. In this study, we aimed to investigate the beneficial effects of statins in the protection of RGCs using a rat model. Glaucomatous injury was induced in rats by chronic ocular hypertension (OHT) achieved after performing a circumlimbal suture. The rats were given either statins such as simvastatin and atorvastatin or a solvent weekly for 6 weeks. Retina sections underwent hematoxylin and eosin, Brn3a, or cleaved casepase-3 staining to evaluate RGC survival. In addition, modulation of glial activation was assessed. While the retinas without statin treatment exhibited increased RGC death due to chronic OHT, statins promoted the survival of RGCs and reduced apoptosis. Statins also suppressed chronic OHT-mediated glial activation in the retina. Our results demonstrate that statins exert neuroprotective effects in rat retinas exposed to chronic OHT, which may support the prospect of statins being a glaucoma treatment.

The potential of BEN815 as an anti-inflammatory, antiviral and antioxidant agent for the treatment of COVID-19.

Background: The corona virus disease 2019 (COVID-19) pandemic has highlighted the fact that there are few effective antiviral agents for treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Although the very recent development of vaccines is an extremely important breakthrough, it remains unclear how long-lived such vaccines will be. The development of new agents therefore remains an important goal. Purpose: Given the multifaceted pathology of COVID-19, a combinatorial formulation may provide an effective treatment. BEN815, a natural nutraceutical composed of extracts from guava leaves (Psidium guajava), green tea leaves (Camellia sinensis), and rose petals (Rosa hybrida), had previously shown to have a therapeutic effect on allergic rhinitis. We investigated whether BEN815 possesses anti-inflammatory, antiviral and antioxidant activities, since the combination of these effects could be useful for the treatment of COVID-19. Study design: We examined the anti-inflammatory effects of BEN815 and its principal active components quercetin and epigallocatechin gallate (EGCG) in lipopolysaccharide (LPS)-induced RAW264.7 cells and in an LPS-challenged mouse model of endotoxemia. We also assessed the antioxidant activity, and antiviral effect of BEN815, quercetin, and EGCG in SARS-CoV-2-infected Vero cells. Methods: The principal active ingredients in BEN815 were determined and quantified using HPLC. Changes in the levels of LPS-induced pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α were measured by ELISA. Changes in the expression levels of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) were analyzed using western blotting. Antioxidant assay was performed using DPPH and ABTS assay. SARS-CoV-2 replication was measured by immunofluorescence staining. Results: BEN815 significantly suppressed the induction of IL-6 and TNF-α as well as COX-2 and iNOS in LPS-induced RAW264.7 cells. In addition, BEN815 protected against LPS-challenged endotoxic shock in mice. Two major constituents of BEN815, quercetin and EGCG, reduced the induction of IL-6 and TNF-α as well as COX-2 and iNOS synthase in LPS-induced RAW264.7 cells. BEN815, quercetin, and EGCG were also found to have antioxidant effects. Importantly, BEN815 and EGCG could inhibit SARS-CoV-2 replications in Vero cells. Conclusion: BEN815 is an anti-inflammatory, antiviral, and antioxidant natural agent that can be used to prevent and improve inflammation-related diseases, COVID-19.

Enhanced repair processes and iron uptake by ischemic preconditioning in the brain during the recovery phase after ischemic stroke.

Ischemic preconditioning (IP) reduces brain damage after subsequent ischemic strokes by activating endogenous protective mechanisms in rodents. Transient ischemic attack (TIA) induces tolerance in the human brain after ischemic strokes; defining mechanisms of IP effects may provide therapeutic targets to improve recovery of patients with ischemic strokes. Iron transported across the blood-brain barrier (BBB) is required for brain functions, including myelination, and its levels should be finely regulated to avoid harmful effects. This study aimed to determine whether IP enhances repair processes by modulating iron metabolism during the post-stroke chronic phase. Male mice were divided into sham and IP groups, and IP was induced 24 h before a transient focal ischemic stroke. Sensorimotor recovery was observed over 8 weeks after the stroke, and brain volumes and levels of proteins related to repair processes and iron metabolism in the ischemic brains were examined 8 weeks after the stroke. There was significantly less ischemic brain atrophy in the IP group than in the sham group, with no differences in sensorimotor recovery between the groups. Levels of tight junction proteins of BBB, neurites outgrowth markers, and myelin sheath proteins and markers for mature oligodendrocytes were significantly increased in the IP group. Iron import proteins, transferrin receptor 1 and DMT1, were also increased in the IP group. These results indicate that IP increases brain repair processes and iron uptake during the chronic phase after an ischemic stroke, and provide new insights to understand the molecular mechanisms of TIA effects on post-stroke recovery.

Borage Oil Treated with Immobilized Lipase Inhibits Melanogenesis.

In the present study, we demonstrated that borage (Borago officinalis L.) seed oil subjected to immobilized lipase pretreatment are enriched with linoleic acid (LNA, 18:2n-6), γ-linolenic acid (GLA, 18:3n-6), and oleic acid (OLA, 18:1n-9). We further showed that lipase-treated borage oil (LT-BOL) regulates the activity and degradation of tyrosinase, an important enzyme implicated in the synthesis of melanin in murine melanocytes, B16F10. LT-BOL and its free fatty acid components reduced the levels of melanin and tyrosinase in melanocytes with GLA exerting similar or stronger effects compared with LNA and OLA. The brightening efficacy of LT-BOL on melanin metabolism in humans was tested by an 8-week, double-blind, randomized clinical trial, which enrolled 21 Korean female adults (mean age 48.57 ± 3.28). Visual evaluation showed that cream containing 1% LT-BOL significantly decreased (p < 0.05) melasma on the treated skin area after 6 and 8 weeks. The analysis of the skin brightness using Chromameter CR-400 confirmed that the brightness of the treated area was significantly increased (p < 0.01) after 4, 6, and 8 weeks. Together, our results suggest that LT-BOL may be suitable as a natural skin whitening cosmeceutical product.

Estrogen deficiency is associated with brain iron deposition via upregulation of hepcidin expression in aged female mice.

The total iron level in the brain increases with age, and excess iron is associated with neurodegenerative diseases; however, the mechanism of brain iron deposition is unknown. In peripheral cells, the expression of hepcidin, a master regulator of iron homeostasis, is regulated by estrogen. This study aimed to determine whether hepcidin was involved in iron deposition in the brain and brain endothelial cells of estrogen-deficient aged female mice. Aged mice showed increased levels of hepcidin and ferritin in the brain and brain microvessels compared with young mice, and these levels were reduced by estrogen replacement in ovariectomized aged mice. In the brain endothelial cell line bEnd.3, the lipopolysaccharide (10 ng/mL)-induced increases of hepcidin mRNA and protein levels, the number of Prussian blue-positive cells, and free radicals were reduced after estrogen treatment. These results suggest that estrogen deficiency with an increase of hepcidin is partly responsible for iron deposition in the brain and brain endothelial cells and that hepcidin can be a target to prevent brain aging and neurodegeneration in postmenopausal women.

Statins Suppress TGF-ß2-Mediated MMP-2 and MMP-9 Expression and Activation Through RhoA/ROCK Inhibition in Astrocytes of the Human Optic Nerve Head.

Purpose: Matrix metalloproteinases (MMPs) are involved in extracellular matrix (ECM) maintenance and remodeling. The present study aimed to determine whether transforming growth factor (TGF)-ß2 regulates MMP-2 and MMP-9 levels and activities in astrocytes derived from the optic nerve head (ONH) and the role of statins in such modulation. Methods: Primary astrocytes cultured from the lamina cribrosa of human donor ONHs were incubated with three types of statins (5 µg/mL) for 1 hour followed by recombinant TGF-ß2 (5 ng/mL) for various periods to test their effects. Levels and activities of MMP-2 and MMP-9 in astrocytes in vitro were determined by western blotting and zymography, respectively. Levels of phosphorylated myosin phosphatase target subunit 1 (MYPT1) in astrocyte lysates were determined by western blotting, and those of phosphorylated myosin light chain (MLC) were determined by western blotting and immunocytochemistry. Results: MMP-2 and MMP-9 levels were upregulated by TGF-ß2 in human ONH astrocytes. Prior incubation with simvastatin, lovastatin, and atorvastatin inhibited TGF-ß2-mediated MMP-2 and MMP-9 expression and activities. Prior incubation with statins downregulated the TGF-ß2-induced phosphorylation of MYPT1 and MLC, which are downstream substrates of RhoA and ROCKs. Conclusions: Statins inhibited the TGF-ß2-mediated regulation of MMP-2 and MMP-9 by inhibiting the RhoA/ROCK signaling pathway. Considering the role of MMP in ECM remodeling, the present findings support the notion that statins positively impact ECM remodeling within the ONH.

Antifibrotic Effects of Sakuraso-Saponin in Primary Cultured Pterygium Fibroblasts in Comparison With Mitomycin C.

Purpose: To investigate the antifibrotic effects of sakuraso-saponin on a primary culture of human pterygium fibroblasts (HPFs) and normal human Tenon fibroblasts (HTFs) as compared to the effects of mitomycin C (MMC). Methods: Samples of HPFs and HTFs were acquired during primary pterygium surgery. Cell toxicity, cell migration, and expression of α-smooth muscle actin (α-SMA) and transforming growth factor-ß (TGF-ß) were evaluated in HPFs and HTFs after treatment with sakuraso-saponin and MMC. To determine the possible mechanisms underlying the antifibrotic effects of sakuraso-saponin, the expression of phosphorylated Smad2/3 was evaluated after treatment with sakuraso-saponin and MMC. Results: MMC (≥200 µg/mL) significantly reduced cell viability in both HPFs and HTFs, whereas sakuraso-saponin (1.0 µg/mL) decreased cell viability in HPFs only. Both sakuraso-saponin (1.0 µg/mL) and MMC (200 µg/mL) treatment significantly reduced the expression of α-SMA and TGF-ß in HPFs (P < 0.05). It is interesting that the expression of α-SMA and TGF-ß after treatment with sakuraso-saponin was significantly lower than that after treatment with MMC (P < 0.05). The expression of phosphorylated Smad2/3 protein was decreased by sakuraso-saponin and MMC in HPFs. Both sakuraso-saponin and MMC inhibited TGF-ß1-induced cell migration as compared to the control in HPFs. Conclusions: Sakuraso-saponin could be more effective than MMC for the reduction of fibrosis in HPFs. Our results might present the basis for its use as a promising candidate drug for adjuvant therapy to prevent recurrent pterygium after surgery.

Repression of adenosine triphosphate-binding cassette transporter ABCG2 by estrogen increases intracellular glutathione in brain endothelial cells following ischemic reperfusion injury.

The adenosine triphosphate-binding cassette efflux transporter ABCG2, which is located in the blood-brain barrier limits the entry of endogenous compounds and xenobiotics into the brain, and its expression and activity are regulated by estrogen. This study was aimed to define the role of ABCG2 in estrogen-mediated neuroprotection against ischemic injury. ABCG2 protein levels before and after ischemic stroke were increased in the brain of female mice by ovariectomy, which were reversed by estrogen replacement. In brain endothelial cell line bEnd.3, estrogen reduced the basal ABCG2 protein level and efflux activity and protected cells from ischemic injury without inducing ABCG2 expression. When bEnd.3 cells were transfected with ABCG2 small interfering RNA, ischemia-induced cell death was reduced, and the intracellular concentration of glutathione, an antioxidant that is transported by ABCG2, was increased. In addition, after ischemic stroke in ovariectomized mice, estrogen prevented the reduction of intracellular glutathione level in brain microvessels. These data suggested that the suppression of ABCG2 by estrogen is involved in neuroprotection against ischemic injury by increasing intracellular glutathione, and that the modulation of ABCG2 activity offers a therapeutic target for brain diseases in estrogen-deficient aged women.

Iron released from reactive microglia by noggin improves myelin repair in the ischemic brain.

We previously reported that the bone morphogenetic protein (BMP) antagonist, noggin, improved the repair process with an increase in the reactive microglia/macrophage population in the ischemic brain. Since BMP plays a role in intracellular iron homeostasis via the hepcidin/ferroportin axis, and iron is required for myelination, this study was aimed to determine whether noggin affected iron status and remyelination in the brain following ischemic stroke. We further examined the effect of blocking the BMP/hepcidin pathway on reactive microglia (BV2) and myelination of oligodendroglial cells (MO3.13) to define the link between microglial iron status and myelin formation. Following the noggin infusion into the ischemic brain of mice, the induction of hepcidin and ferritin protein levels decreased, and the number of myelinated axons and myelin thickness increased at 8 weeks after ischemic stroke. Noggin repressed the increase in hepcidin and ferritin levels in BV2 exposed to lipopolysaccharide (LPS) and oxygen/glucose deprivation and reperfusion (OGD/R). When MO3.13 were exposed to the conditioned media from noggin-treated BV2 (noggin CM) during reperfusion, OGD/R-induced MO3.13 cell death was reduced. Under normal conditions, noggin CM induced myelin production with an increase in ferritin levels in MO3.13, which was reversed by the iron chelator, deferoxamine. These results indicated that noggin altered the iron status in reactive microglia from the iron-storing to the iron-releasing phenotype, which contributed to myelin synthesis by providing iron. We suggest that the BMP/hepcidin pathway can be a target for the regulation of the iron status in microglia to enhance remyelination in the ischemic brain.

Statins reduce TGF-beta2-modulation of the extracellular matrix in cultured astrocytes of the human optic nerve head.

Statins are cholesterol lowering drugs and have shown beneficial effects on glaucoma. With regard to the mechanism of statin action on glaucoma, we investigated the effects of statins on transforming growth factor-beta 2 (TGF-ß2)-induced expression of extracellular matrix (ECM) proteins in human astrocytes of the optic nerve head (ONH) lamina cribrosa (LC). By using primary human ONH astrocytes, we found that both simvastatin and lovastatin inhibited TGF-ß2-mediated expression of ECM proteins such as connective tissue growth factor, collagen I, fibronectin, and plasminogen activator inhibitor-1. Suppression of ECM related proteins is due to inhibition of Smad2/3 activation as statins inhibit TGF-ß2-induced Smad2 phosphorylation and Smad2/3 nuclear accumulation. In ONH astrocytes, TGF-ß2 does not induce MAPK activation. In this study we found an anti-fibrotic effect of statins in human astrocytes of the ONH and identified TGF-ß2 as a mediator of statin action, which may support a beneficial role for statins in blocking glaucomatous axonal damage induced by ECM remodeling.

Resveratrol attenuates peripheral and brain inflammation and reduces ischemic brain injury in aged female mice.

Resveratrol is known to improve metabolic dysfunction associated with obesity. Visceral obesity is a sign of aging and is considered a risk factor for ischemic stroke. In this study, we investigated the effects of resveratrol on inflammation in visceral adipose tissue and the brain and its effects on ischemic brain injury in aged female mice. Mice treated with resveratrol (0.1 mg/kg, p.o.) for 10 days showed reduced levels of interleukin-1ß and tumor necrosis factor-α, as well as a reduction in the size of adipocytes in visceral adipose tissue. Resveratrol also reduced interleukin-1ß and tumor necrosis factor-α protein levels and immunoglobulin G extravasation in the brain. Mice treated with resveratrol demonstrated smaller infarct size, improved neurological function, and blunted peripheral inflammation at 3 days postischemic stroke. These results showed that resveratrol counteracted inflammation in visceral adipose tissue and in the brain and reduced stroke-induced brain injury and peripheral inflammation in aged female mice. Therefore, resveratrol administration can be a valuable strategy for the prevention of age-associated and disease-provoked inflammation in postmenopausal women.

Activation of classical estrogen receptor subtypes reduces tight junction disruption of brain endothelial cells under ischemia/reperfusion injury.

Ischemic stroke, which induces oxidative stress in the brain, disrupts tight junctions (TJs) between brain endothelial cells, resulting in blood-brain barrier (BBB) breakdown and brain edema. Estrogen reduces oxidative stress and protects brain endothelial cells from ischemic insult. The aim of this study was to determine the protective effects of estrogen on TJ disruption and to examine the roles of classical estrogen receptor (ER) subtypes, ERα- and ERß, in estrogen effects in brain endothelial cells (bEnd.3) exposed to oxygen-glucose deprivation/reperfusion (OGD/R) injury. Estrogen pretreatment prevented OGD/R-induced decreases in cell viability and TJ protein levels. ERα- and ERß-specific agonists also reduced TJ disruption. Knockdown of ERα or ERß expression partially inhibited the effects of estrogen, but completely reversed the effects of corresponding ER subtype-specific agonists on the outcomes of OGD/R. During the early reperfusion period, activation of extracellular signal-regulated kinase1/2 and hypoxia-inducible factor 1α/vascular endothelial growth factor was associated with decreased expression of occludin and claudin-5, respectively, and these changes in TJ protein levels were differentially regulated by ER subtype-specific agonists. Our results suggest that ERα and ERß activation reduce TJ disruption via inhibition of signaling molecules after ischemic injury and that targeting each ER subtype can be a useful strategy for protecting the BBB from ischemic stroke in postmenopausal women.

In vitro Effects of Prostaglandin Analogs on Cultured Astrocytes Obtained from the Lamina Cribrosa.

PURPOSE: To evaluate the effects of prostaglandin analogs (PGAs) on cell viability and apoptosis in cultured astrocytes obtained from the lamina cribrosa (LC) of the human optic nerve head (ONH). METHODS: Astrocytes were cultured from LC samples obtained from human donor ONH and treated with three kinds of acid form of PGAs: latanoprost (LAT-A), tafluprost (TAF-A), and bimatoprost (BIM-A) (0.1, 1, 10, 50 and 100 ug/mL). Cell viability was assessed using the WST-1 assay. Cell apoptosis was measured using the deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay. Apoptotic protein expression was evaluated using western blot analysis. RESULTS: ONH astrocytes expressed FP receptor in western blot analysis. In the presence of 0.1 ug/mL of LAT-A, BIM-A, and TAF-A, the cell viability was 85%, 85% and 82%, respectively. WST-1 assay revealed about 50% of cell viability following treatment with 50 ug/mL of all PGAs. After exposing astrocytes to 10 ug/mL of each PGA for 24 hours, apoptotic cells were stained in TUNEL assay. Western blot analysis revealed that the PGAs up-regulated Bax (pro-apoptotic protein) and down-regulated Bcl-xL (anti-apoptotic protein) in the astrocytes. CONCLUSIONS: PGAs affected cell viability in cultured astrocytes obtained from human ONH LC. PGA treatment may induce apoptosis in ONH astrocytes.

Visceral adipose tissue inflammation is associated with age-related brain changes and ischemic brain damage in aged mice.

Visceral adipose tissue is accumulated with aging. An increase in visceral fat accompanied by low-grade inflammation is associated with several adult-onset diseases. However, the effects of visceral adipose tissue inflammation on the normal and ischemic brains of aged are not clearly defined. To examine the role of visceral adipose tissue inflammation, we evaluated inflammatory cytokines in the serum, visceral adipose tissue, and brain as well as blood-brain barrier (BBB) permeability in aged male mice (20 months) underwent sham or visceral fat removal surgery compared with the young mice (2.5 months). Additionally, ischemic brain injury was compared in young and aged mice with sham and visceral fat removal surgery. Interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α levels in examined organs were increased in aged mice compared with the young mice, and these levels were reduced in the mice with visceral fat removal. Increased BBB permeability with reduced expression of tight junction proteins in aged sham mice were also decreased in mice with visceral fat removal. After focal ischemic injury, aged mice with visceral fat removal showed a reduction in infarct volumes, BBB permeability, and levels of proinflammatory cytokines in the ischemic brain compared with sham mice, although the neurological outcomes were not significantly improved. In addition, further upregulated visceral adipose tissue inflammation in response to ischemic brain injury was attenuated in mice with visceral fat removal. These results suggest that visceral adipose tissue inflammation is associated with age-related changes in the brain and contributes to the ischemic brain damage in the aged mice. We suggest that visceral adiposity should be considered as a factor affecting brain health and ischemic brain damage in the aged population.

Antifibrotic effects of pirfenidone on Tenon's fibroblasts in glaucomatous eyes: comparison with mitomycin C and 5-fluorouracil.

PURPOSE: The purpose of this study was to evaluate the antifibrotic effects of pirfenidone (PFD) on primary cultured human Tenon's fibroblasts (HTFs) from primary open-angle glaucoma (POAG) eyes, compared to mitomicin C (MMC) and 5-fluorouracil (5-FU). MATERIALS AND METHODS: Samples of human Tenon's capsule were obtained during respective surgeries from three groups of patients: patients with cataract (CAT group), patients with POAG who underwent glaucoma filtration surgery (GFS) (POAG1 group), and patients with POAG who underwent GFS due to failed bleb of previous GFS (POAG2 group). Cell toxicity, cell migration, and the expression level of α-smooth muscle actin (α-SMA) protein were evaluated in primary cultured HTFs from the three patient groups after treatment (PFD, MMC, or 5-FU). RESULTS: Overall, cell viability after PFD treatment was higher compared to MMC treatment (82.3 ± 5.1 % vs 56.7 ± 3.8 %; p = 0.001) and comparable to 5-FU treatment (82.3 ± 5.1 % vs 85.7 ± 10.7 %, p = 0.214) at the same concentration (0.4 mg/ml). Both 0.3 mg/ml PFD and 0.1 mg/ml MMC inhibited cell migration compared to control (without treatment) cells (p = 0.014 and 0.005, respectively), while 0.2 mg/ml 5-FU showed the highest degree of cell migration among the three agents in the POAG1 group (PFD vs MMC vs 5-FU; 29.5 ± 2.1 % vs 34.5 ± 0.7 % vs 76.0 ± 8.5 %, PFD vs MMC; p = 1.000, PFD vs 5-FU; p = 0.008, MMC vs 5-FU; p = 0.011). PFD (0.1 or 0.3 mg/ml) and MMC (0.05 and 0.1 mg/ml) treatment significantly reduced the protein expression level of α-SMA in the POAG 1 group (all p < 0.05), and the α-SMA protein level following treatment with 0.3 mg/ml PFD was lower than that of 0.1 mg/ml MMC (p = 0.040). CONCLUSION: PFD showed less cytotoxicity compared to MMC. PFD and MMC inhibited cell migration and reduced α-SMA protein expression levels, while 5-FU showed neither inhibition of cell migration nor reduction in α-SMA expression level. These findings indicate PFD as a potential adjunctive antifibrotic agent to prevent bleb failure during GFS.

Estrogen receptor-mediated resveratrol actions on blood-brain barrier of ovariectomized mice.

To test whether resveratrol provides benefits via estrogen receptors (ERs) in the blood-brain barrier of estrogen-deficient females, ovariectomized mice were treated with resveratrol then were subjected to transient middle cerebral artery occlusion (MCAO). Compared with vehicle treatment, resveratrol reduced infarct volume and neurologic deficits after MCAO. Basal tight junction (TJ) protein levels in the brain were increased by resveratrol. After MCAO, blood-brain barrier breakdown reduced levels of TJ proteins, and induction of HIF-1α and VEGF were attenuated by resveratrol. These effects were reversed by the ERs antagonist, ICI182,780. In mouse brain, endothelial cells (bEnd.3) exposed to hypoxia, resveratrol treatment protected the cells against cytotoxicity, increases of paracellular permeability and changes in levels of TJ protein and HIF-1α/VEGF proteins. These effects were reversed by ICI182,780 but not by specific ERα or ERß antagonists, indicating nonspecific ER mediated effects. Altogether, these results showed that neuroprotective effects of resveratrol in ovariectomized mice were mediated by ERs and associated with tightening of blood-brain barrier, suggesting that resveratrol can be an alternative to estrogens to protect the brains of estrogen-deficient females against ischemic insult.

Extracellular signal-regulated kinase1/2-dependent changes in tight junctions after ischemic preconditioning contributes to tolerance induction after ischemic stroke.

Less disruption of the blood-brain barrier (BBB) after severe ischemic stroke is one of the beneficial outcomes of ischemic preconditioning (IP). However, the effect of IP on tight junctions (TJs), which regulate paracellular permeability of the BBB, is not well understood. In the present study, we examined IP-induced changes in TJs before and after middle cerebral artery occlusion (MCAO) in mice, and the association between changes in TJs and tolerance to a subsequent insult. After IP, we found decreased levels of transmembrane TJ proteins occludin and claudin-5, and widened gaps of TJs with perivascular swelling at the ultrastructural level in the brain. An inflammatory response was also observed. These changes were reversed by inhibition of extracellular signal-regulated kinase1/2 (ERK1/2) via the specific ERK1/2 inhibitor U0126. After MCAO, reduced brain edema and inflammatory responses were associated with altered levels of angiogenic factors and cytokines in preconditioned brains. Pretreatment with U0126 reversed the neuroprotective effects of IP against MCAO. These findings suggest that ERK1/2 activation has a pivotal role in IP-induced changes in TJs and inflammatory response, which serve to protect against BBB breakdown and inflammation after ischemic stroke.

Matrix metalloproteinase-8 plays a pivotal role in neuroinflammation by modulating TNF-α activation.

Matrix metalloproteinases (MMPs) play important roles in normal brain development and synaptic plasticity, although aberrant expression of MMPs leads to brain damage, including blood-brain barrier disruption, inflammation, demyelination, and neuronal cell death. In this article, we report that MMP-8 is upregulated in LPS-stimulated BV2 microglial cells and primary cultured microglia, and treatment of MMP-8 inhibitor (M8I) or MMP-8 short hairpin RNA suppresses proinflammatory molecules, particularly TNF-α secretion. Subsequent experiments showed that MMP-8 exhibits TNF-α-converting enzyme (TACE) activity by cleaving the prodomain of TNF-α (A(74)/Q(75), A(76)/V(77) residues) and, furthermore, that M8I inhibits TACE activity more efficiently than TAPI-0, a general TACE inhibitor. Biochemical analysis of the underlying anti-inflammatory mechanisms of M8I revealed that it inhibits MAPK phosphorylation, NF-κB/AP-1 activity, and reactive oxygen species production. Further support for the proinflammatory role of microglial MMP-8 was obtained from an in vivo animal model of neuroinflammatory disorder. MMP-8 is upregulated in septic conditions, particularly in microglia. Administration of M8I or MMP-8 short hairpin RNA significantly inhibits microglial activation and expression/secretion of TNF-α in brain tissue, serum, and cerebrospinal fluid of LPS-induced septic mice. These results demonstrate that MMP-8 critically mediates microglial activation by modulating TNF-α activity, which may explain neuroinflammation in septic mouse brain.