Cilostazol improves the prognosis after hepatectomy in rats with sinusoidal obstruction syndrome.

BACKGROUND AND AIM: Safe radical hepatectomy is important for patients with colorectal liver metastases complicated by sinusoidal obstruction syndrome (SOS) after oxaliplatin-based chemotherapy. This study aimed to investigate the impact of preoperative administration of cilostazol (CZ), an oral selective phosphodiesterase III inhibitor, on hepatectomy in rat SOS model. MATERIAL AND METHODS: Rats were divided into NL (normal liver), SOS (monocrotaline [MCT]-treated), and SOS + CZ (MCT + CZ-treated) groups. MCT or CZ was administered orally, and a 30% partial hepatectomy was performed 48 h after MCT administration. Postoperative survival rates were evaluated (n = 9, for each). Other rats were sacrificed on postoperative days (POD) 1 and 3 and evaluated histologically, immunohistochemically, biochemically, and using transmission electron microscopy (TEM), focusing particularly on SOS findings, liver damage, and liver sinusoidal endothelial cell (LSEC) injury. RESULTS: The cumulative 10-day postoperative survival rate was significantly higher in the SOS + CZ group than in the SOS group (88.9% vs 33.3%, P = 0.001). Total SOS scores were significantly lower in the SOS + CZ group than in the SOS group on both POD 1 and 3. Serum biochemistry and immunohistochemistry showed that CZ reduced liver damage after hepatectomy. TEM revealed that LSECs were significantly preserved morphologically in the SOS + CZ group than in the SOS group on POD 1 (86.1 ± 8.2% vs 63.8 ± 9.3%, P = 0.003). CONCLUSION: Preoperative CZ administration reduced liver injury by protecting LSECs and improved the prognosis after hepatectomy in rats with SOS.

Assaying ADAMTS13 Activity as a Potential Prognostic Biomarker for Sinusoidal Obstruction Syndrome in Mice.

Sinusoidal obstruction syndrome (SOS) is a serious liver disorder that occurs after liver transplantation, hematopoietic stem cell transplantation, and the administration of anticancer drugs. Since SOS is a life-threatening condition that can progress to liver failure, early detection and prompt treatment are required for the survival of patients with this condition. In this study, female CD1 mice were divided into treatment and control groups after the induction of an SOS model using monocrotaline (MCT, 270 mg/kg body weight intraperitoneally). The mice were analyzed at 0, 12, 24, and 48 h after MCT administration, and blood and liver samples were collected for assays and histopathology tests. SOS was observed in the livers 12 h after MCT injection. In addition, immunohistochemical findings demonstrated CD42b-positive platelet aggregations, positive signals for von Willebrand factor (VWF), and a disintegrin-like metalloproteinase with thrombospondin type 1 motifs 13 (ADAMTS13) in the MCT-exposed liver sinusoid. Although ADAMTS13's plasma concentrations peaked at 12 h, its enzyme activity continuously decreased by 75% at 48 h and, inversely and proportionally, concentrations in the VWF-A2 domain, in which the cleavage site of ADAMTS13 is located, increased after MCT injection. These findings suggest that the plasma concentration and activity of ADAMTS13 could be useful biomarkers for early detection and therapeutic intervention in patients with SOS.

A nonsteroidal anti-inflammatory drug, zaltoprofen, inhibits the growth of extraskeletal chondrosarcoma cells by inducing PPARγ, p21, p27, and p53.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor and master transcription factor of adipogenesis-related genes, and has been reported as an antitumor target for chondrosarcomas. Herein, we show that the nonsteroidal anti-inflammatory drug, zaltoprofen, induces the expression of PPARγ at the mRNA and protein levels, following the induction of PPARγ-activating factors, such as Krox20, C/EBPß, and C/EBPα, in human extraskeletal chondrosarcoma H-EMC-SS cells. Upregulation of the cell cycle checkpoint proteins, p21, p27, and p53, was observed upon treatment of H-EMC-SS cells with zaltoprofen, which probably resulted in the inhibition of proliferation of these cells observed in vitro. Zaltoprofen treatment inhibited tumor growth, induced tumor cell apoptosis, and was well tolerated in a mouse model of extraskeletal myxoid chondrosarcoma. Our results provide mechanistic insights into the therapeutic effect of zaltoprofen that should promote further studies on the rational use of this drug for the effective treatment of sarcomas.

Hypoxia-Induced CD36 Expression in Gastric Cancer Cells Promotes Peritoneal Metastasis via Fatty Acid Uptake.

BACKGROUND: The lipid scavenger receptor cluster of differentiation 36 (CD36) has been shown to have a pro-metastatic function in several cancers. Adipose tissue, a favorable site for peritoneal metastasis (PM) from gastric cancer (GC), promotes this process by providing free fatty acids (FFAs); however, the role of CD36 in PM progression from GC remains to be elucidated. MATERIALS AND METHODS: We evaluated CD36 expression in the GC cells under various conditions. CD36 overexpressing (CD36OE) MKN45 cells were prepared and their migration and invasive properties were assessed. A PM mouse model was used to investigate the biological effects of palmitic acid (PA) and CD36. Furthermore, we examined the clinical role of CD36 expression in 82 human PM samples by immunohistochemical staining. RESULTS: Hypoxia markedly increased CD36 expression in GC cells. In normoxia, only CD36OE MKN45 cells treated with PA showed an increase in migration and invasion abilities. An increased expression of active Rac1 and Cdc42 was observed, which decreased following etomoxir treatment. Conversely, hypoxia increased those capacities of both vector and CD36OE MKN45 cells. In a mouse model transplanted with CD36OE MKN45 cells, more peritoneal tumors were observed in the high-fat diet group than those in the normal diet group. In clinical samples, 80% of PM lesions expressed CD36, consistent with hypoxic regions, indicating a significant association with prognosis. CONCLUSION: Our findings indicate that a hypoxia in the peritoneal cavity induces CD36 expression in GC cells, which contributes to PM through the uptake of FFAs.

Oxytocin Dynamics in the Body and Brain Regulated by the Receptor for Advanced Glycation End-Products, CD38, CD157, and Nicotinamide Riboside.

Investigating the neurocircuit and synaptic sites of action of oxytocin (OT) in the brain is critical to the role of OT in social memory and behavior. To the same degree, it is important to understand how OT is transported to the brain from the peripheral circulation. To date, of these, many studies provide evidence that CD38, CD157, and receptor for advanced glycation end-products (RAGE) act as regulators of OT concentrations in the brain and blood. It has been shown that RAGE facilitates the uptake of OT in mother's milk from the digestive tract to the cell surface of intestinal epithelial cells to the body fluid and subsequently into circulation in male mice. RAGE has been shown to recruit circulatory OT into the brain from blood at the endothelial cell surface of neurovascular units. Therefore, it can be said that extracellular OT concentrations in the brain (hypothalamus) could be determined by the transport of OT by RAGE from the circulation and release of OT from oxytocinergic neurons by CD38 and CD157 in mice. In addition, it has recently been found that gavage application of a precursor of nicotinamide adenine dinucleotide, nicotinamide riboside, for 12 days can increase brain OT in mice. Here, we review the evaluation of the new concept that RAGE is involved in the regulation of OT dynamics at the interface between the brain, blood, and intestine in the living body, mainly by summarizing our recent results due to the limited number of publications on related topics. And we also review other possible routes of OT recruitment to the brain.

Inhibitory Effects of Saururus chinensis Extract on Receptor for Advanced Glycation End-Products-Dependent Inflammation and Diabetes-Induced Dysregulation of Vasodilation.

Advanced glycation end-products (AGEs) and the receptor for AGEs (RAGE) are implicated in inflammatory reactions and vascular complications in diabetes. Signaling pathways downstream of RAGE are involved in NF-κB activation. In this study, we examined whether ethanol extracts of Saururus chinensis (Lour.) Baill. (SE) could affect RAGE signaling and vascular relaxation in streptozotocin (STZ)-induced diabetic rats. Treatment with SE inhibited AGEs-modified bovine serum albumin (AGEs-BSA)-elicited activation of NF-κB and could compete with AGEs-BSA binding to RAGE in a dose-dependent manner. Tumor necrosis factor-α (TNF-α) secretion induced by lipopolysaccharide (LPS)-a RAGE ligand-was also reduced by SE treatment in wild-type Ager+/+ mice as well as in cultured peritoneal macrophages from Ager+/+ mice but not in Ager-/- mice. SE administration significantly ameliorated diabetes-related dysregulation of acetylcholine-mediated vascular relaxation in STZ-induced diabetic rats. These results suggest that SE would inhibit RAGE signaling and would be useful for the improvement of vascular endothelial dysfunction in diabetes.

Thrombopoietin accumulation in hepatocytes induces a decrease in its serum levels in a sinusoidal obstruction syndrome model.

Sinusoidal obstruction syndrome (SOS) is a type of fatal hepatic injury, which predominantly occurs following exposure to drugs, such as oxaliplatin, or bone marrow transplantation. Extravasated platelet aggregation (EPA) plays an important role in the development of SOS in rat and mouse models. Furthermore, platelets invading the space of Disse adhere to hepatocytes and are phagocytized in patients with SOS. Aging platelets and platelets in patients with sepsis are phagocytized by hepatocytes through Ashwell­Morell receptors, and thrombopoietin (TPO) is produced by the JAK2­STAT3 signaling pathway. The purpose of the present study was to examine the significance of TPO as a biomarker of SOS. SOS was induced in Crl:CD1(ICR) female mice by intraperitoneal administration of monocrotaline (MCT). TPO levels were measured in the serum and liver tissue. Pathological and immunohistochemical studies of the liver were performed to analyze the expression levels of TPO. TPO mRNA expression levels were measured using reverse transcription­quantitative PCR. In the SOS model, the platelet counts in peripheral blood samples were significantly decreased at 24 and 48 h after MCT treatment as compared with that at 0 h. In addition, a pathological change in hepatic zone 3 was observed in the SOS model group. Furthermore, the protein levels of TPO in liver tissue were significantly increased in the SOS model group compared with those in the control group, which was confirmed by immunohistochemistry. By contrast, serum TPO protein levels were significantly decreased in the SOS model group compared with those in the control group. These results indicated that EPA may induce sinusoidal endothelial fenestration in a mouse model of SOS, preventing TPO from translocating into the blood. In conclusion, serum TPO levels may be reduced in a mouse model of SOS owing to the accumulation in hepatocytes, suggesting that TPO could be a useful biomarker of SOS.

Dual Nature of RAGE in Host Reaction and Nurturing the Mother-Infant Bond.

Non-enzymatic glycation is an unavoidable reaction that occurs across biological taxa. The final products of this irreversible reaction are called advanced glycation end-products (AGEs). The endogenously formed AGEs are known to be bioactive and detrimental to human health. Additionally, exogenous food-derived AGEs are debated to contribute to the development of aging and various diseases. Receptor for AGEs (RAGE) is widely known to elicit biological reactions. The binding of RAGE to other ligands (e.g., high mobility group box 1, S100 proteins, lipopolysaccharides, and amyloid-ß) can result in pathological processes via the activation of intracellular RAGE signaling pathways, including inflammation, diabetes, aging, cancer growth, and metastasis. RAGE is now recognized as a pattern-recognition receptor. All mammals have RAGE homologs; however, other vertebrates, such as birds, amphibians, fish, and reptiles, do not have RAGE at the genomic level. This evidence from an evolutionary perspective allows us to understand why mammals require RAGE. In this review, we provide an overview of the scientific knowledge about the role of RAGE in physiological and pathological processes. In particular, we focus on (1) RAGE biology, (2) the role of RAGE in physiological and pathophysiological processes, (3) RAGE isoforms, including full-length membrane-bound RAGE (mRAGE), and the soluble forms of RAGE (sRAGE), which comprise endogenous secretory RAGE (esRAGE) and an ectodomain-shed form of RAGE, and (4) oxytocin transporters in the brain and intestine, which are important for maternal bonding and social behaviors.

Use of Cocultures to Measure the Blood-Brain Barrier Permeability of Oxytocin.

Primary monkey brain capillary endothelial cell cultures, with rat pericytes and astrocytes, provide an assay system for predicting the ability of oxytocin (OT) to cross the blood-brain barrier (BBB), using a commercially available in vitro BBB kit. The integrity of the in vitro "BBB," which has a high transendothelial electrical resistance (TEER), can be established approximately 4 days after preparations for experiments. Dominant endothelial transport of OT is from the upper (luminal blood side) to lower (abluminal brain side) chambers, dose-dependently. OT is transported by the receptor for advanced glycation end-products (RAGE) in endothelial cells, which is evidenced using the RAGE knockdown system with short hairpin RNA (shRNA) treatment. This in vitro assay system is useful for further assessment of OT transport across the BBB.

Dynamic switch of immunity and antitumor effects of metformin in rat spontaneous esophageal carcinogenesis.

Chronic inflammation contributes to tumor development by creating a local microenvironment that facilitates neoplastic transformation and potentiates the progression of cancer. Esophageal cancer (EC) is an inflammation-associated malignancy with a poor prognosis. The nature of the switch between chronic inflammation of the esophagus and EC-related immunological changes remains unclear. Here, we examined the dynamic alterations of immune cells at different stages of chronic esophagitis, Barrett's esophagus (BE) and EC using an esophageal spontaneous carcinogenesis rat model. We also investigated the anticancer effects of metformin. To stimulate EC carcinogenesis, chronic gastroduodenal reflux esophagitis via esophagojejunostomy was induced in 120 rats in metformin-treated and non-treated (control) groups. After 40 weeks, BE and EC developed in 96.7% and 63.3% of the control group, and in 66.7% and 23.3% of the metformin-treated group, respectively. Flow cytometric analysis demonstrated that the balance of M1/M2-polarized or phospho-Stat3-positive macrophages, regulatory T, cytotoxic T, natural killer (NK), NK T cells, and Th17 T cells was dynamically changed at each stage of the disease and were resolved by metformin treatment. These findings clarify the immunity in esophageal carcinogenesis and suggest that metformin could suppress this disease by improving the immunosuppressive tumor microenvironment and immune evasion.

Soluble receptor for advanced glycation end products protects from ischemia- and reperfusion-induced acute kidney injury.

The full-length receptor for advanced glycation end products (RAGE) is a multiligand pattern recognition receptor. High-mobility group box 1 (HMGB1) is a RAGE ligand of damage-associated molecular patterns that elicits inflammatory reactions. The shedded isoform of RAGE and endogenous secretory RAGE (esRAGE), a splice variant, are soluble isoforms (sRAGE) that act as organ-protective decoys. However, the pathophysiologic roles of RAGE/sRAGE in acute kidney injury (AKI) remain unclear. We found that AKI was more severe, with enhanced renal tubular damage, macrophage infiltration, and fibrosis, in mice lacking both RAGE and sRAGE than in wild-type (WT) control mice. Using murine tubular epithelial cells (TECs), we demonstrated that hypoxia upregulated messenger RNA (mRNA) expression of HMGB1 and tumor necrosis factor α (TNF-α), whereas RAGE and esRAGE expressions were paradoxically decreased. Moreover, the addition of recombinant sRAGE canceled hypoxia-induced inflammation and promoted cell viability in cultured TECs. sRAGE administration prevented renal tubular damage in models of ischemia/reperfusion-induced AKI and of anti-glomerular basement membrane (anti-GBM) glomerulonephritis. These results suggest that sRAGE is a novel therapeutic option for AKI.

RAGE signaling antagonist suppresses mouse macrophage foam cell formation.

The engagement of the receptor for advanced glycation end-products (receptor for AGEs, RAGE) with diverse ligands could elicit chronic vascular inflammation, such as atherosclerosis. Binding of cytoplasmic tail RAGE (ctRAGE) to diaphanous-related formin 1 (Diaph1) is known to yield RAGE intracellular signal transduction and subsequent cellular responses. However, the effectiveness of an inhibitor of the ctRAGE/Diaph1 interaction in attenuating the development of atherosclerosis is unclear. In this study, using macrophages from Ager+/+ and Ager-/- mice, we validated the effects of an inhibitor on AGEs-RAGE-induced foam cell formation. The inhibitor significantly suppressed AGEs-RAGE-evoked Rac1 activity, cell invasion, and uptake of oxidized low-density lipoprotein, as well as AGEs-induced NF-κB activation and upregulation of proinflammatory gene expression. Moreover, expression of Il-10, an anti-inflammatory gene, was restored by this antagonist. These findings suggest that the RAGE-Diaph1 inhibitor could be a potential therapeutic drug against RAGE-related diseases, such as chronic inflammation and atherosclerosis.

Receptor for advanced glycation end-products (RAGE) plays a critical role in retrieval behavior of mother mice at early postpartum.

Receptor for advanced glycation end-products (RAGE) is a pattern recognition molecule belonging to the immunoglobulin superfamily, and it plays a role in the remodeling of endothelial cells under pathological conditions. Recently, it was shown that RAGE is a binding protein for oxytocin (OT) and a transporter of OT to the brain on neurovascular endothelial cells via blood circulation. Deletion of the mouse RAGE gene, Ager (RAGE KO), induces hyperactivity in male mice. Impairment of pup care by mother RAGE KO mice after stress exposure results in the death of neonates 1-2 days after pup birth. Therefore, to understand the role of RAGE during the postpartum period, this study aims to examine parental behavior in female RAGE KO mice and ultrasonic vocalizations in pups. RAGE KO mothers without stress before delivery raised their pups and displayed hyperactivity at postpartum day (PPD) 3. KO dams showed impaired retrieval or interaction behavior after additional stress, such as body restraint stress or exposure to a novel environment, but such impaired behavior disappeared at PPD 7. Postnatal day 3 pups emitted ultrasonic vocalizations at >60 kHz as a part of the mother-pup relationship, but the number and category of calls by RAGE KO pups were significantly lower than wild-type pups. The results indicate that RAGE is important in the manifestation of normal parental behavior in dams and for receiving maternal care by mouse pups; moreover, brain OT recruited by RAGE plays a role in damping of signals of additional external stress and endogenous stress during the early postpartum period. Thus, RAGE-dependent OT may be critical for initiating and maintaining the normal mother-child relationship.

Transport of oxytocin to the brain after peripheral administration by membrane-bound or soluble forms of receptors for advanced glycation end-products.

Oxytocin (OT) is a neuropeptide hormone. Single and repetitive administration of OT increases social interaction and maternal behaviour in humans and mammals. Recently, it was found that the receptor for advanced glycation end-products (RAGE) is an OT-binding protein and plays a critical role in the uptake of OT to the brain after peripheral OT administration. Here, we address some unanswered questions on RAGE-dependent OT transport. First, we found that, after intranasal OT administration, the OT concentration increased in the extracellular space of the medial prefrontal cortex (mPFC) of wild-type male mice, as measured by push-pull microperfusion. No increase of OT in the mPFC was observed in RAGE knockout male mice. Second, in a reconstituted in vitro blood-brain barrier system, inclusion of the soluble form of RAGE (endogenous secretory RAGE [esRAGE]), an alternative splicing variant, in the luminal (blood) side had no effect on the transport of OT to the abluminal (brain) chamber. Third, OT concentrations in the cerebrospinal fluid after i.p. OT injection were slightly higher in male mice overexpressing esRAGE (esRAGE transgenic) compared to those in wild-type male mice, although this did not reach statistical significance. Although more extensive confirmation is necessary because of the small number of experiments in the present study, the reported data support the hypothesis that RAGE may be involved in the transport of OT to the mPFC from the circulation. These results suggest that the soluble form of RAGE in the plasma does not function as a decoy in vitro.

Glycation reaction and the role of the receptor for advanced glycation end-products in immunity and social behavior.

The receptor for advanced glycation end-products (receptor for AGEs, RAGE) is a pattern recognition receptor. The interaction of RAGE with its ligands, such as AGEs, S100 proteins, high mobility group box-1 (HMGB1), and lipopolysaccharides (LPS), is known to play a pivotal role in the propagation of immune responses and inflammatory reactions. The ligand-RAGE interaction elicits cellular responses, for example, in myeloid and lymphoid cells, through distinct pathways by activating NF-κB and Rac1/cdc42, which lead to cytokine production, cell migration, phagocytosis, maturation, and polarization. Recently, oxytocin, a peptide hormone and neuropeptide, was identified as a novel binding molecule for the RAGE; however, it cannot compete with the interaction of RAGE with other ligands or induce RAGE intracellular signaling. The RAGE transports oxytocin from the blood into the brain and regulates brain functions. In this review, we summarize the current understanding of glycation reaction, AGEs, and the RAGE-mediated biological responses as well as the physiological role of RAGE in immunity and social behaviors, particularly, maternal bonding.

Neuroprotective Effects of Endogenous Secretory Receptor for Advanced Glycation End-products in Brain Ischemia.

The receptor for advanced glycation end-products (RAGE) is expressed on human brain endothelial cells (HBEC) and is implicated in neuronal cell death after ischemia. We report that endogenous secretory RAGE (esRAGE) is a splicing variant form of RAGE that functions as a decoy against ischemia-induced neuronal cell damage. This study demonstrated that esRAGE was associated with heparan sulphate proteoglycans on HBEC. The parabiotic experiments between human esRAGE overexpressing transgenic (Tg), RAGE knockout (KO), and wild-type (WT) mice revealed a significant neuronal cell damage in the CA1 region of the WT side of parabiotic WT→WT mice, but not of Tg→WT mice, 7 days after bilateral common carotid artery occlusion. Human esRAGE was detected around the CA1 neurons in the WT side of the parabiotic Tg→WT pair, but not in the KO side of the Tg→KO pair. To elucidate the dynamic transfer of esRAGE into the brain, we used the blood-brain barrier (BBB) system (PharmaCo-Cell) with or without RAGE knockdown in endothelial cells. A RAGE-dependent transfer of esRAGE was demonstrated from the vascular to the brain side. These findings suggested that esRAGE is associated with heparan sulphate proteoglycans and is transferred into the brain via BBB to exert its neuroprotective effects in ischemia.

Aquaporin 1 elicits cell motility and coordinates vascular bed formation by downregulating thrombospondin type-1 domain-containing 7A in glioblastoma.

BACKGROUND: Aquaporin (AQP) 1 expression has been linked with tumor malignancy but its role in glioblastoma (GBM), a lethal glioma, remains to be clarified. METHODS: AQP1 expression was examined in 33 human GBM specimens by immunohistochemistry. GBM cells (U251 and U87) that stably express AQP1 were established and used for cellular proliferation, migration, invasion, and vascular tube formation assays. The GeneChip assay was used to identify differentially expressed genes in AQP1-expressing cells. RESULTS: AQP1 was expressed only in tumor cells. AQP1 dose-dependently accelerated cell migration and invasion, but not proliferation, in GBM cell lines. AQP1 also upregulated cathepsin B, focal adhesion kinase and activities of matrix metalloproteinase 9. AQP1 in GBM cells induced wall thickness of ECV304, vascular endothelial cells, in a contact-dependent manner. Downregulation of thrombospondin type 1 domain containing 7A (THSD7A) was identified in AQP1-expressing GBM cells in vitro, and was negatively correlated with AQP1 expression in human GBM specimens. CONCLUSION: AQP1 is involved in tumor malignancy by facilitating the migration and invasion of GBM cells, and promoting the formation of vascular beds that are characteristic of GBM by downregulating THSD7A.

CD38, CD157, and RAGE as Molecular Determinants for Social Behavior.

Recent studies provide evidence to support that cluster of differentiation 38 (CD38) and CD157 meaningfully act in the brain as neuroregulators. They primarily affect social behaviors. Social behaviors are impaired in Cd38 and Cd157 knockout mice. Single-nucleotide polymorphisms of the CD38 and CD157/BST1 genes are associated with multiple neurological and psychiatric conditions, including autism spectrum disorder, Parkinson's disease, and schizophrenia. In addition, both antigens are related to infectious and immunoregulational processes. The most important clues to demonstrate how these molecules play a role in the brain are oxytocin (OT) and the OT system. OT is axo-dendritically secreted into the brain from OT-containing neurons and causes activation of OT receptors mainly on hypothalamic neurons. Here, we overview the CD38/CD157-dependent OT release mechanism as the initiation step for social behavior. The receptor for advanced glycation end-products (RAGE) is a newly identified molecule as an OT binding protein and serves as a transporter of OT to the brain, crossing over the blood-brain barrier, resulting in the regulation of brain OT levels. We point out new roles of CD38 and CD157 during neuronal development and aging in relation to nicotinamide adenine dinucleotide+ levels in embryonic and adult nervous systems. Finally, we discuss how CD38, CD157, and RAGE are crucial for social recognition and behavior in daily life.