Inverse relationship between platelet Akt activity and hippocampal atrophy: A pilot case-control study in patients with diabetes mellitus.

BACKGROUND: Akt plays diverse roles in humans. It is involved in the pathogenesis of type 2 diabetes mellitus (T2DM), which is caused by insulin resistance. Akt also plays a vital role in human platelet activation. Furthermore, the hippocampus is closely associated with memory and learning, and a decrease in hippocampal volume is reportedly associated with an insulin-resistant phenotype in T2DM patients without dementia. AIM: To investigate the relationship between Akt phosphorylation in unstimulated platelets and the hippocampal volume in T2DM patients. METHODS: Platelet-rich plasma (PRP) was prepared from the venous blood of patients with T2DM or age-matched controls. The pellet lysate of the centrifuged PRP was subjected to western blotting to analyse the phosphorylation of Akt, p38 mitogen-activated protein (MAP) kinase and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Phosphorylation levels were quantified by densitometric analysis. Hippocampal volume was analysed using a voxel-based specific regional analysis system for Alzheimer's disease on magnetic resonance imaging, which proposes the Z-score as a parameter that reflects hippocampal volume. RESULTS: The levels of phosphorylated Akt corrected with phosphorylated p38 MAP kinase were inversely correlated with the Z-scores in the T2DM subjects, whereas the levels of phosphorylated Akt corrected with GAPDH were not. However, this relationship was not observed in the control patients. CONCLUSION: These results suggest that an inverse relationship may exist between platelet Akt activation and hippocampal atrophy in T2DM patients. Our findings provide insight into the molecular mechanisms underlying T2DM hippocampal atrophy.

RasGRP2 Attenuates Oxygen Deprivation-Induced Autophagy in Vascular Endothelial Cells.

Vascular endothelial cells sustain vascular health through barrier and endocrine functions. Insufficient oxygen supply induces endothelial dysfunction in the pathology of various diseases. In addition, oxygen deprivation reportedly induces endothelial dysfunction via autophagy. Ras guanyl-releasing protein 2 (RasGRP2) has guanosine 5'-diphosphate (GDP)/guanosine 5'-triphosphate (GTP) exchange factor activity and activates Rap1 and R-Ras which belong to the small GTPases. RasGRP2 exerts protective effects against vascular endothelial dysfunction. However, the effect of RasGRP2 on hypoxic stress in vascular endothelial cells has not yet been investigated. We examined the protein expression of hypoxia-inducible factor (HIF)-1α, BCL2 interacting protein 3 (BNIP3), and microtubule-associated protein light chain 3ß (LC3ß). We observed that oxygen deprivation increased the expression of HIF-1α, BNIP3 and LC3ß II. RasGRP2 suppressed the induction of HIF-1α and the subsequent increase in LC3ß II. These findings suggest the possibility that RasGRP2 plays a protective role against endothelial dysfunction by suppressing oxygen deprivation-induced autophagy.

Synergy by Ristocetin and CXCL12 in Human Platelet Activation: Divergent Regulation by Rho/Rho-Kinase and Rac.

CXCL12, belonging to the CXC chemokine family, is a weak agonist of platelet aggregation. We previously reported that the combination of CXCL12 and collagen at low doses synergistically activates platelets via not CXCR7 but CXCR4, a specific receptor for CXCL12 on the plasma membrane. Recently, we reported that not Rho/Rho kinase, but Rac is involved in the platelet aggregation induced by this combination. Ristocetin is an activator of the von Willebrand factor that interacts with glycoprotein (GP) Ib/IX/V, which generates thromboxane A2 via phospholipase A2 activation, resulting in the release of the soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of a combination of ristocetin and CXCL12 at low doses on human platelet activation and its underlying mechanisms. Simultaneous stimulation with ristocetin and CXCL12 at subthreshold doses synergistically induce platelet aggregation. A monoclonal antibody against not CXCR7 but CXCR4 suppressed platelet aggregation induced by the combination of ristocetin and CXCL12 at low doses. This combination induces a transient increase in the levels of both GTP-binding Rho and Rac, followed by an increase in phosphorylated cofilin. The ristocetin and CXCL12-induced platelet aggregation as well as the sCD40L release were remarkably enhanced by Y27362, an inhibitor of Rho-kinase, but reduced by NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction. These results strongly suggest that the combination of ristocetin and CXCL12 at low doses synergistically induces human platelet activation via Rac and that this activation is negatively regulated by the simultaneous activation of Rho/Rho-kinase.

Tramadol regulates the activation of human platelets via Rac but not Rho/Rho-kinase.

Tramadol is a useful analgesic which acts as a serotonin and noradrenaline reuptake inhibitor in addition to µ-opioid receptor agonist. Cytoplasmic serotonin modulates the small GTPase activity through serotonylation, which is closely related to the human platelet activation. We recently reported that the combination of subthreshold collagen and CXCL12 synergistically activates human platelets. We herein investigated the effect and the mechanism of tramadol on the synergistic effect. Tramadol attenuated the synergistically stimulated platelet aggregation (300 µM of tramadol, 64.3% decrease, p<0.05). Not morphine or reboxetine, but duloxetine, fluvoxamine and sertraline attenuated the synergistic effect of the combination on the platelet aggregation (30 µM of fluvoxamine, 67.3% decrease, p<0.05; 30 µM of sertraline, 67.8% decrease, p<0.05). The geranylgeranyltransferase inhibitor GGTI-286 attenuated the aggregation of synergistically stimulated platelet (50 µM of GGTI-286, 80.8% decrease, p<0.05), in which GTP-binding Rac was increased. The Rac1-GEF interaction inhibitor NSC23766 suppressed the platelet activation and the phosphorylation of p38 MAPK and HSP27 induced by the combination of collagen and CXCL12. Tramadol and fluvoxamine almost completely attenuated the levels of GTP-binding Rac and the phosphorylation of both p38 MAPK and HSP27 stimulated by the combination. Suppression of the platelet aggregation after the duloxetine administration was observed in 2 of 5 patients in pain clinic. These results suggest that tramadol negatively regulates the combination of subthreshold collagen and CXCL12-induced platelet activation via Rac upstream of p38 MAPK.

The Height and Mobility of Protruding Plaque After Carotid Artery Stenting Are Associated with Postoperative Ischemic Lesions.

BACKGROUND: Tissue protrusion (TP) is a possible cause of cerebral infarction after carotid artery stenting (CAS). Using optical frequency domain imaging (OFDI) and angioscopy, we investigated the relationship between the morphological features of TP and postoperative new ischemic lesions on magnetic resonance imaging (MRI)-diffusion weighted imaging (DWI) after CAS. METHODS: Fifty patients who underwent CAS and subsequent poststenting intravascular evaluation using both OFDI and angioscopy were included. CAS was performed for proximal protection via the femoral artery approach, and intravascular evaluation with OFDI and angioscopy were performed after stent placement. We compared the background and poststenting intravascular findings between patients with and without postoperative new ischemic lesions on MRI-DWI. RESULTS: TP was observed in 42 patients (84%), and postoperative new ischemic lesions on MRI-DWI were observed in 32 patients (64%). The frequency of TP did not differ between the 2 groups, but the height of TP was higher in the DWI-positive group (0.62 mm vs. 0.29 mm, P = 0.0028), and mobile TP was observed only in the DWI-positive group. The height of TP (P = 0.023) was an independent predictor of new periprocedural ischemic brain lesions after CAS, and its cut-off value for mobility was 0.55 mm on the receiver operating characteristic curve (area under the curve, 0.93). CONCLUSIONS: The height of TP on OFDI and mobile-TP on angioscopy after CAS were associated with postoperative new ischemic lesions on MRI-DWI. The intravascular evaluation using OFDI and angioscopy could be helpful for a detailed evaluation of TP.

Relationship between the Responsiveness of Amyloid ß Protein to Platelet Activation by TRAP Stimulation and Brain Atrophy in Patients with Diabetes Mellitus.

Type 2 DM is a risk factor for dementia, including Alzheimer's disease (AD), and is associated with brain atrophy. Amyloid ß protein (Aß) deposition in the brain parenchyma is implicated in the neurodegeneration that occurs in AD. Platelets, known as abundant storage of Aß, are recognized to play important roles in the onset and progression of AD. We recently showed that Aß negatively regulates platelet activation induced by thrombin receptor-activating protein (TRAP) in healthy people. In the present study, we investigated the effects of Aß on the TRAP-stimulated platelet activation in DM patients, and the relationship between the individual responsiveness to Aß and quantitative findings of MRI, the volume of white matter hyperintensity (WMH)/intracranial volume (IC) and the volume of parenchyma (PAR)/IC. In some DM patients, Aß reduced platelet aggregation induced by TRAP, while in others it was unchanged or rather enhanced. The TRAP-induced levels of phosphorylated-Akt and phosphorylated-HSP27, the levels of PDGF-AB and the released phosphorylated-HSP27 correlated with the degree of platelet aggregability. The individual levels of not WMH/IC but PAR/IC was correlated with those of TRAP-stimulated PDGF-AB release. Collectively, our results suggest that the reactivity of TRAP-stimulated platelet activation to Aß differs in DM patients from healthy people. The anti-suppressive feature of platelet activation to Aß might be protective for brain atrophy in DM patients.

Thrombopoietin and collagen in low doses cooperatively induce human platelet activation.

Aim: In acute medicine, we occasionally treat life-threatening conditions such as sepsis and trauma, which cause severe thrombocytopenia. Serum thrombopoietin levels have been reported to increase under the condition of thrombocytopenia related to severity. Collagen is a crucial activator of platelets, and Rho family members, such as Rho/Rho-kinase and Rac, play roles as active molecules involved in the intracellular signaling pathways in platelet activation. The present study aimed to elucidate the effects of thrombopoietin (TPO) on subthreshold low-dose collagen-stimulated human platelets in terms of Rho/Rho-kinase and Rac. Methods: Platelet-rich plasma donated from healthy volunteers was stimulated by the subthreshold low-dose of collagen after pretreatment with TPO and/or NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction, or Y27632, an inhibitor of Rho-kinase. Platelet aggregation was measured using an aggregometer based on laser-scattering methods. Proteins involved in intracellular signaling were analyzed using western blotting, and the secretion of platelet-derived growth factor-AB from activated platelets was determined using an enzyme-linked immunosorbent assay. Results: Under the existence of TPO, the low dose of collagen remarkably elicited the aggregation and platelet-derived growth factor-AB secretion of platelets, which were suppressed by NSC23766 and Y27632. The combination of TPO and collagen considerably induced a transient increase of guanosine triphosphate (GTP)-binding Rac and GTP-binding Rho followed by an increase of phosphorylated cofilin, a Rho-kinase substrate. Conclusion: These results strongly suggest that TPO and collagen in low doses cooperatively potentiate human platelet activation through both Rac and Rho/Rho-kinase mediated pathways.

SERMs (selective estrogen receptor modulator), acting as estrogen receptor ß agonists in hepatocellular carcinoma cells, inhibit the transforming growth factor-α-induced migration via specific inhibition of AKT signaling pathway.

Selective estrogen receptor modulator (SERM) interacts with estrogen receptors and acts as both an agonist or an antagonist, depending on the target tissue. SERM is widely used as a safer hormone replacement therapeutic medicine for postmenopausal osteoporosis. Regarding hepatocellular carcinoma (HCC), accumulating evidence indicates gender differences in the development, and that men are at higher morbidity risk than premenopausal women, suggesting that estrogen protects against HCC. However, it remains unclear whether SERM affects the HCC progression. Previously, we have shown that transforming growth factor (TGF)-α promotes the migration of HCC cells via p38 mitogen-activated protein kinases (MAPK), c-Jun N-terminal kinase and AKT. In the present study, we investigated whether SERM such as tamoxifen, raloxifene and bazedoxifene, affects the HCC cell migration using human HCC-derived HuH7 cells. Raloxifene and bazedoxifene but not tamoxifen, significantly suppressed the TGF-α-induced HuH7 cell migration. ERB041 and DPN, estrogen receptor (ER) ß agonists, inhibited the TGF-α-induced cell migration whereas PPT, an ERα agonist, did not show the suppressive effect on the cell migration. ERB041 attenuated the TGF-α-induced phosphorylation of AKT without affecting the phosphorylation of p38 MAPK and c-Jun N-terminal kinase. Raloxifene and bazedoxifene also inhibited the phosphorylation of AKT by TGF-α. Furthermore, PHTPP, an ERß antagonist, significantly reversed the suppression by both raloxifene and bazedoxifene of the TGF-α-induced cell migration. Taken together, our results strongly indicate that raloxifene and bazedoxifene, SERMs, suppress the TGF-α-induced migration of HCC cells through ERß-mediated inhibition of the AKT signaling pathway.

A Case of Direct Carotid-Cavernous Fistulae Successfully Treated by Bidirectional Double Catheter Technique: A Technical Note.

Objective: Ruptured carotid-cavernous aneurysms (CCAs) are known to result in direct carotid-cavernous fistula (CCF). Although endovascular treatment is recognized as the first-line treatment for direct CCF, obliteration is sometimes difficult because of the high-flow shunt. In this report, we present a case of a direct CCF treated by the combination of transarterial and transvenous approaches. Case Presentation: A 57-year-old woman presented with conjunctival chemosis, exophthalmos, and tinnitus. Ophthalmological examination revealed increased intraocular pressure. DSA demonstrated a direct CCF due to a right ruptured CCA with retrograde shunted flow through the superior ophthalmic vein (SOV), superficial middle cerebral vein, basal vein of Rosenthal, and middle temporal vein. Two microcatheters were guided into the shunt segment from the internal carotid artery and SOV. In addition, a balloon catheter was placed at the neck of the aneurysm to assist coiling. Coil embolization for the CCF was performed using two microcatheters in the opposite direction, which enabled compact and tight packing of the shunt segment with only six coils. The CCF was eliminated. Two-year-follow-up MRA revealed no recurrence. Conclusion: The bidirectional double catheter technique is a useful approach to obliterate a shunt in a short segment with minimal coils.

Trigeminal Neuralgia Caused by Dural Arteriovenous Fistula of the Transverse-Sigmoid Sinus without Vessel Compression at Root Entry Zone.

Objective: The vessel compression at the root entry zone (REZ) of trigeminal nerve is a common cause of trigeminal neuralgia (TN). We report a rare case of TN caused by dural arteriovenous fistula (DAVF) of the transverse-sigmoid sinus without vessel compression at REZ. Case Presentation: A 45-year-old woman presented with right side tinnitus and was diagnosed as a DAVF of the right transverse-sigmoid sinus (Borden Type I). After that, the facial pain in the right maxillary nerve area appeared and was getting worse. DSA revealed an enlargement of the artery of foramen rotundum (AFR) as one of the feeding arteries. MRI revealed no evidence of vascular compression at REZ. The patient was treated with transarterial embolization (TAE) with Onyx via the branches of the middle meningeal artery (MMA) and occipital artery (OA). The AFR decreased in size and the facial pain was improved. However, the DAVF and the facial pain were recurred. Finally, the DAVF was completely embolized with transvenous embolization (TVE). During 1-year follow-up period, the patient remained free of pain without recurrence. Conclusion: The compression of the maxillary nerve by the AFR might result in TN, because the pain diminished after shrinkage of the AFR by the endovascular treatment.

The Role of RASGRP2 in Vascular Endothelial Cells-A Mini Review.

RAS guanyl nucleotide-releasing proteins (RASGRPs) are important proteins that act as guanine nucleotide exchange factors, which activate small GTPases and function as molecular switches for intracellular signals. The RASGRP family is composed of RASGRP1-4 proteins and activates the small GTPases, RAS and RAP. Among them, RASGRP2 has different characteristics from other RASGRPs in that it targets small GTPases and its localizations are different. Many studies related to RASGRP2 have been reported in cells of the blood cell lineage. Furthermore, RASGRP2 has also been reported to be associated with Huntington's disease, tumors, and rheumatoid arthritis. In addition, we also recently reported RASGRP2 expression in vascular endothelial cells, and clarified the involvement of xenopus Rasgrp2 in the vasculogenesis process and multiple signaling pathways of RASGRP2 in human vascular endothelial cells with stable expression of RASGRP2. Therefore, this article outlines the existing knowledge of RASGRP2 and focuses on its expression and role in vascular endothelial cells, and suggests that RASGRP2 functions as a protective factor for maintaining healthy blood vessels.

Intracellular Toxic AGEs (TAGE) Triggers Numerous Types of Cell Damage.

The habitual intake of large amounts of sugar, which has been implicated in the onset/progression of lifestyle-related diseases (LSRD), induces the excessive production of glyceraldehyde (GA), an intermediate of sugar metabolism, in neuronal cells, hepatocytes, and cardiomyocytes. Reactions between GA and intracellular proteins produce toxic advanced glycation end-products (toxic AGEs, TAGE), the accumulation of which contributes to various diseases, such as Alzheimer's disease, non-alcoholic steatohepatitis, and cardiovascular disease. The cellular leakage of TAGE affects the surrounding cells via the receptor for AGEs (RAGE), thereby promoting the onset/progression of LSRD. We demonstrated that the intracellular accumulation of TAGE triggered numerous cellular disorders, and also that TAGE leaked into the extracellular space, thereby increasing extracellular TAGE levels in circulating fluids. Intracellular signaling and the production of reactive oxygen species are affected by extracellular TAGE and RAGE interactions, which, in turn, facilitate the intracellular generation of TAGE, all of which may contribute to the pathological changes observed in LSRD. In this review, we discuss the relationships between intracellular TAGE levels and numerous types of cell damage. The novel concept of the "TAGE theory" is expected to open new perspectives for research into LSRD.

RasGRP2 inhibits glyceraldehyde-derived toxic advanced glycation end-products from inducing permeability in vascular endothelial cells.

Advanced glycation end-products (AGEs) are formed by the non-enzymatic reaction of sugars and proteins. Among the AGEs, glyceraldehyde-derived toxic AGEs (TAGE) are associated with various diseases, including diabetic complications such as diabetic retinopathy (DR). The risk of developing DR is strongly associated with poor glycemic control, which causes AGE accumulation and increases AGE-induced vascular permeability. We previously reported that Ras guanyl nucleotide releasing protein 2 (RasGRP2), which activates small G proteins, may play an essential role in the cell response to toxicity when exposed to various factors. However, it is not known whether RasGRP2 prevents the adverse effects of TAGE in vascular endothelial cells. This study observed that TAGE enhanced vascular permeability by disrupting adherens junctions and tight junctions via complex signaling, such as ROS and non-ROS pathways. In particular, RasGRP2 protected adherens junction disruption, thereby suppressing vascular hyper-permeability. These results indicate that RasGRP2 is an essential protective factor of vascular permeability and may help develop novel therapeutic strategies for AGE-induced DR.

Suppression of Hepatic Stellate Cell Death by Toxic Advanced Glycation End-Products.

Advanced glycation end-products (AGEs) are produced by the non-enzymatic reaction of sugars with proteins. It has been revealed that glyceraldehyde-derived toxic AGEs (TAGE) are elevated in the serum of non-alcoholic steatohepatitis (NASH) patients. NASH causes liver fibrosis and progresses to cirrhosis and hepatocellular carcinoma. However, the impact of TAGE in liver fibrosis caused by extracellular matrix accumulation remains poorly understood. In this study, we examined the effect of TAGE on the activation of hepatic stellate cells that are involved in liver fibrosis. LX-2 cells treated with transforming growth factor-ß1 (TGF-ß1) significantly reduced cell viability by apoptosis. However, the decrease in cell viability with TGF-ß1 treatment was significantly suppressed by TAGE co-treatment. The levels of α-smooth muscle actin (α-SMA) and platelet-derived growth factor (PDGF)-Rß and its ligand PDGF-B were increased in LX-2 cells following TGF-ß1 treatment, suggesting that these cells were activated; however, these increases were unaffected by TAGE co-treatment. Moreover, collagen I level was increased with TGF-ß1 treatment, and this increase was further increased by TAGE co-treatment. These results suggested that the suppression of apoptosis in activated LX-2 cells by TGF-ß1 and TAGE co-treatment is related to an increase in the production of the extracellular matrix such as collagen I. Therefore, it was suggested that TAGE might aggravate the liver fibrosis of chronic hepatitis, such as NASH.

Alterations in Glucose Metabolism Due to Decreased Expression of Heterogeneous Nuclear Ribonucleoprotein M in Pancreatic Ductal Adenocarcinoma.

The prognosis of pancreatic cancer is considerably worse than that of other cancers, as early detection of pancreatic cancer is difficult and due to its hypovascular environment, which involves low blood flow and a low supply of oxygen and nutrients. Moreover, pancreatic cancer demonstrates a mechanism that allows it to survive in a hypovascular environment. However, the detailed mechanism remains elusive. Recently, it has been reported that heterogeneous ribonuclear protein M (HNRNPM) is a splicing factor associated with malignant tumors. Thus, in this study, we investigated the expression and effects of HNRNPM in pancreatic ductal adenocarcinoma (PDA). We observed that HNRNPM expression, which is highly expressed in pancreatic tissues, was reduced in PDA tissues. Additionally, knockdown of HNRNPM under low-glucose conditions that mimic a hypovascular environment was shown to alter glucose metabolism and prolong cell survival by suppressing glucose consumption. These results suggest that the decreased expression of HNRNPM in PDA may be involved in its adaptation to a hypovascular environment.

The inhibition of Bax activation-induced apoptosis by RasGRP2 via R-Ras-PI3K-Akt signaling pathway in the endothelial cells.

Apoptosis of endothelial cells is a very important event in various diseases and angiogenesis. We recently reported that ras guanyl nucleotide releasing protein 2 (RasGRP2), which is a guanine nucleotide exchange factor, was expressed in the human umbilical vein endothelial cells (HUVECs) and that Rap1 activation by its overexpression inhibited apoptosis by suppressing tumor necrosis factor-α induced-reactive oxygen species (ROS) production. However, other signaling pathways and roles of RasGRP2 not mediated via Rap1 are not well understood. Therefore, we compared the Mock (M) and the RasGRP2-stable overexpression (R) immortalized HUVECs using BAM7 and anisomycin, which are apoptosis inducers. BAM7 and anisomycin induced apoptosis without causing ROS production, and such apoptosis was significantly increased in M cells, but not in R cells. RasGRP2 suppressed BAM7- and anisomycin-induced apoptosis, but not via the Rap1 pathway as observed using Rap1 knockdown. Furthermore, RasGRP2 activated not only Rap1 but also R-Ras, and suppressed apoptosis by activating R-Ras-phosphoinositide 3-kinase (PI3K)-Akt signaling pathway. The phosphorylation of Akt by RasGRP2 inhibited Bax translocation by promoting translocation of hexokinase-2 (HK-2) from cytoplasm to mitochondria. Taken together, it was suggested that RasGRP2 suppresses the Bax activation-induced apoptosis by promoting HK-2 translocation to mitochondria via R-Ras-PI3K-Akt signaling pathway.

RASGRP2 Suppresses Apoptosis via Inhibition of ROS Production in Vascular Endothelial Cells.

We have identified ras guanyl releasing protein 2 (rasgrp2) as a blood vessel related gene from Xenopus embryo. In addition, we reported that RASGRP2 is also expressed in human umbilical vein endothelial cells (HUVEC). It is known that RASGRP2 activates Ras-related protein 1 (Rap1). However, the function of RASGRP2 in human vascular endothelium remains unknown. Therefore, we performed functional analysis of RASGRP2 using immortalized HUVEC (TERT HUVEC). We established a stable RASGRP2 overexpressing cell line (TERT HUVEC R) and mock cell line (mock). Furthermore, we compared the activity of Rap1 and the generation of intracellular reactive oxygen species (ROS), which is related to cell death, in both cell lines. Significant increase in Rap1 activity was observed in the TERT HUVEC R compared to the mock. Furthermore, apoptosis by tumor necrosis factor-α (TNF-α) stimulation was significantly more reduced in the TERT HUVEC R than in the mock. In the mock, apoptosis induced by TNF-α stimulation was decreased by pretreatment with diphenyleneiodonium (DPI), which is an inhibitor of NADPH oxidase (NOX). However, in the TERT HUVEC R, apoptosis induced by TNF-α stimulation was not reduced after pretreatment of DPI. Furthermore, there was no reduction in ROS production in the TERT HUVEC R after DPI pretreatment. In addition, the difference in the degree of apoptosis induced by TNF-α stimulation in both cell lines was consistent with the difference in ROS production in the cell lines. From these results, it was suggested that RASGRP2 activates Rap1 and the activated Rap1 suppresses apoptosis via NOX inhibition.

Contribution of the toxic advanced glycation end-products-receptor axis in nonalcoholic steatohepatitis-related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. The main etiologies of HCC are hepatitis B virus and hepatitis C virus (HCV), and non-hepatitis B/non-hepatitis C HCC (NBNC-HCC) has also been identified as an etiological factor. Although the incidence of HCV-related HCC in Japan has decreased slightly in recent years, that of NBNC-HCC has increased. The onset mechanism of NBNC-HCC, which has various etiologies, remains unclear; however, nonalcoholic steatohepatitis (NASH), a severe form of nonalcoholic fatty liver disease, is known to be an important risk factor for NBNC-HCC. Among the different advanced glycation end-products (AGEs) formed by the Maillard reaction, glyceraldehyde-derived AGEs, the predominant components of toxic AGEs (TAGE), have been associated with NASH and NBNC-HCC, including NASH-related HCC. Furthermore, the expression of the receptor for AGEs (RAGE) has been correlated with the malignant progression of HCC. Therefore, TAGE induce oxidative stress by binding with RAGE may, in turn, lead to adverse effects, such as fibrosis and malignant transformation, in hepatic stellate cells and tumor cells during NASH or NASH-related HCC progression. The aim of this review was to examine the contribution of the TAGE-RAGE axis in NASH-related HCC.

In vitro identification of nonalcoholic fatty liver disease-related protein hnRNPM.

AIM: To study the formation of intracellular glyceraldehyde-derived advanced glycation end products (Glycer-AGEs) in the presence of high concentrations of fructose. METHODS: Cells of the human hepatocyte cell line Hep3B were incubated with or without fructose for five days, and the corresponding cell lysates were separated by two-dimensional gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Glycer-AGEs were detected with the anti-Glycer-AGEs antibody. Furthermore, the identification of the proteins that are modified by glyceraldehyde in the presence of high concentrations of fructose was conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The protein and mRNA levels were determined by Western blotting and real-time reverse transcription PCR, respectively. RESULTS: The results of the two-dimensional gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a greater amount of Glycer-AGEs in the sample exposed to high concentrations of fructose than in the control. The detected Glycer-AGEs showed isoelectric points in the range of 8.0-9.0 and molecular weights in the range of 60-80 kDa. The heterogeneous nuclear ribonucleoprotein M (hnRNPM), which plays an important role in regulating gene expression by processing heterogeneous nuclear RNAs to form mature mRNAs, was identified as a modified protein using MALDI-TOF-MS. Increasing the concentration of fructose in the medium induced a concentration-dependent increase in the generated Glycer-AGEs. Furthermore, in an experiment using glyceraldehyde, which is a precursor of Glycer-AGEs, hnRNPM was found to be more easily glycated than the other proteins. CONCLUSION: The results suggest that glyceraldehyde-modified hnRNPM alters gene expression. This change may cause adverse effects in hepatocytes and may serve as a target for therapeutic intervention.

[Polyethylene glycol accelerates loop-mediated isothermal amplification (LAMP) reaction].

Loop-mediated isothermal amplification (LAMP) has several advantages: this technique involves gene amplification under isothermal conditions using only one high-specificity enzyme; the amplification efficiency is so high that large quantities of pyrophosphoric acid are formed as a by-product of DNA synthesis; furthermore, the results can be judged directly on the basis of turbidity. On the other hand, a PCR requires approximately 3.5-4.0 hours. The LAMP method is faster than the PCR method and is also relatively inexpensive. In the present study, we modified the composition of the reaction solutions to reduce the LAMP reaction time; more specifically, a thickener, either polyethylene glycol 8000 or 20000, was added. These results showed that the LAMP method was faster than the original method, and it is able to detect both turbidity and fluorescence. In conclusion, the LAMP reaction could be performed within 20 minutes when reaction mixture supplemented with a thickener was used. This method can be used for tests in various fields such as the diagnosis of hereditary disease and identification of viral infections as point-of-care testing.