Background & Aims Despite high morbidity and mortality of alcoholic liver disease worldwide, the molecular mechanisms underlying alcohol-induced liver cell death are not fully understood. Transglutaminase 2 (TG2) is a cross-linking enzyme implicated in apoptosis. TG2 levels and activity are increased in association with various types of liver injury. However, how TG2 induces hepatic apoptosis is not known. Methods Human hepatic cells or primary hepatocytes from rats or TG2+/+ and TG2−/− mice were treated with ethanol. Mice were administered anti-Fas antibody or alcohol. Liver sections were prepared from patients with alcoholic steatohepatitis. Changes in TG2 levels, Sp1 cross-linking and its activities, expression of hepatocyte growth factor receptor, c-Met, and hepatic apoptosis were measured. Results Ethanol induced apoptosis in hepatic cells, enhanced activity and nuclear accumulation of TG2 as well as accumulation of cross-linked and inactivated Sp1, and reduced expression of the Sp1-responsive gene, c-Met. These effects were rescued by TG2 knockdown, restoration of functional Sp1, or addition of hepatocyte growth factor, whereas apoptosis was reproduced by Sp1 knockdown or TG2 overexpression. Compared with TG2+/+ mice, TG2−/− mice showed markedly reduced hepatocyte apoptosis and Sp1 cross-linking following ethanol or anti-Fas treatment. Treatment of TG2+/+ mice with the TG2 inhibitors putrescine or cystamine blocked anti-Fas–induced hepatic apoptosis and Sp1 silencing. Moreover, enhanced expression of cross-linked Sp1 and TG2 was evident in hepatocyte nuclei of patients with alcoholic steatohepatitis. Conclusions TG2 induces hepatocyte apoptosis via Sp1 cross-linking and inactivation, with resultant inhibition of the expression of c-Met required for hepatic cell viability.

Accentuated sympathetic nerve activity (SNA) is a risk factor for cardiovascular events. In this review, we investigate our working hypothesis that potentiated activity of neurons in the rostral ventrolateral medulla (RVLM) is the primary cause of experimental and essential hypertension. Over the past decade, we have examined how RVLM neurons regulate peripheral SNA, how the sympathetic and renin-angiotensin systems are correlated and how the sympathetic system can be suppressed to prevent cardiovascular events in patients. Based on results of whole-cell patch-clamp studies, we report that angiotensin II (Ang II) potentiated the activity of RVLM neurons, a sympathetic nervous center, whereas Ang II receptor blocker (ARB) reduced RVLM activities. Our optical imaging demonstrated that a longitudinal rostrocaudal column, including the RVLM and the caudal end of ventrolateral medulla, acts as a sympathetic center. By organizing and analyzing these data, we hope to develop therapies for reducing SNA in our patients. Recently, 2-year depressor effects were obtained by a single procedure of renal nerve ablation in patients with essential hypertension. The ablation injured not only the efferent renal sympathetic nerves but also the afferent renal nerves and led to reduced activities of the hypothalamus, RVLM neurons and efferent systemic sympathetic nerves. These clinical results stress the importance of the RVLM neurons in blood pressure regulation. We expect renal nerve ablation to be an effective treatment for congestive heart failure and chronic kidney disease, such as diabetic nephropathy.

SignificanceHepatocellular carcinoma (HCC) is a highly lethal cancer, partly because of its high rate of recurrence, which is caused by the presence of liver cancer stem cells (CSCs). Here, using a selective chemopreventive agent, acyclic retinoid (ACR), as a bioprobe, we identified MYCN, which is mostly recognized as an oncogene in neuroblastoma, as a therapeutic target of ACR for HCC through a selective deletion of MYCN+ liver CSCs. We also demonstrated that the expression of MYCN in HCC served as a prognostic biomarker and positively correlated with recurrence of de novo HCC after curative treatment. Our study highlighted MYCN as a biomarker and therapeutic target in drug discovery for screening chemopreventive agents against the recurrence of HCC.

Glycyrrhizin (GL) has been used in Japan to treat patients with chronic viral hepatitis, as an anti-inflammatory drug to reduce serum alanine aminotransferase levels. GL is also known to exhibit various biological activities, including anti-viral effects, but the anti-hepatitis C virus (HCV) effect of GL remains to be clarified. In this study, we demonstrated that GL treatment of HCV-infected Huh7 cells caused a reduction of infectious HCV production using cell culture-produced HCV (HCVcc). To determine the target step in the HCV lifecycle of GL, we used HCV pseudoparticles (HCVpp), replicon, and HCVcc systems. Significant suppressions of viral entry and replication steps were not observed. Interestingly, extracellular infectivity was decreased, and intracellular infectivity was increased. By immunofluorescence and electron microscopic analysis of GL treated cells, HCV core antigens and electron-dense particles had accumulated on endoplasmic reticulum attached to lipid droplet (LD), respectively, which is thought to act as platforms for HCV assembly. Furthermore, the amount of HCV core antigen in LD fraction increased. Taken together, these results suggest that GL inhibits release of infectious HCV particles. GL is known to have an inhibitory effect on phospholipase A2 (PLA2). We found that group 1B PLA2 (PLA2G1B) inhibitor also decreased HCV release, suggesting that suppression of virus release by GL treatment may be due to its inhibitory effect on PLA2G1B. Finally, we demonstrated that combination treatment with GL augmented IFN-induced reduction of virus in the HCVcc system. GL is identified as a novel anti-HCV agent that targets infectious virus particle release.

Abstract-We compared the electrophysiological properties of neurons in the rostral ventrolateral medulla (RVLM) of neonatal Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), and responses to angiotensin II and its type 1 receptor antagonist candesartan. Using the whole-cell patch-clamp technique, we examined the characteristics of RVLM neurons in brainstem-spinal cord preparations with a preserved sympathetic neuronal network. The baseline membrane potential of irregularly firing neurons was less negative (Ϫ50.1Ϯ0.6 versus Ϫ52.0Ϯ0.6 mV) and the firing rate was faster (3.0Ϯ0.2 versus 2.0Ϯ0. Key Words: brain Ⅲ rostral ventrolateral medulla Ⅲ angiotensin II Ⅲ receptors, angiotensin Ⅲ candesartan Ⅲ hyperpolarization R ostral ventrolateral medulla (RVLM) neurons are located at a pivotal site involved in the baroreflex pathway and play a key role in controlling peripheral sympathetic nerve activity (SNA) and blood pressure (BP). [1][2][3] We have reported impairment of the baroreflex function in hypertension based on recordings of renal SNA (RSNA) in conscious rabbits and rats and on vagal afferent nerve activity. 4,5 We have also confirmed that the angiotensin (Ang) II type 1 (AT 1 ) receptor antagonist candesartan improves the impaired baroreflex in conscious rats with congestive heart failure. 6 Earlier studies examined the responses of BP and SNA to Ang II and Ang II antagonists microinjected into the RVLM of normotensive and hypertensive animals, 7-11 because the RVLM area contains Ang II-immunoreactive nerve terminals and a moderately high density of AT 1 receptors. 12 Microinjection of Ang II increased BP and SNA, 7-10 whereas candesartan reduced BP, RSNA, and heart rate (HR). 11 However, the precise neuronal mechanisms by which RVLM neurons regulate SNA and BP and how these neurons are involved in the development of hypertension have not been fully elucidated. The firing rate of extracellular units in RVLM neurons was faster in adult spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY) in vivo. 13 Iontophoretic application of Ang II increased the extracellular activity of 30% of RVLM neurons in both strains, and the increase was greater in SHR. 14 However, very few studies have compared the intracellular properties of RVLM neurons and responses to Ang II and its antagonist between WKY and SHR. Recently, we determined the intracellular electrophysiological characteristics of RVLM bulbospinal neurons in neonatal WKY using brainstem-spinal cord preparation, in which the neuronal network is preserved from the vagal afferents to the sympathetic efferents exiting the intermediolateral cell column. 15 In the present study, we performed intracellular recordings (whole-cell patch-clamp technique) of RVLM neurons in WKY and SHR during superfusion rather than microinjection of Ang II or candesartan to precisely understand the role of the RVLM neurons. Although BP of SHR increases above that of WKY sometime between 3 and 4 weeks of age, 16 RVLM neuron activity cannot be assumed to be iden...