Autophagy selectively removes abnormal or damaged organelles such as dysfunctional mitochondria. The mitochondrial permeability transition (MPT) is a marker of impaired mitochondrial function that is evident in hepatic ischemia/reperfusion (I/R) injury. However, the relationship between mitochondrial dysfunction and autophagy in I/R injury is unknown. Cultured rat hepatocytes and mouse livers were exposed to anoxia/reoxygenation (A/R) and I/R, respectively. Expression of autophagyrelated protein 7 (Atg7), Beclin-1, and Atg12, autophagy regulatory proteins, was analyzed by western blots. Some hepatocytes were incubated with calpain 2 inhibitors or infected with adenoviruses encoding green fluorescent protein (control), Atg7, and Beclin-1 to augment autophagy. To induce nutrient depletion, a condition stimulating autophagy, hepatocytes were incubated in an amino acid-free and serum-free medium for 3 hours prior to onset of anoxia. For confocal imaging, hepatocytes were coloaded with calcein and tetramethylrhodamine methyl ester to visualize onset of the MPT and mitochondrial depolarization, respectively. To further examine autophagy, hepatocytes were infected with an adenovirus expressing green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) and subjected to A/R. Calpain activity was fluorometrically determined with succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin. A/R markedly decreased Atg7 and Beclin-1 concomitantly with a progressive increase in calpain activity. I/R of livers also decreased both proteins. However, inhibition of calpain isoform 2, adenoviral overexpression, and nutrient depletion all substantially suppressed A/R-induced loss of autophagy proteins, prevented onset of the MPT, and decreased cell death after reoxygenation. Confocal imaging of GFP-LC3 confirmed A/R-induced depletion of autophagosomes, which was reversed by nutrient depletion and adenoviral overexpression. Conclusion: Calpain 2-mediated degradation of Atg7 and Beclin-1 impairs mitochondrial autophagy, and this subsequently leads to MPT-dependent hepatocyte death after A/R. (HEPATOLOGY 2008;47:1725-1736 H epatocyte ischemia during liver transplantation, resection, and shock causes anoxia, depletion of glycolytic substrates, loss of adenosine triphosphate (ATP), and acidosis. 1 When blood flow returns to hepatocytes, the normal oxygen concentration and acidbase balance are restored; however, paradoxically, hepatocyte injury is prominent upon reperfusion. 1 The mechanisms underlying ischemia/reperfusion (I/R) injury are multifactorial, including increased Ca 2ϩ , formation of reactive oxygen species (ROS), activation of injurious catabolic enzymes, and mitochondrial dysfunction. 2 Reperfusion causes onset of the mitochondrial permeability transition (MPT), leading to uncoupling of oxidative phosphorylation and necrotic cell death. 1,[3][4][5] Onset of the MPT also induces large-amplitude swelling of mitochondria and apoptosis by releasing proapoptotic factors that are normally sequestered in the mitochondri...

Polymorphisms in warfarin drug target and metabolizing enzyme genes, in addition to nongenetic factors, were important determinants of warfarin dose requirements.

Hepatocytes that reside in a chronically-injured liver have altered growth responses compared to hepatocytes in normal liver. Transforming growth factor beta (TGFβ) is upregulated in the cirrhotic liver, and cirrhotic hepatocytes, unlike normal hepatocytes exposed to this cytokine, exhibit decreased apoptosis. In fetal hepatocytes, TGFβ also induces epithelial-mesenchymal transition (EMT) and signaling changes in cell survival pathways. Here, chronic murine liver injury was induced by twice-weekly carbon tetrachloride administration for 8 weeks. Normal liver-derived hepatocytes (NLDH) and cirrhotic liver-derived hepatocytes (CLDH) were examined for EMT and the small mothers against decapentaplegic homolog (Smad), phosphatidylinositol-3-kinase (PI3K/Akt), and mitogen activated protein kinase (MAPK) pathways were investigated. Immunofluorescence imaging of cirrhotic livers demonstrated increased vimentin expression, which was confirmed by immunoblot analysis. In vitro, CLDH exhibited increased vimentin and type 1 collagen expression within cellular extensions consistent with EMT. Treatment with TGFβ augmented the EMT response in CLDH. In contrast, untreated NLDH did not display features of EMT but responded to TGFβ with increased vimentin expression and EMT characteristics. In response to PI3K/Akt inhibition, CLDH had decreased basal and insulin-stimulated p-Akt expression and decreased apoptosis compared to NLDH. In both NLDH and CLDH, vimentin expression was dependent on PI3K/Akt activity. CLDH demonstrated increased basal p-extracellular signal-regulated kinase expression that was independent of Smad and PI3K/Akt signaling. Inhibition of the MAPK pathway produced a marked increase in CLDH apoptosis. Conclusion: CLDH have increased vimentin and type 1 collagen expression and morphologic features consistent with EMT. In addition, compared to NLDH, the cellular signaling phenotype of CLDH changes from a MAPK-independent pathway to a MAPK-dependent cell survival pathway. These findings may have clinical implications for chemoprevention of hepatocellular carcinoma in the cirrhotic liver.

Abstract-Angiotensin II (Ang II) promotes vascular smooth muscle growth and may be involved in the initiation and progression of atherosclerosis. To examine whether Ang II receptor expression in vascular tissues is altered in atherosclerosis, male New Zealand White rabbits were fed a high-cholesterol diet (1% cholesterolϩ4% coconut oil mixed with regular chow; hypercholesterolemic group, nϭ12) or regular chow (control group, nϭ8) for 10 weeks. At the end of this period, the serum cholesterol level in the rabbits fed the high-cholesterol diet was higher than that in the control group (3616Ϯ144 versus 30Ϯ1 mg/dL, PϽ0.001). There was no atherosclerosis in the aortas of the control group, whereas 51Ϯ6% of the aorta was covered with atherosclerosis in the hypercholesterolemic group. Total Ang II receptor expression in the atherosclerotic aortic tissues was increased 5-fold in the hypercholesterolemic rabbits (292Ϯ28 versus 51Ϯ32 fmol/mg tissue, meanϮSE, PϽ0.001), and the increased Ang II receptor expression was entirely due to enhanced Ang II type 1 (AT 1 ) receptor expression (289Ϯ38 versus 38Ϯ18 fmol/mg, PϽ0.001), as Ang II type 2 receptor expression was unaltered (7Ϯ5 versus 3Ϯ2 fmol/mg, PϭNS). AT 1 receptors were localized primarily in the media and to some extent in the intima of the atherosclerotic aorta, as determined by immunohistochemistry with specific monoclonal and polyclonal AT 1 receptor antibodies. Increased synthesis of AT 1 receptor mRNA in atherosclerotic tissues was confirmed by reverse transcription-polymerase chain reaction. To evaluate the functional significance of increased AT 1 receptor expression, the constrictor response of aortic rings to Ang II was examined and found to be markedly enhanced in atherosclerotic aortic rings (PϽ0.01 versus control aortic rings). The endotheliumdependent relaxation of aortic rings from hypercholesterolemic rabbits was markedly attenuated (PϽ0.001). This study shows that hypercholesterolemia in rabbits results in atherosclerosis, loss of endothelium-dependent relaxation, and increased Ang II receptor (entirely AT 1 receptor) expression in aortic tissues, which may result in altered vasoreactivity.

Multiple prior studies have identified aldehyde dehydrogenases (ALDH) that are capable of oxidizing retinal to retinoic acid. In this study, we test the hypothesis that the accumulation of intracellular retinoic acid may lead to the suppression of ALDH expression and thus increase cytotoxicity to 4-hydroperoxycyclophosphamide (4-HC) in vitro. Mainly A549, but also other lung cancer cell lines, were used in our experiments, with the former having high levels of two ALDH isozymes expressed. Dose-response and time-course experiments were performed by incubating the cells with all-trans retinoic acid (ATRA) as well as other commercially available retinoids. The results show that incubation of A549 cells with any of the retinoids at pharmacologic doses for Ն48 h results in a significant decrease in ALDH-1A1 and ALDH-3A1 enzyme activity and protein levels but not the corresponding mRNAs. Such a decrease in ALDH activity was seen in all cell lines tested and results in a significant increase in toxicity of 4-HC and acetaldehyde, both of which are substrates for the enzymes. Prior incubation with ATRA also results in increased cytotoxicity, although to a lesser degree, of phenylketophosphamide and melphalan, neither of which is a substrate for ALDHs. These results suggest a post-translational mechanism through which retinoids decrease both ALDH expression, which results in increased cytotoxicity of 4-HC and acetaldehyde, although other previously described effects of these retinoids may contribute to the slight increase in cytotoxicity seen with other chemotherapy agents. These results may have clinical implications in regard to the use of retinoids in lung cancer prevention and treatment.Aldehyde dehydrogenases (ALDH) are a group of enzymes that catalyze the conversion of a broad range of aldehydes to the corresponding acid via a NAD ϩ -dependent irreversible reaction. Two of these enzymes, cytosolic ALDH-1A1 and ALDH-3A1, have been found to be responsible for drug resistance in various tumor types against the antineoplastic drugs collectively known as oxazaphosphorines, which include cyclophosphamide and its active metabolites (Hilton, 1984;Manthey et al., 1990;Sreerama and Sladek, 1993;von Eitzen et al., 1994;Yoshida et al., 1998). Cyclophosphamide is a prodrug that requires cytochrome P450 hydroxylation for activation. Prior to the release of the active alkylating metabolite phosphoramide mustard, cyclophosphamide passes through an aldehyde intermediate, aldophosphamide. ALDH oxidizes aldophosphamide to the inactive metabolite carboxyphosphamide (Manthey et al., 1990). Several inhibitors of ALDH activity have been used to demonstrate the reversal of this drug resistance mechanism. We and others (Bunting et al., 1994;Bunting and Townsend, 1996;Magni et al., 1996;Moreb et al., 1996Moreb et al., , 1998 have successfully shown that overexpression of ALDH-1A1 or ALDH-3A1 in cell lines and normal hematopoietic progenitors results in a significant increase in the resistance to the active metabolites of cyclophospha...

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Abstract-Angiotensinogen (AGT), one of the major components in the renin-angiotensin system, has been linked to hypertension in humans and animals. We have previously systemically administered antisense oligonucleotides and plasmid vectors with DNA that targeted AGT and attenuated hypertension in spontaneously hypertensive rats. The aim of the present study was to prolong the effect of antisense treatment by the use of a recombinant adeno-associated viral (rAAV) vector targeted to AGT. Using a model of lifelong hypertension in which 5-day-old spontaneously hypertensive rats are treated, a single intracardiac injection of rAAV-AGT-antisense (rAAV-AGT-AS) delayed the onset of hypertension for 91 days and significantly attenuated hypertension in adulthood for up to 6 months. Key Words: adeno-associated virus Ⅲ hypertrophy, cardiac Ⅲ antisense Ⅲ gene therapy Ⅲ angiotensinogen A ntisense inhibition for gene therapy of hypertension is a novel approach to provide long-term control of hypertension. 1 Because the antisense strategy can target genes precisely, inhibition of specific proteins can be achieved. Although the genes involved in hypertension are largely unknown, currently used drugs control high blood pressure by inhibiting a few proteins such as angiotensin-converting enzyme (ACE), ␤ 1 -adrenoceptors (␤ 1 -AR), and angiotensin type 1 receptor (AT 1 -R). Therefore, specific gene targeting of these proteins has been our strategy with antisense oligodeoxynucleotides (AS-ODNs). Compared with current drugs, AS-ODNs are more specific and longer lasting and therefore may have certain advantages over current drug therapy. The longer duration of action could provide more consistently effective blood pressure control and thereby increase patient compliance. Despite all the drugs available, only Ϸ29% of all hypertensives have their high blood pressures controlled. Therefore, there is a need to explore a new generation of methods of treatment. Gene therapy, while oversold, still offers an approach that could produce prolonged benefits if based on scientific research. Whereas AS-ODN can reduce high blood pressure in rat models for weeks with a single dose, 1,2 even longer-lasting effects can be achieved with viral vectors. 1 Studies with the retrovirus used to deliver antisense to AT 1 -R or ACE have been shown to produce long-lasting effects in reducing high blood pressure when given in the first 5 days after birth. 3,4 Retroviruses, however, have disadvantages as a vector for therapeutic use. These include the possibility of tumorigenic activity, the vector entering the germ line, and the limitation that retroviruses only infect dividing cells. Adeno-associated virus (AAV) has the potential to provide stable, effective, and very long-lasting delivery of antisense, 5 even in nondividing cells. Because the previous studies have targeted the AT 1 -R for inhibition of the reninangiotensin system (RAS), the purpose of this study was to target angiotensinogen (AGT) mRNA with antisense and deliver it with AAV as a vector. AGT ha...

The atrophy-hypertrophy complex (AHC) refers to the controlled restoration of liver parenchyma following hepatocyte loss. Different types of injury (e.g., toxins, ischemia/reperfusion, biliary obstruction, and resection) elicit the same hypertrophic response in the remnant liver. The AHC involves complex anatomical, histological, cellular, and molecular processes. The signals responsible for these processes are both intrinsic and extrinsic to the liver and involve both physical and molecular events. In patients in whom resection of large liver malignancies would result in an inadequate functional liver remnant, preoperative portal vein embolization may increase the remnant liver sufficiently to permit aggressive resections. Through continued basic science research, the cellular mechanisms of the AHC may be maximized to permit curative resections in patients with potentially prohibitive liver function.