Abstract 102: A Novel Swine Model of Infrarenal Abdominal Aortic Aneurysm

Introduction: A reproducible large animal model for abdominal aortic aneurysms (AAAs) does not exist. This study sought to develop a large animal AAA model in swine using β-aminopropionitrile (BAPN), a lysyl oxidase inhibitor, an enzyme responsible for collagen cross-linking. We hypothesized that elastase/collagenase perfusion and balloon dilatation in combination with BAPN administration would result in AAA formation. Methods: Uncastrated Yorkshire male swine were fed BAPN (0.12g/kg) daily for one week prior to surgery and continued throughout the experiment. After anesthesia, the aorta was exposed from the renal arteries to the aortic bifurcation. The infrarenal aorta was cannulated via the caudal mesenteric artery and an aortic angioplasty was performed to dilate the aorta to 200% of its original diameter. Next, a 30 mL solution consisting of 500 units of elastase and 8,000 units of Type I collagenase was perfused into the infrarenal aorta for 10 minutes. This solution was also then topically applied for 10 minutes. The abdomen was irrigated and closed. Antibiotics and analgesic medications were administered. Results: BAPN-fed swine sacrificed at 28 days (n=5) had a 101% increase in infrarenal aorta diameter compared with day 0 (p=0.008). Swine (n=4) sacrificed at 7 and 14 days showed an increase of 114 and 75%, respectively. Histologically on day 28, collagen decreased by 20% and smooth muscle cell expression decreased by 40% (p=0.0001) in the aortic wall compared with control suprarenal aorta. Infrarenal aortas also showed a strong predilection for M1-polarized macrophages (MCP-1 positive staining, p=0.0165) compared with M2 macrophages (Arg-1 positive staining). MMP2 activity by zymography (p=0.0127) and IL-18 by array was also significantly increased (p=0.0202) on day 28 in the infrarenal AAA. Conclusions: Compared with previous large animal AAA models, this model using conventional techniques including balloon dilatation, elastase/collagenase perfusion, in addition to oral BAPN led to robust AAA with similar molecular and histologic changes to those seen in human AAA. This novel swine AAA model may serve as a much-needed link that will allow for the progression of studies from rodents to humans.

Diabetic KK-Ay mice are highly susceptible to oxidative hepatocellular damage induced by acetaminophen

Despite pathophysiological similarities to alcoholic liver disease, susceptibility to acetaminophen hepatotoxicity in metabolic syndrome-related nonalcoholic steatohepatitis (NASH) has not been well elucidated. In this study, therefore, we investigated acetaminophen-induced liver injury in KK-Ay mice, an animal model of metabolic syndrome. Twelve-week-old male KK-Ay and C57Bl/6 mice were injected intraperitoneally with 300 or 600 mg/kg acetaminophen, and euthanized 6 h later. Liver histology was assessed, and hepatic expression of 4-hydroxy-2-nonenal was detected by immunohistochemistry. Levels of reduced glutathione were determined spectrophotometrically. Phosphorylation of c-Jun NH2-terminal kinase (JNK) was analyzed by Western blotting. Hepatocytes were isolated from both strains by collagenase perfusion, and cell death and oxidative stress were measured fluorometrically by use of propidium iodide and 5-(and-6)-chloromethyl-2′7′-dichloro-dihydrofluorescein diacetate acetyl ester, respectively. Acetaminophen induced more severe necrosis and apoptosis of hepatocytes in KK-Ay mice than in C57Bl/6 mice and significantly increased serum alanine aminotransferase levels in KK-Ay mice. Acetaminophen-induction of 4-hydroxy-2-nonenal in the liver was potentiated, whereas the levels of reduced glutathione in liver were lower in KK-Ay mice. Acetaminophen-induced phosphorylation of JNK in the liver was also enhanced in KK-Ay mice. Exposure to 20 μM tert-butyl hydroperoxide did not kill hepatocytes isolated from C57Bl/6 mice but induced cell death and higher oxidative stress in hepatocytes from KK-Ay mice. These results demonstrated that acetaminophen toxicity is increased in diabetic KK-Ay mice mainly due to enhanced oxidative stress in hepatocytes, suggesting that metabolic syndrome-related steatohepatitis is an exacerbating factor for acetaminophen-induced liver injury.

Norepinephrine modulates the zonally different hepatocyte proliferation through the regulation of transglutaminase activity

A neurotransmitter, norepinephrine (NE), amplifies the mitogenic effect of epidermal growth factor (EGF) in the liver by acting on the α1-adrenergic receptor coupled with G protein, Gαh. However, the molecular mechanism is not well understood. Gαh is known as a transglutaminase 2 (TG2), a cross-linking enzyme implicated in hepatocyte proliferation. We investigated the effect of NE on EGF-induced cell proliferation and TG2 activity using hepatocytes isolated in periportal and perivenous regions of the liver, which differ in proliferative capacity. Periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated by the digitonin-collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, EGF receptor (EGFR) dimerization and phosphorylation, and TG2 activity were measured. NE enhanced EGF-induced DNA synthesis, EGF-induced EGFR dimerization, and its phosphorylation in PVH but not in PPH. [3H]NE binding studies indicated that PVH was found to have a greater affinity and number of receptors than PPH. Furthermore, NE treatment decreased TG2 activity and increased phospholipase C activity in PVH although TG2 level showed no change. These results suggest that NE-induced amplification of EGF-induced DNA synthesis especially in PVH is caused by upregulation of EGFR activation through the switching of function from TG2 to Gαh.

Hepatoprotective role of captopril on paraquat induced hepatotoxicity

Paraquat (PQ) is a highly toxic herbicide that is used in most of the countries without restriction. The cytotoxic effect of PQ is mediated by radicals, which are the products of PQ reduction in cells. The anti-oxidative action of captopril, an angiotensin-converting enzyme inhibitor, appears to be through its ability to scavenge reactive oxygen species. In this study, the heptoprotective effect of captopril against PQ-induced hepatotoxicity was evaluated using primary cultured rat hepatocytes. Hepatocytes were isolated from male Wistar rats using a two-step collagenase perfusion, following incubation in the presence of captopril at 0.1, 0.2, 0.4 and 0.8 mM with or without PQ (5 mM). Hepatoprotective effects of captopril were studied indicating glutathione level intensity, thiobarbituric acid reactive substances (TBARs) formation, lactate dehydrogenase (LDH) leakage and cell viability every 70 min for 210 min. Captopril at 0.2 mM concentration maintained the LDH leakage, glutathione level and cell viability in the presence of 5 mM PQ. In spite of a significant elevation in TBARs formation in the PQ group, captopril did not show any significant protection. In conclusion, our data reveals that incubation of freshly isolated rat hepa-tocytes with captopril (0.2 mM) significantly protected the hepatocytes against the cytotoxicity of PQ ( P < 0.05). Human & Experimental Toxicology (2007) 26, 789— 794

Influence of Preservation Solution on the Isolation and Culture of Human Hepatocytes from Liver Grafts

A major problem for the isolation and transplantation of hepatocytes is the lack of resources for obtaining viable hepatocytes. Improving this situation would enhance hepatic cell transplantation programs. Our objective was to evaluate the influence of the preservation solutions used during organ retrieval on the quality of hepatocytes isolated from liver tissue. We compared the results of the collagenase perfusion technique for isolation of hepatocytes in human livers flushed with University of Wisconsin (UW) and Celsior preservation solutions. Yield (number of viable cells per gram of tissue), cellular viability, efficiency of cells to attach to culture plates and form a monolayer, and drug metabolizing competence of the hepatocytes were measured. Successful isolation was achieved in 63% of the procedures using the UW solution and 100% of the procedures using the Celsior solution. In the UW group, significantly lower cell viability (38 ± 41% vs. 79 ± 14%, p < 0.05), yield of cells (4.0 ± 5.2 × 106 vs. 8.2 ± 5.6 × 106 cells/g, p < 0.05), and protein content at 24 h of culture (0.6 ± 0.6 vs. 1.2 ± 0.3 mg protein per plate, p < 0.05) than in Celsior solution were found. However, similar values of P450 activities were found in both groups. The more successful isolation, better yield, and higher cell viability obtained from human liver grafts preserved in Celsior solution, in comparison to UW solution, suggest Celsior solution as the most appropriate for preserving cadaveric hepatic tissue to be used for hepatocyte harvesting.

Elevation of the adenylate pool in rat cardiomyocytes by S-adenosyl-L-methionine.

Rapid resynthesis of the adenylate pool in cardiac myocytes is important for recovery of contractility and normal function of regulatory mechanisms in the heart. Adenosine and adenine are thought to be the most effective substrates for nucleotide synthesis, but the possibility of using other compounds has been studied very little in cardiomyocytes. In the present study, the effect of S-adenosyl-L-methionine (SAM) on the adenylate pool of isolated cardiomyocytes was investigated and compared to the effect of adenine and adenosine. Adult rat cardiomyocytes were isolated using the collagenase perfusion technique. The cells were incubated in the presence of adenine derivatives for 90 min followed by nucleotide determination by HPLC. The concentrations of adenine nucleotides expressed in nmol/mg of cell protein were initially 22.1 +/- 1.4, 4.0 +/- 0.3 and 0.70 +/- 0.08 for ATP, ADP and AMP, respectively (n = 10, +/- S.E.M.), and the total adenylate pool was 26.8 +/- 1.6. In the presence of 1.25 mM SAM in the medium, the adenylate pool increased by 5.2 +/- 0.4 nmol/mg of cell protein, but only if 1 mM ribose was additionally present in the medium. No changes were observed with SAM alone. A similar increase (by 4.9 +/- 0.6 nmol/mg protein) was observed after incubation with 1.25 mM adenine plus 1 mM ribose, but no increase was observed if ribose was omitted. Adenosine at 0.1 or 1.25 mM concentrations also caused an increase in the adenylate pool (by 5.2 +/- 1.0 and 5.2 +/- 0.9 nmol/mg protein, respectively), which in contrast to the SAM or adenine was independent of the additional presence of ribose. Thus, S-adenosyl-L-methionine could be used as a precursor of the adenylate pool in cardiomyocytes, which is as efficient in increasing the adenylate pool after 90 min of incubation as adenosine or adenine. Nucleotide synthesis from SAM involves the formation of adenine as an intermediate with its subsequent incorporation by adenine phosphoribosyltransferase.

Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells

CD14 is a lipopolysaccharide (LPS) receptor distributed largely in macrophages, monocytes, and neutrophils; however, the role of CD14 in activation of Kupffer cells by LPS remains controversial. The purpose of this study was to determine if different methods used to isolate Kupffer cells affect CD14. Kupffer cells were isolated by collagenase (0.025%) or collagenase-Pronase (0.02%) perfusion and differential centrifugation using Percoll gradients and cultured for 24 h before experiments. CD14 mRNA was detected by RT-PCR from Kupffer cell total RNA as well as from peritoneal macrophages. Western blotting showed that Kupffer cells prepared with collagenase possess CD14; however, it was absent in cells obtained by collagenase-Pronase perfusion. Intracellular calcium in Kupffer cells prepared with collagenase was increased transiently to levels around 300 nM by addition of LPS with 5% rat serum, which contains LPS binding protein. This increase in intracellular calcium was totally serum dependent. Moreover, LPS-induced increases in intracellular calcium in Kupffer cells were blunted significantly (40% of controls) when cells were treated with phosphatidylinositol-specific phospholipase C, which cleaves CD14 from the plasma membrane. However, intracellular calcium did not increase when LPS was added to cells prepared by collagenase-Pronase perfusion even in the presence of serum. These cells were viable, however, because ATP increased intracellular calcium to the same levels as cells prepared with collagenase perfusion. Tumor necrosis factor-α (TNF-α) mRNA was increased in Kupffer cells prepared with collagenase perfusion 1 h after addition of LPS, an effect potentiated over twofold by serum; however, serum did not increase TNF-α mRNA in cells isolated via collagenase-Pronase perfusion. Moreover, treatment with Pronase rapidly decreased CD14 on mouse macrophages (RAW 264.7 cells) and Kupffer cells. These findings indicate that Pronase cleaves CD14 from Kupffer cells, whereas collagenase perfusion does not, providing an explanation for why Kupffer cells do not exhibit a CD14-mediated pathway when prepared with procedures using Pronase. It is concluded that Kupffer cells indeed contain a functional CD14 LPS receptor when prepared gently.