High Fat Diet Induced Hepatic Steatosis Establishes a Permissive Microenvironment for Colorectal Metastases and Promotes Primary Dysplasia in a Murine Model

Non-alcoholic fatty liver disease (NAFLD), which includes steatosis and its progression to non-alcoholic steatohepatitis, is a liver disorder of increasing clinical significance. Here we characterize a murine model of high fat diet-induced NAFLD with progression from liver steatosis to histological features compatible with steatohepatitis and more advanced stages of NAFLD in humans, including chronic portal inflammation, pericellular and bridging fibrosis, Mallory body formation, and bile ductular reaction. Chronic changes induced by the prolonged consumption of a high-fat diet alone culminate in the development of primary liver dysplasias. Importantly, we extend these studies to demonstrate that even the early stages of uncomplicated steatosis provide a permissive microenvironment for the growth of colon cancer cells that are metastatic to the liver. High fat diet-induced steatosis, coupled with a splenic injection model of experimental liver metastasis using syngeneic MC38 colon cancer cells, resulted in an increased number of secondary tumor nodules and metastatic burden in steatotic livers. Metastatic nodules were associated with focal peritumoral areas of infiltrating inflammatory cells and associated apoptotic cell populations. These results suggest that the modulation of specific host factors in the steatotic liver contributes to tumor progression in the microenvironment of NAFLD.Obesity and non-alcoholic steatohepatitis (NASH), have emerged as two independent risk factors for the development of cirrhosis and hepatocellular carcinoma distinct from well established risk factors including viral hepatitis and alcohol consumption.^1,2,3 Nonalcoholic fatty liver disease (NAFLD) can result from a high-fat diet, lack of exercise, hepatitis infection, type II diabetes, or a combination of these factors. NAFLD is commonly associated with central obesity, insulin resistance, and hyperlipidemia, and characterized clinically by the metabolic syndrome.^4,5 Obesity is clinically associated with an increased prevalence of certain malignancies including colon cancer and breast cancer.^6,7 NASH associated with obesity is a recognized risk factor for the development of primary hepatocellular carcinoma.^2,8 Recently, a small case study has identified hepatocellular carcinoma in subset of patients with NAFLD in the absence of cirrhosis, supporting NAFLD as permissive environment for the development of hepatocellular carcinoma.⁹ Further, since systemic disease represents the major cause of cancer mortality, the rising incidence of obesity, NAFLD, and NASH is likely to have an enormous influence on cancer-associated deaths in the United States.In humans, liver steatosis represents an early reversible stage of disease that is histologically characterized by the accumulation of triglycerides in hepatocytes.^10,11 Histologically, the broad term NALFD may include steatosis alone or it can include progressive changes associated with non-alcoholic steatohepatitis such as inflammation, hepatocyte ballooning, necrosis, Mallory’s hyaline, and even fibrosis.^11,12,13 NASH can only be definitively diagnosed by liver biopsy, but it is suspected in patients with obesity and/or syndromes of insulin resistance accompanied by elevated serum aminotransferase levels.^14,15 Naturally occurring genetic mutations such as of the leptin gene (ob/ob), genetically modified mice, environmental dietary models, and toxins such as ethanol have been shown to increase hepatic fat uptake leading to steatosis and steatohepatitis.^16,17Improved murine models that corroborate human disease are important for the understanding of microenvironmental factors that contribute to tumor promotion in the liver. It has been suggested through the use of genetic, as well as dietary mouse models, that obesity-related hepatic steatosis might increase the susceptibility of the liver to malignancy.^18,19,20 It is recognized that other than high-fat diets, alcohol consumption, inflammatory cytokines, oxidative stress, alterations in the extracellular matrix, as well as viral mediators, can participate in development of steatohepatitis in humans and can be contributing risk factors for hepatocellular carcinoma. For example, changes in the inflammatory cytokines and extracellular matrix remodeling proteases have been associated with increased metastatic risk in multiple model systems and organs.^{21,22,23,24,25} Significant steatotic changes and or steatohepatitis cause increases in a number of signaling molecules, including transforming growth factor β and select matrix metalloproteinases that may be important in tumor promotion and growth.^26,27,28The microenvironmental effects of high-fat diet induced hepatic steatosis on tumor growth in our data indicate that early uncomplicated steatosis of the liver microenvironment enhances the number of metastatic foci and tumor burden in an experimental murine model of colorectal cancer metastasis to the liver. We demonstrate that changes in the steatotic liver establish a more susceptible microenvironment for metastasis as compared with normal liver. Additionally, multifocal dysplasia of the liver develops with prolonged consumption of high fat diet concordant with progressive histological changes analogous to NASH in this murine model.     

A Human-Type Nonalcoholic Steatohepatitis Model with Advanced Fibrosis in Rabbits

Nonalcoholic steatohepatitis (NASH) progresses to liver fibrosis and cirrhosis, which can lead to life-threatening liver failure and the development of hepatocellular carcinoma. The aim of the present study was to create a rabbit model of NASH with advanced fibrosis (almost cirrhosis) by feeding the animals a diet supplemented with 0.75% cholesterol and 12% corn oil. After 9 months of feeding with this diet, the rabbits showed high total cholesterol levels in serum and liver tissues in the absence of insulin resistance. The livers became whitish and nodular. In addition, the number of rabbit macrophage antigen-positive cells and the expression of mRNAs for inflammatory cytokines showed a significant increase. Moreover, fibrotic septa composed of collagens and α-smooth muscle actin-positive cells were found between the central and portal veins, indicating alteration of the parenchymal architecture. There was also a marked increase of mRNAs for transforming growth factor-β1 and collagen 1A1. Comprehensive analysis of protein and gene expression revealed an imbalance of the antioxidant system and methionine metabolism. We also found that ezetimibe attenuated steatohepatitis in this model. In conclusion, the present rabbit model of NASH features advanced fibrosis that is close to cirrhosis and may be useful for analyzing the molecular mechanisms of human NASH. Ezetimibe blunted the development of NASH in this model, suggesting its potential clinical usefulness for human steatohepatitis.A high-fat diet is one of the risk factors for metabolic syndrome, which is characterized by obesity, hyperlipidemia, hyperglycemia, and hypertension, and is frequently accompanied by life-threatening arteriosclerosis.¹ A high-calorie, high-fat diet is also considered to cause nonalcoholic fatty liver disease (NAFLD), which covers a spectrum of disorders from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis.^2,3,4,5 Recent clinical studies have shown that NAFLD is one of the common liver diseases that leads to cirrhosis and hepatocellular carcinoma² in a manner similar to the clinical course of chronic viral hepatitis and alcohol abuse.⁶ However, the molecular mechanisms underlying the progression of NAFLD to an advanced stage with active inflammation and fibrosis are not fully understood. We recently reported a rabbit model of steatohepatitis that was generated by feeding the rabbit a high-fat and -cholesterol diet (HFD) supplemented with 20% corn oil and 1.25% (w/w) cholesterol for 8 weeks.⁷ The rabbit showed insulin resistance, accumulation of lipids in hepatocytes, activation of Kupffer cells (liver macrophages), mild fibrosis, and enhanced oxidative stress. Thus, we concluded that this model was useful for analyzing the molecular mechanisms involved in the pathogenesis of human NASH. However, rabbits fed a HFD have a short lifespan attributable to heart failure accompanied by severe arteriosclerosis.⁸ This makes it difficult to study whether advanced fibrosis or even cirrhosis can be caused solely by HFD feeding. We therefore tried to improve the model and produce NASH with advanced fibrosis, which is more similar to the disease observed in humans that gradually develops after several decades. In the present study, this was accomplished by reducing the concentrations of cholesterol and corn oil in the diet and by prolonging the feeding period from 2 to 9 months.NAFLD/NASH is assumed to be most effectively improved by weight control and by restricting lipid and calorie intake, thereby leading to normalized lipid metabolism.^9,10 Nevertheless, drug therapy would be a useful and an easy option because the modern lifestyle habits such as poor diet and lack of regular exercise are difficult to change. Ezetimibe is a relatively new and promising drug candidate for NAFLD/NASH therapy. Ezetimibe selectively inhibits cholesterol absorption via Niemann-Pick C1-like 1 (NPC1L1) protein in the brush border of the small intestine in humans, rodents, rabbits, and other species.^{11,12,13,14,15} It decreases the serum levels of low-density lipoprotein cholesterol and triglycerides (TGs) in humans¹⁶ and reduces plaque formation and improves lipids in a rabbit model of atherosclerosis.¹⁷ Recent reports have indicated that ezetimibe improves liver steatosis and insulin resistance in Zucker obese fatty rats¹⁸ and rats fed a methionine- and choline-deficient diet.¹⁹ Thus, ezetimibe is a potential new therapeutic agent for human NASH.²⁰ In the present study, we also assessed the effect of ezetimibe on the development of NASH in our rabbit model.     

Increased Hepatic Myeloperoxidase Activity in Obese Subjects with Nonalcoholic Steatohepatitis

Inflammation and oxidative stress are considered critical factors in the progression of nonalcoholic fatty liver disease. Myeloperoxidase (MPO) is an important neutrophil enzyme that can generate aggressive oxidants; therefore, we studied the association between MPO and nonalcoholic fatty liver disease. The distribution of inflammatory cells containing MPO in liver biopsies of 40 severely obese subjects with either nonalcoholic steatohepatitis (NASH) (n = 22) or simple steatosis (n = 18) was investigated by immunohistochemistry. MPO-derived oxidative protein modifications were identified by immunohistochemistry and correlated to hepatic gene expression of CXC chemokines and M1/M2 macrophage markers as determined by quantitative PCR. MPO plasma levels were determined by ELISA. The number of hepatic neutrophils and MPO-positive Kupffer cells was increased in NASH and was accompanied by accumulation of hypochlorite-modified and nitrated proteins, which can be generated by the MPO-H₂O₂ system. Liver CXC chemokine expression was higher in patients with accumulation of MPO-mediated oxidation products and correlated with hepatic neutrophil sequestration. Plasma MPO levels were elevated in NASH patients. Interestingly, neutrophils frequently surrounded steatotic hepatocytes, resembling the crown-like structures found in obese adipose tissue. Furthermore, hepatic M2 macrophage marker gene expression was increased in NASH. Our data indicate that accumulation of MPO-mediated oxidation products, partly derived from Kupffer cell MPO, is associated with induction of CXC chemokines and hepatic neutrophil infiltration and may contribute to the development of NASH.The obesity-associated infiltration of neutrophils and macrophages into liver and adipose tissue is increasingly appreciated to be central to the development of chronic diseases such as nonalcoholic fatty liver disease (NAFLD) and insulin resistance.¹ In both liver and adipose tissue, increased levels of free fatty acids are thought to initiate the activation of inflammatory pathways that are responsible for attraction and activation of immune cells.^2,3 Furthermore, recruitment of neutrophils to the liver can be mediated by secretion of the CXC chemokines interleukin (IL)–8 and growth-related oncogene (GRO)-α.^4,5 The inflammatory processes in the liver have been shown to play a particularly important role in the progression of the benign steatotic stage of NAFLD to the more advanced stages of nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis.^6,7Next to inflammation, oxidative stress has been put forward as an important contributor to the progression of NAFLD.^8,9 For example, oxidative stress leading to lipid peroxidation induces the progression of steatosis toward necroinflammation or fibrosis in both animal models of steatohepatitis and humans with steatotic liver disorders.^10,11,12,13 Oxidative stress in the fatty liver can result from cytochrome P450 activity, peroxisomal β-oxidation, mitochondrial electron leak, as well as activities of recruited inflammatory cells.¹⁴One of the principal molecules released after recruitment and activation of phagocytes is myeloperoxidase (MPO), an important enzyme involved in the generation of reactive oxygen species.¹⁵ MPO is highly expressed by neutrophils and as such widely used as a neutrophil marker. In the presence of physiological chloride concentrations, MPO reacts with hydrogen peroxide (H₂O₂, formed by the respiratory burst) to catalyze formation of hypochlorous acid/hypochlorite (HOCl/OCl⁻) and other oxidizing species.¹⁵ These oxidants may contribute to host tissue damage at sites of inflammation through reactions with a wide range of biological substrates, including DNA, lipids, and protein amino groups.¹⁶ In the absence of physiological chloride concentrations, the MPO-H₂O₂ system can also form reactive nitrogen species¹⁷ that may initiate lipid peroxidation or form protein tyrosine residues, another posttranslational modification found in many pathological conditions.¹⁸Besides its abundant presence in neutrophils, where MPO makes up ∼5% of the total cell protein content, lower levels of MPO have been found in monocytes, certain macrophages,^19,20,21 and in a subpopulation of Kupffer cells.²² The presence of MPO in these cells may reflect their inflammatory status, which was recently shown to be modulated in obesity. For example, anti-inflammatory M2 macrophages with tissue-remodeling capacities were largely absent in obese adipose tissue in mice, whereas accumulation of proinflammatory M1 macrophages was associated with obesity and insulin resistance.²³ Moreover, attenuation of the M2 differentiation program of Kupffer cells was associated with hepatic steatosis.²⁴ Interestingly, M1 macrophages are known to generate high amounts of reactive oxygen and nitrogen species.²⁵ Therefore, MPO-containing M1 macrophages/Kupffer cells could play a role in NAFLD.To study the potential contribution of increased MPO activity to the progression of human NAFLD, we quantified inflammatory cells that express MPO and assessed hepatic accumulation of HOCl-modified proteins and nitrated proteins. Furthermore, we determined hepatic gene expression of IL-8 and GRO-α in relation to MPO-derived tissue damage and hepatic neutrophil sequestration to investigate a potential link between MPO activity and neutrophil accumulation. In addition, we analyzed for the first time hepatic gene expression of M1 and M2 macrophage phenotype markers in human NAFLD. Our results support an important role for Kupffer cell MPO-induced oxidative stress in the progression of NAFLD.     

Development of Arterial Blood Supply in Experimental Liver Metastases

In this study, we present a mechanism for the development of arterial blood supply in experimental liver metastases. To analyze the arterialization process of experimental liver metastases, we elucidated a few key questions regarding the blood supply of hepatic lobules in mice. The microvasculature of the mouse liver is characterized by numerous arterioportal anastomoses and arterial terminations at the base of the lobules. These terminations supply one hepatic microcirculatory subunit per lobule, which we call an arterial hepatic microcirculatory subunit (aHMS). The process of arterialization can be divided into the following steps: 1) distortion of the aHMS by metastasis; 2) initial fusion of the sinusoids of the aHMS at the tumor parenchyma interface; 3) fusion of the sinusoids located at the base of the aHMSs, which leads to the disruption of the vascular sphincter (burst pipe); 4) incorporation of the dilated artery and the fused sinusoids into the tumor; and 5) further development of the tumor vasculature (arterial tree) by proliferation, remodeling, and continuous incorporation of fused sinusoids at the tumor–parenchyma interface. This process leads to the inevitable arterialization of liver metastases above the 2000- to 2500-μm size, regardless of the origin and growth pattern of the tumor.It is widely accepted that hepatic metastases and tumors are predominantly supplied by arterial blood, a notion that serves as the basis for hepatic arterial chemotherapy and chemoembolization.^{1,2,3,4,5,6,7} The most cited article on this field dates back to the 1950s.¹ Since then numerous papers have been published using human and experimental materials and different methods such as corrosion casting, confocal and electron microscopy, angiography, radiolabeled microspheres, and in vivo microscopy, have been used to study the blood supply of liver metastases.^{2,3,4,5,6,7,8,9,10,11,12,13,14,15,16} A large proportion of these articles have confirmed the original observation of Breedis and Young,¹ but no mechanism for the development of the arterial blood supply in metastases has ever been presented.^2,3,4,5,6,7 On the other hand, numerous papers, including ours, have emphasized the contribution of the portal vein, either directly or through the sinusoids in the blood supply of hepatic metastases.^{8,9,10,11,12,13,14} This apparent contradiction might result from the observed continuity of the sinusoidal with the tumor vasculature and the presumption that blood flows in an “outside-in” direction from the sinusoids toward the tumor vasculature. Most of the studies dealing with the blood supply of metastases have neglected the importance of arterioportal anastomoses and other interspecies differences in the hepatic microcirculation, which could lead to seriously biased results. According to the observations of Yamamoto et al¹⁵ there are extensive arterioportal anastomoses throughout the vascular tree in rats, whereas a separate arterial and portal tree, without direct arterioportal communication, can be observed in hamster and human liver. Opinions about the presence of arterioportal anastomoses in mice are controversial^10,16; therefore, we have addressed this question first.The classic lobule can be divided into several conical hepatic microcirculatory subunits (HMSs) supplied by a single inlet portal venule. Hepatic arterioles terminate either on the inlet venules or directly on sinusoids. The number of these terminations within a lobule is species-dependent. The blood flow through the inlet venules and terminal arterioles is regulated by sphincters.¹⁷ The most detailed studies on microcirculation of the liver and vessel architecture of liver metastases were performed by corrosion casting. However, in these studies the livers were completely filled with uncolored resin, which made analyzing the three-dimensional organization of the deep interlobular vessels difficult.^10,14,15In the present study, we used a two color corrosion casting technique to analyze the blood supply in liver metastases of experimental tumors in mice. A special filling method was used to prevent the mixing of the “portal and arterial resin” upstream of the hepatic sinusoids. This technique enabled us to analyze separately the contribution of the two vascular systems to the blood supply of liver metastases and to establish the steps of the arterialization process.     

Acidic Sphingomyelinase Controls Hepatic Stellate Cell Activation and in Vivo Liver Fibrogenesis

The mechanisms linking hepatocellular death, hepatic stellate cell (HSC) activation, and liver fibrosis are largely unknown. Here, we investigate whether acidic sphingomyelinase (ASMase), a known regulator of death receptor and stress-induced hepatocyte apoptosis, plays a role in liver fibrogenesis. We show that selective stimulation of ASMase (up to sixfold), but not neutral sphingomyelinase, occurs during the transdifferentiation/activation of primary mouse HSCs into myofibroblast-like cells, coinciding with cathepsin B (CtsB) and D (CtsD) processing. ASMase inhibition or genetic down-regulation by small interfering RNA blunted CtsB/D processing, preventing the activation and proliferation of mouse and human HSCs (LX2 cells). In accordance, HSCs from heterozygous ASMase mice exhibited decreased CtsB/D processing, as well as lower levels of α-smooth muscle actin expression and proliferation. Moreover, pharmacological CtsB inhibition reproduced the antagonism of ASMase in preventing the fibrogenic properties of HSCs, without affecting ASMase activity. Interestingly, liver fibrosis induced by bile duct ligation or carbon tetrachloride administration was reduced in heterozygous ASMase mice compared with that in wild-type animals, regardless of their sensitivity to liver injury in either model. To provide further evidence for the ASMase-CtsB pathway in hepatic fibrosis, liver samples from patients with nonalcoholic steatohepatitis were studied. CtsB and ASMase mRNA levels increased eight- and threefold, respectively, in patients compared with healthy controls. These findings illustrate a novel role of ASMase in HSC biology and liver fibrogenesis by regulating its downstream effectors CtsB/D.Hepatic fibrosis is a wound-healing response of the liver to chronic injury and represents a common and complex clinical challenge worldwide.^1,2,3 Liver fibrosis often reflects progressive liver disease caused by a variety of factors and etiologies including viral infection, cholestasis, alcoholic steatohepatitis, and nonalcoholic steatohepatitis (NASH). In developed countries, nonalcoholic fatty liver disease is associated with increasing rates of obesity and is currently one of the most common forms of chronic liver disease and a major cause of hepatic fibrosis. Nonalcoholic fatty liver disease, which encompasses a spectrum of liver diseases ranging from simple steatosis to NASH, is characterized by oxidative stress, inflammation, hepatocellular death, and fibrosis, which can further progress to cirrhosis and hepatocellular carcinoma.^4,5,6 Thus, the number of individuals at risk for fibrosis and end-stage liver disease is rapidly expanding.⁷In the liver, myofibroblasts are potentially derived from a number of cellular sources including activated hepatic stellate cells (HSCs), which mediate the fibrotic component of the wound-healing response to chronic injury.^1,2,8 Under these conditions, HSCs undergo a phenotypic transformation from quiescent, nonproliferating, retinoid-storing cells, to a proliferating, matrix-producing phenotype similar to myofibroblasts responsible for the progression of liver fibrosis.^9,10 Despite increasing knowledge of the mechanisms that govern this so-called “activation,” the treatment for advanced liver fibrosis is inefficient, and hence the development of novel antifibrotic targets and therapies is essential.Membrane sphingolipids have been implicated in signal transduction cascades regulating proliferation, differentiation, and cell death.^11,12 Among sphingolipids, ceramide has attracted considerable attention, given its central role in sphingolipid metabolism. Cellular ceramide levels increase in response to a variety of stimuli and agents from chemotherapy¹³ to death ligands,^14,15,16,17 radiation,¹⁸ or viral/bacterial infections,^19,20 because of the hydrolysis of sphingomyelin mainly by two specific phosphodiesterases named sphingomyelinases, a plasma membrane-bound neutral sphingomyelinase (NSMase) and an acidic SMase (ASMase).^21,22,23,24 In particular, ASMase is expressed by almost any cell type and is mainly located at the endosomal/lysosomal compartment, although it has also been found at the plasma membrane in specific microdomains, where it serves as a signaling platform by cell surface receptors such as Fas.^14,15,16,17 The placement of ASMase at the crossroad of vesicular trafficking and cellular signaling suggests that ASMase exerts important functions in signal transduction pathways. For instance, tumor necrosis factor (TNF) is a prominent prototype of an ASMase-activating cytokine that is central to the regulation of both the innate and the adaptive immune system as well as cell death.^25,26 Although ASMase has been recognized to play a key role in the pathophysiology of different common diseases,²⁷ its specific contribution to liver fibrosis has not been examined previously.Moreover, the participation of cathepsin D (CtsD) as a target of ASMase-generated ceramide in endolysosomal compartments^28,29 and the observation that cathepsin B (CtsB)-deficient hepatocytes are resistant to TNF-dependent cell death³⁰ imply a functional relationship between ASMase and cathepsins, at least, in cell death regulation. However, this specific link has not been previously addressed in HSC biology and liver fibrosis. Because we have just uncovered a new function of CtsB and CtsD in mediating HSC activation and liver fibrosis,³¹ our aim was to investigate the role of ASMase in hepatic fibrogenesis. In this study, we tested whether ASMase contributes to the activation of mouse HSCs in vitro and to the development of fibrosis in vivo after bile duct ligation (BDL) or chronic carbon tetrachloride (CCl₄) injection. Here, we show that ASMase activity is induced during the transdifferentiation of HSCs into myofibroblasts, whereas HSC activation and proliferation are reduced by genetic or pharmacological antagonism of ASMase. Interestingly, heterozygous ASMase mice were resistant to BDL and chronic CCl₄ administration-induced liver fibrosis compared with wild-type mice, even though their sensitivity to liver injury was markedly different with reduced liver damage in ASMase^+/− mice after BDL but not after CCl₄ administration. In addition, patients with NASH displayed enhanced levels of both CtsB and ASMase mRNA, underscoring the role of the ASMase-cathepsins axis in liver pathology and its relevance for future therapeutic approaches.     

Histamine Regulation in Glucose and Lipid Metabolism via Histamine Receptors : Model for Nonalcoholic Steatohepatitis in Mice

Histamine has been proposed to be an important regulator of energy intake and expenditure. The aim of this study was to evaluate histamine regulation of glucose and lipid metabolism and development of nonalcoholic steatohepatitis (NASH) with a hyperlipidemic diet. Histamine regulation of glucose and lipid metabolism, adipocytokine production, and development of hyperlipidemia-induced hepatic injury were studied in histamine H1 (H1R^−/−) and H2 (H2R^−/−) receptor knockout and wild-type mice. H1R^−/− mice showed mildly increased insulin resistance. In contrast, H2R^−/− mice manifested profound insulin resistance and glucose intolerance. High-fat/high-cholesterol feeding enhanced insulin resistance and glucose intolerance. Studies with two-deoxy-2-[¹⁸F]-fluoro-d-glucose and positron emission tomography showed a brain glucose allocation in H1R^−/− mice. In addition, severe NASH with hypoadiponectinemia as well as hepatic triglyceride and free cholesterol accumulation and increased blood hepatic enzymes were observed in H2R^−/− mice. H1R^−/− mice showed an obese phenotype with visceral adiposity, hyperleptinemia, and less severe hepatic steatosis and inflammation with increased hepatic triglyceride. These data suggest that H1R and H2R signaling may regulate glucose and lipid metabolism and development of hyperlipidemia-induced NASH.Histamine, one of the mediators of inflammation and immunity, is produced from L-histidine by the rate-limiting enzyme histidine decarboxylase (HDC). HDC is expressed in various types of cells, including mast cells, monocytes/macrophages, T lymphocytes, enterochromaffin-like cells, and neuronal cells.^1,2,3 The effects of histamine are mediated through specific histamine receptors, which have been classified into the H1, H2, H3, and H4 subtypes.⁴ The recent development of gene-modified mice lacking HDC or histamine receptors has provided valuable tools to analyze the functions of histamine.⁵ For example, HDC knockout (KO) mice were reported to show clinical features of visceral adiposity, hyperleptinemia, and decreased glucose tolerance.⁶ In addition, it was reported that H1R^−/− mice fed a high-fat diet showed increased fat deposition and leptin resistance and that disruption of the H3R gene in mice resulted in an obese phenotype and glucose intolerance, with elevated blood insulin and leptin levels.^7,8 Taken together, these studies indicate that histamine plays important roles in energy regulation and metabolism.In contrast, insulin resistance/metabolic syndrome, which increases the risk for atherosclerosis and cardiovascular events, is an aggregate of disorders related to obesity or visceral adiposity, insulin resistance, hyperlipidemia, and hypertension.^9,10 Recently, the liver has also been recognized as one of the pathological targets of metabolic syndrome.^11,12 Insulin resistance is associated with increased adiposity and nonalcoholic fatty liver disease, which can lead to the advanced condition known as nonalcoholic steatohepatitis (NASH). Typical morphological features of NASH in humans include steatosis, inflammation and pericellular fibrosis.¹³ Obese and diabetic ob/ob mice develop steatohepatitis, but not fibrosis, in their liver.¹⁴ Other obese and diabetic db/db mice fed a diet lacking methionine and choline exhibited liver fibrosis as a model of NASH.¹⁵ Adiponectin, an adipocytokine secreted from adipocytes,^16,17 was recently reported to be centrally involved in the pathogenesis of metabolic syndrome and nonalcoholic fatty liver disease.^18,19Because the loss of histamine functions has been suggested to result in disturbed energy regulation and metabolism,^6,7,8 we speculated that the functions of histamine might be closely related to the pathogenesis of insulin resistance syndrome, metabolic disturbances, and NASH. In this study, we evaluated the phenotypic differences of wild-type, H1R^−/−, and H2R^−/− mice in terms of glucose metabolism, lipid metabolism, and the expression of adipocytokines, including adiponectin and leptin after high-fat/high-cholesterol diet (HcD). In addition, we examined in vivo glucose uptake using 2-deoxy-2-[¹⁸F]-fluoro-d-glucose ([¹⁸F]FDG) and positron imaging of whole mice bodies.     

The Immunoconjugate “Icon” Targets Aberrantly Expressed Endothelial Tissue Factor Causing Regression of Endometriosis

Endometriosis is a major cause of chronic pain, infertility, medical and surgical interventions, and health care expenditures. Tissue factor (TF), the primary initiator of coagulation and a modulator of angiogenesis, is not normally expressed by the endothelium; however, prior studies have demonstrated that both blood vessels in solid tumors and choroidal tissue in macular degeneration express endothelial TF. The present study describes the anomalous expression of TF by endothelial cells in endometriotic lesions. The immunoconjugate molecule (Icon), which binds with high affinity and specificity to this aberrant endothelial TF, has been shown to induce a cytolytic immune response that eradicates tumor and choroidal blood vessels. Using an athymic mouse model of endometriosis, we now report that Icon largely destroys endometriotic implants by vascular disruption without apparent toxicity, reduced fertility, or subsequent teratogenic effects. Unlike antiangiogenic treatments that can only target developing angiogenesis, Icon eliminates pre-existing pathological vessels. Thus, Icon could serve as a novel, nontoxic, fertility-preserving, and effective treatment for endometriosis.Endometriosis is a gynecological disorder characterized by the presence of functional endometrial tissue outside of the uterus.¹ The disease affects up to 10% of all reproductive-age women and up to 50% of infertile women.^1,2,3 Endometriotic lesions are primarily located on the pelvic peritoneum and ovaries, but can also be found in the colon, pericardium, pleura, lung parenchyma, and brain.² Implants can cause pelvic adhesions, chronic pelvic pain, bowel obstruction, and infertility requiring repetitive, extensive, and expensive medical and surgical treatments.^1,4,5The etiology of the disease has been ascribed to retrograde menstruation, coelomic metaplasia, or both.^1,6,7,8,9 Although its pathogenesis involves a complex interplay of genetic, anatomical, environmental, and immunological factors, there is general agreement that it is associated with a local inflammatory response and that vascularization at the site of ectopic attachment of the lesions is a key determinant in its pathogenesis.^{1,10,11,12,13} Angiogenic agents such as vascular endothelial growth factor and the angiopoietins are likely mediators of endometriotic neovascularization.^1,3,11,12,13Tissue factor (TF), the transmembrane initiator of hemostasis, is not physiologically expressed by endothelial cells but plays a crucial role in embryonic and oncogenic angiogenesis.^14,15,16,17 Recent studies indicate that the type-2 proteinase activated receptor (PAR-2) is intimately involved in TF-mediated signaling and angiogenesis.^15,18,19 Tissue factor may initiate differential signaling pathways in physiological compared with pathological angiogenesis,²⁰ to wit, the physiological pathway is mediated by TF–thrombin–PAR-1 and the pathological pathway by TF/factor FVIIa/PAR-2 signaling.²⁰ These latter vessels display abnormal structure and function and are poorly associated with pericytes causing leakiness and edema.^21,22Our prior studies demonstrated that both TF and PAR-2 are up-regulated in endometria of women with endometriosis compared with unaffected women.²³In this report, we describe the anomalous endothelial expression of TF in ectopic endometrium derived from women with endometriosis. We posited that a novel chimeric immunoconjugate molecule (Icon) will specifically target endothelial TF in ectopic implants leading to their devascularization and atrophy. Icon is composed of a mutated low-coagulation–inducing factor VII (fVII) domain that binds to TF with high affinity and an IgG1 Fc (fVII/IgG1 Fc) effector domain that activates a natural killer cell cytolytic response against TF bearing endothelial cells.^24,25,26,27 This report confirms our hypothesis by demonstrating that Icon largely destroys pre-established human endometriotic lesions in an athymic mouse model without untoward systemic effects, altered fertility, or subsequent teratogenesis.     

Neuroprotective Effect of Oligodendrocyte Precursor Cell Transplantation in a Long-Term Model of Periventricular Leukomalacia

Perinatal white matter injury, or periventricular leukomalacia (PVL), is the most common cause of brain injury in premature infants and is the leading cause of cerebral palsy. Despite increasing numbers of surviving extreme premature infants and associated long-term neurological morbidity, our understanding and treatment of PVL remains incomplete. Inflammation- or ischemia/hypoxia-based rodent models, although immensely valuable, are largely restricted to reproducing short-term features of up to 3 weeks after injury. Given the long-term sequelae of PVL, there is a need for subchronic models that will enable testing of putative neuroprotective therapies. Here, we report long term characterization of a neonatal inflammation-induced rat model of PVL. We show bilateral ventriculomegaly, inflammation, reactive astrogliosis, injury to pre-oligodendrocytes, and neuronal loss 8 weeks after injury. We demonstrate neuroprotective effects of oligodendrocyte precursor cell transplantation. Our findings present a subchronic model of PVL and highlight the tissue protective effects of oligodendrocyte precursor cell transplants that demonstrate the potential of cell-based therapy for PVL.Premature, low-birth weight infants are commonly diagnosed with perinatal white matter damage, which leads to long-term neurological deficits including cerebral palsy.^1,2,3,4 Congenital encephalomyelitis was described almost 150 years ago, a disease in newborn children characterized by pale softened zones of degeneration within the deep white matter surrounding lateral ventricles and is now often referred to as periventricular leukomalacia (PVL).⁵ The white matter damage that occurs in preterm infants has more recently been shown to be also accompanied by significant cerebral-cortex and deep-gray matter abnormalities leading to neurodegeneration and altered neurobehavioral performance.⁶PVL is characterized by selective oligodendrocyte precursor cell (OPC) loss resulting in delayed or disrupted myelination, white matter atrophy and ventriculomegaly, neuronal loss, and cyst and scar formation. Extreme premature birth (approximately 23 to 32 weeks) accompanied by inflammation or infection corresponds to the period when immature dividing and differentiating oligodendrocytes predominate in the cerebral white matter.^7,8,9,10 PVL has a complex etiology. The two most important determinants are cerebral hypoperfusion and maternal intrauterine infection. Periventricular white matter is susceptible to hypoperfusion due to the comparative immaturity of the periventricular vasculature of the preterm infant.^11,12 Early oligodendrocyte lineage cells are vulnerable to the consequences of hypoperfusion and subsequent microglial and astrocytic activation on account of amplified glutamate receptor-mediated responses and lack of efficient antioxidant protection.^{13,14,15,16,17,18} Maternal infection, recently implicated as a causative factor in the pathogenesis of PVL,^{19,20,21,22,23} is believed to initiate an inflammatory/cytokine cascade that results in the release of early-response pro-inflammatory cytokines such as tumor necrosis factor–α (TNF-α), interleukin−1 β (IL-1β) and interleukin−6 (IL-6), and causes damage to immature oligodendrocytes.²⁴ TNF-α appears to have a particularly important role in PVL pathogenesis with in vitro evidence suggesting direct damage to OPCs,^25,26 while IL-1β and IL-6 modulate injury indirectly.^27,28Although cells of the oligodendrocyte lineage are regarded as the primary target in the pathogenesis of PVL, there is increasing evidence that neonatal white matter damage is accompanied by gray matter abnormalities, including neuronal loss, impaired axonal guidance, and altered synaptogenesis.^6,29 Premature newborns affected by PVL often have smaller cerebral cortex and deep gray matter volumes, reduced cortical neurons, and alterations in the orientation of central white matter fiber tracts.^6,30,31,32 Together, these data suggest that developing neurons, like immature oligodendrocyte lineage cells, are also vulnerable to injury caused by inflammatory response or hypoxia.Aspects of PVL have been modeled by ischemia/hypoxia and inflammation-mediated rodent models. Animal models of hypoxia-ischemia have clearly shown that following brain injury there is reduced myelination, enlarged ventricles, loss of neurons and damage to axons and dendrites and altered neurobehavioral performances.^{33,34,35,36,37,38,39} Experimentally induced inflammation has been used in a number of studies to model PVL pathology and test potential treatments.^23,40,41,42 Administration of the endotoxin lipopolysaccharide (LPS), a potent inducer of innate immune response and inflammation,^43,44 either intracerebrally during early neonatal period, intrauterine or peritoneally to a pregnant mother results in inflammation and hypomyelination.^20,22,23,38 Moreover, in animal models of LPS-induced PVL neuronal loss and a reduction in neurite length in the parietal cortex has been observed.^42,45 In vitro evidence suggests LPS is not directly toxic to OPCs, but causes injury through the activation of Toll-like receptor 4-positive microglia, which is a source of pro-inflammatory cytokines, nitric oxide, and free radicals.^44,45,46 Furthermore, it has been shown that LPS-induced inflammation increases the susceptibility of white matter to injury in response to otherwise “harmless” subthreshold hypoxic-ischemic insult.⁴⁷ Current models of PVL are typically short term, with studies using either hypoxia-ischemia or inflammation rarely extending analysis beyond 14 days. There is therefore a need to evaluate the longer-term, subchronic consequences of LPS injury and specifically address whether hypomyelination and neuronal injury are self-limiting or indeed spontaneously repair.Cellular therapeutic strategies are predicated on cell replacement and/or tissue protection independent of specific cellular differentiation. Progenitor cells including OPCs have previously been used for cellular replacement of damaged or lost cells in a variety of CNS injury models where damage to myelin and neuronal loss occur.^{48,49,50,51,52,53} Furthermore, there is accumulating evidence that implicates progenitor cell mediated neuroprotection through a variety of mechanisms including graft derived neurotrophic support independent of directed differentiation.^54,55 Moreover, there is evidence to suggest that OPCs secrete factors that are capable of supporting neuronal survival,^{56,57,58,59,60,61} thus suggesting they may be a potential therapeutic source for replacing lost or damaged cells and protecting healthy tissue following neonatal brain injury. In this study we have examined the subchronic effects of LPS induced injury and then examined the putative neuroprotective effects of OPC transplantation.     

Protective Role of Interleukin-17 in Murine NKT Cell-Driven Acute Experimental Hepatitis

NKT cells are highly enriched within the liver. On activation NKT cells rapidly release large quantities of different cytokines which subsequently activate, recruit, or modulate cells important for the development of hepatic inflammation. Recently, it has been demonstrated that NKT cells can also produce interleukin-17 (IL-17), a proinflammatory cytokine that is also known to have diverse immunoregulatory effects. The role played by IL-17 in hepatic inflammation is unclear. Here we show that during α-galactosylceramide (αGalCer)-induced hepatitis in mice, a model of hepatitis driven by specific activation of the innate immune system via NKT cells within the liver, NK1.1⁺ and CD4⁺ iNKT cells rapidly produce IL-17 and are the main IL-17-producing cells within the liver. Administration of IL-17 neutralizing monoclonal antibodies before αGalCer injection significantly exacerbated hepatitis, in association with a significant increase in hepatic neutrophil and proinflammatory monocyte (ie, producing IL-12, tumor necrosis factor-α) recruitment, and increased hepatic mRNA and protein expression for the relevant neutrophil and monocyte chemokines CXCL5/LIX and CCL2/MCP-1, respectively. In contrast, administration of exogenous recombinant murine IL-17 before α-GalCer injection ameliorated hepatitis and inhibited the recruitment of inflammatory monocytes into the liver. Our results demonstrate that hepatic iNKT cells specifically activated with α-GalCer rapidly produce IL-17, and IL-17 produced after α-GalCer administration inhibits the development of hepatitis.The cytokine interleukin-17A (IL-17) has been increasingly identified as an important regulator of the inflammatory response.^1,2,3 Initially, a new subset of CD4⁺ T cells were considered to be the source of IL-17 and were classified as Th17 cells.^2,3 IL-17 secreted from Th17 cells was implicated as a proinflammatory mediator in a number of experimental models of inflammation, especially those associated with autoimmunity and an adaptive immune response.^4,5,6 However, more recently IL-17 has also been shown to be able to suppress inflammatory responses, mainly in experimental models which are characterized by a more pronounced innate immune response. Specifically, IL-17 has been shown to suppress inflammation in experimental murine models of asthma,⁷ gastritis,⁸ colitis,^9,10 and atherosclerosis.¹¹ However, the role of IL-17 in regulating hepatic inflammation remains unclear. In patients with viral hepatitis, alcoholic liver disease, and autoimmune liver diseases, numbers of IL-17-producing hepatic T cells are increased.¹² In murine models of liver inflammation the role of IL-17 in regulating the inflammatory response remains controversial. In murine T-cell-mediated hepatitis induced by concanavalin A administration, IL-17 has been shown to be both proinflammatory, as well as without a direct inflammation modulating role.^13,14NKT cells are an important component of the innate immune response and are highly enriched within the liver.¹⁵ NKT cells are activated by glycolipid antigens presented in association with the major histocompatibility complex class I–like molecule CD1d expressed on the surface of antigen presenting cells.¹⁶ Activation of NKT cells in this fashion results in the rapid production and release of large amounts of both Th1; eg, interferon (IFN) γ, tumor necrosis factor (TNF) α, and Th2 (eg, IL-4) cytokines.¹⁶ NKT cells have been implicated in human liver disease and are of critical importance in the initiation and development of hepatitis in numerous murine models.^15,17,18 More recently, NKT cells have also been shown to be capable of rapidly producing IL-17 after activation.^19,20,21 To date IL-17 has been reported to be produced mainly by type II (ie, non-invariant) and NK1.1 negative NKT cells^19,22,23; however, within the murine liver most NKT cells express CD4 and NK1.1 and are classified as invariant (iNKT) or type I NKT cells.^15,16α-Galactosylceramide (αGalCer) is a glycolipid, originally isolated from a marine sponge, which specifically activates iNKT cells in both humans and mice after being presented by antigen presenting cells in the context of CD1d.¹⁶ iNKT cells activated in this fashion can in turn transactivate numerous other cell types within the liver, including other components of the innate immune response such as macrophages and NK cells.^24,25 This property of αGalCer has generated interest in developing this compound as an immune stimulating agent for the treatment of human disease, including liver cancers.²⁴ However, αGalCer treatment also induces hepatitis in mice and therefore has been used as an experimental model to study hepatic immune and inflammatory responses which result from the specific activation of iNKT cells and the subsequent downstream stimulation of the hepatic innate immune system.^26,27Therefore, we undertook this series of experiments to determine first whether hepatic NK1.1 positive iNKT cells could also produce IL-17 after specific activation. In addition, given that the adaptive Th17 response develops more slowly, we wanted to determine the role of IL-17, released as part of the early iNKT cell–driven innate hepatic immune response, in the regulation of hepatitis induced by the administration of αGalCer.     

Long-Term Expression of Tissue-Inhibitor of Matrix Metalloproteinase-1 in the Murine Central Nervous System Does Not Alter the Morphological and Behavioral Phenotype but Alleviates the Course of Experimental Allergic Encephalomyelitis

Tissue inhibitors of metalloproteinases (TIMPs) are a family of closely related proteins that inhibit matrix metalloproteinases (MMPs). In the central nervous system (CNS), TIMPs 2, 3, and 4 are constitutively expressed at high levels, whereas TIMP1 can be induced by various stimuli. Here, we studied the effects of constitutive expression of TIMP1 in the CNS in transgenic mice. Transgene expression started prenatally and persisted throughout lifetime at high levels. Since MMP activity has been implicated in CNS development, in proper function of the adult CNS, and in inflammatory disorders, we investigated Timp1-induced CNS alterations. Despite sufficient MMP inhibition, high expressor transgenic mice had a normal phenotype. The absence of compensatory up-regulation of MMP genes in the CNS of Timp1 transgenic mice indicates that development, learning, and memory functions do not require the entire MMP arsenal. To elucidate the effects of strong Timp1 expression in CNS inflammation, we induced experimental allergic encephalomyelitis. We observed a Timp1 dose-dependent mitigation of both experimental allergic encephalomyelitis symptoms and histological lesions in the CNS of transgenic mice. All in all, our data demonstrate that (1) long-term CNS expression of TIMP1 with complete suppression of gelatinolytic activity does not interfere with physiological brain function and (2) TIMP1 might constitute a promising candidate for long-term therapeutic treatment of inflammatory CNS diseases such as multiple sclerosis.Tissue-inhibitor of matrix metalloproteinase-1 (TIMP1), a tightly regulated 28.5-kDa glycoprotein,^1,2 belongs to a family of multifunctional secreted proteins (TIMPs 1 to 4) that regulate the proteolytic activity of matrix metalloproteinases (MMPs). Together, MMPs and TIMPs control the pericellular environment, including the turnover of extracellular matrix proteins, bioavailability of growth factors and cytokines, and shedding of membrane receptors.³ In the central nervous system (CNS), expression of MMP and TIMP genes is highly orchestrated during development and normal brain function.^4,5,6 The spatio-temporal expression pattern of TIMP1 indicates a developmental role in the hippocampus and the cerebellum.^4,7,8,9 TIMP1 was also proposed as a candidate plasticity protein in learning and memory.^10,11 Several MMPs on the other hand are expressed in the developing cerebellum,⁴ and in migrating neural precursors,^12,13 and MMP activity was localized to the growth cone of cultured sympathetic neurons.^14,15,16 In the normal adult CNS, however, MMP expression is low.^17,18 Alterations of the MMP/TIMP ratio are tightly associated with pathological processes and clearly influence the course of various diseases.^12,17,18,19 In autoimmune diseases such as multiple sclerosis and the animal model experimental allergic encephalomyelitis (EAE), elevated activity of several MMPs was detected and suspected to influence the outcome of the disease.^{17,20,21,22,23} Accordingly, synthetic MMP inhibitors blocked the blood-brain-barrier disruption and improved the clinical condition of the animals,^23,24,25 and Timp1 deficient mice revealed increased leukocyte infiltration as well as enhanced spinal cord demyelination.²⁶ Due to serious side-effects, however, only few synthetic MMP-inhibitors have been introduced to human therapy.²⁷Since high levels of Timp1 were expressed by astrocytes around the inflammatory lesions in EAE¹⁷ and to further investigate the role of Timp1 in the CNS, we targeted its expression to astrocytes in a transgenic mouse model.     

Selective Cell Death of Hyperploid Neurons in Alzheimer’s Disease

Aneuploidy, an abnormal number of copies of a genomic region, might be a significant source for neuronal complexity, intercellular diversity, and evolution. Genomic instability associated with aneuploidy, however, can also lead to developmental abnormalities and decreased cellular fitness. Here we show that neurons with a more-than-diploid content of DNA are increased in preclinical stages of Alzheimer’s disease (AD) and are selectively affected by cell death during progression of the disease. Present findings show that neuronal hyperploidy in AD is associated with a decreased viability. Hyperploidy of neurons thus represents a direct molecular signature of cells prone to death in AD and indicates that a failure of neuronal differentiation is a critical pathogenetic event in AD.Understanding the mechanisms underlying generation of neuronal variability and complexity remains a basic challenge to neuroscience. Structural variation in the human genome is likely to be one important mechanism for neuronal diversity and brain disease.¹ The genetic profile of a cell can be permanently altered by chromosomal aneuploidy (i.e., an abnormal number of copies of a genomic region). A combination of multiple different forms of aneuploid cells due to loss or gain of whole chromosomes (mosaic aneuploidy) giving rise to cellular diversity at the genomic level have been described in neurons of the normal and diseased adult human brain.^{2,3,4,5,6,7,8,9,10,11,12}Cells in normal individuals have basically been assumed to contain identical euploid genomes. Still, earlier hypotheses suggested that a number of mammalian somatic tissues are populated by polyploid cells. Adult neurons of mammals were assumed to be postmitotic cells characterized to some extent by a polyploid chromosome complement. Testing this hypothesis in the past through histochemical methods, however, yielded controversial results through technical limitations.^13,14 However, with the recent development of molecular cytogenic techniques, aneuploid cells in the normal developing and mature brain have clearly been identified, indicating that the maintenance of aneuploid neurons in the adult CNS is a widespread, if not universal, property of organization.^{2,3,4,5,6,7,8,9,10,11,12,15}Recent studies of the embryonic brain have shown that approximately one-third of the dividing cells that give rise to the cerebral cortex have genetic variability, manifested as chromosome aneuploidy.^3,7,12 Neurons that constitute the adult brain arise from mitotic neural progenitor cells in the ventricular zone, a proliferating region where aneuploid cells appear to be generated through various chromosome segregation defects initially.^3,10 While a portion of these aneuploid cells apparently die during development,^3,16,17 aneuploid neurons have been identified in the mature brain in all areas assayed^{2,3,4,5,6,7,8,9,10,11,12,15} indicating that aneuploidy does not necessarily impair viability.¹⁸ Aneuploid neurons in the adult have been shown to make distant connections and express markers associated with neural activity, which indicates that these neurons can be integrated into brain circuitry.^4,12Contrary to this physiological consequences of “low-level” aneuploidy potentially contributing to neuronal diversity, aneuploidy above a critical threshold might be detrimental. Genomic instability and imbalances in gene dosage associated with aneuploidy can lead to developmental abnormalities, decreased cellular and organismal fitness, and increased susceptibility to disease.^19,20Aneuploid cells have typically been associated with pathophysiological conditions such as cancer,²¹ and most aneuploid syndromes present brain phenotypes and show a high vulnerability for psychiatric disorders.²² Mental impairment is a characteristic feature of all recognizable autosomal aneuploidy syndromes.Recent studies have shown an increased rate of aneuploid neurons in Alzheimer’s disease (AD), schizophrenia, autism, and ataxia-telangiectasia.^2,5,23,24,25 So far, however, direct evidence for a pathogenetic role for neuronal aneuploidy in these disorders is lacking. In particular, it remains unclear whether neuronal aneuploidy affects cellular viability, thus contributing directly to neurodegeneration and cell death.Here, we analyzed the fate of hyperploid neurons at the conversion from preclinical to mild AD and during further progression to severe stages of the disease. We can show that neurons with a more-than-diploid content of DNA are increased in preclinical stages of AD and are selectively affected by cell death during progression of the disease. Present findings show that neuronal hyperploidy in AD is associated with a decreased viability and directly linked to cell death.     

Mucosal Tolerance Induced by an Immunodominant Peptide from Rat α3(IV)NC1 in Established Experimental Autoimmune Glomerulonephritis

Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture’s disease, can be induced in Wistar Kyoto (WKY) rats by immunization with the noncollagenous domain of the α 3 chain of type IV collagen, α3(IV)NC1. Recent studies have identified an immunodominant peptide, pCol (24-38), from the N-terminus of rat α3(IV)NC1; this peptide contains the major B- and T-cell epitopes in EAG and can induce crescentic nephritis. In this study, we investigated the mechanisms of mucosal tolerance in EAG by examining the effects of the nasal administration of this peptide after the onset of disease. A dose-dependent effect was observed: a dose of 300 μg had no effect, a dose of 1000 μg resulted in a moderate reduction in EAG severity, and a dose of 3000 μg produced a marked reduction in EAG severity accompanied by diminished antigen-specific, T-cell proliferative responses. These results demonstrate that mucosal tolerance in EAG can be induced by nasal administration of an immunodominant peptide from the N-terminus of α3(IV)NC1 and should be of value in designing new therapeutic strategies for patients with Goodpasture’s disease and other autoimmune disorders.Goodpasture’s, or anti-glomerular basement membrane (GBM), disease is an autoimmune disorder characterized by rapidly progressive glomerulonephritis and lung hemorrhage.^1,2 The disease is caused by autoimmunity to a component of the GBM, the non–collagenous domain of the α3 chain of type IV collagen, α3(IV)NC1.^3,4 Epitope mapping studies have localized the immunodominant region for antibody binding to the amino terminal of the α3(IV)NC1 molecule.^5,6 Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture’s disease, can be induced in susceptible strains of rats and mice by immunization with GBM^7,8,9 or with recombinant α3(IV)NC1.^10,11,12 This results in the development of circulating and deposited anti-GBM antibodies, with focal necrotizing crescentic glomerulonephritis and lung hemorrhage. EAG shares many features with the human disease, in that the renal and lung pathology are very similar,¹³ and the anti-GBM antibodies show the same specificity for the main target antigen, α3(IV)NC1.^10,11,12There is now compelling evidence for the role of both humoral and cell-mediated immunity in the pathogenesis of EAG. The pathogenic role of anti-GBM antibodies has been demonstrated in a variety of passive transfer studies.^9,14,15,16 Transfer of disease has been demonstrated using antibodies pooled from the serum of nephritic mice,⁹ antibodies purified from the urine of nephritic rats,¹⁴ monoclonal antibodies derived from rats with EAG,¹⁵ and antibodies eluted from the kidney of nephritic rats.¹⁶ In the latter study, it was shown that deposited anti-GBM antibody has a higher functional affinity for GBM than circulating antibody.The pathogenic role of T cells in EAG has also been demonstrated in several studies. T cells have been shown to be present in the glomeruli of animals with EAG,^11,13 to proliferate in response to α3(IV)NC1,^12,17 and to transfer disease to naive recipients.^9,18 Glomerular T cells from rats with EAG show restricted T-cell receptor CDR3 spectratypes, demonstrating that they are an oligoclonal antigen-driven population.¹⁹ Anti-T-cell immunotherapy has been shown to be effective in preventing or ameliorating disease.^20,21,22,23 Anti-CD4 mAb therapy is effective in the prevention of EAG,²⁰ anti-CD8 mAb therapy is effective in both prevention and treatment of established disease,²¹ and inhibition of T-cell co-stimulation by blockade of either the CD28-B7 pathway²² or the CD154-CD40 pathway²³ has been shown to reduce the severity of glomerulonephritis.Further evidence supporting the role of T-cell-mediated cellular immunity in the pathogenesis of EAG is documented in recent studies demonstrating that synthetic peptides derived from α3(IV)NC1 can induce glomerulonephritis in WKY rats.^24,25,26,27 Recent studies from our group have identified a 15-mer immunodominant peptide, pCol, (24-38) from the N-terminus of rat α3(IV)NC1, which contains the major B- and T-cell epitopes in EAG, and which can induce crescentic nephritis.²⁴ Previous studies by Luo and colleagues²⁵ showed that a 24-mer synthetic peptide, pCol, (28-51) from the N-terminus of α3(IV)NC1 was capable of inducing glomerulonephritis, although this was mild and inconsistent, whereas Wu and colleagues²⁶ showed that a 13-mer peptide, pCol, (28-40) containing a T-cell epitope from α3(IV)NC1, induced severe crescentic glomerulonephritis. In further characterization of this T-cell epitope, it was shown that only three residues were critical for disease induction.²⁷ In addition, it has been reported that peptides containing the T-cell epitope pCol (28-40) not only induced severe glomerulonephritis, but also triggered a diversified anti-GBM antibody response through B-cell epitope spreading, suggesting that the autoantibody response to GBM antigens could be induced by a single nephritogenic T-cell epitope.^28,29,30Mucosal tolerance is a phenomenon whereby peripheral immunological tolerance may be induced by the mucosal administration of autoantigens.^{31,32,33,34,35} The inhibitory effect of orally or nasally administered autoantigens, or immunodominant peptides, has been widely reported in experimental models of autoimmune disease in rodents, including encephalomyelitis,^36,37,38 arthritis,^39,40,41 myasthenia gravis,^42,43 interstitial nephritis,⁴⁴ and glomerulonephritis.^9,45,46 In several of these studies, it has been shown that nasal administration of lower doses of antigen than those given orally has been effective in inducing mucosal tolerance,^36,40,42 and in treating established disease.^37,38,43 Our previous work in EAG has shown that both oral administration of GBM antigen⁴⁵ and nasal administration of recombinant α3(IV)NC1⁴⁶ are effective in preventing the development of crescentic nephritis. However, neither of these studies demonstrated successful treatment of established EAG, which would clearly be more applicable to patients with glomerulonephritis.In the present study, we have examined the effect of nasal administration of an immunodominant peptide, pCol, (24-38) from α3(IV)NC1 after the onset of disease in EAG. We show for the first time that mucosal tolerance and reduction in glomerular injury in established EAG can be induced by nasal administration of an immunodominant peptide. This work may lead to new antigen-specific treatment strategies for patients with anti-GBM disease and other autoimmune disorders.     

After Damage of Large Bile Ducts by Gamma-Aminobutyric Acid,Small Ducts Replenish the Biliary Tree by Amplification of Calcium-Dependent Signaling and de Novo Acquisition of Large Cholangiocyte Phenotypes

Large cholangiocytes secrete bicarbonate in response to secretin and proliferate after bile duct ligation by activation of cyclic adenosine 3′, 5′-monophosphate signaling. The Ca²⁺-dependent adenylyl cyclase 8 (AC8, expressed by large cholangiocytes) regulates secretin-induced choleresis. Ca²⁺-dependent protein kinase C (PKC) regulates small cholangiocyte function. Because γ-aminobutyric acid (GABA) affects cell functions by activation of both Ca²⁺ signaling and inhibition of AC, we sought to develop an in vivo model characterized by large cholangiocyte damage and proliferation of small ducts. Bile duct ligation rats were treated with GABA for one week, and we evaluated: GABA_A, GABA_B, and GABA_C receptor expression; intrahepatic bile duct mass (IBDM) and the percentage of apoptotic cholangiocytes; secretin-stimulated choleresis; and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation and activation of Ca^2+-dependent PKC isoforms and AC8 expression. We found that both small and large cholangiocytes expressed GABA receptors. GABA: (i) induced apoptosis of large cholangiocytes and reduced large IBDM; (ii) decreased secretin-stimulated choleresis; and (iii) reduced ERK1/2 phosphorylation and AC8 expression in large cholangiocytes. Small cholangiocytes: (i) proliferated leading to increased IBDM; (ii) displayed activation of PKCβII; and (iii) de novo expressed secretin receptor, cystic fibrosis transmembrane regulator, Cl⁻/HCO₃⁻ anion exchanger 2 and AC8, and responded to secretin. Therefore, in pathologies of large ducts, small ducts replenish the biliary epithelium by amplification of Ca²⁺-dependent signaling and acquisition of large cholangiocyte phenotypes.Cholangiocytes line the intrahepatic biliary tree,^1,2 a network of interconnecting ducts of different sizes and functions.^1,2 A number of gastrointestinal hormones including secretin modify bile of canalicular origin before reaching the duodenum.^3,4 In humans, cholangiocytes are the target cells in a number of chronic cholestatic liver diseases characterized by biliary proliferation/loss.⁵ Cholangiocytes proliferate or are damaged in animal models of cholestasis including bile duct ligation (BDL) or acute administration of carbon tetrachloride (CCl₄).^4,6,7,8 Secretin receptor (SR, expressed only by large cholangiocytes in rodent liver)^1,2,8,9 is a unique pathophysiological tool for evaluating at the functional level the degree of biliary growth/loss.^6,7,8,9 Whereas enhanced cholangiocyte growth is associated with increased SR expression and secretin-stimulated choleresis, biliary damage leads to decreased functional expression of SR.^7,8The human and rodent biliary epithelium is morphologically and functionally heterogeneous.^1,2,10,11,12 In rat liver, purified small cholangiocytes (≈8 μm in size) derive from small ducts (<15 μm in diameter), whereas large cholangiocytes (≈15 μm in size) originate from large ducts (>15 μm in diameter).^1,2 Whereas the secretory, apoptotic, and proliferative activities of large cholangiocytes are regulated by changes in cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent signaling,^{1,6,8,9,11,13} the function of small cholangiocytes (normally mitotically dormant)^6,8 is regulated by the D-myo-inositol 1,4,5-trisphosphate (IP₃)/Ca²⁺/calmodulin-dependent protein kinase I signaling pathway.^14,15 For example, large (but not small) rodent cholangiocytes express SR,^1,2,10 cystic fibrosis transmembrane regulator (CFTR),^1,2,10 and Cl⁻/HCO₃⁻ exchanger^1,2,10 (recently identified as the Cl⁻/HCO₃⁻ anion exchanger 2 [AE2]),¹⁶ and secrete bile in response to secretin by activation of cAMP⇒protein kinase A (PKA)⇒CFTR⇒Cl⁻/HCO₃⁻ anion AE2.^1,2,9 The Ca²⁺-dependent adenylyl cyclase 8 (AC8, expressed mainly by large cholangiocytes)¹⁷ regulates secretin-stimulated choleresis of large bile ducts.¹⁷ After BDL, large but not small cholangiocytes undergo mitosis (leading to enhanced large duct mass)^6,8,18 by activation of cAMP signaling.^6,8,18 A single dose of CCl₄ to rats induces a functional loss of large cAMP-responsive cholangiocytes, whereas small cholangiocytes (resistant to CCl₄-induced apoptosis) de novo proliferate to compensate for the loss of large biliary mass.⁸ Although some studies suggest that activation of Ca²⁺-dependent signaling may be important in the regulation of small cholangiocyte function,^14,15 the mechanisms by which small cholangiocytes replenish the biliary tree in response to the damage of large bile ducts is unknown.Gamma-aminobutyric acid (GABA) is the chief inhibitory neurotransmitter in the vertebrate central nervous system.¹⁹ In addition to the central nervous system, the liver represents the major site of synthesis and metabolism of GABA.²⁰ GABA actions are mediated by three GABA receptor subtypes (GABA_A, GABA_B, and GABA_C).²¹ Studies have shown that GABAergic activity inhibits hepatic regeneration after partial hepatectomy in rats.²² We have shown that GABA decreases both in vivo and in vitro cholangiocarcinoma growth.²¹ However, no data exist regarding the role of GABA in the regulation of cholangiocyte hyperplasia in cholestasis. Because GABA can affect cell functions by both activation of Ca²⁺ signaling and inhibition of AC activity,²³ we tested the hypothesis that GABA regulates the proliferative, apoptotic, and secretory activities of small and large cholangiocytes by the differential activation/deactivation of Ca²⁺- and cAMP-dependent signaling pathways.     

Generation of a Novel Transgenic Mouse Model for Bioluminescent Monitoring of Survivin Gene Activity in Vivo at Various Pathophysiological Processes : Survivin Expression Overlaps with Stem Cell Markers

Survival has been implicated to play an important role in various pathophysiological processes. However, because of a lack of appropriate animal models, the role and dynamic expression of survivin during pathophysiology are not well defined. We generated a human survivin gene promoter-driven luciferase transgenic mouse model (SPlucTg) so that dynamic survivin gene activity can be monitored during various pathophysiological conditions using in vivo imaging. Our results show that, consistent with survivin positivity in testis, luciferase activity in normal SPlucTg mice was detected in the testis of male mice. Furthermore, similar to the known requirement of transient expression of survivin for pathophysiological responses, we observed a transient luciferase expression in castrated SPlucTg male mice after supplement of androgen. Significantly, it was reported that survivin expression turns on during mouse liver injury and regeneration; a transient and dose-dependent luciferase expression in the mouse liver was observed after administration of carbon tetrachloride into SPlucTg mice. We further demonstrated that luciferase activity closely correlates with endogenous survivin expression. We also demonstrated that only a subset of cells expresses survivin, and its expression overlaps with the expression of several stem cell markers tested. Thus, we have generated a unique animal model for analysis of diverse pathophysiological processes and possible stem cell distribution/activity in vivo.Evidence indicates that survivin, an inhibitor of apoptosis (IAP) family protein and a promoter of cell cycle and mitosis,^1,2,3 plays an important role in cancer initiation⁴ and angiogenesis,^5,6,7 tumor progression, drug/radiation resistance,⁸ and tumor relapse.^3,9 Survivin may also play a role in other pathophysiological processes.^10,11,12 These include embryonic development, hematopoietic cell survival and proliferation, T-cell development, multiple sclerosis, brain pathology, and pathophysiology of other organs including liver, pancreas, gastrointestine, testes, endometrium, and placenta.¹¹ However, studies indicated that the behavior of survivin expression, subcellular localization, and/or gene regulation appear to be different during normal tissue turnover or repair versus cancer initiation and progression.¹¹ Furthermore, in some cases, normal cell division, survival, and development happen in the absence of survivin¹³ or with differential requirement of survivin.¹⁴ Additionally, because of increased expression of survivin in cancer, the interaction of survivin molecules with other proteins may differ from normal cells and/or may exclusively occur in cancer cells.^15,16 This partially explains the observations indicating that whereas abrogation of survivin expression or functions triggers significant apoptosis of cancer cells, it has minimal effects on normal cells and tissues.^{17,18,19,20,21,22,23,24} Thus, survivin appears to be an ideal cancer preventive and therapeutic target.Targeting survivin for cancer treatment might cause little toxicity to normal tissues. Indeed, many natural chemopreventive and chemotherapeutic dietary components, such as resveratrol, silibinin, and sulindac²⁵ as well as selenium,²⁶ retinoid,^27,28 and vitamin D (eg, calcitriol)²⁹ down-regulate survivin expression in cancer cells. This is consistent with a role for survivin in oncogenesis. However, because of the lack of an appropriate in vivo animal model system, the role for survivin either in cancer-related or in other in vivo pathological processes¹¹ remains to be elucidated.Here, we report the generation of a novel transgenic mouse model that expresses the luciferase reporter gene as the human survivin gene surrogate under the control of a human survivin gene promoter. We demonstrated that this new mouse model possesses an active and highly responsive luciferase reporter system, which reflects the dynamic expression of endogenous survivin during pathology. We further show that survivin expression overlaps with several stem cell markers, which suggests that survivin may be a good stem cell marker as well. Our model has several unique features for in vivo imaging in comparison with the model previously reported.³⁰ In our model, we used the human survivin promoter with sufficient length (4.5 kb), instead of using a mouse survivin proximal promoter (0.8 kb; as a note, the design in Xia et al’s report³⁰ is good for studying the relationship between survivin expression and cell cycle/mitosis). Importantly, using the luciferase reporter as a surrogate to monitor survivin gene activity during various pathophysiological processes will increase sensitivity by several orders of magnitude in comparison with using GFP as a reporter for in vivo imaging, although GFP can provide strong signals. Therefore, with our model, it is now possible to monitor endogenous survivin gene activity through in vivo imaging of its surrogate luciferase activity during various pathophysiological processes. This mouse model has the potential to delineate the dynamic expression and role of survivin in cancer and other pathophysiological processes.