Endothelial autophagy is not required for liver regeneration after partial hepatectomy in mice with fatty liver.

BACKGROUND & AIMS: Patients with non-alcoholic fatty liver disease (NAFLD) have impaired liver regeneration. Liver endothelial cells play a key role in liver regeneration. In non-alcoholic steatohepatitis (NASH), liver endothelial cells display a defect in autophagy, contributing to NASH progression. We aimed to determine the role of endothelial autophagy in liver regeneration following liver resection in NAFLD. METHODS: First, we assessed autophagy in primary endothelial cells from wild type mice fed a high fat diet and subjected to partial hepatectomy. Then, we assessed liver regeneration after partial hepatectomy in mice deficient (Atg5lox/lox ;VE-cadherin-Cre+ ) or not (Atg5lox/lox ) in endothelial autophagy and fed a high fat diet. The role of endothelial autophagy in liver regeneration was also assessed in ApoE-/- hypercholesterolemic mice and in mice with NASH induced by methionine- and choline-deficient diet. RESULTS: First, autophagy (LC3II/protein) was strongly increased in liver endothelial cells following hepatectomy. Then, we observed at 40 and 48 h and at 7 days after partial hepatectomy, that Atg5lox/lox ;VE-cadherin-Cre+ mice fed a high fat diet had similar liver weight, plasma AST, ALT and albumin concentration, and liver protein expression of proliferation (PCNA), cell-cycle (Cyclin D1, BrdU incorporation, phospho-Histone H3) and apoptosis markers (cleaved Caspase-3) as Atg5lox/lox mice fed a high fat diet. Same results were obtained in ApoE-/- and methionine- and choline-deficient diet fed mice, 40 h after hepatectomy. CONCLUSION: These results demonstrate that the defect in endothelial autophagy occurring in NASH does not account for the impaired liver regeneration occurring in this setting.

Slc2a10 knock-out mice deficient in ascorbic acid synthesis recapitulate aspects of arterial tortuosity syndrome and display mitochondrial respiration defects.

Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicated GLUT10 in the transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygous Slc2a10 missense mutations did not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient for Slc2a10 as well as Gulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in mouse. Gulo;Slc2a10 double knock-out mice showed mild phenotypic anomalies, which were absent in single knock-out controls. While Gulo;Slc2a10 double knock-out mice did not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. Transforming growth factor beta signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived from Gulo;Slc2a10 double knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined.

Angiopoietin-2 Promotes Pathological Angiogenesis and Is a Therapeutic Target in Murine Nonalcoholic Fatty Liver Disease.

Angiogenesis contributes to the development of nonalcoholic steatohepatitis (NASH) and promotes inflammation, fibrosis, and progression to hepatocellular carcinoma (HCC). Angiopoietin-2 (Ang-2) is a key regulator of angiogenesis. We aimed to investigate the role of Ang-2 and its potential as a therapeutic target in NASH using human samples, in vivo mouse models, and in vitro assays. Serum Ang-2 levels were determined in 104 obese patients undergoing bariatric surgery and concomitant liver biopsy. The effect of the Ang-2/Tie2 receptor inhibiting peptibody L1-10 was evaluated in the methionine-choline deficient (MCD) and streptozotocin-western diet nonalcoholic fatty liver disease mouse models, and in vitro on endothelial cells and bone marrow-derived macrophages. The hepatic vasculature was visualized with µCT scans and scanning electron microscopy of vascular casts. Serum Ang-2 levels were increased in patients with histological NASH compared with patients with simple steatosis and correlated with hepatic CD34 immunoreactivity as a marker of hepatic angiogenesis. Serum and hepatic Ang-2 levels were similarly increased in mice with steatohepatitis. Both preventive and therapeutic L1-10 treatment reduced hepatocyte ballooning and fibrosis in MCD diet-fed mice and was associated with reduced hepatic angiogenesis and normalization of the vascular micro-architecture. Liver-isolated endothelial cells and monocytes from MCD-fed L1-10-treated mice showed reduced expression of leukocyte adhesion and inflammatory markers, respectively, compared with cells from untreated MCD diet-fed mice. In the streptozotocin-western diet model, therapeutic Ang-2 inhibition was able to reverse NASH and attenuate HCC progression. In vitro, L1-10 treatment mitigated increased cytokine production in lipopolysaccharide-stimulated endothelial cells but not in macrophages. Conclusion: Our findings provide evidence for Ang-2 inhibition as a therapeutic strategy to target pathological angiogenesis in NASH.

Placental growth factor inhibition targets pulmonary angiogenesis and represents a therapy for hepatopulmonary syndrome in mice.

Hepatopulmonary syndrome (HPS) is a severe complication of cirrhosis with increased risk of mortality. Pulmonary microvascular alterations are key features of HPS; but underlying mechanisms are incompletely understood, and studies on HPS are limited to rats. Placental growth factor (PlGF), a proangiogenic molecule that is selectively involved in pathological angiogenesis, may play an important role in HPS development; however, its role has never been investigated. In this study, we validated an HPS model by common bile duct ligation (CBDL) in mice, investigated the kinetic changes in pulmonary angiogenesis and inflammation during HPS development, and provide evidence for a novel therapeutic strategy by targeting pathological angiogenesis. Mice with CBDL developed hypoxemia and intrapulmonary shunting on a background of liver fibrosis. Pulmonary alterations included increased levels of proangiogenic and inflammatory markers, which was confirmed in serum of human HPS patients. Increased PlGF production in HPS mice originated from alveolar type II cells and lung macrophages, as demonstrated by immunofluorescent staining. Dysfunctional vessel formation in CBDL mice was visualized by microscopy on vascular corrosion casts. Both prophylactic and therapeutic anti-PlGF (αPlGF) antibody treatment impeded HPS development, as demonstrated by significantly less intrapulmonary shunting and improved gas exchange. αPlGF treatment decreased endothelial cell dysfunction in vivo and in vitro and was accompanied by reduced pulmonary inflammation. Importantly, αPlGF therapy did not affect liver alterations, supporting αPlGF's ability to directly target the pulmonary compartment. CONCLUSION: CBDL in mice induces HPS, which is mediated by PlGF production; αPlGF treatment improves experimental HPS by counteracting pulmonary angiogenesis and might be an attractive therapeutic strategy for human HPS. (Hepatology 2017).

In vitro Phase I- and Phase II-Drug Metabolism in The Liver of Juvenile and Adult Göttingen Minipigs.

PURPOSE: In view of pediatric drug development, juvenile animal studies are gaining importance. However, data on drug metabolizing capacities of juvenile animals are scarce, especially in non-rodent species. Therefore, we aimed to characterize the in vitro biotransformation of four human CYP450 substrates and one UGT substrate in the livers of developing Göttingen minipigs. METHODS: Liver microsomes from late fetal, Day 1, Day 3, Day 7, Day 28, and adult male and female Göttingen minipigs were incubated with a cocktail of CYP450 substrates, including phenacetin, tolbutamide, dextromethorphan, and midazolam. The latter are probe substrates for human CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. In addition, the UGT multienzyme substrate (from the UGT-GloTM assay), which is glucuronidated by several human UGT1A and UGT2B enzymes, was also incubated with the porcine liver microsomes. RESULTS: For all tested substrates, drug metabolism significantly rose postnatally. At one month of age, 60.5 and 75.4% of adult activities were observed for acetaminophen and dextrorphan formations, respectively, while 35.4 and 43.2% of adult activities were present for 4-OH-tolbutamide and 1'-OH-midazolam formations. Biotransformation of phenacetin was significantly higher in 28-day-old and adult females compared with males. CONCLUSIONS: Maturation of metabolizing capacities occurred postnatally, as described in man.

In Vitro Biotransformation of Two Human CYP3A Probe Substrates and Their Inhibition during Early Zebrafish Development.

At present, the zebrafish embryo is increasingly used as an alternative animal model to screen for developmental toxicity after exposure to xenobiotics. Since zebrafish embryos depend on their own drug-metabolizing capacity, knowledge of their intrinsic biotransformation is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this in vitro study was to assess the activity of cytochrome P450 (CYP)-a group of drug-metabolizing enzymes-in microsomes from whole zebrafish embryos (ZEM) of 5, 24, 48, 72, 96 and 120 h post-fertilization (hpf) by means of a mammalian CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR). The same CYP activity assays were performed in adult zebrafish liver microsomes (ZLM) to serve as a reference for the embryos. In addition, activity assays with the human CYP3A4-specific Luciferin isopropyl acetal (Luciferin-IPA) as well as inhibition studies with ketoconazole and CYP3cide were carried out to identify CYP activity in ZLM. In the present study, biotransformation of BOMR was detected at 72 and 96 hpf; however, metabolite formation was low compared with ZLM. Furthermore, Luciferin-IPA was not metabolized by the zebrafish. In conclusion, the capacity of intrinsic biotransformation in zebrafish embryos appears to be lacking during a major part of organogenesis.

Organ data from the developing Göttingen minipig: first steps towards a juvenile PBPK model.

The Göttingen minipig is the most commonly used pig breed in preclinical drug development in Europe and has recently also been explored for physiologically based pharmacokinetic modelling. To develop such a model, not only physiological data from adult animals but also data from juvenile animals are required, especially when using this model for paediatric drug development. Therefore, the aim of our study was to document body and organ weights (brain, heart, lungs, liver, gastrointestinal tract, spleen and kidney), lengths of the small and large intestines and pH values of the gastrointestinal tract in Göttingen minipigs from the foetal stage until the age of 5 months. Postnatal organ and body weights were fitted to regression models to find suitable equations that could be used to estimate organ weights as a function of body weight in the neonatal and juvenile Göttingen minipig. Most organs followed a non-linear growth curve during the first 5 months of life. In general, relative organ weights were the highest during the first week of life, during which the gastric pH was more alkaline than at 28 days of age.

Role of vascular endothelial growth factor in the pathophysiology of nonalcoholic steatohepatitis in two rodent models.

UNLABELLED: The pathophysiology of nonalcoholic steatohepatitis (NASH) should be approached as a multifactorial process. In several stages of NASH, a link between disease progression and hepatic microvasculature changes can be made. In this study we investigated the role of angiogenesis in two mouse models for NASH, and the effect of a preventive and therapeutic antiangiogenic treatment in a diet-induced mouse model for NASH. Protein and RNA levels of angiogenic and inflammatory factors were significantly up-regulated in the liver of C56BL/6 and db/db mice with NASH at different timepoints. To examine the effect of angiogenic factors on the disease progression of NASH, a prevention and treatment study was set up, blocking the placental growth factor (PlGF) or vascular endothelial growth factor receptor 2 (VEGFR2). Our study showed that treatment prevents the progression of NASH by attenuating steatosis and inflammation, both in a preventive and therapeutic setting, thereby confirming the hypothesis that angiogenic factors play an early role in the disease progression from steatosis to NASH. Anti-PlGF (αPlGF) did not significantly improve liver histology. Vascular corrosion casting showed a more disrupted liver vasculature in mice with NASH compared to controls. Treatment with αVEGFR2 showed an improvement of the liver vasculature. Moreover, fat-laden primary hepatocytes treated with αVEGFR2 stored significantly less lipids. CONCLUSION: Our results demonstrate that there is an increased expression of angiogenic factors in the liver in different mouse models for NASH. We found that VEGFR2 blockage attenuates steatosis and inflammation in a diet-induced mouse model for NASH in a preventive and therapeutic setting. Our findings warrant further investigation of the role of angiogenesis in the pathophysiology in NASH.

Analyzing the human liver vascular architecture by combining vascular corrosion casting and micro-CT scanning: a feasibility study.

Although a full understanding of the hepatic circulation is one of the keys to successfully perform liver surgery and to elucidate liver pathology, relatively little is known about the functional organization of the liver vasculature. Therefore, we materialized and visualized the human hepatic vasculature at different scales, and performed a morphological analysis by combining vascular corrosion casting with novel micro-computer tomography (CT) and image analysis techniques. A human liver vascular corrosion cast was obtained by simultaneous resin injection in the hepatic artery (HA) and portal vein (PV). A high resolution (110 µm) micro-CT scan of the total cast allowed gathering detailed macrovascular data. Subsequently, a mesocirculation sample (starting at generation 5; 88 × 68 × 80 mm³) and a microcirculation sample (terminal vessels including sinusoids; 2.0 × 1.5 × 1.7 mm³) were dissected and imaged at a 71-µm and 2.6-µm resolution, respectively. Segmentations and 3D reconstructions allowed quantifying the macro- and mesoscale branching topology, and geometrical features of HA, PV and hepatic venous trees up to 13 generations (radii ranging from 13.2 mm to 80 µm; lengths from 74.4 mm to 0.74 mm), as well as microvascular characteristics (mean sinusoidal radius of 6.63 µm). Combining corrosion casting and micro-CT imaging allows quantifying the branching topology and geometrical features of hepatic trees using a multiscale approach from the macro- down to the microcirculation. This may lead to novel insights into liver circulation, such as internal blood flow distributions and anatomical consequences of pathologies (e.g. cirrhosis).

Welfare Assessment in Pigs Using the Salivary Proteome.

Identifying the potential presence of stress at the pig farm is fundamental since it affects pig welfare. As a result, a reliable and straightforward tool to monitor stress could record the welfare status of the animals. Although numerous methods to assess the welfare of pigs have been developed in the past, no gold standard has been established yet. Recently, the value of saliva as a tool to identify chronic stress in piglets was explored, as it can be collected fast and non-invasively. Since the protein composition, i.e., the proteome of porcine saliva, responds to stress, the affected proteins could be used as salivary stress biomarkers. The present review first defines stress and its relationship with welfare. Next, the porcine gland-specific salivary proteome is characterized. Finally, six potential salivary biomarkers for stress are proposed, i.e., odorant-binding protein, vomeromodulin-like protein, chitinase, lipocalin-1, long palate lung and nasal epithelium protein, and alpha-2-HS-glycoprotein.

The placental growth factor as a target against hepatocellular carcinoma in a diethylnitrosamine-induced mouse model.

BACKGROUND & AIMS: The placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family known to stimulate endothelial cell growth, migration and survival, attract angiocompetent macrophages, and determine the metastatic niche. Unlike VEGF, genetic studies have shown that PlGF is specifically involved in pathologic angiogenesis, thus its inhibition would not affect healthy blood vessels, providing an attractive drug candidate with a good safety profile. METHODS: We assess whether inhibition of PlGF could be used as a potential therapy against hepatocellular carcinoma (HCC), by using PlGF knockout mice and monoclonal anti-PlGF antibodies in a mouse model for HCC. In addition, the effect of PlGF antibodies is compared to that of sorafenib, as well as the combination of both therapies. RESULTS: We have found that both in a transgenic knockout model and in a treatment model, targeting PlGF significantly decreases tumor burden. This was achieved not only by inhibiting neovascularisation, but also by decreasing hepatic macrophage recruitment and by normalising the remaining blood vessels, thereby decreasing hypoxia and reducing the prometastatic potential of HCC. CONCLUSIONS: Considering the favourable safety profile and its pleiotropic effect on vascularisation, metastasis and inflammation, PlGF inhibition could become a valuable therapeutic strategy against HCC.

Osmium tetroxide labeling of (poly)methyl methacrylate corrosion casts for enhancement of micro-CT microvascular imaging.

In order to enhance micro-computer tomography (micro-CT) imaging of corrosion casts of fine vasculature, metals can be added to the casting resin before perfusion. However, perfused metals lead to vasoconstriction or vessel damage resulting in nonphysiologic vascular casts. A novel method for coating methyl methacrylate vascular casts with osmium tetroxide has been developed in order to increase micro-CT contrast without affecting the vascular structure. This technique was verified using corrosion casts of the lung vasculature of New Zealand white rabbits. Osmium tetroxide coating of methyl methacrylate vascular corrosion casts resulted in an increase in overall sample contrast that translated into an increase in the resolution of the vasculature. This method can therefore lead to increased resolution in the characterization of fine vascular structures.

Topographical Anatomy of the Rhesus Monkey (Macaca mulatta)-Part I: Thoracic Limb.

Since the rhesus monkey (Macaca mulatta) is genetically closely related to man, it is generally accepted that its anatomy and physiology are largely similar to that of humans. Consequently, this non-human primate is most commonly used as a model in biomedical research. Not only the validation of the obtained research data, but also the welfare of the captive rhesus monkeys are subject to thorough anatomical knowledge of this species. Unfortunately, anatomical literature on the rhesus monkey is scarce, outdated, and hardly available at present. Furthermore, its anatomy is only illustrated by means of line drawings or black-and-white photographs. Thus, the aim of this study was to describe the anatomy of the thoracic limb of the rhesus monkey topographically, studying the various anatomical structures in relation to each other. In this manuscript, the anatomy of the thoracic limb is described per region. The structures that are visible on the different layers, from the superficial to the deepest layer, are described both in text and in numerous color images. As expected, the anatomy of the rhesus monkey is almost identical to human anatomy. However, some striking differences have been identified. This supports the necessity for an extensive publication on the anatomy of the rhesus monkey.

Topographical Anatomy of the Rhesus Monkey (Macaca mulatta)-Part II: Pelvic Limb.

The rhesus monkey (Macaca mulatta) is a widely used model in biomedical research because its anatomy and physiology bear many similarities to those of humans. Extensive knowledge of the anatomy of this nonhuman primate species is not only required for the correct interpretation of obtained research data but also valuable for the welfare of captive individuals housed in, e.g., zoos. As anatomical publications on the rhesus monkey are hardly available, outdated and provide only line drawings or black-and-white photographs, the anatomy of the rhesus monkey was readdressed in this study. The various anatomical structures are described in relation to each other topographically per hindlimb region. The hip region, the upper limb, the knee, the lower limb and the foot are described from various perspectives. The structures that are visible in the different layers, from the superficial to the deepest layer, were photographed. Although the anatomy of the hindlimbs of rhesus monkeys and humans are remarkably similar, various subtle dissimilarities have been observed. Consequently, an open-access publication that focuses on the anatomy of the rhesus monkey would be highly valued by both biomedical researchers and veterinarians.

Osteology of the Hamadryas Baboon (Papio hamadryas).

Besides living as a free-ranging primate in the horn of Africa and the Arabian Peninsula, the hamadryas baboon has an important place in zoos and can be found in biomedical research centers worldwide. To be valuable as a non-human primate laboratory model for man, its anatomy should be portrayed in detail, allowing for the correct interpretation and translation of obtained research results. Reviewing the literature on the use of the baboon in biomedical research revealed that very limited anatomical works on this species are available. Anatomical atlases are incomplete, use archaic nomenclature and fail to provide high-definition color photographs. Therefore, the skeletons of two male hamadryas baboons were prepared by manually removing as much soft tissues as possible followed by maceration in warm water to which enzyme-containing washing powder was added. The bones were bleached with hydrogen peroxide and degreased by means of methylene chloride. Photographs of the various bones were taken, and the anatomical structures were identified using the latest version of the Nomina Anatomica Veterinaria. As such, the present article shows 31 annotated multipanel figures. The skeleton of the hamadryas baboon generally parallels the human skeleton, but some remarkable differences have been noticed. If these are taken into consideration when evaluating the results of experiments using the hamadryas baboon, justified conclusions can be drawn.

Chronic exposure to multiple stressors alters the salivary proteome of piglets.

Monitoring chronic stress in pigs is not only essential in view of animal welfare but is also important for the farmer, given that stress influences the zootechnical performance of the pigs and increases their susceptibility to infectious diseases. To investigate the use of saliva as a non-invasive, objective chronic stress monitoring tool, twenty-four 4-day-old piglets were transferred to artificial brooders. At the age of 7 days, they were assigned to either the control or the stressed group and reared for three weeks. Piglets in the stressed group were exposed to overcrowding, absence of cage enrichment, and frequent mixing of animals between pens. Shotgun analysis using an isobaric labelling method (iTRAQ) for tandem mass spectrometry performed on saliva samples taken after three weeks of chronic stress identified 392 proteins, of which 20 proteins displayed significantly altered concentrations. From these 20 proteins, eight were selected for further validation using parallel reaction monitoring (PRM). For this validation, saliva samples that were taken one week after the start of the experiment and samples that were taken at the end of the experiment were analysed to verify the profile over time. We wanted to investigate whether the candidate biomarkers responded fast or rather slowly to the onset of chronic exposure to multiple stressors. Furthermore, this validation could indicate whether age influenced the baseline concentrations of these salivary proteins, both in healthy and stressed animals. This targeted PRM analysis confirmed that alpha-2-HS-glycoprotein was upregulated in the stressed group after one and three weeks, while odorant-binding protein, chitinase, long palate lung and nasal epithelium protein 5, lipocalin-1, and vomeromodulin-like protein were present in lower concentrations in the saliva of the stressed pigs, albeit only after three weeks. These results indicate that the porcine salivary proteome is altered by chronic exposure to multiple stressors. The affected proteins could be used as salivary biomarkers to identify welfare problems at the farm and facilitate research to optimise rearing conditions.

Increased intrahepatic resistance in severe steatosis: endothelial dysfunction, vasoconstrictor overproduction and altered microvascular architecture.

Non-alcoholic fatty liver disease can progress to steatohepatitis and fibrosis, and is also associated with impaired liver regeneration. The pathophysiology remains elusive. We recently showed that severe steatosis is associated with an increase in portal pressure, suggesting liver flow impairment. The objective of this study is to directly assess total intrahepatic resistance and its potential functional and structural determinants in an in situ perfusion model. Male Wistar rats fed a control (n = 30) or a methionine-choline-deficient (MCD) diet (n = 30) for 4 weeks were compared. Liver tissue and serum analysis, in vivo haemodynamic measurements, in situ perfusion experiments and vascular corrosion casts were performed. The MCD group showed severe steatosis without inflammation or fibrosis on histology. Serum levels and liver tissue gene expression of interleukin (IL)-6, tumour necrosis factor-α, IL-1ß and interferon-γ, liver tissue myeloperoxidase activity and liver immunohistochemistry with anti-CD68 and anti-α smooth muscle actin were comparable between groups, excluding significant inflammation. Flow-pressure curves were significantly different between groups for all flows (slope values: 0.1636 ± 0.0605 mm Hg/ml/min in controls vs 0.7270 ± 0.0408 mm Hg/ml/min in MCD-fed rats, P < 0.001), indicating an increased intrahepatic resistance, which was haemodynamically significant (portocaval pressure gradient 2.2 ± 1.1 vs 8.2 ± 1.3 mm Hg in controls vs MCD, P<0.001). Dose-response curves to acetylcholine were significantly reduced in MCD-fed rats (P < 0.001) as was the responsiveness to methoxamine (P<0.001). Vascular corrosion casts showed a replacement of the regular sinusoidal anatomy by a disorganized pattern with multiple interconnections and vascular extensions. Liver phosphorylated endothelial NO synthase (eNOS)/eNOS and serum nitrite/nitrate were not increased in severe steatosis, whereas liver thromboxane synthase expression, liver endothelin-1 (ET-1) expression and serum andothelin-1 concentration were significantly increased. Severe steatosis induces a haemodynamically significant increase in intrahepatic resistance, which precedes inflammation and fibrogenesis. Both functional (endothelial dysfunction and increased thromboxane and ET-1 synthesis) and structural factors are involved. This phenomenon might significantly contribute to steatosis-related disease.

Effect of prolyl hydroxylase domain-2 haplodeficiency on the hepatocarcinogenesis in mice.

BACKGROUND & AIMS: The two major primary liver cancers in adults are hepatocellular carcinoma and cholangiocarcinoma. These tumors rapidly outgrow their vascular supply and become hypoxic, resulting in the production of hypoxia inducible factors. Recently, interest has grown in the regulators of these factors. Several reports have been published describing the role of prolyl hydroxylase domains--the key oxygen sensor responsible for the degradation of hypoxia inducible factors--tumor progression and vascularisation. The effect of prolyl hydroxylase domain 2 on the pathogenesis of liver cancer has never been studied. METHODS: A diethylnitrosamine-induced mouse model was used in this study, allowing primary hepatic tumors to occur as a result of chronic liver damage. Several parameters of prolyl hydroxylase domain 2-haplodeficient mice were compared to those of wild type mice, thereby focussing on the expression of angiogenic factors and on the hepatic progenitor cell activation and differentiation. RESULTS: This study shows that inhibiting prolyl hydroxylase domain 2 increases the hepatocarcinogenesis and stimulates the development of cholangiocarcinoma. Furthermore, PHD2 deficiency and the accompanying continuous HIF activation, selected for a more metastatic tumor phenotype. CONCLUSIONS: The effect of prolyl hydroxylase domain 2 deficiency on hepatocarcinogenesis hold a great potential for therapeutic intervention, since hypoxia and the selection for a more aggressive cholangiocarcinoma phenotype might also have a repercussion on patients receiving long-term treatment with anti-angiogenic compounds.

Inhibition of placental growth factor activity reduces the severity of fibrosis, inflammation, and portal hypertension in cirrhotic mice.

UNLABELLED: Placental growth factor (PlGF) is associated selectively with pathological angiogenesis, and PlGF blockade does not affect the healthy vasculature. Anti-PlGF is therefore currently being clinically evaluated for the treatment of cancer patients. In cirrhosis, hepatic fibrogenesis is accompanied by extensive angiogenesis. In this paper, we evaluated the pathophysiological role of PlGF and the therapeutic potential of anti-PlGF in liver cirrhosis. PlGF was significantly up-regulated in the CCl(4) -induced rodent model of liver cirrhosis as well as in cirrhotic patients. Compared with wild-type animals, cirrhotic PlGF(-/-) mice showed a significant reduction in angiogenesis, arteriogenesis, inflammation, fibrosis, and portal hypertension. Importantly, pharmacological inhibition with anti-PlGF antibodies yielded similar results as genetic loss of PlGF. Notably, PlGF treatment of activated hepatic stellate cells induced sustained extracellular signal-regulated kinase 1/2 phosphorylation, as well as chemotaxis and proliferation, indicating a previously unrecognized profibrogenic role of PlGF. CONCLUSION: PlGF is a disease-candidate gene in liver cirrhosis, and inhibition of PlGF offers a therapeutic alternative with an attractive safety profile.

Intussusceptive angiogenesis: a biologically relevant form of angiogenesis.

Angiogenesis, i.e. the development and growth of blood vessels, is a major topic of research as it plays an important role in normal development and in various pathologies. Recent evidence revealed the existence of different mechanisms of blood vessel growth, including sprouting and intussusceptive angiogenesis, vascular mimicry, and blood vessel cooption. The latter two have only been observed in tumor growth, but sprouting and intussusceptive angiogenesis also occur in healthy, physiologically growing tissues. Despite this variety of angiogenic mechanisms, most of the current research is focused on the mechanism of sprouting angiogenesis because this mechanism was first described and because most existing experimental models are related to sprouting angiogenesis. Consequently, the mechanism of intussusceptive angiogenesis is often overlooked in angiogenesis research. Here, the mechanism of intussusceptive angiogenesis is reviewed and the current techniques and models for investigating intussusceptive angiogenesis are summarized. In addition, other mechanisms of vascular growth are briefly reviewed.