Christophe Van Steenkiste

Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease.

Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.

Alteration of protein glycosylation in liver diseases

Chronic liver diseases are a serious health problem worldwide. The current gold standard to assess structural liver damage is through a liver biopsy which has several disadvantages. A non-invasive, simple and non-expensive test to diagnose liver pathology would be highly desirable. Protein glycosylation has drawn the attention of many researchers in the search for an objective feature to achieve this goal. Glycosylation is a posttranslational modification of many secreted proteins and it has been known for decades that structural changes in the glycan structures of serum proteins are an indication for liver damage. The aim of this paper is to give an overview of this altered protein glycosylation in different etiologies of liver fibrosis / cirrhosis and hepatocellular carcinoma. Although individual liver diseases have their own specific markers, the same modifications seem to continuously reappear in all liver diseases: hyperfucosylation, increased branching and a bisecting N-acetylglucosamine. Analysis at mRNA and protein level of the corresponding glycosyltransferases confirm their altered status in liver pathology. The last part of this review deals with some recently developed glycomic techniques that could potentially be used in the diagnosis of liver pathology.

Inhibition of Placental Growth Factor Activity Reduces the Severity of Fibrosis, Inflammation, and Portal Hypertension in Cirrhotic Mice

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 CCl4‐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. (HEPATOLOGY 2011;)

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-1β 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.

Hemodynamic Changes in Splanchnic Blood Vessels in Portal Hypertension

Portal hypertension (PHT) is associated with a hyperdynamic state characterized by a high cardiac output, increased total blood volume, and a decreased splanchnic vascular resistance. This splanchnic vasodilation is a result of an important increase in local and systemic vasodilators (nitric oxide, carbon monoxide, prostacyclin, endocannabinoids, and so on), the presence of a splanchnic vascular hyporesponsiveness toward vasoconstrictors, and the development of mesenteric angiogenesis. All these mechanisms will be discussed in this review. To decompress the portal circulation in PHT, portosystemic collaterals will develop. The presence of these portosystemic shunts are responsible for major complications of PHT, namely bleeding from gastrointestinal varices, encephalopathy, and sepsis. Until recently, it was accepted that the formation of collaterals was due to opening of preexisting vascular channels, however, recent data suggest also the role of vascular remodeling and angiogenesis. These points are also discussed in detail. Anat Rec, 291:699–713, 2008.

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

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. (HEPATOLOGY 2013)

Hepatopulmonary syndrome and portopulmonary hypertension: recent knowledge in pathogenesis and overview of clinical assessment

Hepatopulmonary syndrome and portopulmonary hypertension are cardiopulmonary complications, which are not infrequently seen in patients with liver disease and/or portal hypertension. These entities are both clinically and pathophysiologically different: the hepatopulmonary syndrome is characterized by abnormal pulmonary vasodilation and right‐to‐left shunting resulting in gas exchange abnormalities, whereas portopulmonary hypertension is caused by pulmonary artery vasoconstriction leading to hemodynamic failure. As both hepatopulmonary syndrome and portopulmonary hypertension are associated with significantly increased morbidity and mortality, and as these patients are commonly asymptomatic, all liver transplantation candidates should be actively screened for the presence of these two complications. The aim of is this review is to provide an overview on the hepatopulmonary syndrome and portopulmonary hypertension with primary focus on diagnosis and recent knowledge regarding pathogenesis and therapeutic targets.

Vascular corrosion casting: analyzing wall shear stress in the portal vein and vascular abnormalities in portal hypertensive and cirrhotic rodents

Vascular corrosion casting is an established method of anatomical preparation that has recently been revived and has proven to be an excellent tool for detailed three-dimensional (3D) morphological examination of normal and pathological microcirculation. In addition, the geometry provided by vascular casts can be further used to calculate wall shear stress (WSS) in a vascular bed using computational techniques. In the first part of this study, the microvascular morphological changes associated with portal hypertension (PHT) and cirrhosis in vascular casts are described. The second part of this study consists of a quantitative analysis of the WSS in the portal vein in casts of different animal models of PHT and cirrhosis using computational fluid dynamics (CFD). Microvascular changes in the splanchnic, hepatic and pulmonary territory of portal hypertensive and cirrhotic mice are described in detail with stereomicroscopic examination and scanning electron microscopy. To our knowledge, our results are the first to report the vascular changes in the common bile duct ligation cirrhotic model. Calculating WSS using CFD methods is a feasible technique in PHT and cirrhosis, enabling the differentiation between different animal models. First, a dimensional analysis was performed, followed by a CFD calculation describing the spatial and temporal WSS distributions in the portal vein. WSS was significantly different between sham/cirrhotic/pure PHT animals with the highest values in the latter. Up till now, no techniques have been developed to quantify WSS in the portal vein in laboratory animals. This study showed for the first time that vascular casting has an important role not only in the morphological evaluation of animal models of PHT and cirrhosis, but also in defining the biological response of the portal vein wall to hemodynamic changes. CFD in 3D geometries can be used to describe the spatial and temporal variations in WSS in the portal vein and to better understand the forces affecting mechanotransduction in the endothelium.

Kinetics of angiogenic changes in a new mouse model for hepatocellular carcinoma

BackgroundThe increasing incidence of hepatocellular carcinoma in Western countries has led to an expanding interest of scientific research in this field. Therefore, a vast need of experimental models that mimic the natural pathogenesis of hepatocellular carcinoma (HCC) in a short time period is present. The goal of our study was (1) to develop an efficient mouse model for HCC research, in which tumours develop in a natural background of fibrosis and (2) to assess the time-dependent angiogenic changes in the pathogenesis of HCC.MethodsWeekly intraperitoneal injections with the hepatocarcinogenic compound N-nitrosodiethylamine was applied as induction method and samples were taken at several time points to assess the angiogenic changes during the progression of HCC.ResultsThe N-nitrosodiethylamine-induced mouse model provides well vascularised orthotopic tumours after 25 weeks. It is a representative model for human HCC and can serve as an excellent platform for the development of new therapeutic targets.

Alterations of serum protein N-glycosylation in two mouse models of chronic liver disease are hepatocyte and not B cell driven

N-glycosylation of immunoglobulin G (IgG) has an important impact on the modification of the total serum N-glycome in chronic liver patients. Our aim was to determine the role and magnitude of the alterations in which hepatocytes and B cells are involved in two mouse models of chronic liver disease. Common bile duct ligation (CBDL) and subcutaneous injections with CCl(4) were induced in B cell-deficient and wild-type (WT) mice. IgG depletion was performed with beads covered with protein A/G and the depletions were evaluated by SDS-PAGE and Western blot analysis. N-glycan analysis was performed by improved DSA-FACE technology. Structural analysis of the mouse serum N-glycans was performed by exoglycosidase digests and MALDI-TOF mass spectrometry of permethylated glycans. The alterations seen in B cell-deficient mice closely resembled the alterations in WT mice, in both the CBDL and the CCl(4) models. N-glycan analysis of the IgG fraction in both mouse models revealed different changes compared with humans. Overall, the impact of IgG glycosylation on total serum glycosylation was marginal. Interestingly, the amount of fibrosis present in CBDL B cell-deficient mice was significantly increased compared with CBDL WT mice, whereas the opposite was true for the CCl(4) model as determined by Sirius red staining. However, this had no major effect on the alteration of N-glycosylation of serum proteins. Alterations of total serum N-glycome in mouse models of chronic liver disease are hepatocyte-driven. Undergalactosylation of IgG is not present in mouse models of chronic liver disease.