Anja Geerts

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.

Angiogenesis in chronic liver disease and its complications

Nowadays, liver cancer, cirrhosis and other liver‐related diseases are the fifth most common cause of mortality in the UK. Furthermore, chronic liver diseases (CLDs) are one of the major causes of death, which are still increasing year‐on‐year. Therefore, knowledge about the pathophysiology of CLDs and its complications is of uttermost importance. The goal of this review is to clarify the role of angiogenesis in the disease progression of various liver diseases. Looking closer at the pathophysiology of portal hypertension (PH), fibrosis, cirrhosis, non‐alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC), we find that angiogenesis is a recurring factor in the disease progression. In PH, several factors involved in its pathogenesis, such as hypoxia, oxidative stress, inflammation and shear stress are potential mediators for the angiogenic response. The progression from fibrosis to cirrhosis, the end‐point of CLDs, is distinguished by a prolonged inflammatory and fibrogenic process that leads to an abnormal angioarchitecture distinctive for cirrhosis. In several stages of NASH, a link might be made between the disease progression and hepatic microvasculature changes. HCC is one of the most vascular solid tumours in which angiogenesis plays an important role in its development, progression and metastasis. The close relationship between the progression of CLDs and angiogenesis emphasises the need for anti‐angiogenic therapy as a tool for blocking or slowing down the disease progression. The fact that angiogenesis plays a pivotal role in CLDs gives rise to new opportunities for treating CLDs and its complications.

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 angiogenesis and permeability in the mesenteric microvasculature of rats with cirrhosis and portal hypertension: an in vivo study

Abstract: Background: In vivo evidence for angiogenesis in the splanchnic vasodilation in portal hypertension (PHT) and cirrhosis is lacking. Vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) are mediators of angiogenesis. The present study visualises in vivo structural changes (angiogenesis and vascular hyperpermeability) and examines the presence of VEGF and eNOS in the mesenteric microvasculature of animal models of PHT with and without cirrhosis.

Comparison of three research models of portal hypertension in mice: macroscopic, histological and portal pressure evaluation.

The characterization of mice models of portal hypertension (PHT) is lacking in the literature. Therefore, the aim of the present study was to make a histological approach during development of PHT in two models of cirrhosis with PHT compared with one model of isolated PHT. The model of isolated PHT was developed by partial portal vein ligation (PPVL). Two portal hypertensive cirrhotic mice models were developed either by common bile duct ligation (CBDL) or administration of carbon tetrachloride (CCl4) subcutaneously (twice weekly, 1 ml/kg). These models represent, respectively, a secondary biliary cirrhosis and alcoholic cirrhosis. Mice were killed at several time points to evaluate liver changes by histological and ultrastructural methods. A correlation was made with portal pressure measurements. Histology revealed the absence of fibrosis or cirrhosis in PPVL mice. They developed an isolated portal hypertension. After CBDL induction, the mice developed the characteristics of cirrhosis after 6 weeks, with simultaneous increase in portal pressures. Fifty percent of the mice had ascites at that time point. Sixteen weeks after administration of CCl4, a micronodular cirrhotic aspect of the liver was seen associated with signs of portal hypertension. This is the first descriptive study of three widely used animal models in mice, allowing the study of pathophysiological changes in cirrhosis and portal hypertension. The PPVL in mice leads to a model of isolated portal hypertension. Secondary biliary cirrhosis developed after 6 weeks of common bile duct ligation in 50% of the mice that developed ascites. Subcutaneous injection of CCl4 for 16 weeks induces cirrhosis and poral hypertension, without ascites. Moreover, the present study is the first description of a cirrhotic model in mice developed by subcutaneous injections of CCl4. Well‐described mice models will facilitate use of knock‐out or transgenic mice and lead to a better understanding of the underlying molecular pathways in the field of portal hypertension and cirrhosis.

Role of Placental Growth Factor in Mesenteric Neoangiogenesis in a Mouse Model of Portal Hypertension

BACKGROUND & AIMS Portal hypertension is responsible for the major complications associated with cirrhosis. Angiogenesis has been associated with the pathophysiology of portal hypertension. We investigated the role of placental growth factor (PlGF) and tested the effects of monoclonal antibodies against PlGF (alphaPlGF) in a mouse model of portal hypertension. METHODS Using a mouse model of prehepatic portal hypertension, we measured PlGF levels in the mesenteric tissue at different time points. We used knockout mice and alphaPlGF to determine the role of PlGF in the splanchnic hyperdynamic system and portosystemic collateral formation, examining its effects before and after portal hypertension was induced. RESULTS PlGF was significantly up-regulated in the mesenteric tissue of mice with portal hypertension. Compared with wild-type animals, the vascular density in the mesentery was reduced in PlGF knockout hypertensive mice, preventing collateral formation and attenuation of mesenteric artery flow without affecting portal pressure. In the prevention study, alphaPlGF showed similar findings as in the knockout study. In mice with portal hypertension, administration of alphaPlGF resulted in a 32% decrease in portal pressure, compared with mice given immunoglobulin G(1) (control). CONCLUSIONS Pathologic angiogenesis in the mesenteric tissues of mice with portal hypertension is mediated by PlGF. Blocking PlGF could be an effective strategy for reducing collateral formation and lowering portal pressure; further research into the effects in cirrhosis is warranted.

Evaluation of inflammatory and angiogenic factors in patients with non-alcoholic fatty liver disease

The liver is a major target of injury in obese patients. Non-alcoholic fatty liver disease (NAFLD) is present in 60-90% of obese Americans and can range from simple steatosis to the more severe non-alcoholic steatohepatitis (NASH). The onset of a chronic inflammatory reaction marks the progression from simple steatosis to NASH and the expansion of adipose tissue is strongly associated with angiogenesis. Therefore, we determined the serum concentration of inflammatory [tumor necrosis factor alpha (TNFα) and interleukin 6 (IL6)] and angiogenic [vascular endothelial growth factor A (VEGF)] cytokines and soluble VEGF receptors 1 and 2 (sVEGFR1, sVEGFR2) in the serum of an obese population with simple steatosis and NASH compared to healthy controls. Moreover, we determined the TNFα, IL6, VEGF, VEGFR1 and VEGFR2 gene expression in the liver of these simple steatosis and NASH patients. The population consisted of 30 obese patients, which were diagnosed with simple steatosis and 32 patients with NASH and compared to 30 age-and-sex matched healthy controls. Mean serum TNFα levels were elevated in the serum of simple steatosis and NASH patients compared to healthy controls, reaching significance in NASH patients. IL6 was significantly increased in simple steatosis and NASH patients compared to the healthy controls. VEGF levels were significantly elevated in patients with simple steatosis and borderline significantly elevated in NASH patients compared to the serum levels of healthy control subjects. The concentration of sVEGFR1 was significantly increased in serum of simple steatosis and NASH patients compared to controls. sVEGFR2 concentration was not significantly different in the three groups. TNFα mRNA expression was higher in NASH patients compared to simple steatosis patients. Hepatic gene expression of VEGF, VEGFR1 and VEGFR2 were slightly decreased in NASH patients compared to simple steatosis patients. These data indicate the involvement of inflammatory (TNFα and IL6), angiogenic (VEGF) cytokines and sVEGFR1 in the pathophysiology of NAFLD.

Hypomagnesemia and the risk of new‐onset diabetes after liver transplantation

New‐onset diabetes after transplantation (NODAT) is a frequent complication after liver transplantation and has a negative impact on both patient and graft survival. In analogy with the previous finding of an association between posttransplant hypomagnesemia and NODAT in renal transplant recipients, the relation between both pretransplant and posttransplant hypomagnesemia and NODAT was studied in liver transplant recipients (LTRs). One hundred sixty‐nine adult LTRs (>18 years old) without diabetes who underwent transplantation between 2004 and 2009 were studied (mean age = 52.11 ± 12.6 years, proportion of LTRs who were male = 67.5%, body mass index = 25.5 ± 4.4 kg/m2, proportion receiving tacrolimus = 90.0%). NODAT was defined according to the American Diabetes Association criteria. The association of NODAT with both pretransplant and posttransplant serum magnesium (Mg) was examined. Overall, 52 of 169 patients (30.8%) developed NODAT, and 57.7% of these (30 patients) were treated with antidiabetic drugs. Both pretransplant Mg levels and Mg levels in the first month after transplantation were lower in patients developing NODAT (P = 0.008 and P = 0.001, respectively). A multivariate regression model (adjusted for weight, pretransplant glucose levels, hyperglycemia in the first week after transplantation, gender, hepatitis C, and corticosteroid dosing) demonstrated both pretransplant Mg levels (hazard ratio = 0.844 per 0.1 mg/dL increase, 95% confidence interval = 0.764‐0.932, P = 0.001) and posttransplant Mg levels (hazard ratio = 0.659, 95% confidence interval = 0.518‐0.838, P = 0.001) to be independent predictors of NODAT together with age, biopsy‐proven acute rejection, and cytomegalovirus (CMV) infection in the first year after transplantation. In conclusion, pretransplant hypomagnesemia and early posttransplant hypomagnesemia are independent predictors of new‐onset diabetes after liver transplantation. Other risk factors are age, biopsy‐proven acute rejection, and CMV infection. Liver Transpl 16:1278‐1287, 2010.

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)