Christophe Casteleyn

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;)

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.

The Tonsils Revisited: Review of the Anatomical Localization and Histological Characteristics of the Tonsils of Domestic and Laboratory Animals

This paper gives an overview of the anatomical localization and histological characteristics of the tonsils that are present in ten conventional domestic animal species, including the sheep, goat, ox, pig, horse, dog, cat, rabbit, rat, and pigeon. Anatomical macrographs and histological images of the tonsils are shown. Six tonsils can be present in domestic animals, that is, the lingual, palatine, paraepiglottic, pharyngeal, and tubal tonsils and the tonsil of the soft palate. Only in the sheep and goat, all six tonsils are present. Proper tonsils are absent in the rat, and pigeon. In the rabbit, only the palatine tonsils can be noticed, whereas the pig does not present palatine tonsils. The paraepiglottic tonsils lack in the ox, horse, and dog. In addition, the dog and cat are devoid of the tubal tonsil and the tonsil of the soft palate.

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.

From Vascular Corrosion Cast to Electrical Analog Model for the Study of Human Liver Hemodynamics and Perfusion

Hypothermic machine perfusion (HMP) is experiencing a revival in organ preservation due to the limitations of static cold storage and the need for better preservation of expanded criteria donor organs. For livers, perfusion protocols are still poorly defined, and damage of sinusoidal endothelial cells and heterogeneous perfusion are concerns. In this study, an electrical model of the human liver blood circulation is developed to enlighten internal pressure and flow distributions during HMP. Detailed vascular data on two human livers, obtained by combining vascular corrosion casting, micro-CT-imaging and image processing, were used to set up the electrical model. Anatomical data could be measured up to 5-6 vessel generations in each tree and showed exponential trend lines, used to predict data for higher generations. Simulated flow and pressure were in accordance with literature data. The model was able to simulate effects of pressure-driven HMP on liver hemodynamics and reproduced observations such as flow competition between the hepatic artery and portal vein. Our simulations further indicate that, from a pure biomechanical (shear stress) standpoint, HMP with low pressures should not result in organ damage, and that fluid viscosity has no effect on the shear stress experienced by the liver microcirculation in pressure-driven HMP.

Perfusion characteristics of the human hepatic microcirculation based on three-dimensional reconstructions and computational fluid dynamic analysis

The perfusion of the liver microcirculation is often analyzed in terms of idealized functional units (hexagonal liver lobules) based on a porous medium approach. More elaborate research is essential to assess the validity of this approach and to provide a more adequate and quantitative characterization of the liver microcirculation. To this end, we modeled the perfusion of the liver microcirculation using an image-based three-dimensional (3D) reconstruction of human liver sinusoids and computational fluid dynamics techniques. After vascular corrosion casting, a microvascular sample (±0.134 mm(3)) representing three liver lobules, was dissected from a human liver vascular replica and scanned using a high resolution (2.6 μm) micro-CT scanner. Following image processing, a cube (0.15 × 0.15 × 0.15 mm(3)) representing a sample of intertwined and interconnected sinusoids, was isolated from the 3D reconstructed dataset to define the fluid domain. Three models were studied to simulate flow along three orthogonal directions (i.e., parallel to the central vein and in the radial and circumferential directions of the lobule). Inflow and outflow guidances were added to facilitate solution convergence, and good quality volume meshes were obtained using approximately 9 × 10(6) tetrahedral cells. Subsequently, three computational fluid dynamics models were generated and solved assuming Newtonian liquid properties (viscosity 3.5 mPa s). Post-processing allowed to visualize and quantify the microvascular flow characteristics, to calculate the permeability tensor and corresponding principal permeability axes, as well as the 3D porosity. The computational fluid dynamics simulations provided data on pressure differences, preferential flow pathways and wall shear stresses. Notably, the pressure difference resulting from the flow simulation parallel to the central vein (0-100 Pa) was clearly smaller than the difference from the radial (0-170 Pa) and circumferential (0-180 Pa) flow directions. This resulted in a higher permeability along the central vein direction (k(d,33) = 3.64 × 10(-14) m(2)) in comparison with the radial (k(d,11) = 1.56 × 10(-14) m(2)) and circumferential (k(d,22) = 1.75 × 10(-14) m(2)) permeabilities which were approximately equal. The mean 3D porosity was 14.3. Our data indicate that the human hepatic microcirculation is characterized by a higher permeability along the central vein direction, and an about two times lower permeability along the radial and circumferential directions of a lobule. Since the permeability coefficients depend on the flow direction, (porous medium) liver microcirculation models should take into account sinusoidal anisotropy.

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.

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).

Locations of gut-associated lymphoid tissue in the 3-month-old chicken: a review.

The lymphoid tissue that is associated with the intestinal tract, the so-called gut-associated lymphoid tissue (GALT), is well developed in the chicken. Depending on the location, it is present as aggregations of lymphoid cells, or organized in lymphoid follicles and tonsils. From proximal to distal, the intestinal tract contains a pharyngeal tonsil, diffuse lymphoid tissue and lymphoid follicles in the cervical and thoracic parts of the oesophagus, an oesophageal tonsil, diffuse lymphoid tissue in the proventriculus, a pyloric tonsil, Peyers patches, Meckels diverticulum, two caecal tonsils, diffuse lymphoid tissue in the rectum, the bursa of Fabricius, and diffuse lymphoid tissue in the wall of the proctodeum. The lymphoid tissues are frequently covered by a lympho-epithelium that is infiltrated by lymphoid cells. Such an epithelium often contains M or microfold cells, which are specialized in antigen sampling and transport antigens to the underlying lymphoid tissue. A solid knowledge of the avian GALT could contribute to the development of vaccines to be administered orally. Additionally, immune stimulation via pre- and probiotics is based on the presence of a well-developed intestinal immune system.

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)