Sander Lefere

Hypoxia-regulated mechanisms in the pathogenesis of obesity and non-alcoholic fatty liver disease

The pandemic rise in obesity has resulted in an increased incidence of metabolic complications. Non-alcoholic fatty liver disease is the hepatic manifestation of the metabolic syndrome and has become the most common chronic liver disease in large parts of the world. The adipose tissue expansion and hepatic fat accumulation characteristics of these disorders compromise local oxygen homeostasis. The resultant tissue hypoxia induces adaptive responses to restore oxygenation and tissue metabolism and cell survival. Hypoxia-inducible factors (HIFs) function as master regulators of this hypoxia adaptive response, and are in turn hydroxylated by prolyl hydroxylases (PHDs). PHDs are the main cellular oxygen sensors and regulate HIF proteasomal degradation in an oxygen-dependent manner. HIFs and PHDs are implicated in numerous physiological and pathological conditions. Extensive research using genetic models has revealed that hypoxia signaling is also a key mechanism in adipose tissue dysfunction, leading to adipose tissue fibrosis, inflammation and insulin resistance. Moreover, hypoxia affects liver lipid metabolism and deranges hepatic lipid accumulation. This review summarizes the molecular mechanisms through which the hypoxia adaptive response affects adipocyte and hepatic metabolism, and the therapeutic possibilities of modulating HIFs and PHDs in obesity and fatty liver disease.

Non-alcoholic steatohepatitis induces transient changes within the liver macrophage pool

Kupffer cells (KCs) and monocyte-derived macrophages are implicated in non-alcoholic steatohepatitis (NASH) pathogenesis but their functions remain unclear due to the lack of specific markers to distinguish between the different cell types. Additionally, it is unclear if multiple subsets of KCs are present during NASH. Here, we characterized the liver macrophage subsets during methionine/choline deficient (MCD) diet-induced NASH and recovery. We observed a significant reduced contribution of Ly6CloClec4F+Tim4+KCs to the hepatic macrophage pool in MCD fed mice, which normalized during recovery. Ly6CloClec4F-Tim4- monocyte-derived macrophages increased during MCD feeding and returned to baseline during recovery. Ly6CloClec4F+Tim4- monocyte-derived KCs developed during initial recovery but did not self-renew as their numbers were reduced after full recovery. Initial recovery from MCD diet feeding was further characterized by increased proportions of Ki-67+ proliferating KCs. In conclusion, the hepatic macrophage pool undergoes substantial albeit transient changes during NASH and recovery, with the KC pool being maintained by proliferation and differentiation of short-lived monocyte-derived KCs.

Serum vascular cell adhesion molecule-1 predicts significant liver fibrosis in non-alcoholic fatty liver disease

Background:Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide and is strongly associated with obesity, dyslipidemia and insulin resistance. NAFLD often presents as simple steatosis (NAFL) but can progress to non-alcoholic steatohepatitis (NASH) and fibrosis. Current non-invasive biomarkers are not tailored to identify significant (⩾F2) fibrosis, although recent guidelines recommend a stringent follow-up of this patient population. We and others have reported on the role of pathological angiogenesis in the pathogenesis of NAFLD, highlighting pro-angiogenic factors as potential diagnostic markers.Objective:To investigate the applicability of angiogenic and endothelial dysfunction markers as non-invasive diagnostic tools for NASH or NASH-associated fibrosis in obese patients.Methods:In a prospective cross-sectional study, male patients undergoing bariatric surgery (n=61) and control patients (n=35) were recruited. Serum protein levels and visceral adipose tissue gene expression of endothelial dysfunction and angiogenic markers were analyzed by multiplex bead-based assay and quantitative RT-PCR, respectively. For validation, we recruited a second cohort of patients undergoing bariatric surgery (n=40) and a cohort of NAFLD patients from our outpatient clinic (n=30).Results:We identified serum vascular cell adhesion molecule-1 (VCAM-1) as an independent predictor for ⩾F2 fibrosis (median 14.0 vs 8.7 ng ml−1 in patients with and without significant fibrosis; P<0.0001) with an area under the receiver-operating characteristics (AUROC) curve of 0.80. The cutoff point of 13.2 ng ml−1 showed a sensitivity of 80% and specificity of 83%. In line with these results, VCAM-1 visceral adipose tissue gene expression was also elevated in patients with fibrosis (P=0.030). In the bariatric surgery and clinical validation cohorts, VCAM-1 displayed similar AUROCs of 0.89 and 0.85, respectively.Conclusions:VCAM-1 levels are able to accurately predict significant (⩾F2) fibrosis in NAFLD patients.

What to expect from reliability and validity claims? A pragmatic conception of psychiatric nosology

The reliability and validity of psychiatric diagnoses have always been a major concern. The Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) reliability field trials yielded ambiguous results, with some diagnostic categories scoring well below par. We argue that the emphasis on the reliability of psychiatric diagnoses, which has dominated psychiatric nosology and guided the endeavor of improving the DSM in its consecutive editions, is misguided and lacks in structural validity. In this article, we defend a pragmatic view on psychiatric disease as the most fruitful approach to an understanding of what the categorical distinctions in the DSM (can) represent. Disorders in the DSM are descriptions of clinical pictures and do not necessarily correspond to an identified pathological substrate. Although this is a logical result of the nature of psychiatric disease, it bears important consequences. The various DSM disease categories are not uniform but should be regarded as representing different kinds of disorders, ranging from a separation from normal behavior based on practical grounds to the discrete kind of disorders envisioned by proponents of a strong realistic view. We argue that the explication of kinds of disorders outlined in this article provides interesting perspectives on the problems of reliability and validity that the DSM faces.

Placental growth factor inhibition targets pulmonary angiogenesis and represents a novel 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 αPlGFs 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)

Angiopoietin-2 promotes pathological angiogenesis and is a novel therapeutic target in murine non-alcoholic 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.

Refractory subacute steatohepatitis after biliopancreatic diversion

A 24-year-old woman was referred for evaluation of decompensated cirrhosis. Her past medical history revealed BPD surgery 5 years ago. Postoperatively, she experienced marked weight loss, with a body mass index reduction from 40 to 20.2 kg/m. Physical examination revealed a weak general condition, jaundice, and hepatic encephalopathy. Laboratory evaluation showed a bilirubin level of 11 mg/dL, an albumin of 39 g/L, a creatinine of 0.62 mg/dL, and a platelet count of 109,000/lL. The international normalized ratio was 2.09. Ultrasound and computed tomography were negative for focal liver lesions. She denied alcohol abuse and other causes of cirrhosis were excluded using appropriate clinical, imaging, and laboratory investigations. She was listed for transplantation with an initial Model for End-Stage Liver Disease score of 24 and was transplanted 3 months later. Following transplantation, she was treated with corticosteroids, tacrolimus, and mycofenolate mofetil. The explant liver showed marked pericellular and septal fibrosis, as well as macrovesicular steatosis and sparse lobular infiltrates. The biopsy also revealed cholangitis lenta (Fig. 1). The clinical correlate was a small intestinal bacterial overgrowth (SIBO), as diagnosed by lactulose breath tests. SIBO remained persistently present throughout her course, despite adequate antibiotic therapy with metronidazole, norfloxacine, and amoxicillin-clavulanic acid. She also had consistently and disproportionately high serum bilirubin levels. Dismantling of the BPD was postponed until 8 weeks posttransplantation, attributable to her weak general condition at the time of transplantation. Liver biopsy performed during dismantling already showed signs of recurrent severe steatohepatitis with bilirubinostasis (Fig. 2). She rapidly progressed to cirrhosis within 10 months posttransplantation. She was transplanted again, yet redeveloped subacute hepatic failure only 4 months later, necessitating another retransplantation. Her condition did not improve, and after three failed transplantations we decided in deliberation with the patient not to pursue further therapy. She died shortly thereafter.