Rita Vos

Protection of hepatocyte mitochondrial ultrastructure and function by strict blood glucose control with insulin in critically ill patients

BACKGROUND Maintenance of normoglycaemia by use of insulin reduces morbidity and mortality of patients in surgical intensive care. Studies on mitochondrial function in critical illness or diabetes suggest that effects of intensive insulin therapy on mitochondrial integrity contribute to the clinical benefits. METHODS Enzyme activities of the respiratory-chain complexes and oxidative-stress-sensitive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were measured by spectrophotometry in 36 snap-frozen samples of liver and skeletal muscle obtained after death from patients who had been randomly assigned intensive (normoglycaemia) or conventional (hyperglycaemia) insulin therapy and who were similar in terms of admission diagnoses and causes of death. Mitochondrial ultrastructure was examined by electron microscopy in a random subgroup (n=20). FINDINGS In the liver, hypertrophic mitochondria with an increased number of abnormal and irregular cristae and reduced matrix electron density were observed in seven of nine conventionally treated patients. Only one of 11 patients given intensive insulin treatment had these morphological abnormalities (p=0.005). The effect on ultrastructure was associated with higher activities of respiratory-chain complex I (median 1.53 [IQR 1.14-3.01] vs 0.81 [0.54-1.43] U/g liver; p=0.008) and complex IV (1.69 [1.40-1.97] vs 1.16 [0.97-1.40] U/g; p=0.008) in the intensive group than in the conventional group. There was no detectable difference in GAPDH activity. In skeletal muscle, mitochondrial ultrastructure and function were not affected by intensive insulin therapy. INTERPRETATION Strict glycaemic control with intensive insulin therapy prevented or reversed ultrastructural and functional abnormalities of hepatocyte mitochondria. The lack of effect on skeletal-muscle mitochondria suggests a direct effect of glucose toxicity and glucose control, rather than of insulin, as the likely explanation. RELEVANCE TO PRACTICE Maintenance or restoration of mitochondrial function and cellular energetics is another therapeutic target, in addition to optimisation of cardiac output, systemic oxygen delivery, and regional blood flow, that might improve outcome for critically ill patients. Our findings could help to explain the mechanism underlying the reduction in mortality found when normoglycaemia was maintained with insulin, and further support use of intensive insulin therapy in this setting.

Morphological and biochemical characterization of a human liver in a uPA‐SCID mouse chimera

A small animal model harboring a functional human liver cell xenograft would be a useful tool to study human liver cell biology, drug metabolism, and infections with hepatotropic viruses. Here we describe the repopulation, organization, and function of human hepatocytes in a mouse recipient and the infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) of the transplanted cells. Homozygous urokinase plasminogen activator (uPA)‐SCID mice underwent transplantation with primary human hepatocytes, and at different times animals were bled and sacrificed to analyze plasma and liver tissue, respectively. The plasma of mice that were successfully transplanted contained albumin and an additional 21 human proteins. Liver histology showed progressive and massive replacement of diseased mouse tissue by human hepatocytes. These cells were accumulating glycogen but appeared otherwise normal and showed no signs of damage or death. They formed functional bile canaliculi that connected to mouse canaliculi. Besides mature hepatocytes, human hepatic progenitor cells that were differentiating into mature hepatocytes could be identified within liver parenchyma. Infection of chimeric mice with HBV or HCV resulted in an active infection that did not alter the liver function and architecture. Electron microscopy showed the presence of viral and subviral structures in HBV infected hepatocytes. In conclusion, human hepatocytes repopulate the uPA+/+‐SCID mouse liver in a very organized fashion with preservation of normal cell function. The presence of human hepatic progenitor cells in these chimeric animals necessitates a critical review of the observations and conclusions made in experiments with isolated “mature” hepatocytes. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;41:847–856.)

Clinicopathological study on cholangiolocellular carcinoma suggesting hepatic progenitor cell origin

Cholangiolocellular carcinoma (CLC), a subtype of cholangiocellular carcinoma (CC), is thought to originate from the ductules/canals of Hering, where hepatic progenitor cells (HPCs) are located. We investigated the clinicopathological features of 30 CLCs and their relationship to HPCs. We evaluated the expression of hepatocytic markers (hepatocyte paraffin‐1, canalicular polyclonal carcinoembryonic antigen, and CD10), biliary/HPC markers (keratin [K]7, K19, and neural cell adhesion molecule), the adenosine triphosphate binding cassette transporters: multidrug resistance protein 1, multidrug resistance‐associated protein (MRP)1, MRP3, and breast cancer resistance protein, using immunohistochemistry and electron microscopy. In addition, gene expression profiling of CLC was performed and compared with the profile of hepatocellular carcinoma (HCC) with or without HPC features (K19 expression). In surrounding nontumoral tissue, K7‐positive and K19‐positive HPCs/ductular reaction were observed. More than 90% of the tumor was composed of CLC areas that showed small monotonous and/or anastomosing glands, strongly positive for K7 and K19. Especially at the tumor boundary, all cases showed a HCC‐like trabecular area characterized by canalicular CD10/polyclonal carcinoembryonic antigen expression, and submembranous K7 expression, similar to intermediate hepatocytes. K7‐positive/K19‐positive HPCs were also seen. Out of 30 cases, 19 showed papillary and/or clear glandular formation with mucin production, representing CC areas. These three different areas showed transitional zones with each other. We observed an increased expression of MRP1, MRP3, and breast cancer resistance protein in the tumor. Electron microscopy findings in HCC‐like trabecular areas confirmed the presence of HPCs and intermediate hepatocytes. HPC markers, K7, K19, prominin‐1, receptor for stem cell factor c‐kit, octamer‐4 transcription factor, and leukemia inhibitory factor were upregulated (P < 0.05), while albumin was downregulated in CLC (P = 0.007) toward K19‐negative HCCs. Comparison of CLC with K19‐positive HCCs indicated a high homology. Conclusion: All these findings highly suggest a progenitor cell origin of CLC. (HEPATOLOGY 2008.)

TAK1 Suppresses a NEMO-Dependent but NF-κB-Independent Pathway to Liver Cancer

The MAP3-kinase TGF-beta-activated kinase 1 (TAK1) critically modulates innate and adaptive immune responses and connects cytokine stimulation with activation of inflammatory signaling pathways. Here, we report that conditional ablation of TAK1 in liver parenchymal cells (hepatocytes and cholangiocytes) causes hepatocyte dysplasia and early-onset hepatocarcinogenesis, coinciding with biliary ductopenia and cholestasis. TAK1-mediated cancer suppression is exerted through activating NF-kappaB in response to tumor necrosis factor (TNF) and through preventing Caspase-3-dependent hepatocyte and cholangiocyte apoptosis. Moreover, TAK1 suppresses a procarcinogenic and pronecrotic pathway, which depends on NF-kappaB-independent functions of the I kappaB-kinase (IKK)-subunit NF-kappaB essential modulator (NEMO). Therefore, TAK1 serves as a gatekeeper for a protumorigenic, NF-kappaB-independent function of NEMO in parenchymal liver cells.

Synaptophysin: A novel marker for human and rat hepatic stellate cells.

Synaptophysin is a protein involved in neurotransmitter exocytosis and is a neuroendocrine marker. We studied synaptophysin immunohistochemical expression in 35 human liver specimens (normal and different pathological conditions), in rat models of galactosamine hepatitis and carbon tetrachloride-induced cirrhosis, and in freshly isolated rat stellate cells. Synaptophysin reactivity was present in perisinusoidal stellate cells in both human and rat normal liver biopsies. The number of synaptophysin-reactive perisinusoidal cells increased in pathological conditions. Double staining for alpha-smooth muscle actin and synaptophysin, detected by confocal laser scanning microscopy, unequivocally demonstrated colocalization of both markers in lobular stellate cells. In addition, freshly isolated rat stellate cells expressed synaptophysin mRNA (detected by polymerase chain reaction) and protein. Finally, electron microscopy showed the presence of small electron translucent vesicles, comparable to the synaptophysin-reactive synaptic vesicles in neurons, in stellate cell projections. We conclude that synaptophysin is a novel marker for quiescent as well as activated hepatic stellate cells. Together with the stellate cells expression of neural cell adhesion molecule, glial fibrillary acidic protein, and nestin, this finding raises questions about its embryonic origin and its differentiation. In addition, the presence of synaptic vesicles in stellate cell processes suggests a hitherto unknown mechanism of interaction with neighboring cells.

Determinants of symptoms in functional dyspepsia: gastric sensorimotor function, psychosocial factors or somatisation?

Background: Gastric sensorimotor dysfunction, psychosocial factors and somatisation are all implicated in symptom generation in functional dyspepsia (FD). Aim: To determine the relative contribution of each of these factors to overall dyspeptic symptom severity and weight loss in FD. Methods: In 201 consecutive tertiary care patients with FD (mean age 40.1 (SD 12.6) years), gastric sensorimotor function was studied using barostat (sensitivity, compliance and accommodation). Psychosocial factors (depression and anxiety disorders, positive and negative affect, perceived stress, alexithymia and history of abuse), somatisation and co-morbid irritable bowel syndrome (IBS) and chronic fatigue symptoms were assessed using self-report questionnaires. Variables were correlated with dyspepsia symptom severity (DSS) and weight loss. Hierarchical multiple linear regression was used to identify determinants of DSS and weight loss. Results: Multiple linear regression identified the following determinants of DSS: gastric sensitivity (β = 0.77, p = 0.25), depression (β = 0.12, p = 0.06) and somatisation (β = 0.48, p<0.0001) (controlling for age and occupation, R2 = 0.29, p<0.0001). The effect of depression on DSS is partially mediated by somatisation. Gastric sensitivity (β = 2.87, p = 0.08), history of childhood sexual abuse (β = 9.37, p = 0.0006), depression (β = 0.19, p = 0.24) and somatisation (β = 0.67, p = 0.01) are independent determinants of weight loss (controlling for gender and occupation, R2 = 0.42, p<0.0001). The effect of depression on weight loss is fully mediated by somatisation. Conclusion: Symptom severity and weight loss in FD are determined by psychosocial factors (depression, abuse history) and somatisation, and only to a lesser extent by gastric sensorimotor function. The importance of psychosocial factors and somatisation compared to gastric sensorimotor function is most pronounced in hypersensitive patients.

Hepatic progenitor cells in hepatocellular adenomas

Hepatocellular adenoma is a benign tumor of the liver that has a small but not negligible risk of malignant transformation into hepatocellular carcinoma. In analogy with the established role of oval cells in hepatocarcinogenesis in rodent models, human hepatic progenitor cells may have a function in the development of liver tumors. To investigate this issue, we performed immunohistochemistry on biopsies of 10 consecutively resected hepatocellular adenomas using markers for hepatic progenitor cells. Sections of paraffin-embedded and frozen biopsies were stained using antibodies against cytokeratins 7, 8, 18, and 19, chromogranin-A, OV-6, and neural cell adhesion molecule. Hepatic progenitor cells were observed in five of 10 hepatocellular adenomas. These five tumors also contained cells with an immunohistochemical phenotype intermediate between hepatic progenitor cells and hepatocytes. Hepatic progenitor cells and intermediate hepatocyte-like cells were scattered throughout the tumors with a density that varied from area to area. Ultrastructural examination confirmed the presence of hepatic progenitor cells. Our study shows that hepatic progenitor cells are present in a considerable proportion of hepatocellular adenomas, supporting the hypothesis that human hepatic progenitor cells can play a role in the development of hepatocellular tumors.

Identification of telocytes in the upper lamina propria of the human urinary tract

The upper lamina propria (ULP) area of interstitial cells (IC) has been studied extensively in bladder, but is rather unexplored in the rest of the urinary tract. This cell layer is intriguing because of the localization directly underneath the urothelium, the intercellular contacts and the close relationship with nerve endings and capillaries. In this study, we examine the ULP layer of IC in human renal pelvis, ureter and urethra, and we make a comparison with ULP IC in bladder. Tissue was obtained from normal areas in nephrectomy, cystectomy and prostatectomy specimens, and processed for morphology, immunohistochemistry and electron microscopy. A morphological and immunohistochemical phenotype for the ULP IC was assessed and region‐dependent differences were looked for. The ULP IC in renal pelvis, ureter and urethra had a similar ultrastructural phenotype, which differed somehow from that of bladder IC, that is, thinner and longer cytoplasmic processes, no peripheral actin filaments and presence of dense core granules and microtubules. Together with their immunohistochemical profile, these features are most compatible with the phenotype of telocytes, a recently discovered group of stromal cells. Based on their global ultrastructural and immunohistochemical phenotype, ULP IC in human bladder should also be classified as telocytes. The most striking immunohistochemical finding was the variable expression of oestrogen receptor (ER) and progesterone receptor (PR). The functional relevance of ULP telocytes in the urinary tract remains to be elucidated, and ER and PR might therefore be promising pharmacological research targets.

Relationship between anxiety and gastric sensorimotor function in functional dyspepsia.

Objective: To investigate the relationship between anxiety and gastric sensorimotor function in patients with (hypersensitive) functional dyspepsia (FD). Comorbidity between FD and anxiety disorders is high. In FD, epigastric pain is associated with gastric hypersensitivity and neuroticism, a personality trait related to anxiety. Experimentally induced anxiety in healthy volunteers is associated with changes in sensorimotor function of the proximal stomach. Methods: A total of 139 patients with FD (n = 102 women) underwent a barostat investigation to determine gastric compliance, meal accommodation, and thresholds for discomfort and pain. Anxiety was measured by the State-Trait Anxiety Inventory (STAI) scale (anxiety as a stable personality trait) and the STAI-State scale (momentary anxiety). The anxiety subscale of the Hospital Anxiety and Depression Scale (HADS-A) was filled out to detect comorbid anxiety disorders. Results: Hyper- and normosensitive patients had similar anxiety scores, but gastric compliance was significantly lower in hypersensitive patients (11.4 versus 32.8 ml/mm Hg; p < .001). In the whole patient group, no significant correlations between STAI scores and gastric sensorimotor function were found. In hypersensitive patients (n = 53, 43 women), state anxiety was negatively correlated with discomfort threshold (&rgr; = −.49; p = .001), pain threshold (&rgr; = −.48; p = .02), and gastric compliance (&rgr; = −.46; p = .002). These results were confirmed by multiple linear and logistic regression analyses. Conclusion: In hypersensitive patients with FD, state anxiety is significantly and negatively correlated with discomfort threshold, pain threshold, and compliance. These results strengthen the hypothesis that anxiety is important in FD, especially in hypersensitive patients. FD = functional dyspepsia; STAI = State-Trait Anxiety Inventory; HADS-A = Hospital Anxiety and Depression Scale-Anxiety subscale; FGID = functional gastrointestinal disorders; IBS = irritable bowel syndrome; CNS = central nervous system; MDP = minimal distending pressure; WMW test = Wilcoxon Mann-Whitney test; OR = odds ratio; ANS = autonomic nervous system; EMS = emotional motor system; PAG = periaqueductal grey; LC = locus coeruleus; HPA-axis = hypothalamo-pituitary-adrenal axis; ASI = anxiety sensitivity index; VSI = visceral sensitivity index.

Heparan sulphate proteoglycan expression in human primary liver tumours

Heparan sulphate proteoglycans (HSPGs) play important biological roles in cell–matrix adhesion processes and are essential regulators of growth factor actions (e.g., as co‐receptor for hepatocyte growth factor). Since in liver carcinogenesis, interactions between cells, the matrix, and growth factors play a major role, the aim of this study was to investigate whether the distribution pattern of HSPGs is altered in human primary liver tumours. Twenty‐two primary liver tumours and five normal liver biopsies were studied, using specific monoclonal antibodies against syndecans‐1, ‐2, ‐3, and ‐4; glypican; perlecan; and heparan sulphate chains. Cholangiocarcinomas as well as hepatocellular carcinomas showed an altered immunoreactivity pattern of the different HSPGs in comparison with normal liver parenchyma, probably reflecting the growth regulatory roles of HSPGs. Intracellular positivity for integral membrane HSPGs syndecan‐1 and especially syndecan‐4 was a constant finding in most tumours, suggesting increased synthesis or internalization of these HSPGs. Syndecan‐3 and perlecan expression in tumours was found in an expected distribution pattern. The strong reactivity for syndecan‐3 and perlecan in tumoral stromal vessels might suggest a role for these HSPGs in tumoral angiogenesis. In addition, perlecan probably exerts its known growth factor reservoir function also in the stroma of primary liver tumours.