Effects of ET(A) receptor antagonism on proinflammatory gene expression and microcirculation following hepatic ischemia/reperfusion

BACKGROUND: The objective of this study was to investigate the effect of a specific endothelin(A) receptor antagonist (ET(A)-RA) on mRNA expression of genes encoding vasoactive mediators and proinflammatory cytokines and on the microhemodynamics (assessed by measurement of laser Doppler flow and tissue blood gases) following complete vascular exclusion of the porcine liver. STUDY DESIGN: Sixteen adult German landrace pigs were subjected to 120 min of warm hepatic ischemia by total vascular exclusion. To avoid portal congestion, a passive porto-femoro/jugular bypass was implanted. The animals were divided into 2 groups: the control group received saline solution and the therapy group was given the selective ET(A)-RA BSF 208075. Hepatic microcirculation was evaluated by p(O(2)) and p(CO(2)) measurement with the Paratrend sensor and by laser Doppler flow measurement. Liver tissue samples were collected 1 h after reperfusion and quantitative mRNA expression for prepro-ET-1, pro-IL-1beta, pro-IL-6, pro-TNF-alpha, eNOS was analyzed using the TaqMan system. Additionally, immunohistochemical analysis using a semiquantitative score for ET-1 was performed. Postischemic liver damage was monitored by measurement of liver enzymes and assessed by histological analysis using a semiquantitative scoring classification. RESULTS: Partial oxygen pressure in the hepatic tissue and laser Doppler flow were significantly improved in the therapy group. One hour after reperfusion, quantitative RT-PCR revealed significantly lower expression of prepro-ET-1, eNOS, pro-TNF-alpha, and pro-IL-6 in the therapy group compared to controls. Immunohistochemical analysis demonstrated significantly reduced ET-1 immunostaining after therapy. Furthermore, blockade of ET(A) receptors prevents tissue damage. CONCLUSIONS: Treatment with the selective ET(A)-RA BSF 208075 has protective effects on microcirculation after 120 min liver ischemia and reperfusion. The authors were able to show that ET(A)-RA not only affects the expression of vasoactive genes, but also decreases gene expression of proinflammatory cytokines such as TNF-alpha and IL-6.     

Relationship between the size of the capillary bed and oxidative capacity in various cat skeletal muscles

The size of the capillary bed, assessed by capillary density (CD), capillary per muscle fibre ratio (C/F), total capillary length, surface area and volume was related to the oxidative capacity, assessed by the volume density of mitochondria and O₂max in cat muscles with a different composition of glycolytic and oxidative fibres: predominantly glycolytic gracilis, purely oxidative soleus and gracilis transformed towards oxidative by chronic low frequency (10 Hz) electrical stimulation. Maximal blood flow and lactate output were measured in the muscles during isometric contractions.When capillary supply was estimated by C/F ratio, there was a close correlation between various parameters only in stimulated gracilis. The combined data of all muscles showed a significant correlation between the total volume of mitochondria, O₂max and total capillary surface area. Capillary volume showed a tight correlation with maximal blood flow in both control and stimulated gracilis, but not in soleus. Maximal blood flow was correlated withVO₂max in oxidative muscles (stimulated gracilis and soleus) but not in control glycolytic gracilis. Moreover normal gracilis did not show any relationship between the volume density of mitochondria and the size of the capillary bed. The latter was inversely correlated with the output of lactate which was greater in muscles with a lower C/P ratio.The data on gracilis indicates that the capillary bed can adapt to the increased demand for oxygen and a greater oxidative capacity induced by long-term activity imposed on a glycolytic muscle, while it may be more important for the removal of lactate in the glycolytic muscles under their normal activity. The factors involved in the regulation of blood flow in control soleus — when the morphological size of the vascular bed is not related to blood flow — are discussed.     

Identification of progression markers in B-CLL by gene expression profiling

OBJECTIVE: B-cell chronic lymphocytic leukemia is a heterogeneous disease with a pronounced variation in the clinical course. With the purpose of identifying genes that could be related to disease progression, we have performed gene expression profiling on B-CLL patients with an indolent disease and patients with a progressive disease with need for therapy. MATERIALS AND METHODS: we applied the Affymetrix GeneChip technique to 11 B-CLL patients with stable and 10 patients with clinically progressive disease. Supervised and unsupervised clustering methods with different algorithms were used to identify genes that tend to give a distinction between stable and progressive disease. RESULTS: The supervised learning procedures identified groups of genes with a combined power to discriminate samples from progressive and stable disease with 70-90% accuracy. The gene for protein phosphatase 2 regulatory subunit B' (B56) gamma isoform (PPP2R5C) and the gene for retinoblastoma-like 2 (p130) (RBL2) were included among the best discriminators; both genes were downregulated in progressive as compared to stable B-CLL. In a hierarchical clustering analysis based on gene expression pattern three clinical subcategories could be identified: one with a more severe clinical outcome, a second one with good prognosis, and a third one that was intermediate between the other two groups. CONCLUSIONS: Our application of microarray analysis on a clinically well defined material has identified a number of genes with combined expression patterns related to stable or progressive disease in general. Unsupervised clustering suggested the existence of subclasses of samples in the progressive group that may be identifiable through gene expression patterns.     

Complement activation by both classical and alternative pathways is critical for the effector phase of arthritis

To analyze the role of the classical and alternative pathways of complement activation in the effector phase of arthritis, we have induced arthritis in C3- and factor B (FB)-deficient (C3(-/-) and FB(-/-)) DBA/1J mice using well-defined monoclonal IgG2b and IgG2a antibodies to type II collagen. In control DBA/1J mice, severe swelling of the joints, destruction of cartilage and erosion of bone developed very rapidly with a 100% incidence and a peak on days 7-10. Although 75% of C3(-/-) mice developed arthritis, the clinical severity was very mild and the onset was delayed. Severity of arthritis in FB(-/-) mice ranked intermediate in comparison with C3(-/-) and control mice with an incidence of 100%. Immunohistochemical analysis of the inflamed joints demonstrated substantial reduction in macrophage and neutrophilic leukocyte infiltration in both C3(-/-) and FB(-/-) mice, thereby confirming the clinical findings. We conclude that both the classical and the alternative pathways of complement activation are involved in the effector phase of arthritis.     

Responsiveness to peripherally administered melanocortins in lean and obese mice

To elucidate mechanisms of melanocortin action, we investigated the effects of a melanocortin receptor agonist (melanotetan II [MTII]) in lean C57BL/6J and obese (DIO, ob/ob, UCP1-DTA) mice. MTII administration (100 microg q.i.d. i.p.) for 24 h results in similar weight loss but a more pronounced decrease of food intake in DIO mice. After 4 and 8 days of MTII treatment, however, the reduction in both food intake and body weight is more pronounced in DIO mice than in lean mice. MTII administration for 24 h prevents food deprivation-induced alterations in hypothalamic neuropeptide Y (NPY) and liver adiponectin receptor 1 and adiponectin receptor 2 mRNA expression, but does not alter hypothalamic mRNA expression of melanocortin 4 receptor or adiponectin serum and mRNA expression levels. NPY and agouti gene-related protein (AgRP) mRNA expression after 8 days of MTII is increased to levels comparable to pair-fed mice. In summary, 1) MTII is an effective treatment for obesity and related metabolic defects in leptin-resistant (DIO, UCP1-DTA) and leptin-sensitive (ob/ob) mouse models of obesity; 2) the effects of MTII on food intake and body weight are more pronounced in DIO mice than in lean mice; 3) the tachyphylactic effect after prolonged MTII administration appears to be, at least in part, caused by a compensatory upregulation of NPY and AgRP mRNA levels, whereas decreasing leptin levels may play a very minor role in mediating tachyphylaxis; and 4) alterations in adiponectin receptor mRNA expression after fasting or MTII treatment may contribute to altered insulin sensitivity and needs to be studied further.     

The role of leptin in regulating neuroendocrine function in humans

Eating disorders are a group of disease states including anorexia nervosa, bulimia nervosa and binge eating on one end as well as episodic or chronic overeating resulting in obesity at the other end of the spectrum. These disorders are characterized by decreased and/or increased energy intake and are frequently associated with hormonal and metabolic disorders. The discovery of leptin, an adipocyte-secreted hormone acting in the brain to regulate energy homeostasis, and its subsequent study in human physiology have significantly advanced our understanding of normal human physiology and have provided new opportunities for understanding and possibly treating disease states, such as anorexia and bulimia nervosa. It has been recently discovered that leptin levels above a certain threshold are required to activate the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-thyroid axes in men, whereas the hypothalamic-pituitary-adrenal, renin-aldosterone, and growth hormone-IGF-1 axes may be largely independent of circulating leptin levels in humans. In this review, we summarize the latest findings related to the role of leptin in the regulation of several neuroendocrine axes, such as the hypothalamic-pituitary-gonadal and the hypothalamic-pituitary-thyroid axes in humans and discuss its potential pathophysiologic role in eating disorders.     

Modulation of Rho GTPase activity alleviates chondroitin sulfate proteoglycan-dependent inhibition of neurite extension

The central nervous system (CNS) fails to regenerate after injury. A glial scar forms at the injury site, contributing to regenerative failure partly resulting from the chondroitin sulfate proteoglycans (CSPGs) in the glial scar. The family of Rho GTPases, which includes Cdc42, Rac1, and RhoA, is involved in growth cone dynamics. Although the response of neural cells to the inactivation of Rho when contacting myelin-related substrates, or CSPG, has been investigated, Rac1's and Cdc42's abilities to modulate CSPG-dependent inhibition have yet to be explored. In this study, a stripe assay was utilized to examine the effects of modulating all three Rho GTPases on neurite extension across inhibitory CSPG lanes. Alternating laminin (LN) and CSPG lanes were created and NG108-15 cells and E9 chick dorsal root ganglia (DRGs) were cultured on the lanes. By using the protein delivery agent Chariot, the neuronal response to exposure of constitutively active (CA) and dominant negative (DN) mutants of the Rho GTPases, along with the bacterial toxin C3, was determined by quantifying the percentage ratio of neurites crossing the CSPG lanes. CA-Cdc42, CA-Rac1, and C3 transferase significantly increased the number of neurites crossing into the CSPG lanes compared with the negative controls for both the NG108-15 cells and the E9 chick DRGs. We also show that these mutant proteins require the delivery vehicle, Chariot, to enter the neurons and affect neurite extension. Therefore, activation of Cdc42 and Rac, as well as inhibition of Rho, helps overcome the CSPG-dependent inhibition of neurite extension.