Suman K. Das

Adipose Triglyceride Lipase Contributes to Cancer-Associated Cachexia

Ablation of a gene controlling fat breakdown can protect mice from cancer-associated uncontrolled loss of fat and muscle. 10.1126/science.1198973 Cachexia is a multifactorial wasting syndrome most common in patients with cancer that is characterized by the uncontrolled loss of adipose and muscle mass. We show that the inhibition of lipolysis through genetic ablation of adipose triglyceride lipase (Atgl) or hormone-sensitive lipase (Hsl) ameliorates certain features of cancer-associated cachexia (CAC). In wild-type C57BL/6 mice, the injection of Lewis lung carcinoma or B16 melanoma cells causes tumor growth, loss of white adipose tissue (WAT), and a marked reduction of gastrocnemius muscle. In contrast, Atgl-deficient mice with tumors resisted increased WAT lipolysis, myocyte apoptosis, and proteasomal muscle degradation and maintained normal adipose and gastrocnemius muscle mass. Hsl-deficient mice with tumors were also protected although to a lesser degree. Thus, functional lipolysis is essential in the pathogenesis of CAC. Pharmacological inhibition of metabolic lipases may help prevent cachexia.

miR-192, miR-194, miR-215, miR-200c and miR-141 are downregulated and their common target ACVR2B is strongly expressed in renal childhood neoplasms

Micro RNAs (miRNAs) play an important role during renal development and show a tissue-specific enrichment in the kidney. Nephroblastomas, embryonal renal neoplasms of childhood, are considered to develop from nephrogenic rests (NRs) and resemble morphologically and genetically developing kidney. We therefore investigated the role of kidney-enriched miRNAs in the pathogenesis of nephroblastomas. miR-192, miR-215 and miR-194 had a significantly lower expression in nephroblastomas regardless of the subtype compared with mature kidney measured by quantitative real-time-PCR. miR-141 and miR-200c showed a significantly lower expression in blastema-type and mixed-type tumors. In comparison with NRs, a significantly lower expression of miR-192, miR-194 and miR-215 was identified in blastema-type, mixed-type and stroma-type nephroblastomas and of miR-141 and miR-200c in blastema-type tumors. Kidney parenchyma had a significantly higher expression of miR-192, miR-194, miR-215 and miR-200c compared with NRs. In this study, the activin receptor type 2B (ACVR2B), a member of the transforming growth factor (TGF)-β pathway, was identified as single common target gene for miR-192, miR-215, miR-194, miR-141 and miR-200c in silico for the first time. The interaction between all five miRNAs and ACVR2B was also verified by an in vitro assay. Additionally, a distinct protein expression of ACVR2B was detected in 53 of 55 nephroblastomas paralleled by an upregulation of ACVR2B messenger RNA demonstrated in 25 nephroblastomas of all subtypes. A differential regulation of ACVR2B by miRNAs in NRs and nephroblastomas appears to be an important step in the pathogenesis of nephroblastomas implicating for the first time the TGF-β pathway in this process.

Role of adipose triglyceride lipase (PNPLA2) in protection from hepatic inflammation in mouse models of steatohepatitis and endotoxemia

Hepatic inflammation is a key feature of progressive liver disease. Alterations of fatty acid (FA) metabolism and signaling may play an important role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and its progression to nonalcoholic steatohepatitis (NASH). Moreover, FAs activate peroxisome proliferator‐activated receptor α (PPARα) as a key transcriptional regulator of hepatic FA metabolism and inflammation. Since adipose triglyceride lipase (ATGL/PNPLA2) is the key enzyme for intracellular hydrolysis of stored triglycerides and determines FA signaling through PPARα, we explored the role of ATGL in hepatic inflammation in mouse models of NASH and endotoxemia. Mice lacking ATGL or hormone‐sensitive lipase (HSL) were challenged with a methionine‐choline‐deficient (MCD) diet as a nutritional model of NASH or lipopolysaccharide (LPS) as a model of acute hepatic inflammation. We further tested whether a PPARα agonist (fenofibrate) treatment improves the hepatic phenotype in MCD‐ or LPS‐challenged ATGL‐knockout (KO) mice. MCD‐fed ATGL‐KO mice, although partially protected from peripheral lipolysis, showed exacerbated hepatic steatosis and inflammation. Moreover, ATGL‐KO mice challenged by LPS showed enhanced hepatic inflammation, increased mortality, and torpor, findings which were attributed to impaired PPARα DNA binding activity due to reduced FABP1 protein levels, resulting in impaired nuclear FA import. Notably, liganding PPARα through fenofibrate attenuated hepatic inflammation in both MCD‐fed and LPS‐treated ATGL‐KO mice. In contrast, mice lacking HSL had a phenotype similar to the WT mice on MCD and LPS challenge. Conclusion: These findings unravel a novel protective role of ATGL against hepatic inflammation which could have important implications for metabolic and inflammatory liver diseases. (Hepatology 2014;59:858–869)

A comparison of heparinised saline irrigation solutions in a model of microvascular thrombosis

The use of heparinised irrigation solutions has become common in microvascular surgery, but the concentration of heparin has been determined empirically. A laboratory model of microvascular thrombosis, employing a crush injury, intimal abrasion, and stasis to the rat superficial femoral artery was used to compare heparinised saline irrigation solutions of various concentrations. The solutions included normal saline (Group I, controls) and heparinised normal saline in concentrations of 10 U/ml (Group III), 250 U/ml (Group IV), and 500 U/ml (Group V). Group I animals had a patency rate of 25% at 20 min and 0% at 24 h. Group II showed a patency rate of 75% at 20 min but fell to 37.5% at 24 h. Patency in Group III was 87.5% at 20 min and at 24 h. Group IV had a 100% patency rate at 20 min and at 24 h. Group V animals were 100% patent at 20 min and 87.5% patent at 24 h. The activated partial thromboplastin time was prolonged in animals exposed to 250 U/ml and 500 U/ml of heparinised saline. Patency was significantly improved in animals exposed to 100 U/ml, 250 U/ml and 500 U/ml when compared to the control group (p less than 0.001). These results suggest that topical heparinised saline administration is of benefit in the prevention of microvascular thrombosis. Higher concentrations tested in this study resulted in a significant increase in patency, but also prolonged the activated partial thromboplastin time. 100 U/ml is the ideal concentration of heparinised saline irrigation because it significantly improved patency but did not produce detectable systemic effects in this model.

The role of triglyceride lipases in cancer associated cachexia.

Highlights ► Triglyceride lipases play an important role in the development of cancer associated cachexia. ► Blocking triglyceride lipases might prevent the development of cancer associated cachexia.

Assessment of the lymphocyte response to silicone

The biocompatibility of silicone is once again the focus of increased interest. Long considered inert, silicone has now been reported to be responsible for macrophage inhibition in rats and to possibly cause adjuvant disease in humans, and the related compound silica has elicited an antibody response in mice. The present study evaluates lymphocytic response to silicone as expressed by the demonstration of immunologic memory, or changes in specific lymphocyte subpopulations. Thirty-six female Lewis rats (250 gm body weight) were used as test animals. Group 1 (n = 12) was injected subcutaneously with 2.5 ml Freunds Complete Adjuvant (FCA) alone. Group 2 (n = 12) was injected with 2.5 ml FCA sonicated with silicone gel. Group 3 (n = 6) was injected with 2.5 ml FCA, and at 4 weeks, gel-filled silicone implants were placed subcutaneously. Group 4 (n = 6) was injected with 2.5 ml FCA sonicated with silicone gel, and gel-filled silicone implants were placed at 4 weeks. An additional group of six rats (group 5) served as control for the experimental animals, and a group of four rats (group 6) served as naive control. Groups 1 and 2 were sacrificed at 4 weeks, and splenic lymphocytes were obtained for lymphocyte transformation assays performed against silicone. Assays also were run with the addition of the known mitogens Con A, PHA, LPS, and pokeweed. Cytofluorographic analysis of pan-T, T-helper, T-suppressor, and B-cell populations was performed. Groups 3, 4, 5, and 6 were harvested at 8 months, and splenic lymphocytes were subjected to lymphocyte transformation assay.(ABSTRACT TRUNCATED AT 250 WORDS)

The fate of free autogenous fascial grafts in the rabbit

Fascial grafts were taken from 34 New Zealand rabbits and implanted above and below the cranial periosteum in the same animal. They were placed as four-layered folded grafts and as single-layered grafts. When harvested from 6 to 14 months after transplantation, the multi-layered grafts and the single-layered grafts on bone had maintained their bulk but consisted histologically of only a retained collagen matrix with no viable cellular structure. The one-layered grafts on periosteum, however, retained their cellular make-up with normal vascularity and normal cellular structure when harvested at approximately the same intervals.

The role of various antithrombotic agents in microvascular surgery

The ultimate goal in microvascular surgery is to achieve improved patency rates while reducing complications of systemic antithrombotic agents. Using a described model of microarterial thrombosis, the antithrombotic effects of oral aspirin (ASA) were assessed in rats. ASA-treated animals (30 mg/kg orally) exhibited significantly prolonged mean bleeding times 1 h and 24 h after dosing when compared to controls (p < 0.01). Platelet aggregation profiles also displayed an inhibition of platelet aggregation in the ASA group relative to controls. In the thrombosis model, however, patency rates were significantly improved at 20 min, but all vessels were thrombosed at 24 h.

Apoptosis and fibrosis are early features of heart failure in an animal model of metabolic cardiomyopathy

In previous experiments, we observed signs of cardiac failure in mice overexpressing lipoprotein lipase (LPL) under the control of a muscle specific promotor and in peroxisome proliferators activated receptor alpha (PPARα) knockout mice overexpressing LPL under the control of the same promotor. In our current investigations, we focussed on morphological consequences and changes in mRNA and protein expression in hearts from these animals. mRNA expression was analysed by differential display analysis and Northern blot as well as by cDNA microarray analysis followed by pathway analysis. Protein expression was examined using immunoblot and immunohistochemistry. Fibrosis was determined by chromotrope aniline blue staining for collagen. A distinct increase in the expression of α‐tubulin mRNA was noted in hearts of all mutant mouse strains compared with the control. This result was paralleled by increased α‐tubulin protein expression. Using cDNA microarray analysis, we detected an activation of apoptosis, in particular an increase of caspase‐3 expression in hearts of mice overexpressing LPL but not in PPARα knockout mice overexpressing LPL. This finding was confirmed immunohistochemically. In addition, we identified a distinct interstitial increase in collagen and an increase around blood vessels. In our mouse model, we detect mRNA and protein changes typical for cardiomyopathy even before overt clinical signs of heart failure. In addition, a small but distinct increase in the rate of apoptosis of cardiomyocytes and fibrotic changes contributes to cardiac failure in mice overexpressing LPL, whereas additional deficiency in PPARα seems to protect hearts from these effects.

Cancer Induces Cardiomyocyte Remodeling and Hypoinnervation in the Left Ventricle of the Mouse Heart

Cancer is often associated with cachexia, cardiovascular symptoms and autonomic dysregulation. We tested whether extracardiac cancer directly affects the innervation of left ventricular myocardium. Mice injected with Lewis lung carcinoma cells (tumor group, TG) or PBS (control group, CG) were analyzed after 21 days. Cardiac function (echocardiography), serum levels of TNF-α and Il-6 (ELISA), structural alterations of cardiomyocytes and their innervation (design-based stereology) and levels of innervation-related mRNA (quantitative RT-PCR) were analysed. The groups did not differ in various functional parameters. Serum levels of TNF-α and Il-6 were elevated in TG. The total length of axons in the left ventricle was reduced. The number of dense core vesicles per axon profile was reduced. Decreased myofibrillar volume, increased sarcoplasmic volume and increased volume of lipid droplets were indicative of metabolic alterations of TG cardiomyocytes. In the heart, the mRNA level of nerve growth factor was reduced whereas that of β1-adrenergic receptor was unchanged in TG. In the stellate ganglion of TG, mRNA levels of nerve growth factor and neuropeptide Y were decreased and that of tyrosine hydroxylase was increased. In summary, cancer induces a systemic pro-inflammatory state, a significant reduction in myocardial innervation and a catabolic phenotype of cardiomyocytes in the mouse. Reduced expression of nerve growth factor may account for the reduced myocardial innervation.