Kimberly Viker

Differential expression of lumican and fatty acid binding protein-1: New insights into the histologic spectrum of nonalcoholic fatty liver disease†

The basis of hepatocellular injury and progressive fibrosis in a subset of patients with nonalcoholic fatty liver disease (NAFLD) is poorly understood. We sought to identify hepatic proteins that are differentially abundant across the histologic spectrum of NAFLD. Hepatic protein abundance was measured in liver samples from four groups (n = 10 each) of obese (body mass index >30 kg/m2) patients: (1) obese normal group (normal liver histology), (2) simple steatosis (SS), (3) nonalcoholic steatohepatitis (NASH)‐mild (steatohepatitis with fibrosis stage 0‐1), and (4) NASH‐progressive (steatohepatitis with fibrosis stage 2‐4). Hepatic peptides were analyzed on an API Qstar XL quadrupole time‐of‐flight mass spectrometer using Analyst QS software. Linear trends tests were performed and used to screen for differential abundance. Nine known proteins were expressed with differential abundance between study groups. For seven proteins differential abundance is likely to have been on the basis increased hepatic lipid content and/or inflammation. Lumican, a 40‐kDa keratin sulfate proteoglycan that regulates collagen fibril assembly and activates transforming growth factor‐beta and smooth muscle actin, was expressed similarly in obese normal and SS but was overexpressed in a progressive manner in NASH‐mild versus SS (124%, P < 0.001), NASH‐progressive versus NASH‐mild (156%, P < 0.001) and NASH‐progressive versus obese normal (178%, P < 0.001). Fatty acid binding protein‐1 (FABP‐1), which is protective against the detergent effects of excess free fatty acids, facilitates intracellular free fatty acid transport and is an important ligand for peroxisome proliferator‐activated receptor–mediated transcription, was overexpressed in SS when compared to the obese normal group (128%, P < 0.001), but was paradoxically underexpressed in NASH‐mild versus SS (73%, P < 0.001), NASH‐progressive versus NASH‐mild (81%, P < 0.001), and NASH‐progressive versus obese normal (59%, P < 0.001). Conclusion: Histologically progressive NAFLD is associated with overexpression of lumican, an important mediator of fibrosis in nonhepatic tissues, whereas FABP‐1 is paradoxically underexpressed in NASH, suggesting a new potential mechanism of lipotoxicity in NAFLD. Further studies are needed to determine the biologic basis of lumican and/or FABP‐1 dysregulation in NAFLD. (HEPATOLOGY 2009;49:1375–1384.)

Low Circulating Levels of Dehydroepiandrosterone in Histologically Advanced Nonalcoholic Fatty Liver Disease

The biological basis of variability in histological progression of nonalcoholic fatty liver disease (NAFLD) is unknown. Dehydroepiandrosterone (DHEA) is the most abundant steroid hormone and has been shown to influence sensitivity to oxidative stress, insulin sensitivity, and expression of peroxisome proliferator‐activated receptor alpha and procollagen messenger RNA. Our aim was to determine whether more histologically advanced NAFLD is associated with low circulating levels of DHEA. Serum samples were obtained prospectively at the time of liver biopsy in 439 patients with NAFLD (78 in an initial and 361 in validation cohorts) and in controls with cholestatic liver disease (n = 44). NAFLD was characterized as mild [simple steatosis or nonalcoholic steatohepatitis (NASH) with fibrosis stage 0‐2] or advanced (NASH with fibrosis stage 3‐4). Serum levels of sulfated DHEA (DHEA‐S) were measured by enzyme‐linked immunosorbent assay. Patients with advanced NAFLD had lower plasma levels of DHEA‐S than patients with mild NAFLD in both the initial (0.25 ± 0.07 versus 1.1 ± 0.09 μg/mL, P < 0.001) and validation cohorts (0.47 ± 0.06 versus 0.99 ± 0.04 μg/mL, P < 0.001). A “dose effect” of decreasing DHEA‐S and incremental fibrosis stage was observed with a mean DHEA‐S of 1.03 ± 0.05, 0.96 ± 0.07, 0.83 ± 0.11, 0.66 ± 0.11, and 0.35 ± 0.06 μg/mL for fibrosis stages 0, 1, 2, 3, and 4, respectively. All patients in both cohorts in the advanced NAFLD group had low DHEA‐S levels, with the majority in the hypoadrenal range. The association between DHEA‐S and severity of NAFLD persisted after adjusting for age. A relationship between disease/fibrosis severity and DHEA‐S levels was not seen in patients with cholestatic liver diseases. Conclusion: More advanced NAFLD, as indicated by the presence of NASH with advanced fibrosis stage, is strongly associated with low circulating DHEA‐S. These data provide novel evidence for relative DHEA‐S deficiency in patients with histologically advanced NASH. (HEPATOLOGY 2008;47:484–492.)

Lumican, an extracellular matrix proteoglycan, is a novel requisite for hepatic fibrosis

Lumican, an extracellular matrix proteoglycan was previously shown to be upregulated with increasing severity of nonalcoholic steatohepatitis (NASH). Although lumican is involved in collagen fibrillogenesis in extra-hepatic tissues, little is known about the role of lumican in hepatic disease. We therefore determined lumican expression in etiologies other than clinical NASH. Our results indicated that lumican is upregulated in clinical samples of hepatitis C virus infection, in experimental rodent models of chronic and acute liver injury and could additionally be induced in vitro in response to the pro-fibrotic cytokine transforming growth factor β1 (TGFβ1) and to lipotoxic palmitic acid. Together, these results suggested a role for lumican in hepatic fibrosis. To investigate the functional role of lumican in hepatic fibrosis, lumican null (Null) and wild-type (WT) littermates were administered carbon tetrachloride intra-peritoneally. Serum and liver tissue were analyzed for indices of liver injury, fibrosis, matrix turnover, and proliferation. Hepatic fibrosis was greatly reduced in null animals (P<0.05). Paradoxically, gene expression of fibrosis-related genes such as TGFβ1 and collagen 1 was numerically higher in null animals though statistically insignificant from WT animals. On the other hand, α smooth muscle actin expression (α-SMA), a marker for activated fibroblasts, the main contributors of collagen production was significantly higher (P<0.05) in null animals as compared with WT littermates. Among the matrix metalloproteases (MMP), MMP13 was significantly increased (P<0.05) in null animals. Ultra-structural imaging indicated differences in the organization and spatial distribution of hepatic collagen fibrils of null and WT mice. Cell proliferation was significantly increased (P<0.05) in null animals. We conclude that lumican is a prerequisite for hepatic fibrosis. The protective effect of lumican deficiency in hepatic fibrosis appears to be downstream of collagen production and mediated through the combined effects of impaired collagen fibrillogenesis, increased matrix turnover, and an enhanced proliferative response.

Correlation of Chromosomal Instability, Telomere Length and Telomere Maintenance in Microsatellite Stable Rectal Cancer: A Molecular Subclass of Rectal Cancer

Introduction Colorectal cancer (CRC) tumor DNA is characterized by chromosomal damage termed chromosomal instability (CIN) and excessively shortened telomeres. Up to 80% of CRC is microsatellite stable (MSS) and is historically considered to be chromosomally unstable (CIN+). However, tumor phenotyping depicts some MSS CRC with little or no genetic changes, thus being chromosomally stable (CIN-). MSS CIN- tumors have not been assessed for telomere attrition. Experimental Design MSS rectal cancers from patients ≤50 years old with Stage II (B2 or higher) or Stage III disease were assessed for CIN, telomere length and telomere maintenance mechanism (telomerase activation [TA]; alternative lengthening of telomeres [ALT]). Relative telomere length was measured by qPCR in somatic epithelial and cancer DNA. TA was measured with the TRAPeze assay, and tumors were evaluated for the presence of C-circles indicative of ALT. p53 mutation status was assessed in all available samples. DNA copy number changes were evaluated with Spectral Genomics aCGH. Results Tumors were classified as chromosomally stable (CIN-) and chromosomally instable (CIN+) by degree of DNA copy number changes. CIN- tumors (35%; n=6) had fewer copy number changes (<17% of their clones with DNA copy number changes) than CIN+ tumors (65%; n=13) which had high levels of copy number changes in 20% to 49% of clones. Telomere lengths were longer in CIN- compared to CIN+ tumors (p=0.0066) and in those in which telomerase was not activated (p=0.004). Tumors exhibiting activation of telomerase had shorter tumor telomeres (p=0.0040); and tended to be CIN+ (p=0.0949). Conclusions MSS rectal cancer appears to represent a heterogeneous group of tumors that may be categorized both on the basis of CIN status and telomere maintenance mechanism. MSS CIN- rectal cancers appear to have longer telomeres than those of MSS CIN+ rectal cancers and to utilize ALT rather than activation of telomerase.

Noninvasive diagnosis of acute cellular rejection in liver transplant recipients: A proteomic signature validated by enzyme‐linked immunosorbent assay

The diagnosis of acute cellular rejection (ACR) requires liver biopsy with its attendant expense and risk. Our first aim was to prospectively determine in an exploratory analysis whether there is a serum proteome signature associated with histologically confirmed ACR. Our second aim was to use simpler and faster enzyme‐linked immunosorbent assay (ELISA)‐based assays for proteins identified as differentially abundant in the proteomic analysis to identify patients with ACR in a separate validation cohort. We used sequential high‐abundance protein depletion and isobaric tag for relative and absolute quantitation liquid chromatography–tandem mass spectrometry to characterize the serum proteome in serum samples of patients with or without ACR. Seven of the 41 proteins identified as differentially abundant [serum amyloid A, complement component 4 (C4), fibrinogen, complement component 1q (C1q), complement component 3, heat shock protein 60 (HSP60), and HSP70] could be measured with ELISA‐based assays in a validation cohort consisting of patients with ACR (n = 25) and patients without ACR (n = 21). The mean alanine aminotransferase (ALT) levels in patients with ACR and in patients without ACR were 198 ± 27 and 153 ± 34 U/L, respectively. Among the 7 proteins for which ELISA assays were available, C4 and C1q were both independent predictors of ACR. C4 had the greatest predictivity for differentiating patients with or without ACR. A C4 level ≤ 0.31 g/L had a sensitivity of 97%, a specificity of 62%, a positive predictive value of 74%, and a negative predictive value of 94%. A C4 level ≤ 0.31 g/L and an ALT level ≥ 70 IU/mL together had a sensitivity of 96%, a specificity of 81%, a positive predictive value of 86%, and a negative predictive value of 94%. In summary, in this exploratory analysis, serum C4 and ALT levels were highly predictive of ACR in liver transplant recipients. Confirmation in a prospective, larger, and diverse population is needed. Liver Transpl 17:723‐732, 2011.

Interactions of allelic variance of PNPLA3 with nongenetic factors in predicting nonalcoholic steatohepatitis and nonhepatic complications of severe obesity

Objective: Allelic variation (rs738409C→G) in adiponutrin (patatin‐like phospholipase domain‐containing protein 3, PNPLA3) has been associated with hepatic steatosis and liver fibrosis. The physiologic impact of the PNPLA3 G allele may be exacerbated in patients with severe obesity. In this study, we investigated the interactions of PNPLA3 rs738409 with a broad panel of metabolic and histologic characteristics of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH) in patients with medically complicated obesity.

Prolonged engraftment of human hepatocytes in mice transgenic for the deleted form of human hepatocyte growth factor

Aim:  Small animal models chimeric for human hepatocytes have provided valuable insights into the biology of hepatotropic viral infection and provided a platform for the study of therapeutic agents. Existing models of human hepatocyte transplantation are limited by phenotypic fragility and impaired immunity. We hypothesized that mice transgenic for human hepatocyte growth factor (HGF), a potent human hepatocyte mitogen, would engraft human hepatocytes in the absence of immunodeficiency.

Stanniocalcin-1 Is an Ocular Hypotensive Agent and a Downstream Effector Molecule That Is Necessary for the Intraocular Pressure–Lowering Effects of Latanoprost

Purpose To identify downstream signaling molecules through which intraocular pressure (IOP) is lowered following treatment with the prostaglandin analog latanoprost. Methods Total RNA and protein isolated from primary human Schlemms canal cells (n = 3) treated with latanoprost (free acid; 100 nM) were processed for quantitative PCR and Western blot analysis. IOP was evaluated in stanniocalcin-1 (STC-1−/−) and wild-type mice following treatment with latanoprost or Rho kinase inhibitor Y27632. Human anterior segment pairs (n = 8) were treated with recombinant STC-1 (5, 50, or 500 ng/mL) and pressure was recorded using custom-designed software. The effect of recombinant STC-1 (0.5 mg/mL) on IOP was evaluated in wild-type mice. Tissue morphology was evaluated by light and transmission electron microscopy. Results Increased STC-1 mRNA (4.0- to 25.2-fold) and protein expression (1.9- to 5.1-fold) was observed within 12 hours following latanoprost treatment. Latanoprost reduced IOP in wild-type mice (22.0% ± 1.9%), but had no effect on STC-1−/− mice (0.5% ± 0.7%). In contrast, Y27632 reduced IOP in both wild-type (12.5% ± 1.2%) and in STC-1−/− mice (13.1% ± 2.8%). Human anterior segments treated with STC-1 (500 ng/mL) showed an increase in outflow facility (0.15 ± 0.03 to 0.27 ± 0.09 μL/min/mm Hg) while no change was observed in paired vehicle-treated controls. Recombinant STC-1 reduced IOP in wild-type mice by 15.2% ± 3.0%. No observable morphologic changes were identified between treatment groups when evaluated by microscopy. Conclusions Latanoprost-induced reduction of IOP is mediated through the downstream signaling molecule STC-1. When used by itself, STC-1 exhibits ocular hypotensive properties.

Analogs of the ATP-Sensitive Potassium (KATP) Channel Opener Cromakalim with in Vivo Ocular Hypotensive Activity

ATP-sensitive potassium (KATP) channel openers have emerged as potential therapeutics for the treatment of glaucoma, lowering intraocular pressure (IOP) in animal models and cultured human anterior segments. We have prepared water-soluble phosphate and dipeptide derivatives of the KATP channel opener cromakalim and evaluated their IOP lowering capabilities in vivo. In general, the phosphate derivatives proved to be more chemically robust and efficacious at lowering IOP with once daily dosing in a normotensive mouse model. Two of these phosphate derivatives were further evaluated in a normotensive rabbit model, with a significant difference in activity observed. No toxic effects on cell structure or alterations in morphology of the aqueous humor outflow pathway were observed after treatment with the most efficacious compound, (3S,4R)-2, suggesting that it is a strong candidate for development as an ocular hypotensive agent.

Integration of myocardial scar identified by preoperative delayed contrast-enhanced MRI into a high-resolution mapping system for planning and guidance of VT ablation procedures

Myocardial scarring creates a substrate for reentrant circuits which can lead to ventricular tachycardia. In ventricular catheter ablation therapy, regions of myocardial scarring are targeted to interrupt arrhythmic electrical pathways. Low voltage regions are a surrogate for myocardial scar and are identified by generating an electro anatomic map at the start of the procedure. Recent efforts have focussed on integration of preoperative scar information generated from delayed contrast-enhanced MR imaging to augment intraprocedural information. In this work, we describe an initial feasibility study of integration of a preoperative MRI derived scar maps into a high-resolution mapping system to improve planning and guidance of VT ablation procedures.