Samrina Rehman

A Systems Biology Approach to Deciphering the Etiology of Steatosis Employing Patient-Derived Dermal Fibroblasts and iPS Cells

Non-alcoholic fatty liver disease comprises a broad spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis. As a result of increases in the prevalences of obesity, insulin resistance, and hyperlipidemia, the number of people with hepatic steatosis continues to increase. Differences in susceptibility to steatohepatitis and its progression to cirrhosis have been attributed to a complex interplay of genetic and external factors all addressing the intracellular network. Increase in sugar or refined carbohydrate consumption results in an increase of insulin and insulin resistance that can lead to the accumulation of fat in the liver. Here we demonstrate how a multidisciplinary approach encompassing cellular reprogramming, transcriptomics, proteomics, metabolomics, modeling, network reconstruction, and data management can be employed to unveil the mechanisms underlying the progression of steatosis. Proteomics revealed reduced AKT/mTOR signaling in fibroblasts derived from steatosis patients and further establishes that the insulin-resistant phenotype is present not only in insulin-metabolizing central organs, e.g., the liver, but is also manifested in skin fibroblasts. Transcriptome data enabled the generation of a regulatory network based on the transcription factor SREBF1, linked to a metabolic network of glycerolipid, and fatty acid biosynthesis including the downstream transcriptional targets of SREBF1 which include LIPIN1 (LPIN) and low density lipoprotein receptor. Glutathione metabolism was among the pathways enriched in steatosis patients in comparison to healthy controls. By using a model of the glutathione pathway we predict a significant increase in the flux through glutathione synthesis as both gamma-glutamylcysteine synthetase and glutathione synthetase have an increased flux. We anticipate that a larger cohort of patients and matched controls will confirm our preliminary findings presented here.

Molecular phenotypic descriptors of Dupuytren's disease defined using informatics analysis of the transcriptome.

PURPOSE Dupuytrens disease (DD) is a fibroproliferative disorder of unknown etiopathogenesis, which may cause progressive, permanent contracture of digits. Previous studies provide compelling evidence that genetic alterations play an important role. Macroscopically affected areas demonstrate phenotypic differences between the two structurally distinct fibrotic elements in DD (ie, the nodule and the cord). In this study, we set out to (1) compare gene expression profiles between DD and transverse carpal fascia of control subjects (external control); (2) profile DD cords and nodules from the palm against the unaffected transverse palmar fascia (internal control); and (3) identify biologically important candidate genes from the transcriptome profiles. METHODS RNA samples from DD nodules (n = 4), cords (n = 4), and internal control (n = 4) as well as external control (n = 4) from unaffected individuals were subjected to differential gene expression profile analysis. Changes of more than 2-fold in DD groups and controls were recorded. Quantitative reverse transcriptase-polymerase chain reactions were performed to validate 16 implicated genes, which included developmental control genes, matrix metalloproteinases, and apoptotic genes. RESULTS Several genes associated with DD formation were common across all 6 pairwise analyses. Genes markedly upregulated shared common expression levels across all pairwise analysis studies. Pairs involving the DD nodule arrays were notably distinguishable from all other permutations. The majority of genes dysregulated in the DD cords demonstrated an increase in fold change when compared with the DD nodule tissues. Key collagens, collagenases, metalloproteinases, and inhibitors were identified. Genes involved in cytoskeleton development and lipid metabolism were markedly dysregulated. Confirmations of these alterations were obtained in quantitative reverse transcriptase-polymerase chain reaction. CONCLUSIONS These data demonstrate a gradation in expression of certain genes in DD tissue phenotypes compared with control fascia. Transcriptome profiling is predictive not only of disease but also of disease phenotype. These results indicate a number of important candidate genes associated with DD formation, which may provide clues for molecular mechanisms involved in DD pathogenesis.

Dupuytren's: a systems biology disease

Dupuytrens disease (DD) is an ill-defined fibroproliferative disorder of the palm of the hands leading to digital contracture. DD commonly occurs in individuals of northern European extraction. Cellular components and processes associated with DD pathogenesis include altered gene and protein expression of cytokines, growth factors, adhesion molecules, and extracellular matrix components. Histology has shown increased but varying levels of particular types of collagen, myofibroblasts and myoglobin proteins in DD tissue. Free radicals and localised ischaemia have been suggested to trigger the proliferation of DD tissue. Although the existing available biological information on DD may contain potentially valuable (though largely uninterpreted) information, the precise aetiology of DD remains unknown. Systems biology combines mechanistic modelling with quantitative experimentation in studies of networks and better understanding of the interaction of multiple components in disease processes. Adopting systems biology may be the ideal approach for future research in order to improve understanding of complex diseases of multifactorial origin. In this review, we propose that DD is a disease of several networks rather than of a single gene, and show that this accounts for the experimental observations obtained to date from a variety of sources. We outline how DD may be investigated more effectively by employing a systems biology approach that considers the disease network as a whole rather than focusing on any specific single molecule.

Multi-omic profiles of human non-alcoholic fatty liver disease tissue highlight heterogenic phenotypes

Non-alcoholic fatty liver disease (NAFLD) is a consequence of sedentary life style and high fat diets with an estimated prevalence of about 30% in western countries. It is associated with insulin resistance, obesity, glucose intolerance and drug toxicity. Additionally, polymorphisms within, e.g., APOC3, PNPLA3, NCAN, TM6SF2 and PPP1R3B, correlate with NAFLD. Several studies have already investigated later stages of the disease. This study explores the early steatosis stage of NAFLD with the aim of identifying molecular mechanisms underlying the etiology of NAFLD. We analyzed liver biopsies and serum samples from patients with high- and low-grade steatosis (also pre-disease states) employing transcriptomics, ELISA-based serum protein analyses and metabolomics. Here, we provide a detailed description of the various related datasets produced in the course of this study. These datasets may help other researchers find new clues for the etiology of NAFLD and the mechanisms underlying its progression to more severe disease states.

Mathematical modelling of miRNA mediated BCR.ABL protein regulation in chronic myeloid leukaemia vis-a-vis therapeutic strategies.

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disease resulting from an aberrant BCR.ABL gene and protein. To predict BCR.ABL protein abundance and phosphorylation in individual cells in a population of CML cells, we modelled BCR.ABL protein regulation through associated miRNAs using a systems approach. The model rationalizes the level of BCR.ABL protein heterogeneity in CML cells in correlation with the heterogeneous BCR.ABL mRNA levels. We also measured BCR.ABL mRNA and BCR.ABLp phosphorylation in individual cells. The experimental data were consistent with the modelling results, thereby partly validating the model. Provided it is tested further, the model may be used to support effective therapeutic strategies including the combined application of a tyrosine kinase inhibitor and miRNAs targeting BCR.ABL. It appears able to predict different effects of the two types of drug on cells with different expression levels and consequently different effects on the generation of resistance.

Cytokeratin 15 marks basal epithelia in developing ureters and is upregulated in a subset of urothelial cell carcinomas.

The mammalian ureter contains a water-tight epithelium surrounded by smooth muscle. Key molecules have been defined which regulate ureteric bud initiation and drive the differentiation of ureteric mesenchyme into peristaltic smooth muscle. Less is known about mechanisms underlying the developmental patterning of the multilayered epithelium characterising the mature ureter. In skin, which also contains a multilayered epithelium, cytokeratin 15 (CK15), an acidic intermediate filament protein, marks cells whose progeny contribute to epidermal regeneration following wounding. Moreover, CK15+ precursor cells in skin can give rise to basal cell carcinomas. In the current study, using transcriptome microarrays of embryonic wild type mouse ureters, Krt15, coding for CK15, was detected. Quantitative polymerase chain reaction analyses confirmed the initial finding and demonstrated that Krt15 levels increased during the fetal period when the ureteric epithelium becomes multilayered. CK15 protein was undetectable in the ureteric bud, the rudiment from which the ureter grows. Nevertheless, later in fetal development, CK15 was immunodetected in a subset of basal urothelial cells in the ureteric stalk. Superficial epithelial cells, including those positive for the differentiation marker uroplakin III, were CK15-. Transformation-related protein 63 (P63) has been implicated in epithelial differentiation in murine fetal urinary bladders. In wild type fetal ureters, CK15+ cells were positive for P63, and p63 homozygous null mutant ureters lacked CK15+ cells. In these mutant ureters, sections of the urothelium were monolayered versus the uniform multilayering found in wild type littermates. Human urothelial cell carcinomas account for considerable morbidity and mortality. CK15 was upregulated in a subset of invasive ureteric and urinary bladder cancers. Thus, in ureter development, the absence of CK15 is associated with a structurally simplified urothelium whereas, postnatally, increased CK15 levels feature in malignant urothelial overgrowth. CK15 may be a novel marker for urinary tract epithelial precursor cells.

Understanding Dupuytren's disease using systems biology: a move away from reductionism.

Dupuytrens disease (DD) resides within the poorly understood, yet important category of superficial quasi-neoplastic proliferative fibromatosis (McFarlane et al., 1990). This nodular palmar fibromatosis often causes permanent flexion contracture of the metacarpophalangeal (MCP) and proximal interphalangeal joints (PIPJ) of the digits (Rayan, 2007) leading to loss of function (Horner and Bralliar, 1971; Schroter, 1971; Tubiana, 1971; Rayan, 2007). DD may invade locally within the palmar aponeurosis of the hand (sparingly supplied with blood vessels). DD does not disseminate to other tissues (Seemayer et al., 1980), but, rather behaves as a benign neoplastic disorder: progressive and irreversible with a high rate of recurrence after surgical excision (current gold standard treatment for DD; Bayat and McGrouther, 2006). The increasing severity and aggressive recurrence may lead to amputation of the affected digit (Shaw et al., 2007). Phenotypically, DD is described by its two distinct fibrotic elements: the nodule and cord. Nodules are highly vascularized, soft-tissue masses containing mostly myofibroblasts, while the cords are relatively avascular with a thickened collagen-rich structure (Rayan, 2007; Rehman et al., 2008, 2011). Different schools of thought exist regarding the definition of DD progression; they debate whether the nodule develops into cord or the two fibrotic elements occur independently from a separate precursor cell. Processes associated with DD pathogenesis include cytokines, growth factors, adhesion molecules, and extracellular matrix components (Rehman et al., 2008; Shih et al., 2009). Free radicals and localized ischemia may trigger the proliferation of DD tissue (Murrell et al., 1987). Histology has confirmed the presence of collagens, myofibroblast, and myoglobin proteins in DD, but at widely varying abundances. Pathophysiology of DD is also thought to arise either from a defect in a wound repair process or from an abnormal response to wounding (Shih and Bayat, 2010). Some of these hypotheses are non-molecular or may associate the disease with effects, rather than with a single cause. No animal model exists for the study of DD fibromatosis, yet investigations in animal studies of possibly related diseases (Akai et al., 1997) may be informative (Tart and Dahners, 1989; Kandel et al., 1991; Akai et al., 1997; Hildebrand et al., 2004). Single gene changes have correlated poorly with DD, and DD is nowadays viewed as a complex disease. We propose that DD may be a network disease, such that a systems biology approach may help its understanding.

SupraBiology 2014: Promoting UK-China collaboration on Systems Biology and High Performance Computing

1 Manchester Institute of Biotechnology, School of Computer Science, Faculty of Engineering and Physical Sciences, Manchester Centre for Integrative Systems Biology, The University of Manchester, Manchester, M139PL, United Kingdom. 2 Department of Informatics, Systems and Communication, University of Milan-Bicocca, Milan, 20126, Italy. 3 SYSBIO – Centre of Systems Biology, Milan, 20126, Italy. 4 Manchester Institute of Biotechnology, School of Chemical Engineering and Analytical Sciences, Manchester Centre for Integrative Systems Biology, The University of Manchester, Manchester, M139PL, United Kingdom. 5 School of Computer Science, National University of Defence Technology, Changsha 410073, China * Correspondence: xkliao@nudt.edu.cn, hans.westerhoff@manchester.ac.uk