Madeline Murphy

Suppression Subtractive Hybridization Identifies High Glucose Levels as a Stimulus for Expression of Connective Tissue Growth Factor and Other Genes in Human Mesangial Cells

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mm versus 5 mm) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor β1 (TGF-β1), and addition of TGF-β1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-β1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFβ1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFβ1-driven matrix production within a diabetic milieu.

Desulfovibrio Bacterial Species Are Increased in Ulcerative Colitis

BACKGROUND: Debate persists regarding the role of Desulfovibrio subspecies in ulcerative colitis. Combined microscopic and molecular techniques enable this issue to be investigated by allowing precise enumeration of specific bacterial species within the colonic mucous gel. The aim of this study was to combine laser capture microdissection and quantitative polymerase chain reaction to determine Desulfovibrio copy number in crypt-associated mucous gel in health and in acute and chronic ulcerative colitis. METHODS: Colonic mucosal biopsies were harvested from healthy controls (n = 19) and patients with acute (n = 10) or chronic (n = 10) ulcerative colitis. Crypt-associated mucous gel was obtained by laser capture microdissection throughout the colon. Pan-bacterial 16S rRNA and Desulfovibrio copy number/mm2 were obtained by polymerase chain reaction at each locus. Bacterial copy numbers were interrogated for correlation with location and disease activity. Data were evaluated using a combination of ordinary linear methods and linear mixed-effects models to cater for multiple interactions. RESULTS: Desulfovibrio positivity was significantly increased in acute and chronic ulcerative colitis at multiple levels within the colon, and after normalization with total bacterial signal, the relative Desulfovibrio load was increased in acute colitis compared with controls. Desulfovibrio counts did not significantly correlate with age, disease duration, or disease activity but interlevel correlations were found in adjacent colonic segments in the healthy control and chronic ulcerative colitis groups. CONCLUSION: The presence of Desulfovibrio subspecies is increased in ulcerative colitis and the data presented suggest that these bacteria represent an increased percentage of the colonic microbiome in acute ulcerative colitis.

High Glucose-altered Gene Expression in Mesangial Cells ACTIN-REGULATORY PROTEIN GENE EXPRESSION IS TRIGGERED BY OXIDATIVE STRESS AND CYTOSKELETAL DISASSEMBLY

High extracellular glucose plays a pivotal role in the pathophysiology of diabetic nephropathy. Here we report 200 genes, identified using suppression-subtractive hybridization, that are differentially expressed when human mesangial cells are propagated in high ambient glucose in vitro. The major functional classes of genes identified included modulators and products of extracellular matrix protein metabolism, regulators of cell growth and turnover, and a cohort of actin cytoskeleton regulatory proteins. Actin cytoskeletal disassembly is a prominent feature of diabetic nephropathy. The induction of actin cytoskeleton regulatory gene expression by high glucose was attenuated by the inhibitor of reactive oxygen species generation, carbonyl cyanide m-chlorophenylhydrazone but not by the protein kinase C inhibitor GF 109203X and was not mimicked by the addition of transforming growth factor β. Enhanced expression of actin cytoskeleton regulatory genes was also observed following disruption of the mesangial cell actin cytoskeleton by cytochalasin D. In aggregate, these results suggest that the induction of genes encoding actin cytoskeleton regulatory proteins (a) is a prominent component of the mesangial cell transcriptomic response in diabetic nephropathy and (b) is dependent on oxidative stress, is independent of protein kinase C and transforming growth factor-β, and represents an adaptive response to actin cytoskeleton disassembly.

Lipoxins Attenuate Renal Fibrosis by Inducing let-7c and Suppressing TGFβR1

Lipoxins, which are endogenously produced lipid mediators, promote the resolution of inflammation, and may inhibit fibrosis, suggesting a possible role in modulating renal disease. Here, lipoxin A4 (LXA4) attenuated TGF-β1-induced expression of fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1 in cultured human proximal tubular epithelial (HK-2) cells through a mechanism involving upregulation of the microRNA let-7c. Conversely, TGF-β1 suppressed expression of let-7c. In cells pretreated with LXA4, upregulation of let-7c persisted despite subsequent stimulation with TGF-β1. In the unilateral ureteral obstruction model of renal fibrosis, let-7c upregulation was induced by administering an LXA4 analog. Bioinformatic analysis suggested that targets of let-7c include several members of the TGF-β1 signaling pathway, including the TGF-β receptor type 1. Consistent with this, LXA4-induced upregulation of let-7c inhibited both the expression of TGF-β receptor type 1 and the response to TGF-β1. Overexpression of let-7c mimicked the antifibrotic effects of LXA4 in renal epithelia; conversely, anti-miR directed against let-7c attenuated the effects of LXA4. Finally, we observed that several let-7c target genes were upregulated in fibrotic human renal biopsies compared with controls. In conclusion, these results suggest that LXA4-mediated upregulation of let-7c suppresses TGF-β1-induced fibrosis and that expression of let-7c targets is dysregulated in human renal fibrosis.

Transforming growth factor‐β‐regulated gene transcription and protein expression in human GFAP‐negative lamina cribrosa cells

Primary open‐angle glaucoma (POAG) is a progressive optic neuropathy, which is a major cause of worldwide visual impairment and blindness. Pathological hallmarks of the glaucomatous optic nerve head (ONH) include retinal ganglion cell axon loss and extracellular matrix (ECM) remodeling of the lamina cribrosa layer. Transforming growth factor‐β (TGF‐β) is an important pro‐fibrotic modulator of ECM metabolism, whose levels are elevated in human POAG lamina cribrosa tissue compared with non‐glaucomatous controls. We hypothesize that in POAG, lamina cribrosa (LC) glial cells respond to elevated TGF‐β, producing a remodeled ONH ECM. Using Affymetrix microarrays, we report the first study examining the effect of TGF‐β1 on global gene expression profiles in glial fibrillary acidic acid (GFAP)‐negative LC glial cells in vitro. Prominent among the differentially expressed genes were those with established fibrogenic potential, including CTGF, collagen I, elastin, thrombospondin, decorin, biglycan, and fibromodulin. Independent TaqMan and Sybr Green quantitative PCR analysis significantly validated genes involved in regulation of cell proliferation (platelet‐derived growth factor [PDGF‐α]), angiogenesis (vascular endothelial growth factor [VEGF]), ECM accumulation and degradation (CTGF, IL‐11, and ADAMT‐S5), and growth factor binding (ESM‐1). Bioinformatic analysis of the ESM‐1 promoter identified putative Smad and Runx transcription factor binding sites, and luciferase assays confirmed that TGF‐β1 drives transcription of the ESM‐1 gene. TGF‐β1 induces expression and release of ECM components in LC cells, which may be important in regulating matrix remodeling in the lamina cribrosa. In disease states such as POAG, the LC cell may represent an important pro‐fibrotic cell type and an attractive target for novel therapeutic strategies.

IHG-1 Amplifies TGF-β1 Signaling and Is Increased in Renal Fibrosis

Induced in high glucose-1 (IHG-1) is an evolutionarily conserved gene transcript upregulated by high extracellular glucose concentrations, but its function is unknown. Here, it is reported that the abundance of IHG-1 mRNA is nearly 10-fold higher in microdissected, tubule-rich renal biopsies from patients with diabetic nephropathy compared with control subjects. In the diabetic nephropathy specimens, in situ hybridization localized IHG-1 to tubular epithelial cells along with TGF-beta1 and activated Smad3, suggesting a possible role in the development of tubulointerstitial fibrosis. Supporting this possibility, IHG-1 mRNA and protein expression also increased with unilateral ureteral obstruction. In the HK-2 proximal tubule cell line, overexpression of IHG-1 increased TGF-beta1-stimulated expression of connective tissue growth factor and fibronectin. IHG-1 was found to amplify TGF-beta1-mediated transcriptional activity by increasing and prolonging phosphorylation of Smad3. Conversely, inhibition of endogenous IHG-1 with small interference RNA suppressed transcriptional responses to TGF-beta1. In summary, IHG-1, which increases in diabetic nephropathy, may enhance the actions of TGF-beta1 and contribute to the development of tubulointerstitial fibrosis.

Overexpression of the microRNA miR-433 promotes resistance to paclitaxel through the induction of cellular senescence in ovarian cancer cells

Annually, ovarian cancer (OC) affects 240,000 women worldwide and is the most lethal gynecological malignancy. High‐grade serous OC (HGSOC) is the most common and aggressive OC subtype, characterized by widespread genome changes and chromosomal instability and is consequently poorly responsive to chemotherapy treatment. The objective of this study was to investigate the role of the microRNA miR‐433 in the cellular response of OC cells to paclitaxel treatment. We show that stable miR‐433 expression in A2780 OC cells results in the induction of cellular senescence demonstrated by morphological changes, downregulation of phosphorylated retinoblastoma (p‐Rb), and an increase in β‐galactosidase activity. Furthermore, in silico analysis identified four possible miR‐433 target genes associated with cellular senescence: cyclin‐dependent kinase 6 (CDK6), MAPK14, E2F3, and CDKN2A. Mechanistically, we demonstrate that downregulation of p‐Rb is attributable to a miR‐433‐dependent downregulation of CDK6, establishing it as a novel miR‐433 associated gene. Interestingly, we show that high miR‐433 expressing cells release miR‐433 into the growth media via exosomes which in turn can induce a senescence bystander effect. Furthermore, in relation to a chemotherapeutic response, quantitative real‐time polymerase chain reaction (qRT‐PCR) analysis revealed that only PEO1 and PEO4 OC cells with the highest miR‐433 expression survive paclitaxel treatment. Our data highlight how the aberrant expression of miR‐433 can adversely affect intracellular signaling to mediate chemoresistance in OC cells by driving cellular senescence.

Search for the Bovine Homolog of the Murine Ped Gene and Characterization of Its Messenger RNA Expression During Bovine Preimplantation Development

Abstract In mice, a gene (Ped: preimplantation embryo development) that regulates preimplantation embryonic growth, including cleavage rate and embryo survivability, has been described. The objective of the current study was to identify the bovine homolog of the Ped gene and to characterize the mRNA expression pattern of this gene during bovine preimplantation embryo development. The NCBI GenBank/EBI expressed sequence tags (EST) databases were searched for bovine ESTs that were homologous to the murine Ped gene, and the resulting ESTs were aligned and assembled into a contiguous sequence. The homology of the sequence to the murine Ped gene was confirmed. Primers were designed for the sequence, and the mRNA expression pattern was characterized during bovine preimplantation embryo development in vivo and in vitro. In vitro-produced bovine zygotes were cultured either in vitro, in synthetic oviduct fluid, or in vivo in the ewe oviduct for 1–7 days and processed for quantitative real-time polymerase chain reaction (PCR). Transcript abundance increased at each stage of development. However, the expression levels were consistently higher in in vivo-cultured embryos at all stages, with in vivo-cultured embryos showing a 9-fold increase in relative transcript abundance during culture from the zygote to the blastocyst stage in contrast to just under a 4-fold increase during the same culture period in vitro. The mRNA expression pattern of the gene was investigated in early- and late-cleaving two-cell embryos collected at 25, 28, 32, and ≥36 h postinsemination (pi). Transcript relative abundance was highest in those embryos that had cleaved by 28 hpi and decreased almost 3-fold thereafter. In conclusion, we have identified a potential bovine homolog of the murine Ped gene. We have characterized the mRNA expression pattern of this gene during preimplantation embryo development in cattle and shown that a greater relative abundance of the gene transcript is associated with embryos of higher quality (in vivo cultured) and greater developmental potential (early cleaving).

Simvastatin impairs smad-3 phosphorylation and modulates transforming growth factor β1-mediated activation of intestinal fibroblasts†

Transforming growth factor (TGF) β1, acting through the smad pathway, is critical to fibroblast‐mediated intestinal fibrosis. Simvastatin exhibits antifibrotic properties. This study assessed the effects of simvastatin on TGF‐β1‐mediated intestinal fibroblast activation.

Recapitulation of Embryological Programmes in Renal Fibrosis – The Importance of Epithelial Cell Plasticity and Developmental Genes

Chronic fibrosis represents the final common pathway in progressive renal disease. Myofibroblasts deposit the constituents of renal scar, thus crippling renal function. It has recently emerged that an important source of these pivotal effector cells is the injured renal epithelium. This review concentrates on the process of epithelial-mesenchymal transition (EMT) and its regulation. The role of the developmental gene, gremlin, which is reactivated in adult renal disease, is the subject of particular focus. This member of the cysteine knot protein superfamily is critical to the process of nephrogenesis but quiescent in normal adult kidney. There is increasing evidence that gremlin expression reactivates in diabetic nephropathy, and in the diseased fibrotic kidney per se. Known to antagonize members of the bone morphogenic protein (BMP) family, gremlin may also act downstream of TGF-β in induction of EMT. An increased understanding of the extracellular modulation of EMT and, in particular, of the gremlin-BMP axis may result in strategies that can halt or reverse the devastating progression of chronic renal fibrosis.