Advanced glycation end products cause increased CCN family and extracellular matrix gene expression in the diabetic rodent retina

Aims/hypothesis: Referred to as CCN, the family of growth factors consisting of cystein-rich protein 61 (CYR61, also known as CCN1), connective tissue growth factor (CTGF, also known as CCN2), nephroblastoma overexpressed gene (NOV, also known as CCN3) and WNT1-inducible signalling pathway proteins 1, 2 and 3 (WISP1, -2 and -3; also known as CCN4, -5 and -6) affects cellular growth, differentiation, adhesion and locomotion in wound repair, fibrotic disorders, inflammation and angiogenesis. AGEs formed in the diabetic milieu affect the same processes, leading to diabetic complications including diabetic retinopathy. We hypothesised that pathological effects of AGEs in the diabetic retina are a consequence of AGE-induced alterations in CCN family expression.

Materials and methods: CCN gene expression levels were studied at the mRNA and protein level in retinas of control and diabetic rats using real-time quantitative PCR, western blotting and immunohistochemistry at 6 and 12 weeks of streptozotocin-induced diabetes in the presence or absence of aminoguanidine, an AGE inhibitor. In addition, C57BL/6 mice were repeatedly injected with exogenously formed AGE to establish whether AGE modulate retinal CCN growth factors in vivo.

Results: After 6 weeks of diabetes, Cyr61 expression levels were increased more than threefold. At 12 weeks of diabetes, Ctgf expression levels were increased twofold. Treatment with aminoguanidine inhibited Cyr61 and Ctgf expression in diabetic rats, with reductions of 31 and 36%, respectively, compared with untreated animals. Western blotting showed a twofold increase in CTGF production, which was prevented by aminoguanidine treatment. In mice infused with exogenous AGE, Cyr61 expression increased fourfold and Ctgf expression increased twofold in the retina.

Conclusions/interpolation: CTGF and CYR61 are downstream effectors of AGE in the diabetic retina, implicating them as possible targets for future intervention strategies against the development of diabetic retinopathy.

Differential expression of steroid 5 alpha-reductase isozymes and association with disease severity and angiogenic genes predict their biological role in prostate cancer

The biological role of steroid 5 alpha-reductase isozymes (encoded by the SRD5A1 and SRD5A2 genes) and angiogenic factors that play important roles in the pathogenesis and vascularization of prostate cancer (PC) is poorly understood. The sub-cellular expression of these isozymes and vascular endothelial growth factor (VEGF) in PC tissue microarrays (n=62) was examined using immunohistochemistry. The effect of SRD5A inhibition on the angiogenesis pathway genes in PC was also examined in prostate cell lines, LNCaP, PC3, and RWPE-1, by treating them with the SRD5A inhibitors finasteride and dutasteride, followed by western blot, quantitative PCR, and ELISA chip array techniques. In PC tissues, nuclear SRD5A1 expression was strongly associated with higher cancer Gleason scores (P=0.02), higher cancer stage (P=0.01), and higher serum prostate specific antigen (PSA) levels (P=0.01), whereas nuclear SRD5A2 expression was correlated with VEGF expression (P=0.01). Prostate tumor cell viability was significantly reduced in dutasteride-treated PC3 and RWPE-1 cells compared with finasteride-treated groups. Expression of the angiogenesis pathway genes transforming growth factor beta 1 (TGFB1), endothelin (EDN1), TGF alpha (TGFA), and VEGFR1 was upregulated in LNCaP cells, and at least 7 out of 21 genes were upregulated in PC3 cells treated with finasteride (25 mu M). Our findings suggest that SRD5A1 expression predominates in advanced PC, and that inhibition of SRD5A1 and SRD5A2 together was more effective in reducing cell numbers than inhibition of SRD5A2 alone. However, these inhibitors did not show any significant difference in prostate cell angiogenic response. Interestingly, some angiogenic genes remained activated after treatment, possibly due to the duration of treatment and tumor resistance to inhibitors. Endocrine-Related Cancer (2010) 17 757-770

Elucidating the molecular physiopathology of acute respiratory distress syndrome in severe acute respiratory syndrome patients

Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury. It is a response to various diseases of variable etiology, including SARS-CoV infection. To date, a comprehensive study of the genomic physiopathology of ARDS (and SARS) is lacking, primarily due to the difficulty of finding suitable materials to study the disease process at a tissue level (instead of blood, sputa or swaps). Hereby we attempt to provide such study by analyzing autopsy lung samples from patient who died of SARS and showed different degrees of severity of the pulmonary involvement. We performed real-time quantitative PCR analysis of 107 genes with functional roles in inflammation, coagulation, fibrosis and apoptosis: some key genes were confirmed at a protein expression level by immunohistochemistry and correlated to the degree of morphological severity present in the individual samples analyzed. Significant expression levels were identified for ANPEP (a receptor for CoV), as well as inhibition of the STAT1 pathway, IFNs production and CXCL10 (a T-cell recruiter). Other genes unassociated to date with ARDS/SARS include C1Qb, C5R1, CASP3, CASP9, CD14, CD68, FGF7, HLA-DRA, ICF1, IRF3, MALAT-1, MSR1, NFIL3, SLPI, USP33, CLC, GBP1 and TACI. As a result, we proposed to therapeutically target some of these genes with compounds such as ANPEP inhibitors, SLPI and dexamethasone. Ultimately, this study may serve as a model for future, tissue-based analyses of fibroinflammatory conditions affecting the lung. (C) 2009 Elsevier B.V. All rights reserved.

Schistosome I/Lamides - A new family of bioactive helminth neuropeptides

Here we report the identification of a new family of helminth neuropeptides with members in both nematodes and flatworms, and include preliminary cell biological and functional characterisation of one of the peptides from the trematode parasite of humans, Schistosoma mansoni. Bioinformatics and Rapid Amplification of cDNA Ends (RACE)-PCR were used to identify the completes. mansoni neuropeptide precursor gene Sm-npp-1, which encodes three pentapeptides bearing the motif (A/G)FVR(I/L).NH2. Similar peptides were identified in three other flatworm species and in 15 nematode species. Quantitative PCR (qPCR) and immunocytochemical (ICC) analyses showed that Sm-npp-1 is constitutively expressed in larval and adult worms. ICC and confocal microscopy were employed to localise one of the schistosome NPP-1 peptides (GFVRIamide) in adult worms and schistosomules; antibodies labelled a pair of neurones in the cerebral ganglia that extend posteriorly along the main nerve cords. GFVRIamide displayed no detectable co-localisation with FMRFamide-like peptides (FLPs), nor was it detectable in muscle innervation. Exogenously applied peptide had a significant inhibitory effect on the mobility of whole adult worm pairs at 10(-5) M (n = 9). Finally, we explored Sm-npp-1 function in schistosomules using RNA interference (RNAi); we successfully achieved specific knockdown of the Sm-npp-1 transcript (54.46 +/- 10.41% knockdown, n = 3), but did not detect any clear, aberrant mobility or morphological phenotypes. NPP-1-like peptides are a new family of helminth peptides with a cell-specific expression pattern distinct from FLPs and a modulatory effect on schistosome muscular activity. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Eosinophil peroxidase induces the expression and function of acid-sensing ion channel-3 in allergic rhinitis:in vitro evidence in cultured epithelial cells

BACKGROUND:
Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR.
OBJECTIVES:
The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects.
METHODS:
Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function.
RESULTS:
Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P < 0.02). Immunohistochemistry confirmed significantly higher ASIC-3 protein expression on nasal epithelial cells in AR patients than controls (P < 0.01). Immunoreactivity for EPO+ eosinophils in both nasal epithelium and submucosa was more prominent in AR compared with controls. A mechanism of induction of ASIC-3 expression relevant to AR was suggested by the finding that eosinophil peroxidase (EPO), acting via ERK1/2, induced the expression of ASIC-3 in epithelial cells. Furthermore, using a quantitative functional measure of epithelial cell secretory function in vitro, EPO increased the air-surface liquid depth via an ASIC-dependent chloride secretory pathway.
CONCLUSIONS:
This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.

Androgen deprivation results in time-dependent hypoxia in LNCaP prostate tumours; informed scheduling of the bioreductive drug AQ4N improves treatment response

Androgen withdrawal induces hypoxia in androgen-sensitive tissue; this is important as in the tumour microenvironment hypoxia is known to drive malignant progression. This study examined the time-dependent effect of androgen deprivation therapy (ADT) on tumour oxygenation and investigated the role of ADT-induced hypoxia on malignant progression in prostate tumours. LNCaP xenografted tumours were treated with anti-androgens and tumour oxygenation measured. Dorsal skin fold chambers (DSF) were used to image tumour vasculature in vivo. Quantitative PCR (QPCR) identified differential gene expression following treatment with bicalutamide. Bicalutamide and vehicle-only treated tumours were re-established in vitro and invasion and sensitivity to docetaxel were measured. Tumour growth delay was calculated following treatment with bicalutamide combined with the bioreductive drug AQ4N. Tumour oxygenation measurements showed a precipitate decrease following initiation of ADT. A clinically relevant dose of bicalutamide (2mg/kg/day) decreased tumour oxygenation by 45% within 24h, reaching a nadir of 0.09% oxygen (0.67±0.06 mmHg) by day 7; this persisted until day 14 when it increased up to day 28. Using DSF chambers, LNCaP tumours treated with bicalutamide showed loss of small vessels at days 7 and 14 with revascularization occurring by day 21. QPCR showed changes in gene expression consistent with the vascular changes and malignant progression. Cells from bicalutamide-treated tumours were more malignant than vehicle-treated controls. Combining bicalutamide with AQ4N (50mg/kg; single dose) caused greater tumour growth delay than bicalutamide alone. This study shows that bicalutamide-induced hypoxia selects for cells that show malignant progression; targeting hypoxic cells may provide greater clinical benefit.

Constitutive and Treatment-Induced CXCL8-Signalling Selectively Modulates the Efficacy of Anti-Metabolite Therapeutics in Metastatic Prostate Cancer

Background: The current study was undertaken to characterize the effect of anti-metabolites on inducing CXCL8 signaling and determining whether the constitutive and/or drug-induced CXCL8 signaling in metastatic prostate cancer (CaP) cells modulates their sensitivity to this class of agent.

Methods: The response of metastatic CaP cells to 5-Fluorouracil (5-FU), Pemetrexed or Tomudex was determined using cell count assays, flow cytometry and PARP cleavage analysis. Quantitative-PCR, ELISA and immunoblots were employed to determine effects of drugs or CXCL8 administration on target gene/protein expression.

Results: Administration of 5-FU but not pemetrexed potentiated CXCL8 secretion and increased CXCR1 and CXCR2 gene expression in metastatic PC3 cells. Consistent with this, the inhibition of CXCL8 signaling using a CXCR2 antagonist, AZ10397767, increased the cytotoxicity of 5-FU by 4-fold (P,0.001), and increased 5-FU-induced apoptosis in PC3 cells (P,0.01). In contrast, while administration of AZ10397767 had no effect on the sensitivity of pemetrexed, the CXCR2 antagonist exerted the greatest effect in increasing the sensitivity of PC3 cells to Tomudex, a directed thymidylate synthase (TS) inhibitor. Subsequent experiments confirmed that administration of recombinant human CXCL8 increased TS expression, a response mediated in part by the CXCR2 receptor. Moreover, siRNA-mediated knockdown of the CXCL8-target gene Bcl-2 increased the sensitivity of PC3 cells to 5-FU.

Conclusions: CXCL8 signaling provides a selective resistance of metastatic prostate cancer cells to specific anti-metabolites by promoting a target-associated resistance, in addition to underpinning an evasion of treatment-induced apoptosis. © 2012 Wilson et al.

HOXA/PBX3 knockdown impairs growth and sensitizes cytogenetically normal acute myeloid leukemia cells to chemotherapy

The cytogenetically normal subtype of acute myeloid leukemia (CN-AML) is associated with Intermediate risk which complicates therapeutic options. Lower overall HOX/TALE expression appears to correlate with more favorable prognosis/better response to treatment in some leukemias and solid cancer. The functional significance of the associated gene expression and response to chemotherapy is not known. Three independent microarray datasets obtained from large patient cohorts along with quantitative PCR validation was used to identify a four gene HOXA/TALE signature capable of prognostic stratification. Biochemical analysis was used to identify interactions between the four encoded proteins and targeted knockdown used to examine the functional importance of sustained expression of the signature in leukemia maintenance and response to chemotherapy. An eleven HOXA/TALE code identified in an Intermediate risk (n=315) compared to a Favourable group of patients (n=105) was reduced to a four gene signature of HOXA6, HOXA9, PBX3 and MEIS1 by iterative analysis of independent platforms. This signature maintained the Favorable/Intermediate risk partition and where applicable, correlated with overall survival in CN-AML. We further show that cell growth and function is dependent on maintained levels of these core genes and that direct targeting of HOXA/PBX3 sensitizes CN-AML cells to standard chemotherapy. Together the data support a key role for HOXA/TALE in CN-AML and demonstrate that targeting of clinically significant HOXA/PBX3 elements may provide therapeutic benefit to these patients.

Distinctive Profile of IsomiR Expression and Novel MicroRNAs in Rat Heart Left Ventricle

MicroRNAs (miRNAs) are single-stranded non-coding RNAs that negatively regulate target gene expression through mRNA cleavage or translational repression. There is mounting evidence that they play critical roles in heart disease. The expression of known miRNAs in the heart has been studied at length by microarray and quantitative PCR but it is becoming evident that microRNA isoforms (isomiRs) are potentially physiologically important. It is well known that left ventricular (patho)physiology is influenced by transmural heterogeneity of cardiomyocyte phenotype, and this likely reflects underlying heterogeneity of gene expression. Given the significant role of miRNAs in regulating gene expression, knowledge of how the miRNA profile varies across the ventricular wall will be crucial to better understand the mechanisms governing transmural physiological heterogeneity. To determinine miRNA/isomiR expression profiles in the rat heart we investigated tissue from different locations across the left ventricular wall using deep sequencing. We detected significant quantities of 145 known rat miRNAs and 68 potential novel orthologs of known miRNAs, in mature, mature* and isomiR formation. Many isomiRs were detected at a higher frequency than their canonical sequence in miRBase and have different predicted targets. The most common miR-133a isomiR was more effective at targeting a construct containing a sequence from the gelsolin gene than was canonical miR-133a, as determined by dual-fluorescence assay. We identified a novel rat miR-1 homolog from a second miR-1 gene; and a novel rat miRNA similar to miR-676. We also cloned and sequenced the rat miR-486 gene which is not in miRBase (v18). Signalling pathways predicted to be targeted by the most highly detected miRNAs include Ubiquitin-mediated Proteolysis, Mitogen-Activated Protein Kinase, Regulation of Actin Cytoskeleton, Wnt signalling, Calcium Signalling, Gap junctions and Arrhythmogenic Right Ventricular Cardiomyopathy. Most miRNAs are not expressed in a gradient across the ventricular wall, with exceptions including miR-10b, miR-21, miR-99b and miR-486.

Gelsolin induces colorectal tumor cell invasion via modulation of the urokinase-type plasminogen activator cascade

Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin's influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites.

Oxidised, glycated LDL selectively influences tissue inhibitor of metalloproteinase-3 gene expression and protein production in human retinal capillary pericytes

Aims/hypothesis: Matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitor of metalloproteinases (TIMPs), regulate important biological processes including the homeostasis of the extracellular matrix, proteolysis of cell surface proteins, proteinase zymogen activation, angiogenesis and inflammation. Studies have shown that their balance is altered in retinal microvascular tissues in diabetes. Since LDLs modified by oxidation/glycation are implicated in the pathogenesis of diabetic vascular complications, we examined the effects of modified LDL on the gene expression and protein production of MMPs and TIMPs in retinal pericytes. Methods: Quiescent human retinal pericytes were exposed to native LDL (N-LDL), glycated LDL (G-LDL) and heavily oxidised and glycated LDL (HOG-LDL) for 24 h. We studied the expression of the genes encoding MMPs and TIMPs mRNAs by analysis of microarray data and quantitative PCR, and protein levels by immunoblotting and ELISA. Results: Microarray analysis showed that MMP1, MMP2, MMP11, MMP14 and MMP25 and TIMP1, TIMP2, TIMP3 and TIMP4 were expressed in pericytes. Of these, only TIMP3 mRNA showed altered regulation, being expressed at significantly lower levels in response to HOG- vs N-LDL. Quantitative PCR and immunoblotting of cell/matrix proteins confirmed the reduction in TIMP3 mRNA and protein in response to HOG-LDL. In contrast to cellular TIMP3 protein, analysis of secreted TIMP1, TIMP2, MMP1 and collagenase activity indicated no changes in their production in response to modified LDL. Combined treatment with N- and HOG-LDL restored TIMP3 mRNA expression to a level comparable with that after N-LDL alone. Conclusions/interpretation: Among the genes encoding for MMPs and TIMPs expressed in retinal pericytes, TIMP3 is uniquely regulated by HOG-LDL. Reduced TIMP3 expression might contribute to microvascular abnormalities in diabetic retinopathy. © 2007 Springer-Verlag.

Novel hydrolysis-probe based qPCR assay to detect saxitoxin transcripts of dinoflagellates in environmental samples

Paralytic Shellfish Poisoning (PSP) is a serious human illness caused by ingestion of seafood enriched with paralytic shellfish toxins (PSTs). PSTs are neurotoxic compounds produced by marine dinoflagellates, specifically by Alexandrium spp., Gymnodinium catenatum and Pyrodinium bahamense. Every year, massive monitoring of PSTs and their producers is undertaken worldwide to avoid PSP incidences. Here we developed a sensitive, hydrolysis probe-based quantitative PCR (qPCR) assay to detect a gene essential for PST synthesis across different dinoflagellate species and genera and tested it on cDNA generated from environmental samples spiked with Alexandrium minutum or Alexandrium fundyense cells. The assay was then applied to two environmental sample series from Norway and Spain and the results were complemented with cell counts, LSU-based microarray data and toxin measurements (enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) biosensor method). The overall agreement between the results of the qPCR assay and the complementary data was good. The assay reliably detected sxtA transcripts from Alexandrium spp. and G. catenatum, even though Alexandrium spp. cell concentrations were mostly so low that they could not be quantified microscopically. Agreement between the novel assay and toxin measurements or cell counts was generally good; the few inconsistencies observed were most likely due to disparate residence times of sxtA transcripts and PSTs in seawater, or, in the case of cell counts, to dissimilar sxtA4 transcript numbers per cell in different dinoflagellate strains or species. © 2013 Elsevier B.V.

Differential expression of liver fluke β-tubulin isotypes at selected life cycle stages

We have shown that Fasciola hepatica expresses at least six ß-tubulins in the adult stage of its life cycle, designated F.hep-ß-tub1-6 (Ryan et al., 2008). Here we show that different complements of tubulin isotypes are expressed in different tissues and at different life cycle stages; this information may inform the search for novel anthelmintics. The predominant (as judged by quantitative PCR) isotype transcribed at the adult stage was F.hep-ß-tub1 and immunolocalisation studies revealed that this isotype occurred mainly in mature spermatozoa and vitelline follicles. Quantitative PCR indicated that changes occurred in the transcription levels of ß-tubulin isotypes at certain life cycle stages and may be of importance in the efficacy of benzimidazole-based anthelmintic drugs, but there were no significant differences between the triclabendazole (TCBZ)-susceptible Leon isolate and the TCBZ-resistant Oberon isolate in the transcription levels of each of the isotypes. When three well-characterised isolates with differing susceptibilities to TCBZ were compared, only one amino acid change resulting from a homozygous coding sequence difference (Gly269Ser) in isotype 4 was observed. However, this change was not predicted to alter the overall structure of the protein. In conclusion, these findings indicate that there is tissue-specific expression of tubulin isotypes in the liver fluke but the development of resistance to TCBZ is not associated with changes in its presumed target molecule.

Nilotinib 300mg BID as frontline treatment of CML:Prospective analysis of the Xpert BCR-ABL Monitor system and significance of 3-month molecular response

Sixty patients with early chronic phase CML (ECPCML) received Nilotinib on a phase II study which included a comparison of the Xpert BCR-ABL Monitor™ PCR system with standardized (IS) BCR-ABL1 real-time quantitative PCR (RQ-PCR). 88% patients achieved MMR with 45% achieving MR4.5. At 3 months BCR-ABL1/ABL1 IS >1% and

qPCR utilization for the study of RNA Polymerase I transcription

Ribosome biogenesis is a fundamental cellular process tightly linked to cell growth and proliferation, which requires the coordinated transcription of all three nuclear polymerases. Synthesis of ribosomal RNA (rRNA) by RNA polymerase I (Pol I) has been suggested as a key regulator of ribosome biogenesis, and there is a strong link between transcription of ribosomal RNAs and cellular proliferation. This makes Pol I transcription a valid and attractive target for anticancer therapy. At the moment however there are only a small number of compounds that act as specific inhibitors of Pol I transcription and this makes it very difficult for the development of drugs which would target rRNA transcription and consequently ribosome biogenesis. Therefore, to aid in the development of new inhibitors of Pol I, high-throughput methods to monitor and detect changes in Pol I activity need to be developed. This current study aimed to address the question of whether or not quantitative PCR (qPCR) could be used to detect changes in rRNA production in cells under different conditions that repress Pol I activity i.e. serum starvation and drug treatment. Our results have shown that using primers and a hydrolysis probe designed for the 5’ETS region of the pre-rRNA molecule, rRNA levels in both treated and untreated cells could be determined by using qPCR.
Amplification resulted in formation of a single product and S1 nuclease protection assay confirmed the down-regulation of Pol I transcription. Following serum-starvation and drug treatment there was a dramatic reduction in the amount of 5’ETS transcript quantitated by both Sybr Green chemistry and the use of a fluorescently labelled hydrolysis probe. The optimization of the qPCR strategy will be discussed.