Increased Messenger RNA Levels Research Articles

Isoliquiritigenin pretreatment regulates ER stress and attenuates cisplatin-induced nephrotoxicity in male Wistar rats.

Cisplatin (CP) is a chemotherapeutic drug used to treat solid tumors. However, studies have revealed its nephrotoxic effect. Oxidative stress, endoplasmic reticulum (ER) stress, and mitochondrial dysfunction are involved in CP-induced renal damage. Thus, preconditioning (hormetic effect) of ER stress is a strategy to prevent CP-induced renal damage. On the other hand, isoliquiritigenin (IsoLQ) is recognized as a flavonoid with antioxidant properties and an inducer of ER stress. Therefore, we evaluated the ER stress-inducing capacity of IsoLQ and its possible protective effect against CP-induced nephrotoxicity in adult male Wistar rats. The findings reflected that IsoLQ pretreatment might decrease renal damage by reducing plasma creatinine and blood urea nitrogen levels in animals with CP-induced nephrotoxicity. These may be associated with IsoLQ activating ER stress and unfolded protein response (UPR). We found increased messenger RNA levels of the ER stress marker glucose-related protein 78 kDa (GRP78). In addition, we also found that pretreatment with IsoLQ reduced the levels of CCAAT/enhancer-binding protein-homologous protein (CHOP) and X-box-binding protein 1 (XBP1) in the renal cortex, reflecting that IsoLQ can regulate the UPR and activation of the apoptotic pathway. Moreover, this preconditioning with IsoLQ of ER stress had oxidative stress-regulatory effects, as it restored the activity of glutathione peroxidase and glutathione reductase enzymes. Finally, IsoLQ modifies the protein expression of mitofusin 2 (Mfn-2) and voltage-dependent anion channel (VDAC). In conclusion, these data suggest that IsoLQ pretreatment has a nephroprotective effect; it could functionally regulate the ER and mitochondria and reduce CP-induced renal damage by attenuating hormesis-mediated ER stress.

Epidermal growth factor effect on lipopolysaccharide-induced inflammation in fibroblasts derived from diabetic foot ulcer.

BackgroundDiabetic foot ulcers (DFU) are characterised by high levels of inflammatory mediators, resulting from sustained hyperglycaemic insult and the local microbial biofilm. The intralesional administration of epidermal growth factor (EGF) has emerged as an effective treatment that stimulates granulation and closure of DFU, reducing the risk of amputation. Within the wound, fibroblasts play key roles during the healing process, promoting granulation and contraction. The aim of the present study was to examine the anti-inflammatory effect of EGF in DFU-derived fibroblasts, challenged with lipopolysaccharide (LPS), under hyperglycaemic conditions, recreating in vitro what happens in a clinical scenario.MethodsHealthy skin (HS) and DFU granulation tissue biopsies were used to isolate primary fibroblasts. The effect of LPS on cell proliferation was analysed. Transcriptional expression of toll-like receptor (TLR) pathway mediators (TLR4, TLR2, CD14, MYD88 and NFKB) and pro-inflammatory cytokines (TNF, IL-6 and IL-1B) were measured by semi-quantitative polymerase chain reaction (qPCR), in cells treated with appropriate concentrations of LPS, EGF and their combination. IL-6 protein concentration was quantified by ELISA.ResultsLPS stimulated proliferation of HS-derived fibroblasts, while inhibiting the proliferation of cells derived from DFU at the highest assayed concentration of 1 µg/mL. Regarding the TLR signalling pathway, LPS increased messenger RNA levels of mediators and pro-inflammatory genes, while EGF, alone or in the presence of LPS, downregulated them, except for IL-1B.ConclusionThe results suggest that EGF might elicit an anti-inflammatory response in LPS-challenged fibroblasts, even in a hyperglycaemic milieu. Collectively, our findings contribute to explain newly observed effects of EGF in the clinical arena.Lay SummaryIn this research article, we analyse the putative anti-inflammatory effect of epidermal growth factor (EGF) on fibroblast isolated from diabetic foot ulcer (DFU) granulation tissue. To induce the inflammatory response, the cells were treated with lipopolysaccharide (LPS), simulating the gram-negative bacterial infection that takes place in the wounds of diabetic patients. We studied the expression of genes involved in bacterial recognition receptors signalling pathway and those that code for different pro-inflammatory cytokines.We obtained primary fibroblasts from biopsies of a neuropathic diabetic ulcer and from healthy skin, the former was used as the control. Cells were isolated and grown in high glucose Dulbecco’s Modified Eagle Medium (DMEM) culture medium, to simulate the hyperglycaemic insult. The effect of increasing concentrations of LPS on cell proliferation was analysed. Relative transcriptional expression of genes in the study was quantified by quantitative polymerase chain reaction (qPCR) in cells treated with LPS, EGF or a combination. Untreated cells served to normalise the expression.In the present study, we demonstrated that EGF modulated the primary immune response by reducing the activation of pathogen-recognition receptors and common genes involved in these signalling pathways, even in hyperglycaemic conditions. This effect translated in a decreased expression of pro-inflammatory cytokines. These results contribute to explain our previous observations about the reduction of circulating levels of inflammatory cytokines after local administration of human recombinant EGF in DFU. Further molecular studies should be carried out to fully understand the biological mechanisms elicited by EGF in this clinical scenario.

Antifibrosis treatment by inhibition of VEGF, FGF, and PDGF receptors improves bladder wall remodeling and detrusor overactivity in association with modulation of C-fiber afferent activity in mice with spinal cord injury.

Spinal cord injury (SCI) above the sacral level causes bladder dysfunction and remodeling with fibrosis. This study examined the antifibrotic effects using nintedanib, an inhibitor of vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor receptors, on detrusor overactivity (DO) and bladder fibrosis, as well as the modulation mechanisms of C-fiber afferent pathways. Thirty female C57BL/6 mice were divided into group A (spinal intact), group B (SCI with vehicle), and group C (SCI with nintedanib). At 2 weeks after SCI, vehicle or 50 mg/kg nintedanib was administered subcutaneously for 2 weeks. Then, cystometry was conducted, followed by RT-PCR measurements of fibrosis-related molecules, muscarinic, β-adrenergic, TRP and purinergic receptors in the bladder or L6-S1 dorsal root ganglia (DRG). Trichrome stain and Western blot analysis of transforming growth factor-beta and fibronectin were performed in the bladder. TRPV1 expression in L6 DRG was measured by immunohistochemistry. In cystometry, intercontraction intervals, nonvoiding contractions, voided volume, and voiding efficiency were significantly improved in group C versus group B. RT-PCR, Western blotting, and trichrome staining revealed the fibrotic changes in the bladder of group B, which was improved in group C. Increased messenger RNA levels of TRPV1, TRPA1, P2X2 , and P2X3 in DRG of group B were significantly decreased in group C. TRPV1 immunoreactivity in DRG was increased in group B, but decreased in group C. Nintedanib improves storage and voiding dysfunctions and bladder fibrosis in SCI mice. Also, nintedanib-induced improvement of DO is associated with reduced expression of C-fiber afferent markers, suggesting the modulation of bladder C-fiber afferent activity.

Inflammatory Pre-Conditioning of Adipose-Derived Stem Cells with Cerebrospinal Fluid from Traumatic Brain Injury Patients Alters the Immunomodulatory Potential of ADSC Secretomes.

Immunomodulation by adipose-tissue-derived stem cells (ADSCs) is of special interest for the alleviation of damaging inflammatory responses in central nervous system injuries. The present study explored the effects of cerebrospinal fluid (CSF) from traumatic brain injury (TBI) patients on this immunomodulatory potential of ADSCs. CSF conditioning of ADSCs increased messenger RNA levels of both pro- and anti-inflammatory genes compared to controls. Exposure of phorbol-12-myristate-13-acetate-differentiated THP1 macrophages to the secretome of CSF-conditioned ADSCs downregulated both proinflammatory (cyclooxygenase-2, tumor necrosis factor alpha) and anti-inflammatory (suppressor of cytokine signaling 3, interleukin-1 receptor antagonist, and transforming growth factor beta) genes in these cells. Interleukin-10 expression was elevated in both naïve and conditioned secretomes. ADSC secretome treatment, further, induced macrophage maturation of THP1 cells and increased the percentage of CD11b+, CD14+, CD86+, and, to a lesser extent, CD206+ cells. This, moreover, enhanced the phagocytic activity of CD14+ and CD86+ cells, though independently of pre-conditioning. Secretome exposure, finally, also induced a reduction in the percentage of CD192+ adherent cells in cultures of peripheral blood mononuclear cells (PBMCs) from both healthy subjects and TBI patients. This limited efficacy (of both naïve and pre-conditioned secretomes) suggests that the effects of lymphocyte-monocyte paracrine signaling on the fate of cultured PBMCs are strongest upon adherent cell populations.

Can eosinophils in adipose tissue add fuel to the fire?

In inguinal adipose tissue, beige adipocytes are interspersed among white adipocytes and in close communication with dynamic populations of immune cells. Recent data have demonstrated that anti-inflammatory macrophages (M2) increase thermogenic activity of beige adipocytes, although the mechanism is currently under debate. ILC2, cells via secretion, of methionine enkephalin peptide were found to be able to increase beige thermogenesis. Knights et al. have recently generated a whole-body knock-out of KLF3 (KLF3-/-) to explore its contribution to thermogenesis and weight gain in a diet-induced obesity animal model.

Extracellular Vesicles Released From Articular Chondrocytes Play a Major Role in Cell-Cell Communication.

The purpose of this investigation was to determine the role of extracellular vesicles (EVs), released from articular chondrocytes in a physiological or pathological state, in cell-cell communication with other articular chondrocytes or chondrocyte precursor cells. The conditioned medium from interleukin-1β (IL-1β)-treated human articular chondrocytes stimulated catabolic events and inhibited type II collagen expression in articular chondrocytes to a much greater degree than medium from IL-1β-treated chondrocytes after complete removal of EVs. The vehicle-treated and IL-1β-treated human articular chondrocytes released EVs of similar size; however, the number of EVs released by IL-1β-treated chondrocytes was markedly higher than the number of EVs released from the vehicle-treated cells. Furthermore, our findings demonstrate that similar to medium from IL-1β-treated chondrocytes containing EVs, EVs isolated from medium of IL-1β-treated chondrocytes stimulated catabolic events in articular chondrocytes, whereas EVs isolated from the medium of vehicle-treated chondrocytes inhibited catabolic events and increased messenger RNA levels of aggrecan and type II collagen in IL-1β-treated chondrocytes. Furthermore, the medium containing EVs from vehicle-treated articular chondrocytes or EVs isolated from this medium stimulated chondrogenesis of C3H10T1/2 cells, whereas medium containing EVs from IL-1β-treated chondrocytes or EVs isolated from this medium inhibited chondrogenesis. Our findings suggest that EVs released by articular chondrocytes play a key role in the communication between joint cells and ultimately in joint homeostasis, maintenance, pathology, and repair. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:731-739, 2020.

Inhibition of myeloid differentiation 1 specifically in colon with antisense oligonucleotide exacerbates dextran sodium sulfate-induced colitis.

Myeloid differentiation 1 (MD-1), also known as lymphocyte antigen 86 (Ly86), is a soluble protein homologous to MD-2 and forms a complex with radioprotective 105 (RP105). RP105/MD-1 complex negatively regulates toll-like receptor 4 (TLR4) signaling and is involved in several immune disorders. However, the precise role of MD-1 in inflammatory bowel diseases (IBD) remains poorly understood. To further investigate the involvement of MD-1 in IBD, we inhibited MD-1 in colon with antisense oligonucleotide (AS-ODN) and assessed the effect of MD-1 inhibition on dextran sodium sulfate (DSS)-induced colitis. We discovered that MD-1 protein expression was remarkably decreased in both patients with ulcerative colitis and mice with DSS-induced colitis. For the first time, we showed that oral administration of MD-1 AS-ODN to mice significantly suppressed the MD-1 protein levels in colon rather than systemic tissues. Subsequently, we found that MD-1 AS-ODN treated mice were more susceptible to DSS-induced colitis based on loss of body weight, colon length, histological scores, and disease activity index. MD-1 inhibition also significantly enhanced inflammatory cytokines production such as IL-6 and IL-1β in colons. Finally, mice treated with MD-1 AS-ODN exhibited increased messenger RNA levels of TLR4 and MyD88 after DSS exposure and showed enhanced nuclear factor (NF)-κB activation compared with the control. Taken together, specifically suppression of MD-1 in colon tissues with AS-ODN exacerbates DSS-induced experimental colitis in mice, which is possibly related to activation of TLR4/NF-κB signaling.

Identification of the potential therapeutic target gene UBE2C in human hepatocellular carcinoma: An investigation based on GEO and TCGA databases.

Hepatocellular carcinoma (HCC) ranks the third major cause of cancer-associated mortality globally. Numerous studies have attempted to elucidate the underlying mechanisms of HCC using various biomarkers. In the present study, two expression profiles datasets from Gene Expression Omnibus (GSE76427 and GSE84402) and data associated with liver cancer samples from The Cancer Genome Atlas (TCGA) were downloaded for integrated analysis. Five differentially expressed genes (DEGs) exhibiting high expression, including ubiquitin-conjugating enzyme 2C (UBE2C), topoisomerase II α, pituitary tumor transforming gene 1, glypican-3 and polycomb-repressive complex 1, were selected and considered as candidate genes. Enrichment analysis demonstrated that these genes were associated with Gene Ontology terms of cellular components and molecular functions, including regulation of apoptosis, stabilization of p53 and Anaphase Promoting Complex/Cyclosome (APC/C) (APC/C:Cdc20)-mediated degradation of Securin. The expression profiles of these genes in HCC, other human malignancies and different human HCC cell lines were validated using GSE14520, GSE3500 and TCGA data. The results confirmed the upregulation of UBE2C in tissues from patients with HCC or other human malignancies and human liver cancer cell lines, compared with the expression levels in the corresponding adjacent non-tumor tissues and cell lines, respectively. Patients with HCC who exhibited an increased messenger RNA level of UBE2C exhibited a significantly shorter survival time. The results of the present study suggest that the overexpression of UBE2C may be used as a novel prognostic biomarker of HCC.

Stress-Induced Neuronal Colony Stimulating Factor 1 Provokes Microglia-Mediated Neuronal Remodeling and Depressive-like Behavior

BackgroundChronic stress exposure causes neuronal atrophy and synaptic deficits in the medial prefrontal cortex (PFC), contributing to development of anxiety- and depressive-like behaviors. Concomitantly, microglia in the PFC undergo morphological and functional changes following stress exposure, suggesting that microglia contribute to synaptic deficits underlying behavioral consequences. MethodsMale and female mice were exposed to chronic unpredictable stress (CUS) to examine the role of neuron–microglia interactions in the medial PFC during development of anxiety- and depressive-like behaviors. Thy1-GFP-M mice were used to assess microglia-mediated neuronal remodeling and dendritic spine density in the medial PFC. Viral-mediated knockdown of neuronal colony stimulating factor 1 (CSF1) was used to modulate microglia function and behavioral consequences after CUS. ResultsCUS promoted anxiety- and depressive-like behaviors that were associated with increased messenger RNA levels of CSF1 in the PFC. Increased CSF1 messenger RNA levels were also detected in the postmortem dorsolateral PFC of individuals with depression. Moreover, microglia isolated from the frontal cortex of mice exposed to CUS show elevated CSF1 receptor expression and increased phagocytosis of neuronal elements. Notably, functional alterations in microglia were more pronounced in male mice compared with female mice. These functional changes in microglia corresponded with reduced dendritic spine density on pyramidal neurons in layer 1 of the medial PFC. Viral-mediated knockdown of neuronal CSF1 in the medial PFC attenuated microglia-mediated neuronal remodeling and prevented behavioral deficits caused by CUS. ConclusionsThese findings revealed that stress-induced elevations in neuronal CSF1 provokes microglia-mediated neuronal remodeling in the medial PFC, contributing to synaptic deficits and development of anxiety- and depressive-like behavior.

SIRTUIN 1 ACTIVATOR SRT1720 PROTECTS AGAINST ORGAN INJURY INDUCED BY INTESTINAL ISCHEMIA-REPERFUSION.

Intestinal ischemia-reperfusion (I/R) occurs in various clinical situations and causes local and remote organ injury, especially in the lungs, leading to significant morbidity and mortality. The maintenance of mitochondrial biogenesis is essential for cell survival and is regulated in part by sirtuin 1 (SIRT1), an energy-sensing enzyme. We hypothesized that SIRT1 activation with SRT1720 would reduce local and remote organ injury after intestinal I/R. Intestinal I/R was induced by the occlusion of the superior mesenteric artery of adult male C57BL/6 mice for 45 min, followed by reperfusion for 4 h. SRT1720 or vehicle was injected intravenously at the time of reperfusion. Blood, small intestine, and lung tissues were collected for analysis. The SRT1720 treatment of I/R mice resulted in a 57% increase in protein levels of succinate dehydrogenase, an index of mitochondrial mass, and a 120% increase in messenger RNA levels of mitochondrial transcription factor A, a marker for mitochondrial biogenesis. The microscopic architecture and apoptosis of the gut tissue was improved in the SRT1720-treated I/R mice. SRT1720 decreased intestinal messenger RNA levels of tumor necrosis factor-α by 60% and inducible nitric oxide synthase to baseline after I/R. Systemic inflammation, as determined by serum interleukin-6, was reduced in treated mice. Lung injury, as measured by histological architecture and myeloperoxidase activity, and lung apoptosis were also improved after the SRT1720 treatment. SRT1720 preserved mitochondrial biogenesis and mass, leading to inhibition of inflammation and oxidative stress, thereby protecting against intestinal I/R-induced injury. Thus, the SIRT1-mediated pathway is a promising target for the treatment of intestinal I/R injury.

Melatonin attenuates carbon tetrachloride–induced liver fibrosis via inhibition of necroptosis

We investigated the protective mechanisms of melatonin (MLT) associated with necroptosis signaling and damage-associated molecular patterns, which are mediated by the activation of pattern recognition receptors in liver fibrosis. Rats were given an intraperitoneal injection of carbon tetrachloride (CCl4) dissolved in olive oil (1:3, vol/vol) twice a week (0.5 mL/kg) for 8 weeks. During this period, MLT was administered orally at 2.5, 5, and 10 mg/kg once a day. Chronic CCl4 administration increased hepatic hydroxyproline content and hepatocellular damage. MLT attenuated these increases. The expression levels of transforming growth factor β1 and α-smooth muscle actin that were increased by chronic CCl4 exposure were attenuated by MLT. CCl4 significantly increased receptor-interacting protein 1 (RIP1) expression, the formation of the RIP1 and RIP3 necrosome complex, and the level of mixed lineage kinase domain-like protein in liver tissue, which were attenuated by MLT. MLT also attenuated CCl4-induced increases in serum high-mobility group box 1 (HMGB1) and interleukin 1α, as well as the interaction between HMGB1 receptors for advanced glycation end products (RAGE). The increases in toll-like receptor 4 expression, p38, c-Jun N-terminal kinases phosphorylation, and nuclear factor κB translocation were suppressed by MLT. MLT attenuated the overexpression of RAGE, increased level of early growth response protein 1, and increased messenger RNA level of macrophage inflammatory protein 2. Our findings suggest MLT may prevent liver fibrosis by inhibiting necroptosis-associated inflammatory signaling.

Aberrant expression of the human epidermal growth factor receptor 2 oncogene is not a common feature in osteosarcoma

Human epidermal growth factor receptor 2 expression in osteosarcoma and its relationship to prognosis have been the subject of several conflicting reports, most of them relying on immunohistochemical studies. Because the urgent need of prognostic markers and effective new treatment options for osteosarcoma patients, we evaluated the role of human epidermal growth factor receptor 2 in 2 well-characterized sets of pretherapeutic osteosarcoma samples (46 paraffin-embedded and 46 fresh-frozen biopsy samples) using immunohistochemistry with 2 different antibodies [DAKO A0485 (Glostrup, Denmark) and Novocastra CB11 (Newcastle, UK)] as well as fluorescence in situ hybridization, real-time polymerase chain reaction, and SNP array analyses and correlated our findings with clinicopathological parameters. However, our study failed to detect unequivocal evidence of human epidermal growth factor receptor 2 gene amplification or overexpression of human epidermal growth factor receptor 2 messenger RNA or protein in any of the investigated tumors. Only in a small subset of samples, a moderate increase in messenger RNA levels (13.6%) or focal membranous immunoreactivity (8.7%; A0485) was detected but did not correlate with survival or response to chemotherapy. Cytoplasmic staining was identified more frequently (63%; CB11) but again did not show any association with clinicopathological parameters. In conclusion, our study does not support a role for human epidermal growth factor receptor 2 as a prognostic marker in osteosarcoma.

Activation of interleukin-6-induced glycoprotein 130/signal transducer and activator of transcription 3 pathway in mesenchymal stem cells enhances hepatic differentiation, proliferation, and liver regeneration

Adult bone marrow-derived mesenchymal stem cells (MSCs) exist in all living species and are capable of differentiating into different types of specific cells. In this study, we demonstrate the therapeutic effectiveness of rat MSC transplantation in D-galactosamine (GalN)-induced acute liver injury and identified the novel pathways which are involved in hepatic differentiation of MSCs. In vivo, intraportal transplantation with 5 × 10(6) MSCs at 24 hours after GalN administration resulted in significant reduction in serum levels of alanine aminotransferase, aspartate aminotransferase, and total bilirubin compared to the control group. Engrafted MSCs actively proliferated, differentiated, and further enhanced hepatocyte proliferation activity. In vitro, coculture of MSCs with GalN-induced injured hepatocytes showed efficient differentiation and was evidenced by progressive increase in messenger RNA levels of hepatic markers, including albumin, α-fetoprotein, CCAAT-enhancer binding protein α, α-1-antitryspin, and hepatocyte nuclear factor-3β. Immunofluorescent staining revealed that these cells were positive for albumin, α-fetoprotein, and cytokeratin 18, but not clusters of differentiation 34, cytokeratin 19, or OV6. During hepatic differentiation, signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling were constantly activated, and a gradual down-regulation of β-catenin expression in messenger RNA and protein levels was detected. Hyper-interleukin-6 fusion protein but not interleukin-6 (IL-6) alone caused reduction in β-catenin expression associated with the up-regulation of Wnt-5a in MSCs via activating the glycoprotein 130 (gp130)-mediated STAT3 signaling pathway, which indicates the operation of the trans-signaling mechanism. Activation of IL-6/gp130-mediated STAT3 signaling pathway in MSCs triggered wound healing, cell migration, and proliferation. In conclusion, transplantation of MSCs promotes cell proliferation and organ repair, and activation of IL-6/gp130-mediated STAT3 signaling pathway via soluble IL-6 receptor is crucial in hepatic differentiation of MSCs.

Balance of Profibrotic and Antifibroting Signaling in Nephrogenic Systemic Fibrosis Skin Lesions

Nephrogenic systemic fibrosis (NSF) is an uncommon fibrotic disorder occurring after administration of linear gadolinium contrast agents in patients with severely decreased kidney function. The underlying pathogenetic mechanism of fibrosis remains to be elucidated. Transforming growth factor beta (TGF-beta), a key player in the pathogenesis of fibrotic disorders, has been found to be overexpressed in NSF skin lesions. The aim of this study is to analyze the TGF-beta-SMAD-connective tissue growth factor (CTGF) axis in NSF skin lesions compared with skin specimens from patients with systemic sclerosis, hemodialysis patients without NSF, and healthy controls. Additionally, expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) and antifibrotic tumor necrosis factor alpha (TNF-alpha) were examined. Observational study. Full-thickness skin biopsy specimens from fibrotic lesions or healthy skin were obtained from 10 patients with NSF, 16 patients with systemic sclerosis, 8 non-NSF hemodialysis patients, and 17 healthy participants. Patient diagnosis of NSF, systemic sclerosis, non-NSF hemodialysis patients, and healthy participants, as defined using skin biopsy. Dermal messenger RNA and protein expression of profibrotic TGF-beta, SMAD2, SMAD3, SMAD4, SMAD7, CTGF, TIMP-1, antifibrotic SMAD7, and TNF-alpha were analyzed using real-time reverse transcription-polymerase chain reaction and immunohistologic examination on formalin-embedded tissue. Dermal expression of nearly all parameters differed in hemodialysis patients compared with healthy controls. In comparison to hemodialysis patients and healthy participants, we found increased messenger RNA levels for TGF-beta, the profibrotic receptor-activated SMAD2 and SMAD3, CTGF, and TIMP-1 in NSF and systemic sclerosis lesions. Few differences between NSF and non-NSF hemodialysis patients were observed for common SMAD4, inhibitory SMAD7, and TNF-alpha. Small patient cohort. Our results suggest a profibrotic imbalance in the TGF-beta-SMAD-CTGF axis in NSF skin lesions. Significantly increased dermal expression of TGF-beta and TIMP-1 in non-NSF hemodialysis patients in comparison to healthy participants emphasizes the need for a hemodialysis control group for future investigations and suggests a pre-existing profibrotic situation in the skin of hemodialysis patients.

Stimulation of bikaverin production by sucrose and by salt starvation in Fusarium fujikuroi

The fungus Fusarium fujikuroi (Gibberella fujikuroi mating group C) exhibits a rich secondary metabolism that includes the synthesis of compounds of biotechnological interest, such as gibberellins, bikaverin, and carotenoids. The effect of the carbon source on their production was checked using a two-phase incubation protocol, in which nine different sugars were added upon transfer of the fungus from repressed to appropriate inducing conditions, i.e., nitrogen starvation for gibberellins and bikaverin and illumination for carotenoids production. Most of the carbon sources allowed the synthesis of these metabolites in significant amounts. However, bikaverin production was strongly increased by the presence of sucrose in comparison to other carbon sources, an effect not exhibited for the production of gibberellins and carotenoids. The bikaverin inducing effect was enhanced in the absence of phosphate and/or sulfate. Similar results were also observed in carotenoid-overproducing strains known to be altered in bikaverin production. The induction by salt starvation, but not by sucrose, correlated with an increase in messenger RNA levels of gene bik1, encoding a polyketide synthase of the bikaverin pathway.

Memory deficits and neurochemical changes induced by C-reactive protein in rats: implication in Alzheimer’s disease

C-reactive protein (CRP), an acute phase protein that is released in response to inflammatory stimuli, is implicated in Alzheimer's disease (AD). However, the role of CRP in memory deficits associated with AD remains unclear. Experiments were carried out to determine whether CRP impaired memory and altered neurochemical measures associated with AD. The effects of intra-cerebroventricular administration of CRP or beta-amyloid peptide 25-35 (Abeta(25-35)) on memory performance were evaluated using rat Morris water-maze and step-through passive avoidance tests; the levels of inflammatory cytokines (interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor (TNF-alpha)), endogenous CRP, and markers of the endogenous production of Abeta, including amyloid precursor protein (APP), presenilins (PS-1 and PS-2), and beta-site of APP cleaving enzyme (BACE), were also determined in brain regions using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting analysis. Treatment with CRP (25.6 microg/rat) or Abeta(25-35) (10 microg/rat) 2 weeks ahead produced impairment of long-term memory in both animal tests. Real-time RT-PCR revealed increases in messenger RNA levels of APP, IL-1beta, IL-6, TNF-alpha, and CRP in the cerebral cortex and hippocampus and those of PS-1 and PS-2 in the cerebral cortex produced by treatment with CRP or Abeta(25-35). Immunoblotting analysis showed that while expression of APP was increased in both the cerebral cortex and the hippocampus, expression of IL-1beta, BACE, and TNF-alpha was increased only in the hippocampus. The results suggest that CRP contributes to memory loss and early phase of pathogenesis of AD. CRP can be a novel target for therapeutic intervention of AD.

Microparticles harboring Sonic Hedgehog promote angiogenesis through the upregulation of adhesion proteins and proangiogenic factors

Microparticles (MPs) are small fragments generated from the plasma membrane after cell stimulation or apoptosis. We have recently shown that MPs harboring the morphogen Sonic Hedgehog (MPs(Shh+)) correct endothelial injury by release of nitric oxide from endothelial cells [Agouni, Mostefai, Porro, Carusio, Favre, Richard, Henrion, Martínez and Andriantsitohaina (2007) FASEB J., 21, 2735-2741]. Here, we show that MPs(Shh+) induce the formation of capillary-like structures in an in vitro model using human endothelial cells, although they inhibited cell migration. Besides, MPs(Shh+) regulate cell proliferation. Both cell adhesion and expression of proteins involved in this process such as Rho A and phosphorylation of focal-activated kinase were increased by MPs(Shh+), via a Rho-associated coiled-coil-containing protein kinase inhibitor-sensitive pathway. We demonstrate that MPs(Shh+) increase messenger RNA and protein levels of proangiogenic factors as measured by quantitative reverse transcription-polymerase chain reaction and western blot. In spite of vascular endothelial growth factor expression, conditioned media from endothelial cells treated avec MPs(Shh+) reduces angiogenesis. Interestingly, the effects induced by MPs(Shh+) on the formation of capillary-like structures, expression of adhesion molecules and proangiogenic factors were reversed after silencing of the Shh receptor, using small interfering RNA or when Sonic Hedgehog (Shh) signaling was pharmacologically inhibited with cyclopamine. Taken together, we show that Shh carried by MPs(Shh+) regulate angiogenesis probably through both a direct and an indirect mechanisms, and we propose that MPs harboring Shh may contribute to the generation of a vascular network in pathologies associated with tumor growth.

Preventive effects of curcumin and dexamethasone on lung transplantation-associated lung injury in rats

To investigate potential effects of curcumin or dexamethasone on lung transplantation-associated lung injury. Prospective, randomized, controlled study. Research laboratory. Adult male Sprague-Dawley rats. Sham-operated rats were used as time-matched controls. Experimental rats were subjected to unilateral orthotopic lung transplantation with 4 hrs of cold ischemia followed by 2 hrs (or 24 hrs) of reperfusion. Animals were randomly assigned to vehicle-, curcumin-, or dexamethasone-treated groups. Transplantation-associated lung injury was characterized by an increased alveolar-capillary permeability and myeloperoxidase activity and decreased levels of arterial oxygen tension/inspired oxygen concentration ratio. Pretreatment with curcumin and dexamethasone significantly prevented barrier disruption, lung edema, tissue inflammation, and decreased PaO2 at the early stage of posttransplantation. Nuclear factor-kappaB in transplanted lungs was activated, accompanied by an increase in messenger RNA levels and protein content of tumor necrosis factor-alpha, interleukin-6, and matrix metalloproteinase-9 in lung graft. Those changes were prevented by pretreatment with curcumin and dexamethasone. Curcumin can be an alternative therapy for protecting lung transplantation-associated injury by suppressing nuclear factor-kappaB-mediated expression of inflammatory genes.

Hepatitis C virus replication is inhibited by 22β-methoxyolean-12-ene-3β, 24(4β)-diol (ME3738) through enhancing interferon-β

A derivative of soyasapogenol, 22beta-methoxyolean-12-ene-3beta, 24(4beta)-diol (ME3738), ameliorates liver injury induced by Concanavalin A in mice. We examined whether ME3738 has independent antiviral effects against hepatitis C virus (HCV) using an established HCV replication model that expresses the full-length genotype 1a HCV complementary DNA plasmid (pT7-flHCV-Rz) under the control of a replication-defective adenoviral vector expressing T7 polymerase. Hepatocellular carcinoma (HepG2) cells, human hepatoma (Huh7) cells, or monkey kidney (CV-1) cells were transfected with pT7-flHCV-Rz, and infected with adenoviral vector expressing T7 polymerase. ME3738 or interferon-alpha (IFN-alpha) was added thereafter and then protein and RNA were harvested from the cells at 9 days after infection. HCV-positive and HCV-negative strands were measured by real-time reverse-transcription polymerase chain reaction and HCV core protein expression was measured using an enzyme-linked immunosorbent assay. The messenger RNA levels of innate antiviral response-related genes were assessed using real-time reverse-transcription polymerase chain reaction. ME3738 dose-dependently reduced HCV-RNA and core protein in hepatocyte-derived cell lines. The antiviral effect was more pronounced in HepG2 than in Huh7 cells. ME3738 increased messenger RNA levels of interferon-beta (IFN-beta) and of IFN-stimulated genes (2'-5' oligoadenylate synthetase, myxovirus resistance protein A [MxA]). Interferon-beta knockdown by small interfering RNA abrogated the anti-HCV effect of ME3738. Moreover, the anti-HCV effects were synergistic when ME3738 was combined with IFN-alpha. ME3738 has antiviral effects against HCV. The enhancement of autocrine IFN-beta suggests that ME3738 exerts antiviral action along the type I IFN pathway. This anti-HCV action by ME3738 was synergistically enhanced when combined with IFN-alpha. ME3738 might be a useful anti-HCV drug either with or without IFN-alpha.

Keratin Mutation Predisposes to Mouse Liver Fibrosis and Unmasks Differential Effects of the Carbon Tetrachloride and Thioacetamide Models

Keratins 8 and 18 (K8/K18) are important hepatoprotective proteins. Animals expressing K8/K18 mutants show a marked susceptibility to acute/subacute liver injury. K8/K18 variants predispose to human end-stage liver disease and associate with fibrosis progression during chronic hepatitis C infection. We sought direct evidence for a keratin mutation-related predisposition to liver fibrosis using transgenic mouse models because the relationship between keratin mutations and cirrhosis is based primarily on human association studies. Mouse hepatofibrosis was induced by carbon tetrachloride (CCl(4)) or thioacetamide. Nontransgenic mice, or mice that over express either human Arg89-to-Cys (R89C mice) or wild-type K18 (WT mice) were used. The extent of fibrosis was evaluated by quantitative real-time reverse-transcription polymerase chain reaction of fibrosis-related genes, liver hydroxyproline measurement, and Picro-Sirius red staining and collagen immunofluorescence staining. Compared with control animals, CCl(4) led to similar liver fibrosis but increased injury in K18 R89C mice. In contrast, thioacetamide caused more severe liver injury and fibrosis in K18 R89C as compared with WT and nontransgenic mice and resulted in increased messenger RNA levels of collagen, tissue inhibitor of metalloproteinase 1, matrix metalloproteinase 2, and matrix metalloproteinase 13. Analysis in nontransgenic mice showed that thioacetamide and CCl(4) have dramatically different molecular expression responses involving cytoskeletal and chaperone proteins. Over expression of K18 R89C predisposes transgenic mice to thioacetamide- but not CCl(4)-induced liver fibrosis. Differences in the keratin mutation-associated fibrosis response among the 2 models raise the hypothesis that keratin variants may preferentially predispose to fibrosis in unique human liver diseases. Findings herein highlight distinct differences in the 2 widely used fibrosis models.