Inflammation and oxidative stress are thought to promote tissue damage in multiple sclerosis. Thus, novel therapeutics enhancing cellular resistance to free radicals could prove useful for multiple sclerosis treatment. BG00012 is an oral formulation of dimethylfumarate. In a phase II multiple sclerosis trial, BG00012 demonstrated beneficial effects on relapse rate and magnetic resonance imaging markers indicative of inflammation as well as axonal destruction. First we have studied effects of dimethylfumarate on the disease course, central nervous system, tissue integrity and the molecular mechanism of action in an animal model of chronic multiple sclerosis: myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis in C57BL/6 mice. In the chronic phase of experimental autoimmune encephalomyelitis, preventive or therapeutic application of dimethylfumarate ameliorated the disease course and improved preservation of myelin, axons and neurons. In vitro, the application of fumarates increased murine neuronal survival and protected human or rodent astrocytes against oxidative stress. Application of dimethylfumarate led to stabilization of the transcription factor nuclear factor (erythroid-derived 2)-related factor 2, activation of nuclear factor (erythroid-derived 2)-related factor 2-dependent transcriptional activity and accumulation of NADP(H) quinoline oxidoreductase-1 as a prototypical target gene. Furthermore, the immediate metabolite of dimethylfumarate, monomethylfumarate, leads to direct modification of the inhibitor of nuclear factor (erythroid-derived 2)-related factor 2, Kelch-like ECH-associated protein 1, at cysteine residue 151. In turn, increased levels of nuclear factor (erythroid-derived 2)-related factor 2 and reduced protein nitrosylation were detected in the central nervous sytem of dimethylfumarate-treated mice. Nuclear factor (erythroid-derived 2)-related factor 2 was also upregulated in the spinal cord of autopsy specimens from untreated patients with multiple sclerosis. In dimethylfumarate-treated mice suffering from experimental autoimmune encephalomyelitis, increased immunoreactivity for nuclear factor (erythroid-derived 2)-related factor 2 was detected by confocal microscopy in neurons of the motor cortex and the brainstem as well as in oligodendrocytes and astrocytes. In mice deficient for nuclear factor (erythroid-derived 2)-related factor 2 on the same genetic background, the dimethylfumarate mediated beneficial effects on clinical course, axon preservation and astrocyte activation were almost completely abolished thus proving the functional relevance of this transcription factor for the neuroprotective mechanism of action. We conclude that the ability of dimethylfumarate to activate nuclear factor (erythroid-derived 2)-related factor 2 may offer a novel cytoprotective modality that further augments the natural antioxidant responses in multiple sclerosis tissue and is not yet targeted by other multiple sclerosis therapies.
Oxidative stress is central to the pathology of several neurodegenerative diseases, including multiple sclerosis, and therapeutics designed to enhance antioxidant potential could have clinical value. The objective of this study was to characterize the potential direct neuroprotective effects of dimethyl fumarate (DMF) and its primary metabolite monomethyl fumarate (MMF) on cellular resistance to oxidative damage in primary cultures of central nervous system (CNS) cells and further explore the dependence and function of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway in this process. Treatment of animals or primary cultures of CNS cells with DMF or MMF resulted in increased nuclear levels of active Nrf2, with subsequent up-regulation of canonical antioxidant target genes. DMF-dependent up-regulation of antioxidant genes in vivo was lost in mice lacking Nrf2 [Nrf2(Ϫ/Ϫ)]. DMF or MMF treatment increased cellular redox potential, glutathione, ATP levels, and mitochondrial membrane potential in a concentration-dependent manner. Treating astrocytes or neurons with DMF or MMF also significantly improved cell viability after toxic oxidative challenge in a concentration-dependent manner. This effect on viability was lost in cells that had eliminated or reduced Nrf2. These data suggest that DMF and MMF are cytoprotective for neurons and astrocytes against oxidative stress-induced cellular injury and loss, potentially via up-regulation of an Nrf2-dependent antioxidant response. These data also suggest DMF and MMF may function through improving mitochondrial function. The clinical utility of DMF in multiple sclerosis is being explored through phase III trials with BG-12, which is an oral therapeutic containing DMF as the active ingredient.
MethodsFc:TβRII and transgenic mice. Fc:TβRII has been described previously (11). FVB MMTV-Polyomavirus middle T antigen (MMTV-PyV mT) mice (13) (The Jackson Laboratories, Bar Harbor, Maine, USA) were housed in the Animal Care Facility at Vanderbilt University following The American Association for the Accrediation of Laboratory Animal Care guidelines. Three-week-old transgenic mice were treated twice weekly with Fc:TβRII in PBS (5 mg/kg) by intraperitoneal injection. At 110 days, tissues were harvested and fixed in formalin or were snap-frozen. Serum levels of Fc:TβRII were measured by TGF-βs are potent inhibitors of epithelial cell proliferation. However, in established carcinomas, autocrine/paracrine TGF-β interactions can enhance tumor cell viability and progression. Thus, we studied the effect of a soluble Fc:TGF-β type II receptor fusion protein (Fc:TβRII) on transgenic and transplantable models of breast cancer metastases. Systemic administration of Fc:TβRII did not alter primary mammary tumor latency in MMTV-Polyomavirus middle T antigen transgenic mice. However, Fc:TβRII increased apoptosis in primary tumors, while reducing tumor cell motility, intravasation, and lung metastases. These effects correlated with inhibition of Akt activity and FKHRL1 phosphorylation. Fc:TβRII also inhibited metastases from transplanted 4T1 and EMT-6 mammary tumors in syngeneic BALB/c mice. Tumor microvessel density in a mouse dorsal skin window chamber was unaffected by Fc:TβRII. Therefore, blockade of TGF-β signaling may reduce tumor cell viability and migratory potential and represents a testable therapeutic approach against metastatic carcinomas.
TGF-betas are potent inhibitors of epithelial cell proliferation. However, in established carcinomas, autocrine/paracrine TGF-beta interactions can enhance tumor cell viability and progression. Thus, we studied the effect of a soluble Fc:TGF-beta type II receptor fusion protein (Fc:TbetaRII) on transgenic and transplantable models of breast cancer metastases. Systemic administration of Fc:TbetaRII did not alter primary mammary tumor latency in MMTV-Polyomavirus middle T antigen transgenic mice. However, Fc:TbetaRII increased apoptosis in primary tumors, while reducing tumor cell motility, intravasation, and lung metastases. These effects correlated with inhibition of Akt activity and FKHRL1 phosphorylation. Fc:TbetaRII also inhibited metastases from transplanted 4T1 and EMT-6 mammary tumors in syngeneic BALB/c mice. Tumor microvessel density in a mouse dorsal skin window chamber was unaffected by Fc:TbetaRII. Therefore, blockade of TGF-beta signaling may reduce tumor cell viability and migratory potential and represents a testable therapeutic approach against metastatic carcinomas.
Background-Transforming growth factor (TGF-) is a key mediator of collagen synthesis in the development of lung fibrosis. It has previously been shown that the administration of TGF-antibody and TGF-binding proteoglycan, decorin, reduced bleomycin (BL) induced lung fibrosis in animals. The present study was carried out to investigate whether intratracheal instillation of TGF-soluble receptor (TR) would minimise the BL induced lung fibrosis in hamsters. Methods-The eVect of a recombinant TR (TGF RII) on the lung collagen accumulation was evaluated in a BL hamster model of pulmonary fibrosis. Animals were divided into four groups and intratracheally injected with saline or BL at 6.5 U/4 ml/kg followed by intratracheal instillation of phosphate buVered saline (PBS) or 4 nmol TR in 0.3 ml twice a week. Twenty days after the first intratracheal instillation the hamsters were killed for bronchoalveolar lavage (BAL) fluid, biochemical, and histopathological analyses. Results-Treatment of hamsters with TR after intratracheal instillation of BL significantly reduced BL induced lung fibrosis as shown by decreases in the lung hydroxyproline level and prolyl hydroxylase activity, although they were still significantly higher than those of the saline control. Histopathological examination showed a considerable decrease in BL induced fibrotic lesions by TR treatment. However, TR did not prevent the BL induced increases in total cells and protein in the BAL fluid. Conclusions-These results suggest that TR has antifibrotic potential in vivo and may be useful in the treatment of fibrotic diseases where increased TGF-is associated with excess collagen accumulation. (Thorax 1999;54:805-812)
Constrictive remodeling has been identified as a major contributor to restenosis following angioplasty. Characterization of transforming growth factor-β (TGF-β)-mediated cellular events in the adventitia and their contribution to vascular remodeling, however, has not previously been studied in detail. The balloon catheter denudation model was performed on rat carotid artery, and groups of rats were treated with vehicle or a TGF-β inhibitor, a soluble TGF-β receptor type II (TGF-βR:Fc). Adventitial cell proliferation, which peaked 4 days after injury, was characterized by the de novo formation of several cell layers surrounding the outer adventitia and this process was not dependent upon TGF-β activity. These neoadventitial cells expressed an abundance of collagen type I and a fetal isoform of fibronectin containing the EIIIA domain, and the expression of both proteins was suppressed in the presence of TGF-βR:Fc. Lumenal narrowing was apparent 14 days after injury. Inhibition of TGF-β signaling promoted vessel enlargement. As a result, lumen size did not change despite neointima formation. In conclusion, adventitial fibrosis with abundant collagen matrix deposition but not adventitial cell proliferation is dependent upon endogenous TGF-β activity. Furthermore, inhibition of TGF-β signaling prevents injury-induced reduction in lumen area by promoting vessel enlargement.
Background: α7 nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the central nervous system and are reported to have neuroprotective properties. α7 nAChRs are expressed on astrocytes, which are key regulators of neuroinflammation and oxidative stress in several neurodegenerative diseases. However, the anti-inflammatory and antioxidant properties of astroglial α7 nAChRs are not well studied. Therefore, we evaluated the role of astroglial α7 nAChR activation in neuroinflammation.Methods: Anti-inflammatory and antioxidant effects of α7 nAChR activation were evaluated in an in vitro mouse model of neuroinflammation using lipopolysaccharide (LPS) in primary astrocyte cultures. α7 nAChR anti-inflammatory effects on the NF-κB pathway were evaluated using ELISA, gene expression analysis, immunofluorescence, and western blotting. Antioxidant effect of α7 nAChR activation on expression profiles of canonical Nrf2 target genes was examined by quantitative PCR and western blotting. The role of the Nrf2 pathway in α7 nAChR-mediated anti-inflammatory response was evaluated using Nrf2 knockout astrocytes. Brain ex vivo NF-κB luciferase signals were evaluated after treatment with an α7 nAChR agonist in lipopolysaccharide (LPS)-injected NF-κB luciferase reporter mouse model. Results: Astrocytes treated with the α7 nAChR partial agonist (GTS21) showed significantly reduced LPS-mediated secretion of inflammatory cytokines and this effect was reversed by the α7 nAChR antagonist methyllycaconitine (MLA) and by knockdown of α7 nAChR expression with a short hairpin RNA. Further, α7 nAChR activation blocked LPS-mediated NF-κB nuclear translocation indicating that the observed anti-inflammatory effect may be mediated through inhibition of the NF-κB pathway. Treatment with GTS21 also upregulated canonical Nrf2 antioxidant genes and proteins suggesting antioxidant properties of α7 nAChR in astrocytes. Using an astrocyte conditioned media approach, we demonstrated reduction in neuronal apoptosis when astrocytes were pretreated with GTS21. Finally, in an in vivo neuroinflammation model using LPS in NF-κB luciferase reporter mice, we demonstrated reduction in LPS-induced NF-κB activity and pro-inflammatory cytokines with GTS21 treatment in brain tissue. Conclusion: Our results suggest that activating astroglial α7 nAChRs may have a role in neuroprotection by decreasing inflammation and oxidative stress, and therefore could have therapeutic implication for disease modifying treatments of neurodegenerative diseases.
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