Pathological angiogenesis is the hallmark of diseases such as cancer and retinopathies. Although tissue hypoxia and inflammation are recognized as central drivers of vessel growth, relatively little is known about the process that bridges the two. In a mouse model of ischemic retinopathy, we found that hypoxic regions of the retina showed only modest rates of apoptosis despite severely compromised metabolic supply. Using transcriptomic analysis and inducible loss-of-function genetics, we demonstrated that ischemic retinal cells instead engage the endoplasmic reticulum stress inositol-requiring enzyme 1α (IRE1α) pathway that, through its endoribonuclease activity, induces a state of senescence in which cells adopt a senescence-associated secretory phenotype (SASP). We also detected SASP-associated cytokines (plasminogen activator inhibitor 1, interleukin-6, interleukin-8, and vascular endothelial growth factor) in the vitreous humor of patients suffering from proliferative diabetic retinopathy. Therapeutic inhibition of the SASP through intravitreal delivery of metformin or interference with effectors of senescence (semaphorin 3A or IRE1α) in mice reduced destructive retinal neovascularization in vivo. We conclude that the SASP contributes to pathological vessel growth, with ischemic retinal cells becoming prematurely senescent and secreting inflammatory cytokines that drive paracrine senescence, exacerbate destructive angiogenesis, and hinder reparative vascular regeneration. Reversal of this process may be therapeutically beneficial.
The identification of cancer-associated mutations in the tricarboxylic acid (TCA) cycle enzymes isocitrate dehydrogenases 1 and 2 (IDH1/2) highlights the prevailing notion that aberrant metabolic function can contribute to carcinogenesis. IDH1/2 normally catalyse the oxidative decarboxylation of isocitrate into α-ketoglutarate (αKG). In gliomas and acute myeloid leukaemias, IDH1/2 mutations confer gain-of-function leading to production of the oncometabolite R-2-hydroxyglutarate (2HG) from αKG. Here we show that generation of 2HG by mutated IDH1/2 leads to the activation of mTOR by inhibiting KDM4A, an αKG-dependent enzyme of the Jumonji family of lysine demethylases. Furthermore, KDM4A associates with the DEP domain-containing mTOR-interacting protein (DEPTOR), a negative regulator of mTORC1/2. Depletion of KDM4A decreases DEPTOR protein stability. Our results provide an additional molecular mechanism for the oncogenic activity of mutant IDH1/2 by revealing an unprecedented link between TCA cycle defects and positive modulation of mTOR function downstream of the canonical PI3K/AKT/TSC1-2 pathway.
A systemic vasculitis involving particularly the skin and kidneys has been recently described in swine under the name dermatitis/nephropathy syndrome. Twelve pigs with gross cutaneous lesions typical of this condition were necropsied, and morphologic, immunohistochemical, microbiologic, and epidemiologic characteristics were studied. The pigs were divided into three groups comprising eight pigs with acute lesions, two with chronic lesions, and two with acute lesions kept for sequential skin biopsies. Acute skin lesions consisted of round to irregular, red to purple macules and papules that often coalesced to form large, irregular patches and plaques. With time, the lesions became covered by crusts and faded gradually, sometimes leaving scars. Characteristic distribution included the perineal area of the hindquarters, limbs, dependent parts of the abdomen and thorax, and margins of the ears. In the acute phase of the disease, necrotizing and leucocytoclastic vasculitis of small-caliber blood vessels were observed within the dermis and panniculus and in various extracutaneous locations such as the renal pelvis and synovial membranes. All pigs had macroscopic evidence of pneumonia and generalized lymphadenopathy. Microscopically, they had interstitial pneumonia and perivascular cuffing of mononuclear cells in various tissues including skin. The presence of immunoglobulins and complement was demonstrated by immunofluorescence in and around necrotic vessels of the skin in the early stages. Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) antigens were detected by immunohistochemistry in macrophages located around vessels of the tissues examined (skin and kidneys) in acute and chronic cases. PRRSV RNA was demonstrated by reverse transcription-polymerase chain reaction in lung and spleen homogenates from all pigs. The PRRSV was isolated in cell culture from 11 of the pigs. These findings suggest that PRRSV infection may play a role in the pathogenesis of this systemic vascular disease of swine.
Keratinocytes are responsible for reepithelialization and restoration of the epidermal barrier during wound healing. The influence of sensory neurons on this mechanism is not fully understood. We tested whether sensory neurons influence wound closure via the secretion of the neuropeptide substance P (SP) with a new tissue-engineered wound healing model made of an upper-perforated epidermal compartment reconstructed with human keratinocytes expressing green fluorescent protein, stacked over a dermal compartment, innervated or not with sensory neurons. We showed that sensory neurons secreted SP in the construct and induced a two times faster wound closure in vitro. This effect was partially reproduced by addition of SP in the model without neurons, and completely blocked by a treatment with a specific antagonist of the SP receptor neurokinin-1 expressed by keratinocytes. However, this antagonist did not compromise wound closure compared with the control. Similar results were obtained when the model with or without neurons was transplanted on CD1 mice, while wound closure occurred faster. We conclude that sensory neurons play an important, but not essential, role in wound healing, even in absence of the immune system. This model is promising to study the influence of the nervous system on reepithelialization in normal and pathological conditions.
dHepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of corecontaining lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.
To identify new regulators of innate antiviral immunity, we completed the first genome-wide gene silencing screen assessing the transcriptional response at the interferon-β gene (IFNB1) promoter following Sendai virus (SeV) infection. We identified 237 potential modulator genes for which negative or positive actions of gene products were mapped to the different steps of the antiviral responses from virus sensing, signal propagation/amplification up to the feedback regulation. In the present study, we will report on specific proteins that promote IFNB1 expression and innate antiviral response. The functional genomics screen uncovers a novel link between WNT family members and innate antiviral immunity. We show that virus-induced secretion of WNT2B and WNT9B down regulates IFNB1 and ISG56 expression in a β-catenin (CTNNB1)-dependent mechanism. The antiviral response is drastically reduced by GSK3 inhibitors but completely restored in CTNNB1 knockdown cells. The findings confirm a novel regulation of the innate antiviral response by a canonical WNT/GSK3/CTNNB1 pathway in a negative feedback mechanism. The study identifies novel avenues for therapeutically regulating innate immunity for effective treatment of viral infection and prevention of excessive response in autoimmune diseases.
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