RESUMO
The vascular endothelium from individual organs is functionally specialized, and it displays a unique set of accessible molecular targets. These serve as endothelial cell receptors to affinity ligands. To date, all identified vascular receptors have been proteins. Here, we show that an endothelial lung-homing peptide (CGSPGWVRC) interacts with C16-ceramide, a bioactive sphingolipid that mediates several biological functions. Upon binding to cell surfaces, CGSPGWVRC triggers ceramide-rich platform formation, activates acid sphingomyelinase and ceramide production, without the associated downstream apoptotic signaling. We also show that the lung selectivity of CGSPGWVRC homing peptide is dependent on ceramide production in vivo. Finally, we demonstrate two potential applications for this lipid vascular targeting system: i) as a bioinorganic hydrogel for pulmonary imaging and ii) as a ligand-directed lung immunization tool against COVID-19. Thus, C16-ceramide is a unique example of a lipid-based receptor system in the lung vascular endothelium targeted in vivo by circulating ligands such as CGSPGWVRC.
Assuntos
COVID-19 , Humanos , Ligantes , COVID-19/metabolismo , Ceramidas/metabolismo , Pulmão/metabolismo , Endotélio Vascular/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas de Transporte/metabolismo , Esfingomielina Fosfodiesterase/metabolismoRESUMO
BACKGROUND: Our recent studies suggest that sphingomyelin levels in the plasma membrane influence TF (tissue factor) procoagulant activity. The current study was performed to investigate how alterations to sphingomyelin metabolic pathway would affect TF procoagulant activity and thereby affect hemostatic and thrombotic processes. METHODS: Macrophages and endothelial cells were transfected with specific siRNAs or infected with adenoviral vectors to alter sphingomyelin levels in the membrane. TF activity was measured in factor X activation assay. Saphenous vein incision-induced bleeding and the inferior vena cava ligation-induced flow restriction mouse models were used to evaluate hemostasis and thrombosis, respectively. RESULTS: Overexpression of SMS (sphingomyelin synthase) 1 or SMS2 in human monocyte-derived macrophages suppresses ATP-stimulated TF procoagulant activity, whereas silencing SMS1 or SMS2 increases the basal cell surface TF activity to the same level as of ATP-decrypted TF activity. Consistent with the concept that sphingomyelin metabolism influences TF procoagulant activity, silencing of acid sphingomyelinase or neutral sphingomyelinase 2 or 3 attenuates ATP-induced enhanced TF procoagulant activity in macrophages and endothelial cells. Niemann-Pick disease fibroblasts with a higher concentration of sphingomyelin exhibited lower TF activity compared with wild-type fibroblasts. In vivo studies revealed that LPS+ATP-induced TF activity and thrombin generation were attenuated in ASMase-/- mice, while their levels were increased in SMS2-/- mice. Further studies revealed that acid sphingomyelinase deficiency leads to impaired hemostasis, whereas SMS2 deficiency increases thrombotic risk. CONCLUSIONS: Overall, our data indicate that alterations in sphingomyelin metabolism would influence TF procoagulant activity and affect hemostatic and thrombotic processes.
Assuntos
Hemostáticos , Trombose , Camundongos , Humanos , Animais , Esfingomielinas , Esfingomielina Fosfodiesterase/genética , Células Endoteliais/metabolismo , Trombose/genética , Hemostasia , Trifosfato de AdenosinaRESUMO
BACKGROUND/AIMS: It is unknown whether cancer stem cells respond differentially to treatment compared with progeny, potentially providing therapeutic vulnerabilities. Our program pioneered use of ultra-high single dose radiotherapy, which cures diverse metastatic diseases at a higher rate (90-95%) than conventional fractionation (~65%). Single dose radiotherapy engages a distinct biology involving microvascular acid sphingomyelinase/ceramide signaling, which, via NADPH oxidase-2-dependent perfusion defects, initiates an adaptive tumor SUMO Stress Response that globally-inactivates homologous recombination repair of double stand breaks, conferring cure. Accumulating data show diverse stem cells display heightened-dependence on homologous recombination repair to repair resolve double stand breaks. METHODS: Here we use colorectal cancer patient-derived xenografts containing logarithmically-increased Lgr5+ stem cells to explore whether optimizing engagement of this acid sphingomyelinase dependent biology enhances stem cell dependent tumor cure. RESULTS: We show radioresistant colorectal cancer patient-derived xenograft CLR27-2 contains radioresistant microvasculature and stem cells, whereas radiosensitive colorectal cancer patient-derived xenograft CLR1-1 contains radiosensitive microvasculature and stem cells. Pharmacologic or gene therapy enhancement of single dose radiotherapy-induced acid sphingomyelinase/ceramide-mediated microvascular dysfunction dramatically sensitizes CLR27-2 homologous recombination repair inactivation, converting Lgr5+ cells from the most resistant to most sensitive patient-derived xenograft population, yielding tumor cure. CONCLUSION: We posit homologous recombination repair represents a vulnerability determining colorectal cancer stem cell fate, approachable therapeutically using single dose radiotherapy.
Assuntos
Neoplasias Colorretais , Lesões do Sistema Vascular , Animais , Ceramidas , Neoplasias Colorretais/genética , Modelos Animais de Doenças , Humanos , Células-Tronco Neoplásicas , Esfingomielina Fosfodiesterase/genéticaRESUMO
Antidepressants have been reported to enhance stroke recovery independent of the presence of depressive symptoms. They have recently been proposed to exert their mood-stabilizing actions by inhibition of acid sphingomyelinase (ASM), which catalyzes the hydrolysis of sphingomyelin to ceramide. Their restorative action post-ischemia/reperfusion (I/R) still had to be defined. Mice subjected to middle cerebral artery occlusion or cerebral microvascular endothelial cells exposed to oxygen-glucose deprivation were treated with vehicle or with the chemically and pharmacologically distinct antidepressants amitriptyline, fluoxetine or desipramine. Brain ASM activity significantly increased post-I/R, in line with elevated ceramide levels in microvessels. ASM inhibition by amitriptyline reduced ceramide levels, and increased microvascular length and branching point density in wildtype, but not sphingomyelinase phosphodiesterase-1 ([Smpd1]-/-) (i.e., ASM-deficient) mice, as assessed by 3D light sheet microscopy. In cell culture, amitriptyline, fluoxetine, and desipramine increased endothelial tube formation, migration, VEGFR2 abundance and VEGF release. This effect was abolished by Smpd1 knockdown. Mechanistically, the promotion of angiogenesis by ASM inhibitors was mediated by small extracellular vesicles (sEVs) released from endothelial cells, which exhibited enhanced uptake in target cells. Proteomic analysis of sEVs revealed that ASM deactivation differentially regulated proteins implicated in protein export, focal adhesion, and extracellular matrix interaction. In vivo, the increased angiogenesis was accompanied by a profound brain remodeling response with increased blood-brain barrier integrity, reduced leukocyte infiltrates and increased neuronal survival. Antidepressive drugs potently boost angiogenesis in an ASM-dependent way. The release of sEVs by ASM inhibitors disclosed an elegant target, via which brain remodeling post-I/R can be amplified.
Assuntos
Amitriptilina , Vesículas Extracelulares , Amitriptilina/metabolismo , Amitriptilina/farmacologia , Animais , Antidepressivos/metabolismo , Antidepressivos/farmacologia , Encéfalo/metabolismo , Ceramidas/metabolismo , Ceramidas/farmacologia , Desipramina/metabolismo , Desipramina/farmacologia , Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Fluoxetina/metabolismo , Fluoxetina/farmacologia , Isquemia/metabolismo , Camundongos , ProteômicaRESUMO
Little is known about the maintenance of intestinal stem cells (ISCs) and progenitors during immune-mediated tissue damage or about the susceptibility of transplant recipients to tissue damage mediated by the donor immune system during graft versus host disease (GVHD). We demonstrate here that deficiency of recipient-derived IL-22 increased acute GVHD tissue damage and mortality, that ISCs were eliminated during GVHD, and that ISCs as well as their downstream progenitors expressed the IL-22 receptor. Intestinal IL-22 was produced after bone marrow transplant by IL-23-responsive innate lymphoid cells (ILCs) from the transplant recipients, and intestinal IL-22 increased in response to pretransplant conditioning. However, ILC frequency and IL-22 amounts were decreased by GVHD. Recipient IL-22 deficiency led to increased crypt apoptosis, depletion of ISCs, and loss of epithelial integrity. Our findings reveal IL-22 as a critical regulator of tissue sensitivity to GVHD and a protective factor for ISCs during inflammatory intestinal damage.
Assuntos
Transplante de Medula Óssea/imunologia , Doença Enxerto-Hospedeiro/imunologia , Interleucinas/metabolismo , Intestino Delgado/imunologia , Células-Tronco/metabolismo , Animais , Transplante de Medula Óssea/efeitos adversos , Modelos Animais de Doenças , Citometria de Fluxo , Doença Enxerto-Hospedeiro/mortalidade , Imuno-Histoquímica , Interleucina-23/metabolismo , Interleucinas/genética , Interleucinas/imunologia , Intestino Delgado/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina/metabolismo , Interleucina 22RESUMO
Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch and epidermal growth factor (EGF) signals supporting Lgr5(+) crypt base columnar ISCs for normal epithelial maintenance. However, little is known about the regulation of the ISC compartment after tissue damage. Using ex vivo organoid cultures, here we show that innate lymphoid cells (ILCs), potent producers of interleukin-22 (IL-22) after intestinal injury, increase the growth of mouse small intestine organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both mouse and human intestinal organoids, increasing proliferation and promoting ISC expansion. IL-22 induced STAT3 phosphorylation in Lgr5(+) ISCs, and STAT3 was crucial for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after mouse allogeneic bone marrow transplantation enhanced the recovery of ISCs, increased epithelial regeneration and reduced intestinal pathology and mortality from graft-versus-host disease. ATOH1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independently of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support the intestinal epithelium, activating ISCs to promote regeneration.
Assuntos
Células Epiteliais/citologia , Interleucinas/imunologia , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Regeneração , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Doença Enxerto-Hospedeiro/patologia , Humanos , Imunidade nas Mucosas , Interleucinas/deficiência , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Intestino Delgado/imunologia , Intestino Delgado/patologia , Camundongos , Organoides/citologia , Organoides/crescimento & desenvolvimento , Organoides/imunologia , Celulas de Paneth/citologia , Fosforilação , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Nicho de Células-Tronco , Interleucina 22RESUMO
In liposomal delivery, a big question is how to release the loaded material into the correct place. Here, we will test the targeting and release abilities of our sphingomyelin-consisting liposome. A change in release parameters can be observed when sphingomyelin-containing liposome is treated with sphingomyelinase enzyme. Sphingomyelinase is known to be endogenously released from the different cells in stress situations. We assume the effective enzyme treatment will weaken the liposome making it also leakier. To test the release abilities of the SM-liposome, we developed several fluorescence-based experiments. In in vitro studies, we used molecular quenching to study the sphingomyelinase enzyme-based release from the liposomes. We could show that the enzyme treatment releases loaded fluorescent markers from sphingomyelin-containing liposomes. Moreover, the release correlated with used enzymatic activities. We studied whether the stress-related enzyme expression is increased if the cells are treated with radiation as a stress inducer. It appeared that the radiation caused increased enzymatic activity. We studied our liposomes' biodistribution in the animal tumor model when the tumor was under radiation stress. Increased targeting of the fluorescent marker loaded to our liposomes could be found on the site of cancer. The liposomal targeting in vivo could be improved by radiation. Based on our studies, we propose sphingomyelin-containing liposomes can be used as a controlled release system sensitive to cell stress.
Assuntos
Corantes Fluorescentes , Lipossomos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , Estresse Fisiológico/efeitos da radiação , Animais , Catálise , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ativação Enzimática , Corantes Fluorescentes/química , Lipossomos/química , Camundongos , Imagem Molecular , Neoplasias/radioterapia , Imagem Óptica , Esfingomielinas/química , Coloração e RotulagemRESUMO
BACKGROUND/AIMS: Despite enormous effort, anti-angiogenic drugs have not lived up to the promise of globally-enhancing anti-cancer therapies. Clinically, anti-angiogenic drugs have been used to persistently suppress vascular endothelial growth factor (VEGF) in order to "normalize" dysfunctional neo-angiogenic microvasculature and prevent recruitment of endothelial progenitors. Recently, we showed that a 1h pre-treatment with anti-angiogenic drugs prior to ultra-high single dose radiotherapy and specific chemotherapies transiently de-represses acid sphingomyelinase (ASMase), leading to enhanced cancer therapy-induced, ceramide-mediated vascular injury and tumor response. Here we formally decipher parameters of chemotherapy induction of endothelial sphingolipid signaling events and define principles for optimizing anti-angiogenic chemosensitization. METHODS: These studies examine the antimetabolite chemotherapeutic gemcitabine in soft tissue sarcoma (STS), a clinically-relevant combination. RESULTS: Initial studies address the theoretic problem that anti-angiogenic drugs such as bevacizumab, an IgG with a 3-week half-life, have the potential for accumulating during the 3-week chemotherapeutic cycles currently standard-of-care for STS treatment. We show that anti-angiogenic ASMase-dependent enhancement of the response of MCA/129 fibrosarcomas in sv129/BL6 mice to gemcitabine progressively diminishes as the level of the VEGFR2 inhibitor DC101, an IgG, accumulates, suggesting a short-acting anti-angiogenic drug might be preferable in multi-cycle chemotherapeutic regimens. Further, we show lenvatinib, a VEGFR2 tyrosine kinase inhibitor with a short half-life, to be superior to DC101, enhancing gemcitabine-induced endothelial cell apoptosis and tumor response in a multi-cycle treatment schedule. CONCLUSION: We posit that a single delivery of a short-acting anti-angiogenic agent at 1h preceding each dose of gemcitabine and other chemotherapies may be more efficacious for repeated sensitization of the ASMase pathway in multi-cycle chemotherapy regimens than current treatment strategies.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Antineoplásicos/administração & dosagem , Desoxicitidina/análogos & derivados , Compostos de Fenilureia/administração & dosagem , Quinolinas/administração & dosagem , Sarcoma/tratamento farmacológico , Neoplasias de Tecidos Moles/tratamento farmacológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Desoxicitidina/administração & dosagem , Células Endoteliais/efeitos dos fármacos , Masculino , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Sarcoma/metabolismo , Neoplasias de Tecidos Moles/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , GencitabinaRESUMO
By cleaving sphingomyelin into ceramide, which is an essential component of plasma membrane microdomains, acid sphingomyelinase (Asm) pivotally controls cell signaling. To define how the activation of the Asm/ceramide pathway, which occurs within seconds to minutes upon stress stimuli, influences brain ischemia/reperfusion (I/R) injury, we exposed male and female wildtype mice carrying both alleles of Asm's gene sphingomyelinase phosphodiesterase-1 (Smpd1+/+), heterozygously Asm-deficient mice (Smpd1+/-) and homozygously Asm-deficient mice (Smpd1-/-) of different age (8, 12 or 16 weeks) to 30, 60 or 90 min intraluminal middle cerebral artery occlusion (MCAO). For studying the contribution of brain-invading polymorphonuclear neutrophils (PMN) to I/R injury, PMNs were depleted by delivery of a PMN-specific Ly6G antibody. In male and female mice exposed to 30 min, but not 60 or 90 min MCAO, homozygous Smpd1-/- consistently increased I/R injury, blood-brain barrier permeability and brain leukocyte and PMN infiltration, whereas heterozygous Smpd1+/- reduced I/R injury. Increased abundance of the intercellular leukocyte adhesion molecule ICAM-1 was noted on cerebral microvessels of Smpd1-/- mice. PMN depletion by anti-Ly6G delivery prevented the exacerbation of I/R injury in Smpd1-/- compared with wildtype mice and reduced brain leukocyte infiltrates. Our results show that Asm tempers leukocyte entry into the reperfused ischemic brain, thereby attenuating I/R injury.
Assuntos
Encéfalo/irrigação sanguínea , Infarto da Artéria Cerebral Média/enzimologia , Microvasos/enzimologia , Infiltração de Neutrófilos , Neutrófilos/enzimologia , Traumatismo por Reperfusão/enzimologia , Esfingomielina Fosfodiesterase/deficiência , Animais , Modelos Animais de Doenças , Feminino , Heterozigoto , Homozigoto , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/patologia , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/patologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Esfingomielina Fosfodiesterase/genética , Fatores de TempoRESUMO
Ceramide accumulation is known to accompany acute myocardial ischemia, but its role in the pathogenesis of ischemic heart disease is unclear. In this study, we aimed to determine how ceramides accumulate in the ischemic heart and to determine if cardiac function following ischemia can be improved by reducing ceramide accumulation. To investigate the association between ceramide accumulation and heart function, we analyzed myocardial left ventricle biopsies from subjects with chronic ischemia and found that ceramide levels were higher in biopsies from subjects with reduced heart function. Ceramides are produced by either de novo synthesis or hydrolysis of sphingomyelin catalyzed by acid and/or neutral sphingomyelinase. We used cultured HL-1 cardiomyocytes to investigate these pathways and showed that acid sphingomyelinase activity rather than neutral sphingomyelinase activity or de novo sphingolipid synthesis was important for hypoxia-induced ceramide accumulation. We also used mice with a partial deficiency in acid sphingomyelinase (Smpd1(+/-) mice) to investigate if limiting ceramide accumulation under ischemic conditions would have a beneficial effect on heart function and survival. Although we showed that cardiac ceramide accumulation was reduced in Smpd1(+/-) mice 24h after an induced myocardial infarction, this reduction was not accompanied by an improvement in heart function or survival. Our findings show that accumulation of cardiac ceramides in the post-ischemic heart is mediated by acid sphingomyelinase. However, targeting ceramide accumulation in the ischemic heart may not be a beneficial treatment strategy.
Assuntos
Ceramidas/metabolismo , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Esfingomielina Fosfodiesterase/genética , Animais , Genótipo , Hipóxia/metabolismo , Camundongos , Camundongos Knockout , Mortalidade , Mutação , Isquemia Miocárdica/mortalidade , Isquemia Miocárdica/fisiopatologia , Esfingomielina Fosfodiesterase/deficiência , Disfunção VentricularRESUMO
The survival of macrophages depends on the presence of specific cytokines that activate survival signaling events, as well as suppressing formation of apoptosis-inducing pathways. We have previously shown that macrophages deprived of macrophage colony stimulating factor (M-CSF) produce ceramide that contributes to apoptosis of these cells, a pathway that is suppressed by exposure to oxidized LDL. In this study we have examined macrophages derived from mice lacking acid sphingomyelinase (ASMase) to ask whether these events are altered due to the impaired ability of these cells to break down sphingomyelin and produce ceramide. We found that these cells do survive better than cells from wild type mice, but they still undergo cell death and some ceramide is formed. We show that the ceramide is being produced by a de novo synthetic pathway. Therefore, ceramide production in M-CSF-deprived macrophages arises from a combination of ASMase activity and de novo synthesis.
Assuntos
Ceramidas/biossíntese , Macrófagos/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Lipoproteínas LDL/farmacologia , Fator Estimulador de Colônias de Macrófagos/deficiência , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Transdução de Sinais , Esfingomielina Fosfodiesterase/deficiênciaRESUMO
Sphingolipid signaling in Caenorhabditis elegans is vital for sensing environmental change and effecting appropriate cellular response. Many molecular components in sphingolipid intermediary metabolism are conserved throughout evolution. Here we review use of C. elegans as a model system for conducting sphingolipid-based scientific investigation, which has helped us better understand vital roles these remarkable lipids play in human metabolism and disease.
Assuntos
Caenorhabditis elegans/metabolismo , Esfingolipídeos/metabolismo , Animais , Modelos Biológicos , Transdução de SinaisRESUMO
An unexpected benefit of functional genomic screens is that at times they answer questions that they were not designed to ask. A siRNA screen reported by Swanton et al. in this issue of Cancer Cell reveals that silencing of spindle assembly checkpoint genes facilitates mitotic slippage, resulting in escape from taxane-induced cell death, aneuploidy, and chromosomal instability, hallmarks of taxane resistance. Unexpectedly, the screen disclosed that the sphingolipid ceramide is a key regulator of the taxane-mediated spindle assembly checkpoint and taxane-induced cell death. Ceramide metabolism thus serves as a legitimate target for modulation of taxane effect on tumors.
Assuntos
Morte Celular , Ceramidas/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Paclitaxel/farmacologia , Proteínas Serina-Treonina Quinases/genética , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Instabilidade Cromossômica , Humanos , Mitose , Poliploidia , Dobramento de Proteína , RNA Interferente Pequeno/farmacologia , Fuso Acromático/fisiologia , Taxoides/farmacologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Factor VIIa induces the release of extracellular vesicles (EVs) from endothelial cells (EEVs). Factor VIIa-released EEVs are enriched with microRNA-10a (miR10a) and elicit miR10a-dependent cytoprotective responses. OBJECTIVES: To investigate mechanisms by which FVIIa induces miR10a expression in endothelial cells and sorts miR10a into the EVs. METHODS: Activation of Elk-1 and TWIST1 expression was analyzed by immunofluorescence microscopy and immunoblot analysis. Small interfering RNA silencing approach was used to knock down the expression of specific genes in endothelial cells. EVs secreted from endothelial cells or released into circulation in mice were isolated by centrifugation and quantified by nanoparticle tracking analysis. Factor VIIa or EVs were injected into mice; mice were challenged with lipopolysaccharides to assess the cytoprotective effects of FVIIa or EVs. RESULTS: FVIIa activation of ERK1/2 triggered the activation of Elk-1, which led to the induction of TWIST1, a key transcription factor involved in miR10a expression. Factor VIIa also induced the expression of La, a small RNA-binding protein. Factor VIIa-driven acid sphingomyelinase (ASM) activation and the subsequent activation of the S1P receptor pathway were responsible for the induction of La. Silencing of ASM or La significantly reduced miR10a levels in FVIIa-released EEVs without affecting the cellular expression of miR10a. Factor VIIa-EEVs from ASM knocked-down cells failed to provide cytoprotective responses in cell and murine model systems. Administration of FVIIa protected wild-type but not ASM-/- mice against lipopolysaccharide-induced inflammation and vascular leakage. CONCLUSION: Our data suggest that enhanced cellular expression of miR10a coupled with La-dependent sorting of miR10a is responsible for enriching FVIIa-released EVs with miR10a.
Assuntos
Vesículas Extracelulares , MicroRNAs , Camundongos , Animais , Fator VIIa/metabolismo , Células Endoteliais/metabolismo , Transdução de Sinais , Lipopolissacarídeos/metabolismo , Vesículas Extracelulares/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
Diabetic retinopathy is a microvascular disease that causes blindness. Using acid sphingomyelinase knockout mice, we reported that ceramide generation is critical for diabetic retinopathy development. Here, in patients with proliferative diabetic retinopathy, we identify vitreous ceramide imbalance with pathologic long-chain C16-ceramides increasing and protective very long-chain C26-ceramides decreasing. C16-ceramides generate pro-inflammatory/pro-apoptotic ceramide-rich platforms on endothelial surfaces. To geo-localize ceramide-rich platforms, we invented a three-dimensional confocal assay and showed that retinopathy-producing cytokines TNFα and IL-1ß induce ceramide-rich platform formation on retinal endothelial cells within seconds, with volumes increasing 2-logs, yielding apoptotic death. Anti-ceramide antibodies abolish these events. Furthermore, intravitreal and systemic anti-ceramide antibodies protect from diabetic retinopathy in standardized rodent ischemia reperfusion and streptozotocin models. These data support (1) retinal endothelial ceramide as a diabetic retinopathy treatment target, (2) early-stage therapy of non-proliferative diabetic retinopathy to prevent progression, and (3) systemic diabetic retinopathy treatment; and they characterize diabetic retinopathy as a "ceramidopathy" reversible by anti-ceramide immunotherapy.
Assuntos
Ceramidas , Retinopatia Diabética , Imunoterapia , Ceramidas/metabolismo , Retinopatia Diabética/metabolismo , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/patologia , Retinopatia Diabética/imunologia , Animais , Humanos , Camundongos , Células Endoteliais/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Masculino , Retina/metabolismo , Retina/patologia , Interleucina-1beta/metabolismo , Camundongos Endogâmicos C57BL , Ratos , Apoptose/efeitos dos fármacos , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/metabolismo , Corpo Vítreo/metabolismo , Feminino , Camundongos KnockoutRESUMO
BACKGROUND & AIMS: Adult stem cells have been proposed to be quiescent and radiation resistant, repairing DNA double-strand breaks by nonhomologous end joining. However, the population of putative small intestinal stem cells (ISCs) at position +4 from the crypt base contradicts this model, in that they are highly radiosensitive. Cycling crypt base columnar cells (CBCs) at crypt positions +1-3 recently were defined as an alternative population of ISCs. Little is known about the sensitivity of this stem cell population to radiation. METHODS: Radiation-induced lethality of CBCs was quantified kinetically in Lgr5-lacZ transgenic mice. γ-H2AX, BRCA1, RAD51, and DNA-PKcs foci were used as DNA repair surrogates to investigate the inherent ability of CBCs to recognize and repair double-strand breaks. 5-ethynyl-2'-deoxyuridine and 5-bromo-2'-deoxyuridine incorporation assays were used to study patterns of CBC growth arrest and re-initiation of cell cycling. Apoptosis was evaluated by caspase-3 staining. RESULTS: CBCs are relatively radioresistant, repairing DNA by homologous recombination significantly more efficiently than transit amplifying progenitors or villus cells. CBCs undergo apoptosis less than 24 hours after irradiation (32% ± 2% of total lethality) or mitotic death at 24-48 hours. Survival of CBCs at 2 days predicts crypt regeneration at 3.5 days and lethality from gastrointestinal syndrome. Crypt repopulation originates from CBCs that survive irradiation. CONCLUSIONS: Adult ISCs in mice can cycle rapidly yet still be radioresistant. Importantly, homologous recombination can protect adult stem cell populations from genotoxic stress. These findings broaden and refine concepts of the phenotype of adult stem cells.
Assuntos
Células-Tronco Adultas/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA , Jejuno/efeitos da radiação , Tolerância a Radiação , Animais , Apoptose/efeitos da radiação , Medula Óssea/efeitos da radiação , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Jejuno/patologia , Camundongos , Camundongos TransgênicosRESUMO
Recent research has shed new light on the critical role of tissue microvasculature in regulating the tumor response to radiation and drugs. In this issue of Cancer Cell, Moeller et al.(2005) demonstrate that HIF-1 activation during the course of fractionated radiotherapy initiates pleiotropic adaptive responses in both tumor cells and the microvascular network, radiosensitizing tumor cells but concomitantly conferring tumor radioresistance due to protection of the microvascular endothelium. HIF-1 thus serves as a legitimate target for differential modulation of tissue response to radiation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/metabolismo , Neoplasias/irrigação sanguínea , Neoplasias/radioterapia , Neovascularização Patológica/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Capilares/metabolismo , Endotélio Vascular/efeitos da radiação , Humanos , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Neoplasias/metabolismo , Tolerância a RadiaçãoRESUMO
Acid sphingomyelinase (ASMase) is a key initiator of sphingomyelin/ceramide signal transduction activated by many stress stimuli. Over the past two decades, much progress has been made in defining the clinical relevance of sphingomyelin/ceramide signaling in numerous diseases using ASMase knockout mice. Organs that operate this pathway are numerous and the disease states regulated are diverse, with ceramide generation governing injury in tumor, gut, ovary, brain, lung, heart, liver, and during infection. This chapter emphasizes evolutionary conservation of sphingolipid stress signaling and mammalian adaptations that permit transduction of organotypic responses. Recognition that the sphingomyelin/ceramide transducer calibrates extent of tissue injury, ultimately acting as a molecular switch that determines organ fate, is driving development of new pharmacologic concepts and tools to intervene therapeutically.
Assuntos
Esfingomielina Fosfodiesterase/deficiência , Animais , Ceramidas/metabolismo , Modelos Animais de Doenças , Evolução Molecular , Genótipo , Humanos , Camundongos , Camundongos Knockout , Fenótipo , Transdução de Sinais , Especificidade da Espécie , Esfingomielina Fosfodiesterase/genética , Esfingomielinas/metabolismoRESUMO
The enzyme acid sphingomyelinase catalyzes the hydrolysis of sphingomyelin to ceramide. The importance of the enzyme for cell functions was first recognized in Niemann-Pick disease type A and B, the genetic disorders with a massive accumulation of sphingomyelin in many organs. Studies in the last years demonstrated that the enzyme also has an important role in cell signalling. Thus, the acid sphingomyelinase has a central function for the re-organization of molecules within the cell upon stimulation and thereby for the response of cells to stress and the induction of cell death but also proliferation and differentiation. Here, we discuss the current state of the art of the structure, regulation, and function of the acid sphingomyelinase.
Assuntos
Esfingomielina Fosfodiesterase/fisiologia , Animais , Ativação Enzimática , Humanos , Transdução de Sinais/fisiologia , Esfingomielina Fosfodiesterase/químicaRESUMO
c-Abl, a conserved nonreceptor tyrosine kinase, integrates genotoxic stress responses, acting as a transducer of both pro- and antiapoptotic effector pathways. Nuclear c-Abl seems to interact with the p53 homolog p73 to elicit apoptosis. Although several observations suggest that cytoplasmic localization of c-Abl is required for antiapoptotic function, the signals that mediate its antiapoptotic effect are largely unknown. Here we show that worms carrying an abl-1 deletion allele, abl-1(ok171), are specifically hypersensitive to radiation-induced apoptosis in the Caenorhabditis elegans germ line. Our findings delineate an apoptotic pathway antagonized by ABL-1, which requires sequentially the cell cycle checkpoint genes clk-2, hus-1 and mrt-2; the C. elegans p53 homolog, cep-1; and the genes encoding the components of the conserved apoptotic machinery, ced-3, ced-9 and egl-1. ABL-1 does not antagonize germline apoptosis induced by the DNA-alkylating agent ethylnitrosourea. Furthermore, worms treated with the c-Abl inhibitor STI-571 (Gleevec; used in human cancer therapy), two newly synthesized STI-571 variants or PD166326 had a phenotype similar to that generated by abl-1(ok171). These studies indicate that ABL-1 distinguishes proapoptotic signals triggered by two different DNA-damaging agents and suggest that C. elegans might provide tissue models for development of anticancer drugs.