RESUMO
Oxidative stress plays a pivotal role in the pathogenesis of several neurodegenerative diseases. Retinal degeneration causes irreversible death of photoreceptor cells, ultimately leading to vision loss. Under oxidative stress, the synthesis of bioactive sphingolipid ceramide increases, triggering apoptosis in photoreceptor cells and leading to their death. This study investigates the effect of L-Cycloserine, a small molecule inhibitor of ceramide biosynthesis, on sphingolipid metabolism and the protection of photoreceptor-derived 661W cells from oxidative stress. The results demonstrate that treatment with L-Cycloserine, an inhibitor of Serine palmitoyl transferase (SPT), markedly decreases bioactive ceramide and associated sphingolipids in 661W cells. A nontoxic dose of L-Cycloserine can provide substantial protection of 661W cells against H2O2-induced oxidative stress by reversing the increase in ceramide level observed under oxidative stress conditions. Analysis of various antioxidant, apoptotic and sphingolipid pathway genes and proteins also confirms the ability of L-Cycloserine to modulate these pathways. Our findings elucidate the generation of sphingolipid mediators of cell death in retinal cells under oxidative stress and the potential of L-Cycloserine as a therapeutic candidate for targeting ceramide-induced degenerative diseases by inhibiting SPT. The promising therapeutic prospect identified in our findings lays the groundwork for further validation in in-vivo and preclinical models of retinal degeneration.
Assuntos
Apoptose , Ceramidas , Ciclosserina , Estresse Oxidativo , Esfingolipídeos , Estresse Oxidativo/efeitos dos fármacos , Ciclosserina/farmacologia , Animais , Ceramidas/metabolismo , Ceramidas/farmacologia , Camundongos , Esfingolipídeos/metabolismo , Apoptose/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Serina C-Palmitoiltransferase/metabolismo , Serina C-Palmitoiltransferase/antagonistas & inibidores , Peróxido de Hidrogênio/toxicidade , Peróxido de Hidrogênio/farmacologia , Linhagem Celular , Degeneração Retiniana/metabolismo , Degeneração Retiniana/prevenção & controle , Degeneração Retiniana/patologia , Degeneração Retiniana/tratamento farmacológico , Western Blotting , Inibidores Enzimáticos/farmacologia , Sobrevivência Celular/efeitos dos fármacosRESUMO
BACKGROUND: Traumatic brain injury (TBI) causes neuroinflammation and can lead to long-term neurological dysfunction, even in cases of mild TBI (mTBI). Despite the substantial burden of this disease, the management of TBI is precluded by an incomplete understanding of its cellular mechanisms. Sphingolipids (SPL) and their metabolites have emerged as key orchestrators of biological processes related to tissue injury, neuroinflammation, and inflammation resolution. No study so far has investigated comprehensive sphingolipid profile changes immediately following TBI in animal models or human cases. In this study, sphingolipid metabolite composition was examined during the acute phases in brain tissue and plasma of mice following mTBI. METHODS: Wildtype mice were exposed to air-blast-mediated mTBI, with blast exposure set at 50-psi on the left cranium and 0-psi designated as Sham. Sphingolipid profile was analyzed in brain tissue and plasma during the acute phases of 1, 3, and 7 days post-TBI via liquid-chromatography-mass spectrometry. Simultaneously, gene expression of sphingolipid metabolic markers within brain tissue was analyzed using quantitative reverse transcription-polymerase chain reaction. Significance (P-values) was determined by non-parametric t-test (Mann-Whitney test) and by Tukey's correction for multiple comparisons. RESULTS: In post-TBI brain tissue, there was a significant elevation of 1) acid sphingomyelinase (aSMase) at 1- and 3-days, 2) neutral sphingomyelinase (nSMase) at 7-days, 3) ceramide-1-phosphate levels at 1 day, and 4) monohexosylceramide (MHC) and sphingosine at 7-days. Among individual species, the study found an increase in C18:0 and a decrease in C24:1 ceramides (Cer) at 1 day; an increase in C20:0 MHC at 3 days; decrease in MHC C18:0 and increase in MHC C24:1, sphingomyelins (SM) C18:0, and C24:0 at 7 days. Moreover, many sphingolipid metabolic genes were elevated at 1 day, followed by a reduction at 3 days and an absence at 7-days post-TBI. In post-TBI plasma, there was 1) a significant reduction in Cer and MHC C22:0, and an increase in MHC C16:0 at 1 day; 2) a very significant increase in long-chain Cer C24:1 accompanied by significant decreases in Cer C24:0 and C22:0 in MHC and SM at 3 days; and 3) a significant increase of C22:0 in all classes of SPL (Cer, MHC and SM) as well as a decrease in Cer C24:1, MHC C24:1 and MHC C24:0 at 7 days. CONCLUSIONS: Alterations in sphingolipid metabolite composition, particularly sphingomyelinases and short-chain ceramides, may contribute to the induction and regulation of neuroinflammatory events in the early stages of TBI, suggesting potential targets for novel diagnostic, prognostic, and therapeutic strategies in the future.
Assuntos
Encéfalo , Ceramidas , Esfingolipídeos , Esfingomielina Fosfodiesterase , Esfingosina , Animais , Camundongos , Esfingolipídeos/sangue , Esfingolipídeos/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Ceramidas/sangue , Ceramidas/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielina Fosfodiesterase/sangue , Esfingomielina Fosfodiesterase/genética , Esfingosina/análogos & derivados , Esfingosina/sangue , Esfingosina/metabolismo , Modelos Animais de Doenças , Masculino , Esfingomielinas/sangue , Esfingomielinas/metabolismo , Concussão Encefálica/sangue , Concussão Encefálica/metabolismo , Camundongos Endogâmicos C57BL , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/sangue , Lesões Encefálicas Traumáticas/patologia , Lisofosfolipídeos/sangue , Lisofosfolipídeos/metabolismoRESUMO
BACKGROUND: There is a need to develop biomarkers for diagnosis and prediction of treatment responses in depression and post-traumatic stress disorder (PTSD). METHODS: Cross-sectional study examining correlations between tear inflammatory proteins, meibum and tear sphingolipids, and symptoms of depression and PTSD-associated anxiety. Ninety individuals filled depression (Patient Health Questionnaire 9, PHQ-9) and PTSD-associated anxiety (PTSD Checklist-Military Version, PCL-M) questionnaires. In 40 patients, a multiplex assay system was used to quantify 23 inflammatory proteins in tears. In a separate group of 50 individuals, liquid chromatography-mass spectrometry was performed on meibum and tears to quantify 34 species of sphingolipids, encompassing ceramides, monohexosyl ceramides and sphingomyelins. RESULTS: The mean age of the population was 59.4 ± 11.0 years; 89.0% self-identified as male, 34.4% as White, 64.4% as Black, and 16.7% as Hispanic. The mean PHQ-9 score was 11.1 ± 7.6, and the mean PCL-M score was 44.3 ± 19.1. Symptoms of depression and PTSD-associated anxiety were highly correlated (ρ =0.75, p < 0.001). Both PHQ9 and PCL-M scores negatively correlated with multiple sphingolipid species in meibum and tears. In multivariable models, meibum Monohexosyl Ceramide 26:0 (pmol), tear Ceramide 16:0 (mol%), meibum Monohexosyl Ceramide 16:0 (mol%), and tear Ceramide 26:1 (mol%) remained associated with depression and meibum Monohexosyl Ceramide 16:0 (mol%), meibum Monohexosyl Ceramide 26:0 (pmol), tear Sphingomyelin 20:0 (mol%), and tear Sphingosine-1-Phosphate (mol%) remained associated with PTSD-associated anxiety. CONCLUSIONS: Certain meibum and tear sphingolipid species were related to mental health indices. These interactions present opportunities for innovative diagnostic and therapeutic approaches for mental health disorders.
Assuntos
Biomarcadores , Glândulas Tarsais , Transtornos de Estresse Pós-Traumáticos , Lágrimas , Humanos , Masculino , Estudos Transversais , Feminino , Pessoa de Meia-Idade , Transtornos de Estresse Pós-Traumáticos/metabolismo , Transtornos de Estresse Pós-Traumáticos/diagnóstico , Lágrimas/química , Lágrimas/metabolismo , Biomarcadores/metabolismo , Glândulas Tarsais/metabolismo , Inquéritos e Questionários , Idoso , Cromatografia Líquida , Adulto , Esfingolipídeos/metabolismo , Lipídeos/análise , Depressão/metabolismo , Depressão/diagnósticoRESUMO
Human corneal fibrosis can lead to opacity and ultimately partial or complete vision loss. Currently, corneal transplantation is the only treatment for severe corneal fibrosis and comes with the risk of rejection and donor shortages. Sphingolipids (SPLs) are known to modulate fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to both, transforming growth factor beta (TGF-ß) signaling and corneal fibrogenesis. The aim of this study was to investigate the effects of sphingosine-1-phosphate (S1P) and S1P inhibition on specific TGF-ß and SPL family members in corneal fibrosis. Healthy human corneal fibroblasts (HCFs) were isolated and cultured in EMEM + FBS + VitC (construct medium) on 3D transwells for 4 weeks. The following treatments were prepared in a construct medium: 0.1 ng/mL TGF-ß1 (ß1), 1 µM sphingosine-1-phosphate (S1P), and 5 µM Sphingosine kinase inhibitor 2 (I2). Five groups were tested: (1) control (no treatment); rescue groups; (2) ß1/S1P; (3) ß1/I2; prevention groups; (4) S1P/ß1; and (5) I2/ß1. Each treatment was administered for 2 weeks with one treatment and switched to another for 2 weeks. Using Western blot analysis, the 3D constructs were examined for the expression of fibrotic markers, SPL, and TGF-ß signaling pathway members. Scratch assays from 2D cultures were also utilized to evaluate cell migration We observed reduced fibrotic expression and inactivation of latent TGF-ß binding proteins (LTBPs), TGF-ß receptors, Suppressor of Mothers Against Decapentaplegic homologs (SMADs), and SPL signaling following treatment with I2 prevention and rescue compared to S1P prevention and rescue, respectively. Furthermore, we observed increased cell migration following stimulation with I2 prevention and rescue groups, with decreased cell migration following stimulation with S1P prevention and rescue groups after 12 h and 18 h post-scratch. We have demonstrated that I2 treatment reduced fibrosis and modulated the inactivation of LTBPs, TGF-ß receptors, SPLs, and the canonical downstream SMAD pathway. Further investigations are warranted in order to fully uncover the potential of utilizing SphK I2 as a novel therapy for corneal fibrosis.
Assuntos
Córnea , Fibrose , Lisofosfolipídeos , Transdução de Sinais , Esfingosina , Fator de Crescimento Transformador beta , Humanos , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Esfingosina/farmacologia , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/farmacologia , Córnea/metabolismo , Córnea/patologia , Córnea/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Células Cultivadas , Esfingolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Doenças da Córnea/metabolismo , Doenças da Córnea/patologia , Doenças da Córnea/tratamento farmacológicoRESUMO
Vesicating chemicals like sulfur mustard (SM) or nitrogen mustard (NM) can cause devastating damage to the eyes, skin, and lungs. Eyes, being the most sensitive, have complicated pathologies that can manifest immediately after exposure (acute) and last for years (chronic). No FDA-approved drug is available to be used as medical counter measures (MCMs) against such injuries. Understanding the pathological mechanisms in acute and chronic response of the eye is essential for developing effective MCMs. Here, we report the clinical and histopathological characterization of a mouse model of NM-induced ocular surface injury (entire surface) developed by treating the eye with 2% (w/v) NM solution for 5 min. Unlike the existing models of specific injury, our model showed severe ocular inflammation, including the eyelids, structural deformity of the corneal epithelium and stroma, and diminished visual and retinal functions. We also observed alterations of the inflammatory markers and their expression at different phases of the injury, along with an activation of acidic sphingomyelinase (aSMase), causing an increase in bioactive sphingolipid ceramide and a reduction in sphingomyelin levels. This novel ocular surface mouse model recapitulated the injuries reported in human, rabbit, and murine SM or NM injury models. NM exposure of the entire ocular surface in mice, which is similar to accidental or deliberate exposure in humans, showed severe ocular inflammation and caused irreversible alterations to the corneal structure and significant vision loss. It also showed an intricate interplay between inflammatory markers over the injury period and alteration in sphingolipid homeostasis in the early acute phase.
Assuntos
Traumatismos Oculares , Gás de Mostarda , Humanos , Animais , Camundongos , Coelhos , Mecloretamina/toxicidade , Traumatismos Oculares/induzido quimicamente , Pálpebras , Modelos Animais de Doenças , Gás de Mostarda/toxicidade , Esfingolipídeos , InflamaçãoRESUMO
Corneal haze brought on by fibrosis due to insult can lead to partial or complete vision loss. Currently, corneal transplantation is the gold standard for treating severe corneal fibrosis, which comes with the risk of rejection and the issue of donor tissue shortages. Sphingolipids (SPLs) are known to be associated with fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to Transforming Growth Factor ß (TGF-ß) signaling and corneal fibrogenesis. This study aimed to elucidate the interplay of SPLs, specifically sphingosine-1-phosphate (S1P) signaling, and its' interactions with TGF-ß signaling through detailed analyses of the corresponding downstream signaling targets in the context of corneal fibrosis, in vitro. Healthy human corneal fibroblasts (HCFs) were isolated, plated on polycarbonate membranes, and stimulated with a stable Vitamin C derivative. The 3D constructs were treated with either 5 µM sphingosine-1-phosphate (S1P), 5 µM SPHK I2 (I2; inhibitor of sphingosine kinase 1, one of the two enzymes responsible for generating S1P in mammalian cells), 0.1 ng/mL TGF-ß1, or 0.1 ng/mL TGF-ß3. Cultures with control medium-only served as controls. All 3D constructs were examined for protein expression of fibrotic markers, SPLs, TGF-ßs, and relevant downstream signaling pathways. This data revealed no significant changes in any LTBP (latent TGF-ß binding proteins) expression when stimulated with S1P or I2. However, LTBP1 was significantly upregulated via stimulation of TGF-ß1 and TGF-ß3, whereas LTBP2 was significantly upregulated only with TGF-ß3 stimulation. Significant downregulation of TGF-ß receptor II (TGF-ßRII) following S1P stimulation but significant upregulation following I2 stimulation was observed. Following TGF-ß1, S1P, and I2 stimulation, phospho-SMAD2 (pSMAD2) was significantly downregulated. Furthermore, I2 stimulation led to significant downregulation of SMAD4. Adhesion/proliferation/transcription regulation targets, SRC, FAK, and pERK 1/2 were all significantly downregulated by exogenous S1P, whereas I2 only significantly downregulated FAK. Exogenous TGF-ß3 caused significant upregulation of AKT. Interestingly, both I2 and TGF-ß3 caused significant downregulation of JNK expression. Lastly, TGF-ß1 led to significant upregulation of sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 3 (S1PR3), whereas TGF-ß3 caused significant upregulation of only SphK1. Together with previously published work from our group and others, S1P inhibition exhibits great potential as an efficacious anti-fibrotic modality in human corneal stromal ECM. The current findings shed further light on a very complex and rather incompletely investigated mechanism, and cement the intricate crosstalk between SPLs and TGF-ß in corneal fibrogenesis. Future studies will dictate the potential of utilizing SPLs/TGF-ß signaling modulators as novel therapeutics in corneal fibrosis.
Assuntos
Esfingolipídeos , Fator de Crescimento Transformador beta , Animais , Humanos , Esfingolipídeos/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Substância Própria/metabolismo , Fator de Crescimento Transformador beta3 , Transdução de Sinais , Lisofosfolipídeos/farmacologia , Lisofosfolipídeos/metabolismo , Esfingosina/farmacologia , Esfingosina/metabolismo , Fibrose , Mamíferos , Proteínas de Ligação a TGF-beta LatenteRESUMO
Sphingolipids have emerged as bioactive lipids involved in the regulation of many physiological and pathological processes. In the retina, they have been established to participate in numerous processes, such as neuronal survival and death, proliferation and migration of neuronal and vascular cells, inflammation, and neovascularization. Dysregulation of sphingolipids is therefore crucial in the onset and progression of retinal diseases. This review examines the involvement of sphingolipids in retinal physiology and diseases. Ceramide (Cer) has emerged as a common mediator of inflammation and death of neuronal and retinal pigment epithelium cells in animal models of retinopathies such as glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa. Sphingosine-1-phosphate (S1P) has opposite roles, preventing photoreceptor and ganglion cell degeneration but also promoting inflammation, fibrosis, and neovascularization in AMD, glaucoma, and pro-fibrotic disorders. Alterations in Cer, S1P, and ceramide 1-phosphate may also contribute to uveitis. Notably, use of inhibitors that either prevent Cer increase or modulate S1P signaling, such as Myriocin, desipramine, and Fingolimod (FTY720), preserves neuronal viability and retinal function. These findings underscore the relevance of alterations in the sphingolipid metabolic network in the etiology of multiple retinopathies and highlight the potential of modulating their metabolism for the design of novel therapeutic approaches.
Assuntos
EsfingolipídeosRESUMO
Over 11 million people in the United States alone have some form of age-related macular degeneration (AMD). Oxidative stress, cell death, and the degeneration of retinal pigment epithelial (RPE) cells contribute to AMD pathology. Recent evidence suggests that ceramide (Cer), a cellular sphingolipid mediator that acts as a second messenger to induce apoptosis, might play a role in RPE cell death. The lysosomal breakdown of Cer by acid ceramidase [N-acylsphingosine amidohydrolase (ASAH)1] into sphingosine (Sph) is the major source for Sph 1-phosphate production, which has an opposing role to Cer and provides cytoprotection. Here, we investigated the role of Cer in human RPE-derived ARPE19 cells under hydrogen peroxide-induced oxidative stress, and show that Cer and hexosyl-Cer levels increase in the oxidatively stressed ARPE19 cells, which can be prevented by overexpression of lysosomal ASAH1. This study demonstrates that oxidative stress generates sphingolipid death mediators in retinal cells and that induction of ASAH1 could rescue retinal cells from oxidative stress by hydrolyzing excess Cers.
Assuntos
Ceramidase Ácida/genética , Estresse Oxidativo , Epitélio Pigmentado da Retina/metabolismo , Ceramidase Ácida/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular , Ceramidas/metabolismo , Expressão Gênica , Humanos , Peróxido de Hidrogênio/farmacologia , Hidrólise/efeitos dos fármacos , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacosRESUMO
Inflammation is a common underlying factor in a diversity of ocular diseases, ranging from macular degeneration, autoimmune uveitis, glaucoma, diabetic retinopathy and microbial infection. In addition to the variety of known cellular mediators of inflammation, such as cytokines, chemokines and lipid mediators, there is now considerable evidence that sphingolipid metabolites also play a central role in the regulation of inflammatory pathways. Various sphingolipid metabolites, such as ceramide (Cer), ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), and lactosylceramide (LacCer) can contribute to ocular inflammatory diseases through multiple pathways. For example, inflammation generates Cer from sphingomyelins (SM) in the plasma membrane, which induces death receptor ligand formation and leads to apoptosis of retinal pigment epithelial (RPE) and photoreceptor cells. Inflammatory stress by reactive oxygen species leads to LacCer accumulation and S1P secretion and induces proliferation of retinal endothelial cells and eventual formation of new vessels. In sphingolipid/lysosomal storage disorders, sphingolipid metabolites accumulate in lysosomes and can cause ocular disorders that have an inflammatory etiology. Sphingolipid metabolites activate complement factors in the immune-response mediated pathogenesis of macular degeneration. These examples highlight the integral association between sphingolipids and inflammation in ocular diseases.
Assuntos
Oftalmopatias , Inflamação , Esfingolipídeos , Apoptose , Células Endoteliais/citologia , Células Endoteliais/patologia , Oftalmopatias/fisiopatologia , Humanos , Inflamação/fisiopatologia , Esfingolipídeos/metabolismoRESUMO
Inflammation is a powerful immune countermeasure to tissue damage and infection. The inflammatory response is complex and requires the involvement of myriad signaling pathways and metabolic processes, all governed by a multitude of regulatory systems. Although inflammation is a vital defense against tissue injury and a necessary step in tissue healing, the mechanisms which modulate the initiation, intensity, and duration of this innate immune response can malfunction and result in inappropriate or out-of-control inflammation, even in the absence of an appropriate stimulus. Though the human eye exists in an immune-privileged microenvironment, it is not spared from this. The eye is neither devoid of immune cells nor is it fully sequestered from systemic immune responses, and is therefore fully capable of ruining itself through localized inflammatory dysfunction and systemic inflammatory disease (Taylor AW, Front Immunol 7:37, 2016; Zhou R, Caspi RR, Biol Rep 2, 2010). In fact, a wide range of ocular inflammatory diseases exist and are major causes of blindness in humans. Advances in the understanding of inflammatory processes have revealed new key pathways and molecular factors involved in the mechanisms of inflammation. Lipids and sphingolipids are increasingly being recognized as having important signaling roles in the pathophysiology of ocular inflammatory diseases. What follows below is a discussion of fundamental inflammatory processes, the place of sphingolipids as mediators of said processes, brief descriptions of major inflammatory ocular diseases, and new findings implicating sphingolipids in their pathogenesis.
Assuntos
Oftalmopatias , Inflamação , Transdução de Sinais , Esfingolipídeos/metabolismo , Olho/imunologia , HumanosRESUMO
Sphingosine 1-phosphate (S1P) signaling regulates numerous biological processes including neurogenesis, inflammation and neovascularization. However, little is known about the role of S1P signaling in the eye. In this study, we characterize two sphingosine kinases (SPHK1 and SPHK2), which phosphorylate sphingosine to S1P, and three S1P receptors (S1PR1, S1PR2 and S1PR3) in mouse and rat eyes. We evaluated sphingosine kinase and S1P receptor gene expression at the mRNA level in various rat tissues and rat retinas exposed to light-damage, whole mouse eyes, specific eye structures, and in developing retinas. Furthermore, we determined the localization of sphingosine kinases and S1P receptors in whole rat eyes by immunohistochemistry. Our results unveiled unique expression profiles for both sphingosine kinases and each receptor in ocular tissues. Furthermore, these kinases and S1P receptors are expressed in mammalian retinal cells and the expression of SPHK1, S1PR2 and S1PR3 increased immediately after light damage, which suggests a function in apoptosis and/or light stress responses in the eye. These findings have numerous implications for understanding the role of S1P signaling in the mechanisms of ocular diseases such as retinal inflammatory and degenerative diseases, neovascular eye diseases, glaucoma and corneal diseases.
Assuntos
Proteínas do Olho/biossíntese , Regulação da Expressão Gênica/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Receptores de Lisoesfingolipídeo/biossíntese , Retina/metabolismo , Animais , Bovinos , Imuno-Histoquímica , Lisofosfolipídeos/metabolismo , Camundongos , Ratos , Ratos Sprague-Dawley , Retina/citologia , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
The pathophysiology of human keratoconus (KC), a bilateral progressive corneal disease leading to protrusion of the cornea, stromal thinning, and scarring, is not well-understood. In this study, we investigated a novel sphingolipid (SPL) signaling pathway through which KC may be regulated. Using human corneal fibroblasts (HCFs) and human KC cells (HKCs), we examined the SPL pathway modulation. Both cell types were stimulated by the three transforming growth factor (TGF)-ß isoforms: TGF-ß1 (T1), TGF-ß2 (T2), and TGF-ß3 (T3). All samples were analyzed using lipidomics and real-time PCR. Our data showed that HKCs have increased levels of signaling SPLs, ceramide (Cer), and sphingosine 1-phosphate (S1P). Treatment with T1 reversed the increase in Cer in HKCs and treatment with T3 reversed the increase in S1P. S1P3 receptor mRNA levels were also significantly upregulated in HKCs, but were reduced to normal levels following T3 treatment. Furthermore, stimulation with Cer and S1P led to significant upregulation of fibrotic markers in HCFs, but not in HKCs. Additionally, stimulation with a Cer synthesis inhibitor (FTY720) led to significant downregulation of specific fibrotic markers in HKCs (TGF-ß1, collagen type III, and α smooth muscle actin) without an effect on healthy HCFs, suggesting a causative role of Cer and S1P in fibrogenesis. Overall, this study suggests an association of the SPL signaling pathway in KC disease and its relation with the TGF-ß pathway.
Assuntos
Ceramidas/genética , Ceratocone/genética , Lisofosfolipídeos/biossíntese , Esfingolipídeos/genética , Esfingosina/análogos & derivados , Linhagem Celular , Ceramidas/administração & dosagem , Córnea/metabolismo , Córnea/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Cloridrato de Fingolimode/administração & dosagem , Humanos , Ceratocone/patologia , Lisofosfolipídeos/administração & dosagem , RNA Mensageiro/genética , Transdução de Sinais , Esfingolipídeos/isolamento & purificação , Esfingolipídeos/metabolismo , Esfingosina/administração & dosagem , Esfingosina/biossíntese , Fator de Crescimento Transformador beta1/administração & dosagem , Fator de Crescimento Transformador beta2/administração & dosagem , Fator de Crescimento Transformador beta3/administração & dosagemRESUMO
Retinal degeneration (RD) affects millions of people and is a major cause of ocular impairment and blindness. With a wide range of mutations and conditions leading to degeneration, targeting downstream processes is necessary for developing effective treatments. Ceramide and sphingosine-1-phosphate, a pair of bioactive sphingolipids, are involved in apoptosis and its prevention, respectively. Apoptotic cell death is a potential driver of RD, and in order to understand the mechanism of degeneration and potential treatments, we studied rhodopsin mutant RD model, P23H-1 rats. Investigating this genetic model of human RD allows us to investigate the association of sphingolipid metabolites with the degeneration of the retina in P23H-1 rats and the effects of a specific modulator of sphingolipid metabolism, FTY720. We found that P23H-1 rat retinas had altered sphingolipid profiles that, when treated with FTY720, were rebalanced closer to normal levels. FTY720-treated rats also showed protection from RD compared with their vehicle-treated littermates. Based on these data, we conclude that sphingolipid dysregulation plays a secondary role in retinal cell death, which may be common to many forms of RDs, and that the U.S. Food and Drug Administration-approved drug FTY720 or related compounds that modulate sphingolipid metabolism could potentially delay the cell death.
Assuntos
Cloridrato de Fingolimode/farmacologia , Distrofias Retinianas/metabolismo , Esfingolipídeos/metabolismo , Animais , Vias Biossintéticas , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Cloridrato de Fingolimode/uso terapêutico , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Ratos Sprague-Dawley , Distrofias Retinianas/tratamento farmacológico , Esfingomielina Fosfodiesterase/metabolismoRESUMO
Autosomal-dominant Stargardt-like macular dystrophy [Stargardt3 (STGD3)] results from single allelic mutations in the elongation of very-long-chain fatty acids-like 4 (ELOVL4), whereas recessive mutations lead to skin and brain dysfunction. ELOVL4 protein localizes to the endoplasmic reticulum, where it mediates the condensation reaction catalyzing the formation of very-long-chain (VLC) (C-28 to C-40) fatty acids, saturated and polyunsaturated (PUFA). The defective gene product is truncated at the C terminus, leading to mislocalization and aggregation in other organelles. We hypothesized that the STGD3 truncated mutant may generate mislocalized, and therefore toxic, keto intermediates of fatty acid elongation, thereby contributing to the disease process. Using cell-based and cell-free microsome assays, we found that the truncated protein lacked innate condensation activity. Coexpression of different forms of wild-type and mutant ELOVL4 revealed a large dominant-negative effect of mutant protein on ELOVL4 localization and enzymatic activity, resulting in reduced VLC-PUFA synthesis. The reduction in VLC-PUFA levels in STGD3 and age-related macular degeneration may be a contributing factor to their retinal pathology.
Assuntos
Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Ácidos Graxos Insaturados/metabolismo , Degeneração Macular/congênito , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Adenoviridae , Análise de Variância , Animais , Western Blotting , Retículo Endoplasmático/metabolismo , Genes Dominantes/genética , Células HEK293 , Células HeLa , Humanos , Imuno-Histoquímica , Degeneração Macular/genética , Camundongos , Microssomos/metabolismo , Mutação/genéticaRESUMO
Sphingolipids are essential to cell membrane structure and the development and maintenance of neural tissues. The role of bioactive sphingolipids has been established in numerous cellular events, including cell survival, growth, and apoptosis. Ocular inflammatory and autoimmune diseases involve activation and migration of endothelial cells, neovascularization, and infiltration of immune cells into various tissues. Clinically, the impact and role of sphingolipid-mediated signaling is increasingly being appreciated in the pathogenesis and treatment of diseases ranging from multiple sclerosis to neovascularization in age-related macular degeneration and diabetic retinopathy. In this review, we discuss our current knowledge and understanding of sphingolipid metabolism and signaling associated with the pathogenesis of ocular diseases.
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Neovascularização de Coroide/metabolismo , Degeneração Macular/metabolismo , Doenças Retinianas/metabolismo , Esfingolipídeos/metabolismo , Uveíte/metabolismo , Neovascularização de Coroide/imunologia , Humanos , Degeneração Macular/imunologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Doenças Retinianas/imunologia , Uveíte/imunologiaRESUMO
CONTEXT: Ceramides and sphingolipids have been linked to type 2 diabetes (T2D). The Ceramides and Sphingolipids as Predictors of Incident Dysglycemia (CASPID) study is designed to determine the association of plasma sphingolipids with the pathophysiology of human T2D. OBJECTIVE: A comparison of plasma sphingolipids profiles in Black and White adults with (FH+) and without (FH-) family history of T2D. DESIGN: We recruited 100 Black and White FH- (54 Black, 46 White) and 140 FH+ (75 Black, 65 White) adults. Fasting plasma levels of 58 sphingolipid species, including 18 each from 3 major classes (ceramides, monohexosylceramides, and sphingomyelins, all with 18:1 sphingoid base) and 4 long-chain sphingoid base-containing species, were measured by liquid chromatography/mass spectrometry. RESULTS: Sphingomyelin was the most abundant sphingolipid in plasma (89% in FH-), and was significantly elevated in FH+ subjects (93%). Ceramides and monohexosylceramides comprised 5% and 6% of total sphingolipids in the plasma of FH- subjects, and were reduced significantly in FH+ subjects (3% and 4%, respectively). In FH+ subjects, most ceramide and monohexosylceramide species were decreased but sphingomyelin species were increased. The level of C18:1 species of all 3 classes was elevated in FH+ subjects. CONCLUSION: Elevated levels of sphingomyelin, the major sphingolipids of plasma, and oleic acid-containing sphingolipids in healthy FH+ subjects compared with healthy FH- subjects may reflect heritable elements linking sphingolipids and the development of T2D.
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Diabetes Mellitus Tipo 2 , Esfingolipídeos , Adulto , Humanos , Ceramidas , Diabetes Mellitus Tipo 2/genética , Esfingomielinas , População Branca , População NegraRESUMO
Polyunsaturated fatty acids (PUFAs) generate pro- and anti-inflammatory eicosanoids via three different metabolic pathways. This study profiled tear PUFAs and their metabolites and examined the relationships with dry eye (DE) and meibomian gland dysfunction (MGD) symptoms and signs. A total of 40 individuals with normal eyelids and corneal anatomies were prospectively recruited. The symptoms and signs of DE and MGD were assessed, and tear samples (from the right eye) were analyzed by mass spectrometry. Mann-Whitney U tests assessed differences between medians; Spearman tests assessed correlations between continuous variables; and linear regression models assessed the impact of potential confounders. The median age was 63 years; 95% were male; 30% were White; and 85% were non-Hispanic. The symptoms of DE/MGD were not correlated with tear PUFAs and eicosanoids. DE signs (i.e., tear break-up time (TBUT) and Schirmer's) negatively correlated with anti-inflammatory eicosanoids (11,12-dihydroxyeicosatrienoic acid (11,12 DHET) and 14,15-dihydroxyicosatrienoic acid (14,15, DHET)). Corneal staining positively correlated with the anti-inflammatory PUFA, docosahexaenoic acid (DHA). MGD signs significantly associated with the pro-inflammatory eicosanoid 15-hydroxyeicosatetranoic acid (15-HETE) and DHA. Several relationships remained significant when potential confounders were considered. DE/MGD signs relate more to tear PUFAs and eicosanoids than symptoms. Understanding the impact of PUFA-related metabolic pathways in DE/MGD may provide targets for new therapeutic interventions.
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Síndromes do Olho Seco , Humanos , Masculino , Pessoa de Meia-Idade , Feminino , Síndromes do Olho Seco/tratamento farmacológico , Eicosanoides/metabolismo , Lágrimas/metabolismo , Córnea/metabolismo , Ácidos Docosa-Hexaenoicos , Anti-Inflamatórios/uso terapêuticoRESUMO
Purpose: Fingolimod (FTY720; FT), a structural analog of sphingosine, has potential ocular applications. The goal of this study was to develop an FT-loaded nanoemulsion (NE; FT-NE) formulation for the efficient and prolonged delivery of FT to the posterior segment of the eye through the topical route. Methods: FT-NE formulations were prepared using homogenization followed by the probe sonication method. The lead FT-NE formulations (0.15% and 0.3% w/v loading), comprising soybean oil as oil and Tween® 80 and Poloxamer 188 as surfactants, were further evaluated for in vitro release, surface morphology, filtration sterilization, and stability at refrigerated temperature. Ocular bioavailability following topical application of FT-NE (0.3%) was examined in Sprague-Dawley rats. Results: The formulation, at both dose levels, showed desirable physicochemical characteristics, a nearly spherical shape with homogenous nanometric size distribution, and was stable for 180 days (last time point checked) at refrigerated temperature postfiltration through a polyethersulfone (0.22 µm) membrane. In vitro release studies showed prolonged release over 24 h, compared with the control FT solution (FT-S). In vivo studies revealed that effective concentrations of FT were achieved in the vitreous humor and retina following topical application of FT-NE. Conclusions: The results from these studies demonstrate that the FT-NE formulation can serve as a viable platform for the ocular delivery of FT through the topical route.
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CONTEXT: Sphingolipids are linked to the pathogenesis of type 2 diabetes (T2D). OBJECTIVE: To test the hypothesis that plasma sphingolipid profiles predict incident prediabetes. DESIGN: A case-control study nested in the Pathobiology of Prediabetes in a Biracial Cohort (POP-ABC) study, a 5-year follow-up study. SETTING: Academic health center. PARTICIPANTS: Normoglycemic adults enrolled in the POP-ABC study. Assessments included OGTT, insulin sensitivity and insulin secretion. Participants with incident prediabetes were matched in age, sex, and ethnicity with non-progressors. INTERVENTIONS: We assayed 58 sphingolipid species (ceramides, monohexosyl ceramides, sphingomyelins, and sphingosine) using LC/tandem mass spectrometry in baseline plasma levels from participants and determined association with prediabetes risk. MAIN OUTCOME MEASURE: The primary outcome was progression from normoglycemia to prediabetes, defined as impaired fasting glucose or impaired glucose tolerance. RESULTS: The mean age of participants (N = 140; 50% Black, 50% female) was 48.1 ± 8.69 y, BMI 30.1 ± 5.78â kg/m2, fasting plasma glucose (FPG) 92.7 ± 5.84â mg/dl, and two-hour plasma glucose (2hrPG) 121 ± 23.3â mg/dl. Of the 58 sphingolipid species assayed, higher ratios of sphingomyelin C26:0/C26:1 (OR 2.73 [95% CI 1.172-4.408], P = 0.015) and ceramide C18:0/C18:1 (OR 1.236 [95% CI 1.042-1.466], P = 0.015) in baseline plasma specimens were significantly associated with progression to prediabetes during the 5-year follow-up period, after adjustments for age, race, sex, BMI, FPG, 2hPG, insulin sensitivity, and insulin secretion. CONCLUSIONS: We conclude that the saturated-to-monounsaturated ratios of long-chain ceramide C18:0/C18:1 and very-long-chain sphingomyelin C26:0/C26:1 are potential biomarkers of prediabetes risk among individuals with parental history of T2D.
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PURPOSE: The purpose of this study was to examine ocular surface symptoms, tear metrics, and tear cytokines by Meibomian gland dysfunction (MGD) features. METHODS: Symptom questionnaires and an ocular surface evaluation were performed on 40 individuals with varied MGD signs [Meibomian gland (MG) plugging, eyelid vascularity, meibum quality, and MG dropout]. Tear proteins were extracted off Schirmer strips and analyzed for 23 human inflammation-related proteins. Statistical analysis was performed to examine associations between dry eye metrics inflammatory proteins and MGD features. RESULTS: The study involved 40 South Florida veterans with a mean age of 61 ± 13 years; most individuals were male (95%), White (31%), and non-Hispanic (85%). MGD features differentially related to dry eye signs. Eyelid vascularity, meibum quality, and MG dropout, but not MG plugging, correlated with higher corneal staining and lower tear production. MGD features also differentially related to tear cytokines. Eyelid vascularity most closely related to inflammation with significant correlations for interferon-gamma-γ (r = 0.36, P = 0.02), interleukin-4 (IL-4) (r = 0.43, P = 0.006), IL-17A (r = 0.42, P = 0.007), matrix metalloproteinase-2 (r = 0.39, P = 0.01), C-X-C motif chemokine ligand 5 (Regulated upon Activation, Normal T-Cell Expressed and presumably Secreted [RANTES]) (r = 0.32, P = 0.04), and tumor necrosis factor α (r = 0.36, P = 0.02). The other 3 MGD signs were less related to inflammation. Multivariable models revealed IL-4 to be most closely related to eyelid vascularity (standardized ß = 0.39, P < 0.0001). CONCLUSIONS: Eyelid vascularity was the MGD sign most closely related to inflammatory cytokines, suggesting that different MGD features may be driven by different pathophysiological mechanisms.