RESUMEN
Muscle damage elicits a sterile immune response that facilitates complete regeneration. Here, we used mass spectrometry-based lipidomics to map the mediator lipidome during the transition from inflammation to resolution and regeneration in skeletal muscle injury. We observed temporal regulation of glycerophospholipids and production of pro-inflammatory lipid mediators (for example, leukotrienes and prostaglandins) and specialized pro-resolving lipid mediators (for example, resolvins and lipoxins) that were modulated by ibuprofen. These time-dependent profiles were recapitulated in sorted neutrophils and Ly6Chi and Ly6Clo muscle-infiltrating macrophages, with a distinct pro-resolving signature observed in Ly6Clo macrophages. RNA sequencing of macrophages stimulated with resolvin D2 showed similarities to transcriptional changes found during the temporal transition from Ly6Chi macrophage to Ly6Clo macrophage. In vivo, resolvin D2 increased Ly6Clo macrophages and functional improvement of the regenerating muscle. These results reveal dynamic lipid mediator signatures of innate immune cells and provide a proof of concept for their exploitable effector roles in muscle regeneration.
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Mediadores de Inflamación/inmunología , Lípidos/inmunología , Macrófagos/inmunología , Músculo Esquelético/inmunología , Regeneración/inmunología , Animales , Ácidos Docosahexaenoicos/inmunología , Ácidos Docosahexaenoicos/farmacología , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Perfilación de la Expresión Génica , Ontología de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Metabolismo de los Lípidos/inmunología , Lípidos/análisis , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Músculo Esquelético/lesiones , Músculo Esquelético/fisiopatología , Regeneración/genéticaRESUMEN
In the version of this article initially published, two arrows in the far right plot of Fig. 3c were aimed incorrectly, and the error bars were missing in Fig. 6e,f. In Fig. 3c, the arrow labeled '5-LOX' should be aimed at the plot measuring LXB4, and the arrow labeled 'LTA4H' should be aimed at the plot measuring LTB4. The errors have been corrected in the HTML and PDF versions of the article.
RESUMEN
Tissue regeneration requires inflammatory and reparatory activity of macrophages. Macrophages detect and eliminate the damaged tissue and subsequently promote regeneration. This dichotomy requires the switch of effector functions of macrophages coordinated with other cell types inside the injured tissue. The gene regulatory events supporting the sensory and effector functions of macrophages involved in tissue repair are not well understood. Here we show that the lipid activated transcription factor, PPARγ, is required for proper skeletal muscle regeneration, acting in repair macrophages. PPARγ controls the expression of the transforming growth factor-ß (TGF-ß) family member, GDF3, which in turn regulates the restoration of skeletal muscle integrity by promoting muscle progenitor cell fusion. This work establishes PPARγ as a required metabolic sensor and transcriptional regulator of repair macrophages. Moreover, this work also establishes GDF3 as a secreted extrinsic effector protein acting on myoblasts and serving as an exclusively macrophage-derived regeneration factor in tissue repair.
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Factor 3 de Diferenciación de Crecimiento/metabolismo , Músculo Esquelético/fisiología , Mioblastos/metabolismo , PPAR gamma/metabolismo , Regeneración/fisiología , Animales , Western Blotting , Separación Celular , Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/lesiones , Análisis de Secuencia por Matrices de Oligonucleótidos , Cicatrización de Heridas/fisiologíaRESUMEN
Understanding the factors that influence the biological response to inflammation is crucial, due to its involvement in physiological and pathological processes, including tissue repair/healing, cancer, infections, and autoimmune diseases. We have previously demonstrated that in vivo stretching can reduce inflammation and increase local pro-resolving lipid mediators in rats, suggesting a direct mechanical effect on inflammation resolution. Here we aimed to explore further the effects of stretching at the cellular/molecular level in a mouse subcutaneous carrageenan-inflammation model. Stretching for 10 min twice a day reduced inflammation, increased the production of pro-resolving mediator pathway intermediate 17-HDHA at 48 h postcarrageenan injection, and decreased both pro-resolving and pro-inflammatory mediators (e.g., PGE2 and PGD2 ) at 96 h. Single-cell RNA sequencing analysis of inflammatory lesions at 96 h showed that stretching increased the expression of both pro-inflammatory (Nos2) and pro-resolution (Arg1) genes in M1 and M2 macrophages at 96 h. An intercellular communication analysis predicted specific ligand-receptor interactions orchestrated by neutrophils and M2a macrophages, suggesting a continuous neutrophil presence recruiting immune cells such as activated macrophages to contain the antigen while promoting resolution and preserving tissue homeostasis.
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Inflamación , Neutrófilos , Animales , Ratones , Carragenina/metabolismo , Carragenina/farmacología , Dinoprostona/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Neutrófilos/metabolismo , Análisis de la Célula Individual , Ratones Endogámicos C57BL , TranscriptomaRESUMEN
In response to infection or tissue damage, resident peritoneal macrophages (rpMACs) produce inflammatory lipid mediators from the polyunsaturated fatty acid (PUFA), arachidonic acid (AA). Long-chain acyl-CoA synthetase 4 (ACSL4) catalyzes the covalent addition of a CoA moiety to fatty acids, with a strong preference for AA and other PUFAs containing three or more double bonds. PUFA-CoA can be incorporated into phospholipids, which is the source of PUFA for lipid mediator synthesis. In this study, we demonstrated that deficiency of Acsl4 in mouse rpMACs resulted in a significant reduction of AA incorporated into all phospholipid classes and a reciprocal increase in incorporation of oleic acid and linoleic acid. After stimulation with opsonized zymosan (opZym), a diverse array of AA-derived lipid mediators, including leukotrienes, PGs, hydroxyeicosatetraenoic acids, and lipoxins, were produced and were significantly reduced in Acsl4-deficient rpMACs. The Acsl4-deficient rpMACs stimulated with opZym also demonstrated an acute reduction in mRNA expression of the inflammatory cytokines, Il6, Ccl2, Nos2, and Ccl5 When Acsl4-deficient rpMACs were incubated in vitro with the TLR4 agonist, LPS, the levels of leukotriene B4 and PGE2 were also significantly decreased. In LPS-induced peritonitis, mice with myeloid-specific Acsl4 deficiency had a significant reduction in leukotriene B4 and PGE2 levels in peritoneal exudates, which was coupled with reduced infiltration of neutrophils in the peritoneal cavity as compared with wild-type mice. Our data demonstrate that chronic deficiency of Acsl4 in rpMACs reduces the incorporation of AA into phospholipids, which reduces lipid mediator synthesis and inflammation.
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Ácido Araquidónico/inmunología , Coenzima A Ligasas/inmunología , Inflamación/inmunología , Fosfolípidos/inmunología , Zimosan/biosíntesis , Animales , Coenzima A Ligasas/deficiencia , Ratones , Ratones TransgénicosRESUMEN
Radiation is associated with tissue damage and increased risk of atherosclerosis, but there are currently no treatments and a very limited mechanistic understanding of how radiation impacts tissue repair mechanisms. We uncovered that radiation significantly delayed temporal resolution programs that were associated with decreased efferocytosis in vivo. Resolvin D1 (RvD1), a known proresolving ligand, promoted swift resolution and restored efferocytosis in sublethally irradiated mice. Irradiated macrophages exhibited several features of senescence, including increased expression of p16INK4A and p21, heightened levels of SA-ß-gal, COX-2, several proinflammatory cytokines/chemokines, and oxidative stress (OS) in vitro, and when transferred to mice, they exacerbated inflammation in vivo. Mechanistically, heightened OS in senescent macrophages led to impairment in their ability to carry out efficient efferocytosis, and treatment with RvD1 reduced OS and improved efferocytosis. Sublethally irradiated Ldlr -/- mice exhibited increased plaque necrosis, p16INK4A cells, and decreased lesional collagen compared with nonirradiated controls, and treatment with RvD1 significantly reduced necrosis and increased lesional collagen. Removal of p16INK4A hematopoietic cells during advanced atherosclerosis with p16-3MR mice reduced plaque necrosis and increased production of key intraplaque-resolving mediators. Our results demonstrate that sublethal radiation drives macrophage senescence and efferocytosis defects and suggest that RvD1 may be a new therapeutic strategy to limit radiation-induced tissue damage.
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Aterosclerosis/inmunología , Enfermedades Cardiovasculares/inmunología , Ácidos Docosahexaenoicos/metabolismo , Células Madre Hematopoyéticas/fisiología , Macrófagos/inmunología , Traumatismos por Radiación/inmunología , Cicatrización de Heridas/efectos de la radiación , Animales , Aterosclerosis/genética , Células Cultivadas , Senescencia Celular , Ciclooxigenasa 2/metabolismo , Genes p16 , Humanos , Inflamación , Ratones , Ratones Noqueados , RadiaciónRESUMEN
Ischemic injury initiates a sterile inflammatory response that ultimately participates in the repair and recovery of tissue perfusion. Macrophages are required for perfusion recovery during ischemia, in part because they produce growth factors that aid in vascular remodeling. The input signals governing this pro-revascularization phenotype remain of interest. Here we found that hindlimb ischemia increases levels of resolvin D1 (RvD1), an inflammation-resolving lipid mediator that targets macrophages via its receptor, ALX/FPR2. Exogenous RvD1 enhances perfusion recovery during ischemia, and mice deficient in Alx/Fpr2 have an endogenous defect in this process. Mechanistically, RNA sequencing revealed that RvD1 induces a transcriptional program in macrophages characteristic of a pro-revascularization phenotype. Vascularization of ischemic skeletal muscle, as well as cutaneous wounds, is impaired in mice with myeloid-specific deficiency of Alx/Fpr2, and this is associated with altered expression of pro-revascularization genes in skeletal muscle and macrophages isolated from skeletal muscle. Collectively, these results uncover a role of ALX/FPR2 in revascularization that may be amenable to therapeutic targeting in diseases associated with altered tissue perfusion and repair.
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Ácidos Docosahexaenoicos/metabolismo , Isquemia/inmunología , Neovascularización Fisiológica/inmunología , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismo , Cicatrización de Heridas/inmunología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Técnicas de Inactivación de Genes , Humanos , Isquemia/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/inmunología , Músculo Esquelético/patología , Cultivo Primario de Células , RNA-Seq , Receptores de Formil Péptido/genética , Receptores de Lipoxina/genética , Transducción de Señal/inmunología , Piel/irrigación sanguínea , Piel/inmunología , Piel/lesiones , Piel/patología , Transcripción Genética/inmunologíaRESUMEN
Tissue injury elicits an inflammatory response that facilitates host defense. Resolution of inflammation promotes the transition to tissue repair and is governed, in part, by specialized pro-resolving mediators (SPM). The complete structures of a novel series of cysteinyl-SPM (cys-SPM) were recently elucidated, and proved to stimulate tissue regeneration in planaria and resolve acute inflammation in mice. Their functions in mammalian tissue repair are of interest. Here, nine structurally distinct cys-SPM were screened and PCTR1 uniquely enhanced human keratinocyte migration with efficacy similar to epidermal growth factor. In skin wounds of mice, PCTR1 accelerated closure. Wound infection increased PCTR1 that coincided with decreased bacterial burden. Addition of PCTR1 reduced wound bacteria levels and decreased inflammatory monocytes/macrophages, which was coupled with increased expression of genes involved in host defense and tissue repair. These results suggest that PCTR1 is a novel regulator of host defense and tissue repair, which could inform new approaches for therapeutic management of delayed tissue repair and infection.
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Ácidos Docosahexaenoicos/metabolismo , Mediadores de Inflamación/metabolismo , Piel/metabolismo , Cicatrización de Heridas/fisiología , Infección de Heridas/metabolismo , Animales , Movimiento Celular/fisiología , Humanos , Queratinocitos/metabolismo , RatonesRESUMEN
RATIONALE: Regression of atherosclerosis is an important clinical goal; however, the pathways that mediate the resolution of atherosclerotic inflammation and reversal of plaques are poorly understood. Regulatory T cells (Tregs) have been shown to be atheroprotective, yet the numbers of these immunosuppressive cells decrease with disease progression, and whether they contribute to atherosclerosis regression is not known. OBJECTIVE: We investigated the roles of Tregs in the resolution of atherosclerotic inflammation, tissue remodeling, and plaque contraction during atherosclerosis regression. METHODS AND RESULTS: Using multiple independent mouse models of atherosclerosis regression, we demonstrate that an increase in plaque Tregs is a common signature of regressing plaques. Single-cell RNA-sequencing of plaque immune cells revealed that unlike Tregs from progressing plaques that expressed markers of natural Tregs derived from the thymus, Tregs in regressing plaques lacked Nrp1 expression, suggesting that they are induced in the periphery during lipid-lowering therapy. To test whether Tregs are required for resolution of atherosclerotic inflammation and plaque regression, Tregs were depleted using CD25 monoclonal antibody in atherosclerotic mice during apolipoprotein B antisense oligonucleotide-mediated lipid lowering. Morphometric analyses revealed that Treg depletion blocked plaque remodeling and contraction, and impaired hallmarks of inflammation resolution, including dampening of the T helper 1 response, alternative activation of macrophages, efferocytosis, and upregulation of specialized proresolving lipid mediators. CONCLUSIONS: Our data establish essential roles for Tregs in resolving atherosclerotic cardiovascular disease and provide mechanistic insight into the pathways governing plaque remodeling and regression of disease.
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Aorta/metabolismo , Aterosclerosis/metabolismo , Activación de Macrófagos , Macrófagos/metabolismo , Placa Aterosclerótica , Linfocitos T Reguladores/metabolismo , Animales , Anticuerpos/farmacología , Aorta/efectos de los fármacos , Aorta/inmunología , Aorta/patología , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/inmunología , Aterosclerosis/patología , Células Cultivadas , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Femenino , Mediadores de Inflamación/metabolismo , Subunidad alfa del Receptor de Interleucina-2/antagonistas & inhibidores , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones Noqueados para ApoE , Neuropilina-1/genética , Neuropilina-1/metabolismo , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
OBJECTIVE: Plaque necrosis is a key feature of defective resolution in atherosclerosis. Recent evidence suggests that necroptosis promotes plaque necrosis; therefore, we sought to determine how necroptotic cells (NCs) impact resolution programs in plaques. Approach and Results: To investigate the role(s) of necroptosis in advanced atherosclerosis, we used mice deficient of Mlkl, an effector of necroptosis. Mlkl-/- mice that were injected with a gain-of-function mutant PCSK9 (AAV8-gof-PCSK9) and fed a Western diet for 16 weeks, showed significantly less plaque necrosis, increased fibrous caps and improved efferocytosis compared with AAV8-gof-PCSK9 injected wt controls. Additionally, hypercholesterolemic Mlkl-/- mice had a significant increase in proresolving mediators including resolvin D1 (RvD1) and a decrease in prostanoids including thromboxane in plaques and in vitro. We found that exuberant thromboxane released by NCs impaired the clearance of both apoptotic cells and NCs through disruption of oxidative phosphorylation in macrophages. Moreover, we found that NCs did not readily synthesize RvD1 and that exogenous administration of RvD1 to macrophages rescued NC-induced defective efferocytosis. RvD1 also enhanced the uptake of NCs via the activation of p-AMPK (AMP-activated protein kinase), increased fatty acid oxidation, and enhanced oxidative phosphorylation in macrophages. CONCLUSIONS: These results suggest that NCs derange resolution by limiting key SPMs and impairing the efferocytic repertoire of macrophages. Moreover, these findings provide a molecular mechanism for RvD1 in directing proresolving metabolic programs in macrophages and further suggests RvD1 as a potential therapeutic strategy to limit NCs in tissues. Graphic Abstract: A graphic abstract is available for this article.
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Ácidos Docosahexaenoicos/metabolismo , Ácidos Grasos/metabolismo , Macrófagos/metabolismo , Necroptosis/fisiología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Apoptosis , Femenino , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Necrosis , Fosforilación Oxidativa , Fagocitosis , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Prostaglandinas/metabolismo , Proteínas Quinasas/deficiencia , Proteínas Quinasas/genéticaAsunto(s)
Eritrocitos , Material Particulado , Material Particulado/efectos adversos , Eritrocitos/metabolismo , Eritrocitos/efectos de los fármacos , Humanos , Contaminación del Aire/efectos adversos , Masculino , Contaminantes Atmosféricos/efectos adversos , Contaminantes Atmosféricos/toxicidad , FemeninoRESUMEN
BACKGROUND: N-3 polyunsaturated fatty acid (PUFA) supplementation has been associated with reduced mortality and inflammation in patients with cardiovascular disease. There are limited data on the effects of n-3 PUFA supplementation in patients with peripheral artery disease (PAD). MATERIALS AND METHODS: The OMEGA-PAD II trial was a double-blinded, randomized, placebo-controlled trial to assess the effect of 3 mo of high-dose oral n-3 PUFA supplementation on inflammation, endothelial function, and walking ability in patients with PAD. RESULTS: Twenty-four patients with claudication received 4.4 g/d of fish oil or placebo for 3 mo. Outcomes measured included high-sensitivity C-reactive protein levels, the omega-3 index, endothelial function as measured via flow-mediated vasodilation, walking impairment questionnaire, and a 6-min walk test. Plasma levels of specialized pro-resolving lipid mediators (SPMs) were measured by liquid-chromatography-tandem mass spectrometry. In patients treated with fish oil, the absolute mean omega-3 index significantly increased from baseline (fish oil: 7.2 ± 1.2%, P < 0.001; placebo: -0.4 ± 0.9%, P = 0.31; between-group P < 0.001). Furthermore, there were significant increases in several pathway markers of SPM biosynthesis, including several mono-hydroxyeicosapentaenoic acids and mono-hydroxydocosahexaenoic acids. We also observed significant increases in the SPM lipoxin A5 (fish oil: 0.57 ± 0.70 pg/mL, P = 0.05; placebo: 0.01 ± 0.38 pg/mL, P = 0.93; between-group P = 0.04) and resolvin E3 (fish oil: 154 ± 171 pg/mL, P = 0.04; placebo: 32 ± 54 pg/mL, P = 0.08; between-group P = 0.04). There were no significant changes in high-sensitivity C-reactive protein, flow-mediated vasodilation, walking impairment questionnaire, or 6-min walk test in the fish oil group. CONCLUSIONS: Fish oil increases SPMs in plasma of patients with PAD. Further studies are required to determine whether these early changes translate to clinical improvements in patients with PAD.
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Ácidos Docosahexaenoicos/sangre , Ácido Eicosapentaenoico/sangre , Ácidos Grasos Omega-3/administración & dosificación , Inflamación/dietoterapia , Enfermedad Arterial Periférica/dietoterapia , Administración Oral , Anciano , Anciano de 80 o más Años , Suplementos Dietéticos , Ácidos Docosahexaenoicos/inmunología , Método Doble Ciego , Ácido Eicosapentaenoico/inmunología , Femenino , Humanos , Inflamación/sangre , Inflamación/inmunología , Masculino , Persona de Mediana Edad , Enfermedad Arterial Periférica/sangre , Enfermedad Arterial Periférica/inmunología , Placebos/administración & dosificación , Placebos/efectos adversos , Resultado del TratamientoRESUMEN
OBJECTIVE: Human genetic variants near the FADS (fatty acid desaturase) gene cluster (FADS1-2-3) are strongly associated with cardiometabolic traits including dyslipidemia, fatty liver, type 2 diabetes mellitus, and coronary artery disease. However, mechanisms underlying these genetic associations are unclear. APPROACH AND RESULTS: Here, we specifically investigated the physiological role of the Δ-5 desaturase FADS1 in regulating diet-induced cardiometabolic phenotypes by treating hyperlipidemic LDLR (low-density lipoprotein receptor)-null mice with antisense oligonucleotides targeting the selective knockdown of Fads1. Fads1 knockdown resulted in striking reorganization of both ω-6 and ω-3 polyunsaturated fatty acid levels and their associated proinflammatory and proresolving lipid mediators in a highly diet-specific manner. Loss of Fads1 activity promoted hepatic inflammation and atherosclerosis, yet was associated with suppression of hepatic lipogenesis. Fads1 knockdown in isolated macrophages promoted classic M1 activation, whereas suppressing alternative M2 activation programs, and also altered systemic and tissue inflammatory responses in vivo. Finally, the ability of Fads1 to reciprocally regulate lipogenesis and inflammation may rely in part on its role as an effector of liver X receptor signaling. CONCLUSIONS: These results position Fads1 as an underappreciated regulator of inflammation initiation and resolution, and suggest that endogenously synthesized arachidonic acid and eicosapentaenoic acid are key determinates of inflammatory disease progression and liver X receptor signaling.
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Aorta/enzimología , Enfermedades de la Aorta/enzimología , Aterosclerosis/enzimología , Dislipidemias/enzimología , Ácido Graso Desaturasas/metabolismo , Mediadores de Inflamación/metabolismo , Inflamación/enzimología , Lipogénesis , Animales , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Ácido Araquidónico/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Células Cultivadas , delta-5 Desaturasa de Ácido Graso , Modelos Animales de Enfermedad , Dislipidemias/genética , Dislipidemias/patología , Ácido Eicosapentaenoico/metabolismo , Ácido Graso Desaturasas/genética , Inflamación/genética , Inflamación/patología , Hígado/metabolismo , Receptores X del Hígado/metabolismo , Activación de Macrófagos , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Placa Aterosclerótica , Receptores de LDL/deficiencia , Receptores de LDL/genéticaRESUMEN
The acute inflammatory response requires a coordinated resolution program to prevent excessive inflammation, repair collateral damage, and restore tissue homeostasis, and failure of this response contributes to the pathology of numerous chronic inflammatory diseases. Resolution is mediated in part by long-chain fatty acid-derived lipid mediators called specialized proresolving mediators (SPMs). However, how SPMs are regulated during the inflammatory response, and how this process goes awry in inflammatory diseases, are poorly understood. We now show that signaling through the Mer proto-oncogene tyrosine kinase (MerTK) receptor in cultured macrophages and in sterile inflammation in vivo promotes SPM biosynthesis by a mechanism involving an increase in the cytoplasmic:nuclear ratio of a key SPM biosynthetic enzyme, 5-lipoxygenase. This action of MerTK is linked to the resolution of sterile peritonitis and, after ischemia-reperfusion (I/R) injury, to increased circulating SPMs and decreased remote organ inflammation. MerTK is susceptible to ADAM metallopeptidase domain 17 (ADAM17)-mediated cell-surface cleavage under inflammatory conditions, but the functional significance is not known. We show here that SPM biosynthesis is increased and inflammation resolution is improved in a new mouse model in which endogenous MerTK was replaced with a genetically engineered variant that is cleavage-resistant (Mertk(CR)). Mertk(CR) mice also have increased circulating levels of SPMs and less lung injury after I/R. Thus, MerTK cleavage during inflammation limits SPM biosynthesis and the resolution response. These findings contribute to our understanding of how SPM synthesis is regulated during the inflammatory response and suggest new therapeutic avenues to boost resolution in settings where defective resolution promotes disease progression.
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Mediadores de Inflamación/metabolismo , Inflamación/etiología , Inflamación/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteína ADAM17/metabolismo , Animales , Araquidonato 5-Lipooxigenasa/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Inflamación/patología , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peritonitis/etiología , Peritonitis/metabolismo , Peritonitis/patología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/deficiencia , Proteínas Tirosina Quinasas Receptoras/genética , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Transducción de Señal , Tirosina Quinasa c-MerRESUMEN
OBJECTIVE: Inflammation is a key driver of excessive neointimal hyperplasia within vein grafts. Recent work demonstrates that specialized proresolving lipid mediators biosynthesized from omega-3 polyunsaturated fatty acids, such as resolvin D1 (RvD1), actively orchestrate the process of inflammation resolution. We investigated the effects of local perivascular delivery of RvD1 in a rabbit vein graft model. METHODS: Ipsilateral jugular veins were implanted as carotid interposition grafts through an anastomotic cuff technique in New Zealand white rabbits (3-4 kg; N = 80). RvD1 (1 µg) was delivered to the vein bypass grafts in a perivascular fashion, using either 25% Pluronic F127 gel (Sigma-Aldrich, St. Louis, Mo) or a thin bilayered poly(lactic-co-glycolic acid) (PLGA) film. No treatment (bypass only) and vehicle-loaded Pluronic gels or PLGA films served as controls. Delivery of RvD1 to venous tissue was evaluated 3 days later by liquid chromatography-tandem mass spectrometry. Total leukocyte infiltration, macrophage infiltration, and cell proliferation were evaluated by immunohistochemistry. Elastin and trichrome staining was performed on grafts harvested at 28 days after bypass to evaluate neointimal hyperplasia and vein graft remodeling. RESULTS: Perivascular treatments did not influence rates of graft thrombosis (23%), major wound complications (4%), or death (3%). Leukocyte (CD45) and macrophage (RAM11) infiltration was significantly reduced in the RvD1 treatment groups vs controls at 3 days (60%-72% reduction; P < .01). Cellular proliferation (Ki67 index) was also significantly lower in RvD1-treated vs control grafts at 3 days (40%-50% reduction; P < .01). Treatment of vein grafts with RvD1-loaded gels reduced neointimal thickness at 28 days by 61% vs bypass only (P < .001) and by 63% vs vehicle gel (P < .001). RvD1-loaded PLGA films reduced neointimal formation at 28 days by 50% vs bypass only (P < .001). RvD1 treatment was also associated with reduced collagen deposition in vein grafts at 28 days. CONCLUSIONS: Local perivascular delivery of RvD1 attenuates vein graft hyperplasia without associated toxicity in a rabbit carotid bypass model. This effect appears to be mediated by both reduced leukocyte recruitment and decreased cell proliferation within the graft. Perivascular PLGA films may also impart protection through biomechanical scaffolding in this venous arterialization model. Our studies provide further support for the potential therapeutic role of specialized proresolving lipid mediators such as D-series resolvins in modulating vascular injury and repair.
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Antiinflamatorios/administración & dosificación , Implantación de Prótesis Vascular/métodos , Arteria Carótida Común/cirugía , Ácidos Docosahexaenoicos/administración & dosificación , Oclusión de Injerto Vascular/prevención & control , Venas Yugulares/efectos de los fármacos , Venas Yugulares/trasplante , Neointima , Animales , Implantación de Prótesis Vascular/efectos adversos , Proliferación Celular/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Modelos Animales de Enfermedad , Portadores de Fármacos , Femenino , Geles , Oclusión de Injerto Vascular/patología , Hiperplasia , Venas Yugulares/patología , Poloxámero/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Conejos , Factores de TiempoRESUMEN
Recent evidence suggests that specialized proresolving lipid mediators (SPMs) generated from docosahexaenoic acid (DHA) can modulate the vascular injury response. However, cellular sources for these autacoids within the vessel wall remain unclear. Here, we investigated whether isolated vascular cells and tissues can produce SPMs and assessed expression and subcellular localization of the key SPM biosynthetic enzyme 5-lipoxygenase (LOX) in vascular cells. Intact human arteries incubated with DHA ex vivo produced 17-hydroxy DHA (17-HDHA) and D-series resolvins, as assessed by liquid chromatography-tandem mass spectrometry. Addition of 17-HDHA to human arteries similarly increased resolvin production. Primary cultures of human saphenous vein endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) converted 17-HDHA to SPMs, including resolvin D1 (RvD1) and other D-series resolvins and protectins. This was accompanied by a rapid translocation of 5-LOX from nucleus to cytoplasm in both ECs and VSMCs, potentially facilitating SPM biosynthesis. Conditioned medium from cells exposed to 17-HDHA inhibited monocyte adhesion to TNF-α-stimulated EC monolayers. These downstream effects were partially reversed by antibodies against the RvD1 receptors ALX/FPR2 and GPR32. These results suggest that autocrine and/or paracrine signaling via locally generated SPMs in the vasculature may represent a novel homeostatic mechanism of relevance to vascular health and disease.-Chatterjee, A., Komshian, S., Sansbury, B. E., Wu, B., Mottola, G., Chen, M., Spite, M., Conte, M. S. Biosynthesis of proresolving lipid mediators by vascular cells and tissues.
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Ácidos Docosahexaenoicos/farmacología , Células Endoteliales/metabolismo , Metabolismo de los Lípidos/fisiología , Miocitos del Músculo Liso/metabolismo , Anticuerpos , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/metabolismo , Células Cultivadas , Citocinas/metabolismo , Ácidos Docosahexaenoicos/genética , Ácidos Docosahexaenoicos/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Inflamación/metabolismo , Leucocitos/fisiología , Estructura Molecular , Transporte de Proteínas/fisiología , Receptores de Formil Péptido/genética , Receptores de Formil Péptido/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Lipoxina/genética , Receptores de Lipoxina/metabolismoRESUMEN
Acute inflammation is a host-protective response that is mounted in response to tissue injury and infection. Initiated and perpetuated by exogenous and endogenous mediators, acute inflammation must be resolved for tissue repair to proceed and for homeostasis to be restored. Resolution of inflammation is an actively regulated process governed by an array of mediators as diverse as those that initiate inflammation. Among these, resolvins have emerged as a genus of evolutionarily conserved proresolving mediators that act on specific cellular receptors to regulate leukocyte trafficking and blunt production of inflammatory mediators, while also promoting clearance of dead cells and tissue repair. Given that chronic unresolved inflammation is emerging as a central causative factor in the development of cardiovascular diseases, an understanding of the endogenous processes that govern normal resolution of acute inflammation is critical for determining why sterile maladaptive cardiovascular inflammation perpetuates. Here, we provide an overview of the process of resolution with a focus on the enzymatic biosynthesis and receptor-dependent actions of resolvins and related proresolving mediators in immunity, thrombosis, and vascular biology. We discuss how nutritional and current therapeutic approaches modulate resolution and propose that harnessing resolution concepts could potentially lead to the development of new approaches for treating chronic cardiovascular inflammation in a manner that is not host disruptive.
Asunto(s)
Enfermedades Cardiovasculares/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Trombosis/metabolismo , Animales , Antiinflamatorios/uso terapéutico , Fármacos Cardiovasculares/uso terapéutico , Enfermedades Cardiovasculares/tratamiento farmacológico , Ácidos Docosahexaenoicos/uso terapéutico , Humanos , Metabolismo de los Lípidos , Trombosis/tratamiento farmacológicoRESUMEN
BACKGROUND: Resolvins are lipid mediators generated by leukocytes during the resolution phase of inflammation. They have been shown to regulate the transition from inflammation to tissue repair; however, it is unknown whether resolvins play a role in tissue revascularization following ischemia. METHODS: We used a murine model of hind limb ischemia (HLI), coupled with laser Doppler perfusion imaging, microcomputed tomography, and targeted mass spectrometry, to assess the role of resolvins in revascularization and inflammation resolution. RESULTS: In mice undergoing HLI, we identified resolvin D2 (RvD2) in bone marrow and skeletal muscle by mass spectrometry (n=4-7 per group). We also identified RvD2 in skeletal muscle biopsies from humans with peripheral artery disease. Monocytes were recruited to skeletal muscle during HLI and isolated monocytes produced RvD2 in a lipoxygenase-dependent manner. Exogenous RvD2 enhanced perfusion recovery in HLI and microcomputed tomography of limb vasculature revealed greater volume, with evidence of tortuous arterioles indicative of arteriogenesis (n=6-8 per group). Unlike other treatment strategies for therapeutic revascularization that exacerbate inflammation, RvD2 did not increase vascular permeability, but reduced neutrophil accumulation and the plasma levels of tumor necrosis factor-α and granulocyte macrophage colony-stimulating factor. In mice treated with RvD2, histopathologic analysis of skeletal muscle of ischemic limbs showed more regenerating myocytes with centrally located nuclei. RvD2 enhanced endothelial cell migration in a Rac-dependent manner, via its receptor, GPR18, and Gpr18-deficient mice had an endogenous defect in perfusion recovery following HLI. Importantly, RvD2 rescued defective revascularization in diabetic mice. CONCLUSIONS: RvD2 stimulates arteriogenic revascularization during HLI, suggesting that resolvins may be a novel class of mediators that both resolve inflammation and promote arteriogenesis.
Asunto(s)
Ácidos Docosahexaenoicos/uso terapéutico , Miembro Posterior/irrigación sanguínea , Isquemia/tratamiento farmacológico , Enfermedad Arterial Periférica/tratamiento farmacológico , Animales , Células Cultivadas , Estudios de Cohortes , Ácidos Docosahexaenoicos/farmacología , Femenino , Humanos , Inflamación/diagnóstico , Inflamación/tratamiento farmacológico , Inflamación/fisiopatología , Isquemia/fisiopatología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Persona de Mediana Edad , Enfermedad Arterial Periférica/diagnóstico , Enfermedad Arterial Periférica/fisiopatologíaRESUMEN
Adipose tissue metabolism is a critical regulator of adiposity and whole body energy expenditure; however, metabolic changes that occur in white adipose tissue (WAT) with obesity remain unclear. The purpose of this study was to understand the metabolic and bioenergetic changes occurring in WAT with obesity. Wild-type (C57BL/6J) mice fed a high-fat diet (HFD) showed significant increases in whole body adiposity, had significantly lower VÌ(O2), VÌ(CO2), and respiratory exchange ratios, and demonstrated worsened glucose and insulin tolerance compared with low-fat-fed mice. Metabolomic analysis of WAT showed marked changes in lipid, amino acid, carbohydrate, nucleotide, and energy metabolism. Tissue levels of succinate and malate were elevated, and metabolites that could enter the Krebs cycle via anaplerosis were mostly diminished in high-fat-fed mice, suggesting altered mitochondrial metabolism. Despite no change in basal oxygen consumption or mitochondrial DNA abundance, citrate synthase activity was decreased by more than 50%, and responses to FCCP were increased in WAT from mice fed a high-fat diet. Moreover, Pgc1a was downregulated and Cox7a1 upregulated after 6 wk of HFD. After 12 wk of high-fat diet, the abundance of several proteins in the mitochondrial respiratory chain or matrix was diminished. These changes were accompanied by increased Parkin and Pink1, decreased p62 and LC3-I, and ultrastructural changes suggestive of autophagy and mitochondrial remodeling. These studies demonstrate coordinated restructuring of metabolism and autophagy that could contribute to the hypertrophy and whitening of adipose tissue in obesity.