RESUMEN
BACKGROUND: Dedicator of cytokinesis 8 (DOCK8)-deficient patients have severe eczema, elevated IgE, and eosinophilia, features of atopic dermatitis (AD). OBJECTIVE: We sought to understand the mechanisms of eczema in DOCK8 deficiency. METHODS: Skin biopsy samples were characterized by histology, immunofluorescence microscopy, and gene expression. Skin barrier function was measured by transepidermal water loss. Allergic skin inflammation was elicited in mice by epicutaneous sensitization with ovalbumin (OVA) or cutaneous application of Staphylococcus aureus. RESULTS: Skin lesions of DOCK8-deficient patients exhibited type 2 inflammation, and the patients' skin was colonized by Saureus, as in AD. Unlike in AD, DOCK8-deficient patients had a reduced FOXP3:CD4 ratio in their skin lesions, and their skin barrier function was intrinsically intact. Dock8-/- mice exhibited reduced numbers of cutaneous T regulatory (Treg) cells and a normal skin barrier. Dock8-/- and mice with an inducible Dock8 deletion in Treg cells exhibited increased allergic skin inflammation after epicutaneous sensitization with OVA. DOCK8 was shown to be important for Treg cell stability at sites of allergic inflammation and for the generation, survival, and suppressive activity of inducible Treg cells. Adoptive transfer of wild-type, but not DOCK8-deficient, OVA-specific, inducible Treg cells suppressed allergic inflammation in OVA-sensitized skin of Dock8-/- mice. These mice developed severe allergic skin inflammation and elevated serum IgE levels after topical exposure to Saureus. Both were attenuated after adoptive transfer of WT but not DOCK8-deficient Treg cells. CONCLUSION: Treg cell dysfunction increases susceptibility to allergic skin inflammation in DOCK8 deficiency and synergizes with cutaneous exposure to Saureus to drive eczema in DOCK8 deficiency.
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Eccema , Factores de Intercambio de Guanina Nucleótido , Ratones Noqueados , Piel , Staphylococcus aureus , Linfocitos T Reguladores , Animales , Linfocitos T Reguladores/inmunología , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/inmunología , Eccema/inmunología , Staphylococcus aureus/inmunología , Humanos , Ratones , Piel/inmunología , Piel/patología , Femenino , Masculino , Ratones Endogámicos C57BL , Dermatitis Atópica/inmunologíaRESUMEN
Rationale: Data on the molecular mechanisms that regulate platelet-pulmonary endothelial adhesion under conditions of hypoxia are lacking, but may have important therapeutic implications. Objectives: To identify a hypoxia-sensitive, modifiable mediator of platelet-pulmonary artery endothelial cell adhesion and thrombotic remodeling. Methods: Network medicine was used to profile protein-protein interactions in hypoxia-treated human pulmonary artery endothelial cells. Data from liquid chromatography-mass spectrometry and microscale thermophoresis informed the development of a novel antibody (Ab) to inhibit platelet-endothelial adhesion, which was tested in cells from patients with chronic thromboembolic pulmonary hypertension (CTEPH) and three animal models in vivo. Measurements and Main Results: The protein NEDD9 was identified in the hypoxia thrombosome network in silico. Compared with normoxia, hypoxia (0.2% O2) for 24 hours increased HIF-1α (hypoxia-inducible factor-1α)-dependent NEDD9 upregulation in vitro. Increased NEDD9 was localized to the plasma-membrane surface of cells from control donors and patients with CTEPH. In endarterectomy specimens, NEDD9 colocalized with the platelet surface adhesion molecule P-selectin. Our custom-made anti-NEDD9 Ab targeted the NEDD9-P-selectin interaction and inhibited the adhesion of activated platelets to pulmonary artery endothelial cells from control donors in vitro and from patients with CTEPH ex vivo. Compared with control mice, platelet-pulmonary endothelial aggregates and pulmonary hypertension induced by ADP were decreased in NEDD9-/- mice or wild-type mice treated with the anti-NEDD9 Ab, which also decreased chronic pulmonary thromboembolic remodeling in vivo. Conclusions: The NEDD9-P-selectin protein-protein interaction is a modifiable target with which to inhibit platelet-pulmonary endothelial adhesion and thromboembolic vascular remodeling, with potential therapeutic implications for patients with disorders of increased hypoxia signaling pathways, including CTEPH.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Adhesión Celular/fisiología , Hipoxia/fisiopatología , Circulación Pulmonar/fisiología , Embolia Pulmonar/fisiopatología , Transducción de Señal/fisiología , Animales , Plaquetas/fisiología , Células Cultivadas/fisiología , Células Endoteliales/fisiología , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Modelos AnimalesAsunto(s)
Calcifilaxia/inducido químicamente , Pirazoles/efectos adversos , Quinoxalinas/efectos adversos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Anciano , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Neoplasias de los Conductos Biliares/genética , Biopsia , Calcifilaxia/sangre , Calcifilaxia/diagnóstico , Calcifilaxia/patología , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/genética , Femenino , Humanos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Piel/patología , MusloRESUMEN
BACKGROUND: Non-healing wounds are a major global health concern and account for the majority of non-traumatic limb amputations worldwide. However, compared to standard care practices, few advanced therapeutics effectively resolve these injuries stemming from cardiovascular disease, aging, and diabetes-related vasculopathies. While matrix turnover is disrupted in these injuries, debriding enzymes may promote healing by releasing matrix fragments that induce cell migration, proliferation, and morphogenesis, and plasma products may also stimulate these processes. Thus, we created matrix- and plasma-derived peptides, Comb1 and UN3, which induce cellular injury responses in vitro, and accelerate healing in rodent models of non-healing wounds. However, the effects of these peptides in non-healing wounds in diabetes are not known. Here, we interrogated whether these peptides stimulate healing in a diabetic porcine model highly reminiscent of human healing impairments in type 1 and type 2-diabetes. METHODS: After 3-6 weeks of streptozotocin-induced diabetes, full-thickness wounds were surgically created on the backs of adult female Yorkshire swine under general anesthesia. Comb1 and UN3 peptides or sterile saline (negative control) were administered to wounds daily for 3-7 days. Following sacrifice, wound tissues were harvested, and quantitative histological and immunohistochemical analyses were performed for wound closure, angiogenesis and granulation tissue deposition, along with quantitative molecular analyses of factors critical for angiogenesis, epithelialization, and dermal matrix remodeling. RESULTS: Comb1 and UN3 significantly increase re-epithelialization and angiogenesis in diabetic porcine wounds, compared to saline-treated controls. Additionally, fluorescein-conjugated Comb1 labels keratinocytes, fibroblasts, and vascular endothelial cells in porcine wounds, and Far western blotting reveals these cell populations express multiple fluorescein-Comb1-interacting proteins in vitro. Further, peptide treatment increases mRNA expression of several pro-angiogenic, epithelializing, and matrix-remodeling factors, importantly including balanced inductions in matrix metalloproteinase-2, -9, and tissue inhibitor of metalloproteinases-1, lending further insight into their mechanisms. CONCLUSIONS: Comb1 and UN3 stimulate wound resolution in diabetic Yorkshire swine through upregulation of multiple reparative growth factors and cytokines, especially matrix metalloproteinases and inhibitors that may aid in reversing the proteolytic imbalance characteristic of chronically inflamed non-healing wounds. Together, these peptides should have great therapeutic potential for all patients in need of healing, regardless of injury etiology.
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Diabetes Mellitus Experimental/patología , Matriz Extracelular/química , Especificidad de Órganos/efectos de los fármacos , Péptidos/sangre , Péptidos/farmacología , Cicatrización de Heridas/efectos de los fármacos , Adulto , Inductores de la Angiogénesis/metabolismo , Animales , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Quimiotaxis/efectos de los fármacos , Factor de Crecimiento Epidérmico/metabolismo , Femenino , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sus scrofaRESUMEN
Debridement, the removal of diseased, nonviable tissue, is critical for clinicians to readily assess wound status and prepare the wound bed for advanced therapeutics or downstream active healing. Removing necrotic slough and eschar through surgical or mechanical methods is less specific and may be painful for patients. Enzymatic debridement agents, such as Clostridial collagenase, selectively and painlessly degrade devitalized tissue. In addition to its debriding activities, highly-purified Clostridial collagenase actively promotes healing, and our past studies reveal that extracellular matrices digested with this enzyme yield peptides that activate cellular migratory, proliferative and angiogenic responses to injury in vitro, and promote wound closure in vivo. Intriguingly, while collagenase Santyl® ointment, a sterile preparation containing Clostridial collagenases and other non-specific proteases, is a well-accepted enzymatic debridement agent, its role as an active healing entity has never been established. Based on our previous studies of pure Clostridial collagenase, we now ask whether the mixture of enzymes contained within Santyl® produces matrix-derived peptides that promote cellular injury responses in vitro and stimulate wound closure in vivo. Here, we identify novel collagen fragments, along with collagen-associated peptides derived from thrombospondin-1, multimerin-1, fibronectin, TGFß-induced protein ig-h3 and tenascin-C, generated from Santyl® collagenase-digested human dermal capillary endothelial and fibroblastic matrices, which increase cell proliferation and angiogenic remodeling in vitro by 50-100% over controls. Using an established model of impaired healing, we further demonstrate a specific dose of collagenase from Santyl® ointment, as well as the newly-identified and chemically-synthesized ECM-derived peptides significantly increase wound re-epithelialization by 60-100% over saline-treated controls. These results not only confirm and extend our earlier studies using purified collagenase- and matrix-derived peptides to stimulate healing in vitro and in vivo, but these Santyl®-generated, matrix-derived peptides may also represent exciting new opportunities for creating advanced wound healing therapies that are enabled by enzymatic debridement and potentially go beyond debridement.
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Colagenasas/metabolismo , Matriz Extracelular/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Desbridamiento , Fibroblastos/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Colagenasa Microbiana/metabolismo , Modelos Animales , Péptidos/química , Proteolisis , Regeneración , Cicatrización de HeridasRESUMEN
Microvascular endothelial cell-mural cell interactions are instrumental in modulating both physiological and pathologic angiogenesis. Pericyte-endothelial cell communication through direct physical associations and secreted effectors comprises a bidirectional signal array that regulates vascular maturation and integrity. As endothelial cell proliferation, migration, and morphogenesis are key elements of vascular growth and remodeling during angiogenesis, we have developed novel preclinical systems for studying the roles of endothelial-mural cell dynamics on cell cycle entry and angiogenic activity in vitro. These coculture models not only enable evaluation of endothelial cell-pericyte "cross talk" but also allow for the quantitative analysis of both heterotypic contact-dependent and contact-independent cell cycle progression in either cell population, as well as angiogenic sprouting in three-dimensional vascular networks. Cells actively proliferating in two-dimensional assays can be labeled via incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into their DNA. Additionally, each cell population can be vitally labeled with a variety of cell-specific and/or membrane-permeant lipophilic dyes prior to coculture, such as DiO, or through immunofluorescence of mural or endothelial cell-specific markers after cellular fixation and/or permeabilization. Ultimately, this experimental approach can be used to investigate cellular contact-dependent and soluble mechanisms mediating mural-endothelial cell interactions, which may be instrumental in microvascular development and remodeling in vivo.
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Células Endoteliales/citología , Endotelio Vascular/citología , Neovascularización Fisiológica , Pericitos/citología , Comunicación Celular , Ciclo Celular , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Humanos , Neovascularización PatológicaRESUMEN
PURPOSE: To establish the regulatory roles that pericytes have in coordinating retinal endothelial cell (EC) growth and angiogenic potential. METHODS: Pericytes were derived from donor diabetic (DHuRP) or normal (NHuRP) human retinae, and characterized using vascular markers, coculture, contraction, morphogenesis, and proliferation assays. To investigate capillary "cross-talk," pericyte-endothelial coculture growth, and connexin-43 (Cx43) expression assays were performed. Paracrine effects were examined via treating EC with pericyte-derived conditioned media (CM) in proliferation, angiogenesis, and angiocrine assays. The effects of sphingosine 1-phosphate (S1P) were assessed using receptor antagonists. RESULTS: The DHuRP exhibit unique proliferative and morphologic properties, reflecting distinctive cytoskeletal and isoactin expression patterns. Unlike NHuRP, DHuRP are unable to sustain EC growth arrest in coculture and display reduced Cx43 expression. Further, CM from DHuRP (DPCM) markedly stimulates EC proliferation and tube formation. Treatment with S1P receptor antagonists mitigates DPCM growth-promotion in EC and S1P-mediated pericyte contraction. Angiocrine assays on normal and diabetic pericyte secretomes reveal factors involved in angiogenic control, inflammation, and metabolism. CONCLUSIONS: Effects from the diabetic microenvironment appear sustainable in cell culture: pericytes derived from diabetic donor eyes seemingly possess a "metabolic memory" in vitro, which may be linked to original donor health status. Diabetes- and pericyte-dependent effects on EC growth and angiogenesis may reflect alterations in bioactive lipid, angiocrine, and chemomechanical signaling. Altogether, our results suggest that diabetes alters pericyte contractile phenotype and cytoskeletal signaling, which ultimately may serve as a key, initiating event required for retinal endothelial reproliferation, angiogenic activation, and the pathological neovascularization accompanying proliferative diabetic retinopathy.
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Retinopatía Diabética/metabolismo , Células Endoteliales/fisiología , Neovascularización Patológica , Pericitos/fisiología , Actinas/metabolismo , Western Blotting , Proliferación Celular , Células Cultivadas , Microambiente Celular/fisiología , Técnicas de Cocultivo , Conexina 43/metabolismo , Citoesqueleto/metabolismo , Retinopatía Diabética/fisiopatología , Células Endoteliales/citología , Humanos , Lisofosfolípidos/farmacología , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Retina/citología , Esfingosina/análogos & derivados , Esfingosina/farmacologíaRESUMEN
Uncoupling protein-2 (UCP2) regulates insulin secretion by controlling ATP levels in beta-cells. Although UCP2 deficiency improves glycemic control in mice, increased expression of UCP2 interferes with glucose-stimulated insulin secretion. These observations link UCP2 to beta-cell dysfunction in type 2 diabetes with a perplexing evolutionary role. We found higher residual serum insulin levels and blunted lipid metabolic responses in fasted ucp2(-/-) mice, supporting the concept that UCP2 evolved to suppress insulin effects and to accommodate the fuel switch to fatty acids during starvation. In the absence of UCP2, fasting initially promotes peripheral lipolysis and hepatic fat accumulation at less than expected rates but culminates in protracted steatosis, indicating diminished hepatic utilization and clearance of fatty acids. We conclude that UCP2-mediated control of insulin secretion is a physiologically relevant mechanism of the metabolic response to fasting.
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Ayuno/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Hígado Graso/metabolismo , Canales Iónicos/metabolismo , Movilización Lipídica , Hígado/metabolismo , Proteínas Mitocondriales/metabolismo , Adaptación Fisiológica , Animales , Glucemia/metabolismo , Hígado Graso/genética , Hígado Graso/fisiopatología , Regulación Enzimológica de la Expresión Génica , Insulina/sangre , Canales Iónicos/deficiencia , Canales Iónicos/genética , Movilización Lipídica/genética , Hígado/enzimología , Hígado/fisiopatología , Masculino , Ratones , Ratones Noqueados , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Oxidación-Reducción , Factores de Tiempo , Proteína Desacopladora 2RESUMEN
Fatty liver is vulnerable to conditions that challenge hepatocellular energy homeostasis. Lipid-laden hepatocytes highly express uncoupling protein-2 (UCP2), a mitochondrial carrier that competes with adenosine triphosphate (ATP) synthesis by mediating proton leak. However, evidence for a link between UCP2 expression and susceptibility of liver to acute injury is lacking. We asked whether absence of UCP2 protects ob/ob mice from Fas-mediated acute liver damage. UCP2-deficient ob/ob mice (ob/ob:ucp2-/-) and UCP2-competent littermates (ob/ob:ucp2+/+) received a single dose of agonistic anti-Fas antibody (Jo2). Low-dose Jo2 (0.15 mg/kg intraperitoneally) caused less serum alanine aminotransferase (ALT) elevation and lower apoptosis rates in ob/ob:ucp2-/- mice. High-dose Jo2 (0.40 mg/kg intraperitoneally) proved uniformly fatal; however, ob/ob:ucp2-/- mice survived longer with less depletion of liver ATP stores, indicating that fatty hepatocytes may benefit from lack of UCP2 during Jo2 challenge. Although UCP2 reportedly controls mitochondrial oxidant production, its absence had no apparent effect on fatty liver tissue malondialdehyde levels augmented by Jo2. This finding prompted us to determine UCP2 expression in Kupffer cells, a major source of intrahepatic oxidative stress. UCP2 expression was found diminished in Kupffer cells of untreated ob/ob:ucp2+/+ mice, conceivably contributing to increased oxidative stress in fatty liver and limiting the impact of UCP2 ablation. In conclusion, whereas UCP2 abundance in fatty hepatocytes exacerbates Fas-mediated injury by compromising ATP stores, downregulation of UCP2 in Kupffer cells may account for persistent oxidative stress in fatty liver. Our data support a cell-specific approach when considering the therapeutic effects of mitochondrial uncoupling in fatty liver disease.