RESUMEN
In this study, we report the beneficial effects of a newly identified dermal cell subpopulation expressing the ATP-binding cassette subfamily B member 5 (ABCB5) for the therapy of nonhealing wounds. Local administration of dermal ABCB5+ -derived mesenchymal stem cells (MSCs) attenuated macrophage-dominated inflammation and thereby accelerated healing of full-thickness excisional wounds in the iron-overload mouse model mimicking the nonhealing state of human venous leg ulcers. The observed beneficial effects were due to interleukin-1 receptor antagonist (IL-1RA) secreted by ABCB5+ -derived MSCs, which dampened inflammation and shifted the prevalence of unrestrained proinflammatory M1 macrophages toward repair promoting anti-inflammatory M2 macrophages at the wound site. The beneficial anti-inflammatory effect of IL-1RA released from ABCB5+ -derived MSCs on human wound macrophages was conserved in humanized NOD-scid IL2rγ null mice. In conclusion, human dermal ABCB5+ cells represent a novel, easily accessible, and marker-enriched source of MSCs, which holds substantial promise to successfully treat chronic nonhealing wounds in humans. Stem Cells 2019;37:1057-1074.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Dermis/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Sobrecarga de Hierro/metabolismo , Úlcera de la Pierna/metabolismo , Células Madre Mesenquimatosas/metabolismo , Cicatrización de Heridas , Animales , Línea Celular , Dermis/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Sobrecarga de Hierro/patología , Úlcera de la Pierna/patología , Células Madre Mesenquimatosas/patología , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
The deubiquitination of histone H2A on lysine 119 by 2A-DUB/MYSM1, BAP1, USP16, and other enzymes is required for key cellular processes, including transcriptional activation, apoptosis, and cell cycle control, during normal hematopoiesis and tissue development, and in tumor cells. Based on our finding that MYSM1 colocalizes with γH2AX foci in human peripheral blood mononuclear cells, leukemia cells, and melanoma cells upon induction of DNA double-strand breaks with topoisomerase inhibitor etoposide, we applied a mass spectrometry-based proteomics approach to identify novel 2A-DUB/MYSM1 interaction partners in DNA-damage responses. Differential display of MYSM1 binding proteins significantly enriched after exposure of 293T cells to etoposide revealed an interacting network of proteins involved in DNA damage and replication, including factors associated with poor melanoma outcome. In the context of increased DNA-damage in a variety of cell types in Mysm1-deficient mice, in bone marrow cells upon aging and in UV-exposed Mysm1-deficient skin, our current mass spectrometry data provide additional evidence for an interaction between MYSM1 and key DNA replication and repair factors, and indicate a potential function of 2A-DUB/MYSM1 in DNA repair processes.
Asunto(s)
Daño del ADN , Replicación del ADN , Mapas de Interacción de Proteínas , Transactivadores/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Línea Celular Tumoral , Etopósido/toxicidad , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Proteoma/metabolismo , Proteína de Replicación C/metabolismo , Piel/metabolismo , Piel/efectos de la radiación , Transactivadores/genética , Proteasas Ubiquitina-Específicas/genética , Rayos UltravioletaRESUMEN
Histone modifications critically contribute to the epigenetic orchestration of bone homeostasis-in part, by modifying the access of transcription factors to specific genes involved in the osteogenic differentiation process of bone marrow mesenchymal stem cells (MSCs) and osteoblasts. Based on our previous finding that histone H2A deubiquitinase 2A-DUB/Mysm1 interacts with the p53 axis in hematopoiesis and tissue development, we analyzed the molecular basis of the skeletal phenotype of Mysm1-deficient mice and dissected the underlying p53-dependent and -independent mechanisms. Visible morphologic, skeletal deformations of young Mysm1-deficient mice-including a kinked and truncated tail and shortened long bones-were associated with osteopenia of long bones. On the cellular level, Mysm1-deficient primary osteoblasts displayed reduced potential to differentiate into mature osteoblasts, as indicated by decreased expression of osteogenic markers. Reduced osteogenic differentiation capacity of Mysm1-deficient osteoblasts was accompanied by an impaired induction of osteogenic transcription factor Runx2. Osteogenic differentiation of Mysm1-/- MSCs, however, was not compromised in vitro. In line with defective hematopoietic development of Mysm1-deficient mice, Mysm1-/- osteoclasts had reduced resorption activity and were more prone to apoptosis in TUNEL assays. Skeletal alterations and osteopenia of Mysm1-deficient mice were phenotypically completely rescued by simultaneous ablation of p53 in p53-/-Mysm1-/- double-deficient mice-although p53 deficiency did not restore Runx2 expression in Mysm1-/- osteoblasts on the molecular level but, instead, enhanced proliferation and osteogenic differentiation of MSCs. In summary, our results demonstrate novel roles for Mysm1 in osteoblast differentiation and osteoclast formation, resulting in osteopenia in Mysm1-deficient mice that could be abrogated by the loss of p53 from increased osteogenic differentiation of Mysm1-/-p53-/- MSCs.-Haffner-Luntzer, M., Kovtun, A., Fischer, V., Prystaz, K., Hainzl, A., Kroeger, C. M., Krikki, I., Brinker, T. J., Ignatius, A., Gatzka, M. Loss of p53 compensates osteopenia in murine Mysm1 deficiency.
Asunto(s)
Enfermedades Óseas Metabólicas/genética , Endopeptidasas/genética , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Endopeptidasas/deficiencia , Endopeptidasas/metabolismo , Ratones , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis , Transactivadores , Proteína p53 Supresora de Tumor/metabolismo , Proteasas Ubiquitina-EspecíficasRESUMEN
New biomaterials based on nanoparticles (NPs) carrying polyphenols-rich extracts (Cornus mas) recently showed promising anti-inflammatory activity in psoriasis. We aimed to understand how topically delivered silver and gold nanoparticles complexed with Cornus mas (Ag-NPs-CM, Au-NPs-CM) modulate inflammation in psoriasis at cellular and molecular level. The impact on psoriatic inflammation was assessed in vitro on pro-inflammatory macrophages, by clinical score, high-frequency ultrasonography and immunohistology of psoriasis plaques treated with Ag-NPs-CM, Au-NPs-CM or control. Incubation of pro-inflammatory macrophages with nanoparticles significantly decreased the release of NO, IL-12 and TNF-α. Immunofluorescence confirmed that nanoparticles significantly reduced CD68-positive macrophages and their IL-12 and TNF-α production in human psoriasis plaques. NPs-CM appear to repress NF-κB activation in macrophages, inhibiting the production of pro-inflammatory factors with causal role in psoriasis. Ag and Au NPs-CM represent a novel nanoparticle-based "green" technology which may provide an efficient tool for modern psoriasis therapy, circumventing immunosuppression-related side effects of biologicals.
Asunto(s)
Cornus , Oro/uso terapéutico , FN-kappa B/metabolismo , Extractos Vegetales/uso terapéutico , Psoriasis/tratamiento farmacológico , Plata/uso terapéutico , Administración Cutánea , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Células Cultivadas , Combinación de Medicamentos , Humanos , Inflamación/tratamiento farmacológico , Inflamación/etiología , Interleucina-12/metabolismo , Macrófagos/metabolismo , Nanopartículas del Metal/uso terapéutico , Óxido Nítrico/metabolismo , Pomadas , Psoriasis/complicaciones , Psoriasis/diagnóstico por imagen , Psoriasis/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , UltrasonografíaRESUMEN
Development and homeostasis of the epidermis are governed by a complex network of sequence-specific transcription factors and epigenetic modifiers cooperatively regulating the subtle balance of progenitor cell self-renewal and terminal differentiation. To investigate the role of histone H2A deubiquitinase 2A-DUB/Mysm1 in the skin, we systematically analyzed expression, developmental functions, and potential interactions of this epigenetic regulator using Mysm1-deficient mice and skin-derived epidermal cells. Morphologically, skin of newborn and young adult Mysm1-deficient mice was atrophic with reduced thickness and cellularity of epidermis, dermis, and subcutis, in context with altered barrier function. Skin atrophy correlated with reduced proliferation rates in Mysm1-/- epidermis and hair follicles, and increased apoptosis compared with wild-type controls, along with increases in DNA-damage marker γH2AX. In accordance with diminished α6-Integrinhigh+CD34⺠epidermal stem cells, reduced colony formation of Mysm1-/- epidermal progenitors was detectable in vitro. On the molecular level, we identified p53 as potential mediator of the defective Mysm1-deficient epidermal compartment, resulting in increased pro-apoptotic and anti-proliferative gene expression. In Mysm1-/-p53-/- double-deficient mice, significant recovery of skin atrophy was observed. Functional properties of Mysm1-/- developing epidermis were assessed by quantifying the transepidermal water loss. In summary, this investigation uncovers a role for 2A-DUB/Mysm1 in suppression of p53-mediated inhibitory programs during epidermal development.
Asunto(s)
Endopeptidasas/metabolismo , Epidermis/embriología , Epidermis/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/genética , Atrofia , Endopeptidasas/genética , Epidermis/patología , Expresión Génica , Genotipo , Inmunohistoquímica , Ratones , Ratones Noqueados , Células Madre/metabolismo , Transactivadores , Proteína p53 Supresora de Tumor/genética , Proteasas Ubiquitina-EspecíficasRESUMEN
Defective development and function of CD4(+)CD25(high+)Foxp3(+) regulatory T cells (Tregs) contribute to the pathogenesis of psoriasis and other autoimmune diseases. Little is known about the influence of adhesions molecules on the differentiation of Foxp3(+) Tregs into proinflammatory Th17 cells occurring in lesional skin and blood of psoriasis patients. In the CD18(hypo) PL/J mouse model of psoriasis, reduced expression of CD18/ß2 integrin to 2-16% of wild-type levels is associated with progressive loss of Tregs, impaired cell-cell contact between Tregs and dendritic cells (DCs), as well as Treg dysfunction as reported earlier. In the present investigation, Tregs derived from CD18(hypo) PL/J mice were analyzed for their propensity to differentiate into IL-17-producing Th17 cells in vivo and in in vitro Treg-DC cocultures. Adoptively transferred CD18(hypo) PL/J Tregs were more inclined toward conversion into IL-17-producing Th17 cells in vivo in an inflammatory as well as noninflammatory environment compared with CD18(wt) PL/J Tregs. Addition of neutralizing Ab against CD18 to Treg-DC cocultures in vitro promoted conversion of CD18(wt) PL/J Tregs to Th17 cells in a dose-dependent manner similar to conversion rates of CD18(hypo) PL/J Tregs. Reduced thymic output of naturally occurring Tregs and peripheral conversion of Tregs into Th17 cells therefore both contribute to the loss of Tregs and the psoriasiform dermatitis observed in CD18(hypo) PL/J mice. Our data overall indicate that CD18 expression levels impact Treg development as well as Treg plasticity and that differentiation of Tregs into IL-17-producing Th17 cells is distinctly facilitated by a subtotal deficiency of CD18.
Asunto(s)
Antígenos CD18/genética , Antígenos CD18/metabolismo , Diferenciación Celular/inmunología , Regulación hacia Abajo/inmunología , Psoriasis/inmunología , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Células Th17/patología , Animales , Diferenciación Celular/genética , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Humanos , Inmunofenotipificación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Psoriasis/genética , Psoriasis/patología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Células Th17/metabolismoRESUMEN
IL-17 is a critical factor in the pathogenesis of psoriasis and other inflammatory diseases. The impact of γδ T cells, accounting for an important source of IL-17 in acute murine IL-23- and imiquimod-induced skin inflammation, in human psoriasis is still unclear. Using the polygenic CD18(hypo) PL/J psoriasis mouse model spontaneously developing chronic psoriasiform dermatitis due to reduced CD18/ß2 integrin expression to 2-16% of wild-type levels, we investigated in this study the influence of adhesion molecule expression on generation of inflammatory γδ T cells and analyzed the occurrence of IL-17-producing γδ and CD4(+) T cells at different disease stages. Severity of CD18(hypo) PL/J psoriasiform dermatitis correlated with a loss of skin-resident Vγ5(+) T cells and concurrent skin infiltration with IL-17(+), IL-22(+), and TNF-α(+) γδTCR(low) cells preceded by increases in Vγ4(+) T cells in local lymph nodes. In vitro, reduced CD18 levels promoted expansion of inflammatory memory-type γδ T cells in response to IL-7. Similar to IL-17 or IL-23/p19 depletion, injection of diseased CD18(hypo) PL/J mice with anti-γδTCR Abs significantly reduced skin inflammation and largely eliminated pathological γδ and CD4(+) T cells. Moreover, CD18(hypo) γδ T cells induced allogeneic CD4(+) T cell responses more potently than CD18(wt) counterparts and, upon adoptive transfer, triggered psoriasiform dermatitis in susceptible hosts. These results demonstrate a novel function of reduced CD18 levels in generation of pathological γδ T cells that was confirmed by detection of increases in CD18(low) γδ T cells in psoriasis patients and may also have implications for other inflammatory diseases.
Asunto(s)
Antígenos CD18/metabolismo , Linfocitos T CD4-Positivos/inmunología , Dermatitis/inmunología , Psoriasis/inmunología , Subgrupos de Linfocitos T/inmunología , Traslado Adoptivo , Animales , Antígenos CD18/genética , Comunicación Celular , Proliferación Celular , Células Cultivadas , Enfermedad Crónica , Citocinas/inmunología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Regulación hacia Abajo , Humanos , Mediadores de Inflamación/inmunología , Ratones , Ratones Endogámicos , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismoRESUMEN
Severe angiopathy is a major driver for diabetes-associated secondary complications. Knowledge on the underlying mechanisms essential for advanced therapies to attenuate these pathologies is limited. Injection of ABCB5+ stromal precursors at the edge of nonhealing diabetic wounds in a murine db/db model, closely mirroring human type 2 diabetes, profoundly accelerates wound closure. Strikingly, enhanced angiogenesis was substantially enforced by the release of the ribonuclease angiogenin from ABCB5+ stromal precursors. This compensates for the profoundly reduced angiogenin expression in nontreated murine chronic diabetic wounds. Silencing of angiogenin in ABCB5+ stromal precursors before injection significantly reduced angiogenesis and delayed wound closure in diabetic db/db mice, implying an unprecedented key role for angiogenin in tissue regeneration in diabetes. These data hold significant promise for further refining stromal precursors-based therapies of nonhealing diabetic foot ulcers and other pathologies with impaired angiogenesis.
Asunto(s)
Diabetes Mellitus Tipo 2 , Pie Diabético , Animales , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Pie Diabético/patología , Pie Diabético/terapia , Ratones , Ratones Endogámicos , Neovascularización Patológica/patología , Ribonucleasa Pancreática , Cicatrización de HeridasRESUMEN
Fibroblasts residing in the connective tissues constitute the stem cell niche, particularly in organs such as skin. Although the effect of fibroblasts on stem cell niches and organ aging is an emerging concept, the underlying mechanisms are largely unresolved. We report a mechanism of redox-dependent activation of transcription factor JunB, which, through concomitant upregulation of p16INK4A and repression of insulin growth factor-1 (IGF-1), initiates the installment of fibroblast senescence. Fibroblast senescence profoundly disrupts the metabolic and structural niche, and its essential interactions with different stem cells thus enforces depletion of stem cells pools and skin tissue decline. In fact, silencing of JunB in a fibroblast-niche-specific manner-by reinstatement of IGF-1 and p16 levels-restores skin stem cell pools and overall skin tissue integrity. Here, we report a role of JunB in the control of connective tissue niche and identified targets to combat skin aging and associated pathologies.
Asunto(s)
Comunicación Celular , Fibroblastos/metabolismo , Envejecimiento de la Piel , Piel/metabolismo , Nicho de Células Madre , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , Senescencia Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones Noqueados , Piel/patología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo , Factores de Transcripción/genéticaRESUMEN
Histone modifying enzymes, such as histone deacetylases (HDACs) and polycomb repressive complex (PRC) components, have been implicated in regulating tumor growth, epithelial-mesenchymal transition, tumor stem cell maintenance, or repression of tumor suppressor genes - and may be promising targets for combination therapies of melanoma and other cancers. According to recent findings, the histone H2A deubiquitinase 2A-DUB/Mysm1 interacts with the p53-axis in hematopoiesis and tissue differentiation in mice, in part by modulating DNA-damage responses in stem cell and progenitor compartments. Based on the identification of alterations in skin pigmentation and melanocyte specification in Mysm1-deficient mice, we hypothesized that MYSM1 may be involved in melanoma formation. In human melanoma samples, expression of MYSM1 was increased compared with normal skin melanocytes and nevi and co-localized with melanocyte markers such as Melan-A and c-KIT. Similarly, in melanoma cell lines A375 and SK-MEL-28 and in murine skin, expression of the deubiquitinase was detectable at the mRNA and protein level that was inducible by growth factor signals and UVB exposure, respectively. Upon stable silencing of MYSM1 in A375 and SK-MEL-28 melanoma cells by lentivirally-mediated shRNA expression, survival and proliferation were significantly reduced in five MYSM1 shRNA cell lines analyzed compared with control cells. In addition, MYSM1-silenced melanoma cells proliferated less well in softagar assays. In context with our finding that MYSM1 bound to the c-MET promoter region in close vicinity to PAX3 in melanoma cells, our data indicate that MYSM1 is an epigenetic regulator of melanoma growth and potentially promising new target for tumor therapy.
RESUMEN
Photo-oxidative stress and subsequent lipid peroxidation (LPO) is one of the major mechanisms of UVA-related skin pathology. The skin's protection system against photo-oxidative stress involves low molecular scavengers as well as highly specialised antioxidant enzymes like glutathione peroxidase (GPX). Against repetitive UVA-1 exposures in vitro it is partly adaptive, as recent studies have shown exemplarily for antioxidant enzymes. We now investigated in vitro by repetitively irradiating human dermal fibroblasts with UVA-1 whether this adaptive response might reflect itself in reduced cellular membrane damage, that is, LPO. Our experiments show that the degree of cellular protection against LPO and the adaptive potential of the cells against a repetitive UVA-1 exposure varies from donor-to-donor and depends highly on glutathione.
Asunto(s)
Dermis/efectos de la radiación , Fibroblastos/efectos de la radiación , Glutatión/fisiología , Peroxidación de Lípido/efectos de la radiación , Rayos Ultravioleta , Butionina Sulfoximina/farmacología , Células Cultivadas , Interpretación Estadística de Datos , Dermis/química , Dermis/efectos de los fármacos , Dermis/metabolismo , Fibroblastos/química , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Glutatión/análisis , Glutatión Peroxidasa/fisiología , Humanos , Malondialdehído/análisis , Estrés Oxidativo , Especies Reactivas de Oxígeno/análisis , Fenómenos Fisiológicos de la PielRESUMEN
Mesenchymal stem cells (MSCs) have properties which make them promising for the treatment of chronic non-healing wounds. A major so far unmet challenge is the efficient, safe and painless delivery of MSCs to skin wounds. Recently, a surface carrier of medical-grade silicone coated by plasma polymerisation with a thin layer of acrylic acid (ppAAc) was developed, and shown to successfully deliver MSCs to deepithelialised human dermis in vitro. Here we studied the potential of the ppAAc carrier to deliver human adipose tissue derived MSCs (AT-MSCs) to murine full-thickness excisional skin wounds in vivo. Further we investigate the mechanism of action of MSCs in accelerating wound healing in these wounds. AT-MSCs cultured on ppAAc carriers for 4 days or longer fully retained their cell surface marker expression profile, colony-forming-, differentiation- and immunosuppressive potential. Importantly, AT-MSCs delivered to murine wounds by ppAAc carriers significantly accelerated wound healing, similar to AT-MSCs delivered by intradermal injection. More than 80% of AT-MSCs were transferred from carriers to wounds in 3 days. AT-MSCs were detectable in wounds for at least 5 days after wounding. Carrier delivered AT-MSCs were demonstrated to have the capacity to down-modulate TNF-α-dependent inflammation, increase anti-inflammatory M2 macrophage numbers, and induce TGF-ß(1)-dependent angiogenesis, myofibroblast differentiation and granulation tissue formation, thereby enhancing overall tissue repair.
Asunto(s)
Tejido Adiposo/citología , Células Madre Mesenquimatosas/citología , Cicatrización de Heridas/fisiología , Adipogénesis/fisiología , Animales , Western Blotting , Proliferación Celular , Supervivencia Celular/fisiología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Macrófagos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos C57BL , Osteogénesis/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Uncontrolled macrophage activation is now considered to be a critical event in the pathogenesis of chronic inflammatory diseases such as atherosclerosis, multiple sclerosis, and chronic venous leg ulcers. However, it is still unclear which environmental cues induce persistent activation of macrophages in vivo and how macrophage-derived effector molecules maintain chronic inflammation and affect resident fibroblasts essential for tissue homeostasis and repair. We used a complementary approach studying human subjects with chronic venous leg ulcers, a model disease for macrophage-driven chronic inflammation, while establishing a mouse model closely reflecting its pathogenesis. Here, we have shown that iron overloading of macrophages--as was found to occur in human chronic venous leg ulcers and the mouse model--induced a macrophage population in situ with an unrestrained proinflammatory M1 activation state. Via enhanced TNF-α and hydroxyl radical release, this macrophage population perpetuated inflammation and induced a p16(INK4a)-dependent senescence program in resident fibroblasts, eventually leading to impaired wound healing. This study provides insight into the role of what we believe to be a previously undescribed iron-induced macrophage population in vivo. Targeting this population may hold promise for the development of novel therapies for chronic inflammatory diseases such as chronic venous leg ulcers.