RESUMEN
Uncoupling proteins (UCPs) are mitochondrial inner membrane transporters that mediate free-fatty-acid-induced, purine-nucleotide-inhibited proton leak into the mitochondrial matrix, thereby uncoupling respiratory substrate oxidation from ATP synthesis. The aim of this study was to provide functional evidence that the putative Acucp gene of the free-living protozoan amoeba, A. castellanii, encodes the mitochondrial protein with uncoupling activity characteristic of UCPs and to investigate its role during oxidative stress. We report the sequencing and cloning of a complete Acucp coding sequence, its phylogenetic analysis, and the heterologous expression of AcUCP in the S. cerevisiae strain InvSc1. Measurements of mitochondrial respiratory activity and membrane potential indicate that the heterologous expression of AcUCP causes AcUCP-mediated uncoupling activity. In addition, in a model of oxidative stress with increased reactive oxygen species levels (superoxide dismutase 1 knockout yeasts), AcUCP expression strongly promotes cell survival and growth. The level of superoxide anion radicals is greatly reduced in the ΔSOD1 strain expressing AcUCP. These results suggest that AcUCP targeted to yeast mitochondria causes uncoupling and may act as an antioxidant system. Phylogenetic analysis shows that the A. castellanii UCP diverges very early from other UCPs, but clearly locates within the UCP subfamily rather than among other mitochondrial anion carrier proteins.
RESUMEN
While intrinsic changes in aging hematopoietic stem cells (HSCs) are well characterized, it remains unclear how extrinsic factors affect HSC aging. Here, we demonstrate that cells in the niche-endothelial cells (ECs) and CXCL12-abundant reticular cells (CARs)-highly express the heme-degrading enzyme, heme oxygenase 1 (HO-1), but then decrease its expression with age. HO-1-deficient animals (HO-1-/- ) have altered numbers of ECs and CARs that produce less hematopoietic factors. HSCs co-cultured in vitro with HO-1-/- mesenchymal stromal cells expand, but have altered kinetic of growth and differentiation of derived colonies. HSCs from young HO-1-/- animals have reduced quiescence and regenerative potential. Young HO-1-/- HSCs exhibit features of premature exhaustion on the transcriptional and functional level. HO-1+/+ HSCs transplanted into HO-1-/- recipients exhaust their regenerative potential early and do not reconstitute secondary recipients. In turn, transplantation of HO-1-/- HSCs to the HO-1+/+ recipients recovers the regenerative potential of HO-1-/- HSCs and reverses their transcriptional alterations. Thus, HSC-extrinsic activity of HO-1 prevents HSCs from premature exhaustion and may restore the function of aged HSCs.
Asunto(s)
Hemo-Oxigenasa 1 , Células Madre Mesenquimatosas , Animales , Diferenciación Celular , Células Endoteliales , Células Madre Hematopoyéticas , Hemo-Oxigenasa 1/genéticaRESUMEN
Melanoma-initiating cells (MICs) contribute to the tumorigenicity and heterogeneity of melanoma. MICs are identified by surface and functional markers and have been shown to display cancer stem cell (CSC) properties. However, the existence of MICs that follow the hierarchical CSC model has been questioned by studies showing that single unselected melanoma cells are highly tumorigenic in xenotransplantation assays. Herein, we characterize cells expressing MIC markers (CD20, CD24, CD133, Sca-1, ABCB1, ABCB5, ALDHhigh) in the B16-F10 murine melanoma cell line. We use flow cytometric phenotyping, single-cell sorting followed by in vitro clonogenic assays, and syngeneic in vivo serial transplantation assays to demonstrate that the expression of MIC markers does not select CSC-like cells in this cell line. Previously, our group showed that heme-degrading enzyme heme oxygenase-1 (HO-1) can be upregulated in melanoma and increase its aggressiveness. Here, we show that HO-1 activity is important for non-adherent growth of melanoma and HO-1 overexpression enhances the vasculogenic mimicry potential, which can be considered protumorigenic activity. However, HO-1 overexpression decreases clone formation in vitro and serial tumor initiation in vivo. Thus, HO-1 plays a dual role in melanoma, improving the progression of growing tumors but reducing the risk of melanoma initiation.
Asunto(s)
Hemo-Oxigenasa 1 , Melanoma Experimental , Animales , Línea Celular Tumoral , Separación Celular , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Melanoma Experimental/patología , Proteínas de la Membrana , Ratones , Células Madre Neoplásicas/metabolismoRESUMEN
RATIONALE: Transplantation-accelerated arteriosclerosis is one of the major challenges for long-term survival of patients with solid organ transplantation. Although stem/progenitor cells have been implicated to participate in this process, the cells of origin and underlying mechanisms have not been fully defined. OBJECTIVE: The objective of our study was to investigate the role of c-Kit lineage cells in allograft-induced neointima formation and to explore the mechanisms underlying this process. METHODS AND RESULTS: Using an inducible lineage tracing Kit-CreER;Rosa26-tdTomato mouse model, we observed that c-Kit is expressed in multiple cell types in the blood vessels, rather than a specific stem/progenitor cell marker. We performed allograft transplantation between different donor and recipient mice, as well as bone marrow transplantation experiments, demonstrating that recipient c-Kit+ cells repopulate neointimal smooth muscle cells (SMCs) and leukocytes, and contribute to neointima formation in an allograft transplantation model. c-Kit-derived SMCs originate from nonbone marrow tissues, whereas bone marrow-derived c-Kit+ cells mainly generate CD45+ leukocytes. However, the exact identity of c-Kit lineage cells contributing to neointimal SMCs remains unclear. ACK2 (anti-c-Kit antibody), which specifically binds and blocks c-Kit function, ameliorates allograft-induced arteriosclerosis. Stem cell factor and TGF (transforming growth factor)-ß1 levels were significantly increased in blood and neointimal lesions after allograft transplantation, by which stem cell factor facilitated c-Kit+ cell migration through the stem cell factor/c-Kit axis and downstream activation of small GTPases, MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal-regulated kinase)/MLC (myosin light chain), and JNK (c-Jun N-terminal kinase)/c-Jun signaling pathways, whereas TGF-ß1 induces c-Kit+ cell differentiation into SMCs via HK (hexokinase)-1-dependent metabolic reprogramming and a possible downstream O-GlcNAcylation of myocardin and serum response factor. CONCLUSIONS: Our findings provide evidence that recipient c-Kit lineage cells contribute to vascular remodeling in an allograft transplantation model, in which the stem cell factor/c-Kit axis is responsible for cell migration and HK-1-dependent metabolic reprogramming for SMC differentiation.
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Arteriosclerosis/terapia , Movimiento Celular , Miocitos del Músculo Liso/fisiología , Animales , Aorta/fisiología , Aorta/trasplante , Células Cultivadas , Reprogramación Celular , Ratones , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Regeneración , Factor de Células Madre/metabolismo , Túnica Íntima/citología , Túnica Íntima/fisiologíaRESUMEN
Stem/progenitor cells (SPCs) have been implicated to participate in vascular repair. However, the exact role of SPCs in endothelial repair of large vessels still remains controversial. This study aimed to delineate the cellular heterogeneity and possible functional role of endogenous vascular SPCs in large vessels. Using single-cell RNA-sequencing (scRNA-seq) and genetic lineage tracing mouse models, we uncovered the cellular heterogeneity of SPCs, i.e., c-Kit+ cells in the mouse aorta, and found that endogenous c-Kit+ cells acquire endothelial cell fate in the aorta under both physiological and pathological conditions. While c-Kit+ cells contribute to aortic endothelial turnover in the atheroprone regions during homeostasis, recipient c-Kit+ cells of nonbone marrow source replace both luminal and microvessel endothelial cells in transplant arteriosclerosis. Single-cell pseudotime analysis of scRNA-seq data and in vitro cell experiments suggest that vascular SPCs display endothelial differentiation potential and undergo metabolic reprogramming during cell differentiation, in which AKT/mTOR-dependent glycolysis is critical for endothelial gene expression. These findings demonstrate a critical role for c-Kit lineage cells in aortic endothelial turnover and replacement, and may provide insights into therapeutic strategies for vascular diseases.
Asunto(s)
Linaje de la Célula/genética , Endotelio Vascular/crecimiento & desarrollo , Análisis de la Célula Individual/métodos , Células Madre/metabolismo , Animales , Aorta/crecimiento & desarrollo , Aorta/metabolismo , Diferenciación Celular/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Humanos , Ratones , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-kit/genética , RNA-Seq , Células Madre/citología , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Whilst the survival rates of childhood acute lymphoblastic leukemia (ALL) have increased remarkably over the last decades, the therapy resistance and toxicity are still the major causes of treatment failure. It was shown that overexpression of heme oxygenase-1 (HO-1) promotes proliferation and chemoresistance of cancer cells. In humans, the HO-1 gene (HMOX1) expression is modulated by two polymorphisms in the promoter region: (GT)n-length polymorphism and single-nucleotide polymorphism (SNP) A(-413)T, with short GT repeat sequences and 413-A variants linked to an increased HO-1 inducibility. We found that the short alleles are significantly more frequent in ALL patients in comparison to the control group, and that their presence may be associated with a higher risk of treatment failure, reflecting the role of HO-1 in chemoresistance. We also observed that the presence of short alleles may predispose to develop chemotherapy-induced neutropenia. In case of SNP, the 413-T variant co-segregated with short or long alleles, while 413-A almost selectively co-segregated with long alleles, hence it is not possible to determine if SNPs are actually of phenotypic significance. Our results suggest that HO-1 can be a potential target to overcome the treatment failure in ALL patients.
Asunto(s)
Neutropenia Febril Inducida por Quimioterapia/genética , Resistencia a Antineoplásicos/genética , Hemo-Oxigenasa 1/genética , Polimorfismo de Nucleótido Simple , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Células Cultivadas , Neutropenia Febril Inducida por Quimioterapia/etiología , Niño , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Regiones Promotoras GenéticasRESUMEN
OBJECTIVE: Perivascular adipose tissue (PVAT) plays a vital role in maintaining vascular homeostasis. However, most studies ascribed the function of PVAT in vascular remodeling to adipokines secreted by the perivascular adipocytes. Whether mesenchymal stem cells exist in PVAT and play a role in vascular regeneration remain unknown. Approach and Results: Single-cell RNA-sequencing allowed direct visualization of the heterogeneous PVAT-derived mesenchymal stem cells (PV-ADSCs) at a high resolution and revealed 2 distinct subpopulations, among which one featured signaling pathways crucial for smooth muscle differentiation. Pseudotime analysis of cultured PV-ADSCs unraveled their smooth muscle differentiation trajectory. Transplantation of cultured PV-ADSCs in mouse vein graft model suggested the contribution of PV-ADSCs to vascular remodeling through smooth muscle differentiation. Mechanistically, treatment with TGF-ß1 (transforming growth factor ß1) and transfection of microRNA (miR)-378a-3p mimics induced a similar metabolic reprogramming of PV-ADSCs, including upregulated mitochondrial potential and altered lipid levels, such as increased cholesterol and promoted smooth muscle differentiation. CONCLUSIONS: Single-cell RNA-sequencing allows direct visualization of PV-ADSC heterogeneity at a single-cell level and uncovers 2 subpopulations with distinct signature genes and signaling pathways. The function of PVAT in vascular regeneration is partly attributed to PV-ADSCs and their differentiation towards smooth muscle lineage. Mechanistic study presents miR-378a-3p which is a potent regulator of metabolic reprogramming as a potential therapeutic target for vascular regeneration.
Asunto(s)
Tejido Adiposo/metabolismo , MicroARNs/genética , Músculo Liso Vascular/metabolismo , Factor de Crecimiento Transformador beta1/genética , Remodelación Vascular/genética , Adipocitos/metabolismo , Animales , Diferenciación Celular/genética , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Masculino , Células Madre Mesenquimatosas/metabolismo , Metabolómica/métodos , Ratones , Ratones Endogámicos C57BL , Consumo de Oxígeno , ARN Interferente Pequeño/genética , Distribución Aleatoria , Análisis de Secuencia de ARN , Transducción de Señal/genética , Enfermedades Vasculares/genética , Enfermedades Vasculares/metabolismoRESUMEN
Tissue-engineered vascular grafts with long-term patency are greatly needed in the clinical settings, and smooth muscle cells (SMCs) are a critical graft component. Human mesenchymal stem cells (MSCs) are used for generating SMCs, and understanding the underlying regulatory mechanisms of the MSC-to-SMC differentiation process could improve SMC generation in the clinic. Here, we found that in response to stimulation of transforming growth factor-ß1 (TGFß1), human umbilical cord-derived MSCs abundantly express the SMC markers α-smooth muscle actin (αSMA), smooth muscle protein 22 (SM22), calponin, and smooth muscle myosin heavy chain (SMMHC) at both gene and protein levels. Functionally, MSC-derived SMCs displayed contracting capacity in vitro and supported vascular structure formation in the Matrigel plug assay in vivo More importantly, SMCs differentiated from human MSCs could migrate into decellularized mouse aorta and give rise to the smooth muscle layer of vascular grafts, indicating the potential of utilizing human MSC-derived SMCs to generate vascular grafts. Of note, microRNA (miR) array analysis and TaqMan microRNA assays identified miR-503 and miR-222-5p as potential regulators of MSC differentiation into SMCs at early time points. Mechanistically, miR-503 promoted SMC differentiation by directly targeting SMAD7, a suppressor of SMAD-related, TGFß1-mediated signaling pathways. Moreover, miR-503 expression was SMAD4-dependent. SMAD4 was enriched at the miR-503 promoter. Furthermore, miR-222-5p inhibited SMC differentiation by targeting and down-regulating ROCK2 and αSMA. In conclusion, MSC differentiation into SMCs is regulated by miR-503 and miR-222-5p and yields functional SMCs for use in vascular grafts.
Asunto(s)
Prótesis Vascular , Diferenciación Celular , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , MicroARNs/genética , Músculo Liso Vascular/citología , Neovascularización Fisiológica/fisiología , Animales , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones SCID , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Transducción de Señal , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Myeloid angiogenic cells (MAC) derive from hematopoietic stem/progenitor cells (HSPCs) that are mobilized from the bone marrow. They home to sites of neovascularization and contribute to angiogenesis by production of paracrine factors. The number and function of proangiogenic cells are impaired in patients with diabetes or cardiovascular diseases. Both conditions can be accompanied by decreased levels of heme oxygenase-1 (HMOX1), cytoprotective, heme-degrading enzyme. Our study is aimed at investigating whether precursors of myeloid angiogenic cells (PACs) treated with known pharmaceuticals would produce media with better proangiogenic activity in vitro and if such media can be used to stimulate blood vessel growth in vivo. We used G-CSF-mobilized CD34+ HSPCs, FACS-sorted from healthy donor peripheral blood mononuclear cells (PBMCs). Sorted cells were predominantly CD133+. CD34+ cells after six days in culture were stimulated with atorvastatin (AT), acetylsalicylic acid (ASA), sulforaphane (SR), resveratrol (RV), or metformin (Met) for 48 h. Conditioned media from such cells were then used to stimulate human aortic endothelial cells (HAoECs) to enhance tube-like structure formation in a Matrigel assay. The only stimulant that enhanced PAC paracrine angiogenic activity was atorvastatin, which also had ability to stabilize endothelial tubes in vitro. On the other hand, the only one that induced heme oxygenase-1 expression was sulforaphane, a known activator of a HMOX1 inducer-NRF2. None of the stimulants changed significantly the levels of 30 cytokines and growth factors tested with the multiplex test. Then, we used atorvastatin-stimulated cells or conditioned media from them in the Matrigel plug in vivo angiogenic assay. Neither AT alone in control media nor conditioned media nor AT-stimulated cells affected numbers of endothelial cells in the plug or plug's vascularization. Concluding, high concentrations of atorvastatin stabilize tubes and enhance the paracrine angiogenic activity of human PAC cells in vitro. However, the effect was not observed in vivo. Therefore, the use of conditioned media from atorvastatin-treated PAC is not a promising therapeutic strategy to enhance angiogenesis.
Asunto(s)
Atorvastatina/farmacología , Medios de Cultivo Condicionados/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Antígeno AC133/metabolismo , Antígenos CD34/metabolismo , Aspirina/farmacología , Células Cultivadas , Hemo-Oxigenasa 1/metabolismo , Humanos , Inmunoensayo , Isotiocianatos/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Metformina/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Fenotipo , Resveratrol/farmacología , SulfóxidosRESUMEN
OBJECTIVE: Leptin is an adipokine initially thought to be a metabolic factor. Recent publications have shown its roles in inflammation and vascular disease, to which Sca-1+ vascular progenitor cells within the vessel wall may contribute. We sought to elucidate the effects of leptin on Sca-1+ progenitor cells migration and neointimal formation and to understand the underlying mechanisms. APPROACH AND RESULTS: Sca-1+ progenitor cells from the vessel wall of Lepr+/+ and Lepr-/- mice were cultured and purified. The migration of Lepr+/+ Sca-1+ progenitor cells in vitro was markedly induced by leptin. Western blotting and kinase assays revealed that leptin induced the activation of phosphorylated signal transducer and activator of transcription 3, phosphorylated extracellular signal-regulated kinases 1/2, pFAK (phosphorylated focal adhesion kinase), and Rac1 (ras-related C3 botulinum toxin substrate 1)/Cdc42 (cell division control protein 42 homolog). In a mouse femoral artery guidewire injury model, an increased expression of leptin in both injured vessels and serum was observed 24 hours post-surgery. RFP (red fluorescent protein)-Sca-1+ progenitor cells in Matrigel were applied to the adventitia of the injured femoral artery. RFP+ cells were observed in the intima 24 hours post-surgery, subsequently increasing neointimal lesions at 2 weeks when compared with the arteries without seeded cells. This increase was reduced by pre-treatment of Sca-1+ cells with a leptin antagonist. Guidewire injury could only induce minor neointima in Lepr-/- mice 2 weeks post-surgery. However, transplantation of Lepr+/+ Sca-1+ progenitor cells into the adventitial side of injured artery in Lepr-/- mice significantly enhanced neointimal formation. CONCLUSIONS: Upregulation of leptin levels in both the vessel wall and the circulation after vessel injury promoted the migration of Sca-1+ progenitor cells via leptin receptor-dependent signal transducer and activator of transcription 3- Rac1/Cdc42-ERK (extracellular signal-regulated kinase)-FAK pathways, which enhanced neointimal formation.
Asunto(s)
Antígenos Ly/metabolismo , Movimiento Celular , Leptina/metabolismo , Proteínas de la Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Neointima , Células Madre/metabolismo , Lesiones del Sistema Vascular/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Arteria Femoral/lesiones , Arteria Femoral/metabolismo , Arteria Femoral/patología , Quinasa 1 de Adhesión Focal/metabolismo , Predisposición Genética a la Enfermedad , Masculino , Ratones Noqueados , Músculo Liso Vascular/lesiones , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/trasplante , Neuropéptidos/metabolismo , Fenotipo , Fosforilación , Receptores de Leptina/deficiencia , Receptores de Leptina/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Trasplante de Células Madre , Células Madre/patología , Factores de Tiempo , Regulación hacia Arriba , Lesiones del Sistema Vascular/genética , Lesiones del Sistema Vascular/patología , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Mitochondrial responses under drought within Brassica genus are poorly understood. The main goal of this study was to investigate mitochondrial biogenesis of three cauliflower (Brassica oleracea var. botrytis) cultivars with varying drought tolerance. Diverse quantitative changes (decreases in abundance mostly) in the mitochondrial proteome were assessed by two-dimensional gel electrophoresis (2D PAGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Respiratory (e.g., complex II, IV (CII, CIV) and ATP synthase subunits), transporter (including diverse porin isoforms) and matrix multifunctional proteins (e.g., components of RNA editing machinery) were diversely affected in their abundance under two drought levels. Western immunoassays showed additional cultivar-specific responses of selected mitochondrial proteins. Dehydrin-related tryptic peptides (found in several 2D spots) immunopositive with dehydrin-specific antisera highlighted the relevance of mitochondrial dehydrin-like proteins for the drought response. The abundance of selected mRNAs participating in drought response was also determined. We conclude that mitochondrial biogenesis was strongly, but diversely affected in various cauliflower cultivars, and associated with drought tolerance at the proteomic and functional levels. However, discussed alternative oxidase (AOX) regulation at the RNA and protein level were largely uncoordinated due to the altered availability of transcripts for translation, mRNA/ribosome interactions, and/or miRNA impact on transcript abundance and translation.
Asunto(s)
Brassica/metabolismo , Biogénesis de Organelos , Proteoma/genética , Estrés Fisiológico , Transcriptoma , Complejos de ATP Sintetasa/genética , Complejos de ATP Sintetasa/metabolismo , Sequías , Complejo II de Transporte de Electrones/genética , Complejo II de Transporte de Electrones/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Porinas/genética , Porinas/metabolismo , Proteoma/metabolismoRESUMEN
C57BL/6 is the most often used laboratory mouse strain. However, sometimes it is beneficial to cross the transgenic mice on the C57BL/6 background to the other strain, such as FVB. Although this is a common strategy, the influence of crossing these different strains on homeostatic expression of cytokines is not known. Here we have investigated the differences in the expression of selected cytokines between C57BL/6J and C57BL/6JxFVB mice in serum and skeletal muscle. We have found that only few cytokines were altered by crossing of the strains. Concentrations of IL5, IL7, LIF, MIP-2, and IP-10 were higher in serum of C57BL/6J mice than in C57BL/6JxFVB mice, whereas concentration of G-CSF was lower in C57BL/6J. In the skeletal muscle only the concentration of VEGF was higher in C57BL/6J mice than in C57BL/6JxFVB mice. Concluding, the differences in cytokine expression upon crossing C57BL/6 and FVB strain in basal conditions are not profound.
Asunto(s)
Citocinas/sangre , Animales , Cruzamientos Genéticos , Femenino , Hemo-Oxigenasa 1/fisiología , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Especificidad de la EspecieRESUMEN
The aim of the study was to examine cross-talk interactions of soluble sugars (sucrose, glucose and fructose) and infection caused by Fusarium oxysporum f.sp. lupini on the synthesis of genistein in embryo axes of Lupinus luteus L.cv. Juno. Genistein is a free aglycone, highly reactive and with the potential to inhibit fungal infection and development of plant diseases. As signal molecules, sugars strongly stimulated accumulation of isoflavones, including genistein, and the expression of the isoflavonoid biosynthetic genes. Infection significantly enhanced the synthesis of genistein and other isoflavone aglycones in cells of embryo axes of yellow lupine with high endogenous sugar levels. The activity of ß-glucosidase, the enzyme that releases free aglycones from their glucoside bindings, was higher in the infected tissues than in the control ones. At the same time, a very strong generation of the superoxide anion radical was observed in tissues with high sugar contents already in the initial stage of infection. During later stages after inoculation, a strong generation of semiquinone radicals was observed, which level was relatively higher in tissues deficient in sugars than in those with high sugar levels. Observations of actin and tubulin cytoskeletons in cells of infected embryo axes cultured on the medium with sucrose, as well as the medium without sugar, showed significant differences in their organization.
Asunto(s)
Citoesqueleto/metabolismo , Fusarium/fisiología , Genisteína/metabolismo , Lupinus/metabolismo , Benzoquinonas/metabolismo , Vías Biosintéticas , Fructosa/metabolismo , Expresión Génica , Glucosa/metabolismo , Interacciones Huésped-Patógeno , Lupinus/citología , Lupinus/microbiología , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sacarosa/metabolismo , Superóxidos/metabolismo , Tubulina (Proteína)/metabolismo , beta-Glucosidasa/genética , beta-Glucosidasa/metabolismoRESUMEN
Heme oxygenase-1 (HO-1, HMOX1) degrades heme protecting cells from heme-induced oxidative damage. Beyond its well-established cellular functions, heme has emerged as a stabilizer of G-quadruplexes. These secondary DNA structures interfere with DNA replication. We recently revealed that nuclear HO-1 colocalizes with DNA G-quadruplexes and promotes their removal. Here, we investigate whether HO-1 safeguards cells against replication stress. Experiments were conducted in control and HMOX1-deficient HEK293T cell lines. Immunostaining unveiled that DNA G-quadruplexes accumulated in the absence of HO-1, the effect that was further enhanced in response to δ-aminolevulinic acid (ALA), a substrate in heme synthesis. This was associated with replication stress, as evidenced by an elevated proportion of stalled forks analyzed by fiber assay. We observed the same effects in hematopoietic stem cells isolated from Hmox1 knockout mice and in a lymphoblastoid cell line from an HMOX1-deficient patient. Interestingly, in the absence of HO-1, the speed of fork progression was higher, and the response to DNA conformational hindrance less stringent, indicating dysfunction of the PARP1-p53-p21 axis. PARP1 activity was not decreased in the absence of HO-1. Instead, we observed that HO-1 deficiency impairs the nuclear import and accumulation of p53, an effect dependent on the removal of excess heme. We also demonstrated that administering ALA is a more specific method for increasing intracellular free heme compared to treatment with hemin, which in turn induces strong lipid peroxidation. Our results indicate that protection against replication stress is a universal feature of HO-1, presumably contributing to its widely recognized cytoprotective activity.
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Arterias/metabolismo , Aterosclerosis/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Animales , Arterias/patología , Aterosclerosis/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Fenotipo , Placa Aterosclerótica , Transducción de Señal , Células Madre/metabolismo , Células Madre/patologíaRESUMEN
Vascular endothelium is the layer of cells that line blood vessels and serve as the primary barrier between the blood and the tissues. These cells play many important functions such as regulation of the vascular tone, blood flow and pressure control, maintaining the balance between thrombosis and fibrinolysis, participation in immune system reactions and new blood vessels formation. Disturbance in any of these functions may contribute to the development of different diseases such as for e.g. hypertension, atherosclerosis and deep vein thrombosis, as well as cancer.
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Aterosclerosis/fisiopatología , Endotelio Vascular/metabolismo , Fibrinólisis/fisiología , Hemodinámica/fisiología , Homeostasis/fisiología , Diabetes Mellitus/fisiopatología , Humanos , Hipertensión/fisiopatología , Neoplasias/fisiopatología , Trombosis de la Vena/fisiopatologíaRESUMEN
Endothelium has an immense impact on the tissue regeneration, regulation of atherosclerosis and tumour growth. Therefore, modification of endothelial cell differentiation and function seems a promising target for many therapies. MicroRNAs are small RNA molecules, which recognize and inhibit specific mRNAs. In that way, they can regulate and orchestrate whole signalling pathways. It has been shown that microRNAs can fine-tune endothelial cell functions since they have either pro- and antiangiogenic activity, regulate expression of e.g. adhesion molecules or nitric oxide synthase. Furthermore, microRNAs modulate differentiation of embryonic stem cells to endothelial cells and their further specialization towards specific vascular bed. This review focuses mainly on the influence of microRNA on the angiogenesis and endothelial cell differentiation.
Asunto(s)
Células Endoteliales/citología , Células Endoteliales/metabolismo , MicroARNs/metabolismo , Neovascularización Fisiológica/fisiología , Animales , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Células Madre Embrionarias/citología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Neoplasias/patología , Neoplasias/fisiopatología , Neovascularización Patológica/fisiopatología , Óxido Nítrico Sintasa/metabolismoRESUMEN
Slow-cycling cancer cells (SCC) contribute to the aggressiveness of many cancers, and their invasiveness and chemoresistance pose a great therapeutic challenge. However, in melanoma, their tumor-initiating abilities are not fully understood. In this study, we used the syngeneic transplantation assay to investigate the tumor-initiating properties of melanoma SCC in the physiologically relevant in vivo settings. For this we used B16-F10 murine melanoma cell line where we identified a small fraction of SCC. We found that, unlike human melanoma, the murine melanoma SCC were not marked by the high expression of the epigenetic enzyme Jarid1b. At the same time, their slow-cycling phenotype was a temporary state, similar to what was described in human melanoma. Progeny of SCC had slightly increased doxorubicin resistance and altered expression of melanogenesis genes, independent of the expression of cancer stem cell markers. Single-cell expansion of SCC revealed delayed growth and reduced clone formation when compared to non-SCC, which was further confirmed by an in vitro limiting dilution assay. Finally, syngeneic transplantation of 10 cells in vivo established that SCC were able to initiate growth in primary recipients and continue growth in the serial transplantation assay, suggesting their self-renewal nature. Together, our study highlights the high plasticity and tumorigenicity of murine melanoma SCC and suggests their role in melanoma aggressiveness.
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Melanoma Experimental , Humanos , Animales , Ratones , Trasplante Isogénico , Melanoma Experimental/genética , Melanoma Experimental/tratamiento farmacológico , Línea Celular , Proliferación CelularRESUMEN
Kidneys are pivotal organ in iron redistribution and can be severely damaged in the course of hemolysis. In our previous studies, we observed that induction of hypertension with angiotensin II (Ang II) combined with simvastatin administration results in a high mortality rate or the appearance of signs of kidney failure in heme oxygenase-1 knockout (HO-1 KO) mice. Here, we aimed to address the mechanisms underlying this effect, focusing on heme and iron metabolism. We show that HO-1 deficiency leads to iron accumulation in the renal cortex. Higher mortality of Ang II and simvastatin-treated HO-1 KO mice coincides with increased iron accumulation and the upregulation of mucin-1 in the proximal convoluted tubules. In vitro studies showed that mucin-1 hampers heme- and iron-related oxidative stress through the sialic acid residues. In parallel, knock-down of HO-1 induces the glutathione pathway in an NRF2-depedent manner, which likely protects against heme-induced toxicity. To sum up, we showed that heme degradation during heme overload is not solely dependent on HO-1 enzymatic activity, but can be modulated by the glutathione pathway. We also identified mucin-1 as a novel redox regulator. The results suggest that hypertensive patients with less active HMOX1 alleles may be at higher risk of kidney injury after statin treatment.
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Hemo-Oxigenasa 1 , Hipertensión , Ratones , Animales , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Angiotensina II/metabolismo , Mucina-1/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Simvastatina/efectos adversos , Simvastatina/metabolismo , Riñón/metabolismo , Hierro/metabolismo , Hipertensión/inducido químicamente , Hipertensión/genética , Hipertensión/metabolismo , Hemo/metabolismo , Glutatión/metabolismoRESUMEN
Retroposition, a leading mechanism for gene duplication, is an important process shaping the evolution of genomes. Retrogenes are also involved in the gene structure evolution as a major player in the process of intron deletion. Here, we demonstrate the role of retrogenes in intron gain in mammals. We identified one case of "intronization," the transformation of exonic sequences into an intron, in the primate specific retrogene RNF113B and two independent "intronization" events in the retrogene DCAF12L2, one in the common ancestor of primates and rodents and another one in the rodent lineage. Intron gain resulted from the origin of new splice variants, and both genes have two transcript forms, one with retained intron and one with the intron spliced out. Evolution of these genes, especially RNF113B, has been very dynamic and has been accompanied by several additional events including parental gene loss, secondary retroposition, and exaptation of transposable elements.