RESUMO
Our recent findings indicate that Nrf2 transcriptional activity is essential in maintaining the proper large intestinal structure in adult mice. Here, we aimed to verify whether Nrf2-related intestine abnormalities stemmed from the early weaning or gestational periods. Therefore, we analyzed 4-day-old pups and embryos devoid of Nrf2 transcriptional activity (tKO) and their wild-type counterparts. We found significant changes in the intestinal structure of 4-day-old Nrf2 tKO pups including a longer colon, altered crypt distribution, and enlargement of the goblet cells with a markedly higher level of mucin 2. Tracing back the origin of these alterations, we observed that they appeared as early as day 14.5 of embryonic development, independently of sex. Importantly, in this period, we observed a significant increase in the Nrf2 level and a distinctive, untimely pattern of expression of the proliferation factor Ki67. At the latest stage of embryonic development, we detected a premature drop in the differentiation factor Notch1. We suspect that intestine abnormalities in mice lacking Nrf2 transcriptional activity stem from sex-independent disturbed intestinal cell proliferation and could be further exacerbated by altered differentiation. Summing up, we identified Nrf2 transcriptional activity as an important regulator of intestinal formation. It influences the hindgut cell proliferation and differentiation at different stages of embryonic development.
Assuntos
Intestinos , Fator 2 Relacionado a NF-E2 , Animais , Feminino , Camundongos , Gravidez , Diferenciação Celular , Colo , Células Caliciformes/metabolismo , Mucosa Intestinal , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismoRESUMO
Targeting of the TRAIL-DR4/5 pathway was proposed as a promising approach for specific induction of apoptosis in cancer cells. Clinical trials, however, showed inadequate efficiency of TRAIL as a monotherapy. It is a widely held view that the application of multifunctional molecules or combination therapy may lead to substantial improvement. Here, we demonstrate the effectiveness and safety of a novel chimeric protein, AD-O51.4, which is a TRAIL equipped with positively charged VEGFA-derived effector peptides. The study was performed in multiple cancer cell line- and patient-derived xenografts. A pharmacokinetic profile was established in monkeys. AD-O51.4 strongly inhibits tumor growth, even leading to complete long-term tumor remission. Neither mice nor monkeys treated with AD-O51.4 demonstrate symptoms of drug toxicity. AD-O51.4 exhibits a satisfactory half-life in plasma and accumulates preferentially in tumors. The cellular mechanism of AD-O51.4 activity involves both cytotoxic effects in tumor cells and antiangiogenic effects on the endothelium. The presence of DRs in cancer cells is crucial for AD-O51.4-driven apoptosis execution. The TRAIL component of the fusion molecule serves as an apoptosis inducer and a cellular anchor for the effector peptides in TRAIL-sensitive and TRAIL-resistant cancer cells, respectively. The FADD-dependent pathway, however, seems to be not indispensable in death signal transduction; thus, AD-O51.4 is capable of bypassing the refractoriness of TRAIL. AD-O51.4-driven cell death, which exceeds TRAIL activity, is achieved due to the N-terminally fused polypeptide, containing VEGFA-derived effector peptides. The high anticancer efficiency of AD-O51.4 combined with its safety has led to the entry of AD-O51.4 into toxicological studies.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Células A549 , Animais , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Células HCT116 , Células HT29 , Células Hep G2 , Humanos , Camundongos SCID , Neoplasias/patologia , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Carga Tumoral/efeitos dos fármacosRESUMO
Endothelial dysfunction accompanied by the loss of endothelial cell phenotype plays an essential role in cardiovascular diseases. Here, we report that knockdown of biliverdin reductase (BVR), the enzyme of the heme degradation pathway converting biliverdin to bilirubin, shifts endothelial phenotype of the primary human aortic endothelial cells (HAECs) to mesenchymal-like one. It is reflected by the loss of endothelial markers and angiogenic response, with concomitant acquiring of mesenchymal markers, increased migratory capacity and metalloproteinase activity. BVR-deficiency induces the activity of Nrf2 transcription factor and increases heme oxygenase-1 (HO-1) level, which is accompanied by the reduction of cellular heme content, increase in a free iron fraction and oxidative stress. Accordingly, the phenotype of BVR-deficient cells can be mimicked by hemin or iron overload. Depletion of HO-1 in BVR-deficient ECs abrogates the increase in intracellular free iron and oxidative stress, preventing the loss of endothelial markers. Treatment of BVR-deficient cells with bilirubin does not rescue the endothelial phenotype of HAECs. Unlike BLVRA mRNA level, the expression of HMOX1, HMOX1:BLVRA ratio and HO-1 protein level positively correlate with abdominal aortic aneurysm size in clinical samples. Collectively, the non-enzymatic activity of BVR contributes to the maintenance of healthy endothelial phenotype through the prevention of HO-1-dependent iron-overload, oxidative stress and subsequent endothelial-to-mesenchymal transition (EndMT).
Assuntos
Transdiferenciação Celular/genética , Células Endoteliais/citologia , Mesoderma/citologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Técnicas de Silenciamento de Genes , Humanos , FenótipoRESUMO
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.
Assuntos
Atorvastatina/farmacologia , Meios de Cultivo Condicionados/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Antígeno AC133/metabolismo , Antígenos CD34/metabolismo , Aspirina/farmacologia , Células Cultivadas , Heme Oxigenase-1/metabolismo , Humanos , Imunoensaio , Isotiocianatos/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Metformina/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Fenótipo , Resveratrol/farmacologia , SulfóxidosRESUMO
Subcutaneous injection of the tumor cell suspension is a simple and commonly used tool for studying tumor development in vivo. However, subcutaneous models poorly resemble tumor complexity due to the fast growth not reflecting the natural course. Here, we describe an application of the new spheroid-plug model to combine the simplicity of subcutaneous injection with improved resemblance to natural tumor progression. Spheroid-plug model relies on in vitro formation of tumor spheroids, followed by injection of single tumor spheroid subcutaneously in Matrigel matrix. In spheroid-plug model, tumors grow slower in comparison to tumors formed by injection of cell suspension as assessed by 3D ultrasonography (USG) and in vivo bioluminescence measurements. The slower tumor growth rate in spheroid-plug model is accompanied by reduced necrosis. The spheroid-plug model ensures increased and more stable vascularization of tumor than classical subcutaneous tumor model as demonstrated by 3D USG Power Doppler examination. Flow cytometry analysis showed that tumors formed from spheroids have enhanced infiltration of endothelial cells as well as hematopoietic and progenitor cells with stem cell phenotype (c-Kit(+) and Sca-1(+)). They also contain more tumor cells expressing cancer stem cell marker CXCR4. Here, we show that spheroid-plug model allows investigating efficiency of anticancer drugs. Treatment of spheroid-plug tumors with known antiangiogenic agent axitinib decreased their size and viability. The antiangiogenic activity of axitinib was higher in spheroid-plug model than in classical model. Our results indicate that spheroid-plug model imitates natural tumor growth and can become a valuable tool for cancer research.
Assuntos
Carcinogênese/patologia , Neovascularização Patológica/patologia , Esferoides Celulares/patologia , Animais , Antineoplásicos/farmacologia , Axitinibe , Biomarcadores Tumorais/metabolismo , Carcinogênese/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno/metabolismo , Combinação de Medicamentos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Imidazóis/farmacologia , Indazóis/farmacologia , Injeções Subcutâneas/métodos , Laminina/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica/metabolismo , Proteoglicanas/metabolismo , Receptores CXCR4/metabolismo , Esferoides Celulares/metabolismo , Células Tumorais CultivadasRESUMO
Inflammatory bowel diseases (IBD), encompassing Crohn's disease (CD) and ulcerative colitis (UC), are intimately associated with inflammation and overproduction of reactive oxygen species (ROS). Temporal and inter-individual variabilities in disease activity and response to therapy pose significant challenges to diagnosis and patient care. Discovery and validation of truly integrative biomarkers would benefit from embracing redox metabolomics approaches with prioritization of central regulatory hubs. We here make a case for applying a personalized redox medicine approach that aims to selectively inhibit pathological overproduction and/or altered expression of specific enzymatic sources of ROS without compromising physiological function. To this end, improved 'clinical-omics integration' may help to better understand which particular redox signaling pathways are disrupted in what patient. Pharmacological interventions capable of activating endogenous antioxidant defense systems may represent viable therapeutic options to restore local/systemic redox status, with HIF-1α and NRF2 holding particular promise in this context. Achieving the implementation of clinically meaningful mechanism-based biomarkers requires development of easy-to-use, robust and cost-effective tools for secure diagnosis and monitoring of treatment efficacy. Ultimately, matching redox-directed pharmacological interventions to individual patient phenotypes using predictive biomarkers may offer new opportunities to break the therapeutic ceiling in IBD.
Assuntos
Doenças Inflamatórias Intestinais , Fator 2 Relacionado a NF-E2 , Humanos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/patologia , Oxirredução , Biomarcadores/metabolismoRESUMO
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.
Assuntos
Heme Oxigenase-1 , Hipertensão , Camundongos , Animais , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Angiotensina II/metabolismo , Mucina-1/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Sinvastatina/efeitos adversos , Sinvastatina/metabolismo , Rim/metabolismo , Ferro/metabolismo , Hipertensão/induzido quimicamente , Hipertensão/genética , Hipertensão/metabolismo , Heme/metabolismo , Glutationa/metabolismoRESUMO
BACKGROUND AND AIM: The proper functioning of the gastrointestinal system relies on an intricate crosstalk between a plethora of cell types and signaling pathways. Recently we identified that the lack of NRF2 transcriptional activity (NRF2 tKO) triggers significant colon microscopical alterations, still they do not affect the general functioning of mice. Therefore, in this study, we aimed to address the gender-dependent impact of NRF2 transcriptional deficiency on colon function, and relate them to an established model of inflammatory bowel disease (IBD). METHODS: In the study we subjected 3- and 6-month old mice deficient in IL-10 and NRF2 transcriptional activity and wild-type counterparts to tests assessing colon functionality, and histological analyses. To address the role of estrogens, we attempted to rescue the phenotype by the delivery of 17ß-estradiol through subcutaneous implants. RESULTS: In females, NRF2 transcriptional abrogation, like IL-10 deficiency, triggers a functional and microscopic phenotype, that resembles IBD. The females are significantly more affected by the dysfunctional phenotype, and the functional impairmentdecreases with age. We found that NRF2 transcriptional activity influences 17ß-estradiol level and the estrogen receptors expression and location. Exogenous delivery of 17ß-estradiol normalized colon motility in the NRF2 tKO mice, which is related to enhanced ERß signaling. CONCLUSIONS: Summing up, in this study, we underline that NRF2 transcriptional deficiency or the lack of IL-10 results in pronounced GI functional decline in young females. Mechanistically, we show that the impaired distal colon motility is dependent on ERß signaling. Targeting estrogen signaling seems a promising therapeutic strategy to counteract colonic dysfunction.
Assuntos
Estrogênios , Doenças Inflamatórias Intestinais , Animais , Feminino , Camundongos , Colo/metabolismo , Estradiol/metabolismo , Receptor beta de Estrogênio/genética , Estrogênios/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Receptores de Estrogênio/genéticaRESUMO
Endothelial cells (ECs) play a crucial role in the development and propagation of the severe COVID-19 stage as well as multiorgan dysfunction. It remains, however, controversial whether COVID-19-induced endothelial injury is caused directly by the infection of ECs with SARS-CoV-2 or via indirect mechanisms. One of the major concerns is raised by the contradictory data supporting or denying the presence of ACE2, the SARS-CoV-2 binding receptor, on the EC surface. Here, we show that primary human pulmonary artery ECs possess ACE2 capable of interaction with the viral Spike protein (S-protein) and demonstrate the crucial role of the endothelial glycocalyx in the regulation of the S-protein binding to ACE2 on ECs. Using force spectroscopy method, we directly measured ACE2- and glycocalyx-dependent adhesive forces between S-protein and ECs and characterized the nanomechanical parameters of the cells exposed to S-protein. We revealed that the intact glycocalyx strongly binds S-protein but screens its interaction with ACE2. Reduction of glycocalyx layer exposes ACE2 receptors and promotes their interaction with S-protein. These results indicate that the susceptibility of ECs to COVID-19 infection may depend on the glycocalyx condition.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Células Endoteliais/citologia , Glicocálix/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Endoteliais/metabolismo , Humanos , Ligação Proteica , Artéria Pulmonar/citologiaRESUMO
Abdominal aortic aneurysm (AAA) bears a high risk of rupture and sudden death of the patient. The pathogenic mechanisms of AAA remain elusive, and surgical intervention represents the only treatment option. Heme oxygenase-1 (HO-1), a heme degrading enzyme, is induced in AAA, both in mice and humans. HO-1 was reported to mitigate AAA development in an angiotensin II (AngII)-induced model of AAA in hyperlipidemic ApoE-/- mice. Since the role of hyperlipidaemia in the pathogenesis of AAA remains controversial, we aimed to evaluate the significance of HO-1 in the development and progression of AAA in normolipidemic animals. The experiments were performed in HO-1-deficient mice and their wild-type counterparts. We demonstrated in non-hypercholesterolemic mice that the high-dose of AngII leads to the efficient formation of AAA, which is attenuated by HO-1 deficiency. Yet, if formed, they are significantly more prone to rupture upon HO-1 shortage. Differential susceptibility to AAA formation does not rely on enhanced inflammatory response or oxidative stress. AAA-resistant mice are characterized by an increase in regulators of aortic remodeling and angiotensin receptor-2 expression, significant medial thickening, and delayed blood pressure elevation in response to AngII. To conclude, we unveil a dual role of HO-1 deficiency in AAA in normolipidemic mice, where it protects against AAA development, but exacerbates the state of formed AAA.
Assuntos
Angiotensina II/efeitos adversos , Aneurisma da Aorta Abdominal/metabolismo , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Estresse Oxidativo , Aneurisma/metabolismo , Animais , Doenças Cardiovasculares/metabolismo , Linhagem Celular , Colágeno/metabolismo , Genótipo , Humanos , Hiperlipidemias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/metabolismo , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Receptor Tipo 2 de Angiotensina/metabolismo , Serpina E2/metabolismo , Pele/metabolismo , Suínos , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Inibidor Tecidual de Metaloproteinase-2/metabolismoRESUMO
G-quadruplexes (G4) are stacked nucleic acid structures that are stabilized by heme. In cells, they affect DNA replication and gene transcription. They are unwound by several helicases but the composition of the repair complex and its heme sensitivity are unclear. We found that the accumulation of G-quadruplexes is affected by heme oxygenase-1 (Hmox1) expression, but in a cell-type-specific manner: hematopoietic stem cells (HSCs) from Hmox1-/- mice have upregulated expressions of G4-unwinding helicases (e.g., Brip1, Pif1) and show weaker staining for G-quadruplexes, whereas Hmox1-deficient murine induced pluripotent stem cells (iPSCs), despite the upregulation of helicases, have more G-quadruplexes, especially after exposure to exogenous heme. Using iPSCs expressing only nuclear or only cytoplasmic forms of Hmox1, we found that nuclear localization promotes G4 removal. We demonstrated that the proximity ligation assay (PLA) can detect cellular co-localization of G-quadruplexes with helicases, as well as with HMOX1, suggesting the potential role of HMOX1 in G4 modifications. However, this colocalization does not mean a direct interaction was detectable using the immunoprecipitation assay. Therefore, we concluded that HMOX1 influences G4 accumulation, but rather as one of the proteins regulating the heme availability, not as a rate-limiting factor. It is noteworthy that cellular G4-protein colocalizations can be quantitatively analyzed using PLA, even in rare cells.
RESUMO
Nrf2 (NFE2L2 - nuclear factor (erythroid-derived 2)-like 2) is a transcription factor, which is repressed by interaction with a redox-sensitive protein Keap1 (Kelch-like ECH-associated protein 1). Deregulation of Nrf2 transcriptional activity has been described in the pathogenesis of multiple diseases, and the Nrf2/Keap1 axis has emerged as a crucial modulator of cellular homeostasis. Whereas the significance of Nrf2 in the modulation of biological processes has been well established and broadly discussed in detail, the focus on Keap1 rarely goes beyond the regulation of Nrf2 activity and redox sensing. However, recent studies and scrutinized analysis of available data point to Keap1 as an intriguing and potent regulator of cellular function. This review aims to shed more light on Keap1 structure, interactome, regulation and non-canonical functions, thereby enhancing its significance in cell biology. We also intend to highlight the impact of balance between Keap1 and Nrf2 in the maintenance of cellular homeostasis.
Assuntos
Regulação da Expressão Gênica , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2 , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , OxirreduçãoRESUMO
Surgical intervention is currently the only option for an abdominal aortic aneurysm (AAA), preventing its rupture and sudden death of a patient. Therefore, it is crucial to determine the pathogenic mechanisms of this disease for the development of effective pharmacological therapies. Oxidative stress is said to be one of the pivotal factors in the pathogenesis of AAAs. Thus, we aimed to evaluate the significance of nuclear factor erythroid 2-related factor 2 (Nrf2) transcriptional activity in the development of AAA and to verify if simvastatin, administered as pre- and cotreatment, may counteract this structural malformation. Experiments were performed on mice with inhibited transcriptional activity of Nrf2 (tKO) and wild-type (WT) counterparts. We used a model of angiotensin II- (AngII-) induced AAA, combined with a fat-enriched diet. Mice were administered with AngII or saline for up to 28 days via osmotic minipumps. Simvastatin administration was started 7 days before the osmotic pump placement and then continued until the end of the experiment. We found that Nrf2 inactivation increased the risk of development and rupture of AAA. Importantly, these effects were reversed by simvastatin in tKO mice, but not in WT. The abrupt blood pressure rise induced by AngII was mitigated in simvastatin-treated animals regardless of the genotype. Simvastatin-affected parameters that differed between the healthy structure of the aorta and aneurysmal tissue included immune cell infiltration of the aortic wall, VCAM1 mRNA and protein level, extracellular matrix degradation, TGF-ß1 mRNA level, and ERK phosphorylation, but neither oxidative stress nor the level of Angiotensin II Type 1 Receptor (AT1R). Taken together, the inhibition of Nrf2 transcriptional activity facilitates AAA formation in mice, which can be prevented by simvastatin. It suggests that statin treatment of patients with hypercholesterolemia might have not only a beneficial effect in terms of controlling atherosclerosis but also potential AAA prevention.
Assuntos
Aneurisma da Aorta Abdominal/tratamento farmacológico , Fator 2 Relacionado a NF-E2/metabolismo , Sinvastatina/uso terapêutico , Transcrição Gênica , Angiotensina II , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Aorta/fisiopatologia , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/fisiopatologia , Pressão Sanguínea/efeitos dos fármacos , Colágeno/metabolismo , Elastina/metabolismo , Humanos , Inflamação/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
BACKGROUND: Epidemiologic studies suggest that diabetes is associated with an increased risk of cancer. Concurrently, clinical trials have shown that metformin, which is a first-line antidiabetic drug, displays anticancer activity. The underlying mechanisms for these effects are, however, still not well recognized. METHODS: Methods based on atomic force microscopy (AFM) were used to directly evaluate the influence of metformin on the nanomechanical and adhesive properties of endothelial and cancer cells in chronic hyperglycemia. AFM single-cell force spectroscopy (SCFS) was used to measure the total adhesion force and the work of detachment between EA.hy926 endothelial cells and A549 lung carcinoma cells. Nanoindentation with a spherical AFM probe provided information about the nanomechanical properties of cells, particularly the length and grafting density of the glycocalyx layer. Fluorescence imaging was used for glycocalyx visualization and monitoring of E-selectin and ICAM-1 expression. RESULTS: SCFS demonstrated that metformin attenuates adhesive interactions between EA.hy926 endothelial cells and A549 lung carcinoma cells in chronic hyperglycemia. Nanoindentation experiments, confirmed by confocal microscopy imaging, revealed metformin-induced recovery of endothelial glycocalyx length and density. The recovery of endothelial glycocalyx was correlated with a decrease in the surface expression of E-selectin and ICAM-1. CONCLUSION: Our results identify metformin-induced endothelial glycocalyx restoration as a key factor responsible for the attenuation of adhesion between EA.hy926 endothelial cells and A549 lung carcinoma cells. GENERAL SIGNIFICANCE: Metformin-induced glycocalyx restoration and the resulting attenuation of adhesive interactions between the endothelium and cancer cells may account for the antimetastatic properties of this drug.
Assuntos
Adenocarcinoma Bronquioloalveolar/tratamento farmacológico , Antineoplásicos/farmacologia , Células Endoteliais/efeitos dos fármacos , Hiperglicemia/tratamento farmacológico , Hipoglicemiantes/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Metformina/farmacologia , Células A549 , Adenocarcinoma Bronquioloalveolar/patologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Doença Crônica , Células Endoteliais/patologia , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Glicocálix/efeitos dos fármacos , Glicocálix/metabolismo , Humanos , Hiperglicemia/patologia , Neoplasias Pulmonares/patologia , Microscopia de Força AtômicaRESUMO
Premature senescence, a death escaping pathway for cells experiencing stress, is conducive to aging and cardiovascular diseases. The molecular switch between senescent and apoptotic fate remains, however, poorly recognized. Nrf2 is an important transcription factor orchestrating adaptive response to cellular stress. Here, we show that both human primary endothelial cells (ECs) and murine aortas lacking Nrf2 signaling are senescent but unexpectedly do not encounter damaging oxidative stress. Instead, they exhibit markedly increased S-nitrosation of proteins. A functional role of S-nitrosation is protection of ECs from death by inhibition of NOX4-mediated oxidative damage and redirection of ECs to premature senescence. S-nitrosation and senescence are mediated by Keap1, a direct binding partner of Nrf2, which colocalizes and precipitates with nitric oxide synthase (NOS) and transnitrosating protein GAPDH in ECs devoid of Nrf2. We conclude that the overabundance of this "unrestrained" Keap1 determines the fate of ECs by regulation of S-nitrosation and propose that Keap1/GAPDH/NOS complex may serve as an enzymatic machinery for S-nitrosation in mammalian cells.
Assuntos
Aorta/citologia , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Animais , Aorta/metabolismo , Apoptose , Linhagem Celular , Senescência Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Técnicas de Inativação de Genes , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Masculino , Camundongos , Óxido Nítrico/metabolismo , Nitrosação , Transdução de Sinais , Adulto JovemRESUMO
The breach of proteostasis, leading to the accumulation of protein aggregates, is a hallmark of ageing and age-associated disorders, up to now well-established in neurodegeneration. Few studies have addressed the issue of dysfunctional cell response to protein deposition also for the cardiovascular system. However, the molecular basis of proteostasis decline in vascular cells, as well as its relation to ageing, are not understood. Recent studies have indicated the associations of Nrf2 transcription factor, the critical modulator of cellular stress-response, with ageing and premature senescence. In this report, we outline the significance of protein aggregation in physiological and premature ageing of murine and human endothelial cells (ECs). Our study shows that aged donor-derived and prematurely senescent Nrf2-deficient primary human ECs, but not those overexpressing dominant-negative Nrf2, exhibit increased accumulation of protein aggregates. Such phenotype is also found in the aortas of aged mice and young Nrf2 tKO mice. Ageing-related loss of proteostasis in ECs depends on Keap1, well-known repressor of Nrf2, recently perceived as a key independent regulator of EC function and protein S-nitrosation (SNO). Deposition of protein aggregates in ECs is associated with impaired autophagy. It can be counteracted by Keap1 depletion, S-nitrosothiol reductant or rapamycin treatment. Our results show that Keap1:Nrf2 protein balance and Keap1-dependent SNO predominate Nrf2 transcriptional activity-driven mechanisms in governing proteostasis in ageing ECs.
Assuntos
Fator 2 Relacionado a NF-E2 , Agregados Proteicos , Envelhecimento/genética , Animais , Células Endoteliais/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse OxidativoRESUMO
AIMS: MicroRNA-378a, highly expressed in skeletal muscles, was demonstrated to affect myoblasts differentiation and to promote tumour angiogenesis. We hypothesized that miR-378a could play a pro-angiogenic role in skeletal muscle and may be involved in regeneration after ischaemic injury in mice. METHODS AND RESULTS: Silencing of miR-378a in murine C2C12 myoblasts did not affect differentiation but impaired their secretory angiogenic potential towards endothelial cells. miR-378a knockout (miR-378a-/-) in mice resulted in a decreased number of CD31-positive blood vessels and arterioles in gastrocnemius muscle. In addition, diminished endothelial sprouting from miR-378a-/- aortic rings was shown. Interestingly, although fibroblast growth factor 1 (Fgf1) expression was decreased in miR-378a-/- muscles, this growth factor did not mediate the angiogenic effects exerted by miR-378a. In vivo, miR-378a knockout did not affect the revascularization of the ischaemic muscles in both normo- and hyperglycaemic mice subjected to femoral artery ligation (FAL). No difference in regenerating muscle fibres was detected between miR-378a-/- and miR-378+/+ mice. miR-378a expression temporarily declined in ischaemic skeletal muscles of miR-378+/+ mice already on Day 3 after FAL. At the same time, in the plasma, the level of miR-378a-3p was enhanced. Similar elevation of miR-378a-3p was reported in the plasma of patients with intermittent claudication in comparison to healthy donors. Local adeno-associated viral vectors-based miR-378a overexpression was enough to improve the revascularization of the ischaemic limb of wild-type mice on Day 7 after FAL, what was not reported after systemic delivery of vectors. In addition, the number of infiltrating CD45+ cells and macrophages (CD45+ CD11b+ F4/80+ Ly6G-) was higher in the ischaemic muscles of miR-378a-/- mice, suggesting an anti-inflammatory action of miR-378a. CONCLUSIONS: Data indicate miR-378a role in the pro-angiogenic effect of myoblasts and vascularization of skeletal muscle. After the ischaemic insult, the anti-angiogenic effect of miR-378a deficiency might be compensated by enhanced inflammation.
Assuntos
Isquemia/metabolismo , MicroRNAs/metabolismo , Músculo Esquelético/irrigação sanguínea , Mioblastos Esqueléticos/metabolismo , Neovascularização Fisiológica , Regeneração , Idoso , Animais , Estudos de Casos e Controles , Linhagem Celular , Modelos Animais de Doenças , Feminino , Terapia Genética , Humanos , Claudicação Intermitente/sangue , Claudicação Intermitente/genética , Isquemia/genética , Isquemia/fisiopatologia , Isquemia/terapia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/sangue , MicroRNAs/genética , Pessoa de Meia-IdadeRESUMO
Aging is the most critical risk factor for the development of cardiovascular diseases and their complications. Therefore, the fine-tuning of cellular response to getting older is an essential target for prospective therapies in cardiovascular medicine. One of the most promising targets might be the transcription factor Nrf2, which drives the expression of cytoprotective and antioxidative genes. Importantly, Nrf2 expression correlates with potential lifespan in rodents. However, the effect of Nrf2 activity in vascular diseases might be ambiguous and strongly depend on the cell type. On the one hand, the Nrf2 activity may protect cells from oxidative stress and senescence, on the other hand, total lack of Nrf2 is protective against atherosclerosis development. Therefore, this review aims to discuss the current knowledge on the role played by the transcription factor Nrf2 in cardiovascular diseases and its potential effects on aging.
Assuntos
Envelhecimento/metabolismo , Doenças Cardiovasculares/metabolismo , Sistema Cardiovascular/metabolismo , Senescência Celular , Fator 2 Relacionado a NF-E2/metabolismo , Fatores Etários , Animais , Elementos de Resposta Antioxidante , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Doenças Cardiovasculares/fisiopatologia , Sistema Cardiovascular/patologia , Sistema Cardiovascular/fisiopatologia , Regulação da Expressão Gênica , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Transdução de SinaisRESUMO
AIMS: Nrf2 (nuclear factor erythroid 2-like 2) is a transcription factor known to modulate blood vessel formation. Various experimental settings, however, attribute to Nrf2 either stimulatory or repressive influence on angiogenesis. Our findings unveil the mechanism of Nrf2-dependent vessel formation, which reaches beyond transactivation of gene expression and reconciles previous discrepancies. RESULTS: We provide evidence that growth differentiation factor 15 (GDF-15)- and stromal cell-derived factor 1 (SDF-1)-induced angiogenesis strongly depends on the presence of Nrf2 protein but does not rely on its transcriptional activity. Instead, Nrf2 serves as a protein restraining Keap1 (Kelch-like ECH-associated protein 1), its known transcriptional repressor. Angiogenic response is abrogated in Nrf2-deficient endothelial cells but not in cells expressing dominant negative form or Keap1-binding fragment of Nrf2. Deficiency of Nrf2 protein available for Keap1 leads to the overabundance of RhoGAP1 (Rho GTPase-activating protein 1), the protein regulating cell division cycle 42 (Cdc42) activity. This impairs podosome assembly and disrupts actin rearrangements, thereby preventing angiogenesis. Effects of Nrf2 deficiency can be rescued by concomitant knockdown of RhoGAP1 or Keap1. Importantly, in the established murine model of Nrf2 deficiency, the N-terminal fragment of Nrf2 containing Keap1 binding domain is preserved. Thus, this model can be used to characterize Nrf2 as a transcription factor, but not as a Keap1-sequestering protein. Innovation and Conclusion: To date, the significance of Nrf2 in cell function has been ascribed solely to the regulation of transcription. We demonstrate that Nrf2 serves as a protein tethering Keap1 to allow podosome assembly and angiogenesis. Moreover, we emphasize that the new Nrf2 function of a Keap1 scavenger implies revisiting the interpretation of some of the previous data on the Nrf2-Keap1 system.