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1.
J Biomed Nanotechnol ; 17(9): 1874-1881, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34688333

RESUMO

The efficacy of stem cells for the treatment of renal failure is widely recognized; however, an excessive volume of stem cells can block the capillaries; thus, the potential risks should not be ignored. Stem cell exosomes are secretory extracellular vesicles with a size of 30-150 nm, which have similar functions to stem cells but are much smaller in size. This study aims to investigate the role of human umbilical cord mesenchymal stem cells (UCMSCs)-derived exosomes in the treatment of renal failure caused by ischemia-reperfusion. Fifty 8-week-old female C57 mice underwent bilateral renal ischemia-reperfusion surgery for 30 minutes. After 4 weeks, the treated group received UCMSCs-derived exosomes treatment, and the control group was solely injected with the same amount of PBS. At the age of 16 weeks, the kidney function, kidney damage, inflammatory responses and oxidative stress were measured. Moreover, the effect of UCMSCs-derived exosomes on the phenotype of M1 macrophages was also tested. The results showed that UCMSCsderived exosomes significantly reduced the levels of blood urea nitrogen (BUN), serum creatinine (SCR), and urinary albumin and creatinine (ACR) and 8-isoprostane. UCMSCs-derived exosomes also improved the atrophy of the kidney and glomerulus, decreased kidney pro-inflammatory factors expression (mRNA of II-1ß, II-6, Tnf-α, and Mcp-1) and oxidative stress (malondialdehyde), and increased glutathione level. However, F4/80 immunohistochemistry did not show significant differences between the two groups. In systemic inflammation measurement, UCMSCs-derived exosomes decreased proinflammatory factors TNF-α, IL-6, and IL-1ß levels, and increased anti-inflammatory factor IL-10 level. In vitro experiments showed that UCMSCs-derived exosomes decreased the protein expression level of TNF-α and increased the protein expression level of IL-10 in M1 macrophages. UCMSCs-derived exosomes reduce kidney inflammation and oxidative stress by improving systemic inflammation, and thus reduce kidney damage and improve kidney function. This study shows the potential application value of exosomes in the treatment of renal failure.


Assuntos
Exossomos , Células-Tronco Mesenquimais , Insuficiência Renal , Animais , Feminino , Isquemia/metabolismo , Camundongos , Estresse Oxidativo , Insuficiência Renal/metabolismo , Reperfusão , Cordão Umbilical
2.
Molecules ; 26(19)2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34641427

RESUMO

O-GlcNAcylation is a nutrient-driven post-translational modification known as a metabolic sensor that links metabolism to cellular function. Recent evidences indicate that the activation of O-GlcNAc pathway is a potential pro-survival pathway and that acute enhancement of this response is conducive to the survival of cells and tissues. 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-ß-d-pyranoside (SalA-4g), is a salidroside analogue synthesized in our laboratory by chemical structure-modification, with a phenyl ring containing a para-methoxy group and a sugar ring consisting of N-acetylglucosamine. We have previously shown that SalA-4g elevates levels of protein O-GlcNAc and improves neuronal tolerance to ischemia. However, the specific target of SalA-4g regulating O-GlcNAcylation remains unknown. To address these questions, in this study, we have focused on mitochondrial network homeostasis mediated by O-GlcNAcylation in SalA-4g's neuroprotection in primary cortical neurons under ischemic-like conditions. O-GlcNAc-modified mitochondria induced by SalA-4g demonstrated stronger neuroprotection under oxygen glucose deprivation and reoxygenation stress, including the improvement of mitochondrial homeostasis and bioenergy, and inhibition of mitochondrial apoptosis pathway. Blocking mitochondrial protein O-GlcNAcylation with OSMI-1 disrupted mitochondrial network homeostasis and antagonized the protective effects of SalA-4g. Collectively, these data demonstrate that mitochondrial homeostasis mediated by mitochondrial protein O-GlcNAcylation is critically involved in SalA-4g neuroprotection.


Assuntos
Acetilglucosamina/análogos & derivados , Metabolismo Energético , Isquemia/prevenção & controle , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/química , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Acetilglucosamina/farmacologia , Animais , Glucose/metabolismo , Glicosilação , Homeostase , Isquemia/metabolismo , Isquemia/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Oxigênio/metabolismo , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley
3.
Int J Mol Sci ; 22(17)2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34502391

RESUMO

Extracellular Cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, is released from cells upon hypoxia and cold-stress. The overall absence of extra- and intracellular CIRP is associated with increased angiogenesis, most likely induced through influencing leukocyte accumulation. The aim of the present study was to specifically characterize the role of eCIRP in ischemia-induced angiogenesis together with the associated leukocyte recruitment. For analyzing eCIRPs impact, we induced muscle ischemia via femoral artery ligation (FAL) in mice in the presence or absence of an anti-CIRP antibody and isolated the gastrocnemius muscle for immunohistological analyses. Upon eCIRP-depletion, mice showed increased capillary/muscle fiber ratio and numbers of proliferating endothelial cells (CD31+/CD45-/BrdU+). This was accompanied by a reduction of total leukocyte count (CD45+), neutrophils (MPO+), neutrophil extracellular traps (NETs) (MPO+CitH3+), apoptotic area (ascertained via TUNEL assay), and pro-inflammatory M1-like polarized macrophages (CD68+/MRC1-) in ischemic muscle tissue. Conversely, the number of regenerative M2-like polarized macrophages (CD68+/MRC1+) was elevated. Altogether, we observed that eCIRP depletion similarly affected angiogenesis and leukocyte recruitment as described for the overall absence of CIRP. Thus, we propose that eCIRP is mainly responsible for modulating angiogenesis via promoting pro-angiogenic microenvironmental conditions in muscle ischemia.


Assuntos
Isquemia/patologia , Neovascularização Fisiológica/fisiologia , Proteínas de Ligação a RNA/metabolismo , Animais , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Armadilhas Extracelulares/metabolismo , Inflamação/patologia , Isquemia/metabolismo , Contagem de Leucócitos , Leucócitos/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Músculos/metabolismo , Neutrófilos/metabolismo , Proteínas de Ligação a RNA/fisiologia
4.
J Physiol ; 599(20): 4671-4685, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34472099

RESUMO

Naked mole-rats (NMRs; Heterocephalus glaber) are among the most hypoxia-tolerant mammals. There is evidence that the NMR brain tolerates in vitro hypoxia and NMR brain mitochondria exhibit functional plasticity following in vivo hypoxia; however, if and how these organelles tolerate ischaemia and how ischaemic stress impacts mitochondrial energetics and redox regulation is entirely unknown. We hypothesized that mitochondria fundamentally contribute to in vitro ischaemia resistance in the NMR brain. To test this, we treated NMR and CD-1 mouse cortical brain sheets with an in vitro ischaemic mimic and evaluated mitochondrial respiration capacity and redox regulation following 15 or 30 min of ischaemia or ischaemia/reperfusion (I/R). We found that, relative to mice, the NMR brain largely retains mitochondrial function and redox balance post-ischaemia and I/R. Specifically: (i) ischaemia reduced complex I and II-linked respiration ∼50-70% in mice, vs. ∼20-40% in NMR brain, (ii) NMR but not mouse brain maintained relatively steady respiration control ratios and robust mitochondrial membrane integrity, (iii) electron leakage post-ischaemia was lesser in NMR than mouse brain and NMR brain retained higher coupling efficiency, and (iv) free radical generation during and following ischaemia and I/R was lower from NMR brains than mice. Taken together, our results indicate that NMR brain mitochondria are more tolerant of ischaemia and I/R than mice and retain respiratory capacity while avoiding redox derangements. Overall, these findings support the hypothesis that hypoxia-tolerant NMR brain is also ischaemia-tolerant and suggest that NMRs may be a natural model of ischaemia tolerance in which to investigate evolutionarily derived solutions to ischaemic pathology. KEY POINTS: Ischaemia is highly deleterious to the mammalian brain and this damage is largely mediated by mitochondrial dysfunction. Naked mole-rats are among the most hypoxia-tolerant mammals and their brain tolerates ischaemia ex vivo, but the impact of ischaemia on mitochondrial function is unknown. Naked mole-rat but not mouse brain mitochondria retain respiratory capacity and membrane integrity following ischaemia or ischaemia/reperfusion. Differences in free radical management and respiratory pathway control between species may mediate this tolerance. These results help us understand how natural models of hypoxia tolerance also tolerate ischaemia in the brain.


Assuntos
Mitocôndrias , Ratos-Toupeira , Animais , Encéfalo/metabolismo , Hipóxia/metabolismo , Isquemia/metabolismo , Camundongos
5.
Arterioscler Thromb Vasc Biol ; 41(11): 2756-2769, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34551592

RESUMO

Objective: Angiogenic factor AGGF1 (angiogenic factor with G-patch and FHA [Forkhead-associated] domain 1) promotes angiogenesis as potently as VEGFA (vascular endothelial growth factor A) and regulates endothelial cell (EC) proliferation, migration, specification of multipotent hemangioblasts and venous ECs, hematopoiesis, and vascular development and causes vascular disease Klippel-Trenaunay syndrome when mutated. However, the receptor for AGGF1 and the underlying molecular mechanisms remain to be defined. Approach and Results: Using functional blocking studies with neutralizing antibodies, we identified [alpha]5[beta]1 as the receptor for AGGF1 on ECs. AGGF1 interacts with [alpha]5[beta]1 and activates FAK (focal adhesion kinase), Src (proto-oncogene tyrosine-protein kinase), and AKT (protein kinase B). Functional analysis of 12 serial N-terminal deletions and 13 C-terminal deletions by every 50 amino acids mapped the angiogenic domain of AGGF1 to a domain between amino acids 604-613 (FQRDDAPAS). The angiogenic domain is required for EC adhesion and migration, capillary tube formation, and AKT activation. The deletion of the angiogenic domain eliminated the effects of AGGF1 on therapeutic angiogenesis and increased blood flow in a mouse model for peripheral artery disease. A 40-mer or 15-mer peptide containing the angiogenic domain blocks AGGF1 function, however, a 15-mer peptide containing a single amino acid mutation from -RDD- to -RGD- (a classical RGD integrin-binding motif) failed to block AGGF1 function. Conclusions: We have identified integrin [alpha]5[beta]1 as an EC receptor for AGGF1 and a novel AGGF1-mediated signaling pathway of [alpha]5[beta]1-FAK-Src-AKT for angiogenesis. Our results identify an FQRDDAPAS angiogenic domain of AGGF1 crucial for its interaction with [alpha]5[beta]1 and signaling.


Assuntos
Proteínas Angiogênicas/metabolismo , Células Endoteliais/metabolismo , Membro Posterior/irrigação sanguínea , Integrina alfa5beta1/metabolismo , Isquemia/metabolismo , Neovascularização Fisiológica , Células 3T3-L1 , Indutores da Angiogênese/farmacologia , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/farmacologia , Animais , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Feminino , Quinase 1 de Adesão Focal/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Integrina alfa5beta1/genética , Isquemia/tratamento farmacológico , Isquemia/genética , Isquemia/fisiopatologia , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais , Quinases da Família src/metabolismo
6.
Elife ; 102021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34491199

RESUMO

Extrahepatic tissues which oxidise ketone bodies also have the capacity to accumulate them under particular conditions. We hypothesised that acetyl-coenzyme A (acetyl-CoA) accumulation and altered redox status during low-flow ischaemia would support ketone body production in the heart. Combining a Langendorff heart model of low-flow ischaemia/reperfusion with liquid chromatography coupled tandem mass spectrometry (LC-MS/MS), we show that ß-hydroxybutyrate (ß-OHB) accumulated in the ischaemic heart to 23.9 nmol/gww and was secreted into the coronary effluent. Sodium oxamate, a lactate dehydrogenase (LDH) inhibitor, increased ischaemic ß-OHB levels 5.3-fold and slowed contractile recovery. Inhibition of ß-hydroxy-ß-methylglutaryl (HMG)-CoA synthase (HMGCS2) with hymeglusin lowered ischaemic ß-OHB accumulation by 40%, despite increased flux through succinyl-CoA-3-oxaloacid CoA transferase (SCOT), resulting in greater contractile recovery. Hymeglusin also protected cardiac mitochondrial respiratory capacity during ischaemia/reperfusion. In conclusion, net ketone generation occurs in the heart under conditions of low-flow ischaemia. The process is driven by flux through both HMGCS2 and SCOT, and impacts on cardiac functional recovery from ischaemia/reperfusion.


Assuntos
Ácido 3-Hidroxibutírico/metabolismo , Coração/fisiologia , Isquemia/metabolismo , Animais , Cromatografia Líquida , Ciclo do Ácido Cítrico , Hidroximetilglutaril-CoA Sintase , Corpos Cetônicos , Masculino , Mitocôndrias , Isquemia Miocárdica , Miócitos Cardíacos , Oxirredução , Ratos , Ratos Wistar , Espectrometria de Massas em Tandem
7.
Cells ; 10(7)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34359994

RESUMO

Mitochondria are key players of aerobic respiration and the production of adenosine triphosphate and constitute the energetic core of eukaryotic cells. Furthermore, cells rely upon mitochondria homeostasis, the disruption of which is reported in pathological processes such as liver hepatotoxicity, cancer, muscular dystrophy, chronic inflammation, as well as in neurological conditions including Alzheimer's disease, schizophrenia, depression, ischemia and glaucoma. In addition to the well-known spontaneous cell-to-cell transfer of mitochondria, a therapeutic potential of the transplant of isolated, metabolically active mitochondria has been demonstrated in several in vitro and in vivo experimental models of disease. This review explores the striking outcomes achieved by mitotherapy thus far, and the most relevant underlying data regarding isolated mitochondria transplantation, including mechanisms of mitochondria intake, the balance between administration and therapy effectiveness, the relevance of mitochondrial source and purity and the mechanisms by which mitotherapy is gaining ground as a promising therapeutic approach.


Assuntos
Doença de Alzheimer/terapia , Depressão/terapia , Glaucoma/terapia , Hepatite/terapia , Isquemia/terapia , Mitocôndrias/transplante , Distrofias Musculares/terapia , Neoplasias/terapia , Esquizofrenia/terapia , Trifosfato de Adenosina/biossíntese , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Depressão/genética , Depressão/metabolismo , Depressão/patologia , Modelos Animais de Doenças , Glaucoma/genética , Glaucoma/metabolismo , Glaucoma/patologia , Hepatite/genética , Hepatite/metabolismo , Hepatite/patologia , Humanos , Isquemia/genética , Isquemia/metabolismo , Isquemia/patologia , Fígado/metabolismo , Fígado/patologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação Oxidativa , Esquizofrenia/genética , Esquizofrenia/metabolismo , Esquizofrenia/patologia , Resultado do Tratamento
8.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445616

RESUMO

Neovascularization restores blood flow recovery after ischemia in peripheral arterial disease. The main two components of neovascularization are angiogenesis and arteriogenesis. Both of these processes contribute to functional improvements of blood flow after occlusion. However, discriminating between the specific contribution of each process is difficult. A frequently used model for investigating neovascularization is the murine hind limb ischemia model (HLI). With this model, it is difficult to determine the role of angiogenesis, because usually the timing for the sacrifice of the mice is chosen to be optimal for the analysis of arteriogenesis. More importantly, the occurring angiogenesis in the distal calf muscles is probably affected by the proximally occurring arteriogenesis. Therefore, to understand and subsequently intervene in the process of angiogenesis, a model is needed which investigates angiogenesis without the influence of arteriogenesis. In this study we evaluated the in vivo Matrigel plug assay in genetic deficient mice to investigate angiogenesis. Mice deficient for interferon regulatory factor (IRF)3, IRF7, RadioProtective 105 (RP105), Chemokine CC receptor CCR7, and p300/CBP-associated factor (PCAF) underwent the in vivo Matrigel model. Histological analysis of the Matrigel plugs showed an increased angiogenesis in mice deficient of IRF3, IRF7, and RP105, and a decreased angiogenesis in PCAF deficient mice. Our results also suggest an involvement of CCR7 in angiogenesis. Comparing our results with results of the HLI model found in the literature suggests that the in vivo Matrigel plug assay is superior in evaluating the angiogenic response after ischemia.


Assuntos
Antígenos CD/fisiologia , Membro Posterior/irrigação sanguínea , Fator Regulador 3 de Interferon/fisiologia , Fator Regulador 7 de Interferon/fisiologia , Isquemia/patologia , Neovascularização Patológica/patologia , Fatores de Transcrição de p300-CBP/fisiologia , Animais , Colágeno , Combinação de Medicamentos , Membro Posterior/patologia , Isquemia/metabolismo , Laminina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/metabolismo , Proteoglicanas , Recuperação de Função Fisiológica
9.
Ceska Slov Farm ; 70(3): 102-108, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34418947

RESUMO

In this experimental study, the effects of the combined herbal drug Pancreo-Plant® at a dose of 72 mg/kg and the comparison drug silymarin at a dose of 25 mg/ kg on animal mortality, cytolysis activity, free radical oxidation, and functional activity of the liver in the conditions of acute experimental ischemia have been studied. A pronounced antioxidant effect of the studied agent has been found which was manifested in the reduction of the lipid peroxidation products content, namely thiobarbituric acid products and diene conjugates and normalization of the enzymatic and non-enzymatic chains of endogenous antioxidant protection (reduced glutathione, catalase). In the case of acute liver failure, Pancreo-Plant® exhibited a significant anti-cytolytic effect, restored carbohydrate metabolism and protein-synthetic function of the liver. It was found that the total hepatoprotective activity of the combined herbal drug Pancreo-Plant® exceeded the activity of the comparison drug silymarin.


Assuntos
Antioxidantes , Silimarina , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Isquemia/metabolismo , Peroxidação de Lipídeos , Fígado , Silimarina/metabolismo , Silimarina/farmacologia
10.
Angew Chem Int Ed Engl ; 60(39): 21351-21359, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34228388

RESUMO

The imbalance of Cu+ and Cu2+ in the brain is closely related to neurodegenerative diseases. However, it still lacks of effective analytical methods for simultaneously determining the concentrations of Cu+ and Cu2+ . Herein, we created a novel SERS probe (CuSP) to real-time track and accurately quantify extracellular concentrations of Cu+ and Cu2+ in the live brain. The present CuSP probe demonstrated specific ability for recognition of Cu+ and Cu2+ in a dual-recognition mode. Then, a microarray consisting of 8 CuSP probes with high tempo-spatial resolution and good accuracy was constructed for tracking and simultaneously biosensing of Cu+ and Cu2+ in the cerebral cortex of living brain. Using our powerful tool, it was found that that the concentrations of Cu2+ and Cu+ were increased by ≈4.26 and ≈1.80 times upon ischemia, respectively. Three routes were first discovered for understanding the mechanisms of the increased concentrations of Cu+ and Cu2+ during ischemia.


Assuntos
Técnicas Biossensoriais , Encéfalo/diagnóstico por imagem , Cobre/análise , Isquemia/diagnóstico por imagem , Animais , Encéfalo/metabolismo , Cobre/metabolismo , Íons/análise , Íons/metabolismo , Isquemia/metabolismo , Camundongos , Análise Espectral Raman
11.
Sci Rep ; 11(1): 15313, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321516

RESUMO

Ischemic retinal dystrophies are leading causes of acquired vision loss. Although the dysregulated expression of the hypoxia-responsive VEGF-A is a major driver of ischemic retinopathies, implication of additional VEGF-family members in their pathogenesis has led to the development of multivalent anti-angiogenic tools. Designed as a decoy receptor for all ligands of VEGFR1 and VEGFR2, Aflibercept is a potent anti-angiogenic agent. Notwithstanding, the molecular mechanisms mediating Aflibercept's efficacy remain only partially understood. Here, we used the oxygen-induced retinopathy (OIR) mouse as a model system of pathological retinal vascularization to investigate the transcriptional response of the murine retina to hypoxia and of the OIR retina to Aflibercept. While OIR severely impaired transcriptional changes normally ensuing during retinal development, analysis of gene expression patterns hinted at alterations in leukocyte recruitment during the recovery phase of the OIR protocol. Moreover, the levels of Angiopoietin-2, a major player in the progression of diabetic retinopathy, were elevated in OIR tissues and consistently downregulated by Aflibercept. Notably, GO term, KEGG pathway enrichment, and expression dynamics analyses revealed that, beyond regulating angiogenic processes, Aflibercept also modulated inflammation and supported synaptic transmission. Altogether, our findings delineate novel mechanisms potentially underlying Aflibercept's efficacy against ischemic retinopathies.


Assuntos
Inibidores da Angiogênese/farmacologia , Proteínas do Olho/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Isquemia/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Retina/efeitos dos fármacos , Vasos Retinianos , Inibidores da Angiogênese/uso terapêutico , Animais , Quimiotaxia de Leucócito/genética , Retinopatia Diabética , Modelos Animais de Doenças , Metabolismo Energético/genética , Proteínas do Olho/genética , Ontologia Genética , Redes Reguladoras de Genes , Isquemia/genética , Isquemia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/genética , Oxigênio/metabolismo , Oxigênio/toxicidade , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Retina/metabolismo , Retinopatia da Prematuridade , Transcrição Genética/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/fisiologia
12.
FASEB J ; 35(8): e21765, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34318967

RESUMO

The bioactive lipid intermediate palmitoyl CoA (PCoA) can inhibit mitochondrial ADP/ATP transport, though the physiological relevance of this regulation remains unclear. We questioned whether myocardial ischemia provides a pathological setting in which PCoA regulation of ADP/ATP transport would be beneficial, and secondly, whether the chronically elevated lipid content within the diabetic heart could make mitochondria less sensitive to the effects of PCoA. PCoA acutely decreased ADP-stimulated state 3 respiration and increased the apparent Km for ADP twofold. The half maximal inhibitory concentration (IC50 ) of PCoA in control mitochondria was 22 µM. This inhibitory effect of PCoA on respiration was blunted in diabetic mitochondria, with no significant difference in the Km for ADP in the presence of PCoA, and an increase in the IC50 to 32 µM PCoA. The competitive inhibition by PCoA was localised to the phosphorylation apparatus, particularly the ADP/ATP carrier (AAC). During ischemia, the AAC imports ATP into the mitochondria, where it is hydrolysed by reversal of the ATP synthase, regenerating the membrane potential. Addition of PCoA dose-dependently prevented this wasteful ATP hydrolysis for membrane repolarisation during ischemia, however, this beneficial effect was blunted in diabetic mitochondria. Finally, using 31 P-magnetic resonance spectroscopy we demonstrated that diabetic hearts lose ATP more rapidly during ischemia, with a threefold higher ATP decay rate compared with control hearts. In conclusion, PCoA plays a role in protecting mitochondrial energetics during ischemia, by preventing wasteful ATP hydrolysis. However, this beneficial effect is blunted in diabetes, contributing to the impaired energy metabolism seen during myocardial ischemia in the diabetic heart.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Isquemia , Mitocôndrias Cardíacas/metabolismo , Miocárdio , Palmitoil Coenzima A , Trifosfato de Adenosina/metabolismo , Animais , Respiração Celular , Metabolismo Energético , Isquemia/metabolismo , Isquemia/patologia , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Consumo de Oxigênio , Palmitoil Coenzima A/farmacologia , Palmitoil Coenzima A/fisiologia , Ratos , Ratos Wistar
13.
Molecules ; 26(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200394

RESUMO

Zinc is an effective anti-inflammatory and antioxidant trace element. The aim of this study was to analyse the protective effect of zinc and zinc-prolactin systems as additives of preservation solutions in the prevention of nephron damage caused during ischemia. The study used a model for storing isolated porcine kidneys in Biolasol®. The solution was modified with the addition of Zn at a dose of 1 µg/L and Zn: 1 µg/L with prolactin (PRL): 0.1 µg/L. After 2 h and 48 h of storage, the levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, sodium, potassium, creatinine and total protein were determined. Zinc added to the Biolasol® composition at a dose of 1 µg/L showed minor effectiveness in the protection of nephrons. In turn, Zn2+ added to Biolasol + PRL (PRL: 0.1 µg/L) acted as a prolactin inhibitor. We do not recommend the addition of Zn(II) (1 µg/L) and Zn(II) (1 µg/L) + PRL (0.1 µg/L) to the Biolasol solution.


Assuntos
Isquemia/metabolismo , Rim/metabolismo , Prolactina/metabolismo , Zinco/metabolismo , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Creatinina/metabolismo , Criopreservação/métodos , L-Lactato Desidrogenase/metabolismo , Preservação de Órgãos/métodos , Perfusão/métodos , Potássio/metabolismo , Sódio/metabolismo , Suínos
14.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202056

RESUMO

Mesenchymal stem cells (MSC) are known for their vascular regeneration capacity by neoangiogenesis. Even though, several delivery approaches exist, particularly in the case of intravascular delivery, only limited number of cells reach the targeted tissue and are not able to remain on site. Applicated cells exhibit poor survival accompanied with a loss of functionality. Moreover, cell application techniques lead to cell death and impede the overall MSC function and survival. 3D cell spheroids mimic the physiological microenvironment, thus, overcoming these limitations. Therefore, in this study we aimed to evaluate and assess the feasibility of 3D MSCs spheroids for endovascular application, for treatment of ischemic peripheral vascular pathologies. Multicellular 3D MSC spheroids were generated at different cell seeding densities, labelled with ultra-small particles of iron oxide (USPIO) and investigated in vitro in terms of morphology, size distribution, mechanical stability as well as ex vivo with magnetic resonance imaging (MRI) to assess their trackability and distribution. Generated 3D spheroids were stable, viable, maintained stem cell phenotype and were easily trackable and visualized via MRI. MSC 3D spheroids are suitable candidates for endovascular delivery approaches in the context of ischemic peripheral vascular pathologies.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Esferoides Celulares , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Humanos , Isquemia/diagnóstico , Isquemia/etiologia , Isquemia/metabolismo , Isquemia/terapia , Imageamento por Ressonância Magnética , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Doença Arterial Periférica/diagnóstico , Doença Arterial Periférica/etiologia , Doença Arterial Periférica/metabolismo , Doença Arterial Periférica/terapia , Esferoides Celulares/citologia , Esferoides Celulares/ultraestrutura , Coloração e Rotulagem
15.
Biophys J ; 120(16): 3261-3271, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34297964

RESUMO

Mitochondria exhibit unstable inner membrane potentials (ΔΨm) when subjected to stress, such as during ischemia/reperfusion (I/R). Understanding the mechanism of ΔΨm instability involves characterizing and quantifying this phenomenon in an unbiased and reproducible manner. Here, we describe a simple analytical workflow called "MitoWave" that combines wavelet transform methods and image segmentation to unravel dynamic ΔΨm changes in the cardiac mitochondrial network during I/R. In vitro ischemia was affected by placing a glass coverslip on a monolayer of neonatal mouse ventricular myocytes for 1 h and removing the coverslip to allow for reperfusion, revealing complex oscillatory ΔΨm. MitoWave analysis was then used to identify individual mitochondrial clusters within the cells and track their intrinsic oscillation frequencies over the course of reperfusion. Responses segregated into five typical behaviors were quantified by MitoWave that were corroborated by visual inspection of the time series. Statistical analysis of the distribution of oscillating mitochondrial clusters during reperfusion showed significant differences between the five different outcomes. Features such as the time point of ΔΨm depolarization during I/R, area of mitochondrial clusters, and time-resolved frequency components during reperfusion were determined per cell and per mitochondrial cluster. Mitochondria from neonatal mouse ventricular myocytes subjected to I/R oscillate in the frequency range of 8.6-45 mHz, with a mean of 8.73 ± 4.35 mHz. Oscillating clusters had smaller areas ranging from 49.8 ± 1.2 µm2, whereas nonoscillating clusters had larger areas 66 ± 1.5 µm2. A negative correlation between frequency and mitochondrial cluster area was observed. We also observed that late ΔΨm loss during ischemia correlated with early ΔΨm stabilization after oscillation on reperfusion. Thus, MitoWave analysis provides a semiautomated method to quantify complex time-resolved mitochondrial behavior in an easy-to-follow workflow, enabling unbiased, reproducible quantitation of complex nonstationary cellular phenomena.


Assuntos
Isquemia , Miócitos Cardíacos , Animais , Isquemia/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Reperfusão , Análise Espaço-Temporal
16.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298892

RESUMO

One of the most important mechanisms of preconditioning-mediated neuroprotection is the attenuation of cell apoptosis, inducing brain tolerance after a subsequent injurious ischemia. In this context, the antiapoptotic PI3K/AKT signaling pathway plays a key role by regulating cell differentiation and survival. Active AKT is known to increase the expression of murine double minute-2 (MDM2), an E3-ubiquitin ligase that destabilizes p53 to promote the survival of cancer cells. In neurons, we recently showed that the MDM2-p53 interaction is potentiated by pharmacological preconditioning, based on subtoxic stimulation of NMDA glutamate receptor, which prevents ischemia-induced neuronal apoptosis. However, whether this mechanism contributes to the neuronal tolerance during ischemic preconditioning (IPC) is unknown. Here, we show that IPC induced PI3K-mediated phosphorylation of AKT at Ser473, which in turn phosphorylated MDM2 at Ser166. This phosphorylation triggered the nuclear stabilization of MDM2, leading to p53 destabilization, thus preventing neuronal apoptosis upon an ischemic insult. Inhibition of the PI3K/AKT pathway with wortmannin or by AKT silencing induced the accumulation of cytosolic MDM2, abrogating IPC-induced neuroprotection. Thus, IPC enhances the activation of PI3K/AKT signaling pathway and promotes neuronal tolerance by controlling the MDM2-p53 interaction. Our findings provide a new mechanistic pathway involved in IPC-induced neuroprotection via modulation of AKT signaling, suggesting that AKT is a potential therapeutic target against ischemic injury.


Assuntos
Isquemia/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/fisiologia , Células HEK293 , Humanos , Precondicionamento Isquêmico/métodos , Camundongos , Camundongos Endogâmicos C57BL , Neuroproteção/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/fisiologia , Wortmanina/metabolismo
17.
Am J Physiol Regul Integr Comp Physiol ; 321(2): R112-R124, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34075808

RESUMO

Preeclampsia (PE) is characterized by maternal hypertension, intrauterine growth restriction, and increased cytolytic natural killer cells (cNKs), which secrete interferon γ (IFNγ). However, the precise role of IFNγ in contributing to PE pathophysiology remains unclear. Using the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia, we tested the hypothesis that neutralization of IFNγ in RUPPs will decrease placental reactive oxygen species (ROS) and improve vascular function resulting in decreased MAP and improved fetal growth. On gestation day (GD) 14, the RUPP procedure was performed and on GDs 15 and 18, a subset of normal pregnant rats (NP) and RUPP rats were injected with 10 µg/kg of an anti-rat IFNγ monoclonal antibody. On GD 18, uterine artery resistance index (UARI) was measured via Doppler ultrasound and on GD 19, mean arterial pressure (MAP) was measured, animals were euthanized, and blood and tissues were collected for analysis. Increased MAP was observed in RUPP rats compared with NP and was reduced in RUPP + anti-IFNγ. Placental ROS was also increased in RUPP rats compared with NP rats and was normalized in RUPP + anti-IFNγ. Fetal and placental weights were reduced in RUPP rats, but were not improved following anti-IFNγ treatment. However, UARI was elevated in RUPP compared with NP rats and was reduced in RUPP + anti-IFNγ. In conclusion, we observed that IFNγ neutralization reduced MAP, UARI, and placental ROS in RUPP recipients. These data suggest that IFNγ is a potential mechanism by which cNKs contribute to PE pathophysiology and may represent a therapeutic target to improve maternal outcomes in PE.


Assuntos
Anticorpos Monoclonais/farmacologia , Pressão Arterial/efeitos dos fármacos , Interferon gama/antagonistas & inibidores , Células Matadoras Naturais/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Placenta/irrigação sanguínea , Placenta/efeitos dos fármacos , Pré-Eclâmpsia/prevenção & controle , Artéria Uterina/efeitos dos fármacos , Resistência Vascular/efeitos dos fármacos , Proteínas Angiogênicas/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/fisiopatologia , Retardo do Crescimento Fetal/prevenção & controle , Interferon gama/metabolismo , Isquemia/metabolismo , Isquemia/fisiopatologia , Células Matadoras Naturais/metabolismo , Placenta/metabolismo , Circulação Placentária , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/fisiopatologia , Gravidez , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Artéria Uterina/metabolismo , Artéria Uterina/fisiopatologia
18.
J Physiol ; 599(16): 3853-3878, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34159610

RESUMO

KEY POINTS: Females have lower fatigability than males during single limb isometric and dynamic contractions, but whether sex-differences exist during high-intensity whole-body exercise remains unknown. This study shows that males and females respond similarly to repeated supramaximal whole-body exercise, and that at task failure a large functional reserve remains in both sexes. Using post-exercise ischaemia with repeated exercise, we have shown that this functional reserve depends on the glycolytic component of substrate-level phosphorylation and is almost identical in both sexes. Metaboreflex activation during post-exercise ischaemia and the O2 debt per kg of active lean mass are also similar in males and females after supramaximal exercise. Females have a greater capacity to extract oxygen during repeated supramaximal exercise and reach lower P ETC O 2 , experiencing a larger drop in brain oxygenation than males, without apparent negative repercussion on performance. Females had no faster recovery of performance after accounting for sex differences in lean mass. ABSTRACT: The purpose of this study was to ascertain what mechanisms explain sex differences at task failure and to determine whether males and females have a functional reserve at exhaustion. Exercise performance, cardiorespiratory variables, oxygen deficit, and brain and muscle oxygenation were determined in 18 males and 18 females (21-36 years old) in two sessions consisting of three bouts of constant-power exercise at 120% of V ̇ O 2 max until exhaustion interspaced by 20 s recovery periods. In one of the two sessions, the circulation of both legs was occluded instantaneously (300 mmHg) during the recovery periods. Females had a higher muscle O2 extraction during fatiguing supramaximal exercise than males. Metaboreflex activation, and lean mass-adjusted O2 deficit and debt were similar in males and females. Compared to males, females reached lower P ETC O 2 and brain oxygenation during supramaximal exercise, without apparent negative consequences on performance. After the occlusions, males and females were able to restart exercising at 120% of V ̇ O 2 max , revealing a similar functional reserve, which depends on glycolytic component of substrate-level phosphorylation and its rate of utilization. After ischaemia, muscle O2 extraction was increased, and muscle V ̇ O 2 was similarly reduced in males and females. The physiological response to repeated supramaximal exercise to exhaustion is remarkably similar in males and females when differences in lean mass are considered. Both sexes fatigue with a large functional reserve, which depends on the glycolytic energy supply, yet females have higher oxygen extraction capacity, but reduced P ETC O 2 and brain oxygenation.


Assuntos
Consumo de Oxigênio , Caracteres Sexuais , Adulto , Exercício Físico , Feminino , Humanos , Isquemia/metabolismo , Masculino , Músculo Esquelético/metabolismo , Adulto Jovem
19.
Am J Physiol Renal Physiol ; 321(2): F170-F178, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34180718

RESUMO

Pericytes play an important role in the recovery process after ischemic injury of many tissues. Brain pericytes in the peri-infarct area express macrophage markers in response to injury stimuli and are involved in neovascularization. In the kidney, nerve/glial antigen 2 (NG2)+ pericytes have been found to accumulate after renal injury. These accumulated NG2+ cells are not involved in scar formation. However, the role of accumulated NG2+ cells in injured kidneys remains unknown. Here, using a reversible ischemia-reperfusion (I/R) model, we found that renal NG2+ cells were increased in injured kidneys and expressed macrophage markers (CD11b or F4/80) on day 3 after reperfusion. Isolated NG2+ cells from I/R kidneys also had phagocytic activity and expressed anti-inflammatory cytokine genes, including mannose receptor and IL-10. These macrophage-like NG2+ cells did not likely differentiate into myofibroblasts because they did not increase α-smooth muscle actin expression. Intravenous transfusion of renal NG2+ cells isolated from donor mice on day 3 after reperfusion into recipient mice on day 1 after I/R surgery revealed that NG2+ cell-injected mice had lower plasma blood urea nitrogen, reduced kidney injury molecule-1 mRNA expression, ameliorated renal damage, and reduced cellular debris accumulation compared with PBS-injected mice on day 5 after reperfusion. In conclusion, these data suggest that renal NG2+ cells have an M2 macrophage-like ability and play a novel role in facilitating the recovery process after renal I/R injury.NEW & NOTEWORTHY Brain pericytes have macrophage-like activities after injury. However, such properties of pericytes in peripheral tissues have not been investigated. Here, we provide evidence that nerve/glial antigen 2-positive cells increase after renal injury. The population of nerve/glial antigen 2-positive cells, which does not increase expression of myofibroblast-associated gene, express macrophage markers and anti-inflammatory cytokine genes, have phagocytic activity, and play a role in renal recovery after kidney injury.


Assuntos
Antígenos/metabolismo , Isquemia/metabolismo , Rim/metabolismo , Macrófagos/metabolismo , Proteoglicanas/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Isquemia/patologia , Rim/patologia , Macrófagos/patologia , Masculino , Camundongos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fagocitose/fisiologia , Fenótipo , Traumatismo por Reperfusão/patologia
20.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065421

RESUMO

Dendritic cells (DCs) are unique immune cells that can link innate and adaptive immune responses and Immunometabolism greatly impacts their phenotype. Rapamycin is a macrolide compound that has immunosuppressant functions and is used to prevent graft loss in kidney transplantation. The current study evaluated the therapeutic potential of ex-vivo rapamycin treated DCs to protect kidneys in a mouse model of acute kidney injury (AKI). For the rapamycin single (S) treatment (Rapa-S-DC), Veh-DCs were treated with rapamycin (10 ng/mL) for 1 h before LPS. In contrast, rapamycin multiple (M) treatment (Rapa-M-DC) were exposed to 3 treatments over 7 days. Only multiple ex-vivo rapamycin treatments of DCs induced a persistent reprogramming of mitochondrial metabolism. These DCs had 18-fold more mitochondria, had almost 4-fold higher oxygen consumption rates, and produced more ATP compared to Veh-DCs (Veh treated control DCs). Pathway analysis showed IL10 signaling as a major contributing pathway to the altered immunophenotype after Rapamycin treatment compared to vehicle with significantly lower cytokines Tnfa, Il1b, and Il6, while regulators of mitochondrial content Pgc1a, Tfam, and Ho1 remained elevated. Critically, adoptive transfer of rapamycin-treated DCs to WT recipients 24 h before bilateral kidney ischemia significantly protected the kidneys from injury with a significant 3-fold improvement in kidney function. Last, the infusion of DCs containing higher mitochondria numbers (treated ex-vivo with healthy isolated mitochondria (10 µg/mL) one day before) also partially protected the kidneys from IRI. These studies demonstrate that pre-emptive infusion of ex-vivo reprogrammed DCs that have higher mitochondria content has therapeutic capacity to induce an anti-inflammatory regulatory phenotype to protect kidneys from injury.


Assuntos
Injúria Renal Aguda/tratamento farmacológico , Células Dendríticas/efeitos dos fármacos , Isquemia/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Sirolimo/farmacologia , Injúria Renal Aguda/metabolismo , Transferência Adotiva/métodos , Animais , Citocinas/metabolismo , Células Dendríticas/metabolismo , Células HEK293 , Humanos , Inflamação/metabolismo , Isquemia/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacos
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