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1.
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
2.
Biomed Res Int ; 2021: 9963732, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34545331

RESUMO

Acute kidney injury (AKI) is responsible for significant mortality among hospitalized patients that is especially troubling aged people. An effective self-made Chinese medicine formula, Xiaoyu Xiezhuo Drink (XXD), displayed therapeutic effects on AKI. However, the compositions and underlying mechanisms of XXD remain to be elucidated. In this study, we used the ultra-high-performance liquid chromatography method coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) to investigate the chemical components in XXD. Then, the absorbable components of XXD were identified based on the five principles and inputted into the SwissTargetPrediction and STITCH databases to identify the drug targets. AKI-related targets were collected from the GenCLiP 3, GeneCards, and DisGeNET databases. The crossover genes of XXD and AKI were identified for functional enrichment analysis. The protein-protein interaction (PPI) network of crossover genes was constructed, followed by the identification of hub genes. Subsequently, the effects and potential mechanisms of XXD on AKI predicted by the network pharmacology and bioinformatics analyses were experimentally validated in ischemia-reperfusion (I/R) injury-induced AKI aged mouse models. A total of 122 components in XXD were obtained; among them, 58 components were found that could be absorbed in the blood. There were 800 potential drug targets predicted from the 58 absorbable components in AKI which shared 36 crossover genes with AKI-related targets. The results of functional enrichment analysis indicated that crossover genes mostly associated with the response to oxidative stress and the HIF1 signaling pathway. In the PPI network analysis, 12 hub genes were identified, including ALB, IL-6, TNF, TP53, VEGFA, PTGS2, TLR4, NOS3, EGFR, PPARG, HIF1A, and HMOX1. In AKI aged mice, XXD prominently alleviated I/R injury-induced renal dysfunction, abnormal renal pathological changes, and cellular senescence, inflammation, and oxidative damage with a reduction in the expression level of the inflammatory mediator, α-SMA, collagen-1, F4/80, TP53, VEGFA, PTGS2, TLR4, NOS3, EGFR, PPARG, HIF1A, ICAM-1, TGF-ß1, Smad3, and p-Smad3 and an increase of nephridial tissue p-H3, Ki67, HMOX1, MMP-9, and Smad7 levels. In summary, our findings suggest that XXD has renoprotective effects against AKI in aged mice via inhibiting the TGF-ß1/Smad3 and HIF1 signaling pathways.


Assuntos
Injúria Renal Aguda/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/fisiopatologia , Animais , China , Cromatografia Líquida de Alta Pressão/métodos , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/metabolismo , Isquemia/patologia , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Substâncias Protetoras/farmacologia , Reperfusão , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas em Tandem/métodos
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.
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
5.
Sci Rep ; 11(1): 16274, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381134

RESUMO

We evaluated choroidal congestion using multimodal imaging in pachychoroid neovasculopathy (PNV). In a retrospective case series of 100 eyes of 99 treatment-naïve PNV patients, their clinical records were reviewed and the corresponding multimodal imaging studies were analyzed. We assessed areas of choriocapillaris filling delay which overlapped with dilated outer choroidal vessels, choroidal neovascularization (CNV), and retinal pigment epithelium (RPE) atrophy. The study subjects were 78 men (78.8%) and 21 women (21.2%). The mean patient age was 67.5 ± 10.5 years. On indocyanine green angiography, all eyes showed choriocapillaris filling delay in the early phase. Dilated outer choroidal vessels were demonstrated in all eyes by en face optical coherence tomography. The areas of choriocapillaris filling delay overlapped extensively with that of dilated outer choroidal vessels. All eyes showed CNV localized within the sites of choriocapillaris filling delay. RPE atrophy was noted in 71 eyes (71.0%), and 68 of these (95.8%) had RPE atrophy within the areas showing choriocapillaris filling delay. These findings indicate that chronic choriocapillaris ischemia secondary to vortex vein congestion may lead to CNV development as well as RPE atrophy in eyes with PNV.


Assuntos
Corioide/irrigação sanguínea , Neovascularização de Coroide/etiologia , Isquemia/patologia , Idoso , Atrofia/etiologia , Doença Crônica , Dilatação Patológica , Feminino , Humanos , Isquemia/diagnóstico por imagem , Isquemia/etiologia , Masculino , Pessoa de Meia-Idade , Imagem Multimodal , Epitélio Pigmentado da Retina/patologia , Estudos Retrospectivos
6.
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
7.
PLoS One ; 16(8): e0255075, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34375370

RESUMO

Induced endothelial cells (iECs) generated from neonatal fibroblasts via transdifferentiation have been shown to have pro-angiogenic properties and are a potential therapy for peripheral arterial disease (PAD). It is unknown if iECs can be generated from fibroblasts collected from PAD patients and whether these cells are pro-angiogenic. In this study fibroblasts were collected from four PAD patients undergoing carotid endarterectomies. These cells, and neonatal fibroblasts, were transdifferentiated into iECs using modified mRNA. Endothelial phenotype and pro-angiogenic cytokine secretion were investigated. NOD-SCID mice underwent surgery to induce hindlimb ischaemia in a murine model of PAD. Mice received intramuscular injections with either control vehicle, or 1 × 106 neonatal-derived or 1 × 106 patient-derived iECs. Recovery in perfusion to the affected limb was measured using laser Doppler scanning. Perfusion recovery was enhanced in mice treated with neonatal-derived iECs and in two of the three patient-derived iEC lines investigated in vivo. Patient-derived iECs can be successfully generated from PAD patients and for specific patients display comparable pro-angiogenic properties to neonatal-derived iECs.


Assuntos
Células Endoteliais/patologia , Fibroblastos/patologia , Neovascularização Fisiológica , Doença Arterial Periférica/patologia , Acetilação/efeitos dos fármacos , Animais , Capilares/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Colágeno/farmacologia , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Combinação de Medicamentos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/transplante , Fibroblastos/efeitos dos fármacos , Membro Posterior/irrigação sanguínea , Membro Posterior/patologia , Humanos , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Isquemia/patologia , Isquemia/terapia , Laminina/farmacologia , Lipoproteínas LDL/metabolismo , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Neovascularização Fisiológica/efeitos dos fármacos , Perfusão , Lectinas de Plantas/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteoglicanas/farmacologia
8.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199527

RESUMO

Overactive bladder (OAB) syndrome, including frequency, urgency, nocturia and urgency incontinence, has a significantly negative impact on the quality-of-life scale (QoL) and can cause sufferer withdrawal from social activities. The occurrence of OAB can result from an imbalance between the production of pro-oxidants, such as free radicals and reactive species, and their elimination through protective mechanisms of antioxidant-induced oxidative stress. Several animal models, such as bladder ischemia/reperfusion (I/R), partial bladder outlet obstruction (PBOO) and ovarian hormone deficiency (OHD), have suggested that cyclic I/R during the micturition cycle induces oxidative stress, leading to bladder denervation, bladder afferent pathway sensitization and overexpression of bladder-damaging molecules, and finally resulting in bladder hyperactivity. Based on the results of previous animal experiments, the present review specifically focuses on four issues: (1) oxidative stress and antioxidant defense system; (2) oxidative stress in OAB and biomarkers of OAB; (3) OAB animal model; (4) potential nature/plant antioxidant treatment strategies for urinary dysfunction with OAB. Moreover, we organized the relationships between urinary dysfunction and oxidative stress biomarkers in urine, blood and bladder tissue. Reviewed information also revealed the summary of research findings for the effects of various antioxidants for treatment strategies for OAB.


Assuntos
Antioxidantes/uso terapêutico , Isquemia/tratamento farmacológico , Bexiga Urinária Hiperativa/tratamento farmacológico , Incontinência Urinária/tratamento farmacológico , Humanos , Isquemia/patologia , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/patologia , Incontinência Urinária/patologia
9.
Vet J ; 274: 105714, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34252550

RESUMO

There is mounting evidence that kidney ischaemia/hypoxia plays an important role in feline chronic kidney disease (CKD) development and progression, as well as in human disease and laboratory animal models. Ischaemic acute kidney injury is widely accepted as a cause of CKD in people and data from laboratory species has identified some of the pathways underlying this continuum. Experimental kidney ischaemia in cats results in morphological changes, namely chronic tubulointerstitial inflammation, tubulointerstitial fibrosis, and tubular atrophy, akin to those observed in naturally-occurring CKD. Multiple situations are envisaged that could result in acute or chronic episodes of kidney hypoxia in cats, while risk factors identified in epidemiological studies provide further support that kidney hypoxia contributes to spontaneously occurring feline CKD. This review evaluates the evidence for the role of kidney ischaemia/hypoxia in feline CKD and the proposed mechanisms and consequences of kidney hypoxia. As no effective treatments exist that substantially slow or prevent feline CKD progression, there is a need for novel therapeutic strategies. Targeting kidney hypoxia is one such promising approach, with therapies including those that attenuate the hypoxia-inducible factor (HIF) pathway already being utilised in human CKD.


Assuntos
Injúria Renal Aguda/veterinária , Hipóxia/veterinária , Insuficiência Renal Crônica/veterinária , Injúria Renal Aguda/patologia , Animais , Doenças do Gato/etiologia , Gatos , Hipóxia/patologia , Isquemia/patologia , Isquemia/veterinária , Insuficiência Renal Crônica/etiologia
10.
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
11.
PLoS One ; 16(7): e0246978, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34234356

RESUMO

Donation after circulatory death (DCD) has expanded the donor pool for liver transplantation. However, ischemic cholangiopathy (IC) after DCD liver transplantation causes inferior outcomes. The molecular mechanisms of IC are currently unknown but may depend on ischemia-induced genetic reprograming of the biliary epithelium to mesenchymal-like cells. The main objective of this study was to determine if cholangiocytes undergo epithelial to mesenchymal transition (EMT) after exposure to DCD conditions and if this causally contributes to the phenotype of IC. Human cholangiocyte cultures were exposed to periods of warm and cold ischemia to mimic DCD liver donation. EMT was tested by assays of cell migration, cell morphology, and differential gene expression. Transplantation of syngeneic rat livers recovered under DCD conditions were evaluated for EMT changes by immunohistochemistry. Human cholangiocytes exposed to DCD conditions displayed migratory behavior and gene expression patterns consistent with EMT. E-cadherin and CK-7 expressions fell while N-cadherin, vimentin, TGFß, and SNAIL rose, starting 24 hours and peaking 1-3 weeks after exposure. Cholangiocyte morphology changed from cuboidal (epithelial) before to spindle shaped (mesenchymal) a week after ischemia. These changes were blocked by pretreating cells with the Transforming Growth Factor beta (TGFß) receptor antagonist Galunisertib (1 µM). Finally, rats with liver isografts cold stored for 20 hours in UW solution and exposed to warm ischemia (30 minutes) at recovery had elevated plasma bilirubin 1 week after transplantation and the liver tissue showed immunohistochemical evidence of early cholangiocyte EMT. Our findings show EMT occurs after exposure of human cholangiocytes to DCD conditions, which may be initiated by upstream signaling from autocrine derived TGFß to cause mesenchymal specific morphological and migratory changes.


Assuntos
Transição Epitelial-Mesenquimal , Isquemia/patologia , Transplante de Fígado/efeitos adversos , Animais , Humanos , Masculino , Ratos
12.
Proc Natl Acad Sci U S A ; 118(28)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34244428

RESUMO

The emerging field of regenerative cell therapy is still limited by the few cell types that can reliably be differentiated from pluripotent stem cells and by the immune hurdle of commercially scalable allogeneic cell therapeutics. Here, we show that gene-edited, immune-evasive cell grafts can survive and successfully treat diseases in immunocompetent, fully allogeneic recipients. Transplanted endothelial cells improved perfusion and increased the likelihood of limb preservation in mice with critical limb ischemia. Endothelial cell grafts transduced to express a transgene for alpha1-antitrypsin (A1AT) successfully restored physiologic A1AT serum levels in mice with genetic A1AT deficiency. This cell therapy prevented both structural and functional changes of emphysematous lung disease. A mixture of endothelial cells and cardiomyocytes was injected into infarcted mouse hearts, and both cell types orthotopically engrafted in the ischemic areas. Cell therapy led to an improvement in invasive hemodynamic heart failure parameters. Our study supports the development of hypoimmune, universal regenerative cell therapeutics for cost-effective treatments of major diseases.


Assuntos
Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/terapia , Imunocompetência , Células-Tronco Pluripotentes Induzidas/imunologia , Pneumopatias/imunologia , Pneumopatias/terapia , Transplante de Células-Tronco , Animais , Células Endoteliais/transplante , Insuficiência Cardíaca/terapia , Membro Posterior/irrigação sanguínea , Membro Posterior/patologia , Isquemia/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/transplante , Transplante Homólogo , alfa 1-Antitripsina/metabolismo
13.
Mol Med Rep ; 24(3)2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34212986

RESUMO

Tumor necrosis factor (TNF)­α and TNF receptor 1 (TNF­R1) play diverse roles in modulating the neuronal damage induced by cerebral ischemia. The present study compared the time­dependent changes of TNF­α and TNF­R1 protein expression levels in the hippocampal subfield cornu ammonis 1 (CA1) between adult and young gerbils following transient forebrain ischemia (tFI), via western blot and immunohistochemistry analyses. In adult gerbils, delayed neuronal death of pyramidal neurons, the principal neurons in CA1, was recorded 4 days after tFI; however, in young gerbils, delayed neuronal death was recorded 7 days after tFI. TNF­α protein expression levels gradually increased in both groups following tFI; however, TNF­α expression was higher in young gerbils compared with adult gerbils. TNF­R1 protein expression levels markedly increased in both groups 1 day after tFI. Subsequently, TNF­R1 expression gradually decreased in young gerbils, whereas TNF­R1 expression levels were irregularly altered in adult gerbils following tFI. Notably, TNF­α immunoreactivity significantly increased in pyramidal neurons in both groups 1 day after tFI; however, the patterns altered between both groups. In adult gerbils, TNF­α immunoreactivity was rarely exhibited in pyramidal neurons 4 days after tFI due to neuronal death, suggesting that TNF­α immunoreactivity was newly expressed in astrocytes. In young gerbils, TNF­α immunoreactivity increased in pyramidal neurons 4 days after tFI, and TNF­α immunoreactivity was newly expressed in astrocytes. In addition, TNF­R1 immunoreactivity was exhibited in pyramidal cells of both sham groups, and significantly increased 1 day after tFI; however, the patterns altered between both groups. In adult gerbils, TNF­R1 immunoreactivity was rarely exhibited 4 days after tFI, and astrocytes newly expressed TNF­R1 immunoreactivity. In young gerbils, TNF­R1 immunoreactivity increased in pyramidal neurons 4 days after tFI; however, TNF­R1 immunoreactivity was not reported in pyramidal neurons and astrocytes thereafter. Taken together, the results of the present study suggest that different expression levels of TNF­α and TNF­R1 in ischemic CA1 between adult and young gerbils may be due to age­dependent differences of tFI­induced neuronal death.


Assuntos
Astrócitos/metabolismo , Região CA1 Hipocampal/metabolismo , Gerbillinae/metabolismo , Neurônios/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Isquemia Encefálica/patologia , Morte Celular , Córtex Cerebral/metabolismo , Hipocampo/metabolismo , Isquemia/patologia , Masculino , Neurogênese , Prosencéfalo , Células Piramidais/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/genética
14.
Aging (Albany NY) ; 13(12): 16165-16177, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34114972

RESUMO

The occurrence of hypoxia-ischemia (HI) in the developing brain is closely associated with neuronal injury and even death. However, the underlying molecular mechanism is not fully understood. This study was designed to investigate phosphatase and tensin homolog (PTEN) nuclear translocation and its possible role in rat cortical neuronal damage following oxygen-glucose deprivation (OGD) in vitro. An in vitro OGD model was established using primary cortical neurons dissected from newborn Sprague-Dawley rats to mimic HI conditions. The PTENK13R mutant plasmid, which contains a lysine-to-arginine mutation at the lysine 13 residue, was constructed. The nuclei and cytoplasm of neurons were separated. Neuronal injury following OGD was evidenced by increased lactate dehydrogenase (LDH) release and apoptotic cell counts. In addition, PTEN expression was increased and the phosphorylation of extracellular signal-regulated kinase 1/2 (p-ERK1/2) and activation of nuclear factor kappa B (NF-κB) were decreased following OGD. PTENK13R transfection prevented PTEN nuclear translocation; attenuated the effect of OGD on nuclear p-ERK1/2 and NF-κB, apoptosis, and LDH release; and increased the expression of several anti-apoptotic proteins. We conclude that PTEN nuclear translocation plays an essential role in neuronal injury following OGD via modulation of the p-ERK1/2 and NF-κB pathways. Prevention of PTEN nuclear translocation might be a candidate strategy for preventing brain injury following HI.


Assuntos
Núcleo Celular/metabolismo , Isquemia/metabolismo , Isquemia/patologia , NF-kappa B/metabolismo , Neurônios/patologia , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais , Animais , Hipóxia Celular , Células Cultivadas , Glucose/deficiência , Sistema de Sinalização das MAP Quinases , Neurônios/metabolismo , Oxigênio , Fosforilação , Transporte Proteico , Ratos Sprague-Dawley
15.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068392

RESUMO

Myocardial infarction (MI) remains the leading cause of death in the western world. Despite advancements in interventional revascularization technologies, many patients are not candidates for them due to comorbidities or lack of local resources. Non-invasive approaches to accelerate revascularization within ischemic tissues through angiogenesis by providing Vascular Endothelial Growth Factor (VEGF) in protein or gene form has been effective in animal models but not in humans likely due to its short half-life and systemic toxicity. Here, we tested the hypothesis that PR1P, a small VEGF binding peptide that we developed, which stabilizes and upregulates endogenous VEGF, could be used to improve outcome from MI in rodents. To test this hypothesis, we induced MI in mice and rats via left coronary artery ligation and then treated animals with every other day intraperitoneal PR1P or scrambled peptide for 14 days. Hemodynamic monitoring and echocardiography in mice and echocardiography in rats at 14 days showed PR1P significantly improved multiple functional markers of heart function, including stroke volume and cardiac output. Furthermore, molecular biology and histological analyses of tissue samples showed that systemic PR1P targeted, stabilized and upregulated endogenous VEGF within ischemic myocardium. We conclude that PR1P is a potential non-invasive candidate therapeutic for MI.


Assuntos
Antígeno AC133/metabolismo , Modelos Animais de Doenças , Isquemia/complicações , Infarto do Miocárdio/prevenção & controle , Neovascularização Fisiológica/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Feminino , Isquemia/metabolismo , Isquemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Função Ventricular Esquerda/efeitos dos fármacos
16.
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
17.
Sci Rep ; 11(1): 12806, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140618

RESUMO

The failure of neuroprotective treatment-related clinical trials may be partially caused by unestablished animal models. Existing animal models are less likely to provide occlusion confined to the middle cerebral artery (MCA), making transarterial intervention difficult. We aimed to develop a novel focal stroke model using a microcatheter and zirconium dioxide that is non-magnetic under fluoroscopic guidance, which can monitor MCA occlusion and can improve hemorrhagic complications. Using male Sprague Dawley rats (n = 10), a microcatheter was navigated from the caudal ventral artery to the left internal carotid artery using an X-ray fluoroscopy to establish local occlusion. All rat cerebral angiographies were successful. No rats had hemorrhagic complications. Eight (80%) rats underwent occlusion of the MCA bifurcation by zirconium dioxide. Accidentally, the left posterior cerebral artery was failure embolized in 2 rats (20%). The median operating time was 8 min. All rats of occlusion MCA revealed an incomplete hemiparesis on the right side with neurological deficit score ranging from 1 to 3 (median 1, interquartile range 1-3) at 24 h after the induction of ischemia. Moreover, 2% 2,3,5-triphenyl tetrazolium chloride staining showed that the median infarct volume (mm3) was 280 (interquartile range 267-333) 24 h after the left MCA bifurcation occlusion. We present a novel rat model for focal stroke using a microcatheter and zirconium dioxide which does not affect the MRI. The model is predictable which is well confined within the territory supplied by the MCA, and reproducibility of this model is 80%. Fluoroscopy was able to identify which the MCA occlusion and model success while creating the model. It permitted exclusion of animals with complications from the experiment.


Assuntos
Cateteres , Fluoroscopia , Infarto da Artéria Cerebral Média/patologia , Isquemia/patologia , Zircônio/química , Animais , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Isquemia/complicações , Isquemia/diagnóstico por imagem , Imageamento por Ressonância Magnética , Masculino , Ratos Sprague-Dawley , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/patologia
18.
PLoS One ; 16(6): e0253506, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34143845

RESUMO

BACKGROUND: We developed a jejunal and colonic experimental human ischemia-reperfusion (IR) model to study pathophysiological intestinal IR mechanisms and potential new intestinal ischemia biomarkers. Our objective was to evaluate the safety of these IR models by comparing patients undergoing surgery with and without in vivo intestinal IR. METHODS: A retrospective study was performed comparing complication rates and severity, based on the Clavien-Dindo classification system, in patients undergoing pancreatoduodenectomy with (n = 10) and without (n = 20 matched controls) jejunal IR or colorectal surgery with (n = 10) and without (n = 20 matched controls) colon IR. Secondary outcome parameters were operative time, blood loss, 90-day mortality and length of hospital stay. RESULTS: Following pancreatic surgery, 63% of the patients experienced one or more postoperative complications. There was no significant difference in incidence or severity of complications between patients undergoing pancreatic surgery with (70%) or without (60%, P = 0.7) jejunal IR. Following colorectal surgery, 60% of the patients experienced one or more postoperative complication. Complication rate and severity were similar in patients with (50%) and without (65%, P = 0.46) colonic IR. Operative time, amount of blood loss, postoperative C-reactive protein, length of hospital stay or mortality were equal in both intervention and control groups for jejunal and colon IR. CONCLUSION: This study showed that human experimental intestinal IR models are safe in patients undergoing pancreatic or colorectal surgery.


Assuntos
Cirurgia Colorretal/efeitos adversos , Intestinos/irrigação sanguínea , Isquemia/patologia , Pancreaticoduodenectomia/efeitos adversos , Traumatismo por Reperfusão/patologia , Idoso , Animais , Feminino , Humanos , Isquemia/etiologia , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/patologia , Período Pós-Operatório , Traumatismo por Reperfusão/etiologia , Estudos Retrospectivos
19.
FASEB J ; 35(6): e21612, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33948996

RESUMO

Lipid overload is intimately connected with the change of endothelial epigenetic status which impacts cellular signaling activities and endothelial function. Activating transcription factor 4 (ATF4) is involved in the regulation of lipid metabolism and meanwhile an epigenetic modifier. However, the role of ATF4 in the angiogenesis under lipid overload is not well understood. Here, to induce lipid overload status, we employed high-fat diet (HFD)-induced obese mouse model in vivo and palmitic acid (PA) to stimulate endothelial cells in vitro. Compared with mice fed with normal chow diet (NCD), HFD-induced obese mice showed angiogenic defects evidenced by decline in (1) blood flow recovery after hind limb ischemia, (2) wound healing speed after skin injury, (3) capillary density in injured tissues and matrigel plugs, and (4) endothelial sprouts of aortic ring. ATF4 deficiency aggravated above angiogenic defects in mice while ATF4 overexpression improved the blunted angiogenic response. Mechanistically, lipid overload lowered the H3K4 methylation levels at the regulatory regions of NOS3 and ERK1 genes, leading to reduced angiogenic signaling activity. Methionine adenosyltransferase 2A (MAT2A) is identified as a target of ATF4 and formed complex with ATF4 to direct lysine methyltransferase 2A (MLL1) to the regulatory regions of both genes for the maintenance of the H3K4 methylation level and angiogenic signaling activity. Here, we uncovered a novel metabolic-epigenetic coupling orchestrated by the ATF4-MAT2A axis for angiogenesis. The ATF4-MAT2A axis links lipid overload milieu to altered epigenetic status of relevant angiogenic signaling in endothelial cells, suggesting a potential therapeutic target for angiogenesis impaired by lipid overload.


Assuntos
Fator 4 Ativador da Transcrição/fisiologia , Epigênese Genética , Isquemia/patologia , Lipídeos/efeitos adversos , Metionina Adenosiltransferase/metabolismo , Neovascularização Patológica/patologia , Obesidade/complicações , Animais , Dieta Hiperlipídica , Isquemia/etiologia , Isquemia/metabolismo , Masculino , Metionina Adenosiltransferase/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Neovascularização Patológica/etiologia , Neovascularização Patológica/metabolismo
20.
Methods Mol Biol ; 2262: 361-395, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977490

RESUMO

Animal models have become in recent years a crucial tool to understand the physiological and pathological roles of many cellular proteins. They allow analysis of the functional consequences of [1] complete or partial (time- or organ-limited) removal of specific proteins (knockout animals), [2] the exchange of a wild-type allele for a mutant or truncated version found in human illnesses (knock-in), or [3] the effect of overexpression of a given protein in the whole body or in specific organs (transgenic mice). In this regard, the study of phenotypes in Ras GEF animal models has allowed researchers to find specific functions for otherwise very similar proteins, uncovering their role in physiological contexts such as memory formation, lymphopoiesis, photoreception, or body homeostasis. In addition, mouse models have been used to unveil the functional role of Ras GEFs under pathological conditions, including Noonan syndrome, skin tumorigenesis, inflammatory diseases, diabetes, or ischemia among others. In the following sections, we will describe the methodological approaches employed for Ras GEF animal model analyses, as well as the main discoveries made.


Assuntos
Modelos Animais de Doenças , Marcação de Genes/métodos , Homeostase , Isquemia/patologia , Neoplasias/patologia , Fatores ras de Troca de Nucleotídeo Guanina/metabolismo , Animais , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Isquemia/genética , Isquemia/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neurogênese , Fatores ras de Troca de Nucleotídeo Guanina/antagonistas & inibidores , Fatores ras de Troca de Nucleotídeo Guanina/genética
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