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1.
Dig Dis Sci ; 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34655012

RESUMO

A patient with systemic amyloidosis developed portal hypertension, acute liver failure and multiorgan dysfunction. Extensive testing was unrevealing for paraproteinemia, plasma cell dyscrasia, infectious, or inflammatory conditions. He was transferred to our institution for orthotopic liver transplant evaluation but was ultimately declined given clinical instability and dysautonomia. Post-mortem evaluation revealed extensive amyloid deposition in multiple organs determined to be AL-lambda amyloidosis.

2.
Nat Commun ; 12(1): 4884, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385460

RESUMO

Pathology is practiced by visual inspection of histochemically stained tissue slides. While the hematoxylin and eosin (H&E) stain is most commonly used, special stains can provide additional contrast to different tissue components. Here, we demonstrate the utility of supervised learning-based computational stain transformation from H&E to special stains (Masson's Trichrome, periodic acid-Schiff and Jones silver stain) using kidney needle core biopsy tissue sections. Based on the evaluation by three renal pathologists, followed by adjudication by a fourth pathologist, we show that the generation of virtual special stains from existing H&E images improves the diagnosis of several non-neoplastic kidney diseases, sampled from 58 unique subjects (P = 0.0095). A second study found that the quality of the computationally generated special stains was statistically equivalent to those which were histochemically stained. This stain-to-stain transformation framework can improve preliminary diagnoses when additional special stains are needed, also providing significant savings in time and cost.


Assuntos
Biópsia com Agulha de Grande Calibre/métodos , Aprendizado Profundo , Diagnóstico por Computador/métodos , Nefropatias/patologia , Rim/patologia , Coloração e Rotulagem/métodos , Algoritmos , Corantes/química , Corantes/classificação , Corantes/normas , Diagnóstico Diferencial , Humanos , Nefropatias/diagnóstico , Patologia Clínica/métodos , Patologia Clínica/normas , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e Rotulagem/normas
3.
J Neurosci ; 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34083257

RESUMO

Within the perinatal stroke field, there is a need to establish preclinical models where putative biomarkers for motor function can be examined. In a mouse model of perinatal stroke, we evaluated motor map size and movement latency following optogenetic cortical stimulation against three factors of post-stroke biomarker utility: 1) Correlation to chronic impairment on a behavioral test battery; 2) Amenability to change using a skilled motor training paradigm; 3) Ability to distinguish individuals with potential to respond well to training. Thy1-ChR2-YFP mice received a photothrombotic stroke at postnatal day 7 and were evaluated on a battery of motor tests between days 59-70. Following a cranial window implant, mice underwent longitudinal optogenetic motor mapping both before and after 3 weeks of skilled forelimb training. Map size and movement latency of both hemispheres was positively correlated with impaired spontaneous forelimb use, whereas only ipsilesional hemisphere map size was correlated with performance in skilled reaching. Map size and movement latency did not show groupwise changes with training; however, mice with the smallest pre-training map sizes and worst impairments demonstrated the greatest expansion of map size in response to skilled forelimb training. Overall, motor map size showed utility as a potential biomarker for impairment and training-induced modulation in specific individuals. Future assessment of the predictive capacity of post-stroke motor representations for behavioral outcome in animal models opens the possibility of dissecting how plasticity mechanisms contribute to recovery following perinatal stroke.SIGNIFICANCE STATEMENTWe investigated the utility of two cortical motor representation measures (motor map size and movement onset latency) as potential biomarkers for post-stroke motor recovery in a mouse model of perinatal stroke. Both motor map size and movement latency were associated with functional recovery after perinatal stroke, with map size showing an additional association between training responsiveness and severity of impairment. Overall, both motor map size and movement onset latency show potential as neurophysiological correlates of recovery. As such, future studies of perinatal stroke rehabilitation and neuromodulation should include these measures in order to help explain neurophysiological changes that might be occurring in response to treatment.

4.
Aging (Albany NY) ; 13(6): 8628-8642, 2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33714955

RESUMO

Primary open angle glaucoma (POAG) is the leading cause of irreversible blindness. Dysfunction of the trabecular meshwork (TM), resulting in decreased outflow of aqueous humor and increased intraocular pressure (IOP), plays an important role in the pathogenesis of POAG. However, the underlying mechanisms still remain unclear. In this study, we demonstrated that the eIF2-α/ATF4/CHOP branch of unfolded protein response (UPR) was activated in human trabecular meshwork cells (HTMCs) upon tert-butyl hydroperoxide (TBHP) exposure. Inhibition of ATF4 ameliorated TBHP-induced apoptosis and inflammatory cytokine production, while ectopic expression of ATF4 increased the expression of endothelial leukocyte adhesion molecule (ELAM)-1 and IL-8 in HTMCs. Furthermore, we found that ATF4 inhibition reduced tunicamycin-induced caspase-3 activation, ROS production, ELAM-1 expression, and HTMCs phagocytosis impairment. By an in vivo study in mice, we showed that overexpression of ATF4 in the TM induced C/EBP homologous protein (CHOP) expression and TM cells apoptosis, contributing to inflammatory cytokine production, and probably IOP elevation. More importantly, upregulation of ATF4 and CHOP, and colocalization of ATF4 with ELAM-1 were found in the TM of POAG patients. These results suggest that ATF4 is a critical mediator of oxidative stress and ER stress-induced TM cell dysfunction and apoptosis in POAG.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Apoptose/fisiologia , Glaucoma de Ângulo Aberto/metabolismo , Glaucoma de Ângulo Aberto/patologia , Malha Trabecular/metabolismo , Malha Trabecular/patologia , Animais , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/fisiologia
5.
Angiogenesis ; 24(2): 363-377, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33201372

RESUMO

NADPH oxidase 4 (Nox4) is a major isoform of NADPH oxidases playing an important role in many biological processes. Previously we have shown that Nox4 is highly expressed in retinal blood vessels and is upregulated in oxygen-induced retinopathy (OIR). However, the exact role of endothelial Nox4 in retinal angiogenesis remains elusive. Herein, using endothelial cell (EC)-specific Nox4 knockout (Nox4EC-KO) mice, we investigated the impact of endothelial Nox4 deletion on retinal vascular development and pathological angiogenesis during OIR. Our results show that deletion of Nox4 in ECs led to retarded retinal vasculature development with fewer, blunted-end tip cells and sparser, dysmorphic filopodia at vascular front, and reduced density of vascular network in superficial, deep, and intermediate layers in postnatal day 7 (P7), P12, and P17 retinas, respectively. In OIR, loss of endothelial Nox4 had no effect on hyperoxia-induced retinal vaso-obliteration at P9 but significantly reduced aberrant retinal neovascularization at P17 and decreased the deep layer capillary density at P25. Ex vivo study confirmed that lack of Nox4 in ECs impaired vascular sprouting. Mechanistically, loss of Nox4 significantly reduced expression of VEGF, p-VEGFR2, integrin αV, angiopoietin-2, and p-ERK1/2, attenuating EC migration and proliferation. Taken together, our results indicate that endothelial Nox4 is important for retinal vascular development and contributes to pathological angiogenesis, likely through regulation of VEGF/VEGFR2 and angiopoietin-2/integrin αV/ERK pathways. In addition, our study suggests that endothelial Nox4 appears to be essential for intraretinal revascularization after hypoxia. These findings call for caution on targeting endothelial Nox4 in ischemic/hypoxic retinal diseases.

6.
Free Radic Biol Med ; 158: 53-59, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32682927

RESUMO

Glutathione (GSH) plays critical roles in the inflammatory response by acting as the master substrate for antioxidant enzymes and an important anti-inflammatory agent. In the early phase of the inflammatory response of macrophages, GSH content is decreased due to the down regulation of the catalytic subunit of glutamate cysteine ligase (GCLC). In the current study we investigated the underlying mechanism for this phenomenon. In human THP1-differentiated macrophages, GCLC mRNA had a half-life of 4 h under basal conditions, and it was significantly reduced to less than 2 h upon exposure to lipopolysaccharide (LPS), suggesting an increased decay of GCLC mRNA in the inflammatory response. The half-life of GCLC protein was >10 h under basal conditions, and upon LPS exposure the degradation rate of GCLC protein was significantly increased. The pan-caspase inhibitor Z-VAD-FMK but not the proteasome inhibitor MG132, prevented the down regulation of GCLC protein caused by LPS. Both caspase inhibitor Z-LEVD-FMK and siRNA of caspase-5 abrogated LPS-induced degradation of GCLC protein. In addition, supplement with γ-GC, the GCLC product, efficiently restored GSH content and suppressed the induction of NF-κB activity by LPS. In conclusion, these data suggest that GCLC down-regulation in the inflammatory response of macrophages is mediated through both increased mRNA decay and caspase-5-mediated GCLC protein degradation, and γ-GC is an efficient agent to restore GSH and regulate the inflammatory response.


Assuntos
Glutamato-Cisteína Ligase , Glutationa , Caspases/metabolismo , Regulação para Baixo , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Humanos , Macrófagos/metabolismo
7.
Acad Pathol ; 7: 2374289520914021, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32284966

RESUMO

The following fictional cases are intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, see http://journals.sagepub.com/doi/10.1177/2374289517715040.1.

8.
Transl Stroke Res ; 11(3): 345-364, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31654281

RESUMO

There may be the potential to improve stroke recovery with mesenchymal stem cells (MSCs); however, questions about the efficacy and safety of this treatment remain. To address these issues and inform future studies, we performed a preclinical and clinical systematic review of MSC therapy for subacute and chronic ischemic stroke. MEDLINE, Embase, the Cochrane Register of Controlled Trials, and PubMed were searched. For the clinical review, interventional and observational studies of MSC therapy in ischemic stroke patients were included. For the preclinical review, interventional studies of MSC therapy using in vivo animal models of subacute or chronic stroke were included. Measures of safety and efficacy were assessed. Eleven clinical and 76 preclinical studies were included. Preclinically, MSC therapy was associated with significant benefits for multiple measures of motor and neurological function. Clinically, MSC therapy appeared to be safe, with no increase in adverse events reported (with the exception of self-limited fever immediately following injection). However, the efficacy of treatment was less apparent, with significant heterogeneity in both study design and effect size being observed. Additionally, in the only randomized phase II study to date, efficacy of MSC therapy was not observed. Preclinically, MSC therapy demonstrated considerable efficacy. Although MSC therapy demonstrated safety in the clinical setting, efficacy has yet to be determined. Future studies will need to address the discordance in the continuity of evidence as MSC therapy has been translated from "bench-to-bedside".

9.
J Diabetes Complications ; 33(10): 107410, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31434620

RESUMO

BACKGROUND: To determine if serum pigment epithelium-derived factor (PEDF) levels predict cardiovascular events, renal dysfunction and mortality in the Veterans Affairs Diabetes Study (VADT). METHODS: PEDF was evaluated in relation to subsequent cardiovascular outcomes, mortality, and renal dysfunction (defined as urinary albumin creatinine ratio (ACR) ≥300 mg/g), or chronic kidney disease (CKD) stages 3 (eGFR<60 ml/min) or 4 (eGFR<60 and <30 ml/min respectively). PEDF was measured by ELISA on sera from 881 participants collected a median (range) of 1.7 (0-5.0) years post-baseline, and later, from 832 participants 4.0 (1.5-6.9) years post-baseline. RESULTS: In 743 participants, PEDF was measured at both time-points. PEDF increased over time from (mean ±â€¯SD) 10.5 ±â€¯4.03 to 11.0 ±â€¯4.86 ng/ml (paired t-test p = 0.0092). Lower eGFR (p < 0.01), higher serum creatinine (p < 0.01) and urinary ACR (p < 0.01) were associated with increasing PEDF. Multivariate event time models included either one or two follow-up windows (i.e., between first and second PEDF measures; and, when available, from second PEDF measure until study-end). PEDF tertiles were not associated with cardiovascular events, but were significantly associated with all-cause mortality [HR = 2.00 (1.03, 3.89) comparing first to third tertile] in models adjusted for age, minority status, VADT treatment arm and prior cardiovascular event status. Higher PEDF levels also associated with development of kidney dysfunction with adjusted HRs (95% CI comparing third to first PEDF tertiles: 2.74 (1.71, 4.39) for stage 3 CKD; and 3.84 (95% CI: 1.17, 12.5) for stage 4 CKD. CONCLUSIONS: Over 2-years, higher serum PEDF levels predicted advanced nephropathy in patients with type 2 diabetes.


Assuntos
Doenças Cardiovasculares/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/mortalidade , Nefropatias Diabéticas/sangue , Proteínas do Olho/sangue , Fatores de Crescimento Neural/sangue , Serpinas/sangue , Albuminúria/sangue , Biomarcadores/sangue , Doenças Cardiovasculares/epidemiologia , Diabetes Mellitus Tipo 2/complicações , Angiopatias Diabéticas/sangue , Angiopatias Diabéticas/epidemiologia , Feminino , Taxa de Filtração Glomerular , Humanos , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/epidemiologia , Veteranos
10.
J Clin Med ; 8(6)2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242599

RESUMO

Retinal neuronal injury and degeneration is one of the primary manifestations of diabetic retinopathy, a leading cause of vision loss in working age adults. In pathological conditions, including diabetes and some physiological conditions such as aging, protein homeostasis can become disrupted, leading to endoplasmic reticulum (ER) stress. Severe or unmitigated ER stress can lead to cell death, which in retinal neurons results in irreversible loss of visual function. X-box binding protein 1 (XBP1) is a major transcription factor responsible for the adaptive unfolded protein response (UPR) to maintain protein homeostasis in cells undergoing ER stress. The purpose of this study is to determine the role of XBP1-mediated UPR in retinal neuronal survival and function in a mouse model of type 1 diabetes. Using a conditional retina-specific XBP1 knockout mouse line, we demonstrate that depletion of XBP1 in retinal neurons results in early onset retinal function decline, loss of retinal ganglion cells and photoreceptors, disrupted photoreceptor ribbon synapses, and Müller cell activation after induction of diabetes. Our findings suggest an important role of XBP1-mediated adaptive UPR in retinal neuronal survival and function in diabetes.

11.
Biol Lett ; 15(3): 20190006, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30862308

RESUMO

Damaraland mole rats (DMRs, Fukomys damarensis) are a eusocial fossorial species that spend the majority of their life in densely populated underground burrows, in which they likely experience intermittent periods of elevated CO2 (i.e. hypercapnia). The primary physiological response to hypercapnia in most mammals is to increase depth and rate of breathing (i.e. hyperpnoea), but this response is often blunted in species that inhabit hypercapnic environments. In their natural habitat, DMRs putatively experience a gaseous environment ranging from normocapnic (0.1% CO2) to hypercapnic (6.0% CO2) conditions (Roper et al. 2001 J. Zool. 254, 101-107). As such, we hypothesized that DMRs would exhibit blunted hypercapnic ventilatory and metabolic responses, relative to those of non-fossorial rodent species. To test this hypothesis, we exposed awake, freely behaving DMRs to normoxic normocapnia (21% O2, 0% CO2, balance N2) or graded normoxic hypercapnia (21% O2, 0, 2, 5, 7 and 10% CO2, balance N2), and measured ventilation and metabolism using whole-body plethysmography and indirect calorimetry, respectively. We found that ventilation and metabolism were unchanged during prolonged normocapnia, whereas during graded hypercapnia, ventilation was elevated at 2% CO2 and above. As a result, O2 extraction efficiency at the lungs decreased with increasing hyperpnoea. Conversely, metabolic rate did not increase until 10% CO2, presumably due to the metabolic cost of hyperpnoea. Taken together, our results suggest that despite their fossorial lifestyle, DMRs do not exhibit adaptations in their ventilatory or metabolic responses to environmental hypercapnia.


Assuntos
Hipercapnia , Ratos-Toupeira , Aclimatação , Animais , Dióxido de Carbono , Hipóxia , Pulmão , Respiração
12.
J Comp Physiol B ; 189(2): 319-334, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30725174

RESUMO

Damaraland and naked mole rats are the only eusocial mammalian species and live in densely populated, poorly ventilated underground burrows, within which they likely experience intermittent periods of hypoxia. Naked mole rats are the most hypoxia-tolerant mammal and do not exhibit a hypoxic ventilatory response to acute or chronic hypoxia but instead rely upon a robust hypoxic metabolic response to tolerate reduced environmental O2. Conversely, physiological responses to hypoxia have not been explored in Damaraland mole rats but given their social and environmental similarities to naked mole rats, we hypothesized that they would exhibit similar physiological responses to hypoxia. We predicted that they would rely primarily on metabolic rate depression when O2 is limited and would not exhibit ventilatory responses to acute or chronic hypoxia. To test this hypothesis, we exposed Damaraland mole rats to normoxia (21% O2) or progressive hypoxia (12-5% O2), before and after acclimation to chronic hypoxia (8-10 days at 10% O2), and measured ventilatory, metabolic, and thermoregulatory responses. We found that ventilation increased up to fourfold with progressive hypoxia and body temperature decreased ~ 2 °C; however, a hypoxic metabolic response was absent. Following acclimation to chronic hypoxia, ventilation in 21% O2 was ~ twofold higher than in control animals, indicating the occurrence of ventilatory plasticity to hypoxia, and body temperature and metabolic rate were elevated. However, ventilation was not further augmented in acute hypoxia following acclimation to chronic hypoxia, indicating that ventilatory acclimatization to hypoxia was atypical of other mammals. These results refute our hypothesis and we conclude that Damaraland and naked mole rats have divergent physiological responses to hypoxia.


Assuntos
Metabolismo Basal , Regulação da Temperatura Corporal , Hipóxia/fisiopatologia , Ratos-Toupeira/fisiologia , Ventilação Pulmonar , Animais , Feminino , Masculino
13.
Diabetologia ; 62(3): 531-543, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30612139

RESUMO

AIMS/HYPOTHESIS: Müller glia (MG) are major sources of retinal cytokines, and their activation is closely linked to retinal inflammation and vascular leakage in diabetic retinopathy. Previously, we demonstrated that X-box binding protein 1 (XBP1), a transcription factor activated by endoplasmic reticulum (ER) stress in diabetic retinopathy, is involved in regulation of inflammation in retinal endothelial cells. Now, we have explored the role of XBP1 and ER stress in the regulation of MG-derived proinflammatory factors, and their influence on vascular permeability in diabetic retinopathy. METHODS: MG-specific conditional Xbp1 knockout (Xbp1Müller-/-) mice were generated by crossing Xbp1 flox/flox mice with Müller-Cre transgenic mice. Diabetes was modelled by induction with streptozotocin, and retinal vascular permeability was measured with FITC-conjugated dextran 2 months after induction. Primary Müller cells were isolated from Xbp1Müller-/- and Xbp1Müller+/+ mice and exposed to hypoxia and high levels of glucose. Levels of ER-stress and inflammatory factors were examined by real-time PCR, western blotting or immunohistochemistry. RESULTS: Xbp1Müller-/- mice exhibited normal retinal development and retinal function and expressed similar levels of ER-stress and inflammatory genes to Xbp1Müller+/+ littermates. In diabetes-inducing conditions, compared with Xbp1Müller+/+ mice, Xbp1Müller-/- mice had higher mRNA levels of retinal Vegf (also known as Vegfa) and Tnf-α (also known as Tnf) and ER-stress marker genes Grp78 (also known as Hspa5), Atf4, Chop (also known as Ddit3) and Atf6 and higher protein levels of vascular endothelial growth factor (VEGF), TNF-α, phospho-c-Jun N-terminal kinase (JNK), 78 kDa glucose-regulated protein (GRP78), phospho-eukaryotic translation initiation factor (eIF)2α and activating transcription factor (ATF)6. Retinal vascular permeability was significantly higher in diabetic Xbp1Müller-/- mice than in diabetic Xbp1Müller+/+ mice (p < 0.01). Results obtained in vitro with primary Müller cells isolated from Xbp1Müller-/- mice confirmed higher expression levels of inflammatory and ER-stress markers (but not GRP78) than in cells from Xbp1Müller+/+ mice. Moreover, XBP1-deficient Müller cells were more susceptible to high-glucose- or hypoxia-induced ER stress and inflammation than cells from Xbp1Müller+/+ mice. Inhibition of ER stress with chemical chaperones suppressed hypoxia-induced VEGF and TNF-α production in XBP1-deficient Müller cells. CONCLUSIONS/INTERPRETATION: Our results have revealed an important role of XBP1 and ER stress in MG-driven retinal inflammation, and suggest that targeting ER stress may represent a promising approach for the prevention and treatment of diabetic retinopathy.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Células Ependimogliais/metabolismo , Inflamação/metabolismo , Retina/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Permeabilidade Capilar/fisiologia , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/patologia , Estresse do Retículo Endoplasmático/fisiologia , Células Ependimogliais/patologia , Inflamação/patologia , Camundongos , Retina/patologia
14.
Neural Regen Res ; 13(11): 1861-1870, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30233053

RESUMO

The retina is one of the most energy demanding tissues in the body. Like most neurons in the central nervous system, retinal neurons consume high amounts of adenosine-5'-triphosphate (ATP) to generate visual signal and transmit the information to the brain. Disruptions in retinal metabolism can cause neuronal dysfunction and degeneration resulting in severe visual impairment and even blindness. The homeostasis of retinal metabolism is tightly controlled by multiple signaling pathways, such as the unfolded protein response (UPR), and the close interactions between retinal neurons and other retinal cell types including vascular cells and Müller glia. The UPR is a highly conserved adaptive cellular response and can be triggered by many physiological stressors and pathophysiological conditions. Activation of the UPR leads to changes in glycolytic rate, ATP production, de novo serine synthesis, and the hexosamine biosynthetic pathway, which are considered critical components of Müller glia metabolism and provide metabolic support to surrounding neurons. When these pathways are disrupted, neurodegeneration occurs rapidly. In this review, we summarize recent advance in studies of the UPR in Müller glia and highlight the potential role of the UPR in retinal degeneration through regulation of Müller glia metabolism.

15.
Front Aging Neurosci ; 10: 267, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30245625

RESUMO

p58IPK is an endoplasmic reticulum (ER)-resident chaperone playing a critical role in facilitating protein folding and protein homeostasis. Previously, we have demonstrated that p58IPK is expressed broadly in retinal neurons including retinal ganglion cells (RGCs) and loss of p58IPK results in age-related RGC degeneration. In the present study, we investigate the role of p58IPK in neuroprotection by in vitro and in vivo studies using primary RGC culture and two well-established disease-relevant RGC injury models: retinal ischemia/reperfusion (I/R) and microbead-induced ocular hypertension. Our results demonstrate that in both in vivo models, p58IPK -/- mice exhibit significantly increased RGC loss compared to wild type (WT) mice. In vitro, p58IPK-deficient RGCs show reduced viability and are more susceptible to cell death induced by the ER stress inducer tunicamycin (TM). Overexpression of p58IPK by adeno-associated virus (AAV) significantly diminishes TM-induced cell death in both WT and p58IPK -/- RGCs. Interestingly, we find that loss of p58IPK leads to reduced mRNA expression, but not the protein level, of mesencephalic astrocyte-derived neurotrophic factor (MANF), a neurotrophic factor that resides in the ER. Treatment with recombinant MANF protein protects R28 retinal neural cells and mouse retinal explants from TM-induced cell death. Taken together, our study suggests that p58IPK functions as an endogenous neuroprotectant for RGCs. The mechanisms underlying p58IPK's neuroprotective action and the potential interactions between p58IPK and MANF warrant future investigation.

16.
Adv Exp Med Biol ; 1074: 421-427, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29721972

RESUMO

The molecular chaperone endoplasmic reticulum protein 29 (ERp29) plays a critical role in protein folding, trafficking, and secretion. Though ubiquitously expressed, ERp29 is upregulated in response to ER stress and is found at higher levels in certain cell types such as secretory epithelial cells and neurons. As an ER resident protein, ERp29 shares many structural and functional similarities with protein disulfide isomerases, but is not regarded as part of this family due to several key differences. The broad expression and myriad roles of ERp29 coupled with its upregulation via the unfolded protein response (UPR) upon ER stress have implicated ERp29 in a range of cellular processes and diseases. We summarize the diverse activities of ERp29 in protein trafficking, cell survival and apoptosis, and ER homeostasis and highlight a potential role of ERp29 in neuroprotection in retinal and neurodegenerative diseases.


Assuntos
Proteínas de Choque Térmico/fisiologia , Doenças Neurodegenerativas/metabolismo , Degeneração Retiniana/metabolismo , Apoptose , Reparo do DNA , Estresse do Retículo Endoplasmático , Células Epiteliais/metabolismo , Proteínas do Olho/fisiologia , Junções Comunicantes/fisiologia , Homeostase , Humanos , Terapia de Alvo Molecular , Doenças Neurodegenerativas/prevenção & controle , Doenças Neurodegenerativas/terapia , Neurônios/metabolismo , Dobramento de Proteína , Transporte Proteico , Degeneração Retiniana/prevenção & controle , Degeneração Retiniana/terapia , Resposta a Proteínas não Dobradas
17.
Mol Neurodegener ; 13(1): 16, 2018 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-29615095

RESUMO

BACKGROUND: Aging is the strongest risk factor for neurodegenerative diseases and extended age results in neuronal degeneration and functional decline in the visual system. Among many contributing factors to age-related deterioration of neurons is an insufficient activation of the Unfolded Protein Response (UPR) in the endoplasmic reticulum (ER) in response to cellular stress. X-box binding protein 1 (XBP1) is a major component of the UPR and is essential for maintaining protein homeostasis and reducing cellular stresses. Herein, we investigate the role of XBP1 in maintaining morphological and functional integrity in retinal neurons during adulthood and the early stages of aging. METHODS: The basal and induced levels of XBP1 activation in the retina were measured in young adult and aged mice. Conditional knockout (cKO) of XBP1 in retinal neurons was achieved by crossing XBP1 floxed mice with a retina specific Cre-recombinase line (Chx10-Cre). Retinal morphology, neuronal populations including photoreceptors, bipolar cells, and retinal ganglion cells (RGCs), synaptic structure, and microglial activation were examined with immunohistochemistry and staining of retinal sections. Retinal function was evaluated with light-adapted (photopic) and dark adapted (scotopic) electroretinograms. Retinal mitochondrial function and metabolism was assessed by Seahorse XFe24 Extracellular Flux Analyzer. RESULTS: The retinas of aged wild type (WT) mice display a significantly reduced basal level of Xbp1s and compromised activation of ER stress response. In XBP1 cKO mice, significant structural degeneration of the retina, evidenced by thinning of retinal layers and a loss of RGCs, and functional defects indicated by diminished photopic and scotopic ERG b-waves are observed at the age of 12-14 months. Furthermore, discontinuous and disorganized synaptic laminae, colocalized with activated microglia, in the inner plexiform layer is found in the XBP1 cKO retinas. In addition, cKO mice demonstrate a significant increase in ectopic synapses between bipolar cells and photoreceptors, which is strikingly similar to WT mice at 20-24 months of age. These changes are associated with defective retinal glycolysis while mitochondrial respiratory function appears normal in the cKO retina. CONCLUSIONS: XBP1 cKO mice at 12-14 months of age show significant structural, functional, and metabolic deficits that closely resemble WT mice twice that age. Our findings suggest that the absence of XBP1, a critical component of the UPR, accelerates age-related retinal neurodegeneration.


Assuntos
Envelhecimento/metabolismo , Degeneração Neural/metabolismo , Retina/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Proteína 1 de Ligação a X-Box/metabolismo , Envelhecimento/patologia , Animais , Camundongos , Camundongos Knockout , Degeneração Neural/patologia , Retina/patologia
18.
Cell Death Dis ; 9(5): 467, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29700294

RESUMO

Persistent vascular injury and degeneration in diabetes are attributed in part to defective reparatory function of angiogenic cells. Our recent work implicates endoplasmic reticulum (ER) stress in high-glucose-induced bone marrow (BM) progenitor dysfunction. Herein, we investigated the in vivo role of ER stress in angiogenic abnormalities of streptozotocin-induced diabetic mice. Our data demonstrate that ER stress markers and inflammatory gene expression in BM mononuclear cells and hematopoietic progenitor cells increase dynamically with disease progression. Increased CHOP and cleaved caspase- 3 levels were observed in BM--derived early outgrowth cells (EOCs) after 3 months of diabetes. Inhibition of ER stress by ex vivo or in vivo chemical chaperone treatment significantly improved the generation and migration of diabetic EOCs while reducing apoptosis of these cells. Chemical chaperone treatment also increased the number of circulating angiogenic cells in peripheral blood, alleviated BM pathology, and enhanced retinal vascular repair following ischemia/reperfusion in diabetic mice. Mechanistically, knockdown of CHOP alleviated high-glucose-induced EOC dysfunction and mitigated apoptosis, suggesting a pivotal role of CHOP in mediating ER stress-associated angiogenic cell injury in diabetes. Together, our study suggests that targeting ER signaling may provide a promising and novel approach to enhancing angiogenic function in diabetes.


Assuntos
Apoptose , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Estresse do Retículo Endoplasmático , Neovascularização Fisiológica , Células-Tronco/metabolismo , Animais , Caspase 3/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/patologia , Masculino , Camundongos , Células-Tronco/patologia , Fator de Transcrição CHOP/metabolismo
19.
Front Genet ; 9: 658, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30619478

RESUMO

Normal function of the retinal pigment epithelium (RPE) is essential for maintaining the structural integrity of retinal photoreceptors and the visual process. Sustained oxidative damage of the RPE due to aging and other risk factors contributes to the development of age-related macular degeneration (AMD). The transcription factor NF-E2-related factor 2 (Nrf2) is a central regulator of cellular antioxidant and detoxification responses. Enhancing Nrf2 function protects RPE cells from oxidation-related apoptosis and cell death. Previously, we demonstrated that Nrf2 activation can be induced by endoplasmic reticulum (ER) stress; however, the mechanisms are not fully understood. In the present study, we examined the role of X box-binding protein 1 (XBP1), an ER stress-inducible transcription factor, in regulation of Nrf2 in the RPE. We found that RPE-specific XBP1 conditional knockout (cKO) mice exhibit a significant reduction in Nrf2 mRNA and protein levels, along with decreased expression of major Nrf2 target genes, in the RPE/choroid complex. Using primary RPE cells isolated from XBP1 cKO mice and human ARPE-19 cell line, we confirmed that loss of XBP1 gene or pharmacological inhibition of XBP1 splicing drastically reduces Nrf2 levels in the RPE. Conversely, overexpression of spliced XBP1 results in a modest but significant increase in cytosolic and nuclear Nrf2 protein levels without affecting the transcription of Nrf2 gene. Moreover, induction of ER stress by tunicamycin and thapsigargin markedly increases Nrf2 expression, which is abolished in cells pretreated with XBP1 splicing inhibitors 4µ8C and quinotrierixin. Mechanistic studies indicate that quinotrierixin reduces Nrf2 expression likely through inhibition of protein translation. Finally, we demonstrate that overexpression of Nrf2 protected RPE cells against oxidative injury but appeared to be insufficient to rescue from XBP1 deficiency-induced cell death. Taken together, our results indicate that XBP1 modulates Nrf2 activity in RPE cells and that XBP1 deficiency contributes to oxidative injury of the RPE.

20.
J Appl Physiol (1985) ; 123(4): 844-850, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28751373

RESUMO

While the mechanisms underlying the control of cutaneous vasodilation have been extensively studied, there remains a lack of understanding of the different factors that may modulate cutaneous perfusion during an exercise-induced heat stress. We evaluated the hypothesis that heat shock protein 90 (HSP90) contributes to the heat loss response of cutaneous vasodilation via the activation of nitric oxide synthase (NOS) during exercise in the heat. In 11 young males (25 ± 5 yr), cutaneous vascular conductance (CVC) was measured at four forearm skin sites that were continuously treated with 1) lactated Ringer solution (control), 2) NOS inhibition with 10 mM NG-nitro-l-arginine methyl ester (l-NAME), 3) HSP90 inhibition with 178 µM geldanamycin, or 4) a combination of 10 mM l-NAME and 178 µM geldanamycin. Participants rested in a moderate heat stress (35°C) condition for 70 min. Thereafter, they performed a 50-min bout of moderate-intensity cycling (~52% V̇o2peak) followed by a 30-min recovery period. We showed that NOS inhibition attenuated CVC (~40-50%) relative to the control site during pre- and postexercise rest in the heat (P ≤ 0.05); however, no effect of HSP90 inhibition was observed (P > 0.05). During exercise, we observed an attenuation of CVC with the separate inhibition of NOS (~40-50%) and HSP90 (~15-20%) compared with control (both P ≤ 0.05). However, the effect of HSP90 inhibition was absent in the presence of the coinhibition of NOS (P > 0.05). We show that HSP90 contributes to cutaneous vasodilation in young men exposed to the heat albeit during exercise only. We also show that the HSP90 contribution is due to NOS-dependent mechanisms.NEW & NOTEWORTHY We show that heat shock protein 90 functionally contributes to the heat loss response of cutaneous vasodilation during exercise in the heat, and this response is mediated through the activation of nitric oxide synthase. Therefore, interventions that may activate heat shock protein 90 may facilitate an increase in heat dissipation through an augmentation of cutaneous perfusion. In turn, this may attenuate or reduce the increase in core temperature and therefore the level of heat strain.


Assuntos
Exercício Físico , Proteínas de Choque Térmico HSP90/metabolismo , Temperatura Alta , Óxido Nítrico Sintase/metabolismo , Vasodilatação , Adulto , Pressão Sanguínea , Regulação da Temperatura Corporal , Humanos , Masculino , Fenômenos Fisiológicos da Pele , Adulto Jovem
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