Nuclear Hsp104 safeguards the dormant translation machinery during quiescence.

The resilience of cellular proteostasis declines with age, which drives protein aggregation and compromises viability. The nucleus has emerged as a key quality control compartment that handles misfolded proteins produced by the cytosolic protein biosynthesis system. Here, we find that age-associated metabolic cues target the yeast protein disaggregase Hsp104 to the nucleus to maintain a functional nuclear proteome during quiescence. The switch to respiratory metabolism and the accompanying decrease in translation rates direct cytosolic Hsp104 to the nucleus to interact with latent translation initiation factor eIF2 and to suppress protein aggregation. Hindering Hsp104 from entering the nucleus in quiescent cells results in delayed re-entry into the cell cycle due to compromised resumption of protein synthesis. In sum, we report that cytosolic-nuclear partitioning of the Hsp104 disaggregase is a critical mechanism to protect the latent protein synthesis machinery during quiescence in yeast, ensuring the rapid restart of translation once nutrients are replenished.

Using reporters of different misfolded proteins reveals differential strategies in processing protein aggregates.

The accumulation of misfolded proteins is a hallmark of aging and many neurodegenerative diseases, making it important to understand how the cellular machinery recognizes and processes such proteins. A key question in this respect is whether misfolded proteins are handled in a similar way regardless of their genetic origin. To approach this question, we compared how three different misfolded proteins, guk1-7, gus1-3, and pro3-1, are handled by the cell. We show that all three are nontoxic, even though highly overexpressed, highlighting their usefulness in analyzing the cellular response to misfolding in the absence of severe stress. We found significant differences between the aggregation and disaggregation behavior of the misfolded proteins. Specifically, gus1-3 formed some aggregates that did not efficiently recruit the protein disaggregase Hsp104 and did not colocalize with the other misfolded reporter proteins. Strikingly, while all three misfolded proteins generally coaggregated and colocalized to specific sites in the cell, disaggregation was notably different; the rate of aggregate clearance of pro3-1 was faster than that of the other misfolded proteins, and its clearance rate was not hindered when pro3-1 colocalized with a slowly resolved misfolded protein. Finally, we observed using super-resolution light microscopy as well as immunogold labeling EM in which both showed an even distribution of the different misfolded proteins within an inclusion, suggesting that misfolding characteristics and remodeling, rather than spatial compartmentalization, allows for differential clearance of these misfolding reporters residing in the same inclusion. Taken together, our results highlight how properties of misfolded proteins can significantly affect processing.

Genetically controlled mtDNA deletions prevent ROS damage by arresting oxidative phosphorylation.

Deletion of mitochondrial DNA in eukaryotes is currently attributed to rare accidental events associated with mitochondrial replication or repair of double-strand breaks. We report the discovery that yeast cells arrest harmful intramitochondrial superoxide production by shutting down respiration through genetically controlled deletion of mitochondrial oxidative phosphorylation genes. We show that this process critically involves the antioxidant enzyme superoxide dismutase 2 and two-way mitochondrial-nuclear communication through Rtg2 and Rtg3. While mitochondrial DNA homeostasis is rapidly restored after cessation of a short-term superoxide stress, long-term stress causes maladaptive persistence of the deletion process, leading to complete annihilation of the cellular pool of intact mitochondrial genomes and irrevocable loss of respiratory ability. This shows that oxidative stress-induced mitochondrial impairment may be under strict regulatory control. If the results extend to human cells, the results may prove to be of etiological as well as therapeutic importance with regard to age-related mitochondrial impairment and disease.

Large organellar changes occur during mild heat shock in yeast.

When the temperature is increased, the heat-shock response is activated to protect the cellular environment. The transcriptomics and proteomics of this process are intensively studied, while information about how the cell responds structurally to heat stress is mostly lacking. Here, Saccharomyces cerevisiae were subjected to a mild continuous heat shock (38°C) and intermittently cryo-immobilised for electron microscopy. Through measuring changes in all distinguishable organelle numbers, sizes and morphologies in over 2100 electron micrographs, a major restructuring of the internal architecture of the cell during the progressive heat shock was revealed. The cell grew larger but most organelles within it expanded even more, shrinking the volume of the cytoplasm. Organelles responded to heat shock at different times, both in terms of size and number, and adaptations of the morphology of some organelles (such as the vacuole) were observed. Multivesicular bodies grew by almost 70%, indicating a previously unknown involvement in the heat-shock response. A previously undescribed electron-translucent structure accumulated close to the plasma membrane. This all-encompassing approach provides a detailed chronological progression of organelle adaptation throughout the cellular heat-stress response.

Nuclear envelope budding is a response to cellular stress.

Nuclear envelope budding (NEB) is a recently discovered alternative pathway for nucleocytoplasmic communication distinct from the movement of material through the nuclear pore complex. Through quantitative electron microscopy and tomography, we demonstrate how NEB is evolutionarily conserved from early protists to human cells. In the yeast Saccharomyces cerevisiae, NEB events occur with higher frequency during heat shock, upon exposure to arsenite or hydrogen peroxide, and when the proteasome is inhibited. Yeast cells treated with azetidine-2-carboxylic acid, a proline analog that induces protein misfolding, display the most dramatic increase in NEB, suggesting a causal link to protein quality control. This link was further supported by both localization of ubiquitin and Hsp104 to protein aggregates and NEB events, and the evolution of these structures during heat shock. We hypothesize that NEB is part of normal cellular physiology in a vast range of species and that in S. cerevisiae NEB comprises a stress response aiding the transport of protein aggregates across the nuclear envelope.

An Hsp90 co-chaperone links protein folding and degradation and is part of a conserved protein quality control.

In this paper, we show that the essential Hsp90 co-chaperone Sgt1 is a member of a general protein quality control network that links folding and degradation through its participation in the degradation of misfolded proteins both in the cytosol and the endoplasmic reticulum (ER). Sgt1-dependent protein degradation acts in a parallel pathway to the ubiquitin ligase (E3) and ubiquitin chain elongase (E4), Hul5, and overproduction of Hul5 partly suppresses defects in cells with reduced Sgt1 activity. Upon proteostatic stress, Sgt1 accumulates transiently, in an Hsp90- and proteasome-dependent manner, with quality control sites (Q-bodies) of both yeast and human cells that co-localize with Vps13, a protein that creates organelle contact sites. Misfolding disease proteins, such as synphilin-1 involved in Parkinson's disease, are also sequestered to these compartments and require Sgt1 for their clearance.

Snd3 controls nucleus-vacuole junctions in response to glucose signaling.

Membrane contact sites facilitate the exchange of metabolites between organelles to support interorganellar communication. The nucleus-vacuole junctions (NVJs) establish physical contact between the perinuclear endoplasmic reticulum (ER) and the vacuole. Although the NVJ tethers are known, how NVJ abundance and composition are controlled in response to metabolic cues remains elusive. Here, we identify the ER protein Snd3 as central factor for NVJ formation. Snd3 interacts with NVJ tethers, supports their targeting to the contacts, and is essential for NVJ formation. Upon glucose exhaustion, Snd3 relocalizes from the ER to NVJs and promotes contact expansion regulated by central glucose signaling pathways. Glucose replenishment induces the rapid dissociation of Snd3 from the NVJs, preceding the slow disassembly of the junctions. In sum, this study identifies a key factor required for formation and regulation of NVJs and provides a paradigm for metabolic control of membrane contact sites.

Severe Hip Dysplasia in Skeletally Mature Patients With Spastic Cerebral Palsy: The Technique and Early Outcome of Comprehensive Surgical Correction (Including the Bernese PAO).

BACKGROUND: Treatment of symptomatic hip dysplasia in skeletally mature patients with spastic cerebral palsy (CP) can be challenging. This study examines our technical experience with the Bernese periacetabular osteotomy (PAO) in combination with adjunctive procedures in the treatment of this complex hip deformity. METHODS: Sixteen consecutive patients (18 hips) with symptomatic CP hip dysplasia were treated with a PAO and variable adjunctive procedures and retrospectively reviewed. Two patient (2 hips) were excluded due to insufficient follow-up. The average age at the time of surgery was 17.7 years (range: 13 to 28 y). We compared the preoperative to postoperative changes in radiographic parameters as well as early outcomes as measured by patient assessment of hip pain and function using the modified Harris Hip Score (mHHS). RESULTS: The average time of follow-up was 3.3 years (range: 2.0 to 6.3 y). Tönnis angles decreased from a median of 30 degrees (range: 18 to 45 degrees) preoperatively to a median of 6 degrees (range: -9 to 21 degrees) postoperatively. Lateral center-edge angles increased from a median of -8 degrees (range: -28 to 15 degrees) to a median of 32 degrees (range: 19 to 38 degrees). Anterior center-edge angles increased from a median of 2 degrees (range: -22 to 39 degrees) to a median of 35 degrees (range: 22 to 47 degrees). The extrusion index decreased from a median of 57% preoperatively (range: 35% to 73%) to a median of 21% (range: 11% to 36%) postoperatively.The median mHHS was 62 (range: 37 to 81) preoperatively and 85 (range: 65 to 100) postoperatively. Notably, the pain component of the mHHS improved from 20 (range: 0 to 44) to 42 (range: 30 to 44). Tönnis osteoarthritis grade preoperatively was either 0 (11 hips) or 1 (5 hips) and remained unchanged in 11 hips and increased by 1 grade in 5 hips. CONCLUSIONS: It has been our experience that the Bernese PAO in combination with appropriate adjunctive treatments has provided a very satisfactory surgical approach in the treatment of CP hip dysplasia. In the adolescent and young adult with spastic CP, utilizing the Bernese PAO technique makes it possible to obtain redirection of often a very severe acetabular dysplasia. Adjunctive soft tissue procedures and a proximal femoral osteotomy are frequently necessary to maintain postoperative stability. A notable improvement in the quality of life and function directly attributable to our surgical treatment of their pre-existing problematic hip dysplasia has been consistently noted in early follow-up for our patients. LEVEL OF EVIDENCE: Level IV-therapeutic.

Glucose-dependent insulinotropic peptide and risk of cardiovascular events and mortality: a prospective study.

AIMS/HYPOTHESIS: Evidence that glucose-dependent insulinotropic peptide (GIP) and/or the GIP receptor (GIPR) are involved in cardiovascular biology is emerging. We hypothesised that GIP has untoward effects on cardiovascular biology, in contrast to glucagon-like peptide 1 (GLP-1), and therefore investigated the effects of GIP and GLP-1 concentrations on cardiovascular disease (CVD) and mortality risk. METHODS: GIP concentrations were successfully measured during OGTTs in two independent populations (Malmö Diet Cancer-Cardiovascular Cohort [MDC-CC] and Prevalence, Prediction and Prevention of Diabetes in Botnia [PPP-Botnia]) in a total of 8044 subjects. GLP-1 (n = 3625) was measured in MDC-CC. The incidence of CVD and mortality was assessed via national/regional registers or questionnaires. Further, a two-sample Mendelian randomisation (2SMR) analysis between the GIP pathway and outcomes (coronary artery disease [CAD] and myocardial infarction) was carried out using a GIP-associated genetic variant, rs1800437, as instrumental variable. An additional reverse 2SMR was performed with CAD as exposure variable and GIP as outcome variable, with the instrumental variables constructed from 114 known genetic risk variants for CAD. RESULTS: In meta-analyses, higher fasting levels of GIP were associated with risk of higher total mortality (HR[95% CI] = 1.22 [1.11, 1.35]; p = 4.5 × 10-5) and death from CVD (HR[95% CI] 1.30 [1.11, 1.52]; p = 0.001). In accordance, 2SMR analysis revealed that increasing GIP concentrations were associated with CAD and myocardial infarction, and an additional reverse 2SMR revealed no significant effect of CAD on GIP levels, thus confirming a possible effect solely of GIP on CAD. CONCLUSIONS/INTERPRETATION: In two prospective, community-based studies, elevated levels of GIP were associated with greater risk of all-cause and cardiovascular mortality within 5-9 years of follow-up, whereas GLP-1 levels were not associated with excess risk. Further studies are warranted to determine the cardiovascular effects of GIP per se.

Inhibition of NFAT Signaling Restores Microvascular Endothelial Function in Diabetic Mice.

Central to the development of diabetic macro- and microvascular disease is endothelial dysfunction, which appears well before any clinical sign but, importantly, is potentially reversible. We previously demonstrated that hyperglycemia activates nuclear factor of activated T cells (NFAT) in conduit and medium-sized resistance arteries and that NFAT blockade abolishes diabetes-driven aggravation of atherosclerosis. In this study, we test whether NFAT plays a role in the development of endothelial dysfunction in diabetes. NFAT-dependent transcriptional activity was elevated in skin microvessels of diabetic Akita (Ins2 +/- ) mice when compared with nondiabetic littermates. Treatment of diabetic mice with the NFAT blocker A-285222 reduced NFATc3 nuclear accumulation and NFAT-luciferase transcriptional activity in skin microvessels, resulting in improved microvascular function, as assessed by laser Doppler imaging and iontophoresis of acetylcholine and localized heating. This improvement was abolished by pretreatment with the nitric oxide (NO) synthase inhibitor l-N G-nitro-l-arginine methyl ester, while iontophoresis of the NO donor sodium nitroprusside eliminated the observed differences. A-285222 treatment enhanced dermis endothelial NO synthase expression and plasma NO levels of diabetic mice. It also prevented induction of inflammatory cytokines interleukin-6 and osteopontin, lowered plasma endothelin-1 and blood pressure, and improved mouse survival without affecting blood glucose. In vivo inhibition of NFAT may represent a novel therapeutic modality to preserve endothelial function in diabetes.

Can a Septic Hip Decision Rule Aid in the Evaluation of Suspected Pediatric Musculoskeletal Infections?

BACKGROUND: Musculoskeletal (MSK) infections can be difficult to diagnose in acute care settings. The utility of clinical decision tools for pediatric MSK infections in an emergency department has not been well studied. OBJECTIVE: Our aim was to evaluate the performance of a septic hip clinical decision rule (CDR) in the evaluation of pediatric musculoskeletal infections. METHODS: We performed a retrospective study of children evaluated for an MSK infection in our emergency department from 2014 to 2016. Data collection included demographics, discharge diagnoses, and clinical/laboratory predictors from the CDR. A χ2 analysis and Wilcoxon rank-sum tests compared patients with and without MSK infections. Logistic regression analysis examined the predictors for MSK infections. A receiver operating characteristic (ROC) curve was calculated to evaluate the performance of the predictors. RESULTS: Of 996 evaluations included in the final analysis, 109 (10.9%) had MSK infections. In a multivariable model, an adjusted odds ratio (OR) was significant for fever (OR 3.9, 95% confidence interval [CI] 2.4-6.4), refusal to bear weight/pseudoparalysis (OR 4.4, 95% CI 2.7-7.1), and C-reactive protein (CRP) > 2.0 mg/dL (OR 5.4, 95% CI 3.2-9.1). The probability of infection was 75.1% with five predictors present, 1.9% for zero predictors, and 5.1% if one predictor was present. An ROC curve showed an area under the curve of 0.82, indicating moderate accuracy. CONCLUSIONS: A septic hip CDR demonstrates a low predicted probability of an MSK infection with zero or one clinical predictor present and moderate predictability with all five predictors. Fever, refusal to bear weight/pseudoparalysis, and CRP > 2.0 mg/dL performed best and should alert providers to consider other MSK infections in addition to septic arthritis.

Experience With a Care Process Model in the Evaluation of Pediatric Musculoskeletal Infections in a Pediatric Emergency Department.

OBJECTIVES: Care process models (CPMs) for certain conditions have improved clinical outcomes in children. This study describes the implementation and impact of a CPM for the evaluation of musculoskeletal infections in a pediatric emergency department (ED). METHODS: A retrospective pre-post intervention study was performed to analyze the impact of a musculoskeletal infection CPM. Patients were identified retrospectively through electronic order history for imaging of an extremity or joint and recommended laboratory tests. Clinical outcomes evaluated included hospital length of stay (LOS), time to magnetic resonance imaging (MRI), time to administration of antibiotics, hospital admission rate, and 30-day readmission rate. RESULTS: Musculoskeletal infection evaluations completed in the ED were reviewed from 1 year before implementation (n = 383) and 2 years after implementation (n = 1219) of the CPM. A significant improvement in the time to antibiotic administration for all patients (4.3 vs 3.7 hours, P < 0.05) and for patients with confirmed musculoskeletal infections (9.5 vs 4.9 hours, P < 0.05) was observed after the implementation of the CPM. The overall time to MRI (13.2 vs 10.3 hours, P = 0.29) and hospital LOS (4.7 vs 3.7 days, P = 0.11) were improved for all patients but were not statistically significant. The admission rate and 30-day readmission were similar before and after the implementation of the CPM. CONCLUSIONS: The implementation of a musculoskeletal infection CPM has standardized the approach to the evaluation and diagnosis of musculoskeletal infections resulting in a significant decrease in the time to administer antibiotics and a downward trend in time to MRI and hospital LOS.

In vivo inhibition of nuclear factor of activated T-cells leads to atherosclerotic plaque regression in IGF-II/LDLR-/-ApoB100/100 mice.

AIMS: Despite vast clinical experience linking diabetes and atherosclerosis, the molecular mechanisms leading to accelerated vascular damage are still unclear. Here, we investigated the effects of nuclear factor of activated T-cells inhibition on plaque burden in a novel mouse model of type 2 diabetes that better replicates human disease. METHODS & RESULTS: IGF-II/LDLR-/-ApoB100/100 mice were generated by crossbreeding low-density lipoprotein receptor-deficient mice that synthesize only apolipoprotein B100 (LDLR-/-ApoB100/100) with transgenic mice overexpressing insulin-like growth factor-II in pancreatic ß cells. Mice have mild hyperglycaemia and hyperinsulinaemia and develop complex atherosclerotic lesions. In vivo treatment with the nuclear factor of activated T-cells blocker A-285222 for 4 weeks reduced atherosclerotic plaque area and degree of stenosis in the brachiocephalic artery of IGF-II/LDLR-/-ApoB100/100 mice, as assessed non-invasively using ultrasound biomicroscopy prior and after treatment, and histologically after termination. Treatment had no impact on plaque composition (i.e. muscle, collagen, macrophages). The reduced plaque area could not be explained by effects of A-285222 on plasma glucose, insulin or lipids. Inhibition of nuclear factor of activated T-cells was associated with increased expression of atheroprotective NOX4 and of the anti-oxidant enzyme catalase in aortic vascular smooth muscle cells. CONCLUSION: Targeting the nuclear factor of activated T-cells signalling pathway may be an attractive approach for the treatment of diabetic macrovascular complications.

ORAI channels are critical for receptor-mediated endocytosis of albumin.

Impaired albumin reabsorption by proximal tubular epithelial cells (PTECs) has been highlighted in diabetic nephropathy (DN), but little is known about the underlying molecular mechanisms. Here we find that ORAI1-3, are preferentially expressed in PTECs and downregulated in patients with DN. Hyperglycemia or blockade of insulin signaling reduces the expression of ORAI1-3. Inhibition of ORAI channels by BTP2 and diethylstilbestrol or silencing of ORAI expression impairs albumin uptake. Transgenic mice expressing a dominant-negative Orai1 mutant (E108Q) increases albuminuria, and in vivo injection of BTP2 exacerbates albuminuria in streptozotocin-induced and Akita diabetic mice. The albumin endocytosis is Ca2+-dependent and accompanied by ORAI1 internalization. Amnionless (AMN) associates with ORAIs and forms STIM/ORAI/AMN complexes after Ca2+ store depletion. STIM1/ORAI1 colocalizes with clathrin, but not with caveolin, at the apical membrane of PTECs, which determines clathrin-mediated endocytosis. These findings provide insights into the mechanisms of protein reabsorption and potential targets for treating diabetic proteinuria.

Differential effects of soluble and aggregating polyQ proteins on cytotoxicity and type-1 myosin-dependent endocytosis in yeast.

Huntington's disease develops when the polyglutamine (polyQ) repeat in the Huntingtin (Htt) protein is expanded to over 35 glutamines rendering it aggregation-prone. Here, using Htt exon-1 as a polyQ model protein in a genome-wide screen in yeast, we show that the normal and soluble Htt exon-1 is toxic in cells with defects in type-1 myosin-dependent endocytosis. The toxicity of Htt is linked to physical interactions with type-1 myosins, which occur via the Htt proline-rich region, leading to a reduction in actin patch polarization and clathrin-dependent endocytosis. An expansion of the polyQ stretch from 25 to 103 glutamines, which causes Htt aggregation, alleviated Htt toxicity in cells lacking Myo5 or other components involved in early endocytosis. The data suggest that the proline-rich stretch of Htt interacts with type-1 myosin/clathrin-dependent processes and demonstrate that a reduction in the activity of such processes may result in a positive selection for polyQ expansions.

Glucose-Dependent Insulinotropic Polypeptide Stimulates Osteopontin Expression in the Vasculature via Endothelin-1 and CREB.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone with extrapancreatic effects beyond glycemic control. Here we demonstrate unexpected effects of GIP signaling in the vasculature. GIP induces the expression of the proatherogenic cytokine osteopontin (OPN) in mouse arteries via local release of endothelin-1 and activation of CREB. Infusion of GIP increases plasma OPN concentrations in healthy individuals. Plasma endothelin-1 and OPN concentrations are positively correlated in patients with critical limb ischemia. Fasting GIP concentrations are higher in individuals with a history of cardiovascular disease (myocardial infarction, stroke) when compared with control subjects. GIP receptor (GIPR) and OPN mRNA levels are higher in carotid endarterectomies from patients with symptoms (stroke, transient ischemic attacks, amaurosis fugax) than in asymptomatic patients, and expression associates with parameters that are characteristic of unstable and inflammatory plaques (increased lipid accumulation, macrophage infiltration, and reduced smooth muscle cell content). While GIPR expression is predominantly endothelial in healthy arteries from humans, mice, rats, and pigs, remarkable upregulation is observed in endothelial and smooth muscle cells upon culture conditions, yielding a "vascular disease-like" phenotype. Moreover, the common variant rs10423928 in the GIPR gene is associated with increased risk of stroke in patients with type 2 diabetes.

Systematic study of constitutive cyclooxygenase-2 expression: Role of NF-κB and NFAT transcriptional pathways.

Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system.

A witnessed case of a classic metaphyseal fracture caused during IV line placement in a child: Insight into mechanism of injury.

Recent publications argue that classic metaphyseal fractures are caused by rickets as opposed to trauma. Previous case reports of accidental traumatic classic metaphyseal fractures have been discounted due to lack of identification of the fracture at the time of the traumatic event, and lack of an evaluation for boney metabolic disorders. We report a case of a 20 day old male with a diagnosis of congenital vertical talus who sustained a classic metaphyseal fracture of the distal tibia during manipulation in preparation for intravenous line placement. The mechanics of the event causing the classic metaphyseal fracture were witnessed and accompanied by an audible "pop". Prior x-rays of the tibia demonstrate normal osseous morphology, and an evaluation for boney metabolic disorders was normal. This case identifies a traumatic classic metaphyseal fracture and provides insight into the types of forces necessary to cause such a fracture.

Nuclear factor of activated T cells is activated in the endothelium of retinal microvessels in diabetic mice.

The pathogenesis of diabetic retinopathy (DR) remains unclear but hyperglycemia is an established risk factor. Endothelial dysfunction and changes in Ca2+ signaling have been shown to precede the onset of DR. We recently demonstrated that high extracellular glucose activates the Ca(2+)/calcineurin-dependent transcription factor NFAT in cerebral arteries and aorta, promoting the expression of inflammatory markers. Here we show, using confocal immunofluorescence, that NFAT is expressed in the endothelium of retinal microvessels and is readily activated by high glucose. This was inhibited by the NFAT blocker A-285222 as well as by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. Acute hyperglycemia induced by an IP-GTT (intraperitoneal glucose tolerance test) resulted in increased NFATc3 nuclear accumulation and NFAT-dependent transcriptional activity in retinal vessels of NFAT-luciferase reporter mice. In both Akita (Ins2(+/-) ) and streptozotocin- (STZ-) induced diabetic mice, NFAT transcriptional activity was elevated in retinal vessels. In vivo inhibition of NFAT with A-285222 decreased the expression of OPN and ICAM-1 mRNA in retinal vessels, prevented a diabetes driven downregulation of anti-inflammatory IL-10 in retina, and abrogated the increased vascular permeability observed in diabetic mice. Results identify NFAT signaling as a putative target for treatment of microvascular complications in diabetes.

High glucose enhances store-operated calcium entry by upregulating ORAI/STIM via calcineurin-NFAT signalling.

UNLABELLED: ORAI and stromal interaction molecule (STIM) are store-operated channel molecules that play essential roles in human physiology through a coupling mechanism of internal Ca(2+) store to Ca(2+) influx. However, the roles of ORAI and STIM in vascular endothelial cells under diabetic conditions remain unknown. Here, we investigated expression and signalling pathways of ORAI and STIM regulated by high glucose or hyperglycaemia using in vitro cell models, in vivo diabetic mice and tissues from patients. We found that ORAI1-3 and STIM1-2 were ubiquitously expressed in human vasculatures. Their expression was upregulated by chronic treatment with high glucose (HG, 25 mM D-glucose), which was accompanied by enhanced store-operated Ca(2+) influx in vascular endothelial cells. The increased expression was also observed in the aortae from genetically modified Akita diabetic mice (C57BL/6-Ins2(Akita)/J) and streptozocin-induced diabetic mice, and aortae from diabetic patients. HG-induced upregulation of ORAI and STIM genes was prevented by the calcineurin inhibitor cyclosporin A and NFATc3 siRNA. Additionally, in vivo treatment with the nuclear factor of activated T cells (NFAT) inhibitor A-285222 prevented the gene upregulation in Akita mice. However, HG had no direct effects on ORAI1-3 currents and the channel activation process through cytosolic STIM1 movement in the cells co-expressing STIM1-EYFP/ORAIs. We concluded that upregulation of STIM/ORAI through Ca(2+)-calcineurin-NFAT pathway is a novel mechanism causing abnormal Ca(2+) homeostasis and endothelial dysfunction under hyperglycaemia. KEY MESSAGE: ORAI1-3 and STIM1-2 are ubiquitously expressed in vasculatures and upregulated by high glucose. Increased expression is confirmed in Akita (Ins2(Akita)/J) and STZ diabetic mice and patients. Upregulation mechanism is mediated by Ca(2+)/calcineurin/NFATc3 signalling. High glucose has no direct effects on ORAI1-3 channel activity and channel activation process.