Macular and retinal dysfunction of unknown origin in adults with normal fundi: evidence for an autoimmune pathophysiology.

Here we report the discovery of and phenotypic characterization of a retinal disorder of unknown origin in adults using clinical, electrophysiological and psychophysical techniques, and to seek the presence of circulating retinal autoantibodies in the sera of these patients. Sixteen patients were identified with progressive bilateral visual loss over a period of months. Ten of the patients were male, and the average age was 55.3 years (range from 43 to 76 years). Known causes such as carcinoma-associated retinopathy, acute zonal occult outer retinopathy and hereditary cone dystrophy appeared unlikely. Investigations included electrophysiology, fundus autofluorescence imaging and psychophysical tests. The sera of these patients were analyzed with indirect immunocytochemistry and Western immunoblot analysis on murine (BALB/c) retinal tissue for the presence of retinal autoantibodies. Bilateral visual loss and photophobia progressed over a period of months to years (average 28.7 months, range 3-67) and subsequently stabilized. No abnormality was observed by biomicroscopy, angiography or autofluorescence imaging. Electrophysiology indicated predominant cone-system dysfunction, either macular or generalized, and post-phototransduction involvement in 9 patients (56%). Photopic and scotopic visual fields and dark adaptation kinetics showed both cone and rod system involvement in all cases. Heterogeneous immunohistochemical staining patterns were seen with the sera of these patients as compared with controls. A majority of the affected patients (9/15) stained with an antinuclear pattern. The retinal autoantibodies from the sera of most patients reacted with the retinal proteins of molecular weight between 34 and 40 kDa. The aetiology of this distinctive retinal disorder therefore appears to be mediated through an autoimmune mechanism.

La programmmation foetale de la dysfonction vasculaire pulmonaire chez la souris : rôle des mécanismes épigénétiques = Fetal programming of pulmonary vascular dysfunction in mice : role of epigenetic mechanisms

Rapport de synthèseDes événements pathologiques survenant pendant la période foetale prédisposent la descendance aux maladies cardiovasculaires systémiques. Il existe peu de connaissances au sujet de la circulation pulmonaire et encore moins quant aux mécanismes sous-jacents. La sous-alimentation maternelle pendant la grossesse peut représenter un modèle d'investigation de ces mécanismes, parce que chez l'animal et l'homme elle est associée à une dysfonction vasculaire systémique chez la progéniture. Chez le rat, la diète restrictive pendant la grossesse induit une augmentation du stress oxydatif dans le placenta. Les dérivés de l'oxygène sont connus pour induire des altérations épigénétiques et peuvent traverser la barrière placentaire. Nous avons dès lors spéculé que chez la souris la diète restrictive pendant la grossesse induit une dysfonction vasculaire pulmonaire chez sa progéniture qui serait liée à un mécanisme épigénétique.Pour tester cette hypothèse, nous avons examiné la fonction vasculaire pulmonaire et la méthylation de l'ADN pulmonaire à la fin de 2 semaines d'exposition à l'hypoxie chez la progéniture de souris soumises à une diète restrictive pendant la grossesse et des souris contrôles. Nous avons trouvé que la vasodilatation endothélium-dépendante de l'artère pulmonaire in vitro était défectueuse, et que l'hypertension pulmonaire et l'hypertrophie ventriculaire droite induites par l'hypoxie in vivo étaient exagérées chez la progéniture de souris soumises à une diète restrictive pendant la grossesse. Cette dysfonction vasculaire pulmonaire était associée avec une altération de la méthylation de l'ADN pulmonaire. L'administration d'inhibiteurs de la déacétylase des histones, le Butyrate et la Trichostatine-A à la progéniture de souris soumises à une diète restrictive pendant la grossesse a normalisé la méthylation de l'ADN et la fonction vasculaire pulmonaire. Finalement, l'administration du nitroxyde Tempol aux mères durant la diète restrictive pendant la grossesse a prévenu la dysfonction vasculaire et la dysméthylation chez la progéniture.Ces découvertes démontrent que chez la souris la sous-alimentation pendant la gestation induit une dysfonction vasculaire chez la progéniture qui est causée par un mécanisme épigénétique. Il est possible qu'un mécanisme similaire soit impliqué dans la programmation foetale de la dysfonction vasculaire chez les humains.

Subclinical thyroid dysfunction, cardiac function, and the risk of heart failure. The Cardiovascular Health study.

OBJECTIVES: The goal of this study was to determine whether subclinical thyroid dysfunction was associated with incident heart failure (HF) and echocardiogram abnormalities. BACKGROUND: Subclinical hypothyroidism and hyperthyroidism have been associated with cardiac dysfunction. However, long-term data on the risk of HF are limited. METHODS: We studied 3,044 adults>or=65 years of age who initially were free of HF in the Cardiovascular Health Study. We compared adjudicated HF events over a mean 12-year follow-up and changes in cardiac function over the course of 5 years among euthyroid participants, those with subclinical hypothyroidism (subdivided by thyroid-stimulating hormone [TSH] levels: 4.5 to 9.9, >or=10.0 mU/l), and those with subclinical hyperthyroidism. RESULTS: Over the course of 12 years, 736 participants developed HF events. Participants with TSH>or=10.0 mU/l had a greater incidence of HF compared with euthyroid participants (41.7 vs. 22.9 per 1,000 person years, p=0.01; adjusted hazard ratio: 1.88; 95% confidence interval: 1.05 to 3.34). Baseline peak E velocity, which is an echocardiographic measurement of diastolic function associated with incident HF in the CHS cohort, was greater in those patients with TSH>or=10.0 mU/l compared with euthyroid participants (0.80 m/s vs. 0.72 m/s, p=0.002). Over the course of 5 years, left ventricular mass increased among those with TSH>or=10.0 mU/l, but other echocardiographic measurements were unchanged. Those patients with TSH 4.5 to 9.9 mU/l or with subclinical hyperthyroidism had no increase in risk of HF. CONCLUSIONS: Compared with euthyroid older adults, those adults with TSH>or=10.0 mU/l have a moderately increased risk of HF and alterations in cardiac function but not older adults with TSH<10.0 mU/l. Clinical trials should assess whether the risk of HF might be ameliorated by thyroxine replacement in individuals with TSH>or=10.0 mU/l.

Treatment of ruptured abdominal aortic aneurysm, a permanent challenge or a waste of resources? Prediction of outcome using a multi-organ-dysfunction score.

OBJECTIVES: in a retrospective study, attempts have been made to identify individual organ-dysfunction risk profiles influencing the outcome after surgery for ruptured abdominal aortic aneurysms. METHODS: out of 235 patients undergoing graft replacement for abdominal aortic aneurysms, 57 (53 men, four women, mean age 72 years [s.d. 8.8]) were treated for ruptured aneurysms in a 3-year period. Forty-eight preoperative, 13 intraoperative and 34 postoperative variables were evaluated statistically. A simple multi-organ dysfunction (MOD) score was adopted. RESULTS: the perioperative mortality was 32%. Three patients died intraoperatively, four within 48 h and 11 died later. A significant influence for pre-existing risk factors was identified only for cardiovascular diseases. Multiple linear-regression analysis indicated that a haemoglobin <90 g/l, systolic blood pressure <80 mmHg and ECG signs of ischaemia at admission were highly significant risk factors. The cause of death for patients, who died more than 48 h postoperatively, was mainly MOD. All patients with a MOD score >/=4 died (n=7). These patients required 27% of the intensive-care unit (ICU) days of all patients and 72% of the ICU days of the non-survivors. CONCLUSION: patients with ruptured aortic aneurysms from treatment should not be excluded. However, a physiological scoring system after 48 h appears justifiable in order to decide on the appropriateness of continual ICU support.

Expressed isolated integrin beta1 subunit cytodomain induces endothelial cell death secondary to detachment.

Expression of isolated beta integrin cytoplasmic domains in cultured endothelial cells was reported to induce cell detachment and death. To test whether cell death was the cause or the consequence of cell detachment, we expressed isolated integrin beta1 cytoplasmic and transmembrane domains (CH1) in cultured human umbilical vein endothelial cells (HUVEC), and monitored detachment, viability, caspase activation and signaling. CH1 expression induced dose-dependent cell detachment. At 24 h over 90% of CH1-expressing HUVEC were detached but largely viable (>85%). No evidence of pro-caspase-8,-3, and PARP cleavage or suppression of phosphorylation of ERK, PKB and Ikappa-B was observed. The caspase inhibitor z-VAD did not prevent cell detachment. At 48 h, however, CH1-expressing cells were over 50% dead. As a comparison trypsin-mediated detachment resulted in a time-dependent cell death, paralleled by caspase-3 activation and suppression of ERK, PKB and Ikappa-B phosphoyrylation at 24 h or later after detachment. HUVEC stimulation with agents that strengthen integrin-mediated adhesion (i.e. PMA, the Src inhibitor PP2 and COMP-Ang1) did not prevent CH1-induced detachment. Expression of CH1 in rat carotid artery endothelial cells in vivo caused endothelial cell detachment and increased nuclear DNA fragmentation among detached cells. A construct lacking the integrin cytoplasmic domain (CH2) had no effect on adhesion and cell viability in vitro and in vivo. These results demonstrate that isolated beta1 cytoplasmic domain expression induces caspase-independent detachment of viable endothelial cells and that death is secondary to detachment (i.e. anoikis). They also reveal an essential role for integrins in the adhesion and survival of quiescent endothelial cells in vivo.

Insulin-secreting beta-cell dysfunction induced by human lipoproteins.

Diabetes is associated with significant changes in plasma concentrations of lipoproteins. We tested the hypothesis that lipoproteins modulate the function and survival of insulin-secreting cells. We first detected the presence of several receptors that participate in the binding and processing of plasma lipoproteins and confirmed the internalization of fluorescent low density lipoprotein (LDL) and high density lipoprotein (HDL) particles in insulin-secreting beta-cells. Purified human very low density lipoprotein (VLDL) and LDL particles reduced insulin mRNA levels and beta-cell proliferation and induced a dose-dependent increase in the rate of apoptosis. In mice lacking the LDL receptor, islets showed a dramatic decrease in LDL uptake and were partially resistant to apoptosis caused by LDL. VLDL-induced apoptosis of beta-cells involved caspase-3 cleavage and reduction in the levels of the c-Jun N-terminal kinase-interacting protein-1. In contrast, the proapoptotic signaling of lipoproteins was antagonized by HDL particles or by a small peptide inhibitor of c-Jun N-terminal kinase. The protective effects of HDL were mediated, in part, by inhibition of caspase-3 cleavage and activation of Akt/protein kinase B. In conclusion, human lipoproteins are critical regulators of beta-cell survival and may therefore contribute to the beta-cell dysfunction observed during the development of type 2 diabetes.

Complementary effects of adenosine and angiotensin II in hypoxemia-induced renal dysfunction in the rabbit.

The acute renal effects of hypoxemia and the ability of the co-administration of an angiotensin converting enzyme inhibitor (perindoprilat) and an adenosine receptor antagonist (theophylline) to prevent these effects were assessed in anesthetized and mechanically-ventilated rabbits. Renal blood flow (RBF) and glomerular filtration rate (GFR) were determined by the clearances of para-aminohippuric acid and inulin, respectively. Each animal acted as its own control. In 8 untreated rabbits, hypoxemia induced a significant drop in mean blood pressure (-12 +/- 2%), GFR (-16 +/- 3%) and RBF (-12 +/- 3%) with a concomitant increase in renal vascular resistance (RVR) (+ 18 +/- 5%), without changes in filtration fraction (FF) (-4 +/- 2%). These results suggest the occurrence of both pre- and postglomerular vasoconstriction during the hypoxemic stress. In 7 rabbits pretreated with intravenous perindoprilat (20 microg/kg), the hypoxemia-induced changes in RBF and RVR were prevented. FF decreased significantly (-18 +/- 2%), while the drop in GFR was partially blunted. These results could be explained by the inhibition of the angiotensin-mediated efferent vasoconstriction by perindoprilat. In 7 additional rabbits, co-administration of perindoprilat and theophylline (1 mg/kg) completely prevented the hypoxemia-induced changes in RBF (+ 11 +/- 3%) and GFR (+ 2 +/- 3%), while RVR decreased significantly (-14 +/- 3%). Since adenosine and angiotensin II were both shown to participate, at least in part, in the renal changes induced by hypoxemia, the beneficial effects of perindoprilat and theophylline in this model could be mediated by complementary actions of angiotensin II and adenosine on the renal vasculature.

Effect of atazanavir versus other protease inhibitor-containing antiretroviral therapy on endothelial function in HIV-infected persons: randomised controlled trial.

OBJECTIVE: Impaired endothelial function was demonstrated in HIV-infected persons on protease inhibitor (PI)-containing antiretroviral therapy, probably due to altered lipid metabolism. Atazanavir is a PI causing less atherogenic lipoprotein changes. This study determined whether endothelial function improves after switching from other PI to atazanavir. DESIGN: Randomised, observer-blind, treatment-controlled trial. SETTING: Three university-based outpatient clinics. PATIENTS: 39 HIV-infected persons with suppressed viral replication on PI-containing regimens and fasting low-density lipoprotein (LDL)-cholesterol greater than 3 mmol/l. INTERVENTION: Patients were randomly assigned to continue the current PI or change to unboosted atazanavir. MAIN OUTCOME MEASURES: Endpoints at week 24 were endothelial function assessed by flow-mediated dilation (FMD) of the brachial artery, lipid profiles and serum inflammation and oxidative stress parameters. RESULTS: Baseline characteristics and mean FMD values of the two treatment groups were comparable (3.9% (SD 1.8) on atazanavir versus 4.0% (SD 1.5) in controls). After 24 weeks' treatment, FMD decreased to 3.3% (SD 1.4) and 3.4% (SD 1.7), respectively (all p = ns). Total cholesterol improved in both groups (p<0.0001 and p = 0.01, respectively) but changes were more pronounced on atazanavir (p = 0.05, changes between groups). High-density lipoprotein and triglyceride levels improved on atazanavir (p = 0.03 and p = 0.003, respectively) but not in controls. Serum inflammatory and oxidative stress parameters did not change; oxidised LDL improved significantly in the atazanavir group. CONCLUSIONS: The switch from another PI to atazanavir in treatment-experienced patients did not result in improvement of endothelial function despite significantly improved serum lipids. Atherogenic lipid profiles and direct effects of antiretroviral drugs on the endothelium may affect vascular function. Trial registration number: NCT00447070.

The integrin antagonist cilengitide activates alphaVbeta3, disrupts VE-cadherin localization at cell junctions and enhances permeability in endothelial cells.

Cilengitide is a high-affinity cyclic pentapeptdic alphaV integrin antagonist previously reported to suppress angiogenesis by inducing anoikis of endothelial cells adhering through alphaVbeta3/alphaVbeta5 integrins. Angiogenic endothelial cells express multiple integrins, in particular those of the beta1 family, and little is known on the effect of cilengitide on endothelial cells expressing alphaVbeta3 but adhering through beta1 integrins. Through morphological, biochemical, pharmacological and functional approaches we investigated the effect of cilengitide on alphaVbeta3-expressing human umbilical vein endothelial cells (HUVEC) cultured on the beta1 ligands fibronectin and collagen I. We show that cilengitide activated cell surface alphaVbeta3, stimulated phosphorylation of FAK (Y(397) and Y(576/577)), Src (S(418)) and VE-cadherin (Y(658) and Y(731)), redistributed alphaVbeta3 at the cell periphery, caused disappearance of VE-cadherin from cellular junctions, increased the permeability of HUVEC monolayers and detached HUVEC adhering on low-density beta1 integrin ligands. Pharmacological inhibition of Src kinase activity fully prevented cilengitide-induced phosphorylation of Src, FAK and VE-cadherin, and redistribution of alphaVbeta3 and VE-cadherin and partially prevented increased permeability, but did not prevent HUVEC detachment from low-density matrices. Taken together, these observations reveal a previously unreported effect of cilengitide on endothelial cells namely its ability to elicit signaling events disrupting VE-cadherin localization at cellular contacts and to increase endothelial monolayer permeability. These effects are potentially relevant to the clinical use of cilengitide as anticancer agent.

Caveolin-1 opens endothelial cell junctions by targeting catenins.

AIMS: A fundamental phenomenon in inflammation is the loss of endothelial barrier function, in which the opening of endothelial cell junctions plays a central role. However, the molecular mechanisms that ultimately open the cell junctions are largely unknown.¦METHODS AND RESULTS: Impedance spectroscopy, biochemistry, and morphology were used to investigate the role of caveolin-1 in the regulation of thrombin-induced opening of cell junctions in cultured human and mouse endothelial cells. Here, we demonstrate that the vascular endothelial (VE) cadherin/catenin complex targets caveolin-1 to endothelial cell junctions. Association of caveolin-1 with VE-cadherin/catenin complexes is essential for the barrier function decrease in response to the pro-inflammatory mediator thrombin, which causes a reorganization of the complex in a rope ladder-like pattern accompanied by a loss of junction-associated actin filaments. Mechanistically, we show that in response to thrombin stimulation the protease-activated receptor 1 (PAR-1) causes phosphorylation of caveolin-1, which increasingly associates with β- and γ-catenin. Consequently, the association of β- and γ-catenin with VE-cadherin is weakened, thus allowing junction reorganization and a decrease in barrier function. Thrombin-induced opening of cell junctions is lost in caveolin-1-knockout endothelial cells and after expression of a Y/F-caveolin-1 mutant but is completely reconstituted after expression of wild-type caveolin-1.¦CONCLUSION: Our results highlight the pivotal role of caveolin-1 in VE-cadherin-mediated cell adhesion via catenins and, in turn, in barrier function regulation.

Autophagosome maturation is impaired in Fabry disease.

Fabry disease is a lysosomal storage disorder (LSD) caused by a deficiency in alpha-galactosidase A. The disease is characterized by severe major organ involvement, but the pathologic mechanisms responsible have not been elucidated. Disruptions of autophagic processes have been reported for other LSDs, but have not yet been investigated in Fabry disease. Renal biopsies were obtained from five adult male Fabry disease patients before and after three years of enzyme replacement therapy (ERT) with agalsidase alfa. Vacuole accumulation was seen in renal biopsies from all patients compared with control biopsies. Decreases in the number of vacuoles were seen after three years of ERT primarily in renal endothelial cells and mesangial cells. Measurement of the levels of LC3, a specific autophagy marker, in cultured cells from Fabry patients revealed increased basal levels compared to cells from non-Fabry subjects and a larger increase in response to starvation than seen in non-Fabry cells. Starvation in the presence of protease inhibitors did not result in a significant increase in LC3 in Fabry cells, whereas a further increase in LC3 was observed in non-Fabry cells, an observation that is consistent with impaired autophagic flux in Fabry disease. Overexpression of LC3 mRNA in Fabry fibroblasts compared to control cells is consistent with an upregulation of autophagy. Furthermore, LC3 and p62/SQSTM1 (that binds to LC3) staining in renal tissues and in cultured fibroblasts from Fabry patients supports impairment of autophagic flux. These findings suggest that Fabry disease is linked to a deregulation of autophagy.

Functional analysis of Plasmodium vivax VIR proteins reveals different subcellular localizations and cytoadherence to the ICAM-1 endothelial receptor.

The subcellular localization and function of variant subtelomeric multigene families in Plasmodium vivax remain vastly unknown. Among them, the vir superfamily is putatively involved in antigenic variation and in mediating adherence to endothelial receptors. In the absence of a continuous in vitro culture system for P. vivax, we have generated P. falciparum transgenic lines expressing VIR proteins to infer location and function. We chose three proteins pertaining to subfamilies A (VIR17), C (VIR14) and D (VIR10), with domains and secondary structures that predictably traffic these proteins to different subcellular compartments. Here, we showed that VIR17 remained inside the parasite and around merozoites, whereas VIR14 and VIR10 were exported to the membrane of infected red blood cells (iRBCs) in an apparent independent pathway of Maurer's clefts. Remarkably, VIR14 was exposed at the surface of iRBCs and mediated adherence to different endothelial receptors expressed in CHO cells under static conditions. Under physiological flow conditions, however, cytoadherence was only observed to ICAM-1, which was the only receptor whose adherence was specifically and significantly inhibited by antibodies against conserved motifs of VIR proteins. Immunofluorescence studies using these antibodies also showed different subcellular localizations of VIR proteins in P. vivax-infected reticulocytes from natural infections. These data suggest that VIR proteins are trafficked to different cellular compartments and functionally demonstrates that VIR proteins can specifically mediate cytoadherence to the ICAM-1 endothelial receptor.

Mitochondrial dysfunction increases oxidative stress and decreases chronological life span in fission yeast

Background: Oxidative stress is a probable cause of aging and associated diseases. Reactive oxygen species (ROS) originate mainly from endogenous sources, namely the mitochondria. Methodology/Principal Findings: We analyzed the effect of aerobic metabolism on oxidative damage in Schizosaccharomyces pombe by global mapping of those genes that are required for growth on both respiratory-proficient media and hydrogen-peroxide-containing fermentable media. Out of a collection of approximately 2700 haploid yeast deletion mutants, 51 were sensitive to both conditions and 19 of these were related to mitochondrial function. Twelve deletion mutants lacked components of the electron transport chain. The growth defects of these mutants can be alleviated by the addition of antioxidants, which points to intrinsic oxidative stress as the origin of the phenotypes observed. These respiration-deficient mutants display elevated steady-state levels of ROS, probably due to enhanced electron leakage from their defective transport chains, which compromises the viability of chronologically-aged cells. Conclusion/Significance: Individual mitochondrial dysfunctions have often been described as the cause of diseases or aging, and our global characterization emphasizes the primacy of oxidative stress in the etiology of such processes.