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(1) Background: This study aims to evaluate retinal perfusion by optical coherence tomography angiography (OCTA) in pediatric patients with type 1 diabetes mellitus (T1D) without diabetic retinopathy (DR). (2) Methods: Thirty-one patients affected by T1D were enrolled. All participants were evaluated using OCTA. The foveal avascular zone (FAZ) and superficial and deep macular vessel density (VD) were analyzed. The correlation of these parameters with metabolic factors such as body mass index (BMI), glycated hemoglobin (HbA1c), and the type of insulin therapy (multiple daily injections, MDI vs. continuous subcutaneous insulin infusion, CSII) was determined. (3) Results: None of the OCTA parameters were significantly different between the groups. The patients' HbA1C level did not influence any of the OCTA parameters. The use of MDI tended to reduce the parafoveal and perifoveal deep VD (p = 0.048 and p = 0.021, respectively) compared to CSII. An elevated BMI tended to increase the deep macular (p = 0.005) and perifoveal VD (p = 0.006). (4) Conclusion: VD and FAZ are normal in pubescent children with T1D without signs of DR. Treatment with CSII may be a better choice compared to MDI, as CSII may be protective against retinal microvascular damage. Our results indicate the need for new clinical parameters of glycemic control in addition to HbA1c which could assess the risk of DR.
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Elevated concentrations of palmitate in serum of obese individuals can impair endothelial function, contributing to development of cardiovascular disease. Although several molecular mechanisms of palmitate-induced endothelial dysfunction have been proposed, there is no consensus on what signaling event is the initial trigger of detrimental palmitate effects. Here we report that inhibitors of ER stress or ceramid synthesis can rescue palmitate-induced autophagy impairment in macro- and microvascular endothelial cells. Furthermore, palmitate-induced cholesterol synthesis was reverted using these inhibitors. Similar to cell culture data, autophagy markers were increased in serum of obese individuals. Subsequent lipidomic analysis revealed that palmitate changed the composition of membrane phospholipids in endothelial cells and that these effects were not reverted upon application of above-mentioned inhibitors. However, ER stress inhibition in palmitate-treated cells enhanced the synthesis of trilglycerides and restored ceramide levels to control condition. Our results suggest that palmitate induces ER-stress presumably by shift in membrane architecture, leading to impaired synthesis of triglycerides and enhanced production of ceramides and cholesterol, which altogether enhances lipotoxicity of palmitate in endothelial cells.
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Estrés del Retículo Endoplásmico , Células Endoteliales , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Autofagia/efectos de los fármacos , Triglicéridos/metabolismo , Colesterol/metabolismo , Palmitatos/farmacología , Ceramidas/metabolismoRESUMEN
BACKGROUND: The aim of the study was to detect the changes in retinal and choroidal vasculature via optical coherence tomography angiography (OCTA) by comparing the quantitative OCTA parameters in patients with and without myotonic dystrophies (DM). MATERIAL: The cross-sectional study. Forty-one consecutive patients affected by DMs were enrolled. The inclusion criteria were molecular diagnosis of DM types 1 and 2. To avoid the age effect on microvascular changes and to justify a comparison between DM1 and DM2 patients, two control groups matched for sex and age were established. RESULTS: The vascular density was found to be significantly decreased in the DM groups compared to the controls in the macular, parafoveal and perifoveal zone of superficial capillary plexus (p < 0.001 for the DM1 group, and p = 0.001, p = 0.005 and p = 0.026, respectively, for the DM2 group), as well as in the macular zone in the deep capillary plexus for DM1 (p = 0.002) and deep macular and perifoveal zone for DM2 (p = 0.007, p = 0.001, respectively). The foveal avascular zone showed no significant differences between DM1 and DM2 compared to their control groups (p = 0.320 and p = 0.945, respectively). CONCLUSION: Our results show that DM is associated not only with the classic pigmentary changes but also with superficial and deep retinal microvasculature abnormalities, suggesting that these changes may be related to local hypoperfusion. Optical coherence tomography angiography is a useful tool for the diagnosis and characterization of retinal changes in DM and should be part of the standard evaluation of these patients.
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Purpose: The aim of this study is to measure retinal vessel density and flow rate area by optical coherence tomography angiography (OCTA) in patients with autoimmune diseases taking hydroxychloroquine (HCQ). Methods: The cross-sectional study included 98 patients divided into three groups. Group I included patients with the diagnosis of an autoimmune disease, for whom the introduction of HCQ was planned. Group II implied low-risk patients for retinal toxicity (≤5 years of HCQ use), whereas Group III implied patients that were at high-risk (>5 years of drug use). All patients underwent a computerized visual field, central macular thickness by optical coherence tomography, and OCTA measurements. Results: The vascular density was found to be statistically significantly decreased in the high-risk group compared to the control group in the superficial parafoveal zone (P = 0.030), whereas it was decreased compared to the low-risk and control groups in the deep layers whole (P = 0.006, P = 0.010, respectively) and perifoveal zones (P = 0.003, P = 0.010, respectively). The foveal avascular zone was significantly enlarged in the high-risk group compared to the control (P < 0.018). Retinal flow rates did not show statistically significant differences between the groups (P > 0.05). Conclusion: Patients treated with HCQ for more than 5 appear have a significant loss of vascular density in the parafoveal and perifoveal regions, and FAZ area is significantly increased compared to low-risk patients and controls. These findings indicate that OCTA may be beneficial for monitoring high-risk patients and may stratify their risk of further retinal damage.
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Enfermedades Autoinmunes , Hidroxicloroquina , Humanos , Hidroxicloroquina/efectos adversos , Angiografía con Fluoresceína/métodos , Tomografía de Coherencia Óptica/métodos , Estudios Transversales , Vasos Retinianos/diagnóstico por imagenRESUMEN
Background and Objectives: The aim of the study was to evaluate vision-related quality of life (VR-QOL) and treatment satisfaction (TS) in patients with diabetic retinopathy treated with panretinal photocoagulation (PRP). Material and Methods: The panel study included 95 patients who underwent PRP for diabetic retinopathy. Eligible patients with no history of previous PRP were interviewer-administered the National Eye Institute Visual Function Questionnaire (NEI VFQ-25) and Retinopathy Treatment Satisfaction Questionnaire (RetTSQ) beforehandand one month after the last session of laser application. The study was conducted between June 2017 and June 2019 at tertiary care center in Serbia, Belgrade. We assessed pre- to post-PRP values of the composite score and subscale scores of VFQ-25 and RetTSQ, using a paired samples t-test. Univariate logistic regression was used to analyze the relationship between binary outcomes and potential predictors. Multivariate regression included predictors from univariate analyses that were statistically significant. Results: The mean VFQ-25 composite score was 65.4 ± 17.4 before and 63.3 ± 19.5 after PRP (p = 0.045). Subscale analysis showed that two of the 11 items achieved a significant decrease after laser application (general vision and dependency). The mean RetTSQ score at baseline was 60.0 ± 11.8 and at the exit visit was 60.3 ± 12.3 (p = 0.858). Sub-scale analysis showed significant deterioration for five of the 13 items. Multivariate logistic regression found that significant predictor of VFQ-25 composite score reduction was fewer laser burns (p = 0.002) while significant predictor of RetTSQ total score reduction was presence of hyperlipidaemia (p = 0.021). Conclusion: The use of vision-related quality of life and treatment satisfaction questionnaires in conjunction with clinical examination, appears to provide a more comprehensive overview of an individual's daily well-being following PRP. Laser treatment for diabetic retinopathy leads to deterioration of some of the patients' perceived VR-QOL and TS. Health-care providers should inform patients about their treatment options and together decide which therapeutic method is best for them.
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Diabetes Mellitus , Retinopatía Diabética , Humanos , Calidad de Vida , Retinopatía Diabética/cirugía , Agudeza Visual , Coagulación con Láser/métodos , Satisfacción Personal , Diabetes Mellitus/terapiaRESUMEN
Objective: This study aimed to determine the intravitreal concentration of VEGF in eyes with PDR and to evaluate the effects of previous PRP on its level. Methods: It was a cross-sectional study performed at the Clinical Centre University. It included 90 eyes surgically treated with PPV, divided into three groups, group A - patients with PDR with previous PRP, group B - patients with PDR without previous PRP, and group C - PPV performed due to the indication unrelated to diabetes. A vitreous sample was obtained during PPV, and the VEGF concentration was determined using an Enzyme-linked immunosorbent assay test (ELISA). Shapiro-Wilk, nonparametric tests Kruskal-Wallis, Mann-Whithney U test, ANOVA and Spearman's correlation test were used. Results: The highest vitreous VEGF concentration was in group B - 972.96 (743.33-1149.13) and was higher than in group A - 69.22 (37.33-225.15) and in group C - 19.93 (1.15-32.17) (p<0.001). There was a positive correlation between VEGF vitreous concentration and glucose level in group A patients (Rho=0.410; p=0.027). Conclusion: As a treatment before PPV surgery, PRP showed to be effective in the reduction of VEGF levels, which also highlighted a decrease in complications during and postoperatively. Abbreviations: DRS = Diabetic Retinopathy Study, PDR = proliferative diabetic retinopathy, VEGF = vascular endothelial growth factor, PRP = pan-retinal photocoagulation, PPV = pars plana vitrectomy, HbA1c = glycosylated hemoglobin, ETDRS = Early treatment diabetic retinopathy study, ESR = erythrocyte sedimentation rate, BCVA = best corrected visual acuity, OCT = optical coherent tomography, ILM = internal limiting membrane, PHACO = phacoemulsification, IOL = intraocular lens, ELISA = Enzyme-linked immunosorbent assay test, AUC = area under the curve, DME = diabetic macular oedema, TDR = tractional retinal detachment, VMT = vitreomacular traction.
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Diabetes Mellitus , Retinopatía Diabética , Humanos , Retinopatía Diabética/diagnóstico , Retinopatía Diabética/cirugía , Factor A de Crecimiento Endotelial Vascular , Estudios Transversales , Coagulación con Láser , Vitrectomía , Factores de Crecimiento Endotelial Vascular/uso terapéutico , Rayos LáserRESUMEN
Purpose: The study was conducted to determine the ocular pulse amplitude (OPA) changes, measured with a dynamic contour tonometer (DCT), after surgical retinal detachment repair. Methods: This was a prospective and comparative study. Thirty patients (30 eyes) who had undergone uncomplicated unilateral scleral buckling and encircling procedures for quadrant or half-retinal rhegmatogenous retinal detachment were referred for DCT one day before the surgery was performed, on the 1st, 7th, and 30th postoperative day. Methods of descriptive (arithmetical mean, standard deviation) and analytical statistics (analysis of variance) were used to analyze the data and evaluate the significance of the difference. A value of P less than 0.05 was considered statistically significant. The data were evaluated for normality with the single-sample Kolmogorov-Smirnov test. Results: OPA values decreased significantly after scleral buckling procedures (p < 0.0001), but regained near to preoperative values one month after the surgery. Conclusion: OPA tends to decrease after retinal detachment surgery. Restoring patients' vision with scleral buckling and encircling procedures gives early changes in blood supply to the choroid and ocular nerve, and since OPA is an indirect parameter of choroidal vascularization, measuring these values can help make an insight into ocular hemodynamics.
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Desprendimiento de Retina , Coroides , Humanos , Estudios Prospectivos , Retina , Desprendimiento de Retina/diagnóstico , Desprendimiento de Retina/cirugía , Curvatura de la Esclerótica/métodosRESUMEN
Signaling by the Rho GTPase Rac1 is key to the regulation of cytoskeletal dynamics, cell spreading and adhesion. It is widely accepted that the inactive form of Rac1 is bound by Rho GDI, which prevents Rac1 activation and Rac1-effector interactions. In addition, GDI-bound Rac1 is protected from proteasomal degradation, in line with data showing that Rac1 ubiquitination occurs exclusively when Rac1 is activated. We set out to investigate how Rac1 activity, GDI binding and ubiquitination are linked. We introduced single amino acid mutations in Rac1 which differentially altered Rac1 activity, and compared whether the level of Rac1 activity relates to Rac1 ubiquitination and GDI binding. Results show that Rac1 ubiquitination and the active Rac1 morphology is proportionally increased with Rac1 activity. Similarly, we introduced lysine-to-arginine mutations in constitutively active Rac1 to inhibit site-specific ubiquitination and analyze this effect on Rac1 signaling output and ubiquitination. These data show that the K16R mutation inhibits GTP binding, and consequently Rac1 activation, signaling and-ubiquitination, while the K147R mutation does not block Rac1 signaling, but does inhibits its ubiquitination. In both sets of mutants, no direct correlation was observed between GDI binding and Rac1 activity or -ubiquitination. Taken together, our data show that a strong, positive correlation exists between Rac1 activity and its level of ubiquitination, but also that GDI dissociation does not predispose Rac1 to ubiquitination.
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Movimiento Celular , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Ubiquitinación , Proteína de Unión al GTP rac1/metabolismo , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico/metabolismo , Secuencia de Aminoácidos , Forma de la Célula , Células HEK293 , Humanos , Lisina/metabolismo , Proteínas Mutantes/metabolismo , Mutación/genética , Fenotipo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Ubiquitina/metabolismoRESUMEN
The endothelial monolayer forms a barrier between the lumen of blood vessels and the underlying tissues. Stable VE-cadherin-based adherens junctions are essential for maintaining this barrier, whereas their remodeling is required for angiogenesis in health and disease. Here, we position the ERAD-associated ubiquitin ligase MARCH6 as a determinant of angiogenic sprouting and barrier integrity through its ability to promote the degradation of the rate-limiting cholesterol biosynthetic enzyme squalene epoxidase (SQLE). Accordingly, MARCHF6 ablation in endothelial cells increases SQLE protein and cholesterol load. This leads to altered membrane order, disorganized adherens junctions, decreased endothelial barrier function, and impaired SQLE-dependent sprouting angiogenesis. Akin to MARCHF6 silencing, the overexpression of SQLE impairs angiogenesis. However, angiogenesis is also attenuated when SQLE is silenced, indicating that fine-tuning cholesterol biosynthesis is a determinant of healthy endothelial function. In summary, we propose a mechanistic link between regulation of cholesterol homeostasis by the MARCH6-SQLE axis and endothelial integrity and angiogenesis.
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Colesterol/metabolismo , Homeostasis , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteínas de la Membrana/metabolismo , Neovascularización Fisiológica , Escualeno-Monooxigenasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Uniones Adherentes/metabolismo , Uniones Adherentes/ultraestructura , Antígenos CD/metabolismo , Cadherinas/metabolismo , Silenciador del Gen , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/ultraestructura , HumanosRESUMEN
Endothelial cell-cell contacts are essential for vascular integrity and physiology, protecting tissues and organs from edema and uncontrolled invasion of inflammatory cells. The vascular endothelial barrier is dynamic, but its integrity is preserved through a tight control at different levels. Inflammatory cytokines and G-protein-coupled receptor agonists, such as histamine, reduce endothelial integrity and increase vascular leakage. This is due to elevated myosin-based contractility, in conjunction with phosphorylation of proteins at cell-cell contacts. Conversely, reducing contractility stabilizes or even increases endothelial junctional integrity. Rho GTPases are key regulators of such cytoskeletal dynamics and endothelial cell-cell contacts. In addition to signaling-induced regulation, the expression of junctional proteins, such as occludin, claudins and vascular endothelial cadherin, also controls endothelial barrier function. There is increasing evidence that, in addition to protein phosphorylation, ubiquitylation (also known as ubiquitination) is an important and dynamic post-translational modification that regulates Rho GTPases, junctional proteins and, consequently, endothelial barrier function. In this Review, we discuss the emerging role of ubiquitylation and deubiquitylation events in endothelial integrity and inflammation. The picture that emerges is one of increasing complexity, which is both fascinating and promising given the clinical relevance of vascular integrity in the control of inflammation, and of tissue and organ damage.
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Células Endoteliales/metabolismo , Inflamación/metabolismo , Ubiquitina/metabolismo , Endotelio Vascular/metabolismo , HumanosRESUMEN
RhoGTPases regulate cytoskeletal dynamics, migration and cell-cell adhesion in endothelial cells. Besides regulation at the level of guanine nucleotide binding, they also undergo post-translational modifications, for example ubiquitination. RhoGTPases are ubiquitinated by Cullin RING ligases which are in turn regulated by neddylation. Previously we showed that inhibition of Cullin RING ligase activity by the neddylation inhibitor MLN4924 is detrimental for endothelial barrier function, due to accumulation of RhoB and the consequent induction of contractility. Here we analyzed the effect of pharmacological activation of Cullin RING ligases on endothelial barrier integrity in vitro and in vivo. CSN5i-3 induced endothelial barrier disruption and increased macromolecule leakage in vitro and in vivo. Mechanistically, CSN5i-3 strongly induced the expression and activation of RhoB and to lesser extent of RhoA in endothelial cells, which enhanced cell contraction. Elevated expression of RhoGTPases was a consequence of activation of the NF-κB pathway. In line with this notion, CSN5i-3 treatment decreased IκBα expression and increased NF-κB-mediated ICAM-1 expression and consequent adhesion of neutrophils to endothelial cells. This study shows that sustained neddylation of Cullin RING-ligases leads to activation the NF-κB pathway in endothelial cells, elevated expression of RhoGTPases, Rho/ROCK-dependent activation of MLC and disruption of the endothelial barrier.
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Complejo del Señalosoma COP9/metabolismo , Endotelio Vascular/metabolismo , Inflamación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptido Hidrolasas/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Animales , Ciclopentanos/farmacología , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Neutrófilos/metabolismo , Pirimidinas/farmacología , Ubiquitina/química , Regulación hacia Arriba , Pez CebraRESUMEN
Rho GTPases control both the actin cytoskeleton and adherens junction stability and are recognized as essential regulators of endothelial barrier function. They act as molecular switches and are primarily regulated by the exchange of GDP and GTP. However, posttranslational modifications such as phosphorylation, prenylation, and ubiquitination can additionally alter their localization, stability, and activity. F-box proteins are involved in the recognition of substrate proteins predestined for ubiquitination and subsequent degradation. Given the importance of ubiquitination, we studied the effect of the loss of 62 members of the F-box protein family on endothelial barrier function in human umbilical vein endothelial cells. Endothelial barrier function was quantified by electrical cell impedance sensing and macromolecule passage assay. Our RNA interference-based screen identified FBXW7 as a key regulator of endothelial barrier function. Mechanistically, loss of FBXW7 induced the accumulation of the RhoB GTPase in endothelial cells, resulting in their increased contractility and permeability. FBXW7 knockdown induced activation of the cholesterol biosynthesis pathway and changed the prenylation of RhoB. This effect was reversed by farnesyl transferase inhibitors and by the addition of geranylgeranyl pyrophosphate. In summary, this study identifies FBXW7 as a novel regulator of endothelial barrier function in vitro. Loss of FBXW7 indirectly modulates RhoB activity via alteration of the cholesterol biosynthesis pathway and, consequently, of the prenylation status and activity of RhoB, resulting in increased contractility and disruption of the endothelial barrier.
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Vías Biosintéticas , Colesterol/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Prenilación , Proteína de Unión al GTP rhoB/metabolismo , Transferasas Alquil y Aril/antagonistas & inhibidores , Transferasas Alquil y Aril/metabolismo , Vías Biosintéticas/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Técnicas de Silenciamiento del Gen , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Modelos Biológicos , ARN Interferente Pequeño/metabolismo , Trombina/farmacologíaRESUMEN
Background Dysfunctional endothelium may contribute to the development of cardiovascular complications in chronic kidney disease ( CKD ). Supplementation with active vitamin D has been proposed to have vasoprotective potential in CKD , not only by direct effects on the endothelium but also by an increment of α-Klotho. Here, we explored the capacity of the active vitamin D analogue paricalcitol to protect against uremia-induced endothelial damage and the extent to which this was dependent on increased α-Klotho concentrations. Methods and Results In a combined rat model of CKD with vitamin D deficiency, renal failure induced vascular permeability and endothelial-gap formation in thoracic aorta irrespective of baseline vitamin D, and this was attenuated by paricalcitol. Downregulation of renal and serum α-Klotho was found in the CKD model, which was not restored by paricalcitol. By measuring the real-time changes of the human endothelial barrier function, we found that paricalcitol effectively improved the recovery of endothelial integrity following the addition of the pro-permeability factor thrombin and the induction of a wound. Furthermore, immunofluorescence staining revealed that paricalcitol promoted vascular endothelial-cadherin-based cell-cell junctions and diminished F-actin stress fiber organization, preventing the formation of endothelial intracellular gaps. Conclusions Our results demonstrate that paricalcitol attenuates the CKD -induced endothelial damage in the thoracic aorta and directly mediates endothelial stability in vitro by enforcing cell-cell interactions.
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Aorta Torácica/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Ergocalciferoles/farmacología , Insuficiencia Renal Crónica/metabolismo , Uremia/metabolismo , Deficiencia de Vitamina D/metabolismo , Actinas/efectos de los fármacos , Actinas/metabolismo , Animales , Aorta Torácica/metabolismo , Cadherinas/efectos de los fármacos , Cadherinas/metabolismo , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Glucuronidasa/efectos de los fármacos , Glucuronidasa/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Uniones Intercelulares/efectos de los fármacos , Uniones Intercelulares/metabolismo , Proteínas Klotho , Ratas , Fibras de Estrés/efectos de los fármacosRESUMEN
BACKGROUND: Systemic inflammation, endothelial dysfunction and deficient vascularization of either uterus or myocardium are mechanistic hallmarks of early-onset preeclampsia and heart failure with preserved ejection fraction (HFpEF). HFpEF is especially prevalent in elderly women and preceded in middle age by preclinical left ventricular (LV) diastolic dysfunction. To detect if preeclampsia predisposes to HFpEF at later age, echocardiographic indices of LV function and of LV structure and biomarkers of systemic inflammation and of endothelial dysfunction were compared in middle-aged women with a history of early-onset preeclampsia or uncomplicated pregnancy. METHODS AND FINDINGS: Middle-aged women with a history of early-onset preeclampsia (n = 131) or uncomplicated pregnancy (n = 56) were prospectively recruited 9 to 16 years after pregnancy. Women with a history of preeclampsia had higher body mass index (p = 0.006), blood pressure (p<0.001) and plasma levels of interleukin-6 (p = 0.005) and soluble intercellular adhesion molecule-1 (sICAM-1) (p = 0.014). They had thicker septal (p = 0.001) and posterior (p = 0.003) LV walls and worse diastolic LV function evident from reduced mean mitral annular lengthening velocity (E'mean; p = 0.007) and higher ratio of early diastolic mitral flow velocity (E) over E'mean (E/E'mean; p<0.001). Differences of sICAM-1, E'mean and E/E'mean remained significant after accounting for BMI and blood pressure. CONCLUSIONS: History of preeclampsia predisposes in middle age to worse LV diastolic function, which could increase the likelihood of later HFpEF development. This predisposition derives not only from persistent cardiovascular risk but may also be caused by persistent endothelial dysfunction hindering adequate vascularization in the uterus during pregnancy and in the myocardium in middle age.
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Diástole , Susceptibilidad a Enfermedades , Insuficiencia Cardíaca/etiología , Preeclampsia/fisiopatología , Disfunción Ventricular Izquierda/etiología , Adulto , Edad de Inicio , Estudios de Casos y Controles , Ecocardiografía Doppler , Femenino , Insuficiencia Cardíaca/fisiopatología , Humanos , Persona de Mediana Edad , Embarazo , Estudios Prospectivos , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
OBJECTIVE: Nemaline myopathy (NM) is one of the most common congenital nondystrophic myopathies and is characterized by muscle weakness, often from birth. Mutations in ACTA1 are a frequent cause of NM (ie, NEM3). ACTA1 encodes alpha-actin 1, the main constituent of the sarcomeric thin filament. The mechanisms by which mutations in ACTA1 contribute to muscle weakness in NEM3 are incompletely understood. We hypothesized that sarcomeric dysfunction contributes to muscle weakness in NEM3 patients. METHODS: To test this hypothesis, we performed contractility measurements in individual muscle fibers and myofibrils obtained from muscle biopsies of 14 NEM3 patients with different ACTA1 mutations. To identify the structural basis for impaired contractility, low angle X-ray diffraction and stimulated emission-depletion microscopy were applied. RESULTS: Our findings reveal that muscle fibers of NEM3 patients display a reduced maximal force-generating capacity, which is caused by dysfunctional sarcomere contractility in the majority of patients, as revealed by contractility measurements in myofibrils. Low angle X-ray diffraction and stimulated emission-depletion microscopy indicate that dysfunctional sarcomere contractility in NEM3 patients involves a lower number of myosin heads binding to actin during muscle activation. This lower number is not the result of reduced thin filament length. Interestingly, the calcium sensitivity of force is unaffected in some patients, but decreased in others. INTERPRETATION: Dysfunctional sarcomere contractility is an important contributor to muscle weakness in the majority of NEM3 patients. This information is crucial for patient stratification in future clinical trials. Ann Neurol 2018;83:269-282.
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Actinas/genética , Contracción Muscular/fisiología , Debilidad Muscular/genética , Miopatías Estructurales Congénitas/fisiopatología , Sarcómeros/patología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Debilidad Muscular/fisiopatología , Músculo Esquelético/patología , Miopatías Estructurales Congénitas/genética , Sarcómeros/fisiología , Adulto JovenRESUMEN
RhoGTPases control endothelial cell (EC) migration, adhesion, and barrier formation. Whereas the relevance of RhoA for endothelial barrier function is widely accepted, the role of the RhoA homologue RhoB is poorly defined. RhoB and RhoA are 85% identical, but RhoB's subcellular localization and half-life are uniquely different. Here, we studied the role of ubiquitination for the function and stability of RhoB in primary human ECs. We show that the K63 polyubiquitination at lysine 162 and 181 of RhoB targets the protein to lysosomes. Moreover, we identified the RING E3 ligase complex Cullin-3-Rbx1-KCTD10 as key modulator of endothelial barrier integrity via its regulation of the ubiquitination, localization, and activity of RhoB. In conclusion, our data show that ubiquitination controls the subcellular localization and lysosomal degradation of RhoB and thereby regulates the stability of the endothelial barrier through control of RhoB-mediated EC contraction.
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Proteínas Portadoras/metabolismo , Proteínas Cullin/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Ubiquitinación , Proteína de Unión al GTP rhoB/metabolismo , Proteínas Portadoras/genética , Proteínas Cullin/genética , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Canales de Potasio con Entrada de Voltaje/genética , Proteína de Unión al GTP rhoB/genéticaRESUMEN
PURPOSE: To assess the effect of panretinal laser photocoagulation on ocular pulse amplitude (OPA) in normotensive eyes with proliferative diabetic retinopathy. METHODS: Prospectively, we performed unilateral argon laser panretinal photocoagulation (PRP) in 30 patients with diabetes mellitus type II and previously untreated bilateral proliferative diabetic retinopathy. Before and 7 and 30 days after the treatment, OPA was measured using dynamic contour tonometer. RESULTS: Compared with the untreated contralateral eyes, laser photocoagulation led to a reduction of OPA. Ocular pulse amplitude did not significantly differ in photocoagulated eyes 7 days after the treatment, but there was a significant difference in OPA 30 days after the treatment. The decrease in OPA values was 15% 7 days after PRP and 40% 30 days after PRP. CONCLUSIONS: Ocular pulse amplitude reduction after PRP indirectly informs us about choriocapillary closure, already reported in previous studies.
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Retinopatía Diabética/cirugía , Coagulación con Láser/métodos , Adulto , Anciano , Presión Sanguínea/fisiología , Coroides/irrigación sanguínea , Diabetes Mellitus Tipo 2/complicaciones , Retinopatía Diabética/fisiopatología , Femenino , Frecuencia Cardíaca/fisiología , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Flujo Pulsátil/fisiología , Flujo Sanguíneo Regional/fisiología , Retina/fisiopatología , Tonometría Ocular/métodosRESUMEN
Cholesterol metabolism is subject to complex transcriptional and nontranscriptional regulation. Herein, the role of ubiquitylation is emerging as an important post-translational modification that regulates cholesterol synthesis and uptake. Similar to other post-translational modifications, ubiquitylation is reversible in a process dependent on activity of deubiquitylating enzymes (DUBs). Yet whether these play a role in cholesterol metabolism is largely unknown. As a first step to test this possibility, we used pharmacological inhibition of cellular DUB activity. Short term (2 h) inhibition of DUBs resulted in accumulation of high molecular weight ubiquitylated proteins. This was accompanied by a dramatic decrease in abundance of the LDLR and attenuated LDL uptake into hepatic cells. Importantly, this occurred in the absence of changes in the mRNA levels of the LDLR or other SREBP2-regulated genes, in line with this phenotype being a post-transcriptional event. Mechanistically, we identify transcriptional induction of the E3 ubiquitin ligase IDOL in human and rodent cells as the underlying cause for ubiquitylation-dependent lysosomal degradation of the LDLR following DUB inhibition. In contrast to the established transcriptional regulation of IDOL by the sterol-responsive liver X receptor (LXR) transcription factors, induction of IDOL by DUB inhibition is LXR-independent and occurs in Lxrαß(-/-) MEFs. Consistent with the role of DUBs in transcriptional regulation, we identified a 70-bp region in the proximal promoter of IDOL, distinct from that containing the LXR-responsive element, which mediates the response to DUB inhibition. In conclusion, we identify a sterol-independent mechanism to regulate IDOL expression and IDOL-mediated lipoprotein receptor degradation.
Asunto(s)
Lipoproteínas LDL/metabolismo , Regiones Promotoras Genéticas , Receptores de LDL/metabolismo , Transcripción Genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Absorción Fisiológica/efectos de los fármacos , Sustitución de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Genes Reporteros , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Receptores X del Hígado , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Lisosomas/metabolismo , Ratones , Mutación , Receptores Nucleares Huérfanos/genética , Receptores Nucleares Huérfanos/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Receptores de LDL/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transcripción Genética/efectos de los fármacos , Ubiquitina-Proteína Ligasas/química , Proteasas Ubiquitina-Específicas/antagonistas & inhibidores , Proteasas Ubiquitina-Específicas/genética , Ubiquitinación/efectos de los fármacosRESUMEN
Introduction: Horner's syndrome is an interruption of the sympathetic nervous system at any point along its course between the hypothalamus and the orbit. Horner's syndrome is classically presented as an ipsilateral miosis, subtle ptosis, and facial anhidrosis. Pharmacologic testing is very useful in the diagnosis of Horner's syndrome as it could help to localize the lesioned neuron in the sympathetic pathway, suggesting an etiology. Case Outline: We present a case report of a 41-year-old woman who reported right eyelid drooping immediately after operation of sympathetic chain schwannoma. We performed apraclonidine test for the diagnosis of Horner's syndrome, which produced mydriasis on the affected eye, while there was no significant change of the normal eye. Based on the clinical presentation of anisocoria and one-sided ptosis, and previous medical history of surgical removal of the mediastinal tumor, the patient was diagnosed with a right-sided, partial Horner's syndrome. Conclusion: Timely recognition, exact localization of the lesioned neuron, and referral for urgent imaging studies are important for ophthalmologists in order to prevent and treat life-threatening conditions. Besides its diagnostic value in Horner's syndrome, topical apraclonidine could correct ptosis for the sake of esthetics or when ptosis reduces the superior visual field.
Asunto(s)
Agonistas de Receptores Adrenérgicos alfa 2/administración & dosificación , Clonidina/análogos & derivados , Síndrome de Horner/diagnóstico , Síndrome de Horner/tratamiento farmacológico , Adulto , Anisocoria/tratamiento farmacológico , Blefaroptosis/tratamiento farmacológico , Clonidina/administración & dosificación , Femenino , Humanos , Soluciones OftálmicasRESUMEN
Introduction: Scleroderma (systemic sclerosis) is a severe chronic connective tissue disease, which results in involvement of numerous internal organs. Changes in the eye are the consequences of organ-specific manifestations of scleroderma or adverse effects of immunosuppressive treatment applied. Case report: We reported a 42-year-old woman with systemic sclerosis and acute deterioration of vision in the left eye, with visual acuity 0.9. After thorough clinical examination, including fluorescein angiography and optical coherence tomography, the diagnosis of nonischemic central retinal vein occlusion was made. Further biochemical, rheumatological and immunological investigation, apart from inactive systemic sclerosis, showed normal findings. Therefore, the cause of central retinal vein occlusion could only be attributed to the microvascular changes in systemic sclerosis. After three months, visual acuity deteriorated to 0.6 due to the development of cystoid macular edema. The patient received intravitreal injection of bevacizumab and after a single dose visual acuity improved to 0.9. After a 6- month follow-up, macular edema resolved and visual acuity stabilized. Conclusion: According to our knowledge and current data from the literature, central retinal vein occlusion is a rare vision threatening manifestation of scleroderma. There are only few published case reports on central vein occlusion in scleroderma patients. Examination of the ocular fundus is recommended for evaluation of vascular disease in patients with systemic sclerosis.