RESUMEN
Semicarbazide-sensitive amine oxidase (SSAO) is both a soluble- and membrane-bound transmembrane protein expressed in the vascular endothelial and in smooth muscle cells. In vascular endothelial cells, SSAO contributes to the development of atherosclerosis by mediating a leukocyte adhesion cascade; however, its contributory role in the development of atherosclerosis in VSMCs has not yet been fully explored. This study investigates SSAO enzymatic activity in VSMCs using methylamine and aminoacetone as model substrates. The study also addresses the mechanism by which SSAO catalytic activity causes vascular damage, and further evaluates the contribution of SSAO in oxidative stress formation in the vascular wall. SSAO demonstrated higher affinity for aminoacetone when compared to methylamine (Km = 12.08 µM vs. 65.35 µM). Aminoacetone- and methylamine-induced VSMCs death at concentrations of 50 & 1000 µM, and their cytotoxic effect, was reversed with 100 µM of the irreversible SSAO inhibitor MDL72527, which completely abolished cell death. Cytotoxic effects were also observed after 24 h of exposure to formaldehyde, methylglyoxal and H2O2. Enhanced cytotoxicity was detected after the simultaneous addition of formaldehyde and H2O2, as well as methylglyoxal and H2O2. The highest ROS production was observed in aminoacetone- and benzylamine-treated cells. MDL72527 abolished ROS in benzylamine-, methylamine- and aminoacetone-treated cells (**** p < 0.0001), while ßAPN demonstrated inhibitory potential only in benzylamine-treated cells (* p < 0.05). Treatment with benzylamine, methylamine and aminoacetone reduced the total GSH levels (**** p < 0.0001); the addition of MDL72527 and ßAPN failed to reverse this effect. Overall, a cytotoxic consequence of SSAO catalytic activity was observed in cultured VSMCs where SSAO was identified as a key mediator in ROS formation. These findings could potentially associate SSAO activity with the early developing stages of atherosclerosis through oxidative stress formation and vascular damage.
Asunto(s)
Amina Oxidasa (conteniendo Cobre) , Ratas , Animales , Amina Oxidasa (conteniendo Cobre)/metabolismo , Músculo Liso Vascular/metabolismo , Peróxido de Hidrógeno/farmacología , Piruvaldehído/farmacología , Células Endoteliales/metabolismo , Especies Reactivas de Oxígeno/farmacología , Metilaminas/metabolismo , Bencilaminas/farmacología , Formaldehído/farmacologíaRESUMEN
Vascular smooth muscle cells (VSMCs) are the main stromal cells in the medial layer of the vascular wall. These cells produce the extracellular matrix (ECM) and are involved in many pathological changes in the vascular wall. Semicarbazide-sensitive amine oxidase (SSAO) and lysyl oxidase (LOX) are vascular enzymes associated with the development of atherosclerosis. In the vascular smooth muscle cells, increased SSAO activity elevates reactive oxygen species (ROS) and induces VSMCs death; increased LOX induces chemotaxis through hydrogen peroxide dependent mechanisms; and decreased LOX contributes to endothelial dysfunction. This study investigates the relationship between SSAO and LOX in VSMCs by studying their activity, protein, and mRNA levels during VSMCs passaging and after silencing the LOX gene, while using their respective substrates and inhibitors. At the basal level, LOX activity decreased with passage and its protein expression was maintained between passages. ßAPN abolished LOX activity (** p < 0.01 for 8 vs. 3 and * p < 0.05 for 5 vs. 8) and had no effect on LOX protein and mRNA levels. MDL72527 reduced LOX activity at passage 3 and 5 (## p < 0.01) and had no effect on LOX protein, and mRNA expression. At the basal level, SSAO activity also decreased with passage, and its protein expression was maintained between passages. MDL72527 abolished SSAO activity (**** p < 0.0001 for 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 5 (** p < 0.01) and 8 (**** p < 0.0001), and Aoc3 mRNA levels at passage 8 (* p < 0.05). ßAPN inhibited SSAO activity (**** p < 0.0001 for 5 vs. 3 and 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 3 (* p < 0.05), and Aoc3 mRNA levels at passage 3 (* p < 0.05). Knockdown of the LOX gene (**** p < 0.0001 for Si6 vs. Sictrl and *** p < 0.001 for Si8 vs. Sictrl) and LOX protein (** p < 0.01 for Si6 and Si8 vs. Sictrl) in VSMCs at passage 3 resulted in a reduction in Aoc3 mRNA (#### p < 0.0001 for Si6 vs. Sictrl and ### p < 0.001 for Si8 vs. Sictrl) and VAP-1 protein (# p < 0.05 for Si8 vs. Sictrl). These novel findings demonstrate a passage dependent decrease in LOX activity and increase in SSAO activity in rat aortic VSMCs and show an association between both enzymes in early passage rat aortic VSMCs, where LOX was identified as a regulator of SSAO activity, protein, and mRNA expression.
Asunto(s)
Amina Oxidasa (conteniendo Cobre) , Ratas , Animales , Amina Oxidasa (conteniendo Cobre)/genética , Amina Oxidasa (conteniendo Cobre)/metabolismo , Músculo Liso Vascular/metabolismo , Proteína-Lisina 6-Oxidasa/genética , Proteína-Lisina 6-Oxidasa/metabolismo , Aorta/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Context Incontinence-associated dermatitis (IAD) is a type of moisture-associated dermatitis caused by repeated skin exposure to urine or stool. A product that could mitigate such symptoms would have a significant impact on cost of care and patients' quality of life. Objective This study compared the clinical efficacy of RD1433 and a comparator product (Vaseline®) in preventing and treating experimental IAD skin lesions. Materials and methods For the "prevention" part of the study, skin sites in eight human volunteers were treated daily for 5 d with either RD1433 or Vaseline® immediately prior to synthetic urine exposure. In the "treatment" part, exposure to synthetic urine was substituted for Vaseline® or RD1433 application on the first 2 d to promote the development of skin lesions prior to the application of the products from day three. Product efficacy was quantified by visual scoring and an array of biophysical instruments. Results Both RD1433 and Vaseline® significantly reduced lesion progression when applied as a prophylactic. When applied as a treatment (following establishment of skin lesions), RD1433 demonstrated a statistically significant improvement in several measures of skin function whereas there was no statistically significant improvement following treatment with Vaseline®. Conclusions The findings of this study suggest that RD1433 may be superior to Vaseline® in the prevention and treatment of experimental IAD lesions. Clearly, further work is required to establish the efficacy of RD1433 with patients in a clinical environment.
Asunto(s)
Dermatitis , Sustancias Protectoras/uso terapéutico , Crema para la Piel/uso terapéutico , Incontinencia Urinaria/complicaciones , Administración Tópica , Adulto , Anciano , Anciano de 80 o más Años , Dermatitis/tratamiento farmacológico , Dermatitis/etiología , Dermatitis/prevención & control , Éteres/uso terapéutico , Femenino , Fluorocarburos/uso terapéutico , Humanos , Irritantes , Masculino , Persona de Mediana Edad , Vaselina/uso terapéutico , Politetrafluoroetileno/uso terapéutico , Piel/efectos de los fármacos , Resultado del Tratamiento , OrinaRESUMEN
UNLABELLED: Oxidative stress (OS) plays a central role in the progression of liver disease and in damage to liver by toxic xenobiotics. We have developed methods for noninvasive assessment of hepatic OS defenses by measuring flux through the glutathione (GSH) synthesis pathway. (13) C-labeled GSH is endogenously produced and detected by in vivo magnetic resonance after administration of [2-(13) C]-glycine. We report on a successful first-ever human demonstration of this approach as well as preclinical studies demonstrating perturbed GSH metabolism in models of acute and chronic OS. Human studies employed oral administration of [2-(13) C]-glycine and (13) C spectroscopy on a 3T clinical magnetic resonance (MR) imaging scanner and demonstrated detection and quantification of endogenously produced (13) C-GSH after labeled glycine ingestion. Plasma analysis demonstrated that glycine (13) C fractional enrichment achieved steady state during the 6-hour ingestion period. Mean rate of synthesis of hepatic (13) C-labeled GSH was 0.32 ± 0.18 mmole/kg/hour. Preclinical models of acute OS and nonalcoholic steatohepatitis (NASH) comprised CCl4 -treated and high-fat, high-carbohydrate diet-fed Sprague-Dawley rats, respectively, using intravenous administration of [2-(13) C]-glycine and observation of (13) C-label metabolism on a 7T preclinical MR system. Preclinical studies demonstrated a 54% elevation of GSH content and a 31% increase in flux through the GSH synthesis pathway at 12 hours after acute insult caused by CCl4 administration, as well as a 23% decrease in GSH content and evidence of early steatohepatitis in the model of NASH. CONCLUSION: Our data demonstrate in vivo (13) C-labeling and detection of GSH as a biomarker of tissue OS defenses, detecting chronic and acute OS insults. The methods are applicable to clinical research studies of hepatic OS in disease states over time as well as monitoring effects of therapeutic interventions.
Asunto(s)
Glutatión/biosíntesis , Hígado/metabolismo , Estrés Oxidativo , Adulto , Animales , Biomarcadores/metabolismo , Isótopos de Carbono , Glicina , Humanos , Imagen por Resonancia Magnética , Masculino , Ratas , Ratas Sprague-Dawley , Investigación Biomédica TraslacionalRESUMEN
We have previously shown that systemic administration of S(+)3-chloropropanediol (3-CPD) produces a morphological loss of astrocytes in specific nuclei of the rodent brain that precedes loss of both neurones and endothelial tight junctions. Here, we have evaluated the differential susceptibility of neuronal and astrocytic function to 3-CPD, in order to see if this parallels the morphological selectivity. To do this, we have developed an in vivo method for monitoring astrocyte function over time by giving hourly 20-min bolus challenge exposures to ammonia via an implanted microdialysis probe and measuring the resulting transient increases in the extracellular glutamine : glutamate ratio. These challenge ammonia exposures evoked a stable response for at least 5 h when the probe was implanted in the rat inferior colliculus, but caused no behavioural response or morphological damage. Although 3-CPD produced a rapid and sustained abolition of the ammonia response within 2 h, the field potential response of inferior collicular neurones to sound fell significantly to 75.0 ± 3.9% pre-dose at up to 8 h but then fell markedly, reaching 20.5 ± 3.7% at 2 days. Blood flow in the inferior colliculus also showed only late changes, increasing substantially at 2 days. Astrocyte damage at the EM level was seen from 3 h, followed by loss of astrocytes from 18 h to a minimum of 7 ± 10% control at 3 days. The rapid abolition of the ammonia response suggests that in addition to selective astrocyte death, 3-CPD also produces an earlier impairment of astrocyte function that precedes loss of neuronal function. This initial functional selectivity of 3-CPD provides a potential investigative tool in neurochemical studies of astrocyte-neuronal interactions.