RESUMEN
OBJECTIVE: The relevance of margin positivity on recurrence after resection of intraductal papillary mucinous neoplasms (IPMNs) is poorly defined and represents one reason controversy remains regarding optimal surveillance recommendations. METHODS: Patients undergoing surgery for noninvasive IPMN at 8 academic medical centers from the Central Pancreas Consortium were analyzed. A positive margin was defined as presence of IPMN or pancreatic intraepithelial neoplasia. RESULTS: Five hundred two patients underwent surgery for IPMN; 330 (66%) did not have invasive cancer on final pathology and form the study cohort. Of these, 20% harbored high grade dysplasia. A positive margin was found in 20% of cases and was associated with multifocal disease (P = 0.02). The majority of positive margins were associated with low grade dysplasia. At a median follow-up of 36 months, 34 (10.3%) patients recurred, with 6.7% developing recurrent cystic disease and 3.6% developing invasive cancer. On multivariate analysis, margin positivity was not associated with recurrence of either IPMN or invasive cancer (P > 0.05). No association between margin status and development of recurrence at the margin was found. Only 6% of recurrences developed at the resection margin and median time to recurrence was 22 months. Of note, 18% of recurrences occurred > 5 years following surgery. CONCLUSION: Margin positivity after resection for noninvasive IPMNs is primarily due to low grade dysplasia and is not associated with developing recurrence in the remnant pancreas or at the resection margin. Long-term surveillance is required for all patients, as a significant number of recurrences developed over 5 years after the index operation.
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Adenocarcinoma Mucinoso/patología , Adenocarcinoma Mucinoso/cirugía , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/cirugía , Márgenes de Escisión , Recurrencia Local de Neoplasia/patología , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/cirugía , Centros Médicos Académicos , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Selección de Paciente , Estudios Retrospectivos , Resultado del Tratamiento , Estados UnidosRESUMEN
Light chain (AL) amyloidosis is a disease associated with significant morbidity and mortality arising from multi-organ injury induced by amyloidogenic light chain proteins (LC). There is no available treatment to reverse the toxicity of LC. We previously showed that chaperone glycoprotein clusterin (CLU) and nanoliposomes (NL), separately, restore human microvascular endothelial function impaired by LC. In this work, we aim to prepare PEGylated-nanoliposomal clusterin (NL-CLU) formulations that could allow combined benefit against LC while potentially enabling efficient delivery to microvascular tissue, and test efficacy on human arteriole endothelial function. NL-CLU was prepared by a conjugation reaction between the carboxylated surface of NL and the primary amines of the CLU protein. NL were made of phosphatidylcholine (PC), cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG 2000 carboxylic acid) at 70:25:5 mol%. The protective effect of NL-CLU was tested by measuring the dilation response to acetylcholine and papaverine in human adipose arterioles exposed to LC. LC treatment significantly reduced the dilation response to acetylcholine and papaverine; co-treatment of LC with PEGylated-nanoliposomal CLU or free CLU restored the dilator response. NL-CLU is a feasible and promising approach to reverse LC-induced endothelial damage.
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Proteínas Amiloidogénicas/metabolismo , Amiloidosis/tratamiento farmacológico , Clusterina/administración & dosificación , Endotelio Vascular/efectos de los fármacos , Liposomas/química , Nanopartículas/química , Acetilcolina/química , Arteriolas/efectos de los fármacos , Arteriolas/metabolismo , Colesterol/química , Clusterina/química , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Humanos , Papaverina/química , Tamaño de la Partícula , Fosfatidilcolinas/química , Polietilenglicoles/química , Vasodilatación/efectos de los fármacosRESUMEN
The aggregation and fibril deposition of amyloid proteins have been implicated in a range of neurodegenerative and vascular diseases, and yet the underlying molecular mechanisms are poorly understood. Here, we use a combination of cell-based assays, biophysical analysis, and atomic force microscopy to investigate the potential involvement of oxidative stress in aortic medial amyloid (AMA) pathogenesis and deposition. We show that medin, the main constituent of AMA, can induce an environment rich in oxidative species, increasing superoxide and reducing bioavailable nitric oxide in human cells. We investigate the role that this oxidative environment may play in altering the aggregation process of medin and identify potential posttranslational modification sites where site-specific modification and interaction can be unambiguously demonstrated. In an oxidizing environment, medin is nitrated at tyrosine and tryptophan residues, with resultant effects on morphology that lead to longer fibrils with increased toxicity. This provides further motivation to investigate the role of oxidative stress in AMA pathogenicity.
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Antígenos de Superficie/toxicidad , Aorta/metabolismo , Proteínas de la Leche/toxicidad , Estrés Oxidativo/efectos de los fármacos , Antígenos de Superficie/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteínas de la Leche/metabolismo , Nitratos/metabolismoRESUMEN
CONTEXT: A newly-recognized pathogenic mechanism underlying light chain amyloidosis (AL) involves endothelial dysfunction and cell injury caused by misfolded light chain proteins (LC). Nanoliposomes (NL) are artificial phospholipid vesicles that could attach to misfolded proteins and reduce tissue injury. OBJECTIVE: To test whether co-treatment with NL reduces LC-induced endothelial dysfunction and cell death. METHODS: Abdominal subcutaneous adipose arterioles from 14 non-AL subjects were cannulated; dilator response to acetylcholine and papaverine were measured at baseline and following 1-hour exposure to LC (20 µg/mL, 2 purified from AL subjects' urine, 1 from human recombinant LC [AL-09]) ± NL (phosphatidylcholine/cholesterol/phosphatidic acid 70/25/5 molar ratio) or NL alone. Human aortic artery endothelial cells (HAEC) were exposed to Oregon Green-labeled LC ± NL for 24 hours and intracellular LC and apoptosis (Hoechst stain) were measured. Circular dichroism spectroscopy was performed on AL-09 LC ± NL to follow changes in secondary structure and protein thermal stability. RESULTS: LC caused impaired dilation to acetylcholine that was restored by NL (control - 94.0 ± 1.8%, LC - 65.0 ± 7.1%, LC + NL - 95.3 ± 1.8%, p ≤ 0.001 LC versus control or LC + NL). NL protection was inhibited by L-NG-nitroarginine methyl ester. NL increased the beta sheet structure of LC, reduced endothelial cell internalization of LC and protected against LC-induced endothelial cell death. CONCLUSIONS: LC induced human adipose arteriole endothelial dysfunction and endothelial cell death, which were reversed by co-treatment with NL. This protection may partly be due to enhancing LC protein structure and reducing LC internalization. Nanoliposomes represent a promising new class of agents to ameliorate tissue injury from protein misfolding diseases such as AL.
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Amiloide/química , Amiloidosis/tratamiento farmacológico , Endotelio/efectos de los fármacos , Liposomas/uso terapéutico , Nanopartículas/uso terapéutico , Anciano , Apoptosis/efectos de los fármacos , Endotelio/lesiones , Endotelio/patología , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/patología , Humanos , Liposomas/química , Masculino , Persona de Mediana Edad , Nanopartículas/química , Deficiencias en la Proteostasis/tratamiento farmacológicoRESUMEN
Light chain amyloidosis (AL) involves overproduction of amyloidogenic light chain proteins (LC) leading to heart failure, yet the mechanisms underlying tissue toxicity remain unknown. We hypothesized that LC induces endothelial dysfunction in non-AL human microvasculature and apoptotic injury in human coronary artery endothelial cells (HCAECs). Adipose arterioles (n = 34, 50 ± 3 yr) and atrial coronary arterioles (n = 19, 68 ± 2 yr) from non-AL subjects were cannulated. Adipose arteriole dilator responses to acetylcholine/papaverine were measured at baseline and 1 h exposure to LC (20 µg/ml) from biopsy-proven AL subjects (57 ± 11 yr) without and with antioxidant cotreatment. Coronary arteriole dilation to bradykinin/papaverine was measured post-LC exposure. HCAECs were exposed to 1 or 24 h of LC. LC reduced dilation to acetylcholine (10(-4) M: 41.6 ± 7 vs. 85.8 ± 2.2% control, P < 0.001) and papaverine (81.4 ± 4.6 vs. 94.8 ± 1.3% control, P < 0.01) in adipose arterioles and to bradykinin (10(-6) M: 68.6 ± 6.2 vs. 90.9 ± 1.6% control, P < 0.001) but not papaverine in coronary arterioles. There was an increase in superoxide and peroxynitrite in arterioles treated with LC. Adipose arteriole dilation was restored by cotreatment with polyethylene glycol-superoxide dismutase and tetrahydrobiopterin but only partially restored by mitoquinone (mitochondria-targeted antioxidant) and gp91ds-tat (NADPH oxidase inhibitor). HCAECs exposed to LC showed reduced NO and increased superoxide, peroxynitrite, annexin-V, and propidium iodide compared with control. Brief exposure to physiological amounts of LC induced endothelial dysfunction in human adipose and coronary arterioles and increased apoptotic injury in coronary artery endothelial cells likely as a result of oxidative stress, reduced NO bioavailability, and peroxynitrite production. Microvascular dysfunction and injury is a novel mechanism underlying AL pathobiology and is a potential target for therapy.
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Tejido Adiposo/irrigación sanguínea , Amiloidosis/metabolismo , Apoptosis , Vasos Coronarios/metabolismo , Células Endoteliales/metabolismo , Cadenas Ligeras de Inmunoglobulina/metabolismo , Vasodilatación , Anciano , Amiloidosis/patología , Amiloidosis/fisiopatología , Antioxidantes/farmacología , Arteriolas/metabolismo , Arteriolas/patología , Arteriolas/fisiopatología , Estudios de Casos y Controles , Células Cultivadas , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/patología , Vasos Coronarios/fisiopatología , Relación Dosis-Respuesta a Droga , Células Endoteliales/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Óxido Nítrico/metabolismo , Estrés Oxidativo , Ácido Peroxinitroso/metabolismo , Superóxidos/metabolismo , Factores de Tiempo , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Background The function of medin, one of the most common human amyloid proteins that accumulates in the vasculature with aging, remains unknown. We aim to probe medin's role in cerebrovascular disease by comparing cerebral arterial medin content between cognitively normal and vascular dementia (VaD) patients and studying its effects on endothelial cell (EC) immune activation and neuroinflammation. We also tested whether monosialoganglioside-containing nanoliposomes could reverse medin's adverse effects. Methods and Results Cerebral artery medin and astrocyte activation were measured and compared between VaD and cognitively normal elderly brain donors. ECs were exposed to physiologic dose of medin (5 µmol/L), and viability and immune activation (interleukin-8, interleukin-6, intercellular adhesion molecule-1, and plasminogen activator inhibitor-1) were measured without or with monosialoganglioside-containing nanoliposomes (300 µg/mL). Astrocytes were exposed to vehicle, medin, medin-treated ECs, or their conditioned media, and interleukin-8 production was compared. Cerebral collateral arterial and parenchymal arteriole medin, white matter lesion scores, and astrocyte activation were higher in VaD versus cognitively normal donors. Medin induced EC immune activation (increased interleukin-8, interleukin-6, intercellular adhesion molecule-1, and plasminogen activator inhibitor-1) and reduced EC viability, which were reversed by monosialoganglioside-containing nanoliposomes. Interleukin-8 production was augmented when astrocytes were exposed to medin-treated ECs or their conditioned media. Conclusions Cerebral arterial medin is higher in VaD compared with cognitively normal patients. Medin induces EC immune activation that modulates astrocyte activation, and its effects are reversed by monosialoganglioside-containing nanoliposomes. Medin is a candidate novel risk factor for aging-related cerebrovascular disease and VaD.
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Antígenos de Superficie/toxicidad , Astrocitos/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Arterias Cerebrales/efectos de los fármacos , Demencia Vascular/tratamiento farmacológico , Células Endoteliales/efectos de los fármacos , Gangliósidos/farmacología , Proteínas de la Leche/toxicidad , Nanopartículas , Anciano , Anciano de 80 o más Años , Astrocitos/inmunología , Astrocitos/metabolismo , Astrocitos/patología , Estudios de Casos y Controles , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Arterias Cerebrales/inmunología , Arterias Cerebrales/metabolismo , Arterias Cerebrales/patología , Técnicas de Cocultivo , Demencia Vascular/inmunología , Demencia Vascular/metabolismo , Demencia Vascular/patología , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Humanos , Liposomas , Masculino , Estrés Oxidativo/efectos de los fármacos , Transducción de SeñalRESUMEN
BACKGROUND AND AIMS: Peripheral insulin resistance is associated with several metabolic abnormalities, including elevated serum fatty acids that contribute to vascular injury and atherogenesis. Our goals were to examine whether saturated fatty acids can modify innate immune responses to subclinical concentrations of lipopolysaccharide (LPS) in endothelial cells, and to explore the underlying pathway and determine whether it is modified by high density lipoprotein (HDL) and other factors commonly altered in insulin resistance. METHODS: Physiologic concentrations of palmitic acid were added to human aortic endothelial cells with and without a variety of inhibitors or HDL and measures of cell inflammation and function assessed. RESULTS: Palmitic acid significantly amplified human aortic endothelial cell inflammatory responses to LPS. Similar results were obtained from lipolysis products of triglyceride rich lipoproteins. Metabolism of palmitic acid to ceramide and subsequent activation of PKC-ζ, MAPK and ATF3 appeared critical in amplifying LPS induced inflammation. The amplified response to palmitic acid/LPS was decreased by HDL, dose dependently, and this inhibition was dependent on activation of PI3K/AKT and reduction in ATF3. CONCLUSIONS: These results indicate that endothelial cell innate immune responses are modified by metabolic abnormalities commonly present in insulin resistance and provide evidence for a novel mechanism by which HDL may reduce vascular inflammation.
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Antiinflamatorios/farmacología , Células Endoteliales/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Lipoproteínas HDL/farmacología , Ácido Palmítico/farmacología , Factor de Transcripción Activador 3/metabolismo , Células Cultivadas , Ceramidas/metabolismo , Relación Dosis-Respuesta a Droga , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Ácido Palmítico/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
AIMS: Medin is a common amyloidogenic protein in humans that accumulates in arteries with advanced age and has been implicated in vascular degeneration. Medin's effect on endothelial function remains unknown. The aims are to assess medin's effects on human arteriole endothelial function and identify potential mechanisms underlying medin-induced vascular injury. METHODS AND RESULTS: Ex vivo human adipose and leptomeningeal arterioles were exposed (1 h) to medin (0.1, 1, or 5 µM) without or with FPS-ZM1 [100 µM, receptor for advanced glycation endproducts (RAGE)-specific inhibitor] and endothelium-dependent function (acetylcholine dilator response) and endothelium-independent function (dilator response to nitric oxide donor diethylenetriamine NONOate) were compared with baseline control. Human umbilical vein endothelial cells were exposed to medin without or with FPS-ZM1 and oxidative and nitrative stress, cell viability, and pro-inflammatory signaling measures were obtained. Medin caused impaired endothelial function (vs. baseline response: -45.2 ± 5.1 and -35.8 ± 7.9% in adipose and leptomeningeal arterioles, respectively, each P < 0.05). Dilator response to NONOate was not significantly changed. Medin decreased arteriole and endothelial cell nitric oxide production, increased superoxide production, reduced endothelial cell viability, proliferation, and migration. Medin increased gene and protein expression of interleukin-6 and interleukin-8 via activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). Medin-induced endothelial dysfunction and oxidative stress were reversed by antioxidant polyethylene glycol superoxide dismutase and by RAGE inhibitor FPS-ZM1. CONCLUSIONS: Medin causes human microvascular endothelial dysfunction through oxidative and nitrative stress and promotes pro-inflammatory signaling in endothelial cells. These effects appear to be mediated via RAGE. The findings represent a potential novel mechanism of vascular injury.
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Endotelio Vascular/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antioxidantes/farmacología , Arteriolas/metabolismo , Benzamidas/farmacología , Endotelio Vascular/efectos de los fármacos , Femenino , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inflamación/metabolismo , Masculino , Persona de Mediana Edad , Estrés Oxidativo/efectos de los fármacos , Superóxidos/metabolismo , Vasodilatadores/farmacologíaRESUMEN
We tested whether nanoliposomes containing phosphatidylcholine, cholesterol and phosphatidic acid (NLPA) prevent ß-amyloid 1-42 (Aß42) fibrillation and Aß42-induced human arteriole endothelial dysfunction. NLPA abolished Aß42 fibril formation (thioflavin-T fluorescence/electron microscopy). In ex-vivo human adipose and leptomeningeal arterioles, Aß42 impaired dilator response to acetylcholine that was reversed by NLPA; this protection was abolished by L-NG-nitroarginine methyl ester. Aß42 reduced human umbilical vein endothelial cell NO production that was restored by NLPA. Nanoliposomes prevented Aß42 amyloid formation, reversed Aß42-induced human microvascular endothelial dysfunction and may be useful in Alzheimer's disease.
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Péptidos beta-Amiloides , Arteriolas/patología , Endotelio Vascular/patología , Liposomas/uso terapéutico , Fragmentos de Péptidos , Enfermedades Vasculares/inducido químicamente , Enfermedades Vasculares/prevención & control , Acetilcolina/antagonistas & inhibidores , Acetilcolina/farmacología , Tejido Adiposo/irrigación sanguínea , Colesterol/administración & dosificación , Colesterol/uso terapéutico , Humanos , Técnicas In Vitro , Masculino , Meninges/irrigación sanguínea , Persona de Mediana Edad , NG-Nitroarginina Metil Éster/farmacología , Nanopartículas/uso terapéutico , Óxido Nítrico/biosíntesis , Ácidos Fosfatidicos/administración & dosificación , Ácidos Fosfatidicos/uso terapéutico , Fosfatidilcolinas/administración & dosificación , Fosfatidilcolinas/uso terapéutico , Enfermedades Vasculares/patología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
BACKGROUND: Light chain amyloidosis (AL) is associated with high mortality, especially in patients with advanced cardiovascular involvement. It is caused by toxicity of misfolded light chain proteins (LC) in vascular, cardiac, and other tissues. There is no treatment to reverse LC tissue toxicity. We tested the hypothesis that nanoliposomes composed of monosialoganglioside, phosphatidylcholine, and cholesterol (GM1 ganglioside-containing nanoliposomes [NLGM1]) can protect against LC-induced human microvascular dysfunction and assess mechanisms behind the protective effect. METHODS AND RESULTS: The dilator responses of ex vivo abdominal adipose arterioles from human participants without AL to acetylcholine and papaverine were measured before and after exposure to LC (20 µg/mL) with or without NLGM1 (1:10 ratio for LC:NLGM1 mass). Human umbilical vein endothelial cells were exposed for 18 to 20 hours to vehicle, LC with or without NLGM1, or NLGM1 and compared for oxidative and nitrative stress response and cellular viability. LC impaired arteriole dilator response to acetylcholine, which was restored by co-treatment with NLGM1. LC decreased endothelial cell nitric oxide production and cell viability while increasing superoxide and peroxynitrite; these adverse effects were reversed by NLGM1. NLGM1 increased endothelial cell protein expression of antioxidant enzymes heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 and increased nuclear factor, erythroid 2 like 2 (Nrf-2) protein. Nrf-2 gene knockdown reduced antioxidant stress response and reversed the protective effects of NLGM1. CONCLUSIONS: NLGM1 protects against LC-induced human microvascular endothelial dysfunction through increased nitric oxide bioavailability and reduced oxidative and nitrative stress mediated by Nrf-2-dependent antioxidant stress response. These findings point to a potential novel therapeutic approach for light chain amyloidosis.
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Colesterol/administración & dosificación , Endotelio Vascular/efectos de los fármacos , Gangliósidos/administración & dosificación , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/complicaciones , Fosfatidilcolinas/administración & dosificación , Enfermedades Vasculares/prevención & control , Tejido Adiposo/irrigación sanguínea , Arteriolas/efectos de los fármacos , Arteriolas/fisiología , Supervivencia Celular/fisiología , Combinación de Medicamentos , Células Endoteliales/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/prevención & control , Masculino , Persona de Mediana Edad , NAD(P)H Deshidrogenasa (Quinona)/genética , Factor 2 Relacionado con NF-E2/genética , Nanopartículas/administración & dosificación , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Papaverina/farmacología , Ácido Peroxinitroso/biosíntesis , Interferencia de ARN/fisiología , ARN Interferente Pequeño/fisiología , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo , Transfección , Enfermedades Vasculares/fisiopatología , Vasodilatadores/farmacologíaRESUMEN
GLP-1 receptor (GLP-1R) agonists may improve endothelial function (EF) via metabolic improvement and direct vascular action. The current study determined the effect of GLP-1R agonist exenatide on postprandial EF in type 2 diabetes and the mechanisms underlying GLP-1R agonist-mediated vasodilation. Two crossover studies were conducted: 36 participants with type 2 diabetes received subcutaneous exenatide or placebo for 11 days and EF, and glucose and lipid responses to breakfast and lunch were determined; and 32 participants with impaired glucose tolerance (IGT) or diet-controlled type 2 diabetes had EF measured before and after intravenous exenatide, with or without the GLP-1R antagonist exendin-9. Mechanisms of GLP-1R agonist action were studied ex vivo on human subcutaneous adipose tissue arterioles and endothelial cells. Subcutaneous exenatide increased postprandial EF independent of reductions in plasma glucose and triglycerides. Intravenous exenatide increased fasting EF, and exendin-9 abolished this effect. Exenatide elicited eNOS activation and NO production in endothelial cells, and induced dose-dependent vasorelaxation and reduced high-glucose or lipid-induced endothelial dysfunction in arterioles ex vivo. These effects were reduced with AMPK inhibition. In conclusion, exenatide augmented postprandial EF in subjects with diabetes and prevented high-glucose and lipid-induced endothelial dysfunction in human arterioles. These effects were largely direct, via GLP-1R and AMPK activation.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Células Endoteliales/efectos de los fármacos , Péptidos/farmacología , Receptores de Glucagón/agonistas , Vasodilatación/efectos de los fármacos , Ponzoñas/farmacología , Proteínas Quinasas Activadas por AMP/fisiología , Glucemia/análisis , Células Cultivadas , Estudios Cruzados , Diabetes Mellitus Tipo 2/fisiopatología , Método Doble Ciego , Células Endoteliales/fisiología , Exenatida , Femenino , Receptor del Péptido 1 Similar al Glucagón , Humanos , Masculino , Receptores de Glucagón/fisiología , Triglicéridos/sangreRESUMEN
BACKGROUND: Evidence point to vascular dysfunction and hypoperfusion as early abnormalities in Alzheimer's disease (AD); probing their mechanistic bases can lead to new therapeutic approaches. We tested the hypotheses that ß-amyloid peptide induces endothelial dysfunction and oxidative stress in human microvasculature and that response will be similar between peripheral adipose and brain leptomeningeal arterioles. NEW METHOD: Abdominal subcutaneous arterioles from living human subjects (n=17) and cadaver leptomeningeal arterioles (n=6) from rapid autopsy were exposed to Aß1-42 (Aß) for 1-h and dilation response to acetylcholine/papaverine were measured and compared to baseline response. Adipose arteriole reactive oxygen species (ROS) production and nitrotyrosine content were measured. COMPARISON WITH EXISTING METHODS: Methods described allow direct investigation of human microvessel functional response that cannot be replicated by human noninvasive imaging or post-mortem histology. RESULTS: Adipose arterioles exposed to 2 µM Aß showed impaired dilation to acetylcholine that was reversed by antioxidant polyethylene glycol superoxide dismutase (PEG-SOD) (Aß-60.9 ± 6%, control-93.2 ± 1.8%, Aß+PEGSOD-84.7 ± 3.9%, both p<0.05 vs. Aß). Aß caused reduced dilation to papaverine. Aß increased adipose arteriole ROS production and increased arteriole nitrotyrosine content. Leptomeningeal arterioles showed similar impaired response to acetylcholine when exposed to Aß (43.0 ± 6.2% versus 81.1 ± 5.7% control, p<0.05). CONCLUSION: Aß exposure induced adipose arteriole endothelial and non-endothelial dysfunction and oxidative stress that were reversed by antioxidant treatment. Aß-induced endothelial dysfunction was similar between peripheral adipose and leptomeningeal arterioles. Ex vivo living adipose and cadaver leptomeningeal arterioles are viable, novel and practical human tissue models to study Alzheimer's vascular pathophysiology.
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Tejido Adiposo/irrigación sanguínea , Péptidos beta-Amiloides , Arteriolas/fisiopatología , Células Endoteliales/fisiología , Meninges/irrigación sanguínea , Fragmentos de Péptidos , Abdomen/irrigación sanguínea , Acetilcolina/farmacología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estrés Oxidativo/fisiología , Papaverina/farmacología , Especies Reactivas de Oxígeno/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Vasodilatadores/farmacologíaRESUMEN
UNLABELLED: Misfolded immunoglobulin light chain proteins (LC) in light chain amyloidosis (AL) are toxic to vascular tissues. We tested the hypothesis that chaperone protein clusterin preserves endothelial function and cell survival during LC exposure. METHODS: LC (20 µg/mL) were given to human aortic endothelial cells (EC) for 24-h and clusterin protein/gene expression and secretion were measured. DNA fragmentation was measured with/without recombinant clusterin (Clu, 300 ng/mL). Adipose arterioles (non-AL subjects) were tested for dilator responses to acetylcholine/papaverine at baseline and after 1-h of LC ± Clu. RESULTS: LC reduced EC clusterin secretion, protein and gene expression while increasing DNA fragmentation. Clu attenuated LC-induced DNA fragmentation and restored dilator response to acetylcholine (logEC50: control -7.05 ± 0.2, LC + Clu -6.53 ± 0.4, LC -4.28 ± 0.7, p < 0.05 versus control, LC + Clu). CONCLUSIONS: LC induced endothelial cell death and dysfunction while reducing clusterin protein/gene expression and secretion. Exogenous clusterin attenuated LC toxicity. This represents a new pathobiologic mechanism and therapeutic target for AL amyloidosis.