RESUMEN

The consequences of severe acute viral respiratory syndrome (COVID 19) pandemic include collateral effects, one of which has been the significant reduction in routine hospital work. With widespread reports indicating reduction of cardiac procedures including MI presentation to hospitals, we aimed to analyze the local data over a 10-week period during lockdown in a tertiary cardiac centre Catheter Laboratory in England. METHODS: We conducted a retrospective review of the coronary catheterisation procedures and admissions with MI over the peak COVID-19 pandemic 10-week period (23rd March-30th May) in 2020, compared with the same 10-week period (25th March-2nd June) in 2019. RESULTS: In 2019, 539 patients were admitted to the Cath lab for coronary catheterisation (M = 385:F = 154; mean age 65 years; STEMI = 186, NSTEMI = 192, elective = 161). In 2020, during peak period of COVID19 pandemic in England, a total of 278 patients were admitted for coronary catheterisation over the 10-week period (M = 201:F = 77; mean age 60.5 years; STEMI = 132, NSTEMI = 118, elective = 28). During peak COVID19 pandemic, this represents a 48.4% drop in all coronary catheterisations. The reduction in STEMI was 29% (54 less), in NSTEMI was 38.9% (74 less) and elective procedures dropped by 83% (133 less). CONCLUSION: During peak COVID hospital admission period in England, we report a 48.5% reduction in coronary catheterisation in our tertiary hospital. These results are consistent with reports from other countries, and highlight the worrying potential consequences for these patients arising from delays in presentation with MI, and the challenges for restoring services post-pandemic.

RESUMEN

Scanning tunnelling microscopy observations resolve the structure and dynamics of metallic glass Cu100-xHfx films and demonstrate scanning tunnelling microscopy control of aging at a metallic glass surface. Surface clusters exhibit heterogeneous hopping dynamics. Low Hf concentration films feature an aged surface of larger, slower clusters. Argon ion-sputtering destroys the aged configuration, yielding a surface in constant fluctuation. Scanning tunnelling microscopy can locally restore the relaxed state, allowing for nanoscale lithographic definition of aged sections.

RESUMEN

There is growing interest in the large scale cyclotron production of (99m)Tc via the (100)Mo(p,2n)(99m)Tc reaction. While the use and recycling of cyclotron-irradiated enriched molybdenum targets has been reported previously in the context of (94m)Tc production, to the best of our knowledge, previous recycling studies have been limited to the use of oxide targets. To facilitate reuse of high-power enriched (100)Mo targets, this work presents and evaluates a strategy for recycling of enriched metallic molybdenum. For the irradiated (100)Mo targets in this study, an overall metal to metal recovery of 87% is reported. Evaluation of "new" and "recycled" (100)Mo revealed no changes in the molybdenum isotopic composition (as measured via ICP-MS). For similar irradiation conditions of "new" and "recycled" (100)Mo, (i.e. target thicknesses, irradiation time, and energy), comparable levels of (94g)Tc, (95g)Tc, and (96g)Tc contaminants were observed. Comparable QC specifications (i.e. aluminum ion concentration, pH, and radiochemical purity) were also reported. We finally note that [(99m)Tc]-MDP images obtained by comparing MDP labelled with generator-based (99m)Tc vs. (99m)Tc obtained following the irradiation of recycled (100)Mo demonstrated comparable biodistribution. With the goal of producing large quantities of (99m)Tc, the proposed methodology demonstrates that efficient recycling of enriched metallic (100)Mo targets is feasible and effective.

RESUMEN

We describe two cases of a non-epileptic florid movement disorder presenting as status epilepticus. Both patients presented with florid jerking of the limbs and eyes. Convulsive status epilepticus related to presumed meningitis or encephalitis was suspected in both cases. The patients received treatment for seizures, without resolution of the abnormal movements, resulting ultimately in anaesthetic, intubation and ventilation. EEGs showed no epileptic discharges. The diagnosis was opsoclonus myoclonus syndrome in both. One patient was treated with adrenocorticotropic hormone (40 IU/day), the other with prednisolone (4 mg/kg/day) with rapid resolution of symptoms. Neither patient had an underlying neoplasm or infectious agent identified. To date, neither patient has suffered a relapse of symptoms nor does either show any sign of developmental delay. These cases show that the movements in opsoclonus myoclonus syndrome can be sufficiently florid to mimic convulsive status epilepticus. Video footage of both patients at the time of diagnosis is presented online.

Asunto(s)

Síndrome de Opsoclonía-Mioclonía/diagnóstico , Estado Epiléptico/diagnóstico , Hormona Adrenocorticotrópica/uso terapéutico , Diagnóstico Diferencial , Electroencefalografía , Glucocorticoides/uso terapéutico , Hormonas/uso terapéutico , Humanos , Lactante , Masculino , Movimiento/fisiología , Síndrome de Opsoclonía-Mioclonía/tratamiento farmacológico , Prednisolona/uso terapéutico , Estado Epiléptico/tratamiento farmacológico

RESUMEN

c-Myc is involved in the formation of neointimal hyperplasia. We investigated in vitro, ex vivo and in vivo release of antisense c-myc from cationically modified phosphorylcholine-coated stents, as well as the effects on c-Myc expression and neointima formation in a porcine coronary stent model. In vitro experiments were performed to determine optimal loading of stents with antisense. Stents loaded with labelled antisense were deployed in porcine arteries ex vivo and in vivo. Antisense was detected in the vessel wall directly surrounding the stent of pig carotid and coronary artery up to 48 h after stent deployment. Nuclear uptake was observed in endothelial and vascular smooth muscle cells. Labelled antisense within peripheral tissues in vivo was <1.0% of that within stented arterial segments. Control and antisense loaded stents implanted into 10 pig coronary arteries and analysed at 28 days post-stenting showed that lumen area within the antisense stents was significantly increased (i.e. 30.5% greater, P<0.01), whilst both neointimal area and neointimal thickness were significantly reduced (17.5% and 19.5%, respectively, P<0.01) compared to control stents. Cationically modified phosphorylcholine coated stent-based delivery of c-myc antisense is feasible with minimal systemic delivery and is associated with a reduction of in-stent neointimal hyperplasia in pig coronary arteries.

Asunto(s)

Prótesis Vascular , Materiales Biocompatibles Revestidos/química , ADN sin Sentido/administración & dosificación , Proteínas de Unión al ADN/genética , Oclusión de Injerto Vascular/prevención & control , Fosforilcolina/química , Stents , Factores de Transcripción/genética , Animales , Materiales Biocompatibles Revestidos/administración & dosificación , ADN sin Sentido/genética , Implantes de Medicamentos/administración & dosificación , Implantes de Medicamentos/química , Análisis de Falla de Equipo , Estudios de Factibilidad , Marcación de Gen/métodos , Oclusión de Injerto Vascular/genética , Técnicas In Vitro , Diseño de Prótesis , Porcinos

RESUMEN

In women, heel ultrasound (US) bone mineral density (BMD) has been shown to predict fracture risk, but the usefulness of this screening tool in men is not known. We measured the heel quantitative ultrasound index (QUI( in a convenience sample 185 of men (136 Caucasian, 1 Asian, and 48 African-American) with an average age of 63 yr (range of 25-85) undergoing BMD of the spine and hip by dual X-ray absorptiometry (DXA) to determine whether the heel measurement could predict central BMD. The average DXA T-score was -0.97, -1.20, and -1.61 for the spine, total hip, and femoral neck, respectively. The mean heel US BMD T-score (using the only available T-score, which was defined for Caucasian postmenopausal women) was -0.92. There were significant correlations among the various DXA measurements and the heel US BMD T-score (r = 0.373-0.483, p < 0.001). We defined arbitrarily osteopenia as a spine, total hip, or femoral neck T-score by DXA of < -1.5. We also made two different arbitrary definitions of osteoporosis by DXA: < -2.0 and < -2.5. Using these numbers as disease definitions, we determined the specificity, sensitivity, as well as positive and negative predictive values of using the heel US T-score to predict osteopenia or osteoporosis. Using various cutoffs for the heel T-score, we found that increasing the cutoff toward 0 increased the sensitivity but lowered the specificity. No cutoff was found that provided both good sensitivity and specificity. By analyzing the men by ethnic and age groups, we found that the best set of receiver operating characteristic (ROC) curves was derived from data using heel US to predict osteopenia and osteoporosis in men younger than age 65, although the areas under the ROC curve were approx 0.8. In conclusion, despite a strong correlation between the heel QUI and the spine and hip BMD by DXA, no heel T-score could predict osteopenia or osteoporosis with satisfactory sensitivity and specificity. It is possible that the use of risk factor assessment plus heel QUI might have better predictive value, and further studies are needed to determine whether heel QUI or other US determination is an independent risk factor for fracture in men.

Asunto(s)

Densidad Ósea , Calcáneo/diagnóstico por imagen , Talón/diagnóstico por imagen , Absorciometría de Fotón , Índice de Masa Corporal , Calcáneo/fisiología , Fracturas Óseas/etiología , Cadera/diagnóstico por imagen , Cadera/fisiología , Humanos , Masculino , Persona de Mediana Edad , Osteoporosis/complicaciones , Osteoporosis/diagnóstico , Curva ROC , Estudios Retrospectivos , Sensibilidad y Especificidad , Columna Vertebral/diagnóstico por imagen , Columna Vertebral/fisiología , Ultrasonografía

RESUMEN

Previous studies have shown that inhibition of the proto-oncogene c-myb inhibits neointimal formation in various animal models. However, the temporal and spatial expression of c-Myb in the vessel wall after injury is not known, and the mechanism of action of antisense oligonucleotide (AS-ODN-c-myb) inhibition remains unclear. One potential effect of cell cycle dysregulation by inhibition of c-myb is an increase in the rates of apoptosis. In this study, c-Myb expression after percutaneous transluminal coronary angioplasty (PTCA) injury and induction of apoptosis after AS-ODN-c-myb treatment were determined. Immunohistochemistry and cellular phenotyping were used to localize c-Myb expression in porcine coronary arteries at various time intervals after PTCA. In vitro, the effects of AS-ODN-c-myb on the apoptosis of porcine vascular smooth muscle cells (PVSMCs) and endothelial cells were determined by using a cell-death ELISA and time-lapse video microscopy. In vivo, local delivery of AS-ODN-c-myb was performed after PTCA of pig coronary arteries, and apoptosis was quantified at 6 hours. c-Myb is induced in pig coronary arteries after angioplasty, with maximal expression in inflammatory cells at 18 hours and in vascular smooth muscle cells at 3 to 7 days. In vitro, AS-ODN-c-myb enhanced PVSMCs (6.8+/-0.8% [P=<0.001] versus 0.5% serum) but not endothelial cell apoptosis (1.4+/-0.5% [P=NS] versus 0.5% serum). In vivo, 6 hours after porcine coronary angioplasty and delivery of AS-ODN-c-myb, the proportion of apoptotic cells within the media was 4.2+/-0.8% (PTCA alone), 2.3+/-0.2% (PTCA+vehicle), and 9.0+/-1.1% (PTCA+AS-ODN-c-myb; P<0.05 versus PTCA alone and P<0.01 versus PTCA+saline). c-Myb is expressed after PTCA of pig coronary arteries, and AS-ODN-c-myb induces apoptosis of PVSMCs in vitro and medial cells in vivo.

Asunto(s)

Angioplastia Coronaria con Balón/efectos adversos , Apoptosis , Vasos Coronarios/citología , Vasos Coronarios/metabolismo , Oligonucleótidos Antisentido/farmacología , Proteínas Proto-Oncogénicas c-myb/metabolismo , Animales , Biomarcadores/análisis , Células Cultivadas , Reestenosis Coronaria/etiología , Ensayo de Inmunoadsorción Enzimática , Etiquetado Corte-Fin in Situ , Cinética , Microscopía por Video , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas c-myb/genética , Porcinos

RESUMEN

OBJECTIVE: Restenosis following angioplasty involves processes that may be influenced by local production of cytokines. We investigated the expression of active and total transforming growth factor beta (TGFbeta) following porcine coronary angioplasty (PTCA), and have correlated this with the expression of potential in vivo activators of TGFbeta: mannose-6-phosphate/insulin-like growth factor-II (M6P/IGF-II) receptor and thrombospondin-1. METHODS: Oversized porcine PTCA was performed and the arteries excised after selected intervals. Levels of in situ active and total (active plus latent) TGFbeta were determined using a modified plasminogen activator-inhibitor/luciferase bioassay. RESULTS: Levels of active TGFbeta significantly increased 2 h to 7 days after angioplasty, compared to non-injured controls. Levels returned to baseline by 28 days. Active TGFbeta in tissues adjacent to the injured artery did not change. Total TGFbeta was significantly higher than controls 2-6 h after injury. M6P/IGF-II receptor mRNA was upregulated between 6 h and 3 days after injury, with protein detectable at 3-28 days. Thrombospondin-1 was detected between 1 h and 14 days. CONCLUSIONS: We conclude that balloon injury causes an early rapid increase in levels of active TGFbeta, that correlates with the expression of TGFbeta activators. Thus, TGFbeta is a good potential target for anti-restenotic therapies.

Asunto(s)

Angioplastia Coronaria con Balón , Enfermedad Coronaria/metabolismo , Vasos Coronarios/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Western Blotting , Técnicas para Inmunoenzimas , Receptor IGF Tipo 2/metabolismo , Recurrencia , Porcinos , Trombospondina 1/metabolismo , Factores de Tiempo

RESUMEN

AIMS: To examine the angiographic (quantitative coronary angiography), morphometric, light microscopic (LM) (i.e., histology and immunohistochemical staining) and electron microscopic (EM) findings after implantation of phosphorylcholine (PC)-coated compared to uncoated stents in porcine coronary arteries. METHODS: Forty (25 PC-coated, 15 uncoated) divYsio stents were implanted into the coronary arteries of 20 pigs. Quantitative coronary angiography (QCA) was performed pre-stent and post-implantation in fifteen pigs, at 28 days. Two pigs were killed at 5 days (LM and scanning EM), one pig at 14 days (scanning EM) and 17 pigs at 28 days (LM, scanning EM, transmission EM). At 28 days, thirty-two of 34 stented segments excised were formalin-fixed, of which 30 were embedded in resin and sectioned for morphometry and LM. Remaining stents were examined by TEM and SEM. RESULTS: No angiographically occlusive thrombosis occurred in any of the stents. LM at 5 days showed endothelialization of PC-coated and uncoated stents, which was also confirmed by scanning EM at 14 days. At 28 days, QCA and morphometry showed no significant differences between PC-coated and uncoated stents. A few inflammatory cells were seen in both stent types at 5 days but there was no inflammatory or additional tissue reaction to PC-coated compared to uncoated stents at 28 days. CONCLUSIONS: The divYsio stents, with or without PC coating, performed equally well in terms of acute patency, 28-day QCA and morphometry. The PC coating allows a stent to endothelialize normally and is not associated with specific histological changes. The PC coating on the divYsio stent appears biocompatible.

Asunto(s)

Materiales Biocompatibles Revestidos , Vasos Coronarios , Fosforilcolina , Stents , Animales , Angiografía Coronaria , Vasos Coronarios/ultraestructura , Inmunohistoquímica , Ensayo de Materiales , Distribución Aleatoria , Porcinos

Asunto(s)

Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/metabolismo , Inhibidores Enzimáticos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico Sintasa/farmacología , Proteínas Supresoras de Tumor , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Humanos , Hiperplasia , Músculo Liso Vascular/citología , Óxido Nítrico Sintasa de Tipo III , Porcinos

RESUMEN

OBJECTIVE: Stent thrombosis and in-stent restenosis remain problematic in certain patient sub-groups. c7E3-Fab (ReoPro, abciximab) inhibits the platelet glycoprotein IIb/IIIa receptor as well as the smooth muscle cell alpha(v)beta3 receptor, and thus may influence both processes, especially if high local concentrations could be achieved. We have studied the adsorption and elution characteristics of c7E3-Fab on commercially available polymer-coated stents. We have also investigated the effect of such antibody binding on platelet deposition in vitro, and on antibody deposition into ex vivo human saphenous vein wall to assess whether such stents may influence stent thrombosis and restenosis. METHODS AND RESULTS: Adsorption was measured using a radioisotope technique after immersing segments of polymer-coated stents in c7E3-Fab solutions. Uptake was dependent on antibody concentration and duration of immersion of wire in the solution. After 22 h (at 5 mg ml(-1)), 1146+/-101 ng cm(-1) wire was adsorbed. In an in vitro perfusion circuit, the antibody eluted slowly, with 53% remaining after 12 days washing. To determine the value that such stents might have in clinical practise, adsorption to balloon-mounted stents was assessed at room temperature, using commercially available c7E3-Fab (2 mg ml(-1)). Efficacy of eluting c7E3-Fab was determined by measuring deposition of 111-Indium platelets. Immersing stents in c7E3-Fab for 20 min inhibited platelet deposition by 82.3% compared to controls (P=0.018). Deployment of treated stents in ex vivo saphenous vein resulted in the deposition of c7E3-Fab in the intima and media. CONCLUSIONS: c7E3-Fab can be passively adsorbed onto polymer-coated stents. It elutes slowly and in a predictable manner, significantly inhibiting platelet deposition in vitro. These studies pave the way to developing stent-based delivery of a potent anti-platelet agent that may additionally affect smooth muscle cell activity.

Asunto(s)

Anticuerpos Monoclonales/farmacología , Trombosis Coronaria/prevención & control , Fragmentos Fab de Inmunoglobulinas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Stents , Abciximab , Absorción , Sitios de Unión de Anticuerpos , Trombosis Coronaria/cirugía , Estudios de Evaluación como Asunto , Humanos , Recurrencia

RESUMEN

The aim of the study was to determine the effects of radiographic contrast media (RCM) on proliferation and apoptosis of human vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were exposed for either 1 min or 15 min to RCM (diatrizoate, ioxaglate, iopromide, iotrolan) at an iodine concentration of 250 mgl ml-1. Controls were complete growth medium (CGM) and saturated mannitol (osmotic control). [3H]thymidine incorporation was used to determine cell proliferation 24 h after exposure. Apoptosis was determined at 1 h and 6 h by terminal uridine nick end labelling (TUNEL), time lapse video microscopy (TLVM) and DNA electrophoresis. Mean proliferation rates (%) (+/- SEM) (p-values compared with the CGM control) at 1 min and 15 min, respectively, were: diatrizoate: 31.9 (10.6), 5.8 (1.5) (p < 0.001); ioxaglate: 48.4 (10.9), 20.4 (4.5) (p < 0.001); iopromide: 63.4 (8.7), 58.2 (10.2) (p < 0.05); iotrolan: 84.7 (7.3), 72.8 (12.4) (p = ns); saturated mannitol 50.5 (9.6), 45.9 (10.0) (p < 0.001). Mean apoptotic indices (%) (+/- SEM) at 1 h and 6 h following 1 min exposure, respectively, were: CGM: 0.25 (0.13), 0.23 (0.08); diatrizoate: 2.18 (0.19), 2.69 (0.34) (p < 0.001); ioxaglate: 1.90 (0.23), 1.69 (0.02) (p < 0.05); iopromide: 0.59 (0.04), 0.33 (0.02) (p = ns); iotrolan: 0.30 (0.07), 0.27 (0.1) (p = ns); saturated mannitol 2.11 (0.24), 1.4 (0.1) (p < 0.05). After 15 min exposure, apoptosis rates at both 1 h and 6 h, respectively, were: iotrolan: 0.29 (0.17), 0.51 (0.16) (p = ns); diatrizoate: 3.19 (0.81), 11.66 (1.75) (p < 0.001); ioxaglate: 1.88 (0.14), 2.87 (0.20) (p < 0.05); iopromide: 1.06 (0.11), 1.52 (0.15) (p < 0.05); saturated mannitol 1.62 (0.09), 4.63 (0.74) (p < 0.05). TLVM and DNA electrophoresis confirmed the occurence of apoptosis after exposure to RCM. In conclusion, saturated mannitol and all tested RCM, with the exception of iotrolan, (diatrizoate > ioxaglate > iopromide) reduced proliferation and increased apoptosis of HUVECs. The effects were more pronounced with ionic RCM and seem to depend on osmolality as well as the chemical structure of these agents. Endothelial injury and apoptosis may be responsible for some of the side effects associated with intravascular use of RCM.

Asunto(s)

Medios de Contraste/farmacología , Endotelio Vascular/efectos de los fármacos , Yohexol/análogos & derivados , Venas Umbilicales/efectos de los fármacos , Apoptosis/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Diatrizoato/farmacología , Diuréticos Osmóticos/farmacología , Endotelio Vascular/citología , Humanos , Técnicas In Vitro , Yohexol/farmacología , Ácido Yoxáglico/farmacología , Manitol/farmacología , Ácidos Triyodobenzoicos/farmacología , Venas Umbilicales/citología

RESUMEN

The insertion of vein grafts into the arterial circulation may contribute to vessel wall thickening and accelerated atherosclerosis, a common feature of late vein graft failure. We aimed to develop a model suitable for investigation of the effects of altered haemodynamics on human saphenous vein following its implantation into the arterial circulation. Segments of human saphenous vein obtained from patients undergoing coronary artery bypass surgery were sutured at each end to PTFE and placed into a flow system. Pressure and flow rates to stimulate the arterial and venous systems were achieved. A theoretical model of the flow chamber was created and computational fluid dynamics software (FLOTRAN, Swanson Analysis Systems) was used to determine the flow profile within the model. In summary, a flow model has been developed to investigate the effect of altered haemodynamics on the molecular and pathological changes that occur in vein grafts incorporated into the arterial circulation.

Asunto(s)

Puente de Arteria Coronaria/instrumentación , Puente de Arteria Coronaria/métodos , Hemodinámica , Modelos Cardiovasculares , Velocidad del Flujo Sanguíneo , Presión Sanguínea , Cateterismo/instrumentación , Cateterismo/métodos , Humanos , Técnicas In Vitro , Perfusión/instrumentación , Perfusión/métodos , Politetrafluoroetileno , Vena Safena/fisiopatología , Estrés Mecánico

RESUMEN

OBJECTIVES: Saphenous vein graft failures, resulting from thrombosis and the abnormal proliferation, migration and apoptosis of vascular smooth muscle cells (VSMC) are major limitations of coronary artery bypass surgery. We investigated whether surgical trauma of human saphenous vein induces the early response gene c-fos and causes alterations in rates of proliferation and apoptosis. METHODS: Surgically prepared human vein consisted of distended (at 350 mmHg for 2 min) or non-distended segments of vein maintained in serum free RPMI at 37 degrees C and 5% CO2 for various time intervals. c-fos expression was detected by Northern analysis. Cell proliferation and apoptosis were determined by [3H]thymidine incorporation combined with proliferation cell nuclear antigen (PCNA) immunostaining and TUNEL, respectively. Labelling indices for proliferation and apoptosis were correlated with vessel was thicknesses. RESULTS: Control, freshly isolated vein expressed no c-fos. Surgically prepared vein synthesized c-fos 1 h following harvesting. There was a significant increase in c-fos in distended compared to non-distended vein. c-Fos protein increased in surgically prepared vein 24 h after harvesting. There was a significant increase in vascular cell proliferation in the non-distended compared to the distended vein: mean (S.E.M.) 1279 (218) vs. 863 (155) dpm/microgram DNA, P < 0.05, n = 6. In addition, the apoptotic index was significantly lower in the media of non-distended vs. distended vein 0.82 (0.2) vs. 5.5 (1.5), P < 0.05, n = 5. CONCLUSIONS: These findings demonstrate that surgical preparation of human saphenous vein increases expression of c-fos mRNA and apoptosis and reduces proliferation when compared with non-distended vein. These changes may influence the failure of saphenous vein grafts.

Asunto(s)

Puente de Arteria Coronaria , Endotelio Vascular/lesiones , Genes fos , Músculo Liso Vascular/lesiones , Vena Safena/lesiones , Manejo de Especímenes/efectos adversos , Adulto , Anciano , Apoptosis , Northern Blotting , División Celular , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Femenino , Expresión Génica , Supervivencia de Injerto , Humanos , Inmunohistoquímica , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Masculino , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/ultraestructura , Presión , ARN Mensajero/análisis , Vena Safena/metabolismo , Vena Safena/ultraestructura

RESUMEN

Antisense oligonucleotides are short segments of synthetic DNA designed to contain sequences of bases complementary to the DNA or RNA of a particular target gene of interest. By binding to the target, the antisense oligonucleotides can prevent translation of the gene into protein via different mechanisms including destruction of the AS-ODN-nucleic acid hybrid by RNAse H, steric hindrance of the ribosome causing interference of protein elongation, or blockage of the initiation of protein translation (1). Figure 1 shows the possible mechanisms of action of AS-ODNs. Fig. 1. Possible mechanisms of action of antisense oligonucleotides. 1, Aptameric or specific binding to transcription factors; 2, triplex formation via binding to doublestranded DNA resulting in steric inhibition of transcription of DNA into RNA; 3, specific binding to splice junctions or poly A signals inhibits mRNA maturation; 4, inhibition of transport from the nucleus; 5, specific binding to mRNA causing inhibition of translation via steric hindrance of ribosomal complexes; 6, duplex formation causing RNAse H mediated cleavage of mRNA; 7, aptameric binding to protein causing inhibition of protein function.

RESUMEN

BACKGROUND: Angioplasty initiates a number of responses in the vessel wall including cellular migration, proliferation, and matrix accumulation, all of which contribute to neointima formation and restenosis. Cellular homeostasis within a tissue depends on the balance between cell proliferation and apoptosis. METHODS AND RESULTS: Profiles of apoptosis and proliferation were therefore examined in a porcine PTCA injury model over a 28-day period. Forty-two arteries from 21 pigs, harvested at the site of maximal injury at 1, 6, and 18 hours, and 3, 7, 14, and 28 days after PTCA, were examined (n=3 animals per time point). Uninjured arteries were used as controls. Apoptosis was demonstrated by the terminal uridine nick-end labeling (TUNEL) method, transmission electron microscopy (TEM), and DNA fragmentation. Cells traversing the cell cycle were identified by immunostaining for proliferating cell nuclear antigen (PCNA). Apoptosis was not detected in control vessels at all time points nor at 28 days after PTCA. Apoptotic cells were identified at all early time points with a peak at 6 hours (5.1+/-0.26%; compared to uninjured artery, P<0.001) and confirmed by characteristic DNA ladders and TEM findings. Regional analysis showed apoptosis within the media, adventitia, and neointima peaked at 18 hours, 6 hours, and 7 days after PTCA, respectively. In comparison, PCNA staining peaked at 3 days after PTCA (7.16+/-0.29%; compared to 1.78+/-0.08% PCNA-positive cells in the uninjured artery, P<0.001). Profiles of apoptosis and cell proliferation after PTCA were discordant in all layers of the artery except the neointima. These profiles also differed between traumatized and nontraumatized regions of the arterial wall. Immunostaining with cell-type specific markers and TEM analysis revealed that apoptotic cells included vascular smooth muscle cells (VSMCs), inflammatory cells, and adventitial fibroblasts. CONCLUSIONS: These results suggest that the profile of apoptosis and proliferation after PTCA is regional and cell specific, and attempts to modulate either of these events for therapeutic benefit requires recognition of these differences.

Asunto(s)

Angioplastia Coronaria con Balón/efectos adversos , Apoptosis/fisiología , Vasos Coronarios/lesiones , Actinas/análisis , Animales , Antígenos/análisis , División Celular/fisiología , Núcleo Celular/ultraestructura , Etiquetado Corte-Fin in Situ , Modelos Logísticos , Microscopía Electrónica , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/ultraestructura , Coloración y Etiquetado , Porcinos

RESUMEN

We recently described the pre-steady state enzymatic binding kinetics of apurinic/apyrimidinic endonuclease (AP endo). In this report we describe the domain structure of the enzyme in solution determined by mild protease digestion in the presence and absence of substrate, product, and an efficient competitive inhibitor (HDP). AP endo is a 35.5-kDa protein with a high degree of homology to its prokaryotic counterpart, exonuclease III (Exo III), except for the amino terminus, which is lacking in the prokaryotic enzyme. The entire conserved region plus an additional 20 residues unique to the eukaryotic enzyme was inaccessible to trypsin and V8 protease, indicating that it forms a tight globular structure. In contrast, the amino-terminal 35 residues were readily accessible to all the proteases investigated, leading us to conclude that they associate poorly with the rest of the structure and constitute a highly fluid region. When AP endo was boiled with SDS and cooled prior to the addition of V8 protease, several acidic residues within the globular domain became protease-accessible, indicating rapid renaturation except along the nuclease fold with restoration of globular conformation for the carboxyl two-thirds of the molecule. Of all the proteases tested, only chymotrypsin was able to cleave internal to the globular portion without prior denaturation. Although AP endo cleaved with chymotrypsin retained full enzymatic activity, the activity was lost when the digested peptides were recovered after denaturation by heat and/or boiling in SDS, precipitation, and renaturation or when fragments were recovered from an SDS gel and renatured. Thus, the protein is probably held together strongly by noncovalent interactions that maintain enzymatic function after protease nicking. The three major chymotrypsin cleavage sites, Tyr-144, Leu-179, and Leu-205, became strikingly less accessible to protease digestion in the presence of abasic site-containing DNA. Since the three residues form a spherical triangle on the surface of the molecule on one side of the nuclease fold, there must be multiple means by which DNA containing an abasic site associates with the enzyme. The most likely explanation is that substrate and product, both of which were present during proteolysis, bind differently to the enzyme. Finally, the two cysteine residues thought to be involved in the redox reaction of AP endo with Jun protein were entirely inaccessible to proteolysis even after prolonged exposure of AP endo to reducing agents. Consequently, if AP endo plays a role in the physiological function of Jun, it must undergo major conformational changes in the process. Alternatively, the two cysteines could maintain an appropriate conformation such that other residues participate directly in the redox activity.

Asunto(s)

Liasas de Carbono-Oxígeno/química , Sitios de Unión/fisiología , Quimotripsina/metabolismo , Cisteína/metabolismo , Reparación del ADN/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Proteínas de Unión al ADN/química , Desoxirribonucleasa IV (Fago T4-Inducido) , Humanos , Modelos Moleculares , Oxidación-Reducción , Fragmentos de Péptidos/química , Conformación Proteica , Desnaturalización Proteica , Serina Endopeptidasas/metabolismo

RESUMEN

Idiopathic dilated cardiomyopathy (DCM) is characterised by a severe dysfunction of the heart muscle resulting in terminal heart failure. Its pathogenesis is believed to be multifactorial involving genetic predisposition, viral infection and autoimmunity, but little is known in detail, and there is no curative treatment except transplantation. Interleukin-1 (IL-1) mediates inflammatory responses to infection and injury. It can be produced by several widely-distributed cell types, including macrophages, and is thought to depress myocyte contractility by stimulating nitric oxide synthase. To investigate whether this pro-inflammatory cytokine may be a pathogenic mediator in DCM, IL-1beta mRNA and protein were evaluated in coronary arteries and myocardium from patients undergoing cardiac transplantation for DCM.IL-1beta mRNA was detected by PCR of cDNA and northern blots of mRNA in coronary arteries and myocardium from patients with DCM. By comparison, samples from patients with ischaemic heart disease (IHD) contained much less IL-1beta mRNA. In contrast, mRNA for other cytokines (TNFalpha, IL-6, IL-10, PDGFA) were similar in both pathologies. In DCM, IL-1beta mRNA and protein were localised to infiltrating macrophages in interstitial regions between myocytes, some of the myocytes themselves, and endothelial cells of vessels in the wall of the arteries. These results suggest that local production of the pro-inflammatory cytokine, IL-1beta may play a part in the pathogenesis of DCM.

Asunto(s)

Cardiomiopatía Dilatada/metabolismo , Vasos Coronarios/metabolismo , Interleucina-1/metabolismo , Miocardio/metabolismo , Adolescente , Adulto , Secuencia de Bases , Northern Blotting , Cardiomiopatía Dilatada/etiología , Cardiomiopatía Dilatada/genética , Citocinas/genética , Citocinas/metabolismo , Cartilla de ADN/genética , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Mediadores de Inflamación/metabolismo , Interleucina-1/genética , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

Interleukin-8 (IL-8) is found in patients following cardiopulmonary bypass (CPB). It may contribute to microvascular injury by activating neutrophils. We examined IL-8 mRNA expression in leucocytes in bypass. IL-8 mRNA levels were measured by Northern analysis and densitometry of isolated mononuclear leucocytes and neutrophils from blood samples taken before, during, and 2 and 48 h after CPB. Plasma IL-8 was measured at each time-point by immunoassay. A strong signal for IL-8 mRNA was detected in neutrophils in five of five and, more weakly, in mononuclear leucocytes of three of five patients studied. The signal peaked consistently during, and fell following bypass, usually to undetectable levels by 48 h. There was always a detectable signal in neutrophils preoperatively. Plasma IL-8 increased from undetectable prebypass levels and peaked later (2 h postbypass) in four of five patients. In the other patient, the cytokine remained detectable throughout. These data demonstrate that IL-8 transcription occurs in leucocytes before and during CPB, but suggest that much of the IL-8 detectable in plasma following bypass may derive, not from these leucocytes, but from other cell types. The release of some IL-8 by neutrophils could lead to local positive feedback in neutrophil recruitment and associated endothelial injury.

Asunto(s)

Puente Cardiopulmonar , Interleucina-8/genética , Leucocitos/metabolismo , ARN Mensajero/análisis , Anciano , Humanos , Interleucina-8/sangre , Masculino , Persona de Mediana Edad , Neutrófilos/fisiología

RESUMEN

BACKGROUND: Study of the vascular response to stent implantation has been hampered by difficulties in sectioning metal and tissue without distortion of the tissue stent interface. The metal is often removed before histochemical processing, causing a loss of arterial architecture. Histological and immunohistochemical sections should be 5 microns with an intact tissue stent interface. OBJECTIVES: To identify the most suitable cutting and grinding equipment, embedding resin, and slides for producing thin sections of stented arteries with the stent wires in situ for histological, immunohistochemical, and transmission electron microscopic (TEM) analyses. METHODS: 20 balloon stainless steel stents were implanted in the coronary arteries of 10 pigs. Twenty eight days later the stented arterial segments were excised, formalin fixed, embedded in five different resins (Epon 812, LR white, T9100, T8100, and JB4), and sectioned with two different high speed saws and a grinder for histological, immunohistochemical, and TEM analyses. Five stented human arteries were obtained at necropsy and processed using the best of the reported methods. RESULTS: The Isomet precision saw and grinder/polisher unit reliably produced 5 microns sections with most embedding resins; minimum section thickness with the horizontal saw was 400 microns. Resin T8100, a glycol methacrylate, enabled satisfactory sectioning, grinding, and histological (toluidine blue, haematoxylin and eosin, and trichromatic and polychromatic stains) and immunohistochemical analyses (alpha smooth muscle actin, von Willebrand factor, vimentin, proliferating cell nuclear antigen, and CD68 (mac 387)). T9100 and T8100 embedded stented sections were suitable for ultrastructural examination with TEM. Stented human arterial sections showed preserved arterial architecture with the struts in situ. CONCLUSION: This study identified the optimal methods for embedding, sawing, grinding, and slide mounting of stented arteries to achieve 5 microns sections with an intact tissue metal interface, excellent surface qualities, histological and immunohistochemical staining properties, and suitability for TEM examination. The technique is applicable to experimental and clinical specimens.

Asunto(s)

Enfermedad Coronaria/cirugía , Vasos Coronarios/patología , Stents , Animales , Biopsia , Enfermedad Coronaria/patología , Humanos , Inmunohistoquímica , Microscopía Electrónica , Microtomía/instrumentación , Microtomía/métodos , Manejo de Especímenes , Porcinos