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INTRODUCTION: Imaging fibroid vascularity may predict fibroid growth and aid to determine most appropriate therapy. Microvascular (MV) flow imaging is relatively new and is able to detect slow flow in small vessels. Data on feasibility, reproducibility, and reliability of MV-flow imaging in fibroids is lacking. The purpose of our study was to determine the reproducibility of MV-flow imaging and to explore this technique for clinical practice for assessing blood flow in fibroids. MATERIAL AND METHODS: Thirty patients with one or multiple fibroids (diameter 1.5-12.0 cm) were prospectively included. Transvaginal ultrasound scanning was performed in B-mode, 2D MV-Flow™, 2D and 3D power Doppler mode (HERA W10, Samsung) by two experienced gynecologists at a tertiary care clinic from February to December 2021. The primary outcome was intra- and interobserver agreement of the vascular index (VI) and color score (CS). The following parameters: '2D MV-flow VI', '3DPD VI', '2D MV-flow CS' and '2DPD CS' were measured offline in the center, pseudocapsule, and entire fibroid. Secondary offline outcomes for exploring 2D MV-flow for clinical practice, included (1) ability to discern vascular structures, (2) assessing the degree of vascularity via CS and calculating a VI, and (3) determining penetration depth of the ultrasound signal in both power Doppler and MV-flow imaging. RESULTS: All scans of the 30 included patients were of sufficient quality to analyze. Inter- and intra-observer correlations of all studied parameters were good to excellent, both for 2D MV-flow and 2D power Doppler (intercorrelation coefficient 0.992-0.996). Using 2D MV-flow different vascular structures were visible in detail, in contrary to using 2D and 3D power Doppler. In significantly more fibroids central flow could be visualized using 2D MV-flow (63%) than with 2D power Doppler (13%, p = 0.001). Finally, penetration of the ultrasound signal was deeper using 2D MV-flow (3.92 cm) than with 2D power Doppler (2.95 cm, p = 0.001). CONCLUSIONS: Using 2D MV-flow imaging for determining vascularity is highly reproducible. It has potential added value for clinical practice as it depicts detailed vascular structures and the degree of vascularity, especially in the center of the fibroid.
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There is evidence for increased angiogenesis in the (ectopic) endometrium of adenomyosis patients under the influence of vascular endothelial growth factor (VEGF). VEGF stimulates both angiogenesis and lymph-angiogenesis. However, information on lymph vessels in the (ectopic) endometrium of adenomyosis patients is lacking. In this retrospective matched case-control study, multiplex immunohistochemistry was performed on thirty-eight paraffin embedded specimens from premenopausal women who had undergone a hysterectomy at the Amsterdam UMC between 2001 and 2018 to investigate the evidence for (lymph) angiogenesis in the (ectopic) endometrium or myometrium of patients with adenomyosis versus controls with unrelated pathologies. Baseline characteristics of both groups were comparable. In the proliferative phase, the blood and lymph vessel densities were, respectively, higher in the ectopic and eutopic endometrium of patients with adenomyosis than in the endometrium of controls. The relative number of blood vessels without α-smooth muscle actinin (α SMA) was higher in the eutopic and ectopic endometrium of adenomyosis patients versus controls. The level of VEGF staining intensity was highest in the myometrium but did not differ between patients with adenomyosis or controls. The results indicate increased angiogenesis and lymphangiogenesis in the (ectopic) endometrium affected by adenomyosis. The clinical relevance of our findings should be confirmed in prospective clinical studies.
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Adenomiosis , Endometriosis , Adenomiosis/metabolismo , Adenomiosis/patología , Estudios de Casos y Controles , Endometriosis/patología , Endometrio/metabolismo , Femenino , Humanos , Inmunohistoquímica , Linfangiogénesis , Neovascularización Patológica/metabolismo , Estudios Prospectivos , Estudios Retrospectivos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Uterine disorders are often presented with overlapping symptoms. The microvasculature holds specific information important for diagnosing uterine disorders. Conventional sonography is an established diagnostic technique in gynecology, but is limited by its inability to image the microvasculature. Contrast-enhanced ultrasound (CEUS), is capable of imaging the microvasculature by means of intravascular contrast agents; that is, gas-filled microbubbles. We provide a literature overview on the use of CEUS in diagnosing myometrial and endometrial disorders, that is, fibroids, adenomyosis, leiomyosarcomas and endometrial carcinomas, as well as for monitoring and enhancing the effectiveness of minimally invasive therapies. A systematic literature search with quality assessment was performed until December 2020. In total 34 studies were included, published between 2007 and 2020.The results entail a description of contrast-enhancement patterns obtained from healthy tissue and from malignant and benign tissue; providing a first base for potential diagnostic differentiation in gynecology. In addition it is also possible to determine the degree of myometrial invasion in case of endometrial carcinoma using CEUS. The effectiveness of minimally invasive therapies for uterine disorders can safely and accurately be assessed with CEUS. In conclusion, the abovementioned applications of CEUS are promising and it is worth further exploring its full potential for gynecology by designing innovative and methodologically high-quality clinical studies.
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Medios de Contraste , Leiomioma , Humanos , Leiomioma/diagnóstico por imagen , Microvasos , UltrasonografíaRESUMEN
In the past few decades, cardiac ultrasound has become a widely available, easy-to-use diagnostic tool in many scenarios in acute cardiac care. The introduction of microbubbles extended its diagnostic value. Not long thereafter, several investigators explored the therapeutic potential of contrast ultrasound on thrombus dissolution. Despite large improvements in therapeutic options, acute ST elevation myocardial infarction remains one of the main causes of mortality and morbidity in the western world. The therapeutic effect of contrast ultrasound on thrombus dissolution might prove to be a new, effective treatment strategy in this group of patients. With the recent publication of human studies scrutinising the therapeutic options of ultrasound and microbubbles in ST elevation myocardial infarction, we have entered a new stage in this area of research. This therapeutic effect is based on biochemical effects both at macrovascular and microvascular levels, of which the exact working mechanisms remain to be elucidated in full. This review will give an up-to-date summary of our current knowledge of the therapeutic effects of contrast ultrasound and its potential application in the field of ST elevation myocardial infarction, along with its future developments.
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Medios de Contraste/farmacología , Circulación Coronaria/fisiología , Trombosis Coronaria/terapia , Infarto del Miocardio con Elevación del ST/terapia , Ultrasonografía Intervencional/métodos , Angiografía Coronaria , Trombosis Coronaria/complicaciones , Trombosis Coronaria/diagnóstico , Ecocardiografía , Humanos , Microcirculación , Infarto del Miocardio con Elevación del ST/diagnóstico , Infarto del Miocardio con Elevación del ST/etiologíaRESUMEN
BACKGROUND AIMS: After a myocardial infarction (MI) atherosclerosis is accelerated leading to destabilization of the atherosclerotic plaque. mesenchymal stromal cells are a promising therapeutic option for atherosclerosis. Previously, we demonstrated a novel stem cell delivery technique, with adipose stem cells coupled to microbubbles (i.e., StemBells) as therapy after MI. In this study, we aim to investigate the effect of StemBell therapy on atherosclerotic plaques in an atherosclerotic mouse model after MI. METHODS: MI was induced in atherosclerotic Apolipoprotein E-deficient mice that were fed a high-fat Western diet. Six days post-MI, the mice received either 5â¯×â¯105/100 µL StemBells or vehicle intravenously. The effects of StemBell treatment on the size and stability of aortic root atherosclerotic plaques and the infarcted heart were determined 28 days post-MI via (immuno)histological analyses. Moreover, monocyte subtypes and lipids in the blood were studied. RESULTS: StemBell treatment resulted in significantly increased cap thickness, decreased intra-plaque macrophage density and increased percentage of intra-plaque anti-inflammatory macrophages and chemokines, without affecting plaque size and serum cholesterol/triglycerides. Furthermore, StemBell treatment significantly increased the percentage of anti-inflammatory macrophages within the infarcted myocardium but did not affect cardiac function nor infarct size. Finally, also the average percentage of anti-inflammatory monocytes in the circulation was increased after StemBell therapy. DISCUSSION: StemBell therapy increased cap thickness and decreased intra-plaque inflammation after MI, indicative of stabilized atherosclerotic plaque. It also induced a shift of circulating monocytes and intra-plaque and intra-cardiac macrophages towards anti-inflammatory phenotypes. Hence, StemBell therapy may be a therapeutic option to prevent atherosclerosis acceleration after MI.
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Trasplante de Células Madre Mesenquimatosas/métodos , Infarto del Miocardio/complicaciones , Placa Aterosclerótica/terapia , Animales , Aorta/patología , Apolipoproteínas E/genética , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Femenino , Lípidos/sangre , Macrófagos/patología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Microburbujas , Monocitos/patología , Infarto del Miocardio/patología , Placa Aterosclerótica/etiologíaRESUMEN
Contrast-enhanced ultrasound (CEUS) is an innovative ultrasound technique capable of visualizing both the macro- and microvasculature of tissues. In this prospective pilot study, we evaluated the feasibility of using CEUS to visualize the microvasculature of uterine fibroids and compared CEUS with conventional ultrasound. Four women with fibroids underwent gray-scale ultrasound, sonoelastography and power/color Doppler scans followed by CEUS examination. Analysis of CEUS images revealed initial perfusion of the peripheral rim, that is, a pseudo-capsule, followed by enhancement of the entire lesion through vessels traveling from the exterior to the interior of the fibroid. The pseudo-capsules exhibited slight hyper-enhancement, making a clear delineation of the fibroids possible. The centers of three fibroids exhibited areas lacking vascularization, information not obtainable with the other imaging techniques. CEUS is a feasible technique for imaging and quantifying the microvasculature of fibroids. In comparison with conventional ultrasound imaging modalities, CEUS can provide additional diagnostic information based on the microvasculature.
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Medios de Contraste , Aumento de la Imagen/métodos , Leiomioma/diagnóstico por imagen , Ultrasonografía/métodos , Neoplasias Uterinas/diagnóstico por imagen , Adulto , Estudios de Factibilidad , Femenino , Humanos , Leiomioma/irrigación sanguínea , Microvasos , Persona de Mediana Edad , Proyectos Piloto , Estudios Prospectivos , Neoplasias Uterinas/irrigación sanguínea , Útero/irrigación sanguínea , Útero/diagnóstico por imagenRESUMEN
Infectious myocarditis is the result of an immune response to a microbial infection of the heart. The blood vessels of the heart, both the intramyocardial microvasculature and the large epicardial coronary arteries, play an important role in the pathogenesis of infectious myocarditis. First of all, in addition to cardiomyocytes, endothelial cells of the cardiac (micro)vasculature are direct targets for infection. Moreover, through the expression of adhesion molecules and antigen presenting Major Histocompatibility Complex molecules, the blood vessels assist in shaping the cellular immune response in infectious myocarditis. In addition, damage and dysfunction of the cardiac (micro)vasculature are associated with thrombus formation as well as aberrant regulation of vascular tone including coronary vasospasm. These in turn can cause cardiac perfusion abnormalities and even myocardial infarction. In this review, we will discuss the role of the cardiac (micro)vasculature in the pathogenesis of infectious myocarditis.
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Vasos Coronarios/patología , Infecciones/complicaciones , Miocarditis/patología , Miocitos Cardíacos/patología , Endotelio Vascular/patología , Humanos , Infecciones/patología , Miocarditis/etiologíaRESUMEN
OBJECTIVE: Although lymphocytic myocarditis (LM) clinically can mimic myocardial infarction (MI), they are regarded as distinct clinical entities. However, we observed a high prevalence (32%) of recent MI in patients diagnosed post-mortem with LM. To investigate if LM changes coronary atherosclerotic plaque, we analyzed in autopsied hearts the inflammatory infiltrate and stability in coronary atherosclerotic lesions in patients with LM and/or MI. METHODS: The three main coronary arteries were isolated at autopsy of patients with LM, with MI of 3-6h old, with LM and MI of 3-6h old (LM+MI) and controls. In tissue sections of atherosclerotic plaque-containing coronary segments inflammatory infiltration, plaque stability, intraplaque hemorrhage and thrombi were determined via (immuno)histological criteria. RESULTS: In tissue sections of those coronary segments the inflammatory infiltrate was found to be significantly increased in patients with LM, LM+MI and MI compared with controls. This inflammatory infiltrate consisted predominantly of macrophages and neutrophils in patients with only LM or MI, of lymphocytes in LM+MI and MI patients and of mast cells in LM+MI patients. Moreover, in LM+MI and MI patients this coincided with an increase of unstable plaques and thrombi. Finally, LM and especially MI and LM+MI patients showed significantly increased intraplaque hemorrhage. CONCLUSIONS: This study demonstrates prevalent co-occurrence of LM with a very recent MI at autopsy. Moreover, LM was associated with remodeling and inflammation of atherosclerotic plaques indicative of plaque destabilization pointing to coronary spasm, suggesting that preexistent LM, or its causes, may facilitate the development of MI.
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Enfermedad de la Arteria Coronaria/complicaciones , Hemorragia/etiología , Inflamación/etiología , Linfocitosis/etiología , Infarto del Miocardio/complicaciones , Miocarditis/etiología , Placa Aterosclerótica/patología , Anciano , Autopsia , Enfermedad de la Arteria Coronaria/patología , Vasos Coronarios/patología , Femenino , Hemorragia/patología , Humanos , Inflamación/patología , Linfocitosis/patología , Masculino , Persona de Mediana Edad , Infarto del Miocardio/patología , Miocarditis/patología , Placa Aterosclerótica/complicacionesRESUMEN
BACKGROUND: Adipose-derived stromal cells (ASCs) are a promising new therapeutic option for patients with acute myocardial infarction (AMI). Previously, we found that ASCs coupled to antibody-targeted microbubbles (StemBells [StBs]) improved cardiac function when administered intravenously 7 days post-AMI in rats. In this study, we compared the efficacy of intravenous StB administration at different administration time points following AMI in rats. METHODS: AMI, followed by reperfusion, was induced in four groups of male Wistar rats, which subsequently received an intravenous 1 × 106 StB bolus 1 day post-AMI (StB1; n = 8), 7 days post-AMI (StB7; n = 9), at both time points (StB1+7; n = 7) or neither (Control; n = 7). The effect onrdiac function was determined using echocardiography prior to AMI, 7 days post-AMI and 42 days post-AMI. The effect on infarct size and macrophages in the infarct core were determined (immuno)histochemically 42 days post-AMI. RESULTS: At 42 days post-AMI, all three StB groups had a significantly improved fractional shortening compared with the control group. Between the StB-treated groups, the effects did not differ significantly at 42 days post-AMI. At 7 days post-AMI, the StB1 group had a significantly improved fractional shortening compared with the control and StB7 groups. No significant changes in infarct size or macrophage numbers were found compared with the control group for any StB group. CONCLUSIONS: StB administration resulted in long-term improvement of cardiac function, independent of the time point of administration. When administered at 1 day post-AMI, this improvement was already evident at 7 days post-AMI.
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Tejido Adiposo/citología , Infarto del Miocardio/terapia , Administración Intravenosa , Animales , Células Cultivadas , Ecocardiografía , Masculino , Microburbujas , Infarto del Miocardio/diagnóstico por imagen , Ratas Wistar , Células del Estroma/trasplante , Factores de TiempoRESUMEN
To diagnose lymphocytic myocarditis (LM), immunohistopathological examination of endomyocardial biopsies (EMBs) is used with a cutoff value of at least 14 leukocytes per mm2, composed of CD3- and CD68-positive cells. We hypothesized that a more common leukocyte marker, CD45, instead of CD3 could increase the diagnostic sensitivity. Hearts of mice with acute viral myocarditis (n = 9) and of controls (n = 7) and the EMB sampling area of the left ventricular posterior wall (LVPW) obtained from autopsied hearts of patients diagnosed with LM (n = 18) and controls (n = 6) were stained with anti-CD68, anti-CD3, and anti-CD45. When applying the threshold of at least 14 leukocytes per mm2, 33% of the mice would be diagnosed with LM with the use of CD3+CD68 and 89% with the use of CD45+CD68. In the EMB sampling area of autopsied hearts, using the cutoff value of at least 14 leukocytes per mm2, CD3+CD68 could only confirm 17% of the diagnosis of LM, whereas CD45+CD68 could confirm 50% of the LM cases. Moreover, we compared inflammation in the EMB sampling area of the LVPW to the remaining myocardium of the LVPW and observed a significant increase of CD45+CD68 cells per mm2 in patients with LM. In conclusion, the use of the common leukocyte marker CD45 increases the sensitivity of the diagnosis of LM. Furthermore, the inflammatory infiltrate in the EMB sampling area is significantly increased compared with the remaining LVPW, indicating that the sampling area constitutes the highest chance for histological diagnosis of LM.
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Complejo CD3/análisis , Inmunohistoquímica , Antígenos Comunes de Leucocito/análisis , Linfocitos/inmunología , Miocarditis/inmunología , Miocardio/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Autopsia , Biomarcadores/análisis , Biopsia , Estudios de Casos y Controles , Niño , Modelos Animales de Enfermedad , Femenino , Humanos , Recuento de Leucocitos , Macrófagos/inmunología , Masculino , Ratones Endogámicos C3H , Persona de Mediana Edad , Miocarditis/patología , Miocardio/patología , Valor Predictivo de las Pruebas , Embarazo , Reproducibilidad de los ResultadosAsunto(s)
Colchicina , Miocarditis , Infecciones por Coxsackievirus , Enterovirus Humano B , Humanos , Miocardio , VirosisRESUMEN
High-mechanical-index ultrasound and intravenous microbubbles might prove beneficial in treating microvascular obstruction caused by microthrombi after primary percutaneous coronary intervention for ST-segment elevation myocardial infarction (STEMI). Experiments in animals have revealed that longer-pulse-duration ultrasound is associated with an improvement in microvascular recovery. This trial tested long-pulse-duration, high-mechanical-index ultrasound in STEMI patients. Non-randomly assigned, non-blinded patients were included in this phase 2 trial. The primary endpoint was any side effect possibly related to the ultrasound treatment. The study was aborted after six patients were included; three patients experienced coronary vasoconstriction of the culprit artery, unresponsive to nitroglycerin. Therefore, coronary artery diameter was measured in five pigs. Coronary artery diameters distal to the injury site decreased after application of ultrasound, after balloon injury plus thrombus injection (from 1.89 ± 0.24 mm before to 1.78 ± 0.17 after ultrasound, p = 0.05). Long-pulse-duration ultrasound might cause coronary vasoconstriction distal to the culprit vessel location.
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Vasos Coronarios/fisiopatología , Microvasos/fisiopatología , Infarto del Miocardio/terapia , Terapia por Ultrasonido/efectos adversos , Terapia por Ultrasonido/métodos , Vasoconstricción/fisiología , Adulto , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Porcinos , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: There is a clinical need for immunosuppressive therapy that can treat myocarditis patients in the presence of an active viral infection. In this study we therefore investigated the effects of colchicine, an immunosuppressive drug which has been used successfully as treatment for pericarditis patients, in a mouse model of coxsackievirus B3(CVB3)-induced myocarditis. METHODS: Four groups of C3H mice were included: control mice (n=8), mice infected with CVB3 (1×10(5) PFU, n=10), mice with colchicine administration (2mg/kg i.p, n=5) and mice with combined CVB3 infection and colchicine administration (n=10). After three days, the heart, pancreas and spleen were harvested and evaluated using (immuno)histochemical analysis and CVB3 qPCR. RESULTS: Mice were terminated at day 3 post-virus infection as colchicine treatment rapidly resulted in severe illness and mortality in CVB3-infected mice. Colchicine significantly decreased the number of macrophages in the heart in CVB3-infected mice (p<0.01) but significantly increased the number of neutrophils (p<0.01). In the pancreas, colchicine caused complete destruction of the acini in the CVB3-infected mice and also significantly decreased macrophage (p<0.01) and increased neutrophil numbers (p<0.01). In the spleen, colchicine treatment of CVB3-infected mice induced massive apoptosis in the white pulp and significantly inhibited the virus-induced increase of megakaryocytes in the spleen (p<0.001). Finally, we observed that colchicine significantly increased CVB3 levels in both the pancreas and the heart. CONCLUSIONS: Colchicine treatment in CVB3-induced myocarditis has a detrimental effect as it causes complete destruction of the exocrine pancreas and enhances viral load in both heart and pancreas.
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Colchicina/administración & dosificación , Infecciones por Coxsackievirus/tratamiento farmacológico , Miocarditis/virología , Páncreas/patología , Bazo/patología , Animales , Colchicina/efectos adversos , Colchicina/farmacología , Infecciones por Coxsackievirus/mortalidad , Modelos Animales de Enfermedad , Enterovirus Humano B/fisiología , Corazón/efectos de los fármacos , Corazón/virología , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Miocarditis/tratamiento farmacológico , Miocarditis/mortalidad , Páncreas/efectos de los fármacos , Páncreas/virología , Bazo/efectos de los fármacos , Bazo/virología , Resultado del Tratamiento , Carga Viral/efectos de los fármacosRESUMEN
BACKGROUND: Complement activation contributes significantly to inflammation-related damage in the heart after acute myocardial infarction. Knowledge on factors that regulate postinfraction complement activation is incomplete however. In this study, we investigated whether endogenous C1-inhibitor, a well-known inhibitor of complement activation, is expressed in the heart after acute myocardial infarction. MATERIALS AND METHODS: C1-inhibitor and complement activation products C3d and C4d were analyzed immunohistochemically in the hearts of patients who died at different time intervals after acute myocardial infarction (n=28) and of control patients (n=8). To determine putative local C1-inhibitor production, cardiac transcript levels of the C1-inhibitor-encoding gene serping1 were determined in rats after induction of acute myocardial infarction (microarray). Additionally, C1-inhibitor expression was analyzed (fluorescence microscopy) in human endothelial cells and rat cardiomyoblasts in vitro. RESULTS: C1-inhibitor was found predominantly in and on jeopardized cardiomyocytes in necrotic infarct cores between 12h and 5days old. C1-inhibitor protein expression coincided in time and colocalized with C3d and C4d. In the rat heart, serping1 transcript levels were increased from 2h up until 7days after acute myocardial infarction. Both endothelial cells and cardiomyoblasts showed increased intracellular expression of C1-inhibitor in response to ischemia in vitro (n=4). CONCLUSIONS: These observations suggest that endogenous C1-inhibitor is likely involved in the regulation of complement activity in the myocardium following acute myocardial infarction. Observations in rat and in vitro suggest that C1-inhibitor is produced locally in the heart after acute myocardial infarction.
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Proteínas Inactivadoras del Complemento 1/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , ARN Mensajero/metabolismo , Animales , Línea Celular , Proteína Inhibidora del Complemento C1 , Complemento C3d/metabolismo , Complemento C4b/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Masculino , Mioblastos Cardíacos/metabolismo , Mioblastos Cardíacos/patología , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Miocardio/patología , Necrosis , Fragmentos de Péptidos/metabolismo , ARN Mensajero/genética , Ratas , Ratas Wistar , Estudios Retrospectivos , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Ultrasound and microbubble-targeted delivery (UMTD) is a promising non-viral technique for genetic-based therapy. We found that UMTD of small interfering RNA (siRNA) is more effective than delivery of plasmid DNA (pDNA). UMTD (1 MHz, 0.22 MPa) of fluorescently labeled siRNA resulted in 97.9 ± 1.5% transfected cells, with siRNA localized homogenously in the cytoplasm directly after ultrasound exposure. UMTD of fluorescently labeled pDNA resulted in only 43.0 ± 4.2% transfected cells, with localization mainly in vesicular structures, co-localizing with endocytosis markers clathrin and caveolin. Delivery of siRNA against GAPDH (glyceraldehyde-3-phosphate dehydrogenase) effectively decreased protein levels to 24.3 ± 7.9% of non-treated controls (p < 0.01). In contrast, 24 h after delivery of pDNA encoding GAPDH, no increase in protein levels was detected. Transfection efficiency, verified with red fluorescently labeled pDNA encoding enhanced green fluorescent protein, revealed that of the transfected cells, only 2.0 ± 0.7% expressed the transgene. In conclusion, the difference in localization between siRNA and pDNA after UMTD is an important determinant of the effectiveness of these genetic-based technologies.
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Células Endoteliales/fisiología , Células Endoteliales/efectos de la radiación , Fosfolípidos/efectos de la radiación , Plásmidos/genética , ARN Interferente Pequeño/genética , Sonicación/métodos , Hexafluoruro de Azufre/efectos de la radiación , Transfección/métodos , Animales , Células Cultivadas , Electroporación/métodos , Células Endoteliales/citología , Ondas de Choque de Alta Energía , Microburbujas , Plásmidos/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , PorcinosRESUMEN
In this study, we investigated the effect of secondary Bjerknes forces on targeted microbubbles using high-speed optical imaging. We observed that targeted microbubbles attached to an underlying surface and subject to secondary Bjerknes forces deform in the direction of their neighboring bubble, thereby tending toward a prolate shape. The deformation induces an elastic restoring force, causing the bubbles to recoil back to their equilibrium position; typically within 100 µs after low-intensity ultrasound application. The temporal dynamics of the recoil was modeled as a simple mass-spring system, from which a value for the effective spring constant k of the order 10(-3) Nm(-1) was obtained. Moreover, the translational dynamics of interacting targeted microbubbles was predicted by a hydrodynamic point particle model, including a value of the spring stiffness k of the very same order as derived experimentally from the recoiling curves. For higher acoustic pressures, secondary Bjerknes forces rupture the molecular adhesion of the bubbles to the surface. We used this mutual attraction to quantify the binding force between a single biotinylated microbubble and an avidin-coated surface, which was found to be between 0.9 and 2 nanonewtons (nN). The observation of patches of lipids left at the initial binding site suggests that lipid anchors are pulled out of the microbubble shell, rather than biotin molecules unbinding from avidin. Understanding the effect of ultrasound application on targeted microbubbles is crucial for further advances in the realm of molecular imaging.
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Medios de Contraste , Microburbujas , Ultrasonido/métodos , Avidina/química , Biotina/química , Elasticidad , Procesamiento de Imagen Asistido por Computador/métodos , Lípidos/química , Modelos TeóricosRESUMEN
Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01-2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47(phox) expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47(phox), and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47(phox) in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47(phox) was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Homocisteína/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Relación Dosis-Respuesta a Droga , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , NADPH Oxidasa 2 , NADPH Oxidasa 4 , Óxido Nítrico/metabolismo , Transporte de Proteínas/efectos de los fármacosRESUMEN
BACKGROUND: Clusterin (Apolipoprotein J), a plasma protein with cytoprotective and complement-inhibiting activities, localizes in the infarcted heart during myocardial infarction (MI). Recently, we have shown a protective effect of exogenous clusterin in vitro on ischaemically challenged cardiomyocytes independent of complement. We therefore hypothesized that intravenous clusterin administration would reduce myocardial infarction damage. METHODS: Wistar rats undergoing experimental MI, induced by 40 min ligation of a coronary vessel, were treated with either clusterin (n=15) or vehicle (n=13) intravenously, for 3 days post-MI. After 4 weeks, hearts were analysed. The putative role of megalin, a clusterin receptor, was also studied. RESULTS: Administration of human clusterin significantly reduced both infarct size (with 75 ± 5%) and death of animals (23% vehicle group vs. 0% clusterin group). Importantly, histochemical analysis showed no signs of impaired wound healing in the clusterin group. In addition, significantly increased numbers of macrophages were found in the clusterin group. We also found that the clusterin receptor megalin was present on cardiomyocytes in vitro which, however, was not influenced by ischaemia. Human clusterin co-localized with this receptor in vitro, but not in the human heart. In addition, using a megalin inhibitor, we found that clusterin did not exert its protective effect on cardiomyocytes through megalin. CONCLUSIONS: Our results thus show that clusterin has a protective effect on cardiomyocytes after acute myocardial infarction in vivo, independent of its receptor megalin. This indicates that clusterin, or a clusterin derivate, is a potential therapeutic agent in the treatment of MI.