RESUMEN
Sphingolipids are structural components of cell membrane, displaying several functions in cell signalling. Extracellular vesicles (EV) are lipid bilayer membrane nanoparticle and their lipid composition may be different from parental cells, with a significant enrichment in sphingolipid species, especially in pathological conditions. We aimed at optimizing EV isolation from plasma and describing the differential lipid content of EV, as compared to whole plasma. As pilot study, we evaluated the diagnostic potential of lipidomic signature of circulating EV in patients with a diagnosis of ST-segment-elevation myocardial infarction (STEMI). STEMI patients were evaluated before reperfusion and 24-h after primary percutaneous coronary intervention. Twenty sphingolipid species were quantified by liquid-chromatography tandem-mass-spectrometry. EV-ceramides, -dihydroceramides, and -sphingomyelins increased in STEMI vs. matched controls and decreased after reperfusion. Their levels correlated to hs-troponin, leucocyte count, and ejection fraction. Plasma sphingolipids levels were 500-to-700-fold higher as compared to EV content; nevertheless, only sphingomyelins differed in STEMI vs. control patients. Different sphingolipid species were enriched in EV and their linear combination by machine learning algorithms accurately classified STEMI patients at pre-PCI evaluation. In conclusion, EV lipid signature discriminates STEMI patients. These findings may contribute to the identification of novel biomarkers and signaling mechanisms related to cardiac ischemia.
Asunto(s)
Vesículas Extracelulares/metabolismo , Isquemia Miocárdica/diagnóstico , Infarto del Miocardio con Elevación del ST/diagnóstico , Esfingolípidos/metabolismo , Anciano , Biomarcadores/sangre , Cromatografía Liquida , Diagnóstico Diferencial , Femenino , Humanos , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Isquemia Miocárdica/metabolismo , Intervención Coronaria Percutánea , Proyectos Piloto , Infarto del Miocardio con Elevación del ST/metabolismo , Espectrometría de Masas en TándemRESUMEN
Embryonic stem (ES) cell-derived cardiomyocytes recapitulate cardiomyogenesis in vitro and are a potential source of cells for cardiac repair. However, this requires enrichment of mixed populations of differentiating ES cells into cardiomyocytes. Toward this goal, we have generated bicistronic vectors that express both the blasticidin S deaminase (bsd) gene and a fusion protein consisting of either myosin light chain (MLC)-3f or human alpha-actinin 2A and enhanced green fluorescent protein (EGFP) under the transcriptional control of the alpha-cardiac myosin heavy chain (alpha-MHC) promoter. Insertion of the DNase I-hypersensitive site (HS)-2 element from the beta-globin locus control region, which has been shown to reduce transgene silencing in other cell systems, upstream of the transgene promoter enhanced MLC3f-EGFP gene expression levels in mouse ES cell lines. The alpha-MHC-alpha-actinin-EGFP, but not the alpha-MHC-MLC3f-EGFP, construct resulted in the correct incorporation of the newly synthesized fusion protein at the Z-band of the sarcomeres in ES cell-derived cardiomyocytes. Exposure of embryoid bodies to blasticidin S selected for a relatively pure population of cardiomyocytes within 3 days. Myofibrillogenesis could be monitored by fluorescence microscopy in living cells due to sarcomeric epitope tagging. Therefore, this genetic system permits the rapid selection of a relatively pure population of developing cardiomyocytes from a heterogeneous population of differentiating ES cells, simultaneously allowing monitoring of early myofibrillogenesis in the selected myocytes.
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Aminohidrolasas/genética , Células Madre Embrionarias/citología , Desarrollo de Músculos , Miocitos Cardíacos/citología , Cadenas Ligeras de Miosina/genética , Aminohidrolasas/metabolismo , Animales , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/metabolismo , Vectores Genéticos , Proteínas Fluorescentes Verdes , Humanos , Ratones , Miocitos Cardíacos/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
UNLABELLED: Bone marrow (BM) stem cells can differentiate into multiple cell types, including vascular cells and, possibly, cardiac myocytes. Stem and progenitor cells are mobilized into the peripheral circulation early after myocardial infarction. Experimental evidence suggests that BM-derived cells injected into infarcted hearts can improve cardiac function. However, mechanisms underlying functional improvements remain unclear. Initial randomized, placebo-controlled trials in patients with acute myocardial infarction have provided controversial RESULTS: On the one hand, a modest but significant and sustained improvement in left ventricular function was observed in the Reinfusion of Enriched Progenitor Cells and Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI) study contributing to the better clinical course. Results of other studies were neutral. Differences in the study design, cell processing or timing of cell delivery might explain, in part, different outcomes among studies. Furthermore, studies in patients with chronic ischemic heart disease remain observational, and therapeutic effects using surrogate end-points needs to be demonstrated. Thus, there is a need for further coordinated research with well designed, hypothesis-driven clinical trials, in parallel with fundamental research aimed at understanding the mechanisms underlying the biological and functional effects of BM cell therapy for cardiac repair.
Asunto(s)
Trasplante de Médula Ósea , Infarto del Miocardio/cirugía , Trasplante de Médula Ósea/tendencias , Enfermedad Crónica , Medicina Basada en la Evidencia , Humanos , Isquemia Miocárdica/cirugía , Resultado del TratamientoRESUMEN
Contraction forces developed by cardiomyocytes are transmitted across the plasma membrane through end-to-end connections between the myocytes, called intercalated disks, which enable the coordinated contraction of heart muscle. A component of the intercalated disk, the adherens junction, consists of the cell adhesion molecule, N-cadherin. Embryos lacking N-cadherin die at mid-gestation from cardiovascular abnormalities. We have evaluated the role of N-cadherin in cardiomyogenesis using N-cadherin-null mouse embryonic stem (ES) cells grown as embryoid bodies (EBs) in vitro. Myofibrillogenesis, the spatial orientation of myofibers, and intercellular contacts including desmosomes were normal in N-cadherin-null ES cell-derived cardiomyocytes. The effect of retinoic acid (RA), a stage and dose-dependent cardiogenic factor, was assessed in differentiating ES cells. all-trans (at) RA increased the number of ES cell-derived cardiomyocytes by approximately 3-fold (at 3 x 10(-9) M) in wt EBs. However, this effect was lost in N-cadherin-null EBs. In the presence of supplemented at-RA, the emergence of spontaneously beating cardiomyocytes appeared to be delayed and slightly less efficient in N-cadherin-null compared with wt and heterozygous EBs (frequencies of EBs with beating activity at 5 days: 54+/-18% vs. 96+/-0.5%, and 93+/-7%, respectively; peak frequencies of EBs with beating activity: 83+/-8% vs. 96+/-0.5% and 100%, respectively). In conclusion, cardiomyoyctes differentiating from N-cadherin-null ES cells in vitro show normal myofibrillogenesis and intercellular contacts, but impaired responses to early cardiogenic effects mediated by at-RA. These results suggest that N-cadherin may be essential for RA-induced cardiomyogenesis in mouse ES cells in vitro.
Asunto(s)
Cadherinas/fisiología , Células Madre Embrionarias/fisiología , Miocitos Cardíacos/fisiología , Tretinoina/fisiología , Animales , Cadherinas/genética , Diferenciación Celular , Células Cultivadas , Células Madre Embrionarias/metabolismo , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Confocal , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiología , Miocitos Cardíacos/metabolismo , Miofibrillas/fisiología , Tretinoina/farmacologíaRESUMEN
Following acute myocardial infarction, necrotic cardiac tissue is replaced by scar leading to ventricular remodeling and pump failure. Transplantation of autologous bone marrow-derived cells into the heart, early post-infarct, aims to prevent ventricular remodeling. This strategy has been evaluated in four controlled, randomized clinical trials, which provided mixed results. A transient improvement in ventricular function was observed in one trial, and a modest improvement (the duration of which remains to be determined) in an additional trial, whereas two trials showed negative results. A modest benefit of bone marrow cell transplantation was also observed in patients with chronic ischemic heart disease. Despite mixed results reported so far, cell therapy of heart disease still is in its infancy and has considerable room for improvement.
Asunto(s)
Trasplante de Médula Ósea , Infarto del Miocardio/cirugía , Ensayos Clínicos Controlados Aleatorios como Asunto , HumanosRESUMEN
Vascularized organ allografts are rapidly destroyed by host immune cells that are recruited along chemokine gradients. Among chemokines, Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) CC chemokine ligand (CCL5) and monocyte chemoattractant protein (MCP)-1 (CCL2) are upregulated in rejecting cardiac allografts. To antagonize these chemokines, we constructed adenoviral vectors expressing NH(2)-terminal deletion (8ND) mutants of the respective genes. Using the F344-to-LEW rat model, intragraft gene transfer of chemokine analogs prolonged cardiac allograft survival from 10.1+/-0.7 and 10.4+/-0.7 days using non-coding adenovirus and vehicle alone, respectively, to 17.0+/-0.7 days for 8ND-RANTES (P<0.001) and 14.2+/-0.8 days for 8ND-MCP-1 (P<0.01). 8ND-RANTES reduced graft infiltration by monocytes/macrophages, cluster of differentiation (CD) 8alpha(+) and T-cell receptor alphabeta(+) cells, while 8ND-MCP-1 reduced monocytes/macrophages. In mixed leukocyte reactions in vitro, proliferation of host lymphocytes from regional lymph nodes in response to donor splenocytes was unaffected by 8ND-RANTES gene transfer. Using a two-gene approach, the contribution of 8ND-MCP-1 was negligible, consistent with available evidence that 8ND-RANTES inhibits both RANTES and MCP-1 activities. 8ND-RANTES gene transfer and a short course of low-dose cyclosporine A synergistically prolonged graft survival to 37.8+/-5.5 vs 15.4+/-0.5 days with cyclosporine alone (P<0.001). These results suggest a role for anti-chemokine gene therapy as an adjuvant therapy in heart transplantation.
Asunto(s)
Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL5/antagonistas & inhibidores , Terapia Genética/métodos , Trasplante de Corazón/inmunología , Animales , Quimiocina CCL2/genética , Quimiocina CCL5/genética , Terapia Combinada , Vasos Coronarios , Ciclosporina/uso terapéutico , Citocinas/genética , Citocinas/inmunología , Eliminación de Gen , Supervivencia de Injerto , Inmunosupresores/uso terapéutico , Infusiones Intravenosas , Masculino , Modelos Animales , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas Lew , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transgenes , Trasplante HomólogoRESUMEN
Terminal heart failure can be the cause or the result of major dysfunctions of the organisms. Although, the outcome of the natural history is the same in both situations, it is of prime importance to differentiate the two, as only heart failure as the primary cause allows for successful mechanical circulatory support as bridge to transplantation or towards recovery. Various objective parameters allow for the establishment of the diagnosis of terminal heart failure despite optimal medical treatment. A cardiac index <2.0 l/min, and a mixed venous oxygen saturation <60%, in combination with progressive renal failure, should trigger a diagnostic work-up in order to identify cardiac defects that can be corrected or to list the patient for transplantation with/without mechanical circulatory support.
Asunto(s)
Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/cirugía , Trasplante de Corazón , Corazón Artificial , Circulación Asistida , Humanos , Oxígeno/sangre , Pronóstico , Insuficiencia Renal/etiologíaRESUMEN
AIMS/HYPOTHESIS: The molecular mechanisms of obesity-related insulin resistance are incompletely understood. Macrophages accumulate in adipose tissue of obese individuals. In obesity, monocyte chemoattractant protein-1 (MCP-1), a key chemokine in the process of macrophage accumulation, is overexpressed in adipose tissue. MCP-1 is an insulin-responsive gene that continues to respond to exogenous insulin in insulin-resistant adipocytes and mice. MCP-1 decreases insulin-stimulated glucose uptake into adipocytes. The A-2518G polymorphism in the distal regulatory region of MCP-1 may regulate gene expression. The aim of this study was to investigate the impact of this gene polymorphism on insulin resistance. METHODS: We genotyped the Ludwigshafen Risk and Cardiovascular Health (LURIC) cohort ( n=3307). Insulin resistance, estimated by homeostasis model assessment, and Type 2 diabetes were diagnosed in 803 and 635 patients respectively. RESULTS: Univariate analysis revealed that plasma MCP-1 levels were significantly and positively correlated with WHR ( p=0.011), insulin resistance ( p=0.0097) and diabetes ( p<0.0001). Presence of the MCP-1 G-2518 allele was associated with decreased plasma MCP-1 ( p=0.017), a decreased prevalence of insulin resistance (odds ratio [OR]=0.82, 95% CI: 0.70-0.97, p=0.021) and a decreased prevalence of diabetes (OR=0.80, 95% CI: 0.67-0.96, p=0.014). In multivariate analysis, the G allele retained statistical significance as a negative predictor of insulin resistance (OR=0.78, 95% CI: 0.65-0.93, p=0.0060) and diabetes (OR=0.80, 95% CI: 0.66-0.96, p=0.018). CONCLUSIONS/INTERPRETATION: In a large cohort of Caucasians, the MCP-1 G-2518 gene variant was significantly and negatively correlated with plasma MCP-1 levels and the prevalence of insulin resistance and Type 2 diabetes. These results add to recent evidence supporting a role for MCP-1 in pathologies associated with hyperinsulinaemia.
Asunto(s)
Quimiocina CCL2/genética , Diabetes Mellitus Tipo 2/genética , Resistencia a la Insulina/fisiología , Polimorfismo de Nucleótido Simple/genética , Adenina , Anciano , Estudios de Cohortes , Diabetes Mellitus Tipo 2/sangre , Femenino , Glucosa/metabolismo , Guanina , Homeostasis , Humanos , Hiperinsulinismo/genética , Masculino , Persona de Mediana EdadRESUMEN
Graft vasculopathy is an accelerated form of coronary artery disease that occurs in transplanted hearts. Despite major advances in immunosuppression, the prevalence of the disease has remained substantially unchanged during the last two decades. According to the 'response to injury' paradigm, graft vasculopathy is the result of a continuous inflammatory response to tissue injury initiated by both alloantigen-dependent and independent stress responses. Experimental evidence suggests that these responses may become self-sustaining, as allograft re-transplantation into the donor strain at a later stage fails to prevent disease progression. Histological evidence of endothelitis and arteritis, in association with intima fibrosis and atherosclerosis, reflects the central role of alloimmunity and inflammation in the development of arterial lesions. Experimental results in gene-targeted mouse models indicate that cellular and humoral immune responses are both involved in the pathogenesis of graft vasculopathy. Circulating antibodies against donor endothelium are found in a significant number of patients, but their pathogenic role is still controversial. Alloantigen-independent factors include donor-transmitted coronary artery disease, surgical trauma, ischaemia-reperfusion injury, viral infections, hyperlipidaemia, hypertension, and glucose intolerance. Recent therapeutic advances include the use of novel immunosuppressive agents such as sirolimus (rapamycin), HMG-CoA reductase inhibitors, calcium channel blockers, and angiotensin converting enzyme inhibitors. Optimal treatment of cardiovascular risk factors remains of paramount importance.
Asunto(s)
Enfermedad Coronaria/etiología , Trasplante de Corazón , Complicaciones Posoperatorias/etiología , Formación de Anticuerpos , Angiografía Coronaria , Enfermedad Coronaria/diagnóstico , Enfermedad Coronaria/inmunología , Endotelio Vascular , Humanos , Isoanticuerpos/inmunología , Isoantígenos/inmunología , Complicaciones Posoperatorias/diagnóstico , Complicaciones Posoperatorias/inmunología , Factores de Riesgo , Inmunología del Trasplante , Tolerancia al Trasplante , Trasplante Homólogo/inmunologíaRESUMEN
The author aims to demonstrate, through a textual analysis of Freud's work, how the creation of psychoanalysis as a plausible set of understandings of the human mind has a methodological origin that has sometimes been overlooked: in the Greek concept of techne. Freud, an acknowledged pupil of Brentano, was well versed in Aristotelian rhetoric, and selected this instrument of investigation, dependent on language, from the outset of his efforts to describe, understand and treat the world of the unconscious mind. Working in the tradition of techne Freud actually rehabilitated 'guessing' (zu erraten)--although it became a largely overlooked concept in Freud's work--and so sought to place conjectural reason as the definitive form of knowledge for the investigation and treatment of the mind. This explains why the 1895 'Project' could not succeed and why technique became irreplaceable as the via regia in 'The Interpretation of Dreams'. Its model is founded in Aristotelian rhetoric, whose conception of language was first rediscovered by Nietzsche and was used therapeutically by Freud. Freud's view is apparent in his 1923 definition of psychoanalysis which is compared to the current IPA definition, a definition which, the author suggests, gives a misleading prominence to 'theory' and which shows how far a questionable rationality has removed conjectural reason from the field, to its detriment. From this point of view it is argued that the 'precious conjunction' (Freud) between investigation and treatment has been abandoned, and the concept of historical truth and its significance for psychoanalysis obscured.
Asunto(s)
Trastornos Mentales/historia , Psicoanálisis/historia , Terapia Psicoanalítica/métodos , Austria , Teoría Freudiana/historia , Historia del Siglo XX , Lenguaje , Trastornos Mentales/terapiaRESUMEN
The utility of adenoviral vectors for arterial gene transfer is limited by the brevity of their expression and by inflammatory host responses. As a step toward circumventing these difficulties, we used a rabbit model of in vivo arterial gene transfer to test 3 second-generation vectors: a vector containing a temperature-sensitive mutation in the E2A region, a vector deleted of E2A, and a vector that expresses the immunomodulatory 19-kDa glycoprotein (gp19k) from adenovirus 2. Compared with similar first-generation vectors, the second-generation vectors did not significantly prolong beta-galactosidase transgene expression or decrease inflammation in the artery wall. Although cyclophosphamide ablated the immune and inflammatory responses to adenovirus infusion, it only marginally prolonged transgene expression (94% drop in expression between 3 and 14 days). In experiments performed with "null" adenoviral vectors (no transgene), loss of vector DNA from the arterial wall was also rapid (>99% decrease between 1 hour and 14 days), unrelated to dose, and only marginally blunted by cyclophosphamide. Thus, the early loss of transgene expression after adenoviral arterial gene transfer is due primarily to loss of vector DNA, is not correlated with the presence of local vascular inflammation, and cannot be prevented by use of E2A-defective viruses, expression of gp19k, or cyclophosphamide-mediated immunosuppression. Adenovirus-induced vascular inflammation can be prevented by cyclophosphamide treatment or by lowering the dose of infused virus. However, stabilization of adenovirus-mediated transgene expression in the arterial wall is a more elusive goal and will require novel approaches that prevent the early loss of vector DNA.
Asunto(s)
Adenoviridae/genética , Arterias/metabolismo , Proteínas de Unión al ADN , ADN/metabolismo , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Ciclofosfamida/farmacología , Vectores Genéticos/efectos adversos , Inmunosupresores/farmacología , Masculino , Conejos , Factores de Transcripción/genética , Vasculitis/etiología , beta-Galactosidasa/genéticaRESUMEN
Fas ligand (FasL) is expressed by cells of the arterial wall and is present in human atherosclerotic lesions. However, the role of FasL in modifying the initiation and progression of atherosclerosis is unclear. To investigate the role of arterial FasL expression in the development of atherosclerosis, we first established a model of primary lesion formation in rabbit carotid arteries. In this model, infusion of adenoviral vectors into surgically isolated, nondenuded arteries of hypercholesterolemic rabbits leads to the formation of human-like early atherosclerotic lesions. Expression of FasL in arterial endothelium in this model decreased T-cell infiltration and expression of vascular cell adhesion molecule-1 but did not affect expression of intercellular adhesion molecule-1. Intimal lesions grew more rapidly in FasL-transduced arteries than in arteries transduced with a control adenovirus that did not express a transgene. Total intimal macrophage accumulation was increased in FasL-transduced arteries; however, the proportion of lesion area occupied by macrophages was not elevated. The accelerated lesion growth was primarily due to the accumulation of intimal smooth muscle cells with a synthetic proliferative phenotype. There was no significant apoptosis in FasL-transduced or control arteries and no granulocytic infiltrates. Thus, the net result of elevated FasL expression is to accelerate atherosclerotic lesion growth by increasing lesion cellularity. Vascular expression of FasL may contribute to the progression of atherosclerosis.
Asunto(s)
Arterias/metabolismo , Arteriosclerosis/etiología , Hipercolesterolemia/complicaciones , Hipercolesterolemia/metabolismo , Glicoproteínas de Membrana/metabolismo , Adenoviridae/fisiología , Animales , Apoptosis/fisiología , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , División Celular/efectos de los fármacos , Endotelio Vascular/metabolismo , Proteína Ligando Fas , Expresión Génica , Macrófagos/patología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Conejos , Linfocitos T/fisiología , Transgenes/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
We developed a murine model of arterial gene transfer and used it to test the role of antigen-specific immunity in the loss of adenovirus-mediated transgene expression. Adenoviral vectors encoding either beta-galactosidase (beta-gal) or green fluorescent protein were infused to the lumen of normal common carotids of CD-1 and C57BL/6 mice and atherosclerotic carotids of Apoe(-/-) mice. At 3 days after gene transfer, significant reporter gene expression was detected in all strains. Transgene expression was transient, with expression undetectable at 14 days. Next, a beta-gal-expressing vector was infused into carotids of ROSA26 mice (transgenic for, and therefore tolerant of, beta-gal) and RAG-2(-/-) mice (deficient in recombinase-activating gene [RAG]-2 and therefore lacking in antigen-specific immunity). beta-Gal expression was again high at 3 days but declined substantially (>90%) by 14 days. In vivo labeling with bromodeoxyuridine revealed that carotid endothelial proliferation was increased dramatically by the gene-transfer procedure alone, likely leading to the loss of episomal adenoviral DNA. Gene transfer to normal and atherosclerotic mouse carotids can be accomplished; however, elimination of antigen-specific immune responses does not prevent the early loss of adenovirus-mediated transgene expression. Efforts to prolong adenovirus-mediated transgene expression in the artery wall must be redirected. These efforts will likely include strategies to avoid the consequences of increased cell turnover. Nevertheless, despite the brevity of expression, this mouse model of gene transfer to normal and severely atherosclerotic arteries will likely be useful for investigating the genetic basis of vascular disease and for developing gene therapies. The full text of this article is available at http://www.circresaha. org.
RESUMEN
We developed a murine model of arterial gene transfer and used it to test the role of antigen-specific immunity in the loss of adenovirus-mediated transgene expression. Adenoviral vectors encoding either beta-galactosidase (beta-gal) or green fluorescent protein were infused to the lumen of normal common carotids of CD-1 and C57BL/6 mice and atherosclerotic carotids of Apoe(-/-) mice. At 3 days after gene transfer, significant reporter gene expression was detected in all strains. Transgene expression was transient, with expression undetectable at 14 days. Next, a beta-gal-expressing vector was infused into carotids of ROSA26 mice (transgenic for, and therefore tolerant of, beta-gal) and RAG-2(-/-) mice (deficient in recombinase-activating gene [RAG]-2 and therefore lacking in antigen-specific immunity). beta-Gal expression was again high at 3 days but declined substantially (>90%) by 14 days. In vivo labeling with bromodeoxyuridine revealed that carotid endothelial proliferation was increased dramatically by the gene-transfer procedure alone, likely leading to the loss of episomal adenoviral DNA. Gene transfer to normal and atherosclerotic mouse carotids can be accomplished; however, elimination of antigen-specific immune responses does not prevent the early loss of adenovirus-mediated transgene expression. Efforts to prolong adenovirus-mediated transgene expression in the artery wall must be redirected. These efforts will likely include strategies to avoid the consequences of increased cell turnover. Nevertheless, despite the brevity of expression, this mouse model of gene transfer to normal and severely atherosclerotic arteries will likely be useful for investigating the genetic basis of vascular disease and for developing gene therapies.
Asunto(s)
Adenoviridae/inmunología , Arteriosclerosis/genética , Arteria Carótida Común/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos/inmunología , Animales , Apolipoproteínas E/genética , Arteriosclerosis/patología , Arteriosclerosis/virología , Azacitidina/farmacología , Arteria Carótida Común/patología , Arteria Carótida Común/virología , División Celular , Endotelio Vascular/citología , Epítopos , Expresión Génica , Genes Reporteros , Tolerancia Inmunológica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Tiempo , Transfección , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND: Previous experimental studies have shown that nitric oxide (NO) modulates cardiac function by an abbreviation of systolic contraction and an enhancement of diastolic relaxation. However, the response to NO donors of patients with severe pressure-overload hypertrophy and diastolic dysfunction is unknown. METHODS AND RESULTS: Intracoronary NO donors were given to 17 patients with severe aortic stenosis. A dose-response curve was obtained with nitroglycerin (30, 90, and 150 microg) in 11 patients and sodium nitroprusside (1, 2, and 4 microg/min) in 6. Left ventricular (LV) high-fidelity pressure measurements with simultaneous LV angiograms were performed at baseline and after the maximal dose of NO. The dose-response curve for intracoronary NO donors showed a marked fall in LV end-diastolic pressure, from 23 to 14 mm Hg (-39%; P<0.0001), whereas LV peak systolic pressure fell only slightly, from 206 to 196 mm Hg (-4%; P<0.01). End-diastolic chamber stiffness decreased from 0.12 to 0.07 mm Hg/mL (P<0.0001) and end-systolic stiffness from 1.6 to 1.3 mm Hg/mL (P<0.01). Heart rate, right atrial pressure, LV ejection fraction, the time constant of isovolumic pressure decay (tau), and LV filling rates remained unchanged. CONCLUSIONS: In patients with severe pressure-overload hypertrophy, intracoronary NO donors exert a marked decrease in LV end-diastolic pressure without affecting LV systolic pump function. Thus, the hypertrophied myocardium appears to be particularly susceptible to NO donors, with a marked improvement in diastolic function.
Asunto(s)
Estenosis de la Válvula Aórtica/complicaciones , Diástole/efectos de los fármacos , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Donantes de Óxido Nítrico/uso terapéutico , Óxido Nítrico/fisiología , Nitroglicerina/uso terapéutico , Nitroprusiato/uso terapéutico , Función Ventricular Izquierda/efectos de los fármacos , Adulto , Anciano , Cateterismo Cardíaco , Angiografía Coronaria , Circulación Coronaria/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Hemodinámica , Humanos , Hipertrofia Ventricular Izquierda/fisiopatología , Infusiones Intraarteriales , Masculino , Persona de Mediana Edad , Contracción Miocárdica/efectos de los fármacos , Donantes de Óxido Nítrico/administración & dosificación , Nitroglicerina/administración & dosificación , Nitroprusiato/administración & dosificación , Sístole/efectos de los fármacos , Presión VentricularRESUMEN
OBJECTIVES: The study aimed to evaluate the role of alpha-adrenergic mechanisms during dynamic exercise in both normal and stenotic coronary arteries. BACKGROUND: Paradoxical vasoconstriction of stenotic coronary arteries has been reported during dynamic exercise and may be due to several factors such as alpha-adrenergic drive, a decreased release of nitric oxide, platelet aggregation with release of serotonin, or a passive collapse of the vessel wall. METHODS: Twenty-six patients were studied at rest, during two levels of supine bicycle exercise and after 1.6 mg sublingual nitroglycerin. The alpha-blocker phentolamine was given to 16 patients before exercise, five of whom had also taken a beta-adrenergic-blocker the same morning. Ten patients served as controls. The cross-sectional areas of a normal and a stenotic coronary vessel were determined by biplane quantitative coronary arteriography. RESULTS: In the normal vessel segments, coronary cross-sectional area did not change after phentolamine injection, but increased in all patient groups similarly during exercise. Although coronary vasoconstriction existed in stenotic vessel segments in control patients, phentolamine-treated patients showed exercise-induced vasodilation without difference in patients with and without chronic beta-blockade. CONCLUSIONS: Exercise-induced vasoconstriction of stenotic coronary arteries is prevented by intracoronary administration of phentolamine. There was no difference in coronary vasomotion between patients receiving phentolamine alone and patients receiving phentolamine in addition to a beta-blocker. This finding suggests that exercise-induced vasoconstriction is mediated not only by endothelial dysfunction but also by alpha-adrenergic mechanisms.
Asunto(s)
Antagonistas Adrenérgicos alfa/administración & dosificación , Circulación Coronaria/efectos de los fármacos , Enfermedad Coronaria/tratamiento farmacológico , Prueba de Esfuerzo/efectos de los fármacos , Fentolamina/administración & dosificación , Vasoconstricción/efectos de los fármacos , Antagonistas Adrenérgicos alfa/efectos adversos , Antagonistas Adrenérgicos beta/administración & dosificación , Antagonistas Adrenérgicos beta/efectos adversos , Adulto , Anciano , Atenolol/administración & dosificación , Atenolol/efectos adversos , Cateterismo Cardíaco , Angiografía Coronaria/efectos de los fármacos , Circulación Coronaria/fisiología , Enfermedad Coronaria/fisiopatología , Femenino , Hemodinámica/efectos de los fármacos , Humanos , Masculino , Metoprolol/administración & dosificación , Metoprolol/efectos adversos , Persona de Mediana Edad , Nitroglicerina/administración & dosificación , Nitroglicerina/efectos adversos , Fentolamina/efectos adversos , Premedicación , Vasoconstricción/fisiología , Vasodilatadores/administración & dosificación , Vasodilatadores/efectos adversosRESUMEN
PURPOSE: Adenovirus-mediated arterial gene transfer is a promising tool in the study of vascular biology and the development of vascular gene therapy. However, intraluminal delivery of adenoviral vectors causes vascular inflammation and neointimal formation. Whether these complications could be avoided and gene transfer efficiency maintained by means of delivering adenoviral vectors via the adventitia was studied. METHODS: Replication-defective adenoviral vectors encoding a beta-galactosidase (beta-gal) gene (AdRSVnLacZ) or without a recombinant gene (AdNull) were infused into the lumen or the adventitia of rabbit carotid arteries. Two days after infusion of either AdRSVnLacZ (n = 8 adventitial, n = 8 luminal) or AdNull (n = 4 luminal), recombinant gene expression was quantitated by histochemistry (performed on tissue sections) and with a beta-gal activity assay (performed on vessel extracts). Inflammation caused by adenovirus infusion was assessed 14 days after infusion of either AdNull (n = 6) or vehicle (n = 6) into the carotid adventitia. Inflammation was assessed by means of examination of histologic sections for the presence of neointimal formation and infiltrating T cells and for the expression of markers of vascular cell activation (ICAM-1 and VCAM-1). To measure the systemic immune response to adventitial infusion of adenovirus, plasma samples (n = 3) were drawn 14 days after infusion of AdNull and assayed for neutralizing antibodies. RESULTS: Two days after luminal infusion of AdRSVnLacZ, approximately 30% of luminal endothelial cells expressed beta-gal. Similarly, 2 days after infusion of AdRSVnLacZ to the adventitia, approximately 30% of adventitial cells expressed beta-gal. beta-gal expression was present in the carotid adventitia, the internal jugular vein adventitia, and the vagus nerve perineurium. Elevated beta-gal activity (50- to 80-fold more than background; P <.05) was detected in extracts made from all AdRSVnLacZ-transduced arteries. The amount of recombinant protein expression per vessel did not differ significantly between vessels transduced via the adventitia (17.1 mU/mg total protein [range, 8.1 to 71.5]) and those transduced via a luminal approach (10.0 mU/mg total protein [range, 3.9 to 42.6]). Notably, adventitial delivery of AdNull did not cause neointimal formation. In addition, vascular inflammation in arteries transduced via the adventitia (ie, T-cell infiltrates and ICAM-1 expression) was confined to the adventitia, sparing both the intima and media. Antiadenoviral neutralizing antibodies were present in all rabbits after adventitial delivery of AdNull. CONCLUSION: Infusion of adenoviral vectors into the carotid artery adventitia achieves recombinant gene expression at a level equivalent to that achieved by means of intraluminal vector infusion. Because adventitial gene transfer can be performed by means of direct application during open surgical procedures, this technically simple procedure may be more clinically applicable than intraluminal delivery. Moreover, despite the generation of a systemic immune response, adventitial infusion had no detectable pathologic effects on the vascular intima or media. For these reasons, adventitial gene delivery may be a particularly useful experimental and clinical tool.
Asunto(s)
Adenoviridae/genética , Arteritis/prevención & control , Arterias Carótidas , ADN Viral/genética , Tejido Elástico , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Animales , Anticuerpos Antivirales/sangre , Arteritis/patología , Arterias Carótidas/enzimología , Arterias Carótidas/patología , ADN Recombinante/genética , Tejido Elástico/enzimología , Tejido Elástico/patología , Endotelio Vascular/enzimología , Endotelio Vascular/patología , Regulación Enzimológica de la Expresión Génica , Regulación Viral de la Expresión Génica , Vectores Genéticos/efectos adversos , Histocitoquímica , Infusiones Intraarteriales/efectos adversos , Molécula 1 de Adhesión Intercelular/genética , Venas Yugulares/enzimología , Masculino , Vehículos Farmacéuticos , Conejos , Linfocitos T/patología , Túnica Íntima/patología , Túnica Media/patología , Nervio Vago/enzimología , Molécula 1 de Adhesión Celular Vascular/genética , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
BACKGROUND AND AIM OF THE STUDY: Abnormal passive elastic properties have been reported in patients with severe mitral stenosis and have been attributed to either: (i) chamber atrophy due to unloading; (ii) myocardial fibrosis; (iii) right and left ventricular (LV) interaction; or (iv) internal restrictions due to the rigid mitral valve apparatus. The study aim was to evaluate the effect of percutaneous mitral balloon valvuloplasty (PMV) on passive elastic properties in 19 patients with severe mitral stenosis. Ten patients with normal coronary arteries and LV function served as controls. METHODS: LV high-fidelity pressure measurements and simultaneous biplane LV angiograms were obtained before and after PMV (n = 11). The constant of chamber stiffness (b; ml(-1)) was calculated from the diastolic pressure-volume relationship and the constant of myocardial stiffness (beta) from the diastolic stress-strain relationship. The time constant of relaxation (T; ms) was calculated from the LV pressure decay during isovolumic relaxation. Regional ejection fraction (radial axis system) was determined in six regions of the right anterior oblique (RAO) and left anterior oblique (LAO) angiographic projections. RESULTS: Mitral valve area was increased from 1.0 to 2.2 cm2 after PMV, whereas diastolic pressure gradient was reduced from 14 to 4 mmHg. Global LV ejection fraction (EF) was slightly reduced (57% versus 63%; p<0.05) before valvuloplasty and normalized thereafter. Regional EF increased significantly (p<0.05) in the posterolateral region of the LAO projection after intervention. Myocardial stiffness was increased before, and decreased significantly after balloon valvuloplasty (from 16 to 11; p<0.05). The rate of relaxation and chamber stiffness remained unchanged. CONCLUSIONS: Myocardial stiffness is increased in patients with mitral stenosis, but normalized after successful PMV. The improvement in passive elastic properties after valvuloplasty can be explained by the mobilization of the subvalvular apparatus with an improvement in regional LV function.
Asunto(s)
Cateterismo , Insuficiencia de la Válvula Mitral/fisiopatología , Insuficiencia de la Válvula Mitral/terapia , Función Ventricular Izquierda/fisiología , Adulto , Anciano , Presión Sanguínea/fisiología , Cateterismo Cardíaco , Volumen Cardíaco/fisiología , Diástole/fisiología , Elasticidad , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Coronary anatomy and myocardial blood flow are major determinants of clinical symptomatology and survival in patients with coronary artery disease. While coronary anatomy has been successfully assessed by coronary angiography and intravascular ultrasound imaging, measurements of coronary blood flow are more difficult and their prognostic value has not been definitively evaluated. Measurements of coronary flow reserve (CFR), defined as maximal hyperemic flow divided by resting flow, have been used to assess the functional significance of coronary artery lesions. However, functional assessment of epicardial coronary lesions is limited by several factors, such as diffuse coronary artery disease, small-vessel disease, regional variations in myocardial flow, endothelial dysfunction, and left ventricular hypertrophy. CFR can be measured by several techniques, each one with distinct advantages and limitations, which are discussed in this review. An important distinction is between techniques that measure coronary blood flow (e.g., positron emission tomography) and those that measure blood flow velocity (e.g., Doppler catheters), from which coronary velocity reserve (CVR) is calculated. Although clinical CFR measurements have been possible for over fifteen years, their implementation in patient care has been slow due to several factors including the requirement for a sophisticated technology, the difficult interpretation of CFR results, and the limited knowledge of their prognostic value. While a normal CFR in patients with single vessel coronary disease is associated with a good prognosis, the converse has not been established, i.e., that there is a critical reduction in CFR that requires interventional treatment. A recent study (DEBATE) showed a decrease in the incidence of cardiac events at 6 months after coronary balloon angioplasty in patients with a post-procedural percent diameter stenosis < 35% and a CVR > 2.5. The complex relation between coronary anatomy, myocardial perfusion, and patient outcome have enormous implications for both patient care and health costs, which need to be addressed in future prospective trials.