RESUMEN

BACKGROUND: Chondrocyte-based cell therapy to repair cartilage has been used for >25 years despite current limitations. This work presents a new treatment option for cartilage lesions. HYPOTHESIS: High-quality hyaline cartilage microtissues called Cartibeads are capable of treating focal chondral lesions once implanted in the defect, by complete fusion of Cartibeads among themselves and their integration with the surrounding native cartilage and subchondral bone. STUDY DESIGN: Controlled laboratory study. METHODS: Cartibeads were first produced from human donors and characterized using histology (safranin O staining of glycosaminoglycan [GAG] and immunohistochemistry of collagen I and II) and GAG dosage. Cartibeads from 6 Göttingen minipigs were engineered and implanted in an autologous condition in the knee (4 or 5 lesions per knee). One group was followed up for 3 months and the other for 6 months. Feasibility and efficacy were measured using histological analysis and macroscopic and microscopic scores. RESULTS: Cartibeads revealed hyaline features with strong staining of GAG and collagen II. High GAG content was obtained: 24.6-µg/mg tissue (wet weight), 15.52-µg/mg tissue (dry weight), and 35 ± 3-µg GAG/bead (mean ± SD). Histological analysis of Göttingen minipigs showed good integration of Cartibeads grafts at 3 and 6 months after implantation. The Bern Score of the histological assay comparing grafted versus empty lesions was significant at 3 months (grafted, n = 10; nongrafted, n = 4; score, 3.3 and 5.3, respectively) and 6 months (grafted, n = 11; nongrafted, n = 3; score, 1.6 and 5.1). CONCLUSION: We developed an innovative 3-step method allowing, for the first time, the use of fully dedifferentiated adult chondrocytes with a high number of cell passage (owing to the extensive amplification in culture). Cartibeads engineered from chondrocytes hold potential as an advanced therapy medicinal product for treating cartilage lesions with established efficacy. CLINICAL RELEVANCE: This successful preclinical study, combined with standardized manufacturing of Cartibeads according to good manufacturing practice guidelines, led to the approval of first-in-human clinical trial by the ethics committee and local medical authority. The generated data highlighted a promising therapy to treat cartilage lesions from a small amount of starting biopsy specimen. With our innovative cell amplification technology, very large lesions can be treated, and older active patients can benefit from it.

Asunto(s)

Cartílago Articular , Cartílago Hialino , Humanos , Adulto , Porcinos , Animales , Cartílago Articular/patología , Condrocitos/trasplante , Porcinos Enanos , Ingeniería de Tejidos/métodos , Colágeno , Glicosaminoglicanos , Modelos Animales , Trasplante Autólogo

RESUMEN

The repair of damaged articular cartilage is an unmet medical need. Chondrocyte-based cell therapy has been used to repair cartilage for over 20 years despite current limitations. Chondrocyte dedifferentiation upon expansion in monolayer is well known and is the main obstacle to their use as cell source for cartilage repair. Consequently, current approaches often lead to fibrocartilage, which is biomechanically different from hyaline cartilage and not effective as a long-lasting treatment. Here, we describe an innovative 3-step method to engineer hyaline-like cartilage microtissues, named Cartibeads, from high passage dedifferentiated chondrocytes. We show that WNT5A/5B/7B genes were highly expressed in dedifferentiated chondrocytes and that a decrease of the WNT signaling pathway was instrumental for full re-differentiation of chondrocytes, enabling production of hyaline matrix instead of fibrocartilage matrix. Cartibeads showed hyaline-like characteristics based on GAG quantity and type II collagen expression independently of donor age and cartilage quality. In vivo, Cartibeads were not tumorigenic when transplanted into SCID mice. This simple 3-step method allowed a standardized production of hyaline-like cartilage microtissues from a small cartilage sample, making Cartibeads a promising candidate for the treatment of cartilage lesions.

Asunto(s)

Cartílago Articular , Cartílago Hialino , Animales , Ratones , Cartílago Hialino/metabolismo , Condrocitos/metabolismo , Vía de Señalización Wnt , Células Cultivadas , Ingeniería de Tejidos/métodos , Ratones SCID

RESUMEN

Background: Characterization of the clot occluding the arteries in acute ischemic stroke received ample attention, in terms of elucidating the relationship between the clot composition, its etiology and its amenability for pharmacological treatment and mechanical thrombectomy approaches. Traditional analytical techniques such as conventional 2D histopathology or electron microscopy sample only small parts of the clot. Visualization and analysis in 3D are necessary to depict and comprehend the overall organization of the clot. The aim of this study is to investigate the potential of microCT for characterizing the clot composition, structure, and organization. Methods: In a pilot study, we analyzed with microCT clots retrieved from 14 patients with acute ischemic stroke. The following parameters were analyzed: overall clot density, clot segmentation with various density thresholds, clot volume. Results: Our findings show that human clots are heterogeneous in terms of CT intra-clot density distribution. After fixation in formalin, the clots display a shift toward negative values. On average, we found the mean HU values of red clots retrieved from patients to be -153 HU, with SD = 23.8 HU, for the intermediate clots retrieved from patients -193 HU, SD = 23.7 HU, and for the white clots retrieved from patients -229 HU, SD = 64.8 HU. Conclusion: Our study shows that volumetric and density analysis of the clot opens new perspectives for clot characterization and for a better understanding of thrombus structure and composition.

RESUMEN

OBJECTIVE: Intracranial aneurysms (IAs) are more often diagnosed in women. Hormones and vessel geometry, which influences wall shear stress, may affect pathophysiological processes of the arterial wall. Here, the authors investigated sex-related differences in the remodeling of the aneurysm wall and in intraluminal thrombus resolution. METHODS: A well-characterized surgical side-wall aneurysm model was used in female, male, and ovariectomized rats. Decellularized grafts were used to model highly degenerated and decellularized IA walls and native grafts to model healthy IA walls. Aneurysm growth and thrombus composition were analyzed at 1, 7, 14, and 28 days. Sex-related differences in vessel wall remodeling were compared with human IA dome samples of men and pre- and postmenopausal women. RESULTS: At 28 days, more aneurysm growth was observed in ovariectomized rats than in males or non-ovariectomized female rats. The parent artery size was larger in male rats than in female or ovariectomized rats, as expected. Wall inflammation increased over time in all groups and was most severe in the decellularized female and ovariectomized groups at 28 days compared with the male group. Likewise, in these groups the most elastin fragmentation was seen at 28 days. In female rats, on days 1, 7, and 14, the intraluminal thrombus was mainly composed of red blood cells and fibrin. On days 14 and 28, macrophage and smooth muscle cell invasion inside the thrombus was shown, leading to the removal of red blood cells and deposition of collagen and elastin. On days 14 and 28, similar profiles of thrombus reorganization were observed in male and ovariectomized female rats. However, collagen content in thrombi and vessel wall macrophage content were higher in aneurysms of male rats at 28 days than in those of female rats. On day 28, thrombus coverage by endothelial cells was lower in ovariectomized than in female or male rats. Finally, analysis of human IA domes showed that endothelial cell coverage was lower in men and postmenopausal women than in younger women. CONCLUSIONS: Aneurysm growth and intraluminal thrombus resolution show sex-dependent differences. While certain processes (endothelial cell coverage and collagen deposition) point to a strong hormonal dependence, others (wall inflammation and aneurysm growth) seem to be influenced by both hormones and parent artery size.

RESUMEN

Pathogenesis of intracranial aneurysm is complex and the precise biomechanical processes leading to their rupture are uncertain. The goal of our study was to characterize the aneurysmal wall histologically and to correlate histological characteristics with clinical and radiological factors used to estimate the risk of rupture. A new biobank of aneurysm domes resected at the Geneva University Hospitals (Switzerland) was used. Histological analysis revealed that unruptured aneurysms have a higher smooth muscle cell (SMC) content and a lower macrophage content than ruptured domes. These differences were associated with more collagen in unruptured samples, whereas the elastin content was not affected. Collagen content and type distribution were different between thick and thin walls of unruptured aneurysms. Classification of aneurysm domes based on histological characteristics showed that unruptured samples present organized wall rich in endothelial and SMCs compared with ruptured samples. Finally, aneurysm wall composition was altered in unruptured domes of patients presenting specific clinical factors used to predict rupture such as large dome diameter, dome irregularities, and smoking. Our study shows that the wall of aneurysm suspected to be at risk for rupture undergoes structural alterations relatively well associated with clinical and radiological factors currently used to predict this risk.

Asunto(s)

Aneurisma Roto/patología , Aneurisma Intracraneal/patología , Adulto , Aneurisma Roto/cirugía , Angiografía Cerebral , Colágeno/metabolismo , Elastina/metabolismo , Células Endoteliales/patología , Femenino , Humanos , Inmunohistoquímica , Aneurisma Intracraneal/cirugía , Macrófagos/patología , Angiografía por Resonancia Magnética , Masculino , Persona de Mediana Edad , Músculo Liso Vascular/patología , Factores de Riesgo , Fumar/patología

RESUMEN

Extracellular ATP is a central signaling molecule in inflammatory responses. Pannexin1 (Panx1) channels release ATP in a controlled manner and have been implicated in various inflammatory pathologies, but their role in atherogenesis remains elusive. Using atherosclerosis-susceptible mouse models with ubiquitous deletion of Panx1 (Panx1 -/- Apoe -/- ) or with Cre recombinase-mediated deletion of Panx1 in endothelial cells and monocytes (Tie2-Cre Tg Panx1 fl/fl Apoe -/- ; Panx1 del Apoe -/- ), we identified a novel role for Panx1 in the lymphatic vasculature. Atherosclerotic lesion development in response to high-cholesterol diet was enhanced in Panx1 del Apoe -/- mice, pointing to an atheroprotective role for Panx1 in endothelial and/or monocytic cells. Unexpectedly, atherogenesis was not changed in mice with ubiquitous Panx1 deletion, but Panx1 -/- Apoe -/- mice displayed reduced body weight, serum cholesterol, triglycerides and free fatty acids, suggesting altered lipid metabolism in these Panx1-deficient mice. Mechanistically, Panx1 -/- Apoe -/- mice showed impairment of lymphatic vessel function with decreased drainage of interstitial fluids and reduced dietary fat absorption. Thus, the detrimental effect of Panx1 deletion in endothelial and/or monocytic cells during atherogenesis is counterbalanced by an opposite effect resulting from impaired lymphatic function in ubiquitous Panx1-deficient mice. Collectively, our findings unveil a pivotal role of Panx1 in linking lymphatic function to lipid metabolism and atherosclerotic plaque development.

Asunto(s)

Aterosclerosis/metabolismo , Conexinas/metabolismo , Metabolismo de los Lípidos/fisiología , Vasos Linfáticos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/patología , Peso Corporal/fisiología , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Conexinas/genética , Dieta Alta en Grasa , Grasas de la Dieta/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Líquido Extracelular/metabolismo , Vasos Linfáticos/patología , Masculino , Ratones Noqueados , Monocitos/metabolismo , Monocitos/patología , Proteínas del Tejido Nervioso/genética

RESUMEN

OBJECTIVES: We validate a 4D strategy tailored for 3T clinical systems to simultaneously quantify function and infarct size in wild type mice after ischemia/reperfusion, with improved spatial and temporal resolution by comparison to previous published protocols using clinical field MRI systems. METHODS: C57BL/6J mice underwent 60min ischemia/reperfusion (n=14) or were controls without surgery (n=6). Twenty-four hours after surgery mice were imaged with gadolinium injection and sacrificed for post-mortem MRI and histology with serum also taken for Troponin I levels. The double ECG- and respiratory-triggered 3D FLASH (Fast Low Angle Shot) gradient echo (GRE) cine sequence had an acquired isotropic resolution of 344µm, TR/TE of 7.8/2.9ms and acquisition time 25-35min. The conventional 2D FLASH cine sequence had the same in-plane resolution of 344µm, 1mm slice thickness and TR/TE 11/5.4ms for an acquisition time of 20-25min plus 5min for planning. Left ventricle (LV) and right ventricle (RV) volumes were measured and functional parameters compared 2D to 3D, left to right and for inter and intra observer reproducibility. MRI infarct volume was compared to histology. RESULTS: For the function evaluation, the 3D cine outperformed 2D cine for spatial and temporal resolution. Protocol time for the two methods was equivalent (25-35min). Flow artifacts were reduced (p=0.008) and epi/endo-cardial delineation showed good intra and interobserver reproducibility. Paired t-test comparing ejection volume left to right showed no significant difference for 3D (p=0.37), nor 2D (p=0.30) and correlation slopes of left to right EV were 1.17 (R2=0.75) for 2D and 1.05 (R2=0.50) for 3D. Quantifiable 'late gadolinium enhancement' infarct volume was seen only with the 3D cine and correlated to histology (R2=0.89). Left ejection fraction and MRI-measured infarct volume correlated (R2>0.3). CONCLUSIONS: The 4D strategy, with contrast injection, was validated in mice for function and infarct quantification from a single scan with minimal slice planning.

Asunto(s)

Corazón/diagnóstico por imagen , Imagen por Resonancia Cinemagnética , Miocardio/patología , Animales , Artefactos , Medios de Contraste , Gadolinio/química , Ventrículos Cardíacos/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador , Ratones , Ratones Endogámicos C57BL , Cintigrafía , Daño por Reperfusión , Reproducibilidad de los Resultados

RESUMEN

Selective pharmacological treatments targeting reperfusion injury produced modest protective effects and might be associated with immunosuppression. In order to identify novel and better-tolerated approaches, we focused on the neutralization of receptor activator of nuclear factor kappa-B ligand [RANKL], a cytokine recently shown to activate inflammatory cells (i.e. neutrophils) orchestrating post-infarction injury and repair. Myocardial ischemia (60min) and reperfusion injury was surgically induced in C57Bl/6 mice. In hearts and serum, RANKL was early upregulated during reperfusion. A "one-shot" injection with neutralizing anti-RANKL IgG during ischemia ameliorated myocardial infarct size and function, but not adverse remodeling (determined by Magnetic Resonance Imaging [MRI]) as compared to Vehicle or control IgG. These beneficial effects were accompanied in vivo by reduction in cardiac neutrophil infiltration, reactive oxygen species (ROS) and MMP-9 release. Anti-RANKL IgG treatment suppressed sudden peak of neutrophil granule products in mouse serum early after reperfusion onset. In vitro, RANK mRNA expression was detected in isolated mouse neutrophils. Co-incubation with neutralizing anti-RANKL IgG abrogated RANKL-induced mouse neutrophil degranulation and migration, suggesting a critical role of RANKL in neutrophil-mediated injury. Conversely, anti-RANKL IgG did not affect salvage pathways in cardiac cells (i.e. ERK p42/p44, Akt and STAT-3) or macrophage cardiac infiltration. Finally, treatment with anti-RANKL IgG showed no effect on B and T lymphocyte polarization (in serum, spleen and infarcted myocardium) and circulating chemokines as compared with Vehicle or control IgG. In conclusion, acute treatment with anti-RANKL IgG improved cardiac infarct size and function by potentially impacting on neutrophil-mediated injury and repair.

Asunto(s)

Anticuerpos Monoclonales/farmacología , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Neutrófilos/efectos de los fármacos , Ligando RANK/antagonistas & inhibidores , Disfunción Ventricular/tratamiento farmacológico , Animales , Biomarcadores , Degranulación de la Célula , Citocinas/sangre , Citocinas/metabolismo , Modelos Animales de Enfermedad , Mediadores de Inflamación/sangre , Mediadores de Inflamación/metabolismo , Subgrupos Linfocitarios/patología , Macrófagos/patología , Imagen por Resonancia Magnética , Masculino , Ratones , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/etiología , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Infiltración Neutrófila , Neutrófilos/inmunología , Neutrófilos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Ligando RANK/metabolismo , Troponina I/sangre , Troponina I/metabolismo

RESUMEN

BACKGROUND: New evidence shows that high density lipoproteins (HDL) have protective effects beyond their role in reverse cholesterol transport. Reconstituted HDL (rHDL) offer an attractive means of clinically exploiting these novel effects including cardioprotection against ischemia reperfusion injury (IRI). However, basic rHDL composition is limited to apolipoprotein AI (apoAI) and phospholipids; addition of bioactive compound may enhance its beneficial effects. OBJECTIVE: The aim of this study was to investigate the role of rHDL in post-ischemic model, and to analyze the potential impact of sphingosine-1-phosphate (S1P) in rHDL formulations. METHODS AND RESULTS: The impact of HDL on IRI was investigated using complementary in vivo, ex vivo and in vitro IRI models. Acute post-ischemic treatment with native HDL significantly reduced infarct size and cell death in the ex vivo, isolated heart (Langendorff) model and the in vivo model (-48%, p<0.01). Treatment with rHDL of basic formulation (apoAI + phospholipids) had a non-significant impact on cell death in vitro and on the infarct size ex vivo and in vivo. In contrast, rHDL containing S1P had a highly significant, protective influence ex vivo, and in vivo (-50%, p<0.01). This impact was comparable with the effects observed with native HDL. Pro-survival signaling proteins, Akt, STAT3 and ERK1/2 were similarly activated by HDL and rHDL containing S1P both in vitro (isolated cardiomyocytes) and in vivo. CONCLUSION: HDL afford protection against IRI in a clinically relevant model (post-ischemia). rHDL is significantly protective if supplemented with S1P. The protective impact of HDL appears to target directly the cardiomyocyte.

Asunto(s)

Lipoproteínas HDL/farmacología , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Miocitos Cardíacos/efectos de los fármacos , Animales , Apolipoproteína A-I/farmacología , Apolipoproteína A-I/uso terapéutico , Células Cultivadas , Lipoproteínas HDL/uso terapéutico , Lisofosfolípidos/farmacología , Lisofosfolípidos/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Factor de Transcripción STAT3/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacología , Esfingosina/uso terapéutico

RESUMEN

AIMS: Several intracellular mediators have been implicated as new therapeutic targets against myocardial ischaemia and reperfusion injury. However, clinically effective salvage pathways remain undiscovered. Here, we focused on the potential role of the adaptor protein p66(Shc) as a regulator of myocardial injury in a mouse model of cardiac ischaemia and reperfusion. METHODS AND RESULTS: Adult male p66(Shc) deficient (p66(Shc) (-/-)) and C57Bl/6 wild-type (WT) mice were exposed to 30, 45, or 60 min of ischaemia and reperfusion (5, 15 min, or 24 h). Infarct size, systemic and intracardiac inflammation and oxidants, as well as cytosolic and mitochondrial apoptotic pathways were investigated. Following 30, but not 45 or 60 min of ischaemia, genetic p66(Shc) deficiency was associated with larger infarcts. In WT mice, in vivo p66(Shc) knock down by siRNA with transient protein deficiency confirmed these findings. P66(Shc) inhibition was not associated with any modification in post-infarction inflammation, oxidative burst nor cardiac vessel density or structure. However, in p66(Shc) (-/-) mice activation of the protective and anti-apoptotic Reperfusion Injury Salvage Kinases and Survivor Activating Factor Enhancement pathways were blunted and mitochondrial swelling and cellular apoptosis via the caspase-3 pathway increased compared with WT. CONCLUSIONS: Genetic deletion of p66(Shc) increased susceptibility to myocardial injury in response to short-term ischaemia and reperfusion in mice. Still, additional studies are needed for assessing the role of this pathway in acute coronary syndrome patients.

Asunto(s)

Eliminación de Gen , Daño por Reperfusión Miocárdica/genética , Proteínas Adaptadoras de la Señalización Shc/genética , Animales , Apoptosis/genética , Biomarcadores/metabolismo , Técnicas de Silenciamiento del Gen , Sistema de Señalización de MAP Quinasas/genética , Masculino , Ratones Endogámicos C57BL , Dilatación Mitocondrial/genética , Reperfusión Miocárdica/métodos , Daño por Reperfusión Miocárdica/patología , Miocardio/metabolismo , Factor de Transcripción STAT3/genética , Proteínas Adaptadoras de la Señalización Shc/deficiencia , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Troponina I/metabolismo

RESUMEN

Reactive oxygen species (ROS) participate in the pathogenesis of emphysema. Among ROS-producing enzymes, NOX NADPH oxidases are thought to be responsible for tissue injury associated with several lung pathologies. To determine whether NOX2 and/or NOX1 participate in the development of emphysema, their expression patterns were first studied by immunohistochemistry in the lungs of emphysematous patients. Subsequently, we investigated their contribution to elastase-induced emphysema using NOX2- and NOX1-deficient mice. In human lung, NOX2 was mainly detected in macrophages of control and emphysematous lungs, while NOX1 was expressed in alveolar epithelium and bronchial cells. We observed an elevated number of NOX2-positive cells in human emphysematous lungs, as well as increased NOX2 and NOX1 mRNA expression in mouse lungs following elastase exposure. Elastase-induced alveolar airspace enlargement and elastin degradation were prevented in NOX2-deficient mice, but not in NOX1-deficient mice. This protection was independent of inflammation and correlated with reduced ROS production. Concomitantly, an elevation of sirtuin 1 (SIRT1) level and a decrease of matrix metalloproteinase-9 (MMP-9) expression and activity were observed in alveolar macrophages and neutrophils. We addressed the specific role of macrophage-restricted functional NOX2 in elastase-induced lung emphysema using Ncf1 mutant mice and Ncf1 macrophage rescue mice (Ncf1 mutant mice with transgenic expression of Ncf1 only in CD68-positive mononuclear phagocytes; the MN mouse). Compared to WT mice, the lack of functional NOX2 led to decreased elastase-induced ROS production and protected against emphysema. In contrast, ROS production was restored specifically in macrophages from Ncf1 rescue mice and contributes to emphysema. Taken together, our results demonstrate that NOX2 is involved in the pathogenesis of human emphysema and macrophage-specific NOX2 participates in elastase-induced emphysema through the involvement of SIRT1/MMP-9 pathways in mice.

Asunto(s)

Macrófagos/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Enfisema Pulmonar/metabolismo , Sirtuina 1/metabolismo , Animales , Humanos , Inflamación/patología , Pulmón/patología , Macrófagos/patología , Ratones , NADPH Oxidasa 2 , Neutrófilos/patología , Enfisema Pulmonar/patología , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

SIGNIFICANCE: Phagocytes play a key role in promoting the oxidative stress after ischemic stroke occurrence. The phagocytic NADPH oxidase (NOX) 2 is a membrane-bound enzyme complex involved in the antimicrobial respiratory burst and free radical production in these cells. RECENT ADVANCES: Different oxidants have been shown to induce opposite effects on neuronal homeostasis after a stroke. However, several experimental models support the detrimental effects of NOX activity (especially the phagocytic isoform) on brain recovery after stroke. Therapeutic strategies selectively targeting the neurotoxic ROS and increasing neuroprotective oxidants have recently produced promising results. CRITICAL ISSUES: NOX2 might promote carotid plaque rupture and stroke occurrence. In addition, NOX2-derived reactive oxygen species (ROS) released by resident and recruited phagocytes enhance cerebral ischemic injury, activating the inflammatory apoptotic pathways. The aim of this review is to update evidence on phagocyte-related oxidative stress, focusing on the role of NOX2 as a potential therapeutic target to reduce ROS-related cerebral injury after stroke. FUTURE DIRECTIONS: Radical scavenger compounds (such as Ebselen and Edaravone) are under clinical investigation as a therapeutic approach against stroke. On the other hand, NOX inhibition might represent a promising strategy to prevent the stroke-related injury. Although selective NOX inhibitors are not yet available, nonselective compounds (such as apocynin and fasudil) provided encouraging results in preclinical studies. Whereas additional studies are needed to better evaluate this therapeutic potential in human beings, the development of specific NOX inhibitors (such as monoclonal antibodies, small-molecule inhibitors, or aptamers) might further improve brain recovery after stroke.

Asunto(s)

Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Oxidación-Reducción/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Accidente Cerebrovascular/fisiopatología , Animales , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Antipirina/análogos & derivados , Antipirina/farmacología , Antipirina/uso terapéutico , Azoles/farmacología , Azoles/uso terapéutico , Encéfalo/patología , Edaravona , Humanos , Isoindoles , NADPH Oxidasa 2 , Compuestos de Organoselenio/farmacología , Compuestos de Organoselenio/uso terapéutico , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Accidente Cerebrovascular/terapia

RESUMEN

AIMS: Gap junctions are indispensable for the function of heart and blood vessels by providing electrical coupling and direct cell-to-cell transfer of small signalling molecules. Gap junction channels between neighbouring cells are composed of 12 connexins (Cx). Changes in Cx43 expression, localization, and channel properties in cardiomyocytes contribute to infarction and reperfusion injury of the heart. It is increasingly recognized that deleterious consequences of ischaemia/reperfusion (IR) are modulated by the inflammatory response and endothelial function. The role of the endothelial connexins, i.e. Cx40 and Cx37, in cardiac IR injury is, however, not known. METHODS AND RESULTS: Following 30 min ischaemia and 24 h reperfusion, we found a significant increase in myocardial infarct size in mice with endothelial-specific deletion of Cx40 (Cx40del), but not in Cx37-deficient mice. The cardioprotective effect of endothelial Cx40 was associated with a decrease in neutrophil infiltration. Moreover, beneficial effects of endothelial Cx40 were not observed in isolated Langendorff-perfused hearts, suggesting direct involvement of endothelial-leucocyte interactions in the cardiac injury. Single-dose administration of methotrexate, a CD73 activator, reduced infarct size and neutrophil infiltration into the infarcted myocardium in Cx40del but not in control mice. Similar to Cx40del mice, CD73-deficient mice showed increased sensitivity to cardiac IR injury, which could not be conversed by methotrexate. CONCLUSION: Endothelial Cx40, but not Cx37, is implicated in resistance of the heart to IR injury by activation of the CD73 pathway. Thus, the Cx40-CD73 axis may represent an interesting target for controlling reperfusion damage associated with revascularization in coronary disease.

Asunto(s)

Conexina 43/metabolismo , Conexinas/metabolismo , Endotelio Vascular/metabolismo , Uniones Comunicantes/metabolismo , Isquemia/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , 5'-Nucleotidasa/metabolismo , Animales , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Ratones , Infiltración Neutrófila/fisiología , Proteína alfa-5 de Unión Comunicante

RESUMEN

Myocardial reperfusion injury is mediated by several processes including increase of reactive oxygen species (ROS). The aim of the study is to identify potential sources of ROS contributing to myocardial ischemia-reperfusion injury. For this purpose, we investigated myocardial ischemia/reperfusion pathology in mice deficient in various NADPH oxidase isoforms (Nox1, Nox2, Nox4, as well as Nox1/2 double knockout). Following 30min of ischemia and 24h of reperfusion, a significant decrease in the size of myocardial infarct was observed in Nox1-, Nox2- and Nox1/Nox2-, but not in Nox4-deficient mice. However, no protection was observed in a model of chronic ischemia, suggesting that NOX1 and NOX2-mediated oxidative damage occurs during reperfusion. Cardioprotective effect of Nox1 and Nox2 deficiencies was associated with decrease of neutrophil invasion, but, on the other hand an improved reperfusion injury was also observed in isolated perfused hearts (Langendorff model) suggesting that inflammatory cells were not the major source of oxidative damage. A decrease in global post-reperfusion oxidative stress was clearly detected in Nox2-, but not in Nox1-deficient hearts. Analysis of key signaling pathways during reperfusion suggests distinct cardioprotective patterns: increased phosphorylation was seen for Akt and Erk in Nox1-deficient mice and for Stat3 and Erk in Nox2-deficient mice. Consequently, NOX1 and NOX2 represent interesting drug targets for controlling reperfusion damage associated with revascularization in coronary disease.

Asunto(s)

Glicoproteínas de Membrana/genética , Daño por Reperfusión Miocárdica/genética , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasas/genética , Animales , Citocinas/sangre , Modelos Animales de Enfermedad , Isoenzimas , Macrófagos/patología , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/metabolismo , Miocardio/patología , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasa 1 , NADPH Oxidasa 2 , NADPH Oxidasa 4 , NADPH Oxidasas/metabolismo , Infiltración Neutrófila/genética , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal

RESUMEN

Chemokines trigger leukocyte trafficking and are implicated in cardiovascular disease pathophysiology. Chemokine-binding proteins, called "Evasins" have been shown to inhibit both CC and CXC chemokine-mediated bioactivities. Here, we investigated whether treatment with Evasin-3 (CXC chemokine inhibitor) and Evasin-4 (CC chemokine inhibitor) could influence post-infarction myocardial injury and remodelling. C57Bl/6 mice were submitted in vivo to left coronary artery permanent ligature and followed up for different times (up to 21 days). After coronary occlusion, three intraperitoneal injections of 10 µg Evasin-3, 1 µg Evasin-4 or equal volume of vehicle (PBS) were performed at 5 minutes, 24 hours (h) and 48 h after ischaemia onset. Both anti-chemokine treatments were associated with the beneficial reduction in infarct size as compared to controls. This effect was accompanied by a decrease in post-infarction myocardial leukocyte infiltration, reactive oxygen species release, and circulating levels of CXCL1 and CCL2. Treatment with Evasin-4 induced a more potent effect, abrogating the inflammation already at one day after ischaemia onset. At days 1 and 21 after ischaemia onset, both anti-chemokine treatments failed to significantly improve cardiac function, remodelling and scar formation. At 21-day follow-up, mouse survival was exclusively improved by Evasin-4 treatment when compared to control vehicle. In conclusion, we showed that the selective inhibition of CC chemokines (i.e. CCL5) with Evasin-4 reduced cardiac injury/inflammation and improved survival. Despite the inhibition of CXC chemokine bioactivities, Evasin-3 did not affect mouse survival. Therefore, early inhibition of CC chemokines might represent a promising therapeutic approach to reduce the development of post-infarction heart failure in mice.

Asunto(s)

Antiinflamatorios/administración & dosificación , Infarto del Miocardio/tratamiento farmacológico , Miocardio/metabolismo , Receptores CXCR/administración & dosificación , Receptores de Quimiocina/administración & dosificación , Animales , Proteínas de Artrópodos , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL1/sangre , Vasos Coronarios/cirugía , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/diagnóstico , Miocardio/patología , Estrés Oxidativo/efectos de los fármacos , Proteínas y Péptidos Salivales

RESUMEN

AIMS: Nicotinamide phosphoribosyltransferase (Nampt) is a key enzyme for nicotinamide adenine dinucleotide (NAD(+)) biosynthesis, and recent evidence indicates its role in inflammatory processes. Here, we investigated the potential effects of pharmacological Nampt inhibition with FK866 in a mouse myocardial ischemia/reperfusion model. In vivo and ex vivo mouse myocardial ischemia/reperfusion procedures were performed. RESULTS: Treatment with FK866 reduced myocardial infarct size, neutrophil infiltration, and reactive oxygen species (ROS) generation within infarcted hearts in vivo in a mouse model of ischemia and reperfusion. The benefit of FK866 was not shown in the Langendorff model (ex vivo model of working heart without circulating leukocytes), suggesting a direct involvement of these cells in cardiac injury. Sera from FK866-treated mice showed reduced circulating levels of the neutrophil chemoattractant CXCL2 and impaired capacity to prime migration of these cells in vitro. The release of CXCL8 (human homolog of murine chemokine CXCL2) by human peripheral blood mononuclear cells (PBMCs) and Jurkat cells was also reduced by FK866, as well as by sirtuin (SIRT) inhibitors and SIRT6 silencing, implying a pivotal role for this NAD(+)-dependent deacetylase in the production of this chemokine. INNOVATION: The pharmacological inhibition of Nampt might represent an effective approach to reduce neutrophilic inflammation- and oxidative stress-mediated tissue damage in early phases of reperfusion after a myocardial infarction. CONCLUSIONS: Nampt inhibition appears as a new strategy to dampen CXCL2-induced neutrophil recruitment and thereby reduce neutrophil-mediated tissue injury in mice.

Asunto(s)

Acrilamidas/administración & dosificación , Infarto del Miocardio , Infiltración Neutrófila/efectos de los fármacos , Nicotinamida Fosforribosiltransferasa , Piperidinas/administración & dosificación , Animales , Quimiocina CXCL2/metabolismo , Humanos , Masculino , Ratones , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/enzimología , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/patología , NAD/biosíntesis , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal

RESUMEN

Neutrophilic inflammation might have a pathophysiological role in both carotid plaque rupture and ischemic stroke injury. Here, we investigated the potential benefits of the CXC chemokine-binding protein Evasin-3, which potently inhibits chemokine bioactivity and related neutrophilic inflammation in two mouse models of carotid atherosclerosis and ischemic stroke, respectively. In the first model, the chronic treatment with Evasin-3 as compared with Vehicle (phosphate-buffered saline (PBS)) was investigated in apolipoprotein E-deficient mice implanted of a 'cast' carotid device. In the second model, acute Evasin-3 treatment (5 minutes after cerebral ischemia onset) was assessed in mice subjected to transient left middle cerebral artery occlusion. Although CXCL1 and CXCL2 were upregulated in both atherosclerotic plaques and infarcted brain, only CXCL1 was detectable in serum. In carotid atherosclerosis, treatment with Evasin-3 was associated with reduction in intraplaque neutrophil and matrix metalloproteinase-9 content and weak increase in collagen as compared with Vehicle. In ischemic stroke, treatment with Evasin-3 was associated with reduction in ischemic brain neutrophil infiltration and protective oxidants. No other effects in clinical and histological outcomes were observed. We concluded that Evasin-3 treatment was associated with reduction in neutrophilic inflammation in both mouse models. However, Evasin-3 administration after cerebral ischemia onset failed to improve poststroke outcomes.

Asunto(s)

Aterosclerosis/tratamiento farmacológico , Infarto Encefálico/prevención & control , Lesiones Encefálicas/prevención & control , Enfermedades de las Arterias Carótidas/tratamiento farmacológico , Receptores CXCR , Accidente Cerebrovascular/prevención & control , Animales , Proteínas de Artrópodos , Aterosclerosis/sangre , Aterosclerosis/complicaciones , Infarto Encefálico/sangre , Infarto Encefálico/etiología , Infarto Encefálico/patología , Lesiones Encefálicas/sangre , Lesiones Encefálicas/etiología , Lesiones Encefálicas/patología , Enfermedades de las Arterias Carótidas/sangre , Enfermedades de las Arterias Carótidas/complicaciones , Enfermedades de las Arterias Carótidas/patología , Quimiocina CXCL1/sangre , Ratones , Ratones Noqueados , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/metabolismo , Neutrófilos/patología , Proteínas y Péptidos Salivales , Accidente Cerebrovascular/sangre , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/patología

RESUMEN

Acute atherosclerotic complications, such as myocardial infarction, are often provoked by the rupture of an atherosclerotic plaque and the subsequent thrombotic occlusion of the arterial lumen, which interrupts the blood flow and renders ischemic the downstream peripheral tissue. Several inflammatory mediators (including cytokines, chemokines and matrix metalloproteases) have been shown to orchestrate common pathophysiological mechanisms regulating both plaque vulnerability and myocardial injury. In particular, the selective activation of certain protective intracellular signaling pathways might represent a promising target to reduce the dramatic consequences of an ischemic cardiac event. In the present review we will update evidence on the active role of intracellular kinase cascades (such as mitogen-activated protein kinases [MAPKs], Akt, Janus kinase [JAK]-signal transducer and activator of transcription [STAT]) to reduce the global patient vulnerability for acute myocardial infarction.

RESUMEN

Thrombosis is a rare but serious complication of stent implantation in atherosclerotic arteries, affecting both bare-metal and drug-eluting stents. Diagnostic criteria for stent thrombosis have recently been updated with the time and probability of the event being considered as crucial parameters. To be considered as "definite", the diagnosis of stent thrombosis has to be confirmed by angiography or histology. This statement position has clearly rendered more difficult the clinical assessment of stent thrombosis in randomised clinical trials. Considering these limitations, stent thrombosis represents a dramatic complication for both patients and cardiologists. In coronary plaques, thrombosis is often associated with death, acute coronary syndromes and arrhythmias. For these reasons, the pharmacological improvement of this outcome represents a "hot-topic" field for research. Among several medications, statins have been shown to potentially reduce the incidence of coronary stent thrombosis in humans. However, randomised clinical trials focussing on "definite" diagnosis are still needed to confirm these promising results. In addition, the use of statins in patients implanted with stents in other arteries is largely unexplored. Finally, statin-eluting stents (only tested in pigs) have to be evaluated in other animal models and human beings. Therefore, a clear recommendation on the use of statins to prevent stent thrombosis is not available and caution should be used. The "pleiotropic" anti-atherosclerotic properties of statins might represent a crucial investigation field to pathophysiologically clarify the role of statins in stent complications.

Asunto(s)

Síndrome Coronario Agudo/terapia , Reestenosis Coronaria/prevención & control , Trombosis Coronaria/prevención & control , Stents Liberadores de Fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Stents/efectos adversos , Reestenosis Coronaria/etiología , Reestenosis Coronaria/fisiopatología , Trombosis Coronaria/etiología , Trombosis Coronaria/fisiopatología , Humanos , Paclitaxel/administración & dosificación , Diseño de Prótesis , Sirolimus/administración & dosificación

RESUMEN

Non-alcoholic fatty liver disease (NAFLD) includes a variety of histological conditions (ranging from liver steatosis and steatohepatitis, to fibrosis and hepatocarcinoma) that are characterized by an increased fat content within the liver. The accumulation/deposition of fat within the liver is essential for diagnosis of NAFLD and might be associated with alterations in the hepatic and systemic inflammatory state. Although it is still unclear if each histological entity represents a different disease or rather steps of the same disease, inflammatory processes in NAFLD might influence its pathophysiology and prognosis. In particular, non-alcoholic steatohepatitis (the most inflamed condition in NAFLDs, which more frequently evolves towards chronic and serious liver diseases) is characterized by a marked activation of inflammatory cells and the upregulation of several soluble inflammatory mediators. Among several mediators, cytokines and chemokines might play a pivotal active role in NAFLD and are considered as potential therapeutic targets. In this review, we will update evidence from both basic research and clinical studies on the potential role of cytokines and chemokines in the pathophysiology of NAFLD.

Asunto(s)

Quimiocinas/inmunología , Citocinas/inmunología , Hígado Graso/inmunología , Animales , Hígado Graso/patología , Hígado Graso/fisiopatología , Humanos , Enfermedad del Hígado Graso no Alcohólico