Morphological Study of Gastric Lesions Developing in the Rat Under Several Damaging Conditions: Modifications Induced by Pretreatment with Zinc Acexamate

Lesions developing in the gastric mucosa of the rat after exposure to different gastric damaging agents (100 mg/kg aspirin, and 70% or 100% ethanol) were assessed by scanning electron microscopy. The severity of the lesions was quantified according to morphological criteria. Modifications in the severity of these lesions induced by pretreatment with zinc acexamate were also analyzed. The scanning electron microscope revealed that with the exception of absolute ethanol, which caused distinctive morphological features, lesions found under the different experimental agents shared a common pattern of progression. Ultrastructural lesions on surface epithelial cells preceded further alterations of parietal cells. After the integrity of the epithelial cells was lost, detachment of the parietal cells occurred, probably, through peptic digestion of the connections between cells and their extracellular matrices. Pretreatment of animals with zinc acexamate increased the presence of mucus on the gastric surface and significantly prevented the progression of lesions towards the severest stages. Ultrastructural damage of surface epithelial cells was not influenced by this treatment, but detachment of damaged cells was clearly diminished. These data confirm the protective effect of zinc acexamate against gastric aggressions. Moreover, our studies confirm the notion that mucus secretion and maintenance of continuity on the gastric lumen by surface epithelial cells is of critical importance in preventing the gastric damage induced in these experimental models

Hemostasis-on-a-chip: Impedance spectroscopy meets microfluidics for hemostasis evaluation

In the case of vascular injury, a complex process (of clotting) starts, involving mainly platelets and coagulation factors. This process in healthy humans is known as hemostasis, but when it is deregulated (thrombosis), it can be the cause of important cardiovascular diseases. Nowadays, the aging of the population and unhealthy lifestyles increase the impact of thrombosis, and therefore there is a need for tools to provide a better understanding of the hemostasis mechanisms, as well as more cost-e ective diagnosis and control devices. This study proposes a novel microflow chamber, with interchangeable biomimetic surfaces to evaluate global hemostasis, using reduced amounts of blood sample and reagents, and also a minimized time required to do the test. To validate the performance of this novel device, a study on the new oral anticoagulant Apixaban (APIX) has been performed and compared to previous conventional techniques. The test shows an excellent agreement, while the amount of the required sample has been reduced (only 100 L is used), and the amount of reagent as well. An imprinted electrode embedded in the chamber in order to measure the impedance during the coagulation process. This approach distinguishes the impedance behavior of plasma poor in platelets (PPP) and plasma rich in platelets (PRP) for the first timePostprint (published version

Cell adhesive peptides functionalized on CoCr alloy stimulate endothelialization and prevent thrombogenesis and restenosis

Immobilization of bioactive peptide sequences on CoCr surfaces is an effective route to improve endothelialization, which is of great interest for cardiovascular stents. In this work, we explored the effect of physical and covalent immoblization of RGDS, YIGSR and their equimolar combination peptides on endothelial cells (EC) and smooth muscle cell (SMC) adhesion and on thrombogenicity. We extensively investigated using RT-qPCR, the expression by ECs cultured on functionalised CoCr surfaces of different genes. Genes relevant for adhesion (ICAM-1 and VCAM-1), vascularization (VEGFA, VEGFR-1 and VEGFR-2) and anti-thrombogenicity (tPA and eNOS) were over-expressed in the ECs grown to covalently functionalized CoCr surfaces compared to physisorbed and control surfaces. Pro-thrombogenic genes expression (PAI-1 and vWF) decreased over time. Cell co-cultures of ECs/SMCs found that functionalization increased the amount of adhered ECs onto modified surfaces compared to plain CoCr, independently of the used peptide and the strategy of immobilization. SMCs adhered less compared to ECs in all surfaces. All studied peptides showed a lower platelet cell adhesion compared to TCPS. Covalent functionalization of CoCr surfaces with an equimolar combination of RGDS and YIGSR represented prevailing strategy to enhance the early stages of ECs adhesion and proliferation, while preventing SMCs and platelet adhesion.Postprint (author's final draft

Phosphotyrosine proteins in platelets from patients with storage pool disease: direct relation between granule defects and defective signal transduction

Background and objectives: storage pool diseases (SPD) are heterogeneous disorders associated with an abnormal presence of intraplatelet granules, which cause mild to moderate bleeding diathesis. We investigated signaling through tyrosine phosphorylation of proteins occurring in platelets with total or partial absence of dense- and alpha-granules in response to activation. Design and methods: we included a patient with severe delta-SPD, a patient with severe alpha-SPD or gray platelet syndrome, and six patients with partial deficiency of dense or a-granules. SPD was confirmed by electron microscopy evaluation of platelet ultrastructure. Platelet function was evaluated by bleeding time determination and conventional aggregometry. Platelet suspensions were activated with collagen and thrombin to analyze changes in tyrosine phosphorylation of proteins by electrophoresis and Western-blotting. Results: bleeding times were prolonged in all the patients included. Aggregation responses were slightly decreased in delta-SPD and normal in the rest of patients. Tyrosine phosphorylation in platelets from patients with partial forms of SPD was equivalent to that observed in control platelets, absent in response to collagen and thrombin activation in delta-SPD, and deficient only to thrombin activation in alpha-SPD. Interpretation and conclusions: tyrosine phosphorylation of proteins in activated platelets is highly dependent on the substances contained in the dense-granules and moderately dependent on those contained in the alpha-granules. A minimum amount of intraplatelet granules ensures signaling through tyrosine phosphorylation of proteins

NFkB in the development of endothelial activation and damage in uremia: an in vitro approach

Impaired hemostasis coexists with accelerated atherosclerosis in patients with chronic kidney disease (CKD). The elevated frequency of atherothrombotic events has been associated with endothelial dysfunction. The relative contribution of the uremic state and the impact of the renal replacement therapies have been often disregarded. Plasma markers of endothelial activation and damage were evaluated in three groups of patients with CKD: under conservative treatment (predialysis), on hemodialysis, and on peritoneal dialysis. Activation of p38 MAPK and the transcription factor NFκB was assessed in endothelial cell (EC) cultures exposed to pooled sera from each group of patients. Most of the markers evaluated (VCAM-1, ICAM-1, VWF, circulating endothelial cells) were significantly higher in CDK patients than in controls, being significantly more increased in the group of peritoneal dialysis patients. These results correlated with the activation of both p38 MAPK and NFκB in EC cells exposed to the same sera samples, and also to the peritoneal dialysis fluids. Hemodialysis did not further contribute to the endothelial damage induced by the uremic state observed in predialysis patients, probably due to the improved biocompatibility of the hemodialysis technique in recent years, resulting in lower cellular activation. However, peritoneal dialysis seemed to exert a significant proinflammatory effect on the endothelium that could be related to the high glucose concentrations and glucose degradation products present in the dialysis fluid. Although peritoneal dialysis has been traditionally considered a more physiological technique, our results raise some doubts with respect to inflammation and EC damage

Antioxidant and Anti-Inflammatory Strategies Based on the Potentiation of Glutathione Peroxidase Activity Prevent Endothelial Dysfunction in Chronic Kidney Disease

BACKGROUND/AIMS: Accelerated atherosclerosis in chronic kidney disease (CKD) is preceded by endothelial dysfunction (ED), which exhibits a proinflammatory and prothrombotic phenotype and enhanced oxidative stress. In this study, the effect of several compounds with anti-inflammatory and/or antioxidant properties on uremia-induced endothelial dysfunction has been evaluated in an in vitro model. METHODS: Endothelial cells (ECs) were exposed to sera from uremic patients in the absence and presence of the flavonoids apigenin, genistein and quercetin, the antioxidant enzyme mimetics (AEM) ebselen (glutathione peroxidase mimetic), EUK-134 and EUK-118 (both superoxide dismutase mimetics), and the pharmacological drug N-acetylcysteine (NAC). We explored changes in the expression of adhesion receptors on the cell surface, by immunofluorescence, the production of radical oxygen species (ROS), by fluorescence detection, and the activation of signaling proteins related to inflammation, by both a phosphospecific antibody cell-based ELISA and immunoblotting techniques. RESULTS: Uremic media induced a significantly increased expression of ICAM-1, overproduction of radical oxygen species (ROS) and activation of p38 mitogen activated protein kinase (p38MAPK) and Nuclear Factor kB (NFkB) in ECs. Quercetin, the AEM and NAC showed a significant inhibitory effect on both ICAM-1 expression and ROS generation (p<0.05). All the compounds reduced p38MAPK activation, but only the AEM, especially ebselen, and NAC, both potentiating the glutathione peroxidase pathway, also inhibited NFkB activation. These two compounds were capable of increasing endothelial glutathione levels, especially in response to uremia. CONCLUSION: Our results indicate that the potentiation of the antioxidant pathways can be an effective strategy to improve endothelial dysfunction in uremia and a potential target to reduce the cardiovascular risk in this population

Endothelial damage, inflammation and immunity in chronic kidney disease

Chronic kidney disease (CKD) patients have an accelerated atherosclerosis, increased risk of thrombotic-ischemic complications, and excessive mortality rates when compared with the general population. There is also evidence of an endothelial damage in which the proinflammatory state, the enhanced oxidative stress, or the accumulation of toxins due to their reduced renal clearance in uremia play a role. Further, there is evidence that uremic endothelial cells are both involved in and victims of the activation of the innate immunity. Uremic endothelial cells produce danger associated molecular patterns (DAMPS), which by binding to specific pattern recognition receptors expressed in multiple cells, including endothelial cells, induce the expression of adhesion molecules, the production of proinflammatory cytokines and an enhanced production of reactive oxygen species in endothelial cells, which constitute a link between immunity and inflammation. The connection between endothelial damage, inflammation and defective immunity in uremia will be reviewed here

Reversal of apixaban induced alterations in hemostasis by different coagulation factor concentrates: significance of studies in vitro with circulating human blood

Apixaban is a new oral anticoagulant with a specific inhibitory action on FXa. No information is available on the reversal of the antihemostatic action of apixaban in experimental or clinical settings. We have evaluated the effectiveness of different factor concentrates at reversing modifications of hemostatic mechanisms induced by moderately elevated concentrations of apixaban (200 ng/ml) added in vitro to blood from healthy donors (n = 10). Effects on thrombin generation (TG) and thromboelastometry (TEM) parameters were assessed. Modifications in platelet adhesive, aggregating and procoagulant activities were evaluated in studies with blood circulating through damaged vascular surfaces, at a shear rate of 600 s−1. The potential of prothrombin complex concentrates (PCCs; 50 IU/kg), activated prothrombin complex concentrates (aPCCs; 75 IU/kg), or activated recombinant factor VII (rFVIIa; 270 μg/kg), at reversing the antihemostatic actions of apixaban, were investigated. Apixaban interfered with TG kinetics. Delayed lag phase, prolonged time to peak and reduced peak values, were improved by the different concentrates, though modifications in TG patterns were diversely affected depending on the activating reagents. Apixaban significantly prolonged clotting times (CTs) in TEM studies. Prolongations in CTs were corrected by the different concentrates with variable efficacies (rFVIIa≥aPCC>PCC). Apixaban significantly reduced fibrin and platelet interactions with damaged vascular surfaces in perfusion studies (p<0.05 and p<0.01, respectively). Impairments in fibrin formation were normalized by the different concentrates. Only rFVIIa significantly restored levels of platelet deposition. Alterations in hemostasis induced by apixaban were variably compensated by the different factor concentrates investigated. However, effects of these concentrates were not homogeneous in all the tests, with PCCs showing more efficacy in TG, and rFVIIa being more effective on TEM and perfusion studies. Our results indicate that rFVIIa, PCCs and aPCCs have the potential to restore platelet and fibrin components of the hemostasis previously altered by apixaban

Papel de las plaquetas en la arteriosclerosis

[spa] DE LA TESIS: El término "arteriosclerosis" debe atribuirse a Lobstein, que lo creó en 1833 para referirse a lo que él denominaba: "un aumento del espesor y la dureza de las paredes arteriales". Etimológicamente, la palabra arteriosclerosis se trata de un nombre compuesto por arteria y "sklerosis" que significa espesamiento con induración. Aunque en el lenguaje clínico corriente, se utilizan muy a menudo los conceptos "arterioesclerosis" o "aterosclerosis" de forma indistinta, no es menos cierto que existe una tendencia al empleo del término "aterosclerosis", debido posiblemente a la profusión de la literatura inglesa que prefiere este término. Existe una tendencia a considerar a la arteriosclerosis como una enfermedad íntimamente ligada a la vida moderna y nada más lejos de la realidad, pues se puede demostrar que la arteriosclerosis ha estado presente en todo el curso de la historia del hombre y así ha quedado reflejado en tratados médicos antiguos. Ya desde un principio se sospechó que la arteriosclerosis tenía relación con hábitos corporales o alimenticios, relacionados con el grado de civilización. En la época en que vivimos, la arteriosclerosis es el trastorno arterial considerado como causa principal de muerte en los Estados Unidos y Europa Occidental, propagándose en los demás continentes en relación con su desarrollo (Nat. Heart and Lung Institute, 1971). Las cotas de mortalidad alcanzadas en los últimos años por las enfermedades cardiovasculares se encuentran entre el 30 y 50% del total de defunciones, variando según los países o razas. De todas maneras, es justo señalar que en diversos países (como los EE.UU.), las campañas de educación sanitaria y prevención de los factores de riesgo, han supuesto una discreta disminución en la incidencia de enfermedades cardiovasculares con desenlace fatal. También hay que indicar que si bien los datos de mortalidad son ciertamente alarmantes, deben entenderse como un reflejo indirecto de los avances tecnológicos experimentados en Medicina, que han logrado disminuir la mortalidad por otras causas, lo que ha permitido prolongar la esperanza de vida media del hombre, y en consecuencia, un incremento aparente de enfermedades cardiovasculares, que en buena parte son consecuencia de la que podíamos llamar "arteriosclerosis fisiológica", es decir, el envejecimiento del sistema vascular con el paso de los años.El creciente aumento de la población mayor de 65 años llevará aparejado, entre otras cosas, que la arteriosclerosis supondrá un problema socio-económico de primera magnitud, no tan sólo por la tasa de defunciones que lleva aparejada, sino también por la carga económica que supondrá para el enfermo y para las arcas del Estado. Estas circunstancias hacen que cualquier investigación, tanto sobre el mecanismo etiopatogénico como en lo tocante al tratamiento o la profilaxis de la enfermedad, esté plenamente justificada. La presente tesis realiza un estudio de distintos aspectos en diferentes fases de la producción de la lesión arteriosclerótica: funcionalismo plaquetario, producción de prostaciclina (PGI2) por la pared arterial y por último la morfología del endotelio aórtico y participación de los elementos formes. Para ello, en la primera parte del trabajo se resumen los resultados obtenidos hasta alcanzar un modelo experimental válido (expuesto en la pág. 133), mientras que en la segunda -y que vendría a ser el núcleo central de la tesis -, se ha trabajado sobre dicho modelo. Sin lugar a dudas, el punto de arranque de esta tesis es el desarrollo del modelo experimental. La consecución de un modelo experimental útil precisa de innumerables horas de dedicación de un equipo; en nuestro caso, el modelo de arteriosclerosis en rata utilizado en esta tesis ha sido desarrollado por el equipo del Dr. C. A. Villaverde, entre cuyos colaboradores me encuentro. Los trabajos iniciales, que proceden del año 1975, han dado lugar a varias publicaciones y presentaciones á Congresos.Continuamos la búsqueda utilizado otros agentes lesionantes. La vitamina D2 había sido utilizada por otros autores Nosotros utilizamos también este método en nuestras experiencias, encontrando diferencias con respecto a los resultados obtenidos con la dieta hiperlipídica. Cuando producíamos sobrecarga de vitamina D2 sola, encontrábamos lesiones localizadas en la aorta, de tipo fibroso con calcificación, pero que no podían ser coloreadas con el Sudan por lo que no pueden ser consideradas como ateromatosas. Estas lesiones se producían en un corto espacio de tiempo (5 días) y la mortalidad era muy elevada. Se completa el estudio de un modelo de arteriosclerosis experimental en la rata con lesiones cuya distribución, aspecto y complicaciones guardan una gran similitud con las que se observan en la enfermedad humana. De los resultados aportados por esta tesis, se puede concluir que en este modelo: 1) La dieta inicial de Vitamina D2 provoca rápidamente un aumento en la agregabilidad de las plaquetas al ADP, una caída en el número de plaquetas circulantes y una disminución de la capacidad de producir PGI2 por la pared vascular. Estos fenómenos son muy precoces pudiendo detectarse a las cuatro horas de la 1ª dosis indicando una alteración en los mecanismos de regulación de la agregación plaquetaria. 2) Estas alteraciones funcionales coinciden con lesiones morfológicas detectables por microscopía electrónica de "scanning" que pone de manifiesto alteraciones del endotelio evidentes ya desde las cuatro horas y que persisten hasta el desarrollo de la lesión final. 3) La agravación de las alteraciones precedentes conduce a un aumento de la permeabilidad del endotelio que se pone de manifiesto por el Azul de Evans en el segundo día de tratamiento. 4) A partir del tercer día la pared vascular recupera su capacidad de producción de PGl2. Se alcanzan valores ligeramente superiores a los normales en el 52 día manteniéndose en los límites de la normalidad en los días sucesivos hasta la configuración de las lesiones finales.5) La pared con lesiones arterioscleróticas mantiene niveles de producción de PGI2 similares a los normales. 6) La agregación plaquetaria al Colágeno está disminuida entre el 39 y 52 día. Existe también una pérdida de sensibilidad de las plaquetas al ADP en el 5º día 7) A lo largo del desarrollo de las lesiones experimentales por microscopía electrónica de scanning no se han podido observar plaquetas acumuladas sobre las zonas lesionadas. Estas solo aparecen en la fase final de complicaciones trombóticas. En consecuencia en este modelo no se puede demostrar la participación de las plaquetas en la génesis de la lesión arteriosclerótica según el concepto clásico de Ross y Barker (1976). 8) El seguimiento de las lesiones con el microscopio electrónico de scanning ha permitido demostrar la presencia precoz y constante de elementos de la serie blanca especialmente monocitos y macrófagos que pueden contribuir a la aparición de lesiones a través de otros mecanismos

Primary arrest of circulating platelets on collagen involves phosphorylation of Syk, cortactin and focal adhesion kinase: studies under flow conditions.

After a vessel wall injury, platelets adhere to the subendothelium following a sequence of events: arrest of single platelets on the surface, progression to platelet spreading and final aggregation. Primary arrest of circulating platelets on subendothelial components occurs through platelet glycoprotein (GP) Ib and collagen receptors; then platelets spread and aggregate through a GPIIb-IIIa-dependent mechanism. A series of strategies were applied to analyse the tyrosine-phosphorylation mechanisms occurring at the different stages of platelet adhesion on subendothelial components under flow conditions, with special attention to primary arrest. To evaluate spread platelets, samples were exposed to acetylsalicylic acid, which blocks aggregate formation. To study single platelets in contact, a monoclonal antibody specific for GPIIb-IIIa was used to prevent platelet spreading and further aggregation. This experimental situation was also investigated using blood from two patients with Glanzmann's thrombasthenia (i.e. lacking GPIIb-IIIa). Results demonstrated that blockade of both spreading and aggregation results in significant changes in the tyrosine-phosphorylation patterns. Arrest of single platelets on collagen-rich surfaces resulted in phosphorylation of p125, identified as focal adhesion kinase (FAK), the 80/85 kDa doublet (cortactin), and p72, identified as Syk. Arrest of single platelets on von Willebrand factor as adhesive substrate showed that interaction through GPIb induces Syk phosphorylation, but not that of cortactin and FAK. Our data indicate that the initial arrest of platelets on subendothelial components involves Syk phosphorylation, which seems to be GPIb-dependent, and this is followed by activation and phosphorylation of cortactin and FAK. These processes seem to occur before GPIIb-IIIa becomes activated