RESUMEN
Throughout storage, red blood cells (RBCs) undergo detrimental changes in viability and their ability to effectively transport oxygen. RBC storage lesions are mediated, in part, by a progressive loss of cell deformability, and associated with the release of extracellular vesicles (EVs). Accumulation of EVs during the storage of RBCs correlates with a decrease in RBC surface area to volume ratio. Similarly, the loss of RBC-deformability is associated with loss of RBC surface area to volume ratio. In this study we thus tested whether loss of RBC-deformability is associated with increased RBC-EV production during blood storage. EVs obtained by differential centrifugation of stored RBCs (non-leukoreduced non-irradiated or leukoreduced γ-irradiated RBCs stored 35 or 28 days respectively) were enumerated by high-sensitivity flow cytometry. RBC deformability was quantified, using a cell-flow-properties-analyzer, by measuring the median cell elongation ratio (MER) and percentage of low and high deformable cells in the population (%, LDFC, and HDFC, respectively). The number of EVs was inversely correlated with the MER and positively correlated with the %LDFC with both measures showing highly significant logarithmic dependence with EV levels in stored RBCs. Considering how highly deformable cells did not correlate with EV formation as compared with low deformable RBCs we propose that the formation of EVs is a key factor leading to increased RBC-rigidity.
Asunto(s)
Conservación de la Sangre/métodos , Eritrocitos/metabolismo , Vesículas Extracelulares/metabolismo , HumanosRESUMEN
Nanoparticles (NP) have drawn increasing interest from many fields in medicine and are a relatively new class of biomedical products. Because there are concerns about the health effects of nanoparticles, it is important to understand how nanoparticles interact specifically with red blood cells (RBC), a central object in the blood circulation. As numerous studies that have examined NP/RBC interaction concentrated on the hemolytic potential of nanoparticles, we describe an investigation of hemolytic activity of polystyrene nanoparticles (PS-NP) in protein free medium and its modulation by albumin. We found that treatment of RBCs with PS-NP induces hemolysis (dose and particle size dependent) in plasma free medium but not in full plasma or in buffer, which contain albumin. Critical albumin concentration is 0.05% wt. According to our results hemolytic effect of nanoparticles is strongly modulated by protein concentration in the medium.
Asunto(s)
Albúminas/metabolismo , Eritrocitos/efectos de los fármacos , Hemólisis/efectos de los fármacos , Nanopartículas , Polímeros/farmacología , Hemólisis/fisiología , Humanos , Tamaño de la PartículaRESUMEN
Red blood cell (RBC) aggregation is enhanced in the presence of ongoing inflammation, because of plasma protein effects, especially fibrinogen. Large RBC aggregates, in addition to being a marker of systemic inflammation, may hinder tissue perfusion and oxygenation. Gaucher disease, the most common lysosomal storage disorder, evinces many of the hallmarks of chronic inflammation. Manifestations of Gaucher disease which may be related to microvascular occlusion include avascular necrosis (AVN), bone crisis, and pulmonary hypertension. This study aims to determine whether increased RBC aggregation in non-splenectomized patients with Gaucher disease is due to Gaucher-related inflammation. The Cell Flow Properties Analyzer (CFA) monitors blood under conditions of different shear stress by creating varying pressure gradients. Blood from non-splenectomized patients with Gaucher disease showed only a slight correlation between aggregation parameters and fibrinogen levels, whereas blood from non-splenectomized patients treated with enzyme replacement therapy (ERT) showed marked correlation between aggregation parameters and fibrinogen, as in the control group. These results underscore the hypothesis that RBC aggregation in Gaucher disease is increased by (at least) two mechanisms: a fibrinogen-mediated inflammatory process and another non-inflammatory process that may be induced by elevated glucocerebroside levels in the RBC and/or inhibited by elevated plasma cerebroside levels.
Asunto(s)
Agregación Eritrocitaria , Fibrinógeno/metabolismo , Enfermedad de Gaucher/metabolismo , Glucosilceramidas/metabolismo , Enfermedades Óseas/metabolismo , Enfermedades Óseas/patología , Enfermedad de Gaucher/patología , Humanos , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Inflamación/metabolismo , NecrosisRESUMEN
To explore the contribution of red blood cell (RBC) deformability and interaction with endothelial cells (ECs) to circulatory disorders, these RBC properties were modified by treatment with hydrogen peroxide (H(2)O(2)), and their effects on vascular resistance were monitored following their infusion into rat mesocecum vasculature. Treatment with 0.5 mM H(2)O(2) increased RBC/EC adherence without significant alteration of RBC deformability. At 5.0 mM H(2)O(2), RBC deformability was considerably reduced, inducing a threefold increase in the number of undeformable cells, whereas RBC/EC adherence was not further affected by the increased H(2)O(2) concentration. This enabled the selective manipulation of RBC adherence and deformability and the testing of their differential effect on vascular resistance. Perfusion of RBCs with enhanced adherence and unchanged deformability (treatment with 0.5 mM H(2)O(2)) increased vascular resistance by about 35% compared with untreated control RBCs. Perfusion of 5.0 mM H(2)O(2)-treated RBCs, with reduced deformability (without additional increase of adherence), further increased vascular resistance by about 60% compared with untreated control RBCs. These results demonstrate the specific effects of elevated adherence and reduced deformability of oxidized RBCs on vascular resistance. These effects can be additive, depending on the oxidation conditions. The oxidation-induced changes applied in this study are moderate compared with those observed in RBCs in pathological states. Yet, they caused a considerable increase in vascular resistance, thus demonstrating the potency of RBC/EC adherence and RBC deformability in determining resistance to blood flow in vivo.
Asunto(s)
Adhesión Celular , Células Endoteliales/metabolismo , Deformación Eritrocítica , Eritrocitos/metabolismo , Resistencia Vascular , Adhesión Celular/efectos de los fármacos , Línea Celular Transformada , Relación Dosis-Respuesta a Droga , Deformación Eritrocítica/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Hemorreología , Humanos , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Resistencia Vascular/efectos de los fármacosRESUMEN
Preterm infants are highly susceptible to ischemic damage. This damage is most obvious in the brain, retina, and gastrointestinal tract. Studies focusing on the rheological properties of premature red blood cells (pRBCs) have consistently shown minimal or no RBC aggregation. Previously, measurements of pRBC aggregation kinetics indicated that specific plasma properties are responsible for the decreased RBC aggregation observed in the neonates, but that their specific RBC properties do not affect it. However, the strength of interaction in the pRBC aggregates as a function of medium composition has not been tested. In our previous research, we described clinically relevant parameters, that is, the aggregate resistance to disaggregation by flow. With the help of a cell flow property analyzer (CFA), we can monitor RBC aggregation by direct visualization of its dynamics during flow. We used the CFA to examine pRBC (from 9 premature babies) in the natural plasma and in PBS buffer supplemented with dextran (500 kDa) to distinguish between RBC intrinsic-cellular and plasma factors. pRBCs suspended in the native plasma showed minimal or no aggregation in comparison to normal adult RBC. When we transferred pRBCs from the same sample to the dextran solution, enhanced resistance to disaggregation by flow was apparent.
Asunto(s)
Agregación Eritrocitaria/fisiología , Eritrocitos/citología , Adulto , Dextranos/farmacología , Eritrocitos/efectos de los fármacos , Humanos , Recién Nacido , Recien Nacido Prematuro , Plasma/citología , Sustitutos del Plasma/farmacologíaRESUMEN
Phospholipase A2 hydrolyzes membrane phospholipids releasing arachidonic acid and lysophospholipids. These are key precursors of inflammatory mediators, such as prostaglandins, leukotrienes, thromboxanes and PAF, in numerous inflammatory/allergic diseases, including skin inflammation. Accordingly, inhibition of PLA2 has long been postulated as a potentially potent anti-inflammatory therapy. In the present study we tested the effect of a novel PLA2 inhibitor on contact dermatitis in human subjects. A double-blind, placebo-controlled pilot study was conducted on contact dermatitis patients (n = 11) treated with the inhibitor-containing topical preparation (1% cream). Disease severity was assessed by physicians assessment before treatment (day 0) as well as after 14-days and 30-days. Patients treated with 1% PLA2 inhibitor-containing cream showed a 69.9% reduction in disease score while placebo-treated patients showed a reduction of 36.5% with p = 0.0024. The clear improvement in the disease score of inhibitor-treated patients supports the involvement of PLA2 activity in skin inflammation and the therapeutic prospective of its inhibition.
Asunto(s)
Dermatitis por Contacto/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Fosfolipasas A/antagonistas & inhibidores , Administración Tópica , Adulto , Química Farmacéutica , Método Doble Ciego , Eccema/tratamiento farmacológico , Inhibidores Enzimáticos/administración & dosificación , Femenino , Humanos , Ácido Hialurónico/uso terapéutico , Persona de Mediana Edad , Fosfatidiletanolaminas/uso terapéutico , Fosfolipasas A2 , Proyectos PilotoRESUMEN
BACKGROUND: The pathophysiology of asthma involves the action of inflammatory/allergic lipid mediators formed following membrane phospholipid hydrolysis by phospholipase A(2) (PLA(2)). Cysteinyl leukotrienes are considered potent inducers of bronchoconstriction and airway remodelling. Ovalbumin (OVA) induced bronchoconstriction in rats is associated with increased secretory PLA(2) (sPLA(2)) activation and cysteinyl leukotriene production, together with suppression of cytosolic PLA(2) and prostaglandin E(2). These processes are reversed when the animals are pretreated systemically with an extracellular cell impermeable sPLA(2) inhibitor which also suppresses the early allergic reaction to OVA challenge. In this study we examine the capacity of the sPLA(2) inhibitor to ameliorate inflammatory and allergic manifestations (early and late bronchoconstriction) of OVA induced allergic bronchitis in rats when the inhibitor was administered by inhalation to confine it to the airways. METHODS: Rats sensitised with OVA were treated with the sPLA(2) inhibitor hyaluronic acid-linked phosphatidyl ethanolamine (HyPE). The rats were divided into four groups (n = 10 per group): (1) naïve controls (no sensitisation/no treatment); (2) positive controls (sensitisation + challenge with OVA inhalation and subcutaneous injection of 1 ml saline before each challenge; (3) sensitisation + challenge with OVA and HyPE inhalation before every challenge; and (4) sensitisation + challenge with OVA and treatment with subcutaneous dexamethasone (300 mug) before each challenge as a conventional reference. Another group received no treatment with HyPE during the sensitisation process but only before or after challenge of already sensitised rats. Pulmonary function was assessed and changes in the histology of the airways, levels of cysteinyl leukotrienes in BAL fluid, and the production of nitric oxide (No) and tumour necrosis factor alpha (TNFalpha) by BAL macrophages were determined. RESULTS: Inhalation of HyPE markedly suppressed OVA induced early and late asthmatic reactions as expressed by bronchoconstriction, airway remodelling (histology), cysteinyl leukotriene level in BAL fluid, and production of TNFalpha and NO by BAL macrophages. OVA induced bronchoconstriction in sensitised non-pretreated rats was also inhibited by inhalation of HyPE either before or after the challenge. CONCLUSIONS: These findings confirm the pivotal role of sPLA(2) in the pathophysiology of both the immediate allergic response and the inflammatory asthmatic process. Control of airway sPLA(2) may be a new therapeutic approach to the treatment of asthma.
Asunto(s)
Proteínas Sanguíneas/uso terapéutico , Bronquitis/inducido químicamente , Ovalbúmina/efectos adversos , Hipersensibilidad Respiratoria/inducido químicamente , Administración por Inhalación , Animales , Biomarcadores/análisis , Bronquitis/tratamiento farmacológico , Ratas , Hipersensibilidad Respiratoria/tratamiento farmacológicoRESUMEN
OBJECTIVE: Enhanced red blood cell (RBC) aggregation has an adverse effect on microcirculatory blood flow and tissue oxygenation. It has been previously shown that obesity is associated with increased RBC aggregation. The objectives of the present study were to further characterize obesity-related RBC aggregation and to examine whether the enhanced aggregation is a plasma- or cellular-dependent process. METHODS: Obese (body mass index (BMI)=40+/-6.3 kg/m2, n=22) and nonobese (BMI=24+/-3.4 kg/m2, n=18) individuals were evaluated for inflammation markers and aggregation parameters. Aggregation parameters were derived from the distribution of RBC population into aggregate sizes, and from the variation of the distribution as a function of flow-derived shear stress, using a cell flow properties analyzer. To differentiate plasmatic from cellular factors, we determined the aggregation in the presence of autologous plasma or dextran-500 kDa and calculated the plasma factor (PF) in the obese group. PF ranges from 0 to 1. When the PF=1, the aggregation is all due to plasmatic factors, when PF=0, the altered aggregation depends entirely on cellular factors, whereas 0Asunto(s)
Agregación Eritrocitaria
, Obesidad Mórbida/sangre
, Adulto
, Antropometría
, Índice de Masa Corporal
, Femenino
, Hemorreología
, Humanos
, Mediadores de Inflamación/sangre
, Masculino
, Persona de Mediana Edad
, Obesidad Mórbida/patología
, Estrés Mecánico
RESUMEN
Bone injury occurs in human hemolytic disorders associated with thrombosis, such as beta-thalassemia and sickle cell disease. Exposure of rats to 2-butoxyethanol (BE) has been associated with hemolytic anemia, disseminated thrombosis, and infarction in multiple organs including bone. This rat model apparently mimics acute hemolysis and thrombosis in humans. To elucidate the extent of bone injury, male and female Fischer F344 rats were given 4 daily doses of 250 mg BE/5 ml water/kg of body weight. Tail vertebrae were studied by histopathology and magnetic resonance imaging (MRI). Thrombosis and infarction were seen in both sexes, but females were more severely affected. Lesions were characterized by extensive medullary fat necrosis, granulomatous inflammation, fibroplasia, growth plate degeneration, and new woven bone formation adjacent to necrotic bone trabeculae. MRI mean and standard deviation tissue-density data for both sexes indicated a significant (P < or = 0.05) decrease following 4-days treatment and a significant increase (P < or = 0.05) following an additional 24 days without treatment. Thus, MRI was useful in revealing BE-induced bone injury, which was predominantly necrotic initially and subsequently regenerative with proliferation of connective tissue and bone following postischemia recovery.
Asunto(s)
Modelos Animales de Enfermedad , Hemólisis/efectos de los fármacos , Osteonecrosis/inducido químicamente , Trombosis/inducido químicamente , Animales , Glicoles de Etileno/toxicidad , Femenino , Imagen por Resonancia Magnética , Masculino , Osteonecrosis/patología , Ratas , Ratas Endogámicas F344 , Factores Sexuales , Solventes/toxicidad , Columna Vertebral/efectos de los fármacos , Columna Vertebral/patología , Cola (estructura animal)/efectos de los fármacos , Cola (estructura animal)/patología , Trombosis/patologíaRESUMEN
The pathophysiology of inflammatory bowel disease (IBD) involves the production of diverse lipid mediators, namely eicosanoids, lysophospholipids, and platelet-activating factor, in which phospholipase A2 (PLA2) is the key enzyme. Accordingly, it has been postulated that control of lipid mediator production by inhibition of PLA2 would be useful for the treatment of IBD. This hypothesis was tested in the present study by examining the therapeutic effect of a novel extracellular PLA2 inhibitor (ExPLI), composed of carboxymethylcellulose-linked phosphatidylethanolamine (CMPE), on trinitrobenzenesulfonic acid-induced colitis. Intraperitoneal administration of CMPE suppressed the colitis as measured by mortality rate, intestinal permeability, plasma PLA2 activity, intestinal myeloperoxidase activity, and histological morphometry. Current therapeutic approaches for inflammatory conditions focus on the selective control of a lipid mediator(s) (e.g., prostaglandins or leukotrienes). The present study supports the concept that inclusive control of lipid mediator production by PLA2 inhibition is a plausible approach to the treatment of colitis and introduces the ExPLIs as a prototype of a novel NSAID for the treatment of intestinal inflammation.
Asunto(s)
Celulasa , Colitis/patología , Inhibidores Enzimáticos/farmacología , Glicósido Hidrolasas/farmacología , Fosfatidiletanolaminas/farmacología , Fosfolipasas A/antagonistas & inhibidores , Animales , Colitis/inducido químicamente , Colitis/metabolismo , Colitis/mortalidad , Colon/patología , Combinación de Medicamentos , Mucosa Intestinal/metabolismo , Permeabilidad/efectos de los fármacos , Peroxidasa/metabolismo , Fosfolipasas A/sangre , Fosfolipasas A2 , Ratas , Ratas Endogámicas , Ácido TrinitrobencenosulfónicoRESUMEN
Red blood cell (RBC) aggregation may contribute to occlusion of the coronary microcirculation during myocardial infarction. We studied the effect of thrombolytic therapy on RBC aggregation in patients with acute myocardial infarction (AMI). Compared with patients with myocardial infarction who did not receive thrombolytic therapy, those treated with systemic thrombolysis exhibited significantly reduced RBC aggregation, reduced plasma fibrinogen levels and increased plasma D-dimer levels. Using measurement of RBC aggregation in a standardized dextran-500 solution, reduction in RBC aggregation after thrombolysis was shown to be plasma dependent. Thrombolytic therapy had no direct effect on intrinsic RBC aggregability in patients with AMI. We conclude that thrombolytic therapy has rheologic consequences that may contribute to its overall efficacy. Inhibition of RBC aggregation by thrombolytic therapy may result from the degradation of fibrinogen, a key factor in the formation of RBC aggregates, and from the generation of fibrinogen degradation products capable of disaggregating RBCs.
Asunto(s)
Agregación Eritrocitaria/efectos de los fármacos , Terapia Trombolítica , Anciano , Aspirina/administración & dosificación , Dextranos/farmacología , Agregación Eritrocitaria/fisiología , Femenino , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Fibrinógeno/análisis , Heparina/administración & dosificación , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/sangre , Infarto del Miocardio/tratamiento farmacológico , Plasma , Estreptoquinasa/administración & dosificación , Terapia Trombolítica/métodos , Activador de Tejido Plasminógeno/administración & dosificaciónRESUMEN
Secretory phospholipase A(2) (sPLA(2)) has been reported to be involved in cell proliferation in general and in endothelial cell migration, processes required for capillary formation. Subsequently, we examined the potential control of angiogenesis by sPLA(2) inhibition, using a cell-impermeable sPLA(2) inhibitor composed of N-derivatized phosphatidyl-ethanolamine linked to hyaluronic acid. This inhibitor effectively inhibits the proliferation and migration of human bone marrow endothelial cells in a dose-dependent manner, and suppresses capillary formation induced by growth factors involved in vascularization of tumors and of atherosclerotic plaques. It is proposed that sPLA(2) inhibition introduces a novel approach in the control of cancer development and atherosclerosis.
Asunto(s)
Capilares/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Neovascularización Patológica , Fosfolipasas A/antagonistas & inhibidores , División Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Humanos , Ácido Hialurónico/farmacología , Fosfatidiletanolaminas/farmacologíaRESUMEN
1. In acute respiratory distress syndrome (ARDS) induced by endotoxins, a high production of inflammatory mediators by microvascular lung endothelial cells (LMVEC) can be observed. Activation of cells by endotoxins may result in elevated secretion of phospholipase A(2) (sPLA(2)) which is thought to contribute to tissue damage. The present study was undertaken to investigate the role of sPLA(2) in chemokine production in human lung microvascular endothelial cells (LMVEC) stimulated with the endotoxins lipopolysaccharide (LPS) and lipoteichoic acid (LTA). In particular, we investigated the effects of sPLA(2) inhibitors, specifically, the extracellular PLA(2) inhibitors (ExPLIs), composed of N-derivatized phosphatidyl-ethanolamine linked to polymeric carriers, and LY311727, a specific inhibitor of non-pancreatic sPLA(2). 2. ExPLIs markedly inhibited LPS and LTA induced production and mRNA expression of the neutrophile attracting chemokines IL-8, Gro-alpha and ENA-78, as well as of the adhesion molecules ICAM-1 and E-selectin. Concomitantly, ExPLIs inhibited the LPS-induced activation of NF-kappaB by LPS but not its activation by TNF-alpha or IL-1. 3. Endotoxin mediated chemokine production in LMVEC seems not to involve PLA(2) activity, since LPS stimulation was not associated with activation of intracellular or secreted PLA(2). It therefore seems that the inhibitory effect of the ExPLIs was not due to their PLA(2) inhibiting capacity. This was supported by the finding that the LPS-induced chemokine production was not affected by the selective sPLA(2) inhibitor LY311727. 4. It is proposed that the ExPLIs may be considered a prototype of potent suppressors of specific endotoxin-induced inflammatory responses, with potential implications for the therapy of subsequent severe inflammation.
Asunto(s)
Quimiocinas/metabolismo , Endotelio Vascular/efectos de los fármacos , Ácido Hialurónico/farmacología , Pulmón/citología , Fosfatidiletanolaminas/farmacología , Moléculas de Adhesión Celular/biosíntesis , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Interacciones Farmacológicas , Endotelio Vascular/metabolismo , Humanos , Ácido Hialurónico/química , Técnicas In Vitro , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Fosfatidiletanolaminas/química , Fosfolipasas A/metabolismoRESUMEN
The phospholipid composition of red blood cells (RBC) from 32 haloperidol-treated schizophrenic patients, classified according to the positive and negative syndrome scale (PANSS) as showing either predominantly positive or predominantly negative symptoms, was determined and compared with that of normal controls. While the levels of phosphatidylcholine and phosphatidylserine were similar in all three groups, sphingomyelin (SM) and phosphatidylethanolamine (PE) were, respectively, increased and decreased in RBCs of schizophrenic patients. In both patient groups, the SM/PE ratios correlated directly with the PANSS negative symptom scale scores and inversely with the positive symptom scale scores. However, the inverse changes in the contents of SM and PE were much more expressed in the negative group. It is suggested that a main source of that difference is a higher activity of the polyunsaturated acid-selective phospholipase A(2) in the negative syndrome patients than in the positive syndrome and control groups.
Asunto(s)
Eritrocitos/metabolismo , Fosfolípidos/sangre , Esquizofrenia/sangre , Psicología del Esquizofrénico , Adulto , Depresión/sangre , Depresión/diagnóstico , Depresión/psicología , Femenino , Alucinaciones/sangre , Alucinaciones/diagnóstico , Alucinaciones/psicología , Humanos , Masculino , Persona de Mediana Edad , Escalas de Valoración Psiquiátrica , Valores de Referencia , Esquizofrenia/diagnósticoRESUMEN
To identify clinically relevant parameters of red blood cell (RBC) aggregation, we examined correlations of aggregation parameters with C-reactive protein and fibrinogen in unstable angina (UA), acute myocardial infarction (AMI), and bacterial infection (BI). Aggregation parameters were derived from the distribution of RBC population into aggregate sizes (cells per aggregate) and changing of the distribution by flow-derived shear stress. Increased aggregation was observed in the following order: UA, AMI, and BI. The best correlation was obtained by integration of large aggregate fraction as a function of shear stress. To differentiate plasmatic from cellular factors in RBC aggregation, we determined the aggregation in the presence and absence of plasma and formulated a "plasma factor" (PF) ranging from 0 to 1. In AMI the enhanced aggregation was entirely due to PF (PF = 1), whereas in UA and BI it was due to both plasmatic and cellular factors (0 < or = PF < or = 1). It is proposed that clinically relevant parameters of RBC aggregation should express both RBC aggregate size distribution and aggregate resistance to disaggregation and distinguish between plasmatic and cellular factors.
Asunto(s)
Reacción de Fase Aguda/inmunología , Eritrocitos/citología , Eritrocitos/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Angina Inestable/inmunología , Infecciones Bacterianas/inmunología , Biomarcadores , Proteína C-Reactiva/metabolismo , Agregación Celular/inmunología , Femenino , Fibrinógeno/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/inmunología , Estrés MecánicoRESUMEN
BACKGROUND: This in vitro study aimed to elucidate the extent and kind of involvement of hyaluronic acid (HA) in the currently accepted view of synovial joint lubrication, in which surface-active phospholipids (SAPL) constitute the main boundary lubricant. The integrity of SAPL is apparently threatened by the lysing activity of phospholipase A(2) (PLA(2)). METHODS: The effects of increasing concentrations of HA degraded by free radicals and non-degraded HA on the lysing activity of PLA(2) were examined in vitro. Liposomes (lipid model membrane) containing phosphatidylcholine (PC) were used as the substrate, on the assumption that they are appropriate representatives of SAPL. RESULTS: HA adhered to the phospholipid membrane (liposomes), inhibiting their lysis by PLA(2). However, in its degraded form, HA not only failed to inhibit PLA(2)-lysing activity, but accelerated it. CONCLUSIONS: It is reasonable to assume that HA plays an important indirect role in the steady state of the boundary lubrication process of joints by protecting SAPL from being lysed by PLA(2). However, as excessive loading generates free radicals within the joint (among other effects), the HA that is degraded in this way is incapable of protecting SAPL from lysis by PLA(2). When the rate of degradation exceeds that of synthesis, there will be insufficient replacement of HA and/or SAPL, resulting in denudation of the articular surfaces. These are then exposed to increasing friction, and hence increased danger of degenerative joint changes.
Asunto(s)
Ácido Hialurónico/metabolismo , Fosfolipasas A/metabolismo , Fosfolípidos/metabolismo , Tensoactivos/metabolismo , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Humanos , Ácido Hialurónico/farmacología , Artropatías/metabolismo , Artropatías/fisiopatología , Liposomas/metabolismo , Membrana Sinovial/ultraestructuraRESUMEN
The hydrolysis of cell membrane phospholipids by phospholipase A(2) (PLA(2)) leads to the production of numerous lipid mediators of diverse pathological conditions, mainly inflammatory diseases. These include lysophospholipids and their derivatives, and arachidonic acid and its derivatives (the eicosanoids). Both these groups of mediators are produced predominantly by the secretory PLA(2)s (sPLA(2)s) which hydrolyze the phospholipids of the cell surface membrane. Protection of cell membrane from these 'inflammatory enzymes' can therefore be used for the treatment of inflammatory processes. A prototype of cell-impermeable PLA(2) inhibitors, which protect the cell membrane from different sPLA(2)s without affecting vital phospholipid metabolism, is presented and discussed in the present review.
Asunto(s)
Membrana Celular/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Inflamación/tratamiento farmacológico , Fosfolipasas A/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Ácidos Araquidónicos/antagonistas & inhibidores , Ácidos Araquidónicos/metabolismo , Calcio/metabolismo , Membrana Celular/enzimología , Permeabilidad de la Membrana Celular , Citosol/enzimología , Eicosanoides/biosíntesis , Humanos , Inflamación/enzimología , Lisofosfolípidos/antagonistas & inhibidores , Lisofosfolípidos/metabolismo , Lípidos de la Membrana/metabolismo , Fosfolipasas A/metabolismo , Fosfolípidos/metabolismoRESUMEN
The measurement of intestinal permeability is widely used to assess different aspects of mucosal barrier disorders and related disease states, and has been proposed for evaluation of disease activity. To provide a simple method for assessment of intestinal permeability, we examined the permeation of inulin-fluorescein (InFl) in rat models of small intestinal injury and colitis. Small intestinal or colonic inflammation was induced by either i.p. administration of indomethacin or rectal administration of trinitrobenzene sulfonic acid (TNBS), respectively. For monitoring of intestinal permeability, InFl was administered orally or rectally to rats with small intestinal or colonic inflammation, respectively, and its level in blood was determined by the fluorescence intensity in the plasma. In small intestinal injury, InFl reached its peak in plasma 3 h after oral administration, while in colitis the InFl peak was reached 1 h after rectal administration. The highest permeability was observed at 72 h or 12 h after induction of small intestinal or colonic inflammation, respectively. In small intestinal injury the InFl permeation, as measured by its plasma level prior to sacrifice, was in agreement with intestinal damage evaluated after sacrifice. In colitis, the permeability at 12 h after induction of the disease correlated well with mortality. These findings demonstrate that InFl can be used as a novel, safe and easy-to-use probe for the evaluation of gut permeation and follow-up of gastrointestinal injury.
Asunto(s)
Fluoresceína , Insulina , Mucosa Intestinal/metabolismo , Animales , Fluoresceína/farmacocinética , Insulina/farmacocinética , Permeabilidad , RatasRESUMEN
Red blood cells (RBCs) in the presence of plasma proteins or other macromolecules may form aggregates, normally in rouleaux formations, which are dispersed with increasing blood flow. Experimental observations have suggested that the spontaneous aggregation process involves the formation of linear rouleaux (FLR) followed by formation of branched rouleaux networks. Theoretical models for the spontaneous rouleaux formation were formulated, taking into consideration that FLR may involve both "polymerization," i.e., interaction between two single RBCs (e + e) and the addition of a single RBC to the end of an existing rouleau (e + r), as well as "condensation" between two rouleaux by end-to-end addition (r + r). The present study was undertaken to experimentally examine the theoretical models and their assumptions, by visual monitoring of the spontaneous FLR (from singly dispersed RBC) in plasma, in a narrow gap flow chamber. The results validate the theoretical model, showing that FLR involves both polymerization and condensation, and that the kinetic constants for the above three types of intercellular interactions are the same, i.e., k(ee) = k(er) = k(rr) = k, and for all tested hematocrits (0.625-6%) k < 0.13 +/- 0.03 s(-1).
Asunto(s)
Agregación Eritrocitaria/fisiología , Fenómenos Biofísicos , Biofisica , Citometría de Flujo , Humanos , Técnicas In Vitro , Cinética , Microscopía por Video , Modelos BiológicosRESUMEN
P388D(1) macrophages prelabeled with [(3)H]arachidonic acid (AA) respond to bacterial lipopolysaccharide (LPS) by mobilizing AA in a process that takes several hours and is mediated by the concerted actions of the group IV cytosolic phospholipase A(2) and the group V secretory phospholipase A(2) (sPLA(2)). Here we show that when the LPS-activated cells are prelabeled with [(3)H]oleic acid (OA), they also mobilize and release OA to the extracellular medium. The time and concentration dependence of the LPS effect on OA release fully resemble those of the AA release. Experiments in which both AA and OA release are measured simultaneously indicate that AA is released 3 times more efficiently than OA. Importantly, LPS-stimulated OA release is strongly inhibited by the selective sPLA(2) inhibitors 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propane sulfonic acid and carboxymethylcellulose-linked phosphatidylethanolamine. The addition of exogenous recombinant sPLA(2) to the cells also triggers OA release. These data implicate a functionally active sPLA(2) as being essential for the cells to release OA upon stimulation with LPS. OA release is also inhibited by methyl arachidonyl fluorophosphonate but not by bromoenol lactone, indicating that the group IV cytosolic phospholipase A(2) is also involved in the process. Together, these data reveal that OA release occurs during stimulation of the P388D(1) macrophages by LPS and that the regulatory features of the OA release are strikingly similar to those previously found for the AA release.