In vitro and ex vivo effect of hyaluronic acid on erythrocyte flow properties

<p>Abstract</p> <p>Background</p> <p>Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed <it>in vitro </it>experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the <it>in vitro </it>experiment with <it>ex vivo </it>RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology.</p> <p>Methods</p> <p>Erythrocyte deformability (by filtration through nucleopore membranes) and erythrocyte aggregability (EA) were tested on blood from healthy donors additioned with purified HA. EA was measured by transmitted light and analyzed with a mathematical model yielding two parameters, the aggregation rate and the size of the aggregates. Conformational changes of cytoskeleton proteins were estimated by electron paramagnetic resonance spectroscopy (EPR).</p> <p>Results</p> <p><it>In vitro</it>, erythrocytes treated with HA showed increased rigidity index (RI) and reduced aggregability, situation strongly related to the rigidization of the membrane cytoskeleton triggered by HA, as shown by EPR results. Also, a significant correlation (r: 0.77, p < 0.00001) was found between RI and serum HA in RA patients.</p> <p>Conclusions</p> <p>Our results lead us to postulate the hypothesis that HA interacts with the erythrocyte surface leading to modifications in erythrocyte rheological and flow properties, both <it>ex vivo </it>and <it>in vitro</it>.</p

Co-Regulation of Expression of Phase II Metabolizing Enzymes and Multidrug Resistance-Associated Protein 2

Treatment of experimental animals with prototypical enzyme inducers represents a useful tool to characterize the role of different isozymes in drug metabolism and to improve our knowledge on factors regulating their synthesis at the transcriptional level. The effect of model enzyme inducers on phase II (conjugating) enzyme families, including UDP-glucuronosyltransferase’s and glutathione-S-transferase’s, has been well characterized in rodent liver. More recently, the effect of inducers on the expression of canalicular multidrug resistance-associated protein 2 (Mrp2) has been focused upon. The identification of a number of conjugated drugs as Mrp2 substrates suggests that both the conjugation and transport systems act coordinately to improve drug elimination from the body. We provide evidence about circumstances resulting in the simultaneous upregulation of phase II enzymes and Mrp2 in hepatic and extrahepatic tissues, most likely involving activation of common nuclear receptors (e.g., FXR, PXR). Additionally, we provide an analysis of examples of drug-induced toxicity leading to the simultaneous downregulation of both systems. Potential therapeutic strategies based on the modulation of expression of these systems are also briefly commented upon

Ursodeoxycholate reduces ethinylestradiol glucuronidation in the rat: Role of prevention in estrogen-induced cholestasis

ABSTRACT Ethinylestradiol (EE) administration (5 mg/kg, s.c., daily for 5 days) to rats leads to cholestasis, and its derivative EE 17␤-glucuronide is a likely mediator of this effect. Coadministration of ursodeoxycholate (UDC) was shown to prevent ethinylestradiol-induced cholestasis. The aim of this study was to evaluate the inhibitory effect of UDC on EE glucuronidation in vivo and in vitro as a potential mechanism to explain UDC protection. UDC treatment (25 mg/kg, i.p., daily for 5 days) decreased the biliary excretion of EE 17␤-glucuronide in bile after administration of a trace dose of [ 3 H]EE and reduced microsomal EE 17␤-glucuronidation activity by 20% and expression of UGT2B1, one of the enzymes involved in EE conjugation, by 30%. Glucuronidation kinetic studies were performed in vitro using normal microsomes and isolated hepatocytes in the presence of tauroursodeoxycholate (TUDC), the major endogenous derivative of UDC in the rat. Kinetic enzymatic studies in microsomes showed a noncompetitive inhibition of EE 17␤-glucuronidation by TUDC, which was unique for this bile salt since other endogenous bile salts such as taurocholate, taurochenodeoxycholate, or taurodeoxycholate did not affect the enzyme activity. Studies in isolated hepatocytes confirmed the inhibitory effect of TUDC on EE glucuronidation and indicated that TUDC can reach the enzyme active site in intact cells. In conclusion, both in vivo and in vitro experiments indicate that UDC decreased the metabolic pathways involved in EE glucuronidation, hence decreasing the formation of the cholestatic derivative EE 17␤-glucuronide

Interaction of Mesoporous Silica Nanoparticles with Human Red Blood Cell Membranes: Size and Surface Effects

The interactions of mesoporous silica nanoparticles (MSNs) of different particle sizes and surface properties with human red blood cell (RBC) membranes were investigated by membrane filtration, flow cytometry, and various microscopic techniques. Small MCM-41-type MSNs (∼100 nm) were found to adsorb to the surface of RBCs without disturbing the membrane or morphology. In contrast, adsorption of large SBA-15-type MSNs (∼600 nm) to RBCs induced a strong local membrane deformation leading to spiculation of RBCs, internalization of the particles, and eventual hemolysis. In addition, the relationship between the degree of MSN surface functionalization and the degree of its interaction with RBC, as well as the effect of RBC−MSN interaction on cellular deformability, were investigated. The results presented here provide a better understanding of the mechanisms of RBC−MSN interaction and the hemolytic activity of MSNs and will assist in the rational design of hemocompatible MSNs for intravenous drug delivery and in vivo imaging

Comparison between internal microviscosity of low-density erythrocytes and the microviscosity of hemoglobin solutions: an electron paramagnetic resonance study.

The hypothesis that the internal viscosity of erythrocytes is governed by the intracellular hemoglobin (Hb) concentration is examined. Here viscosity is determined by labeling of the cytoplasmic reduced glutathione with the spin label maleimido-Tempo. Erythrocyte populations with different Hb concentrations in isosmotic conditions were obtained through incomplete lysis, followed by cell resealing, and discontinuous density gradient separation. This procedure maintains normal cell shape and volume. Microviscosity of membrane-free Hb solutions was measured by addition of spin labeled glutathione. It was found that microviscosity values are similar for the erythrocyte cytoplasm and for Hb solutions of equivalent concentrations, showing that the erythrocyte membrane does not have any influence on internal microviscosity. The dependence of the microviscosity on the concentration of Hb solutions was compared with results of macroscopic viscosity obtained by other authors. It is concluded that microviscosity is sensitive to individual properties of the Hb molecule (intrinsic viscosity), but that it is not sensitive to intermolecular interactions. As the microviscosity behavior as a function of Hb concentration is the same in Hb solutions as in the erythrocyte cytoplasm, the inferences regarding macroscopic viscosity in Hb solutions could be translated to the rheological properties of the erythrocyte cytoplasm. Thus, these properties could be predicted from the values of the mean corpuscular Hb concentration

Haemorheological variables as a rheumatoid arthritis activity indicator

Objective: To investigate if blood hyperviscosity in RA patients is due to a reduced erythrocyte deformability and, therefore, turning it into a reliable activity indicator, as well as a therapy follow-up marker for this pathology. Methods: (1) The haemorheological profile consisting of erythrocyte deformability, blood and plasma viscosity, and erythrocyte membrane fluidity was determined in 24 AR patients and 17 healthy controls. (2) A 4 year follow-up was carried on in 16 patients monitoring blood viscosity, erythrocyte deformability and biochemical variables in relation to clinical assessment of disease activity (Disease Activity Score “DAS 28-4”). Results: Erythrocyte deformability and membrane fluidity were impaired in RA patients compared to controls (p<0.001). Blood viscosity was significantly increased and correlated with the cell rigidity index (r=0.85, p<0.0000) in RA patients. The follow-up showed a good correlation between haemorheological parameters and DAS 28-4 during disease evolution. Conclusion: our results support the hypothesis that in RA, blood hyperviscosity is determined by deformability loss, which in turn is due to a membrane rigidization. This could evidenced that a widespread cell membrane damage is expressed through an impaired erythrocyte deformability, turning haemorheological parameters into reliable tools to study disease evolution. The follow-up study enabled us to confirm that erythrocyte deformability is an efficient indicator of rheumatoid arthritis activity.Fil: Luquita, A.. Universidad Nacional de Rosario; ArgentinaFil: Urli, L.. Universidad Nacional de Rosario; ArgentinaFil: Dominighini, A.. Universidad Nacional de Rosario; ArgentinaFil: Svetaz, M. J.. Universidad Nacional de Rosario; ArgentinaFil: Gennaro, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Volpintesta, R.. Universidad Nacional de Rosario; ArgentinaFil: Palatnik, S.. Universidad Nacional de Rosario; ArgentinaFil: Rasia, M.. Universidad Nacional de Rosario; Argentin

Erythrocyte aggregation in rheumatoid arthritis: cell and plasma factor’s role

ncrease in erythrocyte aggregation (EA) is pathognomonic for rheumatoid arthritis (RA), and its estimation through erythrocyte sedimentation rate (ESR) is part of DAS 28-4 activity diagnosis, with low correlation with EA and that does not discriminate the contribution of cell factors that increase aggregation. Objective: To analyse cell and plasma factors that might be involved in EA increase, to understand how RA affects blood components, thus modifying blood fluid behavior. Methodology: One hundred women presenting active RA were compared with age-matched controls (C). EA was measured by transmitted light, obtaining two parameters: 2k2n0, characterizing the aggregation process kinetics and s0/n0, estimating aggregates size. Cell factors assays: erythrocyte deformability, by filtration through nucleopore membranes, cell shape, by microscopy, and membrane fluidity by EPR. Plasma: total proteins and CRP, albumin, fibrinogen (Fb), by gravimetry, and IgG and IgM by single radial immuno-diffusion. Results: AR and C (x ± SE). 2k2n0: 31.83 ± 2.84, 23.75 ± 1.91; s0/n0: 0.92 ± 0.05, 0.87 ± 0.04. Rigidity index (RI): 14.79 ± 4.71, 6.92 ± 1.31. Morphological index: 0.28 ± 0.03, 0.30 ± 0.05, n.s. Fb (mg/dl): 382 ± 80, 299 ± 70. IgG (mg/dl): 1580 ± 219, 1296 ± 158; IgM (mg/dl) 233 ± 28, 183 ± 23; albumin (g/dl) 3.84 ± 0.44, 3.77 ± 0.51 n.s. p < 0.05 accepted. Correlations: 2k2n0 vs. Fb r = 0.66; s0/n0 vs. Fb r = 0.51; 2k2n0 vs. Igs r = 0.65; s0/n0 vs. Igs r = 0.56. 2k2n0 vs. RI r = -0.59; s0/n0 vs. RI = -0.52, p < 0.05. Conclusions: Plasma factors, Igs and Fb increased aggregation, since RI is altered, this reduces the process efficiency regarding aggregation. Patients with active RA present an increased EA, with values modifications associated with the activity index DAS 28-4, thus becoming an RA activity indicator.Fil: Luquita, A.. Universidad Nacional de Rosario; ArgentinaFil: Urli, L.. Universidad Nacional de Rosario; ArgentinaFil: Svetaz, M. J.. Universidad Nacional de Rosario; ArgentinaFil: Gennaro, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Volpintesta, R.. Universidad Nacional de Rosario; ArgentinaFil: Palatnik, S.. Universidad Nacional de Rosario; ArgentinaFil: Rasia, M.. Universidad Nacional de Rosario; Argentin