Characterizing the glycocalyx of poultry spermatozoa: I. Identification and distribution of carbohydrate residues using flow cytometry and epifluorescence microscopy.
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The aim of the present work was to use a battery of lectins to 1) delineate the carbohydrate content of sperm glycocalyx in the turkey and chicken using flow cytometry analysis, and 2) evaluate the distribution of existing sugars over the sperm plasma membrane surface with epifluorescent microscopy. Carbohydrate groups (corresponding lectins) that were investigated included galactose (GS-I, Jacalin, RCA-I, PNA), glucose and/or mannose (Con A, PSA, GNA), N-acetyl-glucosamine (GS-II, s-WGA, STA), N-acetyl-galactosamine (SBA, WFA), fucose (Lotus, UEA-I), sialic acid (LFA, LPA), and N-acetyl-lactosamine (ECA). Spermatozoa were assessed before and after treatment with neuraminidase to remove sialic acid. Mean fluorescence intensity (MnFI) was used as indicator of lectin binding for flow cytometry analysis. Nontreated spermatozoa from both species showed high MnFI when incubated with RCA-I, Con A, LFA, and LPA, as did chicken spermatozoa incubated with s-WGA. Neuraminidase treatment increased the MnFI for most lectins except LFA and LPA, as expected. Differences in MnFI between species included higher values for s-WGA and ECA in chicken spermatozoa and for WFA in turkey spermatozoa. Microscopy revealed segregation of some sugar residues into membrane-specific domains; however, the 2 staining techniques (cell suspension vs fixed preparation) differed in identifying lectin binding patterns, with fixed preparations yielding a high degree of nonspecific binding. We conclude that 1) the glycocalyx of turkey and chicken spermatozoa contains a diversity of carbohydrate groups, 2) these residues are extensively masked by sialic acid, 3) the glycocalyx composition is species-specific, and 4) some glycoconjugates appear to be segregated into membrane-specific domains. Characterization of the poultry sperm glycocalyx is the first step in identifying the physiological impact of semen storage on sperm function. (+info)
Influence of endothelial glycocalyx degradation and surfactants on air embolism adhesion.
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BACKGROUND: Microbubble adherence to endothelial cells is enhanced after damage to the glycocalyx. The authors tested the hypothesis that exogenous surfactants delivered intravascularly have differential effects on the rate of restoration of blood flow after heparinase-induced degradation of the endothelial glycocalyx. METHODS: Air microbubbles were injected into the rat cremaster microcirculation after perfusion with heparinase or saline and intravascular administration of either saline or one of two surfactants. The surfactants were Pluronic F-127 (Molecular Probes, Eugene, OR) and Perftoran (OJSC SPC Perftoran, Moscow, Russia). Embolism dimensions and dynamics were observed using intravital microscopy. RESULTS: Significant results were that bubbles embolized the largest diameter vessels after glycocalyx degradation. Bubbles embolized smaller vessels in the surfactant treatment groups. The incidence of bubble dislodgement and the magnitude of distal displacement were smallest after glycocalyx degradation alone and largest after surfactant alone. The time to bubble clearance and restoration of blood flow was longest with heparinase alone and shortest with Pluronic F-127 alone. CONCLUSIONS: Degradation of the glycocalyx causes air bubbles to adhere to the endothelium more proximally in the arteriolar microcirculation. Surfactants added after glycocalyx degradation and before gas embolization promotes bubble lodging in the distal microcirculation. Surfactants may have a clinical role in reducing embolism bubble adhesion to endothelial cells undergoing glycocalyx disruption. (+info)
The endothelial glycocalyx affords compatibility of Starling's principle and high cardiac interstitial albumin levels.
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OBJECTIVE: To test the role of an oncotic pressure gradient across the endothelial glycocalyx with respect to extravasation of fluid and colloids and development of tissue edema in a whole organ setting. METHODS: We measured filtration in the intact coronary system of isolated guinea pig hearts, comparing colloid-free perfusion and perfusion with 1.67% albumin or 2% hydroxyethylstarch (oncotic pressures 5.30 vs. 11.10 mm Hg, respectively). Heparinase was used to alter the endothelial glycocalyx. RESULTS: Extremely high net organ hydraulic conductivity was obtained with colloid-free perfusion (9.14 microl/min/g tissue). Supplementing perfusate with albumin caused a significant decrease, also vs. hydroxyethylstarch (1.04 vs. 2.67 microl/min/g, p < 0.05). Albumin also lowered edema formation vs. the other perfusion modes (p < 0.05). Stripping the glycocalyx of heparan sulfate reduced the effect of colloids, especially that of albumin. The steady-state concentrations of hydroxyethylstarch and albumin in the mixed interstitial fluid leaving the intact coronary bed averaged about 95% of the intravascular level. Electron and light microscopy indicated that colloid extravasated mainly in the venular sections. CONCLUSION: We propose a low-filtration model for the coronary system with different barrier properties in arteriolar/capillary and venular sections. Arteriolar/capillary: very little fluid and colloid extravasation due to the endothelial surface layer formed by the glycocalyx and albumin plus the endothelial strand barrier; venular: little net extravsation of fluid and colloids despite large pores, because of low hydrostatic and oncotic pressure differences between intra- and extravascular spaces. The latter sites provide physiological access of large solutes (colloids) to the tissue. (+info)
The glycocalyx protects erythrocyte-bound tissue-type plasminogen activator from enzymatic inhibition.
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Coupling tissue-type plasminogen activator (tPA) to carrier red blood cells (RBC) prolongs its intravascular life span and permits its use for thromboprophylaxis. Here, we studied the susceptibility of RBC/tPA to PA inhibitors including plasminogen activator inhibitor-1 (PAI-1) that constrain its activity and may reduce the duration of its effect. Despite lesser spatial and diffusional limitations, soluble tPA was far less effective than RBC/tPA in dissolving clots formed in vitro from blood of wild-type (WT) mice (40 versus 80% lysis at equal doses of tPA). Furthermore, after i.v. injection, soluble tPA lost activity faster in transgenic mice expressing a high level of PAI-1 than in WT mice, whereas the activity of RBC/tPA was unaffected. PAI-1 inactivated soluble tPA at equimolar ratios in vitro, but it had no effect on the amidolytic or fibrinolytic activity of RBC/tPA. RBC/tPA was also more resistant than soluble tPA to in vitro inhibition by other serpins (alpha2-macroglobulin and alpha1-antitrypsin) and pathologically high levels of glucose. However, coupling to RBC did not protect a truncated tPA mutant, Retavase, from plasma inhibitors. Chemical removal of the RBC glycocalyx negated tPA protection from inhibitors: tPA coupled to glycocalyx-stripped RBC bound twice as much 125I-PAI-1 as did tPA coupled to naive RBC, and susceptibility of the bound tPA to inhibition by PAI-1 was restored. Thus, the RBC glycocalyx protects RBC-coupled tPA against inhibition. Resistance to high levels of inhibitors in vivo contributes to the potential utility of RBC/tPA for thromboprophylaxis. (+info)
The endothelial glycocalyx: composition, functions, and visualization.
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This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell-vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging. (+info)
The role of endothelial glycocalyx components in mechanotransduction of fluid shear stress.
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The surface of endothelial cells is decorated with a wide variety of membrane-bound macromolecules that constitute the glycocalyx. These include glycoproteins bearing acidic oligosaccharides with terminal sialic acids (SA), and proteoglycans with their associated glycosaminoglycan that include: heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). In this study, enzymes were used to selectively degrade glycocalyx components from the surface of bovine aortic endothelial cells and the effects of these alterations on fluid shear-induced nitric oxide (NO) and prostacyclin (PGI(2)) production were determined. Depletion of HS, HA, and SA, but not CS, blocked shear-induced NO production. Surprisingly, the same enzyme depletions that blocked NO production had no influence on shear-induced PGI(2) production. The results may be interpreted in terms of a glypican-caveolae-eNOS mechanism for shear-induced NO transduction, with PGI(2) being transduced in basal adhesion plaques that sense the same reaction stress whether the glycocalyx is intact or not. (+info)
Glycocalyx and endothelial (dys) function: from mice to men.
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Located on the luminal surface of vascular endothelial cells, the glycocalyx is composed of a negatively charged mesh of proteoglycans, glycosaminoglycans, glycoproteins and glycolipids and harbors a wide array of enzymes that contribute in regulation of leukocyte-/thrombocyte adherence, with a principal role in plasma and vessel wall homeostasis. Glycocalyx disruption is accompanied by enhanced sensitivity of the vasculature towards atherogenic stimuli which emphasizes that not only the composition of the glycocalyx is important in facilitating these properties but that the contribution of its physical dimension and barrier properties should also be considered. In addition, similarities found between micro-versus macro vascular beds suggest common structural properties throughout the entire vascular bed that might be of importance in protective strategies against vascular perturbation. Collectively, these data lend support to a potential role of the glycocalyx as a first barrier in protection against atherogenic insults. Therefore, it will be a challenge to determine whether glycocalyx volume measurement, systemically or at the individual capillary level, is a feasible surrogate marker for cardiovascular disease, and whether it may prove to be of use to assess the impact of novel interventions aimed at glycocalyx restoration on atherosclerosis progression. (+info)
Microvascular and capillary perfusion following glycocalyx degradation.
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Systemic parameters and microvascular and capillary hemodynamics were studied in the hamster window chamber model before and after hyaluronan degradation by intravenous injection of Streptomyces hyaluronidase (100 units, 40-50 U/ml plasma). Glycocalyx permeation was estimated using fluorescent markers of different molecular size (40, 70, and 2,000 kDa), and electrical charge. Systemic parameters (blood pressure, heart rate, blood gases) and microhemodynamics (vascular tone, velocity, and blood flow) remained statistically unchanged after injection of hyaluronidase, compared with inactivated hyaluronidase. Conversely, capillary hemodynamics were drastically affected. Functional capillary density, the capillaries perfused with red blood cells (RBCs), decreased by 35%, capillary Hct of the remaining functional capillaries increased from 16 to 27%, and penetration of 70-kDa fluorescent marker increased. Furthermore, plasma-only perfused capillaries statistically increased 30 min after hyaluronidase. The decrease in functional capillary density accounted for an increased RBC flux in the remainder of the capillaries, since the same number of RBCs had to traverse a reduced number of capillaries. Flux balances showed a reduction from baseline of 11% for the RBC flux and 20% for the plasma flux after treatment. These discrepancies are within the margin of error of the techniques used and could be explained by accounting for RBC over-velocity compared with plasma. These findings suggest that the decrease in the glycocalyx leads to capillary perfusion impairments. (+info)