TNF-alpha increases entry of macromolecules into luminal endothelial cell glycocalyx.

The endothelial luminal glycocalyx has been largely ignored as a target in vascular pathophysiology even though it occupies a key location. As a model of the inflammatory response, we tested the hypothesis that tumor necrosis factor-alpha (TNF-alpha) can alter the properties of the endothelial apical glycocalyx. In the intact hamster cremaster microcirculation, fluorescein isothiocyanate (FITC)-labeled Dextrans 70, 580, and 2,000 kDa are excluded from a region extending from the endothelial surface almost 0.5 micrometer into the lumen. This exclusion zone defines the boundaries of the glycocalyx. Red blood cells (RBC) under normal flow conditions are excluded from a region extending even farther into the lumen. The cremaster microcirculation was pretreated with topical or intrascrotal applications of TNF-alpha. After infusion of FITC-dextran, FITC-albumin, or FITC-immunoglubulin G (IgG) via a femoral cannula, microvessels were observed with bright-field and fluorescence microscopy to obtain estimates of the anatomic diameters and the widths of fluorescent tracer columns and of the RBC columns (means +/- SE). After 2 h of intrascrotal TNF-alpha exposure, there was a significant increase in access of FITC-Dextrans 70 and 580 to the space bounded by the apical glycocalyx in arterioles, capillaries, and venules, but no significant change in access of FITC-Dextran 2,000. The effects of TNF-alpha could be observed as early as 20 min after the onset of topical application. TNF-alpha treatment also significantly increased the penetration rate of FITC-Dextran 40, FITC-albumin, and FITC-IgG into the glycocalyx and caused a significant increase in the intraluminal volume occupied by flowing RBC. White blood cell adhesion increased during TNF-alpha application, and we used the selectin antagonist fucoidan to attenuate leukocyte adhesion during TNF-alpha stimulation. This did not inhibit the TNF-alpha-mediated increase in permeation of the glycocalyx. These results show that proinflammatory cytokines can cause disruption of the endothelial apical glycocalyx, leading to an increased macromolecular permeation in the absence of an increase in leukocyte recruitment.

Permeation of the luminal capillary glycocalyx is determined by hyaluronan.

The endothelial cell glycocalyx influences blood flow and presents a selective barrier to movement of macromolecules from plasma to the endothelial surface. In the hamster cremaster microcirculation, FITC-labeled Dextran 70 and larger molecules are excluded from a region extending almost 0.5 micrometer from the endothelial surface into the lumen. Red blood cells under normal flow conditions are excluded from a region extending even farther into the lumen. Examination of cultured endothelial cells has shown that the glycocalyx contains hyaluronan, a glycosaminoglycan which is known to create matrices with molecular sieving properties. To test the hypothesis that hyaluronan might be involved in establishing the permeation properties of the apical surface glycocalyx in vivo, hamster microvessels in the cremaster muscle were visualized using video microscopy. After infusion of one of several FITC-dextrans (70, 145, 580, and 2,000 kDa) via a femoral cannula, microvessels were observed with bright-field and fluorescence microscopy to obtain estimates of the anatomic diameters and the widths of fluorescent dextran columns and of red blood cell columns (means +/- SE). The widths of the red blood cell and dextran exclusion zones were calculated as one-half the difference between the bright-field anatomic diameter and the width of the red blood cell column or dextran column. After 1 h of treatment with active Streptomyces hyaluronidase, there was a significant increase in access of 70- and 145-kDa FITC-dextrans to the space bounded by the apical glycocalyx, but no increase in access of the red blood cells or in the anatomic diameter in capillaries, arterioles, and venules. Hyaluronidase had no effect on access of FITC-Dextrans 580 and 2,000. Infusion of a mixture of hyaluronan and chondroitin sulfate after enzyme treatment reconstituted the glycocalyx, although treatment with either molecule separately had no effect. These results suggest that cell surface hyaluronan plays a role in regulating or establishing permeation of the apical glycocalyx to macromolecules. This finding and our prior observations suggest that hyaluronan and other glycoconjugates are required for assembly of the matrix on the endothelial surface. We hypothesize that hyaluronidase creates a more open matrix, enabling smaller dextran molecules to penetrate deeper into the glycocalyx.

Permselectivity of angiogenic microvessels following alteration of the endothelial fiber matrix by oligosaccharides.

The endothelial glycocalyx, which is composed of integral and peripheral glycoconjugates, forms a fibrous matrix that confers macromolecular sieving properties on the microvascular wall. Changes in pore size within the matrix may regulate macromolecular access to the paracellular and/or vesicular transendothelial pathways. We tested the hypothesis that modifications of the endothelial glycocalyx might play a role in the ontogeny of endothelial permselectivity in proliferating microvessels of the chick chorioallantoic membrane (CAM). Accordingly, we evaluated the effects of Dolichos biflorus agglutinin (DBA) or Arachis hypogaea agglutinin (PNA) lectin binding, and N'N'diacetylchitobiose or hydroxyethyl starch polysaccharide (HES) incorporation on CAM endothelial restriction of FITC-dextrans 40 or 150 at Days 4.5 and 5.0 of development. Extravasation of FITC-dextrans was determined by recording their perivascular interstitial intensities. Following DBA, PNA, and N'N'diacetylchitobiose administration, interstitial accumulation of the tracers near first-order pre- and postcapillaries, and surrounding the capillaries, was similar to that of controls at both Days 4.5 and 5.0. At Day 4.5, pretreatment with HES significantly decreased extravasation of FITC-dextran 40. Thus, retention of HES molecules within the glycocalyx might tighten the matrix, and reduce access of dextran 40 to transendothelial pathways across the angiogenic microvessels.

Distribution of anionic sites on microvascular endothelium of the chick chorioallantoic membrane.

It is generally accepted that luminal surfaces of adult microvascular endothelia present an anionic barrier that limits passage of anionic macromolecules. To assess the ontogeny of the barrier, temporal and spatial expression of endothelial anionic sites was evaluated in the chorioallantoic membrane of chicken embryos from days 4.5 to 18 of incubation. After an initial flush, the vessels were perfused with cationic ferritin (CF, 1.0 mg/ml in PBS) for 2 min. Following a second flush to remove unbound CF, the chick chorioallontoic membranes (CAMs) were fixed and processed for electron microscopy. Continuous CF binding was revealed on the luminal endothelium, the junctional clefts and the plasmalemmal vesicles from days 4.5 to 14. However, by day 18, anionic sites had become discontinuous. Prior perfusion with protamine sulfate abolished CF binding and facilitated native ferritin binding. Further ultrastructural evaluation, using peroxidase labeled LFA lectin, revealed sialic acid moieties in patches on the CAM endothelium. Thus, in early chick embryogenesis, the CAM endothelium displays a continuous pattern of luminal anionic sites comprised in part of sialic acid. As the CAM ages, endothelial anionic sites become reduced. That the expression of endothelial anionic domains remained constant despite changes in CAM microvascular permeability in early development (Rizzo et al., 1995a) serves to suggest a minimal role for anionic domains in the development of microvascular permselectivity during normal angiogenesis.

Glycoconjugate expression in the chick embryonic chorioallantoic membrane: comparisons of the chorionic ectoderm and allantoic endoderm.

BACKGROUND: The chorioallantoic membrane (CAM) of the chick embryo expands during embryogenesis to meet the increased oxygen demands during growth and differentiation. Temporal and spatial glycosylation patterns of CAM ectodermal and endodermal proteins likely contribute to differentiation of the functional attributes of the CAM. METHODS: Using lectins for light and electron microscopic observations, we studied the patterns of glycoconjugate expression on the ectoderm and endoderm of the chorioallantoic membrane (CAM) of the chick at days 4.5, 5.0, 5.5, 6.0, and 10 of morphogenesis. For light microscopy, samples of unfixed CAM were incubated with the following FITC lectins: Con A, DBA, GSA-I, GSA-II, PNA, SBA, UEA-I, and WGA. RESULTS: All lectins, except GSA-I and -II, gave positive results. The positive lectins, labeled with HRP, served to ultrastructurally localize PNA, SBA, and WGA, but not DBA binding to the luminal surface of the endoderm. UEA-I and Con A bound similarly except on day 10 when UEA-I no longer bound. On the ectodermal surface, only WGA bound at all times studied. PNA and SBA binding were present from days 5.0 to 6.0 but absent at days 4.5 and 10. DBA binding occurred through day 5.0 but was absent thereafter. UEA-I bound to the ectoderm at days 4.5, 5.0, and 10 but not days 5.5 and 6.0. Con A bound only on days 5.0 and 10. CONCLUSION: That the ultrastructurally similar ectoderm and endoderm of the CAM display functional differences conforms to the hypothesis that differential expression of glycoconjugate microdomains likely contributes to such functional specialization.

Differential lectin binding to microvascular endothelial glycoconjugates during normal angiogenesis in the chick chorioallantoic membrane.

The ontogenesis of endothelial glycoconjugate expression during normal angiogenesis and microvascular development remains unknown. Using intravital fluorescent microscopy, we studied temporal and spatial lectin binding to carbohydrate moieties of luminal microvascular endothelia of the chick embryo chorioallantoic membrane (CAM) during Days 4.5 to 6.0 of the 21-day incubation. We used a battery of eight FITC-lectins (100-200 micrograms/ml). Fluorescent images from precapillary, capillary, and postcapillary segments of the lectin-perfused microvascular units were analyzed by image analysis software to quantitate differences in lectin binding. Results served to indicate a significant decrease in lectin binding to terminal N-acetyl glucosamine, N-acetyl galactosamine, and the N-acetyl galactosamine disaccharide in the glycocalyx of pre- and postcapillary vessels from Day 4.5 to Day 5.0. Lectin binding to N-acetyl glucosamine and N-acetyl galactosamine subsequently increased at Days 5.0 and 6.0. In the capillaries, lectin binding to endothelial galactose, fucose, and sialic acid increased significantly from Day 5.5 to Day 6.0. That these temporal changes in lectin binding to endothelial luminal glycoconjugates coincide with concomitant changes in CAM microvascular permeability (Rizzo et al., in press) serves to suggest a possible association between expression of endothelial glycoconjugates and the ontogeny of microvascular perm-selectivity during normal angiogenesis.

Organic toxicants and mutagens in ashes from eighteen municipal refuse incinerators.

Ashes, obtained from about one-fourth of the operating municipal refuse incinerators in the United States, were analyzed for a range of organic toxicants and mutagens. Thirty percent of the ash samples, which consisted of bottom ash or bottom ash-fly ash mixtures, contained 20-74% organic matter. Thirty percent of the ashes contained direct-acting and/or promutagens which revertedSalmonella typhimurium TA98 or TA100. Sixty percent of the ashes contained more than 5 ng/g of polychlorinated biphenyls. The concentration of tetra- and pentachlorinated biphenyls were higher than the mono-, di-, hepta- and octachlorinated biphenyls. A similar distribution of congeners was seen in polychlorinated dibenzodioxins found in the ashes. The major volatileN-nitroso compounds found in the ashes wereN-nitrosodimethylamine andN-nitrosomorpholine. Other classes of compounds which were found in the ashes included chlorinated benzenes, phthalates, and substituted benzothiophenes.

Hepatic monooxygenase induction and promutagen activation in channel catfish from a contaminated river basin.

To better understand the etiology of cancer in fish from polluted waters, the impact of environmental contaminants on xenobiotic metabolism of channel catfish (Ictalurus punctatus) from a highly polluted water body, Devil's Swamp in southeastern Louisiana, has been investigated. Fish from Devil's Swamp bioaccumulated polynuclear aromatic hydrocarbons (PAH), chlorinated hydrocarbon insecticides (CHI), and polychlorinated biphenyls (PCB) in fat tissue, the latter exceeding 7000 ppb. Reference catfish from the University farm, Ben Hur, were virtually devoid of PAH, CHI, and PCB. Liver microsomal enzymes (MFO) from Devil's Swamp fish were markedly induced. The specific content of cytochromes P450 and b5 and the specific activities of NAD(P)H-cytochrome c reductase were two to three times higher than those of Ben Hur fish. Consistent with this induction, a 9000g supernatant from Devil's Swamp but not Ben Hur fish activated 2-aminofluorene and benzo[a]pyrene (BP) to mutagens in the Ames test. BP metabolism by Devil's Swamp fish liver microsomes was inhibited to a greater extent by alpha-naphthoflavone than was BP metabolism by Ben Hur fish microsomes. This finding indicates that the induced activity in the Devil's Swamp fish liver was the result of P450 isozymes characteristic of PAH/PCB induction. Thus, exposure of fish to environmental pollutants can alter MFO leading to enhanced metabolic activation of promutagens to mutagens.

Comparative mutagenicity of nine brands of coffee to Salmonella typhimurium TA100, TA102, and TA104.

Nine coffee preparations, four caffeinated instant brands, three caffeinated drip coffees, and two decaffeinated coffees, one of which was an instant brand, were evaluated for mutagenicity by the Ames assay using Salmonella typhimurium TA100, TA102, and TA104. All the coffees contained direct-acting mutagens, which reverted the three strains. The inclusion of a rat microsomal enzyme preparation reduced the mutagenic response of the three strains in the presence of some of the coffee samples. Both glyoxal and methylglyoxal, 1,2-dicarbonyls found in the coffees were mutagenic. The concentration of glyoxal, methyglyoxal, diacetyl, and guiacol were measured by gas chromatography/mass spectrometry. Caffeine, furfural, and 5-methylfurfural concentrations were determined by high performance liquid chromatography. Although lower concentrations of methyglyoxal were found in the drip caffeinated coffees, the mutagenic potency of these preparations was higher than the instant coffees on a weight basis especially when TA104 was the indicator organism. Our findings agree with those of other workers who have shown that carbonyl compounds, which were present in all the brands tested, are partially responsible for the mutagenic response of coffee but that additional mutagens are also present.