Automation improves repeatability of retinal oximetry measurements.

PURPOSE: Retinal oximetry is a technique based on spectrophotometry where images are analyzed with software capable of calculating vessel oxygen saturation and vessel diameter. In this study, the effect of automation of measurements of retinal vessel oxygen saturation and vessel diameter is explored. METHODS: Until now, operators have had to choose each vessel segment to be measured explicitly. A new, automatic version of the software automatically selects the vessels once the operator defines a measurement area. Five operators analyzed image pairs from the right eye of 23 healthy subjects with semiautomated retinal oximetry analysis software, Oxymap Analyzer (v2.5.1), and an automated version (v3.0). Inter- and intra-operator variability was investigated using the intraclass correlation coefficient (ICC) between oxygen saturation measurements of vessel segments in the same area of the retina. RESULTS: For semiautomated saturation measurements, the inter-rater ICC was 0.80 for arterioles and venules. For automated saturation measurements, the inter-rater ICC was 0.97 for arterioles and 0.96 for venules. For semiautomated diameter measurements, the inter-rater ICC was 0.71 for arterioles and venules. For automated diameter measurements the inter-rater ICC was 0.97 for arterioles and 0.95 for venules. The inter-rater ICCs were different (p < 0.01) between the semiautomated and automated version in all instances. CONCLUSION: Automated measurements of retinal oximetry values are more repeatable compared to measurements where vessels are selected manually.

Immunohistochemical analysis of L1 cell adhesion molecule and high endothelial venules in breast cancer brain metastasis.

BACKGROUND: The vasculature is a crucial factor in tumor development. Vascular co-option achieved by the L1 cell adhesion molecule (L1CAM) and lymphocyte recruitment inside tumors by high endothelial venules (HEVs) are important prognostic factors in primary breast cancer. Their status in breast cancer brain metastasis is unknown. AIM OF THE STUDY: To explore the status of L1CAMs and HEVs in this tumor compartment. MATERIAL AND METHODS: Thirty resected breast cancer brain metastases were immunohistochemically studied for L1CAM and MECA-79, an HEV marker. Clinicopathological factors were recorded. RESULTS: Age at brain metastasis diagnosis ranged from 37 to 80 years (median 55). The time to brain metastasis development after primary tumor diagnosis ranged from 12 to 187 months (median 57). Median overall survival after brain metastasis diagnosis was 29 months. None of the tumors expressed the factors studied. CONCLUSION: L1CAM and high endothelial venules are not found in breast cancer brain metastasis.

In vivo Imaging of the Cerebral Endothelial Glycocalyx in Mice.

BACKGROUND/AIMS: Endothelial glycocalyx refers to the proteoglycan or glycoprotein layer of vessel walls and has critical physiological functions. Cerebral glycocalyx may have additional functions considering the blood-brain barrier and other features. However, the assessment of it has only been performed ex vivo, which includes processes presumably damaging the glycocalyx layer. Here we visualize and characterize the cerebral endothelial glycocalyx in vivo. METHODS: We visualized and quantified the cerebral endothelial glycocalyx in vivo under a 2-photon microscope by tagging glycocalyx and vessel lumen with wheat germ agglutinin lectin and dextran, respectively. The radial intensity was analyzed to measure the thickness of the cerebral endothelial glycocalyx in each vessel type. RESULTS: Cerebral arteries and capillaries have an intact endothelial glycocalyx, but veins and venules do not. The thickness of the glycocalyx layer in pial arteries, penetrating arteries, and capillaries was different; however, it was not correlated with the vessel diameter within each vessel type. CONCLUSION: We characterized the distribution of the cerebral endothelial glycocalyx in vivo. Compared to the results from ex vivo studies, the layer is thicker, indicating that the layer may be damaged in ex vivo systems. We also observed an inhomogeneous cerebral endothelial glycocalyx distribution that might reflect the functional heterogeneity of the vessel type.

Primary cutaneous follicular helper T-cell lymphoma treated with allogeneic bone marrow transplantation: immunohistochemical comparison with angioimmunoblastic T-cell lymphoma.

Primary cutaneous follicular helper T (TFH)-cell lymphoma has recently been proposed, and is characterized by proliferation of malignant T cells expressing TFH-cell markers, such as CXCL13, accompanied by numerous reactive B cells. We report here a patient whose skin histology showed massive infiltration of both T and B cells, with a proliferation of arborizing high endothelial venules and follicular dendritic cells. Infiltrating T cells were positive for CXCL13, programmed death (PD)-1, inducible T-cell co-stimulator, and BCL-6. Southern blot analyses using DNA from the skin revealed monoclonality of both T and B cells. The patient had marked resistance to treatments, and complete remission was achieved only after allogeneic stem cell transplantation. The present case showed overlapping features with angio-immunoblastic T-cell lymphoma (AITL), although systemic symptoms were not observed. Further study is needed to define the criteria of this provisional entity, representing the cutaneous counterpart of the nodal follicular peripheral T-cell lymphoma or AITL.

Is quality of diet associated with the microvasculature? An analysis of diet quality and retinal vascular calibre in older adults.

It is unknown whether diet quality is associated with microvascular structure. The present study aimed to investigate the relationship between diet quality, reflecting adherence to dietary guidelines, with retinal microvascular calibre in older adults. The dietary data of 2720 Blue Mountains Eye Study participants, aged 50+ years, were collected using a semi-quantitative FFQ. A modified version of the Healthy Eating Index for Australians was developed to determine total diet scores (TDS). Fundus photographs were taken and retinal vascular calibre measured using computer-assisted techniques and summarised. After adjusting for age, sex, BMI, mean arterial blood pressure, smoking, serum glucose, leucocyte count and history of diagnosed stroke or CHD, persons with higher TDS had healthier retinal vessels cross-sectionally, with wider retinal arteriolar calibre (by approximately 3 µm, comparing the highest with the lowest quartile of TDS, Ptrend = 0·0001) and narrower retinal venular calibre (by approximately 2·5 µm; Ptrend = 0·02). In younger subjects aged ≤65 years, increasing TDS (lowest to the highest quartile) was associated with healthier retinal vessels: approximately 4·4 µm wider retinal arteriolar (Ptrend < 0·0001) and approximately 2·3 µm narrower venular calibre (Ptrend = 0·03). After multivariable adjustment, however, baseline TDS were not associated with retinal arteriolar (Ptrend = 0·89) or venular calibre (Ptrend = 0·25), 5 years later. Also, baseline TDS were not associated with the 5-year change in retinal arteriolar (ß = 0·14; P=0·29) or venular calibre (ß = - 0·26; P=0·07). Greater compliance with published dietary guidelines (higher diet quality) was cross-sectionally associated with wider retinal arterioles and narrower venules, indicating better retinal microvascular health.

Novel 3D analysis of Claudin-5 reveals significant endothelial heterogeneity among CNS microvessels.

Tight junctions (TJs) feature critically in maintaining the integrity of the blood-brain barrier (BBB), and undergo significant disruption during neuroinflammatory diseases. Accordingly, the expression and distribution of CLN-5, a prominent TJ protein in central nervous system (CNS) microvessels and BBB determinant, has been shown to parallel physiological and pathophysiological changes in microvascular function. However, efforts to quantify CLN-5 within the CNS microvasculature in situ, by using conventional two-dimensional immunohistochemical analysis of thin sections, are encumbered by the tortuosity of capillaries and distorted diameters of inflamed venules. Herein, we describe a novel contour-based 3D image visualization and quantification method, employing high-resolution confocal z-stacks from thick immunofluorescently-stained thoraco-lumbar spinal cord cryosections, to analyze CLN-5 along the junctional regions of different-sized CNS microvascular segments. Analysis was performed on spinal cords of both healthy mice, and mice experiencing experimental autoimmune encephalomyelitis (EAE), an animal model of the neuroinflammatory disease multiple sclerosis. Results indicated that, under normal conditions, the density of CLN-5 staining (CLN-5 intensity/ endothelial surface area) was greatest in the capillaries and smaller venules, and least in the larger venules. This heterogeneity in junctional CLN-5 staining was exacerbated during EAE, as spinal venules revealed a significant loss of junctional CLN-5 staining that was associated with focal leukocyte extravasation, while adjacent capillaries exhibited neither CLN-5 loss nor infiltrating leukocytes. However, despite only venules displaying these behaviors, both capillaries and venules evidenced leakage of IgG during disease, further underscoring the heterogeneity of the inflammatory response in CNS microvessels. This method should be readily adaptable to analyzing other junctional proteins of the CNS and peripheral microvasculature, and serve to highlight their role(s) in health and disease.

Microvenular hemangioma presenting with numerous bilateral macules, patches, and plaques: a case report and review of the literature.

Microvenular hemangioma (MVH) is a rare, slowly growing, benign vascular tumor that typically presents as a solitary enlarging plaque or nodule on the trunk or the extremities of young to middle-aged adults. A minority of MVH present with multiple lesions that are either gradually or suddenly acquired (eruptive MVH). Herein, we report a case of a 53-year-old woman who progressively developed numerous bilateral MVHs presenting as enlarging, blanching, erythematous to violaceous macules, patches, and plaques over the proximal thighs and axillae. Two biopsies exhibited the irregular branching venules with inconspicuous lumina lacking endothelial atypia and associated with dermal fibrosis characteristic of MVH. Immunophenotypically, the endothelium expressed Wilms Tumor 1, CD31, CD34, and erythrocyte-type glucose transporter protein (GLUT-1) GLUT-1 focally and was negative for Human herpes virus 8 and the lymphatic marker D2-40. In addition, numerous dermal spindle cells expressing CD34 and procollagen, putative fibrocytes, surrounded the thickened dermal collagen bundles and small vessels of MVH implicating a reactive/reparative (proliferative) process due to an unrecognized cutaneous injury. A review of MVH summarizing its clinicopathologic findings and its natural history is presented.

Calibration of glucose oxidase-based test strips for capillary blood measurement with oxygen saturated venous blood samples.

BACKGROUND: Glucose oxidase biosensors are used in self-monitoring blood glucose concentrations. The capillary blood glucose quantitation requires a calibration curve. Due to the limitation in obtaining calibration curve from capillary blood, an alternate approach by using venous blood for neonatal measurement was investigated. METHODS: A signal correlation between oxygen saturated venous blood and capillary blood was derived. The hematocrit effect was studied for different glucose concentrations. The calibrated glucose strips were validated by neonatal intensive care unit (NICU) samples. RESULTS: A simple equation, finger blood signal=1.39∗(oxygen saturated venous blood signal)-31.2 was derived. The rate of change in glucose concentration due to hematocrit effect was low in lower glucose concentration samples. The BeneCheck Glucose Strips were compared with Beckman Coulter analyzer by using 52 NICU samples. More than 95% of test results were within the variation of ±10 mg/dl of bias and ±15% of bias% when glucose concentration is <75 mg/dl and ≥75 mg/dl respectively. CONCLUSIONS: The BeneCheck Glucose Strips can be accurately calibrated with venous blood. The hematocrit effect can also be predicted. Based on this study, BeneCheck Blood Glucose Monitoring System can be suitable for neonatal glucose measurement.

Endothelial VE-cadherin expression in human lungs.

Cell adhesion molecule vascular endothelial cadherin (VE-cadherin) is the major component of endothelial adherence junctions, maintaining endothelial cell integrity. Studies dealing with constitutive VE-cadherin expression patterns in different pulmonary vessel types (arteries, arterioles, capillaries, venules, veins) or with the influence of physiological factors such as age or sex on VE-cadherin expression have not been published yet. Knowledge of constitutive resp. varying expression patterns not only fundamentally contribute to understanding the role of VE-cadherin in the pathogenesis of pulmonary diseases but also help to develop therapies based on immunotargeting. Hence, endothelial VE-cadherin expression was studied in regular lung tissue. Fifty-eight specimens of regular lung tissue (30 females, 28 males between 1 month and 75 years old) were immunohistochemically stained with an antibody against VE-cadherin. There was strong endothelial expression of VE-cadherin in arteries, arterioles, and capillaries but almost no expression in veins and venules. Neither age nor sex had any influence on the expression pattern or staining intensity. There is a vessel type-specific expression pattern for VE-cadherin in regular human lung tissue, which is not influenced by age or sex. Further studies will have to prove whether this is influenced by pathological conditions, e.g., ARDS.

Arterial versus capillary blood gases: a meta-analysis.

A meta-analysis determined whether capillary blood gases accurately reflect arterial blood samples. A mixed effects model was used on 29 relevant studies obtained from a PubMed/Medline search. From 664 and 222 paired samples obtained from the earlobe and fingertip, respectively, earlobe compared to fingertip sampling shows that the standard deviation of the difference is about 2.5x less (or the precision is 2.5x better) in resembling arterial PO(2) over a wide range of arterial PO(2)'s (21-155 mm Hg ). The lower the arterial PO(2), the more accurate it is when predicting arterial PO(2) from any capillary sample (p<0.05). However, while earlobe sampling predicts arterial PO(2) (adjusted r(2)=0.88, mean bias=3.8 mm Hg compared to arterial), fingertip sampling does not (adjusted r(2)=0.48, mean bias=11.5 mm Hg compared to arterial). Earlobe sampling is slightly more accurate compared to fingertip sampling in resembling arterial PCO(2) (arterial versus earlobe, adjusted r(2)=0.94, mean bias=1.9 mm Hg ; arterial versus fingertip, adjusted r(2)=0.95, mean bias=2.2 mm Hg compared to arterial) but both sites can closely reflect arterial PCO(2) (880 total paired samples, range 10-114 mm Hg ). No real difference between sampling from the earlobe or fingertip were found for pH as both sites accurately reflect arterial pH over a wide range of pH (587 total paired samples, range 6.77-7.74, adjusted r(2)=0.90-0.94, mean bias=0.02). In conclusion, sampling blood from the fingertip or earlobe (preferably) accurately reflects arterial PCO(2) and pH over a wide range of values. Sampling blood, too, from earlobe (but never the fingertip) may be appropriate as a replacement for arterial PO(2), unless precision is required as the residual standard error is 6 mm Hg when predicting arterial PO(2) from an earlobe capillary sample.

Differential coronary microvascular exchange responses to adenosine: roles of receptor and microvessel subtypes.

OBJECTIVE: To assess the role of adenosine receptors in the regulation of coronary microvascular permeability to porcine serum albumin (P(s)(PSA)). METHODS: Solute flux was measured in single perfused arterioles and venules isolated from pig hearts using fluorescent dye-labeled probes by microspectro-fluorometry. Messenger RNA, protein, and cellular distribution of adenosine receptors in arterioles and venules were analyzed by RT-PCR, immunoblot, and immunofluorescence. RESULTS: Control venule P(s)(PSA) (10.7 +/- 4.8 x 10(- 7) cm x s(- 1)) was greater than that of arterioles (6.4+/- 2.8 x 10(-7) cm . s(-1); p < .05). Arteriolar P(s)(PSA) decreased (p < .05) with adenosine suffusion over the range from 10(- 8) to 10(-5) M, while venular P(s)(PSA) did not change. The nonselective A(1) and A(2) receptor antagonist, 8-(p-sulfophenyl) theophylline, blocked the adenosine-induced decrease in arteriolar P(s)(PSA). Messenger RNA for adenosine A(1), A(2A), A(2B), and A(3) receptors was expressed in arterioles and venules. Protein for A(1), A(2A), and A(2B), but not A(3), was detected in both microvessel types and was further demonstrated on vascular endothelial cells. CONCLUSION: Arteriolar P(s)(PSA) decreases with adenosine suffusion but not venular P(s)(PSA). Adenosine A(1), A(2A), and A(2B) receptors are expressed in both arterioles and venules. Selective receptor-linked cellular signaling mechanisms underlying the regulation of permeability remain to be determined.

Endomucin, a sialomucin expressed in high endothelial venules, supports L-selectin-mediated rolling.

Lymphocyte homing to lymph nodes is regulated by transient but specific interactions between lymphocytes and high endothelial venules (HEVs), the initial phase of which is mainly governed by the leukocyte adhesion molecule L-selectin, which recognizes sulfated and sialylated O-linked oligosaccharides displayed on sialomucin core proteins. One of the sialomucin proteins, endomucin, is predominantly expressed in vascular endothelial cells of a variety of tissues including the HEVs of lymph nodes; however, whether it functions as a ligand for L-selectin remains to be formally proven. Here we show that the endomucin splice isoform a is predominantly expressed in PNAd+ HEVs and MAdCAM-1+ HEVs, as seen in non-HEV-type vascular endothelial cells. Using affinity purification with soluble L-selectin, we found that HEV endomucin is specifically modified with L-selectin-reactive oligosaccharides and can bind L-selectin as well as an HEV-specific mAb, MECA-79. Our results also indicated that a 90-100 kDa endomucin species is preferentially decorated with L-selectin-reactive sugar chains, whereas an 80 kDa species represents conventional forms expressed in non-HEV-type vascular endothelial cells in lymph nodes. Furthermore, a CHO cell line expressing endomucin together with a specific combination of carbohydrate-modifying enzymes [core-2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT), alpha-1,3-fucosyltransferase VII (FucTVII) and L-selectin ligand sulfotransferase (LSST)] showed L-selectin-dependent rolling under flow conditions in vitro. These results suggest that when endomucin is appropriately modified by a specific set of glycosyltransferases and a sulfotransferase, it can function as a ligand for L-selectin, and that the endomucin expressed in HEVs may represent another sialomucin ligand for L-selectin.

CD44-chondroitin sulfate interactions mediate leukocyte rolling under physiological flow conditions.

CD44 on leukocytes binds to its glycosaminoglycan (GAG) ligand, hyaluronic acid, and mediates the rolling of leukocytes on vascular endothelial cells. We previously reported that the recombinant CD44 protein binds to other GAGs, including chondroitin sulfates (CS), although the physiological significance of this interaction has remained unclear. Here we report that the CD44 expressed on mouse lymphoma BW5147 cells supports cell binding to immobilized CS under static conditions and mediates cell rolling in CS-coated glass capillary tubes under shear stresses ranging from 0.5 to 1.5 dyn/cm(2), which is within the physiological range of forces in venules. Both interactions were completely inhibited by pretreating the cells with an anti-CD44 antibody or by pretreating the CS with chondroitinase ABC, but not hyaluronidase. To address the role of the CD44-CS interaction in vivo, we examined the tissue localization of the CS that interacts with CD44. Interestingly, a recombinant CD44 fusion protein bound to hepatic sinuosoidal endothelial cells where CS was also expressed, as assessed by immunohistochemistry. These findings support the involvement of the CD44-CS interaction in the primary adhesion of lymphocytes to endothelial cells and raise the possibility that this interaction plays a role in the capture of CD44-positive cells, such as activated T cells and certain tumor cells, by the hepatic sinusoidal vasculature.

Lymphocyte-HEV interactions in lymph nodes of a sulfotransferase-deficient mouse.

The interaction of L-selectin expressed on lymphocytes with sulfated sialomucin ligands such as CD34 and GlyCAM-1 on high endothelial venules (HEV) of lymph nodes results in lymphocyte rolling and is essential for lymphocyte recruitment. HEC-GlcNAc6ST-deficient mice lack an HEV-restricted sulfotransferase with selectivity for the C-6 position of N-acetylglucosamine (GlcNAc). HEC-GlcNAc6ST-/- animals exhibit faster lymphocyte rolling and reduced lymphocyte sticking in HEV, accounting for the diminished lymphocyte homing. Isolated CD34 and GlyCAM-1 from HEC-GlcNAc6ST-/- animals incorporate approximately 70% less sulfate than ligands from wild-type animals. Furthermore, these ligands exhibit a comparable reduction of the epitope recognized by MECA79, a function-blocking antibody that reacts with L-selectin ligands in a GlcNAc-6-sulfate-dependent manner. Whereas MECA79 dramatically inhibits lymphocyte rolling and homing to lymph nodes in wild-type mice, it has no effect on HEC-GlcNAc6ST-/- mice. In contrast, in vitro rolling on purified GlyCAM-1 from HEC-GlcNAc6ST-/- mice, although greatly diminished compared with that on the wild-type ligand, is inhibited by MECA79. Our results demonstrate that HEC-GlcNAc6ST contributes predominantly, but not exclusively, to the sulfation of HEV ligands for L-selectin and that alternative, non-MECA79-reactive ligands are present in the absence of HEC-GlcNAc6ST.

Human cerebrospinal fluid central memory CD4+ T cells: evidence for trafficking through choroid plexus and meninges via P-selectin.

Cerebrospinal fluid (CSF) from healthy individuals contains between 1,000 and 3,000 leukocytes per ml. Little is known about trafficking patterns of leukocytes between the systemic circulation and the noninflamed CNS. In the current study, we characterized the surface phenotype of CSF cells and defined the expression of selected adhesion molecules on vasculature in the choroid plexus, the subarachnoid space surrounding the cerebral cortex, and the cerebral parenchyma. Using multicolor flow cytometry, we found that CSF cells predominantly consisted of CD4+/CD45RA-/CD27+/CD69+-activated central memory T cells expressing high levels of CCR7 and L-selectin. CD3+ T cells were present in the choroid plexus stroma in autopsy CNS tissue sections from individuals who died without known neurological disorders. P- and E-selectin immunoreactivity was detected in large venules in the choroid plexus and subarachnoid space, but not in parenchymal microvessels. CD4+ T cells in the CSF expressed high levels of P-selectin glycoprotein ligand 1, and a subpopulation of circulating CD4+ T cells displayed P-selectin binding activity. Intercellular adhesion molecule 1, but not vascular cell adhesion molecule 1 or mucosal addressin cell adhesion molecule 1, was expressed in choroid plexus and subarachnoid space vessels. Based on these findings, we propose that T cells are recruited to the CSF through interactions between P-selectin/P-selectin ligands and intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 in choroid plexus and subarachnoid space venules. These results support the overall hypothesis that activated memory T cells enter CSF directly from the systemic circulation and monitor the subarachnoid space, retaining the capacity to either initiate local immune reactions or return to secondary lymphoid organs.

Expression of the vascular endothelial growth factor (VEGF) family in human endometrial blood vessels.

Endometrial regrowth is associated with intense angiogenesis, for which vascular endothelial growth factor-A (VEGF-A) is an important regulator. However, the expression of other members of the VEGF family is less well documented. The aim of this study was to localize members of the VEGF family (VEGF-A, -B and -C), and their receptors (VEGFR1, 2 and 3) in human endometrial blood vessels. Endometrial biopsies collected from four healthy and fertile women were used for immunohistochemistry assessments. Co-localization of VEGF-family proteins with CD34 stained endothelial structures was determined by image analysis. We demonstrate here the marked expression of VEGF-A as well as VEGFR2 and 3 in capillaries. Arterioles expressed VEGF-B, VEGFR1, 2, and 3 moderately and VEGF-A variably. Venules expressed only VEGFR3 markedly. In contrast, VEGF-C was not expressed in the arterioles, but moderately in the capillaries and weakly in the venules. VEGF-B was expressed in all blood vessels; however, VEGF-B was weakly expressed in capillaries and arterioles and moderately expressed in venules and arterioles. Thus, expression of VEGF-A. B and C and VEGF receptors 1-3 in endometrial blood vessels indicates a highly structured involvement of VEGF in the regulation of angiogenesis in the human endometrium.

Molecular cloning of SLC26A7, a novel member of the SLC26 sulfate/anion transporter family, from high endothelial venules and kidney.

A unique characteristic of endothelial cells from high endothelial venules (HEVEC) in lymphoid organs and chronically inflamed tissues is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. We have previously shown that HEVEC express two functional classes of sulfate transporters: sodium/sulfate cotransporters and sulfate/anion exchangers. Here, we report the molecular cloning from human HEVEC of a 2.9-kb cDNA encoding SLC26A7, a novel member of the SLC26 (solute carrier 26) sulfate/anion exchanger family. SLC26A7 exhibits 30% identity with three known sulfate transporters from the SLC26 family: SLC26A2 (also known as DTDST), SLC26A1 (also known as SAT1), and SLC26A3 (also known as DRA). Northern blot analysis revealed specific expression of SLC26A7 mRNA in kidney. Alternative splicing and polyadenylation of SLC26A7 pre-mRNA in kidney suggest the existence of two protein isoforms, SLC26A7.1 and SLC26A7.2, differing in their carboxy termini.

Novel sulfated lymphocyte homing receptors and their control by a Core1 extension beta 1,3-N-acetylglucosaminyltransferase.

L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to addressins expressed in the high endothelial venules (HEV) of secondary lymphoid organs. Peripheral node addressin recognized by the MECA-79 antibody is apparently part of the L-selectin ligand, but its chemical nature has been undefined. We now identify a sulfated extended core1 mucin-type O-glycan, Gal beta 1-->4(sulfo-->6)GlcNAc beta 1-->3Gal beta 1-->3GalNAc, as the MECA-79 epitope. Molecular cloning of a HEV-expressed core1-beta 1,3-N-acetylglucosaminyltransferase (Core1-beta 3GlcNAcT) enabled the construction of the 6-sulfo sialyl Lewis x on extended core1 O-glycans, recapitulating the potent L-selectin-mediated, shear-dependent adhesion observed with novel L-selectin ligands derived from core2 beta1,6-N-acetylglucosaminyltransferase-I null mice. These results identify Core1-beta 3GlcNAcT and its cognate extended core1 O-glycans as essential participants in the expression of the MECA-79-positive, HEV-specific L-selectin ligands required for lymphocyte homing.

P-selectin glycoprotein ligand-1 supports rolling on E- and P-selectin in vivo.

Selectin-dependent rolling is the earliest observable event in the recruitment of leukocytes to inflamed tissues. Several glycoproteins decorated with sialic acid, fucose, and/or sulfate have been shown to bind the selectins. The best-characterized selectin ligand is P-selectin glycoprotein-1 (PSGL-1) that supports P-selectin- dependent rolling in vitro and in vivo. In vitro studies have suggested that PSGL-1 may also be a ligand for E- and L-selectins. To study the in vivo function of PSGL-1, without the influence of other leukocyte proteins, the authors observed the interaction of PSGL-1-coated microspheres in mouse venules stimulated to express P- and/or E-selectin. Microspheres coated with functional recombinant PSGL-1 rolled in surgically stimulated and tumor necrosis factor alpha (TNFalpha)-stimulated mouse venules. P-selectin deficiency or inhibition abolished microsphere rolling in surgically and TNFalpha-stimulated venules, whereas E-selectin deficiency or inhibition increased microsphere rolling velocity in TNFalpha-stimulated venules. The results suggest that P-selectin-PSGL-1 interaction alone is sufficient to mediate rolling in vivo and that E-selectin-PSGL-1 interaction supports slow rolling.