The microbiota–gut–brain axis (MGBA) involves bidirectional communication between intestinal microbiota and the gastrointestinal (GI) tract, central nervous system (CNS), neuroendocrine/neuroimmune systems, hypothalamic–pituitary–adrenal (HPA) axis, and enteric nervous system (ENS). The intestinal microbiota can influence host physiology and pathology. Dysbiosis involves the loss of beneficial microbial input or signal, diversity, and expansion of pathobionts, which can lead to loss of barrier function and increased intestinal permeability (IP). Colostrum, the first milk from mammals after birth, is a natural source of nutrients and is rich in oligosaccharides, immunoglobulins, growth factors, and anti-microbial components. The aim of this study was to investigate if bovine colostrum (BC) administration might modulate intestinal microbiota and, in turn, behavior in two mouse models, wild-type (WT) and Zonulin transgenic (Ztm)—the latter of which is characterized by dysbiotic microbiota, increased intestinal permeability, and mild hyperactivity—and to compare with control mice. Bioinformatics analysis of the microbiome showed that consumption of BC was associated with increased taxonomy abundance (p = 0.001) and diversity (p = 0.004) of potentially beneficial species in WT mice and shifted dysbiotic microbial community towards eubiosis in Ztm mice (p = 0.001). BC induced an anxiolytic effect in WT female mice compared with WT female control mice (p = 0.0003), and it reduced anxiogenic behavior in Ztm female mice compared with WT female control mice (p = 0.001), as well as in Ztm male mice compared with WT BC male mice (p = 0.03). As evidenced in MGBA interactions, BC supplementation may well be applied for prophylactic approaches in the future. Further research is needed to explore human interdependencies between intestinal microbiota, including eubiosis and pathobionts, and neuroinflammation, and the potential value of BC for human use. The MGH Institutional Animal Care and Use Committee authorized the animal study (2013N000013).

Zonulin is a physiologic epithelial and endothelial permeability modulator. Zonulin increases antigen trafficking from the gut lumen into the bloodstream and in between body compartments, a mechanism linked to many chronic inflammatory diseases. Upon its initial discovery, it was noted that zonulin was not a single protein, but rather a family of structurally and functionally related proteins referred to as the zonulin family proteins (ZFPs). ZFPs are members of the mannose associated serine proteases (MASP) family and are the result of high mutation rates leading to many zonulin polymorphisms. Pre-haptoglobin 2, the precursor of haptoglobin 2, was identified as the first eukaryotic member of the ZFPs, and properdin, a key positive regulator of the alternative pathway, as a second member. In this study, we report two additional proteins that are likely ZFPs. Human coagulation factor X (FX) and CD5 antigen-like (CD5L). Both FX and CD5L recombinant proteins were detected by anti-zonulin antibody in Western immunoblot analysis, and both proteins decreased epithelial barrier competency of Caco-2 cell monolayers as established by the Trans Epithelial Electrical Resistance (TEER) assay. These results indicate that FX and CD5L have structural and functional similarities with previously identified ZFPs and, therefore, can be considered new members of this family of proteins.

The intestinal barrier comprises a single layer of epithelial cells tightly joined to form a physical barrier. Disruption or compromise of the intestinal barrier can lead to the inadvertent activation of immune cells, potentially causing an increased risk of chronic inflammation in various tissues. Recent research has suggested that specific dietary components may influence the function of the intestinal barrier, potentially offering a means to prevent or mitigate inflammatory disorders. However, the precise mechanism underlying these effects remains unclear. Bovine colostrum (BC), the first milk from cows after calving, is a natural source of nutrients with immunomodulatory, anti-inflammatory, and gut-barrier fortifying properties. This novel study sought to investigate the transcriptome in BC-treated Zonulin transgenic mice (Ztm), characterized by dysbiotic microbiota, intestinal hyperpermeability, and mild hyperactivity, applying RNA sequencing. Seventy-five tissue samples from the duodenum, colon, and brain of Ztm and wild-type (WT) mice were dissected, processed, and RNA sequenced. The expression profiles were analyzed and integrated to identify differentially expressed genes (DEGs) and differentially expressed transcripts (DETs). These were then further examined using bioinformatics tools. RNA-seq analysis identified 1298 DEGs and 20,952 DETs in the paired (Ztm treatment vs. Ztm control) and reference (WT controls) groups. Of these, 733 DEGs and 10,476 DETs were upregulated, while 565 DEGs and 6097 DETs were downregulated. BC-treated Ztm female mice showed significant upregulation of cingulin (Cgn) and claudin 12 (Cldn12) duodenum and protein interactions, as well as molecular pathways and interactions pertaining to tight junctions, while BC-treated Ztm males displayed an upregulation of transcripts like occludin (Ocln) and Rho/Rac guanine nucleotide exchange factor 2 (Arhgf2) and cellular structures and interfaces, protein–protein interactions, and organization and response mechanisms. This comprehensive analysis reveals the influence of BC treatment on tight junctions (TJs) and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling pathway gene expressions. The present study is the first to analyze intestinal and brain samples from BC-treated Ztm mice applying high-throughput RNA sequencing. This study revealed molecular interaction in intestinal barrier function and identified hub genes and their functional pathways and biological processes in response to BC treatment in Ztm mice. Further research is needed to validate these findings and explore their implications for dietary interventions aimed at improving intestinal barrier integrity and function. The MGH Institutional Animal Care and Use Committee authorized the animal study (2013N000013).

The gut-brain axis hypothesis suggests that interactions in the intestinal milieu are critically involved in regulating brain function. Several studies point to a gut-microbiota-brain connection linking an impaired intestinal barrier and altered gut microbiota composition to neurological disorders involving neuroinflammation. Increased gut permeability allows luminal antigens to cross the gut epithelium, and via the blood stream and an impaired blood-brain barrier (BBB) enters the brain impacting its function. Pre-haptoglobin 2 (pHP2), the precursor protein to mature HP2, is the first characterized member of the zonulin family of structurally related proteins. pHP 2 has been identified in humans as the thus far only endogenous regulator of epithelial and endothelial tight junctions (TJs). We have leveraged the Zonulin-transgenic mouse (Ztm) that expresses a murine pHP2 (zonulin) to determine the role of increased gut permeability and its synergy with a dysbiotic intestinal microbiota on brain function and behavior. Here we show that Ztm mice display sex-dependent behavioral abnormalities accompanied by altered gene expression of BBB TJs and increased expression of brain inflammatory genes. Antibiotic depletion of the gut microbiota in Ztm mice downregulated brain inflammatory markers ameliorating some anxiety-like behavior. Overall, we show that zonulin-dependent alterations in gut permeability and dysbiosis of the gut microbiota are associated with an altered BBB integrity, neuroinflammation, and behavioral changes that are partially ameliorated by microbiota depletion. Our results suggest the Ztm model as a tool for the study of the cross-talk between the microbiome/gut and the brain in the context of neurobehavioral/neuroinflammatory disorders.

Martinez, Enid; Lan, Jinggang; Miranda-Ribera, Alba; Fiorentino, Maria; Mehta, Nilesh; Fasano, Alessio Author Information

Gastrointestinal infections cause significant morbidity and mortality worldwide. The complexity of human biology and limited insights into host-specific infection mechanisms are key barriers to current therapeutic development. Here, we demonstrate that two-dimensional epithelial monolayers derived from human intestinal organoids, combined with in vivo-like bacterial culturing conditions, provide significant advancements for the study of enteropathogens. Monolayers from the terminal ileum, cecum, and ascending colon recapitulated the composition of the gastrointestinal epithelium, in which several techniques were used to detect the presence of enterocytes, mucus-producing goblet cells, and other cell types following differentiation. Importantly, the addition of receptor activator of nuclear factor kappa-B ligand (RANKL) increased the presence of M cells, critical antigen-sampling cells often exploited by enteric pathogens. For infections, bacteria were grown under in vivo-like conditions known to induce virulence. Overall, interesting patterns of tissue tropism and clinical manifestations were observed. Shigella flexneri adhered efficiently to the cecum and colon; however, invasion in the colon was best following RANKL treatment. Both Salmonella enterica serovars Typhi and Typhimurium displayed different infection patterns, with S. Typhimurium causing more destruction of the terminal ileum and S. Typhi infecting the cecum more efficiently than the ileum, particularly with regard to adherence. Finally, various pathovars of Escherichia coli validated the model by confirming only adherence was observed with these strains. This work demonstrates that the combination of human-derived tissue with targeted bacterial growth conditions enables powerful analyses of human-specific infections that could lead to important insights into pathogenesis and accelerate future vaccine development. IMPORTANCE While traditional laboratory techniques and animal models have provided valuable knowledge in discerning virulence mechanisms of enteric pathogens, the complexity of the human gastrointestinal tract has hindered our understanding of physiologically relevant, human-specific interactions; and thus, has significantly delayed successful vaccine development. The human intestinal organoid-derived epithelial monolayer (HIODEM) model closely recapitulates the diverse cell populations of the intestine, allowing for the study of human-specific infections. Differentiation conditions permit the expansion of various cell populations, including M cells that are vital to immune recognition and the establishment of infection by some bacteria. We provide details of reproducible culture methods and infection conditions for the analyses of Shigella, Salmonella, and pathogenic Escherichia coli in which tissue tropism and pathogen-specific infection patterns were detected. This system will be vital for future studies that explore infection conditions, health status, or epigenetic differences and will serve as a novel screening platform for therapeutic development.

We examined the relationship between zonulin and gastric motility in critical care patients and a translational mouse model of systemic inflammation. Gastric motility and haptoglobin (HP) 2 isoform quantification, proxy for zonulin, were examined in patients. Inflammation was triggered by lipopolysaccharide (LPS) injection in C57Bl/6 zonulin transgenic mouse (Ztm) and wildtype (WT) mice as controls, and gastro-duodenal transit was examined by fluorescein-isothiocyanate, 6 and 12 h after LPS-injection. Serum cytokines and zonulin protein levels, and zonulin gastric-duodenal mRNA expression were examined. Eight of 20 patients [14 years, IQR (12.25, 18)] developed gastric dysmotility and were HP2 isoform-producing. HP2 correlated with gastric dysmotility (r = − 0.51, CI − 0.81 to 0.003, p = 0.048). LPS injection induced a time-dependent increase in IL-6 and KC-Gro levels in all mice (p < 0.0001). Gastric dysmotility was reduced similarly in Ztm and WT mice in a time-dependent manner. Ztm had 16% faster duodenal motility than WT mice 6H post-LPS, p = 0.01. Zonulin mRNA expression by delta cycle threshold (dCT) was higher in the stomach (9.7, SD 1.4) than the duodenum (13.9, SD 1.4) 6H post-LPS, p = 0.04. Serum zonulin protein levels were higher in LPS-injected mice compared to vehicle-injected animals in a time-dependent manner. Zonulin correlated with gastric dysmotility in patients. A mouse model had time-dependent gastro-duodenal dysmotility after LPS-injection that paralleled zonulin mRNA expression and protein levels.

The balanced interplay between epithelial barrier, immune system, and microbiota maintains gut homeostasis, while disruption of this interplay may lead to inflammation. Paracellular permeability is governed by intercellular tight-junctions (TJs). Zonulin is, to date, the only known physiological regulator of intestinal TJs. We used a zonulin transgenic mouse (Ztm) model characterized by increased small intestinal permeability to elucidate the role of a primary impaired gut barrier on microbiome composition and/or immune profile. Ztm exhibit an altered gene expression profile of TJs in the gut compared to wild-type mice (WT): Claudin-15, Claudin-5, Jam-3, and Myosin-1C are decreased in the male duodenum whereas Claudin-15, Claudin-7, and ZO-2 are reduced in the female colon. These results are compatible with loss of gut barrier function and are paralleled by an altered microbiota composition with reduced abundance of the genus Akkermansia, known to have positive effects on gut barrier integrity and strengthening, and an increased abundance of the Rikenella genus, associated to low-grade inflammatory conditions. Immune profile analysis shows a subtly skewed distribution of immune cell subsets toward a pro-inflammatory phenotype with more IL-17 producing adaptive and innate-like T cells in Ztm. Interestingly, microbiota "normalization" involving the transfer of WT microbiota into Ztm, did not rescue the altered immune profile. Our data suggest that a primary impaired gut barrier causing an uncontrolled trafficking of microbial products leads to a latent pro-inflammatory status, with a skewed microbiota composition and immune profile that, in the presence of an environmental trigger, as we have previously described (1), might promote the onset of overt inflammation and an increased risk of chronic disease.

collected for gross and pathologic evaluation, followed by immunohistochemical staining and molecular changes for inflammatory mediators, permeability protein, and RT-PCR.Raman spectroscopy analysis was done to measure biochemical changes according to group.As a result, macroscopic and histological evidences suggested that administration of IND resulted in significant intestinal damage and co-administration of PPZ resulted in worsened IND-induced enteropathy.However, REV significantly mitigated IND-induced enteropathy (p<0.01).Inflammatory cytokines and oxidative stress-associated factors were significantly ameliorated in REV treated group, while these changes were significantly increased in PPZ group.On immunohistochemical staining with ZO-1 and Occludin-1, significantly decreased in either IND or IND + PPZ, but significantly preserved in IND + REV, suggesting REV preserved TJ proteins.ON Raman spectroscopy, significant spectra were noted in IND + REV, signifying RS can be applied in measuring IND-induced intestinal damages.Conclusively, the fact that PPZ aggravated IND-induced small intestinal damages, while improved with REV combination suggested that P-CAB can be applied to secure NSAID-induced intestinal damages, while PPI should be applied only for rescuing gastric damages.

Idiopathic pulmonary fibrosis (IPF) poses challenges to understanding its underlying cellular and molecular mechanisms and the development of better therapies. Previous studies suggest a pathophysiological role for neuraminidase 1 (NEU1), an enzyme that removes terminal sialic acid from glycoproteins. We observed increased NEU1 expression in epithelial and endothelial cells, as well as fibroblasts, in the lungs of patients with IPF compared with healthy control lungs. Recombinant adenovirus-mediated gene delivery of NEU1 to cultured primary human cells elicited profound changes in cellular phenotypes. Small airway epithelial cell migration was impaired in wounding assays, whereas, in pulmonary microvascular endothelial cells, NEU1 overexpression strongly impacted global gene expression, increased T cell adhesion to endothelial monolayers, and disrupted endothelial capillary-like tube formation. NEU1 overexpression in fibroblasts provoked increased levels of collagen types I and III, substantial changes in global gene expression, and accelerated degradation of matrix metalloproteinase-14. Intratracheal instillation of NEU1 encoding, but not control adenovirus, induced lymphocyte accumulation in bronchoalveolar lavage samples and lung tissues and elevations of pulmonary transforming growth factor-β and collagen. The lymphocytes were predominantly T cells, with CD8(+) cells exceeding CD4(+) cells by nearly twofold. These combined data indicate that elevated NEU1 expression alters functional activities of distinct lung cell types in vitro and recapitulates lymphocytic infiltration and collagen accumulation in vivo, consistent with mechanisms implicated in lung fibrosis.

The highly sialylated vascular endothelial surface undergoes changes in sialylation upon adopting the migratory/angiogenic phenotype. We recently established endothelial cell (EC) expression of NEU1 sialidase (Cross, AS et al; J Biol Chem 287:15966-15980, 2012). We asked whether NEU1 might regulate EC capillary-like tube formation on a Matrigel substrate. In human pulmonary microvascular EC (HPMEC)s, prior silencing of NEU1 did not alter tube formation. Infection of HPMECs with increasing MOIs of an adenovirus (Ad) encoding for catalytically-active wild-type (WT) NEU1 dose-dependently impaired tube formation whereas overexpression of either a catalytically-dead NEU1 mutant, NEU1-G68V, or another sialidase, NEU3, did not. NEU1 overexpression also diminished EC adhesion to the Matrigel substrate and restrained EC migration in a wounding assay. In HPMECs, the adhesion molecule, CD31, also known as platelet endothelial cell adhesion molecule (PECAM)-1, was sialylated via α2,6-linkages, as shown by Sambucus nigra agglutinin (SNA) lectin blotting. NEU1 overexpression increased CD31 binding to Arachis hypogaea or peanut agglutinin (PNA) lectin, indicating CD31 desialylation. In the postconfluent state, when CD31 ectodomains are homophilically engaged, NEU1 was recruited to and desialylated CD31. 1 http://www.jbc.org/cgi/doi/10.1074/jbc.M114.555888 The latest version is at JBC Papers in Press. Published on February 18, 2014 as Manuscript M114.555888 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on A uust 5, 2017 hp://w w w .jb.org/ D ow nladed from NEU1 Desialylates CD31 and Impairs Angiogenesis In postconfluent ECs, CD31 was desialylated compared with subconfluent cells, and prior NEU1 silencing completely protected against CD31 desialylation. Prior CD31 silencing and the use of CD31-null ECs each abrogated the NEU1 inhibitory effect on EC tube formation. Sialyltransferase (ST) 6 GAL-I overexpression increased α2,6-linked CD31 sialylation and dose-dependently counter-acted NEU1mediated inhibition of EC tube formation. These combined data indicate that catalyticallyactive NEU1 inhibits in vitro angiogenesis through desialylation of its substrate, CD31. Angiogenesis is a tightly orchestrated process in which pro-angiogenic and anti-angiogenic factors are released to bind to their cognate endothelial cell (EC) surface receptors (1,2). The pro-angiogenic receptor-ligand interactions are coupled to EC disengagement from neighboring ECs and protease-mediated degradation of the underlying EC extracellular matrix (ECM). These signaling and proteolytic events permit ECs to migrate through the ECM toward the proangiogenic stimulus. Proliferative signals expand the EC population which, coupled with ECM remodeling, lead to EC-EC re-engagement with capillary-like tube formation. A number of these pro-angiogenic processes involve altered glycosylation patterns and specific carbohydratemediated recognition events (3-6). The EC surface is highly sialylated (7-9). The sialic acid (SA)-bearing surface structures on endothelia, together with other membrane-bound polyanionic macromolecules, constitute the EC glycocalyx (10,11). This negatively-charged zone can be extended through hydration and acquisition of plasma proteins. The glycocalyx might nonspecifically diminish cell-cell adhesion through either negative charge-mediated repulsion or the masking of specific binding sites. In fact, we found that prior neuraminidase treatment of pulmonary vascular endothelia increases their adhesiveness for resting polymorphonuclear leukocytes >3-fold, independent of E-selectin or intercellular adhesion molecule (ICAM)-1 expression (12). However, SA residues also can be intrinsic to EC surface recognition motifs that participate in specific intermolecular interactions. For example, increased α2,6-sialylation of the EC surface increases its adhesiveness for CD22bearing B cells (13) and lymphocyte functionassociated antigen (LFA)-1 expressing T cells (14). Migrating bovine aortic ECs display a distinct phenotype that includes hyperglycosylation and surface expression of specific glycoproteins compared with contactinhibited ECs (3). After neuraminidase treatment of these migrating ECs, increased terminal sialylation coupled to subterminal galactose residues was revealed through increased binding of Arachis hypogaea or peanut agglutinin (PNA) lectin (15). In another study, pharmacologic blockade of synthesis of hybrid and complex-type oligosaccharides, including sialyl Lewis-X determinants, inhibited capillary tube formation of bovine capillary ECs (4). In still another study, 48% of the 432 glycan-specific genes profiled in human bone marrow derived ECs stimulated by the pro-angiogenic factor, vascular endothelial growth factor (VEGF), were expressed (6). After VEGF stimulation, expression of several sialyltransferase (ST)s, including ST6GAL-I, were increased. Multiple galectins, endogenous lectins that bind galactose residues and regulate angiogenesis (16), were also upregulated. Finally, a number of EC sialoproteins directly participate in the angiogenic process, including vascular endothelial (VE)-cadherin (17), selectins and other adhesion molecules (18,19), CD31 (20), CD44 (21), fibroblast growth factor receptor (FGFR) (22), and αvβ3 integrin (23). Taken together, these combined studies establish a central role for glycan structures, and more specifically, sialylation, as intrinsic to the angiogenic process. SAs comprise a family of 9-carbon sugars, each carboxylated on the C1 position (24,25). These SA residues are almost always positioned at the terminus of glycan chains. The subterminal sugars to which SA is usually coupled are galactose and N-acetylgalactosamine. In most sialoproteins, the C2 position of SA is coupled to underlying galactose via α2,3 or α2,6 linkage and to N-acetylgalactosamine via α2,6 linkage. For only a few sialoproteins, SA is attached to another SA via α2,8 linkage, generating homopolymers of polysialic acid. The sialylation state of a specific glycoprotein is dynamically and coordinately regulated through the opposing catalytic activities of STs (26,27) and neuraminidase/sialidase (NEU)s (28-30). STs use cytidine monophosphate 2 by gest on A uust 5, 2017 hp://w w w .jb.org/ D ow nladed from NEU1 Desialylates CD31 and Impairs Angiogenesis (CMP)-SA as an activated donor to catalyze transfer of SA residues to terminal positions on glycan chains (26,27). More than 15 distinct human ST cDNAs have been cloned and characterized (26,27). These STs can be classified based on 1) the specific SA linkage they synthesize and 2) the sugar to which they transfer the SA. In contrast, NEUs counter-regulate sialylation through hydrolysis of the linkage between terminal SAs and their subterminal sugars (28-30). Although the numerous prokaryote and eukaryote members of the NEU superfamily share little amino acid (aa) sequence homology, they contain conserved motifs including the Asp box, an aa sequence comprised of –S-X-D-X-G-X-TW-, where X represents variable residues, and the –F/Y-R-I-Pmotif (28). More recently, bioinformatic analysis has revealed high conservation of 6 residues essential for catalytic activity, including an Arg triad (R21, R237, and R304), a Tyr/Glu nucleophile pair (Y334 and E218), and an Asp that functions as an acid/base catalyst (D46) (31). Four putative phosphorylation sites at positions 384, 390, 569, and 865 were >90% conserved in all sialidases. More specific to NEU1 in higher mammals, lysosomal localization signals and the residues that participate in NEU1protective protein/cathepsin A (PPCA) interaction were conserved. These highly dissimilar NEUs do share a tertiary conformation in which 6 antiparallel β-sheets are arranged into a 6-blade propeller-like configuration (28-30). Four human sialidases have been identified, NEU1, 2, 3, and 4 (28-30). We recently established that vascular endothelia predominantly express NEU1 (32). NEU1 is a ~45.5kDa (415aa) protein that contains 3 conserved and 2 degenerate Asp box(s) and 1(F/Y)RIP motif (28). In human tissues, it is the most widely expressed and most abundant NEU (29). Amongst the four human NEUs, NEU1 shares the least homology (29) and displays a substrate preference for glycoproteins over other glycoconjugates, including the SA-containing glycolipids or gangliosides (28-30). NEU1 is a lysosomal enzyme that resides in a multi-enzyme complex comprised of NEU1, PPCA, and βgalactosidase (33,34). PPCA is a multifunctional enzyme that functions as an intracellular chaperone and transport protein that targets NEU1 to the lysosome and is absolutely required for proper folding, stability, oligomerization, and activation of NEU1 (34-36). Although NEU1 had been considered primarily a lysosomal enzyme (34,35), it is now known to associate with several surface multi-receptor signaling complexes, including epidermal growth factor receptor (EGFR)/Mucin 1 (MUC1) (37), Toll-like receptor (TLR)4/CD14/MD2 (38), and the elastin receptor complex (39). Although the mechanisms through which NEU1 might be translocated to the plasma membrane are poorly understood (40), it has been detected on the surface of multiple human cell types, including activated lymphocytes (40,41), phorbol 12-myristate 13-acetate differentiated monocytes (42), fibroblasts (43), and most recently, erythrocytes (44). NEU1 is also surface expressed on human lung microvascular endothelia where it restrains EC migration into a wound (32). In the current studies, we have asked whether NEU1 might also regulate EC capillarylike tube formation and whether the sialylated proangiogenic molecule, CD31, also known as platelet endothelial cell adhesion molecule (PECAM)-1 (45-48), might serve as an in vivo NEU1 substrate. EXPERIMENTAL PROCEDURES: EC Culture Human pulmonary microvascular EC (HPMEC)s (Promocell; Heidelberg, Germany), and human pulmonary artery EC (HPAEC)s (Lonza,

Purpose: The acute phase protein, alpha(1)-acid glycoprotein, is expressed in the lung, and influences endothelial cell function. We asked whether it might regulate angiogenesis in human lung microvascular endothelia. Materials and Methods: alpha(1)-acid glycoprotein was isolated from human serum by HPLC ion exchange chromatography. Its effects on endothelial cell functions including capillary-like tube formation on Matrigel, migration in a wounding assay, chemotaxis in a modified Boyden chamber, adhesion, and transendothelial flux of the permeability tracer, C-14-albumin, were tested. Results: alpha(1)-acid glycoprotein dose-dependently inhibited capillary-like tube formation without loss of cell viability. At >= 0.50 mg/mL, it inhibited tube formation > 70%, and at 0.75 mg/mL, > 97%. alpha(1)-acid glycoprotein dose-and time-dependently restrained EC migration into a wound as early as 2 hours, and in washout studies, did so reversibly. It was inhibitory against vascular endothelial growth factor-A and fibroblast growth factor-2-driven migration but failed to inhibit chemotactic responsiveness. When alpha 1-acid glycoprotein was added to preformed tubes, it provoked their almost immediate disassembly. As early as 15 minutes, it induced tube network collapse without endothelial cell-cell disruption. It exerted a biphasic effect on cell adhesion to the Matrigel substrate. At lower concentrations (0.05-0.25 mg/mL), it increased cell adhesion, whereas at higher concentrations (>= 0.75 mg/mL) decreased adhesion. In contrast, it had no effect on transendothelial C-14-albumin flux. Conclusion: alpha 1-acid glycoprotein, at concentrations found under physiological conditions, rapidly inhibits endothelial cell capillary-like tube formation that may be explained through diminished cell adhesion to the underlying matrix and/or reversibly decreased cell migration.

Background: Endothelia express NEU1 sialidase and undergo changes in sialylation during angiogenesis. Results: CD31 is a NEU1 substrate, and NEU1 disrupts endothelial cell capillary-like tube formation. Conclusion: NEU1 works through its substrate, CD31, to dysregulate angiogenesis. Significance: Human NEU1 is the first sialidase found to regulate angiogenesis, and the CD31 sialylation state dictates its ability to influence endothelial cell differentiation and tube formation. The highly sialylated vascular endothelial surface undergoes changes in sialylation upon adopting the migratory/angiogenic phenotype. We recently established endothelial cell (EC) expression of NEU1 sialidase (Cross, A. S., Hyun, S. W., Miranda-Ribera, A., Feng, C., Liu, A., Nguyen, C., Zhang, L., Luzina, I. G., Atamas, S. P., Twaddell, W. S., Guang, W., Lillehoj, E. P., Puché, A. C., Huang, W., Wang, L. X., Passaniti, A., and Goldblum, S. E. (2012) NEU1 and NEU3 sialidase activity expressed in human lung microvascular endothelia. NEU1 restrains endothelial cell migration whereas NEU3 does not. J. Biol. Chem. 287, 15966–15980). We asked whether NEU1 might regulate EC capillary-like tube formation on a Matrigel substrate. In human pulmonary microvascular ECs (HPMECs), prior silencing of NEU1 did not alter tube formation. Infection of HPMECs with increasing multiplicities of infection of an adenovirus encoding for catalytically active WT NEU1 dose-dependently impaired tube formation, whereas overexpression of either a catalytically dead NEU1 mutant, NEU1-G68V, or another human sialidase, NEU3, did not. NEU1 overexpression also diminished EC adhesion to the Matrigel substrate and restrained EC migration in a wounding assay. In HPMECs, the adhesion molecule, CD31, also known as platelet endothelial cell adhesion molecule-1, was sialylated via α2,6-linkages, as shown by Sambucus nigra agglutinin lectin blotting. NEU1 overexpression increased CD31 binding to Arachis hypogaea or peanut agglutinin lectin, indicating CD31 desialylation. In the postconfluent state, when CD31 ectodomains are homophilically engaged, NEU1 was recruited to and desialylated CD31. In postconfluent ECs, CD31 was desialylated compared with subconfluent cells, and prior NEU1 silencing completely protected against CD31 desialylation. Prior CD31 silencing and the use of CD31-null ECs each abrogated the NEU1 inhibitory effect on EC tube formation. Sialyltransferase 6 GAL-I overexpression increased α2,6-linked CD31 sialylation and dose-dependently counteracted NEU1-mediated inhibition of EC tube formation. These combined data indicate that catalytically active NEU1 inhibits in vitro angiogenesis through desialylation of its substrate, CD31.

The microvascular endothelial surface expresses multiple molecules whose sialylation state regulates multiple aspects of endothelial function. To better regulate these sialoproteins, we asked whether endothelial cells (ECs) might express one or more catalytically active sialidases. Human lung microvascular EC lysates contained heat-labile sialidase activity for a fluorogenic substrate, 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MU-NANA), that was dose-dependently inhibited by the competitive sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid but not its negative control. The EC lysates also contained sialidase activity for a ganglioside mixture. Using real time RT-PCR to detect mRNAs for the four known mammalian sialidases, NEU1, -2, -3, and -4, NEU1 mRNA was expressed at levels 2700-fold higher that those found for NEU2, -3, or -4. Western analyses indicated NEU1 and -3 protein expression. Using confocal microscopy and flow cytometry, NEU1 was immunolocalized to both the plasma membrane and the perinuclear region. NEU3 was detected both in the cytosol and nucleus. Prior siRNA-mediated knockdown of NEU1 and NEU3 each decreased EC sialidase activity for 4-MU-NANA by >65 and >17%, respectively, and for the ganglioside mixture by 0 and 40%, respectively. NEU1 overexpression in ECs reduced their migration into a wound by >40%, whereas NEU3 overexpression did not. Immunohistochemical studies of normal human tissues immunolocalized NEU1 and NEU3 proteins to both pulmonary and extrapulmonary vascular endothelia. These combined data indicate that human lung microvascular ECs as well as other endothelia express catalytically active NEU1 and NEU3. NEU1 restrains EC migration, whereas NEU3 does not.

It is well known that the plasminogen-activating (PA) system plays a key role in the bovine mammary gland during tissue remodelling. However, the modulation of the PA cascade after bacterial infections needs to be elucidated. This study examined the effects of Escherichia coli lipopolysaccharide (LPS) on cell viability, the modulation of cell-associated u-PA activity, and the regulation of u-PA and u-PA receptor (u-PAR) RNA expression using the BME-UV1 bovine mammary epithelial cell line. LPS did not affect cell viability, but induced an increase in u-PA activity, with the maximum response after 6 h of incubation. Moreover, u-PA and u-PAR mRNA expression were both up-regulated in BME-UV1 cells after 3 h of incubation with LPS. These data indicated that E. coli LPS led to an increase in u-PA activity and RNA expression of u-PA and u-PAR in BME-UV1 cells, thus strengthening the role of the PA system during pathological processes.

In this paper, the possible involvement of the acute phase protein alpha(1)-acid glycoprotein (AGP) in the local immunomodulation of inflammation was investigated. The dose response of bovine neutrophils to AGP as to mobilization of primary and secondary granules was studied. It was found that AGP fulfils a protective role against spontaneous exocytosis of secondary, but not primary, granules. This downregulatory effect is much more evident when degranulation is challenged with Zymosan activated serum (ZAS). AGP activity is dose-dependent, the acute phase concentration being more active than the physiological one. Carbohydrate moiety of AGP was found to be critical, since experimentally desialylated protein does not maintain its exocytosis-modulatory activity.The fact that AGP may modulate the degranulation of neutrophils confirms the hypothesis that AGP is heavily involved in the fine tuning of neutrophil activity in the inflammatory environment. (C) 2008 Elsevier Ltd. All rights reserved.