Exercise induces dynamic changes in intra-articular metabolism and inflammation associated with remodeling of the infrapatellar fat pad in mice.

We hypothesized that daily exercise promotes joint health by upregulating anti-inflammatory mediators via adaptive molecular and metabolic changes in the infrapatellar fat pad (IFP). We tested this hypothesis by conducting time-resolved analyses between 1 and 14 days of voluntary wheel running exercise in C57BL/6J mice. IFP structure and cellularity were evaluated by histomorphology, picrosirius red collagen staining, and flow cytometry analysis of stromal vascular fraction cells.

Sexually dimorphic metabolic effects of a high fat diet on knee osteoarthritis in mice.

Background: Women have a higher risk of developing osteoarthritis (OA) than men, including with obesity. To better understand this disparity, we investigated sex differences in metabolic and inflammatory factors associated with OA using a diet-induced mouse model of obesity. We hypothesized that 20 weeks of high-fat diet (HFD) would induce sexually dimorphic changes in both systemic and local risk factors of knee OA.

Sex-specific effects of injury and beta-adrenergic activation on metabolic and inflammatory mediators in a murine model of post-traumatic osteoarthritis.

Objective: Metabolic processes are intricately linked to the resolution of innate inflammation and tissue repair, two critical steps for treating post-traumatic osteoarthritis (PTOA). Based on lipolytic and immunoregulatory actions of norepinephrine, we hypothesized that intra-articular β-adrenergic receptor (βAR) stimulation would suppress PTOA-associated inflammation in the infrapatellar fat pad (IFP) and synovium.

Design: We used the βAR agonist isoproterenol to perturb intra-articular metabolism 3.

Imaging mass cytometry reveals tissue-specific cellular immune phenotypes in the mouse knee following ACL injury.

Objective: To develop an imaging mass cytometry method for identifying complex cell phenotypes, inter-cellular interactions, and population changes in the synovium and infrapatellar fat pad (IFP) of the mouse knee following a non-invasive compression injury.

Design: Fifteen male C57BL/6 mice were fed a high-fat diet for 8 weeks prior to random assignment to sham, 0.88 ​mm, or 1.

Sirt3 Promotes Chondrogenesis, Chondrocyte Mitochondrial Respiration and the Development of High-Fat Diet-Induced Osteoarthritis in Mice.

Understanding how obesity-induced metabolic stress contributes to synovial joint tissue damage is difficult because of the complex role of metabolism in joint development, maintenance, and repair. Chondrocyte mitochondrial dysfunction is implicated in osteoarthritis (OA) pathology, which motivated us to study the mitochondrial deacetylase enzyme sirtuin 3 (Sirt3). We hypothesized that combining high-fat-diet (HFD)-induced obesity and cartilage Sirt3 loss at a young age would impair chondrocyte mitochondrial function, leading to cellular stress and accelerated OA.

Evaluation of Galectin Binding by Surface Plasmon Resonance.

Surface plasmon resonance (SPR) instruments, like the BIAcore 3000, are useful for studying the binding between macromolecules in real time. The high sensitivity and low sample consumption in the Biacore enables the measurement of rapid kinetics and low affinities characteristics of many biological interactions. This chapter describes the affinity measurement of Galectins-1, -2 and -3 and their glycoside ligands using this approach.

Fundamentals of OA. An initiative of Osteoarthritis and Cartilage. Obesity and metabolic factors in OA.

Objective: Obesity was once considered a risk factor for knee osteoarthritis (OA) primarily for biomechanical reasons. Here we provide an additional perspective by discussing how obesity also increases OA risk by altering metabolism and inflammation.

Design: This narrative review is presented in four sections: 1) metabolic syndrome and OA, 2) metabolic biomarkers of OA, 3) evidence for dysregulated chondrocyte metabolism in OA, and 4) metabolic inflammation: joint tissue mediators and mechanisms.

Ile73Asn mutation in protein C introduces a new N-linked glycosylation site on the first EGF-domain of protein C and causes thrombosis.

Activated protein C exerts its anticoagulant activity by protein S-dependent inactivation of factors Va and VIIIa by limited proteolysis. We identified a venous thrombosis patient who has plasma protein C antigen level of 63% and activity levels of 44% and 23%, as monitored by chromogenic and clotting assays. Genetic analysis revealed the proband carries compound heterozygous mutations (c.

Detection of Glycoproteins in Polyacrylamide Gels Using Pro-Q Emerald 300 Dye, a Fluorescent Periodate Schiff-Base Stain.

Pro-Q Emerald 300 glycoprotein stain generates a bright-green fluorescent signal upon reacting with periodic acid-oxidized carbohydrate groups on proteins. With this dye it is possible to detect proteins directly in the gel without the need to transfer them to a membrane. This dye is more sensitive than the standard periodic-acid Schiff's base which uses acidic fuchsin dye.

Inorganic Polyphosphate Amplifies High Mobility Group Box 1-Mediated Von Willebrand Factor Release and Platelet String Formation on Endothelial Cells.

Objective- Inorganic polyphosphate (polyP) is known to modulate coagulation, inflammation, and metabolic pathways. It also amplifies inflammatory responses of HMGB1 (high mobility group box 1) in endothelial cells. The objective of this study was to evaluate the effect of polyP on von Willebrand factor (VWF) release from endothelial cells with or without HMGB1.

Circulating soluble P-selectin must dimerize to promote inflammation and coagulation in mice.

Leukocyte adhesion to P-selectin on activated platelets and endothelial cells induces shedding of the P-selectin ectodomain into the circulation. Plasma soluble P-selectin (sP-selectin) is elevated threefold to fourfold in patients with cardiovascular disease. Circulating sP-selectin is thought to trigger signaling in leukocytes that directly contributes to inflammation and thrombosis.

L-selectin mechanochemistry restricts neutrophil priming in vivo.

Circulating neutrophils must avoid premature activation to prevent tissue injury. The leukocyte adhesion receptor L-selectin forms bonds with P-selectin glycoprotein ligand-1 (PSGL-1) on other leukocytes and with peripheral node addressin (PNAd) on high endothelial venules. Mechanical forces can strengthen (catch) or weaken (slip) bonds between biological molecules.

Glycan Bound to the Selectin Low Affinity State Engages Glu-88 to Stabilize the High Affinity State under Force.

Selectin interactions with fucosylated glycan ligands mediate leukocyte rolling in the vasculature under shear forces. Crystal structures of P- and E-selectin suggest a two-state model in which ligand binding to the lectin domain closes loop 83-89 around the Ca coordination site, enabling Glu-88 to engage Ca and fucose. This triggers further allostery that opens the lectin/EGF domain hinge.

P-Selectin Expressed by a Human SELP Transgene Is Atherogenic in Apolipoprotein E-Deficient Mice.

Objective: During inflammation, P-selectin expressed on activated endothelial cells and platelets mediates rolling adhesion of leukocytes. Atherosclerosis-prone mice crossed with P-selectin-deficient (Selp(-/-)) mice develop smaller lesions. Cytokines, such as tumor necrosis factor-α, increase Selp transcripts and augment atherosclerosis in mice.

Motif mimetic of epsin perturbs tumor growth and metastasis.

Tumor angiogenesis is critical for cancer progression. In multiple murine models, endothelium-specific epsin deficiency abrogates tumor progression by shifting the balance of VEGFR2 signaling toward uncontrolled tumor angiogenesis, resulting in dysfunctional tumor vasculature. Here, we designed a tumor endothelium-targeting chimeric peptide (UPI) for the purpose of inhibiting endogenous tumor endothelial epsins by competitively binding activated VEGFR2.

O-glycans direct selectin ligands to lipid rafts on leukocytes.

Palmitoylated cysteines typically target transmembrane proteins to domains enriched in cholesterol and sphingolipids (lipid rafts). P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are O-glycosylated proteins on leukocytes that associate with lipid rafts. During inflammation, they transduce signals by engaging selectins as leukocytes roll in venules, and they move to the raft-enriched uropods of polarized cells upon chemokine stimulation.

Immunodetection of P-selectin using an antibody to its C-terminal tag.

P-selectin is a multi-domain glycoprotein expressed on activated endothelial cells and activated platelets. We previously expressed a recombinant form of P-selectin containing only its N-terminal lectin and EGF domains in CHO-K1 cells and showed that these two domains are sufficient to mediate ligand binding. We have now expressed the same construct in CHO-Lec1 cells that make truncated glycans.

Inter-α inhibitor protein and its associated glycosaminoglycans protect against histone-induced injury.

Extracellular histones are mediators of tissue injury and organ dysfunction; therefore they constitute potential therapeutic targets in sepsis, inflammation, and thrombosis. Histone cytotoxicity in vitro decreases in the presence of plasma. Here, we demonstrate that plasma inter-α inhibitor protein (IAIP) neutralizes the cytotoxic effects of histones and decreases histone-induced platelet aggregation.

Podoplanin requires sialylated O-glycans for stable expression on lymphatic endothelial cells and for interaction with platelets.

O-glycosylation of podoplanin (PDPN) on lymphatic endothelial cells is critical for the separation of blood and lymphatic systems by interacting with platelet C-type lectin-like receptor 2 during development. However, how O-glycosylation controls endothelial PDPN function and expression remains unclear. In this study, we report that core 1 O-glycan-deficient or desialylated PDPN was highly susceptible to proteolytic degradation by various proteases, including metalloproteinases (MMP)-2/9.

Evaluation of galectin binding by surface plasmon resonance.

Surface Plasmon Resonance (SPR) instruments, like the BIAcore 3000, are useful for studying the binding between macromolecules in real time. The high sensitivity and low sample consumption in the BIAcore enables the measurement of rapid kinetics and low affinities characteristic of many biological interactions. This chapter describes the affinity measurement of Galectins-1, -2, and -3 and their glycoside ligands using this approach.

Fucosyltransferase VII improves the function of selectin ligands on cord blood hematopoietic stem cells.

Selectins and their carbohydrate ligands mediate the homing of hematopoietic stem/progenitor cells (HSPCs) to the bone marrow. We have previously shown that ex vivo fucosylation of selectin ligands on HSPCs by α1,3 fucosyltransferase VI (FUT6) leads to improved human cord blood (CB)-HSPC engraftment in non-obese diabetic (NOD)/severe combined immune deficient (SCID) mice. In the present study, we determined whether surface fucosylation with α1,3 fucosyltransferase VII (FUT7), which is primarily expressed by hematopoietic cells, improves the function of selectin ligands on CB-HSPCs in comparison with FUT6.

Quantitative comparison of human parainfluenza virus hemagglutinin-neuraminidase receptor binding and receptor cleavage.

The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN's two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (Kd) values from these experiments with previously determined Michaelis-Menten constants (Kms) for the enzyme activity.

Detection of glycoproteins in polyacrylamide gels using Pro-Q Emerald 300 dye, a fluorescent periodate Schiff-base stain.

Pro-Q Emerald 300 glycoprotein stain generates a bright-green fluorescent signal upon reacting with periodic acid-oxidized carbohydrate groups on proteins. With this dye, it is possible to detect proteins directly in the gel without the need to transfer them to a membrane. This dye is more sensitive than the standard periodic acid Schiff's base which uses acidic fuchsin dye.

Signal-dependent slow leukocyte rolling does not require cytoskeletal anchorage of P-selectin glycoprotein ligand-1 (PSGL-1) or integrin αLβ2.

In inflamed venules, neutrophils roll on P- or E-selectin, engage P-selectin glycoprotein ligand-1 (PSGL-1), and signal extension of integrin α(L)β(2) in a low affinity state to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Cytoskeleton-dependent receptor clustering often triggers signaling, and it has been hypothesized that the cytoplasmic domain links PSGL-1 to the cytoskeleton. Chemokines cause rolling neutrophils to fully activate α(L)β(2), leading to arrest on ICAM-1.