Osteopontin is a ligand for the alpha4beta1 integrin

1998 ◽  
Vol 111 (9) ◽  
pp. 1165-1174 ◽  
Author(s):  
K.J. Bayless ◽  
G.A. Meininger ◽  
J.M. Scholtz ◽  
G.E. Davis
Keyword(s):  
Leukocyte Adhesion ◽  
Bovine Milk ◽  
Vascular Wall ◽  
Intact Protein ◽  
Binding Peptide ◽  
Substrate Adhesion ◽  
Cell Extracts ◽  
Monocytes And Macrophages ◽  
Localize Potential ◽  
Alpha4beta1 Integrin

Recent work has shown that osteopontin expression is upregulated at sites of cardiovascular injury. It has been hypothesized that osteopontin provides an adhesive matrix for endothelial and smooth muscle cells during remodeling of the vascular wall following injury. Osteopontin has also been found to be synthesized by monocytes and macrophages within injury sites. Here, we present data showing that osteopontin can promote leukocyte adhesion through the alpha4beta1 integrin. In the presence of physiologic concentrations of Mg2+ and Ca2+, osteopontin purified from bovine milk promoted cell-substrate adhesion of HL-60 and Ramos cells, two model leukocyte cell lines. As with other adhesive ligands, adhesion to osteopontin required leukocyte activation. Under these conditions, no adhesion to control substrates such as bovine serum albumin was observed. Leukocyte adhesion was inhibited by anti-integrin antibodies directed at either the alpha4 or beta1 integrin subunits but not by control antibodies directed to other integrins. Further adhesion experiments revealed that leukocyte binding to osteopontin was completely inhibited by an alpha4beta1-binding peptide containing the leucine-aspartate-valine (LDV) sequence, while a control, non-binding peptide containing leucine-glutamate-valine (LEV) had minimal effects. Affinity chromatography using either surface labeled HL-60 or Ramos cell extracts revealed that the alpha4beta1 integrin specifically bound to osteopontin. Immunoprecipitation of eluted fractions from these columns positively identified the alpha4beta1 integrin. In order to localize potential alpha4beta1-binding sites within osteopontin, the protein was proteolytically cleaved with thrombin. A 30 kDa N-terminal osteopontin fragment purified using fast protein liquid chromatography promoted alpha4beta1 dependent leukocyte adhesion in a manner similar to that of the intact protein. These data collectively demonstrate that the alpha4beta1 integrin is a new adhesion receptor for osteopontin and that an alpha4beta1 binding site exists in the NH2-terminal thrombin fragment of osteopontin.

scholarly journals Glucose lowering by SGLT2-inhibitor empagliflozin accelerates atherosclerosis regression in hyperglycemic STZ-diabetic mice

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jan Pennig ◽  
Philipp Scherrer ◽  
Mark Colin Gissler ◽  
Nathaly Anto-Michel ◽  
Natalie Hoppe ◽  
...  
Keyword(s):  
Leukocyte Adhesion ◽  
Plasma Cholesterol ◽  
Sglt2 Inhibitor ◽  
Vascular Wall ◽  
High Cholesterol Diet ◽  
Chow Diet ◽  
Diabetic Mice ◽  
Glucose Lowering ◽  
Atherosclerosis Progression ◽  
Glucose Levels

AbstractDiabetes worsens atherosclerosis progression and leads to a defect in repair of arteries after cholesterol reduction, a process termed regression. Empagliflozin reduces blood glucose levels via inhibition of the sodium glucose cotransporter 2 (SGLT-2) in the kidney and has been shown to lead to a marked reduction in cardiovascular events in humans. To determine whether glucose lowering by empagliflozin accelerates atherosclerosis regression in a mouse model, male C57BL/6J mice were treated intraperitoneally with LDLR- and SRB1- antisense oligonucleotides and fed a high cholesterol diet for 16 weeks to induce severe hypercholesterolemia and atherosclerosis progression. At week 14 all mice were rendered diabetic by streptozotocin (STZ) injections. At week 16 a baseline group was sacrificed and displayed substantial atherosclerosis of the aortic root. In the remaining mice, plasma cholesterol was lowered by switching to chow diet and treatment with LDLR sense oligonucleotides to induce atherosclerosis regression. These mice then received either empagliflozin or vehicle for three weeks. Atherosclerotic plaques in the empagliflozin treated mice were significantly smaller, showed decreased lipid and CD68+ macrophage content, as well as greater collagen content. Proliferation of plaque resident macrophages and leukocyte adhesion to the vascular wall were significantly decreased in empagliflozin-treated mice. In summary, plasma glucose lowering by empagliflozin improves plaque regression in diabetic mice.

scholarly journals Nitric oxide function in atherosclerosis

1997 ◽  
Vol 6 (1) ◽  
pp. 3-21 ◽  
Author(s):  
K. E. Matthys ◽  
H. Bult
Keyword(s):  
Nitric Oxide ◽  
Leukocyte Adhesion ◽  
Atherosclerotic Lesion ◽  
Vascular Wall ◽  
Early Event ◽  
No Production ◽  
Oxygen Intermediates ◽  
Atherosclerotic Lesions ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Atherosclerosis is a chronic inflammatory process in the intima of conduit arteries, which disturbs the endothelium-dependent regulation of the vascular tone by the labile liposoluble radical nitric oxide (NO) formed by the constitutive endothelial nitric oxide synthase (eNOS). This defect predisposes to coronary vasospasm and cardiac ischaemia, with anginal pain as the typical clinical manifestation. It is now appreciated that endothelial dysfunction is an early event in atherogenesis and that it may also involve the microcirculation, in which atherosclerotic lesions do not develop. On the other hand, the inflammatory environment in atherosclerotic plaques may result in the expression of the inducible NO synthase (iNOS) isozyme. Whether the dysfunction in endothelial NO production is causal to, or the result of, atherosclerotic lesion formation is still highly debated. Most evidence supports the hypothesis that constitutive endothelial NO release protects against atherogenesis e.g. by preventing smooth muscle cell proliferation and leukocyte adhesion. Nitric oxide generated by the inducible isozyme may be beneficial by replacing the failing endothelial production but excessive release may damage the vascular wall cells, especially in combination with reactive oxygen intermediates.

scholarly journals Crossing the Vascular Wall: Common and Unique Mechanisms Exploited by Different Leukocyte Subsets during Extravasation

2015 ◽  
Vol 2015 ◽  
pp. 1-23 ◽  
Author(s):  
Michael Schnoor ◽  
Pilar Alcaide ◽  
Mathieu-Benoit Voisin ◽  
Jaap D. van Buul
Keyword(s):  
Rheumatoid Arthritis ◽  
Current Knowledge ◽  
Leukocyte Adhesion ◽  
Vascular Wall ◽  
Point Of View ◽  
Regulatory Mechanisms ◽  
Integrin Receptors ◽  
Endothelial Monolayers ◽  
Leukocyte Extravasation ◽  
Endothelial Adhesion Molecules

Leukocyte extravasation is one of the essential and first steps during the initiation of inflammation. Therefore, a better understanding of the key molecules that regulate this process may help to develop novel therapeutics for treatment of inflammation-based diseases such as atherosclerosis or rheumatoid arthritis. The endothelial adhesion molecules ICAM-1 and VCAM-1 are known as the central mediators of leukocyte adhesion to and transmigration across the endothelium. Engagement of these molecules by their leukocyte integrin receptors initiates the activation of several signaling pathways within both leukocytes and endothelium. Several of such events have been described to occur during transendothelial migration of all leukocyte subsets, whereas other mechanisms are known only for a single leukocyte subset. Here, we summarize current knowledge on regulatory mechanisms of leukocyte extravasation from a leukocyte and endothelial point of view, respectively. Specifically, we will focus on highlighting common and unique mechanisms that specific leukocyte subsets exploit to succeed in crossing endothelial monolayers.

scholarly journals Supplementation with Bovine Milk or Soy Beverages Recovers Bone Mineralization in Young Growing Rats Fed an Insufficient Diet, in Contrast to an Almond Beverage

2019 ◽  
Vol 3 (11) ◽  
Author(s):  
Julie A Cakebread ◽  
Olivia A M Wallace ◽  
Marlena C Kruger ◽  
Mark H Vickers ◽  
Alison J Hodgkinson
Keyword(s):  
Amino Acids ◽  
Bone Density ◽  
Bone Mineralization ◽  
Bovine Milk ◽  
Biomechanical Properties ◽  
Intact Protein ◽  
Sprague Dawley ◽  
Uht Milk ◽  
Growing Rats ◽  
Sustainable Products

ABSTRACT Background Nondairy beverages, produced from soy, rice, oat, almond, or coconut, are increasingly being used as alternatives to dairy milk, with the perception that they are healthier and/or more sustainable products than dairy products. Objective The aim of this study was to compare the effects of supplementing either bovine milk, soy, or almond-based beverages to young, growing rats fed an intact-protein diet or a diet that had protein substituted with amino acids (AA-diet). Methods Three-week-old male Sprague-Dawley rats were randomly assigned to 5 groups (n = 10/group) and fed ad libitum for 4 wk. Two control groups were fed either standard AIN-93G food [20% casein (CN) protein] or AIN-93G with amino acids (AAs) equivalent to CN protein, and water to drink. Three treatment groups were fed AIN-93G AA and supplemented with either bovine ultra-heat treatment (UHT) milk or soy or almond UHT beverages. Rat weight gain and food intakes were recorded. During week 4, body composition was assessed using DEXA to determine lean soft tissue, fat, and bone mass. At trial end, bone biomechanical properties and blood plasma mineral concentrations were measured. Results At the end of the trial, animals supplemented with almond beverage were lightest (P > 0.05), with higher plasma calcium concentrations (P > 0.05) and lower bone mineral content (BMC) and bone density (P > 0.05) than animals supplemented with milk or soy beverage. Soy-supplemented animals had similar BMC and bone density compared with milk-supplemented animals, although the soy group gained most weight (P > 0.05) and had the highest fat:lean ratio (P > 0.05) compared with other groups. Conclusions In the model tested, supplementing rats with bovine UHT milk and soy UHT beverage provided favorable bone health outcomes. Conversely, almond UHT beverage was not an effective supplement and could be detrimental to bone mineralization and strength outcomes.

Protein mass measurement combined with mass spectrometric sequencing of protein digests for detection and characterization of protein modifications1

2006 ◽  
Vol 84 (7) ◽  
pp. 986-997 ◽  
Author(s):  
Chengjie Ji ◽  
Zhengping Wang ◽  
Liang Li
Keyword(s):  
Mass Spectrometry ◽  
Gel Electrophoresis ◽  
Protein Modification ◽  
Mass Spectrometric ◽  
Maldi Ms ◽  
Intact Protein ◽  
Protein Mass ◽  
Cell Extracts ◽  
Peptide Mass

A method for the characterization of modifications of low molecular weight proteins (<20 kDa) extracted from a microorganism based on the use of multiple separation tools and mass spectrometric techniques is described. In this method, intact proteins from cell extracts are first separated and fractionated by liquid chromatography (LC). Individual fractions are then analyzed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) to provide intact protein mass information. The fractions are further characterized by using trypsin digestion and LC electrospray ionization (ESI) MS/MS analysis of the resultant peptides to identify the proteins. Gel electrophoresis of a fraction is also carried out to estimate the molecular masses of the proteins. The gel bands are identified by in-gel digestion and peptide mass mapping and sequencing using MALDI-MS and MALDI-MS/MS. The combined information generated from these experiments is interpreted for detecting and characterizing modified proteins. This method has been developed and applied to the analysis of posttranslational modifications (PTMs) of low-mass proteins (5–20 kDa) extracted from a relatively well-characterized microorganism, Escherichia coli. Using this method, not only previously reported PTMs involving acetylation, methylation, oxidation, and the removal of signal peptides, but also two novel PTMs, namely loss of N-terminal Met-Thr-Met (MTM) and hydroxylation of arginine, were identified. It is envisaged that this method should be applicable to other relatively simple microorganisms for the discovery of new PTMs.Key words: top-down proteomics, protein modification, HPLC, gel electrophoresis, tandem mass spectrometry.

Thromboxane prostanoid receptor stimulation induces shedding of the transmembrane chemokine CX3CL1 yet enhances CX3CL1-dependent leukocyte adhesion

2010 ◽  
Vol 298 (6) ◽  
pp. C1469-C1480 ◽  
Author(s):  
Soumitra Tole ◽  
Anne M. Durkan ◽  
Yi-Wei Huang ◽  
Guang Ying Liu ◽  
Alexander Leung ◽  
...  
Keyword(s):  
Cell Surface ◽  
Vascular Endothelial Cells ◽  
Leukocyte Adhesion ◽  
Vascular Inflammation ◽  
Vascular Wall ◽  
Mononuclear Leukocytes ◽  
Key Factor ◽  
Factor Α ◽  
And Function ◽  
Adherent Leukocytes

In atherosclerosis, chemokines recruit circulating mononuclear leukocytes to the vascular wall. A key factor is CX3CL1, a chemokine with soluble and transmembrane species that acts as both a chemoattractant and an adhesion molecule. Thromboxane A2 and its receptor, TP, are also critical to atherogenesis by promoting vascular inflammation and consequent leukocyte recruitment. We examined the effects of TP stimulation on processing and function of CX3CL1, using CX3CL1-expressing human ECV-304 cells and primary human vascular endothelial cells. TP agonists promoted rapid shedding of cell surface CX3CL1, which was inhibited by pharmacological inhibitors or specific small interfering RNA targeting tumor necrosis factor-α-converting enzyme (TACE). Because it reduced cell surface CX3CL1, we predicted that TP stimulation would inhibit adhesion of leukocytes expressing the CX3CL1 cognate receptor but, paradoxically, saw enhanced adhesion. We questioned whether the enhanced ability of the remaining membrane-associated CX3CL1 to bind targets was caused by changes in its lateral mobility. Using fluorescence recovery after photobleaching, we found that plasmalemmal CX3CL1 was initially tethered but ultimately mobilized by TP agonists. TP stimulation provoked clustering of transmembrane CX3CL1 at sites of contact with adherent leukocytes. These data demonstrate that TP stimulation induces two distinct effects: a rapid cleavage of surface CX3CL1, thereby releasing the soluble chemoattractant, plus mobilization of the remaining transmembrane CX3CL1 to enhance the avidity of interactions with adherent leukocytes. The dual effect of TP allows CX3CL1 to recruit leukocytes to sites of vascular inflammation while enhancing their adhesion once recruited.

Disulphide arrangement in bovine caseins: localization of intrachain disulphide bridges in monomers of κ- and αs2-casein from bovine milk

1994 ◽  
Vol 61 (4) ◽  
pp. 485-493 ◽  
Author(s):  
Lone K. Rasmussen ◽  
Peter Højrup ◽  
Torben E. Petersen
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Bovine Milk ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequence Analysis ◽  
Naturally Occurring ◽  
Localize Potential

SummaryNaturally occurring monomeric κ-casein and αs2-casein in bovine milk were purified by ion-exchange chromatography in order to localize potential intrachain disulphide bridges. Enzymic cleavage of the proteins followed by mass spectrometry and amino acid sequence analysis of cystine-containing peptides revealed the presence of an intrachain disulphide bond in both proteins.

scholarly journals Activation of phosphotyrosine phosphatase activity by reduction of cell-substrate adhesion

1993 ◽  
Vol 90 (23) ◽  
pp. 11177-11181 ◽  
Author(s):  
P A Maher
Keyword(s):  
Phosphatase Activity ◽  
Cellular Proteins ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Phosphotyrosine Phosphatase ◽  
Protein Tyrosine ◽  
Substrate Adhesion ◽  
Cell Extracts ◽  
Cell Substrate ◽  
Cell Substrate Adhesion

Treatment of chicken embryo fibroblasts (CEFs) with trypsin results in a dose- and time-dependent loss of phosphotyrosine from cellular proteins. A similar, but less marked, reduction in protein tyrosine phosphorylation occurs upon incubation of CEFs in phosphate-buffered saline (PBS). The decrease in the phosphotyrosine content of proteins following treatment with trypsin or PBS, as determined by immunoblotting of cell extracts with anti-phosphotyrosine antibodies, corresponds with a loss of phosphotyrosine antibody immunoreactivity at focal contacts, as detected by immunofluorescence microscopy. The recovery of phosphotyrosine in cellular proteins occurs within 30 min following removal of trypsin, even in the presence of the protein synthesis inhibitor cycloheximide, indicating that the loss of phosphotyrosine-containing proteins is not due to their degradation by trypsin. Pretreatment of CEFs with inhibitors of protein-tyrosine-phosphatases greatly reduces the loss of phosphotyrosine from proteins brought about by trypsin. In addition, phosphotyrosine phosphatase activity is increased in extracts prepared from trypsin-treated CEFs. The loss of phosphotyrosine from proteins following treatment with trypsin or PBS is not specific to CEFs but is also observed in established fibroblast lines. Taken together these results suggest that the activity of one or more phosphotyrosine phosphatases is regulated by cell-substrate adhesion.

scholarly journals Immunological Aspects of Atherosclerosis

2014 ◽  
pp. S335-S342 ◽  
Author(s):  
A. KRÁLOVÁ ◽  
I. KRÁLOVÁ LESNÁ ◽  
R. POLEDNE
Keyword(s):  
Atherosclerotic Lesion ◽  
Vascular Wall ◽  
Atherosclerotic Plaques ◽  
M2 Macrophages ◽  
Environmental Signals ◽  
Ldl Particles ◽  
Monocytes And Macrophages ◽  
Tissue Changes ◽  
Inflammatory Macrophages ◽  
M1 And M2 Macrophages

Atherosclerosis is a degenerative inflammatory disease of the vascular wall, which is characterized by the formation of atherosclerotic plaques that contain lipids, activated smooth muscle cells, immune cells, foam cells, a necrotic core and calcified sites. In atherosclerosis pathology, monocytes and macrophages play the most important role by accumulating redundant LDL particles in their oxidized form and producing proinflammatory cytokines. Atherosclerotic plaque macrophages reveal distinct phenotypes that are distinguished into M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages. Numerous environmental signals (cytokines, microbial cell molecules) that are received by macrophages drive their polarization, but it must be determined whether this classification reflects different macrophage subtypes or plasticity and phenotypic tissue changes, but the balance between subsets is crucial. M1 macrophages are dominant in symptomatic atherosclerotic plaques, while M2 macrophages are more frequent in asymptomatic plaques. Nevertheless, a positive correlation of both M1 and M2 macrophages with atherosclerotic lesion severity was also observed.

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