Tag: hematoxylin

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Osteopontin and β3 Integrin Are Coordinately Expressed in Regenerating Endothelium In Vivo and Stimulate Arg-Gly-Asp–Dependent Endothelial Migration In Vitro

Osteopontin is an Arg-Gly-Asp (RGD)–containing acidic glycoprotein postulated to mediate cellular adhesion and migration in a growing number of normal and pathological conditions through interaction with integrin molecules. In this report, we have investigated the potential contributions of osteopontin and one of its receptors, the αvβ3 integrin, to endothelial regenerative processes by using both in vivo and in vitro models. In vivo, uninjured rat arterial endothelium had undetectable levels of osteopontin and β3-integrin mRNA by in situ hybridization. After balloon catheter denudation, osteopontin mRNA levels correlated temporally and spatially with active endothelial proliferation and migration, with the highest levels observed at the wound edge between 8 hours and 2 weeks after injury, declining to uninjured levels at 6 weeks, when regeneration was complete. Osteopontin protein levels, as determined by immunocytochemistry, paralleled the time course of mRNA expression. Likewise, β3-integrin mRNA and protein levels were substantially elevated in regenerating endothelial cells but were not detectable in uninjured or healed endothelium. In vitro, rat smooth muscle cell–derived and bacterial expressed mouse recombinant osteopontins both stimulated the adhesion and directed migration of bovine aortic endothelial cells through interactions with the αvβ3 receptor. Structural mutants of osteopontin confirmed the importance of the RGD domain for both adhesion and migration of endothelial cells through αvβ3. These data suggest important roles for osteopontin and β3 integrin in regenerating endothelium.

http://circres.ahajournals.org/content/77/4/665.full

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Endothelial Responses to Oxidized Lipoproteins Determine Genetic Susceptibility to Atherosclerosis in Mice

Background—Oxidized LDL has been found within the subendothelial space, and it exhibits numerous atherogenic properties, including induction of inflammatory genes. We examined the possibility that variations in endothelial response to minimally modified LDL (MM-LDL) constitute one of the genetic components in atherosclerosis.

Methods and Results—By a novel explant technique, endothelial cells (ECs) were isolated from the aorta of inbred mouse strains with different susceptibilities to diet-induced atherosclerosis. Responses to MM-LDL were evaluated by examining the expression of inflammatory genes involved in atherosclerosis, including monocyte chemotactic protein-1 (MCP-1) and macrophage-colony–stimulating factor (M-CSF), an oxidative stress gene, heme oxygenase-1 (HO-1), and other, noninflammatory, genes. ECs from the susceptible mouse strain C57BL/6J exhibited dramatic induction of MCP-1, M-CSF, and HO-1, whereas ECs from the resistant strain C3H/HeJ showed little or no induction. In contrast, ECs from the 2 strains responded similarly to lipopolysaccharide.

Conclusions—These data provide strong evidence that genetic factors in atherosclerosis act at the level of the vessel wall.

http://circ.ahajournals.org/content/102/1/75.long

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Homocysteine induces monocyte chemoattractant protein-1 expression by activating NF-B in THP-1 macrophages

Homocysteinemia is an independent risk factor for cardiovascular disorders. The recruitment of monocytes is an important event in atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that stimulates monocyte migration into the intima of arterial walls. The objective of the present study was to investigate the effect of homocysteine on MCP-1 expression in macrophages and the underlying mechanism of such effect. Human monocytic cell (THP-1)-derived macrophages were incubated with homocysteine. By nuclease protection assay and ELISA, homocysteine (0.05–0.2 mM) was shown to significantly enhance the expression of MCP-1 mRNA (up to 2.6-fold) and protein (up to 4.8-fold) in these cells. Homocysteine-induced MCP-1 expression resulted in increased monocyte chemotaxis. The increase in MCP-1 expression was associated with activation of nuclear factor (NF)-κB due to increased phosphorylation of the inhibitory protein (IκB-α) as well as reduced expression of IκB-α mRNA in homocysteine-treated cells. In conclusion, our results demonstrate that homocysteine, at pathological concentration, stimulates MCP-1 expression in THP-1 macrophages via NF-κB activation.

http://ajpheart.physiology.org/content/280/6/H2840.long

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IL-8 release and neutrophil activation by Clostridium difficile toxin-exposed human monocytes

Neutrophil infiltration is central to the pathogenesis of Clostridium difficile toxin A-induced enterocolitis. This study examines whether monocyte activation by C. difficile toxins is instrumental in initiating neutrophil activation and recruitment. Human monocytes were exposed to low concentrations of highly purified C. difficile toxins, and the conditioned media were harvested for cytokine and functional assays. Monocytes exposed to C. difficile toxin A (10−10 M) or toxin B (10−12 M) released 100 and 20 times basal levels, respectively, of the neutrophil chemoattractant interleukin-8 (IL-8). Reverse transcriptase-polymerase chain reaction demonstrated a marked increase in IL-8 mRNA expression by monocytes 3 h after toxin exposure. Conditioned media from toxin A- and toxin B-treated monocytes stimulated neutrophil migration (324 and 245% of control, respectively). This effect was completely blocked by IL-8 antiserum. These media also upregulated neutrophil CD11b/CD18 and endothelial cell intercellular adhesion molecule-1 expression. C. difficile toxins, at low concentrations, potently activate monocytes to release factors, including IL-8, that facilitate neutrophil extravasation and tissue infiltration. Our findings indicate a major role for toxin-mediated monocyte and macrophage activation in C. difficile colitis.

http://ajpgi.physiology.org/content/273/6/G1333.full?sid=1956e877-f6e9-4240-9ef5-8be744ec4a2c

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Homocysteine induces monocyte chemoattractant protein-1 expression by activating NF-kappaB in THP-1 macrophages.

Homocysteinemia is an independent risk factor for cardiovascular disorders. The recruitment of monocytes is an important event in atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that stimulates monocyte migration into the intima of arterial walls. The objective of the present study was to investigate the effect of homocysteine on MCP-1 expression in macrophages and the underlying mechanism of such effect. Human monocytic cell (THP-1)-derived macrophages were incubated with homocysteine. By nuclease protection assay and ELISA, homocysteine (0.05-0.2 mM) was shown to significantly enhance the expression of MCP-1 mRNA (up to 2.6-fold) and protein (up to 4.8-fold) in these cells. Homocysteine-induced MCP-1 expression resulted in increased monocyte chemotaxis. The increase in MCP-1 expression was associated with activation of nuclear factor (NF)-kappaB due to increased phosphorylation of the inhibitory protein (IkappaB-alpha) as well as reduced expression of IkappaB-alpha mRNA in homocysteine-treated cells. In conclusion, our results demonstrate that homocysteine, at pathological concentration, stimulates MCP-1 expression in THP-1 macrophages via NF-kappaB activation.

http://ajpheart.physiology.org/content/280/6/H2840.long

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Adenoviral Gene Transfer of Fortilin Attenuates Neointima Formation Through Suppression of Vascular Smooth Muscle Cell Proliferation and Migration

Fortilin, a recently characterized nuclear antiapoptotic factor structurally distinct from inhibitor of apoptosis proteins (IAPs) and Bcl-2 family member proteins, has been suggested to be involved in cell survival and regulation of apoptosis within the cardiovascular system. In this continued investigation, we characterized the influence of adenovirus-mediated fortilin (Ad-fortilin) gene delivery on vascular remodeling after experimental angioplasty.

http://circ.ahajournals.org/content/107/1/98.long

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Loss of responsiveness to chemotactic factors by deletion of the C-terminal protein interaction site of angiomotin

We have recently identified a novel protein, named angiomotin, by its ability to bind the angiogenesis inhibitor angiostatin in the yeast two-hybrid system. Angiomotin belongs to a family with two other members, AmotL-1 and -2 characterized by coiled-coil and C-terminal PDZ binding domains. Here we show that the putative PDZ binding motif of angiomotin serves as a protein recognition site and that deletion of three amino acids in this site results in inhibition of chemotaxis. Furthermore, endothelial cells expressing mutant angiomotin failed to migrate and form tubes in an in vitro tube formation assay. To study the effect of angiomotin on embryonic angiogenesis, we generated transgenic mice expressing wild-type angiomotin and the C-terminal deletion mutant driven by the endothelial cell-specific receptor tyrosine kinase (TIE) promoter. Expression of mutant angiomotin in endothelial cells inhibited migration into the neuroectoderm and intersomitic regions resulting in death at embryonic day 9.5. In contrast, mice expressing wild-type angiomotin developed normally and were fertile. These results suggest that the putative PDZ binding motif of angiomotin plays a critical role in regulating the responsiveness of endothelial cells to chemotactic cues.

http://jcs.biologists.org/content/116/18/3803.long