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The subendothelial extracellular matrix modulates NF-kappaB activation by flow: a potential role in atherosclerosis.

Orr AW, Sanders JM, Bevard M, Coleman E, Sarembock IJ, Schwartz MA - J. Cell Biol. (2005)

Bottom Line: Flow-induced NF-kappaB activation is downstream of conformational activation of integrins, resulting in new integrin binding to the subendothelial extracellular matrix and signaling.Whereas endothelial cells plated on fibronectin or fibrinogen activate NF-kappaB in response to flow, cells on collagen or laminin do not.Furthermore, altering the extracellular matrix to promote p38 activation in cells on fibronectin suppresses NF-kappaB activation, suggesting a novel therapeutic strategy for treating atherosclerosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA.

ABSTRACT
Atherosclerotic plaque forms in regions of the vasculature exposed to disturbed flow. NF-kappaB activation by fluid flow, leading to expression of target genes such as E-selectin, ICAM-1, and VCAM-1, may regulate early monocyte recruitment and fatty streak formation. Flow-induced NF-kappaB activation is downstream of conformational activation of integrins, resulting in new integrin binding to the subendothelial extracellular matrix and signaling. Therefore, we examined the involvement of the extracellular matrix in this process. Whereas endothelial cells plated on fibronectin or fibrinogen activate NF-kappaB in response to flow, cells on collagen or laminin do not. In vivo, fibronectin and fibrinogen are deposited at atherosclerosis-prone sites before other signs of atherosclerosis. Ligation of integrin alpha2beta1 on collagen prevents flow-induced NF-kappaB activation through a p38-dependent pathway that is activated locally at adhesion sites. Furthermore, altering the extracellular matrix to promote p38 activation in cells on fibronectin suppresses NF-kappaB activation, suggesting a novel therapeutic strategy for treating atherosclerosis.

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Shear stress–induced NF-κB activation is matrix specific. (A) BAE cells plated on Coll I, LN, FN, or FG for 4 h were sheared for 60 min, and p65 localization was assessed by immunocytochemistry. The percentage of cells showing nuclear staining for NF-κB was determined. Values are means ± SD (>100 cells counted per condition per experiment; n = 3). *, P < 0.05; **, P < 0.01. A representative p65 stain for these conditions is shown on the right. (B) Endothelial cells were grown on different matrix proteins and sheared for 0, 5, 15, 30, and 60 min. Phosphorylation of p65 on Ser536 was assessed by immunoblotting total cell lysates with a phosphorylation site-specific antibody. Values are means ± SD following normalization for total p65 (n = 4). A representative blot is shown for each condition. (C) Human umbilical vein endothelial cells were plated on different matrix proteins and sheared for 5 h, and the level of ICAM-1 expression was assessed by Western blotting. Densitometric quantification was normalized to actin. n = 3; *, P < 0.05. Immunocytometric staining for ICAM-1 surface expression is shown on the right.
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fig1: Shear stress–induced NF-κB activation is matrix specific. (A) BAE cells plated on Coll I, LN, FN, or FG for 4 h were sheared for 60 min, and p65 localization was assessed by immunocytochemistry. The percentage of cells showing nuclear staining for NF-κB was determined. Values are means ± SD (>100 cells counted per condition per experiment; n = 3). *, P < 0.05; **, P < 0.01. A representative p65 stain for these conditions is shown on the right. (B) Endothelial cells were grown on different matrix proteins and sheared for 0, 5, 15, 30, and 60 min. Phosphorylation of p65 on Ser536 was assessed by immunoblotting total cell lysates with a phosphorylation site-specific antibody. Values are means ± SD following normalization for total p65 (n = 4). A representative blot is shown for each condition. (C) Human umbilical vein endothelial cells were plated on different matrix proteins and sheared for 5 h, and the level of ICAM-1 expression was assessed by Western blotting. Densitometric quantification was normalized to actin. n = 3; *, P < 0.05. Immunocytometric staining for ICAM-1 surface expression is shown on the right.

Mentions: To test whether or not shear stress–induced NF-κB activation depends on the underlying matrix, bovine aortic endothelial (BAE) cells plated on glass slides coated with Coll I, LN, FN, or FG for 4 h were subjected to laminar shear stress (12 dyne/cm2) in a parallel plate flow chamber. NF-κB is normally held inactive in the cytoplasm through interactions with IκB, but stimulus-induced IκB degradation results in NF-κB nuclear targeting and initiation of transcription (Chen and Greene, 2004; Yamamoto and Gaynor, 2004). As previously reported for endothelial cells under standard conditions (Scatena et al., 1998; Klein et al., 2002; Guo et al., 2004), cells on FN or FG showed enhanced nuclear translocation of NF-κB after flow; in contrast, cells on LN responded poorly and cells on Coll showed a higher baseline under static conditions, which decreased in response to flow (Fig. 1 A).


The subendothelial extracellular matrix modulates NF-kappaB activation by flow: a potential role in atherosclerosis.

Orr AW, Sanders JM, Bevard M, Coleman E, Sarembock IJ, Schwartz MA - J. Cell Biol. (2005)

Shear stress–induced NF-κB activation is matrix specific. (A) BAE cells plated on Coll I, LN, FN, or FG for 4 h were sheared for 60 min, and p65 localization was assessed by immunocytochemistry. The percentage of cells showing nuclear staining for NF-κB was determined. Values are means ± SD (>100 cells counted per condition per experiment; n = 3). *, P < 0.05; **, P < 0.01. A representative p65 stain for these conditions is shown on the right. (B) Endothelial cells were grown on different matrix proteins and sheared for 0, 5, 15, 30, and 60 min. Phosphorylation of p65 on Ser536 was assessed by immunoblotting total cell lysates with a phosphorylation site-specific antibody. Values are means ± SD following normalization for total p65 (n = 4). A representative blot is shown for each condition. (C) Human umbilical vein endothelial cells were plated on different matrix proteins and sheared for 5 h, and the level of ICAM-1 expression was assessed by Western blotting. Densitometric quantification was normalized to actin. n = 3; *, P < 0.05. Immunocytometric staining for ICAM-1 surface expression is shown on the right.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171897&req=5

fig1: Shear stress–induced NF-κB activation is matrix specific. (A) BAE cells plated on Coll I, LN, FN, or FG for 4 h were sheared for 60 min, and p65 localization was assessed by immunocytochemistry. The percentage of cells showing nuclear staining for NF-κB was determined. Values are means ± SD (>100 cells counted per condition per experiment; n = 3). *, P < 0.05; **, P < 0.01. A representative p65 stain for these conditions is shown on the right. (B) Endothelial cells were grown on different matrix proteins and sheared for 0, 5, 15, 30, and 60 min. Phosphorylation of p65 on Ser536 was assessed by immunoblotting total cell lysates with a phosphorylation site-specific antibody. Values are means ± SD following normalization for total p65 (n = 4). A representative blot is shown for each condition. (C) Human umbilical vein endothelial cells were plated on different matrix proteins and sheared for 5 h, and the level of ICAM-1 expression was assessed by Western blotting. Densitometric quantification was normalized to actin. n = 3; *, P < 0.05. Immunocytometric staining for ICAM-1 surface expression is shown on the right.
Mentions: To test whether or not shear stress–induced NF-κB activation depends on the underlying matrix, bovine aortic endothelial (BAE) cells plated on glass slides coated with Coll I, LN, FN, or FG for 4 h were subjected to laminar shear stress (12 dyne/cm2) in a parallel plate flow chamber. NF-κB is normally held inactive in the cytoplasm through interactions with IκB, but stimulus-induced IκB degradation results in NF-κB nuclear targeting and initiation of transcription (Chen and Greene, 2004; Yamamoto and Gaynor, 2004). As previously reported for endothelial cells under standard conditions (Scatena et al., 1998; Klein et al., 2002; Guo et al., 2004), cells on FN or FG showed enhanced nuclear translocation of NF-κB after flow; in contrast, cells on LN responded poorly and cells on Coll showed a higher baseline under static conditions, which decreased in response to flow (Fig. 1 A).

Bottom Line: Flow-induced NF-kappaB activation is downstream of conformational activation of integrins, resulting in new integrin binding to the subendothelial extracellular matrix and signaling.Whereas endothelial cells plated on fibronectin or fibrinogen activate NF-kappaB in response to flow, cells on collagen or laminin do not.Furthermore, altering the extracellular matrix to promote p38 activation in cells on fibronectin suppresses NF-kappaB activation, suggesting a novel therapeutic strategy for treating atherosclerosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA.

ABSTRACT
Atherosclerotic plaque forms in regions of the vasculature exposed to disturbed flow. NF-kappaB activation by fluid flow, leading to expression of target genes such as E-selectin, ICAM-1, and VCAM-1, may regulate early monocyte recruitment and fatty streak formation. Flow-induced NF-kappaB activation is downstream of conformational activation of integrins, resulting in new integrin binding to the subendothelial extracellular matrix and signaling. Therefore, we examined the involvement of the extracellular matrix in this process. Whereas endothelial cells plated on fibronectin or fibrinogen activate NF-kappaB in response to flow, cells on collagen or laminin do not. In vivo, fibronectin and fibrinogen are deposited at atherosclerosis-prone sites before other signs of atherosclerosis. Ligation of integrin alpha2beta1 on collagen prevents flow-induced NF-kappaB activation through a p38-dependent pathway that is activated locally at adhesion sites. Furthermore, altering the extracellular matrix to promote p38 activation in cells on fibronectin suppresses NF-kappaB activation, suggesting a novel therapeutic strategy for treating atherosclerosis.

Show MeSH
Related in: MedlinePlus