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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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Coll/FN mixed matrices. BAE cells were plated on increasing amounts of FN with or without precoating with 10 μg/ml Coll. (A) Cells were sheared for 30 min and nuclear p65 accumulation was assessed as previously described. The abscissa represents the amount of FN adsorbed to the coverslips under these conditions. Values reflect the increase in p65 nuclear translocation relative to cells under static conditions. n = 4. (B) Cells were treated as in A, and p65 phosphorylation on Ser536 was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p65. Values represent the increase in p65 phosphorylation relative to cells under static conditions. n = 3. (C) Cells were exposed to shear stress for 5 min and p38 phosphorylation was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p38, and data was expressed as the fold increase in p38 phosphorylation compared with cells under static conditions. n = 3.
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fig5: Coll/FN mixed matrices. BAE cells were plated on increasing amounts of FN with or without precoating with 10 μg/ml Coll. (A) Cells were sheared for 30 min and nuclear p65 accumulation was assessed as previously described. The abscissa represents the amount of FN adsorbed to the coverslips under these conditions. Values reflect the increase in p65 nuclear translocation relative to cells under static conditions. n = 4. (B) Cells were treated as in A, and p65 phosphorylation on Ser536 was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p65. Values represent the increase in p65 phosphorylation relative to cells under static conditions. n = 3. (C) Cells were exposed to shear stress for 5 min and p38 phosphorylation was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p38, and data was expressed as the fold increase in p38 phosphorylation compared with cells under static conditions. n = 3.

Mentions: Cells in vivo are usually exposed to multicomponent matrices of varying composition. To test if Coll can show dominant effects in this system, cells were plated on increasing concentrations of FN in either the absence or presence of an underlying Coll matrix. The amount of FN adsorbed to the coverslips was assayed in separate experiments. In the absence of Coll, p65 nuclear translocation and Ser536 phosphorylation displayed a dose-dependent increase as FN concentration increased (Fig. 5, A and B). The dose-dependent effect reached a maximum between 4 and 6 μg of deposited FN, which corresponds to 20 μg/ml in the coating solution. When examined on coverslips first coated with Coll, the amount of FN necessary to elicit NF-κB activation was notably higher, and maximal p65 activation was reduced even at the highest doses of FN. These results support the notion that Coll signaling actively suppresses the FN-dependent activation of NF-κB by flow. However, suppression of signaling may work in the opposite direction because FN deposition inhibited flow-induced p38 phosphorylation in cells on Coll in a dose-dependent manner (Fig. 5 C).


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)

Coll/FN mixed matrices. BAE cells were plated on increasing amounts of FN with or without precoating with 10 μg/ml Coll. (A) Cells were sheared for 30 min and nuclear p65 accumulation was assessed as previously described. The abscissa represents the amount of FN adsorbed to the coverslips under these conditions. Values reflect the increase in p65 nuclear translocation relative to cells under static conditions. n = 4. (B) Cells were treated as in A, and p65 phosphorylation on Ser536 was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p65. Values represent the increase in p65 phosphorylation relative to cells under static conditions. n = 3. (C) Cells were exposed to shear stress for 5 min and p38 phosphorylation was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p38, and data was expressed as the fold increase in p38 phosphorylation compared with cells under static conditions. n = 3.
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Related In: Results  -  Collection

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fig5: Coll/FN mixed matrices. BAE cells were plated on increasing amounts of FN with or without precoating with 10 μg/ml Coll. (A) Cells were sheared for 30 min and nuclear p65 accumulation was assessed as previously described. The abscissa represents the amount of FN adsorbed to the coverslips under these conditions. Values reflect the increase in p65 nuclear translocation relative to cells under static conditions. n = 4. (B) Cells were treated as in A, and p65 phosphorylation on Ser536 was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p65. Values represent the increase in p65 phosphorylation relative to cells under static conditions. n = 3. (C) Cells were exposed to shear stress for 5 min and p38 phosphorylation was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p38, and data was expressed as the fold increase in p38 phosphorylation compared with cells under static conditions. n = 3.
Mentions: Cells in vivo are usually exposed to multicomponent matrices of varying composition. To test if Coll can show dominant effects in this system, cells were plated on increasing concentrations of FN in either the absence or presence of an underlying Coll matrix. The amount of FN adsorbed to the coverslips was assayed in separate experiments. In the absence of Coll, p65 nuclear translocation and Ser536 phosphorylation displayed a dose-dependent increase as FN concentration increased (Fig. 5, A and B). The dose-dependent effect reached a maximum between 4 and 6 μg of deposited FN, which corresponds to 20 μg/ml in the coating solution. When examined on coverslips first coated with Coll, the amount of FN necessary to elicit NF-κB activation was notably higher, and maximal p65 activation was reduced even at the highest doses of FN. These results support the notion that Coll signaling actively suppresses the FN-dependent activation of NF-κB by flow. However, suppression of signaling may work in the opposite direction because FN deposition inhibited flow-induced p38 phosphorylation in cells on Coll in a dose-dependent manner (Fig. 5 C).

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