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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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FNIII-1C activates p38 and blocks shear stress–induced NF-κB activation. (A) BAE cells on FN were treated with the FNIII-1C or FNIII-2C peptides for 4 h. Then, cells were either left under static conditions or sheared for 30 min and phosphorylation of p38 was assessed by Western blotting with phosphorylation site-specific antibodies (n = 5). (B) Cells were plated on FN, treated with FNIII-1C for 4 h, and stained for phosphorylated p38 and β1 integrin (n = 3). (C) BAE cells were plated on FN, incubated overnight, and treated with 10 μM FNIII1C, 10 μM of heat-denatured FNIII1C, or 10 μM FNIII2C for 4 h. Cells were sheared for 30 min, and NF-κB nuclear translocation was assessed by immunocytochemistry (n = 3–5). (D) Cells were pretreated with FN peptides as in C, followed by incubation with the p38 inhibitor SB202190 (1 μM) for 1 h before onset of shear stress. Cells were sheared for 30 min and nuclear translocation of p65 was assessed. Values are means ± SD (>100 cells counted per condition per experiment; n = 3–8). **, P < 0.01; ***, P < 0.001. (E) Cells were pretreated with the FN peptides as indicated, incubated with SB202190, and sheared for 30 min. p65 phosphorylation at Ser536 was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p65. Values are means ± SD from four to seven experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig7: FNIII-1C activates p38 and blocks shear stress–induced NF-κB activation. (A) BAE cells on FN were treated with the FNIII-1C or FNIII-2C peptides for 4 h. Then, cells were either left under static conditions or sheared for 30 min and phosphorylation of p38 was assessed by Western blotting with phosphorylation site-specific antibodies (n = 5). (B) Cells were plated on FN, treated with FNIII-1C for 4 h, and stained for phosphorylated p38 and β1 integrin (n = 3). (C) BAE cells were plated on FN, incubated overnight, and treated with 10 μM FNIII1C, 10 μM of heat-denatured FNIII1C, or 10 μM FNIII2C for 4 h. Cells were sheared for 30 min, and NF-κB nuclear translocation was assessed by immunocytochemistry (n = 3–5). (D) Cells were pretreated with FN peptides as in C, followed by incubation with the p38 inhibitor SB202190 (1 μM) for 1 h before onset of shear stress. Cells were sheared for 30 min and nuclear translocation of p65 was assessed. Values are means ± SD (>100 cells counted per condition per experiment; n = 3–8). **, P < 0.01; ***, P < 0.001. (E) Cells were pretreated with the FN peptides as indicated, incubated with SB202190, and sheared for 30 min. p65 phosphorylation at Ser536 was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p65. Values are means ± SD from four to seven experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: The selective activation of NF-κB in atherosclerosis-prone regions of the vasculature, together with the importance of NF-κB target genes in atherosclerosis, suggest that inhibition of this transcription factor could have therapeutic value. However, NF-κB has important roles in cell survival and immune protection, thus, global inhibition of the pathway is likely to be deleterious. In contrast, local inactivation of NF-κB within cell–ECM adhesions in atherosclerotic plaque could be beneficial. Although FN does not normally activate p38, a peptide derived from the first type III repeat in FN, termed III-1C, alters the structure of the FN matrix and stimulates p38 activation (Yi and Ruoslahti, 2001; Klein et al., 2003). When cells on FN were treated with this peptide, activated p38 colocalized with integrins at sites of adhesion (Fig. 7, A and B). To test whether or not FNIII-1C might also suppress NF-κB activation, BAE cells on FN were treated for 4 h with FNIII-1C or as controls with heat denatured III-1C or the analogous peptide from the second type III repeat, FNIII-2C. Cells were sheared for 30 min and NF-κB was assayed. FNIII-1C blocked shear stress–induced NF-κB nuclear translocation and phosphorylation, whereas FNIII-2C and heat-denatured FNIII-1C had no effect (Fig. 7, B and D). Furthermore, NF-κB activation was rescued by p38 inhibition in cells treated with FNIII-1C (Fig. 7, C and D). These results show that the proatherosclerotic FN ECM can be modified to locally activate p38 and thereby suppress NF-κB.


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)

FNIII-1C activates p38 and blocks shear stress–induced NF-κB activation. (A) BAE cells on FN were treated with the FNIII-1C or FNIII-2C peptides for 4 h. Then, cells were either left under static conditions or sheared for 30 min and phosphorylation of p38 was assessed by Western blotting with phosphorylation site-specific antibodies (n = 5). (B) Cells were plated on FN, treated with FNIII-1C for 4 h, and stained for phosphorylated p38 and β1 integrin (n = 3). (C) BAE cells were plated on FN, incubated overnight, and treated with 10 μM FNIII1C, 10 μM of heat-denatured FNIII1C, or 10 μM FNIII2C for 4 h. Cells were sheared for 30 min, and NF-κB nuclear translocation was assessed by immunocytochemistry (n = 3–5). (D) Cells were pretreated with FN peptides as in C, followed by incubation with the p38 inhibitor SB202190 (1 μM) for 1 h before onset of shear stress. Cells were sheared for 30 min and nuclear translocation of p65 was assessed. Values are means ± SD (>100 cells counted per condition per experiment; n = 3–8). **, P < 0.01; ***, P < 0.001. (E) Cells were pretreated with the FN peptides as indicated, incubated with SB202190, and sheared for 30 min. p65 phosphorylation at Ser536 was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p65. Values are means ± SD from four to seven experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig7: FNIII-1C activates p38 and blocks shear stress–induced NF-κB activation. (A) BAE cells on FN were treated with the FNIII-1C or FNIII-2C peptides for 4 h. Then, cells were either left under static conditions or sheared for 30 min and phosphorylation of p38 was assessed by Western blotting with phosphorylation site-specific antibodies (n = 5). (B) Cells were plated on FN, treated with FNIII-1C for 4 h, and stained for phosphorylated p38 and β1 integrin (n = 3). (C) BAE cells were plated on FN, incubated overnight, and treated with 10 μM FNIII1C, 10 μM of heat-denatured FNIII1C, or 10 μM FNIII2C for 4 h. Cells were sheared for 30 min, and NF-κB nuclear translocation was assessed by immunocytochemistry (n = 3–5). (D) Cells were pretreated with FN peptides as in C, followed by incubation with the p38 inhibitor SB202190 (1 μM) for 1 h before onset of shear stress. Cells were sheared for 30 min and nuclear translocation of p65 was assessed. Values are means ± SD (>100 cells counted per condition per experiment; n = 3–8). **, P < 0.01; ***, P < 0.001. (E) Cells were pretreated with the FN peptides as indicated, incubated with SB202190, and sheared for 30 min. p65 phosphorylation at Ser536 was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p65. Values are means ± SD from four to seven experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: The selective activation of NF-κB in atherosclerosis-prone regions of the vasculature, together with the importance of NF-κB target genes in atherosclerosis, suggest that inhibition of this transcription factor could have therapeutic value. However, NF-κB has important roles in cell survival and immune protection, thus, global inhibition of the pathway is likely to be deleterious. In contrast, local inactivation of NF-κB within cell–ECM adhesions in atherosclerotic plaque could be beneficial. Although FN does not normally activate p38, a peptide derived from the first type III repeat in FN, termed III-1C, alters the structure of the FN matrix and stimulates p38 activation (Yi and Ruoslahti, 2001; Klein et al., 2003). When cells on FN were treated with this peptide, activated p38 colocalized with integrins at sites of adhesion (Fig. 7, A and B). To test whether or not FNIII-1C might also suppress NF-κB activation, BAE cells on FN were treated for 4 h with FNIII-1C or as controls with heat denatured III-1C or the analogous peptide from the second type III repeat, FNIII-2C. Cells were sheared for 30 min and NF-κB was assayed. FNIII-1C blocked shear stress–induced NF-κB nuclear translocation and phosphorylation, whereas FNIII-2C and heat-denatured FNIII-1C had no effect (Fig. 7, B and D). Furthermore, NF-κB activation was rescued by p38 inhibition in cells treated with FNIII-1C (Fig. 7, C and D). These results show that the proatherosclerotic FN ECM can be modified to locally activate p38 and thereby suppress NF-κB.

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