2 Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908
3 Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908
4 Internal Medicine, University of Virginia, Charlottesville, VA 22908
5 Mellon Prostate Cancer Research Center, University of Virginia, Charlottesville, VA 22908
Correspondence to M.A. Schwartz: maschwartz{at}virginia.edu
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Introduction |
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The NF-B family of transcription factors are involved in numerous cellular processes, including differentiation, inflammation, proliferation, and apoptosis, and are postulated to contribute to atherogenesis (Collins and Cybulsky, 2001; Kutuk and Basaga, 2003). Current data implicate NF-
B as a key regulator of shear stressinduced inflammatory gene expression. NF-
B dimers, particularly the p50/p65 heterodimer, bind to a shear stress responsive element found in the promoter of several atherogenic genes, including ICAM-1, VCAM-1, and MCP-1 (monocyte chemotactic protein-1), which regulate monocyte recruitment, as well as PDGF, which stimulates smooth muscle growth and migration (Resnick et al., 1993; Khachigian et al., 1995; Huo and Ley, 2001). NF-
B expression and activity, as well as ICAM-1 and VCAM-1 expression, are elevated in atherosclerosis-prone regions before or in the absence of fatty streak formation, indicating that they are very early events in atherosclerotic progression (Brand et al., 1996; Nakashima et al., 1998; Iiyama et al., 1999; Wilson et al., 2000). In vitro, disturbed shear stimulates prolonged NF-
B activation and NF-
Bdependent gene expression; in contrast, acute onset of laminar shear stress activates NF-
B but only transiently (Lan et al., 1994; Khachigian et al., 1995; Mohan et al., 1997). We hypothesize that these differences in signaling pathways induced by laminar versus disturbed flow are due in part to differences in adaptation mechanisms, such that changes in flow velocity and direction associated with disturbed flow prevents down-regulation of the responses so that signals activated transiently by laminar flow are sustained in disturbed flow. Thus, data from both in vivo and in vitro models suggest that NF-
B contributes to the initiation of atherosclerosis by fluid shear stress.
Studies in vitro have shown that acute onset of laminar shear triggers conversion of integrins to a high affinity state, followed by their binding to the subendothelial ECM (Tzima et al., 2001). Resultant integrin signaling mediates activation of NF-B through the small GTPase Rac (Tzima et al., 2002). Endothelial cells normally reside on a basement membrane comprised mainly of collagen (Coll) IV and laminin (LN). Coll binds primarily integrins
2ß1 and
1ß1, whereas LN binds mainly
6ß1 and
6ß4 (Belkin and Stepp, 2000; Heino, 2000). Ligation of these integrins is associated with a quiescent cell phenotype, consistent with the low turnover observed in endothelial cells in vivo (Schwartz and Assoian, 2001). Inflammation or injury can trigger the deposition of transitional ECM proteins such as fibronectin (FN) and fibrinogen (FG) into the subendothelial matrix (Sechler et al., 1998). In endothelial cells, FN primarily ligates
5ß1 whereas FG ligates
vß3, although other integrins may also be involved. Signals from
5ß1 and
vß3 are associated with enhanced endothelial cell proliferation and migration, processes important for injury-induced endothelial remodeling (Schwartz and Assoian, 2001). Many studies have shown that different integrins transduce distinct signals (Schwartz and Assoian, 2001). The involvement of integrins in shear stressdependent signaling thus suggests that the composition of the subendothelial ECM may modulate cellular responses to fluid flow. Therefore, we investigated the role of the subendothelial ECM in the endothelial response to fluid shear stress.
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Results |
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ECM remodeling in vivo
The distinct effects of different ECM proteins in flow-induced NF-B activation in vitro prompted us to ask whether or not these effects were relevant to plaque development in vivo. To test whether FN and FG deposition are associated with focal expression of NF-kB target genes in atherosclerosis-prone regions of the vasculature, these proteins were examined in mice. Serial sections from atherosclerosis-susceptible sites were stained for ECM proteins, for ICAM-1 and VCAM-1 as markers of NF-
B activation, and for the leukocyte integrin Mac-2 as a measure of monocyte recruitment. Atherosclerosis-resistant C57/B6 mice and atherosclerosis-prone apolipoprotein E (ApoE) null mice were fed either a normal chow diet or a high fat, Western diet for 10 wk. Sites associated with atherosclerotic plaque formation, such as the innominate artery and the carotid arteries, were then analyzed by immunohistochemistry (IHC; Nakashima et al., 1994).
In C57/B6 mice fed the low fat, chow diet, sites that are susceptible to atherosclerosis in other models demonstrated focal increases in ICAM-1 and VCAM-1 staining and increased staining for FN, although no Mac-2 staining was apparent (Fig. 2 A). We did not detect any change in FG (Fig. 2 A) or LN (not depicted) staining and no staining for FN in regions outside these zones. ApoE null mice fed a chow diet have elevated cholesterol and develop atherosclerotic lesions over the course of their lifespan (Daugherty, 2002). In these mice, similar colocalization of ICAM-1, VCAM-1, FN, and FG was observed when nearby sections were stained (Fig. 2 B). FN has been reported in well-defined fatty streaks (Stenman et al., 1980; Labat-Robert et al., 1985; Shekhonin et al., 1987; Tanouchi et al., 1992), but these data are the first evidence that FN is deposited before fatty streak development. In contrast, no changes in PECAM staining were evident (unpublished data). VCAM-1 staining in vascular smooth muscle cells is indicative of smooth muscle activation and is associated with atherosclerosis, which is consistent with our interpretation of these regions as early atherogenesis (Li et al., 1993).
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Coll-specific p38 signaling inhibits shear-induced NF-B activation
In addition to NF-B, p38 MAP kinase is also involved in stimulating inflammatory protein expression in a variety of systems. However, p38 shows differential effects on NF-
B, either enhancing or suppressing NF-
B depending on the stimulus (Schwenger et al., 1998; Alpert et al., 1999; Bowie and O'Neill, 2000; Bradbury et al., 2001). To examine p38 in this system, BAE cells were plated on different ECM proteins for 4 h and effects of shear were tested. Western blotting with an antibody against the phosphorylated and activated form of p38 showed that shear stress stimulated a transient increase in p38 phosphorylation in cells on Coll but not on other ECM proteins (Fig. 3 A). This result is consistent with previous reports showing that the Coll-binding integrin
2ß1 stimulates p38 activation (Ivaska et al., 1999). To test if this Coll-specific p38 activation mediates inhibition of shear stressinduced NF-
B activation, cells were preincubated with the p38 inhibitor SB202190 (1 µM for 1 h) before fluid flow. Inhibition of p38 with SB202190 restored shear stressinduced NF-
B nuclear translocation and NF-
B phosphorylation in cells on Coll but had no effect on cells on FN (Fig. 3, B and C). To confirm this result, endothelial cells were transiently transfected with a dominant-negative mutant of p38 (p38-AGF). This construct did not alter baseline levels of NF-
B activity but enhanced flow-induced NF-
B nuclear translocation in cells on Coll (Fig. 3 D). Thus, selective activation of p38 mediates suppression of NF-
B in cells on Coll.
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Flow-induced NF-B activation on mixed matrices
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-B 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-
B 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).
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These results appear paradoxical but could be explained if the inhibitory p38 signal from Coll occurs locally within a specific compartment. To test this hypothesis, endothelial cells on Coll or FN were sheared for 5 min and active p38 was localized. Cells on Coll showed colocalization of phospho-p38 with ß1 integrins, which was increased by shear stress (Fig. 6 A). Cells on FN showed only low levels of phospho-p38 staining, which is consistent with results from Western blotting.
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Altering the FN matrix to inhibit NF-B
The selective activation of NF-B in atherosclerosis-prone regions of the vasculature, together with the importance of NF-
B target genes in atherosclerosis, suggest that inhibition of this transcription factor could have therapeutic value. However, NF-
B 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-
B within cellECM 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-
B 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-
B was assayed. FNIII-1C blocked shear stressinduced NF-
B nuclear translocation and phosphorylation, whereas FNIII-2C and heat-denatured FNIII-1C had no effect (Fig. 7, B and D). Furthermore, NF-
B 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-
B.
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Discussion |
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The role of the extracellular matrix as a key regulator of the behavior of advanced atherosclerosis is widely accepted, with ECM remodeling thought to affect both the size and stability of the fibrous atherosclerotic plaque (Katsuda and Kaji, 2003). However, a critical role for the subendothelial matrix in development of atherosclerosis has not been suspected. Our data show that deposition of FN and FG into the subendothelial matrix in regions of disturbed flow precedes fatty streak formation in vivo. These proteins in the subendothelial matrix promote activation of NF-B by shear stress in vitro, whereas the normal basement membrane component Coll suppresses NF-
B through
2ß1-dependent p38 activation. Importantly, p38 is activated locally within adhesion sites and prevents the shear stressinduced activation of IKKß and NF-
B but not the global activation of NF-
B by cytokines. Finally, we demonstrate that FN matrix can be altered to stimulate p38 and suppress shear stressinduced NF-
B activation, suggesting that FN is a possible target for therapeutic intervention.
ECM remodeling in atherogenesis
The normal subendothelial ECM is a basement membrane whose major integrin-binding components are type IV Coll and LNs (Yurchenco and O'Rear, 1994). Injury, inflammation, or angiogenesis result in degradation of the basement membrane and deposition of transitional ECM proteins such as FN and FG. We show here that the presence of transitional matrix proteins in the subendothelial matrix correlates with NF-B activation and target gene expression in regions of disturbed shear stress. These data are consistent with the idea that transitional matrix deposition regulates inflammatory gene expression in early atherogenesis; however, the causal relationships in vivo between ECM and NF-
B remain to be elucidated. Effects of disturbed flow on FN matrix deposition could involve changes in endothelial permeability to plasma proteins (Friedman and Fry, 1993; Phelps and DePaola, 2000; Himburg et al., 2004), changes in endothelial expression of ECM genes (Brooks et al., 2002), or changes in integrin expression or function (Brooks et al., 2002). Identifying the mechanisms involved in local matrix remodeling at atherosclerosis-prone sites in arteries will be an interesting direction for future work.
We also observed marked increases in both the ECM proteins FN and FG and the inflammatory markers ICAM-1 and VCAM-1 in atherosclerosis-susceptible regions of arteries from ApoE null mice fed a Western diet compared with mice on a chow diet, suggesting that the Western diet may further increase ECM remodeling. This effect would be predicted to further enhance endothelial responses to disturbed flow, thus defining a positive feedback loop that would contribute to disease progression.
Inhibition of NF-B activation by locally activated p38
Although p38 enhances NF-B activity in many systems (Carter et al., 1999; Korus et al., 2002), there are several stimuli for which activation of p38 inhibits NF-
B, including vitamin C, salicylate, indomethacin, sorbitol, hydrogen peroxide, and arsenite (Schwenger et al., 1998; Alpert et al., 1999; Bowie and O'Neill, 2000; Bradbury et al., 2001). Drosophila melanogaster p38 isoforms have been shown to limit antimicrobial peptide production, a known target of D. melanogaster NF-
B (Han et al., 1998). In none of these cases has the mechanism by which active p38 represses NF-
B activation been elucidated. Binding of integrin
2ß1 to Coll also activates p38 (Ivaska et al., 1999). Consistent with our model in which shear stress stimulates integrin activation and ligand binding (Jalali et al., 2001; Tzima et al., 2001; Katsumi et al., 2004), we found that shear stress stimulated
2ß1-dependent p38 activation in cells on Coll. Activation of p38 inhibited both IKKß and NF-
B activation. Importantly, both active p38 and active IKKß localized to sites of adhesion. This colocalization correlated with the integrin specificity of the functional interaction because Coll did not inhibit cytokine activation of NF-
B and global activation of 38 did not inhibit shear stress activation of NF-
B. Therefore, the data provide a striking example where signaling specificity is conferred by compartmentalization. Future work will be required to elucidate the pathway by which p38 inhibits IKKß in this system
Altering the FN matrix
Selective inhibition of NF-B activation by shear stress offers potential benefits for treatment of atherosclerosis, but it is essential that inhibitors act locally both to be effective and to avoid global inhibition of NF-
B, which would likely have undesirable side effects. A peptide from the first type III repeat of FN that was reported to trigger p38 activation in cells on FN induced local activation of p38 at sites of adhesion on a FN matrix and inhibited shear stressinduced activation of NF-
B. The effects of FNIII-1C are poorly understood. FNIII-1C has been reported to trigger assembly of soluble FN into fibrils and increase matrix FN (Morla and Ruoslahti, 1992), inhibit deposition of matrix FN (Bourdoulous et al., 1998), and alter its conformation without changing the total amount of matrix (Klein et al., 2003). Changes in FN matrix structure could conceivably alter signaling through a previously bound receptor, perhaps by changing its spatial organization, or could expose new cell binding sites or block existing cell binding sites required to prevent p38 activation. FNIII-1C binding to FN masks the EDA domain present in certain forms of FN (Klein et al., 2003), which is consistent with this last hypothesis. Curiously, mice engineered to lack the EDA domain of FN show reduced atherosclerosis, suggesting a potential role for this interaction in atherogenesis (Tan et al., 2004).
FNIII-1C itself is not a likely candidate for therapy, at least as an injected reagent, because it binds to soluble FN in the plasma and has been reported to induce assembly of this FN into fibrils, an event that could trigger thromboses or other deleterious events. Thus, further investigation into the mechanisms of ECM remodeling and p38 activation will be needed to develop more suitable methods for altering subendothelial ECM or cellular responses to it. Nevertheless, these results point toward an entirely novel strategy for treating atherosclerosis. The substrate dependence for proatherosclerotic gene expression may also have important implications in determining suitable signaling-based therapeutic approaches and designing bioengineered vascular grafts.
We conclude that transitional matrix proteins, such as FN and FG, are required for the flow-induced activation of NF-B and suggest that deposition of FN and FG into the subendothelial matrix at atherosclerosis-prone sites in vivo may regulate the early changes in inflammatory gene expression associated with atherogenesis. Furthermore, this shear stressinduced inflammatory signal appears to be inhibited by the endogenous Coll basement membrane through integrin
2ß1dependent activation of p38. Exploiting this fact, we show that alteration of the FN matrix with the FNIII-1C peptide prevents the shear stressinduced activation of NF-
B through localized p38 signaling. These results suggest a model in which subendothelial matrix remodeling mediates atherosclerotic progression through integrin-dependent mechanotransduction. Further work in which the subendothelial matrix or integrin signals are altered will be required to rigorously test this new model.
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Materials and methods |
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Immunocytochemistry
Cells were fixed with PBS containing 2% formaldehyde, permeabilized with 0.2% Triton X-100 when applicable, and blocked overnight in PBS containing 1% BSA and 10% goat serum. Primary antibodies were incubated with cells in blocking buffer as follows: rabbit anti-p65 (Santa Cruz Biotechnology, Inc.; 1:200 for 1 h), rabbit anti-ICAM (Santa Cruz Biotechnology, Inc.; 1:100 for 1 h), rabbit antiphospho-IKK (Cell Signaling Technology; 1:100 overnight), rabbit antiphospho-p38 (Cell Signaling Technology; 1:100 overnight), and mouse anti-ligated ß1 integrins (12G10; 1 µg/ml for 1 h). Cells were incubated in 1 µg/ml Alexa 488conjugated goat antirabbit IgG or Alexa 568conjugated goat antimouse IgG (Molecular Probes). Slides were mounted with Fluoromount G and images were taken using the 60x oil immersion objective on a microscope (model DiaPhot; Nikon) equipped with a CoolSnap video camera (Photometrics) using the Inovision ISEE software program.
Immunoblotting
Cell lysis and immunoblotting were performed as previously described (Orr et al., 2002). Rabbit antiphospho-p38 (Cell Signaling Technology), mouse anti-p38 (Santa Cruz Biotechnology, Inc.), rabbit antiphospho-p65 (Ser536; Cell Signaling Technology), rabbit anti-p65 (Santa Cruz Biotechnology, Inc.), rabbit antiphospho-IKK/ß (Cell Signaling Technologies), and rabbit anti-IKK
/ß (Santa Cruz Biotechnology, Inc.) were all used at 1:1000 dilutions.
Animals
Six male ApoE-deficient mice on a C57BL/6 background (Jackson ImmunoResearch Laboratories), 812 wk old and weighing 1820 g, were used in these experiments. Four mice were fed a Western-type atherogenic diet (TD 88137; Harlan-Teklad; containing 21% fat by weight, 0.15% by weight cholesterol, and 19.5% by weight casein without sodium cholate) for 10 wk before killing. Control mice were fed a chow diet during this time. Comparable wild-type C57/B6 mice were fed the chow diet during the same time frame.
Vessel harvest
At 20 wk old (10 wk on diet), mice were perfused with 4% formaldehyde and the innominate, left carotid, right carotid, and short segments of the descending abdominal aorta near the renal arteries and the iliac bifurcation were processed for paraffin embedding.
IHC
5-µm paraffin sections were obtained for IHC. IHC for adhesion molecules VCAM-1, ICAM-1, Mac-2, and PECAM-1 (Santa Cruz Biotechnology, Inc.) was performed as previously described (McPherson et al., 2001). After microwave antigen retrieval with antigen unmasking solution (Vector Laboratories), rabbit anti-FN (1:400; Sigma-Aldrich) and rabbit anti-LN (1:500; Sigma-Aldrich) were applied. Detection of antibodies was done with Vectastain Elite Kit (Vector Laboratories). Visualization was done with DAB (Deko Corp). Goat anti-FG (1 µg/ml; Accurate Chemical) was applied as for the other antigens, except that no antigen retrieval was required. Images were acquired using the 20 or 40x objective on a microscope (model BX51; Olympus) equipped with a digital camera (model DP70; Olympus) using the ImagePro Plus software program in the Academic Computing Health Sciences Center at the University of Virginia.
Online supplemental material
In Fig. 4, we demonstrate that incubation with the 2ß1 integrinblocking antibody R2-8C8 can block shear-induced p38 activation on Coll and rescue NF-
B activation in cells on Coll, presumably by preventing the interaction of newly activated
2ß1 with Coll. To demonstrate that this effect was not due to changes in cell adhesiveness, we show that short-term incubation with R2-8C8 does not affect the organization of the actin cytoskeleton (Fig. S1). To show that this effect was specific to Coll, we also prevented the interaction between
6ß1 and LN with the
6ß1-blocking antibody GoH3, which did not rescue flow-induced NF-
B activation (Fig. S2). Finally, we show that flow-induced p38 activation on Coll prevents flow-dependent activation of NF-
B. To determine if this effect was specific for flow, the ability of TNF
to activate NF-
B in cells on different matrix proteins or overexpressing a constitutively active MKK3 construct (MKK3-EE) was assessed (Fig. S3). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200410073/DC1.
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Acknowledgments |
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This work was supported by U.S. Public Health Service grant RO1 HL75092 to M.A. Schwartz, American Heart Association Mid-Atlantic Affiliate fellowship 0325654U to A.W. Orr, and National Institutes of Health grant 1RO1HL66264 to I.J. Sarembock.
Submitted: 13 October 2004
Accepted: 4 March 2005
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