Transforming Growth Factor- Mediates Nuclear Factor B Activation in Strained Arteries
http://www.100md.com
Catherine A. Lemarié, Pierre-Louis Thara
参见附件。
the Inserm U689, Centre de Recherche Cardiovasculaire Inserm Lariboisière, 41, Boulevard de la Chapelle, 75010 Paris, France.
Abstract
Mechanical factors regulate both blood vessel growth and the development and progression of vascular disease. Acting on apoptotic and inflammatory signaling, the transcription factor nuclear factor B (NF-B) is a likely mediator of these processes. Nevertheless, pressure-dependent NF-B activation pathways remain mostly unknown. Here we report that high intraluminal pressure induces reactive oxygen species (ROS) in arteries and that inhibition of NADPH oxidase prevents both the generation of ROS and the activation of NF-B associated with high pressure. We also identify the epidermal growth factor receptor (EGFR) as a ROS-dependent signaling intermediate. In arteries from EGFR mutant mice (waved-2), pressure fails to activate NF-B. Moreover, using vessels from EGFR ligand-deficient mice, we show that transforming growth factor (TGF)-, but neither heparin-binding EGF-like growth factor nor epiregulin, transduces NF-B activation by high pressure. Preventing the release of the active form of TGF- also abolishes NF-B induction by strain. The role of TGF- signaling in vascular remodeling is substantiated in vivo; angiotensin II-induced activation of NF-B and associated cell proliferation and wall thickening are much reduced in TGF-–mutant mice compared with wild-type, despite equivalent hypertension in both groups. Conversely, apoptotic cells are detected only in vessels from hypertensive TGF-–mutant mice, outlining the role of NF-B in cell survival. Finally, the NF-B activation pathway contrasts with that of extracellular signal-regulated kinase 1/2, which is activated by stretch through the EGFR but does not implicate TGF-. Hence, our data identify TGF- as a potential specific target to modulate mechanosensitive NF-B activation and associated vascular remodeling.
Key Words: signal transduction EGFR NF-B hypertension vascular remodeling
Introduction
Arteries are under permanent physiological strain caused by shear stress and blood pressure. In turn, these stresses are major determinants of vessel morphology and composition; prolonged or chronic changes in the mechanical environment lead to adaptative restructuring of the vessel wall. Although beneficial in the context of development, vascular remodeling can also contribute directly to the progression of vascular disease in pathological conditions involving alterations in hemodynamic load.1 Case-in-point, there is a clear association between oscillatory blood flow patterns and atherosclerotic lesion localization,2 and blood pressure itself is correlated with increased risk factors for atherosclerosis.3 Likewise, pulse wave velocity, an index of hypertension-induced vascular remodeling, is an independent predictor of cardiovascular events.4 Intracellular signaling cascades induced by mechanical factors thus constitute attractive targets in the treatment of vascular diseases. In this respect, the nuclear factor B (NF-B) pathway is particularly interesting because it drives the expression of several factors involved in inflammation, cell survival, and vascular remodeling.5
NF-B is induced in cultured endothelial cells exposed to transient laminar or oscillatory shear stress,6–9 and in whole arteries, NF-B activity is greatest in areas of disturbed flow associated with a high propensity for atherosclerosis.10 Mediators of shear-sensitive NF-B activation include integrins,11 the platelet-endothelial cell adhesion molecule 1/cadherin/vascular endothelial growth factor receptor 2 complex,12 Ras,13 Rac1,14 and Akt.15 In comparison, pathways and consequences of pressure-dependent NF-B activation have been much less studied. We previously showed that activation of NF-B in whole vessels exposed to high intraluminal pressure is important for vascular cell survival.16 Moreover, in a model of aortic banding, expression of intracellular cell adhesion molecule 1 and vascular cell adhesion molecule 1 increased only in vessels exposed to high pressure, concomitant with induction of NF-B. In vessels at normal pressure, NF-B activity and adhesion molecule expression were minimal despite elevated circulating levels of angiotensin II.17 These studies suggest a critical role for NF-B in the vascular response to strain. Nevertheless, the pathway involved in NF-B activation in this context remains mostly unknown.
Recent studies indicate that the epidermal growth factor (EGF) receptor (EGFR) participates in the signaling events and cellular responses initiated by various stimuli, including mechanical stretch.18 On the one hand, the EGFR has been characterized as a downstream component of oxidative stress signaling, which is directly or indirectly activated by strain.19 It has also been shown that stretch activates membrane metalloproteinases implicated in the cleavage of EGFR ligand heparin-binding EGF-like growth factor (HB-EGF), leading to the activation of EGFR.20 On the other hand, a recent study established the concept that receptors may be activated directly by mechanical stretch without implication of their ligands.21 Although it is documented that NF-B can be activated by agonist stimulation of the EGFR in vitro,22–24 there is no indication thus far that the same pathway is induced in response to changes in the mechanical environment. The aim of our study was therefore to investigate the role of EGFR and its ligands as potential activators of the NF-B pathway in conditions of elevated cell strain.
Materials and Methods
Organ Culture
Mouse left and right carotid arteries were isolated, canulated at both extremities, and immersed in an organ culture bath filled with Dulbecco’s modified Eagle’s medium (GIBCO BRL) supplemented with 5% fetal calf serum as described previously.16 Each arterial segment was connected to a closed perfusion circuit consisting of a 3-port reservoir, a peristaltic pump (Alitea), and a pressure chamber enabling the application of a controlled intraluminal hydrostatic pressure. Vessels were perfused from the proximal to the distal end, at a flow set to renew the medium within the intraluminal space while creating minimal shear forces (0.5 dyne/cm2). Organ culture of the carotid segments was performed under sterile conditions in an incubator containing 5% CO2 at 37°C. Arterial segments were kept at 80 mm Hg or 150 mm Hg during 24 hours. More details appear in the online data supplement available at http://circres.ahajournals.org.
Some segments were treated with the NADPH oxidase inhibitor apocynin (100 μmol/L, Sigma), the flavin inhibitor diphenylene iodonium (DPI) (50 μmol/L, Calbiochem), the EGFR kinase inhibitor AG1478 (1 μmol/L, Calbiochem), or the metalloproteinase inhibitor GM6001 (30 μmol/L, Calbiochem). The same protocol was used for C57BL/6 mice; waved-2 mice (Wa2; The Jackson Laboratories) that display a point mutation in the tyrosine kinase domain of EGFR, reducing its ability to phosphorylate substrates by 90%25; transforming growth factor (TGF)- deficient waved-1 mice (Wa1; B6.Cg-Tgfawa1)26,27 (the Jackson Laboratories, Bar Harbor, Me); epiregulin-deficient mice (obtained from D.W. Threadgill, University of North Carolina, Chapel Hill)28; and HB-EGF–deficient mice (obtained from D.C. Lee, University of North Carolina, Chapel Hill).29
Details regarding Western blotting, immunohistochemistry, and luminescence assay methods have been described previously16,30,31 and appear in the online data supplement. Antibodies used for Western blot were: anti-IB antibody, anti–p65-NF-B, and anti–extracellular signal-regulated kinase (ERK)2 from Santa Cruz; anti–phospho-p65-NF-B (Ser536), anti–phospho-EGFR (Tyr1068), anti-EGFR, and anti–phospho-ERK1/2 from Cell Signaling Technology; anti–-actin (Sigma); and anti–TGF-1–50 (Bachem). For immunohistochemistry, anti–phospho-p65-NF-B (Ser529; Rockland), anti–HB-EGF (R&D systems), anti–caspase-3, and anti–cleaved caspase-3 (Cell Signaling Technology) were used. -galactosidase activity was determined using a LacZ staining kit (Invivogen), and apoptosis was detected by the TUNEL method with ApopDetek kits (Enzo Diagnostic). Vessel reactive oxygen species (ROS) were visualized with dihydroethidine (Sigma) and quantified using L-012 (Wako) as described recently.30,31
Results
High Pressure Induces NF-B via ROS Production
We investigated the mechanosensitive NF-B activation pathway in mouse carotid arteries maintained at normal (80 mm Hg) or high (150 mm Hg) intraluminal pressure for 24 hours. Using confocal microscopy, we detected increased superoxide production throughout the vessel wall of arteries at high pressure compared with arteries at normal pressure (Figure 1A). Treatment with apocynin prevented the strain-induced production of ROS, indicating that they were generated through NADPH oxidase.32 We confirmed these observations using a quantitative luminescence technique and found a 9-fold increase in ROS production in vessels maintained at high pressure compared with normotensive vessels (P<0.05). This effect was abolished in vessels treated with apocynin or DPI (Figure 1A).
Activation of the NF-B pathway in vessels submitted to high pressure was evidenced by decreased levels of the NF-B inhibitor IB (P<0.01 versus vessels at normal pressure) and increased phosphorylation of the p65 subunit of NF-B, demonstrated by Western blot (P<0.001; data not shown) and immunohistochemistry (Figure 1B). In vessels at 150 mm Hg, 57±8% of cells stained positive for phospho-p65, compared with only 11±5% in vessels at 80 mm Hg (P<0.001). Apocynin treatment completely prevented the activation of NF-B associated with elevated pressure (Figure 1B). Equivalent results were found using the flavin inhibitor DPI, confirming that treatment with antioxidants abolished the activation of NF-B (data not shown). Although we also found ERK1/2 to be activated in carotid arteries at high pressure (P<0.05 versus 80 mm Hg), it was not affected by treatment with apocynin (data not shown). Our results therefore indicate a specific role of ROS in the activation of NF-B but not ERK1/2 in vessels exposed to chronic stretch.
EGFR Transduces Activation of NF-B by Strain
ROS have been shown to stimulate signaling cascades through direct or indirect activation of the EGFR. We observed that high intraluminal pressure induced the phosphorylation of EGFR in whole vessels (P<0.01 versus 80 mm Hg). Apocynin significantly inhibited mechanosensitive EGFR phosphorylation, showing that ROS act upstream of the activation of EGFR (Figure 2B). This was confirmed using the EGFR kinase inhibitor AG1478, which also prevented the phosphorylation of EGFR in vessels at 150 mm Hg (Figure 2B) but did not affect strain-induced ROS production (Figure 2A). High pressure also failed to induce the phosphorylation of EGFR in vessels from Wa2 mice, which have an inactivating mutation in the EGFR kinase domain25,33 (Figure 2B). More importantly, in vessels treated with AG1478 or obtained from Wa2 mice, the absence of EGFR kinase activity coincided with the loss of pressure-dependent NF-B activation, such that IB protein levels and phosphorylation of NF-B were equivalent in vessels at 80 and 150 mm Hg (Figure 2C and 2D). These results revealed for the first time a key role of EGFR in the induction of the NF-B pathway by mechanical strain. The Wa2 mutation and AG1478 also abolished pressure-induced phosphorylation of ERK1/2 (data not shown), corroborating previous studies.19,20
Activation of EGFR by High Intraluminal Pressure Depends on TGF- Release
One pathway potentially implicated in the activation of EGFR involved transactivation by the angiotensin type 1 (AT1) receptor. Based on the notion that high intraluminal pressure stimulates angiotensin II (Ang II) release in isolated vessels,34 that the AT1 receptor itself may be activated directly by stretch,21 and that Ang II can transactivate the EGFR through ROS,35 we verified whether strain-induced NF-B might depend on activation of the angiotensin receptor. Treatment of vessels with the active form of the AT1 antagonist candesartan did not affect NF-B pathway activation by high intraluminal pressure whatsoever (data not shown), disproving this hypothesis.
We therefore focused on EGFR ligands TGF-, HB-EGF, and epiregulin as potential transducers of strain. These ligands are expressed as transmembrane precursors from which the soluble active peptides are released by proteolytic cleavage.36 We first verified that these ligands are expressed in cultured vessels and found positive staining corresponding to TGF-, HB-EGF, and epiregulin in smooth muscle and endothelial cells of arteries at both pressure settings (Figure 3A). Likewise, we verified and confirmed that the EGFR was expressed throughout the vascular wall (data not shown). To investigate the role of each of these ligands on NF-B activation, we used TGF- mutant Wa1 mice,26 HB-EGF knockout mice,29 and epiregulin-deficient mice.28 Compared with arteries from wild-type mice, where high pressure induced a significant increase of EGFR phosphorylation (P<0.01), we found no evidence of EGFR activation in vessels of Wa1 mice (figure 3B). In contrast, absence of epiregulin did not hamper EGFR phosphorylation associated with strain. In HB-EGF knockout mice, the baseline phosphorylation of EGFR was very elevated at normal pressure, and no further increase in phosphorylation could be detected at high pressure. Nevertheless, mechanosensitive activation of NF-B was observed in arteries from HB-EGF knockout mice, comparable to that of epiregulin knockout and wild-type animals (Figure 3C). In vessels of TGF- mutant mice, on the other hand, high pressure failed to induce IB degradation (Figure 3C) or NF-B phosphorylation (Figure 3C). These results uphold the concept that cells can transduce mechanical forces through autocrine/paracrine EGFR activation. More to the point, our data show that TGF- is a major mediator of stress signaling in the vascular wall, leading to NF-B activation. In comparison, pressure-dependent ERK1/2 activation was not blunted in TGF- mutant mice (data not shown).
TGF- activation relies on ectodomain shedding by cleavage of the membrane-bound pro-form, and indeed we detected greater levels of cleaved TGF- in the culture medium of vessels maintained at high pressure than at normal pressure (Figure 4B). In line with evidence implicating the disintegrin and metalloproteinase ADAM17 (also known as the tumor necrosis factor converting enzyme TACE) in the release of soluble TGF-,37,38 we found TACE to be abundantly expressed in smooth muscle and endothelial cells of cultured arteries, so much so that it did not appear to be upregulated by pressure (Figure 4A). Treatment of arteries with the nonspecific inhibitor of metalloproteinases GM6001 both reduced baseline TGF- shedding and prevented mechanosensitive TGF- release (Figure 4B). GM6001 also blocked the phosphorylation of EGFR (Figure 4C) and the activation of NF-B (Figure 4D) stimulated by high intraluminal pressure. These results suggest that through ROS production, pressure induces metalloproteinase-dependent release of TGF- that binds EGFR, leading to NF-B activation. Pressure-induced ERK1/2 was not sensitive to GM6001 (data not shown).
Functional Role of Pressure-Induced TGF-
We previously showed that inhibition of NF-B induced apoptosis in the vascular wall.16 Having found that blocking ROS, TGF-, and EGFR prevents the activation of NF-B in the current study, we hypothesized that this signaling pathway might be important for vascular cell survival. We investigated cell death in vessels from wild-type mice treated with apocynin and AG1478 and in vessels from mice deficient for HB-EGF, TGF-, and epiregulin. TUNEL staining, indicative of cell apoptosis, was increased in vessels treated with apocynin both at 80mm Hg and 150 mm Hg compared with untreated vessels (P<0.001 and P<0.05 versus untreated controls at equivalent pressure, respectively). Comparable results were found in vessels treated with AG1478 (P<0.05). Among EGFR ligand–deficient animals, enhanced TUNEL staining was found only in arteries of Wa1 mice (P<0.01 versus vessels from wild-type mice at equivalent pressure) (Figure 5). These observations were confirmed by staining for cleaved caspase-3 (data not shown).
In Vivo Role of TGF- in Hypertensive Remodeling
To bolster our in vitro results, we used an in vivo model of hypertension where wild-type and Wa1 mice were fitted with minipumps delivering Ang II for 1 week, producing equivalent pressure rises (and associated aortic wall stretch) in both strains (wild-type: 106.5±3 to 142.9± 5.6 mm Hg; Wa1: 105.7±2.3 to 138.5±6.4 mm Hg). No significant morphological differences were found between untreated wild-type and Wa1 mice. However, Ang II–induced hypertension only activated NF-B significantly in the aortic wall of wild-type mice (P<0.001 versus untreated control and P<0.001 versus Wa1 treated mice) (Figure 6A). Concomitantly, wall thickness (P<0.001) and vascular cell number (P<0.01) were enhanced only in wild-type animals (Figure 6B). In TGF-–deficient mice, no pressure-dependent changes in NF-B phosphorylation, cell proliferation, or vascular wall thickening were observed (Figure 6). On the other hand, TUNEL staining was detected exclusively in vessels of hypertensive Wa1 mice (P<0.01 versus wild-type Ang II–infused mice and P<0.001 versus untreated Wa1) (Figure 6A). These latter observations were confirmed by staining for cleaved caspase-3 (data not shown). Hence, the in vivo results support the premise that changes in the mechanical environment can be transduced through the release of TGF-, leading to NF-B activation and vascular remodeling.
Discussion
The present study reveals that the EGFR ligand TGF- plays a crucial role in the activation of NF-B by mechanical strain both ex vivo and in vivo. Soluble, active TGF- appears to be released through metalloproteinase activity, itself dependent on ROS production. The contribution of TGF- to this pathway is unique, as deletion of the other EGFR ligands, HB-EGF and epiregulin, is ineffective in preventing pressure-induced NF-B activation. Finally, TGF- is specific of the NF-B pathway; ERK1/2 activation in stretched vessels also implicates the EGFR but not TGF- or ROS.
We previously demonstrated that high pressure activates NF-B, but the pathway implicated in this process was mostly unknown. ROS had been found to be involved in the acute activation of NF-B in vascular smooth muscle cells exposed to cyclic stretch,39 and this was recently validated in the whole vessels exposed to high intraluminal pressure.40 Our results showing the absence of NF-B activation in apocynin and DPI-treated vessels further confirmed these observations. What remained to be elucidated was how pressure-induced ROS led to NF-B signaling.
Growing evidence suggested that ROS can stimulate signal transduction cascades through direct or indirect activation of the EGFR,19,41,42 and indeed we found EGFR phosphorylation to be enhanced in arteries after prolonged exposure to elevated stretch, an effect abolished by apocynin. The EGFR is ubiquitously expressed in various tissues and is thus positioned to influence a wide range of signal transduction pathways.43 Recent studies indicated that the EGFR could participate in the activation of ERK1/2 initiated by cyclic stretch in cultured smooth muscle cells20 or by acute steady stretch in coronary arteries.19 To the best of our knowledge, however, this is the first report to describe mechanosensitive induction of NF-B via the EGFR. In addition, although other studies have implicated EGF, HB-EGF, TGF-, and epiregulin as upstream mediators of EGFR phosphorylation leading to ERK1/2 activation,20,44 ours is the first to highlight a mechanosensitive pathway of NF-B activation involving TGF-. Pressure-dependent ERK1/2 activation was not affected by TGF- depletion, underscoring the unique character of the NF-B activation pathway. In resting cells, TGF- exists as a pro-form anchored in the plasma membrane. Cleavage of TGF- occurs via the activation of a protease (TACE), which is directly activated by ROS.38,45 Although we could not see an effect of pressure on TACE expression, baseline levels being very high to start with, there was a clear implication for this enzyme in both strain-dependent TGF- release and NF-B induction.
The ERK1/2 signaling pathway studied here in parallel with that of NF-B differs in several aspects from previously described modes of activation. Although we found that strain-induced ERK1/2 induction required EGFR activity, it clearly bypassed the need for ROS, unlike what was reported for bovine arteries exposed to acute stretch.19 Divergent kinetics (acute versus prolonged stimulus) or species could explain such differences. In addition, we found that stimulation of ERK1/2 in vessels exposed to high intraluminal pressure did not rely on metalloproteinase-dependent ligand shedding. This contrasts with a recent work that showed that application of mechanical compressive stress on smooth muscle cells in culture activates ERK1/2 via the cleavage of HB-EGF and its binding to the EGFR.20 Cultured smooth muscle cell phenotypic modulation, caused by loss of the native, tri-dimensional extracellular matrix environment as well as absence of basal strain, may account for these conflicting observations.46 Based on previous studies conducted in whole vessels, our observations suggest that the strain-induced ERK1/2 activation pathway is likely to involve focal adhesion kinase and c-src as upstream kinases,46 which may directly phosphorylate the EGFR.19
Our interest in the NF-B pathway resides in the important role ascribed to this transcription factor, which regulates genes involved in inflammatory, survival, and remodeling processes.47 We previously showed that blocking the nuclear translocation of NF-B leads to vascular cell apoptosis in arteries maintained at 80 mm Hg and more markedly so in vessels at 150 mm Hg.16 Here we found that inhibiting the different steps identified in the NF-B activation pathway produced equivalent results. In arteries treated with apocynin or AG1478 or in vessels from Wa1 mice, absence of NF-B activity was associated with increased apoptosis, which was equally high in vessels maintained at 80 and 150 mm Hg.
Drawing a parallel with our ex vivo experiments, we investigated the role of TGF- in vascular transformations associated with Ang II–induced hypertension. Increased NF-B activity in hypertensive animals coincided with increased vascular cell numbers, accompanied by thickened vessels. Remarkably, these effects were absent in TGF-–deficient animals, suggestive of a crucial role of hypertension-induced TGF- and NF-B in the remodeling response. Moreover, vascular cell apoptosis could be detected in vessels of hypertensive Wa1 mice but not in their wild-type counterparts, drawing a parallel with our ex vivo findings. This is all the more relevant given that pressure-induced NF-B was not affected by angiotensin AT1 receptor blockade in cultured arteries (data not shown), implying that the in vivo effects of Ang II treatment on the vascular wall may have been caused by the hypertensive state of the animals rather than by Ang II itself.
In summary, we hereby identify a pathway through which high blood pressure may induce vascular remodeling. More generally, our results uphold the concept that mechanical forces can be transduced via ROS-dependent autocrine/paracrine EGFR activation and, acting through the NF-B pathway, may regulate cell proliferation and synthetic activity. In this context, our data suggest that TGF- is a major mediator of stress signaling.
Acknowledgments
We thank D.W. Threadgill and D.C. Lee for providing us with epiregulin- and HB-EGF-deficient mice, respectively.
Sources of Funding
This work is supported by the European Vascular Genomics Network (http://www.evgn.org), a Network of Excellence supported by the European Community’s sixth Framework Programme for Research Priority 1 "Life sciences, genomics and biotechnology for health" (Contract N° LSHM-CT-2003–503254).
Disclosures
None.
Footnotes
Original received November 4, 2005; first resubmission received February 9, 2006; second resubmission received April 28, 2006; revised second resubmission received June 23, 2006; accepted July 6, 2006.
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the Inserm U689, Centre de Recherche Cardiovasculaire Inserm Lariboisière, 41, Boulevard de la Chapelle, 75010 Paris, France.
Abstract
Mechanical factors regulate both blood vessel growth and the development and progression of vascular disease. Acting on apoptotic and inflammatory signaling, the transcription factor nuclear factor B (NF-B) is a likely mediator of these processes. Nevertheless, pressure-dependent NF-B activation pathways remain mostly unknown. Here we report that high intraluminal pressure induces reactive oxygen species (ROS) in arteries and that inhibition of NADPH oxidase prevents both the generation of ROS and the activation of NF-B associated with high pressure. We also identify the epidermal growth factor receptor (EGFR) as a ROS-dependent signaling intermediate. In arteries from EGFR mutant mice (waved-2), pressure fails to activate NF-B. Moreover, using vessels from EGFR ligand-deficient mice, we show that transforming growth factor (TGF)-, but neither heparin-binding EGF-like growth factor nor epiregulin, transduces NF-B activation by high pressure. Preventing the release of the active form of TGF- also abolishes NF-B induction by strain. The role of TGF- signaling in vascular remodeling is substantiated in vivo; angiotensin II-induced activation of NF-B and associated cell proliferation and wall thickening are much reduced in TGF-–mutant mice compared with wild-type, despite equivalent hypertension in both groups. Conversely, apoptotic cells are detected only in vessels from hypertensive TGF-–mutant mice, outlining the role of NF-B in cell survival. Finally, the NF-B activation pathway contrasts with that of extracellular signal-regulated kinase 1/2, which is activated by stretch through the EGFR but does not implicate TGF-. Hence, our data identify TGF- as a potential specific target to modulate mechanosensitive NF-B activation and associated vascular remodeling.
Key Words: signal transduction EGFR NF-B hypertension vascular remodeling
Introduction
Arteries are under permanent physiological strain caused by shear stress and blood pressure. In turn, these stresses are major determinants of vessel morphology and composition; prolonged or chronic changes in the mechanical environment lead to adaptative restructuring of the vessel wall. Although beneficial in the context of development, vascular remodeling can also contribute directly to the progression of vascular disease in pathological conditions involving alterations in hemodynamic load.1 Case-in-point, there is a clear association between oscillatory blood flow patterns and atherosclerotic lesion localization,2 and blood pressure itself is correlated with increased risk factors for atherosclerosis.3 Likewise, pulse wave velocity, an index of hypertension-induced vascular remodeling, is an independent predictor of cardiovascular events.4 Intracellular signaling cascades induced by mechanical factors thus constitute attractive targets in the treatment of vascular diseases. In this respect, the nuclear factor B (NF-B) pathway is particularly interesting because it drives the expression of several factors involved in inflammation, cell survival, and vascular remodeling.5
NF-B is induced in cultured endothelial cells exposed to transient laminar or oscillatory shear stress,6–9 and in whole arteries, NF-B activity is greatest in areas of disturbed flow associated with a high propensity for atherosclerosis.10 Mediators of shear-sensitive NF-B activation include integrins,11 the platelet-endothelial cell adhesion molecule 1/cadherin/vascular endothelial growth factor receptor 2 complex,12 Ras,13 Rac1,14 and Akt.15 In comparison, pathways and consequences of pressure-dependent NF-B activation have been much less studied. We previously showed that activation of NF-B in whole vessels exposed to high intraluminal pressure is important for vascular cell survival.16 Moreover, in a model of aortic banding, expression of intracellular cell adhesion molecule 1 and vascular cell adhesion molecule 1 increased only in vessels exposed to high pressure, concomitant with induction of NF-B. In vessels at normal pressure, NF-B activity and adhesion molecule expression were minimal despite elevated circulating levels of angiotensin II.17 These studies suggest a critical role for NF-B in the vascular response to strain. Nevertheless, the pathway involved in NF-B activation in this context remains mostly unknown.
Recent studies indicate that the epidermal growth factor (EGF) receptor (EGFR) participates in the signaling events and cellular responses initiated by various stimuli, including mechanical stretch.18 On the one hand, the EGFR has been characterized as a downstream component of oxidative stress signaling, which is directly or indirectly activated by strain.19 It has also been shown that stretch activates membrane metalloproteinases implicated in the cleavage of EGFR ligand heparin-binding EGF-like growth factor (HB-EGF), leading to the activation of EGFR.20 On the other hand, a recent study established the concept that receptors may be activated directly by mechanical stretch without implication of their ligands.21 Although it is documented that NF-B can be activated by agonist stimulation of the EGFR in vitro,22–24 there is no indication thus far that the same pathway is induced in response to changes in the mechanical environment. The aim of our study was therefore to investigate the role of EGFR and its ligands as potential activators of the NF-B pathway in conditions of elevated cell strain.
Materials and Methods
Organ Culture
Mouse left and right carotid arteries were isolated, canulated at both extremities, and immersed in an organ culture bath filled with Dulbecco’s modified Eagle’s medium (GIBCO BRL) supplemented with 5% fetal calf serum as described previously.16 Each arterial segment was connected to a closed perfusion circuit consisting of a 3-port reservoir, a peristaltic pump (Alitea), and a pressure chamber enabling the application of a controlled intraluminal hydrostatic pressure. Vessels were perfused from the proximal to the distal end, at a flow set to renew the medium within the intraluminal space while creating minimal shear forces (0.5 dyne/cm2). Organ culture of the carotid segments was performed under sterile conditions in an incubator containing 5% CO2 at 37°C. Arterial segments were kept at 80 mm Hg or 150 mm Hg during 24 hours. More details appear in the online data supplement available at http://circres.ahajournals.org.
Some segments were treated with the NADPH oxidase inhibitor apocynin (100 μmol/L, Sigma), the flavin inhibitor diphenylene iodonium (DPI) (50 μmol/L, Calbiochem), the EGFR kinase inhibitor AG1478 (1 μmol/L, Calbiochem), or the metalloproteinase inhibitor GM6001 (30 μmol/L, Calbiochem). The same protocol was used for C57BL/6 mice; waved-2 mice (Wa2; The Jackson Laboratories) that display a point mutation in the tyrosine kinase domain of EGFR, reducing its ability to phosphorylate substrates by 90%25; transforming growth factor (TGF)- deficient waved-1 mice (Wa1; B6.Cg-Tgfawa1)26,27 (the Jackson Laboratories, Bar Harbor, Me); epiregulin-deficient mice (obtained from D.W. Threadgill, University of North Carolina, Chapel Hill)28; and HB-EGF–deficient mice (obtained from D.C. Lee, University of North Carolina, Chapel Hill).29
Details regarding Western blotting, immunohistochemistry, and luminescence assay methods have been described previously16,30,31 and appear in the online data supplement. Antibodies used for Western blot were: anti-IB antibody, anti–p65-NF-B, and anti–extracellular signal-regulated kinase (ERK)2 from Santa Cruz; anti–phospho-p65-NF-B (Ser536), anti–phospho-EGFR (Tyr1068), anti-EGFR, and anti–phospho-ERK1/2 from Cell Signaling Technology; anti–-actin (Sigma); and anti–TGF-1–50 (Bachem). For immunohistochemistry, anti–phospho-p65-NF-B (Ser529; Rockland), anti–HB-EGF (R&D systems), anti–caspase-3, and anti–cleaved caspase-3 (Cell Signaling Technology) were used. -galactosidase activity was determined using a LacZ staining kit (Invivogen), and apoptosis was detected by the TUNEL method with ApopDetek kits (Enzo Diagnostic). Vessel reactive oxygen species (ROS) were visualized with dihydroethidine (Sigma) and quantified using L-012 (Wako) as described recently.30,31
Results
High Pressure Induces NF-B via ROS Production
We investigated the mechanosensitive NF-B activation pathway in mouse carotid arteries maintained at normal (80 mm Hg) or high (150 mm Hg) intraluminal pressure for 24 hours. Using confocal microscopy, we detected increased superoxide production throughout the vessel wall of arteries at high pressure compared with arteries at normal pressure (Figure 1A). Treatment with apocynin prevented the strain-induced production of ROS, indicating that they were generated through NADPH oxidase.32 We confirmed these observations using a quantitative luminescence technique and found a 9-fold increase in ROS production in vessels maintained at high pressure compared with normotensive vessels (P<0.05). This effect was abolished in vessels treated with apocynin or DPI (Figure 1A).
Activation of the NF-B pathway in vessels submitted to high pressure was evidenced by decreased levels of the NF-B inhibitor IB (P<0.01 versus vessels at normal pressure) and increased phosphorylation of the p65 subunit of NF-B, demonstrated by Western blot (P<0.001; data not shown) and immunohistochemistry (Figure 1B). In vessels at 150 mm Hg, 57±8% of cells stained positive for phospho-p65, compared with only 11±5% in vessels at 80 mm Hg (P<0.001). Apocynin treatment completely prevented the activation of NF-B associated with elevated pressure (Figure 1B). Equivalent results were found using the flavin inhibitor DPI, confirming that treatment with antioxidants abolished the activation of NF-B (data not shown). Although we also found ERK1/2 to be activated in carotid arteries at high pressure (P<0.05 versus 80 mm Hg), it was not affected by treatment with apocynin (data not shown). Our results therefore indicate a specific role of ROS in the activation of NF-B but not ERK1/2 in vessels exposed to chronic stretch.
EGFR Transduces Activation of NF-B by Strain
ROS have been shown to stimulate signaling cascades through direct or indirect activation of the EGFR. We observed that high intraluminal pressure induced the phosphorylation of EGFR in whole vessels (P<0.01 versus 80 mm Hg). Apocynin significantly inhibited mechanosensitive EGFR phosphorylation, showing that ROS act upstream of the activation of EGFR (Figure 2B). This was confirmed using the EGFR kinase inhibitor AG1478, which also prevented the phosphorylation of EGFR in vessels at 150 mm Hg (Figure 2B) but did not affect strain-induced ROS production (Figure 2A). High pressure also failed to induce the phosphorylation of EGFR in vessels from Wa2 mice, which have an inactivating mutation in the EGFR kinase domain25,33 (Figure 2B). More importantly, in vessels treated with AG1478 or obtained from Wa2 mice, the absence of EGFR kinase activity coincided with the loss of pressure-dependent NF-B activation, such that IB protein levels and phosphorylation of NF-B were equivalent in vessels at 80 and 150 mm Hg (Figure 2C and 2D). These results revealed for the first time a key role of EGFR in the induction of the NF-B pathway by mechanical strain. The Wa2 mutation and AG1478 also abolished pressure-induced phosphorylation of ERK1/2 (data not shown), corroborating previous studies.19,20
Activation of EGFR by High Intraluminal Pressure Depends on TGF- Release
One pathway potentially implicated in the activation of EGFR involved transactivation by the angiotensin type 1 (AT1) receptor. Based on the notion that high intraluminal pressure stimulates angiotensin II (Ang II) release in isolated vessels,34 that the AT1 receptor itself may be activated directly by stretch,21 and that Ang II can transactivate the EGFR through ROS,35 we verified whether strain-induced NF-B might depend on activation of the angiotensin receptor. Treatment of vessels with the active form of the AT1 antagonist candesartan did not affect NF-B pathway activation by high intraluminal pressure whatsoever (data not shown), disproving this hypothesis.
We therefore focused on EGFR ligands TGF-, HB-EGF, and epiregulin as potential transducers of strain. These ligands are expressed as transmembrane precursors from which the soluble active peptides are released by proteolytic cleavage.36 We first verified that these ligands are expressed in cultured vessels and found positive staining corresponding to TGF-, HB-EGF, and epiregulin in smooth muscle and endothelial cells of arteries at both pressure settings (Figure 3A). Likewise, we verified and confirmed that the EGFR was expressed throughout the vascular wall (data not shown). To investigate the role of each of these ligands on NF-B activation, we used TGF- mutant Wa1 mice,26 HB-EGF knockout mice,29 and epiregulin-deficient mice.28 Compared with arteries from wild-type mice, where high pressure induced a significant increase of EGFR phosphorylation (P<0.01), we found no evidence of EGFR activation in vessels of Wa1 mice (figure 3B). In contrast, absence of epiregulin did not hamper EGFR phosphorylation associated with strain. In HB-EGF knockout mice, the baseline phosphorylation of EGFR was very elevated at normal pressure, and no further increase in phosphorylation could be detected at high pressure. Nevertheless, mechanosensitive activation of NF-B was observed in arteries from HB-EGF knockout mice, comparable to that of epiregulin knockout and wild-type animals (Figure 3C). In vessels of TGF- mutant mice, on the other hand, high pressure failed to induce IB degradation (Figure 3C) or NF-B phosphorylation (Figure 3C). These results uphold the concept that cells can transduce mechanical forces through autocrine/paracrine EGFR activation. More to the point, our data show that TGF- is a major mediator of stress signaling in the vascular wall, leading to NF-B activation. In comparison, pressure-dependent ERK1/2 activation was not blunted in TGF- mutant mice (data not shown).
TGF- activation relies on ectodomain shedding by cleavage of the membrane-bound pro-form, and indeed we detected greater levels of cleaved TGF- in the culture medium of vessels maintained at high pressure than at normal pressure (Figure 4B). In line with evidence implicating the disintegrin and metalloproteinase ADAM17 (also known as the tumor necrosis factor converting enzyme TACE) in the release of soluble TGF-,37,38 we found TACE to be abundantly expressed in smooth muscle and endothelial cells of cultured arteries, so much so that it did not appear to be upregulated by pressure (Figure 4A). Treatment of arteries with the nonspecific inhibitor of metalloproteinases GM6001 both reduced baseline TGF- shedding and prevented mechanosensitive TGF- release (Figure 4B). GM6001 also blocked the phosphorylation of EGFR (Figure 4C) and the activation of NF-B (Figure 4D) stimulated by high intraluminal pressure. These results suggest that through ROS production, pressure induces metalloproteinase-dependent release of TGF- that binds EGFR, leading to NF-B activation. Pressure-induced ERK1/2 was not sensitive to GM6001 (data not shown).
Functional Role of Pressure-Induced TGF-
We previously showed that inhibition of NF-B induced apoptosis in the vascular wall.16 Having found that blocking ROS, TGF-, and EGFR prevents the activation of NF-B in the current study, we hypothesized that this signaling pathway might be important for vascular cell survival. We investigated cell death in vessels from wild-type mice treated with apocynin and AG1478 and in vessels from mice deficient for HB-EGF, TGF-, and epiregulin. TUNEL staining, indicative of cell apoptosis, was increased in vessels treated with apocynin both at 80mm Hg and 150 mm Hg compared with untreated vessels (P<0.001 and P<0.05 versus untreated controls at equivalent pressure, respectively). Comparable results were found in vessels treated with AG1478 (P<0.05). Among EGFR ligand–deficient animals, enhanced TUNEL staining was found only in arteries of Wa1 mice (P<0.01 versus vessels from wild-type mice at equivalent pressure) (Figure 5). These observations were confirmed by staining for cleaved caspase-3 (data not shown).
In Vivo Role of TGF- in Hypertensive Remodeling
To bolster our in vitro results, we used an in vivo model of hypertension where wild-type and Wa1 mice were fitted with minipumps delivering Ang II for 1 week, producing equivalent pressure rises (and associated aortic wall stretch) in both strains (wild-type: 106.5±3 to 142.9± 5.6 mm Hg; Wa1: 105.7±2.3 to 138.5±6.4 mm Hg). No significant morphological differences were found between untreated wild-type and Wa1 mice. However, Ang II–induced hypertension only activated NF-B significantly in the aortic wall of wild-type mice (P<0.001 versus untreated control and P<0.001 versus Wa1 treated mice) (Figure 6A). Concomitantly, wall thickness (P<0.001) and vascular cell number (P<0.01) were enhanced only in wild-type animals (Figure 6B). In TGF-–deficient mice, no pressure-dependent changes in NF-B phosphorylation, cell proliferation, or vascular wall thickening were observed (Figure 6). On the other hand, TUNEL staining was detected exclusively in vessels of hypertensive Wa1 mice (P<0.01 versus wild-type Ang II–infused mice and P<0.001 versus untreated Wa1) (Figure 6A). These latter observations were confirmed by staining for cleaved caspase-3 (data not shown). Hence, the in vivo results support the premise that changes in the mechanical environment can be transduced through the release of TGF-, leading to NF-B activation and vascular remodeling.
Discussion
The present study reveals that the EGFR ligand TGF- plays a crucial role in the activation of NF-B by mechanical strain both ex vivo and in vivo. Soluble, active TGF- appears to be released through metalloproteinase activity, itself dependent on ROS production. The contribution of TGF- to this pathway is unique, as deletion of the other EGFR ligands, HB-EGF and epiregulin, is ineffective in preventing pressure-induced NF-B activation. Finally, TGF- is specific of the NF-B pathway; ERK1/2 activation in stretched vessels also implicates the EGFR but not TGF- or ROS.
We previously demonstrated that high pressure activates NF-B, but the pathway implicated in this process was mostly unknown. ROS had been found to be involved in the acute activation of NF-B in vascular smooth muscle cells exposed to cyclic stretch,39 and this was recently validated in the whole vessels exposed to high intraluminal pressure.40 Our results showing the absence of NF-B activation in apocynin and DPI-treated vessels further confirmed these observations. What remained to be elucidated was how pressure-induced ROS led to NF-B signaling.
Growing evidence suggested that ROS can stimulate signal transduction cascades through direct or indirect activation of the EGFR,19,41,42 and indeed we found EGFR phosphorylation to be enhanced in arteries after prolonged exposure to elevated stretch, an effect abolished by apocynin. The EGFR is ubiquitously expressed in various tissues and is thus positioned to influence a wide range of signal transduction pathways.43 Recent studies indicated that the EGFR could participate in the activation of ERK1/2 initiated by cyclic stretch in cultured smooth muscle cells20 or by acute steady stretch in coronary arteries.19 To the best of our knowledge, however, this is the first report to describe mechanosensitive induction of NF-B via the EGFR. In addition, although other studies have implicated EGF, HB-EGF, TGF-, and epiregulin as upstream mediators of EGFR phosphorylation leading to ERK1/2 activation,20,44 ours is the first to highlight a mechanosensitive pathway of NF-B activation involving TGF-. Pressure-dependent ERK1/2 activation was not affected by TGF- depletion, underscoring the unique character of the NF-B activation pathway. In resting cells, TGF- exists as a pro-form anchored in the plasma membrane. Cleavage of TGF- occurs via the activation of a protease (TACE), which is directly activated by ROS.38,45 Although we could not see an effect of pressure on TACE expression, baseline levels being very high to start with, there was a clear implication for this enzyme in both strain-dependent TGF- release and NF-B induction.
The ERK1/2 signaling pathway studied here in parallel with that of NF-B differs in several aspects from previously described modes of activation. Although we found that strain-induced ERK1/2 induction required EGFR activity, it clearly bypassed the need for ROS, unlike what was reported for bovine arteries exposed to acute stretch.19 Divergent kinetics (acute versus prolonged stimulus) or species could explain such differences. In addition, we found that stimulation of ERK1/2 in vessels exposed to high intraluminal pressure did not rely on metalloproteinase-dependent ligand shedding. This contrasts with a recent work that showed that application of mechanical compressive stress on smooth muscle cells in culture activates ERK1/2 via the cleavage of HB-EGF and its binding to the EGFR.20 Cultured smooth muscle cell phenotypic modulation, caused by loss of the native, tri-dimensional extracellular matrix environment as well as absence of basal strain, may account for these conflicting observations.46 Based on previous studies conducted in whole vessels, our observations suggest that the strain-induced ERK1/2 activation pathway is likely to involve focal adhesion kinase and c-src as upstream kinases,46 which may directly phosphorylate the EGFR.19
Our interest in the NF-B pathway resides in the important role ascribed to this transcription factor, which regulates genes involved in inflammatory, survival, and remodeling processes.47 We previously showed that blocking the nuclear translocation of NF-B leads to vascular cell apoptosis in arteries maintained at 80 mm Hg and more markedly so in vessels at 150 mm Hg.16 Here we found that inhibiting the different steps identified in the NF-B activation pathway produced equivalent results. In arteries treated with apocynin or AG1478 or in vessels from Wa1 mice, absence of NF-B activity was associated with increased apoptosis, which was equally high in vessels maintained at 80 and 150 mm Hg.
Drawing a parallel with our ex vivo experiments, we investigated the role of TGF- in vascular transformations associated with Ang II–induced hypertension. Increased NF-B activity in hypertensive animals coincided with increased vascular cell numbers, accompanied by thickened vessels. Remarkably, these effects were absent in TGF-–deficient animals, suggestive of a crucial role of hypertension-induced TGF- and NF-B in the remodeling response. Moreover, vascular cell apoptosis could be detected in vessels of hypertensive Wa1 mice but not in their wild-type counterparts, drawing a parallel with our ex vivo findings. This is all the more relevant given that pressure-induced NF-B was not affected by angiotensin AT1 receptor blockade in cultured arteries (data not shown), implying that the in vivo effects of Ang II treatment on the vascular wall may have been caused by the hypertensive state of the animals rather than by Ang II itself.
In summary, we hereby identify a pathway through which high blood pressure may induce vascular remodeling. More generally, our results uphold the concept that mechanical forces can be transduced via ROS-dependent autocrine/paracrine EGFR activation and, acting through the NF-B pathway, may regulate cell proliferation and synthetic activity. In this context, our data suggest that TGF- is a major mediator of stress signaling.
Acknowledgments
We thank D.W. Threadgill and D.C. Lee for providing us with epiregulin- and HB-EGF-deficient mice, respectively.
Sources of Funding
This work is supported by the European Vascular Genomics Network (http://www.evgn.org), a Network of Excellence supported by the European Community’s sixth Framework Programme for Research Priority 1 "Life sciences, genomics and biotechnology for health" (Contract N° LSHM-CT-2003–503254).
Disclosures
None.
Footnotes
Original received November 4, 2005; first resubmission received February 9, 2006; second resubmission received April 28, 2006; revised second resubmission received June 23, 2006; accepted July 6, 2006.
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