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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2366.)
© 2000 American Heart Association, Inc.

Vascular Biology

Pulsatile Stretch–Induced Extracellular Signal–Regulated Kinase 1/2 Activation in Organ Culture of Rabbit Aorta Involves Reactive Oxygen Species

Stéphanie Lehoux; Bruno Esposito; Régine Merval; Laurent Loufrani; Alain Tedgui

From INSERM U541 and IFR "Circulation," Hôpital Lariboisière, Paris, France.

Correspondence to Dr Alain Tedgui, INSERM U541, 41 Boul de la Chapelle, 75010 Paris, France. E-mail tedgui{at}infobiogen.fr

	Abstract

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Abstract—Increased steady intraluminal pressure in blood vessels activates the extracellular signal–regulated kinase (ERK)1/2 pathway. However, signal transduction of pulsatile stretch has not been elucidated. Using an organ culture model of rabbit aorta, we studied ERK1/2 activation by pulsatility in vessels maintained at 80 mm Hg for 24 hours. ERK1/2 activity was evaluated by in-gel kinase assays and by Western blot. Compared with control aortas without pulsatility, aortas submitted to a pulsatile 10% variation in vessel diameter displayed a significant increase in ERK1/2 activity (207±12%, P<0.001), which remained high after removal of the endothelium. Unlike steady overstretch, pulsatile stretch–induced activation of ERK1/2 was not modified by herbimycin A, a Src family tyrosine kinase inhibitor, but was reduced by other tyrosine kinase inhibitors, tyrphostin A48 and genistein (162±27% and 144±14%, respectively). Conversely, ERK1/2 activity was markedly decreased in pulsatile vessels treated with staurosporine (114±18%) although neither of the more specific protein kinase C inhibitors, Ro-31-8220 or Gö-6976, blocked ERK1/2 activation (209±24% and 238±34%, respectively), whereas staurosporine had no effect on steady overstretch–induced ERK1/2 activation. Pulsatility induced superoxide anion generation, which was prevented by the NADPH oxidase inhibitor diphenyleneiodonium. Furthermore, polyethylene glycol–superoxide dismutase completely abolished ERK1/2 activation by pulsatility (114±12%). Finally, ERK1/2 and O₂^- levels in freshly isolated vessels were equivalent to the levels found in pulsatile vessels. In conclusion, pulsatile stretch activates ERK1/2 in the arterial wall via pathways different from those induced by steady overstretch. Pulsatility might be considered a physiological stimulus that maintains a certain degree of ERK1/2 activation via oxygen-derived free radical production.

Key Words: mechanical stress • pulsatility • signal transduction • extracellular signal–related kinase • tyrosine kinase

	Introduction

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Blood vessels are permanently exposed to mechanical forces in the form of pulsatile stretch and shear stress. In vitro experiments in cell culture models have been useful in identifying different transduction pathways activated in response to increased shear stress or stretch, including the mitogen-activated protein (MAP) kinase cascade, which lies upstream from gene expression and protein synthesis.^{1 2} In endothelial cells, physiological levels of shear stress stimulate the MAP kinase extracellular signal–regulated kinase (ERK)1/2,^{3 4} whereas cyclic mechanical strain activates ERK1/2 and c-Jun N-terminal kinase in vascular smooth muscle cells (VSMCs).^{5 6} The pathways leading to MAP kinase activation are diverse and, depending on cell type or origin and on culture conditions, may include G proteins, various tyrosine kinases, calcium, or free radicals.^{1 3 4 7 8} One of the key determinants of VSMC response to cyclic strain may be the type and density of extracellular matrix on which the cells are plated,⁹ with emphasis on the importance of considering extracellular matrix–cell interactions when trying to elucidate the vascular response to mechanical stress.

Hitherto, very little data have been available concerning the effects of pulsatility on the vessel wall, and signal transduction pathways elicited by cyclic stretch at time points spanning beyond the first hours after application of the stimulus remain unknown. Courtman et al¹⁰ recently reported that in vivo chronic prevention of normal pulsatility leads to apoptosis of VSMCs and atrophy of the arterial wall; hence, pulsatility was identified as a crucial mechanical stimulus for vascular cells besides steady stretch and shear stress. Therefore, the aim of the present study was to identify some signal transduction pathways initiated by cyclic stretch by using an organ culture model of the aorta. This model was chosen as the best in vitro representation of the vessel in its in vivo environment, where multiple cell types and the extracellular matrix participate in the response to mechanical stimuli. We undertook to investigate (1) the effects of pulsatility on ERK1/2 activity and (2) the transduction cascades involved in ERK1/2 activation by pulsatility.

	Methods

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Organ Culture
Male New Zealand White rabbits (2 to 2.5 kg) were anesthetized with sodium pentobarbital (30 mg/kg IV). Arterial segments from the descending thoracic aorta were isolated and cannulated under pressure as previously described.¹¹ Isolated aortic segments were connected to a closed perfusion circuit consisting of a 3-port reservoir, a peristaltic pump (Masterflex, Cole Palmer Instrument Co), and a pressure chamber, which allowed the application of a controlled intraluminal hydrostatic pressure.¹¹ Vessel segments were immersed in a bath filled with culture medium identical to that used in the intraluminal compartment, consisting of DMEM containing antibiotics (100 IU/L penicillin, 100 mg/L streptomycin, and 10 µg/L amphotericin B) and supplemented with 20% FCS (Boehringer-Mannheim). Organ culture of the aortic segments was carried out under sterile conditions in an incubator containing 5% CO₂ at 37°C. Except for experiments evaluating the kinetics of ERK1/2 activation by cyclic strain, in which vessels were in culture for 5 to 120 minutes, all segments used were maintained in organ culture for 24 hours. All experiments were approved by the local ethics committee.

Each aorta was divided into 3 segments, which were arbitrarily assigned different culture conditions, such that for each complete aorta there was 1 control nonpulsatile segment whose ERK1/2 activity was set at 100%, 1 pulsatile (10%) segment, and 1 segment in a third condition described below.

Experimental Protocol
In the first experiments establishing the effects of pulsatile stretch on ERK1/2 activation, some vessels were maintained at an intraluminal pressure of 80 mm Hg, and others were maintained at 150 mm Hg. Because effects of pulsatile pressure were greater in aortas kept at 80 mm Hg than at 150 mm Hg, all subsequent experiments were carried out at the lower level of pressure. Furthermore, at this pressure (80 mm Hg), vessel diameter was equivalent to that observed in vivo.

Flow was applied via a pulsatile pump (0.75 Hz). Pulsatile conditions were fixed at a flow of 40 mL/min, which remained well below the physiological flow rate (200 mL/min) but which nevertheless produced an important (10%) increase in vessel diameter at each pulse. In vessels maintained at 80 mm Hg, pulsatility caused intraluminal pressure to oscillate between 70 and 90 mm Hg. In comparison, control nonpulsatile vessels were perfused at 0.2 mL/min to ensure renewal of culture medium within the aorta but to avoid any pulsatility (0.5%) effects.

For vessels maintained at a flow of 40 mL/min, various strategies were used to modify pulsatility. The baseline pulsatility for perfused vessels approximated 10%. Pulsatility was abated by partial clamping of the output cannula between the vessel and the pump. This reduced the pulsatility to 2% of vessel diameter while maintaining high flow. On the other hand, pulsatility was enhanced to 20% by increasing the inner diameter of the cannula attached to the pump from 3.2 to 4.8 mm. In all cases, care was taken to ensure that the pulsatile frequency remained at 0.75 Hz and that flow within the vessel remained at 40 mL/min despite the different pulsatility settings. Because we found that ERK1/2 activation was maximal for a pulsatility of 10%, all subsequent experiments were performed with this setting. To investigate a potential role for flow in ERK1/2 activation, vessels were exposed to 10% pulsatility, but tubing occlusion at the pump was reduced such that the forward motion of the intraluminal culture medium became null.

To establish whether the endothelium was involved in the response to pulsatility, a Fogarty catheter (F3) was inserted in some aortic segments, and the endothelium was removed by gently pushing and pulling the balloon back and forth 3 times within the vessel, as previously described.¹¹

Different pharmacological inhibitors were added to the culture medium of pulsatile (10%) aortic segments for study of the potential signaling pathways triggered by mechanical strain. A role for tyrosine kinases was investigated with the use of 2 nonspecific inhibitors, tyrphostin A48 (50 µmol/L, LC Laboratories Europe) and genistein (10 µmol/L, Calbiochem), or with the use of herbimycin A (500 nmol/L, New England Biolabs), a Src-family tyrosine kinase inhibitor. At the concentrations indicated, these inhibitors were able to inhibit platelet-derived growth factor-AB (50 ng/L)–stimulated ERK1/2 activation in aortic ring segments (data not shown). The protein kinase inhibitor staurosporine (1 µmol/L LC Laboratories Europe) was also tested for its effects on pulsatility-induced ERK1/2 activation, along with a more specific protein kinase C (PKC) inhibitor, Ro-31-8220 (400 nmol/L and 5 µmol/L, Calbiochem), and an inhibitor selective for Ca²⁺-dependent PKC isoforms, Gö-6976 (400 nmol/L, Calbiochem). At the concentration used, staurosporine inhibited by 70% phorbol 12,13-dibutyrate (1 µmol/L, Sigma)–induced contraction of fresh aortic rings, whereas Ro-36-8220 and Gö-6976 inhibited pressure-induced myogenic tone in mesenteric arteries (data not shown). A role for protein kinase A (PKA) in the vascular response to pulsatility was studied with use of a specific inhibitor, H-89 (10 µmol/L). Finally, the potential contribution of oxygen-derived free radicals in the vascular response to pulsatility was elucidated by use of polyethylene glycol–superoxide dismutase (PEG-SOD, 40 U/mL, Sigma Chemical Co), which, unlike SOD alone, penetrates cells. Heat-inactivated PEG-SOD was used as a negative control. Also, some vessels were incubated with diphenyleneiodonium chloride (10 µmol/L, Sigma) to determine whether NADPH oxidase was responsible for oxygen-derived free radical production.

Tissue Extraction
Frozen vessel segments were pulverized in liquid nitrogen. The powders were resuspended in ice-cold lysis buffer containing 20 mmol/L Tris-HCl (pH 7.5), 5 mmol/L EGTA, 150 mmol/L NaCl, 20 mmol/L glycerophosphate, 10 mmol/L NaF, 1 mmol/L sodium orthovanadate, 1% Triton X-100, 0.1% Tween 20, 1 µg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, 0.5 mmol/L N-tosyl-L-phenylalanine chloromethyl ketone, and 0.5 mmol/L N()-p-tosyl-L-lysine chloromethyl ketone at a ratio of 0.3 mL/10 mg wet wt. Extracts were incubated on ice for 15 minutes and then centrifuged (14 000g, 15 minutes, 4°C). The detergent-soluble supernatant fractions were retained, and protein concentrations in samples were equalized by a Bio-Rad protein assay.

In-Gel ERK1/2 Assays
Kinase assays in myelin basic protein (MBP)-containing gels were performed as described previously.¹² Briefly, Laemmli sample buffer was added to lysed tissue samples, and samples were boiled for 3 minutes and loaded on a 9% SDS-polyacrylamide gel containing 0.5 mg/mL MBP (Sigma). Gels were subsequently incubated in 50 µCi [-³²P]ATP (Amersham), and MBP phosphorylation was assessed by autoradiography. Alternatively, ERK2 was first immunoprecipitated from 75 µg of lysed tissue samples by use of anti-ERK2 antibodies (2.5 µg, Transduction Laboratories) and protein A/G agarose (Santa Cruz) diluted in lysis buffer. Immunoprecipitates were then processed as indicated for whole lysed tissue samples.¹²

Immunoblotting
Lysates containing equal amounts of protein (20 µg) were electrophoresed on polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk in TBST (20 mmol/L Tris [pH 8.0], 150 mmol/L NaCl, and 0.1% Tween 20) for 1 hour and were then incubated with anti-ERK1 monoclonal antibodies (Transduction Laboratories), anti-ERK2 polyclonal antibodies (Santa Cruz), or anti–phospho p38 antibodies (New England BioLabs) at a dilution of 1:1000 in TBST for an additional hour, followed by incubation with the appropriate secondary antibodies. An enhanced chemiluminescence system was used as the detection method (Amersham). Blots were washed and subjected to autoradiography. This technique confirmed that relative ERK1/2 protein content did not vary between experimental conditions.

Assessment of Pulsatility
To determine the degree of pulsatility generated in our vessels, aortic segments were set up in the perfusion circuit but placed in an organ bath fitted with an ultrasonic microdimensiometer (Application Electronique Montreuil), which was used for continuous measurement of aortic diameter as described earlier.¹³ The aortic diameter was determined from the transit time of a pair of echoes given by the proximal and distal walls. Pulsatility was expressed as the change in vessel diameter as a percentage of mean vessel diameter.

In Situ Detection of Superoxide in Cultured Vessels
The oxidative fluorescent dye hydroethidine (HE) was used to evaluate in situ production of superoxide by use of a method described by Miller et al.¹⁴ HE is freely permeable to cells and in the presence of superoxide anion is oxidized to ethidium bromide, where it is trapped by intercalating with the DNA. Ethidium bromide is excited at 488 nm with an emission spectrum of 610 nm.

Unfixed frozen ring segments were cut into 30-µm-thick sections and placed on a glass slide. HE (2x10^-6 mol/L) was topically applied to each tissue section, and coverslips were applied. Slides were incubated in a light-protected humidified chamber at 37°C for 30 minutes. Images were obtained with a Bio-Rad MRC-1024 confocal microscope equipped with a krypton/argon laser. Segments obtained from control vessels and pulsatile vessels cultured with or without PEG-SOD were processed and imaged in parallel. Laser settings were identical for the acquisition of images from all specimens. Fluorescence was detected with a 585-nm long-pass filter.

Data Analysis
For in-gel kinase assays, percent activity of ERK1/2 was expressed relative to protein content of the samples tested or to autoradiographic density of ERK1/2 in corresponding Western blots. Results of both variants of estimation were highly consistent. Results are expressed as mean±SE. A 1-way ANOVA was constructed with data on ERK1/2 activity. Comparisons between segments originating from the same aorta were performed by Student paired t test. Statistical significance was accepted for values of P<0.05.

	Results

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Assessment of Pulsatility
Aortic diameter was determined from the transit time of a pair of ultrasonic echoes given by the proximal and distal walls of cannulated aorta. Figure 1 displays typical vessel diameter readings for different settings of pulsatility. Vessel diameter at 80 mm Hg approximated in vivo conditions. Pulsatility was calculated as a percentage of mean vessel diameter.

View larger version (36K): [in this window] [in a new window]   Figure 1. Changes in diameter of aortic segments maintained at 80 mm Hg measured with an ultrasonic microdimensiometer. Vessels were perfused at either 0.2 or 40 mL/min, producing a pulsatility of 0.5% or 10%, respectively. Pulsatility of vessels perfused at 40 mL/min was reduced to 2% by partial clamping of the output cannula between the vessel and the pump.

Activation of ERK1/2 by Pulsatility
We have previously observed that steady stretch induces a biphasic activation of ERK1/2, which has a transient peak at 5 minutes and resumes at 24 hours.⁸ Similar results were obtained in vessels kept at 80 mm Hg and submitted to pulsatile strain, except that the initial onset of ERK1/2 activation was slower and reached a maximum at 20 minutes (211±58% increase, P<0.05) before returning to baseline at 2 hours. At 24 hours, effects of pulsatility on ERK 1/2 activity were assessed for 2 pressure settings, 80 and 150 mm Hg. Control conditions (80 mm Hg, no pulsatility) were assigned as 100% ERK1/2 activity. As revealed in the in-gel assay (Figure 2B), baseline ERK1/2 activity in nonpulsatile vessels differed considerably between aortas maintained at normal (80 mm Hg) or high (150 mm Hg) pressure (100% and 262±22%, respectively; P<0.001), showing the role of steady stretch in ERK1/2 activation as previously reported.¹² Nonetheless, for both pressure settings, ERK1/2 was further activated by pulsatility, although the increase was clearly more marked for aortas kept at 80 mm Hg (107% increase in ERK1/2 activity, P<0.001) than at 150 mm Hg (22% increase in ERK1/2 activity, P<0.05; Figure 2). These results were confirmed by Western blot, where a shift in protein content to the band corresponding to phosphorylated ERK2 was observed (Figure 2A). All subsequent experiments were conducted in vessels maintained at 80 mm Hg for 24 hours.

View larger version (64K): [in this window] [in a new window]   Figure 2. Pulsatility (10%) activates ERK1/2 in aortic segments kept at 80 or 150 mm Hg for 24 hours. A, Representative Western blot with use of anti-ERK1 antibodies cross-reactive with ERK1 and ERK2. Note "band shift" to an apparently higher molecular mass of phosphorylated ERK2 (P-ERK2). B, Representative autoradiogram of in-gel kinase assay of ERK1 and ERK2 activity with MBP used as substrate. C, Quantification of in-gel ERK1/2 assays. Results are representative of 4 to 8 separate experiments. *P<0.05 and ***P<0.001 vs no pulsatility (0.5%) at equivalent intraluminal pressure.

Activation of ERK1/2 by Pulsatility: Shear Stress or Cyclic Stretch?
Different strategies were used to assess the relative contribution of shear stress or pulsatility to the observed ERK1/2 activation. A first set of experiments revealed that endothelial cell removal with use of a balloon catheter did not prevent vessel ERK1/2 activation by pulsatility, which remained elevated at 219±9% of control (P<0.001). Furthermore, ERK1/2 activity remained at baseline levels (97±8% of control) in aortic segments maintained at 40 mL/min but with a pulsatility of 2% instead of 10% (Figure I, which can be accessed online at http://atvb.ahajournals.org). We also determined that subjecting aortic segments to pulsatility without flow activated ERK1/2 to the same extent as pulsatility with flow, producing a 179±13% increase in ERK1/2 activity compared with the control condition (P<0.01, Figure I). Hence, the main motor of ERK1/2 activation was unlikely to be shear stress but rather pulsatility. Interestingly, ERK1/2 activation by pulsatile stretch was not further enhanced when pulsatility was increased from 10% to 20% while maintaining similar flow (187±32%, P<0.05; Figure I).

Pathways of ERK1/2 Activation
Several possible routes of ERK1/2 activation were investigated by use of various inhibitors. As depicted in Figure 3, the broad spectrum PKC inhibitor staurosporine completely abolished ERK1/2 activation by pulsatility (114±18%, P<0.05 versus pulsatility without staurosporine), although neither Ro-31-8220 (400 nmol/L), a specific protein kinase C inhibitor, nor Gö-6976 (400 nmol/L), a PKC inhibitor selective for calcium-dependent isoforms of the kinase, prevented ERK1/2 activation by pulsatile stretch (209±24% and 238±34% of control, respectively; P<0.01). Because Ro-31-8220 is a highly selective inhibitor of PKC, PKCßI, PKCßII, PKC, PKC, PKC, and PKC (with EC₅₀ values ranging from 15 to 100 nmol/L) but inhibits PKC with a higher EC₅₀ value (1 µmol/L),¹⁵ we excluded the possibility that PKC was involved in ERK1/2 activation by pulsatile pressure by using Ro-31-8220 at 5 µmol/L; ERK1/2 activity remained elevated (228±40% of control). Because staurosporine might have a potency for PKA inhibition similar to that for PKC inhibition, we tested the effects of the more specific PKA inhibitor H-89. Stretch-induced ERK1/2 activation was not modified by this compound (219±35, P<0.01). The Src-family tyrosine kinase inhibitor herbimycin A also failed to attenuate ERK1/2 activation in vessels exposed to pulsatility (219±11%, P<0.01), whereas the more general tyrosine kinase inhibitors, tyrphostin A48 and genistein, reduced ERK1/2 activation by cyclic stretch (to 162±27% and 144±14% of control, respectively; P<0.05 versus pulsatility without inhibitors; Figure 4).

View larger version (74K): [in this window] [in a new window]   Figure 3. Effect of PKC inhibitors on ERK1/2 activation by 10% pulsatility in aortic segments kept at 80 mm Hg for 24 hours. Top, Representative in-gel ERK1/2 assay obtained from nonpulsatile segments and pulsatile segments cultured in the absence or presence of Ro-31-8220 (Ro, 400 nmol/L), Gö-6976 (Gö, 400 nmol/L), or staurosporine (Stauro, 1 mmol/L). Only Stauro prevents ERK1/2 activation by pulsatile stretch. Bottom, Quantification of in-gel ERK1/2 assays. Results are representative of 6 to 8 separate experiments. **P<0.01 vs no pulsatility (0.5%); §P<0.05 vs 10% pulsatility without treatment.

View larger version (76K): [in this window] [in a new window]   Figure 4. Effect of tyrosine kinase inhibitors on ERK1/2 activation by 10% pulsatility in aortic segments kept at 80 mm Hg for 24 hours. Top, Representative in-gel ERK1/2 assay obtained from nonpulsatile segments and pulsatile segments cultured in the absence or presence of herbimycin A (Herb, 500 nmol/L), tyrphostin A48 (Tyr, 50 mmol/L), or genistein (Gen, 10 µmol/L). Tyr and Gen, but not Herb, partially block ERK1/2 activation by pulsatile stretch. Bottom, Quantification of in-gel ERK1/2 assays. Results are representative of 6 to 8 separate experiments. **P<0.01 vs no pulsatility (0.5%); §P<0.05 vs 10% pulsatility without treatment.

Finally, to evaluate the potential role of superoxide anions in pulsatility-induced ERK1/2 activation, vessels were incubated with PEG-SOD. As shown in Figure 5, incubating vessels with PEG-SOD prevented ERK1/2 activation by pulsatility (114±12%, P<0.05 versus pulsatility without PEG-SOD). In contrast, heat-inactivated PEG-SOD had no such inhibitory effect (309±23%). In fact, superoxide anions generated by pulsatility activated not only ERK1/2 but also p38, as determined by a Western blot probed with an antibody recognizing the activated form of this kinase (Figure II, which can be accessed online at http://atvb.ahajournals.org). In further support of these findings, ring segments from cultured aortas were exposed to HE, an oxidative fluorescent dye, to evaluate in situ production of superoxide anions. Figure 6 demonstrates that vessels submitted to pulsatility in the absence of PEG-SOD showed positive markings of O₂^- production, whereas control nonpulsatile vessels or pulsatile vessels treated with PEG-SOD did not. We established that NADPH oxidase was the source of O₂^- production, inasmuch as hydroethidine staining was negative in pulsatile vessels treated with diphenyleneiodonium chloride (data not shown).

View larger version (74K): [in this window] [in a new window]   Figure 5. PEG-SOD completely prevents ERK1/2 activation by 10% pulsatility in aortic segments kept at 80 mm Hg for 24 hours. Top, Representative in-gel ERK1/2 assay obtained from nonpulsatile segments and pulsatile segments cultured in the absence or presence of PEG-SOD (40 IU/mL) or heat-inactivated PEG-SOD. Bottom, Quantification of in-gel ERK1/2 assays. Results are representative of 6 separate experiments. **P<0.01 vs no pulsatility (0.5%); §§P<0.01 vs pulsatility (10%) without treatment.

View larger version (41K): [in this window] [in a new window]   Figure 6. Evaluation of in situ production of superoxide with use of HE, an oxidative fluorescent dye. Ring segments from cultured aortas from nonpulsatile vessels (A) or pulsatile vessels without (B) or with (C) PEG-SOD treatment were exposed to HE, which is oxidized to ethidium bromide in the presence of superoxide. Ethidium bromide was excited at 488 nm with an emission spectrum of 610 nm and detected by confocal microscopy. Superoxide production, revealed by ethidium bromide staining, is enhanced in vessels exposed to pulsatility compared with nonpulsatile vessels, and this effect is prevented by PEG-SOD treatment. Results are representative of 4 separate experiments.

	Discussion

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The present study reveals that ERK1/2 activity is increased in aortas cultured under pulsatile conditions compared with aortas not exposed to pulsatility. Even though pulsatile vessels were concurrently exposed to a flow of 40 mL/min, endothelial cell removal did not affect ERK1/2 activation by pulsatility. Also, applying flow to vessels in the absence of pulsatility failed to activate ERK1/2. These results led us to conclude that the pulsatile nature of the stimulus, and not the flow-induced shear effect, was responsible for increasing ERK1/2 activity levels in aortas. Hence, pulsatility appears to be a major mechanical factor responsible for activating ERK1/2 in the vascular wall.

Multiple pathways of ERK1/2 activation by cyclic strain have been reported in endothelial cells. Ikeda et al¹⁶ found ERK1/2 activation to be calcium independent and completely blocked by genistein and partly inhibited by the PKC inhibitor calphostin C and also to be blocked by phosphatidylinositol 3-kinase inhibition,¹⁷ whereas Wung et al¹⁸ found ERK1/2 activation to occur downstream from H₂O₂. However, apart from a recent study demonstrating that ERK1/2 activation by cyclic stretch is abated in VSMCs stably transfected with a dominant-negative Ras plasmid compared with normal cells,¹⁹ there is still very little information available regarding the effects of cyclic strain on VSMCs, let alone in the whole vessel. Yet, studying signal transduction of cyclic strain in the whole vessel is particularly relevant in light of the finding that extracellular matrix composition is a key determinant of cell response to cyclic stretch; mechanical strain activates ERK in neonatal rat VSMCs cultured on pronectin, but not laminin, and it increases DNA synthesis in cells cultured on collagen, fibronectin, or vitronectin, but not on elastin or laminin.^{6 9} Furthermore, the VSMC phenotype is likely to influence cell signaling, and we have shown that organ culture at 80 mm Hg imparts a certain degree of mechanical strain, which is necessary to maintain the expression of VSMC marker proteins, h-caldesmon and filamin.⁸ Some studies have used in vivo models to elucidate MAP kinase cascades stimulated in arteries by hypertension²⁰ or to identify pathways leading to vascular remodeling in nonpulsatile vessels,¹⁰ but in vivo experiments are complicated by the possible involvement of nonvascular cells and by hormonal or neuronal input, such that precise experimental conditions cannot be controlled as readily as in vitro. Hence, we believe that using a whole organ model of the aorta, consisting of quiescent cells enmeshed within a complex extracellular matrix, allows for an evaluation of vascular responses that is truest to what occurs in vivo (at least at the level of the VSMCs, which are most abundant in this preparation) while a tightly controlled experimental environment is maintained.

We investigated potential pathways of ERK1/2 activation by cyclic stretch in the aorta by inhibiting various mediators that have been reported to lie upstream from ERK1/2 activation or to be stimulated by cyclic stretch in cell culture experiments. PKC, which is activated in VSMC and endothelial cell cultures in response to cyclic stretch^{21 22} and which mediates endothelial cell proliferation stimulated by cyclic strain,²³ was not involved in ERK1/2 activation by pulsatile pressure in our vessels, as demonstrated by use of the 2 selective PKC inhibitors, Ro-31-8220 and Gö-6976. Nevertheless, the protein kinase inhibitor staurosporine reduced ERK1/2 activity in vessels under pulsatile stretch. Hence, a role for PKA, another target of staurosporine that has been described to be an upstream activator of ERK1/2^{24 25} and that is likely to be stimulated by cyclic stretch,^{21 26 27 28 29} was evaluated. In our experiments, PKA inhibition did not prevent the activation of ERK1/2 by pulsatility. Finally, ERK1/2 activation by pulsatile stretch was partly inhibited by tyrosine kinase inhibition with tyrphostin A48 or genistein, which have been implicated in signal transduction of cyclic stretch,³⁰ but was not affected by the Src-family tyrosine kinase inhibitor herbimycin A, also accountable for transduction of cyclic strain.³⁰

Interestingly, we have previously demonstrated that steady stretch stimulates ERK1/2 via the Src-family tyrosine kinase pathway,¹² whereas cyclic stretch appeared to involve a different set of tyrosine kinases that were insensitive to herbimycin A. On the other hand, ERK1/2 activation by pulsatile stretch was partly inhibited by tyrphostin A48 or genistein, whereas these inhibitors had little or no effect on steady stretch–induced ERK1/2 activation in aortic organ cultures.⁸ Also, staurosporine completely abolished ERK1/2 activation by pulsatility, whereas at an equal concentration, it did not affect steady stretch–induced ERK1/2 activation.⁸ These results, combined with the observation that acute activation of ERK1/2 by pulsatility is of slower onset than that stimulated by steady strain, strongly imply that mechanotransduction of cyclic and steady stretch involves different signaling cascades, leading to the activation of ERK1/2.

We did not see a gradient in cyclic stretch–induced ERK1/2 activation; ERK1/2 activity remained equally high in aortas submitted to a 10% or to a 20% variation in diameter compared with control aortas, whose diameters varied by 0.5%, or with clamped aortas, which had a 2% variation in diameter. Nevertheless, this did not coincide with the maximal threshold of ERK1/2 activation in the aorta, inasmuch as ERK1/2 activity in vessels kept at 80 mm Hg with 10% pulsatility was below that observed in segments submitted to high pressure (150 mm Hg). Increasing the amplitude of cyclic stretch has been shown to induce either a graduated or a single-step response in various cell types,^{31 32} but interestingly, oxygen radical production is consistently reported in cells exposed to a 10% to 12% cyclic stretch.³³ Indeed, it is documented that imposing a 10% cyclic stretch on coronary artery cells induces the production of O₂^-, whereas a 6% stretch has no significant effect.³⁴ These data suggest that 2% pulsatility was too low to induce O₂^- generation, and they coincide well with our finding that free radical scavenging with the use of PEG-SOD prevented the activation of ERK1/2 in pulsatile vessels. Indeed, PEG-SOD also blocked the activation of another MAP kinase, p38, whose acute activation in cultured VSMCs exposed to cyclic stretch has recently been documented.¹⁹ In our experiments, cyclic stretch stimulated O₂^- generation in the vessel wall via diphenyleneiodonium chloride–sensitive NADPH oxidase, as demonstrated by use of histological staining with HE, leading to enhanced ERK1/2 activity. In further agreement with our results, oxidant-induced MAP kinase activation in VSMCs was found to be sensitive to genistein and tyrphostin and to bypass PKC.³⁵ Nonetheless, there is evidence that PKC activation might precede O₂^- generation³⁴ or that it transduces O₂^- signaling to ERK1/2,³⁶ although this was not the case in our setup. Differences in experimental models (organ versus cell culture) or in species could account for these disparities.

In cell culture experiments, cells under cyclic stretch are usually referred to as stimulated and are compared with cells at rest. However, cyclic stretch is likely to reflect a physiological state. In fact, a certain degree of steady stretch is in itself necessary to maintain cell phenotype; in an organ culture, we have shown that there is a rapid loss of VSMC marker proteins in vessels left in no-stretch conditions, whereas maintaining an intraluminal pressure of 80 mm Hg delays loss of these proteins.¹² Furthermore, preventing aortic pulsatility in vivo leads to medial cell loss and vascular remodeling.¹⁰ In freshly isolated aortas, we observed that ERK1/2 activity and O₂^- levels were equivalent to the levels found in vessels cultured during 24 hours of pulsatility (data not shown). Although we have not investigated it in the present study, it is not improbable that reduced ERK1/2 activity in nonpulsatile vessels may participate in marker protein loss. Therefore, some degree of ERK1/2 activity (and O₂^- generation) may be a hallmark of normal vessel function.

In summary, a 10% pulsatile stretch, but not shear stress, activates ERK1/2 in the aorta. The signaling pathway is dependent on the release of oxidated free radicals via NADPH oxidase, and it involves non-PKC staurosporine-sensitive kinases as well as tyrosine kinases. Interestingly, cyclic stretching of the aorta, which presumably reflects physiological conditions, produces an increase in ERK1/2 activity, which occurs via a pathway distinct from that observed after steady stretch of the same vessel at high pressure.

	Acknowledgments

 
This work was supported by a BIOMED-2 grant, "Cellular and Molecular Mechanisms of Resistance Artery Remodeling." S. Lehoux is the recipient of a fellowship from the Fondation de la Recherche Médicale (France). The authors acknowledge the assistance of Daniel Henrion and Véronique Springhetti in performing confocal microscopic analysis.

Received December 17, 1999; accepted April 5, 2000.

	References

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