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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:1528.)
© 2003 American Heart Association, Inc.

Vascular Biology

Cholesterol Dependence of Vascular ERK1/2 Activation and Growth in Response to Stretch

Role of Endothelin-1

Asad Zeidan; Jonas Broman; Per Hellstrand; Karl Swärd

From the Division of Molecular and Cellular Physiology, Department of Physiological Sciences, Lund University, Biomedical Centre, Lund, Sweden.

Correspondence to Karl Swärd, PhD, Department of Physiological Sciences, Lund University, BMC F12, SE-221 84 Lund, Sweden. E-mail karl.sward{at}mphy.lu.se

	Abstract

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Objective— Stretch-induced growth of the vascular wall plays a role in hypertension and neointima formation. Its signal pathways involve integrins, cytoskeleton, membrane receptors, and ion channels, some of which are organized in cholesterol-rich, membrane domains such as lipid rafts or caveolae. This study tested the role of rafts/caveolae in stretch-induced vascular growth by manipulation of membrane cholesterol contents.

Methods and Results— Growth and protein synthesis were induced by mechanical stretch of rat portal veins in vitro. Sucrose gradient centrifugation showed stretch-induced tyrosine phosphorylation primarily in fractions containing caveolin-1. Disruption of membrane caveolae with use of methyl-ß-cyclodextrin (mßcd) reduced weight gain, protein synthesis, and DNA synthesis to levels in unstretched, control veins. These effects were partially reversed by restoration of cellular cholesterol contents. Inhibited growth was associated with abolished activation of extracellular signal–regulated kinase (ERK) 1/2 in response to stretch and endothelin-1 (ET-1) but not to angiotensin II. Inhibition of ET-1 type A (ET_A) receptors by RF139317 or endothelin-converting enzyme by phosphoramidone abolished stretch-induced ERK1/2 activation, which was, however, unaffected by removal of the endothelium.

Conclusions— Stretch-induced growth signaling in vascular smooth muscle depends on cholesterol-rich, membrane microdomains by a mechanism involving ET_A receptors that respond to endogenous ET-1 production.

Key Words: hypertrophy • portal vein • caveolae • ERK1/2 • cyclodextrin

	Introduction

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Blood vessels subjected to increased wall stress undergo structural and biochemical changes, which result in hypertrophy/hyperplasia of vascular smooth muscle. This leads to clinically important alterations in structure and function, as seen in hypertension and graft restenosis.^1–3 A complex interaction of smooth muscle and the endothelium, which involves mechanical stimuli such as pressure and flow as well as growth factors and the extracellular matrix, controls vascular growth and differentiation.^4,5

See page 1481

The sequence of events that cause stretch-induced vascular growth is largely unknown, although several illuminating observations have been made. Extracellular matrix components and integrins, as well as the cytoskeleton, play important roles as mechanical sensors.^6,7 Outside-in signaling by integrins depends on Src family kinases,⁸ and inhibition of Src decreases extracellular signal–regulated kinase (ERK) 1/2 activation and protein synthesis in blood vessels exposed to increased wall stress.^9,10 In parallel with integrin signaling, growth factors produced in response to stretch, eg, angiotensin II (Ang II), endothelin-1 (ET-1), and platelet derived growth factor, act in a paracrine manner.^11–14 Activation of the protein kinases Raf, MEK, and ERK1/2 is essential for the resulting growth response.^{9,10,12,15,16}

The distribution of growth factor receptors and Src in the membrane is influenced by the presence of cholesterol-rich, membrane domains.^17–20 Lipid rafts are aggregates of cholesterol and glycosphingolipids that lack a defined structure.¹⁹ Caveolae have a similar composition and appear as 50- to 100-nm, flask-shaped membrane invaginations.²⁰ The protein caveolin binds cholesterol and stabilizes caveolae.^21,22 Purified rafts/caveolae contain receptors, G proteins, and ERK1/2 in addition Src.²⁰ It is therefore reasonable to hypothesize that these specialized membrane domains play a role in stretch signaling. Indeed, evidence for signaling of shear stress through rafts/caveolae has been obtained in endothelial cells.^23–25 Such mechanotransduction in the endothelium might modulate contractility and also possibly growth and differentiation of the underlying vascular smooth muscle cells.⁵ A direct effect of stretch on smooth muscle growth and differentiation is, however, also present,⁴ and investigation of the role(s) of lipid rafts/caveolae in this response is expected to elucidate mechanisms of vascular hypertrophy.

We have investigated the role of membrane cholesterol in stretch-dependent vascular growth of the rat portal vein in vitro. This vessel has spontaneous activity similar to the myogenic tone of precapillary resistance vessels, and because of its longitudinally oriented smooth muscle cells, the vessel wall might be conveniently stretched by weight loading. Culture under load causes greater contractility, protein synthesis, and cell cross-sectional area relative to unloaded, control veins.¹⁰ The stretch response promotes expression of differentiation-related proteins and depends on endogenous production of Ang II and cytoskeletal dynamics.¹⁶ Here, we tested whether stretch increases the phosphotyrosine (P-Tyr) contents in caveolin-containing membrane microdomains and examined the effects on growth of cholesterol depletion by cyclodextrin, which disrupts rafts and caveolae.^19,22

	Methods

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Animals, Dissection, and Cholesterol Depletion
Female Sprague-Dawley rats were killed by cervical dislocation, as approved by the Animal Ethics Committee, Lund University. Portal veins were dissected under sterile conditions and cut longitudinally into halves. For cholesterol depletion, strips were incubated with 5 mmol/L methyl-ß-cyclodextrin (mßcd) or vehicle in Krebs’ solution (in mmol/L: NaCl 135.5, KCl 5.9, CaCl₂ 2.5, MgCl₂ 1.2, HEPES 11.6, and glucose 11.5, pH 7.4) for 1 to 3 hours at 37°C. The 1-hour treatment was used as the standard incubation time, because longer incubations impaired contractility. Cholesterol was reloaded into depleted strips by cholesterol-saturated mßcd (water-soluble cholesterol, Sigma) in Krebs’ solution for 3 hours at 37°C. Before culture, strips were gently blotted and weighed as described.¹⁰

Organ Culture
After cholesterol depletion, strips were transferred to culture medium (Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium [1:1, vol/vol], containing 2% dialyzed fetal calf serum, 10 nmol/L insulin, 50 U/mL penicillin, and 50 µg/mL streptomycin) and either left undistended or loaded with a 0.6-g gold weight, which represents a preload approximately optimal for active force development The passive length-tension relation is quite steep, so that a 10% shortening from the loaded length eliminates most of the passive force.¹⁰ The culture period was 24 hours, after which the strips were again weighed. No effects of mßcd on growth were observed with longer culture (3 days), presumably reflecting recovery from cholesterol depletion.

Mechanical Recording
Strips were mounted,¹⁰ immersed in 0.4 mL Krebs’ solution, and stretched to a passive tension of 2 mN. After equilibration (45 minutes), experimental protocols were started. Contraction was elicited by replacing NaCl with KCl to a final concentration of 140 mmol/L. The cross-sectional area was determined from the length and wet weight.

Protein and DNA Synthesis
For analysis of protein and DNA synthesis rates, [methyl-³H]thymidine or L-[4,5-³H]leucine (Amersham), at activities of 0.2 and 1 µCi/mL, respectively, were added to the culture flasks, and incorporation was determined as described previously.¹⁰

Immunoblotting
Tissue was equilibrated for 1 hour in normal Krebs’ solution with or without mßcd, loaded for 5 or 10 minutes in culture medium, and then snap-frozen. ERK phosphorylation was determined by Western blotting.¹⁰ Caveolin-1 was detected with the 6B6 antibody²⁶ (a gift from Dr J. Vinten, University of Copenhagen, Copenhagen, Denmark), and tyrosine phosphorylation (P-Tyr), with PY 20 (Transduction Laboratories). Bands were visualized by enhanced chemiluminescence (Lumi-Glo, New England Biolabs or Pierce West-pico). Phosphorylated ERK1/2 was normalized to the total amount of ERK1/2.

Tissue Fractionation by Sucrose Gradient Centrifugation
A detergent-free method to purify caveolae was adopted from that of Song et al.²⁷ Tissues were pulverized in LN₂ and suspended in 1 mL of 500 mmol/L Na₂CO₃ with protease inhibitors (5 µg/mL, Sigma), pH 11. The homogenate was adjusted to 45% sucrose with 1 mL of 90% sucrose in MBS (25 mmol/L MES and 0.15 mol/L NaCl, pH 6.5) and overlayered with 4 mL of 35% sucrose and 4 mL of 5% sucrose, both in MBS with 250 mmol/L Na₂CO₃. After centrifugation at 39 000 rpm for 22 hours (SW 41, Beckman Instruments), ten 1-mL fractions were collected. For detection of P-Tyr, protein was precipitated with 20% trichloroacetic acid, washed with acetone, and solubilized in sample buffer. When stretched and control veins were compared, the volumes loaded on the gel were adjusted to give equal protein contents in fraction 5. Protein synthesis rates are stretch dependent, but measurable differences in overall protein composition between loaded and unloaded veins do not appear during organ culture for several days,¹⁶ and hence, increases in P-Tyr during a 10-minute loading period represent increased phosphorylation of preformed proteins and not increased protein contents.

Electron Microscopy
Veins were fixed (1 to 2 hours) in 2% glutaraldehyde and 1% depolymerized paraformaldehyde in phosphate-buffered saline (pH 7.4, 300 mOsm). After being rinsed in phosphate-buffered saline, veins were postfixed in 1% OsO₄ for 60 minutes, rinsed, dehydrated in graded ethanol (70%, 95%, and 100%), and embedded in mounting medium (Durcupan ACM, Fluka). Ultrathin sections were cut, mounted on copper mesh grids, and counterstained with uranyl acetate and lead citrate. Sections were examined in a transmission electron microscope, and photomicrographs were obtained with a CCD camera (Gatan 791 MultiScan). Caveolae, including completely and partially invaginated vesicles (50- to 100-nm diameter) and those with dilated necks found within 100 nm of the plasma membrane, were counted on coded photomicrographs. Membrane length was determined with commercially available software (ImageJ). The photomicrographs used for calculation of caveolar density were obtained randomly and in a similar way for all conditions, but because we concentrated on areas containing either caveolae or their remnants, we cannot exclude a bias toward regions rich in caveolae.

Statistics
Data are presented as mean±SE. Student’s t test was used to test statistical significance. For multiple comparisons, 1-way ANOVA was used. P<0.05 was considered significant.

	Results

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Stretch Rapidly Increases P-Tyr in Caveolin-1–Containing Membrane Domains
Mechanical stimuli rapidly increase P-Tyr of cellular proteins. To investigate whether stretch increases P-Tyr in caveolae of the intact rat portal vein, a detergent-free method for purification of caveolae by sucrose gradient fractionation was used. Ten minutes of stretch increased P-Tyr of several high-molecular-weight proteins (Figure 1A and 1B,a). P-Tyr increased almost exclusively in the fraction containing the caveolar marker caveolin-1 (Figure 1A). Comparisons (fold differences) of P-Tyr of several protein bands in fraction 5 of stretched versus unstretched veins are shown in Figure 1B,a. Caveolin-1 contents and protein composition in the fractions were the same (Figure 1B,b and Methods). This indicates that the stretch signal involves P-Tyr of several proteins that are located in caveolae.

View larger version (42K): [in this window] [in a new window]   Figure 1. Stretch increases P-Tyr in caveolin-1–containing membrane domains, and cholesterol depletion collapses smooth muscle caveolae. Caveolae were purified by sucrose density fractionation from control and stretched (10 minutes), nondenuded portal veins. After sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transfer to nitrocellulose, membranes were immunoblotted (IB) with anti–P-Tyr (A, top) and anti–caveolin-1 (A, bottom) antibodies. Loading of the gel was adjusted to give identical amounts of protein in fraction 5. The fold increases in P-Tyr of selected bands in the caveolin-1 fraction, compared with unloaded controls, are shown in B,a. Molecular-weight estimates are indicated below bars. The fold increase for smaller proteins (P43, P65) was determined after longer exposure. Blots in A are representative of 3 independent experiments. Densitometry demonstrated similar contents of caveolin-1 in fraction 5 from loaded (Lo) and unloaded (Unlo) veins (B,b). C,a–C,c show representative smooth muscle membrane areas from a control vein (C,a) and veins treated for 1 (C,b) or 2 (C,c) hours with mßcd (5 mmol/L). Arrows point to caveolae and caveolar remnants. SR indicates sarcoplasmic reticulum. Scale indicated in C,a is identical in C,b and C,c.

Cholesterol Depletion Disrupts Caveolar Structure
Cholesterol was extracted with mßcd (5 mmol/L, 1 to 3 hours),^22,28 and membrane profiles were examined by electron microscopy. Caveolae were found as clusters at the cell ends and also along the longitudinal cell axes (Figure 1C,a). After incubation with mßcd for 1 hour, areas of flattened caveolae with dilated openings were present (Figure 1C,b), but in some regions, caveolae appeared normal. After longer treatment (2 to 3 hours), caveolae were more extensively flattened and dilated, and no caveolae of normal appearance were found (Figure 1C,c). The density of caveolae, irrespective of structure, was 3.8±0.4/µm membrane in controls versus 3.8±0.8, 4.3±0.2, and 1.5±0.3 (P<0.01) in preparations treated for 1, 2, and 3 hours, respectively (data from 8 photomicrographs per condition in 2 experiments).

Effect of Cholesterol Depletion on Stretch-Induced Growth and Protein Synthesis
Portal veins were cultured for 24 hours under loaded and unloaded conditions after cholesterol depletion for 1 hour. The weight of loaded, control vessels increased under these conditions,^10,16 whereas unloaded veins lost weight (Figure 2A). Depletion of cellular cholesterol resulted in weight loss of loaded veins, similar to unloaded controls. Cholesterol-depleted, unloaded veins behaved as unloaded controls.

View larger version (16K): [in this window] [in a new window]   Figure 2. Cholesterol depletion abolishes stretch-induced growth of rat portal vein. Portal vein strips, treated as indicated, were subjected to mechanical loading in culture, and wet weight at the end of culture was recorded and normalized to weight before culture (A). Results are mean±SE of 5 to 13 experiments. In B and C, rat portal veins, treated as indicated, were incubated with [3H]leucine and [3H]thymidine for 24 hours. Protein and DNA synthesis rates were measured by scintillation counting. Results are mean±SE of 5 or 6 experiments. *P<0.05 vs loaded controls, P<0.05 vs loaded and cholesterol (chol)-depleted preparations. DPM indicates disintegrations per minute.

The disruption of caveolar structure with mßcd can be reversed by incubation with cholesterol-saturated mßcd (1 mmol/L cholesterol:5 mmol/L mßcd).²⁸ Incubation with cholesterol-saturated mßcd without prior depletion did not affect weight gain in either loaded or unloaded veins (see online Figure I; available at http://atvb.ahajournals.org), demonstrating that mßcd itself has no effect on growth besides that caused by the extraction of cholesterol. Incubation with cholesterol-saturated mßcd for 3 hours after depletion (1 hour) partially restored the stretch-induced weight gain during the following culture (Figure 2A).

Protein and DNA syntheses were measured by [³H]leucine and [³H]thymidine incorporation, respectively, during culture (Figure 2B and 2C). Cholesterol depletion reduced protein and DNA syntheses in loaded veins toward the levels of unloaded controls. Synthesis recovered significantly, but not completely, when depleted veins were incubated with cholesterol-saturated mßcd before culture. Cholesterol-saturated mßcd alone did not influence leucine incorporation in loaded and unloaded veins, again excluding the possibility of nonspecific effects of mßcd on growth (see online Figure I; available at http://atvb.ahajournals.org). Neither protein synthesis nor DNA synthesis was affected by cholesterol depletion in unloaded veins. Extraction with mßcd had no effect on force in response to high-K⁺ depolarization in fresh strips (13±2 vs 11±2 mN/mm²; n=10, P=NS) or in veins cultured under load after extraction (8±3 vs 6±1 mN/mm²; n=10, P=NS). However, the amplitude of the regular spontaneous activity characteristic of the portal vein declined after cholesterol depletion (not shown).

Effect of Cholesterol Depletion on Stretch-Induced ERK1/2 Activation
Stretch-induced growth of the portal vein requires ERK1/2 phosphorylation,¹⁰ which is likely to be represented by the P-Tyr labeling in the 43-kDa region of the caveolar fraction in Figure 1A and 1B. Veins were therefore stretched for 5 minutes, and ERK1/2 phosphorylation was determined by Western blotting. We first verified that mßcd did not have any nonspecific effect on ERK1/2 activation by using cholesterol-saturated mßcd (Figure 3A). Cholesterol depletion, on the other hand, inhibited ERK1/2 phosphorylation in stretched veins by 70% (Figure 3B). Significant recovery of stretch activation was observed after replenishing the cholesterol in depleted veins (Figure 3B).

View larger version (26K): [in this window] [in a new window]   Figure 3. Stretch-induced ERK1/2 phosphorylation (P-ERK1/2) is inhibited by cholesterol depletion. Phosphorylation of ERK1/2 was determined at the end of a 5-minute period in the absence or presence of load and expressed relative to phosphorylation in loaded, control veins. A demonstrates lack of effect of cholesterol (chol)-saturated mßcd (1 mmol/L cholesterol:5 mmol/L mßcd) on ERK1/2 activation by stretch. B demonstrates inhibition of ERK1/2 phosphorylation in stretched veins by cholesterol depletion. Cholesterol replenishment after prior depletion is indicated by mßcd-chol. Below the bar graphs, representative Western blots of P-ERK1/2 and total ERK1/2 are shown. Results are mean±SE of 3 (A) or 6 (B) experiments. *P<0.05 vs loaded controls, P<0.vs loaded and cholesterol-depleted preparations.

Effect of Cholesterol Depletion on ERK1/2 Activation by Ang II and ET-1
Part of the stretch-induced ERK1/2 activation in the portal vein depends on endogenous release of Ang II.¹⁶ We therefore investigated the effect of cholesterol depletion on responses to Ang II. Addition of Ang II (10 µmol/L, 5 minutes) increased ERK1/2 phosphorylation in loaded and unloaded control as well as cholesterol-depleted veins. The effects of load and Ang II were additive (Figure 4). Although cholesterol depletion inhibited stretch-induced activation of ERK1/2, the additional response to Ang II in both loaded and unloaded veins was unaffected.

View larger version (30K): [in this window] [in a new window]   Figure 4. Cholesterol depletion does not impair Ang II-induced ERK1/2 phosphorylation (P-ERK1/2). Rat portal vein strips, treated as indicated, were stretched and/or stimulated with 10 µmol/L Ang II for 5 minutes followed by snap-freezing and Western blotting. Results are mean±SE of 4 experiments. Western blots from a typical experiment of P-ERK1/2 and total ERK1/2 are shown below.

Addition of ET-1 increased ERK1/2 phosphorylation in both stretched and unstretched preparations, whereas the ET-1 type A (ET_A) receptor blocker RF139317 (1 µmol/L) reduced ERK1/2 phosphorylation of both stretched and unstretched preparations below their respective control values (Figure 5). This suggests that endogenous ET-1 production contributes to ERK1/2 activity, as also shown by the finding that the endothelin-converting enzyme inhibitor phosphoramidone (PA, 10 µmol/L) inhibited activation of ERK1/2 by stretch (loaded control, 100%; unloaded, 39±4%; loaded plus PA, 40±4%; unloaded plus PA, 34±2%; n=4 for all, P<0.05 for effect of PA). Although ERK1/2 activation due to endogenous production of Ang II and ET-1 seems to play a role in the stretch response, their dependence on membrane cholesterol differs, because mßcd completely inhibited activation of ERK1/2 by exogenous ET-1 as well as by stretch (Figure 5).

View larger version (29K): [in this window] [in a new window]   Figure 5. Role of ET-1 receptors in stretch-induced ERK1/2 phosphorylation (P-ERK1/2). Portal vein strips were treated with 10 nmol/L ET-1, the ETA receptor antagonist RF139317, and mßcd for 5 minutes followed by snap-freezing and Western blotting. Results are mean±SE of 4 to 8 experiments of 2 different designs, both including loaded and unloaded veins, which allowed direct comparisons. Western blots of P-ERK1/2 and total ERK1/2 from typical experiments are shown below the bar graph. *P<0.05 vs loaded controls, P<0.05 for indicated comparisons.

Role of the Endothelium
In view of the reported role of caveolae as mechanosensors in endothelial cells, the endothelium was removed, and the effect on stretch-induced ERK1/2 activation was examined. Scraping of the luminal vessel surface abolished endothelial nitric oxide synthase labeling, whereas stretch-induced ERK1/2 activation remained unaffected (Figure 6A).

View larger version (27K): [in this window] [in a new window]   Figure 6. Endothelial denudation does not influence stretch-induced ERK1/2 activation, and cholesterol depletion does not displace ERK1/2 from caveolae. Summarized data and Western blots of ERK1/2 phosphorylation (P-ERK1/2), total ERK1/2, and endothelial nitric oxide synthase (eNOS) from a typical experiment are shown in A. Results are mean±SE of 3 independent experiments. *P<0.05 vs loaded controls. B and C show the presence of ERK1/2 in caveolin (Cav-1)-containing fraction after sucrose density fractionation of control and cholesterol-depleted veins, respectively. Blots are representative of 3 independent experiments.

Effect of Cholesterol Extraction on Subcellular Distribution of ERK1/2
With the use of sucrose gradient fractionation, ERK1/2 was found to copurify with the caveolar marker protein caveolin-1 (Figure 6B). Cholesterol depletion did not appear to alter the distribution of either caveolin-1 or ERK1/2 (Figure 6C).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The observation that stretch increases P-Tyr preferentially in membrane microdomains containing caveolin-1 prompted us to examine the growth response after manipulation of caveolae. The effects of mßcd are specific for cholesterol extraction, because cholesterol-saturated mßcd had no effect on the growth response to stretch. The partial recovery of growth after reloading with cholesterol after depletion is likely due to incomplete restoration of cholesterol contents, because delivery of cholesterol is several-fold less effective than depletion by cyclodextrins.²⁹

Even though cholesterol depletion influences caveolar structure and much evidence suggests that this affects signaling mechanisms located in lipid rafts/caveolae, the possibility remains that its functional consequences are linked, at least partly, to altered membrane fluidity. Removal of cholesterol from the membrane will increase its stiffness and thus perhaps the degree of mechanical deformation in response to an applied load. It is notable that limited extraction (1 hour) of cholesterol caused only regional disruption of caveolae yet inhibited growth completely. This might indicate that the effects on growth do not involve the caveolar structure per se but rather the liquid-ordered state normally present in either caveolae or rafts,¹⁷ which might influence mechanical forces acting on resident proteins.

Strain on the extracellular matrix and membrane integrins, which are attached to the cytoskeleton, influences gene expression, which accordingly is affected by manipulation of either matrix/integrin interactions,⁶ formation and receptor binding of endogenous growth factors,^12,14,16 or cytoskeletal integrity.^7,16 The present study implicates cholesterol-rich, membrane domains in the sequence. Mechanical stress is transmitted from contractile and cytoskeletal filaments to the plasma membrane, where adherens junctions constitute specific anchorage sites.³⁰ Even though cholesterol depletion might influence signaling pathways involving the cytoskeleton, our results demonstrate that active force generated by a stimulus independent of caveolar signaling was unaffected by cholesterol depletion, indicating that both the myofilaments and the force transmission from them function normally.

ERK1/2 activation by stretch in the rat portal vein is partly inhibited by candesartan, a blocker of the type 1 angiotensin (AT₁) receptors, and by captopril, an inhibitor of angiotensin-converting enzyme, showing that endogenous Ang II plays an important role in ERK1/2 activation.¹⁶ Selective MEK inhibition by PD 98059 reduces ERK phosphorylation by 70% and completely inhibits stretch-sensitive growth in the portal vein.¹⁰ Thus, the reduced ERK1/2 phosphorylation after cholesterol depletion is sufficient to inhibit the growth response. Because AT₁ receptors translocate to caveolae on ligand binding,¹⁸ we speculated that ERK1/2 activation was inhibited at this level. However, exogenous Ang II still activated ERK1/2 in the portal vein after cholesterol depletion. In cultured vascular smooth muscle cells, transactivation of epidermal growth factor receptors by Ang II was inhibited by cholesterol depletion, whereas c-Src activation and intracellular Ca²⁺ response to Ang II were unaffected.³¹ These results suggest that pathways downstream of the AT₁ receptor might have a different dependence on cholesterol and/or caveolin-1.

In the portal vein, ERK1/2 copurified with the caveolar marker protein caveolin-1. Cholesterol depletion did not, however, affect the position of either caveolin-1 or ERK1/2 in the sucrose gradient, suggesting that the inhibition of stretch-mediated ERK1/2 activation after cholesterol depletion was not due to disrupted membrane targeting of ERK1/2. The unimpaired ERK1/2 response to Ang II also suggests that the function of the kinase was unaffected by cholesterol depletion.

The role of ET-1 in vascular growth seems to be partially linked to that of Ang II, because Ang II infusion causes increased ET-1 expression, and the ensuing hypertension is corrected by an ET_A antagonist.³² Endogenous ET-1 release might thus account for some of the vascular effects ascribed to Ang II. In our experiments, both an ET_A receptor antagonist (RF139317) and an endothelin-converting enzyme inhibitor reduced ERK1/2 phosphorylation in stretched veins. A small effect of RF139317 in unloaded veins was also observed, suggesting that basal ERK1/2 phosphorylation is associated with constitutive ET-1 signaling. Stretch activated ERK1/2 when portal veins were bathed with a high ET-1 concentration, implying that whereas ET-1 is critical, it is not the only relevant factor for activation of ERK1/2 by stretch. Using a similar approach, we have shown that a saturating concentration of Ang II does not eliminate stretch-dependent growth in the portal vein.¹⁶ Whereas ERK1/2 activation by exogenous ET-1 was highly sensitive to cholesterol depletion, this was not the case for Ang II. It is therefore unlikely that ET-1 acts downstream of Ang II, but rather in parallel, while possibly being released via a common mechanism. The cholesterol sensitivity of ERK 1/2 activation by ET-1 is consistent with a similar sensitivity of contractile responses²⁸ and with a reported localization of ET_A receptors in caveolae.³³

ERK1/2 activation by stretch was found to be insensitive to removal of the endothelium, and thus, the stretch-sensing mechanisms that affect gene expression in smooth muscle cells seem to primarily reside in these cells themselves. Substantial evidence indicates that endothelial cells are sensitive to shear stress in a manner dependent on intact caveolae.^23–25 Thus, mechanical sensing in the vascular wall involves 2 separate modalities, wall tension and shear stress, which are transmitted through similar cholesterol-rich structures in entirely different cell types. This similarity argues that a role for caveolae in mechanotransduction might be a general phenomenon.

	Acknowledgments

 
This study was supported by the Swedish Research Council (VR71X-28), the Heart-Lung Foundation, the Royal Physiographic Society, the Crafoord Foundation, and the Magnus Bergvall Foundation.

Received April 10, 2003; accepted July 4, 2003.

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