Published online before print August 11, 2005, doi: 10.1161/01.RES.0000181432.73920.b1
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© 2005 American Heart Association, Inc.
Integrative Physiology |
Reduced Wall Compliance Suppresses Akt-Dependent Apoptosis Protection Stimulated by Pulse Perfusion
From the Division of Cardiology, Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Md.
Correspondence to David A. Kass, MD, Ross 835, 720 Rutland Ave, Johns Hopkins Medical Institutions, Baltimore, MD 21205. E-mail dkass{at}jhmi.edu
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Abstract |
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Reduced arterial compliance and increased pulse pressure are common and major risk factors for cardiovascular disease. Here, we reveal a novel mechanism whereby loss of wall distensibility blunts endothelial cell protection to oxidant stress–induced apoptosis. Bovine aortic endothelial cells cultured in compliant or stiff silastic tubes were pulse perfused by arterial pressure/flow waveforms generated by a servo-pump. Pulse perfusion induced time-dependent Akt activation peaking >6-fold after 2 hours in compliant tubes and a similar time course but half the magnitude in stiff tubes. This was accompanied by quantitatively similar disparities in phosphoinositide-3 kinase activation and in Akt-stimulated suppressors of apoptosis: glycogen synthase kinase-3ß, forkhead, and Bad. Cells perfused in compliant tubes had twice the protection against H2O2-stimulated apoptosis than those in stiffer tubes. This protection was lost by pretreatment with an Akt inhibitor and restored in cells transfected with myristoylated Akt yet perfused in stiff tubes. Shear and stretch Akt signaling coupled to different upstream pathways as inhibition of vascular endothelial growth factor receptor 2 (VEGF2R) or disruption of caveolae blocked steady and pulse flow–mediated activation, yet did not suppress phosphorylated Akt induced by pulse perfusion in compliant tubes (concomitant stretch). Unlike Akt, reactive oxygen species, activated nuclear factor
B, and suppression of H2O2-stimulated c-Jun-N-terminal kinase activity were similar in pulse-perfused compliant and stiff tubes. Thus, cyclic endothelial cell stretch by pulse perfusion enhances Akt-dependent antiapoptosis above that induced by steady or phasic shear stress and, unlike the latter, signals via a VEGF2R/caveolae-independent pathway. Enhancing this stretch pathway may prove useful for improving endothelial function in stiff arteries.
Key Words: apoptosis • vascular biology • stiffness • artery pulse • endothelium
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Introduction |
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Reduced arterial distensibility is a common feature of vascular aging and is attributable in part to structural changes within the vessel wall and abnormal endothelial function that impairs the synthesis and release of vasoactive molecules.1 A major consequence of vascular stiffening is loss of normal buffering of the arterial pulse, resulting in systolic hypertension, widening of the arterial pulse pressure, and increasing phasic shear stress. Importantly, these changes increase the risk of developing heart failure, coronary artery disease, and stroke.2–4 Among the potential mechanisms by which such risks are conferred are changes in the endothelial response to the mechanical forces imposed by pulsatile perfusion. As recently reviewed, shear stress alone potently stimulates release of vasorelaxant mediators such as NO, enhances endothelial survival, and counters cell adhesion and thrombosis.5–7 Endothelial cell stretch also stimulates NO release,8,9 hyperpolarization,10 signaling kinases,9 and oxidant stress.11 With vascular stiffening, shear forces become more pulsatile, whereas stretch stimulation declines. Yet, little is known regarding how such changes influence endothelial mechanosignaling and in particular impact cytoprotection against stress-induced apoptosis.
A primary regulator of shear/stretch vasomotor response and antiapoptotic effects of endothelial mechanostimulation is the serine/threonine kinase Akt.12–15 Steady shear stress activates phosphoinositide-3 kinase (PI3K) to phosphorylate the inner membrane lipid phosphotidylinositol 4,5 bisphosphate. The resulting phospholipid products recruit Akt and 3-phosphoinositide–dependent protein kinase 1 in proximity to the inner membrane, leading to phosphorylation/activation of Akt.15–17 Activated Akt in turn phosphorylates NO synthase (NOS), contributing to sustained NO release and stimulating kinases and transcription factors to regulate vasculogenesis, cytoprotection, and apoptosis.15,16 Cyclic stretch also enhances Akt activity,18 and when both stimuli are combined, as with pulsatile perfusion in a compliant conduit, Akt and NOS phosphorylation are further increased.19 Such in vitro findings are consistent with in vivo data showing that increasing pulse perfusion in normal compliant vessels induces vasodilation partly because of increased NOS stimulation.20,21 In contrast, Akt and NOS phosphorylation are suppressed in endothelial cells exposed to pulsatile perfusion without normal concomitant cyclic stretch.19 Here, we tested the hypothesis that Akt serves as the central compliance-sensitive switch that controls endothelial protection against oxidant stress–induced apoptosis. The results reveal a potential therapeutic avenue whereby stiff arteries could be protected despite a loss of normal distensibility.
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Materials and Methods |
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Cell Culture
Bovine aortic endothelial cells (BAECs; passage 5 to 7; Corriel Cell Repositories) were cultured in DMEM with 10% FBS. Custom-designed silastic cylindrical tubes (4-mm ID, 20-cm long; Specialty Manufacturing) were made at 2 wall compliances to yield either 6% to 7% or 1% to 2% radial strain at a pulse pressure of 70 mm Hg and mean perfusion pressure of 90 mm Hg. Tubes were precoated with 0.01% fibronectin (Sigma) and seeded with
2 to 4x105 endothelial cells/mL. Tubes were gently rotated at 10 rpm to facilitate generation of a confluent monolayer and cultured for 48 hours as described previously.22 Medium was switched to DMEM with 2% FCS overnight before study.
Pulse Perfusion Apparatus
Pulsatile flow was generated using a real-time servo-pump system as described previously.22 This system generates physiologic pulse pressures and flows within the tubes at a mean rate of 250 mL/min (8 dyne/cm2) and pulse pressure of 70 mm Hg. Mean perfusion pressure was maintained at 90 mm Hg by a fixed distal resistor in the flow circuit. The apparatus was computer controlled to maintain the mechanical stimuli over the course of the study. Perfusate was DMEM with 95% O2 and 5% CO2 to provide a pH of 7.37 at 37°C. Nonpulsatile flow was also generated by this system.
Modulation of Akt Activation
The role of Akt activation was studied by pretreating cells respectively with a selective Akt inhibitor (1L-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate; Calbiochem; Akti, 5 or 25 µmol/L added 1 hour before pulse perfusion) or transfection with 2 µg constitutively myristoylated Akt1cDNA, (Upstate Biotechnology) using Lipofectamin and Plus reagents (Invitrogen). Transfection efficacy was determined in separate studies using a plasmid encoding ß-galactosidase and averaged 19.3±3.3%. The role of vascular endothelial growth factor receptor 2 (VEGFR2) activation was tested by pretreatment (1 hour) with the VEGFR2 tyrosine kinase inhibitor (VTI; 4-[(4'-chloro-2'-fluoro)phenylamino]-6,7-dimethoxyquinazoline; 10 µmol/L; Calbiochem). Caveolae-dependent signaling was assessed using the cholesterol depletion agent ß-cyclodextrin (10 mmol/L; Sigma), which disrupts caveolae.
Immunoblotting Analysis
Immunoblots were performed on cells lysed in 50 mmol/L Tris-HCl, pH 7.4, 1% Nonidet P-40 (NP-40), 0.1% sodium dodecyl sulfate (SDS), 150 mmol/L NaCl, 0.5% sodium-deoxycholate, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF) 1 mmol/L Na3VO4, 1 mmol/L NaF, and proteinase inhibitors. Protein concentration was measured by bicinchoninic acid (BCA) assay and 18 µg of protein separated on NuPAGE 4% to 12% Bis-Tris Gel (Invitrogen) and transferred onto Bio-Rad Trans-Blot Nitrocellulose membrane. Polyclonal antibodies against Akt p-Akt(Ser473), glycogen synthase kinase (GSK-3ß, p-GSK-3ß(Ser9), Forkhead transcription factor) FOXO-1 and p-FOXO-1(Ser256), Myc-tag, Bad and p-Bad(Ser136) (Cell Signaling), and c-Jun-N-terminal kinase p-JNK and JNK1 (Santa Cruz Biotechnology) were used following manufacturer protocols. Horseradish peroxidase–conjugated goat anti-rabbit IgG was the secondary antibody (Sigma). Reactions were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Inc) and exposure to autoradiographic film. Signaling was quantified from scanned films using Scion NIH Image software (Scion).
PI3K and Akt Kinase Activity
PI3K activity was evaluated by competitive ELISA (Echelon Biosciences). Cells were washed 3x in iced buffer (in mmol/L: 137 NaCl, 20 Tris-HCl, pH 7.4, 1 CaCl2, 1MgCl, and 0.1 Na3VO4), lysed by addition of 1% NP-40 and 1 mmol/L PMSF, and lysates centrifuged for 10 minutes at 13 000 rpm at 4°C. Supernatant protein concentration was measured by BCA assay (Pierce, Inc) and PI3K pulled down by anti-PI3K p85 antibody (Upstate Biotechnology) and protein A/G beads (Santa Cruz Biotechnology) from 100-µg cell lysates. PI3K activity was assessed using phosphatidylinositol 4,5 bisphosphate as the substrate.
Akt activity was measured using GSK-3 fusion protein as substrate (Cell Signaling). Then 150-µg cell lysates were incubated with immobilized Akt antibody bead slurry overnight at 4°C and immunoprecipitated Akt suspended in kinase buffer supplemented with ATP and GSK3 fusion protein and incubated for 30 minutes at room temperature. The reaction was stopped and samples run on SDS gels to detect GSK-3 phosphorylation using p-GSK-3
/ß(Ser21/9) antibody.
Detection of Endothelial Reactive Oxygen Species Generation
Endothelial cells exposed to 2 hours of steady or pulsatile shear stress in stiff and compliant tubes were removed by trypsin and washed 2x with Krebs-Ringer Bicarbonate buffer (Sigma). Cells were then resuspended in 2 to 3 mL of fresh buffer, loaded with 2',7'-dichloro-dihydrofluorescein diacetate (DCF; 10 µmol/L; Molecular Probes), and incubated for 5 minutes at 37°C in the dark. Cells were then pelleted, washed twice, and resuspended in 1 mL of fresh buffer. Reactive oxygen species (ROS) generation was detected by monitoring DCF fluorescence using a FACScan flow cytometer (Becton Dickinson). Cells exposed solely to 100 µmol/L H2O2 for 30 minutes were used as a control.
Apoptosis Assay
Apoptosis was induced in BAECs by incubation with 800 µmol/L H2O2 (Sigma). Nonperfused cells were directly stimulated with H2O2 for 4 hours, whereas cells within tubes were initially perfused for 25 minutes, followed by 800 µmol/L H2O2 treatment in the perfusion media and additional pulse perfusion for 95 minutes. After 2 hours total perfusion time, cells remained exposed to H2O2 for a total of 4 hours. Apoptotic cells were identified by cell-sorting analysis based on Annexin V–fluorescein isothiocyanate staining (BD Pharmingen) following manufacturer instructions. Analysis was performed using FACScan flow cytometer and CELLQuest software (Becton Dickinson) within 1 hour of staining.
Statistics
Results are shown as mean±SD. Statistical significance was evaluated using an ANOVA, with different pulse conditions serving as a grouping factor.
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Akt and PI3K Activation by Pulse Perfusion Depends on Wall Compliance
Akt expression and phosphorylation were minimal in non–flow-stimulated endothelial cells. Furthermore, growth on the distensible silastic tube material yielded identical levels of total and phosphorylated Akt (p-Akt) as those observed in cells cultured in standard plastic dishes (Figure 1A). Thus, growth on the compliant material did not itself influence Akt in the absence of perfusion. Figure 1B shows example and summary data for the time course of Akt phosphorylation in response to pulsatile perfusion in compliant and stiff tubes. Data are shown as p-Akt normalized to total Akt. There was a consistently greater response in compliant tubes at all time points. The peak response was observed at 1 to 2 hours, similar to results reported to steady (nonpulsatile) shear stress,12 reflecting a 5- to 6-fold increase over baseline. After 2 hours of pulse perfusion, Akt phosphorylation in compliant tubes was twice that in stiff tubes. Based on this analysis, subsequent mechanistic studies focused on this 2-hour time point.
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P<0.05 vs PPc. C, PI3KOur previous study found that inhibition of PI3K activity by Wortmannin eliminated the differential response of p-Akt to pulse perfusion associated with wall compliance.19 However, this did not prove that PI3K activation was itself influenced by the cyclic distension of the cells, but only that its activation was central to Akt phosphorylation. To more directly test the role of PI3K signaling, we determined PI3K activity (Figure 1C) and found it to be nearly twice the level in cells pulse perfused in compliant tubes compared with stiff tubes (P<0.05). Additional confirmation was performed by immunoprecipitating PI3K and then probing for p-Akt. Compliant and stiff perfused tubes showed increased PI3K/p-Akt coprecipitation over nonstimulated controls, but the level in compliant tubes was nearly 2x that in stiff tubes.
Akt Controls Compliance-Sensitive GSK3ß, FOXO-1, and Bad Phosphorylation
GSK3ß, forkhead (FOXO-1), and Bad are all downstream targets of Akt that, when phosphorylated, play important roles in enhancing cell survival. Because loss of wall compliance dramatically blunted p-Akt in response to pulsatile flow, we next determined whether wall compliance similarly regulated activation of GSK3ß, FOXO-1, and Bad and further tested the specific role of Akt activation to this signaling by using a selective Akt inhibitor or cell transfection with myrAkt. Figure 2A shows the efficacy of myrAkt transfection and importantly reveals that basal Akt and secondary targets (eg, GSK3ß and FOXO-1) were not stimulated ariori by this transfection. However, transfection led to similar levels of Akt activation in pulse-perfused stiff tubes as observed in compliant tubes (Figure 2B). The opposite was achieved in compliant tubes if cells were first incubated with a selective Akt inhibitor.
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Pulse perfusion–mediated changes in GSK3ß, FOXO-1, and Bad phosphorylation each varied depending on wall compliance, and this correlated with the changes in Akt (Figure 2C). GSK-3ß, FOXO-1, and Bad phosphorylation rose 5.74±1.44, 2.7±0.75, and 5.22±0.5-fold, respectively, in perfused compliant tubes (P<0.01 versus static control). These responses were 50% lower in pulse-perfused stiff tubes, which was similar to the results with constant (nonpulsatile) flow. Inhibiting Akt in cells perfused within compliant tubes reduced phosphorylation of each protein in response to pulse perfusion (P<0.01 versus untreated). In contrast, enhancing Akt activation in cells perfused within stiff tubes augmented phosphorylation of all 3 enzymes to levels very similar to those observed with cells perfused in compliant tubes.
Wall Compliance Influences Apoptosis Protection by Pulsatile Perfusion
Given the directionally similar disparities in Akt, GSK3ß, FOXO-1, and Bad phosphorylation associated with wall compliance, we tested whether this translated into varying protection against H2O2-stimulated apoptosis. Incubation with H2O2 resulted in marked apoptosis (Figure 3A). Cells were modestly protected against apoptosis by exposure to constant shear stress, but the protection was markedly enhanced by pulse perfusion (identical mean shear stress) in compliant tubes. In contrast, pulse-perfused cells in stiff tubes revealed protection similar to those exposed to steady shear. Marked inhibition of Akt activity in compliant tubes resulted in levels of apoptosis in nonperfused controls, whereas myrAkt-transfected cells perfused in stiff tubes displayed antiapoptosis protection as in cells perfused in compliant tubes.
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Pulse perfusion in compliant and stiff tubes activated Akt, raising the question of whether the 2-fold quantitative disparity was indeed responsible for different apoptosis protection. To test this, we performed studies using a lower dose of Akti (5 µmol/L; the IC50) in cells cultured in compliant tubes. Figure 3B shows that p-Akt after pulse perfusion of such cells declined by 50%, matching that of cells perfused in stiff tubes. The magnitude of apoptosis protection in these cells (Figure 3A, far right) was nearly identical to that in nontreated cells perfused in stiff tubes. Thus, the quantitative disparity in Akt activation could explain protection differences to oxidant-stimulated apoptosis attributable to wall compliance.
Wall Compliance Does Not Alter Perfusion-Related ROS, JNK, or Nuclear Factor B Stimulation
Shear stress itself stimulates production of ROS such as H2O2,23 which can contribute to flow-mediated dilation24 and inhibit apoptosis at low levels.25 However, higher levels of ROS generation exacerbate apoptosis. We therefore determined whether wall compliance altered ROS stimulation by pulsatile perfusion. Figure 4A shows that ROS (DCF fluorescence) was similar in steady versus pulsatile-perfused cells in compliant or stiff tubes.
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The mitogen-activated kinase JNK is stimulated by oxidant stress and cytokines and can play a role in endothelial dysfunction. Because JNK activation is suppressed by constant shear stress,26–28 we tested whether wall compliance influences pulse perfusion-mediated suppression of JNK triggered by 100 µmol/L H2O2 (1 hour; Figure 4B). H2O2-induced JNK activation declined similarly when cells were pre-exposed for 2 hours of pulsatile perfusion in stiff and compliant tubes. Thus, this pathway was influenced primarily by mean shear rather than the presence or absence of concomitant stretch. Finally, shear stress stimulates endothelial NOS (eNOS) via Akt13 and PKA29,30 phosphorylation and enhances eNOS promoter activity by stimulating nuclear factor B (NF-B).31 Because NOS can provide antiapoptotic protection, we tested whether wall compliance differentially alters NF-B activation. As shown in Figure 4C, NF-B p65 subunit phosphorylation rose with steady shear stress and with pulsatile perfusion in cells in compliant and stiff tubes, but the level was similar for each condition.
VEGFR2 and Caveolae Transduce Shear but not Stretch-Stimulated Akt Activation
The preceding findings were compatible with an additive effect of shear stress and stretch on Akt activation that could derive from a common signaling cascade or involve separate inputs. To test this further, we examined the role of nonligand activation of the VEGF2R, which is thought to play a major role in steady shear stress–induced activation of NOS and Akt.16 Inhibiting VEGF2R tyrosine kinase by VTI blocked Akt activation by steady shear stress and pulsatile perfusion in stiff tubes (Figure 5A). However, in compliant tubes, p-Akt stimulated by pulse perfusion was unaltered (Figure 5B).
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Because the VEGF2R colocalizes with caveolae, and lack of caveolin-1 can eliminate VEGF2R-coupled signaling,32,33 we further tested the effects of disrupting caveolae by ß-cyclodextrin (Figure 5C and 5D). Cyclodextrin blocked the p-Akt response to steady shear and pulsatile flow in stiff tubes, but had no effect on the p-Akt response in pulse-perfused compliant tubes. Thus, VEGF2R/caveolae are centrally involved in shear stress–induced Akt activation (constant or phasic being similar), but a phasic stretch component present in compliant tubes involves a separate pathway.
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Discussion |
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We have shown that wall compliance serves as an important signaling switch for endothelial Akt activation, with pulsatile perfusion providing greater cytoprotection to ROS-stimulated apoptosis in cells cultured within compliant conduits. This is coupled with a greater activation of PI3K–Akt and thereby GSK-3ß, FOXO-1, and Bad phosphorylation. Targeted manipulation of Akt converts one phenotype to the other, supporting its role as a central switch to the mechanical stimulation. In contrast, other mechanisms involved with shear stress–mediated endothelial protection, such as enhanced NF-
B, JNK suppression, or ROS generation, were influenced by mean shear rate but not whether shear was phasic or constant nor whether concomitant cell stretch occurred. Finally, we provide novel evidence that shear and stretch signaling are independent activators of Akt, and that the former but not latter is coupled ligand-independent VEGF2R activation within caveolae. These results provide important new insights into the beneficial effects of vascular mechanosignaling and the role of wall compliance to endothelial stress protection and highlight a novel mechanism whereby arterial stiffening can increase vascular disease risk. Akt is a ubiquitously expressed kinase that regulates mechanically driven and receptor–ligand signaling. Akt phosphorylation is primarily linked to PI3K activation by G-protein– and integrin-coupled signaling with subsequent tyrosine kinase activity.34,35 The majority of information regarding Akt mechanostimulation stems from studies using constant shear stress. Dimmeler et al first reported the time course and magnitude of Akt activation by shear stress and demonstrated an important antiapoptotic effect associated with its activation.12 The present study found a near-identical time course and apoptosis protection with phasic (nonreversing) shear stress in the absence of cyclic stretch, whereas pulse perfusion accompanied by phasic cell stretch (compliant tubes) enhanced both phenomena.
Jin et al16 recently reported that nonligand VEGFR2 activation by steady shear stress plays a central role in stimulating eNOS via a PI3K/Akt pathway. VEGF stimulation of the VEGF2R also induces inside-out integrin activation that can alter PI3K/Akt signaling via the extracellular matrix.15,36 Our data extend these observations, providing the first evidence that VEGF2R–Akt activation responds to an integrated shear stimulus because steady and nonreversing phasic shear triggered similar p-Akt responses and were similarly blocked by VTI. Furthermore, this signaling appears targeted to caveolae, consistent with the localization of the VEGF2R to these structures.32,37 Most strikingly, p-Akt induced by pulse perfusion in compliant tubes was unaltered by either intervention, supporting an independent pathway involved with stretch-stimulated Akt and apoptosis protection and likely different from inside-out VEGF2R–integrin activations reported with ligand stimulation.36 Furthermore, the finding that the level of p-Akt in this setting was unchanged means that the 2 stimuli (shear and stretch) are not simply additive, and that stretch appears the more potent signal at least under the present experimental conditions. The current findings regarding stretch-induced p-Akt differs somewhat from data reported using vascular smooth muscle cells, in which absence of caveolin-1 or ß-cyclodextrin treatment eliminated Akt activation.33 This may relate to cell-specific caveolae-localized receptors and thus signaling. Future studies will be needed to fully define the stretch Akt response in pulse-perfused endothelial cells and identify members of this signaling cascade that are VEGF2R/caveolae independent.
GSK-3ß, FOXO-1, and Bad are all antiapoptosis regulatory proteins activated by Akt. Reduction in their phosphorylation enhances apoptosis in response to varying stimuli,15,38 whereas the opposite affords cytoprotection.39 Distal targets of GSK-3ß that modulate cell death include initiation factor eIF2B,40 the microtubule-associated protein Tau, transcription factors CREB, c-myc, c-jun, ß-catenin,41 and mitochondrial permeability transition,42 and GSK-3ß inactivation by Akt leads to antiapoptotic effects. FOXO-1 transcription factors regulate expression of proapoptotic genes such as Fas and Bim, and cell cycle regulators such as p27kip, p130, cyclinD, and GADD45.38,43 FOXO-1 is also inactivated via Akt-mediated phosphorylation promoting FOXO-1 nuclear exclusion and thus inhibition of its gene transcription. Bad binds to and thereby inhibits the antiapoptotic protein Bcl-2, and its phosphorylation by Akt leads to dissociation from Bcl-2 and antiapoptotic effects.15 It is likely that stimulation of these distal Akt targets were important to the apoptotic protection from tube compliance.
Although the present results strongly support a role for Akt/GSK3ß/FOXO-1/Bad in mediating apoptosis protection by wall compliance, other signaling could also be involved. In this regard, several additional factors linked to shear stress mediation of apoptosis were examined. Oxidant stress is induced by shear23,44 as well as cyclic stretch.11 Produced at low levels, it can potentially protect against apoptosis by upregulation of thioredoxin-1 expression.25 However, higher levels of ROS can exacerbate apoptosis. Our findings of similar ROS increases in pulse-perfused stiff and compliant tubes are consistent with the study of Sillaci et al,45 who reported increased ROS generation by nonreversing pulsatile shear and no further augmentation by the superposition of cyclic stretch. However, cell stretch can itself stimulate ROS,11,46 so this phenomena likely depends on the presence of concomitant shear stress.
JNK is an important regulator of the endothelial response to shear stress and can contribute to normal homeostatic pathways and pathophysiologic ones. For example, stimulation of PI3K/Akt results in NOS activation, which, in turn, transiently activates JNK47 but is not associated with enhanced apoptosis.48 However, when JNK activation is more sustained (ie, 60 minutes) by H2O2 or cytokine stimulation, shear stress can reduce JNK activity by augmenting glutathione reductase and inhibiting apoptosis signal–related kinase-1.26–28 It was intriguing to speculate that this beneficial effect might be further enhanced by pulse perfusion in compliant tubes. However, perfusion-dependent suppression of JNK activation also appeared insensitive to whether flow was constant or phasic or whether cell stretch accompanied shear or not. Whether this result further predicts that loss of wall compliance does not alter perfusion-dependent modulation of adhesion molecule expression26 or vascular inflammation remains to be determined.
Vascular stiffening is a major health care problem affecting a growing proportion of the adult aging population. Recent advances in mechanistic understanding of stiffening and the ability to measure it in humans are changing its role in clinical medicine. New pharmacological therapies may more directly enhance arterial distensibility by modifying glycation cross-links in structural molecules in the vessel wall49 or modulating endothelial-regulated vascular tone.1 The current data provide insights into the endothelial signaling coupled to reduced wall compliance and support a novel therapeutic approach, enhancing Akt mechanostimulation even without altering compliance, that may also prove effective.
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL-47511 and AG-18324 (D.A.K.).
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Footnotes |
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*These authors contributed equally to this work.
This manuscript was sent to Donald Heistad, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received March 23, 2005; revision received July 8, 2005; accepted August 3, 2005.
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