Cyclic Strain-Mediated Regulation of Vascular Endothelial Occludin and ZO-

【摘要】  Objective- The vascular endothelium constitutes a highly effective fluid/solute barrier through the regulated apposition of intercellular tight junction complexes. Because endothelium-mediated functions and pathology are driven by hemodynamic forces (cyclic strain and shear stress), we hypothesized a dynamic regulatory link between endothelial tight junction assembly/function and hemodynamic stimuli. We, therefore, examined the effects of cyclic strain on the expression, modification, and function of 2 pivotal endothelial tight junction components, occludin and ZO-1.

Methods and Results- For these studies, bovine aortic endothelial cells were subjected to physiological levels of equibiaxial cyclic strain (5% strain, 60 cycles/min, 24 hours). In response to strain, both occludin and ZO-1 protein expression increased by 2.3±0.1-fold and 2.0±0.3-fold, respectively, concomitant with a strain-dependent increase in occludin (but not ZO-1) mRNA levels. These changes were accompanied by reduced occludin tyrosine phosphorylation (75.7±8%) and increased ZO-1 serine/threonine phosphorylation (51.7±9% and 82.7±25%, respectively), modifications that could be completely blocked with tyrosine phosphatase and protein kinase C inhibitors (dephostatin and rottlerin, respectively). In addition, there was a significant strain-dependent increase in endothelial occludin/ZO-1 association (2.0±0.1-fold) in parallel with increased localization of both occludin and ZO-1 to the cell-cell border. These events could be completely blocked by dephostatin and rottlerin, and they correlated with a strain-dependent reduction in transendothelial permeability to FITC-dextran.

Conclusions- Overall, these findings indicate that cyclic strain modulates both the expression and phosphorylation state of occludin and ZO-1 in vascular endothelial cells, with putative consequences for endothelial tight junction assembly and barrier integrity.

The objective of this study was to investigate the effects of cyclic strain on the expression, modification, and function of 2 pivotal endothelial tight junction components: occludin and ZO-1. Our findings indicate that cyclic strain modulates the expression and phosphorylation of both proteins with consequences for their association and subcellular localization at the cell-cell border and, ultimately, for endothelial barrier integrity.

【关键词】  occludin ZO endothelium cyclic strain permeability

Introduction

The vascular endothelial monolayer or endothelium is a dynamic cellular interface between the vessel wall and bloodstream. In addition to regulating the physiological effects of humoral and mechanical stimuli on blood vessel tone and remodeling, the endothelium participates in immune and inflammatory reactions and presents a nonthrombogenic surface for blood flow. 1,2 Moreover, the vascular endothelium constitutes a highly effective barrier, which regulates fluid and solute balance in addition to movement of molecular/cellular components between bloodstream and tissues. 3-5 As such, regulation of endothelial barrier integrity (or permeability) is crucial for vascular homeostasis and is a central pathophysiologic mechanism of many vascular processes, including wound healing, angiogenesis, and vascular diseases. 5-7 Barrier function is maintained by the regulated apposition of tight junction and adherens protein complexes between adjacent endothelial cells. The organization of these protein complexes is controlled by a number of physiological/pharmacological mediators. The proteins that form tight junctions linking adjacent vascular endothelial cells include occludin, claudin family members, junctional adhesion molecules 1 to 3, cingulin, 7H6, spectrin, and linker proteins, such as the zonula occludens family members (ZO-1/2/3), the latter linking tight junction proteins to each other and the actin cytoskeleton. 3,8-11 Adherens junctions contain the transmembrane cadherins and their cytoskeletal linkers, catenins. 12,13 Although spatially and biochemically distinct, functional interaction between adherens junctions and tight junctions has been demonstrated. 4 See page 10

Among the physiological stimuli that impact on the endothelium, mechanical or hemodynamic forces associated with blood flow are of central importance. These include cyclic circumferential strain, caused by a transmural force acting perpendicularly to the vessel wall, and shear stress, the frictional force of blood dragging against cells. These forces have a profound impact on endothelial cell metabolism and can induce qualitative and quantitative changes in endothelial gene expression leading to changes in cell fate. 14-16 Vascular pathologies exhibiting altered vessel hemodynamic loading with associated remodeling (eg, atherosclerosis, restenosis, retinopathy, inflammatory lung disease, sepsis, edema, and systemic carcinomas) frequently correlate with compromised endothelial barrier integrity. 6,7,17,18 As such, one can hypothesize a dynamic regulatory association between endothelial permeability and hemodynamic stimuli. Indeed, because tight junction components are intimately coupled to the hemodynamically responsive actin cytoskeleton, 19 force-dependent modulation of tight junction assembly and properties is a highly likely process, albeit very poorly understood. In support of this, 2 earlier studies have indicated that shear stress may putatively regulate endothelial occludin expression and phosphorylation, 20,21 thereby implicating hemodynamic force as a putative physiological (and pathological) regulator of vascular endothelial permeability.

This article investigates this hypothesis via a detailed examination of the precise effects of cyclic strain on vascular endothelial tight junction assembly and function at the molecular and subcellular levels. Emphasis is placed on cyclic strain-dependent modulation of the expression, phosphorylation, association (coimmunprecipitation), and subcellular localization in bovine aortic endothelial cells (BAECs) of occludin and ZO-1, 2 of the most critical elements of the tight junction complex, in parallel with the subsequent consequences of strain on endothelial barrier function. To our knowledge, this article is the first definitive attempt to investigate these physiologically important events in depth with respect to cyclic circumferential strain.

Methods

Methods are available online at http://atvb.ahajournals.org.

Results

Cyclic Strain-Dependent Increase in Occludin Expression and Phosphorylation in BAECs

After exposure of BAECs to cyclic strain (5%, 24 hours), occludin protein expression increased by 2.3±0.1-fold ( Figure 1 a). Messenger RNA levels for occludin were also significantly increased by 2.6±0.4-fold in response to strain ( Figure 1 a graph inset). Phosphorylation of occludin was also monitored in total BAEC lysates by IP as described above using phosphospecific antibodies. In response to strain, tyrosine phosphorylation of occludin decreased by 75.7±8% ( Figure 1 b). This is in distinct contrast to relatively small decreases in threonine ( Figure 1 c) and serine ( Figure 1 d) phosphorylation of occludin observed in response to strain (16.3±6% and 30±5%, respectively).

Figure 1. Effect of cyclic strain on occludin expression and phosphorylation in BAECs. BAECs were exposed to cyclic strain (5%, 24 hours) and monitored for (a) occludin protein expression by Western blotting. Phosphorylation of occludin was also monitored in control and strained BAECs by IP and Western blotting using (b) phosphotyrosine-specific, (c) threonine-specific, and (d) serine-specific antibodies. Representative blots are shown above each graph. Also included in (b) is a control blot monitoring strain-dependent change in total occludin protein (ie, lower blot). Histograms represent fold change in band intensity relative to unstrained controls and are averaged from 3 independent experiments, ±SEM; * P 0.05 vs unstrained controls. With respect to (a), densitometric intensity of both bands has been combined. With respect to (b), (c), and (d), histograms are normalized to changes in occludin protein expression. Histogram inset in (a) represents fold change in occludin mRNA levels, as monitored by real-time PCR and is averaged from 3 independent experiments, ±SEM; * P 0.05 vs unstrained control.

Cyclic Strain-Dependent Increase in ZO-1 Expression and Phosphorylation in BAECs

After exposure of BAECs to cyclic strain (5%, 24 hours), ZO-1 protein expression increased by 2.0±0.3-fold ( Figure 2 a). No significant change in mRNA levels for ZO-1 was evident after strain ( Figure 2 a graph inset). After cyclic strain, phosphorylation of ZO-1 was also monitored in total BAEC lysates by IP as described above using phosphospecific antibodies. In response to strain, tyrosine phosphorylation of ZO-1 remained unchanged ( Figure 2 b). By contrast, threonine ( Figure 2 c) and serine ( Figure 2 d) phosphorylation of ZO-1 increased significantly in response to strain (51.7±9% and 82.7±25%, respectively).

Figure 2. Effect of cyclic strain on ZO-1 expression and phosphorylation in BAECs. BAECs were exposed to cyclic strain (5%, 24 hours) and monitored for (a) ZO-1 protein expression by Western blotting. Phosphorylation of ZO-1 was also monitored in control and strained BAECs by IP and Western blotting using (b) phosphotyrosine-specific, (c) threonine-specific, and (d) serine-specific antibodies. Representative blots are shown above each graph. Also included in (b) is a control blot monitoring strain-dependent change in total ZO-1 protein (ie, lower blot). Histograms represent fold change in band intensity relative to unstrained controls and are averaged from 3 independent experiments, ±SEM; * P 0.05 vs unstrained controls. With respect to (a), densitometric intensity of both bands has been combined. With respect to (b), (c), and (d), histograms are normalized to changes in ZO-1 protein expression. Histogram inset in (a) represents fold change in ZO-1 mRNA levels, as monitored by real-time PCR and is averaged from 3 independent experiments, ±SEM.

Cyclic Strain-Dependent Occludin/ZO-1 Association and Subcellular Localization

After exposure of BAECs to cyclic strain (5%, 24 hours), association of occludin and ZO-1 was monitored in total BAEC lysates by IP as described above. In response to strain, the level of occludin detected in anti-ZO-1 immunoprecipitates was seen to increase by 2.0±0.1-fold ( Figure 3 a). Moreover, inclusion of cyclohexamide (2 µg/mL) to block protein synthesis was seen to reduce this increase to 1.6±0.2-fold, suggesting that &40% of the association is a direct consequence of new protein synthesis (data not shown). Subcellular localization of occludin and ZO-1 within BAEC monolayers was also monitored by immunocytochemistry. Occludin immunoreactivity was observed within the cell nucleus and cytosol ( Figure 3 b, part i) but became more concentrated along the cell border in response to chronic strain ( Figure 3 b, part ii). Moreover, ZO-1 immunoreactivity, which exhibited a discontinuous and jagged localization pattern at the cell-cell border in unstrained cells ( Figure 3 b, part iii), became significantly more continuous and well defined along the cell-cell border after strain ( Figure 3 b, part iv).

Figure 3. Effect of cyclic strain on occludin and ZO-1 association and subcellular localization in BAECs. After exposure of BAECs to cyclic strain (5%, 24 hours), (a) association of occludin and ZO-1 were monitored by IP and Western blotting. Representative blot is shown above graph. Also included in (a) is a control blot monitoring strain-dependent change in total ZO-1 protein (ie, lower blot). Histogram represents fold change in band intensity relative to unstrained control and is averaged from 3 independent experiments, ±SEM; * P 0.05 vs unstrained controls. (b) Subcellular localization of occludin and ZO-1 were monitored by immunocytochemistry. Occludin: (i) control vs (ii) strain. ZO-1: (iii) control vs (iv) strain. Both proteins were monitored using standard fluorescent microscopy ( x 1000). White arrows and white zoom boxes highlight plasma membrane localization. Images are representative of 3 individual sets of experiments.

Cyclic Strain Decreases BAEC Transendothelial Permeability to FITC-Dextran

Because transendothelial permeability cannot be directly monitored in Bioflex plates after strain, both control and "strain-conditioned" BAECs were trypsinized and replated into Transwell-Clear plates at a density sufficient to reach confluence within 24 hours. BAEC monolayer permeability to 40 kDa FITC-dextran was subsequently monitored as described above. Results indicate that cyclic strain significantly reduces BAEC permeability to FITC-dextran, with control cells showing a 2.5±1.0-fold higher level of FITC-dextran in the subluminal chamber after 2 hours relative to strained BAECs ( Figure 4 ). Moreover, although this experimental paradigm necessitates testing transendothelial permeability 24 hours after the cessation of strain, we have monitored a number of strain-induced changes in occludin/ZO-1 properties (eg, subcellular localization) and confirmed that they fully persist 24 hours after passage from Bioflex plates into Transwell-Clear plates (data not shown).

Figure 4. Effect of cyclic strain on BAEC transendothelial permeability. After exposure of BAECs to cyclic strain (5%, 24 hours), control and "strain-conditioned" BAECs were trypsinized and replated into Transwell-Clear plates and monitored for permeability to 40 kDa FITC-dextran. Data points are shown as total subluminal fluorescence at a given time point (from 0 to 120 minutes) expressed as a percentage of total abluminal fluorescence at t=0 minutes (ie, % TEE of FD40 or % transendothelial exchange of FITC-dextran 40 kDa). Results are averaged from 3 independent experiments, ±SEM; P 0.05 (unstrained vs strained).

Cyclic Strain-Dependent Occludin/ZO-1 Association Is Attenuated by Tyrosine Phosphatase and Protein Kinase C Blockade

The initial investigation confirmed that strain-induced modification of occludin tyrosine phosphorylation and ZO-1 serine/threonine phosphorylation could be completely reversed by treatment of BAECs with dephostatin (tyrosine phosphatase inhibitor) and rottlerin (protein kinase C inhibitor), respectively (Figure Ia and Ib, available online at http://atvb.ahajournals.org). BAECs were subsequently exposed to cyclic strain (5%, 24 hours) in the absence or presence of either inhibitor, and occludin/ZO-1 association was monitored in total BAEC lysates by IP as described above. Results indicated that strain-induced occludin/ZO-1 association could be blocked by 68.4±44% and 87.7±30% after treatment with dephostatin and rottlerin (Figure Ic and Id), respectively.

Cyclic Strain-Dependent Subcellular Localization of Occludin and ZO-1 Is Attenuated by Tyrosine Phosphatase and PKC Blockade

After BAEC exposure to cyclic strain (5%, 24 hours) in the absence or presence of dephostatin, subcellular localization of occludin was monitored by immunocytochemistry. Likewise, subcellular localization of ZO-1 was monitored in BAECs after strain in the absence or presence of rottlerin. Results indicated that cyclic strain-induced localization of occludin to the cell-cell border was completely ablated by dephostatin treatment ( Figure 5a through 5 f). Moreover, the continuous and well-defined organization of ZO-1 immunoreactivity initially observed along the plasma membrane in response to cyclic strain was completely ablated by rottlerin treatment, reverting to a discontinuous and jagged localization pattern along the cell-cell border ( Figure 5g through 5 j).

Figure 5. Effect of tyrosine phosphatase and PKC inhibition on cyclic strain-induced occludin and ZO-1 subcellular localization in BAECs. After exposure of BAECs to cyclic strain (5%, 24 hours), localization of occludin and ZO-1 were monitored by immunocytochemistry. Occludin: untreated (a) control vs (b and c) strain and dephostatin-treated (d) control vs (e and f) strain. ZO-1: untreated (g) control vs (h) strain and rottlerin-treated (i) control vs (j) strain. Both proteins were monitored using confocal fluorescent microscopy ( x 1200; fields at x 200 are also shown for c and f). White arrows and white zoom boxes highlight plasma membrane localization. Images are representative of 3 individual sets of experiments.

Discussion

The vascular endothelium constitutes a highly effective fluid and solute barrier maintained by the regulated apposition of tight junction protein complexes between adjacent endothelial cells. Because disruption of vascular endothelial barrier integrity is a central feature of many homeostatic and pathophysiological processes (eg, atherosclerosis, sepsis, etc) and frequently correlates with a perturbation in vessel hemodynamic loading and remodeling, 6,7,17,18 we hypothesized a dynamic regulatory association between vascular endothelial permeability and hemodynamic stimuli. In this article, we investigate this hypothesis via examination of the precise effects of cyclic strain on vascular endothelial tight junction assembly and function with particular emphasis on occludin and ZO-1, pivotal components of the tight junction complex.

Our initial investigations clearly demonstrate that chronic cyclic strain (5%, 24 hours) of BAECs significantly upregulates protein expression of occludin and ZO-1 in parallel with an increase in mRNA levels for occludin (but not ZO-1). This latter result suggests that the strain-dependent increase in ZO-1 protein levels is most likely because of increased protein translation and/or decreased ZO-1 protein turnover. Interestingly, immunoblots indicated that both proteins migrated as 2 bands, a finding reflected in other studies. 20,33-35 Hyperphosphorylation and differential expression of occludin and ZO-1, respectively, have been proposed as reasons for this. Moreover, expression of both bands increased in response to strain for each protein. Strain-induced changes in the phosphorylation state of both proteins were also observed. The most prominent changes were decreased tyrosine phosphorylation of occludin and increased serine/threonine phosphorylation of ZO-1. Interestingly, although both proteins appear as a doublet on Western blots, the phosphorylation changes observed for occludin and ZO-1 appear to be restricted to the lower molecular weight band in both instances (data not shown). Mechanoregulation of vascular endothelial tight junction protein expression has also been confirmed in 2 recent, albeit contrasting, studies. In a report by Demaio et al, 20 exposure of BAECs to shear stress reduced occludin mRNA and protein expression in parallel with an increase in tyrosine phosphorylation and endothelial permeability (monitored as hydraulic conductivity). Interestingly, no change in ZO-1 expression was observed in this study. In a more recent article, Conklin et al 21 demonstrate shear stress-induced upregulation of occludin mRNA. The contrast between these studies may reflect differences in the shearing paradigms used. Overall, however, these results concur with our own observations in confirming that tight junction protein expression (and, therefore, permeability) in vascular endothelial cells are subject to regulation by hemodynamic forces.

The composition and integrity of tight junctions can vary immensely depending on environmental and humoral factors. Tight junction assembly is a highly regulated process, which involves a complex network of signaling pathways that include G-proteins, integrins, tyrosine phosphatase, protein kinase C, phospholipase C, and calmodulin. 36-38 Moreover, junctional integrity is regulated by cytoskeletal tension, alterations in junctional protein association (ie, interaction), and linkage between junctional proteins and the actin cytoskeleton, all of which help to govern intercellular cleft size and degree of fluid/solute permeability (for review, see references 3-5 ). As such, one would expect changes in subcellular localization of occludin and ZO-1 at the cell-cell border where tight junctions form, coupled with parallel changes in occludin/ZO-1 association (ie, co-IP), to accompany a change in endothelial barrier function, as evidenced previously by numerous studies. 9,39-43 With this in mind, we decided to broaden our initial investigations by examining occludin/ZO-1 association and subcellular localization (ie, measurable indices of tight junction assembly) in our cyclic strain model. Our results clearly indicate a significant strain-dependent increase in occludin/ZO-1 association. Furthermore, occludin, normally observed within the nucleus and cytosol of unstrained cells, exhibited increased immunoreactivity along the cell-cell border in response to strain, whereas ZO-1 became significantly more continuous and linearly distributed along the plasma membrane. Localization of the latter protein appeared more dramatic, an observation most likely due to the fact that most cellular ZO-1 is present near the cell border, albeit in a discontinuous and jagged localization pattern in unstrained cells. In contrast, occludin appears to exhibit a lower degree of membrane localization in BAECs relative to other cell types (eg, microvascular endothelial cells). In parallel with these observations, cyclic strain significantly reduced BAEC transendothelial permeability to FITC-dextran (ie, a measurable index of barrier function). When viewed collectively, these data lead us to conclude that cyclic strain upregulates endothelial occludin/ZO-1 expression and tight junction assembly, putatively leading to increased barrier integrity. Consistent with this conclusion, a recent study by Shin et al 44 demonstrated reduced transendothelial permeability to albumin after exposure of human umbilical vein endothelial cells to chronic pulse pressure (cyclic pressure), an important hemodynamic component of pulsatile blood flow (ie, in addition to, but distinct from, cyclic strain). To our knowledge, this is the only other existing study reporting on the putative role of pulsatile force in the modulation of endothelial barrier function.

Much attention has focused on the role of phosphorylation in the assembly and function of tight junctions. Indeed, numerous studies have identified kinase/phosphatase-dependent mechanisms leading to modulation of junctional protein association and subcellular distribution (for review, see references 3-5 ). Recent articles by Rao et al 39 and Sheth et al, 43 for example, demonstrate that oxidative stress-induced disruption of tight junctions result from increased tyrosine phosphorylation of occludin and ZO-1 leading to their subsequent dissociation from the actin cytoskeleton, reduction in occludin/ZO-1 association, and cellular redistribution of both proteins. A recent report by Kale et al 45 indicating that tyrosine phosphorylation of occludin reduces its association with ZO-1 has also been published, whereas shear stress-induced increases in transendothelial permeability have recently been demonstrated to correlate with increased tyrosine phosphorylation of occludin. 20 In view of these observations, we decided to reconcile the cyclic strain-dependent modification of occludin and ZO-1 phosphorylation state observed in the present study with the strain-dependent modulation of occludin/ZO-1 association and plasma membrane localization. We began by demonstrating that the strain-induced tyrosine dephosphorylation of occludin and serine/threonine phosphorylation of ZO-1 could be blocked by selective pharmacological inhibition of tyrosine phosphatase (dephostatin) and PKC (rottlerin: highest specificity for PKC 24 ), respectively. Additional investigations demonstrated that treatment of BAECs with dephostatin and rottlerin completely blocked strain-induced localization of occludin and ZO-1, respectively, to the cell-cell border. In control experiments, we demonstrated that, after cyclic strain, rottlerin affected neither occludin membrane localization nor phospho-serine/threonine levels, whereas dephostatin had no significant effect on either ZO-1 membrane localization or phospho-Tyr levels (data not shown). Moreover, strain-dependent increases in occludin/ZO-1 association could also be significantly attenuated after BAEC treatment with either dephostatin or rottlerin. With respect to the former inhibitor, occludin/ZO-1 association was significantly elevated above baseline in unstrained cells after inhibitor treatment. This may reflect an indirect, albeit uncharacterized, increase in ZO-1 serine/threonine phosphorylation levels in response to dephostatin treatment, which could putatively lead to an elevation in protein-protein association above unstrained baseline. Irrespective of this, dephostatin treatment led to a clear reduction in strain-induced occludin/ZO-1 association to unstrained inhibitor-treated levels. Therefore, based on these overall findings, we can conclude that cyclic strain upregulates tight junction assembly, with putative consequences for barrier integrity, most likely via tyrosine phosphatase- and PKC-dependent modulation of occludin and ZO-1, respectively.

The specific mechanistic roles of these phosphorylation events in tight junction assembly, however, remain unclear. Perturbation of the interaction between proximal membrane occludin domains leading to activation of intracellular signaling cascades and subsequent alteration in the phosphorylation state of tight junction proteins is an attractive and highly probable model of barrier regulation by extracellular stimuli. 3,5 Consistent with the current study, previous articles have clearly demonstrated that reduction in endothelial barrier integrity is usually accompanied by increased tyrosine phosphorylation of occludin leading to loss of junctional protein association and membrane localization. 39,43,45 Published evidence also indicates that binding of ZO-1 (and ZO-2/3) to the COOH-terminal cytoplasmic tail of occludin is important for targeting of the latter to the tight junction, as well as for its cytoskeletal tethering, 46 a process that is almost certainly sensitive to the phosphorylation state of the junctional and cytoskeletal components involved. 5,25,39,45 Also consistent with the current study, articles by Lohmann et al 47 and Wachtel et al 48 have demonstrated that pharmacological blockade of tyrosine phosphatase significantly reduces vascular endothelial barrier integrity in blood brain barrier and peripheral vasculature, respectively. Although clearly central to tight junction regulation, additional investigation will be necessary to clarify the precise mechanistic roles of these phosphorylation events in this process.

In addition to the investigations described in this article, preliminary attempts have also been made to confirm a causal relationship between the strain-induced modulation of occludin and ZO-1 expression/properties and transendothelial permeability. Treatment of BAECs with either dephostatin or rottlerin was found to reverse the strain-mediated reduction in transendothelial permeability by 89% and 56%, respectively (ie, results taken at t=240 minutes in transwell permeability assay). This suggests a causal link between changes in occludin/ZO-1 biochemical properties and endothelial barrier function in response to cyclic strain. Because investigations using pharmacological inhibitors are often fraught with nonspecific side effects, however, future investigations will use dominant-negative phosphorylation mutants of occludin/ZO-1, in conjunction with small interfering RNA strategies to selectively knock down tyrosine phosphatase and PKC function, to definitively reconfirm these findings. Considering the wealth of existing studies correlating changes in permeability to alteration in tight junction protein expression and phosphorylation in response to different stimuli, 5,35,40,42 we are confident that this relationship will be confirmed. Additional investigations will also concentrate on detailed characterization of the mechanotransduction pathway(s) upstream of the protein expression and post-translational modification events described in this article and, in particular, will focus on the putative roles of integrin- and vascular endothelial growth factor-mediated signaling pathways in these events.

In summary, this article describes the role of cyclic strain, a hemodynamic force component of pulsatile blood flow, in endothelial tight junction regulation. Our findings clearly indicate that cyclic strain modulates the expression, phosphorylation, association, and cellular distribution of occludin and ZO-1, pivotal components of intercellular tight junctions. Moreover, these findings correlate with, and appear to be putatively causal of, strain-dependent reduction of endothelial barrier integrity. To our knowledge, this is the first in-depth investigation of this physiologically significant phenomenon, and, as such, it enhances our overall understanding of how hemodynamic forces regulate vascular endothelial functions and behavior.

Acknowledgments

This research was supported by grants from the Higher Education Authority Programme for Research in Third Level Institutions (Cycle III), Wellcome Trust (to P.A.C.), Health Research Board of Ireland (to P.A.C., P.M.C.), and Enterprise Ireland/Science Foundation Ireland (to P.M.C.).

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作者单位：Nora T. Collins; Philip M. Cummins; Olga C. Colgan; Gail Ferguson; Yvonne A. Birney; Ronan P. Murphy; Gerardene Meade; Paul A. CahillFrom the Vascular Health Research Centre (N.T.C., P.M.C., O.C.C., G.F., Y.A.B., R.P.M., P.A.C.), Faculty of Science and Health, Dublin City University, Glasnevin, and