Asynchronous Shear Stress and Circumferential Strain Reduces Endothelial NO Synthase and Cyclooxygenase-2 but Induces Endothelin-1 Gene Expr
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《动脉硬化血栓血管生物学》
From the Biomolecular Transport Dynamics Laboratory, Department of Bioengineering (M.B.D., D.E.B.) and Center for Molecular Toxicology and Carcinogenesis (J.P.V.) at The Pennsylvania State University, Fenske Lab, University Park; and Department of Biomedical Engineering (J.M.T.), City College of New York/CUNY, New York.
ABSTRACT
Objective— Endothelium-derived vasoactive agents NO, endothelin-1 (ET-1), and prostacyclin (PGI2) not only regulate vascular tone but also influence atherogenic processes, including smooth muscle migration and proliferation, as well as monocyte and platelet adhesion. Complex hemodynamics characterized by the temporal phase angle between mechanical factors circumferential strain and wall shear stress (stress phase angle ) have been implicated in regions prone to pathologic development, such as atherosclerosis and intimal hyperplasia, in coronary and peripheral arteries where the mechanical forces are highly asynchronous (SPA=–180°). We determined the gene expression of endothelial NO synthase (eNOS), ET-1, and cyclooxygenase-2 (COX-2) affected by asynchronous hemodynamics (SPA=–180°) relative to normal hemodynamics (SPA=0°) in bovine aortic endothelial cells.
Methods and Results— Quantitative competitive RT-PCR analysis showed that eNOS production (at 5 and 12 hours) and COX-2 production (at 5 hours) were reduced at the gene expression level by asynchronous hemodynamics (SPA=–180°) compared with synchronous hemodynamics (SPA=0°), whereas ET-1 exhibited an opposite trend (at 5 and 12 hours). NO, ET-1, and PGI2 secretion followed their respective gene expression profiles after 5 and 12 hours.
Conclusion— Together, these data suggest that highly asynchronous mechanical force patterns (SPA=–180°) can elicit proatherogenic vasoactive responses in endothelial cells at the gene expression level, indicating a novel mechanism that induces cardiovascular pathology.
Asynchronous hemodynamic circumferential strain and wall shear stress occur in pathologic regions. Vasoactive gene expression of eNOS, ET-1, and COX-2 in endothelial cells exhibited a pathologic profile during asynchronous (SPA=–180°) hemodynamics. Asynchronous mechanical force patterns (SPA=–180°) can elicit proatherogenic responses in endothelial cells, indicating a novel mechanism that induces cardiovascular pathology.
Key Words: hemodynamics ? shear stress and strain ? coronary arteries ? eNOS ? ET-1 ? COX-2
Introduction
Endothelial dysfunction is a primary event in development of atherosclerosis, vasospasm, and thrombosis.1,2 Endothelium-derived vasoactive agents such as NO, endothelin-1 (ET-1), and prostacyclin (PGI2) not only regulate vascular tone but also influence atherogenic processes, including smooth muscle migration and proliferation, as well as monocyte and platelet adhesion.3 Endothelial cells (ECs) are a primary target for injuries such as hyperlipidemia, diabetes mellitus, and hypertension2 but also serve as sensors and transducers of the most notable hemodynamic forces: wall shear stress (WSS) and circumferential stress that is induced by circumferential strain (CS ). WSS and CS are widely believed to be important hemodynamic mediators of vascular regulation, atherosclerosis, and remodeling.4–7
Most previous studies of the role of vascular mechanical forces in atherogenesis have emphasized fluid shear stress (WSS) by itself (ie, no pressure or strain).4–7 A few recent studies have noted the importance of complex hemodynamics that include simultaneous flow, pressure, and diameter variation (stretch) in modulating cardiovascular function.6–9 Blood vessel ECs in vivo are subjected to simultaneous pulsatile CS and WSS that act approximately in perpendicular directions. The temporal phase angle between pressure and flow (impedance phase angle) generated by global wave reflection in the circulation and the local inertial effects of blood flow cause time lags to occur between CS and WSS. The temporal phase angle between CS and WSS characterizes the complex, time-varying mechanical force pattern on the EC monolayer. We will refer to the temporal phase angle between CS and WSS as the stress phase angle (SPA)6,7 (Figure 1). A typical transverse arterial cross-section is shown with major vascular cells and hemodynamic features (Figure 1, left). Q in the axial direction induces WSS on the EC-lined vascular wall. P imposes a normal (radial) stress on the wall from the lumen. Changes in pressure distend (strain) the wall and EC monolayer, resulting in uniform CS attributable to the axisymmetric tubular geometry. ECs experience pulsatile WSS and CS simultaneously; however, their magnitude and temporal phase may vary throughout the vasculature and in disease conditions. SPA is used to characterize the dynamic mechanical stimuli experienced by the EC monolayer. Regions of the circulation prone to pathologic development, such as atherosclerosis and intimal hyperplasia, include the outer wall of the abdominal aortic bifurcation,8 the coronary arteries,9 and the distal anastomosis of undersized end-to-end vascular grafts that are characterized by large negative SPA values relative to regions that are typically spared (veins, small arteries, high shear regions in large arteries). It is interesting to note that the most disease-prone arteries of circulation, the coronary arteries, experience complex hemodynamics that allow for the most extreme SPA in the cardiovascular system (SPA close to –180°).9 Hypertension has also been shown to further asynchronize pulsatile WSS and CS, thus creating a more negative SPA in both pathologic and normopathic regions.9 Atheroprotective vasoactive gene expression in ECs has been shown to be induced during steady shear stress (SS; >10 dyne/cm2)10–15 and simultaneous synchronous pulsatile shear stress and CS (SPA=0°).16–18
Figure 1. Representation of the SPA on vascular cells attributable to mechanical effects from hemodynamic features blood flow and pressure (P) with physiological significance. The SPA is used to characterize the dynamic mechanical stimuli experienced by the EC monolayer.
In the present study, we investigated the effects of asynchronous hemodynamics (SPA=–180°) on vasoactive gene expression and corresponding secretion for endothelial NO synthase (eNOS), ET-1, and cyclooxygenase-2 (COX-2). We report that asynchronous hemodynamic conditions (SPA=–180°) induce an atherogenic vasoactive gene expression profile compared with normopathic hemodynamics (SPA=0°). COX-2 and eNOS mRNA levels and corresponding metabolite secretions of PGI2 and NO are reduced by asynchronous mechanical forces (SPA=–180°), whereas ET-1 shows an opposite trend. Our results demonstrate a novel mechanism by which simultaneous mechanical forces, when imposed in a unique pulsatile temporal pattern, can induce a proatherogenic biomolecular response in ECs. This is the first study to demonstrate the influence of SPA on gene expression. The underlying biomolecular mechanisms that mediate these new phenomena will be the subject of future studies.
Materials and Methods
Cell Culture and Hemodynamic Conditions
Primary bovine aortic ECs (BAECs) were isolated from fresh aortas as described previously6 (information available online at http://atvb.ahajournals.org). Cultured tubes were grown in an incubator for 3 to 4 days until confluence before experiment. Cell attachment was verified via visualization through the silicone tubing and cell counting before RNA extraction before and after the experiment. Experimental hemodynamic conditions did not appear to influence cell attachment and cell number. The experimental design focused on isolating the effect of SPA from other hemodynamic factors. A novel simulator was used to impose asynchronous (SPA=–180°) and synchronous (SPA=0°) hemodynamic conditions as described previously.7 Briefly, the sinusoidal WSS and CS waveforms were the same in both hemodynamic conditions; however, the SPA was different. Concurrent controls allowed us to examine the nonoscillatory mechanical effects present in the pulsatile cases such as mean pressure, mean CS, and mean WSS. The purpose of these controls was to demonstrate that cells in this preparation have biomolecular responses to mechanical forces that have been observed in other studies. Relevant hemodynamic parameters are (Table I, available online at http://atvb.ahajournals.org): WSS=10±10 dyne/cm2; CS=(Dmax–Dmin)/Dmean=4±4% ); P=70±20 mm Hg, all at frequency of f=1 Hz, and an unsteadiness parameter =4.3 (=D/2(2f/)1/2; where is the kinematic viscosity). The pressure, diameter, and flow waveforms for the SPA=0° and the SPA=–180° case are shown in supplemental Figure IA and IB (available online at http://atvb.ahajournals.org). The total media volume=175 mL, maximum Reynolds number (U/ =460, pH=7.2±0.05, all at 37°C for 5 and 12 hours. Each experiment consisted of a total of 10 individual tubes with separate media lines: 4 incubator controls, static control (SC), and pressurized control (PC), 2 SS (with mean pressure and mean stretch) controls, and 6 sinusoidal WSS cases (SPA=0° or –180°; Table I). These were repeated 3x.
RNA Extraction and Quantitative RT-PCR Analysis
Total RNA was isolated from cells using Trizol (Life Technologies) following the protocol of the manufacturer. Total RNA extracts were frozen at –80°C until analyses were performed. Quantitative competitive RT-PCR used recombinant RNA as an internal standard to negate tube-to-tube variability in amplification efficiency.19–22 Primers for eNOS, ET-1, COX-2, and hypoxanthine-guanine phosphoribosyltransferase (HPRT) are described at http://atvb.ahajournals.org. Results are expressed in relative terms (number of target molecules per number of HPRT molecules). Figure II (available online at http://atvb.ahajournals.org) depicts the invariance of HPRT gene expression during asynchronous and synchronous hemodynamics.
NO, PGI2, and ET-1 Media Secretions
Media was collected and stored at –80°C before media assays. NO concentration was quantified using a fluorometric assay.23 ET-1 and PGI2 (actually 6-keto PGF1, major stable PGI2 byproduct) concentrations were determined via enzyme immunoassay kits (Cayman Chemicals) following the protocol of the manufacturer. Results were normalized to cell number in all experiments.
Statistics Analysis
The data are presented as mean±SEM. Then a 2-factor ANOVA model was used with the Tukey method on a 95% CI for all experiments. The inset table in Results figure indicates pairwise significant differences: P<0.05 by the shaded boxes and asterisk for 5 versus 12 hours. Note that –180° and 0° refer to SPA.
Results
Asynchronous WSS and CS (SPA=–180°) Reduces eNOS Production at the Gene Expression Level
SS induced more than a 2-fold increase in eNOS mRNA levels from 5 to 12 hour, indicating that the BAECs were responsive to SS (Figure 2A). SS also showed a significant increase in eNOS mRNA levels compared with SC at 12 hours (Figure 2A). SS appeared to increase eNOS mRNA levels compared with SPA=–180° at 12 hours, but this effect did not reach statistical significance (Figure 2A). SS with mean pressure and stretch (SS) exerted an opposite trend compared with constant CS with mean pressure and stretch (PC) by significantly inducing eNOS mRNA relative to SC, whereas PC reduced mRNA levels at 12 hours (Figure 2A). Interestingly, pulsatile asynchronous hemodynamics (SPA=–180°) significantly reduced (2-fold) eNOS mRNA levels compared with pulsatile normopathic hemodynamics (SPA=0°) at both 5 and 12 hours (Figure 2A).
Figure 2. A, Asynchronous hemodynamics (SPA=–180°) reduces eNOS production at the gene expression level. BAECs were exposed to hemodynamic conditions for 5 and 12 hours with concurrent controls. eNOS mRNA was quantitated with competitive RT-PCR normalized to HPRT mRNA. Results are expressed in relative terms (number of eNOS molecules per number of HPRT molecules; n=5). B, Asynchronous hemodynamics (SPA=–180°) reduces NO secretion compared with synchronous hemodynamics (SPA=0°). NO concentration was evaluated by fluorogenic assay and normalized to cell number. Results are expressed in μmol/106 cells (n=5).
Asynchronous WSS and CS (SPA=–180°) Reduce NO Secretion
SS induced significant NO production at 5 and 12 hours compared with SC, indicating that the BAECs were responsive to SS (Figure 2B). PC did not significantly induce NO secretion at either 5 or 12 hours (Figure 2B). Asynchronous hemodynamics (SPA=–180°) significantly reduced (2-fold) NO secretion compared with synchronous hemodynamics (SPA=0°) at 5 hours (Figure 2B). At 12 hours, SPA=–180° reduced NO secretion compared with SPA=0°, but the effect was not significant (P=0.20). SS and SPA=–180° showed similar NO secretion levels at 5 and 12 hours (Figure 2B). These results indicate that asynchronous hemodynamics (SPA=–180°) reduce NO secretion compared with normal hemodynamics (SPA=0°).
Asynchronous WSS and CS (SPA=–180°) Induces ET-1 Gene Expression
SS reduces ET-1 mRNA levels relative to SCs in ECs at 5 and 12 hours, although not statistically significant (Figure 3A). PC also showed a reduction in ET-1 mRNA levels compared with SC at 5 and 12 hours that was not significant (Figure 3A). Pulsatile asynchronous hemodynamics (SPA=–180°) very significantly augmented ET-1 mRNA levels (>3-fold) compared with synchronous hemodynamics (SPA=0°) at 12 hours (Figure 3A). In fact, the SPA=–180° case significantly induced ET-1 mRNA levels relative to all other conditions at 12 hours (Figure 3A).
Figure 3. A, Asynchronous hemodynamics (SPA=–180°) reduces ET-1 production at the gene expression level. BAECs were exposed to hemodynamic conditions for 5 and 12 hours with concurrent controls. ET-1 mRNA was quantitated with competitive RT-PCR normalized to HPRT mRNA. Results are expressed in relative terms (number of ET-1 molecules per number of HPRT molecules; n=5). B, Asynchronous hemodynamics (SPA=–180°) induces ET-1 secretion compared with synchronous hemodynamics (SPA=0°). ET-1 concentration was evaluated by immunoassay and normalized to cell number. Results are expressed in μmol/106 cells (n=5).
Asynchronous Hemodynamics (SPA=–180°) Induce ET-1 Secretion
SS significantly reduced ET-1 secretion levels compared with SC at 5 and 12 hours (Figure 3B). PC and SC levels of ET-1 secretion were not significantly different, but both static cases, SC and PC, displayed significantly higher ET-1 secretion levels compared with dynamic conditions at 5 and 12 hours (Figure 3B). Asynchronous hemodynamics (SPA=–180°) significantly augmented ET-1 secretion (2-fold) compared with synchronous hemodynamics (SPA=0°) at 12 hours (Figure 3B).
Asynchronous WSS and CS (SPA=–180°) Reduces COX-2 Production at the Gene Expression Level at 5 hours, but Increases It at 12 hours
SS did not significantly induce COX-2 mRNA levels compared with SC at 5 and 12 hours (Figure 4A). COX-2 mRNA expression for PC was not significant compared with SS or SC at 5 or 12 hours as well (Figure 4A). However, the SPA=–180° case significantly reduced COX-2 mRNA levels (>4-fold) compared with all cases at 5 hours (Figure 4A). Unexpectedly, synchronous (SPA=0°) and asynchronous (SPA=–180°) hemodynamics showed an opposite trend at 12 hours compared with 5 hours: the SPA=–180° case was increased (4-fold) approaching basal COX-2 mRNA expression levels, whereas the SPA=0° case was reduced (6-fold) falling below basal COX-2 mRNA levels (Figure 4A). These results indicate that SPA has a dramatic, but opposite, effect on COX-2 production at the gene expression level at 5 and 12 hours.
Figure 4. A, Asynchronous hemodynamics (SPA=–180°) reduces COX-2 production at the gene expression level. BAECs were exposed to hemodynamic conditions for 5 and 12 hours with concurrent controls. COX-2 mRNA was quantitated with competitive RT-PCR normalized to HPRT mRNA. Results are expressed in relative terms (number of COX-2 molecules per number of HPRT molecules; n=5). B, Asynchronous hemodynamics (SPA=–180°) induces PGI2 secretion compared with synchronous hemodynamics (SPA=0°). PGI2 concentration was evaluated by immunoassay and normalized to cell number. Results are expressed in μmol/106 cells (n=5).
Asynchronous Hemodynamics (SPA=–180°) Reduce PGI2 Secretion Compared With Synchronous Hemodynamics (SPA=0°) at 5 Hours but Increases It at 12 Hours
SS did induce elevated PGI2 levels that were significant compared with SC at 5 and 12 hours (Figure 4B). PC reduced PGI2 secretion compared with SC at 5 and 12 hours, but the differences were insignificant (Figure 4B). Asynchronous hemodynamics (SPA=–180°) reduced PGI2 levels significantly (>3-fold) compared with synchronous (SPA=0°) at 5 hours (Figure 4B). As with the gene expression results (Figure 4B), unexpectedly, synchronous SPA=0° and asynchronous SPA=–180° showed an opposite trend at 12 hours compared with 5 hours.
Discussion
Most previous studies of the role of vascular mechanical forces in atherogenesis have emphasized fluid shear stress by itself (ie, no pressure or strain),4–7 with low mean and reversing oscillatory shear stress generally being associated with the location of the disease. Yet the most disease prone arteries, the coronary arteries, do not have a particularly low mean shear stress, and reversal is not that prominent.4–7 However, the SPA is most negative in the coronary arteries as well as the outer wall of bifurcations and other sites of atherogenesis. Hypertension, a major risk factor for several cardiovascular diseases, also induces a more negative SPA yet is not known to affect shear stress levels.4–7
In the present study, we showed that the occurrence of concomitant WSS and CS elicits significantly different vasoactive gene expression and biomolecule secretion compared with simple SS or stretch in BAECs. Most significantly, we demonstrated that asynchronous hemodynamics (SPA= –180°) evoke proatherogenic vasoactive gene expression profiles relative to synchronous hemodynamics (SPA=0°) for eNOS (at 5 and 12 hours), ET-1 (at 5 and 12 hours), and COX-2 (at 5 hours). Finally, we observed that the vasoactive biomolecule secretion associated with these genes (NO, ET-1, and PGI2) also exhibit proatherogenic responses for the SPA=–180° condition.
Our results in Figure 2A and 2B are consistent with previous reports showing that SS and pulsatile shear stress (without stretch) increase eNOS mRNA levels and NO secretion in BAECs and that pulsatile shear exerts a greater influence than SS.10,11,16,17,24,25 In previous experiments that examined the effects of shear stress alone, eNOS mRNA was upregulated in SS during time periods of 12 hours.10,11 In an experiment in an elastic tube with oscillatory WSS of 6±6 dynes/cm2 with ±4% CS at 100±30 mm Hg and SPA undefined but probably near 0°, eNOS mRNA was upregulated at 24 hours compared with steady WSS and SC.16,17 Although our NO secretion levels followed eNOS mRNA levels for SS and SPA=0°, eNOS gene expression levels were more responsive than NO metabolite levels at 5 and 12 hours (Figures 2 and 3). It is interesting to note that SS alone induced eNOS mRNA levels, whereas stretch alone reduced eNOS mRNA levels (Figure 2A). These data indicate that these separate (constant) mechanical forces have opposing regulatory roles in gene expression that are altered under physiological pulsatile conditions. Most importantly, pulsatile asynchronous hemodynamics (SPA=–180°) reduced eNOS mRNA levels compared with pulsatile synchronous hemodynamics (SPA=0°) at 5 and 12 hours (Figure 2A). NO secretion showed similar trends (Figure 2B). The eNOS gene expression trends are consistent with observations in vivo in regions expected to have large negative SPA, such as the opposite wall of a flow divider, that have decreased eNOS mRNA levels compared with more positive SPA regions, such as along the flow divider.24 The glycocalyx of the EC may play a role in the signal transduction of the SPA because it has been shown to be shear stress sensor for NO.40,41 Because reduction of eNOS expression or NO availability is an event that can contribute to atherogenesis,2,26,39 asynchronous hemodynamics (SPA=–180°) in vitro, in concert with other risk factors, might be used to simulate the pathogenesis of atherosclerosis by inducing proatherogenic gene and biomolecule responses for studies to mitigate or reverse pathology.
Our results demonstrate that asynchronous hemodynamics (SPA=–180°) induce the greatest ET-1 gene expression level compared with all other hemodynamic cases at 12 hours (Figure 3A). ET-1 secretion was elevated at SPA=–180° compared with SPA=0° as well, but that was not the condition with the highest ET-1 secretion (Figure 3B). In an experiment in an elastic tube with ±4% CS, 5±2 dynes/cm2, and undefined SPA (likely near 0°), Tsukurov et al18 showed a similar decrease in ET-1 mRNA expression at 48 hours. In the present study, SS insignificantly reduced ET-1 mRNA levels compared with SC (Figure 3A). This is not inconsistent with other studies that have shown ET-1 mRNA expression to increase within the first hour of exposure to 20 dynes/cm2 SS then transiently decrease.12,13 SS did significantly reduce ET-1 secretion compared SC (Figure 3B), perhaps because of alterations in membrane fluidity.27–30 Others have shown that cyclic strain alone increased ET-1 mRNA levels <±20% CS at 1 Hz out to 6 hours of exposure, but longer time points showed no further increase in mRNA expression.31 Although SS and CS (or PC) alone exert insignificant effects on ET-1 mRNA levels, combined pulsatile SS and CS with SPA=–180° exhibits proatherogenic ET-1 gene responses (Figure 3A). Because ET-1 is involved in monocyte recruitment and SMC proliferation and migration in atherosclerosis,1,4,5,32,33 asynchronous hemodynamics can be used to induce atherogenic ET-1 gene expression and secretion for studies that attempt to mitigate proatherogenic ET-1 expression. Delerive et al34 proposed that ET-1 gene expression is downregulated by peroxisome proliferator–activated receptors, partly by interfering with activated protein-1 activity and potentially by signal transducer activator of transcription and nuclear factor B (NF-B) signaling pathways. Additional studies are necessary to delineate in detail the molecular mechanisms involved in the positive regulation of ET-1 gene expression in complex pathologic hemodynamics.
SS with mean pressure and strain did not significantly induce COX-2 mRNA levels compared with SC at 5 and 12 hours (Figure 4A). This is not inconsistent with Okahara et al,15 who showed a peak increase from basal COX-2 mRNA levels at 6 hours then a drop to basal levels at 12 hours at an SS of 24 dynes/cm2. However, SS did significantly induce PGI2 secretion compared with SC at 5 and 12 hours (Figure 4B). Previous experiments have shown that elevated PGI2 production induced by shear stress14,35,36 is mediated by increased arachidonic acid release and a combination of increased mRNA expression of cyclooxygenases and PGI2 synthase.15 Because arachidonic acid is membrane bound and shear stress alters membrane fluidity,27–30 SS may exert more immediate effects on PGI2 release than on gene expression. Similar to SS, cyclic CS at physiological levels of ±10% and 1 Hz showed an increase in PGI2 secretion by 2.5-fold compared with SCs.37 Thus, SS and CS alone upregulate PGI2 secretion levels, yet combined pulsatile SS and CS with asynchronous SPA=–180° induces proatherogenic PGI2 inhibition.
Asynchronous hemodynamics (SPA=–180°) significantly reduced COX-2 mRNA levels compared with all other cases at 5 hours (Figure 4A). PGI2 secretion was also significantly lower for SPA=–180° versus SPA=0° at this time point (Figure 4B). It was unexpected that synchronous (SPA=0°) and asynchronous (SPA=–180°) hemodynamics would show a significant opposite trend in expression at 12 hours compared with 5 hours (Figure 4A). However, similar alterations occurred for PGI2 secretion at 12 hours compared with 5 hours (Figure 4B). It may be that COX-2 mRNA expression at different SPA exhibits a classic type-I gene response, an early transient increase followed by decline, as observed in previous SS response data.15 Asynchronous hemodynamics (SPA=–180°) may delay the early transient increase in COX-2 mRNA levels until 12 hours, whereas at SPA=0°, there may be a rapid early transient increase in COX-2 mRNA levels at 5 hours (Figure 4A). Further studies are required to test this hypothesis. COX-2 regulation occurs via many mechanisms. COX-2 transcription has been shown to involve 3 cis-acting elements in the human COX-2 promoter region, namely an NF-B binding site, an NF–interleukin-6–binding site, and a cAMP-responsive element (CRE), where the shear stress response of the COX-2 promoter was dependent on CRE.38 Post-transcriptional regulation under SS has been shown in the 3'-untranslated region of the COX-2 gene.38
A limitation of our study was the focus on the endothelium and lack of an intact vessel and consequent myogenic response. We could not determine the dilation response or true vasodilator effect to asynchronous hemodynamics, although endothelium-dependent and -independent responses play a normal role in epicardial and endocardial vessels.42 Furthermore, there are many factors that the SPA may affect; however, we focused only on several genes, time points, cell types, and many other details that will be the subject of future experiments.
The mechanotransduction mechanisms that mediate the effects of SPA on ECs are not known. However, it is clear that a nonlinear interaction between stretch and shear is involved because the responses are not simply the summation of the stretch response and the shear response. One possibility is that the strain energy of the plasma membrane or the actin cortical web (that is, a nonlinear function of CS and axial strain ) mediates mechanotransduction.43 Another possibility is that the nonlinearity is associated with a mechanochemical pathway that requires a threshold level of stimulation. Then, for example, when stretch and shear are in-phase, the threshold may be exceeded during a time interval around the maximum stretch and shear, whereas the maximum stretch or shear by itself would not provide stimulation exceeding the threshold. These and other possible mechanisms for the SPA effect remain to be explored in future studies.
In conclusion, the results from the present study demonstrate that physiological asynchronous hemodynamics (SPA=–180°) have a dramatic influence on endothelial function. Asynchronous SPA induces an atherogenic profile of eNOS, ET-1, and COX-2 gene expression and release of NO, ET-1, and PGI2. Together, these data suggest that local hemodynamic conditions characterized by the SPA can modulate local vasoautoregulatory and atherogenic responses. To further test the relative importance of low mean and reversing oscillatory shear stress versus asynchronous WSS and CS, future studies should compare gene expression and secretion profiles for low mean and oscillatory shear stress with synchronous and asynchronous hemodynamics.
Acknowledgments
This work was supported by National Institutes of Health National Heart, Lung, and Blood Institute grant ROI-HL35549. We thank the Center for Carcinogenesis and Molecular Toxicology at Penn State University.
References
Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993; 362: 801–809.
Shimokawa H. Primary endothelial dysfunction: atherosclerosis. J Mol Cell Cardiol. 1999; 31: 23–37.
Vanhoutte PM. Endothelial dysfunction and atherosclerosis. Eur Heart J. 1997; 18 (suppl E): E19–E29.
Davies PF, Shi C, Depaola N, Helmke BP, Polacek DC. Hemodynamics and the focal origin of atherosclerosis: a spatial approach to endothelial structure, gene expression, and function. Ann N Y Acad Sci. 2001; 947: 7–16.
Gimbrone MA Jr. Vascular endothelium, hemodynamic forces, and atherogenesis. Am J Pathol. 1999; 155: 1–5.
Qiu Y, Tarbell JM. Interaction between wall shear stress and circumferential strain affects endothelial cell biochemical production. J Vasc Res. 2000; 37: 147–157.
Dancu M, Tarbell J. Large negative stress phase angle (SPA) atteneutates nitric oxide production in bovine aortic endothelial cells. J Biomech Eng. In press.
Lee CS, Tarbell JM. Wall shear rate distribution in an abdominal aortic bifurcation model: effects of vessel compliance and phase angle between pressure and flow waveforms. J Biomech Eng. 1997; 119: 333–342.
Qiu Y, Tarbell JM. Numerical simulation of pulsatile flow in a compliant curved tube model of a coronary artery. J Biomech Eng. 2000; 122: 77–85.
Ranjan V, Xiao Z, Diamond SL. Constitutive NOS expression in cultured endothelial cells is elevated by fluid shear stress. Am J Physiol. 1995; 269: H550–H555.
Uematsu M, Ohara Y, Navas JP, Nishida K, Murphy TJ, Alexander RW, Nerem RM, Harrison DG. Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress. Am J Physiol. 1995; 269: C1371–C1378.
Malek AM, Izumo S. Mechanism of endothelial cell shape change and cytoskeletal remodeling in response to fluid shear stress. J Cell Sci. 1996; 109: 713–726.
Malek AM, Izumo S. Control of endothelial cell gene expression by flow. J Biomech. 1995; 28: 1515–1528.
Frangos JA, Eskin SG, McIntire LV, Ives CL. Flow effects on prostacyclin by cultured human endothelial cells. Science. 1985; 227: 1477–1479.
Okahara K, Sun B, Kambayashi J. Upregulation of prostacyclin synthesis-related gene expression by shear stress in vascular endothelial cells. Arterioscler Thromb Vasc Biol. 1998; 18: 1922–1926.
Ziegler T, Bouzourene K, Harrison VJ, Brunner HR, Hayoz D. Influence of oscillatory and unidirectional flow environments on the expression of endothelin and nitric oxide synthase in cultured endothelial cells. Arterioscler Thromb Vasc Biol. 1998; 18: 686–692.
Ziegler T, Silacci P, Harrison VJ, Hayoz D. Nitric oxide synthase expression in endothelial cells exposed to mechanical forces. Hypertension. 1998; 32: 351–355.
Tsukurov OI, Kwolek CJ, L’Italien GJ, Benbrahim A, Milinazzo BB, Conroy NE, Gertler JP, Orkin RW, Abbott WM. The response of adult human saphenous vein endothelial cells to combined pressurized pulsatile flow and cyclic strain, in vitro. Ann Vasc Surg. 2000; 14: 260–267.
Vanden Heuvel JP, Tyson FL, Bell DA. Construction of recombinant RNA templates for use as internal standards in quantitative RT-PCR. BioTechniques. 1993; 14: 395–398.
Gilliland G, Perrin S, Blanchard K, Bunn HF. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A. 1990; 87: 2725–2729.
Vanden Heuvel JP, Clark GC, Kohn MC, Tritscher AM, Greenlee WF, Lucier GW, Bell DA. Dioxin-responsive genes: examination of dose-response relationships using quantitative reverse transcriptase-polymerase chain reaction. Cancer Res. 1994; 54: 62–68.
Zachar V, Thomas RA, Goustin AS. Nucleic Acids Res. 1993; 21: 2017–2018.
Misko TP, Schilling RJ, Salvemini D, Moore WM, Currie MG. A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem. 1993; 214: 11–16.
Huige L, Forstermann TW. Physiological mechanisms regulating the expression of endothelial-type NO synthase. Nitric Oxide. 2002; 7: 132–147.
Benbrahim A, L’Italien GJ, Milinazzo BB, Warnock DF, Dhara S, Gertler JP, Orkin RW, Abbott WM. A compliant tubular device to study the influences of wall strain and fluid shear stress on cells of the vascular wall. J Vasc Surg. 1994; 20: 184–194.
Vanhoutte PM. Endothelial dysfunction and inhibition of converting enzyme. Eur Heart J. 1998; 19 (suppl J): J7–J15.
Mallipattu SK, Haidekker MA, Von Dassow P, Latz MI, Frangos JA. Evidence for shear-induced increase in membrane fluidity in the dinoflagellate Lingulodinium polyedrum. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2002; 188: 409–416.
Haidekker MA, L’Heureux N, Frangos JA. Fluid shear stress increases membrane fluidity in endothelial cells: a study with DCVJ fluorescence. Am J Physiol Heart Circ Physiol. 2000; 278: H1401–H1406.
Butler PJ, Tsou TC, Li JY, Usami S, Chien S. Rate sensitivity of shear-induced changes in the lateral diffusion of endothelial cell membrane lipids: a role for membrane perturbation in shear-induced MAPK activation. FASEB J. 2002; 16: 216–218.
Butler PJ, Norwich G, Weinbaum S, Chien S. Shear stress induces a time- and position-dependent increase in endothelial cell membrane fluidity. Am J Physiol Cell Physiol. 2001; 280: C962–C969.
Wang ZG, Li G, Wu J, Du W, Pu L, Zhang H, Wang D, Sumpio BE. Enhanced patency of venous Dacron grafts by endothelial cell sodding. Ann Vasc Surg. 1993; 7: 429–436.
Palumbo R, Gaetano C, Melillo G, Toschi E, Remuzzi A, Capogrossi MC. Shear stress downregulation of platelet-derived growth factor receptor-? and matrix metalloprotease-2 is associated with inhibition of smooth muscle cell invasion and migration. Circulation. 2000; 102: 225–230.
Kohler TR, Jawien A. Flow affects development of intimal hyperplasia after arterial injury in rats. Arterioscler Thromb. 1992; 12: 963–971.
Delerive P, Martin-Nizard F, Chinetti G, Trottein F, Fruchart JC, Najib J, Duriez P, Staels B. Peroxisome proliferator-activated receptor activators inhibit thrombin-induced endothelin-1 production in human vascular endothelial cells by inhibiting the activator protein-1 signaling pathway. Circ Res. 1999; 85: 394–402.
Grabowski EF, Jaffe EA, Weksler BB. Prostacyclin production by cultured endothelial cell monolayers exposed to step increases in shear stress. J Lab Clin Med. 1985; 105: 36–43.
Grabowski EF, Naus GJ, Weksler BB. Prostacyclin production in vitro by rabbit aortic endothelium: correction for unstirred diffusional layers. Blood. 1985; 1047–1052.
Carosi JA, Eskin SG, McIntire LV. Cyclical strain effects on production of vasoactive materials in cultured endothelial cells. J Cell Physiol. 1992; 151: 29–36.
Inoue H, Taba Y, Miwa Y, Yokota C, Miyagi M, Sasaguri T. Transcriptional and posttranscriptional regulation of cyclooxygenase-2 expression by fluid shear stress in vascular endothelial cells. Arterioscler Thromb Vasc Biol. 2002; 22: 1415–1420.
Sorop O, Spaan JA, Sweeney TE, VanBavel E. Effect of steady versus oscillating flow on porcine coronary arterioles: involvement of NO and superoxide anion. Circ Res. 2003; 92: 1344–1351.
Florian JA, Kosky JR, Ainslie K, Pang Z, Dull RO, Tarbell JM. Heparan sulfate proteoglycan is a mechanosensor on endothelial cells. Circ Res. 2003; 93: e136–e142.
Mochizuki S, Vink H, Hiramatsu O, Kajita T, Shigeto F, Spaan JA, Kajiya F. Role of hyaluronic acid glycosaminoglycans in shear-induced endothelium-derived nitric oxide release. Am J Physiol Heart Circ Physiol. 2003; 285: H722–H726.
Sorop O, Spaan JA, VanBavel E. Pulsation-induced dilation of subendocardial and subepicardial arterioles: effect on vasodilator sensitivity. AmJ Physiol Heart Circ Physiol. 2002; 282: H311–H319.
Fung YC. Biomechanics. 2nd ed. New York, NY: Springer-Verlag; 1993: 300–306.(Michael B. Dancu; Daniell)
ABSTRACT
Objective— Endothelium-derived vasoactive agents NO, endothelin-1 (ET-1), and prostacyclin (PGI2) not only regulate vascular tone but also influence atherogenic processes, including smooth muscle migration and proliferation, as well as monocyte and platelet adhesion. Complex hemodynamics characterized by the temporal phase angle between mechanical factors circumferential strain and wall shear stress (stress phase angle ) have been implicated in regions prone to pathologic development, such as atherosclerosis and intimal hyperplasia, in coronary and peripheral arteries where the mechanical forces are highly asynchronous (SPA=–180°). We determined the gene expression of endothelial NO synthase (eNOS), ET-1, and cyclooxygenase-2 (COX-2) affected by asynchronous hemodynamics (SPA=–180°) relative to normal hemodynamics (SPA=0°) in bovine aortic endothelial cells.
Methods and Results— Quantitative competitive RT-PCR analysis showed that eNOS production (at 5 and 12 hours) and COX-2 production (at 5 hours) were reduced at the gene expression level by asynchronous hemodynamics (SPA=–180°) compared with synchronous hemodynamics (SPA=0°), whereas ET-1 exhibited an opposite trend (at 5 and 12 hours). NO, ET-1, and PGI2 secretion followed their respective gene expression profiles after 5 and 12 hours.
Conclusion— Together, these data suggest that highly asynchronous mechanical force patterns (SPA=–180°) can elicit proatherogenic vasoactive responses in endothelial cells at the gene expression level, indicating a novel mechanism that induces cardiovascular pathology.
Asynchronous hemodynamic circumferential strain and wall shear stress occur in pathologic regions. Vasoactive gene expression of eNOS, ET-1, and COX-2 in endothelial cells exhibited a pathologic profile during asynchronous (SPA=–180°) hemodynamics. Asynchronous mechanical force patterns (SPA=–180°) can elicit proatherogenic responses in endothelial cells, indicating a novel mechanism that induces cardiovascular pathology.
Key Words: hemodynamics ? shear stress and strain ? coronary arteries ? eNOS ? ET-1 ? COX-2
Introduction
Endothelial dysfunction is a primary event in development of atherosclerosis, vasospasm, and thrombosis.1,2 Endothelium-derived vasoactive agents such as NO, endothelin-1 (ET-1), and prostacyclin (PGI2) not only regulate vascular tone but also influence atherogenic processes, including smooth muscle migration and proliferation, as well as monocyte and platelet adhesion.3 Endothelial cells (ECs) are a primary target for injuries such as hyperlipidemia, diabetes mellitus, and hypertension2 but also serve as sensors and transducers of the most notable hemodynamic forces: wall shear stress (WSS) and circumferential stress that is induced by circumferential strain (CS ). WSS and CS are widely believed to be important hemodynamic mediators of vascular regulation, atherosclerosis, and remodeling.4–7
Most previous studies of the role of vascular mechanical forces in atherogenesis have emphasized fluid shear stress (WSS) by itself (ie, no pressure or strain).4–7 A few recent studies have noted the importance of complex hemodynamics that include simultaneous flow, pressure, and diameter variation (stretch) in modulating cardiovascular function.6–9 Blood vessel ECs in vivo are subjected to simultaneous pulsatile CS and WSS that act approximately in perpendicular directions. The temporal phase angle between pressure and flow (impedance phase angle) generated by global wave reflection in the circulation and the local inertial effects of blood flow cause time lags to occur between CS and WSS. The temporal phase angle between CS and WSS characterizes the complex, time-varying mechanical force pattern on the EC monolayer. We will refer to the temporal phase angle between CS and WSS as the stress phase angle (SPA)6,7 (Figure 1). A typical transverse arterial cross-section is shown with major vascular cells and hemodynamic features (Figure 1, left). Q in the axial direction induces WSS on the EC-lined vascular wall. P imposes a normal (radial) stress on the wall from the lumen. Changes in pressure distend (strain) the wall and EC monolayer, resulting in uniform CS attributable to the axisymmetric tubular geometry. ECs experience pulsatile WSS and CS simultaneously; however, their magnitude and temporal phase may vary throughout the vasculature and in disease conditions. SPA is used to characterize the dynamic mechanical stimuli experienced by the EC monolayer. Regions of the circulation prone to pathologic development, such as atherosclerosis and intimal hyperplasia, include the outer wall of the abdominal aortic bifurcation,8 the coronary arteries,9 and the distal anastomosis of undersized end-to-end vascular grafts that are characterized by large negative SPA values relative to regions that are typically spared (veins, small arteries, high shear regions in large arteries). It is interesting to note that the most disease-prone arteries of circulation, the coronary arteries, experience complex hemodynamics that allow for the most extreme SPA in the cardiovascular system (SPA close to –180°).9 Hypertension has also been shown to further asynchronize pulsatile WSS and CS, thus creating a more negative SPA in both pathologic and normopathic regions.9 Atheroprotective vasoactive gene expression in ECs has been shown to be induced during steady shear stress (SS; >10 dyne/cm2)10–15 and simultaneous synchronous pulsatile shear stress and CS (SPA=0°).16–18
Figure 1. Representation of the SPA on vascular cells attributable to mechanical effects from hemodynamic features blood flow and pressure (P) with physiological significance. The SPA is used to characterize the dynamic mechanical stimuli experienced by the EC monolayer.
In the present study, we investigated the effects of asynchronous hemodynamics (SPA=–180°) on vasoactive gene expression and corresponding secretion for endothelial NO synthase (eNOS), ET-1, and cyclooxygenase-2 (COX-2). We report that asynchronous hemodynamic conditions (SPA=–180°) induce an atherogenic vasoactive gene expression profile compared with normopathic hemodynamics (SPA=0°). COX-2 and eNOS mRNA levels and corresponding metabolite secretions of PGI2 and NO are reduced by asynchronous mechanical forces (SPA=–180°), whereas ET-1 shows an opposite trend. Our results demonstrate a novel mechanism by which simultaneous mechanical forces, when imposed in a unique pulsatile temporal pattern, can induce a proatherogenic biomolecular response in ECs. This is the first study to demonstrate the influence of SPA on gene expression. The underlying biomolecular mechanisms that mediate these new phenomena will be the subject of future studies.
Materials and Methods
Cell Culture and Hemodynamic Conditions
Primary bovine aortic ECs (BAECs) were isolated from fresh aortas as described previously6 (information available online at http://atvb.ahajournals.org). Cultured tubes were grown in an incubator for 3 to 4 days until confluence before experiment. Cell attachment was verified via visualization through the silicone tubing and cell counting before RNA extraction before and after the experiment. Experimental hemodynamic conditions did not appear to influence cell attachment and cell number. The experimental design focused on isolating the effect of SPA from other hemodynamic factors. A novel simulator was used to impose asynchronous (SPA=–180°) and synchronous (SPA=0°) hemodynamic conditions as described previously.7 Briefly, the sinusoidal WSS and CS waveforms were the same in both hemodynamic conditions; however, the SPA was different. Concurrent controls allowed us to examine the nonoscillatory mechanical effects present in the pulsatile cases such as mean pressure, mean CS, and mean WSS. The purpose of these controls was to demonstrate that cells in this preparation have biomolecular responses to mechanical forces that have been observed in other studies. Relevant hemodynamic parameters are (Table I, available online at http://atvb.ahajournals.org): WSS=10±10 dyne/cm2; CS=(Dmax–Dmin)/Dmean=4±4% ); P=70±20 mm Hg, all at frequency of f=1 Hz, and an unsteadiness parameter =4.3 (=D/2(2f/)1/2; where is the kinematic viscosity). The pressure, diameter, and flow waveforms for the SPA=0° and the SPA=–180° case are shown in supplemental Figure IA and IB (available online at http://atvb.ahajournals.org). The total media volume=175 mL, maximum Reynolds number (U/ =460, pH=7.2±0.05, all at 37°C for 5 and 12 hours. Each experiment consisted of a total of 10 individual tubes with separate media lines: 4 incubator controls, static control (SC), and pressurized control (PC), 2 SS (with mean pressure and mean stretch) controls, and 6 sinusoidal WSS cases (SPA=0° or –180°; Table I). These were repeated 3x.
RNA Extraction and Quantitative RT-PCR Analysis
Total RNA was isolated from cells using Trizol (Life Technologies) following the protocol of the manufacturer. Total RNA extracts were frozen at –80°C until analyses were performed. Quantitative competitive RT-PCR used recombinant RNA as an internal standard to negate tube-to-tube variability in amplification efficiency.19–22 Primers for eNOS, ET-1, COX-2, and hypoxanthine-guanine phosphoribosyltransferase (HPRT) are described at http://atvb.ahajournals.org. Results are expressed in relative terms (number of target molecules per number of HPRT molecules). Figure II (available online at http://atvb.ahajournals.org) depicts the invariance of HPRT gene expression during asynchronous and synchronous hemodynamics.
NO, PGI2, and ET-1 Media Secretions
Media was collected and stored at –80°C before media assays. NO concentration was quantified using a fluorometric assay.23 ET-1 and PGI2 (actually 6-keto PGF1, major stable PGI2 byproduct) concentrations were determined via enzyme immunoassay kits (Cayman Chemicals) following the protocol of the manufacturer. Results were normalized to cell number in all experiments.
Statistics Analysis
The data are presented as mean±SEM. Then a 2-factor ANOVA model was used with the Tukey method on a 95% CI for all experiments. The inset table in Results figure indicates pairwise significant differences: P<0.05 by the shaded boxes and asterisk for 5 versus 12 hours. Note that –180° and 0° refer to SPA.
Results
Asynchronous WSS and CS (SPA=–180°) Reduces eNOS Production at the Gene Expression Level
SS induced more than a 2-fold increase in eNOS mRNA levels from 5 to 12 hour, indicating that the BAECs were responsive to SS (Figure 2A). SS also showed a significant increase in eNOS mRNA levels compared with SC at 12 hours (Figure 2A). SS appeared to increase eNOS mRNA levels compared with SPA=–180° at 12 hours, but this effect did not reach statistical significance (Figure 2A). SS with mean pressure and stretch (SS) exerted an opposite trend compared with constant CS with mean pressure and stretch (PC) by significantly inducing eNOS mRNA relative to SC, whereas PC reduced mRNA levels at 12 hours (Figure 2A). Interestingly, pulsatile asynchronous hemodynamics (SPA=–180°) significantly reduced (2-fold) eNOS mRNA levels compared with pulsatile normopathic hemodynamics (SPA=0°) at both 5 and 12 hours (Figure 2A).
Figure 2. A, Asynchronous hemodynamics (SPA=–180°) reduces eNOS production at the gene expression level. BAECs were exposed to hemodynamic conditions for 5 and 12 hours with concurrent controls. eNOS mRNA was quantitated with competitive RT-PCR normalized to HPRT mRNA. Results are expressed in relative terms (number of eNOS molecules per number of HPRT molecules; n=5). B, Asynchronous hemodynamics (SPA=–180°) reduces NO secretion compared with synchronous hemodynamics (SPA=0°). NO concentration was evaluated by fluorogenic assay and normalized to cell number. Results are expressed in μmol/106 cells (n=5).
Asynchronous WSS and CS (SPA=–180°) Reduce NO Secretion
SS induced significant NO production at 5 and 12 hours compared with SC, indicating that the BAECs were responsive to SS (Figure 2B). PC did not significantly induce NO secretion at either 5 or 12 hours (Figure 2B). Asynchronous hemodynamics (SPA=–180°) significantly reduced (2-fold) NO secretion compared with synchronous hemodynamics (SPA=0°) at 5 hours (Figure 2B). At 12 hours, SPA=–180° reduced NO secretion compared with SPA=0°, but the effect was not significant (P=0.20). SS and SPA=–180° showed similar NO secretion levels at 5 and 12 hours (Figure 2B). These results indicate that asynchronous hemodynamics (SPA=–180°) reduce NO secretion compared with normal hemodynamics (SPA=0°).
Asynchronous WSS and CS (SPA=–180°) Induces ET-1 Gene Expression
SS reduces ET-1 mRNA levels relative to SCs in ECs at 5 and 12 hours, although not statistically significant (Figure 3A). PC also showed a reduction in ET-1 mRNA levels compared with SC at 5 and 12 hours that was not significant (Figure 3A). Pulsatile asynchronous hemodynamics (SPA=–180°) very significantly augmented ET-1 mRNA levels (>3-fold) compared with synchronous hemodynamics (SPA=0°) at 12 hours (Figure 3A). In fact, the SPA=–180° case significantly induced ET-1 mRNA levels relative to all other conditions at 12 hours (Figure 3A).
Figure 3. A, Asynchronous hemodynamics (SPA=–180°) reduces ET-1 production at the gene expression level. BAECs were exposed to hemodynamic conditions for 5 and 12 hours with concurrent controls. ET-1 mRNA was quantitated with competitive RT-PCR normalized to HPRT mRNA. Results are expressed in relative terms (number of ET-1 molecules per number of HPRT molecules; n=5). B, Asynchronous hemodynamics (SPA=–180°) induces ET-1 secretion compared with synchronous hemodynamics (SPA=0°). ET-1 concentration was evaluated by immunoassay and normalized to cell number. Results are expressed in μmol/106 cells (n=5).
Asynchronous Hemodynamics (SPA=–180°) Induce ET-1 Secretion
SS significantly reduced ET-1 secretion levels compared with SC at 5 and 12 hours (Figure 3B). PC and SC levels of ET-1 secretion were not significantly different, but both static cases, SC and PC, displayed significantly higher ET-1 secretion levels compared with dynamic conditions at 5 and 12 hours (Figure 3B). Asynchronous hemodynamics (SPA=–180°) significantly augmented ET-1 secretion (2-fold) compared with synchronous hemodynamics (SPA=0°) at 12 hours (Figure 3B).
Asynchronous WSS and CS (SPA=–180°) Reduces COX-2 Production at the Gene Expression Level at 5 hours, but Increases It at 12 hours
SS did not significantly induce COX-2 mRNA levels compared with SC at 5 and 12 hours (Figure 4A). COX-2 mRNA expression for PC was not significant compared with SS or SC at 5 or 12 hours as well (Figure 4A). However, the SPA=–180° case significantly reduced COX-2 mRNA levels (>4-fold) compared with all cases at 5 hours (Figure 4A). Unexpectedly, synchronous (SPA=0°) and asynchronous (SPA=–180°) hemodynamics showed an opposite trend at 12 hours compared with 5 hours: the SPA=–180° case was increased (4-fold) approaching basal COX-2 mRNA expression levels, whereas the SPA=0° case was reduced (6-fold) falling below basal COX-2 mRNA levels (Figure 4A). These results indicate that SPA has a dramatic, but opposite, effect on COX-2 production at the gene expression level at 5 and 12 hours.
Figure 4. A, Asynchronous hemodynamics (SPA=–180°) reduces COX-2 production at the gene expression level. BAECs were exposed to hemodynamic conditions for 5 and 12 hours with concurrent controls. COX-2 mRNA was quantitated with competitive RT-PCR normalized to HPRT mRNA. Results are expressed in relative terms (number of COX-2 molecules per number of HPRT molecules; n=5). B, Asynchronous hemodynamics (SPA=–180°) induces PGI2 secretion compared with synchronous hemodynamics (SPA=0°). PGI2 concentration was evaluated by immunoassay and normalized to cell number. Results are expressed in μmol/106 cells (n=5).
Asynchronous Hemodynamics (SPA=–180°) Reduce PGI2 Secretion Compared With Synchronous Hemodynamics (SPA=0°) at 5 Hours but Increases It at 12 Hours
SS did induce elevated PGI2 levels that were significant compared with SC at 5 and 12 hours (Figure 4B). PC reduced PGI2 secretion compared with SC at 5 and 12 hours, but the differences were insignificant (Figure 4B). Asynchronous hemodynamics (SPA=–180°) reduced PGI2 levels significantly (>3-fold) compared with synchronous (SPA=0°) at 5 hours (Figure 4B). As with the gene expression results (Figure 4B), unexpectedly, synchronous SPA=0° and asynchronous SPA=–180° showed an opposite trend at 12 hours compared with 5 hours.
Discussion
Most previous studies of the role of vascular mechanical forces in atherogenesis have emphasized fluid shear stress by itself (ie, no pressure or strain),4–7 with low mean and reversing oscillatory shear stress generally being associated with the location of the disease. Yet the most disease prone arteries, the coronary arteries, do not have a particularly low mean shear stress, and reversal is not that prominent.4–7 However, the SPA is most negative in the coronary arteries as well as the outer wall of bifurcations and other sites of atherogenesis. Hypertension, a major risk factor for several cardiovascular diseases, also induces a more negative SPA yet is not known to affect shear stress levels.4–7
In the present study, we showed that the occurrence of concomitant WSS and CS elicits significantly different vasoactive gene expression and biomolecule secretion compared with simple SS or stretch in BAECs. Most significantly, we demonstrated that asynchronous hemodynamics (SPA= –180°) evoke proatherogenic vasoactive gene expression profiles relative to synchronous hemodynamics (SPA=0°) for eNOS (at 5 and 12 hours), ET-1 (at 5 and 12 hours), and COX-2 (at 5 hours). Finally, we observed that the vasoactive biomolecule secretion associated with these genes (NO, ET-1, and PGI2) also exhibit proatherogenic responses for the SPA=–180° condition.
Our results in Figure 2A and 2B are consistent with previous reports showing that SS and pulsatile shear stress (without stretch) increase eNOS mRNA levels and NO secretion in BAECs and that pulsatile shear exerts a greater influence than SS.10,11,16,17,24,25 In previous experiments that examined the effects of shear stress alone, eNOS mRNA was upregulated in SS during time periods of 12 hours.10,11 In an experiment in an elastic tube with oscillatory WSS of 6±6 dynes/cm2 with ±4% CS at 100±30 mm Hg and SPA undefined but probably near 0°, eNOS mRNA was upregulated at 24 hours compared with steady WSS and SC.16,17 Although our NO secretion levels followed eNOS mRNA levels for SS and SPA=0°, eNOS gene expression levels were more responsive than NO metabolite levels at 5 and 12 hours (Figures 2 and 3). It is interesting to note that SS alone induced eNOS mRNA levels, whereas stretch alone reduced eNOS mRNA levels (Figure 2A). These data indicate that these separate (constant) mechanical forces have opposing regulatory roles in gene expression that are altered under physiological pulsatile conditions. Most importantly, pulsatile asynchronous hemodynamics (SPA=–180°) reduced eNOS mRNA levels compared with pulsatile synchronous hemodynamics (SPA=0°) at 5 and 12 hours (Figure 2A). NO secretion showed similar trends (Figure 2B). The eNOS gene expression trends are consistent with observations in vivo in regions expected to have large negative SPA, such as the opposite wall of a flow divider, that have decreased eNOS mRNA levels compared with more positive SPA regions, such as along the flow divider.24 The glycocalyx of the EC may play a role in the signal transduction of the SPA because it has been shown to be shear stress sensor for NO.40,41 Because reduction of eNOS expression or NO availability is an event that can contribute to atherogenesis,2,26,39 asynchronous hemodynamics (SPA=–180°) in vitro, in concert with other risk factors, might be used to simulate the pathogenesis of atherosclerosis by inducing proatherogenic gene and biomolecule responses for studies to mitigate or reverse pathology.
Our results demonstrate that asynchronous hemodynamics (SPA=–180°) induce the greatest ET-1 gene expression level compared with all other hemodynamic cases at 12 hours (Figure 3A). ET-1 secretion was elevated at SPA=–180° compared with SPA=0° as well, but that was not the condition with the highest ET-1 secretion (Figure 3B). In an experiment in an elastic tube with ±4% CS, 5±2 dynes/cm2, and undefined SPA (likely near 0°), Tsukurov et al18 showed a similar decrease in ET-1 mRNA expression at 48 hours. In the present study, SS insignificantly reduced ET-1 mRNA levels compared with SC (Figure 3A). This is not inconsistent with other studies that have shown ET-1 mRNA expression to increase within the first hour of exposure to 20 dynes/cm2 SS then transiently decrease.12,13 SS did significantly reduce ET-1 secretion compared SC (Figure 3B), perhaps because of alterations in membrane fluidity.27–30 Others have shown that cyclic strain alone increased ET-1 mRNA levels <±20% CS at 1 Hz out to 6 hours of exposure, but longer time points showed no further increase in mRNA expression.31 Although SS and CS (or PC) alone exert insignificant effects on ET-1 mRNA levels, combined pulsatile SS and CS with SPA=–180° exhibits proatherogenic ET-1 gene responses (Figure 3A). Because ET-1 is involved in monocyte recruitment and SMC proliferation and migration in atherosclerosis,1,4,5,32,33 asynchronous hemodynamics can be used to induce atherogenic ET-1 gene expression and secretion for studies that attempt to mitigate proatherogenic ET-1 expression. Delerive et al34 proposed that ET-1 gene expression is downregulated by peroxisome proliferator–activated receptors, partly by interfering with activated protein-1 activity and potentially by signal transducer activator of transcription and nuclear factor B (NF-B) signaling pathways. Additional studies are necessary to delineate in detail the molecular mechanisms involved in the positive regulation of ET-1 gene expression in complex pathologic hemodynamics.
SS with mean pressure and strain did not significantly induce COX-2 mRNA levels compared with SC at 5 and 12 hours (Figure 4A). This is not inconsistent with Okahara et al,15 who showed a peak increase from basal COX-2 mRNA levels at 6 hours then a drop to basal levels at 12 hours at an SS of 24 dynes/cm2. However, SS did significantly induce PGI2 secretion compared with SC at 5 and 12 hours (Figure 4B). Previous experiments have shown that elevated PGI2 production induced by shear stress14,35,36 is mediated by increased arachidonic acid release and a combination of increased mRNA expression of cyclooxygenases and PGI2 synthase.15 Because arachidonic acid is membrane bound and shear stress alters membrane fluidity,27–30 SS may exert more immediate effects on PGI2 release than on gene expression. Similar to SS, cyclic CS at physiological levels of ±10% and 1 Hz showed an increase in PGI2 secretion by 2.5-fold compared with SCs.37 Thus, SS and CS alone upregulate PGI2 secretion levels, yet combined pulsatile SS and CS with asynchronous SPA=–180° induces proatherogenic PGI2 inhibition.
Asynchronous hemodynamics (SPA=–180°) significantly reduced COX-2 mRNA levels compared with all other cases at 5 hours (Figure 4A). PGI2 secretion was also significantly lower for SPA=–180° versus SPA=0° at this time point (Figure 4B). It was unexpected that synchronous (SPA=0°) and asynchronous (SPA=–180°) hemodynamics would show a significant opposite trend in expression at 12 hours compared with 5 hours (Figure 4A). However, similar alterations occurred for PGI2 secretion at 12 hours compared with 5 hours (Figure 4B). It may be that COX-2 mRNA expression at different SPA exhibits a classic type-I gene response, an early transient increase followed by decline, as observed in previous SS response data.15 Asynchronous hemodynamics (SPA=–180°) may delay the early transient increase in COX-2 mRNA levels until 12 hours, whereas at SPA=0°, there may be a rapid early transient increase in COX-2 mRNA levels at 5 hours (Figure 4A). Further studies are required to test this hypothesis. COX-2 regulation occurs via many mechanisms. COX-2 transcription has been shown to involve 3 cis-acting elements in the human COX-2 promoter region, namely an NF-B binding site, an NF–interleukin-6–binding site, and a cAMP-responsive element (CRE), where the shear stress response of the COX-2 promoter was dependent on CRE.38 Post-transcriptional regulation under SS has been shown in the 3'-untranslated region of the COX-2 gene.38
A limitation of our study was the focus on the endothelium and lack of an intact vessel and consequent myogenic response. We could not determine the dilation response or true vasodilator effect to asynchronous hemodynamics, although endothelium-dependent and -independent responses play a normal role in epicardial and endocardial vessels.42 Furthermore, there are many factors that the SPA may affect; however, we focused only on several genes, time points, cell types, and many other details that will be the subject of future experiments.
The mechanotransduction mechanisms that mediate the effects of SPA on ECs are not known. However, it is clear that a nonlinear interaction between stretch and shear is involved because the responses are not simply the summation of the stretch response and the shear response. One possibility is that the strain energy of the plasma membrane or the actin cortical web (that is, a nonlinear function of CS and axial strain ) mediates mechanotransduction.43 Another possibility is that the nonlinearity is associated with a mechanochemical pathway that requires a threshold level of stimulation. Then, for example, when stretch and shear are in-phase, the threshold may be exceeded during a time interval around the maximum stretch and shear, whereas the maximum stretch or shear by itself would not provide stimulation exceeding the threshold. These and other possible mechanisms for the SPA effect remain to be explored in future studies.
In conclusion, the results from the present study demonstrate that physiological asynchronous hemodynamics (SPA=–180°) have a dramatic influence on endothelial function. Asynchronous SPA induces an atherogenic profile of eNOS, ET-1, and COX-2 gene expression and release of NO, ET-1, and PGI2. Together, these data suggest that local hemodynamic conditions characterized by the SPA can modulate local vasoautoregulatory and atherogenic responses. To further test the relative importance of low mean and reversing oscillatory shear stress versus asynchronous WSS and CS, future studies should compare gene expression and secretion profiles for low mean and oscillatory shear stress with synchronous and asynchronous hemodynamics.
Acknowledgments
This work was supported by National Institutes of Health National Heart, Lung, and Blood Institute grant ROI-HL35549. We thank the Center for Carcinogenesis and Molecular Toxicology at Penn State University.
References
Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993; 362: 801–809.
Shimokawa H. Primary endothelial dysfunction: atherosclerosis. J Mol Cell Cardiol. 1999; 31: 23–37.
Vanhoutte PM. Endothelial dysfunction and atherosclerosis. Eur Heart J. 1997; 18 (suppl E): E19–E29.
Davies PF, Shi C, Depaola N, Helmke BP, Polacek DC. Hemodynamics and the focal origin of atherosclerosis: a spatial approach to endothelial structure, gene expression, and function. Ann N Y Acad Sci. 2001; 947: 7–16.
Gimbrone MA Jr. Vascular endothelium, hemodynamic forces, and atherogenesis. Am J Pathol. 1999; 155: 1–5.
Qiu Y, Tarbell JM. Interaction between wall shear stress and circumferential strain affects endothelial cell biochemical production. J Vasc Res. 2000; 37: 147–157.
Dancu M, Tarbell J. Large negative stress phase angle (SPA) atteneutates nitric oxide production in bovine aortic endothelial cells. J Biomech Eng. In press.
Lee CS, Tarbell JM. Wall shear rate distribution in an abdominal aortic bifurcation model: effects of vessel compliance and phase angle between pressure and flow waveforms. J Biomech Eng. 1997; 119: 333–342.
Qiu Y, Tarbell JM. Numerical simulation of pulsatile flow in a compliant curved tube model of a coronary artery. J Biomech Eng. 2000; 122: 77–85.
Ranjan V, Xiao Z, Diamond SL. Constitutive NOS expression in cultured endothelial cells is elevated by fluid shear stress. Am J Physiol. 1995; 269: H550–H555.
Uematsu M, Ohara Y, Navas JP, Nishida K, Murphy TJ, Alexander RW, Nerem RM, Harrison DG. Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress. Am J Physiol. 1995; 269: C1371–C1378.
Malek AM, Izumo S. Mechanism of endothelial cell shape change and cytoskeletal remodeling in response to fluid shear stress. J Cell Sci. 1996; 109: 713–726.
Malek AM, Izumo S. Control of endothelial cell gene expression by flow. J Biomech. 1995; 28: 1515–1528.
Frangos JA, Eskin SG, McIntire LV, Ives CL. Flow effects on prostacyclin by cultured human endothelial cells. Science. 1985; 227: 1477–1479.
Okahara K, Sun B, Kambayashi J. Upregulation of prostacyclin synthesis-related gene expression by shear stress in vascular endothelial cells. Arterioscler Thromb Vasc Biol. 1998; 18: 1922–1926.
Ziegler T, Bouzourene K, Harrison VJ, Brunner HR, Hayoz D. Influence of oscillatory and unidirectional flow environments on the expression of endothelin and nitric oxide synthase in cultured endothelial cells. Arterioscler Thromb Vasc Biol. 1998; 18: 686–692.
Ziegler T, Silacci P, Harrison VJ, Hayoz D. Nitric oxide synthase expression in endothelial cells exposed to mechanical forces. Hypertension. 1998; 32: 351–355.
Tsukurov OI, Kwolek CJ, L’Italien GJ, Benbrahim A, Milinazzo BB, Conroy NE, Gertler JP, Orkin RW, Abbott WM. The response of adult human saphenous vein endothelial cells to combined pressurized pulsatile flow and cyclic strain, in vitro. Ann Vasc Surg. 2000; 14: 260–267.
Vanden Heuvel JP, Tyson FL, Bell DA. Construction of recombinant RNA templates for use as internal standards in quantitative RT-PCR. BioTechniques. 1993; 14: 395–398.
Gilliland G, Perrin S, Blanchard K, Bunn HF. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A. 1990; 87: 2725–2729.
Vanden Heuvel JP, Clark GC, Kohn MC, Tritscher AM, Greenlee WF, Lucier GW, Bell DA. Dioxin-responsive genes: examination of dose-response relationships using quantitative reverse transcriptase-polymerase chain reaction. Cancer Res. 1994; 54: 62–68.
Zachar V, Thomas RA, Goustin AS. Nucleic Acids Res. 1993; 21: 2017–2018.
Misko TP, Schilling RJ, Salvemini D, Moore WM, Currie MG. A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem. 1993; 214: 11–16.
Huige L, Forstermann TW. Physiological mechanisms regulating the expression of endothelial-type NO synthase. Nitric Oxide. 2002; 7: 132–147.
Benbrahim A, L’Italien GJ, Milinazzo BB, Warnock DF, Dhara S, Gertler JP, Orkin RW, Abbott WM. A compliant tubular device to study the influences of wall strain and fluid shear stress on cells of the vascular wall. J Vasc Surg. 1994; 20: 184–194.
Vanhoutte PM. Endothelial dysfunction and inhibition of converting enzyme. Eur Heart J. 1998; 19 (suppl J): J7–J15.
Mallipattu SK, Haidekker MA, Von Dassow P, Latz MI, Frangos JA. Evidence for shear-induced increase in membrane fluidity in the dinoflagellate Lingulodinium polyedrum. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2002; 188: 409–416.
Haidekker MA, L’Heureux N, Frangos JA. Fluid shear stress increases membrane fluidity in endothelial cells: a study with DCVJ fluorescence. Am J Physiol Heart Circ Physiol. 2000; 278: H1401–H1406.
Butler PJ, Tsou TC, Li JY, Usami S, Chien S. Rate sensitivity of shear-induced changes in the lateral diffusion of endothelial cell membrane lipids: a role for membrane perturbation in shear-induced MAPK activation. FASEB J. 2002; 16: 216–218.
Butler PJ, Norwich G, Weinbaum S, Chien S. Shear stress induces a time- and position-dependent increase in endothelial cell membrane fluidity. Am J Physiol Cell Physiol. 2001; 280: C962–C969.
Wang ZG, Li G, Wu J, Du W, Pu L, Zhang H, Wang D, Sumpio BE. Enhanced patency of venous Dacron grafts by endothelial cell sodding. Ann Vasc Surg. 1993; 7: 429–436.
Palumbo R, Gaetano C, Melillo G, Toschi E, Remuzzi A, Capogrossi MC. Shear stress downregulation of platelet-derived growth factor receptor-? and matrix metalloprotease-2 is associated with inhibition of smooth muscle cell invasion and migration. Circulation. 2000; 102: 225–230.
Kohler TR, Jawien A. Flow affects development of intimal hyperplasia after arterial injury in rats. Arterioscler Thromb. 1992; 12: 963–971.
Delerive P, Martin-Nizard F, Chinetti G, Trottein F, Fruchart JC, Najib J, Duriez P, Staels B. Peroxisome proliferator-activated receptor activators inhibit thrombin-induced endothelin-1 production in human vascular endothelial cells by inhibiting the activator protein-1 signaling pathway. Circ Res. 1999; 85: 394–402.
Grabowski EF, Jaffe EA, Weksler BB. Prostacyclin production by cultured endothelial cell monolayers exposed to step increases in shear stress. J Lab Clin Med. 1985; 105: 36–43.
Grabowski EF, Naus GJ, Weksler BB. Prostacyclin production in vitro by rabbit aortic endothelium: correction for unstirred diffusional layers. Blood. 1985; 1047–1052.
Carosi JA, Eskin SG, McIntire LV. Cyclical strain effects on production of vasoactive materials in cultured endothelial cells. J Cell Physiol. 1992; 151: 29–36.
Inoue H, Taba Y, Miwa Y, Yokota C, Miyagi M, Sasaguri T. Transcriptional and posttranscriptional regulation of cyclooxygenase-2 expression by fluid shear stress in vascular endothelial cells. Arterioscler Thromb Vasc Biol. 2002; 22: 1415–1420.
Sorop O, Spaan JA, Sweeney TE, VanBavel E. Effect of steady versus oscillating flow on porcine coronary arterioles: involvement of NO and superoxide anion. Circ Res. 2003; 92: 1344–1351.
Florian JA, Kosky JR, Ainslie K, Pang Z, Dull RO, Tarbell JM. Heparan sulfate proteoglycan is a mechanosensor on endothelial cells. Circ Res. 2003; 93: e136–e142.
Mochizuki S, Vink H, Hiramatsu O, Kajita T, Shigeto F, Spaan JA, Kajiya F. Role of hyaluronic acid glycosaminoglycans in shear-induced endothelium-derived nitric oxide release. Am J Physiol Heart Circ Physiol. 2003; 285: H722–H726.
Sorop O, Spaan JA, VanBavel E. Pulsation-induced dilation of subendocardial and subepicardial arterioles: effect on vasodilator sensitivity. AmJ Physiol Heart Circ Physiol. 2002; 282: H311–H319.
Fung YC. Biomechanics. 2nd ed. New York, NY: Springer-Verlag; 1993: 300–306.(Michael B. Dancu; Daniell)