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(Circulation Research. 1997;81:320-327.)
© 1997 American Heart Association, Inc.

Articles

Platelet-Derived Growth Factor Ligand and Receptor Expression in Response to Altered Blood Flow In Vivo

J. Sheppard Mondy, Volkhard Lindner, Jody K. Miyashiro, Bradford C. Berk, Richard H. Dean, , Randolph L. Geary

From the Department of Surgery (J.S.M., R.H.D., R.L.G.), The Bowman Gray School of Medicine, Winston-Salem, NC; the Maine Medical Center Research Institute (V.L.), Portland; and the University of Washington School of Medicine (J.K.M., B.C.B.), Seattle.

Correspondence to Randolph L. Geary, MD, Division of Surgical Sciences, The Bowman Gray School of Medicine, Wake Forest University, Medical Center Blvd, Winston-Salem, NC 27157. E-mail rgeary{at}bgsm.edu

	Abstract

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Abstract Blood flow and the tractive force shear stress are important determinants of artery caliber, and reduced shear predisposes arteries to intimal thickening and atherosclerosis. The molecular basis for shear-induced changes in artery wall structure is poorly defined. A number of factors associated with normal and pathological artery wall remodeling are induced by shear stress in endothelial cell cultures. These include platelet-derived growth factor (PDGF), a potent mitogen, chemoattractant, and vasoconstrictor. To determine whether similar changes occur in vivo, we examined the effects of reduced blood flow on endothelial cell PDGF expression and proliferation in the rat carotid artery. Branches of the right internal and external carotid arteries were ligated, reducing common carotid artery blood flow from 8.0±0.6 to 0.5±0.1 mL/min while increasing flow in the left carotid from 7.1±0.6 to 10.8±0.7 mL/min. Shear stress following the procedure was 1.4±0.2 and 33.4±1.1 dyne/cm² in carotids with reduced blood flow (RF) and increased blood flow (IF), respectively. Arteries were harvested 6, 24, 48, or 72 hours after ligation, perfusion-fixed, and opened longitudinally. Endothelial cell proliferation (bromodeoxyuridine [BrdU] labeling) was assessed en face at 24, 48, and 72 hours; expression of mRNA for PDGF-A and -B chains and PDGF - and ß-receptors (in situ hybridization) was determined at 6, 48, and 72 hours after unilateral flow reduction. RF induced endothelial cell proliferation, which peaked at 48 hours (RF BrdU labeling: 24 hours, 0.4±0.2%; 48 hours, 7.2±2.0%; and 72 hours, 4.1±0.6%; n=5). PDGF-B expression increased in RF compared with IF endothelium within 48 hours and persisted at 72 hours (percent labeling [RF/IFx100]: 6 hours, 76±20%; 48 hours, 395±179%; and 72 hours, 208±44%; n=3). PDGF-A expression was similarly increased in RF endothelium. In contrast, expression of PDGF - and ß-receptors was undetectable in RF and IF endothelium at all times. We conclude that endothelial cell PDGF ligand expression is induced by reduced shear stress in vivo and may play an important role in flow-mediated remodeling and atherogenesis.

Key Words: endothelial cell • shear stress • blood flow • platelet-derived growth factor • proliferation

	Introduction

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Changes in the fluid mechanical force associated with altered blood flow (shear stress) has direct effects on artery wall structure during growth and development¹ and on the progression of atherosclerosis and intimal hyperplasia.^{2 3 4} At sites within the vascular tree where the endothelium is constantly exposed to low shear stress, such as in the carotid bulb and at vessel branch ostia, intimal thickening and atherosclerosis develop prematurely.^{5 6 7} This predilection for intimal pathology has been attributed to dysfunctional endothelial cell gene expression due to chronic perturbation by reduced or disturbed shear stress. A link between low shear and atherogenesis may be the overexpression of certain growth factors (or underexpression of inhibitors) by endothelial cells at these sites.⁸

Recent in vitro experiments have begun to demonstrate a wide variety of genes induced or inhibited by the application of shear stress.⁹ When confluent endothelial cells are subjected to steady shear, transcription of a number of genes associated with endothelial cell activation increases. These include vasoactive molecules,^{10 11} adhesion molecules,¹² growth factors,^{13 14 15 16} and intracellular signaling molecules.^{17 18} Whether similar patterns of gene expression occur in vivo in response to altered shear stress is unclear. The change from static conditions to steady shear in culture may invoke a very different response than low oscillating shear stress in vivo.

PDGF has been extensively characterized in models of atherosclerosis and arterial injury. It is a potent mitogen for vascular SMCs in vitro and promotes SMC migration, proliferation, and intimal hyperplasia in vivo.^{19 20 21} PDGF ligands consist of homodimers or heterodimers of A and B chains that selectively bind and dimerize - or ß-receptor subunits to induce intracellular signaling.²² PDGF-A and -B chains can both be induced in cultured endothelial cells by shear stress,^{14 16 23 24} and Resnick and colleagues^{15 25 26} have recently identified unique promoter sequences in the PDGF-A and -B genes that can induce transcription in response to shear. The effects of altered shear stress on endothelial cell PDGF expression in normal arteries has not been defined.

We herein report an en face approach for exploring changes in endothelial cell gene expression in response to altered blood flow and shear stress in vivo. Partial ligation of rat common carotid artery outflow acutely reduced blood flow in the ipsilateral common carotid while increasing flow in the contralateral carotid artery. The endothelium was then studied using a method recently adapted by Lindner and colleagues^{27 28} for studies of arterial injury. Monolayers of endothelium were prepared for in situ hybridization and immunohistochemistry at various times after unilateral flow reduction. Endothelial cell proliferation and PDGF-A and -B chain expression increased in RF arteries, whereas PDGF - and ß-receptor expression were undetectable. These results confirm the in vitro observation that shear stress alters endothelial cell PDGF ligand expression and support a role for PDGF in shear-mediated atherogenesis.

	Materials and Methods

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Animal Model
Forty-six male Sprague-Dawley rats (300 g, Harlan Sprague Dawley, Indianapolis, Ind) underwent acute RF in one common carotid artery. Anesthesia was induced with a mixture of ketamine hydrochloride, xylazine, and acepromazine maleate (50, 5, and 1 mg/kg IP, respectively), and the neck was shaved and prepared aseptically. The internal and external carotid artery branches were exposed bilaterally while leaving both common carotid arteries undisturbed. The right internal carotid artery was ligated at its origin, and the external carotid artery was ligated beyond its first small branch(es) with a 6-0 polypropylene suture. Antegrade flow in the right common carotid artery was maintained only through the small ascending pharyngeal or superior thyroid branches of the external carotid artery. Anatomic common carotid artery outflow was maintained on the left.

The degree of RF was determined in 8 animals that underwent surgical exposure of common carotid arteries. These animals were used only for hemodynamic determinations because the surgical manipulation required to apply flow probes to the common carotid artery would likely alter vessel wall proliferation and gene expression. Volume flow (mL/min) was measured with a transit-time flowmeter (Transonic Laboratories) immediately before and after the procedure. The diameter of each common carotid artery was measured using a dissecting microscope and eyepiece reticule before and after flow reduction. Shear stress (dyne/cm²) was then estimated using a modification of the Hagen-Poiseuille formula

where is shear, µ is the viscosity of blood (0.035 poise), and Q is volume blood flow.³ Blood pressure and heart rate were measured before and after ligation of the right carotid branches using an intra-arterial catheter inserted via the femoral artery and connected to a transducer (Stratham P23) and line chart recorder (Grass).

For studies of endothelial cell proliferation and PDGF expression, groups of animals were euthanized at 6, 24, 48, or 72 hours after flow reduction. Deep anesthesia was induced with pentobarbital (150 mg/kg IM), the abdominal aorta was cannulated, and animals were perfused at 100 mm Hg with lactated Ringer's solution (until clear of blood) followed by 4% paraformaldehyde. The heart and both carotid arteries were removed en bloc, cleaned of adventitia, and immersed in 4% paraformaldehyde (in situ samples) or 70% ethanol (immunohistochemistry samples) for an additional 24 hours.

All animal care and procedures were performed in accordance with state and federal laws. Animal protocols were approved by the Bowman Gray Animal Care and Use Committee and conformed to guidelines set forth by the American Association for Accreditation of Laboratory Animal Care and by the National Institutes of Health (publication No. 86-23, Guide for the Care and Use of Laboratory Animals).

Cell Proliferation (BrdU Labeling)
Carotid arteries removed from animals at 24, 48, and 72 hours after flow reduction were studied (n=5 per time point). BrdU was administered 24 hours before necropsy as a subcutaneous pellet (150 mg/kg, Boehringer-Mannheim) to label nuclei of cells entering S phase. Animals were infused intra-arterially with 50 mL of 0.5% silver nitrate (Fisher Scientific) to stain endothelial cell borders. After perfusion/fixation, arteries were opened longitudinally and pinned flat onto polytetrafluoroethylene sheets, rehydrated in graded alcohols, and rinsed in 0.3% hydrogen peroxide and then 10% pepsin (Sigma Chemical Co) in 0.1N HCl for 30 minutes, followed by 1.5N HCl for 15 minutes at 37°C. Specimens were then washed in 0.1 mol/L borax buffer (pH 8.5) and then Tris-buffered saline (pH 7.6). The primary anti-BrdU antibody (Boehringer) was then applied at a dilution of 1:40 and localized with a biotinylated secondary antibody (Vector Laboratories) and tertiary avidin-biotin-complex reaction (Vector) with the diaminobenzidine chromogen (Sigma). Specimens were mounted onto glass slides and coverslipped, and the endothelial surface was examined en face at x600 by light microscopy.

Endothelial cells were counted using a standard sampling protocol perpendicular to the direction of flow at four sites evenly spaced along the length of each artery. Individual endothelial cells were defined by silver staining, and proliferating cells were defined by brown nuclear BrdU staining. At least 1000 endothelial cells were counted per artery, and the number of labeled cells were expressed as a percentage of total endothelial cells counted.

In Situ Hybridization (PDGF Expression)
In situ hybridization was performed on en face endothelial cell preparations from arteries removed at 6 hours (n=6), 48 hours (n=9), and 72 hours (n=6) after unilateral flow reduction using a method recently described in the rat carotid balloon injury model.²⁷ Briefly, carotid arteries were opened longitudinally after perfusion/fixation and pinned onto polytetrafluoroethylene cards. Initially, 3 animals were studied at 48 hours after flow reduction for PDGF-B chain expression. Subsequently, 6 animals were studied at each of 6-, 48-, and 72-hour time points. Carotid arteries were divided in half so that two of the four PDGF riboprobes were used in each animal, providing at least 3 animals per probe per time point.

Specimens were incubated with proteinase K for 15 minutes at 37°C (1 µg/mL Boehringer) and then hybridization buffer for 2 hours at 37°C (0.3 mol/L NaCl, 20 mmol/L Tris, pH 7.5, 5 mmol/L EDTA, 1x Denhardt's solution, 10% dextran sulfate, 10 mmol/L dithiothreitol, and 50% formamide). Sense and antisense riboprobes were created using T3 and T7 polymerases (Promega) from previously characterized rat cDNAs for PDGF-A, -B, -receptor, and ß-receptor genes.^{28 29} After labeling with [³⁵S]UTP, riboprobes were applied overnight in hybridization buffer at 55°C. Subsequent steps were as previously described.³⁰ The Hautchen procedure for transferring endothelial cell monolayers onto glass slides was performed after hybridization,³¹ and slides were then coated with autoradiographic emulsion (Kodak, NTB2), exposed for 3 weeks, developed (Kodak, D-19), and counterstained with hematoxylin.

To estimate the degree of mRNA expression in each sample, a computer-assisted grain-counting strategy was used. Slides were examined using dark-field microscopy, and 20 images were captured at x200 magnification for analysis. Nonoverlapping images were captured using a sampling protocol similar to that described above for cell proliferation and imported into an image analysis program (IP Lab Spectrum for the Macintosh, Signal Analytics Corp).³² Color images were segmented by pixel value, and limits were created for each image that allowed for optimal separation of white silver grains from the dark background (Fig 1). An estimate of gene expression was then calculated by measuring the image area occupied by silver grains (expressed as percentage of total image area). For consistency in labeling, hybridizations for individual riboprobes were batched and run simultaneously after a single labeling for each riboprobe.

View larger version (180K): [in this window] [in a new window]   Figure 1. Analysis of in situ hybridization. En face photomicrograph of common carotid endothelium hybridized with an antisense 35S-labeled riboprobe for PDGF-B 48 hours after reducing blood flow (A). This image was captured by video microscopy and imported into a computerized image-analysis program (see "Materials and Methods"). Silver grains, indicating riboprobe binding, were quantified after segmenting the image by pixel value (B). Grains were assigned white and background green. The percentage of total image area occupied by silver grains was then calculated and used to estimate mRNA expression.

The agreement between hand counts of grain density and computer-assisted counts was validated as follows: Twenty en face dark-field images were selected at random from IF and RF carotid arteries labeled for PDGF-A or -B chain. The number of silver grains in each image was then counted five times by hand and five times by computer. Each image was resegmented and thresholded before each computer-assisted measurement. Computer-assisted counts were highly correlated with hand counts (r=.91, P<.0001), and mean intraobserver variability was 16% and 10%, respectively.

Statistical Analysis
Within each time point, intra-animal comparisons were made (right versus left) by paired Student's t test. Comparisons among time points were made using an unpaired Student's t test. Results are expressed as the mean±SEM, and significance was assumed at P<.05.

	Results

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Animals tolerated the unilateral flow-reduction procedure well. There were no deaths, and all common carotid arteries were patent at the time of necropsy. No thrombus was visible on gross or microscopic inspection of the lumen surface of each common carotid artery, and few adherent platelets or leukocytes were seen on microscopic examination. RF did not result in endothelial cell denudation, which was seen in <10% of IF and RF arteries, and when present, <1% of the endothelial cell surface was denuded.

Hemodynamic Measurements
Acute changes in blood flow and vessel diameter were measured, and common carotid artery shear stress was estimated after the flow-reduction procedure. Blood flow was decreased by 94±2% in RF arteries, while a compensatory increase occurred in IF arteries (Fig 2). Carotid diameter measurements did not change appreciably immediately after flow reduction (data not shown), so that calculated shear stress mirrored changes in blood flow. Estimated mean shear stress in RF common carotid arteries fell from 28.4±1.3 to 1.4±0.2 dyne/cm² after unilateral flow reduction, which is well below the normal range of mean arterial shear stress^{6 33} but within a range reported for atherosclerosis-prone regions of human arteries.³⁴ In the contralateral IF common carotid artery, estimated shear stress increased from 27.0±0.8 to 33.4±1.1 dyne/cm². Blood pressure and heart rate were not altered by the procedure (P=NS).

View larger version (22K): [in this window] [in a new window]   Figure 2. Hemodynamic effects of unilateral flow reduction. Bar chart shows common carotid artery blood flow (mL/min) before and after unilateral flow reduction as measured with a transit-time flowmeter (mean±SEM, n=8). *P<.05 vs preligation.

Endothelial Cell Proliferation
Endothelial cell proliferation was determined from the BrdU labeling indexes calculated at each time point for individual RF and IF carotid arteries. Flow reduction significantly increased proliferation in RF arteries at 48 hours compared with RF arteries at 24 hours. The increase in proliferation persisted at 72 hours (Fig 3). In contrast, proliferation in IF arteries increased only modestly by 48 and 72 hours. A modest increase in endothelial cell proliferation (1.2% at 48 hours, n=2) occurred in rats undergoing a sham operation, with no change in blood flow in either carotid artery, indicating that anesthesia and surgery may account for some of the increase observed in IF arteries.

View larger version (15K): [in this window] [in a new window]   Figure 3. Endothelial cell proliferation. Bar chart shows endothelial cell BrdU labeling (%) in RF and IF arteries over time. Proliferation increased within 48 hours in response to RF and persisted at 72 hours. Proliferation increased only slightly in IF arteries from the same animals (mean±SEM, n=5). *P<.05 for IF vs RF.

The pattern of proliferation was not uniform in the endothelium of RF common carotid arteries. Labeled endothelial cells were frequently clustered with adjacent groups of unlabeled cells. These islands of labeled cells were generally oriented along the axis of blood flow. A similar pattern was noted for the few labeled endothelial cells in IF arteries (Fig 4).

View larger version (164K): [in this window] [in a new window]   Figure 4. Endothelial cell proliferation. En face photomicrographs of carotid artery endothelium 48 hours after flow reduction. Left, IF artery. Right, RF artery. Nuclei of proliferating endothelial cells are labeled with an anti-BrdU antibody (brown staining), and endothelial cells are outlined by silver staining (original magnification x400).

PDGF Ligand and Receptor mRNA Expression
Expression of both PDGF-A and -B chain mRNA increased in RF arteries. After determining that proliferation was maximal 48 hours after RF, we performed an initial hybridization for PDGF-B chain in carotid arteries of 3 animals at this time point (Fig 5). The signal-to-noise ratio was very high for this hybridization (low background), and labeling was 7-fold higher in RF than in IF arteries (percent area labeled, 21.8±2.4 [RF] versus 2.7±0.6 [IF]; P<.001). A second experiment was then performed to determine the change in PDGF ligand and receptor expression over time. PDGF-B chain expression was similar in RF and IF arteries at 6 hours and then increased at 48 and 72 hours in RF compared with IF arteries (Fig 6). The temporal expression of PDGF-A chain mirrored that for PDGF-B chain in RF arteries (Fig 6). Expression of PDGF - and ß-receptors was not detectable in endothelial cells from RF or IF arteries at each of the three time points (Fig 7). This is in agreement with a previous study using riboprobes generated from these same rat cDNAs in which PDGF receptor expression was undetectable in rat carotid endothelial cells under normal flow conditions but readily detectable in rat endothelial cells and SMCs after injury.²⁹

View larger version (164K): [in this window] [in a new window]   Figure 5. PDGF-B mRNA expression. Photomicrographs of Hautchen preparations of common carotid artery endothelium from 3 individual rats (animals A, B, and C) 48 hours after unilateral flow reduction. The endothelium of each common carotid artery was hybridized with a 35S riboprobe for rat PDGF-B. Left, IF control carotid arteries. Right, RF arteries.

View larger version (14K): [in this window] [in a new window]   Figure 6. Time course of PDGF ligand expression after flow reduction. Bar chart shows the relative expression of PDGF-A and -B chains by the endothelial cells of RF arteries. Labeling extent in RF endothelium was expressed as a percentage of labeling in IF arteries from the same animal (mean±SEM, n=3).

View larger version (105K): [in this window] [in a new window]   Figure 7. PDGF receptor mRNA expression. Photomicrographs of Hautchen preparations of common carotid artery endothelium hybridized with a 35S riboprobe for rat PDGF -receptor (A and B) and ß-receptor (C and D) 48 hours after right carotid artery flow reduction. Panels A and C depict the left carotid endothelium (IF), and panels B and D depict the respective right carotid endothelium (RF) of individual animals. Labeling was no greater than background and was not altered by a change in blood flow. Labeling is representative of other time points as well (6 hours and 72 hours).

The pattern of PDGF ligand expression, much like that described above for endothelial cell proliferation (BrdU labeling), tended to be more pronounced in clusters of endothelial cells aligned in the direction of blood flow. The demarcation among these groups of cells was less distinct than for BrdU labeling, as some PDGF expression was seen in adjacent endothelial cells (Figs 1 and 5). Double-labeling experiments were not performed to determine the spatial relationship between endothelial cell replication and PDGF expression.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study is the first report of acute flow-induced endothelial cell PDGF expression in normal arteries and demonstrates that a change from normal to low shear stress will induce endothelial cell PDGF-A and -B chain expression without inducing either PDGF receptor. These data support the concept of the endothelium functioning as a mechanotransducer, converting force (shear stress) into a biochemical signal (PDGF) to the underlying artery wall. Endothelial cell replication in RF arteries corresponded to the temporal pattern of PDGF expression. Whether PDGF expression is induced in replicating cells or in quiescent neighboring cells remains to be defined. The association between reduced shear stress and increased endothelial cell PDGF expression fits with the paradigm that low shear stress promotes intimal thickening and atherogenesis.

Low shear stress colocalizes with sites within the vascular tree prone to develop atherosclerosis,^{9 34} suggesting that quiescence within the artery wall is lost partially because of the loss of inhibitory signals from the endothelium. The prototypical inhibitor is nitric oxide produced by type-3 nitric oxide synthase, which is constitutively expressed by endothelium under normal and high shear conditions but inhibited by low shear stress.³⁵ Nitric oxide, in addition to its vasodilator properties, inhibits SMC growth and intimal hyperplasia,^{36 37} platelet adhesion,³⁸ and PDGF expression by endothelial cells.³⁹ Vasodilators are often SMC growth inhibitors,^{36 40 41 42} and vasoconstrictors are often SMC mitogens.⁴³ At any given moment, the endothelium likely expresses a complex balance of growth inhibitor/vasodilators and mitogen/vasoconstrictors. Other atherogenic stimuli such as endothelial cell adhesion molecules may also be increased in atherosclerosis-prone regions and induced by low shear stress.^{44 45}

Rat large-vessel endothelium normally does not express PDGF ligands or receptors,²⁹ and our finding of increased ligand expression without detectable receptor expression in RF arteries suggests a paracrine effect in response to altered flow. PDGF expression can be induced within rat endothelium by mechanical trauma, and localized expression of both ligands occurs within 8 hours after balloon injury at the edge of the wounded endothelium. Endothelial cell PDGF expression persists as long as 6 weeks after balloon injury, whereas endothelial cell proliferation at the wounded edge subsides within days.²⁹ Since PDGF receptor expression in rat arteries is limited to medial and intimal SMCs, endothelial cell PDGF ligand expression may play a paracrine role in the injury response as well.

The differential effects of chronic low shear–mediated PDGF-A chain and -B chain expression on the normal artery wall are not known, and blocking experiments will be required to determine their respective roles. However, we can speculate on the basis of the extensive literature describing the effects of PDGF in arterial injury. The role of PDGF-B in the rat carotid balloon injury model has been well described. Injury induces platelet adhesion, degranulation, and release of PDGF-BB. Thrombocytopenia or an antibody to PDGF will inhibit intimal proliferation without blocking medial SMC replication.^{20 46} Infused PDGF-B will increase intimal thickening after injury with minimal effects on replication.^{19 47} Together, these data strongly suggest that PDGF-B promotes SMC migration in vivo. PDGF-B is also a weak mitogen and alters SMC collagen expression after injury.^{19 47} In culture, PDGF-B is a potent mitogen and enhances rat SMC migration, although effects can be altered by selective blockade of receptor isoforms. PDGF-B can bind each of the receptor dimers. If the ß-receptor is blocked, PDGF-B inhibits baboon SMC migration by -receptor binding, whereas either receptor can signal PDGF-B–induced proliferation.⁴⁸ Recent in vivo data support this concept. Antisense oligonucleotides targeting PDGF ß-receptor expression inhibit PDGF-B–induced intimal hyperplasia in the rat carotid artery.⁴⁹ The role of PDGF-A, which binds only to the -receptor, is less clear. Anti–A chain neutralizing antibodies do not affect intimal hyperplasia in the rat despite increased PDGF-A expression by medial SMCs early after injury.^{50 51} In baboons, as in the present study, the A chain is induced by reduced shear in endothelialized graft neointima, and expression correlates with proliferation and intimal hyperplasia.⁵² However, the B chain is not induced in the baboon graft model, so species differences may complicate this issue further. In summary, flow-induced PDGF-B chain expression may play a role in SMC migration, proliferation, and possibly vasoconstriction,⁴³ each of which have been implicated in the pathogenesis of atherosclerosis.⁵³ The impact of PDGF-A chain is less well defined.

How shear stress induces transcription of the PDGF ligands is also distinct. The PDGF-B gene promoter was the first described to contain a unique cis-acting response element (SSRE) subsequently identified in the promoter of a number of other shear-responsive genes.^{12 15 54} More recently, this same group has shown that nuclear factor-B binds to the SSRE and plays a functional role in regulating shear-dependent gene expression.^{25 54} The PDGF-A gene promoter does not contain the SSRE sequence but has an alternate SSRE, which is activated by binding of the early response gene, EGR-1. This sequence is also found in the promoter regions of several other endothelial cell genes induced by shear (tissue factor and transforming growth factor-ß₁).²⁶ Shear inhibitory elements have also been identified in genes downregulated by shear stress, such as the preproendothelin-1.^{54 55} Further studies are needed to define the role of individual regulatory elements in the chronic homeostasis of endothelial cell gene expression in normal and pathological flow environments in vivo.

The effect of reduced shear stress on endothelial cell PDGF expression in vessels was not predicted by previous in vitro studies because comparisons were made between cells exposed to physiological shear stress and those maintained under static conditions with no shear stress. Studies in vitro have not reported the effects of abnormally low shear stress (<5 dyne/cm²) compared with physiological shear stress as in the present study. Moreover, responses in vitro have varied in direction, duration, and magnitude.^{14 15 16 23 24 56} These variable responses to shear stress in culture may reflect differences in species, levels of shear stress, culture conditions, or cell growth state and underscore the difficulty of modeling in vivo fluid mechanical environments.

The present model is similar to the rabbit carotid model so well characterized by Langille and colleagues^{1 44 45 57} and shares many of its attributes. The rat common carotid artery is straight, does not taper appreciably, and has no side branches. As a result, the pattern of blood flow is undisturbed and uniform along its length so that the entire endothelium experiences the same magnitude of altered shear. In leaving patent only the ascending pharyngeal and superior thyroidal artery branches, the magnitude of common carotid artery flow reduction is greater than that after external carotid artery ligation in rabbits^{1 44 45 57} or after internal carotid artery ligation in rats⁴ (94%, 70%, and 35% reduction, respectively). The resulting shear stresses, estimated at 1.4 dyne/cm² in RF carotids and 33.4 dyne/cm² in IF carotids, are similar to those reported by Ku et al³⁴ in the atherosclerosis-prone human carotid bulb (-0.5 to 4 dyne/cm²) and in the atherosclerosis-resistant carotid flow divider (10 to 41 dyne/cm²), respectively.

The en face approach is a powerful tool for studies of endothelial cell behavior because it provides more information than can be obtained from vessel cross sections. Both the pattern and magnitude of change within the entire endothelium can be ascertained by using this technique, as illustrated by the clustering of cell proliferation and PDGF expression in the present study. Patchy changes in endothelial cell proliferation and gene expression have previously been reported in both undisturbed arteries and in response to changes in blood flow. An association between increased endothelial cell proliferation and the ostia of branch vessels has long suggested a relationship between shear stress, endothelial cell turnover, and atherogenesis.⁵⁸ However, it is intriguing that focal endothelial cell proliferation occurs spontaneously in regions remote from branch ostia. Schwartz and Benditt⁵⁹ labeled rats with tritiated thymidine for 24 hours and then prepared Hautchen autoradiograms of the entire aortic endothelium. Although overall proliferation was low (0.55%), many labeled cells were grouped into clusters with proliferation as high as 60% in fields containing an average of 143 cells. The labeling scheme used in the present study was similar to that used by Schwartz and Benditt, and BrdU labeling indexes for both carotid arteries at 24 hours were identical to their findings in the normal rat aorta (0.5%). Although proliferating cells at this time point were often clustered, these were small groups of cells compared with clusters seen at 48 and 72 hours in RF carotids (Fig 4).

The regional heterogeneity of both proliferation and PDGF expression in RF carotids suggests that cell populations exist within the endothelium that are less responsive to altered blood flow. The genetic basis for flow-responsive and -unresponsive endothelial cell phenotypes could be central to the control of vessel wall remodeling in response to altered blood flow. Langille and colleagues^{1 44 45 57} have reported similar patterns of endothelial cell responses to altered blood flow. For instance, VCAM expression increased in response to reduced flow in the rabbit carotid endothelium, and islands of VCAM-expressing cells were surrounded by endothelium free of staining.⁴⁵ They speculated that clusters of endothelial cells responding to altered shear stress may be of a different clonal origin than the surrounding cells. Such clonal selection might be related to the observations of Schwartz and Benditt,⁵⁹ who observed that centers of high endothelial cell turnover appear spontaneously within the undisturbed endothelium. Daughter cells accumulating at these sites may represent clones that vary in their ability to sense and respond to altered shear stress.

In summary, an acute reduction in blood flow and shear stress results in both endothelial cell proliferation and PDGF ligand expression in normal rat arteries. These data suggest that low shear stress alters the quiescent state within the vessel wall by changing the balance of growth factors and inhibitors expressed by the endothelium. PDGF and other atherogenic stimuli induced by low shear stress may explain, in part, the regional susceptibility to lesion formation within the human arterial tree.

	Selected Abbreviations and Acronyms

 

BrdU = bromodeoxyuridine IF = increased blood flow PDGF = platelet-derived growth factor RF = reduced blood flow SMC = smooth muscle cell SSRE = shear stress response element VCAM = vascular cell adhesion molecule

	Acknowledgments

 
This study was supported in part by an American Heart Association Grant-In-Aid (Dr Lindner) and National Institutes of Health grant HL-44721 (Dr Berk). Dr Berk is an Established Investigator of the American Heart Association, and Dr Miyashiro is supported by the National Institutes of Health (T32 HL-07090). The authors would like to thank Deanna Brown for expertise in immunohistochemistry and Michelle Gammons for preparing the manuscript.

	Footnotes

 
This manuscript was sent to Howard E. Morgan, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received September 4, 1996; accepted May 28, 1997.

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