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Circulation Research. 1996;79:45-53

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(Circulation Research. 1996;79:45-53.)
© 1996 American Heart Association, Inc.

Articles

Acute Reductions in Blood Flow and Shear Stress Induce Platelet-Derived Growth Factor-A Expression in Baboon Prosthetic Grafts

Larry W. Kraiss, Randolph L. Geary, Erney J.R. Mattsson, Selina Vergel, Y.P. Tina Au, Alexander W. Clowes

the Division of Vascular Surgery (L.W.K.), Department of Surgery and the Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah School of Medicine, Salt Lake City; the Department of General Surgery (R.L.G.), Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC; the Department of Surgery (E.J.R.M.), Malmo (Sweden) General Hospital; and the Department of Surgery (S.V., Y.P.T.A., A.W.C.), University of Washington, Seattle.

Correspondence to Alexander W. Clowes, MD, Department of Surgery, Box 356410, University of Washington, Seattle, WA 98195-6410. E-mail clowes@u.washington.edu.


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
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Abrupt reductions in fluid shear stress induce subendothelial smooth muscle cells (SMCs) to proliferate in experimental prosthetic grafts. Platelet-derived growth factor (PDGF), an important SMC mitogen, is expressed by cultured endothelial cells and modulated by shear stress. We hypothesized that this growth factor would be modulated by changes in shear stress in vivo. Bilateral aortoiliac prosthetic grafts were implanted into five baboons. High flow was generated by construction of femoral arteriovenous fistulas on both sides. Two months later, one of the fistulas was ligated, reducing shear stress in the upstream graft by 78±6%. Four days after fistula ligation, all grafts were removed and analyzed. As previously reported, SMC proliferation in low-flow grafts exceeded that in high-flow grafts, although the neointimal area was similar. mRNA levels for PDGF-A were significantly increased in low-flow grafts compared with high-flow grafts. In situ hybridization and immunohistochemical studies localized the increased PDGF-A mRNA and protein to the luminal endothelium and subjacent SMCs. Abrupt reductions in blood flow and fluid shear stress may induce accelerated neointimal thickening by a PDGF-A–mediated mechanism, since endothelial expression of this gene is temporally and anatomically associated with neointimal SMC proliferation.


Key Words: smooth muscle cell • endothelium • hemodynamics • in situ hybridization • immunohis-tochemistry


*    Introduction
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 *Introduction
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The structure of the blood vessel wall is highly responsive to the effects of two physical forces: pressure-dependent wall stress and flow-dependent shear stress. These factors interact to regulate both the mass of the vessel wall and its luminal diameter.1 The fluid shear stress applied to the luminal surface is proportional to blood flow and viscosity and is inversely proportional to the cube of the vessel radius. In large and medium-sized mammalian arteries, shear stress appears to be closely regulated between 5 and 20 dyne/cm2.1 Large arteries respond to acute changes in flow and shear stress by changing diameter; they dilate in response to increased flow2 3 and constrict in response to low flow.4 The overall effect of these vasoactive responses to changes in flow is to return near-wall shear stress to the physiological range.

Low shear stress also promotes intimal hyperplasia and cellular proliferation in the walls of vein5 6 7 and endothelialized prosthetic grafts.8 9 10 In particular, an abrupt decrease in blood flow in prosthetic grafts implanted in baboons results in a marked proliferative response within 4 days by neointimal SMCs—a response that precedes any increase in neointimal area. Most of these proliferating SMCs are located in the first few layers just beneath the luminal endothelium.10 Since prosthetic grafts cannot constrict, neointimal expansion may also be interpreted as an adaptive measure to reduce luminal diameter and return near-wall shear stress to a physiological range.11

The vasoactive responses of arteries to changes in flow and fluid shear stress are endothelium dependent.12 13 Thus, it is logical to consider the endothelial cell as the sensor of changes in fluid shear stress.14 Vascular SMCs are the effector cells in vasomotor changes (through contraction or relaxation) as well as the development of intimal hyperplasia (through proliferation and synthesis of extracellular matrix). We have hypothesized that changes in fluid shear stress modulate endothelial expression of genes that might be responsible for these adaptive changes by vascular SMCs.15

PDGF is a potent mitogen for SMCs and is secreted not only by platelets but also by endothelial cells, SMCs, and macrophages.16 PDGF is also a potent vasoconstrictor.17 We have previously demonstrated the expression and production of various PDGF isoforms by the neointimal cells lining prosthetic grafts implanted in baboons.15 18 Steady state PDGF mRNA expression by endothelial cells in vitro is regulated by shear stress,19 20 21 and a putative shear stress-responsive element has been identified in the 5'-flanking region of the PDGF-B gene.22 Since PDGF is particularly localized to prosthetic graft neointimal endothelium and is regulated by shear stress in vitro, we hypothesized that this mitogen is induced by acute changes in flow in vivo and participates in the resulting growth response.

In the present study, we show that PDGF-A mRNA and protein are upregulated in the neointima of low-flow prosthetic grafts compared with high-flow grafts. This growth factor induction occurs at a time when SMCs are known to be proliferating but before any measurable increase in neointimal area can be detected.


*    Materials and Methods
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 *Materials and Methods
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Chemicals
All chemicals were purchased from Sigma Chemical Co unless otherwise noted. BrdU was obtained from Boehringer Mannheim.

Baboon Prosthetic Graft Model of Intimal Hyperplasia
All surgical procedures were performed with the animals under general inhalational halothane anesthesia in the facilities of the University of Washington Regional Primate Research Center according to protocols approved by the institutional animal care committee. Sedation before surgery and for other procedures was obtained with an intramuscular injection of a mixture of ketamine (10 mg/kg) and xylazine (3 mg/kg).

Five male baboons (Papio cynocephalus) weighing {approx}10 kg were used in the present study. At the initial surgery, bilateral aortoiliac unwrapped polytetrafluoroethylene grafts (internal diameter, 4 mm; internodal distance, 60 µm; W.L. Gore & Associates, Inc) 6 to 8 cm in length were placed, and the native vessels were ligated to divert all pelvic and lower-extremity flow through the grafts. Bilateral femoral arteriovenous fistulas were also constructed to generate high flow through the proximal grafts.10

Eight weeks later, the grafts were studied by duplex scanning (Acuson 128), and the mean shear stress ({tau}, in dynes per square centimeter) for each graft was calculated according to a modification of the Hagen-Poiseuille equation8 :

(E1)
where {eta} is blood viscosity (taken to be 0.035 poise), VTAV is time-averaged velocity (in centimeters per second) in the graft over at least six cardiac cycles, and r (in centimeters) is graft radius. VTAV and r are measured by duplex ultrasound scanning. The graft on the side with the highest calculated shear stress was chosen for flow reduction, and the fistula below this graft was ligated.10 Hemodynamic variables in ketamine-sedated animals are not significantly different from those in resting unsedated animals.8

Four days later, the grafts were again studied by duplex scanning. The animals were then euthanized by barbiturate overdose and systemically perfused at physiological pressure with lactated Ringer's solution, and the grafts were removed. The animals received a total of three intramuscular injections of BrdU (30 mg/kg) at 17, 9, and 1 hour before necropsy. Representative cross sections from the midportion of the 6- to 8-cm-long grafts (approximately seven diameters from the proximal anastomosis) were immersion-fixed in 10% buffered formalin or methacarn solution and then paraffin-embedded for later morphometry or immunohistochemistry. The neointimal tissue was scraped from the remainder of the grafts, immediately homogenized in denaturing solution D (4 mol/L guanidinium thiocyanate, 25 mmol/L sodium citrate, pH 7.0, 0.5% sarcosyl, and 0.1 mol/L 2-mercaptoethanol),23 and stored at -70°C.

Northern Analysis
Total RNA was isolated from neointimal tissue according to the acid guanidinium thiocyanate-phenol-chloroform extraction method described by Chomczynski and Sacchi.23 The samples were then separated by electrophoresis in 1.2% agarose gels (10 to 15 µg per lane) and transferred to nylon blotting membranes (Zeta-Probe, Bio-Rad). After baking the membranes at 80°C for 2 hours, the blots were hybridized overnight at 42°C with radiolabeled probes.

cDNA probes for human PDGF-A,24 PDGF-B,25 PDGFR-{alpha},26 PDGFR-ß,27 and GAPDH28 were radiolabeled with [32P]CTP by nick-translation.

Autoradiograms were prepared by exposing the blots to ra-diographic film (Kodak). The blots were also analyzed by phosphorimaging (PhosphorImager model 400S, Molecular Dynamics).29 Quantitative data are reported as a ratio of the signal from the low-flow graft to the high-flow graft signal from the same animal.

In Situ Hybridization
A 1.28-kb human PDGF-A cDNA fragment24 and a 1.5-kb human PDGFR-{alpha} cDNA fragment26 (gifts from J.N. Wilcox, Emory University, Atlanta, Ga) were transcribed into an antisense riboprobe using a commercial kit (Promega) and labeled with [35S]UTP (Amersham). The transcription mixture contained 1 µg of linearized cDNA template, 250 µCi [35S]UTP (>1000 Ci/mmol), 2.5 mmol/L each of ATP, CTP, and GTP, 40 U RNasin, 10 mmol/L dithiothreitol, 40 mmol/L Tris-HCl (pH 7.9), and 15 U SP6 or 19 U T7 polymerase. After incubation for 75 minutes at 37°C, the cDNA was digested with 1 U DNase and incubated at 37°C for an additional 15 minutes. The labeled probe was separated from free nucleotides using a Sephadex G-50 column. Activity of the probes ranged from 2 to 4x105 cpm/µL. Probes were stored at 4°C and used within 24 hours of synthesis.

As described previously by O'Brien et al,30 10% formalin-fixed histological sections were deparaffinized and digested with 5 µg/mL proteinase K (Sigma) in RNase buffer (500 mmol/L NaCl and 10 mmol/L Tris-HCl, pH 8.0) for 40 minutes at 37°C. After three washes with 0.5x SSC (1x SSC contains 150 mmol/L NaCl and 15 mmol/L sodium citrate, pH 7.0), the slides were incubated in prehybridization buffer (50% formamide, 0.3 mol/L NaCl, 20 mmol/L Tris-HCl, pH 8.0, 5 mmol/L EDTA, 1x Denhardt's solution, 10% dextran sulfate, and 10 mmol/L dithiothreitol) for 2 hours at 50°C. Each cross section was then covered with labeled probe, and the slides were incubated overnight at 50°C. During prehybridization and hybridization, the slides were kept in a chamber moistened with buffer (4x SSC and 50% formamide). The following day, the slides were washed three times in 0.5x SSC and digested with ribonuclease A (20 µg/mL) for 30 minutes at 37°C. Three high-stringency washes with 0.1x SSC and 0.5% Tween 20 followed, each for 40 minutes at 37°C. The specimens were then washed three times in 2x SSC, once in distilled H2O, air-dried, and dipped in a 50% solution of NTB2 photographic emulsion (Kodak) for autoradiography. The slides were exposed in the dark at 4°C for 3 weeks, then developed, and counterstained with hematoxylin and eosin.

Immunohistochemistry
Primary antibodies were a rabbit polyclonal antibody to PDGF-A (Santa Cruz Biotechnology, Inc), a monoclonal antibody to PDGF-B (PGF-007, Mochida Pharmaceutical), a monoclonal antibody to BrdU (Boehringer Mannheim), a monoclonal antibody to smooth muscle {alpha}-actin (Boehringer Mannheim), and a polyclonal antibody to factor VIII-related antigen (Dako).

Paraffin-embedded histological cross sections taken from the central portions of the grafts were fixed in either 10% buffered formalin or methacarn solution and mounted on positively charged microscope slides (Superfrost Plus, Curtin Matheson). Immunohistochemical procedures were performed according to the avidin-biotin-peroxidase method (Vector Laboratories).31 32 Primary antibodies were applied to the slides overnight at 4°C, except for the antibody against BrdU, which was applied for 1 hour at room temperature. Biotinylated secondary antibodies were then applied for 30 minutes. Bound antibody complexes were visualized using diaminobenzidine with or without NiCl2 as the chromogen. Controls were either nonimmune rabbit serum (for polyclonal antibodies) or irrelevant monoclonal antibodies.

Morphometric Analysis and Determination of SMC Proliferative Rates
Midgraft neointimal areas were measured on histological cross sections using a camera lucida and a computer-linked digitizing pad. The number of neointimal cells on histological cross section positively stained for BrdU33 were counted and divided by the total number of neointimal nuclei per section. The results are reported as percent nuclei stained.

Statistical Analysis
Differences in shear stress, mRNA expression, SMC proliferation, and neointimal area between high- and low-flow grafts were evaluated by a pairwise nonparametric test (Wilcoxon) using a microcomputer-based statistical software package (Statview, Abacus Concepts). Significance was assumed at P<=.05.


*    Results
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 up arrowMaterials and Methods
 *Results
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Fistula Ligation Causes a Significant Reduction in Mean Shear Stress
One femoral arteriovenous fistula in each animal was ligated at 8 weeks. Mean shear stress in the ipsilateral upstream graft before fistula ligation was 47.3±12.0 dyne/cm2. Four days after flow reduction, shear stress in these grafts had fallen to 9.4±4.1 dyne/cm2 (P=.04) (Fig 1Down).



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  		Figure 1. Changes in shear stress (±SE) in reduced-flow (fistula ligated) and high-flow (fistula left intact) prosthetic aortoiliac grafts (n=5 pairs). Center-stream blood flow velocity (centimeters per second) and the diameter of each graft (centimeters) were measured by duplex scanning immediately before unilateral fistula ligation and again 4 days later, immediately before graft removal. Shear stress was then calculated according to the Hagen-Poiseuille equation (see "Materials and Methods").

Calculated shear stress at the same time points in the contralateral (control) grafts not subjected to fistula ligation was 41.1±7.9 and 29.7±6.7 dyne/cm2 (P=.08). Although it appears that a trend toward decreased flow was also present in these grafts, the percent reduction in shear stress in grafts above ligated fistulas was significantly greater than in those grafts above intact arteriovenous fistulas (78±6% versus 24±11%, P=.04).

Reduced Shear Stress Induces SMC Proliferation 4 Days Later
In flow-reduced grafts, mean neointimal area was 1.6±0.7 mm2 compared with 1.6±0.6 mm2 in grafts that remained at high flow (P=.72). The SMC proliferative rate (BrdU labeling index) was 2.3±1.0% in flow-reduced grafts and 0.9±0.4% in high-flow grafts (P=.27). This trend, although not statistically significant, is consistent with data previously reported by our group10 indicating that SMC proliferation is induced by flow reduction and precedes any apparent increase in neointimal area (Fig 2Down).



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  		Figure 2. Morphometric analysis of midgraft cross sections from reduced-flow and high-flow grafts (n=5 pairs) 4 days after unilateral fistula ligation. SMC proliferative rates (percent cells labeled±SE) in the graft neointima were determined by counting BrdU-labeled cells on histological cross sections. Neointimal areas (square millimeters±SE) for the same sections were determined using a camera lucida and computer-linked digitizing pad.

Reduced Shear Stress Induces PDGF-A
Flow reduction caused statistically significant increases in neointimal levels of mRNA for PDGF-A (2.9-fold) and GAPDH (2.1-fold) (Figs 3Down and 4). The PDGF-A transcript frequently appears as a triplet.16 This pattern has been demonstrated previously in baboon prosthetic graft material.34 Increases in the expression of PDGFR-{alpha} (2.1-fold) and PDGFR-ß (1.7-fold) were also apparent, but these differences were not statistically significant. The increased expression of GAPDH mRNA in a low-shear (or zero-shear) environment in vitro has been previously observed.21 22



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  		Figure 3. Induction of PDGF-A and GAPDH mRNA in a reduced-flow graft 4 days after ipsilateral fistula ligation. Neointima from reduced-flow and high-flow (fistula left intact) grafts (R and H, respectively) was collected at the time of graft removal for isolation of total cellular RNA. Equal amounts of RNA (10 µg, determined by spectrophotometry) from each graft were loaded into the respective lanes. The membrane was sequentially hybridized with cDNA probes for PDGF-A and then GAPDH. Subsequent hybridizations were made with cDNA probes for PDGFR-{alpha} and PDGFR-ß (data not shown). Similar results were obtained from the other four animals studied.

PDGF-B mRNA was detected in the prosthetic graft only in small amounts and was not affected by flow reduction (data not shown). The relatively low expression of PDGF-B mRNA in baboon prosthetic graft neointimal tissue is consistent with previous reports.15 18 PDGF-B protein is nearly undetectable in extracts of baboon neointimal tissue, whereas PDGF-A is relatively plentiful.15

In situ hybridization studies with a cDNA probe for PDGF-A were performed to localize the areas of increased expression. Fig 5Down illustrates the results obtained using tissue from the same animal depicted in Fig 3Up. Studies from the other four animals were consistent with these findings (data not shown). There is obviously much more signal for PDGF-A in the low-flow graft (Fig 5ADown) than the high-flow graft (Fig 5BDown). It is also important to note that the increased signal for PDGF-A was particularly prominent over the luminal cells, which were identified as endothelial cells by anti–factor VIII staining.18




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  		Figure 5. In situ hybridization analysis of PDGF-A expression in reduced-flow (A) and high-flow (B) grafts shown in Figs 3 and 6UpUp. Formalin-fixed, paraffin-embedded, histological cross sections from the central portions of the grafts were processed as described in "Materials and Methods." Samples from both grafts taken from each animal were analyzed in the same experiment. A 35S-labeled antisense riboprobe to PDGF-A mRNA was then hybridized to the cross sections overnight. After they were washed, the slides were coated with photographic emulsion and exposed in the dark for 3 weeks at 4°C, then developed, and counterstained with hematoxylin and eosin. Black grains overlying the cells and nuclei represent hybridized probe. Arrows indicate the endothelial lining. Original magnification, x400.

It also appears that the difference in PDGF-A expression between high- and low-flow grafts in the in situ hybridization studies (Fig 5Up) is greater than the Northern analysis (Figs 3 and 4UpDown) would suggest. The histological studies indicate that PDGF-A induction occurs at or just beneath the luminal surface. This region represents a relatively small proportion of the total neointima, yet the material submitted for Northern analysis represents the entire neointima. Thus, the intensity of PDGF-A induction as measured by Northern analysis is muted by inclusion of a relatively large amount of tissue in which there is no change in PDGF-A expression.



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  		Figure 4. Quantitative analysis of PDGF-A, PDGFR-{alpha}, PDGFR-ß, and GAPDH mRNA induction in reduced-flow grafts (n=5) 4 days after ipsilateral fistula ligation. After initial preparation of autoradiograms, the Northern blots described in Fig 3Up were scanned using a PhosphorImager model 400S (Molecular Dynamics). High- and low-flow grafts from the same animal were always analyzed on the same blot, but separate blots were prepared for each animal. For each probe (PDGF-A, PDGFR-{alpha}, PDGFR-ß, and GAPDH), the ratio of the activity in the reduced-flow grafts (±SE) is compared with the activity in the high-flow grafts. High-flow graft activity for each probe is arbitrarily set at 1 and indicated by the dotted line. Low-flow graft activity represents fold induction over high-flow graft activity.

To characterize further the change in PDGF-A expression with changes in blood flow, histological cross sections from low- and high-flow grafts were stained with antibodies against PDGF-A (Fig 6Down). The immunohistochemical results mirrored those obtained by in situ hybridization: the low-flow graft neointima (Fig 6ADown) stained much more intensely than the high-flow tissue (Fig 6BDown). The increased production of PDGF-A appeared to be highly concentrated in the luminal endothelial cells.




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  		Figure 6. Immunohistochemical localization of PDGF-A protein in reduced-flow (A) and high-flow (B) grafts shown in Figs 3 and 5UpUp. Histological cross sections were cut from the same paraffin blocks used in Fig 5Up and processed as described in "Materials and Methods." Samples from both grafts taken from each animal were analyzed in the same experiment. A polyclonal antibody against PDGF-A was applied to each slide overnight at 4°C. Bound primary antibody was then visualized using the avidin-biotin peroxidase system (black reaction product occupying the cytoplasm). The slides were then counterstained with hematoxylin and eosin. Arrows indicate the endothelial lining. Original magnification, x400.


*    Discussion
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 *Discussion
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In baboon prosthetic grafts, reduced shear stress enhances growth of the neointimal lining by stimulating SMC proliferation.10 Reduction of blood flow by closing the fistula changes the physical forces acting on the luminal surface but does not cause thrombosis or mechanical damage to the endothelium or mural SMCs. Thus, the response of the vessel wall to reduced shear stress might be more physiological than pathophysiological. In this model, one prominent feature of the response to reduced shear stress is induction of the SMC mitogen, PDGF-A, and possibly its receptor, PDGFR-{alpha}.

In this model, we have used nondistensible prosthetic grafts to isolate the effects of flow-dependent shear stress ({tau}) from pressure-dependent wall stress (S). S is defined as wall tension (T) divided by wall thickness (t). T is defined by Laplace's law as the product of intraluminal pressure (P) and luminal radius (r), leading to the following expression for wall stress (S):

(E2)
Since there was no difference in neointimal area between high- and low-flow grafts and since we assume that the grafts were manufactured at constant thickness, wall thickness (t) remained constant throughout the experiment. The grafts are nondistensible; therefore, the luminal radius also remained constant. We have previously documented that there is no significant difference in intraluminal pressure between high- and low-flow grafts in this model9 ; thus, the low-flow graft is not selectively exposed to higher intraluminal pressures as a result of fistula ligation. Since all the variables influencing wall stress (S) were the same in both high- and low-flow grafts, this model has isolated wall stress from shear stress.

The Potential Role of PDGF in the Vessel Wall's Response to Shear Stress
The apparent flow-related change in GAPDH expression complicates the interpretation of the change in PDGF-A expression as measured by Northern analysis. We addressed this problem in two ways. First, these blots were probed for the presence of the endothelial isoform of NO synthase. Expression of the endothelial isoform of NO synthase was elevated in high-flow grafts and suppressed in low-flow grafts—a pattern exactly the opposite of PDGF-A (E.J.R. Mattsson, L.W. Kraiss, A.W. Clowes, unpublished data, 1996). This finding greatly reduces the possibility that the increased expression of PDGF-A in the low-flow grafts resulted from unequal mRNA loading. Second, the in situ hybridization studies, which are not subject to "loading error," corroborate the Northern analysis.

Studies of cultured endothelial monolayers show that shear stress induces PDGF gene expression.19 20 21 22 These observations lead to the conclusion that shear stress should stimulate SMC growth. But this is exactly the opposite of what is observed in vivo: SMC proliferation and intimal thickening are inhibited by high shear stress.5 6 7 8 9 How can these disparate results be reconciled?

First, cultured endothelial cells and endothelial cells overlying a graft or artery may respond differently to shear stress. The dynamic interactions with other vessel wall components, such as the internal elastic lamina and medial SMCs, may be critical. Second, the usual in vitro comparison of zero shear to some level of shear stress is somewhat artificial since endothelial cells in vivo are rarely, if ever, found in a zero-flow environment. Third, the presence of other formed elements in blood may participate in the in vivo response to changes in shear stress in ways that cannot be adequately modeled in vitro. Finally, the overall reaction of the vessel to changes in shear stress is probably determined by the net effect of a number of different responses induced in the endothelial cell. It is doubtful that any single mitogen or growth inhibitor operates in isolation in the vessel wall. Thus, in vitro studies that examine the regulation of a single molecule or gene by shear stress yield valuable, but incomplete, information about the overall effects of shear stress on blood vessels.

PDGF is expressed in apparently normal quiescent vessels35 36 as well as in vessels in which significant growth is occurring. Its detection in normal unperturbed vessels suggests that PDGF may be important for cellular functions other than proliferation. Indeed, we have found that closure of the femoral arteriovenous fistula induces PDGF-A expression in iliac arteries as well as in the neointimas of aortoiliac prosthetic grafts. The expression of PDGF-A is associated with a decrease in the iliac artery diameter (E.J.R. Mattsson, L.W. Kraiss, A.W. Clowes, unpublished data, 1996). Perhaps PDGF helps to maintain vasoconstrictive tone in situations in which no obvious cell proliferation is occurring. It may also function as one of the as-yet-uncharacterized mediators of endothelium-dependent vasoconstriction in response to reduced flow.17

The shear-responsive pattern of PDGF-A expression in prosthetic graft neointima, particularly in the endothelium, suggests that it might function as a mitogen in this tissue. If PDGF-A is to have any biological function, the target tissue must express a relevant form of the PDGF receptor. For PDGF-A, the relevant PDGFR is the {alpha} isoform.37 We have demonstrated the presence of mRNA for this receptor isoform in baboon prosthetic graft neointima and provided some evidence for its regulation by different flow conditions. We have not yet demonstrated the presence of PDGFR-{alpha} protein in extracts of mature quiescent baboon neointima.15 Since reagents for the immunohistochemical detection of PDGFR-{alpha} remain unavailable, we cannot state with certainty that the relevant receptor for PDGF-A is expressed on the surface of neointimal SMCs. To determine whether PDGF actually mediates shear-induced neointimal growth in this model, we will ultimately need to perform experiments in which PDGF activity is blocked.

Is PDGF-A directly regulated by shear stress? The answer to this question is complicated. In vitro, steady state mRNA levels for PDGF-A in human umbilical vein endothelial cells are shear responsive.19 20 However, in bovine aortic endothelial cells, PDGF-A is not induced by exposure to shear stress (although PDGF-B was strongly stimulated).21 A putative shear stress–responsive element (GAGACC) has been identified in the 5'-flanking region of the PDGF-B gene and other shear-sensitive genes (c-fos, c-jun, transforming growth factor-ß, and tissue plasminogen activator)22 but not PDGF-A. However, the expression of a gene may still be directly regulated by shear stress despite the absence of this particular sequence, since there are other nucleotide sequences38 or other mechanisms39 that confer shear responsiveness. Alternatively, PDGF-A expression may be a secondary event in response to the induction (or perhaps decline) of some other shear-responsive factor.

The Role of the Endothelium in Vascular Adaptation to Changes in Blood Flow
The endothelium, being interposed between the flowing blood and the SMCs in the intima or media, appears to transduce the physical stimulus of shear stress into a biological signal directed at other components of the vessel wall. In response to increased flow and shear stress, blood vessels dilate; this response is abolished if the vessel is denuded of endothelium.12 This loss of endothelium is associated with reduced or absent release of NO by the vessel and can be restored by adding back endothelial cells to the test system.40 These observations and many others support the conclusion that NO is the major, shear-dependent, endothelium-dependent vasodilator. The vasoconstrictive response to reduced shear stress is just as dependent on the presence of endothelium,4 13 but the molecular mediators of this response are undefined.

In addition to its effects on lumen diameter, shear stress appears to regulate intimal mass in normal vessels,41 42 atherosclerotic vessels,43 44 arterialized vein grafts,5 6 7 and endothelialized prosthetic grafts.8 9 10 One common feature in all of these animal studies is that intimal hyperplasia is suppressed by high shear stress and enhanced by low shear stress. These vessels are usually covered with endothelium, and we assume that these cells respond to shear by generating biochemical signals that affect SMC growth and intimal thickening as well as luminal diameter. Many of the factors that regulate vasoconstriction and vasorelaxation might also influence SMC growth, and factors that control SMC growth might also regulate vasomotor function.45 Many growth-regulatory molecules, including PDGF and epidermal growth factor, are vasoconstrictors.17 46 47 Conversely, many molecules known primarily for their vasoactive effects regulate the growth of SMCs, at least in culture. Among these factors are endothelin-1,48 angiotensin II,49 the catecholamines,50 serotonin,51 prostacyclin,52 53 and especially NO.40 54 55 A general pattern has emerged for these multifunctional molecules: vasoconstrictors are usually mitogens for SMCs, whereas vasodilators tend to be growth inhibitors. This pattern holds for those molecules whose expression is also known to be shear sensitive, such as PDGF,19 20 21 22 endothelin-1,56 prostacyclin,57 and NO.58 59

The growth-regulatory effects of these multifunctional molecules can be isolated from their vasoactive effects when studied in endothelialized prosthetic grafts, since the rigid synthetic material prevents the usual vasomotor response to changes in shear stress. Shear stress on the endothelium can only be increased if the graft lumen is narrowed by an increase in wall mass. Low shear stress conditions may stimulate endothelium to upregulate the expression of vasoconstrictive growth factors, downregulate the expression of vasorelaxant growth inhibitors, or both. Because the prosthetic graft cannot acutely reduce its luminal diameter (and therefore immediately increase near-wall shear stress), the net effect of this change in growth-regulatory balance is likely to be SMC growth and neointimal thickening.

The simplest explanation for the effects of shear stress on the vessel wall is that there are a small number of molecules that produce both the acute (vasoactive) and chronic (changes in wall mass) changes in wall structure. Given the multifunctional nature of many growth-regulatory and vasoactive molecules, it is attractive to postulate that the acute and chronic effects of flow reduction result from increased expression of molecules with vasoconstrictive and growth-promoting actions. Similarly, the acute and chronic effects of increased flow might be mediated by increased expression of molecules with vasodilatory and growth-inhibitory actions.

Perhaps the acute and reversible vasoactive response to changes in flow occur by relatively short and transient expression of these multifunctional molecules. If the change in flow and shear stress is more sustained, then expression of these vasoactive growth regulators may similarly be more prolonged, leading to changes in the growth state and therefore the structure of the vessel. Such a simple model inadequately explains the response of normal blood vessels to changes in flow but might help explain the effects of flow and shear stress on diseased vessels or prosthetic grafts.

In normal blood vessels exposed to chronically reduced flow, fixed reductions in luminal diameter occur without increases in wall mass.1 60 If vasoconstrictive growth factors mediate this process, why is there no increase in wall mass? The normal vessel's acute response to increased flow is vasodilation mediated by NO,12 61 but increases in wall mass are observed at later times in these vessels if the increased flow persists.62 NO is not known to stimulate the cellular proliferation and matrix synthesis that must have occurred for wall mass to have increased. Thus, the ultimate response to sustained alterations in shear stress in normal vessels probably arises through the interaction of multiple molecules with opposing vasoactive and growth effects.

The previously described paradigm might explain the effects of shear stress in noncompliant diseased vessels, arterialized vein grafts, or prosthetic graft neointima. In general, these vessels display overexpression of vasoconstrictive growth-promoting influences (such as PDGF) relative to vasodilatory growth-inhibitory influences. Atherosclerotic arteries and arterialized vein grafts do not relax as well as normal arteries in response to vasodilatory stimuli that normally induce the release of NO.63 64 These findings suggest that the growth-promoting influence of vasoconstrictive factors in these vessels might be inadequately counterbalanced by growth-inhibitory vasodilators. In contrast to normal vessels, these diseased or artificial vessels develop increased wall mass in response to low flow.5 6 7 43 44 Alternatively, increased flow and shear stress may inhibit intimal thickening and induce these "abnormal" vessels to express more vasodilators and growth inhibitors than they do under normal flow conditions.65 66 67 68

Summary
Abrupt reductions in shear stress induce SMC proliferation and the subsequent neointimal thickening in endothelialized prosthetic grafts in baboons.10 The data from the present study are consistent with our previous report. Further, a significant induction of endothelial PDGF-A mRNA and protein expression coincides both temporally and spatially with neointimal SMC growth. These results are consistent with the hypothesis that endothelial cells regulate neointimal SMC growth in a shear-dependent fashion, perhaps via PDGF-dependent pathways.


*    Selected Abbreviations and Acronyms
 
BrdU = bromodeoxyuridine
PDGF = platelet-derived growth factor
PDGFR = PDGF receptor
SMC = smooth muscle cell


*    Acknowledgments
 
This study was supported in part by grants from the National Institutes of Health, US Public Health Service, to A.W. Clowes (HL-30946) and to the Regional Primate Research Center (RR-00166). Dr Mattsson was the recipient of a National Institutes of Health Fogarty International Fellowship (1FO5-TWO-5037-01) and also received assistance from the Swedish Medical Research Council and the Svenska Lakaresallskapet. The able technical assistance of Kim Cantwell-Gab, RVT, is acknowledged, as is the valuable advice of Marina Ferguson (Biosupport Inc, Redmond, Wash) concerning the in situ hybridization studies. We thank Monika Clowes for her assistance in preparing the illustrations. The prosthetic grafts were generous gifts of W.L. Gore & Associates (Flagstaff, Ariz), and polypropylene suture was given by Davis & Geck (Danbury, Conn). Quantitative phosphorimaging is provided as a service of the Markey Molecular Medicine Center at the University of Washington.

Received January 22, 1996; accepted April 5, 1996.


*    References
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