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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2245-2249.)
© 1997 American Heart Association, Inc.

Articles

Increased Blood Flow Induces Regression of Intimal Hyperplasia

Erney J. R. Mattsson; Ted R. Kohler; Selina M. Vergel; ; Alexander W. Clowes

From the Department of Surgery, University of Washington School of Medicine, Seattle, Wash.

Correspondence to Alexander W. Clowes, MD, Department of Surgery, University of Washington School of Medicine, HSB 442, Box 356410, Seattle, WA 98195-6410. E-mail clowes{at}u.washington.edu

	Abstract

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Abstract We have previously shown that high shear stress inhibits growth of developing neointima in a primate model of polytetrafluoroethylene (PTFE) graft healing. We used this model to test the hypothesis that increased shear stress can cause atrophy of an established neointima. High porosity PTFE grafts were inserted into the aorto-iliac circulation bilaterally in baboons. These grafts develop neointimal hyperplasia comprising smooth muscle cells and a luminal surface of confluent endothelium. Neointima was allowed to develop for 2 months. At that time 8 animals were sacrificed. In eight other animals blood flow in one of two grafts was increased by construction of a femoral arterio-venous fistula. These animals were sacrificed 2 months later (4 months after graft placement). At four months, intimal cross sectional area was smaller on the high shear stress side compared to the contralateral, normal shear stress side (2.53±0.75 versus 6.83±0.65 mm², P<.05). Neointima from grafts exposed to 2 months normal shear stress followed by 2 months of high shear stress had regressed when compared to normal-shear stress grafts studied at 2 months (2.53±0.75 versus 4.56±0.68 mm², P<.05). Morphometric analysis using transmission electron microscopy revealed that the decrease in intimal cross sectional area was attributable to a loss of both smooth muscle cells and matrix. Endothelial nitric oxide synthase was induced in high-flow graft intima. These observations support the conclusion that elevated shear stress can cause vessel wall atrophy. This process might be mediated by nitric oxide.

Key Words: intimal hyperplasia • regression • nitric oxide synthase • vascular graft

	Introduction

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Hemodynamic forces are important modulators of vessel wall structure. The arterial wall diameter responds to changes in blood flow (shear stress)^{1 2} while the vessel wall thickness is regulated by wall tension, which varies with the intraluminal pressure.³ It is not known if shear stress can also affect wall thickness. In synthetic or venous bypass grafts, low shear stress promotes intimal hyperplasia and high shear stress inhibits it. In relatively rigid, porous PTFE grafts (60 µm internodal distance) placed in the aorto-iliac circulation of baboons, we have found that the luminal surface is entirely covered with endothelium at 2 weeks by transmural capillary ingrowth from perigraft tissue and ingrowth from the adjacent vessels. Standard porosity grafts (30 µm internodal distance) heal only by ingrowth from the ends. Over a period of two to three months, smooth muscle cells (SMCs), presumably derived from pericytes, proliferate and secrete extracellular matrix which contributes to intimal mass. The resulting neointima consists of a confluent endothelium with underlying SMCs in a collagen- and elastin-rich matrix.^{4 5} We have used this healed graft to define the effects of changes in shear stress on vascular tissue. In this experimental graft model, neointimal thickening is inhibited in the presence of high blood shear stress⁶ and can be induced to increase if shear stress is switched from high to normal levels.⁵ Neointimal expansion in response to a reduction in shear stress might be an adaptive measure regulating luminal diameter to keep shear stress in a physiological range even though vasoconstriction is not possible in rigid grafts.

There may be a significant connection between vasomotor function and the regulation of vessel wall mass. The molecular signals that induce vasodilation under some circumstances inhibit SMC growth (nitric oxide, prostacyclin) while those that induce vasoconstriction are often mitogens (endothelin, angiotensin II). Nitric oxide (NO) is a potent vasodilator⁷ and inhibits SMC proliferation in vitro and in vivo.^{8 9} NO can also induce cell death.¹⁰ In normal vessels, NO is generated through the enzymatic activity of the endothelial constitutive NO synthase (cNOS).¹¹ Increased flow induces cNOS, and NO synthesis might have an effect on wall structure over the long term apart from causing vasodilation. We have investigated the possibility that increases in blood flow might cause atrophy of the vascular structure when vasodilation is not possible.

	Methods

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Animal Model
Anesthesia was induced in young male baboons (Papio cynocephalus) with intramuscular ketamine hydrochloride (10 mg/kg) and maintained with inhaled halothane. The animals were anticoagulated with heparin intravenously (100 U/kg, Elkins-Sinn, Inc), and antibiotics were administered intramuscularly (25 mg/kg Cefazolin sodium, Bristol-Myers Squibb). Bilateral aorto-iliac PTFE grafts (unwrapped, 4 mm internal diameter, 6 to 8 cm in length, internodal distance 60 µm, W.L. Gore and Associates, Inc) were implanted in an end-to-side fashion using 6-0 polypropylene suture (Davis and Geck).⁵ The native vessels were ligated, thereby diverting all flow through the grafts.

The grafts were allowed to heal under normal flow conditions for 2 months. Eight animals were sacrificed at this timepoint (2 months NF). In another eight animals the blood flow was increased through one of the grafts by creating a one centimeter, side-to-side arterio-venous fistula between the femoral artery and vein in the groin. These animals were euthanatized after another 2 months (2 months NF + 2 months HF, and 4 months NF).

Blood flow was monitored before and after graft placement 2 and 4 months postoperatively. The duplex scanning (Acuson 128) was used while the animals were under ketamine sedation. Data obtained from implantable Doppler crystals and telemetric monitoring have shown that ketamine does not alter blood flow.¹² Mean shear stress was calculated according to the modified Hagen-Poiseuille equation: =4Q/r³, where is blood viscosity (0.035 poise), Q is volume flow (mL/sec), and r is the vessel radius in centimeters.

Graft Retrieval and Morphology
All animals received bromodeoxyuridine (BrdU, 30 mg/kg) intramuscularly 17, 9, and 1 hour prior to sacrifice to determine the proliferation index. Specimens from the midportion of the grafts were immersed in methyl Carnoy's fixative or formalin for immunohistochemistry and morphometry. The intimal tissue was separated from the remaining portions of the grafts and was immediately added to 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), homogenized and stored at -70°C for later analysis of ribonucleic acid (RNA). Immunohistochemistry was performed with antibodies directed against smooth muscle -actin (Boehringer Mannheim), the endothelial form of cNOS (Transduction Lab), BrdU (Boehringer Mannheim), and von Willebrand factor (vWF) (Dako Corp). The sections were incubated one hour at room temperature with the primary antibody (BrdU, vWF, -actin) or overnight at 4° (cNOS). Biotinylated secondary antibodies (Vector Laboratories, Inc), avidin-biotinylated peroxidase, and diaminobenzidine/NiCl₂ were used as the chromogen and methyl green as the counterstain for nuclei. The number of cells positively stained for BrdU were counted and divided by the total number of nuclei per section to obtain a BrdU labeling index.

Morphometry
For morphometric measurements midgraft cross sections were stained with hematoxylin and eosin and projected onto a computerized digitizing pad with a camera lucida. To evaluate the relative contribution of SMCs and matrix, the percentage of the neointimal area occupied by SMCs was assessed. Paraffin embedded neointimas were reembedded in Epon for preparation of thin sections. Continuous photographs (8x10 in) were taken from the lumen to the outer surface of the intima at each of 4 quadrants of every graft using transmission electron microscopy. These were taken at an original magnification of x3000 and printed at a final magnification of x9000. A grid with 108 points, 2 cm apart was placed over the photograph and the number of points overlying SMCs were counted. This grid pattern was chosen to yield optimum sampling distribution.¹³ The percentage of neointimal area occupied by matrix was calculated as 100 minus the percentage occupied by SMCs.

Northern Analysis
Total RNA was extracted as described by Chomczynski and Sacchi.¹⁴ The samples were separated by electrophoresis and transferred to nylon blotting membranes (Zeta-Probe, Bio-Rad). Partial cDNA probes for human cNOS (0.9 kb) and the inducible isoform of NOS (iNOS; 0.3 kb) were kindly provided to us by Dr James Liao (Brigham and Womens Hospital, Boston). These probes were labeled with ³²P by nick translation, added to the hybridization solution (0.15 mol/L Na₂HPO₄, pH 7.2, 0.28 mol/L NaCl, 7.8% SDS, 1 mol/L EDTA, 50% formamide, 10% polyethylene glycol and 200 mg/mL of denatured salmon sperm DNA) and incubated at 42°C for 20 to 24 hours. Signals were detected by autoradiography (Kodak). To compare the intensity of signals on the same membrane a computerized phosphoimaging technique was used (PhosphorImager model 400S, Molecular Dynamics). As a control for equal loading between lanes, the membrane was rehybridized with a probe for 28S ribosomal RNA.

Statistics and Animal Care
Comparisons were made within animals between the side with high flow and the side with normal flow (paired students t test) and between animals from 2 and 4 months (Wilcoxon signed rank test). A statistically significant difference was considered at a value of P<.05. Data are expressed as mean±SEM.

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

	Results

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The blood flow in the aortoiliac grafts was 103±11 mL/min after graft placement. At 4 months the blood flow and mean shear stress were 603±64 mL/min and 60±4 dynes/cm² on the side of the fistula and 80±21 mL/min and 9±2dynes/cm² on the control side. The diameter of the vascular grafts remained constant under all conditions. There were no significant differences in blood pressure between grafts with normal and high blood flow.

The neointima in all grafts consisted of matrix and SMCs staining positive for -actin covered with a luminal lining of endothelium (vWF positive). The intimal area in the high shear stress grafts (2 months NF + 2 months HF) was significantly less than in the normal shear stress grafts (2 months NF or 4 months NF). The intimal thickening in normal shear stress grafts at 4 months was significantly larger than at 2 months (Fig 1 and Table). These results demonstrate that neointimal thickening regressed from 2 to 4 months as a response to increased shear stress, while the grafts subjected to normal shear stress thickened even further during the same period. No differences in the fraction of the intimal area occupied by SMCs were observed between groups by transmission electron microscopy (Table). The BrdU index for grafts from 2 months normal flow was significantly higher than for all other grafts excised after 4 months (Table). There was no significant differences in BrdU index between high-flow and normal-flow grafts at 4 months. As previously noted the majority of the proliferating SMCs were located near the lumen of the graft.⁵

View larger version (53K): [in this window] [in a new window]   Figure 1. Neointimal thickening in grafts subjected to (A) 2 months of normal flow, (B) 4 months of normal flow, and (C) 2 months of normal followed by 2 months of high flow. Arrowheads delineate the neointimal thickening; bar=1 mm.

View this table: [in this window] [in a new window]   Table 1. Morphology of Grafts

Northern blotting with cNOS demonstrated equal loading and a 4.3 kb transcript with a 4-fold increase of the message in the high flow grafts in comparison to aorta and normal flow grafts at 2 and 4 months (Fig 2). No expression of iNOS was observed. This difference was confirmed by immunohistochemistry. The endothelium at the luminal surface of high flow grafts exhibited increased staining for cNOS compared to the endothelium in normal flow grafts (Fig 3).

View larger version (88K): [in this window] [in a new window]   Figure 2. Northern blot demonstrating the expression of cNOS (4.3 kB) in the aorta and the intimas of 2 months normal plus 2 months high flow grafts, 2 months normal flow grafts, and 4 months normal flow grafts. The blot was also probed for 28 S ribosomal RNA. NF indicates normal flow; HF, high flow.

View larger version (79K): [in this window] [in a new window]   Figure 3. Immunohistochemical staining of graft intima with monoclonal antibodies directed against the endothelial form of constitutive nitric oxide synthase. A, 2 months normal flow (NF); B, 2 months normal flow followed by 2 months high flow (HF). Arrow indicates endothelial layer; bar=10 µm.

	Discussion

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These experiments were designed to test the hypothesis that increased shear stress could cause atrophy of an established neointima in PTFE grafts, a circumstance where vasodilation, the normal response to increased blood flow, can not occur. Neointimal thickening decreased markedly in response to an increase in blood flow due to loss of both SMCs and matrix. This decrease in neointimal mass was associated with increased expression of cNOS in the overlying endothelium.

Flow Effects on Intimal Thickening in PTFE Grafts
These observations extend previous work performed in this and other laboratories demonstrating that a decrease in shear stress is associated with an increase in intimal thickening and high shear stress with inhibition of intimal thickening in PTFE grafts. These experimental grafts have increased porosity, which allows them to heal by ingrowth of capillaries along their entire length. A complete endothelium with underlying smooth muscle cells forms by two weeks. Conventional grafts used clinically form such a neointima only at the ends where endothelium grows a few centimeters from the native vessel onto the PTFE surface. The effects of flow on neointimal growth in this model stand in contrast to the findings in normal blood vessels. Langille and O'Donnell¹⁵ have shown that a decrease in blood flow leading to a decrease in luminal diameter is initially reversible but later becomes fixed due to changes in wall structure. For example, SMC proliferation and cell death (apoptosis) are increased during the massive remodeling that occurs in the infrarenal aorta of sheep following parturition when blood flow decreases by 95%.^{16 17} However, in pathological states, the response of the blood vessel to flow may be markedly altered. Kohler and Jawien¹⁸ demonstrated that decreased blood flow causes increased vessel wall mass in injured rat carotid arteries. Increased wall thickening may have resulted from the denuded vessel's inability to vasoconstrict in response to decreased shear. In vein grafts and synthetic grafts, intimal thickening tends to appear in regions covered by endothelium. In both kinds of arterial substitutes, increased shear stress inhibits intimal thickening and a reduction of shear stress induces it.^{19 20 21}

In previous studies, we have observed SMC proliferation in the intima underlying the endothelium, but not deep in the intima or in the matrix even though there are substantial numbers of macrophages and other inflammatory cells surrounding the graft fabric.⁵ These findings suggest that the endothelium regulates this SMC growth. This concept is further supported by the observation that PTFE graft intima formed under conditions of high shear stress can be induced to thicken by switching from high to normal shear stress. Under these circumstances, SMC proliferation begins by 4 days and continues for one month. Analysis by BrdU labeling confirms that most proliferating cells are in the sub-endothelial region. Signals from the endothelium might cause this response since the switch in blood flow does not cause endothelial denudation or thrombosis. cNOS production is decreased²² and PDGF-A expression is increased.²³ SMCs may be responding both to a loss of growth inhibition and an increase in mitogenic stimulation. Proof that intimal growth is regulated by endothelial factors is still lacking.

Mediators of Flow-Induced Atrophy
We found that a switch from normal to high shear stress caused the intima overlying the graft to atrophy. Atrophy is the result of decreased cell mass (cell death or decreased proliferation) or decreased extracellular matrix (decreased synthesis or increased proteolysis) or both. In this study the percentage of intimal volume composed of SMCs (ca 20%) did not change. This relationship between SMC and matrix has been remarkably constant in graft intima under all flow conditions we have studied. The percentage of SMCs found in the intima was also the same as that found in intima of injured rat arteries under low or high flow.¹⁸ This result suggests a fundamental relationship between the SMCs and the amount of surrounding matrix they produce.

Many flow-regulated genes may be involved in wall atrophy including ones that are either induced or suppressed by high flow. We found an association between atrophy and increased cNOS expression. There are data to suggest that the metabolic product, NO, is one of the atrophy-inducing molecules. Numerous studies indicate that NO is the major physiological regulator of vasovascular tone and diameter. NO is also an important factor in vascular remodeling.²⁴ The addition of pharmacological amounts of L-arginine to the diet enhances NO production and inhibits intimal thickening in injured and atherosclerotic arteries and vein grafts.^{25 26 27} Intimal hyperplasia after injury is inhibited either by the administration of synthetic donors of NO or by transfer of cNOS gene into SMCs.^{8 9} NO also inhibits cell proliferation in vitro and can cause cell death under some circumstances.^{28 29 30 31} This molecule might also affect protein synthesis and degradation.^{32 33} Thus, NO could mediate flow-induced atrophy.

Can the observations of this study be generalized to other vessels? Similar effects of blood flow have been observed not only in the baboon but also in dogs and monkeys.^{34 35 36} It seems likely that increased shear stress may induce atrophy in rigid, diseased vessels. This process might be mediated by vasodilators like NO. For example, focal, high-grade stenoses are frequently associated with poststenotic dilatation of the relatively normal, distal segments. Vessel atrophy occurs in the dilated poststenotic aorta distal to experimental aortic coarctation.³⁷ High blood flow through a pathological stenosis might also cause atrophy of the tissue near the luminal surface. Thus, increased shear at the site of luminal narrowing may contribute to plaque rupture both by increasing the stress upon the wall and causing atrophy of the fibrous cap.^{38 39 40}

In summary, these studies demonstrate that elevated blood flow increases cNOS expression and atrophy of neointima in rigid vascular grafts. These findings may help us understand how vessel wall mass is regulated in advanced arteriosclerosis.

	Acknowledgments

 
These studies were supported by a Fogarty Award (E.J.M.) and NIH grant HL-30946 and RR00166. We thank W. L. Gore & Associates and Davis & Geck, Inc, for supplying the grafts and sutures. We thank Dr James Liao for providing us with a cDNA probe for human cNOS and iNOS, Holly Lea for technical assistance in the animal operations, and Monika Clowes, Rene Collman, and Stephanie Lara for assistance with the transmission electron microscopy.

Received February 4, 1997; accepted April 4, 1997.

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