Opioid growth factor inhibits intimal hyperplasia in balloon-injured rat carotid artery

Opioid growth factor inhibits intimal hyperplasia in balloon-injured rat carotid artery Ian S. Zagon, PhD,a Frank M. Essis, Jr, MS,a Michael F. Verderame, PhD,b Dean A. Healy, MD,c Robert G. Atnip, MD,c and Patricia J. McLaughlin, DEd,a Hershey, Pa Objective: The endogenous opioid [Met5]-enkephalin (opioid growth factor [OGF]) is a tonically active, receptormediated inhibitory growth peptide in developing and adult vasculature. This study was designed to determine the role of OGF in neointimal hyperplasia. Methods: The carotid artery in adult male Sprague-Dawley rats was denuded with balloon catheterization. OGF (10 mg/kg), the opioid antagonist naltrexone (NTX; 30 mg/kg), or saline solution (0.2 mL) was injected intraperitoneally daily for 28 days into the rats, and restenosis of the carotid artery was examined with morphometric analysis using Optimas software. Proliferation of the neointima and media was measured by radioactive thymidine incorporation over 3 hours. The presence of OGF and its receptor, OGFr, were examined with immunofluorescence microscopy. Results: OGF depressed DNA synthesis in the intima and media from 16% to 78% of control levels in the first 2 weeks after deendothelialization, whereas NTX exposure elevated DNA synthesis by 21% to 89%. OGF action was receptor-mediated. In the month after injury the thickness of the intima in OGF-treated rats was decreased by 18% to 31% from control values, whereas intimal thickness was increased in the NTX group by 10% to 31%. Luminal area was almost 25% greater than control values in the OGF group, but was reduced 17% by NTX. OGF and the OGF receptor were detected in the carotid artery with immunohistochemistry. Conclusions: These results demonstrate for the first time that a native opioid system modulates repair of vascular injury. OGF is a constitutively active peptide that has a receptor-mediated action in the negative regulation of neointimal growth, a major cause of restenosis. (J Vasc Surg 2003;37:636-43.)

The wall of arteries is a complex, multicellular structure that is important in regulation of inflammation, coagulation, and regional blood flow.1,2 Vascular smooth muscle cells (SMC), mainly located in the tunica media of the artery, are important modulators of vascular tone and blood pressure in some blood vessels. Injury to the arterial wall results in migration, proliferation, and deposition of extracellular matrix material by SMCs, leading to intimal hyperplasia.1-3 This neointimal SMC proliferative response contributes to the pathogenesis of several cardiovascular disorders, including atherosclerosis.4 Neointimal hyperplasia is not only a major cause of early restenosis after intervention in the cardiovascular system but is associated with hemodynamically significant restenosis at the endarterectomy site in patients undergoing carotid endarterectomy and is a major factor in graft failure in the coronary and peripheral arterial systems.5,6 Many growth factors influence proliferation of vascular SMCs in vitro and in vivo,7-10 From the Departments of Neuroscience and Anatomy,a Medicine,b and Surgery,c The Pennsylvania State University College of Medicine. Supported in part by the Laverty Foundation. Competition of interest: none. Presented at the American Society of Cell Biology, Washington, DC, December 11-15, 1999. Reprint requests: Ian S. Zagon, PhD, Department of Neuroscience and Anatomy, H-109, The Pennsylvania State University, The M. S. Hershey Medical Center, 500 University Dr, Hershey, PA 17033 (e-mail: [email protected]). Copyright © 2003 by The Society for Vascular Surgery and The American Association for Vascular Surgery. 0741-5214/2003/$30.00 ⫹ 0 doi:10.1067/mva.2003.165

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but a clear understanding of this process is needed to design strategies for prevention and treatment. Endogenous opioid peptides are potent regulators of growth11-16 and neuromodulators17 and may participate in pial artery dilation.18 One native opioid peptide, [Met5]enkephalin, encoded by the preproenkephalin gene, is a negative growth regulator in development, cellular renewal, wound healing, cancer, and angiogenesis.12 To distinguish the role of [Met5-enkephalin as a growth factor in neural and nonneural cells and tissues and in prokaryotes and eukaryotes, this peptide was termed opioid growth factor (OGF).12 This endogenous opioid peptide is a constitutively active, autocrine expressed, and secreted inhibitory growth factor with broad action and function. OGF displays activity at physiologically relevant concentrations, and it does not elicit physical dependence, tolerance, or withdrawal. This peptide has a direct, rapid, prolonged, stereospecific, receptor-mediated, noncytotoxic, reversible influence on growth in vitro and in prokaryotic and eukaryotic organisms. At least three biological processes, ie, cell proliferation, cell migration, and tissue organization, are associated with OGF activity. Blockade of the interaction of OGF with its receptor, OGFr, with antibody neutralization, opioid antagonist (naltrexone [NTX]), or antisense technology enhances cell replication, migration, and organizational cues, suggesting that OGF is tonically active. OGFr has been cloned and sequenced, and human OGFr has a chromosomal location at 20q13.3.12 Although OGF has receptor-mediated action that is pharmacologically similar to characteristics of an opioid receptor (eg, naloxone [NAL] reversible, stereospecificity), the receptor has no

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homology to classical opioid receptors but is distinctly different in structure.12 Gene and protein expression for OGF have been documented in endothelial cells, smooth muscle cells, and fibroblasts in the neonatal and adult rat aorta,13,14 and this autocrine-produced peptide inhibits DNA synthesis in the newborn rat aorta.14 Moreover, OGF is a tonically active negative growth regulator in measures of angiogenesis15,16 and has a differential effect on arteries and veins.16 The question can be raised as to whether endogenous opioids, specifically OGF, have a role in wound repair of vasculature. Using the well-documented balloon catheterization model of the common carotid artery in the rat,3 and recognizing the limitations of this model,3 the effects of excess agonist (OGF) and continuous blockade of opioid-receptor interactions (NTX) during remodeling on DNA synthesis were evaluated. Receptor mediation of OGF action also was evaluated during healing of injured carotid artery. The influence of OGF and NTX on cross-sectional morphometric analysis of intimal area, ratio of intima-media area, and percent luminal narrowing were examined. Finally, to ascertain the presence of OGF and OGFr in the carotid artery, immunohistochemistry was used. METHODS Animals and surgery. The arterial injury model used in this study followed the technique of Clowes et al.19,20 Adult male Sprague-Dawley AVF rats, weighing 350 to 450 g, were obtained from Charles River Laboratories (Wilmington, Mass). Rats were anesthetized with intraperitoneal injection of xylazine (10 mg/kg) and ketamine (50 mg/kg), and the common, internal, and external carotid arteries were exposed bilaterally through a midline neck incision. Arteriotomy was performed in the left external carotid artery to introduce a 2F Fogarty balloon catheter into the common carotid artery and advance it to the aortic arch. The balloon was inflated and passed three times with gentle resistance along the length of the carotid artery to denude the endothelium and injure the innermost layers of the medial smooth muscle of the common carotid artery. The balloon catheter was removed and the external carotid artery was ligated. Drug treatment. Immediately after balloon angioplasty, rats were randomly assigned to groups receiving daily (9:00-10:00 AM) intraperitoneal injections of [Met5]enkephalin (OGF, 10 mg/kg), OGF (10 mg/kg) and NAL (20 mg/kg), NAL (20 mg/kg), NTX (30 mg/kg), or an equivalent volume of sterile water (control). A dose of 10 mg/kg of OGF markedly depresses DNA synthesis in developing vascular cells.14 Although the pharmacokinetics of this dosage of OGF are not known, a study21 monitoring OGF with high-pressure liquid chromatography in rats showed that injections of 40 mg/kg of OGF into adult rats persisted for at least 14 hours. Extrapolation to a dosage of 10 mg/kg OGF could suggest that this peptide is present for at least 3 to 4 hours. NTX at 30 mg/kg was selected because this dose blocks morphine-induced anti-nociception for at least 24 hours.22 A dose of 10 mg/kg of the

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short-acting (effective duration of action, 1 hour) and lowpotency opioid antagonist NAL was used because it blocks the effects of OGF without having any influence on vascular growth (eg, DNA synthesis) when administered alone.14 Autoradiography. At least 6 rats from each treatment group were examined at 1, 2, 4, 7, 14, and 28 days after surgery. On the day of collection, rats were injected with [3H]-thymidine (0.5 mCi per rat per injection; 20 Ci/ mmol, New England Nuclear, Boston, Mass) every hour for 3 hours (three injections, for a total of 1.5 mCi). Animals were anesthetized with 0.9 mL of sodium pentobarbital, and 10% neutral buffered formalin solution was perfused through the left ventricle at 120 mm Hg of pressure. Arteries were removed and fixed with immersion in buffered formalin solution for 18 hours. The specimens were embedded in paraffin, and sections (8 ␮m in cross section) were processed for autoradiography. After a 14day incubation at 4° C, the tissues were developed and counterstained with hematoxylin-eosin. Labeling indexes. With sections taken from the center of the injured segment, at least 300 cells were counted in each section from five sections per rat (six rats per group per time point). The labeling index was determined as the ratio of labeled cells to total number of cells; only cells with nuclei in the intima and media were counted. Labeled cells were those cells with four or more grains (background was less than or equal to one grain per cell). Morphometric analysis. Images of the cross sections of the carotid arteries were captured with a Sony CCD camera attached to an Olympus BH-2 microscope with Adobe Photoshop 4.0. The thickness of the intima and of the media were measured independently with Optimas 6.0 software according to previous methods.8,23-25 For each section, four random noncontiguous microscopic fields were examined, and the mean thickness of the intima and media were determined; at least two nonadjacent sections per carotid artery, six rats per group per time point were used. The ratio of the thickness of intima to thickness of media was calculated at each age to control for the plane of section, interanimal variation, and variation in arteries. On day 14 after de-endothelialization, the areas of the intima and media were measured in two sections per animal, and 3 animals per group, with the Optimas program. Medial area was calculated by subtracting the area defined by the internal elastic lamina from the area defined by the external elastic lamina, and intimal area was determined as the area defined by the luminal surface and the internal elastic lamina.8 In addition, the ratio of area of intima to area of media was calculated. Luminal opening. To determine whether the vessel size had changed because of alterations in the outer wall rather than the lumen, area and perimeter measurements of the inside and outside dimensions of vessels were taken from cross sections of the wounded arteries at 14 and 28 days after surgery with at least two nonadjacent sections per carotid artery, six rats per group. Immunohistochemistry. To examine the localization of OGF and OGFr in carotid arteries, fresh tissue was

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Fig 1. Histograms of labeling indexes in intima and media of carotid arteries after injury. Adult male rats received daily injections of NTX (30 mg/kg), OGF (10 mg/kg), or equivalent volume of sterile water (control) beginning at the time of injury. Data represent mean ⫾ SEM for five sections per animal, six animals per group; at least 300 cells were counted for each section. Dotted line, Baseline cell turnover in an uninjured rat carotid artery. *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001.

harvested from naive rats and immunohistochemical procedures were performed as described.14 Tissues were incubated for 16 to 18 hours at 4° C with primary antibodies (1:100) to either OGF (CO-172) or OGFr (AO-440); characterization of each polyclonal antibody has been published.14 Secondary antibodies were rhodamine-conjugated goat anti-rabbit immunoglobulin G (1:100). Control sections were those stained with antibodies preabsorbed with an excess of the respective antigen and sections stained with only secondary antibody. To identify SMCs and to examine for the presence of endothelial cells, carotid arteries at 14 days after balloon catheterization and daily injection of OGF, NTX, or sterile water were stained with a monoclonal mouse anti-human smooth muscle actin antibody (clone 1A4) or rabbit antihuman von Willebrand factor (DAKO Corp, Carpinteria, Calif). Statistics. All experiments were performed by investigators blinded to the data. Data on labeling indexes and morphometric analysis were compared within each time point with one-way analysis of variance, and subsequent comparisons were made with Newman-Keuls multiple comparisons tests (GraphPad Prism, San Diego, Calif).

RESULTS DNA synthesis. The role of endogenous opioids on DNA synthesis after balloon angioplasty was examined over 28 days. Labeling indexes in the intima and media from animals injected over the long term with either OGF or NTX are presented in Fig 1. On days 1 and 2 after surgery there was no appearance of an intima at the light microscopic level of resolution. At 4 days after injury and for at least an additional 10 days, prolonged NTX treatment elevated DNA synthesis by 35% to 62% from control levels. OGF had a profound effect of depressing DNA synthesis, with decrease from control values of 78% at 4 days, 60% at 7 days, and 32% at 14 days after injury. At 4 weeks after balloon catheterization, labeling index in the carotid artery was at baseline value (1.40% ⫾ 0.02%) for all groups of rats. Changes in DNA synthesis in the media of the carotid artery of animals subjected to denuding of the endothelium were recorded within 1 day of injury. In NTX-treated animals, the number of cells incorporating radioactive thymidine increased, ranging from 21% to 89% over control levels. In animals receiving OGF, labeling index was decreased, ranging from 16% to 67% of control values during the first 2 weeks after injury. At 4 weeks after injury,

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labeling index in all groups was comparable with control values (less than 1%). The opioid antagonist NTX significantly increased thymidine incorporation within 24 hours of surgery, with NTX-treated rats having 89% more cells undergoing DNA synthesis than control counterparts. The NTX group had a significantly higher labeling index than did the control group through the first week after injury, ranging from 21% to 89% greater. At 1 month after balloon catheterization, the level of DNA synthesis in the media was comparable between the control group and NTX-treated rats. Receptor mediation. To inquire whether the effects of OGF on DNA synthesis in the injured carotid artery were receptor mediated in the intima and media, specimens at 4 days after injury from animals receiving OGF, OGF and the short-acting opioid antagonist NAL, and NAL alone were examined (Fig 2). OGF inhibited DNA synthesis in the intima and media by 78% and 16%, respectively, from control values, whereas neither OGF and NAL nor NAL alone had any effect on labeling index. Morphometric analysis. The structural repercussions of treatment with OGF and NTX on the carotid artery after balloon catheterization were analyzed at days 4, 7, 14, and 28 after injury (Figs 3 to 5). Gross changes in the thickness of the media were not noted between groups, but the intima of the OGF-exposed group was subnormal by 31% at 7 days , 26% at 14 days, and 18% at 28 days. The thickness of the intima of the NTX-treated group was increased from control levels by 31% at 7 days, 25% at 14 days, and 10% at 28 days. The ratio of intima thickness to media thickness in the repairing carotid artery in rats subjected to OGF was subnormal, by 25% at 7 days, 20% at 14 days, and 15% at 28 days (Fig 3). The ratio of intima to media thickness in the carotid artery of rats treated with NTX was elevated from control values, by 28% at 7 days, 16% at 14 days, and 11% at 28 days after injury (Fig 3). Results of immunohistochemistry revealed that the neointima in the OGF, NTX, and control groups at 14 days after injury stained with antibodies to smooth muscle actin (data not shown), arguing that the cells of the media, not the adventitial elements, make a major contribution to vascular remodeling. The luminal border of the wounded carotid artery was not composed of cells stained with von Willebrand factor, suggesting that these neointimal cells were derived from the SMCs.3 At 14 days after wounding of the carotid artery, calculation of the ratio of intima-media area showed a 79% reduction from control values (2.6 ⫾ 0.2) for the OGF group and a 62% increase in the NTX-treated specimens; these differences were statistically significant (P ⬍ .01). At the conclusion of the experiments 1 month after injury, the area and circumference of the lumen in the OGF group were increased 23% and 28%, respectively, from control levels (Fig 4). The NTX group had a significantly smaller lumen compared with the control group in area and circumference, with reductions of 17% and 4%, respectively. Expression of OGF and OGFr. Assessment of OGF and OGFr expression in the carotid artery showed that both peptide and receptor had a similar pattern of immunoreac-

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Fig 2. Histograms of labeling indexes in intima and media of carotid arteries 4 days after injury. Adult male rats received daily injections of OGF, 10 mg/kg; OGF, 10 mg/kg, and NAL, 20 mg/kg; NAL alone, 20 mg/kg; or equivalent volume of sterile water (control). Data represent mean ⫾ SEM for five sections per animal, six animals per group; at least 300 cells were counted for each section. *P ⬍ .05; ***P ⬍ .001.

tivity in the carotid artery (Fig 6). The cytoplasm of endothelial, smooth muscle, and fibroblast cells was immunoreactive, and staining extended into the cell processes (Fig 6, A, C). Little immunoreactivity was recorded in the nuclei of these cell types. No staining was detected in control specimens processed with antibody preabsorbed with respective antigens or in samples incubated with secondary antibody only (Fig 6, B, D). DISCUSSION Modulation of a native opioid-receptor system has a profound effect on cell replication after injury of an artery. In a well-documented model of arterial wounding,3,19,20 application of exogenous OGF depressed DNA synthesis during the course of healing, suggesting that endogenous OGF (as shown at immunohistochemistry to be present in these carotid arteries) acted as a negative growth regulator, a function of OGF reported earlier in the homeostasis of cellular renewal of neonatal rat aorta.14 OGF had a recep-

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Fig 3. Ratio of intima to media thickness in carotid artery after injury. Animals received daily injections of NTX, 30 mg/kg; OGF, 10 mg/kg; or equivalent volume of sterile water (control) beginning at the time of injury. Data represent mean ⫾ SEM for five sections per animal, six animals per group. *P ⬍ .05; **P ⬍ .01.

Fig 4. Area and circumference of lumen of carotid artery at 28 days after injury. Animals received daily injections of NTX, 30 mg/kg; OGF, 10 mg/kg; or equivalent volume of sterile water (control) beginning at the time of injury. Data represent mean ⫾ SEM of measurements from five sections per animal, six animals per group. ***P ⬍ .001.

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Fig 5. Photomicrographs of cross sections through rat carotid artery 14 days after balloon angioplasty. Animals received daily injections of sterile water in equivalent volume to OGF or NTX (control; A and B); OGF, 10 mg/kg (C and D); or NTX, 30 mg/kg (E and F) beginning at the time of injury. Sections were stained with hematoxylin-eosin. ADV, Adventitia; MED, media; NEO, neointima; LU, lumen. Bar, A, C, E, 1.8 mm; B, D, F, 0.6 mm.

tor-mediated effect on DNA synthesis in the carotid artery subjected to denudation of the endothelium, because the low-potency, short-acting opioid antagonist NAL neutralized OGF when given concomitantly, but NAL given once at this dosage alone had no effect. This finding was consonant with the observation of OGFr in the carotid artery at immunohistochemistry. Exposure of the injured carotid artery to the potent, long-acting opioid antagonist NTX with a dosage that blocks opioid-receptor interaction11 increased DNA synthesis levels; since the duration of opioid-receptor blockade rather than dosage is a determinant of opioid-induced modulation of growth,22 other opioid antagonists (eg, NAL) would be expected to have a similar effect, given the appropriate conditions. Although NTX blocks classical (␮, ␦, ␬) and nonclassical (OGFr) receptors, only OGF is related to cell replication.14 Therefore OGF must be a tonically active and constitutively expressed growth factor, because displacement of OGF from the cellular elements in the injured intima and media undergo-

ing DNA synthesis by NTX resulted in increased cell replication. Thus, although the native opioid peptide OGF has a role in vasculogenesis, angiogenesis, and homeostatic cellular renewal of blood vessels, and does so at the locus of OGFr,14-16 we now see for the first time that the OGFOGFr axis is important in regulation of DNA synthesis for repair of injured vasculature. The total effect that the OGF-OGFr axis exerts on cell replication is sizable. The activity of endogenous OGF on cell replication can be estimated by summation of the effects of opioid-receptor blockade with NTX and those of exogenously administered OGF. Specifically, an increase in radioactive thymidine incorporation in only 3 hours ranged from 35% to 62% in the intima during the first 2 weeks after injury, and from 21% to 89% in the media over the same period. The capacity of exogenous OGF, presumably acting by saturating sites where endogenous peptide is absent, must be given consideration. Exposure to OGF decreased DNA synthesis by 32% to 78% in the intima and by 16% to

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Fig 6. Photomicrographs of immunohistochemical preparations of cross sections through uninjured carotid artery. Preparations were stained with antibodies to OGF (A and B) or OGFr (C and D), with immunoreactivity denoted by arrow. Control sections were stained with antibodies preabsorbed with pure antigens (B and D). LU, Lumen; INT, intima; MED, media; ADV, adventitia. Bar, 0.5 mm.

67% in the media during the first 2 weeks after balloon catheterization. Thus summing the effects of endogenous and exogenous OGF activity yields a cumulative effect on growth as great as 146% in the injured intima and media within 3 hours of [3H]-thymidine administration. These calculations confirm that the OGF-OGFr axis has a profound and important regulatory effect in governing the equilibrium of cellular duplication after de-endothelialization. Given that we may only be looking at a daily 3-hour to 4-hour window of OGF exposure estimated from previous information,21 more extended exposure and elimination of intermittent repercussions from OGF treatment could yield even greater changes. Thus an abundance or deficiency in OGF could have dramatic consequences on DNA synthesis of cells in the blood vessel during repair. Alterations in DNA synthesis induced with manipulation of OGF-OGFr interfacing were manifested in changes in the magnitude of wound healing of the carotid artery. In OGF-treated animals the area of the lumen was approximately 25% larger than in the control group within 1 month of injury. This increase in area of the lumen of the carotid artery in animals subjected to OGF beginning at the time of balloon catheterization was due to reduction in the thickness and area of the intima as a result of cumulative reduction in cell proliferation and possibly diminishment in differentiation processes in the reestablished intima. Thus exposure to OGF in the process of wound repair of this

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artery diminished cell replication without altering luminal dimensions, suggesting enhancement of hemodynamic processes. In the case of animals in which OGF-OGFr interactions were continuously blocked by the opioid antagonist NTX, the area of the lumen was decreased by 17%. This reduction in luminal area was the result of notable increases in neointimal wall thickness and area. These data indicate that disruption of OGF-OGFr interaction can exacerbate events related to neointimal proliferation and lead to diminished luminal size, thereby restricting blood flow. At least four distinct processes contribute to restenosis: proliferation of SMCs, migration of SMCs from the vessel media to the intima, deposition of extracellular matrix material by SMCs, and contraction of neointimal tissue by SMCs.19,20,26-28 With application of this knowledge to OGF modulation of blood vessel repair, our studies clearly demonstrate that the OGF-OGFr axis has a profound influence on DNA synthesis of cells positive for smooth muscle actin, putative SMCs. We found the temporal sequence of an increase in cell replication in the media preceding that in the intima was consistent with the results of other investigators.3,19 Moreover, OGF-OGFr interactions can now be seen to affect cell replication in both the media (giving rise to cells feeding the intima) and the intima, and to do so in a manner consonant with the timetable of migration and cellular repopulation. Only the magnitude of DNA synthesis in the media and intima of rats treated with OGF or NTX deviated from control levels. These results infer that cell proliferation is a prime target for OGF activity in regard to blood vessel repair. Although cell migration in the corneal epithelium is influenced by OGF,29 it remains to be seen whether cell migration from media to intima in the injured artery is also affected by the OGF-OGFr axis. From studies in other cells and tissues, it is known that OGF is not cytotoxic29 and does not involve necrotic or apoptotic pathways.30 In fact, OGF appears to be a cytostatic agent and to act on the cell cycle by prolonging the G0/G1 phase.31 NTX increases the number of cells in DNA synthesis by shortening the timetable of the cell cycle, particularly the G0/G1 phase and recruitment of cells into the S phase, but does not alter survival time.31 Thus OGF appears to be a tonically active inhibitory growth factor that is targeted to cell proliferative events and does not decrease cells through cytotoxicity. To date, many growth factors have been reported to also act through proliferative pathways, but many stimulate vascular smooth muscle proliferation (eg, IGF-1, acidic-fibroblast growth factor, basic-fibroblast growth factor, platelet-derived growth factor).3,32 However, only a few (eg, heparin, retinoblastoma protein) inhibit vascular proliferation,33,34 and the mechanisms of these compounds need to be ascertained before comparisons and relationships can be drawn to OGF. Our data extend knowledge that the OGF-OGFr axis is important in vasculogenesis and angiogenesis,14-16 but manipulation of this system in the course of vessel repair may have dramatic consequences on vessel architecture that will ultimately contribute to enhancing circulatory properties.

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It appears that the inhibitory capabilities of the autocrine loop involved with OGF-OGFr interfacing on cell proliferation is disrupted by injury and that sufficient OGF cannot be made available to maintain equilibrium for cell replication. Although animal paradigms such as the rat carotid injury model have limitations, eg, lack of atherosclerotic plaque, they do offer insight into the pathophysiologic mechanisms of intimal hyperplasia.3 Thus OGF treatment to restore negative regulation may be of therapeutic benefit in such circumstances. Further studies are needed. We thank Mr. Sprague W. Hazard III for technical assistance in performing some of the immunohistochemical procedures, and Mr. James D. Wylie for some of the morphometric analysis. We are grateful to Dr. James P. Bartek, Department of Surgery, The Pennsylvania State University, for performing some of the balloon catheterization procedures. REFERENCES 1. Ross R. The pathogenesis of atherosclerosis: A perspective for the 1990s. Nature 1993;362:801-9. 2. Ross R. Atherosclerosis: An inflammatory disease. N Engl J Med 1999; 340:115-26. 3. Kraiss LW, Clowes AW. Response of the arterial wall to injury and intimal hyperplasia. In: Sidawy AN, Sumpio BE, DePalma RG, editors. The basic science of vascular disease. Armonk, NY: Futura Publishing; 1997. p 289-317. 4. Ip JH, Fuster V, Badimon L, Taubman MB, Chesebro JH. Syndromes of accelerated atherosclerosis: Role of vascular injury and smooth muscle proliferation. J Am Coll Cardiol 1990;15:1667-87. 5. Lafont A, Guzman LA, Whitlow PL, Goormastic M, Cornhill JF, Chisolm GM. Restenosis after experimental angioplasty: Intimal, medial, and adventitial changes associated with constrictive remodeling. Circ Res 1995;76:996-1002. 6. Healy D, Zierler R, Nicholls S, Clowes A, Primozich J, Bergelin R, et al. Long-term follow up and clinical outcome of carotid restenosis. J Vasc Surg 1989;10:662-9. 7. Ferns GA, Raines EW, Sprugel KH, Motani AS, Reidy MA, Ross R. Inhibition of neointima smooth muscle accumulation after angioplasty by an antibody to PDGF. Science 1991;253:1129-32. 8. Smith JD, Bryant SR, Couper LL, Vary PH, Gotwals PJ, Koteliansky VE, et al. Soluble transforming growth factor-beta type II receptor inhibits negative remodeling, fibroblast transdifferentiation, and intimal lesion formation but not endothelial growth. Circ Res 1999;84:121222. 9. Niemann-Jonsson A, Ares MPS, Yan S-Q, Bu D-X, Fredrikson GN, Branen L, et al. Increased rate of apoptosis in intimal arterial smooth muscle cells through endogenous activation of TNF receptors. Arterioscler Thromb Vasc Biol 2001;21:1909-20. 10. DeYoung MB, Clifford T, Dichek DA. Plasminogen activator inhibitor type 1 increases neointima formation in balloon-injured rat carotid arteries. Circulation 2001;104:1972-77. 11. Zagon IS, McLaughlin PJ. Opioid growth factor receptor in the developing nervous system. In: Zagon IS, McLaughlin PJ, editors. Receptors in the developing nervous system. Vol 1: Growth factors and hormones, London: Chapman and Hall; 1993. p 39-62. 12. Zagon IS, Verderame MF, McLaughlin PJ. The biology of the opioid growth factor receptor (OGFr). Brain Res Rev 2002;38:351-76. 13. Wu Y, McLaughlin PJ, Zagon IS. Ontogeny of the opioid growth factor, [Met5]-enkephalin, preproenkephalin gene expression, and the ␨ opioid receptor in the developing and adult aorta of rat. Dev Dynamics 1998;211:327-37.

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14. Zagon IS, Wu Y, McLaughlin PJ. Opioid growth factor-dependent DNA synthesis in the neonatal rat aorta. Am J Physiol 1996;270:R2232. 15. Blebea J, Mazo JE, Kihara TR, Vu JH, McLaughlin PJ, Atnip RC, et al. Opioid growth factor modulates angiogenesis. J Vasc Surg 2000;32: 364-73. 16. Blebea J, Vu JH, Assadnia S, McLaughlin PJ, Atnip RC, Zagon IS. Differential effects of vascular growth factors on arterial and venous angiogenesis. J Vasc Surg 2002;35:532-8. 17. Akil H, Watson SJ, Young E, Lewis ME, Katchaturian H, Walter JM. Endogenous opioids: Biology and function. Annu Rev Neurosci 1984; 7:233-55. 18. Armstead WM. Role of opioids in hypoxic pial artery dilation is stimulus duration dependent. Am J Physiol 1998;275:H861-7. 19. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. I: Smooth muscle growth in the absence of endothelium. Lab Invest 1983;49:327-33. 20. Clowes AW, Reidy MA, Clowes MM. Mechanism of stenosis after arterial injury. Lab Invest 1983;49:208-15. 21. Zagon IS, Wylie JD, Hurst WJ, McLaughlin PJ. Transplacental transfer of the opioid growth factor, [Met5]-enkephalin, in rats. Brain Res Bull 2001;55:341-6. 22. Zagon IS, McLaughlin PJ. Duration of opiate receptor blockade determines tumorigenic response in mice with neuroblastoma: A role for endogenous opioid systems in cancer. Life Sci 1984:35:409-16. 23. Capron L, Janet J, Heudes D, Joseph-Monrose D, Bruneval P. Repeated balloon injury of rat aorta: A model of neointima with attenuated inhibition by heparin. Arterioscler Thromb Vasc Biol 1997;17:164956. 24. Buchwald AB, Unterberg C, Nebendahl K, Grone H-J, Wiegand V. Low-molecular-weight heparin reduces neointimal proliferation after coronary stent implantation in hypercholesterolemic minipigs. Circulation 1992;86:531-7. 25. Olivetti G, Anversa P, Melissari M, Loud AV. Morphometric study of early postnatal development of the thoracic aorta in the rat. Circ Res 1980;47:417-24. 26. Clowes AW, Clowes MM, Reidy MA. Kinetics of cellular proliferation after arterial injury. III: Endothelial and smooth muscle growth in chronically denuded vessels. Lab Invest 1986;54:295-303. 27. Clowes AW, Schwartz SM. Significance of quiescent smooth muscle migration in the injured rat carotid artery. Circ Res 1985;56:139-45. 28. Fingerle J, Au YP, Clowes AW, Reidy MA. Intimal lesion formation in rat carotid arteries after endothelial denudation in absence of medial injury. Arteriosclerosis 1990;10:1082-7. 29. Zagon IS, Sassani JW, McLaughlin PJ. Opioid growth factor modulates corneal epithelial growth in tissue culture. Am J Physiol 1995;268: R942-50. 30. Zagon IS, Wylie, JD, McLaughlin PJ. Opioid growth factor (OGF) does not mediate growth through apoptotic and/or necrotic pathways [abstract]. Society Neuroscience 2002, 428.4. 31. Zagon IS, Roesener CD, Verderame MF, Ohlsson-Wilhelm BM, Levin RJ, McLaughlin. Opioid growth factor regulates the cell cycle of human neoplasias. Int J Oncol 2000;17:1053-61. 32. Newby AC, George SJ. Proposed roles for growth factors in mediating smooth muscle proliferation in vascular pathologies. Cardiovasc Res 1993;27:1173-83. 33. Chang MW, Barr E, Seltzer J, Jiang Y-Q, Nabel GJ, Nabel EG, et al. Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product. Science 1995;267:518-22. 34. Welt FGP, Woods TC, Edelman ER. Oral heparin prevents neointimal hyperplasia after arterial injury: Inhibitory potential depends on type of vascular injury. Circulation 2001;104:3121-4. Submitted Jun 14, 2002; accepted Sep 17, 2002.