Orally active endothelin receptor antagonist BMS-182874 suppresses neointimal development in balloon-injured rat carotid arteries

Vascular smooth muscle cell (SMC) proliferation is an important component in the development of restenosis. Because endothelin (ET) has been reported to act as an SMC mitogen, we postulated that the orally active ETA receptor antagonist BMS-182874 would suppress the development of the intimal lesion that develops in rat carotid arteries after balloon injury. Using cultured rat aortic SMC, we noted that ET-1-stimulated increases in [3H]thymidine incorporation were blocked by BMS-182874. To determine the effect of the drug on intimal lesion formation, we treated rats with BMS-182874 (100 mg/kg orally, p.o.) or vehicle once daily for 3 weeks, beginning 1 week before balloon injury. Two weeks after injury, drug-treated rats had a 35% decrease in lesion area and a 34% decrease in the lesion/media ratio as compared with the vehicle-treated rats. In situ hybridization (ISH) analysis of balloon-injured rat carotid arteries showed an increase in ETA receptor mRNA. These data support the concept that ETA receptor activation contributes to intimal lesion formation by promotion of SMC proliferation and suggest a potential use for ETA receptor antagonists in the amelioration of hyperproliferative vascular diseases, including restenosis.     

In vivo adenoviral vector-mediated gene transfer into balloon-injured rat carotid arteries

Homozygotes for the dsy1 desynaptic mutant of maize show massive failure of chiasma maintenance during diplotene and diakinesis. Although some chiasmata persist until anaphase I in most microsporocytes expressing this mutant, homozygotes are completely or nearly completely sterile, owing apparently to disjunctive irregularities. Pachytene synaptic errors and some synaptic failure also are found, but recombination nodules are common in homologously synapsed regions, and equational separation of a heterozygous knob into univalents or open arms at diakinesis clearly demonstrates that chiasma failure occurs following crossing-over. A wider than normal synaptonemal complex central region and uniform apparent weakness of central region cross connections to spreading procedures strongly suggest the presence of a genetic lesion in a synaptonemal complex central region component. The dsy1 mutant may provide an especially important source of material for molecular studies on the nature of chiasma maintenance mechanism.     

Adenovirus-mediated gene transfer of the human TIMP-1 gene inhibits smooth muscle cell migration and neointimal formation in human saphenous vein

Neointimal formation involving smooth muscle cell (SMC) migration and proliferation is a common feature of atherosclerosis, restenosis after angioplasty, and vein graft intimal thickening. Extracellular matrix remodeling by metalloproteinase (MMP) enzymes is an essential component of neointimal formation and therefore MMPs are a potential target for localized gene therapy. To evaluate this concept using human tissue, we used the highly reproducible organ culture model of neointimal formation in human saphenous vein to investigate the effect of adenovirus-mediated gene transfer of tissue inhibitor of metalloproteinase 1 (TIMP-1) and the bacterial LacZ gene (RAd35) as a control. Incubating veins with 100 microl of RAd35 (1.2 x 10(10) pfu/ml) led to expression of LacZ in 39 +/- 7% of surface cells but had no effect on SMC proliferation, migration, or neointimal formation. Similar infection with RAdTIMP-1 increased explanation of TIMP-1 in surface cells and significantly inhibited neointimal formation and SMC migration after 14 days by 54% and 78%, respectively (n = 6, p < 0.05 Student's paired t test). No effect on SMC proliferation or deleterious effect on cell viability was observed. A specific MMP inhibitory effect was detected using in situ zymography. These data confirm the importance of MMPs in neointimal formation and highlight the potential for application of TIMP gene therapy.     

Local expression of C-type natriuretic peptide markedly suppresses neointimal formation in rat injured arteries through an autocrine/paracrine loop

BACKGROUND: In vivo gene transfer into injured arteries may provide a new means to facilitate molecular understanding of and to treat the intractable fibroproliferative arterial diseases. Selection of an optimal molecule to be transferred will be a key to successful gene therapy in the future. We tested the hypothesis that a secreted multifactorial molecule should act more efficiently through an autocrine/paracrine loop to suppress neointimal formation elicited in injured arteries than a simple growth-inhibiting molecule that might be expressed inside cells. METHODS AND RESULTS: We constructed an adenoviral vector (AdCACNP) expressing C-type natriuretic peptide (CNP), a secreted stimulator of membrane-bound guanyl cyclase. AdCACNP directs cells to secrete large quantities of biologically active CNP. Serum-stimulated DNA synthesis and cell proliferation were only moderately suppressed in arterial smooth muscle cells infected with AdCACNP in vitro. However, when AdCACNP was applied to balloon-injured rat carotid arteries in vivo, neointimal formation was markedly reduced (90% reduction) in an infection-site-specific manner without an increase in plasma CNP level. CONCLUSIONS: Our results showed that CNP, a secreted multifactorial molecule, was indeed effective in suppressing fibroproliferative response in injured arteries and suggest that the potent antiproliferation effect may not be the most critical factor for the effective suppression of neointimal formation. An adenovirus-mediated expression of CNP could be an effective and site-specific form of molecular intervention in proliferative arterial diseases.     

Adenovirus-mediated in vivo gene transfer and expression in normal rat pancreas

Growth hormone (GH) secretion declines during normal aging along with reproductive activity in mammalian species. Various behavioral changes also occur in aged animals. In these experiments we have studied the effects of GH administration on behavioral and endocrine alterations exhibited by aged (18 months old) female rats of the Sprague-Dawley strain. Animals were selected showing at least 2 weeks of cornified vaginal smears (constant estrous) and treated with GH (0.1 mg/kg SC) daily for 8 weeks. Vaginal smears performed during the drug treatment revealed a recovery of estrous cycle in 60% of animals. GH treatment was also followed by an increased acquisition of shuttle-box active avoidance behavior and a facilitated retention of passive avoidance response. Compared to saline-injected controls, female rats treated with GH also exhibited a decrease of novelty-induced excessive grooming. The endocrine pattern of GH-treated aged female rats revealed a decrease in plasma prolactin levels and an increase in luteinizing hormone and 17 beta-estradiol levels as compared to those of control animals. These results support the concept that behavioral and endocrine alterations occurring in aging are not irreversible and that GH may interfere with these changes probably by means of its trophic action on different target organs.     

Adenovirus-mediated in vivo gene transfer in guinea pig middle ear mucosa

This article describes a study designed to assess the feasibility of using recombinant adenovirus for delivering therapeutic peptides in vivo in the guinea pig middle ear cleft. A recombinant adenoviral vector AdCMVsp1 LacZ containing the Escherichia coli beta-galactosidase was injected into the middle ear space. Qualitative assessment of cell middle ear transfection was performed on day 2 by light microscopy study, after injecting a multiplicity of infection (MOI) ranging from 0 to 1000. At an MOI of 30, 30% of the promontory area epithelial cells were stained. An MOI of 50 stained 60% of the cells and an MOI of 100 or more stained more than 90% of the cells. The duration of cell transfection was studied after injecting an MOI of 50. The percentage of stained cells was 60% on day 2, 10% on day 7, and 0% on day 14. Middle ear mucosal inflammation, consisting of a granulocytic infiltrate, was observed when an MOI above 50 was used. Even at a high MOI (500), no staining could be found in the cochlea, in the facial nerve, in the brain, or in visceral organs. These data suggest that recombinant adenovirus vectors can be used to transfer genes in the middle ear. This method appears to be safe, and may be envisaged as a short-duration treatment to transfer genes in vivo in the treatment of middle ear diseases.     

Adenovirus-mediated gene transfer is augmented in basilar and carotid arteries of heritable hyperlipidemic rabbits

OBJECTIVE: Regional presynaptic dopaminergic function and its regulation by dopamine agonists in different stages of PD can be measured by L-[11C]dopa and PET. In the current investigation, we studied the effects of therapeutic apomorphine on L-[11C]dopa uptake in patients with early and advanced PD. BACKGROUND: With disease progression and chronic dopamine agonist treatment, motor response complications supervene in a majority of PD patients. It is assumed that both presynaptic and postsynaptic changes in the dopaminergic system act to modify dopaminergic efficacy. METHODS: Patients with early and advanced stages of PD were included in the study. All patients were investigated twice with PET and L-[11C]dopa drug free and during a subsequent standardized therapeutic apomorphine infusion. RESULTS: Subregional analysis of the striatum showed differences in the effects of apomorphine infusion on the L-[11C]dopa influx rate in the two patient categories. In patients with early and uncomplicated PD, apomorphine infusion decreased the L-[11C]dopa influx rate. This decrease was most pronounced in the dorsal part of the putamen. In advanced PD patients, apomorphine did not affect the striatal L-[11C]dopa influx rate. CONCLUSIONS: We suggest that in mild and stable PD an upregulated presynaptic inhibitory feedback regulation, particularly in the dorsal putamen, acts to maintain congruity within the dopaminergic system in response to antiparkinsonian medication. However, this inhibitory feedback regulation is diminished with the progression of nigrostriatal degeneration and chronic dopamine agonist treatment.     

Stimulated activation of platelet-derived growth factor receptor in vivo in balloon-injured arteries: a link between angiotensin II and intimal thickening

BACKGROUND: Growth factors such as platelet-derived growth factor (PDGF) have been postulated to be important mediators of neointimal formation in balloon-injured artery. Binding of growth factors to their receptors activates intrinsic receptor tyrosine kinase, resulting in tyrosine phosphorylation of receptors themselves and cellular substrate proteins. We investigated in vivo activities of growth factors by determining the extent of tyrosine phosphorylation of growth factor receptors and substrate proteins in injured artery. METHODS AND RESULTS: Rat balloon-injured carotid artery was analyzed for phosphotyrosine content of PDGF alpha- and beta-receptors, epidermal growth factor (EGF) receptors, and insulin receptor substrate-1 (IRS-1) by immunoprecipitation and anti-phosphotyrosine Western blot. The development of intimal thickening after deendothelializing balloon catheterization of rat carotid artery was accompanied by transient twofold to threefold increases in the extent of tyrosyl phosphorylation of PDGF alpha- and beta-receptors but not EGF receptor or IRS-1. The AT1 angiotensin II (Ang II) receptor antagonist TCV-116 markedly inhibited both tyrosyl phosphorylation of PDGF alpha- and beta-receptors and intimal thickening. The AT1 antagonist reduced mRNA levels of both PDGF-A and -B chains in injured arteries. CONCLUSIONS: The present study provides direct evidence for increased PDGF activities in injured artery in situ and the involvement of Ang II in stimulated activation of PDGF receptors. These results are consistent with the pathogenetic role for PDGF in intimal thickening.     

Gene transfer in utero biologically engineers a patent ductus arteriosus in lambs by arresting fibronectin-dependent neointimal formation

Closure of the ductus arteriosus requires prenatal formation of intimal cushions, which occlude the vessel lumen at birth. Survival of newborns with severe congenital heart defects, however, depends on ductal patency. We used a gene transfer approach to create a patent ductus arteriosus by targeting the fibronectin-dependent smooth muscle cell migration required for intimal cushion formation. Fetal lamb ductus arteriosus was transfected in utero with hemagglutinating virus of Japan liposomes containing plasmid encoding 'decoy' RNA to sequester the fibronectin mRNA binding protein. Fibronectin translation was inhibited and intimal cushion formation was prevented. We thus established the essential role of fibronectin-dependent smooth muscle cell migration in intimal cushion formation in the intact animal and the feasibility of incorporating biological engineering in the management of congenital heart disease.     

Gene transfer of human prostacyclin synthase prevents neointimal formation after carotid balloon injury in rats

BACKGROUND AND PURPOSE: A disordered proliferative process in the vascular wall is thought to underlie the pathogenesis of restenosis after percutaneous transluminal angioplasty and carotid endarterectomy. A growth inhibitory property of overexpressed prostacyclin (PGI2) synthase (PGIS) was recently implicated in the pathological proliferation of vascular smooth muscle cells (VSMC) in vitro. Here, we investigated the effects of increased PGI2 synthesis on the pathological proliferation of VSMCs. METHODS: The cDNA encoding human PGIS was transfected into endothelium-denuded rat carotid arteries after arterial balloon injury with the use of hemagglutinating virus Japan (HVJ). HVJ liposome vector complex without PGIS cDNA was used for vehicle control. The level of 6-keto PGF1alpha, a stable hydrolyzed metabolite of PGI2, the histological distribution of the immunoreactivity for human PGIS and the ratio of neointimal/medial area were analyzed. RESULTS: In the analyses of 6-keto PGF1alpha, the level in the carotid arteries was significantly elevated 3 days after PGIS expression-vector transfection compared with that in the arteries after vehicle transfection. Seven days after human PGIS expression-vector transfection, the PGIS cDNA-transfected neointimal cells were strongly positive for human PGIS immunoreactivity in 81% sections examined. Fourteen days after the injury, the ratio of neointimal/medial area was 1.2+/-0.4 in the PGIS expression-vector transfected group, which was significantly smaller than that of the vehicle control group, 1.7+/-0.5; P<0.01. CONCLUSIONS: It was thus demonstrated that the gene transfer of human PGIS expression-vector into rat carotid arteries resulted in the increased production of human PGI2 in the vascular wall, the expression of human PGIS in the developing neointima and significantly inhibited the neointimal formation generated after balloon injury.     

Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant

Angiogenesis, the sprouting of capillaries from pre-existing blood vessels, is a fundamental process in the formation of the vascular system during embryonic development. In adulthood, angiogenesis takes place during corpus luteum formation and in pathological conditions such as wound healing, diabetic retinopathy, and tumor-igenesis. Vascularization is essential for solid tumour growth and is thought to be regulated by tumour cell-produced factors, which have a chemotactic and mitogenic effect on endothelial cells. Vascular endothelial growth factor (VEGF), a homodimeric glycoprotein of relative molecular mass 45,000, is the only mitogen, however, that specifically acts on endothelial cells, and it may be a major regulator of tumour angiogenesis in vivo. Its expression has been shown to be upregulated by hypoxia, and its cell-surface receptor, Flk-1, is exclusively expressed in endothelial cells. Here we investigate the biological relevance of the VEGF/Flk-1 receptor/ligand system for angiogenesis using a retrovirus encoding a dominant-negative mutant of the Flk-1/VEGF receptor to infect endothelial target cells in vivo, and find that tumour growth is prevented in nude mice. Our results emphasize the central role of the Flk-1/VEGF system in angiogenesis in general and in the development of solid tumours in particular.     

p53 gene transfer to the injured rat carotid artery decreases neointimal formation

PURPOSE: We studied the effect of adenovirus-mediated p53 gene transfer on the injured rat carotid artery to determine its ability to decrease the formation of neointima. METHODS: In vivo gene transfer was used in isolated segments of balloon-injured rat carotid arteries. Genetically modified adenovirus containing the gene encoding for wild-type p53 (AdWTp53) was applied in three concentrations: 8 x 10(10), 1.6 x 10(10), and 8 x 10(9) pfu/mL. Control rats received either adenovirus null (AdNull), 8 x 10(10) pfu/mL, or Medium-199 solution (vehicle). Expression of p53 was determined 4 days after gene transfer by Western blotting. Neointimal formation was assessed after 14 days by harvesting carotid arteries and determining the intima/media (I/M) ratio based on cross-sectional area measurement. Simultaneously, immunohistochemistry was done to detect the presence of p53 on smooth muscle cell nuclei. RESULTS: P53 expression was confirmed by Western blotting. There was a significant reduction in neointimal formation on all treated animals compared with controls. The highest dose of AdWTp53 (8 x 10(10) pfu/mL) resulted in a near-total arrest of neointimal formation (I/M = 0.09 +/- 0.03, mean +/- SEM) with P <. 0001 versus vehicle (I/M = 2.23 +/- 0.15) or AdNull (I/M = 2.12 +/-. 12). The intermediate dose of AdWTp53 (1.6 x 10(10) pfu/mL) resulted in an I/M value of 1.04 +/- 0.18, with P <.001 versus vehicle and P =.001 versus AdNull. The lowest dose (8 x 10(9) pfu/mL) resulted in an I/M value of 1.12 +/- 0.18, with P <.001 versus vehicle and P <. 002 versus AdNull. The immunohistochemistry was positive for the presence of p53 in rats infected with AdWTp53. CONCLUSIONS: Adenovirus-mediated gene transfer of p53 protein significantly decreases the formation of neointima in the rat carotid injury model. This may represent a potential therapy for restenosis in humans.     

Adenovirus-mediated regulable target gene expression in vivo

To regulate expression of a transferred gene in response to an exogenous compound, we have combined a high capacity adenoviral vector devoid of all viral coding sequences with a regulatory system that can be used to express a target gene in vivo in a selected site and at a desired time. This system uses a chimeric transactivator, GLp65, which consists of a mutated progesterone receptor-ligand binding domain fused to the GAL4 DNA binding domain and part of the activation domain of the human p65 protein, a component of the NF-kappaB complex. In the presence of the antiprogestin mifepristone, this chimeric regulator binds to a target gene containing the 17-mer GAL4 binding site, resulting in an efficient ligand-inducible transactivation of the target gene. We inserted the regulator GLp65 and a regulable human growth hormone target gene containing the 17-mer GAL4 binding site into the same adenoviral vector. To obtain tissue-specific expression of the target gene, we coupled the regulator to a liver-specific promoter. Infection of HepG2 cells and experimental mice with the adenovirus resulted in consistently high induction levels of human growth hormone in the presence of mifepristone whereas the transgene expression was undetectable in the absence of the ligand. Taken together, our regulable adenoviral vector represents an important tool for transgene regulation that can be used for potentially diverse applications, ranging from tissue-specific gene expression in transgenic animals to human gene therapy.     

Adenovirus-mediated expression of PML suppresses growth and tumorigenicity of prostate cancer cells

Our previous studies demonstrated that the promyelocytic leukemia gene, PML, encodes a growth and transformation suppressor. Overexpression of PML inhibits cancer cell growth in vitro and in vivo. In this study, we further explored the possibility of applying PML as a potential agent for developing prostate cancer gene therapy using an adenovirus delivery system. We have constructed and produced the recombinant PML-adenovirus, Ad-PML, in which the full-length PML cDNA is driven by the strong cytomegalovirus promoter. In LNCaP, DU145, and PC-3 prostate cancer cell lines, an infection efficiency of 90% can be achieved at a concentration of 2, 10, and 100 multiplicity of infection (MOI), respectively. Western blotting and immunofluorescence staining demonstrated that the AD-PML-infected cells expressed a high level of PML protein. The protein expression peaked at days 3-4 postinfection, and a detectable level of PML was found at day 18 after viral infection. To test the effect of Ad-PML on the growth of prostate cancer cells, the DU145 and LNCaP cells were infected with 10 and 2 MOI of Ad-PML. We found that the growth rate of the Ad-PML-infected DU145 and LNCaP cells were significantly inhibited. A tumorigenicity test in nude mice showed that the Ad-PML-treated DU145 cells failed to form tumors. Most importantly, direct injection of Ad-PML into DU145-induced tumors was able to repress tumor growth in nude mice by 64%. Taken together, these data indicate that PML is a tumor growth suppressor in prostate cancer and that Ad-PML may be a potential candidate for human prostate cancer therapy.     

Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo

PURPOSE: The aim of this study was to determine whether heparan sulfate proteoglycans (HSPGs) from the normal arterial wall inhibit neointimal formation after injury in vivo and smooth muscle cell (SMC) phenotype change and proliferation in vitro. METHODS: Arterial HSPGs were extracted from rabbit aortae and separated by anion-exchange chromatography. The effect of HSPGs, applied in a periadventitial gel, on neointimal formation was assessed 14 days after balloon catheter injury of rabbit carotid arteries. Their effect on SMC phenotype and proliferation was measured by point-counting morphometry of the cytoplasmic volume fraction of myofilaments (Vvmyo) and 3H-thymidine incorporation in SMCs in culture. RESULTS: Arterial HSPGs (680 microg) reduced neointimal formation by 35% at 14 days after injury (P=.029), whereas 2000 microg of the low-molecular-weight heparin Enoxaparin was ineffective. HSPGs at 34 microg/mL maintained subconfluent primary cultured SMCs with the same high Vvmyo (52.1%+/-13.8%) after 5 days in culture as did cells freshly isolated from the arterial wall (52.1%+/-15.1%). In contrast, 100 microg/mL Enoxaparin was ineffective in preventing phenotypic change over this time period (Vvmyo 38.9%+/-14.6%, controls 35.9%+/-12.8%). HSPGs also inhibited 3H-thymidine incorporation into primary cultured SMCs with an ID50 value of 0.4 microg/mL compared with a value of 14 microg/mL for Enoxaparin (P< .01). CONCLUSION: When used periadventitially in the rabbit arterial injury model, natural arterial HSPGs are effective inhibitors of neointimal formation. In vitro, the HSPGs maintain SMCs in a quiescent state by inhibiting phenotypic change and DNA synthesis. This study suggests that HSPGs may be a natural agent for the treatment of clinical restenosis.     

Transplanted oligodendrocyte progenitor cells expressing a dominant-negative FGF receptor transgene fail to migrate in vivo

The proliferation, migration, survival, and differentiation of oligodendrocyte progenitor cells, precursors to myelin-forming oligodendrocytes in the CNS, are controlled by a number of polypeptide growth factors in vitro. The requirement and roles for individual factors in vivo, however, are primarily unknown. We have used a cell transplantation approach to examine the role of fibroblast growth factor (FGF) in oligodendrocyte development in vivo. A dominant-negative version of the FGF receptor-1 transgene was introduced into oligodendrocyte progenitors in vitro, generating cells that were nonresponsive to FGF but responsive to other mitogens. When transplanted into the brains of neonatal rats, mutant cells were unable to migrate and remained within the ventricles. These results suggest a role for FGF signaling in establishing a motile phenotype for oligodendrocyte progenitor cell migration in vivo and illustrate the utility of a somatic cell mutagenesis approach for the study of gene function during CNS development in vivo.     

Adenovirus-mediated retinoblastoma gene therapy suppresses spontaneous pituitary melanotroph tumors in Rb+/- mice

The retinoblastoma gene (RB) is the prototypic tumor suppressor. Studies to date have demonstrated cancer suppression with tumor cells reconstituted with RB ex vivo and implanted into immunodeficient mice, as well as with germline transmission of a human RB transgene into tumor-prone Rb +/- mice. To mimic the therapy of cancer more closely, spontaneous pituitary melanotroph tumors arising in immunocompetent Rb +/- mice were treated with a recombinant adenovirus carrying RB cDNA. Intratumoral RB gene transfer decreased tumor cell proliferation, reestablished innervation by growth-regulatory dopaminergic neurons, inhibited the growth of tumors, and prolonged the life spans of treated animals.     

Stimulation of activin A expression in rat aortic smooth muscle cells by thrombin and angiotensin II correlates with neointimal formation in vivo

Vasoactive GTP-binding protein-coupled receptor agonists (e.g., angiotensin II [AII] and alpha-thrombin) stimulate the production of mitogenic factors from vascular smooth muscle cells. In experiments to identify mitogens secreted from AII- or alpha-thrombin-stimulated rat aortic smooth muscle (RASM) cells, neutralizing antibodies directed against several growth factors (e.g., PDGF and basic fibroblast growth factor [basic FGF]) failed to inhibit the mitogenic activity of conditioned media samples derived from the cells. In this report, we found that polyclonal neutralizing antibodies directed against purified human placental basic FGF reduced the mitogenic activity of AII-stimulated RASM cell-conditioned media and in immunoblot experiments identified a 26-kD protein (14 kD under reducing conditions) that was distinct from basic FGF. After purification from RASM cell-conditioned medium, amino acid sequence analysis identified the protein as activin A, a member of the TGF-beta superfamily. Increased activin A expression was observed after treatment of the RASM cells with AII, alpha-thrombin, and the protein kinase C agonist PMA. In contrast, PDGF-BB or serum caused only a minor induction of this protein. Although activin A alone only weakly stimulated RASM cell DNA synthesis, it demonstrated a potent comitogenic effect in combination with either EGF or heparin-binding EGF-like growth factor in the RASM cells, increasing DNA synthesis by up to fourfold. Furthermore, in a rat carotid injury model, activin A mRNA was upregulated within 6 h after injury followed by increases in immunoreactive protein detected in the expanding neointima 7 and 14 d later. Taken together, these results indicate that activin A is a vascular smooth muscle cell-derived factor induced by vasoactive agonists that may, either alone or in combination with other vascular derived growth factors, have a role in neointimal formation after arterial injury.     

Probucol suppresses oxidant stress in hypertensive arteries. Immunohistochemical evidence

Oxy-free radicals may be involved in the pathogenesis of accelerated atherosclerosis in hypertension. We evaluated the direct antioxidant potential of probucol in hypertensive arteries by studying the spatial immunohistochemical distribution of three primary antioxidant enzymes (AEs). Nineteen normocholesterolemic New Zealand White rabbits were divided into two groups: normotensive controls (NT; n = 6) and 13 animals rendered hypertensive by surgical coarctation of abdominal aorta. The hypertensive group was subdivided into hypertensive alone (HT; n = 8) and hypertensive treated with 1% probucol (PO) for 9 weeks (HT-P; n = 5). Blood pressure rose significantly in both hypertensive groups (P < .005). At autopsy, both hypertensive groups showed similarly significant increases in mean arterial intima-media thickness (IMT) whether or not they were treated with probucol. However, only HT rabbits revealed significant increases in the intima-media depth penetration of glutathione peroxidase, superoxide dismutase, and catalase AEs. By contrast, in HT-P animals probucol produced significant reductions of immunostaining of all three AEs compared to the HT group (P < .05). Additionally, specific macrophage immunostaining revealed that the arterial wall of HT rabbits had numerous (10 to 12 per high power field) subintimal and medial macrophages as compared to the HT-P animals (1 to 2 per high power field). The blood pressure level correlated significantly with IMT in all three groups, but with depth penetration of the three AEs only in the NT and HT groups. Probucol, therefore, appears to act in concert with the native arterial antioxidant enzymes as a potent free radical scavenger to reduce oxidative stress and thus attenuate the macrophage invasive response in hypertensive arteries.