Cardiovascular Research

Cardiovascular Research 2000 47(3):436-445; doi:10.1016/S0008-6363(00)00091-2
© 2000 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Oxidative stress as a signaling mechanism of the vascular response to injury

The redox hypothesis of restenosis

Luciano C.P. Azevedo^a,b, Marcelo de A. Pedro^a, Liliete C. Souza^a,c, Heraldo P. de Souza^a,b, Mariano Janiszewski^a,b, Protásio L. da Luz^a and Francisco R.M. Laurindo^a,*

^aHeart Institute (InCor), University of São Paulo Medical School, Av. Eneas Carvalho Aguiar, 44, subsolo, CEP 05403-000, São Paulo, Brazil
^bEmergency Medicine Department, University of São Paulo Medical School, São Paulo, Brazil
^cFederal University of Santa Catarina, Florianópolis, Brazil

^* Corresponding author. Tel.: +55-11-853-7887; fax: +55-11-282-2354 expfrancisco{at}incor.usp.br

Received 8 February 2000; accepted 4 April 2000

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results and discussion References

 
The prominent role of redox processes in tissue injury and in vascular cell signaling suggest their involvement in the repair reaction to vessel injury, which is a key determinant of restenosis post-angioplasty. Experimental studies showed a protective effect of superoxide dismutase or antioxidants on vasospasm, neointimal thickening or remodeling after balloon injury. It was also shown that oxidized thiols induce chelatable metal-dependent amplification of the vascular repair reaction. Ongoing or completed clinical trials show a promising effect of the antioxidant probucol against restenosis. However, few studies addressed the molecular physiological mechanisms underlying the redox hypothesis of restenosis. We recently showed evidence for marked oxidative stress early after balloon injury, with superoxide production mediated primarily by non-endothelial NAD(P)H oxidase-type flavoenzyme(s). This effect was closely related to the degree of injury. There is evidence supporting a role for such early redox processes in apoptotic cell loss and NF-kappa B activation. We present new data on the time course of oxidative stress after balloon injury of intact rabbit iliac arteries. Our data show that despite substantial neointimal growth and lumen narrowing, superoxide production and glutathione levels are unaltered at day 14 and 28 after balloon injury. At day 7 after injury, the peak neointimal proliferation in this model, there was significant decrease of vascular superoxide dismutase activity, without clear evidence of spontaneous superoxide production. Thus, oxidative stress after injury is likely to be an early transient event, which parallels the inflammatory and proliferative phases of the vascular response. We propose that such early redox processes act as dose-dependent signal transducers of gene programs that affect the final repair.

KEYWORDS Angioplasty; Restenosis; Free radicals; Endothelial factors; Atherosclerosis

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results and discussion References

 
The most immediate implication of the study of vascular repair mechanisms is understanding restenosis post-angioplasty. Indeed, restenosis may be viewed as a variable combination of (a) lumen obstruction by the original atheroma, (b) a de novo proliferative and remodeling process with neoplastic features and (c) scar formation, i.e., a homeostatic repair of the vessel wall [1,2]. In addition, the vascular repair reaction has broader significance as a prototype of several common themes underlying physiological adaptations or pathological alterations occuring in a variety of conditions, e.g., flow-induced structural remodeling, hypertension and atherosclerosis [3]. The mechanisms of vascular repair have been actively pursued with integrative as well as reductive approaches. Such innumerable studies have defined thrombotic and inflammatory mediators underlying the early inflammatory phase [1–3], which is also accompanied by massive vascular cell death [4] and matrix degradation [5,6]. A myriad of candidate growth factors and cytokines underlying the subsequent proliferative phase has also been described [1,2]. The late phase of resolutive lesion stabilization is governed by residual proliferation, cell migration [7], apoptosis [4,8], remodeling and extracellular matrix secretion [5,9].

The role of oxidative stress as a mediator of vascular response to injury has not been explored in depth and is yet unclear. Such a role can be anticipated from the well established importance of reactive oxygen and nitrogen-derived species, as well as other redox-active intermediates in normal vascular function. In addition, the importance of redox-dependent signaling in vascular cell growth, apoptosis and senescence is increasingly apparent [10–13]. The aim of the present article is to review evidence suggesting the occurrence and possible implications of oxidative stress after vascular injury — i.e., the redox hypothesis of restenosis [14]. Emphasis will be given to recent data from our laboratory studying the mechanisms of early redox changes after injury. In addition, new data focusing on the time course of oxidative stress throughout the vascular repair reaction will be presented.

1.1 Evidences of oxidative stress after experimental vascular injury
One of the earliest evidences of reactive oxygen species (ROS) production after angioplasty was provided by the observation that superoxide dismutase infusion completely prevented early vasoconstriction and abrogated the growth of mural thrombi after overdistention injury of canine coronary arteries [15]. Catalase, deferoxamine and dimethylthiourea were without effect, suggesting that superoxide could be a primary species underlying those effects. Nunes et al. [16,17] provided evidence of oxidative stress after balloon injury in porcine coronary arteries. This study, as well as many analogous ones, utilized the technique of lucigenin-amplified chemiluminescence (which we will term lucigenin reductase activity, as outlined in the Discussion), taken as an index of superoxide production. The augmented luminescence signals in arterial segments excised 14 days after injury suggested sustained redox imbalance during the repair reaction. Moreover, the proliferating myocyte was suggested as the major cellular source of oxidative stress, with a negligible role of the endothelium [17]. Studies examining the effects of alpha-tocopherol in experimental models of balloon injury showed a decrease in neointimal proliferation [18,19] or a reduction in vascular caliber loss [18]. In the porcine model, the combination of vitamins C and E was shown to improve vessel wall architecture without a clear effect on neointimal size, a finding taken as evidence for a role of redox processes in vascular remodeling [16,17]. Other studies depicted decreases in neointimal size induced by probucol in hyper and normocholesterolemic rabbit or pig models [20–24] or L-cysteine in rats [25], as well as a decrease in macrophage infiltration in the rabbit vessel wall induced by the novel antioxidant and lipid-lowering compound nicanartine [26]. The mechanisms underlying the protective effects of probucol have been studied in normocholesterolemic rabbits and involve a significant decrease in neointimal proliferation rates in apparent correlation with decreased expression of platelet-derived growth factor A chain [23] or diminished activation of mitogen-activated protein kinases and protein C-kinase [24]. In contrast to probucol, beta-carotene was ineffective in the rabbit atherosclerotic balloon injury model [27], whereas in the same model, recent data suggest that administration of red wine decreases monocyte adhesion and slightly decreases neointimal thickening [28]. Another intriguing finding was the interaction between citomegaloviral infection, production of reactive oxygen species and redox-dependent activation of the transcription factor NF-kappaB in smooth muscle cells [29], in a way that antioxidants inhibited replication of such virus putatively involved in the restenosis process [2,8]. Our laboratory performed a reverse-mode test of the redox hypothesis of vascular repair by assessing the effects of superimposed disulfide-induced oxidative stress on the response to balloon overdistention injury of intact rabbit iliac arteries. A single early infusion of oxidized glutathione or cystine induced a marked amplification of neointimal thickening and cell proliferation indexes assessed 14 days later [30]. This amplifying effect was completely prevented by metal chelators (Fig. 1), raising the possibility of increased metalloproteinase activation mediated by disulfides [6]. Taken together, the above described studies provided a theoretical, yet indirect, basis for some clinical studies with antioxidants.

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Neointimal thickening (open bars, intimal/medial area ratio) and intimal proliferation (filled bars, PCNA/cyclin immunoreactivity) assessed at day 14 after balloon injury of intact iliac arteries in control rabbits and in those given a single infusion of glutathione disulfide (GSSG, 6.5 µmol/kg) immediately after injury. The marked amplification in the vascular response induced by GSSG was completely prevented by co-infusion of the metal chelators 1,10-orthophenanthroline (OP, 50.0 nmol/kg) or N-(CBZ) Pro-Leu-Gly hydroxamic acid (HXA, 9.2 µmol/kg). HXA alone led to a significant decrease in neointimal thickening, though not of cell proliferation, vs. control. *P<0.05 vs. control rabbits and P<0.05 vs. GSSG-exposed group; n=5 rabbits per group. Reproduced from Ref. [30].

 
1.2 Clinical studies with antioxidants in restenosis
A summary of randomized studies (Refs. [31–38]) designed to assess the effects of antioxidants in clinical restenosis is shown in Table 1. These trials suggest that among the compounds tested so far, probucol has shown more consistent results, whereas multivitamins have shown little effect. In contrast with the above discussed data obtained in experimental models, the mechanisms of the anti-restenotic effect of probucol appear to involve improvement of vascular remodeling rather than decrease in neointimal thickening [39]. From the practical standpoint, the use of probucol may be somewhat limited by the apparent need for prolonged therapy before the procedure, as well as by untoward effects in blood lipids, which might render other atheromas susceptible to progression [14]. From the mechanistic standpoint, the contribution of these data to the redox hypothesis of restenosis relies on the yet unclear specificity of probucol as an antioxidant.

View this table: [in this window] [in a new window]   Table 1 A summary of randomized clinical trials of antioxidants in restenosis

 
1.3 Characterization of early oxidative stress after injury
Despite the reasonable number of undergoing or completed clinical trials, surprisingly few studies addressed the mechanisms underlying the redox hypothesis of vascular repair. In particular, there is little information on the pattern, time course and molecular physiological mechanisms underlying such processes. The pronounced early protection against vasospasm by superoxide dismutase [15] and the late consequences of transient early exposure to disulfides [30] suggested a prominence of early vascular superoxide production and related oxidative events. This was further suggested by the lasting protection afforded by a single administration of a nitric oxide protein adduct immediately after arterial injury in rabbits [40].

We recently sought to characterize vascular oxidative stress early after injury. Ex vivo injury performed in arterial rings yielded a massive oxygen-dependent peak of lucigenin-amplified luminescence which decayed exponentially and was proportional to the degree of injury (Ref. [41], data shown in Table 2). More specific techniques (including electron paramagnetic resonance spectroscopy) provided unambiguous evidence for oxidative stress and superoxide production. Significant glutathione depletion was also demonstrated (Table 2). Studies with pharmacological inhibitors indicated that the enzymatic source of superoxide after vascular injury could be ascribed to a flavoprotein-dependent source, since the signals were abolished by dyphenylene iodonium, but not by several other putatively inhibitor compounds. This suggests NAD(P)H oxidase activity as the major free radical source, a finding in line with a number of studies suggesting this(ese) yet non-characterized vascular enzyme(s) as a major source of superoxide [42–45]. Gentle removal of endothelium prior to injury scarcely affected the amount of luminescence. Since it is clear that cultured endothelial cells generate ROS by a NAD(P)H oxidase activity [46], it is likely that the overall contribution of the endothelium to ROS generation is relatively dwarfed by the greater amount of smooth muscle cells or adventitial fibroblasts [44]. Endothelium-dependent oxidative stress, however, could be important for its later functional regeneration. The role of NAD(P)H oxidase after injury is further suggested by the report of increased expression of the phagocyte-simile NADPH oxidase subunit p47phox in neointimal and selected medial cells up to 21 days after vascular injury in rats [43].

View this table: [in this window] [in a new window]   Table 2 Oxidative stress and evolution of the repair reaction after balloon injury of intact rabbit iliac arteries

 
We recently gained insight into the molecular physiology of vascular NAD(P)H oxidase activity by examining its sensitivity to thiol oxidizing reagents [47]. The oxidant diamide or the cell-impermeable alkylator p-chloro-mercury phenylsulfonate were able to almost completely abolish NAD(P)H-driven oxidase activity of either vascular homogenates or intact rings. Fig. 2 shows that diamide also induced reversible inhibition of injury-triggered lucigenin reductase activity, presumably through the same mechanism. Interestingly, the oxidant 5,5-dithio-bis-nitrobenzoic acid significantly blocked NADPH-driven oxidase activity, but had no effect on NADH-driven signals. Thus, maintenance of NAD(P)H oxidase reactive thiols in the reduced state appears to be necessary for its function, a fact that adds further complexity to the elaboration of working paradigms of vascular oxidative stress.

View larger version (9K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Marked increase in lucigenin reductase activity of ex vivo segments of rabbit iliac artery immediately after balloon injury. Arteries were incubated in Krebs–HEPES buffer with 0.25 mmol/l lucigenin and luminescence assessed immediately after balloon insuflation. Further studies (not shown) indicated the generation of superoxide radicals after injury. Incubation of vessel segments with the thiol oxidant diamide (1.0 mmol/l) led to significant inhibition of luminescence signals. Diamide-induced inhibition was reversed by N-acetylcysteine (3.0 mmol/l), whereas the later was ineffective by itself. These results further support the possible role of regulatory thiol groups in vascular oxidase activity described by our laboratory [47].

 
1.4 Redox signaling after injury: potential afferent and efferent pathways
One of the key questions concerning vascular redox stress after injury is its significance as a real mediator of the vascular response, as opposed to being just a harmless marker of cell disruption. Lipid peroxidation [48] and redox-active metal release [49], for instance, are known consequences of mechanical tissue disintegration, which might or might not be deleterious. A definitive answer to this question is unavailable at present, in part due to lack of efficient and specific modulators of in vivo oxidative stress.

It is still unclear whether the afferent trigger of early superoxide production is the mechanical stimulus itself, which has been shown to activate the NADPH oxidase in single fibroblasts [50], or else signaling molecules such as ERKs and p38MAPK, which are involved in growth factor-induced ROS production [10–13]. It is conceivable that some of these afferents are shared with those involved in physiological or pathological ROS production due to shear stress [46,51] or pulsatile stretch [52].

The efferent pathways of oxidative stress can be multiple, considering the array of signaling molecules influenced by redox processes (reviewed in Refs. [10–13]), the apparent majority of them involving cell effector targets modulated by thiol-disulfide redox chemistry and/or redox-active metals. Surprisingly, not many studies directly focused on redox signals after vascular injury. Our study showed early activation of the transcription factor NF-kappaB after injury, which was decreased by the flavoenzyme (NADPH oxidase) inhibitor dyphenylene iodonium [41]. Probucol-inhibitable activation of MAP kinases and protein-C kinase has been reported after arterial injury in rabbits [24]. Pollman et al. [4] showed very early redox-dependent activation of stress-activated protein kinases (SAPK) after injury. Since their data showed massive vascular apoptotic cell loss after injury, which was prevented by thiol antioxidants [4], it is likely that the early oxidative stress has the potential to activate the apoptosis signaling program. Finally, vascular oxidative stress induced by leukocyte activation is a likely occurrence, given their documented expression of adhesion molecules [53] and superoxide production [53,54] early after balloon injury in man. Sustained activation of leukocytes was documented after arterial injury in rabbits and could be involved in oxidative stress and related signaling events [55].

1.5 Time course of vascular oxidative stress throughout the repair reaction
Although our studies defined oxidative stress early after balloon injury and prior studies suggested a sustained increase in lucigenin reductase activity, the time course of oxidative stress in the different phases of the vessel repair reaction is unknown. The aim of the novel data reported herein was to assess the redox status of intact rabbit arteries submitted to overdistention injury in the later phases of the repair reaction. In order to provide such results an integrated perspective, data reported previously (Refs. [30,41,56]) will be reproduced here. Although strict comparison among such different series should be performed with caution, the reproducibility of our parameters has been consistent among those studies.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results and discussion References

 
Male NZW rabbits were submitted to overdistention injury of the intact left iliac artery in vivo as described previously [30,56]. Vessels were harvested in the control condition, immediately after injury, and 7, 14, or 28 days later. The 3.0-mm-gauge balloon provided an average 42% overdistention, as assessed by arteriography in separate experiments. Neointimal thickening was expressed by the histological intimal/medial area ratio of specimens collected after in situ perfusion fixation with formalin; other indexes of neointimal size have yielded comparable results (data not shown). Vessel wall proliferation was assessed as described in Refs. [30,56] by immunoreactivity to PCNA/cyclin and quantified as number of reactive cells per mm². In vivo arteriography of the lower abdominal aorta was performed in anesthetized rabbits at specified time intervals after injury through a carotid access. Simultaneous imaging of the injured and control artery allowed expression of results as the % change vs. control. Recent data (Brito F Jr, daLuz PL, Laurindo, FRM: unpublished observations) showed that absolute diameter measurements with quantitative arteriography yielded comparable results.

In another series of studies [41], the lucigenin reductase activity was assessed immediately after injury of ex vivo arterial segments as described. In the present studies, a similar concentration of lucigenin (0.25 mmol/l) was used to probe oxidative stress in arteries from rabbits sacrificed 7, 14, or 28 days after prior in vivo injury. In separate experiments, more specific indexes of superoxide production were obtained through studies using a 0.005 mmol/l lucigenin concentration or the novel superoxide-sensitive probe coelenterazine (0.005 mmol/l, from Prolume, NY, USA), as described in Ref. [57]. Due to the possibility of coelenterazine oxidation by redox-active metals and scavenging of superoxide by nitric oxide, studies were performed both in the absence or presence of the metal chelators DTPA (0.1 mM) and diethylthiocarbamate (0.1 mM), as well as the nitric oxide synthase inhibitor L-NMMA (1.0 mM), as described for the analogous probe CLA [58]. NAD(P)H-driven oxidase activity was assessed in vascular rings as described in Refs. [41,59], through the lucigenin reductase technique at 0.005 and 0.25 mmol/l concentration. Glutathione levels were assessed [60,61] in whole vessel homogenates through NADPH consumption after reaction with DTNB, with recycling assay after incubation with N-ethylmaleimide. Superoxide dismutase activity of whole vessel homogenates was determined according to previously described techniques [62]. The rate of inhibition of cytochrome-c reduction induced by xanthine oxidase-derived superoxide was monitored spectrophotometrically at 550 nm at room temperature. Each assay was performed in a pool of normal and control iliac arteries and results were normalized for protein concentration (Bradford method). Statistical analysis was performed through t test or one-way ANOVA, with a 0.05 significance level. The studies described herein conformed with the Guide for the Care and Use of Laboratory Animals, published by NIH (Publication No. 85–23, revised 1996).

	3 Results and discussion

Top Abstract 1 Introduction 2 Methods 3 Results and discussion References

 
Table 2 depicts the time course of the vascular response and oxidative stress after balloon injury. The time course of neointimal thickening and cell proliferation indexes are similar to those described in analogous models in other species [1,2,8,9]. The present model, on the other hand, differs, e.g., from the pig coronary artery model by exhibiting a defined lumen narrowing whose extent does not correlate with neointimal thickening, a finding in line with previous reports underscoring the role of remodeling in experimental restenosis models [9]. At day 28 after injury, such narrowed vessel segments can still dilate in response to nitrates, but the relative stenosis vs. controls is not reversed (data not shown).

After the massive increase in lucigenin reductase activity immediately after balloon dilation, the injured segments examined 24 or 48 h later show an average 10 to 15-fold luminescence increase vs. uninjured controls (data not shown), although results showed some variability, probably due to non-uniform contamination with inflammatory cells. Day 7 after injury signals the peak in neointimal proliferative activity in this model, although neointimal size is still small. The lucigenin reductase activity (0.25 mmol/l) was significantly increased vs. normal, although the profound glutathione depletion documented immediately after injury was not significant any more. These results were similar to those assessed at day 4 after injury (data not shown). Despite substantial neointimal thickening, minor residual proliferation and an accelerated rate of caliber loss between day 7 and day 14, injured segments show little evidence of enhanced superoxide production and oxidative stress at day 14 after injury. This lack of difference was even more pronounced at day 28 after injury. Therefore, oxidative stress appears to be an early event, which peaks immediately after injury and is sustained in a way that roughly parallels the early inflammatory and proliferative phase of vascular repair.

Increases in lucigenin reductase activity were evident at day 7 following injury. This index is an uniform marker of vascular dysfunction associated, e.g., with hypercholesterolemia, diabetes and hypertension, and has been taken as a measure of increased superoxide production [16,17,59,63,64]. In fact, the altered vascular function in those circumstances has often been improved by the administration of superoxide dismutase or other antioxidants [65]. The significance of this method, however, has been debated, since redox cycling of lucigenin in the presence of a reductase and reduced dinucleotides can produce superoxide [57,66,67]. Other observations suggested that lower doses of lucigenin induced sufficiently low redox cycling to allow detection of endogenous superoxide production [68,69]. (The term lucigenin reductase activity was coined [57] as a way to express this uncertainty regarding the role of endogenous superoxide anions).

Therefore, further studies were conducted in order to clarify the significance of increased lucigenin reductase activity at day 7 following arterial injury. Those results are depicted in Table 3. The data showed similar rates of spontaneous superoxide radical generation in injured vs. control arteries, as assessed by ex vivo assays with low-dose lucigenin or coelenterazine. The results with coelenterazine were comparable in the presence or absence of L-NMMA and metal chelators (data not shown, see Methods). In each of these assays, addition of 0.25 mmol/l lucigenin at the end of the experiment elicited the expected increased signals in injured arteries vs. controls (not shown). In apparent contrast to those findings, measurements of total vascular SOD activity depicted a significant decrease in injured vs. control arteries. Accordingly, the 0.25 mmol/l lucigenin reductase activity assessed after incubation with the SOD inhibitor diethyldithiocarbamate showed no increase in injured vessels, vs. substantial increase in normal vessels, so that the two assays yielded comparable results. Table 4 depicts measurements of NAD(P)H oxidase activity in control and injured vessel rings; increase in enzyme activity was only detected with high-dose (0.25 mmol/l) lucigenin. Taken together, these data suggest that the increased lucigenin (0.25 mM) reductase activity of injured vessels may reflect at least in part a deficiency in superoxide dismutase activity, which allowed the redox cycling and/or electron transfer reactions detected by lucigenin to become more conspicuous. This proposal is in line with data obtained in mutant SOD-deficient E. coli [70]. The relevance and the mechanisms underlying the observed decrease in superoxide dismutase activity are yet unknown. In addition, it is clear from our data that NAD(P)H oxidase activity, a major known source of spontaneous superoxide generation [41–46,59], appears to exhibit at day 7 a secondary or more complex role in comparison with that demonstrated immediately after injury [41].

View this table: [in this window] [in a new window]   Table 3 Redox parameters in injured or control rabbit iliac artery segments 7 days after balloon injurya

 

View this table: [in this window] [in a new window]   Table 4 Vascular NADH/NADPH-driven oxidase activity in arterial rings 7 days after balloon injurya

 
Do our data indicate a sustained oxidative stress at day 7 after injury? The formal, but non-categorical answer to this question is that the deficiency in SOD activity defines a state of oxidative stress, even though no clear differences in spontaneous superoxide release were detected under our assay conditions. Since the coelenterazine studies performed in the presence or absence of L-NMMA yielded similar results, it is unlikely that an increased production of superoxide was masked by its increased scavenging by putative high-output nitric oxide from the inducible [71] or endothelial [72] synthase. On the other hand, one should consider that present limitations in the methods for superoxide quantification in biological samples, particularly the low specificity of chemiluminescence, may obscure minor differences in superoxide production. Therefore, subtle, transient or localized free radical production cannot be excluded in our injured vessels. Further insights into these questions should require studies with a large variety of other techniques, as well as improvements in EPR methods that allow reliable superoxide detection in whole vessel rings.

Our results indicate that direct roles of oxidative stress as a signaling mechanism of tissue repair are likely to involve mainly cell proliferation or early apoptosis [4] rather than events related to the late resolutive phases. One of the potential implications of this ‘hit-and-run’ behavior is in the design of therapeutic schedules regarding trials of antioxidants in restenosis, since it is suggested that concentrated antioxidant coverage immediately and early after injury may be essential. Accordingly, if redox imbalance is to play a role as a mediator of late phases of vascular repair, it probably does so by more indirect mechanisms, which may include (a) Early activation of transcription factors such as NF-kappaB that may have a lasting effect on the gene program of vascular repair [41,73–75]; (b) Early induction of hemodynamic and/or structural changes that alter the setpoint of signaling programs regulating, e.g., senescence and apoptosis; (c) Lasting cellular protein or lipid modifications such as oxidation and degradation [76]; (d) Exacerbation of early cell death [4], with accompanying consequences concerning the late regeneration of vascular cell population. Therefore, our data cannot be interpreted as evidence against any role of oxidative stress in the late phase of vascular repair. Finally, although our findings clearly show that the presence of a vascular scar is not a sufficient condition for oxidative stress, it is possible that in the human atherosclerotic plaque a sustained increase in oxidative stress may be triggered by injury due to persistent macrophage infiltration [50], as well as increases in xanthine oxidase [77] or inducible nitric oxide synthase activities [71].

3.1 Concluding remarks
Oxidative stress clearly occurs as part of the vascular reaction to injury. There is suggestive evidence that superoxide production via NAD(P)H oxidase activity plays a role in the early signaling program of the vascular repair reaction. On the other hand, our data show that oxidative stress is a ‘hit-and-run’ phenomenon, not necessarily present in vessels undergoing more advanced phases of vascular response, despite significant growth of neointimal tissue. Considering factors such as (a) strict dependence of early oxidative stress on the degree of injury [41]; (b) transient nature of the redox imbalance; (c) sustained augmentation of late vascular response after early transient oxidative stress [30]; (d) accompanying activation of transcription factors and significant influence of oxidative stress on early cell loss after vascular balloon injury, we propose that a major role of oxidative stress as a signal effector of the vascular repair may be to act as a ‘dose-dependent’ sensor of the degree of injury. Further studies are necessary in order to clarify whether and how early redox processes may affect late resolutive phases of vascular repair. Although the vascular repair reaction is only a part of the restenosis process, such results may help not only to unravel clinically relevant effectors of the vascular caliber loss but also to design better antioxidant strategies to counteract this challenging problem.

Time for primary review 30 days.

	Acknowledgements

 
Supported by grants from: FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), FINEP (Financiadora de Estudos e Projetos), PRONEX (Programa de Núcleos de Excelência), Fundação EJ Zerbini. The authors would like to thank Leonora Loppnow for her great technical assistance.

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