Journal of Vascular Surgery
Volume 45, Issue 6, Supplement , Pages A64-A73, June 2007

The role of nitric oxide in the pathophysiology of intimal hyperplasia

Division of Vascular Surgery, Northwestern University, Chicago, Ill.

Received 18 January 2007; accepted 11 February 2007.

Article Outline

Since its discovery, nitric oxide (NO) has emerged as a biologically important molecule and was even named Molecule of the Year by Science magazine in 1992. Specific to our interests, NO has been implicated in the regulation of vascular pathology. This review begins with a summary of the molecular biology of NO, from its discovery to the mechanisms of endogenous production. Next, we turn our attention to describing the arterial injury response of neointimal hyperplasia, and we review the role of NO in the pathophysiology of neointimal hyperplasia. Finally, we review the literature regarding NO-based therapies. This includes the development of inhalational-based NO therapies, systemically administered L-arginine and NO donors, NO synthase gene therapy, locally applied NO donors, and NO-releasing prosthetic materials. By reviewing the current literature, we emphasize the tremendous clinical potential that NO-based therapies can have on the development of neointimal hyperplasia.

 

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Nitric oxide: The basics 

Since its discovery 20 years ago, nitric oxide (NO) has emerged as an important signaling molecule in a myriad of pathways, including the nervous, immune, and cardiovascular systems. It was even named Molecule of the Year by Science magazine in 1992. This review focuses on the actions of NO in the vasculature and, more specifically, as a treatment to prevent the development of neointimal hyperplasia that arises after vascular interventions.

Nitric oxide was initially discovered in an attempt to reconcile the apparent conflict in the actions of acetylcholine in vivo and in vitro. Although acetylcholine is normally a potent vasodilator in vivo, during preparations of vessels for in vitro studies, denuding of the endothelial layer by accidental rubbing caused loss of responsiveness to acetylcholine, yielding conflicting results.1 It was suspected that the endothelial cells were releasing a substance that aided in acetylcholine-induced vasodilation and that this substance was subsequently lost from the denuded in vitro preparations. This molecule was termed endothelial-derived relaxation factor. Eventually, NO was identified as the endothelial-derived relaxation factor responsible for mediating vasodilation in response to acetylcholine, and it was the loss of the endothelial-derived NO that was confounding the in vitro studies of acetylcholine activity.2

Once it was determined that NO existed in cells, researchers endeavored to discover how it was produced and regulated. The mechanism of synthesis was shortly discovered to proceed through oxidation of one of the amidine nitrogens of L-arginine, yielding NO and L-citrulline (Fig 1).3 Upon its synthesis, NO was shown to be a potent stimulator of guanylate cyclase to form cyclic guanylate monophosphate (cGMP) and cause relaxation of vascular smooth muscle cells (VSMC).4, 5 These investigations into the role of NO as a signaling molecule in the cardiovascular system earned Drs Robert F. Furchgott, Louis J. Ignarro, and Ferid Murad a Nobel Prize in Physiology and Medicine in 1998.6, 7, 8

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  • Fig 1. 

    Production of nitric oxide (NO) by nitric oxide synthase (NOS). NADP+, nicotinamide adenine dinucleotide phosphate; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; HEME, hemoglobin; H4B, tetrahydrobiopterin; O2, oxygen; Ca2+, calcium; CaM, calmodulin.

While the role of NO in the vasculature was being determined, focus was also aimed at understanding the regulation of NO synthesis in the cell. To this end, three forms of NO synthase (NOS) were discovered by researchers: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS).9 These isoforms are clearly distinct but share a number of similarities. They all require the cofactors nicotinamide adenine dinucleotide phosphate hydrogen, flavin adenine dinucleotide, flavin mononucleotide, and tetrahydrobiopterin to catalyze the enzyme reaction.9

In general, eNOS and nNOS are constitutively expressed enzymes, and NO production is predominately regulated by intracellular calcium fluxes that permit calmodulin binding, which activates the enzyme.9 In contrast, iNOS is transcriptionally regulated and is not normally produced by most cells.9, 10 Typically, iNOS is expressed in response to cellular stress and generates 100-fold to 1000-fold more NO than its constitutive counterparts that are involved in physiologic regulation.10, 11

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Arterial injury response: Neointimal hyperplasia 

Neointimal hyperplasia is an exaggerated healing process that occurs in the vessel wall after injury. It is responsible for restenosis, limiting the success of many vascular interventions including bypass grafting, endarterectomy, and balloon angioplasty with or without stenting. The development of neointimal hyperplasia is a complex process initiated by injury and exposure of the VSMC to circulating blood elements. The process is further characterized by platelet aggregation, leukocyte chemotaxis, VSMC proliferation and migration, extracellular matrix (ECM) changes, and, finally, endothelial cell proliferation.

As mentioned, the initiating event in neointimal hyperplasia is endothelial denudation. Disrupting the overlying layer of endothelium exposes the underlying VSMC to circulating blood elements, which then activates a cascade of events culminating in neointimal hyperplasia.12 Rat common carotid arteries lacking endothelium reliably produce neointimal hyperplasia. To systematically study this response, Alexander Clowes, MD, established the rat carotid artery balloon injury model, which is still consistently used in the investigation of the pathophysiology of neointimal hyperplasia.12

Within seconds after endothelial loss, platelets aggregate and adhere to the site of injury.13 This was demonstrated by removal of the endothelium from the carotid artery of a rat using a balloon catheter, which subsequently led to the rapid accumulation of platelets on the exposed subendothelium as measured by scanning electron microscopy.13 These platelets remained adhered for 6, 24, and 48 hours and up to 4 and 7 days after injury.13 The platelets formed a monolayer in most regions and discharged the contents of their dense granules.14 Among the substances released were platelet-derived growth factor (PDGF) that caused VSMC migration and proliferation in the injured wall.14, 15

After platelet aggregation and adherence, leukocyte chemotaxis occurs. Leukocytes are paramount in the secretion of many cytokines and growth factors that influence the subsequent events in neointimal hyperplasia (Table I).16 One example is macrophages that produce PDGF and interleukin-1 (IL-1), which both promote VSMC proliferation and are released in the arterial wall during the repair process.17, 18 The central role of leukocyte chemotaxis is demonstrated by the novel model for inflammatory neointimal hyperplasia that consists of leukocyte-derived myeloperoxidase in the presence of its substrate hydrogen peroxide. The infusion of myeloperoxidase (200 nM) and hydrogen peroxide (1 mM) into an isolated rat common carotid artery, followed by 1-hour incubation, elicited a neointimal hyperplasia not seen in the arteries infused with phosphate buffered saline.19 It was postulated that in this model, the myeloperoxidase and hydrogen peroxide were able to induce neointimal hyperplasia without mechanical injury to the rat common carotid artery by compromising NO signaling through an as-yet-unknown mechanism.20

Table I. Cellular sources of growth factors and cytokines involved in the pathogenesis of fibroproliferative vasculopathies
Growth factors, cytokinesCell source
LeukocytesMonocytes, macrophagesMast cells
IGF-1 X
PDGF XX
TGF-α X
TGF-βXXX
VEGFXXX
EGF X
FGF X
TNF-αXXX
IL-1βXXX
IL-4X X
IL-6 XX
IL-8XXX
IL-10 XX
IL-18XXX
MCPXXX

IGF, Insulin-like growth factor; PDGF, platelet-derived growth factor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; EGF, epidermal growth factor; FGF, fibroblast growth factor; TNF, tumor necrosis factor; IL, interleukin, MCP, macrophage chemoattractant protein. Adapted from Mitra et al.16

In conjunction with leukocyte chemotaxis, VSMC proliferation and migration are the fundamental characteristics of neointimal hyperplasia. This proliferation is, in part, initially mediated by the release of basic fibroblast growth factor (bFGF), which is a potent mitogen for VSMC in vivo.21 Proliferation begins as early as 24 hours after injury and continues for several weeks.22 Under the influence of platelet-derived growth factor (PDGF), VSMC begin migrating to the intima between 1 to 3 days after injury.23

The ECM generally acts as a barrier to VSMC migration from the media to the intima; however, after injury the ECM is modified to allow for the movement of cells. Migration and matrix reconfiguration are associated with an increase in the expression and activity of matrix-degrading enzymes. Two examples, plasminogen activator, which lyses clots and activates matrix-degrading enzymes, and matrix metalloproteinase, which degrades collagen and elastin, are upregulated after injury.24, 25

While some elements of the ECM undergo degradation, other elements seem to be upregulated. After balloon angioplasty, rat carotid arteries demonstrated increased transforming growth factor-β (TGF-β), which correlated with increased fibronectin, type I collagen, and type III collagen gene expression.26 Gene transfer of TGF-β also resulted in the production of procollagen, collagen, and proteoglycans.27

Cells producing certain ECM components may also be targets for regulation. This is demonstrated by the targeted reduction of cGMP-dependent protein kinase I expression in response to injury in proliferating VSMC simultaneously expressing osteopontin, an ECM protein.28

Finally, to restore the protective barrier of the endothelium disrupted by injury, endothelial proliferation must occur. Concurrent with VSMC proliferation and migration, endothelial regeneration begins through stimulation by bFGF ≤24 hours of injury.29 This regeneration begins from the ends of the denuded area and approaches the center within several weeks, restoring endothelial continuity.12

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Effects of nitric oxide on neointimal hyperplasia 

All the aforementioned events lead to the development of neointimal hyperplasia. The next question is: what is the role of NO in the formation of neointimal hyperplasia? Studies conducted with NOS knockout mice and NOS inhibitors demonstrate the importance of NO in regulating neointimal hyperplasia. The eNOS knockout mice that underwent external carotid artery ligation displayed an impaired vascular remodeling, accompanied with an increase in wall thickness and a hyperplastic response of the arterial wall.30 Endothelium-derived relaxing factor activity, as assayed by acetylcholine-induced relaxation, was also absent in eNOS knockout mice, producing hypertension.31 The eNOS knockout mice have specifically been shown to have an exacerbated carotid artery ligation-induced expression of stromal cell-derived factor-1α, a molecule involved in recruitment of circulating VSMC progenitor cells into the neointima.32 The mice lacking eNOS also demonstrated an increase in circulating stem cell antigen-1+ cells, which has been shown to be a progenitor to VSMC.32 This suggested that constitutive eNOS inhibited stromal cell-derived factor-1α expression and provided an important vasculoprotective mechanism for intact endothelium to limit VSMC proliferation and recruitment in response to vascular injury.32

Finally, rats that received pharmacologic NOS inhibition with L-nitro-1-arginine and cyclooxygenase inhibition with indomethacin synergistically were found to have increased intimal thickening after balloon catheter injury of the left carotid artery.33 Together, these studies demonstrate that a primary defect in the NOS/NO pathway promotes abnormal remodeling and neointimal hyperplasia, and verifies the critical role for endogenous NO in maintaining a normal vascular environment (Fig 2).

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  • Fig 2. 

    Role of nitric oxide (NO) in neointimal hyperplasia. Nitric oxide works to both stimulate (below) and inhibit (above) elements of the vasculature that ultimately results in overall inhibition of neointimal hyperplasia. VSMC, vascular smooth muscle cells.

Inhibition of platelet aggregation and adhesion 

Nitric oxide acts through several mechanisms to protect the vasculature. One of the initial mechanisms is the ability of NO to prevent platelet aggregation. In 1987, Radomski et al34 discovered that NO contributed to the nonadhesive properties of the vascular endothelium by demonstrating that the adhesion of unstimulated and thrombin-stimulated platelets washed and labeled with indium-111 was lower in the presence of exogenous NO. After balloon catheter denudation of the rat carotid artery, Yan et al35 measured iNOS expression, platelet adhesion, and blood flow. The results showed that upregulation of iNOS in vivo not only prevented the adherence of platelets to the injured site but also preserved blood flow.35 Because injury results in endothelial denudation and loss of eNOS, upregulation of iNOS represents a protective mechanism against platelet adherence that compensates for the loss of the endothelium.

Inhibition of leukocyte chemotaxis 

After arterial injury, leukocytes accumulate and produce several growth factors and cytokines that stimulate VSMC proliferation and migration. Nitric oxide has been shown to inhibit the inflammatory infiltration and, hence, inhibit the accumulation of VSMC. This has been verified by disruption of the NOS/NO pathways. Inhibitors of NO production, NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME), when infused into a cat mesenteric preparation, increased leukocyte adherence and emigration as measured by intravital video microscopy.36 In addition, when compared with wild-type mice, iNOS-deficient mice have a higher number of rolling and adherent leukocytes in postcapillary venules of the cremaster muscle and the sinusoids as well as postsinusoidal venules of the hepatic microcirculation in response to an endotoxin infusion as seen by intravital video microscopy.37 These results suggest that NO production after iNOS upregulation that follows an injury may function as a homeostatic regulator of leukocyte recruitment and may play a role in regulation of vascular inflammation.

Inhibition of vascular smooth muscle cell proliferation and migration 

Early studies demonstrated that NO modulates VSMC proliferation through a cGMP-mediated mechanism.38 Further research has revealed some of the details behind this inhibition. One mechanism is through the induction of a G0/G1 cell cycle arrest that prevents cells from entering the synthesis phase of the cell cycle required for proliferation.39 This was demonstrated by Sarkar et al39 in the treatment of cultured rat aortic VSMC with the NO donors S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione. The introduction of the NO donors resulted in a 50% reduction in the fraction of cells in the S and G2+M phases and an increase in the G1 fraction, suggesting that NO inhibited entry into S phase and caused accumulation in G1 phase.39

Subsequently, Ishida et al40 used SNAP-treated VSMC to demonstrate that NO halted the G1/S transition by a p21-mediated inhibition of the phosphorylation of the retinoblastoma protein by cyclin-dependent kinase 2. Gene transfer of iNOS into VSMC has also demonstrated a p53-independent and cGMP-independent increase in expression of p21, a cyclin-dependent kinase inhibitor known to inhibit cell cycle progression.41 Further verification by Tanner et al42 demonstrated that NO has been shown to alter the expression or activity of many cell cycle regulatory proteins, including cyclin A, cyclin-dependent kinase 2, and p21.40

Nitric oxide also retards the VSMC migration that, in addition to VSMC proliferation, plays a key role in neointimal hyperplasia. Dubey et al43 showed that the NO donors sodium nitroprusside and SNAP inhibited angiotensin II–induced rat VSMC migration in vitro, as measured by a modified Boyden Chamber. Similarly, stimulating iNOS expression using IL-1β increased NO production and inhibited angiotensin II–stimulated rat aortic VSMC migration.43 In fact, many NO donors, including diethylamine NONO-ate (DETA/NO), spermine NONO-ate (SPER/NO), and S-nitrosoglutathione, all exhibited concentration-dependent inhibition of both the number of migrating VSMC and the maximal distance migrated.44 This inhibition was reversible upon removal of the NO donors.

Stimulation of vascular smooth muscle cell apoptosis 

Apoptosis of VSMC has an important role in the prevention of neointimal hyperplasia. Although the regulation of VSMC apoptosis is not fully understood, NO has been shown to induce apoptosis. Pollman et al45 showed that the administration of NO donors SNAP or sodium nitroprusside to cultured rabbit VSMC caused apoptosis in a dose-dependent fashion as measured by propidium iodide staining. Also in 1996, Nishio et al46 exposed rabbit VSMC to SNAP and showed a dose-dependent increase in apoptosis by NO as measured by terminal deoxynucleotide transferase-mediated dUTP biotin nick end-labeling (TUNEL). They also showed that addition of cGMP inhibitor KT5823 had no effect on NO-mediated apoptosis, indicating a cGMP-independent pathway.46

In 1998, a complimentary DNA (cDNA) construct expressing iNOS was transfected into rat and human VSMC by lipofection, resulting in expression of the iNOS protein with full catalytic activity to generate massive NO in proportion to the cDNA used.47 Overexpression of iNOS led to marked inhibition of DNA synthesis and induction of apoptosis in VSMC as measured by internucleosomal DNA fragmentation by agarose gel electrophoresis, positive staining for TUNEL, and the appearance of hypodiploid cells in flow cytometry analysis.47 In addition, apoptosis was markedly suppressed when the NOS inhibitor L-NMMA was administered to the iNOS-transfected VSMC, further solidifying the role of NO in regulation of apoptosis.47

Extracellular matrix changes 

As mentioned previously, the ECM acts as a barrier to VSMC migration from the media to the intima. Nitric oxide inhibits VSMC migration in part by regulating matrix metalloproteinase (MMP) activity, the enzymes responsible for degrading the basement membrane and the ECM.48 Interestingly, when a replication-deficient adenovirus containing bovine eNOS was transfected into cultured rat aortic VSMC, the NO produced decreased the activity of MMP-2 and MMP-9 while increasing the activity of the tissue inhibitor of metalloproteinases-2.48 DNA chip studies of L-NAME–induced hypertensive rat, showed a significant modulation of 28 known genes at both 15 and 30 days after treatment.49 The functional classification of the genes highlighted three major biologic pathways modulated in the aortic media during L-NAME administration: genes regulating cell proliferation, genes of the NO/cGMP signaling pathway, and genes involved in ECM remodeling.49

This matrix reconfiguration involves both degradation and deposition of matrix elements. In grafts treated with the NO donor SPER/NO, expression of insulin-like growth factor (IGF), bFGF, thrombospondins, fibronectin, and tenascin was reduced.50 L-arginine, the substrate for NO production, significantly diminished hyaluronan synthase expression (one of the two enzymes responsible for making hyaluronan, a key component of the ECM).51 Another NO donor, DETA/NO, caused cell cycle arrest associated with overexpression of TGFβ-1 and an increase in synthesis of collagen I and III in human coronary VSMC.52

Conversely, studies have demonstrated inhibition of collagen levels with NO. In vitro, NO has been demonstrated to directly inhibit basal type I collagen levels and decrease the endothelin-induced responses of proliferation, protein synthesis, and ECM production as measured by cell counts and enzyme-linked immunosorbent assay.53 Two additional NO donors, SNAP and sodium nitroprusside, also inhibited total protein and collagen synthesis within VSMC.54 Thus, NO produces a complex effect on the ECM that ultimately creates an environment limiting the migration of VSMC.

Stimulation of endothelial cell proliferation 

Nitric oxide has been demonstrated to play a dual role in regulating cells after arterial injury. While NO inhibits VSMC proliferation, it stimulates endothelial regeneration.55 Nitric oxide appears to trigger capillary endothelial cell growth and differentiation through cGMP-dependent gene transcription,56 and is also is a component of the pathways underlying vascular endothelial growth factor (VEGF)-activated endothelial cell proliferation.57 Vascular endothelial growth factor induces the expression of eNOS and promotes the release of NO through activation of the MAPK cascade.57 Furthermore, NOS inhibition with L-NAME blocks angiogenesis induced by VEGF and substance P in rabbit cornea.58, 59 In eNOS-deficient mice, the angiogenic response to hind limb ischemia was impaired and could not be overcome by administration of VEGF.60 Of interest was that although VEGF-induced angiogenesis is mediated by NO, the capillary growth stimulated by FGF-2 can be both NO-independent and inhibited by NO.61, 62 Although different proposed mechanisms exist, the end result is the stimulation of endothelial cell proliferation by NO.

Inhibition of endothelial cell apoptosis 

In a further attempt to maintain the integrity of the endothelial layer, NO also suppresses endothelial cell apoptosis. Using cultured human umbilical vein endothelial cells that had been subjected to shear stress in the presence of NO, Dimmeler et al demonstrated that NO prevented apoptosis.63 Similarly, Tzeng et al64 showed that an adenoviral vector overexpressing iNOS caused an inhibition of lipopolysaccharide-induced apoptosis in cultured sheep arterial endothelial cells by reducing caspase-3-like protease activity. In further support, DeMeester et al65 showed a similar inhibition of endothelial cell apoptosis by NO in cells cultured from a porcine model. Taken together, these results suggest that NO production favors healing of the injured vasculature by inhibiting further apoptosis in order to rapidly cover the injured site. Once endothelial cell regeneration is complete, the stimulus for platelet adherence and leukocyte chemotaxis is removed, and the cascade of cytokine and growth factor release leading to neointimal hyperplasia is abolished.

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Nitric oxide-based therapies 

Given the role of NO in maintaining a normal vascular environment, many investigators have hypothesized that replacement of NO at the site of injury would prevent development of neointimal hyperplasia. Here we attempt to summarize the gamut of NO-based therapies that have been investigated to date (Table II).66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87

Table II. Therapeutic effects of nitric oxide on neointimal hyperplasia
MethodNO source% InhibitionSpeciesModelTime periodReference, year
Inhalational NONO43RatBalloon angioplasty2 wkLee,66 1996
Systemic L-arginineL-arginine39RabbitBalloon angioplasty2 wkMcNamara,67 1993
L-arginine65RatBalloon angioplasty2 wkChen,68 1999
Systemic NO donorsMolsidomine32PigBalloon angioplasty3 wkGroves,69 1995
Linsidomine, molsidomine18HumanCoronary angioplasty6 moLablanche,70 1997
NO-releasing aspirin23MouseBalloon angioplasty3 wkNapoli,71 2001
Molsidomine0HumanCoronary angioplasty6 moWohrle,72 2003
NOS gene therapyAdenoviral eNOS70RatBalloon angioplasty2 wkvon der Leyen,73 1995
Adenoviral eNOS37RatBalloon angioplasty2 wkChen,74 1998
Adenoviral eNOS72RatBalloon angioplasty2 wkJanssens,75 1998
Adenoviral eNOS28PigCoronary angioplasty3 wkVarenne,76 1998
Adenoviral iNOS>95RatBalloon angioplasty6 wkShears,77 1998
Adenoviral iNOS52PigBalloon angioplasty3 wk
Adenoviral iNOS30PigVein graft3 wkKibbe,78 2001
Adenoviral iNOS37PigCoronary angioplasty4 wkWang,79 2001
Local NO donorsNO-albumin77RabbitBalloon angioplasty2 wkMarks,80 1995
L-arginine37RabbitIliac injury4 wkSchwarzacher,81 1997
SPER/NO41RabbitVein graft4 wkChaux,50 1998
SNAP36RabbitVein graft4 wkFulton,82 1998
SPER/NO73RatBalloon angioplasty2 wkKaul,83 2000
SIN-146PigCoronary angioplasty8 wkHarnek,84 2003
NO-releasing prostheticsNO eluting stent0PigCoronary stent4 wkYoon,85 2002
NO eluting stent32RabbitAortic stent4 wkDo,86 2004
NO eluting stent80PigBalloon angioplasty4 wkHou,87 2005

NO, Nitric oxide; iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide synthase; IH, intimal hyperplasia; SPER/NO, spermine/NO; SNAP, S-nitroso-N-acetylpenicillamine; SIN-1, 3-morpholino-sydnonimine.

Inhalational nitric oxide 

In 1996, Lee et al examined the effects of inhalational NO on neointimal hyperplasia after balloon injury of the rat carotid artery.66 Compared with control rats, there was a 43% reduction in the intima-to-media (I/M) area ratio in rats receiving inhalational NO for 2 weeks. Rats breathing 80 ppm NO for 7 days showed no difference in the I/M area ratio. Thus, the entire 14 days was required to inhibit neointimal hyperplasia. Unfortunately, when Rich et al88 measured the concentration of NO in the effluent artery of isolated perfused rat lungs ventilated with NO, they observed that lungs perfused with whole blood demonstrated undetectable levels of NO due to the rapid inactivation of NO by hemoglobin. Thus, this study challenged the study done by Lee et al and suggested that if adequate levels of NO in the circulation from inhalation cannot be achieved, then the differences observed were not from differences in NO concentrations.

Administration of L-arginine systemically 

McNamara et al67 administered the NO precursor L-arginine to rabbits from 2 days before to 2 weeks after balloon injury to the thoracic aorta. Animals receiving L-arginine demonstrated a 39% decrease in neointimal hyperplasia compared with the control. This reduction was reversed by coadministration of the NO inhibitor L-NAME, indicating that these effects were specific to NO. Subsequently, Chen et al68 administered L-arginine (2.5 mg/mL) in the drinking water of rats that underwent balloon injury of the carotid artery 2 weeks before injury and for 2 weeks thereafter. Animals treated with L-arginine showed a 65% reduction of the I/M area ratio and a 26% reduction in the intimal cell proliferation compared with controls. Despite these successes in animal models, no human trials have conclusively demonstrated the clinical efficacy of systemic L-arginine administration on inhibiting neointimal hyperplasia.

Administration of nitric oxide donors systemically 

In addition to L-arginine, many studies have also used the systemic administration of NO donors. Groves et al69 gave the oral NO donor molsidomine every 8 hours for 48 hours total in a pig carotid balloon angioplasty model and showed a 32% reduction in neointimal hyperplasia at 21 days. However, this was only in circumstances where the internal elastic lamina remained intact after angioplasty. There was no significant influence on neointimal hyperplasia in more severe injury. It is possible that the antiproliferative effects of orally administered NO were overwhelmed when injury was severe and, therefore, were not associated with a reduction in intimal thickening. Despite this fact, Napoli et al71 demonstrated that mice receiving a NO-releasing aspirin derivative experienced less restenosis after percutaneous transluminal coronary angioplasty. Unfortunately, administration of systemic NO has not consistently demonstrated inhibition of neointimal hyperplasia in human studies.

The Angioplastic Coronaire Corvasal Diltiazem (ACCORD) study showed that patients undergoing angioplasty who received NO from intravenous linsidomine while an inpatient, followed by oral molsidomine as an outpatient for a total of 6 months, had a statistically significant improvement in angiographic results, with a 10% reduction in luminal diameter.70 In a separate study, Wohrle et al72 did demonstrate a slight improvement in anginal status in patients receiving high-dose oral molsidomine for 6 months after coronary angioplasty; however, this did not translate into an effect on angiographic restenosis rate. In addition to the conflicting results in human clinical trials, NO administered systemically can have the undesirable effects of vasodilation, hypotension, headaches, and increased bleeding complications, ultimately limiting its clinical application.

Nitric oxide synthase gene therapy 

Developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease and, as such, much effort has been invested in studying the effects of NOS gene transfer on neointimal hyperplasia. In 1995, von der Leyen73 used a highly efficient Sendai virus/liposome to transfer eNOS to rat carotid arteries after balloon injury and demonstrated a 70% reduction in neointimal hyperplasia at 2 weeks. Chen et al74 used a retrovirus to transfect VSMC with eNOS and used the carotid artery balloon injury model to demonstrate that neointimal hyperplasia was inhibited by 37% at 2 weeks after injury. Even in the larger porcine model, more representative of the human system, Varenne et al76 found that adenovirus-mediated transfer of eNOS to VSMC restored NO production in the injured coronary arteries of pigs and significantly reduced luminal narrowing.

Gene transfer of iNOS has demonstrated similar effects on neointimal hyperplasia. Shears et al77 showed that adenoviral delivery of human iNOS to balloon-injured rat carotid arteries resulted in a 95% reduction in intimal thickening at 2 weeks. This protective effect was reversed by continuous infusion of an iNOS-selective inhibitor. In the more clinically relevant model of the pig iliac artery balloon injury model, a 52% reduction in the I/M area ratio was observed after iNOS gene delivery.77 Kibbe et al78 performed iNOS gene transfer in a porcine model of vein bypass grafting that similarly showed a 30% decrease in the I/M area ratio at 21 days compared with control. These results were supported by Wang et al,79 who demonstrated that iNOS gene transfer inhibits neointimal hyperplasia by 37% in the porcine coronary stent model.

To directly compare adenoviral-mediated overexpression of both iNOS and eNOS, Cooney et al89 studied human coronary artery smooth muscle cells and human umbilical vein endothelial cells infected with adenoviral vectors encoding eNOS or iNOS. Interestingly, proliferation was diminished to a similar degree in AdeNOS-infected and AdiNOS-infected cells compared with noninfected cells in both human coronary artery smooth muscle cells and human umbilical vein endothelial cells.89 Apoptosis was not detected in either cell type with either of the isoforms. For the first time, these results suggested a similar effect of both isoforms on endothelial and vascular smooth muscle cell biology despite the obvious differences between the NOS isoforms.

Local application of nitric oxide donors 

Many investigators have studied the effects of locally delivered NO on sites of arterial injury. Marks et al80 investigated the impact of a polythiolated form of bovine serum albumin (pS-BSA) modified to carry several S-nitrosothiol groups (pS-NO-BSA) on arterial balloon injury in rabbits. Locally delivered pS-NO-BSA reduced the development of neointimal hyperplasia by 77% and also inhibited platelet deposition after denudation of the artery. Furthermore, Schwarzacher et al81 showed at 4 weeks after balloon injury that delivery of intramural L-arginine in rabbits enhanced local NO generation and inhibited neointimal hyperplasia by 37%. Vein grafts treated with L-arginine polymer were also demonstrated by Kown et al90 to increase NO levels and reduce neointimal hyperplasia in a rabbit vein graft model.

Other NO-donors such as SPER/NO, when locally applied, have also shown inhibition of neointimal hyperplasia: 41% in rabbit vein graft models after 4 weeks and 73% in rat balloon angioplasty models after 2 weeks.50, 83 Furthermore, inhibition of neointimal hyperplasia has been observed by other locally delivered NO donors, such as molsidomine on a hydrogel-coated angioplasty balloon catheter in pigs, SNAP gel on rabbit vein grafts, and 3-morpholino-sydnonimine after pig percutaneous transluminal coronary angioplasty.91, 82, 84

Prosthetic materials that release nitric oxide 

Each of the mentioned therapies have some limitations in their clinical applicability because of systemic side effects, safety concerns, complicated delivery schemes, or inability to concentrate NO at the site of injury for prolonged periods of time. To circumvent these problems, recent research has been directed at administering NO through NO-releasing prosthetic materials. In fact, NO-releasing prosthetic materials gained widespread attention recently in The New England Journal of Medicine.92

As early as 2002, Yoon et al85 used the NO donor sodium nitroprusside incorporated into a metallic coil stent coated with a polyurethane polymer to investigate the effect on neointimal hyperplasia in the porcine coronary artery stent injury model. The stented arteries did release increased NO and demonstrated increased local cGMP levels, but no difference in neointimal area was observed between control and treatment groups after 4 weeks.88 Two subsequent studies by Hou et al87 and Do et al86 did demonstrate a reduction in neointimal hyperplasia with application of a NO-eluting stent. In 2004, Do et al deployed stents containing bioerodable microspheres of the NO donor N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino) ethanamine into the aortas of rabbits. Nitric oxide release was sustained for 3 weeks, and the I/M area ratio in the NO-treated group was reduced by 46% and 32% relative to controls at 7 and 28 days, respectively.

One year later, Hou et al also demonstrated a reduction in neointimal proliferation when they used silicone-containing sodium nitroprusside to coat the interior of a self-expanding polytetrafluoroethylene-covered stent. After implanting the stent and inducing balloon overstretch injury to the pig carotid artery, the mean neointimal area was reduced from 2.4 mm2 for control stents to 0.49 mm2 for NO-eluting stents, which resulted in a 24% reduction of angiographic vessel narrowing. These studies demonstrate the importance of the NO donor used as well as the polymer.

Several investigators have recently used NO-releasing molecules to modify prosthetic bypass grafts. Because this research is in its infancy, focus has been more on the biomechanics of NO release and the effect of these alterations on the prosthetic materials. Smith et al93 were the first to use diazeniumdiolates in polymers and incorporate those polymers into vascular grafts. These grafts did spontaneously release NO. Unfortunately, the coating process changed the architecture of the graft, which potentially limited the long-term biocompatibility of the technique.

Another method developed by Zhang et al94 incorporated diazeniumdiolated silica nanoparticles into vascular grafts by embedding them into hydrophobic matrices that were then used to coat the inside lining of extracorporeal venovenous circuits in the rabbit model. Unfortunately, the diazeniumdiolates used in these studies demonstrated leaching, and measurable levels of nitrosamines, a well-known class of carcinogens, were formed. To address this issue, investigators have attempted techniques such as coating grafts with layers of a diazeniumdiolate-containing polyvinyl chloride or covalently binding diazeniumdiolates into a polyurethane backbone to prevent leaching and further optimize the properties of the NO-releasing prosthetic materials.95, 96, 97

The S-nitrosothiols are another class of NO donors that have been used in the development of NO-releasing polymers. Several polymers have been developed with the S-nitrosothiol covalently linked to the polymer to prevent leaching or reaction by-products. Bohl and West98 used S-nitrosothiols to create a NO-releasing hydrogel, speculating that this material could be used to coat the vessel wall or vascular grafts. Unfortunately, the limitation of using both diazeniumdiolates and S-nitrosothiols is the finite reservoir of NO available. New materials are being developed that rely on S-nitrosothiols or nitrite, or both, that is already circulating in the blood as an unlimited endogenous source of NO.99, 100 Once the biomechanics of these NO-releasing prosthetic materials are perfected, attention can then be turned to their influence on neointimal hyperplasia.

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Conclusion 

Nitric oxide-based therapies have tremendous potential to have a significant impact on the development of neointimal hyperplasia in the clinical arena. Here we describe the basics of NO, the arterial injury response, and the pathophysiologic role of NO in the development of neointimal hyperplasia, and lastly the clinical applications of NO-based therapies. Overall, we believe that NO-based pharmacologic approaches tailored to locally deliver a set concentration of NO over a specific period of time will prove to be a valuable therapy in vascular surgery.

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 Competition of interest: Dr Kibbe is a paid carotid stenting proctor for Abbott.

PII: S0741-5214(07)00313-8

doi:10.1016/j.jvs.2007.02.027

Journal of Vascular Surgery
Volume 45, Issue 6, Supplement , Pages A64-A73, June 2007

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