In-stent restenosis of coronary arteries is the major reason for the recurrence of occlusion after successful percutaneous coronary intervention (PCI) and occurs in 20–50% of patients.1 The development of in-stent restenosis depends on complex cellular interactions within the vessel wall involving endothelial cells (ECs), smooth muscle cells (SMCs), fibroblasts, lymphocytes, and macrophages.2 Vessel injury from balloon angioplasty and stenting cause the release of soluble mediators that initiate cellular activation, migration, proliferation, extracellular matrix production and secretion via autocrine or paracrine mechanisms. The recent development of intracoronary stents coated with drugs that inhibit in-stent restenosis rely on the fact that cell proliferation and cell migration play key roles in this process. Sirolimus- (Rapamune®,Wyeth Ayerst Laboratories, Madison, New Jersey) eluting stents (Cordis Corporation, Johnson & Johnson, Miami, Florida), and paclitaxel- (Taxol®, Bristol Myers Squibb Company, Princeton, New Jersey) eluting stents (Boston Scientific Corporation, Maple Grove, Minnesota) are clinically effective inhibitors of in-stent restenosis when utilized with concomitant antiplatelet therapy.3–6 This article focuses on how the molecular mechanisms of these two drug-eluting stents differ specifically in their course of action towards ECs and SMCs. Much of the data available are derived from cellular studies and animal models and are translated to humans since it has been difficult to study these cellular processes in vivo in humans. Pathogenesis of In-Stent Restenosis Neointimal hyperplasia, defined as the uncontrolled proliferation of SMC within a blood vessel intima with concomitant deposition of extracellular matrix molecules, e.g., proteoglycans, is an important mechanism contributing to restenosis within stents.7,8 Local inflammation and arterial injury after stent deployment and adjunctive balloon angioplasty augments neointimal growth of cells.9,10 The earliest step in the process of in-stent restenosis, before SMC proliferation, is platelet deposition and aggregation.11 Platelets release multiple growth and migratory-promoting factors in addition to those released from injured vascular cells and surrounding extracellular matrix such as thrombin, platelet-derived growth factor (PDGF), interleukin (IL)-1, insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), vascular endothelial cell growth factor (VEGF), and others.12–14 This complex interplay of growth factors then regulate SMC migration and proliferation through cell surface receptors and intracellular signaling molecules inducing early response genes necessary for cells to leave their quiescent state and enter the cell cycle.15 Positive regulatory proteins, e.g. cyclins and cyclin-dependent kinases (CDK), and negative regulators, e.g., cell cycle inhibitors, both tightly control cell cycle events. Progression from one cell cycle phase into the next is regulated by the formation and activation of cyclin-CDK complexes, predominantly cyclin D-cdk4/6 and cyclin E-cdk2.15 Endogenous cyclin-dependent kinase inhibitors (CKI) attenuate the activity of cyclin-CDK complexes, leading to G1 arrest, thereby functioning in-growth regulation and wound repair.16 One example, the CKI p27kip1, which is constitutively expressed in normal arteries, becomes down-regulated after injury and up-regulated later during arterial repair. Its protein levels are inversely correlated with vascular SMC proliferation.17 In SMC, p27kip1 and p21cip1 are the two important CKIs that regulate CDK activity, and overexpression of p27kip1 inhibits intimal cell proliferation and neointimal development after vessel injury.18 Cellular Mechanism of Sirolimus The drug sirolimus, also known as rapamycin, gained popularity during the early 1990s as an immunosuppressive agent; an oral form was FDA-approved in 1999 for prophylaxis of organ rejection in patients receiving renal transplants.19 Sirolimus inhibits T-lymphocyte activation and proliferation in response to antigen and cytokine (IL-2, IL-4, and IL-15) stimulation by a mechanism distinct from other immunosuppressants.20 Sirolimus is lipophilic and crosses cell membranes to bind the FK binding protein-12 (FKBP-12), generating an active complex.21 The sirolimus:FKBP-12 complex binds and inhibits mammalian Target Of Rapamycin (TOR).22 Inhibition of TOR by sirolimus suppresses cytokine-driven T-cell proliferation by inhibiting progression through the cell cycle via the following mechanisms: a) directly reducing CDK levels and CDK activity, b) increasing concentration of CKI p27kip1, and c) reducing phosphorylation of the retinoblastoma (Rb) protein, thus activating this cell cycle inhibitor. The net effect is the arrest of cells at the cell cycle restriction point (G0), thereby preventing progression from the G1 to the S phase. The mechanism of inhibition is cytostatic rather than cytotoxic since the affected cells remain viable. Sirolimus Effects on Smooth Muscle Cell Proliferation In normal blood vessels, medial layer SMCs are quiescent and remain at the G0/G1 phase of the cell cycle.23 Upon vessel injury, vascular SMCs are stimulated to divide by released mitogens and exit the G1 phase, entering into the S phase. Similar to lymphocytes, SMCs are subject to the antiproliferative effects of sirolimus,24 including inhibition of growth factor-stimulated DNA synthesis.25 Sirolimus effects correlate with reduced Rb protein phosphorylation at the G1/S transition and decreased CDK activity,24 thus sirolimus-FKBP-12-TOR complexes inhibit vascular SMC proliferation. SMC proliferation requires both gene transcription to make key mRNA species, and increased mRNA translation to generate cell cycle proteins. An intracellular signaling pathway initiated by growth factor receptors is essential for the activation of translation and involves the p70/p85-kDa S6 kinases (p70S6).26 Upon SMC activation, following growth factor stimulation or balloon injury, p70S6 kinase phosphorylates ribosomal protein S6, leading to the activation of transcription factors, particularly the eukaryotic initiation factor 4E (eIF4E).27 Sirolimus inhibits p70S6 kinase and blocks protein up-regulation of cyclins and CDK,28 thereby arresting cells, suggesting that TOR is key for SMC cell cycle progression29 (Figure 1 – pathway P). Growth factors also down-regulate cellular inhibitors of cell cycle progression. The CKI p27kip1 regulates cell entry into the S phase,30 and in T-lymphocytes, sirolimus inhibits growth factor-induced down-regulation of p27Kip1, thereby preventing the activation of cyclin-CDK complexes.31 However, coronary artery SMC growth factor- or balloon injury-induced down-regulation of p27Kip1 is not inhibited by sirolimus29 (Figure 1). Vascular injury in p27-deficient knockout mice confirmed that the lack of p27Kip1 does not affect intimal hyperplasia after injury.32 Sirolimus reduces intimal hyperplasia in both p27 knockout mice and wild-type mice, suggesting that SMC proliferation in response to vessel injury occurs by a p27Kip1-independent mechanism.32 Sirolimus-coated stents applied to rabbit iliac arteries33 or porcine coronaries34 resulted in 30–45% less neointimal formation. This associated with a marked reduction in strut-associated inflammation. Histologic analysis shows less SMC colonization adjacent to sirolimus-coated stent struts compared to uncoated stents and no focal medial necrosis or intimal hemorrhage.34 Sirolimus Effects on Smooth Muscle Cell Migration SMC migration is also important for coronary restenosis after PTCA.35 Quiescent, differentiated SMC possess abundant contractile actin and myosin filaments that await signals for activation. Upon vessel injury, the release of potent chemo-attractants, e.g., PDGF, stimulate phenotypic change and SMC migration from media to intima.36 FGF-2, abundant in the vessel wall, is released from injured EC and underlying extracellular matrix, and although a less potent SMC chemo-attractant, it is required for PDGF-directed SMC migration via a calcium/calmodulin-dependent kinase mechanism.37 Sirolimus inhibits growth factor-induced human SMC migration.38,39 The CKI p27kip1 is important for sirolimus effects on SMC migration since SMCs containing p27kip1 are inhibited from migrating, and cells lacking this inhibitor continue to migrate 40 (Figure 1 – pathway M). Sirolimus Effects on Endothelial Cell Proliferation Endothelium response to injury after balloon angioplasty and stenting is one of replacement and re-growth, a process termed endothelial regeneration. Most common, a denuding type of injury removes or damages ECs and underlying extracellular matrix is exposed.41 The response to injury is dependent on damage extent. Denuded endothelium accompanied by widespread injury to underlying layers during angioplasty and stenting results in aggressive platelet and leukocyte responses. Platelets are seen early covering the denuded areas,42 followed by adherence of neutrophils, and then massive infiltration of macrophages into the intima matrix, a process greatly exaggerated when angioplasty is performed in the presence of hypercholesterolemia.43 Replacement and regrowth of the injured endothelium requires growth factors such as FGF, VEGF, PDGF, and others derived from ECs or circulating cells (e.g. platelets and infiltrating monocytes).44 Enlargement and spreading of adjacent ECs, no longer contact-inhibited, into the denuded area is followed by cell migration and mitogenesis by 24 hours.45 Completion time of endothelial re-establishment depends upon the denudation degree, ranging from 2 days for small areas, to weeks for extensive injury.46 Growth factors responsible for endothelial regeneration may be present in the vessel wall and released upon injury or denudation, or induced by cell activation, protein synthesis, and proliferation.46 For example, FGF-2 is released from cells by membrane disruption following vessel injury or by proteases from stores in the extracellular matrix,47,48 serving as a potent autocrine and paracrine inducer of EC migration and proliferation.49 In catheter denudation models of carotid arteries, released FGF-2 induces EC replication at the wound edges.50 Re-endothelialization is completed at 10 weeks after FGF-2 treatment, but in the absence of FGF-2, regeneration ceases before cellular cover is reached. Exposure of normal intact endothelium to FGF-2 has little effect on proliferation,50 emphasizing FGF-2 importance in wound repair. Sirolimus potently inhibits growth factor-stimulated proliferation of human ECs.51 Similar to SMCs, FGF-2-induced EC proliferation requires activation of downstream p70S6 kinase, and sirolimus inhibits EC proliferation by preventing its activation52,53 (Figure 2 – pathway P). Two signaling cascades important for EC proliferation induced by growth factor receptors are: 1) Ras-p42/p44 MAP (Mitogen Activated Protein) kinase pathway and 2) Phosphatidylinositol-3 Kinase (PI3 Kinase)-p70 S6 kinase pathway. A mutant p70S6 kinase resistant to sirolimus expressed in vascular EC reverses the inhibitory effect on DNA synthesis, confirming that sirolimus acts via TOR and p70S6 kinase.53 Yet, sirolimus does not block EC growth factor-induced Ras-p42/p44 MAP Kinase activity54 (Figure 2 – pathway M). VEGF, via its receptor, stimulates EC proliferation by activating PI3 kinase.55 Mutated VEGF receptors lacking intracellular PI3 kinase binding sites, or synthetic PI3 kinase inhibitors, inhibit VEGF-induced p70S6 kinase activation and cell growth.56,57 However, inhibition of Ras protein or MAP kinase signaling does not inhibit VEGF-induced EC growth stimulation.57 Thus, VEGF, like FGF-2, also stimulates EC proliferation via a sirolimus-sensitive PI3 Kinase-p70S6 kinase signaling pathway, and not the Ras-p42/p44 MAP kinase pathway. Shear stress from oscillatory fluid flow also induces DNA synthesis in vascular ECs. The signaling pathway mediating this growth factor-independent phenomenon in ECs also involves the activation of PI3 Kinase-p70S6 kinase and CDK expression; sirolimus abrogates this effect.58,59 Inhibition of MAP-kinase does not affect oscillatory flow-induced proliferation.58,59 Shear stress and blood flow disturbances from denuded endothelium after PTCA and stenting may associate with cell proliferation via this sirolimus-sensitive signaling pathway. Sirolimus Effects on Endothelial Cell Migration VEGF and FGF-2 both stimulate vascular EC migration.55 In response to FGF-2 ECs change morphology, elongate, and undergo intracellular re-organization that correlates with cell migration near the wound edge.60 Endothelial wounding releases FGF-2, which increases EC-derived plasminogen activator production, an important protease for macrovascular and microvascular EC migration.49 No published studies demonstrate sirolimus inhibition of stimulated endothelial migration. Porcine studies suggest re-endothelialization is intact after sirolimus-eluting stents are implanted, similar to uncoated stents.34 Intracellular signaling involved in EC migration suggests a pathway not inhibited by sirolimus (Figure 2M). Mutated VEGF receptors lacking PI3-kinase binding sites resulted in inhibition of p70S6 kinase and cell growth.57 These mutated VEGF receptors were able to stimulate cell migration,57 suggesting that EC migration is not mediated via the PI3-Kinase-p70S6 kinase pathway, and EC migration is not sensitive to sirolimus. Supporting this hypothesis, EC migration in response to mechanical injury is accompanied by activation of the Ras-p42/p44 MAP-kinase signaling pathway, specifically at the wound edge.61 ECs genetically deficient in FGF-2 neither migrate nor activate Ras-p42/p44 MAP kinase in response to mechanical wounding.61 In this model, EC migration required a Ras-p42/p44 MAP-kinase pathway (Figure 2M), however FGF-2-induced proliferation (Figure 2P) was not inhibited. Recently, endothelialization was assessed after delivery of sirolimus-coated stents into porcine coronary arteries. Stent implantation resulted in disruption of luminal ECs. At 3 days post-implantation, stent struts were covered by matrix, and by 14 days, sirolimus-eluting stents were completely covered with an endothelial layer over the neointima.62 Tight junctions were observed by electron microscopy between ECs from sirolimus-treated vessels, supporting the fact that sirolimus does not affect EC migration and vessel re-endothelialization. Cellular Mechanism of Paclitaxel Paclitaxel is a lipophilic molecule that inhibits SMC proliferation and migration, yet by a different mechanism compared to sirolimus. Paclitaxel is a microtubule-stabilizing agent with potent antiproliferative and antimigratory activity for multiple cell types, including fibroblasts, epithelial cells and tumor cells.63 The drug does not inhibit polymerization of tubulin into microtubules, but enhances microtubule assembly into stable polymerized structures. This decreases the concentration of tubulin required for new microtubule formation.64 Microtubules are components of the cytoskeleton and mitotic spindle, being required for both cell division and motility. The activation of MAP-kinase signaling by growth factors associates with microtubule depolymerization, and is inhibited by paclitaxel.65 By stabilizing cytoplasmic microtubules and blocking microtubule disassembly, paclitaxel prevents DNA synthesis initiated by growth factors.66 Paclitaxel-inhibited cells remain at the G0/G1 and G2/M interfaces of the cell cycle.67 Cells exposed to paclitaxel undergo apoptosis or cell death.68 In tissue, paclitaxel associates with mitotic arrest and cellular necrosis.69 Paclitaxel Effects on Smooth Muscle Cells Migration and Proliferation The antiproliferative effect of paclitaxel towards vascular cells was demonstrated using cultured SMCs. Exposure of human arterial SMCs, or co-cultures of SMCs and ECs, with paclitaxel for 24 hours or even 20 minutes, caused complete and prolonged inhibition of SMC growth for 14 days.70 The presence of growth factors or endothelium did not attenuate the effects of paclitaxel. In rabbits, paclitaxel significantly reduces neointimal formation, vessel thickness and stenosis after balloon angioplasty.70 It prevents vascular SMC proliferation and neointimal SMC accumulation in the carotid artery after balloon dilatation and endothelial wounding.71 Inhibition of coronary intimal hyperplasia by paclitaxel-eluting stents was complicated in animal models because of local cytotoxic effects. Histological findings revealed incomplete healing in the paclitaxel-eluting stents consisting of decreased medial wall thickness, persistent intimal fibrin deposition, intra-intimal hemorrhage, increased intimal and adventitial inflammation, late presence of inflammatory cells, e.g., macrophages, and cell necrosis.72–74 Neointimal thickness was reduced with moderate-dose paclitaxel, however neointimal growth was no longer suppressed at 90 days, and SMCs were no longer growth-inhibited.73 Thus, paclitaxel delayed but did not inhibit in-stent neointimal growth in the rabbit model. In addition, histological findings showed incomplete healing and a local arterial toxic effect from paclitaxel. Incomplete healing may delay re-endothelialization and prolong the period of vessel wall thrombogenicity, as has been suggested after brachytherapy.75,76 The mechanism of increased thrombogenicity may be secondary to delayed re-endothelialization with paclitaxel treatment. Paclitaxel Effects on Endothelial Cell Migration and Proliferation Similar to SMCs, paclitaxel has strong anti-proliferative effects towards ECs,77 and microtubules are important structures during EC migration.78 EC proliferation, motility, invasiveness, and cord formation using a basement membrane model system are all inhibited by paclitaxel in dose-dependent manners.
79 The anti-angiogenic activity of paclitaxel is not linked to its cytotoxic effect, since drug concentrations that inhibit human EC migration and invasiveness do not affect EC proliferation; low, non-toxic doses of paclitaxel produce significant decrements in migration and new vessel formation.80
Re-endothelialization requires both EC migration and proliferation. In the presence of paclitaxel, denudation alone sufficiently promoted proliferation of ECs within an area bordering a wound, however these cells are unable to migrate.81 ECs further away from the wound edge fail to proliferate after paclitaxel treatment since their ability to migrate is inhibited, suggesting that proliferation for these cells is dependent on migration.81 Also, human ECs can undergo apoptosis after exposure to low concentrations of paclitaxel.82 These effects may be specific for proliferating endothelium in denuded areas after PTCA and stenting, in contrast to quiescent and contact-inhibited endothelium away from the wound that is resistant to apoptosis.
In the rabbit iliac artery following implantation of paclitaxel-eluting stents, endothelium regrowth in the stent after denudation was lower compared to uncoated stents.72 In contrast, EC regrowth occurred with or without paclitaxel, when endothelial denudation was absent.83 Unfortunately, endothelium denudation is clinically difficult to prevent during stent implantation.
In addition to endothelial regrowth from surrounding areas after denudation, circulating progenitor cells possibly contribute to re-endothelialization. Recent studies suggest that circulating vascular progenitor cells may mediate vascular repair and restenosis.84 Functional endothelial progenitor cells appear to play a protective role in attenuating restenosis.84,86 It is unknown how paclitaxel- or sirolimus-eluting stents affect EC progenitor cell homing and function.
Coronary artery stent placement has been a significant advance in the percutaneous treatment of atherosclerotic disease, but in-stent restenosis remains an important limitation. The recent introduction of drug-eluting stents with antiproliferative and antimigratory properties, such as sirolimus and paclitaxel, appear to be efficacious clinically in inhibiting restenosis by inhibiting SMCs. These lipophilic drugs act locally in the vessel wall and do not result in significant systemic distribution, thus limiting adverse effects. Sirolimus is a cytostatic inhibitor of SMC and EC proliferation via specific intracellular protein interactions with FKBP-12, subsequently inhibiting TOR, p70 S6K, and cyclin-CDK complexes. Different intracellular signaling pathways activate the migration of ECs and SMCs. The migration of ECs utilizes a Ras-p42/p44 MAP-kinase signaling pathway, which is not inhibited by sirolimus. Paclitaxel is a cytotoxic inhibitor of SMCs and ECs via non-specific inhibition of microtubule disassembly. The models in Figure 3 demonstrate how differential effects of these two drugs on EC may delay the process of re-endothelialization during stent-induced vessel injury and endothelial denudation. The different mechanisms may translate into different clinical efficacy and possible adverse events. Future studies comparing these two drug-eluting stent systems will be required to correlate cellular mechanisms with human experience.
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