17beta-Estradiol and Restenosis: A Novel Vaso-Protective Role for Estrogen?

The possible role of estrogen in coronary artery disease (CAD) has been extensively studied. Prospective randomized clinical trials have not substantiated a beneficial role for estrogen in CAD. The Heart and Estrogen/Progestin Study follow-up (HERS II) did not demonstrate a reduced risk of CAD after 6.8 years of hormone replacement therapy.¹ The Women’s Health Initiative (WHI) trial has concluded that overall health risks exceeded benefits with the use of hormone replacement therapy for a mean treatment period of 5.2 years.² There is no longer any support for systemic estrogens in CAD in clinical practice. The only remaining area of interest, therefore, is local delivery. The vaso-protective effect of estrogen reaches beyond its effects on plasma lipids and atherosclerosis. Estrogen exerts a marked effect on the vascular endothelium, smooth muscle cells (SMC), and other components involved in tissue repair following arterial injury. Development of restenosis. Restenosis is a complex process involving the interaction of various cellular components and growth factors. Endothelial damage, activation of platelets, migration and proliferation of SMC, inflammatory cellular infiltration, and extracellular matrix synthesis are all constitutive. Disruption of endothelium occurs following angioplasty and the endothelial dysfunction persists or even worsens with time.⁴ Besides predisposing to abnormal vascular reactivity, endothelial dysfunction is an independent predictor of restenosis.⁴ Platelets activated following injury enhance thrombin generation and also release several growth factors⁵ which induce proliferation of SMC. Migration of SMC without proliferation is also important in the formation of neointima; as much as 50% of SMC migrating into the intima do not proliferate.⁶ Inflammatory cellular infiltration contributes to neointima formation⁷ and correlates directly with the extent of neointimal proliferation.^8,9 Extracellular matrix formation, initially predominant in proteoglycans such as chondroitin, dermatan and heparan sulfate, and subsequently replaced by collagen and elastin, is important in the remodeling process.¹⁰Estrogen and response to injury. In the last decade, several experiments have provided compelling evidence demonstrating the inhibitory effects of 17beta-estradiol on neointimal proliferation (Table 1). In a study on male rabbits fed on a high-cholesterol diet, 17beta-estradiol therapy significantly inhibited intimal thickening associated with graft atherosclerosis without influencing serum cholesterol levels.¹¹ Subsequent experiments studied the effect of 17beta-estradiol following iatrogenic arterial injury such as balloon injury,^12-19 endoluminal wire-induced injury,^20-22 and application of an external cuff.²³ Systemic treatment with 17beta-estradiol successfully inhibited neointimal formation in all the studies, irrespective of the mode of injury. In a model without iatrogenic injury, ovariectomized sheep showed marked intimal thickening at the aortoiliac bifurcation, an effect attenuated in ovariectomized sheep on 17beta-estradiol replacement.²⁴Vaso-protective effects of estrogen following arterial injury. The beneficial effect of estrogen following injury appears to involve multiple underlying mechanisms. Estrogen exerts favorable effects on SMC migration and proliferation, and enhances endothelial recovery following injury. Estrogen also exerts an influence on inflammatory cellular infiltration. These effects possibly contribute to the inhibition of neointimal formation (Figure 1). Effect on SMC. The migration of SMC and their proliferation play a fundamental role in neointimal formation and are thus potential targets for manipulation. In cell culture assays, 17beta-estradiol was successful in inhibiting migration of SMC.^25,26 In addition to the effect on SMC migration, treatment with 17beta-estradiol decreases growth factor-induced SMC proliferation.^23,26,27 Direct receptor-mediated effects may be important in the anti-proliferative response of 17beta-estradiol. The interaction of estrogen with estrogen receptor (ER) leads to binding of the ER to and activation of “estrogen response elements” of target genes regulating protein synthesis.²⁸ Estrogen may also exert its anti-proliferative effect on SMC by regulation of endothelial nitric oxide synthase (eNOS),^29,30 or of prostaglandin cyclo-oxygenase and prostacyclin synthase.³¹ Other possible mechanisms include stimulation of cAMP synthesis and inhibition of vascular SMC growth by cAMP-derived adenosine via A(2) adenosine receptor,³² and down-regulation of Sp-1 (a transcription factor that binds to the PDGF-A gene promoter site).³³ Additionally, 17beta-estradiol inhibits SMC-mediated migration of adventitial fibroblasts by an ER-dependent mechanism.³⁴Effect on endothelium. Treatment with estrogen enhances structural and functional recovery of endothelium following arterial injury. Two weeks following injury, arteries from estrogen-treated animals showed significantly greater re-endothelialized area compared to control, accompanied by increased nitric oxide (NO) production.¹⁶ In another study, isolated ring segments from estrogen-treated rats following injury showed significantly greater endothelium-dependent vascular relaxation to acetylcholine.¹⁷ The enhanced reendothelialization and functional recovery observed with estrogen may be related to a direct angiogenic effect on endothelial cells,³⁵ basic fibroblast growth factor release,³⁶ vascular endothelial growth factor expression,¹⁶ inhibition of endothelial cell apoptosis³⁷ and eNOS expression.^29,30 Other potential mechanisms by which estrogen may regulate NO production include prolongation of eNOS mRNA half-life by suppression of cytokine-induced down-regulation of eNOS mRNA, enhancing cell membrane localization of eNOS, increasing the availability of cofactor (biopterin, calmodulin, and intracellular calcium) and substrate (L-arginine), and increasing the bioavailability of NO by virtue of its antioxidant effects as reviewed in detail elsewhere.³⁸Effect on inflammation. Nitric oxide-mediated inhibition plays an important role in the effect of estrogen therapy on the inflammatory response. Nitric oxide is of importance in the regulation of leukocyte adhesion.³⁹ Estrogen induces up-regulation of neuronal-type NOS protein in neutrophils from both women and men, leading to decreased adhesiveness of neutrophils.⁴⁰ Besides, estrogen suppresses neutrophil chemotaxis by a receptor-mediator effect.⁴¹ Estrogen also reduces monocyte chemoattractant protein-1 levels and macrophage accumulation.⁴² Hormonal therapy in postmenopausal women leads to reduction in E-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 levels.⁴³Effect on extracellular matrix. Estrogen therapy is associated with decreased collagen synthesis in the aorta of atherosclerotic rabbits.⁴⁴ Male hypertensive rats, upon treatment with estrogen, showed significantly lower aortic medial thickness and absolute weight of elastin and collagen as compared to untreated or progesterone-treated rats.⁴⁵ The effect of estrogen-treatment on extracellular matrix synthesis following arterial injury has not been studied, although, with the increasing use of stents (which effectively neutralize negative or positive remodeling) during percutaneous coronary interventions (PCI), the remodeling process may likely be of less importance. Estrogen receptors. The presence of ER has been documented in human coronary artery SMC and endothelial cells,^46,47 and their functional capacity has been demonstrated.⁴⁸ Subsequently, the identification of ERbeta in humans, with a very high homology to the classical ER (ERalpha), was reported.⁴⁹ Treatment with estrogen has been shown to inhibit the neointimal response to injury in ERalpha-knock-out²¹ and ERbeta-knock-out mice.²² A study on vascular SMC proliferation in double knockout mice has led to speculation about whether inhibition of SMC proliferation in double knock-out mice is caused by a receptor-independent mechanism or an unidentified receptor responsive to estrogen.⁵⁰ Potential clinical applications. In retrospective studies, a significant reduction in adverse events after PCI was observed with estrogen replacement therapy (ERT), although no effect on target vessel revascularization (TVR) was noted.^51,52 Patients on ERT in the CAVEAT 1 study showed significantly lower late-loss and restenosis following directional atherectomy, but not after balloon angioplasty.⁵³ A recent study of patients undergoing intracoronary stenting has reported lower rates of TVR with ERT.⁵⁴ An important concern that arises in the context of clinical application is a gender-specific effect of estrogen.¹⁵ However, several studies have documented estrogen-responsiveness in the vasculature of male gender.^11,26,42,45 The presence of ERbeta and the increased expression of ERbeta mRNA induced by arterial injury has been demonstrated in male rats.⁵⁵ Healthy men treated with estrogen demonstrated enhanced endothelial-dependent, flow-mediated vasodilatation which correlated with estrogen levels.⁵⁶ However, systemic therapy in males may be associated with side effects such as gynecomastia, decreased growth or testicular atrophy.^11,45,56 Local delivery of 17beta-estradiol. An attractive alternative approach is the local delivery of estrogen at the site of angioplasty. Locally delivered estrogen would effectively circumvent the adverse effects associated with systemic therapy and would thus be applicable in both women and men. Systemic adverse effects following local delivery of 17beta-estradiol are unlikely, as 17beta-estradiol that could possibly enter the circulation is rapidly eliminated, mostly by the liver.⁵⁷ Local delivery of 17beta-estradiol and injury response. Animal experiments (porcine model) on local delivery of estrogen have yielded promising results. In an experiment assessing structural and functional endothelial recovery after balloon angioplasty, enhanced reendothelialization and improved endothelium- dependent vascular reactivity (with up-regulation of eNOS expression) were observed at the site of coronary angioplasty as late as 4 weeks following local delivery of estrogen.⁵⁸ In this study, 17beta-estradiol-treated vessels showed nearly complete reendothelialization (90% complete) compared to a significantly lower extent of reendothelialization (69% complete) in untreated vessels. Enhanced functional recovery of endothelium in 17beta-estradiol-treated vessels was evident as demonstrated by significantly higher eNOS expression (in 17beta-estradiol-treated vessels) and the absence of paradoxical vasoconstriction (in 17beta-estradiol-treated vessels) to intracoronary acetylcholine infusion, while paradoxical vasoconstriction was marked in untreated vessels. Another experiment reports the effect of local delivery of 17b-estradiol on neointimal formation after balloon angioplasty.⁵⁹ In this study, local delivery of 17beta-estradiol was associated with a significantly lower neointimal area, neointima/media area ratio, restenotic index and morphologic percent stenosis at 28 days. Furthermore, by immunohistochemistry, a significantly lower SMC proliferation at 7 days was evident in 17beta-estradiol-treated vessels. Compared to untreated vessels, those treated with local delivery of 17beta-estradiol demonstrated 50% lower neointimal formation, with no effect on remodeling. This observation would be especially advantageous in the prevention of in-stent restenosis, where neointimal proliferation (and not remodeling) is the fundamental underlying mechanism. This effect on in-stent restenosis has been reported in an experiment where after initial pre-dilatation, catheter-based local delivery of 17beta-estradiol was performed, followed by bare metal stent implantation.⁶⁰ In this study, at the end of 28 days, 17beta-estradiol-treated arteries demonstrated angiographically significant greater minimal lumen diameter and lower percent diameter stenosis and late loss compared to controls. Histological analysis showed significantly lower percent morphologic stenosis, neointimal thickness and inflammation score in 17beta-estradiol-treated arteries. By immunohistochemistry, significantly fewer proliferating SMC and greater eNOS expression in 17beta-estradiol-treated arteries were noted. No adverse effects were attributable to local delivery of estrogen. New et al. have reported the effects of 17beta-estradiol-eluting stent in a porcine coronary artery model.⁶¹ A 40% reduction in neointimal area was seen with 17beta-estradiol-eluting stents compared to control stents at 30 days. Complete endothelialization around the stent struts was noted with 17beta-estradiol-eluting stents at 30 days (as well as control stents). In addition, 17beta-estradiol-eluting stents did not demonstrate any increase in intimal fibrin, inflammatory response, adventitial fibrosis or any other features of a tissue reaction. It may be worth noting that the 40% and 50% reduction in neointimal area observed with locally delivered 17b-estradiol is similar to the reduction in neointimal area reported in experimental studies with rapamycin^62,63 and paclitaxel.^64,65 Moreover, it is of potential interest that 17beta-estradiol delivered locally promotes reendothelialization after arterial injury, an effect thus far not demonstrated with rapamycin or paclitaxel. Mode of local delivery of 17beta-estradiol. 17beta-estradiol can be delivered intramurally either by a local delivery catheter, or by way of a drug-eluting stent. The InfusaSleeve catheter (LocalMed Inc., Palo Alto, California) has been used safely to deliver 17beta-estradiol locally without significant injury.^58–60 Stent-based delivery of 17b-estradiol has been performed successfully using a phosphorylcholine-coated stainless steel stent.⁶¹ The drug stent was prepared in the cardiac catheterization laboratory by immersion of a phosphorylcholine coated stainless steel stent (BiodivYsio DD Stent, Biocompatibles, Surrey, U.K.) in a solution of 17beta-estradiol, followed by drying at room temperature. A novel method of loading 17beta-estradiol onto a metallic stent surface by plasma polymerization was recently reported.⁶⁶ The method involves activation of stainless steel surface with silane, followed by treatment with acrylic acid and subsequent covalent linking of 17beta-estradiol to the resultant stent surface. The authors of this method were able to demonstrate sustained release of 17beta-estradiol in aqueous buffer in vitro upon evaluation by ultraviolet-visible spectrophotometry and high performance liquid chromatography. Conclusion. Available experimental data clearly demonstrate the ability of estrogen to inhibit neointimal response to injury.^11–24 A few clinical studies have suggested that ERT has the potential to decrease TVR following PCI.^53,54 Estrogen exerts a favorable effect on many of the mechanisms underlying restenosis, leading to enhanced reendothelialization and improved endothelial function, inhibition of SMC proliferation and suppression of inflammatory cellular infiltration. However, systemic estrogen therapy may be associated with limiting adverse effects. An attractive alternative approach is the local delivery of estrogen during intervention. An interesting proposition would be the use of 17beta-estradiol-eluting stents to deliver estrogen locally. The extent of reduction in neointimal area observed in experiments with locally-delivered 17beta-estradiol is similar to that reported in experiments with rapamycin and paclitaxel. Furthermore, locally delivered 17beta-estradiol promotes reendothelialization, an effect thus far not demonstrated with rapamycin or paclitaxel. Finally, controlled randomized trials will determine whether the reported promising experimental results can be translated into clinical benefits.

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