Selective clearance of macrophages in atherosclerotic plaques by autophagy.

OBJECTIVES: The purpose of this study was to investigate whether stent-based delivery of an inhibitor of mammalian target of rapamycin (mTOR) can selectively clear macrophages in rabbit atherosclerotic plaques. BACKGROUND: Current pharmacologic approaches to stabilize atherosclerotic plaques have only partially reduced the incidence of acute coronary syndromes and sudden death. Macrophages play a pivotal role in plaque destabilization, whereas smooth muscle cells (SMC) promote plaque stability. METHODS: Stents eluting the mTOR inhibitor everolimus were implanted in atherosclerotic arteries of cholesterol-fed rabbits. In addition, in vitro experiments using explanted atherosclerotic segments and cultured macrophages as well as SMC were performed. RESULTS: Stents eluting everolimus led to a marked reduction in macrophage content without altering the amount of SMC compared with polymer control stents. In vitro studies showed that everolimus treatment induced inhibition of translation in both cultured macrophages and SMC. However, cell death occurred only in macrophages and was characterized by bulk degradation of long-lived proteins, processing of microtubule-associated protein light chain 3, and cytoplasmic vacuolization, which are all markers of autophagy. Everolimus-induced autophagy was mediated by mTOR inhibition, because cell viability was not affected using tacrolimus, an mTOR-independent everolimus analog. Moreover, mTOR gene silencing was associated with selective induction of macrophage cell death. Autophagic macrophage cell death was confirmed by transmission electron microscopy both in cultured cells and in atherosclerotic explants. CONCLUSIONS: Stent-based delivery of everolimus selectively cleared macrophages in rabbit atherosclerotic plaques by autophagy, an mTOR inhibition-dependent and novel mechanism to induce cell death in mammalian cells.

Effects of cytochalasin D-eluting stents on intimal hyperplasia in a porcine coronary artery model.

OBJECTIVE: To investigate whether cytochalasin D-eluting stents (CDES) suppress intimal hyperplasia in porcine coronary arteries and to compare the efficacy of paclitaxel and cytochalasin D as inhibitors of vascular smooth muscle cell (SMC) proliferation and platelet aggregation in vitro. METHODS: Rabbit platelet-rich plasma and SMC cultures derived from rabbit aortas were exposed to 10(-8)-10(-5) M cytochalasin D or paclitaxel. Stents directly coated with 2 microg cytochalasin D (low-dose CDES, n=12) and bare stents (n=12) were randomly deployed in the right and left coronary artery of 12 pigs. Six weeks later, neointima was studied using quantitative coronary angiography (QCA) and morphometry. To examine a ten-fold higher dose, polybutyl methacrylate/polyvinyl acetate-coated stents were loaded with 20 microg cytochalasin D. High-dose CDES (n=10) and polymer-only stents (n=11) were deployed in 11 pigs. RESULTS: After 7 days, cytochalasin D (IC(50) 9.9+/-0.4 10(-8) M) and paclitaxel (IC(50) 1.1+/-0.4 10(-8) M) inhibited SMC proliferation in vitro (n=4). In contrast, cytochalasin D (10(-6)-10(-5) M, n=5), but not paclitaxel, attenuated platelet shape change and aggregation induced by ADP. In vivo QCA showed less late lumen loss in low-dose CDES (0.08+/-0.07 vs. 0.32+/-0.08 mm, P=0.05), but morphometry demonstrated only a tendency toward a decreased intimal area. High-dose CDES inhibited both late lumen loss (0.31+/-0.08 vs. 0.91+/-0.06 mm, P<0.01) and intimal area (1.57+/-0.20 vs. 2.46+/-0.22 mm(2), P<0.01). Immunohistochemistry revealed that CDES suppressed peri-strut macrophage recruitment (CD68, P=0.04) and cell proliferation (Ki67, P=0.03) as compared to polymer-only stents without interfering with endothelial cell recovery or the density of alpha-SMC actin staining. Thromboses or edge effects were not observed in either study. CONCLUSIONS: CDES inhibited in-stent hyperplasia. The reduction (39%) with 20 mug CDES was equivalent to that reported for paclitaxel-eluting stents in pigs. Interference with platelet aggregation, SMC migration, SMC proliferation, and leukocyte recruitment could contribute to the benefit. The data indicate that targeting of actin microfilaments has a potential to suppress in-stent restenosis.

Use of a tacrolimus-eluting stent to inhibit neointimal hyperplasia in a porcine coronary model.

In-stent restenosis remains an unresolved problem which occurs in 5-20% of patients undergoing coronary stenting within the first 3-6 months. Neointimal formation is the main contributor to in-stent restenosis. Stent-induced arterial injury and peri-strut inflammation are involved in the process of neointimal formation by activating cytokines and growth factors which induce smooth muscle cell dedifferentiation, migration, and proliferation. Histopathological studies found that neointimal hyperplasia is principally composed of smooth muscle cells, inflammatory cells, and extracellular matrix. Stent-based delivery of anti-proliferative and/or anti-inflammatory agents have shown beneficial effects on neointimal hyperplasia in experimental studies and clinical trials. Tacrolimus (FK506) is a water-insoluble macrolide immunosuppressant discovered in 1984. It has been widely used in reducing the incidence and severity of allograft rejection after organ transplantation. It has also been used to treat other inflammatory conditions such as atopic dermatitis. In this study, we evaluated the efficacy of stent-based delivery of tacrolimus on inflammation and neointimal formation in an overstretched coronary stent model.

Stent-mediated methylprednisolone delivery reduces macrophage contents and in-stent neointimal formation.

OBJECTIVE: Corticosteroids have a wide range of biological effects. Stent-based methylprednisolone delivery could effectively suppress peri-strut inflammation and neointima induced by a polymer matrix. We tested the safety and efficacy of local stent-mediated methylprednisolone delivery using a biological coating on in-stent neointimal formation in a porcine coronary stent model. METHODS: Stainless steel coronary stents were dip-coated in a biological polymer/ methylprednisolone solution, resulting in total load of 530 mug methylprednisolone per stent. In-vitro drug release was performed. Stainless steel bare stents, polymer-only and methylprednisolone-coated stents (MP) were implanted in coronary arteries of pigs with a stent/artery ratio of 1.2 : 1. Histopathologic evaluation, morphometry and immunohistochemistic staining were analyzed at 4-week follow-up. RESULTS: In-vitro drug release studies showed sustained release up to 10 weeks. In vivo the vascular response of polymer-only-coated stents was comparable with the bare stents. No increased peri-strut inflammation and neointimal hyperplasia were observed. The in-stent neointimal formation of methylprednisolone-coated stents was significantly reduced compared with the bare and polymer-only-coated stents (bare, 1.92+/-0.73; polymer-only, 2.14+/-1.50; MP, 1.01+/-0.47 mm, P=0.019). The macrophage content of methylprednisolone-coated stents (bare, 30.74+/-48.67; polymer-only, 19.55+/-24.60; MP, 1.16+/-3.33/mm, P=0.072) was dramatically decreased. However, there were no significant difference among the three group in terms of the proliferating cells expressed by proliferation cell nuclear antigens. CONCLUSION: Stent-based local methylprednisolone delivery could effectively decrease both vascular macrophage infiltration and in-stent neointimal hyperplasia.

Histopathologic evaluation of a novel-design nitinol stent: the Biflex stent.

BACKGROUND: Optimalization and improvement in stent material, stent design and deployment may alleviate the problem of restenosis after stenting. The Biflex stent is a novel-design stent made of nitinol; the vascular response after deployment in rabbit iliac arteries was evaluated. METHODS AND RESULTS: Normocholesterolemic New Zealand white rabbits (n = 8) were used. Iliac arteries were randomized to receive either a stainless steel control stent or a nitinol stent and rabbits were euthanized at 30 days after implantation. All animals survived and there were no adverse events. Vessels were harvested and prepared for histopathologic analysis and histomorphometry. Stents were well opposed to the vessel wall and thrombi were absent. The lumen area and the area within the internal elastic lamina were significantly larger in the nitinol stent group as opposed to the control group (3.8 +/- 0.1 vs 3.3 +/- 0.1 mm, p = 0.009 and 4.6 +/- 0.1 vs 4.1 +/- 0.2 mm, p = 0.03, respectively). There were no differences in injury score, neointimal area, medial area, area within the external elastic lamina and amount of inflammatory cells. Staining for alpha-smooth muscle cell actin and endothelium did not show any differences between the two groups as assessed semiquantitatively. CONCLUSION: This nitinol stent with a novel design demonstrated acceptable biocompatibility in iliac arteries of normocholesterolemic rabbits with minimal foreign-body reaction and minimal neointimal formation.

Addition of cytochalasin D to a biocompatible oil stent coating inhibits intimal hyperplasia in a porcine coronary model.

BACKGROUND: Polymer-based, drug-eluting stents, are currently under extensive investigation in the conquest against in-stent restenosis. Concern remains, however, about potential long-term lack of biocompatibility of the polymers used in these studies. Therefore, this study aimed to evaluate in porcine coronary arteries (1) the in vivo biocompatibility of a new natural, eicosapentaenoic acid oil stent-coating and (2) the efficacy of this coating in preventing in-stent restenosis when cytochalasin D--an inhibitor of actin filament formation, that interferes with cell proliferation and migration--was added. METHODS AND RESULTS: To assess in vivo biocompatibility of the oil coating, 15 bare and 15 oil-coated stents were randomly deployed in coronary arteries of 15 pigs. No difference in tissue response, regarding inflammation or proliferation, was seen between both groups at five days or at four weeks follow-up. To evaluate the efficacy of the coating in preventing in-stent restenosis by adding a potential anti-restenotic drug, stents were dip-coated in 20 mg cytochalasin D/ml oil solution, resulting in 93 +/- 18 microg cytochalasin D/stent load (n = 3). In vitro drug release studies showed sustained release up to four weeks. Next, 11 oil-coated and 11 cytochalasin D-loaded stents were randomly implanted in coronary arteries of 11 pigs. At four weeks, a 39% decrease in neointimal hyperplasia (p < 0.05, ANCOVA, with injury as covariate) was found in cytochalasin D-loaded stents compared to oil-coated stents. CONCLUSIONS: This new natural oil stent-coating shows excellent biocompatibility to vascular tissue. Local cytochalasin D delivery from this stent-platform significantly inhibits neointimal hyperplasia in a porcine coronary model.

A three-dimensional quantitative analysis of restenosis parameters after balloon angioplasty: comparison between semi-automatic computer-assisted planimetry and stereology.

Semi-automatic computer-assisted planimetry is often used for the quantification of restenosis parameters after balloon angioplasty although it is a time-consuming method. Moreover, slicing the artery to enable analysis of two-dimensional (2-D) images leads to a loss of information since the vessel structure is three-dimensional (3-D). Cavalieri's principle uses systematic random sampling allowing 3-D quantification. This study compares the accuracy and efficiency of planimetry versus point-counting measurements on restenosis parameters after balloon angioplasty and investigates the use of Cavalieri's principle for 3-D volume quantification. Bland and Altman plots showed good agreement between planimetry and point counting for the 2-D and 3-D quantification of lumen, internal elastic lamina (IEL) and external elastic lamina (EEL), with a slightly smaller agreement for intima and media. Mean values and induced coefficients of variation were similar for both methods for all parameters. Point counting induced a 6% error in its 3-D quantification, which is negligible in view of the biological variation (>90%) among animals. However, point counting was 3 times faster compared to planimetry, improving its efficiency. This study shows that combining Cavalieri's principle with point counting is a precise and efficient method for the 3-D quantification of restenosis parameters after balloon angioplasty.