Comparative vascular responses three months after paclitaxel and everolimus-eluting stent implantation in streptozotocin-induced diabetic porcine coronary arteries

Abstract

Background: Diabetes remains a significant risk factor for restenosis/thrombosis following stenting. Although vascular healing responses following drug-eluting stent (DES) treatment have been characterized previously in healthy animals, comparative assessments of different DES in a large animal model with isolated features of diabetes remains limited. We aimed to comparatively assess the vascular response to paclitaxel-eluting (PES) and everolimus-eluting (EES) stents in a porcine coronary model of streptozotocin (STZ)-induced type I diabetes.

Method: Twelve Yucatan swine were induced hyperglycemic with a single STZ dose intravenously to ablate pancreatic β-cells. After two months, each animal received one XIENCE V® (EES) and one Taxus Liberte (PES) stent, respectively, in each coronary artery. After three months, vascular healing was assessed by angiography and histomorphometry. Comparative in vitro effects of everolimus and paclitaxel (10-5 M-10-12 M) after 24 hours on carotid endothelial (EC) and smooth muscle (SMC) cell viability under hyperglycemic (42 mM) conditions were assayed by ELISA. Caspase-3 fluorescent assay was used to quantify caspase-3 activity of EC treated with everolimus or paclitaxel (10-5 M, 10-7 M) for 24 hours.

Results: After 3 months, EES reduced neointimal area (1.60 ± 0.41 mm, p < 0.001) with trends toward reduced % diameter stenosis (11.2 ± 9.8%, p = 0.12) and angiographic late-loss (0.28 ± 0.30 mm, p = 0.058) compared to PES (neointimal area: 2.74 ± 0.58 mm, % diameter stenosis: 19.3 ± 14.7%, late loss: 0.55 ± 0.53 mm). Histopathology revealed increased inflammation scores (0.54 ± 0.21 vs. 0.08 ± 0.05), greater medial necrosis grade (0.52 ± 0.26 vs. 0.0 ± 0.0), and persistently elevated fibrin scores (1.60 ± 0.60 vs. 0.63 ± 0.41) with PES compared to EES (p < 0.05). In vitro, paclitaxel significantly increased (p < 0.05) EC/SMC apoptosis/necrosis at high concentrations (≥ 10-7 M), while everolimus did not affect EC/SMC apoptosis/necrosis within the dose range tested. In ECs, paclitaxel (10-5 M) significantly increased caspase-3 activity (p < 0.05) while everolimus had no effect.

Conclusion: After 3 months, both DES exhibited signs of delayed healing in a STZ-induced diabetic swine model. PES exhibited greater neointimal area, increased inflammation, greater medial necrosis, and persistent fibrin compared to EES. Differential effects of everolimus and paclitaxel on vascular cell viability may potentially be a factor in regulating delayed healing observed with PES. Further investigation of molecular mechanisms may aid future development of stent-based therapies in treating coronary artery disease in diabetic patients.

Figure 1

Histological assessment of coronary artery tissue stented with PES and EES in STZ-induced type I diabetic swine. Histopathological scoring (mean ± standard deviation, n = 11) revealed delayed neointimal healing in response to PES after 90 days. Both A) inflammation and B) fibrin were significantly increased in response to PES (Taxus) (p < 0.05) compared to EES (Xience V). C) No significant differences in neointimal immaturity were found between PES and EES. ^* p < 0.05

Figure 2

Histopathology of coronary artery tissue stented with PES and EES in STZ-induced type I diabetic swine. Representative histopathological microscopy images (20x) of coronary arteries stented with EES (Xience V) and PES (Taxus) and stained with H&E after 90 day implantation

Figure 3

Effects of paclitaxel and everolimus on SMC and EC apoptosis and necrosis. SMC or EC were cultured in vitro under hyperglycemic conditions (42 mM) in 24-well plates and allowed to grow to 80% confluence. Cells were then treated with growth media alone (control), DMSO vehicle control, or everolimus or paclitaxel over a range of concentrations (10^-5 M–10^-12 M) for 24 hours. A Cell Death Detection ELISA^PLUS kit was then used to detect histone-associated nucleosome concentration in cell media supernatants to determine overall necrosis, while concentration in cell lysates was used to determine relative induction of apoptosis. Data expressed as fold change relative to control (mean ± standard deviation, n = 3). A) Paclitaxel significantly increased SMC apoptosis and necrosis in a dose-dependent manner at concentrations of 1 μM and greater. B) Paclitaxel significantly increased EC apoptosis and necrosis in a dose-dependent manner at concentrations of 0.1 μM and greater. Everolimus did not increase SMC or EC apoptosis or necrosis at any dose tested. ^* p < 0.05 vs. control

Figure 4

Effects of paclitaxel and everolimus on caspase-3 activity in EC. EC were cultured in vitro under hyperglycemic conditions (42 mM) and allowed to grow to 80% confluence. Cells were then treated with growth media alone (control), or everolimus or paclitaxel (10^-5 M, 10^-7 M) for 24 hours. A caspase-3 fluorescent assay kit was used to quantify caspase-3 activity. Data expressed as fold change relative to control (mean ± standard deviation, n = 3). Paclitaxel increased EC caspase-3 activity in a dose-dependent manner while everolimus had no effect. ^* p < 0.05 vs. control