Polymeric stent materials dysregulate macrophage and endothelial cell functions: implications for coronary artery stent

Abstract

Background: Biodegradable polymers have been applied as bulk or coating materials for coronary artery stents. The degradation of polymers, however, could induce endothelial dysfunction and aggravate neointimal formation. Here we use polymeric microparticles to simulate and demonstrate the effects of degraded stent materials on phagocytic activity, cell death and dysfunction of macrophages and endothelial cells.

Methods: Microparticles made of low molecular weight polyesters were incubated with human macrophages and coronary artery endothelial cells (ECs). Microparticle-induced phagocytosis, cytotoxicity, apoptosis, cytokine release and surface marker expression were determined by immunostaining or ELISA. Elastase expression was analyzed by ELISA and the elastase-mediated polymer degradation was assessed by mass spectrometry.

Results: We demonstrated that poly(D,L-lactic acid) (PLLA) and polycaprolactone (PCL) microparticles induced cytotoxicity in macrophages and ECs, partially through cell apoptosis. The particle treatment alleviated EC phagocytosis, as opposed to macrophages, but enhanced the expression of vascular cell adhesion molecule (VCAM)-1 along with decreased nitric oxide production, indicating that ECs were activated and lost their capacity to maintain homeostasis. The activation of both cell types induced the release of elastase or elastase-like protease, which further accelerated polymer degradation.

Conclusions: This study revealed that low molecule weight PLLA and PCL microparticles increased cytotoxicity and dysregulated endothelial cell function, which in turn enhanced elastase release and polymer degradation. These indicate that polymer or polymer-coated stents impose a risk of endothelial dysfunction after deployment which can potentially lead to delayed endothelialization, neointimal hyperplasia and late thrombosis.

Keywords: Dysfunction; Elastase; Endothelial cell; Poly(L-lactic acid); Polycaprolactone; Stent.

Figure 1

SEM images of microparticles: (a) stainless steel, (b) PLLA and (c) PCL. Scale bar: 150 (a and b) or 37.5 (c) μm.

Figure 2

Cytotoxicity, phagocytosis and apoptosis by interaction with microparticles: (a) Cell number of each group indicated by Hoechst florescence intensity; (b) cytotoxicity measured by lactate dehydrogenase released from damaged cells and normalized to no polymer control; (c) phagocytosis of fluorescent-tagged E. coli particles by activated macrophages and ECs. The fluorescence intensity emitted from E. coli particles was normalized to the corresponding cell number measured by Hoechst staining; (d) apoptosis in untreated (left) and PCL microparticle-treated ECs (right), blue: Hoechst, green: Annexin V; (e) quantification of apoptosis presented as the ratio of Annexin V-positive cells relative to the total cell number. *p<0.05 versus no polymer control; ^†p<0.05 versus SS control; ^‡p<0.05 between PLLA and PCL groups connected by line.

Figure 3

Microparticle-induced EC activation : (a) Merged images of APC-VCAM staining (red) and phase contrast; (b) VCAM-1 positive cells (%) relative to the total cell number in each sample. *p<0.05 versus no polymer control; ^†p<0.05 versus SS control.

Figure 4

Microparticle-mediated macrophage activation: (a) TNF-α release into cell culture media which was determined by ELISA; (b) An intracellular superoxide level which was stained by DHE and normalized to the corresponding cell number measured by Hoechst staining.

Figure 5

EC dysfunction induced by microparticles was determined by quantitating the amount of nitric oxide in supernatant (Greiss reagent). The absorbance was measured at 540nm and normalized to the cell number measured by Hoechst staining. *p<0.05 versus no polymer control.

Figure 6

Elastase production from cells after incubation with microparticles. Elastase production in cell culture media was determined by reacting with elastase substrate and comparing with the elastase standard at 415 nm. The absorbance value was normalized to the corresponding cell number measured by Hoechst staining. *p<0.05 versus no polymer control; ^†p<0.05 versus SS control.

Figure 7

Elastase-mediated polyester degradation. Mass spectrum of PLLA microparticle and degradation products. PLLA particles were suspended in DPBS without (a) or with (b) elastase and incubated at 37°C for 7 days. Degradation products in filtered solutions were confirmed by LC-MS with an acetonitrile-H₂O mobile phase system (positive ion mode).