Reduction of in-stent restenosis risk on nickel-free stainless steel by regulating cell apoptosis and cell cycle

Abstract

High nitrogen nickel-free austenitic stainless steel (HNNF SS) is one of the biomaterials developed recently for circumventing the in-stent restenosis (ISR) in coronary stent applications. To understand the ISR-resistance mechanism, we have conducted a comparative study of cellular and molecular responses of human umbilical vein endothelial cells (HUVECs) to HNNF SS and 316L SS (nickel-containing austenitic 316L stainless steel) which is the stent material used currently. CCK-8 analysis and flow cytometric analysis were used to assess the cellular responses (proliferation, apoptosis, and cell cycle), and quantitative real-time PCR (qRT-PCR) was used to analyze the gene expression profile of HUVECs exposed to HNNF SS and 316L SS, respectively. Flow cytometry analysis revealed that 316L SS could activate the cellular apoptosis more efficiently and initiate an earlier entry into the S-phase of cell cycle than HNNF SS. At the molecular level, qRT-PCR results showed that the genes regulating cell apoptosis and autophagy were overexpressed on 316L SS. Further examination indicated that nickel released from 316L SS triggered the cell apoptosis via Fas-Caspase8-Caspase3 exogenous pathway. These molecular mechanisms of HUVECs present a good model for elucidating the observed cellular responses. The findings in this study furnish valuable information for understanding the mechanism of ISR-resistance on the cellular and molecular basis as well as for developing new biomedical materials for stent applications.

Figure 1. Adhesion of HUVECs to 316L SS substrate, HNNF SS substrate and Type-IV collagen-coated culture dish (referred as control).

Figure 2. Optical images of HUVECs grown on different surfaces of materials.

Culture for 3 days in culture medium (A), 316L SS (B) and HNNF SS (C), and for 7 days in culture medium (D), 316L SS (E) and HNNF SS (F). Scale Bars = 100 µm.

Figure 3. Fluorescent images of HUVECs grown on different surfaces of materials.

Culture for 3 days in culture medium (A), 316L SS (B) and HNNF SS (C), and for 7 days in culture medium (D), 316L SS (E) and HNNF SS (F). Scale Bars = 100 µm.

Figure 4. Measurement of HUVEC proliferation.

(A) Relative growth rates of HUVECs on 316L SS and HNNF SS after 1-, 3-, 5- and 7-day growth with respect to the control of HUVECs (100%). (B) Growth curve of HUVECs on surfaces of 316L SS and HNNF SS, and in culture medium in a 7-day period.

Figure 5. Effect of 316L SS and HNNF SS on cell cycle progression of HUVECs.

(A) Flow cytometric data showing the cell cycle distributions of HUVECs cultured on 316L SS and HNNF SS, respectively, without FBS for 22 h (top panel), and cell cycle distributions of HUVECs cultured with the whole culture medium for 24 h after cell synchronization (bottom panel). (B) Histographic representations of the cell cycle distributions of HUVECs.

Figure 6. Flow cytometric analysis of apoptosis of HUVECs.

Cells were cultured in culture medium, on surfaces of 316L SS and HNNF SS for 7 days.

Figure 7. Gene expression profiles of HUVECs.

Cells grew in culture medium, on surfaces of 316L SS and HNNF SS for 7 days determined using qRT-PCR method.

Figure 8. Schematic diagram of pathways proposed in 316L SS and HNNF SS induced cell apoptosis.

The proposed model shows that 316L SS and HNNF SS cause cell apoptosis through extrinsic apoptosis pathway by the ligation of Fas ligand to Fas receptor activating caspase-8 and caspase-3, whereas HNNF SS reduces the activation of caspase-8 and caspase-3 compared with 316L SS.