In vivo distribution of c-myc antisense oligodeoxynucleotides local delivered by gelatin-coated platinmn-iridium stents in rabbits and its effect on apoptosis

摘要

Background Post-stenting restenosis is a significant clinical problem, involving vascular smooth muscle cells(VSMCs) proliferation and apoptosis. It is reported that c-myc antisense oligodeoxynucleotides (ASODNs) local delivered by catheter can inhibit VSMCs proliferation. This study was designed to assess tissue distribution of c-myc ASODN local delivered using gelatin-coated platinum-iridium (Pt-Ir) stents, and its effect on apoptosis of VSMCs. Methods Gelatin-coated Pt-Ir stents that had absorbed caroboxyfluorescein-5-succimidyl ester (FAM) labeled c-myc ASODNs (550 μg per stent) were implanted into the right carotid arteries of 6 rabbits. Tissue samples were obtained at 45 minutes, 2 hours, and 6 hours. Tissue distribution of c- myc ASODNs was assessed by fluorescence microscopy. In addition, 32 rabbits were randomly divided into two groups. Rabbits in the control group (n=16) were implanted with gelatin-coated Pt-Ir stents, and those in the treatment group (n=16) were implanted with gelatin-coated stents that had absorbed c-myc ASODNs. 7, 14, 30, or 90 days (n=4, respectively, for each group) after the stenting procedure, the stented segments were harvested, and histopathological examinations were performed to calculate neointimal area and mean neointimal thickness. The expression of c-myc was assessed using in situ hybridization (ISH) and immunohistochemical methods. Apoptotic VSMCs were detected using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and transmission electron microscope (TEM). Results According to fluorescence microscopic results, FAM-labeled c-myc ASODNs were concentrated in the target vessel media at the 45 minutes time point, and then dispersed to the adventitia. Morphometric analysis showed that neointimal area and mean neointimal thickness increased continuously up to 90 days after stent implantation, but that total neointimal area and mean neointimal thickness were less in the treatment group than in the control group at all time points ( P＜ 0.0001). At day 7 and day 14 after stenting, there were no detectable apoptotic cells in either group. However, apoptotic cells were present in the neointima 30 and 90 days after stenting, and the number of apoptotic cells was less at 30 days than at 90 days. Meanwhile, c-myc ASODNs appeared to induce apoptosis in more cells in the treatment group than that in the control group. Typical apoptotic VSMCs were observable under TEM. The expression of c-myc was positive in the control group and negative or weakly positive in the c-myc ^SODN treatment group, according to both ISH and immunohistochemical examination. Conclusion Gelatin-coated Pt-Ir stent mediated local delivery of c-myc ASODNs is feasible. The localization of c-myc ASODN is primarily in the target vessel walls, c-myc ASODNs can inhibit VSMCs proliferation and induce its apoptosis after local delivery in vivo.