Experiment Study Of Inhibiting Effects Of Zoledronate On Vascular Intimal Hyperplasia

Objective:The development of surgical treatment of obstructive arterial diseases represent an important achievement in the field of vascular surgery. However, restenosis due to intimal hyperplasia remains the major obstacle to satisfactory long term patency of open and catheter based treatment of obstructive arterial diseases. Bisphosphonates had also been found to exhibit the effects of inhibiting development of atherosclerosis and neointimal hyperplasia. These effects were mostly attributed to the transient systemic inactivation of monocytes and macrophages.Bisphosphonates not only bind tightly to hydroxyapatite in the bone but also concentrate in the arterial wall of both healthy and especially atheromatous rabbits for at least several days or a week. Thus, this leads to the hypothesis that bisphosphonates might exert direct effects on vascular smooth muscle cells, which are the major cellular components of vascular wall. In the present study, we investigated the effects of a bisphosphonate, zoledronate, on the proliferation, adhesion, migration and microstructure of vascular smooth muscle cells from Sprague-Dawley rats. Furthermore, we investigated whether systemic or local delivery of zoledronate would be sufficient to prevent intimal hyperplasia.Methods:(1) The vascular smooth muscle cells were isolated from the thoracic aorta of Sprague-Dawley rats. Cell numbers were counted by using a Beckman Coulter Z2 Series counter. Cell cycle distribution was assayed by flow cytometry. Cell apoptosis was detected by Hoechst 33258 staining and Double staining of annexin V and propodium iodide. Cell adhesion and migration was detected by commercially available Transwell. Western blot analysis was applied to test the phosphorylation of focal adhesion kinase. The ultrastructure changes of vascular smooth muscle cell treated with zoledronate were examined under an inverted phase contrast microscope and a transmission electron microscope.(2) Twenty-four male Sprague-Dawley rats were assigned into four groups:non-treated group, systemic zoledronate-treated group, local collagen-treated group and local zoledronate-treated group. All four groups underwent balloon injury to the right common carotid artery. The left uninjured carotid arteries of the non-treated group were considered as normal artery controls. Twenty-one days after arterial injury and treatment, the right and left common carotid arteries were fixed, sectioned, stained and measured by computer-aided image analysis.Results:(1) It was shown that zoledronate suppressed vascular smooth muscle cells proliferation after 48 h cultivation in a dose dependent manner, most obviously at concentrations above 10?M. Cell cycle analysis indicated that zoledronate inhibited the proliferation of vascular smooth muscle cells via cell cycle arrest at S/G2/M phase. This inhibition was not associated with cell death. In a modified Boyden chamber model, it was shown that zoledronate dose-dependently inhibited vascular smooth muscle cells adhesion to collagen and migration stimulated by platelet-derived growth factor-BB. Western blot analysis suggested that zoledronate significantly inhibited the phosphorylation of focal adhesion kinase. Furthermore, we observed that more and more vascular smooth muscle cells changed from a bipolar appearance to a globular shape under inverted light microscope as zoledronate concentration increased from 0.1?M to 100?M. Images under transmission electron microscope confirmed this morphological change, and many electron density bodies (lysosomes) were obseved within zoledronate treated vascular smooth muscle cells.(2) At 3 weeks, there was a 59% reduction of the intima/media area ratio in the systemic zoledronate-treated group compared with the non-treated group (P<0.01). There was an 87% reduction of the intima /media area ratio in the local zoledronate-treated group compared with the local collagen-treated group (P<0.01). Conclusion:(1) Zoledronate inhibited the proliferation, adhesion and migration of vascular smooth muscle cells.(2) Both systemic and local delivery of zoledronate correspond to a significant reduction in intimal hyperplasia seen at 3 weeks.