Differential Effect Of Zoledronic Acid On Quiescent Vs. Proliferating Vascular Smooth Muscle Cells

Abstract

Introduction: Restenosis following arterial interventions is primarily due to phenotypic alteration vascular smooth-muscle cells. Zoledronic Acid (ZA), the most potent member of the nitrogen containing Bisphosphonates (BP), has proven to be effective in treating bone resorption and bone malignancies in humans. BP are known to exert their effects by inhibiting the post-translational modification of the Ras-related GTP binding proteins (e.g. Rap1A) by an isoprenoid lipid (prenylation) which are part of the mevalonate pathway. These experiments were designed to compare the relative sensitivity and the dose response of actively proliferating vs. quiescent cultured human vascular smooth cells to ZA treatment. Finally, to test whether ZA will modulate the prenylation of Rap-1A, an important mediator of multiple different cellular activities. Methods: Human aortic smooth muscle cells (HASMC) were cultured under growth condition until they reached ∼60% confluence. Cells were switched to quiescent media for 48 hours to achieve growth arrest. After which, cells were divided into two groups; proliferating cells group which was fed with growth media, or the quiescent cells group which continued to be fed with quiescent media. Both groups were supplemented with 0, 1, 10, 100μM ZA. Relative metabolic activity and cytotoxicity were measured by WST-1 and Cyto-tox fluor assays respectively at 48 hours. The effect of ZA on proliferating vs. quiescent HASMC migration was assessed by modified Boyden chamber. The relative prenylation of Rap-1A protein (index of mevolanate pathway activity) was evaluated by western blotting. Data was expressed as percent of the untreated control cells (0 μM ZA) from the same group. Results: Proliferating HASMC treated with 1μMZA did not differ in their metabolic activity or cytotoxicity index compared to untreated cells (Metabolic activity: p=0.99, Cytotoxicity: p=0.19). In contrast at 10 and 100μMZA treatment remarkably decreased metabolic activity (10μM p=0.03, 100μM p=0.03) and increased cytotoxicity in proliferating cells compared to quiescent cells (10μM p=0.007, 100μM p=0.03). The decreased metabolic activity and increased cytotoxicity correlated with the appearance of unprenylated Rap-1A protein. Further more ZA was more effective in inhibiting cellular migration in actively proliferating cells compared to quiescent cells at all ZA concentrations (1μM: p<0.002, 10μM: p<0.002, 100μM: p<0.002). Conclusions: These data suggest that ZA is more effective in inhibiting migration and promoting cytotoxicity in proliferating rather than quiescent HASMC. Since the expression of unprenylated RAP-1A correlated with inhibition of migration and increased cellular toxicity and reduced metabolic activity, this suggest that mevolante pathway may play a role in the inhibition of these crucial cellular functions. ZA might have a favorable therapeutic profile as a candidate drug to prevent intimal hyperplasia.