Effects of Tacrolimus or Sirolimus on the adhesion of vascular wall cells: Controlled in-vitro comparison study

Abstract

In drug eluting stents the cytostatic drugs Sirolimus or Tacrolimus are used to inhibit blood vessel restenosis by limiting the proliferation of smooth muscle cells. However, the cytostatic activity of both drugs was shown to be not cell specific and could also affect the stent endothelialisation, respectively. Currently, only limited in vitro data are available about the impact of Sirolimus and Tacrolimus on endothelial cell proliferation over a broad concentration range. To answer this question the following study was performed.

Commercially obtained HUVEC were expanded with DMEM cell culture medium (GIBCO, Germany) supplemented with 5 vol% fetal calf serum on non-coated regular polystyrene-based 24-multiwell plates. For drug testings 2×10⁴ cells/cm² were seeded and grown for 24 h until 30–40% of the multiwell surfaces were covered and then exposed to Sirolimus (1.0×10^–11 – 1.0×10^–5 mol/l) or Tacrolimus (2.0×10^–8 – 6.2×10^–5 mol/l), both dissolved in DMSO. 12, 24 and 48 h after adding the drugs cell numbers per area were quantified by counting the cells in six wells with four fields of view per well, representing 0.6 mm², using a confocal laser microscope.

After 48 h of cell growth in the drug-free cell culture medium, the HUVEC number increased from 2.0×10⁴ to 3.55×10⁴ cells/cm² (mean cell doubling time: 53.6 h, n = 6). At lower concentrations (≤2.0×10^–6 mol/l) Tacrolimus reduced the number of adherent HUVEC significantly less than Sirolimus (p < 0.05). However, at higher concentrations (≥2.07×10^–5 mol/l) the effect of Tacrolimus on the number of adherent endothelial cells was significantly greater than that of Sirolimus (p < 0.05). At the highest concentration applied (6.22×10^–5 mol/l), Tacrolimus induced detachment of all HUVECs within 12 h after drug application. The number of adherent HUVEC decreased only slightly (about 9%) after Sirolimus application at the highest concentration (1.09×10^–5 mol/l).

These data show that in a non-flow model the cytostatic drug Tacrolimus reduced the number of adherent endothelial cells less than Sirolimus, as long as the drug concentration did not surpass 10^–6 mol/l. At the limits of solubility, Sirolimus (1×10^–5 mol/l) reduced the number of adherent endothelial cells less than Tacrolimus (6×10^–5 mol/l), which induced detachment of endothelial cells.