An in vitro arterial model for testing cardiovascular drugs
Cardiovascular drug-eluting stents (DESs) have been used to prevent restenosis occurring after the implantation of bare metal stents (BMSs). Recent studies, however, have indicated that DESs can result in late in-stent thrombosis (LST) with a probability comparable to that of BMSs. LST can be best prevented by promoting endothelial cell (EC) growth in the stented region. One way of achieving this is to deliver a drug that can facilitate EC growth, in addition to preventing smooth muscle cell (SMC) hyperplasia. Thus, there is a need to screen for new, LST-preventing DES drugs.^ To expedite the screening process for candidate drugs, we have developed an in vitro EC-SMC co-culture platform. Transforming growth factor-β1 (TGF-β1) and heparin were introduced to SMC cultures to up-regulate the SMC differentiation markers, α-smooth muscle actin (SMA) and calponin. Increased SMA and calponin levels were detected in the EC-SMC co-cultures (heterotypic model), substantially exceeding the levels in TGF-β1 and heparin-treated SMC mono-cultures (homotypic model). The expression levels increased further on pre-treating the SMCs with TGF-β1 and heparin. These expression levels were tested using immunocytochemistry followed by confocal microscopy and corroborated via in-cell westerns. Establishment of the EC-SMC cell culture model is the first step toward the development of a robust, in vitro test bed for evaluating cardiovascular therapeutics.^ In addition, we tested the drugs probucol and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) in EC and SMC monocultures as a first level screening for further testing. Probucol was tested both in suspension format, in concentrations ranging from 1x10-7 to 1x10-5 M, and in controlled release format in a polyurethane (PU) matrix in strengths ranging from 0 to 10% w/w. AICAR was tested, in concentrations ranging from 1x10-3 to 4x10-3 M, in suspension format alone. Both drugs statistically decreased the proliferation of SMCs. Probucol prevented EC apoptosis in vitro and, in controlled release format, 5% w/w probucol eluting PU resulted in significantly higher proliferation as compared to 2% probucol eluting PU and PU control.^ Keywords: Endothelial denudation, in vitro EC-SMC co-culture model, TGF-β1, α-smooth muscle actin (SMA), calponin.^
Kinam Park, Purdue University.