Sirolimus blocks the accumulation of hyaluronan (HA) by arterial smooth muscle cells and reduces monocyte adhesion to the ECM

Abstract

Sirolimus (SRL), an inhibitor of human arterial smooth muscle cell (ASMC) proliferation and migration, prevents in-stent restenosis (ISR). Little is known about the effect of SRL on the extracellular matrix (ECM) component, hyaluronan, a key macromolecule in neointimal hyperplasia and inflammation. In this study, we investigated SRL regulation of the synthesis of hyaluronan by cultured human ASMC and the effect of SRL on hyaluronan mediated monocyte adhesion to the ECM. Hyaluronan production on a per cell basis was significantly inhibited by SRL at 4 days and remained so through 10 days. This reduction was correlated with reduced levels of hyaluronan synthase mRNAs while hyaluronan degradation rates were unchanged. Poly I:C, a viral mimetic, caused increased hyaluronan accumulation by ASMC cell layers and this increase was inhibited by SRL. The inhibition was paralleled by a reduction in hyaluronan-dependent monocyte adhesion to the ECM. This study demonstrates that SRL not only regulates the proliferation of ASMC but reduces the production of hyaluronan by these cells. This alteration in ECM composition results in reduced monocyte adhesion to the ECM in cultures of ASMC. Alterations in hyaluronan accumulation may contribute to the inhibition of ISR that is achieved by SRL.

Figure 1

Inhibition of proliferation in ASMC by SRL. A. One hundred ng/ml SRL inhibited ASMC growth in 2% FBS over a 10-day period. ASMC were seeded at 3× 10⁴ per well on day 0 in 10% FBS medium and then changed on day 1 to 2% FBS medium with or without 100 ng/ml SRL. The cells were fed every two days. Closed circles, control, open circles, SRL. B. Reversal of SRL inhibition of growth. After 8-days exposure to SRL in 2% FBS medium, ASMC were fed 10% FBS medium without SRL. (Open triangles, cells previously grown in 2 ng/ml SRL, open squares, 100 ng/ml). Some cells were removed from the dishes with trypsin and EDTA after SRL treatment, and re-seeded on tissue culture plastic. Closed circles, controls, closed triangles, cells grown in 2 ng/ml SRL, closed squares, 100 ng/ml. After a lag of 3–5 days, cells in all treatments showed similar growth rates.

Figure 2

Inhibition of hyaluronan (HA) accumulation by ASMC treated with SRL. One hundred ng/ml SRL was fed to ASMC in 2% FBS every two days for times up to 10 days. Hyaluronan levels, on a per-cell basis, are presented as percentages of the controls. A. Hyaluronan accumulation in the medium during the previous 48 hours. B. Hyaluronan accumulation in the cell layer during the entire experiment prior to the time point. **P<0.01 vs control. One of three similar experiments in each of which, n = 3.

Figure 3

ASMC exposed to 2 or 100 ng/ml SRL for two days contained significantly less mRNA for the three hyaluronan synthases (HAS1–3) than controls. *P<0.05, **P<0.01 vs control. One of two similar experiments in each of which, n = 3.

Figure 4

SRL inhibits hyaluronan (HA) accumulation induced by poly I:C. Zero or one hundred ng/ml SRL was fed to ASMC in 2% FBS for 2 days. They were then treated for 24 hours with fresh control or SRL media containing 0 or 20 µg/ml poly I:C. SRL reduced total hyaluronan in cultures with or without poly I:C. SRL also caused a significant reduction in poly I:C induced cell layer associated hyaluronan. Solid bars, medium; open bars, cell layer. *P<0.05, **P<0.01 vs control. ⁺⁺P<0.01 vs cells treated with poly I:C alone. One of three similar experiments in each of which, n = 4.

Figure 5

SRL inhibits hyaluronidase sensitive monocyte adhesion induced by poly I:C. ASMC were grown in 2% FBS medium with or without 100 ng/ml SRL for 9 days. The cells were seeded in a 96 well tray in the same (fresh) media and allowed to attach and grow for 24 hours. Parallel wells were seeded in 24 well trays for cell counts. Then, fresh control or SRL media containing 0 or 20 µg/ml poly I:C were added and the cells were incubated for a further 24 hours. Monocyte adhesion was assessed after 90 minutes at 4°C and hyaluronidase insensitive binding was subtracted from the total to obtain hyaluronidase sensitive binding. Monocyte adhesion on a per-cell basis was significantly increased above controls by poly I:C treatment and this increase was significantly inhibited by SRL (*P<0.05, poly I:C alone vs poly I:C plus SRL.). One of three similar experiments in each of which, n = 4.