Pang, Vincent
(2017)
Investigating the effects and mechanisms of HMG-CoA reductase inhibitor simvastatin on the human myometrium.
PhD thesis, University of Nottingham.
Abstract
Preterm birth is responsible for 28% of all early neonatal deaths worldwide (Liu et al., 2015). The primary cause of preterm birth is preterm labour, occurring in 40 – 45% of all preterm deliveries (Goldenberg et al., 2008). Current management alleviates symptoms without disease prevention, due largely to incomplete mechanistic understanding. Emerging evidence suggests a role for cholesterol in the regulation of uterine contractility (Taggart et al., 2000; Kendrick et al., 2004; Smith et al., 2005), with evidence that cholesterol-manipulation agents can affect myometrial contractions. Further research into the effects of cholesterol modulation on myometrial contractility may reveal possible new tocolytics in the treatment of preterm labour.
In this study, we determined the effects and mechanisms of simvastatin, a cholesterol-lowering drug, on the human myometrium, using myometrial biopsies obtained from term non-labouring women undergoing scheduled caesarean section delivery. Biopsies were either dissected into individual tissue strips for isometric tension recordings, or disaggregated into myometrial cells and cultured for in-vitro experiments. Myometrial biopsies were also obtained from women undergoing elective hysterectomy for benign conditions but were only prepared for isometric tension recordings. Tissue strips from non-pregnant women were used to determine the effects of simvastatin exposure on spontaneous uterine contractility only.
Firstly, the effects of independent simvastatin exposure on spontaneous myometrial contractility were assessed. This was followed by investigating the effects of cholesterol depletion and addition, using methyl-β-cyclodextrin (MCD) and water-soluble cholesterol respectively. The effects of simvastatin on myometrial cell total cholesterol concentration was also assessed. The effects of nitric oxide (NO) inhibition via Nω-nitro-L-arginine methyl ester (L-NAME), and evidence for the presence of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase pathway, by replenishment with mevalonate and geranyl-geranyl pyrophosphate (GGPP) in the presence of simvastatin were also determined. Evidence of simvastatin-induced ion channel modulation was investigated using broad inhibition of voltage-operated potassium (K+) and calcium (Ca2+)-operated K+ channels, specific inhibition of small-conductance (SK) and large-conductance (BK) Ca2+-operated K+ channels, and activation of voltage-operated Ca2+ channels (VOCC). In cultured myometrial cells, the effects of simvastatin exposure on intracellular oxytocin-induced Ca2+ release, and cell permeability to the Hoechst 33342 dye tracer molecule were investigated. Finally, changes in cholesterol-containing lipid-raft structure caveolae, via its cholesterol-binding protein caveolin-1 expression, in myometrial cells after simvastatin exposure over time was determined.
Simvastatin caused a cumulative reduction of spontaneous myometrial contractions in tissues obtained from pregnant and non-pregnant women. Co-incubation with MCD inhibited simvastatin-mediated effects, but replenishment with water-soluble cholesterol did not. Furthermore, no change in total cholesterol concentration was observed after simvastatin exposure in myometrial cells over time. L-NAME-induced inhibition of endothelial nitric oxide synthase (eNOS) partially prevented simvastatin-mediated effects, but not replenishment with mevalonate or GGPP. Broad inhibition of voltage-operated K+ and Ca2+-operated K+ channels inhibited simvastatin’s effects on myometrial contractility. Specific inhibition of SK and BK channels, and activation of VOCCs also appeared to inhibit simvastatin-mediated effects. Furthermore, myometrial cells exposed to simvastatin overnight caused a reduction in oxytocin-induced Ca2+ release. However, myometrial cells showed no change in permeability to Hoechst 33342 dye tracer after simvastatin exposure. Finally, a gradual reduction in caveolin-1 expression was observed after simvastatin exposure over time.
Our findings have shown for the first time, that independent exposure to simvastatin results in the cumulative reduction of spontaneous contractions in human myometrial tissue. This effect appears to be cholesterol-independent, involving several different mechanisms not associated with inhibition of cholesterol or the HMG-CoA reductase pathway. However, these effects were observed at concentrations (3.3nM – 330μM) higher than those found in human serum (1 – 15nM) after therapeutic doses (0.1 – 1mg/kg). Additional experiments are therefore required to determine whether these effects are observed at therapeutic concentrations. Furthermore, the complexity of simvastatin’s mechanisms requires further investigations before its potential as a possible tocolytic can be assessed.
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