Cardiovascular pharmacology
Low nanomolar thapsigargin inhibits the replication of vascular smooth muscle cells through reversible endoplasmic reticular stress

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Abstract

Thapsigargin (TG), an inhibitor of Ca2+ ATPase pumps in the endoplasmic reticulum (ER), inhibits replication of human vascular smooth muscle cell (hVSMC) at low nM concentrations. TG blocks replication of other cell types through promotion of ER stress (ERS). In order to determine whether ERS may mediate the cytostatic effect of TG in hVSMCs, the effect of TG on ERS in hVSMCs was studied by assessing markers of ERS: Immunoglobulin Heavy Chain Binding Protein (BiP), growth inhibitory transcription factor, GADD153, phosphorlylated eukaryotic initiation factor 2α (p-eIF2α) and phosphorlylated protein kinase R (p-PKR). hVSMCs derived from saphenous veins were rendered quiescent with serum-free medium for 96 h incubated with 10 nM TG at 37° C for 24 h, then washed free of TG and incubated with 10% foetal calf serum (FCS) for a further 24 h. At selected times, BiP, GADD153, p-eIF2α, p-PKR and cyclin D1 expression was assessed. TG promoted a marked increase in BiP and GADD153, but suppressed cyclin D1 mRNA and protein expression. Under serum-free conditions p-eIF2α and p-PKR expression was not enhanced by TG. 15–24 h After removal of TG all these factors returned to levels seen in control cells. These data demonstrate that the inhibitory effect of 10 nM TG on hVSMC replication is mediated through induction of ERS and associated factors that cessate replication and is reversible. These observations have implications in the aetiology and treatment of diseases that include atherogenesis, vein graft failure and restenosis.

Introduction

Despite the success of coronary artery bypass graft surgery using autologous saphenous veins, late vein graft failure occurs in 50% of patients within 10 years (Jeremy et al., 2002, Jeremy et al., 2004, Jeremy et al., 2007, Jeremy and Thomas, 2010). In vein grafts vascular smooth muscle cells (VSMCs) proliferate to form the neointima, upon which atherogenesis is superimposed (Jeremy et al., 1997, Wan et al., 2004, Wan et al., 2007, Shukla and Jeremy, 2012). No intervention has hitherto proved clinically effective in preventing late vein graft failure (Jeremy and Thomas, 2010, Shukla and Jeremy, 2012). The concensus, therefore, is that inhibition of neointima formation would reduce vein graft failure.

One strategic approach has been proposed to treat saphenous veins with drugs that inhibit VSMC replication in the interim between excision from the leg to implantation (Schachner et al., 2005, Murphy et al., 2007, Jeremy and Thomas, 2010). One candidate drug for this approach is thapsigargin since pre-incubation with low concentrations (10 nM) of the drug for 1 h inhibits human VSMC (hVSMC) replication for up to 14 days (Birkett et al., 1999, George et al., 1997, Shukla et al., 2005, Shukla et al., 2006). In hVSMCs, 10 nM thapsigargin depletes Ca2+ pools, inhibits entry into the S-phase of the cell cycle (Shukla et al., 1997, Shukla et al., 2005). This can be considered to be low concentrations of thapsigargin since at 10 nM it does not elicit calcium mobilisation (Birkett et al., 1999). The precise mechanisms of action of 10 nM thapsigargin on human VSMC replication have not been fully clarified, however.

It is known, however, that thapsigargin in micromolar concentrations promotes endoplasmic reticular stress (ER stress) in cell types other than VSMCs, which results is the unfolded protein response (UPR) (Kass and Orrenius, 1999, Zhao and Ackerman, 2006, Szegezdi et al., 2006, Boyce and Yuan, 2006). The UPR is a pro-survival response designed to restore normal ER function that alters translational events including the cessation of cell replication (Brewer et al., 1999, Brewer, 2000). Prolonged UPR, however, elicits apoptosis and cell death (Brewer et al., 1999, Brewer, 2000). On initiation of ER stress, the ER chaperone glucose-response protein, 78 kDa/Immunoglobulin Heavy Chain Binding Protein (BiP) and the growth inhibitory transcription factor, GADD153, a non-ER resident protein are rapidly transcribed (Lee, 2005, Oyadomari and Mori, 2004). ERS is linked to the translational machinery by protein kinase R (PKR) and eukaryotic initiation factor 2α (eIF2α), both of which block cell replication (Zhang et al., 2001, Prostko et al., 1995, Srivastava et al., 1995, Aktas et al., 1995).

Studies were therefore undertaken to determine whether pre-exposure of cells to 10 nM thapsigargin elicits ERS in human VSMCs, whether this is reversible and results in apoptosis. Human VSMCs were rendered quiescent and then incubated with 10 nM thapsigargin for 24 h in serum-free conditions and replication triggered by add-back of serum and thapsigargin concomitantly removed. The expression of BiP, GADD153 and the phosphorylation of PKR and eIF2α and cyclin D1 expression was then assessed biochemically at selected time points over this time scale.

Section snippets

Cell culture

Saphenous veins were obtained from patients undergoing coronary artery bypass graft surgery, Ethical Committee approval and patient consent having been obtained. Veins were placed in medium RPMI 1640 (Gibco BRL; Paisley, Scotland) containing 2% amphotericin (Gibco BRL and 0.4% heparin (Sigma Chemical Co, Poole Dorset, UK)). VSMCs were then grown in Dulbecco's Minimum Essential Medium-Glutamax without sodium pyruvate (DMEM; Gibco BRL), containing 100 units penicillin (Sigma), 100 mg/ml

Results

Following quiescence for 96 h, 10 nM thapsigargin elicited a marked upregulation of BiP within 6 h above that in cycling cells (Fig. 1). Following the removal of thapsigargin and simultaneous addition of 10% FCS, BiP levels diminished such that at 24 h it was not significantly different from cycling cells (Fig. 1). In cells not treated with thapsigargin, FCS alone elicited no significant change in BiP levels (Fig. 1).

GADD153 protein levels were low in cycling cells and no induction was observed

Discussion

The objective of the present study was to clarify the mechanisms underlying the cytostatic effect of a low (non-apoptotic) concentration of thapsigargin (10 nM) on ER stress in isolated hVSMC. Levels of both BiP and GADD153 (standard indices of ER stress) levels were low in untreated cycling cells and no induction was observed after serum withdrawal or after serum add-back. Following the addition of 10 nM thapsigargin to quiescent human hVSMC, however, BiP and GADD153 proteins were rapidly

Acknowledgement

This work was supported by funding from the NIHR Biomedical Research Unit in Cardiovascular Medicine.

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