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Notes: Aim: Heat shock protein (HSP) expression has been widely implicated in atherogenesis. Vein graft failure following coronary artery bypass graft surgery (CABG) involves medial thickening and neointima formation, a phenomenon mediated by vascular smooth muscle cell (VSMC) proliferation. Superimposed atherogenesis can then result in vein graft failure in as many as 50% of cases within ten years after surgery. In order to explore the possible role of HSP in mediating vein graft disease, the effect of HSP 65 kDA on the proliferation of VSMCS obtained from human saphenous vein was investigated. The role of calcium was also studied using thapsigargin which blocks the release of calcium from intracellular storage pools. Methods: VSMCs were grown from human saphenous vein using standard culture methods. Cells were grown to confluence using DMEM + 10% Foetal Calf serum (FCS). When confluent, cells were trypsinised and cultured onto 96-well plates and rendered quiescent with 0.4% FCS. HSP 65kDA, over a range of concentrations (with and without 10 nM thapsigargin) was added to the cells and proliferation stimulated with 10% FCS and further incubated for 48 hours. Proliferation was assessed by the uptake of 5-bromo-2′ deoxyurindine (Brdu) using colorimetric ELISA and cell counts from which% changes in proliferation were calculated. Results: HSP elicited a dose-dependent increase in the proliferation of VSMCs (〈link href="#t6"〉table 1) an effect completely inhibited by the presence of 10 nM thapsigargin (〈link href="#t6"〉table 1). This concentration of thapsigargin has previously been shown to inhibit VSMC proliferation through depletion of intracellular calcium pools.〈tabular xml:id="t6"〉1〈title type="main"〉 Effect of HSP 65 kDa on VSMC Proliferation (% Change Relative to Zero) [± SEM; n = 6 ]: A) Without Thapsigargin and B) with Thapsigargin *p 〈 0.05 , Compared to Zero; # p 〈 0.05 , A vs. B 〈table frame="topbot"〉〈tgroup cols="7" align="left"〉〈colspec colnum="1" colname="col1" align="left"/〉〈colspec colnum="2" colname="col2" align="center"/〉〈colspec colnum="3" colname="col3" align="center"/〉〈colspec colnum="4" colname="col4" align="center"/〉〈colspec colnum="5" colname="col5" align="center"/〉〈colspec colnum="6" colname="col6" align="center"/〉〈colspec colnum="7" colname="col7" align="center"/〉〈thead valign="bottom"〉 [HSP].(μg/ml) 0 0.001 0.01 0.1 1 10 〈tbody valign="top"〉A0 +46 ± 10* +61 ± 14* +65 ± 18* +60 ± 12* +55 ± 10* B0 −2 ± 0.2# −4 ± 0.4# −10 ± 4# −18 ± 4# −15 ± 3# Conclusions: Since HSP 65 kDa stimulates the proliferation of VSMCs, they may play a role in graft thickening and neointima formation as well as superimposed atherogenesis. As this may result in late vein graft failure, further investigations into the role of HSP expression in vein graft disease is warranted. The inhibition of this HSP-mediated effect by thapsigargin consolidates the crucial role of calcium in mediating VSMC proliferation and that inhibition of this event represents a potential therapeutic strategy for the prevention of vein graft thickening.

Notes: Abstract Urinary bladder hypertrophy and hyperplasia are well recognised in diabetic cystopathy. The urinary bladder is known to synthesise endothelin-1 (ET-1), a potent vasoconstrictor peptide with mitogenic properties. Using diabetic New Zealand White (NZW) rabbits, we investigated the potential role of ET receptor subtypes (ETA and ETB) on the proliferation of bladder smooth muscle cells (SMC). Diabetes mellitus was induced in adult male NZW rabbits. After 6 months, control (n=6) and diabetic (n=6) bladders were removed and SMC from the dome and bladder neck were grown using standard explant methodology. At passage two, the cells were made quiescent and then further incubated in foetal calf serum (FCS), control age-matched rabbit serum (CRS) or diabetic rabbit serum (DRS) in the presence or absence of ETA-antagonist (BQ123) or ETB-antagonist (BQ788). SMC proliferation was then measured with 5-bromo-2′deoxy-uracil 24 h later and by cell counting (using a haemocytometer) at 48 h. Neither BQ123 nor BQ788 influenced detrusor or bladder neck SMC proliferation in FCS or CRS. However, in the presence of DRS, BQ123 and BQ788 significantly inhibited diabetic detrusor and bladder neck SMC proliferation at 30 and 100 nmol/l (P 〈 0.03 and P 〈 0.01, respectively). Cell counts were also significantly reduced from the diabetic detrusor and bladder neck (P 〈 0.01 and P 〈 0.03 with BQ123 and BQ788, respectively). These results suggest that ET may play a pathophysiological role in the bladder SMC hyperplasia associated with diabetes mellitus.