Phospholipase C delta as a potential modulator of vascular smooth muscle cell differentiation status H Abdel-Hadi, LS Harrington, AD Hughes & JS Lymn.Department of Clinical Pharmacology, NHLI Division, Faculty of Medicine, Imperial College London.	Print Abstract Search PubMed for: Abdel-Hadi H Harrington LS Hughes AD Lymn JS

Phospholipase C (PLC) consists of a number of subfamilies of enzymes which show differential expression in vascular smooth muscle (VSM). A number of studies have determined that PLC₁ is the predominant isoform in differentiated, contractile VSM (Lymn et al, 2000) while we have previously demonstrated that the activity of this isoform is regulated by the monomeric G-protein RhoA (Hodson et al, 1998). Taken together these data suggest an association between PLC₁, the actin cytoskeleton and contractility. The aim of this study was to determine whether PLC₁ expression is a marker for differentiation and if there is a functional association with the actin cytoskeleton.

This study used third passage VSM cells derived from human saphenous vein by an explant technique. The tissue was surplus to requirements following CABG and its use conformed to the guidelines set down by our local ethics committee. VSM cells were re-differentiated by prolonged serum deprivation. The temporal effect of serum deprivation on VSM cell DNA synthesis was measured by means of [³H]-thymidine incorporation while protein expression was measured by western blotting techniques. Serum-deprived VSM cells were then treated with either cytochalasin D (10-100nM) or colchicine (0.1-2 µM) for 2 hours and visualisation of protein expression and intracellular localisation was performed using confocal microscopy. Protein expression is expressed as mean ± SEM of the % change with respect to DNA synthesis studies demonstrated a significant decrease in [³H]-thymidine incorporation over the first three days in culture but no significant change thereafter. Both SM -actin and PLC₁ expression remained unchanged until 7 days post-serum deprivation at which point they were significantly elevated (SM -actin = 359±213%, PLC₁ = 322±144%; n=8; p<0.05). In contrast PLC₁ expression changed throughout the course of serum-deprivation reaching 69±10% of control values by day 7 (n=8, p<0.05). Prolonged serum deprivation resulted in the formation of long fibres of SM -actin, which traversed the length of the cell and was associated with cellular translocation of PLC₁ from the perinuclear region to the cytoplasm. Treatment of these serum-deprived VSM cells with cytochalasin D resulted in a dose-dependent breakdown in the actin cytoskeleton and was associated with a specific loss in PLC₁ expression while PLC₁ expression and cellular localisation were unaffected. In contrast treatment of these cells with cochicine resulted in a dose-dependent breakdown in the microtubule system but was not associated with any change in PLC₁ expression or localisation.

These data suggest that an increase in PLC₁ expression occurs as a post-growth arrest phenomenon and may represent a marker for differentiation status in VSM. Cellular localisation and hence, function of,
PLC₁ is critically and specifically associated with the organisation of the actin cytoskeleton and may therefore play a role in maintaining contractility.

Hodson et al(1998) BBA 1403:97-101.
Lymn et al (2000) NIPS, 15:41-45.