Oxidatively modified low density lipoproteins (LDL) play a central role in atherogenesis (Steinberg, 2002) while dietary antioxidant vitamins are protective against this process (Carr et al. 2000). We have shown that vitamin C attenuates oxidised LDL-induced cell death by apoptosis (Siow et al. 1999a) in vascular smooth muscle cells (SMC). The antioxidant stress protein heme oxygenase-1 (HO-1) catabolises heme to generate the vasodilator carbon monoxide and antioxidant biliverdin, and can be induced by highly oxidised LDL (oxLDL) in SMC (Siow et al. 1999b). We have now investigated whether vitamin C modulates levels of the endogenous cellular antioxidant glutathione (GSH) and HO-1 protein expression in human aortic SMC treated with moderately-oxidized LDL (mLDL), a species containing higher levels of lipid hydroperoxides, and the involvement of mitogen activated protein kinases (MAPK) in HO-1 induction.
Human smooth muscle cells were cultured from aortic explants obtained with ethical approval. Confluent SMC cultures were pretreated with vitamin C (24 h, 100 µM) prior to treatment in the absence of vitamin C with human native LDL (nLDL), mLDL or oxLDL (0-300 µg ml-1) for 0-24 h. In some experiments, cells were treated with MAPK inhibitors, SP600125 (20 µM, c-Jun NH2-terminal kinase), U0126 (1 µM, MEK), SB203580 (2 µM, p38 MAPK) for 30 min prior to LDL treatments.
Western blot analysis revealed that mLDL and oxLDL induced HO-1 expression in a dose- and time-dependent manner. HO-1 was not detected in control or nLDL treated SMC, but mLDL enhanced HO-1 expression to a significantly (P < 0.01, n = 3, Student’s unpaired t test) greater extent (mean ± S.D., 240±67%) than oxLDL. Intracellular GSH levels, measured using a fluorometric assay, were unaltered by nLDL (28.6 ± 3.6 nmol (mg protein)-1) but elevated by mLDL to a significantly (P < 0.01, n = 3) higher level (43.4 ± 1.6 nmol (mg protein)-1) than oxLDL. Vitamin C (55 ± 5%) and MAPK inhibitors (SP600125: 41 ± 21%, U0126: 56 ± 19%, SB203580: 54 ± 10%) significantly (P < 0.01, n = 3) attenuated induction of HO-1 by mLDL. Vitamin C pretreatment also significantly (P < 0.05, n = 3) attenuated elevation of GSH levels mediated by mLDL (19 ± 4%) and oxLDL (17 ± 3%).
These findings suggest that HO-1 induction in SMC by mLDL is mediated by MAPK signalling cascades, that GSH levels and HO-1 expression can be differentially enhanced in response to mLDL and oxLDL and that vitamin C affords protection against the more atherogenic moderately oxidized species of LDL.
This work was supported by the Wellcome Trust and Guy’s & Thomas’ Charitable Foundation.