gms | German Medical Science

We recently demonstrated that oxidized low density lipoprotein (OxLDL) induces proliferation in human endothelial cells (JASN 2000;11:1819-1825). Since the small GTPase RhoA is a known regulator of the cell cycle (Seasholtz, Circ Res 2001;89:488-495), we investigated the impact of OxLDL on RhoA activity, and its role in OxLDL-induced cell cycle regulation.

We oxidized human LDL by Cu++. Proliferation of HUVEC was quantified by the colorimetric MTT assay. Cell cycle regulation was studied by western blot analysis. p27KIP1 expression was downregulated by transient transfection with antisense oligonucleotides. Activation of RhoA was detected immunohistochemically by its translocation from the cytosol to the cell membrane. The role of RhoA and the RhoA signalling pathway in OxLDL induced endothelial cell proliferation was investigated in cells transfected with a dominant negative RhoA mutant and by using the Rho-kinase inhibitor Y27632.

Our experiments demonstrate a concentration-dependent proliferative effect of OxLDL in HUVEC (maximal increase by 250 - 300 % at 5 µg/ml), associated with downregulation of p27KIP1 and cell cycle progression. Downregulation of p27KIP1 expression, using an antisense strategy, significantly increased the OxLDL-induced proliferative response in HUVEC. Concomitantly, OxLDL caused a significant activation of the small GTPase RhoA in HUVEC. Importantly, cells transfected with a dominant negative RhoA mutant showed an almost completely abolished proliferative response to OxLDL. HUVEC pre-incubated with the Rho-kinase-inhibitor Y27632 also showed a significantly decreased proliferative response to OxLDL stimulation.

In summary, these results support our hypothesis, that the RhoA/Rho-kinase pathway is involved in OxLDL-induced cell cycle regulation. OxLDL-induced cell proliferation may contribute to intimal hyperplasia in atherosclerosis and other diseases associated with increased levels of oxidized lipoprotein.