Homocysteine induces reactive oxygen species-dependent apoptosis of arterial smooth muscle cells coinciding with (peri)nuclear NOX4 translocation

Elevated levels of homocysteine (hcy) form a vascular risk factor. It has been shown that hcy induces cell death in vascular smooth muscle cells (smcs). Recent studies have shown that nadph oxidase (nox)-mediated reactive oxygen species (ros) play a role in hcy-induced apoptosis in cardiomyocytes and endothelial cells. In this study we have analyzed the role of hcy on the different nox isoforms in arterial smcs. Human arterial smcs isolated from the human umbilical cord were incubated with 100 μm hcy during 24 hours and were analyzed for cell viability using facs analysis and caspase 3 activity. Diphenylene iodonium (dpi) and nox4 small interference rna (sirna) were used to reduce nadph oxidase activity and to analyze nox4 in hcy-reduced cell viability. The effects of hcy on the expression of different nox isoforms and nox-mediated ros production were then studied using digital-imaging microscopy. Incubation of isolated arterial smcs with 100 μm hcy during 24 hours induced a significant increase in single Annexin v positive cells and caspase 3 activity. Furthermore, hcy resulted in a significant shift of nox4 from the cytoplasm to (peri)nuclear regions coinciding with significantly increased ros production at these sites. No effects of hcy on expression levels, nor location of nox1 and nox2 were found. Inhibition of nox-mediated ros production using dpi and knock-down of nox4 using sirna led to significant decreased apoptosis, nox4 expression and nitrotyrosine levels. hcy induced apoptosis of human arterial smcs in which (peri)nuclear nox4-mediated ros plays a central role.