The effects of angiotensin II and the oxidative stress mediator 4-hydroxynonenal on human osteoblast-like cell growth: possible relevance to otosclerosis

Otosclerosis is a complex disease characterized by an abnormal bone turnover of the otic capsule resulting in conductive hearing loss. Recent findings have shown that angiotensin II (Ang II), a major effector peptide of the renin–angiotensin system, plays an important role in the pathophysiology of otosclerosis, most likely by its proinflammatory effects on the bone cells. Because reactive oxygen species play a role both in inflammation and in the cellular signaling pathway of Ang II, the appearance of protein adducts of the “second messenger of free radicals,” the aldehyde 4-hydroxynonenal (HNE), in otosclerotic bone has been analyzed. Immunohistochemical analysis of HNE-modified proteins in tissue samples of the stapedial bones performed on 15 otosclerotic patients and 6 controls revealed regular HNE–protein adducts present in the subperiosteal parts of control bone specimens, whereas irregular areas of a pronounced HNE–protein adduct presence were found within stapedial bone in cases of otosclerosis. To study possible interference by HNE and Ang II in human bone cell proliferation, differentiation, and induction of apoptosis we used an in vitro model of osteoblast-like cells. HNE interacted with Ang II in a dose-dependent manner, both by forming HNE–Ang II adducts, as revealed by immunoblotting, and by modifying its effects on cultured cells. Namely, treatment with 0.1nM Ang II and 2.5μM HNE stimulated proliferation, whereas treatment with 10μM HNE or in combination with Ang II (0.1, 0.5, and 1nM) decreased cell proliferation. Moreover, 10μM HNE alone and with Ang II (except if 1nM Ang II was used) increased cellular differentiation and apoptosis. HNE at 5μM did not affect differentiation nor significantly change apoptosis. On the other hand, when cells were treated with lower concentrations of HNE and Ang II we observed a decrease in cellular differentiation (combination of 1.0 or 2.5μM HNE with 0.1nM Ang II) and decrease in apoptosis (0.1 and 0.5nM Ang II). Cellular necrosis was increased with 5 and 10μM HNE if given alone or combined with Ang II, whereas 0.5nM Ang II and combination of 1μM HNE with Ang II (0.1 and 0.5nM) reduced necrosis. These results indicate that HNE and Ang II might act mutually dependently in the regulation of bone cell growth and in the pathophysiology of otosclerosis. ► HNE–protein adducts were detected in normal and otosclerotic stapes. ► Various distribution patterns of HNE–protein adducts were seen in otosclerosis. ► Angiot