Docosahexaenoic acid (DHA)-induced heme oxygenase-1 attenuates cytotoxic effects of DHA in vascular smooth muscle cells

Abstract

Objective: Docosahexaenoic acid (DHA), a member of n-3 polyunsaturated fatty acids (n-3 PUFA) is a potent regulator of molecular events implicated in cardiovascular health. In a previous study we found that Ca(2+)-dependent oxidative stress is the central and initial event responsible for induction of unfolded protein response (UPR), cell cycle arrest and apoptosis in DHA treated primary human smooth muscle cells isolated from small pulmonary artery (hPASMC). In the present study we examined the impact of heme oxygenase (HO)-1, induced by DHA, on DHA-induced oxidative stress, UPR, cell proliferation and apoptosis in hPASMC.

Methods & results: DHA led to a time- and concentration-dependent increase in HO-1 mRNA and protein levels in hPASMC. The DHA-induced HO-1 upregulation could be attenuated by preincubation of cells with a strong antioxidant Tempol or by siRNA-mediated depletion of nuclear factor erythroid 2-related factor-2 (Nrf2). In DHA-treated hPASMC, depletion of HO-1 by siRNA-mediated silencing resulted in increased levels of reactive oxygen species (ROS) and increased duration of UPR, the latter revealed by monitoring of spliced X-box binding protein 1 (XBP-1) variant. Moreover, HO-1 silencing augmented apoptosis in DHA-treated hPASMC as found by increased numbers of cleaved caspase-3-positive cells. HO-1 silencing did not affect proliferation of hPASMC exposed to DHA.

Conclusion: Our results indicate that DHA-induced, ROS-dependent upregulation of HO-1 attenuates oxidative stress, UPR and apoptosis in DHA-treated hPASMC.

Keywords: ATF6; Apoptosis; CAT; CO; DHA; ER; FCS; GPX; HO; HO-1; Heme oxygenase; Human aortic smooth muscle cell; IRE1a; Keap1; Kelch-like ECH-associated protein; Nrf2; PERK; RFU; ROS; UPR; X-box binding protein 1; XBP-1; activating transcription factor 6; carbon monoxide; catalase; docosahexaenoic acid; eIF-2a; endoplasmatic reticulum; eukaryotic initiation factor 2 alpha; fetal calf serum; glutathione peroxidase; hASMC; hPASMC; human pulmonary artery smooth muscle cells; inositol-requiring enzyme 1alpha; n-3 PUFA; n-3 polyunsaturated fatty acid; nuclear factor erythroid 2-related factor-2; protein kinase RNA-like endoplasmic reticulum kinase; reactive oxygen species; relative fluorescent units; unfolded protein response.