title: Docosahexaenoic acid-induced unfolded protein response, cell cycle arrest, and apoptosis in vascular smooth muscle cells are triggered by Ca²⁺-dependent induction of oxidative stress.
abstract: Proliferation of vascular smooth muscle cells is a characteristic of pathological vascular remodeling and represents a significant therapeutic challenge in several cardiovascular diseases. Docosahexaenoic acid (DHA), a member of the n-3 polyunsaturated fatty acids, was shown to inhibit proliferation of numerous cell types, implicating several different mechanisms. In this study we examined the molecular events underlying the inhibitory effects of DHA on proliferation of primary human smooth muscle cells isolated from small pulmonary artery (hPASMCs). DHA concentration-dependently inhibited hPASMC proliferation, induced G1 cell cycle arrest, and decreased cyclin D1 protein expression. DHA activated the unfolded protein response (UPR), evidenced by increased mRNA expression of HSPA5, increased phosphorylation of eukaryotic initiation factor 2α, and splicing of X-box binding protein 1. DHA altered cellular lipid composition and led to increased reactive oxygen species (ROS) production. DHA-induced ROS were dependent on both intracellular Ca(2+) release and entry of extracellular Ca(2+). Overall cellular ROS and mitochondrial ROS were decreased by RU360, a specific inhibitor of mitochondrial Ca(2+) uptake. DHA-induced mitochondrial dysfunction was evidenced by decreased mitochondrial membrane potential and decreased cellular ATP content. DHA triggered apoptosis as found by increased numbers of cleaved caspase-3- and TUNEL-positive cells. The free radical scavenger Tempol counteracted DHA-induced ROS, cell cycle arrest, induction of UPR, and apoptosis. We conclude that Ca(2+)-dependent oxidative stress is the central and initial event responsible for induction of UPR, cell cycle arrest, and apoptosis in DHA-treated hPASMCs.
authors.affiliation: Institute of Molecular Biology and Biochemistry, Center for Molecular Medicine, Austria.
authors.forename: Slaven
authors.initials: S
authors.lastname: Crnkovic
authors.forename: Monika
authors.initials: M
authors.lastname: Riederer
authors.forename: Margarete
authors.initials: M
authors.lastname: Lechleitner
authors.forename: Seth
authors.initials: S
authors.lastname: Hallström
authors.forename: Roland
authors.initials: R
authors.lastname: Malli
authors.forename: Wolfgang F
authors.initials: WF
authors.lastname: Graier
authors.forename: Jörg
authors.initials: J
authors.lastname: Lindenmann
authors.forename: Helmut
authors.initials: H
authors.lastname: Popper
authors.forename: Horst
authors.initials: H
authors.lastname: Olschewski
authors.forename: Andrea
authors.initials: A
authors.lastname: Olschewski
authors.forename: Saša
authors.initials: S
authors.lastname: Frank
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects*; D002478:Cells, Cultured; D017931:DNA Primers; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005434:Flow Cytometry; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018384:Oxidative Stress; D010766:Phosphorylation; D060888:Real-Time Polymerase Chain Reaction; D056811:Unfolded Protein Response / Q000187:drug effects*
chemical_list: D017931:DNA Primers; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D004281:Docosahexaenoic Acids; D002118:Calcium