Zinc regulates Nox1 expression through a NF-κB and mitochondrial ROS dependent mechanism to induce senescence of vascular smooth muscle cells.

The role of oxidative stress and inflammation in the development and progression of cardiovascular diseases (CVD) is well established. Increases in oxidative stress can further exacerbate the inflammatory response and lead to cellular senescence. We previously reported that angiotensin II (Ang II) and zinc increase reactive oxygen species (ROS) and cause senescence of vascular smooth muscle cells (VSMCs) and that senescence induced by Ang II is a zinc-dependent process. Zinc stimulated NADPH oxidase (Nox) activity; however, the role of Nox isoforms in zinc effects was not determined. Here, we show that downregulation of Nox1, but not Nox4, by siRNA prevented both Ang II- and zinc-induced senescence in VSMCs. On the other hand, overexpression of Nox1 induced senescence, which was associated with reduced proliferation, reduced expression of telomerase and increased DNA damage. Zinc increased Nox1 protein expression, which was inhibited by chelation of zinc with TPEN and by overexpression of the zinc exporters ZnT3 and ZnT10. These transporters work to reduce cytosolic zinc, suggesting that increased cytosolic zinc mediates Nox1 upregulation. Other metals including copper, iron, cobalt and manganese failed to upregulate Nox1, suggesting that this pathway is zinc specific. Nox1 upregulation was inhibited by actinomycin D (ACD), an inhibitor of transcription, by inhibition of NF-κB, a known Nox1 transcriptional regulator and by N-acetyl cysteine (NAC) and MitoTEMPO, suggesting that NF-κB and mitochondrial ROS mediate zinc effects. Supporting this idea, we found that zinc increased NF-κB activation in the cytosol, stimulated the translocation of the p65 subunit to the nucleus, and that zinc accumulated in mitochondria increasing mitochondrial ROS, measured using MitoSox. Further, zinc-induced senescence was reduced by inhibition of NF-κB or reduction of mitochondrial ROS with MitoTEMPO. NF-κB activity was also reduced by MitoTEMPO, suggesting that mitochondrial ROS is upstream of NF-κB. Our data demonstrate that altered zinc distribution leading to accumulation of zinc in the mitochondria increases mitochondrial ROS production causing NF-κB activation which in turn upregulates Nox1 expression inducing senescence of VSMCs.

Salazar G ，Huang J ，Feresin RG ，Zhao Y ，Griendling KK ... -  《-》

Reactive oxygen species derived from NADPH oxidase 1 and mitochondria mediate angiotensin II-induced smooth muscle cell senescence.

Cellular senescence has emerged as an important player in both physiology and pathology. Excessive reactive oxygen species (ROS) is known to mediate cellular senescence. NADPH oxidases are major sources for ROS production in the vascular wall; the roles of different NADPH oxidase isoforms in cellular senescence remain unclear, however. We investigated the roles of two NADPH oxidase isoforms in mitochondrial dysfunction during angiotensin II (Ang II)-induced cellular senescence of human aortic vascular smooth muscle cells (VSMCs). Ang II (10(-7)M) stimulated ROS generation, exhibiting early increases between 30 and 60min and sustained increases between 24h and 72h, and induced VSMCs senescence after 48h or 72h treatment as assessed with senescence-associated β-galactosidase activity and the expression of two cell cycle inhibitors, p21 and p16. ROS scavengers and membrane-permeable catalase (catalase-PEG) reduced Ang II-stimulated cellular senescence. Furthermore, small interfering RNA (siRNA) of NADPH oxidase catalytic subunit Nox1, but not that of another isoform Nox4, inhibited Ang II-induced cellular senescence. Nox1 siRNA inhibited both early and sustained ROS increases induced by Ang II. In addition, a mitochondrial-specific antioxidant, mitoQ10, effectively inhibited Ang II-induced ROS increases and cellular senescence. Ang II decreased ATP synthesis and induced mitochondrial membrane depolarization, which were attenuated by pre-treating cells with Nox1 siRNA, mitoQ10 or catalase-PEG. The effect of Ang II on the mitochondrial regulator peroxisome-proliferator-activated receptor gamma coactivator-1α (PGC-1α) and its downstream genes was examined. Ang II stimulated S570 phosphorylation of PGC-1α with concomitant decreases in catalase and uncoupling protein-2 (UCP-2) levels between 12h and 72h, which were inhibited by Nox1 siRNA. Knockdown of both catalase and UCP-2 mimicked Ang II-induced VSMC senescence. These results suggested that Ang II-stimulated Nox1 activation mediates mitochondrial dysfunction, probably by decreasing PGC-1α activity and increasing mitochondrial oxidative stress, and leads to cellular senescence of VSMCs.

Tsai IC ，Pan ZC ，Cheng HP ，Liu CH ，Lin BT ，Jiang MJ ... -  《-》

Angiotensin II requires zinc and downregulation of the zinc transporters ZnT3 and ZnT10 to induce senescence of vascular smooth muscle cells.

Patrushev N ，Seidel-Rogol B ，Salazar G 《PLoS One》

SAA1 increases NOX4/ROS production to promote LPS-induced inflammation in vascular smooth muscle cells through activating p38MAPK/NF-κB pathway.

Yu MH ，Li X ，Li Q ，Mo SJ ，Ni Y ，Han F ，Wang YB ，Tu YX ... -  《BMC Molecular and Cell Biology》

SQSTM1/p62 and PPARGC1A/PGC-1alpha at the interface of autophagy and vascular senescence.

Salazar G ，Cullen A ，Huang J ，Zhao Y ，Serino A ，Hilenski L ，Patrushev N ，Forouzandeh F ，Hwang HS ... -  《-》