Hironori Uekita

LOX-1-MT1-MMP axis is crucial for RhoA and Rac1 activation induced by oxidized low-density lipoprotein in endothelial cells

AIMS RhoA and Rac1 activation plays a key role in endothelial dysfunction. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a major receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells (ECs). Membrane type 1 matrix metalloproteinase (MT1-MMP) has been shown to be involved in atherogenesis. This study was conducted to investigate the role of the LOX-1-MT1-MMP axis in RhoA and Rac1 activation in response to ox-LDL in ECs. METHODS AND RESULTS Ox-LDL induced rapid RhoA and Rac1 activation as well as MT1-MMP activity in cultured human aortic ECs. Inhibition of LOX-1 prevented ox-LDL-dependent RhoA and Rac1 activation. Knockdown of MT1-MMP by small interfering RNA prevented ox-LDL-induced RhoA and Rac1 activation, indicating that MT1-MMP is upstream of RhoA and Rac1. Fluorescent immunostaining revealed the colocalization of LOX-1 and MT1-MMP, and the formation of a complex of LOX-1 with MT1-MMP was detected by immunoprecipitation. Blockade of LOX-1 or MT1-MMP prevented RhoA-dependent endothelial NO synthase protein downregulation and cell invasion, Rac1-mediated NADPH oxidase activity, and reactive oxygen species generation. CONCLUSION The present study provides evidence that the LOX-1-MT1-MMP axis plays a crucial role in RhoA and Rac1 activation signalling pathways in ox-LDL stimulation, suggesting that this axis may be a promising target for treating endothelial dysfunction.

Role of LOX‐1 in monocyte adhesion‐triggered redox, Akt/eNOS and Ca2+ signaling pathways in endothelial cells

This study was conducted to examine the role of lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) in monocyte adhesion‐induced redox‐sensitive, Akt/eNOS and Ca2+ signaling pathways in endothelial cells (ECs). LOX‐1 was blocked by an antibody‐neutralizing LOX‐1 TS92 or small interfering RNA. In cultured human aortic ECs, monocyte adhesion activated Rac1 and p47phox, and increased NADPH oxidase activity and reactive oxygen species (ROS) generation within 30 min and NF‐κB phosphorylation within 1 h, resulting in redox‐sensitive gene expression. Akt and eNOS phosphorylation was induced 15 min after adding monocytes and returned to control level after 30 min, whereas NO production was not altered by monocyte adhesion. Blockade of LOX‐1 blunted the monocyte adhesion‐triggered redox‐sensitive signaling pathway and Akt/eNOS phosphorylation in ECs. Both endothelial intracellular Ca2+ mobilization and Ca2+ influx caused by monocyte attachment were markedly attenuated by pretreatment of ECs with TS92. This suggests that LOX‐1 is involved in redox‐sensitive, Akt/eNOS and Ca2+ signaling pathways in monocyte adhesion to ECs independent of oxidized low‐density lipoprotein (ox‐LDL). Furthermore, blockade of Ca2+ inhibited monocyte adhesion‐triggered Rac1 and p47phox activation and ROS generation in ECs, whereas Ca2+ signaling was suppressed by blockade of NADPH oxidase and ROS generation. Finally, TS92 blocked the monocyte adhesion to ECs stimulated with or without tumor necrosis factor‐α or ox‐LDL. We provide evidence that LOX‐1 plays a role in redox‐sensitive, Akt/eNOS and Ca2+ signaling pathways in monocyte adhesion to ECs independent of the ox‐LDL–LOX‐1 axis. J. Cell. Physiol. 220: 706–715, 2009.

Preventive effects of pravastatin on thrombin-triggered vascular responses via Akt/eNOS and RhoA/Rac1 pathways in vivo

AIMS Small GTPases RhoA and Rac1 play crucial roles in endothelial dysfunction and reactive oxygen species (ROS) generation. We reported evidence that in thrombin-stimulated endothelial cells, rapid geranylgeranylation is an essential process for full activation of unprocessed RhoA, which is blocked by statin. In this study, we examined the effects of intravenous administration of pravastatin on thrombin-triggered vascular responses in vivo, as well as on the lipid modification of unprocessed forms of RhoA and Rac1 and their activation induced by thrombin. METHODS AND RESULTS Thrombin (50 U/kg) was intravenously injected with or without 0.3 mg/kg pravastatin into Wistar and spontaneously hypertensive rats. Coadministration of pravastatin prevented thrombin-induced impaired endothelium-dependent coronary vasodilation and down-regulated Akt/endothelial nitric oxide synthase (eNOS) phosphorylation within 1 h, as well as the down-regulation of eNOS protein expression within 4 h. In addition, thrombin increased Rac1/p47(phox)-dependent NAD(P)H oxidase activities of rat aortas within 1 h, resulting in ROS generation, which was prevented by the coadministration of pravastatin. Furthermore, the coadministration of pravastatin prevented thrombin-induced conversion of unprocessed RhoA and Rac1 into the geranylgeranylated forms as well as GTP-loading and membrane translocation within 1 h. CONCLUSION Intravenous injection of pravastatin prevents impaired NO-dependent vasodilation and Rac1/NAD(P)H oxidase-mediated-ROS generation by blocking the down-regulation of Akt/eNOS pathways and the full activation of unprocessed RhoA and Rac1 in vivo.

Involvement of membrane type 1-matrix metalloproteinase (MT1-MMP) in RAGE activation signaling pathways

An advanced glycation end products (AGE)/a receptor for AGE (RAGE) axis plays a key role in diabetic vascular complications. Membrane type 1‐matrix metalloproteinase (MT1‐MMP) has been shown to function not only as a proteolytic enzyme but also as a signaling molecule. In this study, we investigated the role of MT1‐MMP in the AGE/RAGE‐triggered signaling pathways in cultured rabbit smooth muscle cells (SMCs) and the molecular interaction between RAGE and MT1‐MMP in vitro and in vivo. In SMCs, AGE‐activated Rac1 and p47phox within 1 min, NADPH oxidase activity and reactive oxygen species (ROS) generation within 5 min, and NF‐κB phosphorylation within 15 min, thereby inducing redox‐sensitive molecular expression. Silencing of RAGE by small‐interfering RNA (siRNA) blocked the AGE‐induced signaling pathways. AGE‐induced geranylgeranyl transferase I (GGTase I) activity, Rac1·p47phox activation, NADPH oxidase activity, ROS generation, and molecular expression were also markedly attenuated by silencing of MT1‐MMP. An inhibitor of GGTase I mimicked the effects of MT1‐MMP‐specific siRNA. Fluorescent immunohistochemistry revealed that MT1‐MMP was partially co‐localized with RAGE in SMCs, and RAGE was found to form a complex with MT1‐MMP in both cultured SMCs and the aortae of diabetic rats by immunoprecipitation. Furthermore, MT1‐MMP and RAGE formed a complex in the aortic atherosclerotic lesions of hyperlipidemic rabbits. We show that MT1‐MMP plays a crucial role in RAGE‐activated NADPH oxidase‐dependent signaling pathways and forms a complex with RAGE in the vasculature, thus suggesting that MT1‐MMP may be a novel therapeutic target for diabetic vascular complications. J. Cell. Physiol. 226: 1554–1563, 2011.