Tohru Fukai

Publications of Tohru Fukai

• Nadph Oxidase 2 Regulates Bone Marrow Microenvironment Following Hindlimb Ischemia: Role in Reparative Mobilization of Progenitor Cells.

  Authors: Norifumi Urao, Ronald D McKinney, Tohru Fukai, Masuko Ushio-Fukai

  Stem cells (Dayton, Ohio). 01/2012;

  Bone marrow (BM) microenvironment, which is regulated by hypoxia and proteolytic enzymes, is crucial for stem/progenitor cell function and mobilization involved in postnatal neovascularization. WeBone marrow (BM) microenvironment, which is regulated by hypoxia and proteolytic enzymes, is crucial for stem/progenitor cell function and mobilization involved in postnatal neovascularization. We demonstrated that NADPH oxidase2 (Nox2)-derived reactive oxygen species (ROS) are involved in post-ischemic mobilization of BM cells and revascularization. However, role of Nox2 in regulating BM microenvironment in response to ischemic injury remains unknown. Here we show that hindlimb ischemia of mice increases ROS production in both the endosteal and central region of BM tissue in situ, which is almost completely abolished in Nox2 knockout (KO) mice. This Nox2-dependent ROS production is mainly derived from Gr-1(+) myeloid cells in BM. In vivo injection of hypoxyprobe reveals that endosteum at the BM is hypoxic with high expression of HIF-1α in basal state. Following hindlimb ischemia, hypoxic areas and HIF-1α expression are expanded throughout the BM, which is inhibited in Nox2 KO mice. This ischemia-induced alteration of Nox2-dependent BM microenvironment is associated with an increase in VEGF expression and Akt phosphorylation in BM tissue, thereby promoting Lin(-) progenitor cell survival and expansion, leading to their mobilization from BM. Furthermore, hindlimb ischemia increases proteolytic enzymes MT1-MMP expression and MMP-9 activity in BM, which is inhibited in Nox2 KO mice. In summary, Nox2-dependent increase in ROS play a critical role in regulating hypoxia expansion and proteolytic activities in BM microenvironment in response to tissue ischemia. This in turn promotes progenitor cell expansion and reparative mobilization from BM, leading to post-ischemic neovascularization and tissue repair.

• Nitroglycerin drives endothelial nitric oxide synthase activation via the phosphatidylinositol 3-kinase/protein kinase B pathway.

  Authors: Mao Mao, Varadarajan Sudhahar, Kristine Ansenberger-Fricano, Denise C Fernandes, Leonardo Y Tanaka, Tohru Fukai, Francisco R M Laurindo, Ronald P Mason, Jeannette Vasquez-Vivar, Richard D Minshall, Krisztian Stadler, Marcelo G Bonini

  Free radical biology & medicine. 01/2012; 52(2):427-35.

  Nitroglycerin (GTN) has been clinically used to treat angina pectoris and acute heart episodes for over 100 years. The effects of GTN have long been recognized and active research has contributed toNitroglycerin (GTN) has been clinically used to treat angina pectoris and acute heart episodes for over 100 years. The effects of GTN have long been recognized and active research has contributed to the unraveling of numerous metabolic routes capable of converting GTN to the potent vasoactive messenger nitric oxide. Recently, the mechanism by which minute doses of GTN elicit robust pharmacological responses was revisited and eNOS activation was implicated as an important route mediating vasodilation induced by low GTN doses (1-50nM). Here, we demonstrate that at such concentrations the pharmacologic effects of nitroglycerin are largely dependent on the phosphatidylinositol 3-kinase, Akt/PKB, and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signal transduction axis. Furthermore, we demonstrate that nitroglycerin-dependent accumulation of 3,4,5-InsP(3), probably because of inhibition of PTEN, is important for eNOS activation, conferring a mechanistic basis for GTN pharmacological action at pharmacologically relevant doses.

• Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelial cells.

  Authors: Jin Oshikawa, Seok-Jo Kim, Eiji Furuta, Cristiana Caliceti, Gin-Fu Chen, Ronald D McKinney, Frank Kuhr, Irena Levitan, Tohru Fukai, Masuko Ushio-Fukai

  American journal of physiology. Heart and circulatory physiology. 11/2011; 302(3):H724-32.

  p66Shc, a longevity adaptor protein, is demonstrated as a key regulator of reactive oxygen species (ROS) metabolism involved in aging and cardiovascular diseases. Vascular endothelial growth factorp66Shc, a longevity adaptor protein, is demonstrated as a key regulator of reactive oxygen species (ROS) metabolism involved in aging and cardiovascular diseases. Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that ROS derived from Rac1-dependent NADPH oxidase are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. However, a role of p66Shc in VEGF signaling and physiological responses in ECs is unknown. Here we show that VEGF promotes p66Shc phosphorylation at Ser36 through the JNK/ERK or PKC pathway as well as Rac1 binding to a nonphosphorylated form of p66Shc in ECs. Depletion of endogenous p66Shc with short interfering RNA inhibits VEGF-induced Rac1 activity and ROS production. Fractionation of caveolin-enriched lipid raft demonstrates that p66Shc plays a critical role in VEGFR2 phosphorylation in caveolae/lipid rafts as well as downstream p38MAP kinase activation. This in turn stimulates VEGF-induced EC migration, proliferation, and capillary-like tube formation. These studies uncover a novel role of p66Shc as a positive regulator for ROS-dependent VEGFR2 signaling linked to angiogenesis in ECs and suggest p66Shc as a potential therapeutic target for various angiogenesis-dependent diseases.

• Localized cysteine sulfenic acid formation by vascular endothelial growth factor: role in endothelial cell migration and angiogenesis.

  Authors: Nihal Kaplan, Norifumi Urao, Eiji Furuta, Seok-Jo Kim, Masooma Razvi, Yoshimasa Nakamura, Ronald D McKinney, Leslie B Poole, Tohru Fukai, Masuko Ushio-Fukai

  Free radical research. 07/2011; 45(10):1124-35.

  Reactive oxygen species (ROS) are important mediators for VEGF receptor 2 (VEGFR2) signalling involved in angiogenesis. The initial product of Cys oxidation, cysteine sulfenic acid (Cys-OH), is a keyReactive oxygen species (ROS) are important mediators for VEGF receptor 2 (VEGFR2) signalling involved in angiogenesis. The initial product of Cys oxidation, cysteine sulfenic acid (Cys-OH), is a key intermediate in redox signal transduction; however, its role in VEGF signalling is unknown. We have previously demonstrated IQGAP1 as a VEGFR2 binding scaffold protein involved in ROS-dependent EC migration and post-ischemic angiogenesis. Using a biotin-labelled Cys-OH trapping reagent, we show that VEGF increases protein-Cys-OH formation at the lamellipodial leading edge where it co-localizes with NADPH oxidase and IQGAP1 in migrating ECs, which is prevented by IQGAP1 siRNA or trapping of Cys-OH with dimedone. VEGF increases IQGAP1-Cys-OH formation, which is prevented by N-acetyl cysteine or dimedone, which inhibits VEGF-induced EC migration and capillary network formation. In vivo, hindlimb ischemia in mice increases Cys-OH formation in small vessels and IQGAP1 in ischemic tissues. In summary, VEGF stimulates localized formation of Cys-OH-IQGAP1 at the leading edge, thereby promoting directional EC migration, which may contribute to post-natal angiogenesis in vivo. Thus, targeting Cys-oxidized proteins at specific compartments may be the potential therapeutic strategy for various angiogenesis-dependent diseases.

• Superoxide dismutases: role in redox signaling, vascular function, and diseases.

  Authors: Tohru Fukai, Masuko Ushio-Fukai

  Antioxidants & redox signaling. 06/2011; 15(6):1583-606.

  Excessive reactive oxygen species Revised abstract, especially superoxide anion (O₂•-), play important roles in the pathogenesis of many cardiovascular diseases, including hypertension andExcessive reactive oxygen species Revised abstract, especially superoxide anion (O₂•-), play important roles in the pathogenesis of many cardiovascular diseases, including hypertension and atherosclerosis. Superoxide dismutases (SODs) are the major antioxidant defense systems against (O₂•-), which consist of three isoforms of SOD in mammals: the cytoplasmic Cu/ZnSOD (SOD1), the mitochondrial MnSOD (SOD2), and the extracellular Cu/ZnSOD (SOD3), all of which require catalytic metal (Cu or Mn) for their activation. Recent evidence suggests that in each subcellular location, SODs catalyze the conversion of (O₂•-), H2O2, which may participate in cell signaling. In addition, SODs play a critical role in inhibiting oxidative inactivation of nitric oxide, thereby preventing peroxynitrite formation and endothelial and mitochondrial dysfunction. The importance of each SOD isoform is further illustrated by studies from the use of genetically altered mice and viral-mediated gene transfer. Given the essential role of SODs in cardiovascular disease, the concept of antioxidant therapies, that is, reinforcement of endogenous antioxidant defenses to more effectively protect against oxidative stress, is of substantial interest. However, the clinical evidence remains controversial. In this review, we will update the role of each SOD in vascular biologies, physiologies, and pathophysiologies such as atherosclerosis, hypertension, and angiogenesis. Because of the importance of metal cofactors in the activity of SODs, we will also discuss how each SOD obtains catalytic metal in the active sites. Finally, we will discuss the development of future SOD-dependent therapeutic strategies.

• Unexpected role of the copper transporter ATP7A in PDGF-induced vascular smooth muscle cell migration.

  Authors: Takashi Ashino, Varadarajan Sudhahar, Norifumi Urao, Jin Oshikawa, Gin-Fu Chen, Huan Wang, Yuqing Huo, Lydia Finney, Stefan Vogt, Ronald D McKinney, Edward B Maryon, Jack H Kaplan, Masuko Ushio-Fukai, Tohru Fukai

  Circulation research. 09/2010; 107(6):787-99.

  Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copperCopper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1 (copper transporter 1) but also by the copper exporter ATP7A (Menkes ATPase), whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts, which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A small interfering RNA or CTR small interfering RNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor prolysyl oxidase (Pro-LOX) in lipid raft fraction, as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based x-ray fluorescence microscopy at neointimal VSMCs in wire injury model. These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge, as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis.

• Protective role of extracellular superoxide dismutase in renal ischemia/reperfusion injury.

  Authors: Markus P Schneider, Jennifer C Sullivan, Paul F Wach, Erika I Boesen, Tatsuo Yamamoto, Tohru Fukai, David G Harrison, David M Pollock, Jennifer S Pollock

  Kidney international. 08/2010; 78(4):374-81.

  Extracellular superoxide dismutase (SOD3) is highly expressed in renal tissues and a critical regulator of vascular function. We hypothesized that deletion of SOD3 would attenuate recovery of renalExtracellular superoxide dismutase (SOD3) is highly expressed in renal tissues and a critical regulator of vascular function. We hypothesized that deletion of SOD3 would attenuate recovery of renal blood flow (RBF) and increase oxidative stress and injury following renal ischemia/reperfusion (I/R). To test this, we evaluated SOD expression and activity, basal superoxide production, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in kidneys from male and female wild-type (WT) and SOD3-knockout mice. RBF, measured using an ultrasonic flow probe, and histological indices of oxidative stress and injury were assessed after 1 h of ischemia. Following ischemia, RBF was attenuated in kidneys from male, but not female, knockout mice compared with their WT counterparts. Total SOD activity was significantly reduced in male knockout compared with WT male mice but was similar in female mice of both genotypes, suggesting upregulated SOD1 activity. Basal superoxide production and NADPH oxidase activity were unrelated to the differences in RBF. After 24 h, kidneys from both genders of knockout mice were found to have more oxidative stress (3-nitrotyrosine immunohistochemistry) and renal cast formation than those from WT mice. Thus, our study found a key role for SOD3 in regulating renal I/R injury.

• Loss of fibulin-5 binding to beta1 integrins inhibits tumor growth by increasing the level of ROS.

  Authors: Marie K Schluterman, Shelby L Chapman, Grzegorz Korpanty, Kiyoshi Ozumi, Tohru Fukai, Hiromi Yanagisawa, Rolf A Brekken

  Disease models & mechanisms. 03/2010; 3(5-6):333-42.

  Tumor survival depends in part on the ability of tumor cells to transform the surrounding extracellular matrix (ECM) into an environment conducive to tumor progression. Matricellular proteins areTumor survival depends in part on the ability of tumor cells to transform the surrounding extracellular matrix (ECM) into an environment conducive to tumor progression. Matricellular proteins are secreted into the ECM and impact signaling pathways that are required for pro-tumorigenic activities such as angiogenesis. Fibulin-5 (Fbln5) is a matricellular protein that was recently shown to regulate angiogenesis; however, its effect on tumor angiogenesis and thus tumor growth is currently unknown. We report that the growth of pancreatic tumors and tumor angiogenesis is suppressed in Fbln5-null (Fbln5(-/-)) mice compared with wild-type (WT) littermates. Furthermore, we observed an increase in the level of reactive oxygen species (ROS) in tumors grown in Fbln5(-/-) animals. Increased ROS resulted in elevated DNA damage, increased apoptosis of endothelial cells within the tumor, and represented the underlying cause for the reduction in angiogenesis and tumor growth. In vitro, we identified a novel pathway by which Fbln5 controls ROS production through a mechanism that is dependent on beta1 integrins. These results were validated in Fbln5(RGE/RGE) mice, which harbor a point mutation in the integrin-binding RGD motif of Fbln5, preventing its interaction with integrins. Tumor growth and angiogenesis was reduced in Fbln5(RGE/RGE) mice, however treatment with an antioxidant rescued angiogenesis and elevated tumor growth to WT levels. These findings introduce a novel function for Fbln5 in the regulation of integrin-induced ROS production and establish a rationale for future studies to examine whether blocking Fbln5 function could be an effective anti-tumor strategy, alone or in combination with other therapies.

• IQGAP1 is involved in post-ischemic neovascularization by regulating angiogenesis and macrophage infiltration.

  Authors: Norifumi Urao, Masooma Razvi, Jin Oshikawa, Ronald D McKinney, Rupal Chavda, Wadie F Bahou, Tohru Fukai, Masuko Ushio-Fukai

  PloS one. 01/2010; 5(10):e13440.

  Neovascularization is an important repair mechanism in response to ischemic injury and is dependent on inflammation, angiogenesis and reactive oxygen species (ROS). IQGAP1, an actin-binding scaffoldNeovascularization is an important repair mechanism in response to ischemic injury and is dependent on inflammation, angiogenesis and reactive oxygen species (ROS). IQGAP1, an actin-binding scaffold protein, is a key regulator for actin cytoskeleton and motility. We previously demonstrated that IQGAP1 mediates vascular endothelial growth factor (VEGF)-induced ROS production and migration of cultured endothelial cells (ECs); however, its role in post-ischemic neovascularization is unknown. Ischemia was induced by left femoral artery ligation, which resulted in increased IQGAP1 expression in Mac3(+) macrophages and CD31(+) capillary-like ECs in ischemic legs. Mice lacking IQGAP1 exhibited a significant reduction in the post-ischemic neovascularization as evaluated by laser Doppler blood flow, capillary density and α-actin positive arterioles. Furthermore, IQGAP1(-/-) mice showed a decrease in macrophage infiltration and ROS production in ischemic muscles, leading to impaired muscle regeneration and increased necrosis and fibrosis. The numbers of bone marrow (BM)-derived cells in the peripheral blood were not affected in these knockout mice. BM transplantation revealed that IQGAP1 expressed in both BM-derived cells and tissue resident cells, such as ECs, is required for post-ischemic neovascularization. Moreover, thioglycollate-induced peritoneal macrophage recruitment and ROS production were inhibited in IQGAP1(-/-) mice. In vitro, IQGAP1(-/-) BM-derived macrophages showed inhibition of migration and adhesion capacity, which may explain the defective macrophage recruitment into the ischemic tissue in IQGAP1(-/-) mice. IQGAP1 plays a key role in post-ischemic neovascularization by regulating, not only, ECs-mediated angiogenesis but also macrophage infiltration as well as ROS production. Thus, IQGAP1 is a potential therapeutic target for inflammation- and angiogenesis-dependent ischemic cardiovascular diseases.

• Extracellular SOD-derived H2O2 promotes VEGF signaling in caveolae/lipid rafts and post-ischemic angiogenesis in mice.

  Authors: Jin Oshikawa, Norifumi Urao, Ha Won Kim, Nihal Kaplan, Masooma Razvi, Ronald McKinney, Leslie B Poole, Tohru Fukai, Masuko Ushio-Fukai

  PloS one. 01/2010; 5(4):e10189.

  Reactive oxygen species (ROS), in particular, H(2)O(2), is essential for full activation of VEGF receptor2 (VEGFR2) signaling involved in endothelial cell (EC) proliferation and migration.Reactive oxygen species (ROS), in particular, H(2)O(2), is essential for full activation of VEGF receptor2 (VEGFR2) signaling involved in endothelial cell (EC) proliferation and migration. Extracellular superoxide dismutase (ecSOD) is a major secreted extracellular enzyme that catalyzes the dismutation of superoxide to H(2)O(2), and anchors to EC surface through heparin-binding domain (HBD). Mice lacking ecSOD show impaired postnatal angiogenesis. However, it is unknown whether ecSOD-derived H(2)O(2) regulates VEGF signaling. Here we show that gene transfer of ecSOD, but not ecSOD lacking HBD (ecSOD-DeltaHBD), increases H(2)O(2) levels in adductor muscle of mice, and promotes angiogenesis after hindlimb ischemia. Mice lacking ecSOD show reduction of H(2)O(2) in non-ischemic and ischemic limbs. In vitro, overexpression of ecSOD, but not ecSOD-DeltaHBD, in cultured medium in ECs enhances VEGF-induced tyrosine phosphorylation of VEGFR2 (VEGFR2-pY), which is prevented by short-term pretreatment with catalase that scavenges extracellular H(2)O(2). Either exogenous H(2)O(2) (<500 microM), which is diffusible, or nitric oxide donor has no effect on VEGF-induced VEGFR2-pY. These suggest that ecSOD binding to ECs via HBD is required for localized generation of extracellular H(2)O(2) to regulate VEGFR2-pY. Mechanistically, VEGF-induced VEGFR2-pY in caveolae/lipid rafts, but non-lipid rafts, is enhanced by ecSOD, which localizes at lipid rafts via HBD. One of the targets of ROS is protein tyrosine phosphatases (PTPs). ecSOD induces oxidation and inactivation of both PTP1B and DEP1, which negatively regulates VEGFR2-pY, in caveolae/lipid rafts, but not non-lipid rafts. Disruption of caveolae/lipid rafts, or PTPs inhibitor orthovanadate, or siRNAs for PTP1B and DEP1 enhances VEGF-induced VEGFR2-pY, which prevents ecSOD-induced effect. Functionally, ecSOD promotes VEGF-stimulated EC migration and proliferation. In summary, extracellular H(2)O(2) generated by ecSOD localized at caveolae/lipid rafts via HBD promotes VEGFR2 signaling via oxidative inactivation of PTPs in these microdomains. Thus, ecSOD is a potential therapeutic target for angiogenesis-dependent cardiovascular diseases.

• Mitochondrial Thioredoxin. Novel Regulator for NADPH Oxidase and Angiotensin II- Induced Hypertension.

  Authors: Tohru Fukai

  Hypertension. 07/2009;

• Extracellular SOD and Aged Blood Vessels.

  Authors: Tohru Fukai

  American journal of physiology. Heart and circulatory physiology. 06/2009;

  No abstract Key words: ecSOD, aging, endothelial function, superoxide.

• Copper and angiogenesis: unravelling a relationship key to cancer progression.

  Authors: Lydia Finney, Stefan Vogt, Tohru Fukai, David Glesne

  Clinical and experimental pharmacology & physiology. 01/2009; 36(1):88-94.

  1. Angiogenesis, the formation of new capillaries from existing vasculature, is a critical process in normal physiology as well as several physiopathologies. A desire to curb the supportive role1. Angiogenesis, the formation of new capillaries from existing vasculature, is a critical process in normal physiology as well as several physiopathologies. A desire to curb the supportive role angiogenesis plays in the development and metastasis of cancers has driven exploration into anti-angiogenic strategies as cancer therapeutics. Key to this, angiogenesis additionally displays an exquisite sensitivity to bioavailable copper. Depletion of copper has been shown to inhibit angiogenesis in a wide variety of cancer cell and xenograft systems. Several clinical trials using copper chelation as either an adjuvant or primary therapy have been conducted. Yet, the biological basis for the sensitivity of angiogenesis remains unclear. Numerous molecules important to angiogenesis regulation have been shown to be either directly or indirectly influenced by copper, yet a clear probative answer to the connection remains elusive. 2. Measurements of copper in biological systems have historically relied on techniques that, although demonstrably powerful, provide little or no information as to the spatial distribution of metals in a cellular context. Therefore, several new approaches have been developed to image copper in a biological context. One such approach relies on synchrotron-derived X-rays from third-generation synchrotrons and the technique of high resolution X-ray fluorescence microprobe (XFM) analysis. 3. Recent applications of XFM approaches to the role of copper in regulating angiogenesis have provided unique insight into the connection between copper and cellular behaviour. Using XFM, copper has been shown to be highly spatially regulated, as it is translocated from perinuclear areas of the cell towards the tips of extending filopodia and across the cell membrane into the extracellular space during angiogenic processes. Such findings may explain the heightened sensitivity of this cellular process to this transition metal and set a new paradigm for the kinds of regulatory roles that the spatial dynamics of cellular transition metals may play.

• Novel mechanism for regulation of extracellular SOD transcription and activity by copper: Role of antioxidant-1.

  Authors: Shinichi Itoh, Kiyoshi Ozumi, Ha Won Kim, Osamu Nakagawa, Ronald D McKinney, Rodney J Folz, Igor N Zelko, Masuko Ushio-Fukai, Tohru Fukai

  Free radical biology & medicine. 11/2008;

  Extracellular superoxide dismutase (SOD3), a secretory copper-containing antioxidant enzyme, plays an important role in various oxidative stress-dependent cardiovascular diseases. Although cofactorExtracellular superoxide dismutase (SOD3), a secretory copper-containing antioxidant enzyme, plays an important role in various oxidative stress-dependent cardiovascular diseases. Although cofactor copper is required for SOD3 activity, it remains unknown whether it can regulate SOD3 transcription. We previously demonstrated that SOD3 activity requires the copper chaperone antioxidant-1 (Atox1), involved in copper delivery to SOD3 at the trans-Golgi network (TGN). Here we show that copper treatment in mouse fibroblasts significantly increases mRNA and protein levels of SOD3, but not SOD1, which is abolished in Atox1-deficient cells. Copper promotes Atox1 translocation to the nucleus. Promoter deletion analysis identifies copper- and Atox1-response elements (REs) at the SOD3 promoter. Gel-shift and ChIP assays reveal that Atox1 directly binds to the Atox1 RE in a copper-dependent manner in vitro and in vivo. Adenovirus-mediated reexpression in Atox1(-/-) cells of nucleus-targeted Atox1 (Atox1-NLS), but not TGN-targeted Atox1 (Atox1-TGN), increases SOD3 transcription without affecting SOD3 activity. Importantly, reexpression of both Atox1-NLS and Atox1-TGN together, but not either alone, in Atox1(-/-) cells increases SOD3 activity. SOD3 transcription is positively regulated by copper through the transcription factor function of Atox1, whereas the full activity of SOD3 requires both the copper chaperone and the transcription factor functions of Atox1. Thus, Atox1 is a potential therapeutic target for oxidant stress-dependent cardiovascular disease.

• Role of Menkes ATPase in Angiotensin II-Induced Hypertension. A Key Modulator for Extracellular Superoxide Dismutase Function.

  Authors: Zhenyu Qin, Maria Carolina Gongora, Kiyoshi Ozumi, Shinichi Itoh, Kamran Akram, Masuko Ushio-Fukai, David G Harrison, Tohru Fukai

  Hypertension. 10/2008;

  The extracellular superoxide dismutase (SOD3), a secretory copper-containing enzyme, regulates angiotensin II (Ang II)-induced hypertension by modulating levels of extracellular superoxide anion. TheThe extracellular superoxide dismutase (SOD3), a secretory copper-containing enzyme, regulates angiotensin II (Ang II)-induced hypertension by modulating levels of extracellular superoxide anion. The present study was designed to determine the role of the copper transporter Menkes ATPase (MNK) in Ang II-induced SOD3 activity and hypertension in vivo. Here we show that chronic Ang II infusion enhanced systolic blood pressure and vascular superoxide anion production in MNK mutant (MNK(mut)) mice as compared with those in wild-type mice, which are associated with impaired acetylcholine-induced endothelium-dependent vasorelaxation in MNK(mut) mice. These effects in MNK(mut) mice are rescued by infusion of the SOD mimetic Tempol. By contrast, norepinephrine-induced hypertension, which is not associated with an increase in vascular superoxide anion production, is not affected in MNK(mut) mice. Mechanistically, basal and Ang II infusion-induced increase in vascular SOD3-specific activity is significantly inhibited in MNK(mut) mice. Coimmunoprecipitation analysis reveals that Ang II stimulation promotes association of MNK with SOD3 in cultured vascular smooth muscle cell and in mouse aortas, which may contribute to SOD3-specific activity by increasing copper delivery to SOD3 through MNK. In summary, MNK plays an important role in modulating Ang II-induced hypertension and endothelial function by regulating SOD3 activity and vascular superoxide anion production and becomes a potential therapeutic target for oxidant stress-dependent cardiovascular diseases.

• Loss of Extracellular Superoxide Dismutase Leads to Acute Lung Damage in the Presence of Ambient Air. A Potential Mechanism Underlying Adult Respiratory Distress Syndrome.

  Authors: Maria Carolina Gongora, Heinrich E Lob, Ulf Landmesser, Tomasz J Guzik, W David Martin, Kiyoski Ozumi, Susan M Wall, David Scott Wilson, Niren Murthy, Michael Gravanis, Tohru Fukai, David G Harrison

  The American journal of pathology. 10/2008;

  The extracellular superoxide dismutase 3 (SOD3) is highly expressed in both blood vessels and lungs. In different models of pulmonary injury, SOD3 is reduced; however, it is unclear whether thisThe extracellular superoxide dismutase 3 (SOD3) is highly expressed in both blood vessels and lungs. In different models of pulmonary injury, SOD3 is reduced; however, it is unclear whether this contributes to lung injury. To study the role of acute SOD3 reduction in lung injury, the SOD3 gene was deleted in adult mice by using the Cre-Lox technology. Acute reduction of SOD3 led to a fivefold increase in lung superoxide, marked inflammatory cell infiltration, a threefold increase in the arterial-alveolar gradient, respiratory acidosis, histological changes similar to those observed in adult respiratory distress syndrome, and 85% mortality. Treatment with the SOD mimetic MnTBAP and intranasal administration of SOD-containing polyketal microparticles reduced mortality, prevented the histological alterations, and reduced lung superoxide levels. To understand how mice with the SOD3 embryonic deletion survived without lung injury, gene array analysis was performed. These data demonstrated the up-regulation of 37 genes and down-regulation of nine genes, including those involved in cell signaling, inflammation, and gene transcription in SOD3(-/-) mice compared with either mice with acute SOD3 reduction or wild-type controls. These studies show that SOD3 is essential for survival in the presence of ambient oxygen and that acute loss of this enzyme can lead to severe lung damage. Strategies either to prevent SOD3 inactivation or to augment its levels might prove useful in the treatment of acute lung injury.

• Role of nox2-based NADPH oxidase in bone marrow and progenitor cell function involved in neovascularization induced by hindlimb ischemia.

  Authors: Norifumi Urao, Hyoe Inomata, Masooma Razvi, Ha Won Kim, Kishore Wary, Ronald McKinney, Tohru Fukai, Masuko Ushio-Fukai

  Circulation research. 08/2008; 103(2):212-20.

  Bone marrow (BM) is the major reservoir for endothelial progenitor cells (EPCs). Postnatal neovascularization depends on not only angiogenesis but also vasculogenesis, which is mediated throughBone marrow (BM) is the major reservoir for endothelial progenitor cells (EPCs). Postnatal neovascularization depends on not only angiogenesis but also vasculogenesis, which is mediated through mobilization of EPCs from BM and their recruitment to the ischemic sites. Reactive oxygen species (ROS) derived from Nox2-based NADPH oxidase play an important role in postnatal neovascularization; however, their role in BM and EPC function is unknown. Here we show that hindlimb ischemia of mice significantly increases Nox2 expression and ROS production in BM-mononuclear cells (BMCs), which is associated with an increase in circulating EPC-like cells. Mice lacking Nox2 show reduction of ischemia-induced flow recovery, ROS levels in BMCs, as well as EPC mobilization from BM. Transplantation of wild-type (WT)-BM into Nox2-deficient mice rescues the defective neovascularization, whereas WT mice transplanted with Nox2-deficient BM show reduced flow recovery and capillary density compared to WT-BM transplanted control. Intravenous infusion of WT- and Nox2-deficient BMCs into WT mice reveals that neovascularization and homing capacity are impaired in Nox2-deficient BMCs in vivo. In vitro, Nox2-deficient c-kit+Lin- BM stem/progenitor cells show impaired chemotaxis and invasion as well as polarization of actins in response to stromal derived factor (SDF), which is associated with blunted SDF-1-mediated phosphorylation of Akt. In conclusion, Nox2-derived ROS in BM play a critical role in mobilization, homing, and angiogenic capacity of EPCs and BM stem/progenitor cells, thereby promoting revascularization of ischemic tissue. Thus, NADPH oxidase in BM and EPCs is potential therapeutic targets for promoting neovascularization in ischemic cardiovascular diseases.

• Role of protein tyrosine phosphatase 1B in vascular endothelial growth factor signaling and cell-cell adhesions in endothelial cells.

  Authors: Yoshimasa Nakamura, Nikolay Patrushev, Hyoe Inomata, Dolly Mehta, Norifumi Urao, Ha Won Kim, Masooma Razvi, Vidisha Kini, Kalyankar Mahadev, Barry J Goldstein, Ronald McKinney, Tohru Fukai, Masuko Ushio-Fukai

  Circulation research. 06/2008; 102(10):1182-91.

  Vascular endothelial growth factor (VEGF) binding induces phosphorylation of VEGF receptor (VEGFR)2 in tyrosine, which is followed by disruption of VE-cadherin-mediated cell-cell contacts ofVascular endothelial growth factor (VEGF) binding induces phosphorylation of VEGF receptor (VEGFR)2 in tyrosine, which is followed by disruption of VE-cadherin-mediated cell-cell contacts of endothelial cells (ECs), thereby stimulating EC proliferation and migration to promote angiogenesis. Tyrosine phosphorylation events are controlled by the balance of activation of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Little is known about the role of endogenous PTPs in VEGF signaling in ECs. In this study, we found that PTP1B expression and activity are markedly increased in mice hindlimb ischemia model of angiogenesis. In ECs, overexpression of PTP1B, but not catalytically inactive mutant PTP1B-C/S, inhibits VEGF-induced phosphorylation of VEGFR2 and extracellular signal-regulated kinase 1/2, as well as EC proliferation, whereas knockdown of PTP1B by small interfering RNA enhances these responses, suggesting that PTP1B negatively regulates VEGFR2 signaling in ECs. VEGF-induced p38 mitogen-activated protein kinase phosphorylation and EC migration are not affected by PTP1B overexpression or knockdown. In vivo dephosphorylation and cotransfection assays reveal that PTP1B binds to VEGFR2 cytoplasmic domain in vivo and directly dephosphorylates activated VEGFR2 immunoprecipitates from human umbilical vein endothelial cells. Overexpression of PTP1B stabilizes VE-cadherin-mediated cell-cell adhesions by reducing VE-cadherin tyrosine phosphorylation, whereas PTP1B small interfering RNA causes opposite effects with increasing endothelial permeability, as measured by transendothelial electric resistance. In summary, PTP1B negatively regulates VEGFR2 receptor activation via binding to the VEGFR2, as well as stabilizes cell-cell adhesions through reducing tyrosine phosphorylation of VE-cadherin. Induction of PTP1B by hindlimb ischemia may represent an important counterregulatory mechanism that blunts overactivation of VEGFR2 during angiogenesis in vivo.

• Novel role of antioxidant-1 (Atox1) as a copper-dependent transcription factor involved in cell proliferation.

  Authors: Shinichi Itoh, Ha Won Kim, Osamu Nakagawa, Kiyoshi Ozumi, Susan M Lessner, Hiroki Aoki, Kamran Akram, Ronald D McKinney, Masuko Ushio-Fukai, Tohru Fukai

  The Journal of biological chemistry. 05/2008; 283(14):9157-67.

  Copper plays a fundamental role in regulating cell growth. Many types of human cancer tissues have higher copper levels than normal tissues. Copper can also induce gene expression. However,Copper plays a fundamental role in regulating cell growth. Many types of human cancer tissues have higher copper levels than normal tissues. Copper can also induce gene expression. However, transcription factors that mediate copper-induced cell proliferation have not been identified in mammals. Here we show that antioxidant-1 (Atox1), previously appreciated as a copper chaperone, represents a novel copper-dependent transcription factor that mediates copper-induced cell proliferation. Stimulation of mouse embryonic fibroblasts (MEFs) with copper markedly increased cell proliferation, cyclin D1 expression, and entry into S phase, which were completely abolished in Atox1(-/-) MEFs. Promoter analysis and EMSA revealed that copper stimulates the Atox1 binding to a previously undescribed cis element in the cyclin D1 promoter. The ChIP assay confirms that copper stimulates Atox1 binding to the DNA in vivo. Transfection of Atox1 fused to the DNA-binding domain of Gal4 demonstrated a copper-dependent transactivation in various cell types, including endothelial and cancer cells. Furthermore, Atox1 translocated to the nucleus in response to copper through its highly conserved C-terminal KKTGK motif and N-terminal copper-binding sites. Finally, the functional role of nuclear Atox1 is demonstrated by the observation that re-expression of nuclear-targeted Atox1 in Atox1(-/-) MEFs rescued the defective copper-induced cell proliferation. Thus, Atox1 functions as a novel transcription factor that, when activated by copper, undergoes nuclear translocation, DNA binding, and transactivation, thereby contributing to cell proliferation.

• Extracellular superoxide dismutase (ecSOD) in vascular biology: an update on exogenous gene transfer and endogenous regulators of ecSOD.

  Authors: Zhenyu Qin, Krzysztof J Reszka, Tohru Fukai, Neal L Weintraub

  Translational research : the journal of laboratory and clinical medicine. 03/2008; 151(2):68-78.

  Extracellular superoxide dismutase (ecSOD) is the major extracellular scavenger of superoxide (O(2)(.-)) and a main regulator of nitric oxide (NO) bioactivity in the blood vessel wall, heart, lungs,Extracellular superoxide dismutase (ecSOD) is the major extracellular scavenger of superoxide (O(2)(.-)) and a main regulator of nitric oxide (NO) bioactivity in the blood vessel wall, heart, lungs, kidney, and placenta. Involvement of O(2)(.-) has been implicated in many pathological processes, and removal of extracellular O(2)(.-) by ecSOD gene transfer has emerged as a promising experimental technique to treat vascular disorders associated with increased oxidant stress. In addition, recent studies have clarified mechanisms that regulate ecSOD expression, tissue binding, and activity, and they have provided new insight into how ecSOD interacts with other factors that regulate vascular function. Finally, studies of a common gene variant in humans associated with disruption of ecSOD tissue binding suggest that displacement of the enzyme from the blood vessel wall may contribute to vascular diseases. The purpose of this review is to summarize recent research findings related to ecSOD function and gene transfer and to stimulate other investigations into the role of this unique antioxidant enzyme in vascular pathophysiology and therapeutics.