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

Department of Pharmacology, Center for Lung and Vascular Biology, University of Illinois, Chicago, IL 60612, USA.
AJP Heart and Circulatory Physiology (Impact Factor: 3.84). 11/2011; 302(3):H724-32. DOI: 10.1152/ajpheart.00739.2011
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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 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.

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Available from: Cristiana Caliceti, Feb 16, 2015
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    • "In the last decade the scientific community clarified the importance of low levels of ROS as key signaling molecules in physiological functions such as the regulation of cell signaling , proliferation, and differentiation [3]. Different authors provided scientific evidences regarding a sequential and direct link between Notch and Wnt signaling pathways in tuning endothelial cells (ECs) and cardiomyocytes functions and vascular morphogenesis [4]. "
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    ABSTRACT: Reactive oxygen species (ROS), traditionally viewed as toxic by-products that cause damage to biomolecules, now are clearly recognized as key modulators in a variety of biological processes and pathological states. The development and regulation of the cardiovascular system require orchestrated activities; Notch and Wnt/ β -catenin signaling pathways are implicated in many aspects of them, including cardiomyocytes and smooth muscle cells survival, angiogenesis, progenitor cells recruitment and differentiation, arteriovenous specification, vascular cell migration, and cardiac remodelling. Several novel findings regarding the role of ROS in Notch and Wnt/ β -catenin modulation prompted us to review their emerging function in the cardiovascular system during embryogenesis and postnatally.
    02/2014; 2014:318714. DOI:10.1155/2014/318714
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    • "Phosphorylation of p66Shc on Ser36 has been shown to mediate cellular aging, damage, and apoptosis, and to be induced by oxidative stress [41]. We found that stimulation of HUVEC with TNFα induced a 2- to 3-fold increase in p66Shc phosphorylation on Ser36 (Figure 2, A and B), which was almost completely blocked by pretreatment of cells with the specific JNK inhibitor (Figure 2, C and D) and only partially reduced by pretreatment of cells with the p38 inhibitor (Figure S3A), in line with previous results obtained in bovine aortic endothelial cells exposed to H2O2 [41] and in HUVEC treated with the Vascular Endothelial Growth Factor [42]. Specific isoforms of the protein kinase C (PKC) family have also been proposed as mediators of ROS triggered stress signals. "
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    ABSTRACT: Endothelial cells participate in inflammatory events leading to atherogenesis by regulating endothelial cell permeability via the expression of VE-Cadherin and β-catenin and leukocyte recruitment via the expression of E-Selectins and other adhesion molecules. The protein p66(Shc) acts as a sensor/inducer of oxidative stress and may promote vascular dysfunction. The objective of this study was to investigate the role of p66(Shc) in tumor necrosis factor TNFα-induced E-Selectin expression and function in human umbilical vein endothelial cells (HUVEC). Exposure of HUVEC to 50 ng/ml TNFα resulted in increased leukocyte transmigration through the endothelial monolayer and E-Selectin expression, in association with augmented phosphorylation of both p66(Shc) on Ser(36) and the stress kinase c-Jun NH2-terminal protein kinase (JNK)-1/2, and higher intracellular reactive oxygen species (ROS) levels. Overexpression of p66(Shc) in HUVEC resulted in enhanced p66(Shc) phosphorylation on Ser(36), increased ROS and E-Selectin levels, and amplified endothelial cell permeability and leukocyte transmigration through the HUVEC monolayer. Conversely, overexpression of a phosphorylation-defective p66(Shc) protein, in which Ser(36) was replaced by Ala, did not augment ROS and E-Selectin levels, nor modify cell permeability or leukocyte transmigration beyond those found in wild-type cells. Moreover, siRNA-mediated silencing of p66(Shc) resulted in marked reduction of E-Selectin expression and leukocyte transmigration. In conclusion, p66(Shc) acts as a novel intermediate in the TNFα pathway mediating endothelial dysfunction, and its action requires JNK-dependent phosphorylation of p66(Shc) on Ser(36).
    PLoS ONE 12/2013; 8(12):e81930. DOI:10.1371/journal.pone.0081930 · 3.23 Impact Factor
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    • "In this study, we confirmed the increase in human catalase mRNA in aorta (Figure S1A) and catalase protein expression in the cultured ECs in Cat-Tg mice (Figure S1B). Furthermore, VEGF-induced increase in intracellular oxidation state, as measured by DCF-DA fluorescence analysis [38], [39], was significantly inhibited in cultured ECs derived from Cat-Tg mice, as compared to WT-ECs (Figure S1C). "
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    ABSTRACT: Background Reactive oxygen species (ROS) play an important role in angiogenesis in endothelial cells (ECs) in vitro and neovascularization in vivo. However, little is known about the role of endogenous vascular hydrogen peroxide (H2O2) in postnatal neovascularization. Methodology/Principal Findings We used Tie2-driven endothelial specific catalase transgenic mice (Cat-Tg mice) and hindlimb ischemia model to address the role of endogenous H2O2 in ECs in post-ischemic neovascularization in vivo. Here we show that Cat-Tg mice exhibit significant reduction in intracellular H2O2 in ECs, blood flow recovery, capillary formation, collateral remodeling with larger extent of tissue damage after hindlimb ischemia, as compared to wild-type (WT) littermates. In the early stage of ischemia-induced angiogenesis, Cat-Tg mice show a morphologically disorganized microvasculature. Vascular sprouting and tube elongation are significantly impaired in isolated aorta from Cat-Tg mice. Furthermore, Cat-Tg mice show a decrease in myeloid cell recruitment after hindlimb ischemia. Mechanistically, Cat-Tg mice show significant decrease in eNOS phosphorylation at Ser1177 as well as expression of redox-sensitive vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1) in ischemic muscles, which is required for inflammatory cell recruitment to the ischemic tissues. We also observed impaired endothelium-dependent relaxation in resistant vessels from Cat-Tg mice. Conclusions/Significance Endogenous ECs-derived H2O2 plays a critical role in reparative neovascularization in response to ischemia by upregulating adhesion molecules and activating eNOS in ECs. Redox-regulation in ECs is a potential therapeutic strategy for angiogenesis-dependent cardiovascular diseases.
    PLoS ONE 03/2013; 8(3):e57618. DOI:10.1371/journal.pone.0057618 · 3.23 Impact Factor
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