Angiopoietin-1 Reduces H2O2-Induced Increases in Reactive Oxygen Species and Oxidative Damage to Skin Cells  Nesreen S. Ismail, Elke A. Pravda, Dan Li, Shou-Ching Shih, Susan M. Dallabrida  Journal of Investigative Dermatology  Volume 130, Issue 5, Pages 1307-1317 (May 2010) DOI: 10.1038/jid.2009.431 Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Ang1 prevents H2O2-induced increases in superoxides in skin cells. (a) HaCaT and (b) HeMn cells were given 100μM H2O2 and 200nM ang1, collagen III (coll III), vitronectin (VN), or fibronectin (FN) matrices or vehicle controls (Con.) (30minutes), and the ·O2− levels were measured (n=6 per group with studies performed in triplicate, mean±SD). In graphs, each matrix is paired by color with its appropriate vehicle control. (a, b) H2O2 increased ·O2− levels in HaCaT and HeMn cells (*P≤0.0000004). Ang1 prevented H2O2-induced increases in ·O2− (**P=0.00001 (HaCaT), #P≤0.004 (HeMn)) (a) as did fibronectin (***P=0.003, HaCaT). Collagen III and vitronectin had no significant effect on ·O2− levels. (a, b) Superoxide scavenger, tiron, eliminated the signal, indicating signal specificity. Journal of Investigative Dermatology 2010 130, 1307-1317DOI: (10.1038/jid.2009.431) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Ang1 reduces H2O2-induced increases in superoxides in skin cells. (a) HaCaT and (b) HeMn cells were given 100μM H2O2 (15minutes), ang1 or collagen III was added (15minutes) (n=6 per group with studies performed in duplicate, mean±SD), and the ·O2− levels were measured. In graphs, each matrix is paired by color with its appropriate vehicle control (Con.). (a, b) H2O2 increased ·O2− levels in HaCaT and HeMn cells (*P≤0.0006). Ang1 reduced H2O2-induced increases in ·O2− in HaCaT and HeMn cells (**P≤0.007) after ROS damage was initiated. (a, b) Tiron almost completely eliminated the signal. Journal of Investigative Dermatology 2010 130, 1307-1317DOI: (10.1038/jid.2009.431) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Ang1 blocks H2O2-induced increases in nitrotyrosinylation in skin cells. (a) HaCaT and (b) HeMn cells were given 200nM ang1, collagen III, or vehicle controls (Con.), and 100μM H2O2 was added (30minutes) (n=6 per group with studies performed in duplicate, mean±SD). In graphs, each matrix is paired by color with its appropriate vehicle control. Ang1, but not collagen III, reduced protein nitrotyrosinylation in (a) HaCaT (*P=0.001) and (b) HeMn (**P=0.03) cells. Journal of Investigative Dermatology 2010 130, 1307-1317DOI: (10.1038/jid.2009.431) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Ang1 reduces H2O2-induced DNA damage to skin cells. (a) HaCaT and (b) HeMn cells were given 200nM ang1 or vehicle control, and 100μM H2O2 was added (30minutes). Fluorescent immunocytochemistry with confocal microscopy imaging was conducted (bar=20μm). OxyDNA staining shows DNA damage (green). Nuclei were stained (DAPI, blue). Merged images show an overlap of 8OHDG with DAPI. Graphs show the 8OHDG signal normalized to cell number. Ang1 reduced H2O2-induced 8OHDG DNA damage to (a) HaCaT (*P=0.0003) and (b) HeMn (**P=0.01) cells. Journal of Investigative Dermatology 2010 130, 1307-1317DOI: (10.1038/jid.2009.431) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 MGTP integrin subunit profiles. MGTP real-time PCR was conducted using (a) HaCaT and (b) HeMn cells. Absolute mRNA levels of each integrin subunit were normalized to 18S mRNA (n=3 per group, studies performed in duplicate, mean±SD). (a) HaCaT cells expressed β4>β1>α2>αv>α6>β8>β5>α3>β6>>α4>α1>β7 mRNA. (b) HeMn cells expressed β1>β3>β5>α3>β8>αv>>α1>β7>α2>α6>α7 mRNA. Neither skin cell expressed tie2 mRNA. Journal of Investigative Dermatology 2010 130, 1307-1317DOI: (10.1038/jid.2009.431) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Ang1 increases skin cell viability. (a) HaCaT and (b) HeMn cells were given 200nM ang1, collagen III, or a vehicle control (Con.) (90minutes), and viability was assessed (n=6 per group with studies performed in duplicate, mean±SD). In graphs, each matrix is paired by color with its appropriate vehicle control. Ang1 increased (a) HaCaT (*P=0.00003) and (b) HeMn (**P=0.03) survival, whereas collagen III had no such effect. Journal of Investigative Dermatology 2010 130, 1307-1317DOI: (10.1038/jid.2009.431) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Ang1 activates skin cell Akt and MAPKp42/44 signaling. (a) HaCaT and (b) HeMn cells were given 200nM ang1 or vehicle control (45minutes), and signaling was assessed by western blotting (n=4 per group with studies performed in duplicate, mean+SD). Ang1 phosphorylated akt and MAPKp42/44 in (a) HaCaT and (b) HeMn cells (*P≤0.004, **P=0.0001, ***P=0.002). Journal of Investigative Dermatology 2010 130, 1307-1317DOI: (10.1038/jid.2009.431) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Ang1 promotes skin cell adhesion. (a) HaCaT and (b) HeMn cells were added to immobilized 200nM ang1 or vehicle control (45minutes), and adhesion was assessed (n=4 per group with studies performed in triplicate, mean±SD). Bar=200μm. Ang1 increased adhesion to (a) HaCaT (*P=0.02) and (b) HeMn (***P=0.0001) cells. EDTA reduced (a) HaCaT (**P=0.0004) and (b) HeMn (#P=0.0002) cell adhesions to ang1. Journal of Investigative Dermatology 2010 130, 1307-1317DOI: (10.1038/jid.2009.431) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Select integrin antibodies disrupt Ang1 adhesion to skin cells. (a) HaCaT and (b) HeMn cells were added to immobilized 200nM ang1 or vehicle control (45minutes), and adhesion was assessed (n=4 per group with studies performed in duplicate, mean±SD). Ang1 promoted (a) HaCaT and (b) HeMn cell adhesion (*P≤0.00001). In (a) HaCaT cells, integrin subunit antibodies α3, α4, αv, β1, β4, and αvβ6 reduced adhesion to ang1, and in (b) HeMn cells, αv, β1, and αvβ5 reduced adhesion to ang1 (#P≤0.006, ##P≤0.0006, ###P=0.0000002, ####P=0.03). (a) In HaCaTs, α6 antibody increased adhesion to ang1 (^P=0.006). Journal of Investigative Dermatology 2010 130, 1307-1317DOI: (10.1038/jid.2009.431) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 10 Select integrin antibodies abrogate effects of Ang1 on skin cell superoxides. (a) HaCaT and (b) HeMn cells were preincubated±function-blocking integrin antibodies. Then, 100μM H2O2 and 200nM ang1 or vehicle control (30minutes) were added, and the ·O2− levels were measured (n=6 per group with studies performed in duplicate, mean±SD). In (a) HaCaT cells, H2O2 increased ·O2− levels (^P=0.00006), and ang1 prevented H2O2-induced increases in ·O2− (*P≤0.02). Integrin antibodies to αv (#P=0.04) or β1 (##P=0.001) abrogated ang1's effect on reducing ·O2−. Integrin α1 antibody did not affect ang1's effect on lowering ·O2− levels (*P≤0.02). In (b) HeMn cells, H2O2 increased ·O2− levels (**P≤0.004), and ang1 reduced this increase in ·O2− (*P≤0.02). Integrin antibodies αv (**P≤0.004) and β1 (###P=0.0006) disrupted ang1's capacity to lower ·O2− levels, whereas with integrin antibody α2, ang1 was still able to reduce ·O2− levels (***P=0.05). Journal of Investigative Dermatology 2010 130, 1307-1317DOI: (10.1038/jid.2009.431) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions