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Daniel J. Wong, MD, Daniel Y. Lu, MD, Clinton D

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1 Ephrin type-B receptor 4 activation reduces neointimal hyperplasia in human saphenous vein in vitro 
Daniel J. Wong, MD, Daniel Y. Lu, MD, Clinton D. Protack, MD, PhD, Go Kuwahara, MD, PhD, Hualong Bai, MD, Nirvana Sadaghianloo, MD, George Tellides, MD, PhD, Alan Dardik, MD, PhD  Journal of Vascular Surgery  Volume 63, Issue 3, Pages (March 2016) DOI: /j.jvs Copyright © Terms and Conditions

2 Fig 1 Ephrin-B2/Fc reduces neointimal thickness in human saphenous vein organ culture. Representative photomicrographs of (A) saphenous vein at day 0, or the same vein after 14 days in organ culture and (B) control or (C) treated with 2 μg/mL ephrin-B2/Fc (elastin von Gieson stain; scale bar, 100 μm). D, Bar graph shows mean neointima/media ratio for vein rings, day 0 or 14, in matched samples treated without or with ephrin-B2/Fc (n = 19). *P = .001; **P = .048 vs day 0 and P = .017 vs day 14 untreated by paired t-test. E, Graph shows the neointima/media ratio in matched vein segments after 14 days. F, Bar graph shows proportion of Ki-67-positive nuclei in vein rings after 3 days in organ culture. P = .396 day 3 untreated vs day 0; P = .798 day 3 treated vs day 0; P = .384 day 3 untreated vs treated by paired t-test (n = 3). G, Bar graph shows densitometry of Western blot analysis of cleaved caspase-3 expression, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in vein rings, day 0 or 3. P = .079 day 3 untreated vs day 0; P = .055 day 3 treated vs day 0; P = .827 day 3 untreated vs treated by paired t-test (n = 3). The range bars in the graphs indicate the standard error. a.u., Arbitrary units. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

3 Fig 2 Messenger RNA (mRNA) expression in human saphenous vein organ culture. A, Bar graph shows relative number of ephrin type-B receptor 4 (Eph-B4) mRNA transcripts at day 0 or 14, without or with ephrin-B2/Fc, in matched samples (n = 7). *P = .005; **P = .015 vs day 0 and P = .599 v. day 14 untreated. B, Bar graph shows relative number of ephrin-B2 mRNA transcripts at day 0 or 14, without or with ephrin-B2/Fc in matched samples (n = 7). *P < .001; **P = .001 vs day 0 and P = .740 vs day 14 untreated. C, Bar graph shows relative number of osteopontin mRNA transcripts at day 0 or 14, without or with ephrin-B2/Fc in matched samples (n = 7). *P = .044, P = .078 vs day 0, and P = .746 vs day 14 untreated. D, Bar graph shows relative number of matrix metalloproteinase 2 (MMP-2) mRNA transcripts at day 0 or 14, without or with ephrin-B2/Fc, in matched samples (n = 7). P = .763 day 0 vs day 14 untreated, P = .547 day 0 vs day 14 treated, and P = .391 day 14 untreated vs treated. E, Bar graph shows relative number of collagen 1 (ColA1) mRNA transcripts at day 0 or 14, without or with ephrin-B2/Fc in matched samples (n = 7). P = .895 day 0 vs day 14 untreated, P = .833 day 0 vs day 14 treated, and P = .906 day 14 untreated vs treated. Paired t-tests were used for all comparisons. All expression is in arbitrary units relative to the control. The range bars indicate the standard error. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

4 Fig 3 Ephrin-B2/Fc stimulates ephrin type B receptor 4 (Eph-B4) phosphorylation and downstream signaling in human umbilical vein endothelial cells (HUVEC). A, Immunoprecipitation analysis of tyrosine-phosphorylated Eph-B4 (110 kDa) in HUVECs treated without or with ephrin-B2/Fc. *Immunoglobulin G (IgG) heavy chain; **immunoglobulin G light chain. IB, Immunoblot; IP, immunoprecipitation. B, Phosphorylated (ser473) and total Akt expression in HUVECs treated with ephrin-B2/Fc at 0, 5, and 15 minutes, representative Western blot analysis and bar graph show relative densitometry (n = 6). P = vs 15 minutes by unpaired t-test). C, Phosphorylated (ser1177) and total endothelial nitric oxide (NO) synthase (eNOS) expression in HUVECs treated with ephrin-B2/Fc at 0, 5, and 15 minutes. Representative Western blot analysis and bar graph show relative densitometry (n = 6). P = vs 15 minutes by unpaired t-test. D, Phosphorylated (tyr14) and total caveolin-1 (Cav-1) expression in HUVECs treated with ephrin-B2/Fc at 0, 15, and 30 minutes. Representative Western blot analysis and bar graph show relative densitometry (n = 6). *P = vs 30 minutes by unpaired t-test. E, Representative immunofluorescence images of HUVEC (left panel) without and (right panel) with ephrin-B2/Fc (2 μg/mL) at 5 min (n = 3). Red, caveolin-1 (Cav-1); green, Eph-B4; blue, 4′,6-diamidino-2-phenylindole (DAPI). The arrows show colocalization of Eph-B4 and caveolin-1. Scale bar, 25 μm. F, Bar graph shows relative Eph-B4 messenger RNA transcript numbers, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in HUVEC treated without or with ephrin-B2/Fc (2 μg/mL) for 12 hours (n = 3). P = .745 by unpaired t-test. The range bars in the graphs indicate the standard error. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

5 Fig 4 Ephrin-B2/Fc stimulates nitric oxide (NO) release but not proliferation in human umbilical vein endothelial cells (HUVEC). A, Bar graph shows NO release in HUVECs treated without or with ephrin-B2/Fc (2 μg/mL) for 30 minutes, normalized to total protein (n = 6) *P = .007 vs 30 minutes untreated by unpaired t-test. Two bars for time 0 are shown, matched to each of the two time 30 runs. B, Bar graph shows relative cell numbers in HUVECs stimulated with serum or ephrin-B2/Fc for 72 hours (n = 3). *P < .05 by unpaired t-test. The range bars in the graphs indicate the standard error. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

6 Fig 5 Adventitial delivery of ephrin-B2/Fc stimulates endothelial ephrin type B receptor 4 (Eph-B4) phosphorylation. Immunofluorescence of human saphenous vein treated on its adventitial surface with Pluronic gel (Sigma-Aldrich, St. Louis, Mo), without or with ephrin-B2/Fc (2 μg/mL), and placed in a bioreactor; analysis of matched samples. A, Saphenous vein before initiation of arterial flow. B, Saphenous vein treated with Pluronic gel alone for 24 hours. C, Saphenous vein treated with Pluronic gel containing ephrin-B2/Fc for 24 hours. Red, phosphotyrosine; green, Eph-B4; blue, 4′,6-diamidino-2-phenylindole (DAPI). The arrows show colocalization of Eph-B4 and phosphotyrosine in the endothelium; *indicates the vessel lumen (n = 3). Scale bar, 50 μm. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

7 Fig 6 Schematic of ephrin type B receptor 4 (Eph-B4) signal transduction. Ephrin-B2 ligand (ephrin-B2/Fc in the experiments) binds to the Eph-B4 receptor, activating it by dimerization and phosphorylation. Activated Eph-B4 stimulates additional downstream signaling pathways, including caveolin-1, Akt, and endothelial nitric oxide (NO) synthase (eNOS) signaling among others, leading to additional functions in the blood vessel. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

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