PirB limits F4/80-positive cell infiltration during vein graft adaptation.

<p>(<b>A</b>) Representative H&E image of PirB WT and KO mouse vein grafts. Scale bar, 100 µm. Arrowheads show vein graft wall. n = 7. (<b>B</b>) Representative image of smooth muscle alpha-actin (SMA) staining of PirB WT and KO mouse vein grafts. n = 4. (<b>C</b>) Representative images of PirB WT and KO vein grafts with immunohistochemical staining for F4/80. n = 4. (<b>D</b>) Bar graph shows summary of morphological analysis of wall thickness of PirB WT and KO grafts. *, p<0.0001, t-test; n = 7. (<b>E</b>) Bar graph shows summary of densitometry of F4/80 staining. *, p = 0.0211, t-test; n = 4. (<b>F</b>) Bar graph shows summary of mRNA transcript expression in macrophages derived from PirB WT (▪) or PirB KO (□) mice. *, p<0.02, t-test. n = 6. (<b>G</b>) Bar graph shows summary of mRNA transcript expression in macrophages derived from PirB WT (▪) or PirB KO (□) mice. *, p<0.03, t-test. n = 6. (<b>H</b>) Bar graph shows mean densitometry of TIMP1 immunoreactivity in vein grafts derived from PirB WT (▪) or PirB KO (□) mice. n.s., not significant; p = 0.7859, t-test; n = 3. (<b>I</b>) Bar graph shows mean densitometry of TIMP2 immunoreactivity in vein grafts derived from PirB WT (▪) or PirB KO (□) mice. n.s., not significant; p = 0.8941, t-test; n = 3. (<b>J</b>) Bar graph shows mean densitometry of MMP2 activity in vein grafts derived from PirB WT (▪) or PirB KO (□) mice. n.s., not significant; p = 0.2252, t-test; n = 3. (<b>K</b>) Bar graph shows mean densitometry of MMP14 activity in vein grafts derived from PirB WT (▪) or PirB KO (□) mice. n.s., not significant; p = 0.6569, t-test; n = 4. (<b>L</b>) Bar graph shows mean densitometry of MMP9 activity in vein grafts derived from PirB WT (▪) or PirB KO (□) mice. *, p = 0.0109, t-test; n = 3.</p

Nogo-B limits F4/80-positive cell infiltration during vein graft adaptation.

<p>(<b>A</b>) Representative ultrasound images of mouse vein grafts. Nogo KO grafts (right panel) had significantly thicker graft walls compared to those of Nogo WT grafts (left panel). *, lumen. (<b>B</b>) Representative H&E image of WT and Nogo KO mouse vein grafts. Scale bar, 100 µm. (<b>C</b>) Representative immunofluorescence images of Nogo staining in WT and KO mouse vein grafts; no staining is present in Nogo KO vein grafts. Scale bar, 50 µm. (<b>D</b>) Representative image of smooth muscle alpha-actin (SMA) staining of Nogo WT and KO mouse vein grafts. (<b>E</b>) Representative images of Nogo WT and KO vein grafts with immunohistochemical staining for F4/80. Arrowheads show vein graft wall. n = 20, 25. (<b>F</b>) Line graph shows the time course of vein graft wall thickening as determined by ultrasound imaging. Nogo KO grafts had approximately 40 percent thicker walls compared to those of wild type grafts. *, p = 0.0041; ANOVA. (<b>G</b>) Bar graph shows summary of morphological analysis of wall thickness of Nogo WT and KO grafts. *, p = 0.0061; t-test; n = 5. (<b>H</b>) Bar graph shows summary of densitometry of F4/80 staining. *, p = 0.0011; t-test. n = 20, 25. (<b>I</b>) Bar graphs show summary of proliferation and apoptosis indices in mouse vein grafts. *, p<0.0001, ANOVA; post-hoc testing p<0.05; n = 8, 16, 8, 32 in each of the 4 groups, respectively.</p

Increased Nogo-B and PirB expression in mouse and human vein grafts.

<p>(<b>A</b>) Representative immunohistochemical staining images in mouse (top row) and human (bottom row) vein (left), vein graft (center), and aorta (right). n = 4. Scale bar, 40 µm. (<b>B</b>) Representative Western blot analysis showing Nogo-B and PirB up-regulation in individual mouse vein grafts; n = 2 matched specimens are shown of n = 4 samples. (<b>C</b>) Representative Western blot analysis showing Nogo-B and PirB up-regulation in individual human vein grafts; n = 4 unmatched specimens are shown. (<b>D</b>) Bar graph shows densitometry of Nogo-B bands shown in Panels (B) and (C) during mouse and human vein graft adaptation. *, p<0.05; t-test (vein vs. vein graft). (<b>E</b>) Bar graph shows densitometry of PirB bands shown in Panels (B) and (C) during mouse and human vein graft adaptation. *, p<0.05; t-test (vein vs. vein graft). (<b>F</b>) Bar graph shows summary of gene expression during mouse vein graft adaptation; the number of mRNA transcripts expressed during vein graft adaptation is compared to the number expressed in the wild type pre-implantation inferior vena cava (IVC), denoted by the red broken line. n = 4. ▪, Nogo WT; □, Nogo KO vein grafts. (<b>G</b>) Bar graph shows Nogo-B and PirB mRNA transcripts during human vein graft adaptation. Analysis of n = 8 veins, 6 vein grafts, and 5 arterial human specimens. *, p<0.05; t-test (vein vs. vein graft).</p

PirB mediates adhesion in vitro and in vivo.

<p>(<b>A</b>) Representative immunofluorescence image showing lack of PirB signal in the pre-implantation vein. IVC, inferior vena cava. Scale bar, 20 µm. (<b>B</b>) PirB positive cells localized to the luminal surface and the interface between the medial and adventitial layers. *, vessel lumen; red arrowhead, PirB positive cells; I, intimal layer; I+M, intima-medial layer; Ad, adventitial layer. (<b>C–E</b>) PirB-positive cells on the vein graft luminal vessel surface (<b>C</b>) did not co-localize with CD31-positive endothelial cells (<b>E</b>). *, vessel lumen; white arrows, PirB-positive cells. (<b>F–H</b>) PirB-positive cells in between the medial and adventitial layers (<b>F</b>) co-localize with F4/80-positive cells (<b>G,H</b>). I+M, intima-medial layer; Ad, adventitial layer. n = 4. (<b>I</b>) Bar graph shows macrophage adhesion to bovine serum albumin (BSA) or fibronectin. Macrophages were derived from PirB WT (▪) or PirB KO (□) mice. n.s., not significant. n = 8. *, p<0.0001; ANOVA; post-hoc testing p<0.05. (<b>J</b>) Bar graph shows macrophage adhesion to endothelial cells. Macrophages were derived from PirB WT (▪) or PirB KO (□) mice and activated with TNF-α. *, p = 0.0499, t-test; n = 3.</p

Mouse vein graft configurations.

<p>Mouse vein graft configurations.</p

PirB mediates other macrophage-mediated vascular functions.

<p>(<b>A</b>) Line graph shows mean deep muscle perfusion (ratio ischemic:control leg), 0–21 days after induction of ischemia in PirB WT (▪) or PirB KO (□) mice. *, p<0.0001, ANOVA; n = 10. (<b>B</b>) Bar graph show summary of capillary density, at baseline (p = 0.5350, t-test; n = 3) or 14 days after induction of ischemia (*, p<0.0001, t-test; n = 3) in PirB WT (▪) or PirB KO (□) mice. (<b>C</b>) Bar graphs show mean VEGF-A mRNA (*, p = 0.001, t-test; n = 5) 72 hours after induction of ischemia in PirB WT (▪) and PirB KO (□) mice. (<b>D</b>) Bar graphs show mean VEGF-A protein secretion by PirB WT (▪) and PirB KO macrophages (□) (*, p = 0.0003, t-test; n = 6). (<b>E</b>) Representative photomicrographs showing F4/80 staining (black arrowheads) in gastrocnemius muscle 3 days after induction of ischemia in PirB WT (▪) or PirB KO (□) mice. Right panel shows bar graph summarizing mean number of F4/80-positive cells per hpf. *, p = 0.0016, t-test; n = 3.</p

Histological assessment demonstrated active vessel remodeling and neo-vessel formation at 12 months after Implantation.

<p>Histologic analysis of TEVG was performed by Hematoxylin and Eosin staining at 5x (top) and 20x (bottom). At 4 months, there is a thin endothelium and some cellular infiltration at the periphery of the TEVG. By 8 months, tissue ingrowth at the periphery has increased. By 12 months, tissue ingrowth has increased at the luminal and peripheral surfaces, with increasing cellularity within the graft. (n = 5 in each group.)</p

Expression of endothelial markers indicate functional endothelium in TEVG.

<p><b>A.</b> Whole mount staining of native aorta and TEVG. VE-cadherin is a marker of cellular borders of endothelial cells (green). Endothelial nitric oxide synthase (eNOS) is a marker of a functional endothelium (red). DAPI is a nuclear stain (blue). <b>B.</b> Real-time PCR analysis of ephrinB2 and eNOS. Ephrin-B2, a marker of arterial vessels. (n = 5–10 in each group)</p

Endothelialization and smooth muscle cell (SMC) differentiation in TEVG neotissue at 6 months.

TEVG ECs and SMCs were mature and well-organized, which reflected the native carotid artery (CA) in area, distribution, and density. Representative photomicrographs are shown for vWF (A, E), α- smooth muscle actin (SMA) (B, F), calponin (C, G), and myosin heavy chain (MHC) (D, H) for native CA (A-D) and nanofiber PCL/CS TEVGs (E-H). The scale bar represents a length of 20μm for A and E, and 100μm otherwise.</p

Expression of vascular cell markers in TEVG was similar to that of native aorta.

<p>A. Immuno-labeling of vascular cell markers on cross-sections of TEVG and native aorta. Left panel: von Willebrand factor (vWF, green), an endothelial cell marker and CD68 (red), a macrophage marker. Middle panel: CD31 (red), an endothelial cell marker; alpha-smooth muscle actin (α-SMA, green), a marker of smooth muscle cell and myofibroblasts. Right panel: CD31 (red) and collagen type I (Col-1, green). DAPI is for nucleus staining (blue). <b>B.</b> Immune-labeling of intracellular adhesion molecule (ICAM) as a marker of endothelial cells.</p