Accelerated Reendothelialization, Increased Neovascularization and Erythrocyte Extravasation after Arterial Injury in BAMBI^−/− Mice

<div><p>Background</p><p>Intimal injury rapidly activates TGFβ and enhances vascular repair by the growth of endothelial (EC) and vascular smooth muscle cells (VSMC). The response to the TGFβ family of growth factors can be modified by BAMBI (BMP, Activin, Membrane Bound Inhibitor) acting as a non-signaling, competitive antagonist of TGFβ type I receptors such as ALK 1 and 5. In vivo the effect of BAMBI will depend on its cell-specific expression and of that of the ALK type receptors. We recently reported EC restricted BAMBI expression and genetic elimination of BAMBI resulting in an in vitro and in vivo phenotype characterized by endothelial activation and proliferation involving alternative pathway activation by TGFβ through ALK 1.</p> <p>Methodology/Principal Findings</p><p>To test the hypothesis that BAMBI modulates arterial response to injury via its effects on endothelial repair and arterial wall neovascularization we used a model of femoral arterial denudation injury in wild type (WT) and BAMBI^−/− mice. Arterial response was evaluated at 2 and 4 weeks after luminal endothelial denudation of femoral arteries. The BAMBI^−/− genotype mice showed accelerated luminal endothelial repair at 2 weeks and a highly unusual increase in arterial wall neovascularization compared to WT mice. The exuberant intimal and medial neovessel formation with BAMBI^−/− genotype was also associated with significant red blood cell extravasation. The bleeding into the neointima at 2 weeks transiently increased it’s area in the BAMBI^−/−genotype despite the faster luminal endothelial repair in this group. Vascular smooth muscle cells were decreased at 2 weeks in BAMBI^−/− mice, but comparable to wild type at 4 weeks.</p> <p>Conclusions/Significance</p><p>The absence of BAMBI results in a highly unusual surge in arterial wall neovascularization that surprisingly mimiks features of intra-plaque hemorrhage of advanced atheroma in a mechanical injury model. This suggests important effects of BAMBI on arterial EC homeostasis that need to be further studied in a model of inflammatory atherosclerosis.</p> </div

BAMBI deficiency results in neo-intimal neovasculariztion with proliferating endothelial cells.

<p>Proliferation of endothelial cells was evaluated by double staining for the proliferation marker Ki67 (green) and the endothelial cell marker vWF (red); co-localization of both signals showing as yellow (overlay) The lamina elastica interna shows typical green autofluorescence. At two weeks double positive cells (white arrows) were restricted to the luminal surface in the BAMBI^−/− mice, whereas at 4 weeks double positive cells also appeared deep in the neointima in a pattern consistent with ongoing neo-vessel formation (original magnification ×1000).</p

Smooth muscle cells in neointima and media from BAMBI^+/+ and BAMBI ^−/− mice.

<p>Staining for alphaSMA in the femoral arteries from BAMBI^+/+ (A) and BAMBI^−/− (B) mice 2 and 4 weeks after injury. (Original magnification ×400 and ×1000; bar = 50 µm; Ni: neointima, Me: media and Ad: adventitia). (C) and (D) alphaSMA positive areas in media and neointima of the femoral arteries. Data are mean ± SEM, n = 5–9, *P<0.05 compared to respective wild type.</p

BAMBI deficiency accelerates reendothelialization and increased neo-intimal neovascularization and accumulation of erythrocytes.

<p>(<b>A</b>) Endothelial cells of femoral arteries were stained by isolectin B4 in BAMBI^+/+ and BAMBI^−/− mice 2 and 4 weeks after intimal denudation. (Ni: neointima, Me: media and Ad: adventitia. Black arrows indicate lectin positive luminal endothelial cells and intimal microvessels, original magnification ×400 upper panel and ×1000 lower panel; bar = 50 µm). (<b>B</b>) Reendothelialization was also determined by staining with von Willebrand factor (black arrows) magnification ×1000.(<b>C</b>) Fibrin and erythrocytes were visualized by Carstairs’ staining of femoral arteries at the same time point (Original magnification ×400 upper panel and ×1000 lower panel; bar = 50 µm; black arrows indicate RBC infiltration). (<b>D</b>) Quantification of the reendothelialization of the femoral arteries. (<b>E</b>) Microvascular density in the different layers of the femoral arteries 4 weeks after injury. (<b>F</b>) Erythrocytes accumulation in neointima 2 and 4 weeks after injury. Data are mean ± SEM, n = 5–9, *<i>P</i><0.05 compared to respective wild type.</p

Deficiency of BAMBI increases early neo-intimal hyperplasia after intima denudation.

<p>Representative pictures of femoral arteries from BAMBI^+/+ (<b>A</b>) and BAMBI^−/− (<b>B</b>) mice at 2 and 4 weeks after injury. (Hematoxylin and eosin stains. Original magnification ×400 and ×1000; bar = 50 µm; Ni: neointima, Me: media and Ad: adventitia). (<b>C</b>). Morphometric analysis of the femoral arteries. Data are mean ± SEM; n = 5–9; *<i>P</i><0.05 compared to respective wild type.</p

Electron microscopy pictures show an activated endothelial phenotype in tissues from BAMBI^−/− as compared to BAMBI^+/+ mice.

<p><b>A</b>. Myocardium of the BAMBI<b>⁻</b>^/<b>−</b> mice is notable for the number of endothelial cells per capillary cross-section and the prominent nuclei. In BAMBI^+/+ mice myocardial capillaries show one endothelial cell per cross-section, while those from BAMBI<b>⁻</b>^/<b>−</b> mice show two nuclei per cross-section in 70% of the capillaries (scale bar 2 µm). <b>B</b> and <b>C</b>. glomeruli from BAMBI<b>⁻</b>^/<b>−</b> mice show a prominent number of endothelial cells, which also appear swollen as compared to those from BAMBI^+/+ mice. The glomerular capillary lumen is almost obliterated by the endothelial cells in the BAMBI<b>⁻</b>^/<b>−</b> mice <i>vs.</i> BAMBI^+/+ (low magnification).The activated endothelial phenotype in the BAMBI<b>⁻</b>^/<b>−</b> glomeruli is also apparent at higher magnification (bottom row). <b>D.</b> Peritubular capillaries of BAMBI<b>⁻</b>^/<b>−</b> mice also show a prominent and swollen endothelial cells impinging on the capillary lumen as compared to BAMBI ^+/+ mice.</p

Albumin creatinine ratio, and BUN.

<p>Urinary albumin to creatinine ratios from BAMBI^+/+ and BAMBI<b>⁻</b>^/<b>−</b> mice at baseline and two weeks after unilateral nephrectomy. BUN levels from BAMBI^+/+ and BAMBI<b>⁻</b>^/<b>−</b> mice at baseline and 2 weeks after unilateral nephrectomy. Values are means ± SEM for the number of animals indicated in brackets.</p

<i>In vivo</i> angiogenesis by modified-matrigel implantation assay is enhanced in BAMBI^−/− mice in comparison to their BAMBI^+/+ littermates.

<p><b>A. </b><i>In vivo</i> angiogenesis was evaluated under basal conditions and in the presence of VEGF and FGF in the implants in BAMBI^+/+ and BAMBI<b>⁻</b>^/<b>−</b> mice. In-growth of endothelial cells was evaluated after 12 days by isolectin B4 fluorescence determination. Results are means ± SEM from 4 animals per group. # <i>P</i><0.05 <i>vs</i>. respective basal values; * <i>P</i><0.05 BAMBI<b>⁻</b>^/<b>−</b><i>vs</i>. BAMBI^+/+. <b>B.</b> Comparable experiments except that TGFβ ± LY364947 or LY364947 alone were added to the matrigel implants. Results are means ± SEM from 5 mice per group. * <i>P</i><0.05 comparing respective basal <i>vs.</i> growth factor stimulated values and # <i>P</i><0.05 for group comparison of BAMBI^+/+ and BAMBI<b>⁻</b>^/<b>−</b> mice in the same condition.</p

Expression of BAMBI in different mouse organs.

<p><b>A.</b> Levels of mRNA (relative copy number) were determined in different organs from BAMBI^+/+ and BAMBI<b>⁻</b>^/<b>−</b> mice. Two different primer pairs were used, spanning either exons 2–3 (top panel) or exons 1–2 (bottom panel) in order to exclude transcripts from a potential second start site in the BAMBI gene. In the tissues from the BAMBI<b>⁻</b>^/<b>−</b> mice, and using either primer pair, the levels of mRNA for BAMBI were indistinguishable from the RT minus or blank controls. Results are means ± SEM from triplicate determinations performed in two sets of mice. <b>B.</b> Immunofluorescence staining for BAMBI performed on different organs from BAMBI^+/+ and BAMBI<b>⁻</b>^/<b>−</b> mice. Organs are by rows: A: lung; B: heart; C: liver; D: kidney (g indicates glomeruli); E: femoral artery and vein; F: veins with accompagnying lymphatic vessel (LYVE-1 staining). Magnification 400x for A, B, C and F. 200x for D and E. BAMBI staining in red (first column A–F BAMBI^+/+ mouse tissues), second column endothelial markers in green (MECA32, BS 4 Isolectin ) and the merge in the third column in yellow. BAMBI co-localizes with endothelial markers in all tissues, but is negative in tissues from BAMBI<b>⁻</b>^/<b>−</b> mice (Inserts first column row A–D). Lymphatic endothelium stains with Lyve-1 antibody, but not with BAMBI antibody or MECA 32 (bottom row).</p

Kidney weights per body weight and glomerular and endothelial areas in the unilateral nephrectomy specimens (day 0) and in the remaining kidneys after 14 days of compensatory hypertrophy (day 14).

<p><b>A.</b> Kidney weights per body weight are comparable between BAMBI^+/+ and BAMBI<b>⁻</b>^/<b>−</b> mice and increase to a comparable degree during the 14 days of compensatory hypertrophy <b>B</b> and <b>C</b>. Glomerular areas <b>B.</b> and endothelial (isolectin B4 positive) areas <b>C</b>. are larger in BAMBI<b>⁻</b>^/<b>−</b> than in BAMBI^+/+ mice in the unilateral nephrectomy specimens and the differences become even bigger after 14 days of compensatory renal hypertrophy. Data are mean ± SEM, <i>n</i> = 5 mice per group.</p