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Accelerated reendothelialization, increased neovascularization and erythrocyte extravasation after arterial injury in BAMBI-/- mice.

Guillot N, Kollins D, Badimon JJ, Schlondorff D, Hutter R - PLoS ONE (2013)

Bottom Line: The exuberant intimal and medial neovessel formation with BAMBI(-/-) genotype was also associated with significant red blood cell extravasation.Vascular smooth muscle cells were decreased at 2 weeks in BAMBI(-/-) mice, but comparable to wild type at 4 weeks.This suggests important effects of BAMBI on arterial EC homeostasis that need to be further studied in a model of inflammatory atherosclerosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Mount Sinai School of Medicine, New York, New York, United States of America.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

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BAMBI deficiency accelerates reendothelialization and increased neo-intimal neovascularization and accumulation of erythrocytes.(A) 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) Reendothelialization was also determined by staining with von Willebrand factor (black arrows) magnification ×1000.(C) 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). (D) Quantification of the reendothelialization of the femoral arteries. (E) Microvascular density in the different layers of the femoral arteries 4 weeks after injury. (F) Erythrocytes accumulation in neointima 2 and 4 weeks after injury. Data are mean ± SEM, n = 5–9, *P<0.05 compared to respective wild type.
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pone-0058550-g002: BAMBI deficiency accelerates reendothelialization and increased neo-intimal neovascularization and accumulation of erythrocytes.(A) 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) Reendothelialization was also determined by staining with von Willebrand factor (black arrows) magnification ×1000.(C) 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). (D) Quantification of the reendothelialization of the femoral arteries. (E) Microvascular density in the different layers of the femoral arteries 4 weeks after injury. (F) Erythrocytes accumulation in neointima 2 and 4 weeks after injury. Data are mean ± SEM, n = 5–9, *P<0.05 compared to respective wild type.

Mentions: Histo-morphometric analysis of arteries at 2 and 4 weeks after endothelial denudation revealed increased neointima formation without a change in media or total vessel size in the BAMBI−/− compared to WT mice; resulting in a significantly greater intima/media ratio at 2 weeks (Figure 1A–C). Staining for BAMBI in WT mice confirmed its presence restricted to endothelial cells and also showed, that all regenerating endothelial cells at two weeks after denudation expressed BAMBI (Figure S2). Reendothelialization was significantly accelerated with BAMBI−/− genotype as measured by morphometric evaluation of isolectin staining showing 79% of the luminal circumference covered by endothelial cells at 2 weeks after injury in BAMBI−/− mice compared to 52% in WT mice (P<0.05) (Figure 2A and D). Comparable results were obtained with staining of endothelial cells by von Willebrand factor (Figure 2B). By morphometric quantification 71.4±6.7% of luminal circumference were covered at 2 weeks in BAMBI−/− mice (n = 6 ) versus 52±3% in wild type mice (n = 5; P<0.04). At 4 weeks, reendothelialization was almost complete in both groups, no longer showing a significant difference with 92% in BAMBI−/− and 84% in WT mice (Figure 2A, B and D, P = NS). Enhanced proliferation of endothelial cells contributed to the accelerated reendothelialization in the BAMBI−/− mice at 2 weeks as evaluated by KI-67 staining for proliferating luminal cells (7.7×10−3±1.6 KI-67 positive cells per micrometer of luminal surface in BAMBI+/+ mice versus 15.9×10−3±2.7 in BAMBI−/− mice; n = 4–6 per group; P<0.04). At 4 weeks after denudation the number of KI67 positive luminal cells was no longer significantly different between BAMBI+/+ and BAMBI−/− mice (not shown). The endothelial nature of the KI-67 positive luminal cells was confirmed by double staining with vWF (Figure 3). Of note, enhanced endothelial growth was not limited to macro-vascular endothelial cells repairing the luminal surface. We also observed neovascularization in the media and neointima with BAMBI−/− genotype, two compartments of the arterial wall where neovascularization is normally minimal or undetectable in this model as demonstrated by the absence of neovessels in the WT mice after endothelial denudation (Figure 2A and B and quantified in Fig. 2E). The contribution of proliferating endothelial cells to neo-vessel formation in the neo-intima at 4 weeks is illustrated by the presence of KI67/vWF double positive endothelial cells within the neointima of BAMBI−/− mice (Figure 3). These findings are consistent with an activated EC phenotype in BAMBI−/− mice, and would support the hypothesis of an inhibitory function of BAMBI on EC proliferation in response to TGFβ through ALK 1 and alternative signaling, as recently reported by us [9]. Thus the accelerated reendothelialization and marked neovascularization in BAMBI−/− mice could potentially be explained by the enhanced alternative pathway response to the TGFβ generated at the site of vascular injury [3], [16]–[18].


Accelerated reendothelialization, increased neovascularization and erythrocyte extravasation after arterial injury in BAMBI-/- mice.

Guillot N, Kollins D, Badimon JJ, Schlondorff D, Hutter R - PLoS ONE (2013)

BAMBI deficiency accelerates reendothelialization and increased neo-intimal neovascularization and accumulation of erythrocytes.(A) 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) Reendothelialization was also determined by staining with von Willebrand factor (black arrows) magnification ×1000.(C) 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). (D) Quantification of the reendothelialization of the femoral arteries. (E) Microvascular density in the different layers of the femoral arteries 4 weeks after injury. (F) Erythrocytes accumulation in neointima 2 and 4 weeks after injury. Data are mean ± SEM, n = 5–9, *P<0.05 compared to respective wild type.
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pone-0058550-g002: BAMBI deficiency accelerates reendothelialization and increased neo-intimal neovascularization and accumulation of erythrocytes.(A) 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) Reendothelialization was also determined by staining with von Willebrand factor (black arrows) magnification ×1000.(C) 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). (D) Quantification of the reendothelialization of the femoral arteries. (E) Microvascular density in the different layers of the femoral arteries 4 weeks after injury. (F) Erythrocytes accumulation in neointima 2 and 4 weeks after injury. Data are mean ± SEM, n = 5–9, *P<0.05 compared to respective wild type.
Mentions: Histo-morphometric analysis of arteries at 2 and 4 weeks after endothelial denudation revealed increased neointima formation without a change in media or total vessel size in the BAMBI−/− compared to WT mice; resulting in a significantly greater intima/media ratio at 2 weeks (Figure 1A–C). Staining for BAMBI in WT mice confirmed its presence restricted to endothelial cells and also showed, that all regenerating endothelial cells at two weeks after denudation expressed BAMBI (Figure S2). Reendothelialization was significantly accelerated with BAMBI−/− genotype as measured by morphometric evaluation of isolectin staining showing 79% of the luminal circumference covered by endothelial cells at 2 weeks after injury in BAMBI−/− mice compared to 52% in WT mice (P<0.05) (Figure 2A and D). Comparable results were obtained with staining of endothelial cells by von Willebrand factor (Figure 2B). By morphometric quantification 71.4±6.7% of luminal circumference were covered at 2 weeks in BAMBI−/− mice (n = 6 ) versus 52±3% in wild type mice (n = 5; P<0.04). At 4 weeks, reendothelialization was almost complete in both groups, no longer showing a significant difference with 92% in BAMBI−/− and 84% in WT mice (Figure 2A, B and D, P = NS). Enhanced proliferation of endothelial cells contributed to the accelerated reendothelialization in the BAMBI−/− mice at 2 weeks as evaluated by KI-67 staining for proliferating luminal cells (7.7×10−3±1.6 KI-67 positive cells per micrometer of luminal surface in BAMBI+/+ mice versus 15.9×10−3±2.7 in BAMBI−/− mice; n = 4–6 per group; P<0.04). At 4 weeks after denudation the number of KI67 positive luminal cells was no longer significantly different between BAMBI+/+ and BAMBI−/− mice (not shown). The endothelial nature of the KI-67 positive luminal cells was confirmed by double staining with vWF (Figure 3). Of note, enhanced endothelial growth was not limited to macro-vascular endothelial cells repairing the luminal surface. We also observed neovascularization in the media and neointima with BAMBI−/− genotype, two compartments of the arterial wall where neovascularization is normally minimal or undetectable in this model as demonstrated by the absence of neovessels in the WT mice after endothelial denudation (Figure 2A and B and quantified in Fig. 2E). The contribution of proliferating endothelial cells to neo-vessel formation in the neo-intima at 4 weeks is illustrated by the presence of KI67/vWF double positive endothelial cells within the neointima of BAMBI−/− mice (Figure 3). These findings are consistent with an activated EC phenotype in BAMBI−/− mice, and would support the hypothesis of an inhibitory function of BAMBI on EC proliferation in response to TGFβ through ALK 1 and alternative signaling, as recently reported by us [9]. Thus the accelerated reendothelialization and marked neovascularization in BAMBI−/− mice could potentially be explained by the enhanced alternative pathway response to the TGFβ generated at the site of vascular injury [3], [16]–[18].

Bottom Line: The exuberant intimal and medial neovessel formation with BAMBI(-/-) genotype was also associated with significant red blood cell extravasation.Vascular smooth muscle cells were decreased at 2 weeks in BAMBI(-/-) mice, but comparable to wild type at 4 weeks.This suggests important effects of BAMBI on arterial EC homeostasis that need to be further studied in a model of inflammatory atherosclerosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Mount Sinai School of Medicine, New York, New York, United States of America.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

Show MeSH
Related in: MedlinePlus