BMP9 and BMP10 are critical for postnatal retinal vascular remodeling - PubMed (original) (raw)

BMP9 and BMP10 are critical for postnatal retinal vascular remodeling

Nicolas Ricard et al. Blood. 2012.

Abstract

ALK1 is a type I receptor of the TGF-β family that is involved in angiogenesis. Circulating BMP9 was identified as a specific ligand for ALK1 inducing vascular quiescence. In this work, we found that blocking BMP9 with a neutralizing antibody in newborn mice significantly increased retinal vascular density. Surprisingly, Bmp9-KO mice did not show any defect in retinal vascularization. However, injection of the extracellular domain of ALK1 impaired retinal vascularization in Bmp9-KO mice, implicating another ligand for ALK1. Interestingly, we detected a high level of circulating BMP10 in WT and Bmp9-KO pups. Further, we found that injection of a neutralizing anti-BMP10 antibody to Bmp9-KO pups reduced retinal vascular expansion and increased vascular density, whereas injection of this antibody to WT pups did not affect the retinal vasculature. These data suggested that BMP9 and BMP10 are important in postnatal vascular remodeling of the retina and that BMP10 can substitute for BMP9. In vitro stimulation of endothelial cells by BMP9 and BMP10 increased the expression of genes involved in the Notch signaling pathway (Jagged1, Dll4, Hey1, Hey2, Hes1) and decreased apelin expression, suggesting a possible cross-talk between these pathways and the BMP pathway.

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Figures

Figure 1

Anti-BMP9 treatment increases vascularization of the retina. (A-C) Visualization of blood vessels by isolectin B4 (iB4) staining of OF1 retinas at P6 after treatment with BMP9-blocking antibodies (5 mg/kg) or recombinant ALK1ecd (5 mg/kg) injected at P1 and P3. (D) Number of branching points at the capillary plexus and at the front of migration. (E) Bar graphs show the relative distance covered by the vascular plexus calculated as the ratio of the vascular radius over the retinal radius in percentage. (F-H) Pericyte coverage visualized by NG2 immunostaining of OF1 retinas at P6. Panels A through H show data from 2 litters containing 10 PBS treated pups, 9 anti-BMP9 treated pups and 5 ALK1ecd treated pups. Error bars show SEM. **P < .01 versus PBS group. (A-C) Scale bar = 200 μm.

Figure 2

_Bmp9_-KO mice have a normal vascularized retina. (A-B) Visualization of blood vessels by isolectin B4 (iB4) staining of WT and _Bmp9_-KO retinas at P6. (C) Bar graphs show the relative distance covered by the vascular plexus calculated as the ratio of the vascular radius over the retinal radius in percentage at P5 (n = 6), P6 (n = 7), P7 (n = 9), and P10 (n = 4) from 2 litters. (D) Number of branching points at the capillary plexus and at the front of migration of P6 pups (n = 3 in each group). (E-F) Pericyte coverage was visualized by NG2 immunostaining. Error bars show SEM. (A-B) Scale bar = 200 μm.

Figure 3

ALK1ecd treatment in _Bmp9_-KO mice increases vascularization of the retina. (A-B) Visualization of blood vessels by isolectin B4 (iB4) staining of retinas at P5 from _Bmp9_-KO mice treated or not with ALK1ecd (5 mg/kg) injected at P1 and P3. (C) Bar graphs show the relative distance covered by the vascular plexus calculated as the ratio of the vascular radius over the retinal radius in percentage. (D-E) Higher magnifications of the retinas show that ALK1ecd treatment in _Bmp9_-KO mice lead to larger and multilayered blood vessels (see also z scan in panels F and G). Panels A through G show data from 2 litters containing 8 PBS treated _Bmp9_-KO mice and 7 ALK1ecd treated _Bmp9_-KO mice. Error bars show SEM. (A-B) Scale bar = 200 μm. (D-E) Scale bar = 25 μm.

Figure 4

BMP10 expression in WT and _Bmp9_-KO mice plasma. (A-B) BMP10 levels were measured from pooled diluted plasma (1/20) taken from WT OF1 mice at the indicated developmental stages (A) and from WT (n = 9) and _Bmp9_-KO (n = 14) P5 pups (B) with a murine BMP10 ELISA. Error bars show SEM.

Figure 5

Anti-BMP10 treatment disrupts vascularization of the retina of _Bmp9_-KO mice. Visualization of blood vessels by isolectin B4 (iB4) staining of retinas at P5 from _Bmp9_-KO (A-B) or WT (C-D) mice treated with PBS or anti-BMP10 antibody (15 mg/kg) injected at P1 and P3. (E-F) Bar graphs show the relative distance covered by the vascular plexus calculated as the ratio of the vascular radius over the retinal radius in percentage in _Bmp9_-KO mice (E) and in WT mice (F). (G-H) Higher magnifications of the retinas show that anti-BMP10 treatment in _Bmp9_-KO mice lead to larger and multilayered blood vessels (see also z scan in panels I and J). Panels A, B, E, G, H, I, and J show data from 4 litters containing 12 PBS-treated _Bmp9_-KO pups and 7 anti-BMP10 treated pups. Panels C, D, and F show data from 2 litters containing 6 PBS treated WT pups and 6 anti-BMP10 treated pups. Error bars show SEM. **P < .01 versus PBS group. (A-D) Scale bar = 200 μm. (G-H) Scale bar = 20 μm.

Figure 6

BMP9 and BMP10 regulated genes in endothelial cells. mRNA fold changes in Dll4, Jag1, Hey1, Hey 2, Hes1, and apelin mRNA levels in hPAEcs stimulated with 0.5 ng/mL BMP9 or BMP10 at different time points (1, 2, 4, and 6 hours). Expression of HPRT was used as the normalization control. Data pooled from 3 individual experiments, each containing 2 replicates. Error bars show SEM.

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BMP9 and BMP10 are critical for postnatal retinal vascular remodeling - PubMed (original) (raw)