A mouse model of hereditary hemorrhagic telangiectasia generated by transmammary-delivered immunoblocking of BMP9 and BMP10

Abstract

Hereditary hemorrhagic telangiectasia (HHT) is a potentially life-threatening genetic vascular disorder caused by loss-of-function mutations in the genes encoding activin receptor-like kinase 1 (ALK1), endoglin, Smad4, and bone morphogenetic protein 9 (BMP9). Injections of mouse neonates with BMP9/10 blocking antibodies lead to HHT-like vascular defects in the postnatal retinal angiogenesis model. Mothers and their newborns share the same immunity through the transfer of maternal antibodies during lactation. Here, we investigated whether the transmammary delivery route could improve the ease and consistency of administering anti-BMP9/10 antibodies in the postnatal retinal angiogenesis model. We found that anti-BMP9/10 antibodies, when intraperitoneally injected into lactating dams, are efficiently transferred into the blood circulation of lactationally-exposed neonatal pups. Strikingly, pups receiving anti-BMP9/10 antibodies via lactation displayed consistent and robust vascular pathology in the retina, which included hypervascularization and defects in arteriovenous specification, as well as the presence of multiple and massive arteriovenous malformations. Furthermore, RNA-Seq analyses of neonatal retinas identified an increase in the key pro-angiogenic factor, angiopoietin-2, as the most significant change in gene expression triggered by the transmammary delivery of anti-BMP9/10 antibodies. Transmammary-delivered BMP9/10 immunoblocking in the mouse neonatal retina is therefore a practical, noninvasive, reliable, and robust model of HHT vascular pathology.

Figure 1. Transmammary transfer of BMP9 and BMP10 blocking Abs into the circulation of mouse neonates.

(A–D) ELISAs were performed to measure IgG2a (A,C) and anti-BMP9 Ab (B,D) levels in the serum of P6 neonates treated at P3 with vehicle (PBS), isotype control IgGs (IgG2a/2b), or anti-BMP9/10 Abs. Neonates were treated during lactation either from dams injected i.p. with (A,B), or by direct i.p. injections of (C,D), the different Abs or vehicle. Data represent mean ± s.e.m. (n = 5–7 pups per group from 2 dams); ****P < 0.0001, ANOVA.

Figure 2. Transmammary transfer of BMP9 and BMP10 blocking Abs leads to abnormal hypervascularization in neonatal retinas.

(A–C”) Representative images of fluorescent isolectin B4-stained retinas either from P6 neonates fed for 3 days by dams injected on P3 with PBS (A), control IgG2a/b Abs (B), or BMP9/10 blocking Abs (C–C”). (C’,C”) are higher magnification images of the relevant boxed areas in C. a, artery; v, vein; scale bars, 500 μm. (D–L) Higher magnification showing retinal vasculature fields between an artery and a vein (Plexus, D–F), or at the front of an artery (G–I) or a vein (J–L) from neonates treated as in (A–C). Scale bars, 100 μm. (M–O) Scatter plots showing the vascular density on retinal ‘petals’ at the plexus (M), artery front (N), or vein front (O). Data represent mean ± s.e.m. (n = 6 pups per group from 2 dams); ****P < 0.0001, ANOVA and Kruskal-Wallis test. Arrows indicate AVMs, defined as direct shunts between an artery and a vein.

Figure 3. Transmammary transfer of BMP9 and BMP10 blocking Abs induces AVMs in neonatal retinas.

(A,B) Representative images of blue latex-perfused retinal vasculature of P6 neonates fed for 3 days by dams injected on P3 with control IgG2a/b Abs (A) or BMP9/10 blocking Abs (B). Arrows in B indicate AVMs. Scale bars, 500 μm. (C) Scheme depicting the method employed for the quantification of the number of latex dye-positive vessels. (D) Histogram showing the total number of vascular crosses. (E) Histogram showing the number of vascular crosses per concentric circle on circles 1 to 3. Data represent mean ± s.e.m. (n = 5–7 pups per group from 2 dams); ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05; ANOVA and Kruskal-Wallis test (D,E).

Figure 4. Gene expression changes in BMP9/10-immunoblocked retinas and ALK1-Fc-treated HUVECs.

(A) RNA-Seq heat map displaying differently expressed genes in mouse whole retinas following transmammary transfer of anti-BMP9/10 or control IgG2a/2b Abs (n = 6 pups per group from 1 dam). (B) HUVECs were treated or not (Ctrl) with ALK1-Fc (1 μg/mL, 24 h). Cell extracts were then analyzed by WB using Abs directed against the indicated proteins. (C) Densitometric analyses and quantification of phospho-Smad1/5/8, ID1, and ANG2 relative levels in three independent experiments as in (B). (D) ECs isolated from retinas of pups fed for 3 days by dams injected on P3 with control IgG2a/b Abs (Ctrl) or BMP9/10 blocking Abs (anti-BMP9/10) were analyzed for Id1 and Angpt2 mRNA levels by RT-qPCR. The results are expressed as relative levels of the control condition (n = 3 determinations, n = 6 pups per group from 1 dam). Data in (C,D) are mean ± s.e.m.; ***P < 0.001, **P < 0.01, *P < 0.05, unpaired Student’s t-test.