Deletion of Rbpj from postnatal endothelium leads to abnormal arteriovenous shunting in mice

Abstract

Arteriovenous malformations (AVMs) are tortuous vessels characterized by arteriovenous (AV) shunts, which displace capillaries and shunt blood directly from artery to vein. Notch signaling regulates embryonic AV specification by promoting arterial, as opposed to venous, endothelial cell (EC) fate. To understand the essential role of endothelial Notch signaling in postnatal AV organization, we used inducible Cre-loxP recombination to delete Rbpj, a mediator of canonical Notch signaling, from postnatal ECs in mice. Deletion of endothelial Rbpj from birth resulted in features of AVMs by P14, including abnormal AV shunting and tortuous vessels in the brain, intestine and heart. We further analyzed brain AVMs, as they pose particular health risks. Consistent with AVM pathology, we found cerebral hemorrhage, hypoxia and necrosis, and neurological deficits. AV shunts originated from capillaries (and possibly venules), with the earliest detectable morphological abnormalities in AV connections by P8. Prior to AV shunt formation, alterations in EC gene expression were detected, including decreased Efnb2 and increased Pai1, which encodes a downstream effector of TGFβ signaling. After AV shunts had formed, whole-mount immunostaining showed decreased Efnb2 and increased Ephb4 expression within AV shunts, suggesting that ECs were reprogrammed from arterial to venous identity. Deletion of Rbpj from adult ECs led to tortuosities in gastrointestinal, uterine and skin vascular beds, but had mild effects in the brain. Our results demonstrate a temporal requirement for Rbpj in postnatal ECs to maintain proper artery, capillary and vein organization and to prevent abnormal AV shunting and AVM pathogenesis.

Keywords: Arteriovenous; Cerebrovascular; Endothelial cell; Mouse; Notch; Rbpj.

Fig. 1.

Endothelial deletion of Rbpj from birth results in abnormal AV shunts in P14 brains. (A,B) 2PEFM images of cerebrovascular casts. (A) Capillaries (dashed outline) lie between arteries (a) and veins (v) in controls. (B) In mutants, enlarged AV connections (dashed outline) directly connect arteries to veins. (C-F) Microspheres lodged in control brain capillaries (C) and very few circulated to the lungs (E). Some microspheres were retained in Rbpj^iΔEC brain (D), but most bypassed the brain and lodged in lung capillaries (F). Insets show merged fluorescence and brightfield (bf). (G) The diameter of AV connections was increased in Rbpj^iΔEC brains, as compared with controls. ***P=0.0001. N=6 controls (39 AV connections), N=6 mutants (40 AV connections). (H) Quantification of microsphere (FluoSphere) fluorescence, represented as percentage of lung/(lung+brain) fluorescence. **P=0.0066. N=5 controls, N=5 mutants. (I,J) Brightfield images of 1 mm sagittal brain slices show evidence of hemorrhage in mutants (J) as compared with control (I). N=3 controls, N=3 mutants. P-values were according to Student's t-test. nc, neocortex; dg, dentate gyrus. Scale bars: 100 μm in A,B,I,J; 5 mm in C-F.

Fig. 2.

Endothelial deletion of Rbpj from birth results in large, irregular vessels and increased vascular density in P14 brains. (A,B) 3D reconstructions derived from 2PEFM for mGFP for control (A) and Rbpj^iΔEC (B) forebrain. Bracket indicates vessel entanglement in the mutant. (C,D) 2PEFM 3D reconstructions from control (C) and mutant (D) cerebellum. Asterisks highlight avascular regions; arrow indicates enlarged vessel. N=3 controls, N=3 mutants. (E-H) Sagittal sections through immature forebrain (E,F) and cerebellum (G,H) show increased vascular density in Rbpj^iΔEC brains (F,H) compared with controls (E,G). Insets show CreER^T2-negative cells that expressed mTomato. (I) Area of mGFP-positive endothelium was increased in Rbpj^iΔEC brains. *P<0.0421, according to Student's t-test. N=5 controls, N=5 mutants. (J) Schematics indicating regions shown in A-D in sagittal brain slices. Scale bars: 200 μm.

Fig. 3.

Endothelial deletion of Rbpj from birth results in gross and histopathological abnormalities in P14 brains. (A) Dorsal view of brain (left) indicating region shown in B,C; the dashed line indicates the plane of section in D-I. The sagittal view (right) indicates regions of cerebellum shown in D-I (at different magnifications). (B,C) Whole-mount brain in dorsal view. The cerebellum (outlined) is abnormal in the mutant (C; 40/40 mice), as compared with the control (B, 0/40 mice). (D-G) H&E staining indicates area of necrosis (asterisk in E), disrupted Purkinje cells (arrows in G) and reduced molecular layer (E,G) in Rbpj^iΔEC cerebellum. F,G are magnified regions from D,E. N=8 controls, N=6 mutants. (H,I) Immunostaining against pimonidazole HCl (anti-Hypoxyprobe-1) indicates hypoxic cells in Rbpj^iΔEC cerebellum. Insets show perfused vasculature highlighted by biotinylated tomato lectin. N=3 controls, N=3 mutants. ml, molecular layer; gcl, granule cell layer. Scale bars: 2 mm in B,C; 200 μm in D,E,H,I; 50 μm in F,G.

Fig. 4.

Endothelial deletion of Rbpj from birth results in vascular abnormalities resembling AVMs in the intestine and heart at P14. (A,B) Live imaging of intestine. Mesentery arteries (red arrowheads) and veins (blue arrows) appear enlarged in the mutant (B) compared with the control (A). N=3 controls, N=4 mutants. (C-F) H&E staining of intestine show a complex network of enlarged, tortuous vessels in the mutant mesentery (C,D; red arrowheads, arteries; blue arrows, veins) and submucosa (E,F; yellow arrows, capillaries). (G,H) Ventral view of heart. Vessels on the ventricle surface appear enlarged in Rbpj^iΔEC mice (H). N=12 controls, N=8 mutants. (I,J) H&E staining of ventricular myocardium shows enlarged vessels (arrows) in the mutant (J). (K) Quantification of vessel size in intestine mesentery, submucosa and heart. The inner (lumenal) perimeter of vessels was measured by tissue section. Mesentery: **P=0.0061; N=5 controls (78 vessels), N=5 mutants (77 vessels). Submucosa: *P=0.0301; N=5 controls (112 vessels), N=5 mutants (92 vessels). Myocardium: *P=0.0305; N=5 controls (97 vessels), N=5 mutants (94 vessels). P-values were according to Student's t-test. Scale bars: 5 mm in A,B; 100 μm in C,D; 50 μm in E,F; 2 mm in G,H; 200 μm in I,J.

Fig. 5.

Endothelial deletion of Rbpj from birth leads to illness and death in P14 mice. (A) Kaplan–Meier survival curve indicating 50% probability of survival for P14 Rbpj^iΔEC mice. ***P<0.0001. N=24 controls, N=43 mutants. (B) Overall body size was compromised in Rbpj^iΔEC mice. (C) Total body weight was reduced in P14 Rbpj^iΔEC mice, but not in P7 Rbpj^iΔEC mice. P7: P=0.217 (not significant); N=42 controls, N=29 mutants. P14: ***P<0.0001; N=48 controls, N=49 mutants. P-values were according to Student's t-test.

Fig. 6.

Endothelial deletion of Rbpj from 6 weeks leads to mild brain abnormalities and to vascular abnormalities resembling AVMs in gastrointestinal tract, uterus and skin at 12 weeks. TAM administered at 6 weeks; tissue harvested at 12 weeks. (A,B) Whole brain, dorsal view (ant, anterior; post, posterior). Gross abnormalities were not observed in mutant brain (B) as compared with control (A). N=4 controls, N=5 mutants. (C,D) Whole-mount mGFP fluorescence, produced by mT/mG. Capillaries (dashed outline) lie at the artery-to-vein (a-v) interface in control (C) and mutant (D) brains. (E-H) H&E staining shows scattered dilated blood vessels in mutant cerebellum (F, arrows) and hypoxic-ischemic neurons in mutant striatum (H, arrows). N=5 controls, N=6 mutants. (I,J) An entanglement of vessels (outlined) connects the IVC to the gastrointestinal system in 0/4 controls and 6/6 mutants. (K-N) Vessels associated with non-gravid/non-post-partum uterus and skin were enlarged and tortuous in mutants (L,N). (O) Quantification of vessel diameter from brain, uterus and skin. Brain AV connections: P=0.4476; N=3 controls (30 AV connections), N=8 mutants (77 AV connections). Uterus: *P=0.0177; N=3 controls (27 vessels), N=4 mutants (40 vessels). Skin: **P=0.0026; N=4 controls (4 vessels), N=6 mutants (6 vessels). P-values were according to Student's t-test. Scale bars: 5 mm in A,B,M,N; 100 μm in C,D; 200 μm in E,F; 50 μm in G,H; 2 mm in I-L.

Fig. 7.

Following endothelial Rbpj deletion from birth, brain AV connections are abnormal by P8, lack αSMA and do not proliferate. (A-H) Whole-mount timecourse images showing brain AV connections (arrows). At P8, Rbpj^iΔEC AV connections (F) appear different than in controls (E). For each time point, N=3 controls, N=3 mutants, with at least seven fields imaged from each brain. (I-L) Whole-mount αSMA immunostaining. Arrows point to AV connections, which lacked αSMA staining in both control and mutant. For P7, N=4 controls, N=3 mutants; for P14, N=3 controls, N=3 mutants. (M-O) BrdU incorporation in ECs from P5-P7 was unchanged in mutant (N) versus control (M) brain (as quantified in O). Arrow indicates EC nucleus. P=0.9752, according to Student's t-test. N=4 controls (29 fields), N=5 mutants (33 fields). a, artery; v, vein. Scale bars: 100 μm in A-L; 25 μm in M,N.

Fig. 8.

Altered gene expression in brains following endothelial deletion of Rbpj from birth. (A) Quantitative RT-PCR analysis on purified P7 brain ECs. Hey1 (P=0.0538), Hey2 (**P=0.0029) and Efnb2 (**P=0.0063) decreased; Ephb4 (P=0.1795), Vegfa (P=0.3118), Smad4 (P=0.2686), Tgfb1 (P=0.0806) and Rasa1 (P=0.2372) did not change significantly; and Pai1 (*P=0.0198) increased in the mutant. For each gene, N=6 mutant and control pairs; except Pai1 and Rasa1, N=4 pairs. Quantitative PCRs were run in triplicate. P-values were according to Student's t-test. (B-G) Whole-mount β-gal staining on P14 brains. (B,C) Coup-TFII^fx-nlacZ was expressed in veins (v), venules and the venous portion of AV connections in control (B) and mutant (C) brains. Arrowheads indicate AV shunts. N=4 controls, N=6 mutants. (D) In controls, Efnb2^tau-lacZ was expressed by arterial (a) ECs, including the arterial portion of AV connections. (E) In mutants, Efnb2^tau-lacZ expression was retained to a certain level in arteries and arterioles but was absent from AV shunts (arrowheads). N=7 controls, N=8 mutants. (F) In controls, Ephb4^tau-lacZ was expressed by veins but not arteries. (G) In mutants, Ephb4^tau-lacZ was misexpressed in AV shunts and arterioles (arrowheads). N=10 controls, N=5 mutants. Scale bars: 100 μm.