Unlabelled

Liver vasculature is crucial for adequate hepatic functions. Global deletion of Notch signaling in mice results in liver vascular pathologies. However, whether Notch in endothelium is essential for hepatic vascular structure and function remains unknown. To uncover the function of endothelial Notch in the liver, we deleted Rbpj, a transcription factor mediating all canonical Notch signaling, or Notch1 from the endothelium of postnatal mice. We investigated the hepatic vascular defects in these mutants. The liver was severely affected within 2 weeks of endothelial deletion of Rbpj from birth. Two-week old mutant mice had enlarged vessels on the liver surface, abnormal vascular architecture, and dilated sinusoids. Vascular casting and fluorosphere passage experiments indicated the presence of porto-systemic shunts. These mutant mice presented with severely necrotic liver parenchyma and significantly larger hypoxic areas, likely resulting from vascular shunts. We also found elevated levels of VEGF receptor 3 together with reduced levels of ephrin-B2, suggesting a possible contribution of these factors to the generation of hepatic vascular abnormalities. Deletion of Rbpj from the adult endothelium also led to dilated sinusoids, vascular shunts, and necrosis, albeit milder than that observed in mice with deletion from birth. Similar to deletion of Rbpj, loss of endothelial Notch1 from birth led to similar hepatic vascular malformations within 2 weeks.

Conclusions

Endothelial Notch signaling is essential for the development and maintenance of proper hepatic vascular architecture and function. These findings may elucidate the molecular pathogenesis of hepatic vascular malformation and the safety of therapeutics inhibiting Notch. (Hepatology 2016;64:1302-1316).

Upregulation of Notch signaling is associated with brain arteriovenous malformation (bAVM), a disease that lacks pharmacological treatments. Tetracycline (tet)-regulatable endothelial expression of constitutively active Notch4 (Notch4*tetEC) from birth induced bAVMs in 100% of mice by P16. To test whether targeting downstream signaling, while sustaining the causal Notch4*tetEC expression, induces AVM normalization, we deleted Rbpj, a mediator of Notch signaling, in endothelium from P16, by combining tet-repressible Notch4*tetEC with tamoxifen-inducible Rbpj deletion. Established pathologies, including AV connection diameter, AV shunting, vessel tortuosity, intracerebral hemorrhage, tissue hypoxia, life expectancy, and arterial marker expression were improved, compared with Notch4*tetEC mice without Rbpj deletion. Similarly, Rbpj deletion from P21 induced advanced bAVM regression. After complete AVM normalization induced by repression of Notch4*tetEC, virtually no bAVM relapsed, despite Notch4*tetEC re-expression in adults. Thus, inhibition of endothelial Rbpj halted Notch4*tetEC bAVM progression, normalized bAVM abnormalities, and restored microcirculation, providing proof of concept for targeting a downstream mediator to treat AVM pathologies despite a sustained causal molecular lesion.

Arteriovenous (AV) malformation (AVM) is a devastating condition characterized by focal lesions of enlarged, tangled vessels that shunt blood from arteries directly to veins. AVMs can form anywhere in the body and can cause debilitating ischemia and life-threatening hemorrhagic stroke. The mechanisms that underlie AVM formation remain poorly understood. Here, we examined the cellular and hemodynamic changes at the earliest stages of brain AVM formation by time-lapse two-photon imaging through cranial windows of mice expressing constitutively active Notch4 (Notch4*). AVMs arose from enlargement of preexisting microvessels with capillary diameter and blood flow and no smooth muscle cell coverage. AV shunting began promptly after Notch4* expression in endothelial cells (ECs), accompanied by increased individual EC areas, rather than increased EC number or proliferation. Alterations in Notch signaling in ECs of all vessels, but not arteries alone, affected AVM formation, suggesting that Notch functions in the microvasculature and/or veins to induce AVM. Increased Notch signaling interfered with the normal biological control of hemodynamics, permitting a positive feedback loop of increasing blood flow and vessel diameter and driving focal AVM growth from AV connections with higher blood velocity at the expense of adjacent AV connections with lower velocity. Endothelial expression of constitutively active Notch1 also led to brain AVMs in mice. Our data shed light on cellular and hemodynamic mechanisms underlying AVM pathogenesis elicited by increased Notch signaling in the endothelium.