Cerebral Cavernous Malformation-1 Protein Controls DLL4-Notch3 Signaling Between the Endothelium and Pericytes

Abstract

Background and Purpose— Cerebral cavernous malformation (CCM) is a neurovascular dysplasia characterized by conglomerates of enlarged endothelial channels in the central nervous system, which are almost devoid of pericytes or smooth muscle cells. This disease is caused by loss-of-function mutations in CCM1 , CCM2 , or CCM3 genes in endothelial cells, making blood vessels highly susceptible to angiogenic stimuli. CCM1 - and CCM3 -silenced endothelial cells have a reduced expression of the Notch ligand Delta-like 4 (DLL4) resulting in impaired Notch signaling and irregular sprouting angiogenesis. This study aimed to address if DLL4, which is exclusively expressed on endothelial cells, may influence interactions of endothelial cells with pericytes, which express Notch3 as the predominant Notch receptor. Methods— Genetic manipulation of primary human endothelial cells and brain pericytes. Transgenic mouse models were also used. Results— Endothelial cell–specific ablation of Ccm1 and Ccm2 in different mouse models led to the formation of CCM-like lesions, which were poorly covered by periendothelial cells. CCM1 silencing in endothelial cells caused decreased Notch3 activity in cocultured pericytes. DLL4 proteins stimulated Notch3 receptors on human brain pericytes. Active Notch3 induced expression of PDGFRB2, N-Cadherin , HBEGF , TGFB1, NG2 , and S1P genes. Notch3 signaling in pericytes enhanced the adhesion strength of pericytes to endothelial cells, limited their migratory and invasive behavior, and enhanced their antiangiogenic function. Pericytes silenced for Notch3 expression were more motile and could not efficiently repress angiogenesis. Conclusions— The data suggest that Notch signaling in pericytes is important to maintain the quiescent vascular phenotype. Deregulated Notch signaling may, therefore, contribute to the pathogenesis of CCM.

Pericytes of the neurovascular unit: key functions and signaling pathways

“…CCM is characterized by raspberry-like lesions of microvessels that exhibit pericyte deficiency and enlarged endothelial cells with pinocytotic vesicles and poorly developed tight and adherens junctions, making the vessels prone to hemorrhaging 99,103 . This phenotype is present in sporadic and familial CCM including loss-of-function mutations in CCM1-3 104 .…”

“…This phenotype is present in sporadic and familial CCM including loss-of-function mutations in CCM1-3 104 . An analogous vascular phenotype develops in mice with endothelial cell-specific inducible ablation of Ccm1 ( iCcm1 ΔEC/EC ) 99,103 and Ccm2-3 99 . Disrupted TGFβ signaling at the BBB is reported in studies using iCcm1 ΔEC/EC mice, human brain tissue of CCM subjects, and cultured human brain vascular cells 99,103 and contributes to CCM by inducing structural instability of capillaries.…”

“…An analogous vascular phenotype develops in mice with endothelial cell-specific inducible ablation of Ccm1 ( iCcm1 ΔEC/EC ) 99,103 and Ccm2-3 99 . Disrupted TGFβ signaling at the BBB is reported in studies using iCcm1 ΔEC/EC mice, human brain tissue of CCM subjects, and cultured human brain vascular cells 99,103 and contributes to CCM by inducing structural instability of capillaries. Human CCM3 also binds to paxillin, a scaffolding protein, and CCM3 and paxillin co-localize in mouse cerebral pericytes 105 .…”

“…Several studies using transgenic models, human tissue, and/or cultured brain vascular cells report CCM is associated with disruptions in TGFβ and Notch signaling at the BBB 99,103 .…”

Exosomes in stroke pathogenesis and therapy

“…In addition to neurons, exosomes released by cortical neurons transferred miR-124 to astrocytes and increased the expression of the excitatory amino acid transporter GLT-1 in astrocytes, suggesting a role for neuronal exosomes in regulatIn addition, activation of the Notch signaling pathway between cerebral endothelial cells and pericytes is required for cerebral angiogenesis and BBB integrity (72). For example, Delta-like 4 (Dll4), a membrane-bound Notch ligand expressed by cerebral endothelial cells, stimulates Notch3 receptors on pericytes to keep the cerebral vascular structure quiescent (73). Exosomes released from human microvascular endothelial cells (HMECs) and human umbilical vein endothelial cells (HVECs) contain Dll4 proteins and have been shown to regulate development of angiogenesis (74,75).…”