Constitutively active endothelial Notch4 causes lung arteriovenous shunts in mice

Laboratory for Accelerated Vascular Research, Division of Vascular Surgery, San Francisco, CA 94143-0507, USA.
AJP Lung Cellular and Molecular Physiology (Impact Factor: 4.08). 11/2009; 298(2):L169-77. DOI: 10.1152/ajplung.00188.2009
Source: PubMed


Lung arteriovenous (AV) shunts or malformations cause significant morbidity and mortality in several distinct clinical syndromes. For most patients with lung AV shunts, there is still no optimal treatment. The underlying molecular and cellular etiology for lung AV shunts remains elusive, and currently described animal models have insufficiently addressed this problem. Using a tetracycline-repressible system, we expressed constitutively active Notch4 (Notch4*) specifically in the endothelium of adult mice. More than 90% of mice developed lung hemorrhages and respiratory insufficiency and died by 6-7 wk after gene expression began. Vascular casting and fluorescent microsphere analysis showed evidence of lung AV shunts in affected mice. Cessation of Notch4* expression reversed these pathophysiological effects. Assessment of the vascular morphology revealed enlarged, tortuous vessels in the lungs that resembled arteriovenous malformations. By using whole lung organ culture, we demonstrated the effects of constitutively active Notch4 on the lung vasculature to be a primary lung phenomenon. Together, our results indicate the importance of Notch signaling in maintaining the lung vasculature and offer a new, reliable model with which to study the pathobiology of lung arteriovenous shunts and malformations.

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    • "Adult endothelial Rbpj deletion leads to enlarged, tortuous vessels in mature vascular beds associated with the gastrointestinal tract, uterus and skin. Similarly, activation of the Notch4* mutation from birth results in brain AV shunts (Murphy et al., 2008), whereas Notch4* activation post-weaning leads to AV shunts in the gastrointestinal tract, uterus, skin and lung (Carlson et al., 2005; Miniati et al., 2010) but not in the brain (Carlson et al., 2005). Our results suggest that Rbpj is required in postnatal endothelium during two distinct temporal windows, but selectively in organs such as the brain. "
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    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.
    Full-text · Article · Sep 2014 · Development
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    • "Mice were anesthetized with ketamine (0.1 mg/g) and xylazine (0.01 mg/g). After we confirmed that mice were well sedated, they were given 50 µl of heparin (50 mg/ml) subcutaneously.[15] The trachea was surgically exposed and cannulated with a 20G Terumo Surflo Catheter (Terumo Corporation, Tokyo, Japan). "
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    ABSTRACT: Liver dysfunction and cirrhosis affect vasculature in several organ systems and cause impairment of organ functions, thereby increasing morbidity and mortality. Establishment of a mouse model of hepatopulmonary syndrome (HPS) would provide greater insights into the genetic basis of the disease. Our objectives were to establish a mouse model of lung injury after common bile duct ligation (CBDL) and to investigate pulmonary pathogenesis for application in future therapeutic approaches. Eight-week-old Balb/c mice were subjected to CBDL. Immunohistochemical analyses and real-time quantitative reverse transcriptional polymerase chain reaction were performed on pulmonary tissues. The presence of HPS markers was detected by western blot and microarray analyses. We observed extensive proliferation of CD31-positive pulmonary vascular endothelial cells at 2 weeks after CBDL and identified 10 upregulated and 9 down-regulated proteins that were associated with angiogenesis. TNF-α and MMP-9 were highly expressed at 3 weeks after CBDL and were less expressed in the lungs of the control group. We constructed a mouse lung injury model by using CBDL. Contrary to our expectation, lung pathology in our mouse model exhibited differences from that of rat models, and the mechanisms responsible for these differences are unknown. This phenomenon may be explained by contrasting processes related to TNF induction of angiogenic signaling pathways in the inflammatory phase. Thus, we suggest that our mouse model can be applied to pulmonary pathological analyses in the inflammatory phase, i.e., to systemic inflammatory response syndrome, acute lung injury, and multiple organ dysfunction syndrome.
    Full-text · Article · Apr 2014 · PLoS ONE
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    • "In cultured cells, NOTCH4 is reported to signal in response to ligand [38] [39] but little else is known about its mechanism of signal transduction. Studies in mice show that endothelial specific expression of constitutively active NOTCH4 (encoded by the int3 allele) causes dramatic vascular changes including lack of small branched vessels, loss of vessel integrity [40] [41] and arterial shunts [42] [43]. Induction of int3 expression in mature vessel endothelium (postnatally), causes expression of the Notch target gene EphrinB2 and increased smooth muscle layers, resulting in arterialisation of venous vessels [40]. "
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    ABSTRACT: Notch4 is a divergent member of the Notch family of receptors that is primarily expressed in the vasculature. Its expression implies an important role for Notch4 in the vasculature; however, mice homozygous for the Notch4(d1) knockout allele are viable. Since little is known about the role of Notch4 in the vasculature and how it functions, we further investigated Notch4 in mice and in cultured cells. We and found that the Notch4(d1) allele is not null as it expresses a truncated transcript encoding most of the NOTCH4 extracellular domain. In cultured cells, NOTCH4 did not signal in response to ligand. Moreover, NOTCH4 inhibited signalling from the NOTCH1 receptor. This is the first report of cis-inhibition of signalling by another Notch receptor. The NOTCH4 extracellular domain also inhibits NOTCH1 signalling when expressed in cis, raising the possibility that reported Notch4 phenotypes may not be due to loss of NOTCH4 function. To better address the role of NOTCH4 in vivo, we generated a Notch4 null mouse in which the entire coding region was deleted. Notch4 null mice exhibited slightly delayed vessel growth in the retina, consistent with our novel finding that NOTCH4 protein is expressed in the newly formed vasculature. These findings indicate a role of NOTCH4 in fine-tuning the forming vascular plexus.
    Preview · Article · Mar 2014 · Biochimica et Biophysica Acta
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