Patrick A. Murphy

Endothelial Notch4 signaling induces hallmarks of brain arteriovenous malformations in mice

Brain arteriovenous malformations (BAVMs) can cause devastating stroke in young people and contribute to half of all hemorrhagic stroke in children. Unfortunately, the pathogenesis of BAVMs is unknown. In this article we show that activation of Notch signaling in the endothelium during brain development causes BAVM in mice. We turned on constitutively active Notch4 (int3) expression in endothelial cells from birth by using the tetracycline-regulatable system. All mutants developed hallmarks of BAVMs, including cerebral arteriovenous shunting and vessel enlargement, by 3 weeks of age and died by 5 weeks of age. Twenty-five percent of the mutants showed signs of neurological dysfunction, including ataxia and seizure. Affected mice exhibited hemorrhage and neuronal cell death within the cerebral cortex and cerebellum. Strikingly, int3 repression resolved ataxia and reversed the disease progression, demonstrating that int3 is not only sufficient to induce, but also required to sustain the disease. We show that int3 expression results in widespread enlargement of the microvasculature, which coincided with a reduction in capillary density, linking vessel enlargement to Notchs known function of inhibiting vessel sprouting. Our data suggest that the Notch pathway is a molecular regulator of BAVM pathogenesis in mice, and offer hope that their regression might be possible by targeting the causal molecular lesion.

Notch4 normalization reduces blood vessel size in arteriovenous malformations.

Normalization of Notch expression restores enlarged blood vessels to microvessels through EphB4-mediated reprogramming of arterial endothelial cells. Reducing Inflation Arteriovenous malformations (AVMs) are a class of vascular abnormalities in which arteries connect directly with veins, thus bypassing the capillary beds and diverting blood flow away from tissues. In these vascular diseases, blood vessels, particularly the veins, become inflated in size and eventually rupture, resulting in hemorrhage and ischemia. AVMs, which can be found in any tissue, are particularly problematic in the brain, where surgical options are limited, and they often result in stroke or death. In a tour-de-force study, Murphy et al. now show that dialing down Notch4 receptor signaling in established AVMs in mouse brain reduces the size of enlarged blood vessels, resulting in restoration of blood flow to capillary beds and the reversal of hypoxia in mouse brain tissue. The Notch receptor is a master regulator of arteriovenous development and is up-regulated in AVMs in human brain. Overexpression of a constitutively active form of Notch4 in endothelial cells lining blood vessel walls is sufficient to induce AVMs in mice. In their new work, Murphy et al. first wanted to establish whether correction of Notch4 signaling could induce the regression of AVMs. Using their mouse brain AVM model, they obtained four-dimensional imaging data of the mouse brain vasculature viewed through a window cut into the cranium with two-photon fluorescence microscopy. When Notch4 signaling was normalized, they found regression of enlarged AVMs, which became similar in size to capillaries. This shrinkage in size enabled blood flow to return to oxygen-deprived tissues in the mouse brain. Surprisingly, the authors discovered that AVM regression was not induced by loss of endothelial cells, thrombotic occlusion, or vessel rupture. Rather, it required reprogramming of arterial endothelial cells in the enlarged AVM vessels to a venous endothelial cell specification. This reprogramming was activated by a decrease in Notch4 receptor signaling, which prompted arterial endothelial cells to start expressing the venous marker EphB4. These findings suggest that strategies to manipulate Notch receptor signaling in blood vessel endothelial cells may help to shrink AVMs and may be a new approach to treating AVMs and other vascular diseases. Abnormally enlarged blood vessels underlie many life-threatening disorders including arteriovenous (AV) malformations (AVMs). The core defect in AVMs is high-flow AV shunts, which connect arteries directly to veins, “stealing” blood from capillaries. Here, we studied mouse brain AV shunts caused by up-regulation of Notch signaling in endothelial cells (ECs) through transgenic expression of constitutively active Notch4 (Notch4*). Using four-dimensional two-photon imaging through a cranial window, we found that normalizing Notch signaling by repressing Notch4* expression converted large-caliber, high-flow AV shunts to capillary-like vessels. The structural regression of the high-flow AV shunts returned blood to capillaries, thus reversing tissue hypoxia. This regression was initiated by vessel narrowing without the loss of ECs and required restoration of EphB4 receptor expression by venous ECs. Normalization of Notch signaling resulting in regression of high-flow AV shunts, and a return to normal blood flow suggests that targeting the Notch pathway may be useful therapeutically for treating diseases such as AVMs.

Endothelial Notch signaling is upregulated in human brain arteriovenous malformations and a mouse model of the disease.

Brain arteriovenous malformations (BAVMs) can cause lethal hemorrhagic stroke and have no effective treatment. The cellular and molecular basis for this disease is largely unknown. We have previously shown that expression of constitutively-active Notch4 receptor in the endothelium elicits and maintains the hallmarks of BAVM in mice, thus establishing a mouse model of the disease. Our work suggested that Notch pathway could be a critical molecular mediator of BAVM pathogenesis. Here, we investigated the hypothesis that upregulated Notch activation contributes to the pathogenesis of human BAVM. We examined the expression of the canonical Notch downstream target Hes1 in the endothelium of human BAVMs by immunofluorescence, and showed increased levels relative to either autopsy or surgical biopsy controls. We then analyzed receptor activity using an antibody to the activated form of the Notch1 receptor, and found increased levels of activity. These findings suggest that Notch activation may promote the development and even maintenance of BAVM. We also detected increases in Hes1 and activated Notch1 expression in our mouse model of BAVM induced by constitutively active Notch4, demonstrating molecular similarity between the mouse model and the human disease. Our work suggests that activation of Notch signaling is an important molecular candidate in BAVM pathogenesis and further validates that our animal model provides a platform to study the progression as well as the regression of the disease.

Constitutively active Notch4 receptor elicits brain arteriovenous malformations through enlargement of capillary-like vessels

Significance Brain arteriovenous malformations are focal lesions of enlarged, tangled vessels that shunt blood from arteries directly to veins. They can cause ischemia, hemorrhage, disability, and death, particularly in young people, accounting for 50% of childhood stroke. The molecular etiology of the disease remains poorly understood, hindering the development of therapeutic treatments. Here, we report that, in an animal model, the lesion arises from the enlargement of capillary-like vessels. Notch signaling in the endothelium of microvasculature and veins is critical for the disease initiation by increasing cell areas but not proliferation. Blood flow mediates disease progression by a positive feedback of increasing flow and vessel diameter. Our data shed light on the mechanism underlying the pathogenesis of this devastating disease. 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.

Alternative Splicing of Endothelial Fibronectin Is Induced by Disturbed Hemodynamics and Protects Against Hemorrhage of the Vessel Wall

Objective— Abnormally low-flow conditions, sensed by the arterial endothelium, promote aneurysm rupture. Fibronectin (FN) is among the most abundant extracellular matrix proteins and is strongly upregulated in human aneurysms, suggesting a possible role in disease progression. Altered FN splicing can result in the inclusion of EIIIA and EIIIB exons, generally not expressed in adult tissues. We sought to explore the regulation of FN and its splicing and their possible roles in the vascular response to disturbed flow. Approach and Results— We induced low and reversing flow in mice by partial carotid ligation and assayed FN splicing in an endothelium-enriched intimal preparation. Inclusion of EIIIA and EIIIB was increased as early as 48 hours, with negligible increases in total FN expression. To test the function of EIIIA and EIIIB inclusion, we induced disturbed flow in EIIIAB −/− mice unable to include these exons and found that they developed focal lesions with hemorrhage and hypertrophy of the vessel wall. Acute deletion of floxed FN caused similar defects in response to disturbed flow, consistent with a requirement for the upregulation of the spliced isoforms, rather than a developmental defect. Recruited macrophages promote FN splicing because their depletion by clodronate liposomes blocked the increase in endothelial EIIIA and EIIIB inclusion in the carotid model. Conclusions— These results uncover a protective mechanism in the inflamed intima that develops under disturbed flow, by showing that splicing of FN mRNA in the endothelium, induced by macrophages, inhibits hemorrhage of the vessel wall.

Tumor angiogenesis in the absence of fibronectin or its cognate integrin receptors.

Binding of α5β1 and αvβ3/β5 integrin receptors on the endothelium to their fibronectin substrate in the extracellular matrix has been targeted as a possible means of blocking tumor angiogenesis and tumor growth. However, clinical trials of blocking antibodies and peptides have been disappointing despite promising preclinical results, leading to questions about the mechanism of the inhibitors and the reasons for their failure. Here, using tissue-specific and inducible genetics to delete the α5 and αv receptors in the endothelium or their fibronectin substrate, either in the endothelium or globally, we show that both are dispensable for tumor growth, in transplanted tumors as well as spontaneous and angiogenesis-dependent RIP-Tag-driven pancreatic adenocarcinomas. In the nearly complete absence of fibronectin, no differences in vascular density or the deposition of basement membrane laminins, ColIV, Nid1, Nid2, or the TGFβ binding matrix proteins, fibrillin-1 and -2, could be observed. Our results reveal that fibronectin and the endothelial fibronectin receptor subunits, α5 and αv, are dispensable for tumor angiogenesis, suggesting that the inhibition of angiogenesis induced by antibodies or small molecules may occur through a dominant negative effect, rather than a simple functional block.

Integrin-targeted cancer immunotherapy elicits protective adaptive immune responses

Certain RGD-binding integrins are required for cell adhesion, migration, and proliferation and are overexpressed in most tumors, making them attractive therapeutic targets. However, multiple integrin antagonist drug candidates have failed to show efficacy in cancer clinical trials. In this work, we instead exploit these integrins as a target for antibody Fc effector functions in the context of cancer immunotherapy. By combining administration of an engineered mouse serum albumin/IL-2 fusion with an Fc fusion to an integrin-binding peptide (2.5F-Fc), significant survival improvements are achieved in three syngeneic mouse tumor models, including complete responses with protective immunity. Functional integrin antagonism does not contribute significantly to efficacy; rather, this therapy recruits both an innate and adaptive immune response, as deficiencies in either arm result in reduced tumor control. Administration of this integrin-targeted immunotherapy together with an anti–PD-1 antibody further improves responses and predominantly results in cures. Overall, this well-tolerated therapy achieves tumor specificity by redirecting inflammation to a functional target fundamental to tumorigenic processes but expressed at significantly lower levels in healthy tissues, and it shows promise for translation.

Mouse Models of Cerebral Arteriovenous Malformation

Arteriovenous (AV) malformation (AVM) is a vascular anomaly capable of both hemorrhagic and ischemic insults, leading to seizures, headaches, stroke, and even death.1 BAVM prevalence is estimated at 0.05%,2 often occurring in young people between 20 and 40 years of age.3 BAVMs account for 50% of hemorrhagic stroke in children4 and 1% to 2% of all strokes in the population.5 Brain AVMs (BAVMs) can cause life-threatening intracerebral hemorrhage (Figure 1).6 Fifty percent of patients are first diagnosed on intracerebral hemorrhage,1 with 1% and 5% annual hemorrhage rate for previously unruptured and ruptured AVMs, respectively.7,8 After BAVM rupture, reported mortality rates range from to 15% to 29%,7 and long-term morbidity rates range from 16% to 56%.1,9 Thus, BAVM is defined by vascular features and accompanying neurological deficits.1 Figure 1. Features of human brain arteriovenous malformation (AVM). A , An AVM is visualized on the lateral temporal surface of a human brain. 5, 6 are landmarks placed by surgeon; 40 shows Brocas area; 48 shows Wernickes area. B , Left internal carotid artery (ICA) angiography (lateral view) reveals a left lateral temporal AVM with a large feeding artery and draining vein. C , Cartoon of this subtype (lateral view), indicating feeding arteries and draining veins. ATA indicates anterior temporal artery. Reprinted from Lawton6 with permission of the publisher. Copyright

Alternative RNA splicing in the endothelium mediated in part by Rbfox2 regulates the arterial response to low flow

Low and disturbed blood flow drives the progression of arterial diseases including atherosclerosis and aneurysms. The endothelial response to flow and its interactions with recruited platelets and leukocytes determine disease progression. Here, we report widespread changes in alternative splicing of pre-mRNA in the flow-activated murine arterial endothelium in vivo. Alternative splicing was suppressed by depletion of platelets and macrophages recruited to the arterial endothelium under low and disturbed flow. Binding motifs for the Rbfox-family are enriched adjacent to many of the regulated exons. Endothelial deletion of Rbfox2, the only family member expressed in arterial endothelium, suppresses a subset of the changes in transcription and RNA splicing induced by low flow. Our data reveal an alternative splicing program activated by Rbfox2 in the endothelium on recruitment of platelets and macrophages and demonstrate its relevance in transcriptional responses during flow-driven vascular inflammation.