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 Notch's 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.angiogenesis ͉ cell signaling ͉ endothelial cell ͉ stroke ͉ cerebrovascular

Infant rats were infected intranasally with mixtures of streptomycin-sensitive and streptomycin-resistant strains of Haemophilus influenzae type b and cultures of nasopharyngeal washings, blood, and cerebrospinal fluid were obtained. If from independent action, as opposed to cooperative interaction of intranasally inoculated organisms. The results also suggested that the meninges were invaded by the hematogenous route. Intranasal inoculation of infant rats with Haemophilus influenzae type b results in bacteremia and meningitis and has proved to be a useful experimental model of the human disease (1-3). These studies provided evidence that the pathogenesis of meningitis involved three sequential events: nasopharyngeal colonization, bacteremia, and central nervous system invasion, although direct invasion of the meninges by contiguous spread from the nasopharynx was not precluded. The studies also revealed that an inoculum of at least 1000 organisms was a prerequisite for the induction of bacteremia and meningitis. This observation might be explained as follows. First, it is possible that every organism in the initial inoculum behaves in a manner that is uninfluenced by the other bacteria present (independent action) (4-6). Each organism has a low probability of successfully overcoming the host defenses; however, the probability is not zero. As the dose of organisms is increased, it becomes progressively more likely that one of them will establish a local or distant focus of infection. Under these circumstances, the bacteria recovered from blood or cerebrospinal fluid (CSF), for example, would be descended from a few organisms, perhaps even a single organism of the original inoculum (4-7). Alternatively, organisms en masse may possess properties that are denied to single bacteria (cooperative action). When the inoculum attains a critical magnitude, infection ensues (8, 9). Under these circumstances, bacteria cultured from the blood or CSF would be descendents of many different organisms of the original inoculum.The following study was designed to test the theory of independent action compared to that of cooperative action in the pathogenesis of H. influenzae type b, bacteremia, and meningitis. The results also provided data relevant to the relationship between nasopharyngeal colonization, bacteremia, and invasion of the central nervous system. MATERIALS AND METHODSThe Eagan strain of H. influenzae type b and its one-step streptomycin-resistant mutant, the media used for its growth, the methods of intranasal inoculation, the technique of blood and CSF sampling, and the quantitation of bacteria in blood and CSF were identical to those used previously (1-3). Natural litters of 5-day-old rats (Sprague-Dawley, strain COBS/CD) were obtained from Charles River Laboratories, Wilmington, MA. Inocula containing an approximately equal number of the streptomycin-sensitive (SMS) and streptomycin-resistant (SMR) variants were prepared as follows. Each variant was grown overnight on solid medium at 370. A few colonies ...

Summary High-flow through abnormal blood vessels underlies many life-threatening diseases. The ability to safely and non-invasively normalize these vessels by molecular intervention holds promise to treat these devastating conditions. Here we studied high-flow AV shunts caused by upregulation of Notch signaling via endothelial expression of constitutively-active Notch4 (Notch4*). Using 4D, two-photon imaging with cellular resolution in live mice, we found that normalizing Notch signaling by turning off Notch4* promptly converted large caliber, high-flow AV shunts to capillary-like vessels. The process was initiated by vessel narrowing without the loss of endothelial cells. Restoration of venous receptor EphB4 is an underlying mechanism, as EphB4 expression was recovered upon Notch normalization and required for the vessel regression. The structural regression of the high-flow AV shunts returned shunting flow to perfusing vessels, reversing tissue hypoxia and dysfunction. Our data provide direct, in vivo evidence that a single genetic manipulation in Notch pathway can exert dominant effects over hemodynamics leading to safe degeneration of the high-flow AV shunts at the core of AV Malformations.

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 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.

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.

Objective: Alterations in extracellular matrix quantity and composition contribute to atherosclerosis, with remodeling of the subendothelial basement membrane to a fibronectin-rich matrix preceding lesion development. Endothelial cell interactions with fibronectin prime inflammatory responses to a variety of atherogenic stimuli; however, the mechanisms regulating early atherogenic fibronectin accumulation remain unknown. We previously demonstrated that oxidized LDL (oxLDL) promotes endothelial pro inflammatory gene expression by activating the integrin α5β1, a classic mediator of fibronectin fibrillogenesis. Approach and Results: We now show that oxLDL drives robust endothelial fibronectin deposition and inhibiting α5β1 (blocking antibodies, α5 knockout cells) completely inhibits oxLDL-induced fibronectin deposition. Consistent with this, inducible endothelial-specific α5 integrin deletion in ApoE knockout mice significantly reduces atherosclerotic plaque formation, associated with reduced early atherogenic inflammation. Unlike TGFβ-induced fibronectin deposition, oxLDL does not induce fibronectin expression (mRNA, protein) or the endothelial-to-mesenchymal transition phenotype. In addition, we show that cell-derived and plasma-derived fibronectin differentially affect endothelial function, with only cell-derived fibronectin capable of supporting oxLDL-induced VCAM-1 expression despite plasma fibronectin deposition by oxLDL. The inclusion of EIIIA and EIIIB domains in cell-derived fibronectin mediates this effect, as EIIIA/EIIIB knockout endothelial cells show diminished oxLDL-induced inflammation. Furthermore, our data suggests that EIIIA/EIIIB-positive cellular fibronectin is required for maximal α5β1 recruitment to focal adhesions and fibronectin fibrillogenesis. Conclusions: Taken together, our data demonstrate that endothelial α5 integrins drives oxLDL-induced fibronectin deposition and early atherogenic inflammation. Additionally, we show that α5β1-dependent endothelial fibronectin deposition mediates oxLDL-dependent endothelial inflammation and fibronectin fibrillogenesis.

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 48hrs, 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, since 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.

No abstract