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Cerebral Vascular Abnormalities in a Murine Model of Hereditary Hemorrhagic Telangiectasia
Abstract
Hereditary hemorrhagic telangiectasia type 1 (HHT1) is an autosomal dominant vascular dysplasia caused by mutations in the endoglin gene and characterized by dilated vessels and arteriovenous malformations (AVMs). To understand the etiology of this disorder, we evaluated the cerebral vasculature of endoglin heterozygous (Eng+/-) mice, which represent the only animal model of HHT1.
The cerebral vasculature of Eng+/- and Eng+/+ mice from C57BL/6 (B6) and 129/Ola (129) strains with a differential susceptibility to HHT1 was studied with corrosion casting. Casts were observed by scanning electron microscopy to detect malformations and evaluate arterial diameters and orientation of endothelial nuclei. Measurements were taken to assess relative constriction at arteriolar branching points and downstream relative dilatation.
Three of 10 Eng+/- mice demonstrated abnormal vascular findings including AVMs, while none of 15 Eng+/+ mice did. The incidence of relative constriction at arteriolar branching points was significantly less in both Eng+/- groups than in their Eng+/+ counterparts. The occurrence of relative dilatation was significantly greater in B6-Eng+/- than in B6-Eng+/+ mice. Endothelial nuclei were significantly rounder and deviated more from the direction of blood flow in Eng+/- than in Eng+/+ mice.
Eng+/- mice showed significant structural alterations in cerebral blood vessels, indicating that the level of endoglin on endothelium is critical for maintenance of normal vasculature. Since endoglin haploinsufficiency is associated with HHT1, such changes in arteriolar structures might occur in HHT1 patients and predispose them to AVMs and their sequelae.
... More research is required to determine if these changes actually contribute to the formation of AVMs in HHT patients [70]. In another mouse model that was stimulated with VEGF (vascular endothelial growth factor), 89% of the Eng +/í mice showed vascular abnormalities and none were observed in the controls [71]. ...
... In another mouse model that was stimulated with VEGF (vascular endothelial growth factor), 89% of the Eng +/í mice showed vascular abnormalities and none were observed in the controls [71]. The lung, liver and intestine showed dilation in some vessels with no external signs in one particular animal [70]. Another animal that had an ear telangiectasis showed an AVM nidus that resembled those in human and canine brain [70]. ...
... The lung, liver and intestine showed dilation in some vessels with no external signs in one particular animal [70]. Another animal that had an ear telangiectasis showed an AVM nidus that resembled those in human and canine brain [70]. Despite the fact that Eng +/í and Acvrl1 +/í mice are the best available HHT models at present, past studies do not show the HHT features at high frequency [69,72,73]. ...
Arteriovenous malformations (AVMs) are congenital lesions that cause brain haemorrhage in children and young adults. Current treatment modalities include surgery, radiosurgery and embolization. These treatments are generally effective only for small AVMs. Over one third of AVMs cannot be treated safely and effectively with existing options. Several animal models have been developed with the aims of understanding AVM pathophysiology and improving treatment. No animal model perfectly mimics a human AVM. Each model has limitations and advantages. Models contribute to the understanding of AVMs and hopefully to the development of improved therapies. This paper reviews animal models of AVMs and their advantages and disadvantages.
... Genetic mouse models of bAVM have been mostly developed by manipulating Eng or Alk1 genes. Eng +/− or Alk1 +/− heterozygous knockout (KO) mice are viable and show HHT phenotypes during adulthood, while Eng -/-or Alk1 -/-homozygous KO mice are embryonic lethal [38,[54][55][56][57][58][59]. bAVMs, including AV shunts and niduses of dilated vessels, occurred in only 30% of Eng +/− mice aged 25 to 40 weeks with incomplete penetrance [56]. ...
... Eng +/− or Alk1 +/− heterozygous knockout (KO) mice are viable and show HHT phenotypes during adulthood, while Eng -/-or Alk1 -/-homozygous KO mice are embryonic lethal [38,[54][55][56][57][58][59]. bAVMs, including AV shunts and niduses of dilated vessels, occurred in only 30% of Eng +/− mice aged 25 to 40 weeks with incomplete penetrance [56]. Brain AVMs did not develop effectively in mice with haploinsufficiency. ...
Arteriovenous malformations (AVMs) are abnormal connections of vessels that shunt blood directly from arteries into veins. Rupture of brain AVMs (bAVMs) can cause life-threatening intracranial bleeding. Even though the majority of bAVM cases are sporadic without a family history, some cases are familial. Most of the familial cases of bAVMs are associated with a genetic disorder called hereditary hemorrhagic telangiectasia (HHT). The mechanism of bAVM formation is not fully understood. The most important advances in bAVM basic science research is the identification of somatic mutations of genes in RAS-MAPK pathways. However, the mechanisms by which mutations of these genes lead to AVM formation are largely unknown. In this review, we summarized the latest advance in bAVM studies and discussed some pathways that play important roles in bAVM pathogenesis. We also discussed the therapeutic implications of these pathways.
... If fact, ENG level in ENG +/mouse was about 50% and 3 of 10 mice developed vascular abnormalities including AVM-like structure. 65) Severity and heterogeneity of symptomatology might be associated with the other epigenetic factors such as environment, blood pressure, oxygenation, shear stress, and hormonal levels. 60) Homozygous mutation of ALK1 -/in mice is also lethal at E10.5-11.5, exhibiting severe vascular abnormalities. ...
... 62) Heterozygous mice (ENG +/-) without stimuli developed less often abnormal microvessel formation than heterozygous mice (ENG +/-) stimulated by VEGF. 65,66) ENG +/mice developed severer cerebrovascular dysplasia than ALK1 +/mice stimulated by VEGF. 67) Inflammatory cells are often found in and around AVMs. Dysmorphic vessels developed in ENG +/and ALK1 +/mice at the capillary levels, but no AV shunts developed. ...
Brain arteriovenous malformations (bAVMs) represent a high risk of intracranial hemorrhages, which are substantial causes of morbidity and mortality of bAVMs, especially in children and young adults. Although a variety of factors leading to hemorrhages of bAVMs are investigated extensively, their pathogenesis is still not well elucidated. The author has reviewed the updated data of genetic aspects of bAVMs, especially focusing on clinical and experimental knowledge from hereditary hemorrhagic telangiectasia, which is the representative genetic disease presenting with bAVMs caused by loss-of-function in one of the two genes: endoglin and activin receptor-like kinase 1. This knowledge may allow us to infer the pathogensis of sporadic bAVMs and in the development of new medical therapies for them.
... Eng or Alk1 knockouts exhibit embryonic vascular defects, including dilated and fused artery-vein pairs and die in utero. 42,43 Eng +/− or Alk1 +/− heterozygous mice are viable and develop characteristics of HHT during adulthood; [10][11][12][13] however, features of BAVM, including AV shunts, niduses of dilated vessels, and rounded, misaligned EC nuclei, occur in 30% of Eng +/− mice aged 25-40 weeks, similar to BAVM incidence in HHT1 patients. 12 Thus, loss of one allele of Eng or Alk1 is sufficient to induce BAVM in adult mice, but with incomplete penetrance. ...
... 42,43 Eng +/− or Alk1 +/− heterozygous mice are viable and develop characteristics of HHT during adulthood; [10][11][12][13] however, features of BAVM, including AV shunts, niduses of dilated vessels, and rounded, misaligned EC nuclei, occur in 30% of Eng +/− mice aged 25-40 weeks, similar to BAVM incidence in HHT1 patients. 12 Thus, loss of one allele of Eng or Alk1 is sufficient to induce BAVM in adult mice, but with incomplete penetrance. ...
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 Broca's area; 48 shows Wernicke's 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 © 2014, Thieme Medical Publishers.
AVM is characterized by high-flow AV connections that shunt blood directly from arteries to veins, displacing intervening capillaries with a nidus of enlarged and tortuous vessels. BAVM clinical characteristics include (1) AV shunting, the presence of direct connections between arteries and veins, displacing intervening capillaries; (2) abnormally high blood flow through the feeding artery, AV …
... Eng heterozygous mice developed normally, but older animals displayed clinical signs of HHT, such as nosebleeds and telangiectasia [64,93]. These animals develop AVMs of the brain as early as twenty-five weeks of age [94,95]. Such vascular morphology is correlated with a poorly organized and underdeveloped smooth muscle layer, a seventy percent reduction in smooth muscle coverage and an increase in mast cell-driven inflammation [95]. ...
... Although it appears that ALK1 and ENG share some transduction pathways, the observed differences between mutants for these receptors are not clearly understood, both in animal models and in patients with HHT. In humans, heterozygosity for ENG results in more pulmonary and cerebral AVMs, while heterozygosity for ACVRL1 results in more hepatic AVMs [7,94]. Furthermore, mouse models demonstrate that Eng mutants do not display the degree of dilatation observed in Acvrl1 mutants, show vascular shunts later than Acvrl1 mutants, and do not display the downregulation of arterial EPHRINB2 [107]. ...
Arteriovenous malformations occur when abnormalities of vascular patterning result in the flow of blood from arteries to veins without an intervening capillary bed. Recent work has revealed the importance of the Notch and TGF-β signaling pathways in vascular patterning. Specifically, Notch signaling has an increasingly apparent role in arterial specification and suppression of branching, whereas TGF-β is implicated in vascular smooth muscle development and remodeling under angiogenic stimuli. These physiologic roles, consequently, have implicated both pathways in the pathogenesis of arteriovenous malformation. In this review, we summarize the studies of endothelial signaling that contribute to arteriovenous malformation and the roles of genes implicated in their pathogenesis. We further discuss how endothelial signaling may contribute to vascular smooth muscle development and how knowledge of signaling pathways may provide us targets for medical therapy in these vascular lesions.
... The MCA was imaged at five contiguous, non-overlapping segments. For quantitative analysis, the area of 80–100 ECN was determined from the MCA and five branching arteries per animal [49] using ImageJ. Nuclei located at the edge of the vessel or near a branching point were excluded. ...
... These vessels appeared ragged and irregular in sedentary mice but in exercised mice had a smoother, more uniform structure. Vascular casts also revealed welldefined imprints of the ECN (endothelial cell nuclei) which were quantified as previously described [49]. Exercised mice had ECN areas that were ,50% greater than sedentary animals (p,0.005, ...
Healthy brain aging and cognitive function are promoted by exercise. The benefits of exercise are attributed to several mechanisms, many which highlight its neuroprotective role via actions that enhance neurogenesis, neuronal morphology and/or neurotrophin release. However, the brain is also composed of glial and vascular elements, and comparatively less is known regarding the effects of exercise on these components in the aging brain. Here, we show that aerobic exercise at mid-age decreased markers of unhealthy brain aging including astrocyte hypertrophy, a hallmark of brain aging. Middle-aged female mice were assigned to a sedentary group or provided a running wheel for six weeks. Exercise decreased hippocampal astrocyte and myelin markers of aging but increased VEGF, a marker of angiogenesis. Brain vascular casts revealed exercise-induced structural modifications associated with improved endothelial function in the periphery. Our results suggest that age-related astrocyte hypertrophy/reactivity and myelin dysregulation are aggravated by a sedentary lifestyle and accompanying reductions in vascular function. However, these effects appear reversible with exercise initiated at mid-age. As this period of the lifespan coincides with the appearance of multiple markers of brain aging, including initial signs of cognitive decline, it may represent a window of opportunity for intervention as the brain appears to still possess significant vascular plasticity. These results may also have particular implications for aging females who are more susceptible than males to certain risk factors which contribute to vascular aging.
... Recent genetic studies provided evidence that the development of AVMs is underpinned by a deficiency in the linear ENG-ALK1-SMAD4 pathway [15][16][17]. In mouse models of HHT, the induction of AVMs requires heterozygous [15,18] or homozygous deletion of the Alk1, Eng, or Smad4 gene [16,17,19]. Furthermore, additional factors such as wounding or angiogenic stimulation, in addition to the genetic deletion of HHT genes, are necessary for inducing AVMs in adult mice [17,20,21]. ...
Brain arteriovenous malformations (BAVMs) are a critical concern in hereditary hemorrhagic telangiectasia (HHT) patients, carrying the risk of life-threatening intracranial hemorrhage. While traditionally seen as congenital, the debate continues due to documented de novo cases. Our primary goal was to identify the precise postnatal window in which deletion of the HHT gene Endoglin (Eng) triggers BAVM development. We employed SclCreER(+);Eng2f/2f mice, enabling timed Eng gene deletion in endothelial cells via tamoxifen. Tamoxifen was given during four postnatal periods: P1–3, P8–10, P15–17, and P22–24. BAVM development was assessed at 2–3 months using latex dye perfusion. We examined the angiogenic activity by assessing vascular endothelial growth factor receptor 2 (VEGFR2) expression via Western blotting and Flk1-LacZ reporter mice. Longitudinal magnetic resonance angiography (MRA) was conducted up to 9 months. BAVMs emerged in 88% (P1–3), 86% (P8–10), and 55% (P15–17) of cases, with varying localization. Notably, the P22–24 group did not develop BAVMs but exhibited skin AVMs. VEGFR2 expression peaked in the initial 2 postnatal weeks, coinciding with BAVM onset. These findings support the “second hit” theory, highlighting the role of early postnatal angiogenesis in initiating BAVM development in HHT type I mice.
... Genetically engineered mouse models are ubiquitous in basic and translational research and have offered insight into the molecular and genetic basis of bAVMs, especially in regard to HHT [62,63]. Aimed at studying the etiology of HHT1, a mouse model was developed harboring a heterozygous knockout of endoglin (Eng +/-) to evaluate changes in cerebral vasculature [62]. ...
Arteriovenous malformations (AVMs) are an anomaly of the vascular system where feeding arteries are directly connected to the venous drainage network. While AVMs can arise anywhere in the body and have been described in most tissues, brain AVMs are of significant concern because of the risk of hemorrhage which carries significant morbidity and mortality. The prevalence of AVM’s and the mechanisms underlying their formation are not well understood. For this reason, patients who undergo treatment for symptomatic AVM’s remain at increased risk of subsequent bleeds and adverse outcomes. The cerebrovascular network is delicate and novel animal models continue to provide insight into its dynamics in the context of AVM’s. As the molecular players in the formation of familial and sporadic AVM’s are better understood, novel therapeutic approaches have been developed to mitigate their associated risks. Here we discuss the current literature surrounding AVM’s including the development of models and therapeutic targets which are currently being investigated.
... Genetically engineered mouse models are ubiquitous in basic and translational research and have offered insight into the molecular and genetic basis of bAVMs, especially in regard to HHT [62,63]. Aimed at studying the etiology of HHT1, a mouse model was developed harboring a heterozygous knockout of endoglin (Eng +/− ) to evaluate changes in cerebral vasculature [62]. ...
Arteriovenous malformations (AVMs) are an anomaly of the vascular system where feeding arteries are directly connected to the venous drainage network. While AVMs can arise anywhere in the body and have been described in most tissues, brain AVMs are of significant concern because of the risk of hemorrhage which carries significant morbidity and mortality. The prevalence of AVM's and the mechanisms underlying their formation are not well understood. For this reason, patients who undergo treatment for symptomatic AVM's remain at increased risk of subsequent bleeds and adverse outcomes. The cerebrovascular network is delicate and novel animal models continue to provide insight into its dynamics in the context of AVM's. As the molecular players in the formation of familial and sporadic AVM's are better understood, novel therapeutic approaches have been developed to mitigate their associated risks. Here we discuss the current literature surrounding AVM's including the development of models and therapeutic targets which are currently being investigated.
... Treatment methods for brain AVM are limited (Raper et al., 2020); therefore, it is important to understand the mechanisms of disease pathogenesis in order to develop novel therapies. Molecular and genetic studies from human tissue and animal models have revealed several signaling pathways involved in brain AVM pathogenesis, namely TGFβ (Bourdeau et al., 1999;Satomi et al., 2003;Park et al., 2009), Ras/Raf/MEK/ERK (Nikolaev et al., 2018;Fish et al., 2020), and Notch (Murphy et al., 2014;Nielsen et al., 2014). Collectively, these data support the emerging view that diverse mechanisms underlie brain AVM pathologies and diverse therapies must be developed to treat brain AVM patients safely and effectively. ...
Pericytes, like vascular smooth muscle cells, are perivascular cells closely associated with blood vessels throughout the body. Pericytes are necessary for vascular development and homeostasis, with particularly critical roles in the brain, where they are involved in regulating cerebral blood flow and establishing the blood-brain barrier. A role for pericytes during neurovascular disease pathogenesis is less clear—while some studies associate decreased pericyte coverage with select neurovascular diseases, others suggest increased pericyte infiltration in response to hypoxia or traumatic brain injury. Here, we used an endothelial loss-of-function Recombination signal binding protein for immunoglobulin kappa J region (Rbpj)/Notch mediated mouse model of brain arteriovenous malformation (AVM) to investigate effects on pericytes during neurovascular disease pathogenesis. We tested the hypothesis that pericyte expansion, via morphological changes, and Platelet-derived growth factor B/Platelet-derived growth factor receptor β (Pdgf-B/Pdgfrβ)-dependent endothelial cell-pericyte communication are affected, during the pathogenesis of Rbpj mediated brain AVM in mice. Our data show that pericyte coverage of vascular endothelium expanded pathologically, to maintain coverage of vascular abnormalities in brain and retina, following endothelial deletion of Rbpj. In Rbpj-mutant brain, pericyte expansion was likely attributed to cytoplasmic process extension and not to increased pericyte proliferation. Despite expanding overall area of vessel coverage, pericytes from Rbpj-mutant brains showed decreased expression of Pdgfrβ , Neural (N)-cadherin , and cluster of differentiation ( CD)146 , as compared to controls, which likely affected Pdgf-B/Pdgfrβ-dependent communication and appositional associations between endothelial cells and pericytes in Rbpj-mutant brain microvessels. By contrast, and perhaps by compensatory mechanism, endothelial cells showed increased expression of N-cadherin . Our data identify cellular and molecular effects on brain pericytes, following endothelial deletion of Rbpj, and suggest pericytes as potential therapeutic targets for Rbpj/Notch related brain AVM.
... 58 The first cerebrovascular AVM Eng±heterozygous mouse mutant was developed in 1999 and alterations of this core HHT gene model are still in practice. [59][60][61] Subsequent Eng± and Alk1± mouse models were supplemented with VEGF overexpression vectors to induce angiogenesis and investigate the underpinnings of AVM formation. 62,63 Interestingly, these mouse models have been utilized to identify novel defenses against hemorrhage secondary to AVM such as Bevacizumab (a monoclonal antibody against VEGF-A) or Lenalidomide (by decreasing dysplastic vessels and increasing mural cell coverage in the brain AVM). ...
BACKGROUND
Research of William Hunter's hypothesized (then discovered) arteriovenous varix (now arteriovenous malformation [AVM]) has developed exponentially over the previous quarter‐millennium. Virchow and Luschka's subsequent contributions (nearly 100 years later) by identifying an AVM of the brain and its congenital nature were 2 of the first significant developments made in the field. AVMs present as an erroneous connection (known as a fistula) between an artery and a vein that bypasses the capillary circulation. The arteries and arterioles contributing to the malformation are known as feeders which connect to the draining veins via a plexiform vascular network known as a nidus. Prior to the design of a synthetic anastomosis coupled with vessel ligation by Spetzler et al, animal models were largely based on embolization or study of the normal anatomy. The animal and early genetic models have been reported on at length and numerous times across the literature, but novel developments spanning the previous decade have ushered in a technological revolution of vascular modeling that warrants discussion and analysis.
METHODS
Parameterization of a PubMed query to include all literature including the words “brain,” “arteriovenous malformation,” and “model” yielded 489 articles. After extraction of relevant literature and full‐text screening, 41 articles were chosen for detailed review.
RESULTS
Technological innovations outside of neurosurgery have greatly impacted the development of novel AVM models in the form of 3D flow models printed into silicon models and combined with advanced imaging technology such as 4D flow magnetic resonance imagin. Technological developments in preservation solutions, catheterization tools, and imaging technologies have also allowed for advent of the cerebrovascular placental model for testing of treatments such as radiosurgery, glue embolization, coiling, as well as histological assessment of tissue directly after intervention.
CONCLUSION
We review the breadth of AVM models in the literature over the last almost 5 decades.
... The impairment of the TGFβ signaling pathway is associated with cerebrovascular disease, including AVMs and aneurysms [25]. In particular, the haploinsufficiency of TGFβ receptors such as Eng-or Alk1 with a focal VEGF-A overexpression caused the development of HHT type 1 or 2-associated AVMs in mice [37,38]. Meanwhile, enhanced activity of MMPs (e.g., MMP-14) has been observed in bAVMs and may be involved in the sENG production via ENG cleavage [9,[39][40][41]. ...
Increased soluble endoglin (sENG) has been observed in human brain arteriovenous malformations (bAVMs). In addition, the overexpression of sENG in concurrence with vascular endothelial growth factor (VEGF)-A has been shown to induce dysplastic vessel formation in mouse brains. However, the underlying mechanism of sENG-induced vascular malformations is not clear. The evidence suggests the role of sENG as a pro-inflammatory modulator, and increased microglial accumulation and inflammation have been observed in bAVMs. Therefore, we hypothesized that microglia mediate sENG-induced inflammation and endothelial cell (EC) dysfunction in bAVMs. In this study, we confirmed that the presence of sENG along with VEGF-A overexpression induced dysplastic vessel formation. Remarkably, we observed increased microglial activation around dysplastic vessels with the expression of NLRP3, an inflammasome marker. We found that sENG increased the gene expression of VEGF-A, pro-inflammatory cytokines/inflammasome mediators (TNF-α, IL-6, NLRP3, ASC, Caspase-1, and IL-1β), and proteolytic enzyme (MMP-9) in BV2 microglia. The conditioned media from sENG-treated BV2 (BV2-sENG-CM) significantly increased levels of angiogenic factors (Notch-1 and TGFβ) and pERK1/2 in ECs but it decreased the level of IL-17RD, an anti-angiogenic mediator. Finally, the BV2-sENG-CM significantly increased EC migration and tube formation. Together, our study demonstrates that sENG provokes microglia to express angiogenic/inflammatory molecules which may be involved in EC dysfunction. Our study corroborates the contribution of microglia to the pathology of sENG-associated vascular malformations.
... TGFβ signaling pathway is essential for vessel development and the maintenance [35,36], and impairment of the signaling is associated with cerebrovascular disease, including AVMs and aneurysms [25]. In particular, the TGFβ receptor, Eng-or Alk1-hap-loinsufficiency with a focal VEGF-A overexpression disrupts TGFβ signaling causing development of HHT type 1 or 2-associated AVMs in mice [37,38]. Meanwhile, enhanced activity of MMPs (e.g., MMP-14) is observed in bAVMs and involved in the sENG production by ENG cleavage [9,[39][40][41]. ...
Increased soluble endoglin (sENG) were observed in human brain arteriovenous malformations (bAVMs), and overexpression of sENG with vascular endothelial growth factor (VEGF)-A induced dysplastic vessel formation in mouse brain. However, the underlying mechanism of sENG-induced vascular malformations is not clear. While evidence suggests the role of sENG as a pro-inflammatory modulator, increased microglial accumulation and inflammations were observed in bAVMs. Therefore, we hypothesized that microglia mediate sENG-induced inflammation and endothelial cell (EC) dysfunction in bAVMs. In this study, we confirmed that sENG with VEGF-A overexpression induced dysplastic vessel formation. Remarkably, there were increased microglial activation around dysplastic vessels with expression of NLRP3, inflammasome marker. We found that sENG increased the gene expression of VEGF-A, pro-inflammatory cytokines/inflammasome mediators (TNF-α, IL-6, NLRP3, ASC, Caspase-1, and IL-1β), and proteolytic enzyme (MMP-9) in BV2 microglia. The conditioned media from sENG-treated BV2 (BV2-sENG-CM) significantly increased angiogenic factors (Notch-1 and TGFβ) and pERK1/2 in ECs while it decreased IL-17RD, an anti-angiogenic mediator. Finally, the BV2-sENG-CM significantly increased EC migration and tube formation. Together, our study demonstrates that sENG provokes microglia to release angiogenic/inflammatory responses which may be involved in EC dysfunction. Our study suggests the contribution of microglia in the pathology of sENG-associated vascular malformations.
... Further researchers were able to generate heterozygous Eng+/-and Alk1+/-mice. These living mice models were capable of producing vascular anomalies in several organs, but not brain AVM [43]. Further advances using Cre-Lox technique were able to create target inducible knock-out Eng-iKO or Alk1-iKO mice that together with an external noxa or stimuli could develop vascular malformations [44]. ...
Background
Brain arteriovenous malformations (bAVM) are one of the most complex vascular lesions in humans. Their understanding and treatment have been possible through the use of different experimental models. The aim of this scoping review was to systematically map the existing experimental models used for bAVM research and training.
Methods
A scoping review was conducted, and a search process was performed in 8 electronic databases from inception to April 30th, 2020. Study selection included all types of research articles that used any kind of experimental model for AVM study. Selection and data extraction were performed by independent reviewers.
Results
The initial search retrieved 954 articles which were reduced to 177 articles after the whole inclusion / exclusion process. We identified 9 main AVM experimental models, divided in in vivo: transgenic, rete mirabile, carotid-jugular fistula, carotid-jugular plexus fistula, arteriovenous shunt and cornea; or in vitro: 3D cast, computer generated and biological graft. First developed models were dedicated to study the hemodynamic effects and then followed by endovascular testing using the swine rete mirabile. The latest developments have come with transgenic models, allowing the manipulation and creation of AVMs in rodent brains, giving a huge step in the understanding of genetic origin, angiogenic mechanisms or potential therapeutic targets for the future.
Conclusions
There is no unique model that could account for all features of bAVM. We expect a continuous development of more accurate models that could lead to optimize and develop new treatment strategies for increasing the cure rate of this disease.
... Analysis of a large number of mice over a period of one-year established disease prevalence at 72% in 129/Ola, intermediate in backcrosses (36%), and low in C57BL/6 (7%) [121]. Multiple signs of HHT were detected, such as ear telangiectasia, hemorrhage, dilated vessels, liver and lung congestion, brain and heart ischemia, and even cerebral AVMs [121,122]. Disease sequelae included stroke, fatal hemorrhage, and congestive heart failure. Interestingly, 129/Ola inbred mice had previously been shown to carry significant alterations in liver and lung vasculature, such as portal shunting and reduction/truncation of peripheral vessels, when compared to C57BL/6 mice [123,124]. ...
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetic disorder that presents with telangiectases in skin and mucosae, and arteriovenous malformations (AVMs) in internal organs such as lungs, liver, and brain. Mutations in ENG (endoglin), ACVRL1 (ALK1), and MADH4 (Smad4) genes account for over 95% of HHT. Localized telangiectases and AVMs are present in different organs, with frequencies which differ among affected individuals. By itself, HHT gene heterozygosity does not account for the focal nature and varying presentation of the vascular lesions leading to the hypothesis of a “second-hit” that triggers the lesions. Accumulating research has identified a variety of triggers that may synergize with HHT gene heterozygosity to generate the vascular lesions. Among the postulated second-hits are: mechanical trauma, light, inflammation, vascular injury, angiogenic stimuli, shear stress, modifier genes, and somatic mutations in the wildtype HHT gene allele. The aim of this review is to summarize these triggers, as well as the functional mechanisms involved.
... Aging and pathological states disrupt endothelial cell morphology and function. ECN surface area is decreased in middle-aged and aged mice (Latimer et al. 2011), shrinks in rat models of hypertension (Arribas et al. 1997), and takes on irregular shapes and orientations in cases of mouse cerebral vascular dysfunction (Junichiro et al. 2003). Dysfunction of endothelial cells is associated with neurodegenerative disease pathology, including Alzheimer's disease, Parkinson's disease, and stroke (Iadecola and Davisson 2008;Lange-Asschenfeldt and Kojda 2008;Steinert et al. 2010;Erickson and Banks 2013). ...
Exercise is beneficial to brain health, and historically, the advantageous effects of exercise on the brain have been attributed to neuronal plasticity. However, it has also become clear that the brain vascular system also exhibits plasticity in response to exercise. This plasticity occurs in areas involved in movement, such as the motor cortex. This experiment aimed to further characterize the effects of exercise on structural vascular plasticity in the male rat motor cortex, by specifically identifying whether features of angiogenesis, the growth of new capillaries, or changes in vessel diameter were present. Male rats in the exercise group engaged in a 5-week bout of voluntary wheel running, while a second group of rats remained sedentary. After the exercise regimen, vascular corrosion casts, resin replicas of the brain vasculature, were made for all animals and imaged using a scanning electron microscope. Results indicate sprouting angiogenesis was the primary form of structural vascular plasticity detected in the motor cortex under these aerobic exercise parameters. Additionally, exercised rats displayed a slight increase in capillary diameter and expanded endothelial cell nuclei diameters in this region.
... While the genetic pathogenesis of arteriovenous malformations is largely unknown, AVMs can also be associated with hereditary lesions [101]. Hereditary hemorrhagic telangiectasia, for example, could be studied through modifications in TGFβ signaling in human endothelial cells used for organoid vascularization [102][103][104]. Increased expression of VEGF-A mRNA has been identified in the microarray analysis of AVM tissue [105]. ...
Our group has recently created a novel in-vivo human brain organoid vascularized with human iPSC-derived endothelial cells. In this review article, we discuss the challenges of creating a perfused human brain organoid model in an immunosuppressed rodent host and discuss potential applications for neurosurgical disease modeling.
... Unlike the bat wing, however, the cerebral cortex has dense microvessels and a mean capillary diameter of 5-7 mm, which is similar between rodents and humans, according to three-dimensional scanning electron microscopy (SEM) microvascular corrosion casts. [74][75][76][77][78][79] Just like distinctions are emerging from different types of pericytes and mural cells, capillaries should be separated into several sub-types depending on their location, distance from descending arteries or ascending veins, and endothelial and/or pericyte phenotypes. 25,80 However, it is usually difficult to categorize the capillary phenotype based on visual inspection of morphology apart from branch positions. ...
The cerebral microvasculature consists of pial vascular networks, parenchymal descending arterioles, ascending venules and parenchymal capillaries. This vascular compartmentalization is vital to precisely deliver blood to balance continuously varying neural demands in multiple brain regions. Optical imaging techniques have facilitated the investigation of dynamic spatial and temporal properties of microvascular functions in real time. Their combination with transgenic animal models encoding specific genetic targets have further strengthened the importance of optical methods for neurovascular research by allowing for the modulation and monitoring of neuro vascular function. Image analysis methods with three-dimensional reconstruction are also helping to understand the complexity of microscopic observations. Here, we review the compartmentalized cerebral microvascular responses to global perturbations as well as regional changes in response to neural activity to highlight the differences in vascular action sites. In addition, microvascular responses elicited by optical modulation of different cell-type targets are summarized with emphasis on variable spatiotemporal dynamics of microvascular responses. Finally, long-term changes in microvascular compartmentalization are discussed to help understand potential relationships between CBF disturbances and the development of neurodegenerative diseases and cognitive decline.
... HHT phenotypes arise in patients due to mutations (most commonly missense mutations) that lead to haploinsufficiency [10]. In contrast, mouse models of HHT often utilize null genetic backgrounds because loss of one allele of Alk1, Eng or Smad4 does not result in consistent presence of AVMs in predictable locations [21,26,[53][54][55]. Furthermore, HHT patients harbor germline mutations, which manifest from gestation and remain throughout adulthood. ...
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder that leads to abnormal connections between arteries and veins termed arteriovenous malformations (AVM). Mutations in TGFβ pathway members ALK1, ENG and SMAD4 lead to HHT. However, a Smad4 mouse model of HHT does not currently exist. We aimed to create and characterize a Smad4 endothelial cell (EC)-specific, inducible knockout mouse (Smad4f/f;Cdh5-CreERT2) that could be used to study AVM development in HHT. We found that postnatal ablation of Smad4 caused various vascular defects, including the formation of distinct AVMs in the neonate retina. Our analyses demonstrated that increased EC proliferation and size, altered mural cell coverage and distorted artery–vein gene expression are associated with Smad4 deficiency in the vasculature. Furthermore, we show that depletion of Smad4 leads to decreased Vegfr2 expression, and concurrent loss of endothelial Smad4 and Vegfr2 in vivo leads to AVM enlargement. Our work provides a new model in which to study HHT-associated phenotypes and links the TGFβ and VEGF signaling pathways in AVM pathogenesis.
Electronic supplementary material
The online version of this article (10.1007/s10456-018-9602-0) contains supplementary material, which is available to authorized users.
... The dilation of arterioles as an initial step for AVM formation was first described for cerebral AVMs in endoglin heterozygous mice. 90 Here, rounder endothelial nuclei shape and irregular cellular orientation deviating from that of the blood flow decreased arteriolar constriction and shear stress, suggested to be the cause for arteriolar dilation. Recently, an elegant study by Jin et al 76 showed that AVMs originate in the arterioles using a mouse model for HHT1. ...
Correct organization of the vascular tree requires the balanced activities of several signaling pathways that regulate tubulogenesis and vascular branching, elongation, and pruning. When this balance is lost, the vessels can be malformed and fragile, and they can lose arteriovenous differentiation. In this review, we concentrate on the transforming growth factor (TGF)-β/bone morphogenetic protein (BMP) pathway, which is one of the most important and complex signaling systems in vascular development. Inactivation of these pathways can lead to altered vascular organization in the embryo. In addition, many vascular malformations are related to deregulation of TGF-β/BMP signaling. Here, we focus on two of the most studied vascular malformations that are induced by deregulation of TGF-β/BMP signaling: hereditary hemorrhagic telangiectasia (HHT) and cerebral cavernous malformation (CCM). The first of these is related to loss-of-function mutation of the TGF-β/BMP receptor complex and the second to increased signaling sensitivity to TGF-β/BMP. In this review, we discuss the potential therapeutic targets against these vascular malformations identified so far, as well as their basis in general mechanisms of vascular development and stability.
... Eng (ENG human ortholog) (4,18,82) and Acvrl1 (ACVRL1 human ortholog) (101, 154) knockout mice generally have normal vasculogenesis but die at midgestation from defective angiogenesis, including poor vascular smooth muscle development, and endothelial remodeling. Some mice heterozygous for Eng (4,18,82,133) or Acvrl1 (142) mutations exhibited vascular phenotypes similar to HHT, including brain AVMlike vascular lesions (for a review, see Marchuk et al. [88]). ...
CEREBROVASCULAR MALFORMATIONS AFFECT more than 3% of the population, exposing them to a lifetime risk of hemorrhagic stroke, seizures, and focal neurological deficits. Cerebral cavernous malformations (CCMs) exhibit an immature vessel wall, a brittle hemorrhagic tendency, and epileptogenesis, whereas arteriovenous malformations (AVMs) lack capillary beds and manifest apoplectic bleeding under high-flow conditions. There are also more benign venous anomalies, capillary malformations, and lesions with mixed and transitional features. Advances have been made toward understanding the natural history, radiological and pathological correlates, and clinical management. Yet, mechanisms of lesion genesis and clinical manifestations remain largely unknown, and the clinical behavior in individual patients is highly unpredictable. Lesion pathogenesis likely involves abnormal assembly or maintenance of blood vessels, resulting in dysmorphic vessel phenotypes. Familial CCM disease is in part caused by mutations in a cytoskeletal-related protein that is likely integral to interendothelial cell connectivity and maturation of the vascular wall. Rare familial forms of AVM disease have been correlated with two different transforming growth factor-β receptor components, possibly causing disturbance in signaling during vascular assembly. Relevance of these mechanisms to the more common and otherwise identical sporadic CCM and AVM lesions is being explored. In this report, basic mechanisms of vasculogenesis and angiogenesis and how they possibly relate to the common cerebrovascular malformation lesions are reviewed. Novel concepts are discussed related to the cellular, molecular, and genetic substrates in CCM and AVM as well as to how this knowledge can be applied to predict, explain, and possibly modify clinical disease manifestations.
... These mice die during gestation likely from severe impairments in vascular development ( Li et al. 1999, Urness et al. 2000. Alterations in a single copy of endoglin or ALK-1 in mice result in spontaneous vascular dysplasia, telangiectasia and random hemorrhages ( Bourdeau et al. 1999, Satomi et al. 2003, Srinivasan et al. 2003, Torsney et al. 2003, Park et al. 2008). Small vessel malformations are observed in these mice together with disappearance of vascular smooth muscle cells. ...
... The presence of mutations in the genes of endoglin and activin receptor-like kinase 1 predisposes to aberrant TGF-beta signaling and is associated with HHT. These mutations commonly lead to loss of function in the genes associated with TGF-beta signaling [35]. It has also been shown that genes that code for angiopoietins (ANGPT1 and ANGPT2) and their receptor TIE-2 have a crucial role in angiogenesis and vascular stability and there are reports available that in AVM, there is an imbalance of TIE-2-angiopoietin system [36]. ...
Vascular malformations are developmental congenital abnormalities of the vascular system which may involve any segment of the vascular tree such as capillaries, veins, arteries, or lymphatics. Arteriovenous malformations (AVMs) are congenital vascular lesions, initially described as “erectile tumors,” characterized by atypical aggregation of dilated arteries and veins. They may occur in any part of the body, including the brain, heart, liver, and skin. Severe clinical manifestations occur only in the brain. There is absence of normal vascular structure at the subarteriolar level and dearth of capillary bed resulting in aberrant arteriovenous shunting. The causative factor and pathogenic mechanisms of AVMs are unknown. Importantly, no marker proteins have been identified for AVM. AVM is a high flow vascular malformation and is considered to develop because of variability in the hemodynamic forces of blood flow. Altered local hemodynamics in the blood vessels can affect cellular metabolism and may trigger epigenetic factors of the endothelial cell. The genes that are recognized to be associated with AVM might be modulated by various epigenetic factors. We propose that AVMs result from a series of changes in the DNA methylation and histone modifications in the genes connected to vascular development. Aberrant epigenetic modifications in the genome of endothelial cells may drive the artery or vein to an aberrant phenotype. This review focuses on the molecular pathways of arterial and venous development and discusses the role of hemodynamic forces in the development of AVM and possible link between hemodynamic forces and epigenetic mechanisms in the pathogenesis of AVM.
... Both Eng +/− and Alk1 +/− haploinsufficient mice could be successfully generated. These mice develop vascular lesions in various organs, but spontaneous lesions in the brain were modest in Eng +/− mice and minimal in Alk1 +/− mice [20,22]. A research group headed by Su et al. induced cerebral microvascular dysplasia by transferring virusmediated VEGF gene to the brain of Eng +/− or Alk1 +/− adult mice [23][24][25]. ...
Brain arteriovenous malformation (AVM) is an important cause of hemorrhagic stroke. The etiology is largely unknown and the therapeutics are controversial. A review of AVM-associated animal models may be helpful in order to understand the up-to-date knowledge and promote further research about the disease. We searched PubMed till December 31, 2014, with the term “arteriovenous malformation,” limiting results to animals and English language. Publications that described creations of AVM animal models or investigated AVM-related mechanisms and treatments using these models were reviewed. More than 100 articles fulfilling our inclusion criteria were identified, and from them eight different types of the original models were summarized. The backgrounds and procedures of these models, their applications, and research findings were demonstrated. Animal models are useful in studying the pathogenesis of AVM formation, growth, and rupture, as well as in developing and testing new treatments. Creations of preferable models are expected.
... Early studies in small animals use surgical anastomoses of the carotid artery to the jugular vein, connecting the intracranial arterial circulation to the extracranial venous circulation, to mimic the changes in cerebral blood flow [30,31,60]. Genetic mouse models of AVMs include those for hereditary hemorrhagic telangiectasia (HHT; Rendu-Osler-Weber syndrome) with mutations in endoglin or activin receptor-like kinase 1 (Alk1) [172][173][174][175][176][177][178] or proteins regulated by Alk1 [62]. Other AVM models have mutations in Notch signaling, responsible for arterial versus venous endothelial specification [179][180][181]. ...
Peripheral artery disease (PAD) is a broad disorder encompassing multiple forms of arterial disease outside of the heart. As such, PAD development is a multifactorial process with a variety of manifestations. For example, aneurysms are pathological expansions of an artery that can lead to rupture, while ischemic atherosclerosis reduces blood flow, increasing the risk of claudication, poor wound healing, limb amputation, and stroke. Current PAD treatment is often ineffective or associated with serious risks, largely because these disorders are commonly undiagnosed or misdiagnosed. Active areas of research are focused on detecting and characterizing deleterious arterial changes at early stages using non-invasive imaging strategies, such as ultrasound, as well as emerging OPEN ACCESS Int. J. Mol. Sci. 2015, 16 11132 technologies like photoacoustic imaging. Earlier disease detection and characterization could improve interventional strategies, leading to better prognosis in PAD patients. While rodents are being used to investigate PAD pathophysiology, imaging of these animal models has been underutilized. This review focuses on structural and molecular information and disease progression revealed by recent imaging efforts of aortic, cerebral, and peripheral vascular disease models in mice, rats, and rabbits. Effective translation to humans involves better understanding of underlying PAD pathophysiology to develop novel therapeutics and apply non-invasive imaging techniques in the clinic.
... While endoglin deficient mice are embryonically lethal around embryonic day (E)10.5 and show defects in cardiac development and impaired maturation of blood vessels in the yolk sac (Bourdeau et al., 1999;Arthur et al., 2000), endoglin heterozygous mice are vital. However, adult endoglin heterozygous mice show impaired angiogenesis, AVMs and display cerebral vascular abnormalities (Satomi et al., 2003;van Laake et al., 2006). Choi et al. (2014) specifically deleted endoglin in endothelial and smooth muscle cells using the SM22α-Cre mouse model. ...
Hereditary hemorrhagic telangiectasia (HHT) or Rendu–Osler–Weber disease is a rare genetic vascular disorder known for its endothelial dysplasia causing arteriovenous malformations and severe bleedings. HHT-1 and HHT-2 are the most prevalent variants and are caused by heterozygous mutations in endoglin and activin receptor-like kinase 1, respectively. An undervalued aspect of the disease is that HHT patients experience persistent inflammation. Although endothelial and mural cells have been the main research focus trying to unravel the mechanism behind the disease, wound healing is a process with a delicate balance between inflammatory and vascular cells. Inflammatory cells are part of the mononuclear cells (MNCs) fraction, and can, next to eliciting an immune response, also have angiogenic potential. This biphasic effect of MNC can hold a promising mechanism to further elucidate treatment strategies for HHT patients. Before MNC are able to contribute to repair, they need to home to and retain in ischemic and damaged tissue. Directed migration (homing) of MNCs following tissue damage is regulated by the stromal cell derived factor 1 (SDF1). MNCs that express the C-X-C chemokine receptor 4 (CXCR4) migrate toward the tightly regulated gradient of SDF1. This directed migration of monocytes and lymphocytes can be inhibited by dipeptidyl peptidase 4 (DPP4). Interestingly, MNC of HHT patients express elevated levels of DPP4 and show impaired homing toward damaged tissue. Impaired homing capacity of the MNCs might therefore contribute to the impaired angiogenesis and tissue repair observed in HHT patients. This review summarizes recent studies regarding the role of MNCs in the etiology of HHT and vascular repair, and evaluates the efficacy of DPP4 inhibition in tissue integrity and repair.
... The authors showed that Lrg1 directly interacts with the endothelial-specific TGFβ co-receptor endoglin/CD105, thereby regulating TGFβ-induced differential activation of either smad1/5 or smad2/3 and, through this, regulating endothelial proliferation of retinal and brain endothelial cells. At this point, the contribution of Lrg1 to brain angiogenesis and BBB differentiation is unknown but endothelial Lrg1 deficiency might interfere with the proper formation of the brain vasculature, similar to the defects observed in endoglin/ CD105 heterozygous mice, which represent a model of hereditary hemorrhagic telangiectasia type 1 (Satomi 2003). ...
The blood-brain barrier (BBB) is essential for maintaining homeostasis within the central nervous system (CNS) and is a prerequisite for proper neuronal function. The BBB is localized to microvascular endothelial cells that strictly control the passage of metabolites into and out of the CNS. Complex and continuous tight junctions and lack of fenestrae combined with low pinocytotic activity make the BBB endothelium a tight barrier for water soluble moleucles. In combination with its expression of specific enzymes and transport molecules, the BBB endothelium is unique and distinguishable from all other endothelial cells in the body. During embryonic development, the CNS is vascularized by angiogenic sprouting from vascular networks originating outside of the CNS in a precise spatio-temporal manner. The particular barrier characteristics of BBB endothelial cells are induced during CNS angiogenesis by cross-talk with cellular and acellular elements within the developing CNS. In this review, we summarize the currently known cellular and molecular mechanisms mediating brain angiogenesis and introduce more recently discovered CNS-specific pathways (Wnt/β-catenin, Norrin/Frizzled4 and hedgehog) and molecules (GPR124) that are crucial in BBB differentiation and maturation. Finally, based on observations that BBB dysfunction is associated with many human diseases such as multiple sclerosis, stroke and brain tumors, we discuss recent insights into the molecular mechanisms involved in maintaining barrier characteristics in the mature BBB endothelium.
... To date, there are no animal models that truly reflect the human AVMs. Experimental models of brain AVMs have been established in rats, cats and dogs [18][19][20][21]. Especially, rat models have been most extensively investigated. ...
Arteriovenous malformations (AVMs) are a type of high-flow vascular malformations that most commonly occurs in the head and neck. They are present at birth but are usually clinically asymptomatic until later in life. The pathogenesis of AVMs remains unclear and therapeutic approaches to AVMs are unsatisfied. In order to provide a tool for studying the pathogenesis and therapies of this disease, we established and studied a xenograft animal model of human AVMs.
Fresh human AVMs specimens harvested from 4 patients were sectioned (5x5x5 mm) and xenografted subcutaneously in 5 immunologically naive nude mice (Athymic Nude-Foxn1nu). Each mouse had four pieces specimens in four quadrants along the back. The grafts were observed weekly for volume, color and texture. The grafts were harvested at every 30 days intervals for histologic examination. All grafts (n = 20) were sectioned and stained for hematoxylin and eosin (H&E). Comparative pathologic evaluation of the grafts and native AVMs were performed by two blinded pathologists. Immunohistochemical examination of human-specific nuclear antigen, vascular endothelial growth factor receptor-2 (VEGFR-2) and Ki-67 was performed.
Clinical characteristics and pathologic diagnosis of native human derived AVMs were confirmed. 85% (n = 17) of AVM xenografts survived although the sizes decreased after implantation. Histological examination demonstrated numerous small and medium-size vessels and revealed structural characteristics matching the native AVMs tissue.76.5% (n = 13) of the surviving xenografts were positive for Ki-67 and human-specific nuclear antigen suggesting survival of the human derived tissue, 52.9% (n = 9) were positive for VEGFR-2.
This preliminary xenograft animal model suggests that AVMs can survive in the nude mouse. The presence of human-specific nuclear antigen, VEGFR-2, and Ki-67 demonstrates the stability of native tissue qualities within the xenografts.
... Here, we show that endoglin associates with ALK-1 and potentiates ALK-1/Smad1-dependent signaling. Several lines of evidence in vivo support this functional collaboration between endoglin and ALK-1: (i) Mutations in endoglin or ALK-1 genes give rise to a common phenotype of vascular dysplasia in humans known as HHT1 or HHT2, respectively (Marchuk and Lux, 2001) that strongly suggest the existence of a common signaling pathway; (ii) both, endoglin and ALK-1 null mice die at day E10-11 due to cardiovascular abnormalities (Bourdeau et al., 1999;Li et al., 1999;Arthur et al., 2000;Oh et al., 2000); and (iii) haploinsufficient endoglin or ALK-1 null mice show telangiectasia and arteriovenous malformations that resemble the HHT phenotype in humans (Bourdeau et al., 1999;Satomi et al., 2003;Srinivasan et al., 2003;Torsney et al., 2003). ...
Transforming growth factor-beta (TGF-beta) signaling in endothelial cells is able to modulate angiogenesis and vascular remodeling, although the underlying molecular mechanisms remain poorly understood. Endoglin and ALK-1 are components of the TGF-beta receptor complex, predominantly expressed in endothelial cells, and mutations in either endoglin or ALK-1 genes are responsible for the vascular dysplasia known as hereditary hemorrhagic telangiectasia. Here we find that the extracellular and cytoplasmic domains of the auxiliary TGF-beta receptor endoglin interact with ALK-1 (a type I TGF-beta receptor). In addition, endoglin potentiates TGF-beta/ALK1 signaling, with the extracellular domain of endoglin contributing to this functional cooperation between endoglin and ALK-1. By contrast, endoglin appears to interfere with TGF-beta/ALK-5 signaling. These results suggest that the functional association of endoglin with ALK-1 is critical for the endothelial responses to TGF-beta.
... Young adult pigs (Sus scrofa; = 5) and adult chinchillas (Chinchilla laniger; = 6) were used. In the anesthetized animal, a transcardial [5,6] or aortic perfusion [7] of the head and upper body was carried out, clamping off lower body vessels. The right atrium was incised to allow venous return of perfusate. ...
The mammalian ear has an extraordinary capacity to detect very low-level acoustic signals from the environment. Sound pressures as low as a few μ Pa (-10 dB SPL) can activate cochlear hair cells. To achieve this sensitivity, biological noise has to be minimized including that generated by cardiovascular pulsation. Generally, cardiac pressure changes are transmitted to most peripheral capillary beds; however, such signals within the stria vascularis of the cochlea would be highly disruptive. Not least, it would result in a constant auditory sensation of heartbeat. We investigate special adaptations in cochlear vasculature that serve to attenuate cardiac pulse signals. We describe the structure of tortuous arterioles that feed stria vascularis as seen in corrosion casts of the cochlea. We provide a mathematical model to explain the role of this unique vascular anatomy in dampening pulsatile blood flow to the stria vascularis.
... Similar to clinical symptoms of HHT, age of onset, severity, and location of HHT-like vascular abnormalities in Eng +/and Alk1 +/mice are highly variable, and only 30-70% of heterozygotes displayed a detectable HHT-like phenotype (100,(104)(105)(106). While the heterozygous Eng or Alk1 knockout mice are excellent resources for identifying genetic factors (e.g. ...
Hereditary Haemorrhagic Telangiectasia (HHT, Osler–Weber–Rendu syndrome) exemplifies diseases which have catalysed advances
in the understanding of fundamental pathophysiological mechanisms. The hallmark of HHT is the development of abnormal blood
vessels, involving the lung in approximately 50% of cases. This chapter will focus on the molecular mechanisms that underlie
their generation. While not all clinical problems in HHT can be directly attributed to the presence of abnormal vessels, the
emergent data suggesting non-vascular sequelae of the underlying gene mutations are beyond the scope of this chapter.
KeywordsOsler-Weber-Rendu-haemoptysis-epistaxis-arteriovenous malformation-endoglin-angiogenesis
... Patients with hereditary hemorrhagic telangiectasia (OMIM#187300) have a much higher prevalence of bAVM than does the generation population, on the order of ≈1000fold higher in hereditary hemorrhagic telangiectasia-1 caused by endoglin (ENG) haploinsufficiency and ≈100fold higher in hereditary hemorrhagic telangiectasia-2 with activin receptor-like kinase (ALK1) haploinsufficiency. 2 Vascular lesions developed in various organs in Eng +/−6 and Alk1 +/− adult mice, 7 but spontaneous lesions in the brain are quite modest, and are only seen in older mice. 8,9 Our group demonstrated greatly increased brain microvascular dysplasia after virally mediated VEGF stimulation in both Eng +/− and Alk1 +/− mice. 10 Somatic conditional deletion of Alk1 in adult mice induced arteriovenous fistulas and hemorrhage in the lung and gastrointestinal tract, but not in the skin or brain. ...
Vascular endothelial growth factor (VEGF) expression is elevated in human brain arteriovenous malformations (bAVM). We have developed a bAVM model in the adult mouse by focal Alk1 gene deletion and human VEGF stimulation. We hypothesized that once the abnormal vasculature has been established, tonic VEGF stimulation is necessary to maintain the abnormal phenotype, and VEGF antagonism by bevacizumab (Avastin) would reduce vessel density and attenuate the dysplastic vascular phenotype.
Angiogenesis and bAVM were induced by injection of adeno-associated viral vector expressing human VEGF alone into the brain of wild-type mice or with adenoviral vector expressing Cre recombinase (Ad-Cre) into Alk1(2f/2f) mice. Six weeks later, bevacizumab or trastuzumab (Herceptin, bevacizumab control) was administered. Vessel density, dysplasia index, vascular cell proliferation and apoptosis, and human IgG were assessed (n=6/group).
Compared with trastuzumab (15 mg/kg), administration of 5, 10, and 15 mg/kg of bevacizumab to adeno-associated viral vector expressing human VEGF treated wild-type mice reduced focal vessel density (P<0.05); administration of 5 mg/kg bevacizumab decreased proliferating vascular cells (P=0.04) and increased TUNEL-positive vascular cells (P=0.03). More importantly, bevacizumab (5 mg/kg) treatment reduced both vessel density (P=0.01) and dysplasia index (P=0.02) in our bAVM model. Human IgG was detected in the vessel wall and in the parenchyma in the angiogenic foci of bevacizumab-treated mice.
We provide proof-of-principle that, once abnormal AVM vessels have formed, VEGF antagonism may reduce the number of dysplastic vessels and should be evaluated further as a therapeutic strategy for the human disease.
TGF-β/ALK1/ENG signaling pathway maintains quiescent state of endothelial cells, but at the same time, it regulates neutrophil functions. Importantly, mutations of this pathway lead to a rare autosomal disorder called Hereditary Hemorrhagic Telangiectasia (HHT), characterized with abnormal blood vessel formation (angiogenesis). As neutrophils are potent regulators of angiogenesis, we investigated how disturbed TGF-β/ALK1/ENG signaling influences angiogenic properties of these cells in HHT. We could show for the first time that not only endothelial cells, but also neutrophils isolated from such patients are ENG/ALK1-deficient. This deficiency obviously stimulates proangiogenic switch of such neutrophils. Elevated proangiogenic activity of HHT neutrophils is mediated by the increased spontaneous degranulation of gelatinase granules, resulting in high release of matrix-degrading MMP9. In agreement, therapeutic disturbance of this process using Src tyrosine kinase inhibitors impaired proangiogenic capacity of such neutrophils. Similarly, inhibition of MMP9 activity resulted in significant impairment of neutrophil-mediated angiogenesis. All in all, deficiency in TGF-β/ALK1/ENG signaling in HHT neutrophils results in their proangiogenic activation and disease progression. Therapeutic strategies targeting neutrophil degranulation and MMP9 release and activity may serve as a potential therapeutic option for HHT.
Blood vessels form tubular networks that ramify throughout the organism and are essential for transport of metabolites into and out of tissues. The mature blood vasculature consists of a hierarchical circuit of interconnected vessels of various sizes, including arteries, arterioles, capillaries, venules, and veins, which are connected to the heart and allow closed blood circulation between the heart and all other body tissues. The lymphatic vasculature is a parallel, open network that absorbs extracellular fluids, immune cells, and intestinal lipids and returns them to the venous circulation. In this chapter, we will describe cellular and molecular mechanisms controlling development of the vascular and lymphatic systems.
Cerebrovascular malformations are a major cause of intracranial hemorrhage in young adults and children. They are resource-intensive to manage with invasive treatment modalities, and there is no current medical therapy option available for patients. A better understanding of the genetics and biology of cerebrovascular malformations is needed to elucidate common biologic mechanisms and potential therapeutic targets. Different cerebrovascular malformations have varying genetic etiology but share commonalities in signaling pathways affecting angiogenesis, vascular remodeling, inflammation, and response to injury. Inherited mutations underlie hereditary hemorrhagic telangiectasia (HHT), cerebral cavernous malformations (CCMs), capillary malformation-arteriovenous malformation (CM-AVM), and venous malformations. Somatic mutations have now been identified in most cerebrovascular malformations, including most recently for sporadic brain AVM, further suggesting common pathways affecting downstream ERK signaling as a potential therapeutic target. Additional modifier genes and other factors are emerging as important predictors explaining the variable expressivity of the disease and may shed light on the progression of these related vascular malformations. In this chapter, we focus on the vascular biology and genetics of brain AVMs and CCMs; other cerebrovascular malformations are also discussed briefly.
Background:
Brain arteriovenous malformations (BAVMs) are vascular malformations composed of tangles of abnormally developed vasculature without capillaries. Abnormal shunting of arteries and veins is formed, resulting in high-pressure vascular channels, which potentially lead to rupture. BAVMs are generally considered a congenital disorder. But clinical evidence regarding involution, regrowth, and de novo formation argue against the static condition of this disease. Recently, the presence of the somatic activating KRAS mutations in more than half of BAVM cases was reported, suggesting the role of KRAS function in the pathogenesis.
Methods:
KRAS mutation in codon35 (G→A, G12D; G→T, G12V) was examined by a digital polymerase chain reaction analysis using genome purified from paraffin-embedded slides of human BAVMs. We also examined protein expression of KRAS G12D in lesions to corroborate results from digital polymerase chain reaction analysis.
Results:
We detected codon35 G→A mutation in 15 (39.5%) among 38 samples and codon35 G→T mutation in 10 (27.0%) among 37 samples we could assess mutations. There were no samples positive for both codon35 G→A and G→T mutation. The ratio of codon35 G→A mutation ranged from 0.60% to 12.28% and that of G→T was from 1.20% to 8.99%. We next examined protein expression of KRAS G12D in BAVM lesions in immunohistochemistry. A KRAS G12D mutant was detected mainly in endothelial cells of dilated vessels in lesions.
Conclusions:
KRAS mutations in codon35 were detected in about two thirds of specimens examined. KRAS function may actively contribute to the pathobiology of BAVM and can become a therapeutic target.
Intracranial AVMs are complex lesions requiring multidisciplinary expertise for optimal patient outcomes. While the optimal treatment depends on AVM-specific factors such as rupture status, size, location within the brain, and the presence of deep venous drainage, the treating neurosurgeon must be able to synthesize the risks and benefits of multiple treatment options to best determine the safest treatment modality for each patient. In this chapter we present current review on best practices for the treatment of intracranial AVMs.
Background
Brain arteriovenous malformations (BAVM) represent a congenital anomaly of the cerebral vessels with a prevalence of 10–18/100 000. BAVM is the leading aetiology of intracranial haemorrhage in children. Our objective was to identify gene variants potentially contributing to disease and to better define the molecular aetiology underlying non-syndromic sporadic BAVM.
Methods
We performed whole-exome trio sequencing of 100 unrelated families with a clinically uniform BAVM phenotype. Pathogenic variants were then studied in vivo using a transgenic zebrafish model.
Results
We identified four pathogenic heterozygous variants in four patients, including one in the established BAVM-related gene, ENG, and three damaging variants in novel candidate genes: PITPNM3, SARS and LEMD3, which we then functionally validated in zebrafish. In addition, eight likely pathogenic heterozygous variants (TIMP3, SCUBE2, MAP4K4, CDH2, IL17RD, PREX2, ZFYVE16 and EGFR) were identified in eight patients, and 16 patients carried one or more variants of uncertain significance. Potential oligogenic inheritance (MAP4K4 with ENG, RASA1 with TIMP3 and SCUBE2 with ENG) was identified in three patients. Regulation of sma- and mad-related proteins (SMADs) (involved in bone morphogenic protein (BMP)/transforming growth factor beta (TGF-β) signalling) and vascular endothelial growth factor (VEGF)/vascular endotheliual growth factor recepter 2 (VEGFR2) binding and activity (affecting the VEGF signalling pathway) were the most significantly affected biological process involved in the pathogenesis of BAVM.
Conclusions
Our study highlights the specific role of BMP/TGF-β and VEGF/VEGFR signalling in the aetiology of BAVM and the efficiency of intensive parallel sequencing in the challenging context of genetically heterogeneous paradigm.
Arteriovenous malformations are the most dangerous vascular malformations and extremely difficult to treat. While most of them are sporadic, some are associated with autosomal dominant disorders, such as hereditary hemorrhagic telangiectasia, PTEN hamartoma tumor syndrome, and capillary malformation-arteriovenous malformation. Although important advances have been made in the diagnosis and treatment of arteriovenous malformations, the pathogenic mechanisms remain poorly understood. Yet, this is an essential step towards the development of targeted therapies. Here, we discuss the most recent insights on arteriovenous malformations, on the basis of studies on arteriovenous differentiation in animal models, and the monogenic disorders with a predisposition to arteriovenous malformations.
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal-dominant, highly penetrant and variable multisystem disorder due to maldevelopment of capillaries. The condition used to be called Osler–Weber–Rendu syndrome. The major features are epistaxis; mucocutaneous telangiectasias; and visceral arteriovenous malformations (especially in the brain, gastrointestinal tract, liver and lung). The causes defined thus far are in genes involved in signaling through the transforming growth factor β/bone morphogenetic protein pathway in endothelial cells. Early diagnosis, clinical screening and aggressive management can greatly reduce morbidity and mortality.
Hereditary hemorrhagic telangiectasia (HHT), also known as Osler-Rendu-Weber disease, is an autosomal dominant disorder that results from multi-system vascular dysplasia. It is not a rare condition, but is under-recognized, and is characterized by the presence of mucocutaneous telangiectases and arteriovenous malformations (AVMs) of the brain, lung, liver, and spinal cord. Neurological manifestations may develop due to paradoxical embolisms from a pulmonary AVM or hemorrhage of AVMs of the brain and spinal cord. This article summarizes the clinical features of HHT as well as its treatment, and also emphasizes the need for a high index of suspicion for this disease in patients with characteristic clinical manifestations.
Neither the pathogenesis nor the natural history of brain arteriovenous malformations (BAVMs) and dural arteriovenous fistulas (DAVFs) has as yet been fully elucidated. BAVMs are generally considered to be congenital vascular disorders, while some factors after birth may still influence their genesis or growth. Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia characterized by dilated vessels and AVMs, and is also known as a genetic disease associated with BAVMs. Three genes, endoglin, ALK-1 and SMAD-4 are associated with HHT. The natural history of BAVMs is influenced by angioarchitecture and prior hemorrhage. DAVFs are considered as an acquired lesions；however, their pathogenesis is not clear. The natural history of DAVFs is influenced by not only by the lesion’s angioarchitecture such as its cortical venous drainage, but also by the patient’s symptomatology. Furthermore DAVF can alter its angioarchitecture with proliferative changes in the arterial component and occlusive changes in venous drainage. © 2013 Japanese Congress of Neurological Surgeons. All rights reserved.
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder that predisposes patients to develop direct connections between arteries and veins, or arteriovenous malformations (AVMs). Although the genes responsible for the majority of HHT cases have been known for nearly 20 years, molecular and cellular mechanisms underlying pathogenesis are poorly understood, and the genetic and/or environmental factors that confer variability to age of onset and expressivity of HHT remain unknown. Herein, we review the genetics and genotype/phenotype correlations associated with HHT and summarize data from animal and cell culture models that lend insight into disease mechanism. At present, therapies available to HHT patients for treatment of visceral AVMs are primarily surgical, although antiangiogenic agents show some efficacy in treatment of telangiectasias, epistaxis, and liver AVMs. In light of new mechanistic data on disease pathogenesis, we consider possible approaches for development of more targeted therapeutics for HHT patients.
Background and purpose:
Endoglin deficiency causes hereditary hemorrhagic telangiectasia-1 and impairs myocardial repair. Pulmonary arteriovenous malformations in patients with hereditary hemorrhagic telangiectasia-1 are associated with a high incidence of paradoxical embolism in the cerebral circulation and ischemic brain injury. We hypothesized that endoglin deficiency impairs stroke recovery.
Methods:
Eng heterozygous (Eng+/-) and wild-type mice underwent permanent distal middle cerebral artery occlusion (pMCAO). Pial collateral vessels were quantified before pMCAO. Infarct/atrophic volume, vascular density, and macrophages were quantified in various days after pMCAO, and behavioral function was assessed using corner and adhesive removal tests on days 3, 15, 30, and 60 after pMCAO. The association between ENG 207G>A polymorphism and brain arteriovenous malformation rupture and surgery outcome was analyzed using logistic regression analysis in 256 ruptured and 157 unruptured patients.
Results:
After pMCAO, Eng+/- mice showed larger infarct/atrophic volumes at all time points (P<0.05) and showed worse behavior performance (P<0.05) at 15, 30, and 60 days when compared with wild-type mice. Eng+/- mice had fewer macrophages on day 3 (P=0.009) and more macrophages on day 60 (P=0.02) in the peri-infarct region. Although Eng+/- and wild-type mice had similar numbers of pial collateral vessels before pMCAO, Eng+/- mice had lower vascular density in the peri-infarct region (P=0.05) on day 60 after pMCAO. In humans, ENG 207A allele has been associated with worse outcomes after arteriovenous malformation rupture or surgery of patients with unruptured arteriovenous malformation.
Conclusions:
Endoglin deficiency impairs brain injury recovery. Reduced angiogenesis, impaired macrophage homing, and delayed inflammation resolution could be the underlying mechanism.
Patients harboring brain arteriovenous malformation (bAVM) are at life-threatening risk of rupture and intracranial hemorrhage (ICH). The pathogenesis of bAVM has not been completely understood. Current treatment options are invasive, and ≈ 20 % of patients are not offered interventional therapy because of excessive treatment risk. There are no specific medical therapies to treat bAVMs. The lack of validated animal models has been an obstacle for testing hypotheses of bAVM pathogenesis and testing new therapies. In this review, we summarize bAVM model development and bAVM pathogenesis and potential therapeutic targets that have been identified during model development.
Although some cases of vein of Galen aneurysmal malformation (VGAM) present initial clinical symptoms such as cardiopulmonary disturbance in the neonatal period, pial arteriovenous fistula is very seldom present as a clinical symptom immediately after birth. A neonatal patient, the first-born to his family, presented with tachypnea postpartum. This baby had a family history of hereditary hemorrhagic telangiectasia. A cerebral MR image revealed multiple macrocerebral arteriovenous fistulas (MCAVFs), resulting in a large partially thrombosed venous pouch within the cerebral cortex. Trans-arterial embolizations of the main two fistulas were performed using N-butyl cyanoacrylate (NBCA) with tantalum powder six months after birth. Post-embolization angiography confirmed the obliteration of the fistulas and magnetic resonance imaging (MRI) revealed thrombosis and reduction in size of the venous component. His tachypnea disappeared completely. There were no neurological complications due to the treatment. The prognosis of multiple MCAVFs mainly depends on the presence of medullar signs and symptoms and a delay before treatment. Pure glue endovascular intervention, as used in our case, is considered to be the first therapeutic choice to decrease the risk of neurological consequences.
Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystemic vascular dysplasia and recurrent haemorrhage. Linkage for some families has been established to chromosome 9q33-q34. In the present study, endoglin, a transforming growth factor beta (TGF-beta) binding protein, was analysed as a candidate gene for the disorder based on chromosomal location, expression pattern and function. We have identified mutations in three affected individuals: a C to G substitution converting a tyrosine to a termination codon, a 39 base pair deletion and a 2 basepair deletion which creates a premature termination codon. We have identified endoglin as the HHT gene mapping to 9q3 and have established HHT as the first human disease defined by a mutation in a member of the TGF-beta receptor complex.
129/Ola mice resemble WEHI 129J mice in that around 70% of the individuals in any given population resist a primary infection with Schistosoma mansoni. Squashed-organ autoradiographic tracking of [75Se]selenomethionine-labelled parasites has shown that the kinetics of worm migration in 129/Ola mice follows the expected pattern, and that all rodents harbour essentially similar numbers of worms on day 14 post-infection. Combined lung and liver worm recovery techniques have revealed, however, that segregation of mice into 'permissive' and 'non-permissive' individuals can first be detected on day 20. 'Non-permissive' mice are characterized by the absence of schistosome eggs at all times in the liver parenchyma and, in consequence, lack the attendant manifestations of pathology; they do, however, harbour a few stunted worms in the liver and significant numbers of adult schistosomes in the pulmonary vasculature. Histological analysis of sectioned lung tissue from such animals indicates that some lung-located schistosomes feed, pair and lay eggs. Nevertheless, eosinophil-enriched inflammatory reactions develop around such worms and the parasites themselves exhibit various manifestations of trauma, ranging from minor vacuolation to gut herniation and extrusion. The phenomenon of 'non-permissiveness' thus involves retardation of worm development in the liver and, in consequence, relocation of the parasites to the lungs, where they become subject to host effector responses.
Changes in shape and cytoskeletal structure of vascular endothelial cells induced by fluid-imposed shear stress were studied in vivo and in vitro. In in vivo experiments, aortic tissue specimens including the flow dividers of the branching of the left subclavian artery and the aortic intercostal ostium were obtained and their endothelial cell shapes were observed using the scanning electron microscope. It was found that the shape and orientation of endothelial cells were strongly affected by blood flow conditions. In in vitro cell culture experiments, porcine aortic endothelial cells were cultured on glass coverslips and extracellular matrix coated (ECM coated) glass coverslips and exposed to shear stress using a parallel plate flow chamber. When we applied a shear stress of 2 Pa for 24 hrs, the rearrangement of F-actin filaments occurred within 3 hrs and preceded the cell shape change in the early stage after shear exposure. The endothelial cells on ECM-coated coverslips exhibited more elongated cell shapes even under no-flow conditions. After exposure to shear stress, the endothelial cells on ECM-coated glass showed more retarded elongation and orientation to the direction of flow than those on no-coated glass, suggesting that the anchorage to the substrate was enhanced by ECMs.
Endoglin (CD105), a component of the TGF-beta 1 receptor complex, is the target gene for the dominantly inherited vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1). We have identified a novel endoglin splice site mutation, leading to an in-frame deletion of exon 3, in a new-born from a family with HHT. Expression of normal and mutant endoglin proteins was analyzed in umbilical vein endothelial cells from this baby and in activated monocytes from the affected father. In both samples, only normal dimeric endoglin (160 kD) was observed at the cell surface, at 50% of control levels. Despite an intact transmembrane region, mutant protein was only detectable by metabolic labeling, as an intracellular homodimer of 130 kD. In monocytes from three clinically affected HHT1 patients, with known mutations creating premature stop codons in exons 8 and 10, surface endoglin was also reduced by half and no mutant was detected. Overexpression into COS-1 cells of endoglin cDNA truncated in exons 7 and 11, revealed their intracellular expression, inability to be secreted and to form heterodimers at the cell surface. These results indicate that mutated forms of endoglin are transiently expressed intracellularly and not likely to act as dominant negative proteins, as proposed previously. A reduction in the level of functional endoglin is thus involved in the generation of HHT1, and associated arteriovenous malformations.
Our goal was to describe the prevalence and types of cerebral vascular malformations (CVMs) seen with MR imaging in patients with hereditary hemorrhagic telangiectasia (HHT).
We reviewed retrospectively the brain MR images of 184 consecutive patients with HHT. Catheter angiography was performed in 17 patients with CVMs detected on MR images.
MR imaging revealed 63 CVMs in 42 patients. Classic arteriovenous malformations (n = 10) had a conspicuous network of vessels with flow voids and enlarged adjacent pial vessels. Apparent venous malformations (n = 5) were best seen after administration of contrast material as a prominent vessel coursing through normal brain parenchyma. Indeterminate vascular malformations (n = 48) had a spectrum of appearances characterized by variable combinations of heterogeneous signal intensity, enhancement, or hemosiderin. Angiography in 17 patients revealed 47 CVMs. Forty-six were arteriovenous malformations (AVMs), including 25 CVMs not seen with MR imaging and 21 CVMs that by MR criteria included 8 AVMs and 13 indeterminate vascular malformations. Angiography confirmed 1 venous malformation seen with MR imaging but failed to detect 3 indeterminate lesions revealed by MR imaging.
MR imaging of a large cohort of consecutive patients with HHT revealed a CVM prevalence of 23% (42/184). Most CVMs (48/63) have an atypical appearance for vascular malformations on MR images. Angiographic correlation suggests that MR imaging underestimates the prevalence of CVMs and that the majority of indeterminate CVMs, despite their variable MR appearance, are AVMs.
Endoglin is a transforming growth factor-beta (TGF-beta) binding protein expressed on the surface of endothelial cells. Loss-of-function mutations in the human endoglin gene ENG cause hereditary hemorrhagic telangiectasia (HHT1), a disease characterized by vascular malformations. Here it is shown that by gestational day 11.5, mice lacking endoglin die from defective vascular development. However, in contrast to mice lacking TGF-beta, vasculogenesis was unaffected. Loss of endoglin caused poor vascular smooth muscle development and arrested endothelial remodeling. These results demonstrate that endoglin is essential for angiogenesis and suggest a pathogenic mechanism for HHT1.
Endoglin (CD105), an accessory protein of the TGF-beta receptor superfamily, is highly expressed on endothelial cells. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is associated with mutations in the Endoglin gene, leading to haploinsufficiency. To generate a disease model and ascertain the role of endoglin in development, we generated mice lacking 1 or both copies of the gene. Endoglin null embryos die at gestational day 10.0-10.5 due to defects in vessel and heart development. Vessel formation appears normal until hemorrhage occurs in yolk sacs and embryos. The primitive vascular plexus of the yolk sac fails to mature into defined vessels, and vascular channels dilate and rupture. Internal bleeding is seen in the peritoneal cavity, implying fragile vessels. Heart development is arrested at day 9.0, and the atrioventricular canal endocardium fails to undergo mesenchymal transformation and cushion-tissue formation. These data suggest that endoglin is critical for both angiogenesis and heart valve formation. Some heterozygotes, either with an inbred 129/Ola or mixed C57BL/6-129/Ola background, show signs of HHT, such as telangiectases or recurrent nosebleeds. In this murine model of HHT, it appears that epigenetic factors and modifier genes, some of which are present in 129/Ola, contribute to disease heterogeneity.
Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal-dominant vascular dysplasia with a high prevalence of cerebrovascular malformations (CVMs), mostly manifested as arteriovenous malformations (AVMs). The natural history and bleeding risk of these CVMs is unknown. The authors investigated the risk of bleeding in conjunction with clinical and radiological features in patients with HHT and proven CVMs.
Intravenous digital subtraction (DS) angiography was used to screen 196 patients with HHT for the presence of CVMs. Patients with abnormal results on DS angiography were asked to undergo a conventional cerebral angiographic study. All patients with a proven CVM were assessed by a neurologist. The bleeding risk was retrospectively and prospectively calculated for patients with AVMs only, as well as for the whole cohort of patients with CVMs. Twenty-four patients (12.2%; 16 female and eight male), aged 14 to 66 years (mean 35.4 years) with one or more CVMs were identified. Fifteen patients (62.5%) had a CVM and a pulmonary AVM. Eleven patients (45.8%) exhibited no neurological signs of their CVM; six (25%) had headache or migraine; four (16.7%) had seizures; and three (12.5%) had an intracranial hemorrhage. Twenty-two patients had at least one AVM (with a total of 28 AVMs), whereas two patients only had telangiectases. Twenty-seven AVMs were small (96%), 36% were located in eloquent areas of the brain, and 82% had superficial venous drainage. One third of the patients had multiple CVMs. The bleeding risk for patients with at least one AVM ranged from 0.41 to 0.72% per year, and for the whole cohort the range was 0.38 to 0.69% per year. Calculation of the bleeding risk as determined by lesion-years ranged from 0.36 to 0.56% per year for patients with AVMs and from 0.27 to 0.46% per year for all patients with CVMs.
Patients with HHT have a high risk of harboring a CVM, especially in the presence of a pulmonary AVM. These CVMs are mostly low-grade AVMs (Spetzler-Martin Grade I or II), are frequently multiple, and have a lower risk of bleeding than that associated with sporadic AVMs. Female patients are more often affected than male patients. The inherent low sensitivity of DS angiography screening for CVMs may yield false negative results.
Hereditary hemorrhagic telangiectasia type 1 (HHT1) is associated with mutations in the ENDOGLIN gene which normally codes for a polypeptide of 653 amino acids expressed at the cell surface as a dimeric glycoprotein. To maximize the detection of potential mutant proteins, we analyzed by pulse-chase experiments the expression of large truncation mutants in endothelial cells from newborns with HHT1. A mutant truncated at residue 490 (Delta490) and the Delta517 mutant, previously suggested to act as dominant negative, were undetectable. Proteins Delta471 and Delta571 were barely detectable as transient monomers of 62 and 72 kDa. A de novo 13 bp deletion in exon 11 encoded a monomeric protein of 70 kDa (Delta557), present at low levels in activated monocytes. Six novel missense mutants and DeltaS411 were expressed only as the 80 kDa intracellular precursor of surface endoglin, suggesting impaired processing. All nine novel mutations reported failed to be expressed other than intracellularly. Several constructs of endoglin were expressed in COS-1 cells; only the full-length protein was processed to the cell surface. Recombinant Delta586, corresponding to the complete extracellular domain, was secreted as monomeric and dimeric glycosylated species. Our studies show that all HHT1 mutants analyzed, although expressed to various degrees in COS-1 cells, are either undetectable, present at low levels as transient intracellular forms, or expressed as partially glycosylated precursors in endogenous cells. These mutants do not form heterodimers with normal endoglin and do not interfere with its normal trafficking to the cell surface, further supporting the haploinsufficiency model.
Endoglin is a transforming growth factor–β (TGF-β) binding protein expressed on the surface of endothelial cells. Loss-of-function
mutations in the human endoglin gene ENGcause hereditary hemorrhagic telangiectasia (HHT1), a disease characterized by vascular malformations. Here it is shown that
by gestational day 11.5, mice lacking endoglin die from defective vascular development. However, in contrast to mice lacking
TGF-β, vasculogenesis was unaffected. Loss of endoglin caused poor vascular smooth muscle development and arrested endothelial
remodeling. These results demonstrate that endoglin is essential for angiogenesis and suggest a pathogenic mechanism for HHT1.
Hereditary hemorrhagic telangiectasia (HHT) is a dominantly inherited vascular disorder that is heterogeneous in terms of age of onset and clinical manifestations, Endoglin is the gene mutated in HHT1, which is associated with a higher prevalence of pulmonary arteriovenous malformations than HHT2, where ALK-1 is the mutated gene. Endoglin is constitutively expressed on endothelial cells and inducible on peripheral blood activated monocytes so that protein levels can be measured by metabolic labeling and immunoprecipitation. We report the analysis of umbilical vein endothelial cells in 28 newborns from 24 families with a clinical diagnosis of HHT. Reduced levels of endoglin were observed in umbilical vein endothelial cells in 15/28 subjects and in activated monocytes of all clinically affected relatives tested, suggesting that these individuals had HHT1. No mutant protein was expressed at the cell surface in any of these cases, and a transient intracellular species was seen in samples of only two families, supporting a haploinsufficiency model. Quantitative multiplex PCR fragment analysis was established for the endoglin gene and revealed six mutations that were confirmed by automated DNA sequencing. An additional 10 mutations were identified in newborns by sequencing all exons. Of the 16 mutations, 10 were novel, three had been independently identified in related families, and three were previously known. Our data confirm that endoglin levels correlate with the presence or absence of mutation in HHT1 families, allowing the early identification of affected newborns that should be screened clinically to avoid serious complications of this disorder, such as cerebral arteriovenous malformations.
Vascular casts of the rat aorta were made using modified Batson's No. 17 anatomical corrosion compound. The outline of the silver-stained endothelial cell boundaries were clearly visible on the cast when examined using both scanning electron and light microscopy. The physical properties of the injectate were determined. The exothermic nature of the polymerization of the injectate did not grossly damage the endothelial cells. A method was developed for recovery, without damage, of the arterial tissue surrounding the cast. The technique of vascular casting appears to be a powerful tool for the study of the arterial endothelium which avoids shrinkage artifacts and maintains 3-dimensional geometry.
Vascular casts were made of rabbit aortas by infusing Batson's No. 17 anatomical corrosion compound into the artery at physiological pressure. The arterial tissue was then digested with sodium hydroxide and the cast viewed by scanning electron microscopy (SEM). Outlines of the endothelial cells and their silver stained boundaries were clearly visible. Cell nuclei and fine surface detail were also discernible. In EDTA damaged arteries, injured endothelial cells and platelets could also be observed in the vascular casts.
Arterial branch sites have very high intramural stresses at physiologic intraluminal pressures; the same sites have a predilection for atherosclerosis. The effect of intramural stress on endothelial cell morphology was investigated. Five rabbits had permanent casts placed around a segment of the abdominal aorta-left renal artery branch area during controlled hypotension, thus reducing intramural stress without narrowing the lumen. These five animals, and three normal rabbits, were sacrificed after 4-8 weeks, and the vessels were perfused with buffered 2.5% glutaraldehyde for 2 h at 100 mm Hg pressure. The aortas were examined by scanning electron microscopy. In normal aortas, the distal region of the ostia of the left renal and celiac arteries just beyond the flow divider displayed many morphologically altered endothelial cells ranging from spindle shape to cobble-stone shape. The same aortic area of casted rabbits, as well as the straight abdominal aorta in all rabbits, showed a smooth surface of endothelial cells with intact cell borders and no morphologically altered cells. At branch sites, the occurrence of morphologically altered endothelial cells may be due to increased intramural stress. When intramural stress is reduced, the morphology of branch endothelial cells changes to resemble that of the unbranched regions. In conclusion, endothelial cell morphology changes in response to changes in intramural stress.
The present paper states very briefly the main steps leading to the technique of scanning electron microscopy (SEM) of vascular corrosion casts. From the terms presently used (injection method, microcorrosion cast, injection replica, vascular corrosion cast, vascular cast) the use of "vascular corrosion cast" for lymphatic and blood vessels is recommended. Specification and pretreatment (kind, volume, dosage of anticoagulants, vasoactive substances and spasmolytica used) of the animals examined are referenced as they are available from the literature. The recommendation is given to pay more attention to these parameters than done so far. The steps necessary for producing reasonable and suitable vascular corrosion casts are critically described. Special attention is paid to the physical and chemical properties of the casting media and their significance for polymerization, shrinkage, casting quality, corrosion resistance, and thermal and spatial stability. Emphasis is also focused on the advantages of cutting the vascular corrosion casts embedded in an ice block by a band saw, a self constructed multi-blade cutting device or a mini wheel-saw placed in the chamber of a cryomicrotome. From the drying methods presently used freeze-drying is stressed because of minimal specimen damage. To render casts conductive in most cases sputter-coating is sufficient. It is recommended to run the SEM with 5-10 kV since the resolution received still reveals all details the casting media presently can replicate. Further the application of scanning electron microscopy of vascular corrosion casts in fully differentiated normal tissue, in pathologic tissue as well as in developing tissues and organs is stated. Lastly possibilities and conditions are discussed under which SEM of vascular corrosion casts can serve to quantify vascular structures in order to make the technique more than pure descriptive.
The integrity of the hepatic portal vasculature was examined, relative to the resistance to Schistosoma mansoni observed in 68% of 129/Ola mice. The passage of microspheres to the lungs, following their injection via the superior mesenteric vein, indicated the presence of shunts in the majority of both naive and infected mice. There was a negative association between shunting of microspheres to the lungs and paucity of liver worms at 28/35 days post-infection. Schistosomula accumulated in the livers of resistant mice at a slower rate than in susceptible animals, and after day 21 relocated to the lungs. Many lung schistosomula injected via the superior mesenteric passed immediately to the lungs; the shunts thus greatly reduce the probability of trapping in the liver. Some parasites migrated back from the lungs, successfully lodged in the liver and began to feed on blood. Latex infusion demonstrated the location of large intrahepatic connections between the portal and hepatic veins. We suggest that as these liver worms grow, migrating upstream into progressively larger vessels, they reach the connections, pass out of the hepatic portal system, and relocate to the lungs. The presence of the natural shunts thus accounts for the resistant status of the mice.
Vascular endothelial cells appear to be aligned with the flow in the immediate vicinity of the arterial wall and have a shape which is more ellipsoidal in regions of high shear and more polygonal in regions of low shear stress. In order to study quantitatively the nature of this response, bovine aortic endothelial cells grown on Thermanox plastic coverslips were exposed to shear stress levels of 10, 30, and 85 dynes/cm2 for periods up to 24 hr using a parallel plate flow chamber. A computer-based analysis system was used to quantify the degree of cell elongation with respect to the change in cell angle of orientation and with time. The results show that (i) endothelial cells orient with the flow direction under the influence of shear stress, (ii) the time required for cell alignment with flow direction is somewhat longer than that required for cell elongation, (iii) there is a strong correlation between the degree of alignment and endothelial cell shape, and (iv) endothelial cells become more elongated when exposed to higher shear stresses.
PRECAPILLARY arteriovenous anastomoses are a normal part of the microcirculatory bed of skin, muscle, lung, heart, intestine, liver, spleen, kidney, ear, and eye.1,2 These have been labeled "preferential channels"3 or "throughfare channels" by Chambers and Zweifach.4 Whether similar anastomoses occur in the brain parenchyma has been a subject of debate. Rowbotham and Little5-9 observed precapillary arteriovenous anastomoses in the pial circulatory system but not in the brain parenchyma of man, while Pfeifer10,11 and Solnitsky12 saw them in both.
Benda and Brownell13 and Swank and Hain14 injected particles up to 60μ in diameter into the carotid arteries of dogs and observed that they passed to the cerebral veins. Because the average capillary diameter has been found to be 5.8μ by Ueda,15 the passage of large emboli from artery to vein means that there is a shunt bypassing the capillaries or that
The objective of the study was (1) to measure systematically the orientation, morphology, and population density of endothelial nuclei of the canine thoracic aorta and its major branches and (2) to obtain evidence in a chronic in vivo preparation that altered flow patterns do indeed change patterns of nuclear orientation. For this purpose, a segment of the descending thoracic aorta was removed, opened longitudinally, and reclosed to form a tube with a new longitudinal axis 90° from the original vessel axis. The new segment was then sutured back in place. The animals were killed at suitable postoperative periods. Endothelial nuclear patterns were studied from en face photomicrographs of preparations stained with Evans blue dye. Results indicated: (1) In uniform vessel segments, e.g., middle and lower descending thoracic aorta, the nuclei were oriented parallel to the axis of the blood vessel, and the ratio of major to minor axes of the nucleus was large. The flow in these regions is known to be stable. (2) Nonaxial, less-ordered nuclear orientation with smaller ratios of major to minor axes were found in entrance regions of many major arteries and in the ascending aorta. (3) In chronic studies in which the flow pattern was altered, the nuclear pattern realigned in the direction of flow within 10 days after surgery.
The production of micro-corrosion casts suitable for scanning electron microscopy (SEM) is described in this tutorial. The casts are produced by filling an internal luminal system or space with a liquid medium which becomes solid in situ. The surrounding tissue is then removed (corroded) and the resulting replica is dried, rendered conductive, and examined in the SEM. An historical review describes the evolution of the technique and provides perspective for the SEM applications. The criteria which should be fulfilled by the injection medium are listed, and the procedures used for those media which have proven successful, together with their inherent artefacts are described in detail. The two groups of media most commonly used are the rubber compounds and the polymer resins. The latter so faithfully replicate luminal surfaces that a distinction between arteries and veins can be made on the basis of endothelial cell impressions on the surface of the replica. A review of currently used non-SEM techniques provides a comprehensive analysis of the methods used to determine success with SEM micro-corrosion casting, as well as complementary methods for the visualization of vascular and alveolar systems. The paper is illustrated mainly with material from the rat brain vascular system.
A quantitative study of the en face size and shape of endothelial cells from aortic intercostal ostia has been carried out in rabbits. Photomicrographs were taken from vascular casts of the rabbit aorta and the endothelial cell outlines were analyzed quantitatively using a digitizer and digital computer. The morphology of the endothelial cells was described using 8 calculated parameters (area, perimeter, length, width, angle of orientation, width: length ratio, axis-intersection ratio and shape index). Marked changes in cell morphology were found in the regions proximal and distal to ostia as well as around flow dividers. Cells on the aorta are aligned with the flow direction, and the endothelial cells within the ostia have an angle of orientation of approximately 45 deg to the axis of the vessel. The results obtained to date suggest that endothelial cell morphology and orientation around a branch vessel may be a natural marker or indicator of the detailed features of blood flow.
We reinvestigated the still controversial existence of arterial pathways by-passing glomeruli within kidneys of rats from weaning to more than 12 months old (i.e., body weight ranging from 39 g to 643 g). For this purpose, the arterial injection of microspheres 7.5 micrometer to 17 micrometer in diameter was combined to corrosion-replication of the arterial bed of a vasodilated perfused kidney preparation. This procedure allowed easy detection of arterial by-passes with light microscope and detailed observation with scanning electron microscope. Our results clearly demonstrate the existence of various categories of arterial by-passes throughout renal cortex regardless of age. Some of them had never been described before. These vascular by-passes were found with increased frequency from superficial to juxtamedullary cortex. In the latter area, frequency was not age-dependent, and approximately 10% (range 4-22%) of juxtamedullary glomeruli were involved. Data derived from previous microsphere studies would suggest that these structures are (partially) nonfunctioning in basal physiological conditions, but more information is needed to assess their possible functional role in the rat.
Quantitative studies were done with the scanning electron microscope (SEM) on aortic endothelial cells from ten rabbits. Of these, five were plastic casts and five were dehydrated with three different, but standard, techniques. The results indicated that all forms of dehydration caused significant shrinkage artefacts and that these were different in different directions in both the thoracic and abdominal aorta. The greatest shrinkage was found with the critical point drying technique, 45% in the abdominal aorta and 31% in the thoracic aorta. In the abdominal aorta this shrinkage was mainly due to a shrinkage in length (36%) rather than a shrinkage in width (15%). In comparison, in the thoracic aorta critical point drying resulted in a 15% shrinkage in length and a 19% shrinkage in width. Air drying and alcohol dehydration caused considerable shrinkage (29% and 18% respectively in the thoracic aorta, 29% nad 36% respectively in the abdominal aorta). Directional differences were also found with these techniques, for instance alcohol dehydration in the thoracic aorta resulted in 0% shrinkage in length and 18% shrinkage in width.
A family with central nervous system (CNS) arteriovenous malformations (AVMs) and hereditary hemorrhagic telangiectasia (HHT) is reported. A 46-year-old man had an intracerebral hemorrhage. Cerebral angiography showed one AVM and two angiomas. The HHT was diagnosed because of the concomitant existence of cutaneous telangiectasia. The patient's brother had HHT and paraplegia since the age of 21. Magnetic resonance imaging revealed an old spinal cord hemorrhage. The patient's son with HHT had an intracerebral hemorrhage at age 6. Angiograms showed two AVMs and one angioma. Familial CNS AVMs with HHT are extremely rare. The loci for human leukocyte antigen of the affected cases with HHT were evaluated, and the management of CNS AVMs with HHT is discussed.
Six patients with vascular malformation of the brain in hereditary hemorrhagic telangiectasia (HHT) were reviewed to determine clinical and radiographic characteristics of these lesions. There were two patients with arteriovenous fistula (AVF), three with arteriovenous malformation (AVM), and one with multiple AVMs associated with AVF. Seizures were the most common presenting symptom (seen in three patients), and two of them had intracerebral hematomas (ICH). In the remainder, the malformations were incidentally found in the course of evaluation of other diseases. Their locations were variable, but the majority was superficially confined to the cerebral cortex. Arterial supply was from mostly one feeding artery that was a cortical branch of either anterior, middle, or posterior cerebral artery. In six of nine malformations, the venous drainage was through a superficial cerebral vein into either the superior sagittal sinus or transverse sinus. Direct surgery was done on two patients with ICH, artificial embolization on one, and stereotactic radiosurgery on one. The cerebral vascular malformations in HHT are not infrequent, and in particular the importance of computed tomography and cerebral angiography should be recognized in patients with pulmonary AVF associated with HHT.
Seventeen patients with symptomatic cerebral arteriovenous malformations (AVMs) were diagnosed between 1980 and 1990 in the Leeward Islands of the Netherlands Antilles. Five patients had multiple AVMs. The annual incidence of symptomatic AVMs was 1.1/100,000. The mean age of presentation was 35 years. In 6 patients cerebral AVMs were associated with hereditary hemorrhagic telangiectasia (HHT); 4 of these patients had multiple AVMs. We conclude that HHT is frequently encountered in Netherlands Antillians with symptomatic and multiple cerebral AVMs.
Pulmonary arteriovenous malformations (PAVMs) occur in up to 27% of patients with hereditary haemorrhagic telangiectasia (HHT) and are associated with a rate of paradoxical cerebral embolism at presentation of up to 36%. At least two different loci have been shown for HHT. Mutations in endoglin have been found in some families and the locus designated ORW1. In other families this locus has been excluded. In this paper we confirm that in families linked to ORW1 there is a prevalence of PAVMs among affected members of 29.2%, compared to a prevalence of 2.9% in families in which this locus has been excluded (chi 2 = 19.2, p < 0.001). This information can be used to decide how to screen HHT patients for PAVMs.
The activin receptor-like kinase 1 gene (ALK-1) is the second locus for the autosomal dominant vascular disease hereditary hemorrhagic telangiectasia (HHT). In this paper we present the genomic structure of the ALK-1 gene, a type I serine-threonine kinase receptor expressed predominantly in endothelial cells. The coding region is contained within nine exons, spanning < 15 kb of genomic DNA. All introns follow the GT-AG rule, except for intron 6, which has a TAG/gcaag 5' splice junction. The positions of introns in the intracellular domain are almost identical to those of the mouse serine-threonine kinase receptor TSK-7L. By sequencing ALK-1 from genomic DNA, mutations were found in six of six families with HHT either shown to link to chromosome 12q13 or in which linkage of HHT to chromosome 9q33 had been excluded. Mutations were also found in three of six patients from families in which available linkage data were insufficient to allow certainty with regard to the locus involved. The high rate of detection of mutations by genomic sequencing of ALK-1 suggests that this will be a useful diagnostic test for HHT2, particularly where preliminary linkage to chromosome 12q13 can be established. In two cases in which premature termination codons were found in genomic DNA, the mutant mRNA was either not present or present at barely detectable levels. These data suggest that mutations in ALK-1 are functionally null alleles.
The human cerebral cortex is supplied by vessels that arise from the pial arteries. These vessels give rise to a dense vascular network that is highly interconnected. Cortical arteries have been classified in different categories. Both their angioarchitectonic pattern and anatomical structures involved in their regulation are not fully understood.
Twelve fresh human brains were studied by scanning electron microscopy of vascular corrosion casts.
Four types of arterial vessels in the cerebral cortex--short, middle, long, and transcortical--were identified. The cortical vascular network was formed by several interconnected clusters of vessels, which were arranged in four vascular layers parallel to the pial surface and characterized by different vascular densities. The greatest vascular density corresponded to the middle and deep vascular layers. Circular constrictions were found at the origin of cortical arteries and at their branching sites, probably related to vascular sphincters. Connections between cortical arteries were observed at their initial course. Plastic strips, occasionally related to constrictions, were observed around both middle and long cortical arteries. Other plastic structures, morphologically similar to pericytes, were found around capillary vessels.
The blood supply to the human cerebral cortex depends on the short, middle, and long cortical arteries, which give rise to a highly anastomosed capillary network. There exist vascular connections between pial arteries and occasionally between cortical arteries. Blood flow autoregulation is probably mediated by smooth muscle cells at the arteriolar level and by pericytes at the capillary level, through endothelial connections.
Endoglin (CD105) is expressed on the surface of endothelial and haematopoietic cells in mammals and binds TGFbeta isoforms 1 and 3 in combination with the signaling complex of TGFbeta receptors types I and II. Endoglin expression increases during angiogenesis, wound healing, and inflammation, all of which are associated with TGFbeta signaling and alterations in vascular structure. The importance of endoglin for normal vascular architecture is further indicated by the association of mutations in the endoglin gene with the inherited disorder Hereditary Haemorrhagic Telangiectasia Type 1 (HHT1), a disease characterised by bleeding from vascular malformations. In order to study the role of endoglin in vivo in more detail and to work toward developing an animal model of HHT1, we have derived mice that carry a targeted nonsense mutation in the endoglin gene. Studies on these mice have revealed that endoglin is essential for early development. Embryos homozygous for the endoglin mutation fail to progress beyond 10.5 days postcoitum and fail to form mature blood vessels in the yolk sac. This phenotype is remarkably similar to that of the TGFbeta1 and the TGFbeta receptor II knockout mice, indicating that endoglin is needed in vivo for TGFbeta1 signaling during extraembryonic vascular development. In addition, we have observed cardiac defects in homozygous endoglin-deficient embryos, suggesting endoglin also plays a role in cardiogenesis. We anticipate that heterozygous mice will ultimately serve as a useful disease model for HHT1, as some individuals have dilated and fragile blood vessels similar to vascular malformations seen in HHT patients.
Endoglin is predominantly expressed on endothelium and is mutated in hereditary hemorrhagic telangiectasia (HHT) type 1 (HHT1). We report the analysis of endoglin in tissues of a newborn (family 2), who died of a cerebral arteriovenous malformation (CAVM), and in a lung specimen surgically resected from a 78-year-old patient (family 5), with a pulmonary AVM (PAVM). The clinically affected father of the newborn revealed a novel mutation that was absent in his parents and was identified as a duplication of exons 3 to 8, by quantitative multiplex polymerase chain reaction. The corresponding mutant protein (116-kd monomer) and the missense mutant protein (80-kd monomer) present in family 5 were detected only as transient intracellular species and were unreactive by Western blot analysis and immunostaining. Normal endoglin (90-kd monomer) was reduced by 50% on peripheral blood-activated monocytes of the HHT1 patients. When analyzed by immunostaining and densitometry, presumed normal blood vessels of the newborn lung and brain and vessels adjacent to the adult PAVM showed a 50% reduction in the endoglin/PECAM-1 ratio. A similar ratio was observed in the CAVM and PAVM, suggesting that all blood vessels of HHT1 patients express reduced endoglin in situ and that AVMs are not attributed to a focal loss of endoglin.
The activin receptor-like kinase 1 (ALK1) is a type I receptor for transforming growth factor-beta (TGF-beta) family proteins. Expression of ALK1 in blood vessels and mutations of the ALK1 gene in human type II hereditary hemorrhagic telangiectasia patients suggest that ALK1 may have an important role during vascular development. To define the function of ALK1 during development, we inactivated the ALK1 gene in mice by gene targeting. The ALK1 homozygous embryos die at midgestation, exhibiting severe vascular abnormalities characterized by excessive fusion of capillary plexes into cavernous vessels and hyperdilation of large vessels. These vascular defects are associated with enhanced expression of angiogenic factors and proteases and are characterized by deficient differentiation and recruitment of vascular smooth muscle cells. The blood vessel defects in ALK1-deficient mice are reminiscent of mice lacking TGF-beta1, TGF-beta type II receptor (TbetaR-II), or endoglin, suggesting that ALK1 may mediate TGF-beta1 signal in endothelial cells. Consistent with this hypothesis, we demonstrate that ALK1 in endothelial cells binds to TGF-beta1 and TbetaR-II. Furthermore, the ALK1 signaling pathway can inhibit TGF-beta1-dependent transcriptional activation mediated by the known TGF-beta1 type I receptor, ALK5. Taken together, our results suggest that the balance between the ALK1 and ALK5 signaling pathways in endothelial cells plays a crucial role in determining vascular endothelial properties during angiogenesis.
Cerebral arteriovenous malformations (AVMs) are occasionally associated with hereditary hemorrhagic telangiectasia (HHT), which is characterized by the presence of multiple mucocutaneous telangiectasia, epistaxis, and familial inheritance. We analyzed the angiographic and clinical characteristics of patients with cerebral AVMs related to HHT.
Among 638 patients with cerebral AVMs, we identified 14 patients with HHT. The AVMs were classified as those with nidi of 1 cm or less (micro AVMs), those with nidi between 1 and 3 cm (small AVMs), and those of the fistulous type (arteriovenous fistulas [AVFs]).
A total of 28 AVMs were found; seven of 14 patients had multiple AVMs. The 28 AVMs were categorized as 12 micro AVMs, eight small AVMs, and eight AVFs. All except one micro AVM were asymptomatic, whereas all small AVMs were symptomatic. Three of eight AVFs were asymptomatic. All 28 AVMs were located on the cortex. All micro AVMs and AVFs had single feeders and single draining veins, whereas the small AVMs had multiple feeders in all lesions and single draining veins in six of eight lesions.
Multiple, cortical, micro AVMs or AVFs harboring single feeding arteries and single draining veins should raise clinical suspicion of HHT-related AVMs.
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder because of mutations in the genes coding for endoglin (HHT1) or ALK-1 (HHT2). The disease is associated with haploinsufficiency and a murine model was obtained by engineering mice that express a single Endoglin allele. Of a total of 171 mice that were observed for 1 year, 50 developed clinical signs of HHT. Disease prevalence was high in 129/Ola strain (72%), intermediate in the intercrosses (36%), and low in C57BL/6 backcrosses (7%). Most mice first presented with an ear telangiectasia and/or recurrent external hemorrhage. One-third of mice with HHT showed severe vascular abnormalities such as dilated vessels, hemorrhages, liver and lung congestion, and/or brain and heart ischemia. Disease sequelae included stroke, hydrocephalus, fatal hemorrhage, and congestive heart failure. Thus the murine model reproduces the multiorgan manifestations of the human disease. Levels of circulating latent transforming growth factor (TGF)-beta1 were significantly lower in the 129/Ola than in the C57BL/6 strain. Intercrosses and 129/Ola mice expressing reduced endoglin also showed lower plasma TGF-beta1 levels than control. These data suggest that modifier genes involved in the regulation of TGF-beta1 expression act in combination with a single functional copy of endoglin in the development of HHT.
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia. Most cases are caused by mutations in the endoglin gene on chromosome 9 (HHT type 1) or the activin receptor-like kinase 1 gene on chromosome 12 (HHT type 2), which leads to telangiectases and arteriovenous malformations (AVM) of the skin, mucosa, and viscera. Epistaxis is the most frequent presentation. Visceral involvement includes pulmonary, gastrointestinal, and cerebral AVMs, which have been reported predominantly in adults. The purpose of this article is to describe 9 children who presented with intracranial hemorrhage (ICH) secondary to cerebral AVM. None of these children was suspected of having HHT before the incident, despite family histories of the disease.
We report the first case of an ICH secondary to a cerebral AVM in a neonate confirmed to have HHT type 1 by molecular analysis. We also describe a series of 8 additional cases of ICH secondary to cerebral AVM in children presumed to have HHT. Examination of multiple affected members from each of these families, using well-accepted published criteria, confirmed the diagnosis of HHT. In addition, genetic linkage studies and/or mutation analysis identified endoglin as the disease-causing gene in 6 of these families. Autopsy, imaging studies, and/or surgery confirmed the presence of cerebral AVMs and ICH in all 9 cases.
Our report shows that infants and children with a family history of HHT are at risk for sudden and catastrophic ICH. A preemptive diagnosis may potentially identify and prevent more serious sequelae.
A prospective study of retinopathy of prematurity (ROP) of 505 infants who weighed <1701 g at birth was undertaken in the mid-1980s. This cohort was traced at 10 to 12 years of age to determine how low birth weight alone and ROP might influence their ophthalmic outcome.
Outcome measures were 1) visual functions (visual acuity, contrast sensitivity, stereoacuity, perimetry, and color vision), 2) presence of strabismus, and 3), measurements of eye size and the dimensions of its components including refractive state. A total of 169 11-year-olds who were born at term were recruited as control subjects and examined under the same conditions.
A total of 448 of the original cohort were traced, and 254 consented to a further examination. Compared with the control group, the follow-up cohort differed significantly with reduced visual functions and increased incidence of both myopia and strabismus. Compared with published data, eye size was smaller in the low birth weight cohort. To summarize the ophthalmic data, we defined ophthalmic morbidity as visual acuity below 0.0 log units or the presence of strabismus, myopia, color vision defect, or visual field defect. The rate of ophthalmic morbidity was 50.8% (n = 129/254) in the study cohort compared with 19.5% (n = 33/169) in the control group. The highest rate of ophthalmic morbidity was associated with severe ROP (stages 3/4), although those with no ROP had a less favorable outcome than the control group.
This study shows that low birth weight children are at increased risk of visual impairments compared with children who are born at full term. Visual impairments are associated with low birth weight per se and severe ROP. Regressed mild ROP is only a risk factor for strabismus. The functional significance of these deficits is largely unknown.
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