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We have previously demonstrated that deletion of activin receptor-like kinase 1 (Alk1) or endoglin in a fraction of endothelial cells (ECs) induces brain arteriovenous malformations (bAVMs) in adult mice upon angiogenic stimulation. Here, we addressed three related questions: (1) could Alk1- mutant bone marrow (BM)-derived ECs (BMDECs) cause bAVMs? (2) is Alk1- ECs clonally expended during bAVM development? and (3) is the number of mutant ECs correlates to bAVM severity? For the first question, we transplanted BM from PdgfbiCreER;Alk12f/2f mice (EC-specific tamoxifen-inducible Cre with Alk1-floxed alleles) into wild-type mice, and then induced bAVMs by intra-brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor and intra-peritoneal injection of tamoxifen. For the second question, clonal expansion was analyzed using PdgfbiCreER;Alk12f/2f;confetti+/- mice. For the third question, we titrated tamoxifen to limit Alk1 deletion and compared the severity of bAVM in mice treated with low and high tamoxifen doses. We found that wild-type mice with PdgfbiCreER;Alk12f/2f BM developed bAVMs upon VEGF stimulation and Alk1 gene deletion in BMDECs. We also observed clusters of ECs expressing the same confetti color within bAVMs and significant proliferation of Alk1- ECs at early stage of bAVM development, suggesting that Alk1- ECs clonally expanded by local proliferation. Tamoxifen dose titration revealed a direct correlation between the number of Alk1- ECs and the burden of dysplastic vessels in bAVMs. These results provide novel insights for the understanding of the mechanism by which a small fraction of Alk1 or endoglin mutant ECs contribute to development of bAVMs.

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.

Pazopanib (Votrient) is an orally administered tyrosine kinase inhibitor that blocks VEGF receptors potentially serving as anti-angiogenic treatment for hereditary hemorrhagic telangiectasia (HHT). We report a prospective, multi-center, open-label, dose-escalating study [50 mg, 100 mg, 200 mg, and 400 mg], designed as a proof-of-concept study to demonstrate efficacy of pazopanib on HHT-related bleeding, and to measure safety. Patients, recruited at 5 HHT Centers, required ≥ 2 Curacao criteria AND [anemia OR severe epistaxis with iron deficiency]. Co-primary outcomes, hemoglobin (Hgb) and epistaxis severity, were measured during and after treatment, and compared to baseline. Safety monitoring occurred every 1.5 weeks. Seven patients were treated with 50 mg pazopanib daily. Six/seven showed at least 50% decrease in epistaxis duration relative to baseline at some point during study; 3 showed at least 50% decrease in duration during Weeks 11 and 12. Six patients showed a decrease in ESS of > 0.71 (MID) relative to baseline at some point during study; 3/6 showed a sustained improvement. Four patients showed > 2 gm improvement in Hgb relative to baseline at one or more points during study. Health-related QOL scores improved on all SF-36 domains at Week 6 and/or Week 12, except general health (unchanged). There were 19 adverse events (AE) including one severe AE (elevated LFTs, withdrawn from dosing at 43 days); with no serious AE. In conclusion, we observed an improvement in Hgb and/or epistaxis in all treated patients. This occurred at a dose much lower than typically used for oncologic indications, with no serious AE. Further studies of pazopanib efficacy are warranted.

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.

An abnormally high number of macrophages are present in human brain arteriovenous malformations (bAVM) with or without evidence of prior hemorrhage, causing unresolved inflammation that may enhance abnormal vascular remodeling and exacerbate the bAVM phenotype. The reasons for macrophage accumulation at the bAVM sites are not known. We tested the hypothesis that persistent infiltration and pro-inflammatory differentiation of monocytes in angiogenic tissues increase the macrophage burden in bAVM using two mouse models and human monocytes. Mouse bAVM was induced through deletion of AVM causative genes, Endoglin (Eng) globally or Alk1 focally, plus brain focal angiogenic stimulation. An endothelial cell and vascular smooth muscle cell co-culture system was used to analyze monocyte differentiation in the angiogenic niche. After angiogenic stimulation, the Eng-deleted mice had fewer CD68(+) cells at 2 weeks (P = 0.02), similar numbers at 4 weeks (P = 0.97), and more at 8 weeks (P = 0.01) in the brain angiogenic region compared with wild-type (WT) mice. Alk1-deficient mice also had a trend toward more macrophages/microglia 8 weeks (P = 0.064) after angiogenic stimulation and more RFP(+) bone marrow-derived macrophages than WT mice (P = 0.01). More CD34(+) cells isolated from peripheral blood of patients with ENG or ALK1 gene mutation differentiated into macrophages than those from healthy controls (P

Hereditary hemorrhagic telangiectasia (HHT), a genetic bleeding disorder leading to systemic arteriovenous malformations (AVMs), is caused by loss-of-function mutations in the ALK1/ENG/Smad1/5/8 pathway. Evidence suggests that HHT pathogenesis strongly relies on overactivated PI3K/Akt/mTOR and VEGFR2 pathways in endothelial cells (ECs). In the BMP9/10-immunoblocked (BMP9/10ib) neonatal mouse model of HHT, we report here that the mTOR inhibitor, sirolimus, and the receptor tyrosine kinase inhibitor, nintedanib, could synergistically fully block, but also reversed, retinal AVMs to avert retinal bleeding and anemia. Sirolimus plus nintedanib prevented vascular pathology in the oral mucosa, lungs, and liver of the BMP9/10ib mice, as well as significantly reduced gastrointestinal bleeding and anemia in inducible ALK1-deficient adult mice. Mechanistically, in vivo in BMP9/10ib mouse ECs, sirolimus and nintedanib blocked the overactivation of mTOR and VEGFR2, respectively. Furthermore, we found that sirolimus activated ALK2-mediated Smad1/5/8 signaling in primary ECs --including in HHT patient blood outgrowth ECs--and partially rescued Smad1/5/8 activity in vivo in BMP9/10ib mouse ECs. These data demonstrate that the combined correction of endothelial Smad1/5/8, mTOR, and VEGFR2 pathways opposes HHT pathogenesis. Repurposing of sirolimus plus nintedanib might provide therapeutic benefit in patients with HHT., Introduction Hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber syndrome, is an autosomal dominant genetic disease with a prevalence of approximately 1 in 5,000 individuals (1). HHT is a hemorrhagic [...]

In endothelial cells, two type I receptors of the transforming growth factor β (TGF-β) family, ALK1 and ALK5, coordinate to regulate embryonic angiogenesis in response to BMP9/10 and TGF-β. Whereas TGF-β binds to and activates ALK5, leading to Smad2/3 phosphorylation and inhibition of endothelial cell proliferation and migration, BMP9/10 and TGF-β also bind to ALK1, resulting in the activation of Smad1/5. SnoN is a negative regulator of ALK5 signaling through the binding and repression of Smad2/3. Here we uncover a positive role of SnoN in enhancing Smad1/5 activation in endothelial cells to promote angiogenesis. Upon ligand binding, SnoN directly bound to ALK1 on the plasma membrane and facilitated the interaction between ALK1 and Smad1/5, enhancing Smad1/5 phosphorylation. Disruption of this SnoN-Smad interaction impaired Smad1/5 activation and up-regulated Smad2/3 activity. This resulted in defective angiogenesis and arteriovenous malformations, leading to embryonic lethality at E12.5. Thus, SnoN is essential for TGF-β/BMP9-dependent biological processes by its ability to both positively and negatively modulate the activities of Smad-dependent pathways.

ObjectiveVessels in brain arteriovenous malformations are prone to rupture. The underlying pathogenesis is not clear. Hereditary hemorrhagic telangiectasia type 2 patients with activin receptor-like kinase 1 (Alk1) mutation have a higher incidence of brain arteriovenous malformation than the general population. We tested the hypothesis that vascular endothelial growth factor impairs vascular integrity in the Alk1-deficient brain through reduction of mural cell coverage.Methods and resultsAdult Alk1(1f/2f) mice (loxP sites flanking exons 4-6) and wild-type mice were injected with 2×10(7) PFU adenovious-cre recombinase and 2×10(9) genome copies of adeno-associated virus-vascular endothelial growth factor to induce focal homozygous Alk1 deletion (in Alk1(1f/2f) mice) and angiogenesis. Brain vessels were analyzed 8 weeks later. Compared with wild-type mice, the Alk1-deficient brain had more fibrin (99±30×10(3) pixels/mm(2) versus 40±13×10(3); P=0.001), iron deposition (508±506 pixels/mm(2) versus 6±49; P=0.04), and Iba1(+) microglia/macrophage infiltration (888±420 Iba1(+) cells/mm(2) versus 240±104 Iba1(+); P=0.001) after vascular endothelial growth factor stimulation. In the angiogenic foci, the Alk1-deficient brain had more α-smooth muscle actin negative vessels (52±9% versus 12±7%, P

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.08.022 Byline: Young Jae Lee, Alexandra McPherron, Susan Choe, Yasuo Sakai, Roshantha A. Chandraratna, Se-Jin Lee, S. Paul Oh Keywords: GDF11; ACVR2; CYP26A1; Retinoic acid; Vertebral patterning; RAR inhibitor Abstract: Mice deficient in growth differentiation factor 11 (GDF11) signaling display anterior transformation of axial vertebrae and truncation of caudal vertebrae. However, the in vivo molecular mechanisms by which GDF11 signaling regulates the development of the vertebral column have yet to be determined. We found that Gdf11 and Acvr2b mutants are sensitive to exogenous RA treatment on vertebral specification and caudal vertebral development. We show that diminished expression of Cyp26a1, a retinoic acid inactivating enzyme, and concomitant elevation of retinoic acid activity in the caudal region of Gdf11.sup.-/- embryos may account for this phenomenon. Reduced expression or function of Cyp26a1 enhanced anterior transformation of axial vertebrae in wild-type and Acvr2b mutants. Furthermore, a pan retinoic acid receptor antagonist (AGN193109) could lessen the anterior transformation phenotype and rescue the tail truncation phenotype of Gdf11.sup.-/- mice. Taken together, these results suggest that GDF11 signaling regulates development of caudal vertebrae and is involved in specification of axial vertebrae in part by maintaining Cyp26a1 expression, which represses retinoic acid activity in the caudal region of embryos during the somitogenesis stage. Article History: Received 20 March 2010; Revised 20 August 2010; Accepted 20 August 2010

Arteriovenous malformations (AVMs) are vascular anomalies where arteries and veins are directly connected through a complex, tangled web of abnormal arteries and veins instead of a normal capillary network. AVMs in the brain, lung, and visceral organs, including the liver and gastrointestinal tract, result in considerable morbidity and mortality. AVMs are the underlying cause of three major clinical symptoms of a genetic vascular dysplasia termed hereditary hemorrhagic telangiectasia (HHT), which is characterized by recurrent nosebleeds, mucocutaneous telangiectases, and visceral AVMs and caused by mutations in one of several genes, including activin receptor--like kinase 1 (ALK1). It remains unknown why and how selective blood vessels form AVMs, and there have been technical limitations to observing the initial stages of AVM formation. Here we present in vivo evidence that physiological or environmental factors such as wounds in addition to the genetic ablation are required for Alk1-deficient vessels to develop to AVMs in adult mice. Using the dorsal skinfold window chamber system, we have demonstrated for what we believe to be the first time the entire course of AVM formation in subdermal blood vessels by using intravital bright-field images, hyperspectral imaging, fluorescence recordings of direct arterial flow through the AV shunts, and vascular casting techniques. We believe our data provide novel insights into the pathogenetic mechanisms of HHT and potential therapeutic approaches., Introduction Arteriovenous malformation (AVM) refers to a vascular anomaly wherein arteries and veins are directly connected through a complex, tangled web of abnormal arteries and veins by one or more [...]

Activin receptor patterning is discussed relative to foregut organogenesis. Mutations of activin receptors ActRHA and Act RIIb can be seen to disrupt the development of posterior foregut-derived organs. The conclusion is that activin-receptor mediated signaling regulates axial patterning, cell differentiation, and function of foregut-derived organs.

Research has been conducted on activin type IIA and type IIB receptors. The authors report that the results of genetic and biochemical studies have demonstrated that these receptors mediate growth and differentiation factor 11 signal in patterning axial vertebrate.

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-[Beta]1, TGF-[Beta] type II receptor (T[Beta]R-II), or endoglin, suggesting that ALK1 may mediate TGF-[Beta]1 signal in endothelial cells. Consistent with this hypothesis, we demonstrate that ALK1 in endothelial cells binds to TGF-[Beta]1 and T[Beta]R-II. Furthermore, the ALK1 signaling pathway can inhibit TGF-[Beta]1-dependent transcriptional activation mediated by the known TGF-[Beta]1 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.

Aims Chemokine CXCL12 (stromal derived factor 1: SDF1) has been shown to play important roles in various processes of cardiovascular development. In recent avian studies, CXCL12 signalling has been implicated in guidance of cardiac neural crest cells for their participation in the development of outflow tract and cardiac septum. The goal of this study is to investigate the extent to which CXCL12 signalling contribute to the development of aortic arch and pulmonary arteries in mammals. Methods and results Novel Cxcl12-LacZ reporter and conditional alleles were generated. Using whole mount X-gal staining with the reporter allele and vascular casting techniques, we show that the domain branching pattern of pulmonary arteries in Cxcl12-null mice is completely disrupted and discordant with that of pulmonary veins and airways. Cxcl12-null mice also displayed abnormal and superfluous arterial branches from the aortic arch. The early steps of pharyngeal arch remodelling in Cxcl12-null mice appeared to be unaffected, but vertebral arteries were often missing and prominent aberrant arteries were present parallel to carotid arteries or trachea, similar to aberrant vertebral artery or thyroid ima artery, respectively. Analysis with computed tomography not only confirmed the results from vascular casting studies but also identified abnormal systemic arterial supply to lungs in the Cxcl12-null mice. Tie2-Cre mediated Cxcr4 deletion phenocopied the Cxcl12-null phenotypes, indicating that CXCR4 is the primary receptor for arterial patterning, whereas Cxcl12 or Cxcr4 deletion by Wnt1-Cre did not affect aortic arch patterning. Conclusion CXCL12-CXCR4 signalling is essential for the correct patterning of aortic arches and pulmonary arteries during development. Superfluous arteries in Cxcl12-null lungs and the aortic arch infer a role of CXCL12 in protecting arteries from uncontrolled sprouting during development of the arterial system. [ABSTRACT FROM AUTHOR]

Angiogenesis in the developing central nervous system (CNS) is regulated by neuroepithelial cells, although the genes and pathways that couple these cells to blood vessels remain largely uncharacterized. Here, we have used biochemical, cell biological and molecular genetic approaches to demonstrate that ß8 integrin (Itgb8) and neuropilin 1 (Nrp1) cooperatively promote CNS angiogenesis by mediating adhesion and signaling events between neuroepithelial cells and vascular endothelial cells. ß8 integrin in the neuroepithelium promotes the activation of extracellular matrix (ECM)-bound latent transforming growth factor ß (TGFß) ligands and stimulates TGFß receptor signaling in endothelial cells. Nrp1 in endothelial cells suppresses TGFß activation and signaling by forming intercellular protein complexes with ß8 integrin. Cell type-specific ablation of ß8 integrin, Nrp1, or canonical TGFß receptors results in pathological angiogenesis caused by defective neuroepithelial cell-endothelial cell adhesion and imbalances in canonical TGFß signaling. Collectively, these data identify a paracrine signaling pathway that links the neuroepithelium to blood vessels and precisely balances TGFß signaling during cerebral angiogenesis. [ABSTRACT FROM AUTHOR]