Activin Receptor-like Kinase 1 Signaling Research Articles

Shear Stress and Sub-Femtomolar Levels of Ligand Synergize to Activate ALK1 Signaling in Endothelial Cells.

Endothelial cells (ECs) respond to concurrent stimulation by biochemical factors and wall shear stress (SS) exerted by blood flow. Disruptions in flow-induced responses can result in remodeling issues and cardiovascular diseases, but the detailed mechanisms linking flow-mechanical cues and biochemical signaling remain unclear. Activin receptor-like kinase 1 (ALK1) integrates SS and ALK1-ligand cues in ECs; ALK1 mutations cause hereditary hemorrhagic telangiectasia (HHT), marked by arteriovenous malformation (AVM) development. However, the mechanistic underpinnings of ALK1 signaling modulation by fluid flow and the link to AVMs remain uncertain. We recorded EC responses under varying SS magnitudes and ALK1 ligand concentrations by assaying pSMAD1/5/9 nuclear localization using a custom multi-SS microfluidic device and a custom image analysis pipeline. We extended the previously reported synergy between SS and BMP9 to include BMP10 and BMP9/10. Moreover, we demonstrated that this synergy is effective even at extremely low SS magnitudes (0.4 dyn/cm2) and ALK1 ligand range (femtogram/mL). The synergistic response to ALK1 ligands and SS requires the kinase activity of ALK1. Moreover, ALK1's basal activity and response to minimal ligand levels depend on endocytosis, distinct from cell-cell junctions, cytoskeleton-mediated mechanosensing, or cholesterol-enriched microdomains. However, an in-depth analysis of ALK1 receptor trafficking's molecular mechanisms requires further investigation.

An integrated docking and molecular dynamics simulation approach to discover potential inhibitors of activin receptor-like kinase 1.

Activin receptor-like kinase 1 (ALK1) is a transmembrane receptor involved in crucial signaling pathways associated with angiogenesis and vascular development. Inhibition of ALK1 signaling has emerged as a promising therapeutic strategy for various angiogenesis-related diseases, including cancer and hereditary hemorrhagic telangiectasia. This study aimed to investigate the potential of phytoconstituents as inhibitors of ALK1 using a combined approach of virtual screening and molecular dynamics (MDs) simulations. Phytoconstituents from the IMPPAT 2.0 database underwent virtual screening to identify potential inhibitors of ALK1. The compounds were initially filtered based on physicochemical parameters, following Lipinski's rules and the PAINS filter. Subsequently, compounds demonstrating high binding affinities in docking analysis were further analyzed. Additional assessments, including ADMET, PAINS, and PASS evaluations, were conducted to identify more potent hits. Through interaction analysis, a phytoconstituent, Candidine, exhibited appreciable affinity and specific interactions with the ALK1 active site. To validate the results, MD simulations and principal components analysis were performed. The MD simulations demonstrated that Candidine stabilized the ALK1 structure and reduced conformational fluctuations. In conclusion, Candidine shows promising potential as binding partners of ALK1. These findings provide a foundation for further exploration and development of Candidine as a lead molecule for therapeutic interventions targeting ALK1-associated diseases.

Thrombosis in hereditary hemorrhagic telangiectasia

Thrombosis in hereditary hemorrhagic telangiectasia

Regulation of Blood-Brain Barrier Transporters by Transforming Growth Factor-β/Activin Receptor-Like Kinase 1 Signaling: Relevance to the Brain Disposition of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors (i.e., Statins).

Our laboratory has shown that activation of transforming growth factor- β (TGF- β )/activin receptor-like kinase 1 (ALK1) signaling can increase protein expression and transport activity of organic anion transporting polypeptide 1a4 (Oatp1a4) at the blood-brain barrier (BBB). These results are relevant to treatment of ischemic stroke because Oatp transport substrates such as 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (i.e., statins) improve functional neurologic outcomes in patients. Advancement of our work requires determination if TGF- β /ALK1 signaling alters Oatp1a4 functional expression differently across brain regions and if such disparities affect central nervous system (CNS) statin disposition. Therefore, we studied regulation of Oatp1a4 by the TGF- β /ALK1 pathway, in vivo, in rat brain microvessels isolated from cerebral cortex, hippocampus, and cerebellum using the ALK1 agonist bone morphogenetic protein-9 (BMP-9) and the ALK1 inhibitor 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride 193189. We showed that Oatp1a4 protein expression and brain distribution of three currently marketed statin drugs (i.e., atorvastatin, pravastatin, and rosuvastatin) were increased in cortex relative to hippocampus and cerebellum. Additionally, BMP-9 treatment enhanced Oatp-mediated statin transport in cortical tissue but not in hippocampus or cerebellum. Although brain drug delivery is also dependent upon efflux transporters, such as P-glycoprotein and/or Breast Cancer Resistance Protein, our data showed that administration of BMP-9 did not alter the relative contribution of these transporters to CNS disposition of statins. Overall, this study provides evidence for differential regulation of Oatp1a4 by TGF- β /ALK1 signaling across brain regions, knowledge that is critical for development of therapeutic strategies to target Oatps at the BBB for CNS drug delivery. SIGNIFICANCE STATEMENT: Organic anion transporting polypeptides (Oatps) represent transporter targets for brain drug delivery. We have shown that Oatp1a4 statin uptake is higher in cortex versus hippocampus and cerebellum. Additionally, we report that the transforming growth factor- β /activin receptor-like kinase 1 agonist bone morphogenetic protein-9 increases Oatp1a4 functional expression, but not efflux transporters P-glycoprotein and Breast Cancer Resistance Protein, in cortical brain microvessels. Overall, this study provides critical data that will advance treatment for neurological diseases where drug development has been challenging.

ALK1 signaling is required for the homeostasis of Kupffer cells and prevention of bacterial infection.

Macrophages are highly heterogeneous immune cells that fulfill tissue-specific functions. Tissue-derived signals play a critical role in determining macrophage heterogeneity. However, these signals remain largely unknown. The BMP receptor activin receptor–like kinase 1 (ALK1) is well known for its role in blood vessel formation; however, its role within the immune system has never been revealed to our knowledge. Here, we found that BMP9/BMP10/ALK1 signaling controlled the identity and self-renewal of Kupffer cells (KCs) through a Smad4-dependent pathway. In contrast, ALK1 was dispensable for the maintenance of macrophages located in the lung, kidney, spleen, and brain. Following ALK1 deletion, KCs were lost over time and were replaced by monocyte-derived macrophages. These hepatic macrophages showed significantly reduced expression of the complement receptor VSIG4 and alterations in immune zonation and morphology, which is important for the tissue-specialized function of KCs. Furthermore, we found that this signaling pathway was important for KC-mediated Listeria monocytogenes capture, as the loss of ALK1 and Smad4 led to a failure of bacterial capture and overwhelming disseminated infections. Thus, ALK1 signaling instructs a tissue-specific phenotype that allows KCs to protect the host from systemic bacterial dissemination.

P149 DECREASED COLONIC ACTIVIN RECEPTOR-LIKE KINASE 1 DISRUPTS EPITHELIAL BARRIER INTEGRITY AND IS ASSOCIATED WITH A POOR CLINICAL OUTCOME IN CROHN’S DISEASE

Abstract Background and Aims Intestinal epithelial cell (IEC) barrier dysfunction is critical to the development of Crohn’s disease (CD). Mechanisms controlling colonocyte differentiation and barrier defects are understudied in CD. We recently reported increased expression of microRNA-31-5p (miR-31-5p) in colonic IECs of CD patients compared to non-IBD (NIBD) controls. In this study we identify and characterize the miR-31-5p target gene Activin Receptor-Like Kinase 1 (ALK1) as a key regulator of colonocyte maturation and IEC barrier integrity, specifically the functional impact of aberrant ALK1 signaling on colonocyte differentiation, barrier integrity, and clinical disease outcomes in CD. Methods MiR-31-5p target genes were identified in CD patients by integrative analysis of RNA- and small RNA-sequencing data from colonic mucosa and confirmed by quantitative RT-PCR (qPCR) in isolated colonic IECs. Functional characterization of ALK1 in colonic IECs was performed ex vivo using 2- or 3-dimensional cultured human primary colonic IECs. The impact of altered colonic ALK1 signaling for the risk of surgery and endoscopic relapse was evaluated by a multivariate regression analysis and a Kaplan-Meier estimator among CD patients. Results Integrative analysis of RNA- and small RNA-sequencing and follow-up qPCR identified ALK1 as a candidate target of miR-31-5p in the colonic IECs of CD patients. A 3’-UTR reporter assay with site-directed mutagenesis confirmed the direct regulation of ALK1 expression by miR-31-5p in HEK293T cells. Increased activation of ALK1 restricted the proliferation of primary colonic IECs in an EdU proliferation assay and down-regulated the expression of stemness-related genes, such as LGR5 and ASCL2. Activated ALK1 signaling directed the fate of human colonic IEC differentiation toward colonocytes. Down-regulated ALK1 signaling was associated with increased stemness-related gene expression and decreased colonocyte-specific gene expression in the isolated colonic IECs of CD patients compared to non-IBD controls. Activation of ALK1 did not affect colonic IEC migration in a wound healing assay, but enhanced epithelial barrier integrity in a trans-epithelial electrical resistance (TEER)-based permeability assay. Lower colonic ALK1 expression was associated with higher risks of surgery and endoscopic relapse in CD patients. Conclusion Decreased colonic ALK1 signaling disrupts colonic IEC barrier integrity and is associated with a poor clinical outcome in CD patients.

P149 DECREASED COLONIC ACTIVIN RECEPTOR-LIKE KINASE 1 DISRUPTS EPITHELIAL BARRIER INTEGRITY AND IS ASSOCIATED WITH A POOR CLINICAL OUTCOME IN CROHN’S DISEASE

Intestinal epithelial cell (IEC) barrier dysfunction is critical to the development of Crohn’s disease (CD). Mechanisms controlling colonocyte differentiation and barrier defects are understudied in CD. We recently reported increased expression of microRNA-31-5p (miR-31-5p) in colonic IECs of CD patients compared to non-IBD (NIBD) controls. In this study we identify and characterize the miR-31-5p target gene Activin Receptor-Like Kinase 1 (ALK1) as a key regulator of colonocyte maturation and IEC barrier integrity, specifically the functional impact of aberrant ALK1 signaling on colonocyte differentiation, barrier integrity, and clinical disease outcomes in CD.

Treatment with low-dose tacrolimus inhibits bleeding complications in a patient with hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) can be found in patients suffering from a loss-of-function mutation of the gene encoding for the activin receptor-like kinase 1 (ALK-1), a bone morphogenetic protein (BMP) type 1 receptor. Interestingly, ALK-1 mutations also lead to hereditary hemorrhagic telangiectasia (HHT), an autosomal dominant disease characterized by arteriovenous malformations (AVMs) leading to potentially life-threatening bleeding complications such as epistaxis. Current therapeutic options for both diseases are limited and often only temporary or accompanied by severe side effects. Here, we report of a patient with a mutation of the ALK-1 gene suffering from both HHT and PAH. Recently, it was shown that tacrolimus increased ALK-1 signaling and had beneficial effects in selected end-stage PAH patients. We thus hypothesized that treatment with tacrolimus may prevent disease progression in this patient. Surprisingly, treatment with low-dose tacrolimus dramatically improved his HHT-associated epistaxis but did not attenuate progression of PAH.

Functional Expression of Organic Anion Transporting Polypeptide 1a4 Is Regulated by Transforming Growth Factor-β/Activin Receptor-like Kinase 1 Signaling at the Blood-Brain Barrier.

Central nervous system (CNS) drug delivery can be achieved by targeting drug uptake transporters such as Oatp1a4. In fact, many drugs that can improve neurologic outcomes in CNS diseases [3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (i.e., statins)] are organic anion transporting polypeptide (OATP) transport substrates. To date, transport properties and regulatory mechanisms of Oatp1a4 at the blood-brain barrier (BBB) have not been rigorously studied. Such knowledge is critical to develop Oatp1a4 for optimization of CNS drug delivery and for improved treatment of neurological diseases. Our laboratory has demonstrated that the transforming growth factor-β (TGF-β)/activin receptor-like kinase 1 (ALK1) signaling agonist bone morphogenetic protein 9 (BMP-9) increases functional expression of Oatp1a4 in rat brain microvessels. Here, we expand on this work and show that BMP-9 treatment increases blood-to-brain transport and brain exposure of established OATP transport substrates (i.e., taurocholate, atorvastatin, and pravastatin). We also demonstrate that BMP-9 activates the TGF-β/ALK1 pathway in brain microvessels as indicated by increased nuclear translocation of specific Smad proteins associated with signaling mediated by the ALK1 receptor (i.e., pSmad1/5/8). Furthermore, we report that an activated Smad protein complex comprised of phosphorylated Smad1/5/8 and Smad4 is formed following BMP-9 treatment and binds to the promoter of the Slco1a4 gene (i.e., the gene that encodes Oatp1a4). This signaling mechanism causes increased expression of Slco1a4 mRNA. Overall, this study provides evidence that Oatp1a4 transport activity at the BBB is directly regulated by TGF-β/ALK1 signaling and indicates that this pathway can be targeted for control of CNS delivery of OATP substrate drugs.

Abstract 11814: Pulmonary Hypertension With Altered eNOS and ALK-1 Signaling in the Autoimmuno-disease Mice

Background: Pulmonary artery hypertension is often associated with connective tissue disease and major determinant of prognosis. MRL/lpr mouse, which has hypergammaglobulinemia and products various autoimmuno antibody, develops vasculitis and nephritis spontaneously and is widely used as the model of lupus nephritis or Sjogren syndrome. In this study, we examined the right ventricular pressure overload, pathological features and the signaling molecules in the lung tissues of MRL/lpr mice. Methods: We used the female MRL/lpr mice at the age of 12 to 18 weeks. We measured right ventricular systolic pressure (RVSP) by a micro-manometer catheter and the ratio of right ventricular weight to left ventricular weight including ventricular septum (RV/LV). Lung sections were stained with α-smooth muscle actin, and the medial smooth muscle area was expressed as a percentage of the external area of the vessel. Levels of endothelial nitric oxide synthase (eNOS), survivin, endothelin-1 (ET-1), and activin receptor-like kinase-1 (ALK-1) were analyzed by Western blotting. Results: RVSP in the MRL/lpr mice was significantly higher (29.8 ± 7.2 vs. 19.2 ± 1.9 mmHg, P < 0.05) than that in WT C57/Bl6 mice. RV/LV was increased in the MRL/lpr mice compared to the WT C57/Bl6 mice (0.33 ± 0.58 vs. 0.20 ± 0.67, P < 0.05). The medial smooth muscle area of MRL/lpr mice were larger than that of WT C57/bl6 (56.3 ± 4.0 vs. 39.5 ± 6.1%, P < 0.05). Western blot analysis revealed the markedly elevated levels of ET-1 and survivin as well as decreased ALK-1 expression and eNOS phosphorylation in the lung tissue of MRL/lpr mice. Conclusion: We demonstrated that MRL/lpr mice were complicated with pulmonary hypertension. The possible mechanisms include the vasoconstriction enhanced by increased ET-1 level and impaired vasodilatation caused by decreased eNOS phosphorylation and ALK-1 expression. In addition, upregulation of survivin promoted the medial wall thickening of pulmonary vessels. MRL/lpr mice appeared to be a useful model in considering the mechanism of pulmonary hypertension associated with connective tissue diseases.

Inhibition of ALK1 signaling with dalantercept combined with VEGFR TKI leads to tumor stasis in renal cell carcinoma.

Treatment of metastatic renal cell carcinoma (mRCC) with agents that block signaling through vascular endothelial growth factor receptor 2 (VEGFR2) induces disease regression or stabilization in some patients; however, these responses tend to be short-lived. Therefore, development of combination therapies that can extend the efficacy of VEGFR antagonists in mRCC remains a priority.We studied murine xenograft models of RCC that become refractory to treatment with the VEGFR tyrosine kinase inhibitor (TKI) sunitinib. Dalantercept is a novel antagonist of Activin receptor-like kinase 1 (ALK1)/Bone morphogenetic protein (BMP) 9 signaling. Dalantercept inhibited growth in the murine A498 xenograft model which correlated with hyperdilation of the tumor vasculature and an increase in tumor hypoxia. When combined with sunitinib, dalantercept induced tumor necrosis and prevented tumor regrowth and revascularization typically seen with sunitinib monotherapy in two RCC models. Combination therapy led to significant downregulation of angiogenic genes as well as downregulation of endothelial specific gene expression particularly of the Notch signaling pathway.We demonstrate that simultaneous targeting of molecules that control distinct phases of angiogenesis, such as ALK1 and VEGFR, is a valid strategy for treatment of mRCC. At the molecular level, combination therapy leads to downregulation of Notch signaling.

Alk1 controls arterial endothelial cell migration in lumenized vessels.

Heterozygous loss of the arterial-specific TGFβ type I receptor, activin receptor-like kinase 1 (ALK1; ACVRL1), causes hereditary hemorrhagic telangiectasia (HHT). HHT is characterized by development of fragile, direct connections between arteries and veins, or arteriovenous malformations (AVMs). However, how decreased ALK1 signaling leads to AVMs is unknown. To understand the cellular mis-steps that cause AVMs, we assessed endothelial cell behavior in alk1-deficient zebrafish embryos, which develop cranial AVMs. Our data demonstrate that alk1 loss has no effect on arterial endothelial cell proliferation but alters arterial endothelial cell migration within lumenized vessels. In wild-type embryos, alk1-positive cranial arterial endothelial cells generally migrate towards the heart, against the direction of blood flow, with some cells incorporating into endocardium. In alk1-deficient embryos, migration against flow is dampened and migration in the direction of flow is enhanced. Altered migration results in decreased endothelial cell number in arterial segments proximal to the heart and increased endothelial cell number in arterial segments distal to the heart. We speculate that the consequent increase in distal arterial caliber and hemodynamic load precipitates the flow-dependent development of downstream AVMs.

BMP9 Induces Cord Blood-Derived Endothelial Progenitor Cell Differentiation and Ischemic Neovascularization via ALK1.

Modulating endothelial progenitor cells (EPCs) is essential for therapeutic angiogenesis, and thus various clinical trials involving EPCs are ongoing. However, the identification of environmental conditions and development of optimal methods are required to accelerate EPC-driven vasculogenesis. We evaluated gene expression profiles of cord blood-derived EPCs and endothelial cells to identify the key factors in EPC→endothelial cell differentiation and to show that transforming growth factor-β family members contribute to EPC differentiation. The expression levels of activin receptor-like kinase 1 (ALK1) and its high-affinity ligand, bone morphogenetic protein 9 (BMP9) were markedly changed in EPC→endothelial cell differentiation. Interestingly, BMP9 induced EPC→endothelial cell differentiation and EPC incorporation into vessel-like structures by acting on ALK1 expressed on EPCs in vitro. BMP9 also induced neovascularization in mice with hindlimb ischemia by increasing vessel formation and the incorporation of EPCs into vessels. Conversely, neovascularization was impaired when ALK1 signaling was blocked. Furthermore, EPCs exposed to either short- or long-term BMP9 stimulation demonstrated these functions in EPC-mediated neovascularization. Collectively, our results indicated that BMP9/ALK1 augmented vasculogenesis and angiogenesis, and thereby enhanced neovascularization. Thus, we suggest that BMP9/ALK1 may improve the efficacy of EPC-based therapies for treating ischemic diseases.

DART Study: A phase II randomized trial of dalantercept plus axitinib versus placebo plus axitinib in advanced clear cell renal cell carcinoma (RCC): Results from Part 1.

407 Background: Activin receptor-like kinase 1 (ALK1) is a novel target in angiogenesis involved in blood vessel maturation and stabilization. Concurrent targeting of ALK1 and vascular endothelial growth factor (VEGF) signaling results in dual angiogenic blockade and augmented inhibition of tumor growth in RCC xenograft models. Dalantercept (Dal) is an ALK1 receptor-fusion protein that acts as a ligand trap and has demonstrated monotherapy activity with an acceptable safety profile in a completed phase I study. Methods: Part 1 of this study evaluated the safety, tolerability, and preliminary activity of escalating dose levels of Dal plus fixed dose axitinib in pts with advanced RCC. 3-6 pts each received Dal (0.6, 0.9, or 1.2 mg/kg) SC Q3W and axitinib 5 mg PO BID. Key eligibility: predominantly clear cell RCC, 1 prior VEGFR TKI, ≤3 prior tx. Results: As of September 12, 2014, 29 pts were enrolled in three cohorts (n=6, 9, 14) at dose levels of 0.6, 0.9 and 1.2 mg/kg, respectively. At the 1.2 mg/kg dose level, 1 DLT of grade 3 abdominal and back pain occurred (n=1) in addition to more Dal associated edema events including peripheral edema (n=6), fluid overload (n=1), ascites (n=1), and pleural effusion (n=1). The 0.9 mg/kg dose level was well tolerated (3 pts with low grade edema; no pleural effusions or ascites) and selected for Part 2. Frequent AEs regardless of attribution: fatigue, diarrhea, dysphonia, nausea, peripheral edema, creatinine rise, epistaxis, hypertension, arthralgia, hand-foot rash, and cough. 28 pts were evaluable for response by RECIST 1.1. The ORR was 25% (n=7) and 31.3% (n=5) among pts who received ≥ 2 prior txs (n=16), including VEGF targeted tx, mTOR inhibitors, and immune tx. Of those evaluable for disease control at 6 mos. (n=25, 3 active pts have not reached 6 mos.), 52% (n=13) remained progression free. PFS data are maturing and will be presented. Conclusions: The combination of dalantercept and axitinib is well tolerated and associated with encouraging activity in pts with prior VEGF, mTOR, and immune therapies. Part 2 of this study randomizes pts to dalantercept + axitinib vs. placebo + axitinib and is actively accruing patients. Clinical trial information: NCT01727336.

Activin Receptor-Like Kinase 1: a Novel Anti-angiogenesis Target from TGF-β Family

Anti-angiogenic therapy represents a very promising approach in cancer treatment, as most tumors needs to be supplied by a functional vascular network in order to grow beyond the local boundaries and metastatize. The accessibility of vessels to drug delivery and the broad spectrum of cancers treatable with the same compound have arisen interest in research of suitable molecules, with several, especially targeting the VEGF pathway, entered in clinical trials and approved by the Food and Drug Administration. Despite good results, the major hurdle resides in the limited duration of an effective clinical response before tumors start to grow again. Thus, researchers are looking for different alternative targets for a combined and parallel multi-targeting of angiogenic signaling circuits. Activin Receptor-like kinase 1 (ALK1) is a TGF-β type I receptor with high affinity for the BMP9 member of Bone Morphogenic Proteins superfamily: it is expressed mainly, even if not exclusively, on endothelial cells and seems to be involved in the regulatory phase of angiogenesis. Despite a non-completely elucidated mechanism, the targeting of this pathway, both by a soluble ALK1-Fc receptor developed by Acceleron Pharma and by a fully human monoclonal antibody developed by Pfizer, has achieved encouraging results. After having briefly summarized the state of the art of anti-angiogenic therapy, we will first review existing evidence about the molecular mechanisms of ALK1 signaling and we will then analyse in detail the pre-clinical and clinical data available about these two drugs.

Specificity and Structure of a High Affinity Activin Receptor-like Kinase 1 (ALK1) Signaling Complex

Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-β superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-β family ligands BMP9 and BMP10 as well as the three type II TGF-β family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies.

Crossveinless 2 regulates bone morphogenetic protein 9 in human and mouse vascular endothelium

AbstractThe importance of morphogenetic proteins (BMPs) and their antagonists in vascular development is increasingly being recognized. BMP-4 is essential for angiogenesis and is antagonized by matrix Gla protein (MGP) and crossveinless 2 (CV2), both induced by the activin receptor like-kinase 1 (ALK1) when stimulated by BMP-9. In this study, however, we show that CV2 preferentially binds and inhibits BMP-9 thereby providing strong feedback inhibition for BMP-9/ALK1 signaling rather than for BMP-4/ALK2 signaling. CV2 disrupts complex formation involving ALK2, ALK1, BMP-4, and BMP-9 required for the induction of both BMP antagonists. It also limits VEGF expression, proliferation, and tube formation in ALK1-expressing endothelial cells. In vivo, CV2 deficiency translates into a dysregulation of vascular BMP signaling, resulting in an abnormal endothelium with increased endothelial cellularity and expression of lineage markers for mature endothelial cells. Thus, mutual regulation by BMP-9 and CV2 is essential in regulating the development of the vascular endothelium.

ALK1 Signaling Inhibits Angiogenesis by Cooperating with the Notch Pathway

Activin receptor-like kinase 1 (ALK1) is an endothelial-specific member of the TGF-β/BMP receptor family that is inactivated in patients with hereditary hemorrhagic telangiectasia (HHT). How ALK1 signaling regulates angiogenesis remains incompletely understood. Here we show that ALK1 inhibits angiogenesis by cooperating with the Notch pathway. Blocking Alk1 signaling during postnatal development in mice leads to retinal hypervascularization and the appearance of arteriovenous malformations (AVMs). Combined blockade of Alk1 and Notch signaling further exacerbates hypervascularization, whereas activation of Alk1 by its high-affinity ligand BMP9 rescues hypersprouting induced by Notch inhibition. Mechanistically, ALK1-dependent SMAD signaling synergizes with activated Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2, thereby repressing VEGF signaling, tip cell formation, and endothelial sprouting. Taken together, these results uncover a direct link between ALK1 and Notch signaling during vascular morphogenesis that may be relevant to the pathogenesis of HHT vascular lesions.

Phase I study of ACE-041, a novel inhibitor of vascular maturation, in patients with advanced solid tumors or relapsed/refractory multiple myeloma.

TPS179 Background: Activin receptor-like kinase 1 (ALK1) is a type I receptor belonging to the TGFβ superfamily of proteins that is transiently expressed on arterial endothelial cells during development and in response to injury or disease (Seki et al. Circ. Res. 2003). ALK1 signaling plays a pivotal role in the maturation phase of angiogenesis and is therefore critical to vasculature development. ACE-041 is a soluble receptor fusion protein consisting of the ligand-binding extracellular domain of human ALK1 linked to a human IgG1-Fc region. ACE-041 binds with high affinity to BMP9 and BMP10 but does not bind to either TGFβ1, 2 or 3; VEGF or bFGF. By inhibiting their interaction with ALK1, ACE-041 blocks ALK1-mediated signaling, prohibiting vascular endothelial cell maturation and further vascular development. Unlike other angiogenesis inhibitors that act on the initial proliferative phase, ACE-041 blocks a critical and convergent step in the maturation phase of angiogenesis. In vitro and in vivo studies demonstrated that ACE-041 inhibited vascular maturation regardless of which growth factor triggered the proliferative phase (e.g., VEGF, bFGF). ACE-041 has also been shown to inhibit tumor vascular development, resulting in potent antitumor activity in a variety of solid and hematological tumor models (Mitchell et al., MCT 2010, in press). Methods: A phase I study of ACE-041 is ongoing in the United States to evaluate the safety, tolerability, pharmacokinetics, and anti- tumor activity of ACE-041 in patients with advanced solid tumors or relapsed/refractory multiple myeloma. Cohorts of 3 to 6 patients will be enrolled at escalating dose levels and will receive ACE-041 subcutaneously once every 3 weeks for a total of 4 doses or until disease progression. Dose escalation is determined following review of safety data through 21 days from at least 3 patients at each dose level. The safety and efficacy data from this phase I first-in-human study of ACE-041 will be evaluated to plan additional monotherapy and combination studies in patients with solid tumors and hematologic malignancies. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Acceleron Pharma Acceleron Pharma Acceleron Pharma Acceleron Pharma

Activin receptor-like kinase 1 modulates transforming growth factor-beta 1 signaling in the regulation of angiogenesis.

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.