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Osteopenia is frequently observed in patients with iron overload, especially in those with HFE-dependent hereditary hemochromatosis (HH). Interestingly, not all mouse models of HH show bone loss, suggesting that iron overload alone may not suffice to induce bone loss. In this study, the bone phenotypes of Hjv -/- and hepatocyte-specific Alk2- and Alk3-deficient mice as additional mouse models of HH were investigated to further clarify, how high iron levels lead to bone loss and which signaling mechanisms are operational. Neither male nor female 12-week-old Hjv -/- mice had an altered trabecular or cortical bone mass or bone turnover, despite severe iron overload. Male 12-month-old Hjv -/- mice even presented with a higher femoral trabecular bone volume compared to wildtype mice. Similarly, female mice with hepatocyte-specific Alk2 or Alk3 deficiency did not show an altered bone phenotype at 3, 6, and 12 months of age. Male hepatocyte-specific Alk3-deficient mice also had a normal trabecular bone mass at all ages analyzed, despite showing increased bone resorption and decreased bone formation parameters. Interestingly, hepatocyte-specific Alk2-deficient mice showed reduced femoral trabecular bone at 6 months of age due to suppressed bone formation. Cortical thickness at the femur was reduced in both, 6-month-old male hepatocyte-specific Alk2- and Alk3-deficient mice. Raising hepatocyte-specific Alk2-deficient male mice on an iron-deficient diet rescued the bone phenotype. Taken together, despite iron overload, trabecular bone microarchitecture was not altered in mice deficient of Hjv or Alk3. Only male hepatocyte-specific Alk2-deficient mice showed site-specific lower trabecular and cortical bone mass at the femur, which was dependent on iron. Thus, bone loss does not correlate with the extent of iron overload in these mouse models, but may relate to the amount of iron-loaded macrophages, as precursors of osteoclasts, in the bone marrow., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Activin receptor type 1 (ACVR1; ALK2) and activin receptor like type 1 (ACVRL1; ALK1) are transforming growth factor beta family receptors that integrate extracellular signals of bone morphogenic proteins (BMPs) and activins into Mothers Against Decapentaplegic homolog 1/5 (SMAD1/SMAD5) signaling complexes. Several activating mutations in ALK2 are implicated in fibrodysplasia ossificans progressiva (FOP), diffuse intrinsic pontine gliomas, and ependymomas. The ALK2 R206H mutation is also present in a subset of endometrial tumors, melanomas, non-small lung cancers, and colorectal cancers, and ALK2 expression is elevated in pancreatic cancer. Using DNA-encoded chemistry technology, we screened 3.94 billion unique compounds from our diverse DNA-encoded chemical libraries (DECLs) against the kinase domain of ALK2. Off-DNA synthesis of DECL hits and biochemical validation revealed nanomolar potent ALK2 inhibitors. Further structure-activity relationship studies yielded center for drug discovery (CDD)-2789, a potent [NanoBRET (NB) cell IC 50 : 0.54 μM] and metabolically stable analog with good pharmacological profile. Crystal structures of ALK2 bound with CDD-2281, CDD-2282, or CDD-2789 show that these inhibitors bind the active site through Van der Waals interactions and solvent-mediated hydrogen bonds. CDD-2789 exhibits high selectivity toward ALK2/ALK1 in KINOMEscan analysis and NB K192 assay. In cell-based studies, ALK2 inhibitors effectively attenuated activin A and BMP-induced Phosphorylated SMAD1/5 activation in fibroblasts from individuals with FOP in a dose-dependent manner. Thus, CDD-2789 is a valuable tool compound for further investigation of the biological functions of ALK2 and ALK1 and the therapeutic potential of specific inhibition of ALK2., Competing Interests: Competing interests statement:A provisional patent was submitted by R.J., D.M., K.M.B., F.L., J.-Y.L., Z.T. D.W.Y., S.S.P., and M.M.M. from Baylor College of Medicine.

Reversing cancer cachexia remains a challenge. Genetic biomarkers for the early detection of the disease have been explored in order to enable the implementation of preventive measures. We therefore genotyped candidate genes based on cachexia phenotype and quantified adiponectin and GDF-15 levels in cachectic patients with gastrointestinal cancer. Patients with a diagnosis of gastrointestinal cancer were divided into a cachectic and a non-cachectic group after the start of chemotherapy. A control group (no cancer) was also included. We genotyped the following single nucleotide polymorphisms (SNPs) by quantitative PCR: FOXO3 (rs1935949), FOXO3 (rs4946935), ACVR2B (rs2268757), and SELP (rs6136). In addition, we quantified adiponectin and GDF-15 levels by ELISA. The rs2268757 SNP in the ACVR2B gene was associated with the weight loss phenotype in cachectic patients with gastrointestinal cancer (non-cachectic, P = 0.004). Plasma adiponectin levels were higher in cachectic patients compared to controls (P = 0.01) and non-cachectic patients (P = 0.004). GDF-15 was also elevated in cachectic patients compared to controls (P < 0.0001) and non-cachectic patients (P = 0.001). Analysis by sex showed elevated adiponectin levels in men (control, P = 0.01) and cachectic women (control, P = 0.04; non-cachectic, P = 0.01), as well as elevated GDF-15 levels in men (control, P = 0.002) and cachectic women (control, P = 0.002; non-cachectic, P = 0.007). However, there was no significant difference in the levels of these cytokines between cachectic men and women. The results suggest the rs2268757 SNP in the ACVR2B gene, adiponectin, and GDF-15 as potential biomarkers of cachexia in gastrointestinal cancer., Competing Interests: Declarations Ethics approval and consent to participate This study was approved by the Research Ethics Committee of the University of Ribeirão Preto (UNAERP) (protocol number 5.968.150), São Paulo, Brazil. Written informed consent was obtained from all participants in the study. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Increased endothelial cell proliferation is a hallmark of arteriovenous malformations (AVMs) in hereditary hemorrhagic telangiectasia (HHT). Here, we report a cyclin-dependent kinase 6 (CDK6)-driven mechanism of cell cycle deregulation involved in endothelial cell proliferation and HHT pathology. Specifically, endothelial cells from the livers of HHT mice bypassed the G1/S checkpoint and progressed through the cell cycle at an accelerated pace. Phosphorylated retinoblastoma (pRB1)-a marker of G1/S transition through the restriction point-accumulated in endothelial cells from retinal AVMs of HHT mice and endothelial cells from skin telangiectasia samples from HHT patients. Mechanistically, inhibition of activin receptor-like kinase 1 signaling increased key restriction point mediators, and treatment with the CDK4/6 inhibitors palbociclib or ribociclib blocked increases in pRB1 and retinal AVMs in HHT mice. Palbociclib also improved vascular pathology in the brain and liver, and slowed cell cycle progression in endothelial cells and endothelial cell proliferation. Endothelial cell-specific deletion of CDK6 was sufficient to protect HHT mice from AVM pathology. Thus, clinically approved CDK4/6 inhibitors might have the potential to be repurposed for HHT., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Hereditary hemorrhagic telangiectasia (HHT) is an inherited disorder of vascular malformations characterized by mucocutaneous telangiectases and arteriovenous malformations (AVMs) in internal organs. HHT is caused by inheritance of a loss of function mutation in one of three genes. Although individuals with HHT are haploinsufficient for one of these genes throughout their entire body, rather than exhibiting a systemic vascular phenotype, vascular malformations occur as focal lesions in discrete anatomic locations. The inconsistency between genotype and phenotype has provoked debate over whether haploinsufficiency or a different mechanism gives rise to the vascular malformations. We previously showed that HHT-associated skin telangiectases develop by a two-hit mutation mechanism in an HHT gene. However, somatic mutations were identified in only half of the telangiectases, raising the question whether a second-hit somatic mutation is a necessary (required) event in HHT pathogenesis. Here, we show that another mechanism for the second hit is loss of heterozygosity across the chromosome bearing the germline mutation. Secondly, we investigate the two-hit mutation mechanism for internal organ AVMs, the source of much of the morbidity of HHT. Here, we identified somatic molecular genetic events in eight liver telangiectases, including point mutations and a loss of heterozygosity event. We also identified somatic mutations in one pulmonary AVM and two brain AVMs, confirming that mucocutaneous and internal organ vascular malformations undergo the same molecular mechanisms. Together, these data argue that bi-allelic loss of function in an HHT gene is a required event in the pathogenesis of HHT-associated vascular malformations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Human spermatogonial stem cells (SSCs) have significant applications in reproductive medicine and regenerative medicine because of their great plasticity. Nevertheless, it remains unknown about the functions and mechanisms of long non-coding RNA (LncRNA) in regulating the fate determinations of human SSCs. Here we have demonstrated that LncRNA ACVR2B-as1 (activin A receptor type 2B antisense RNA 1) controls the self-renewal and apoptosis of human SSCs by interaction with ALDOA via glycolysis activity. LncRNA ACVR2B-as1 is highly expressed in human SSCs. LncRNA ACVR2B-as1 silencing suppresses the proliferation and DNA synthesis and enhances the apoptosis of human SSCs. Mechanistically, our ChIRP-MS and RIP assays revealed that ACVR2B-as1 interacted with ALDOA in human SSCs. High expression of ACVR2B-as1 enhanced the proliferation, DNA synthesis, and glycolysis of human SSCs but inhibited their apoptosis through up-regulation of ALDOA. Importantly, overexpression of ALDOA counteracted the effect of ACVR2B-as1 knockdown on the aforementioned biological processes. Collectively, these results indicate that ACVR2B-as1 interacts with ALDOA to control the self-renewal and apoptosis of human SSCs by enhancing glycolysis activity. This study is of great significance because it sheds a novel insight into molecular mechanisms underlying the fate decisions of human SSCs and it may offer innovative approaches to address the etiology of male infertility., (© 2024. The Author(s).)

Circular RNA (circRNA) is thought to mediate the occurrence and development of human cancer and usually acts as a tiny RNA (miRNA) sponge to regulate downstream gene expression. However, it is not clear whether and how circACVR2A (hsa&#95;circ&#95;0001073) is involved in the progression of HCC. The purpose of this study is to clarify the potential role and molecular mechanism of circACVR2A in regulating the progression of hepatocellular carcinoma cells (HCC). The abundance of related proteins in circACVR2A, microRNA (miR511-5p) and PI3K-Akt signaling pathway was determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) or Western blotting. Cell viability, invasion and apoptosis were analyzed by CCK-8, Transwell analysis and Tunel staining, respectively. The interaction between circACVR2A and microRNA was evaluated by double luciferase reporter gene assay. The results showed that circACVR2A was highly expressed in hepatocellular carcinoma cell lines. Our in vivo and in vitro data showed that circACVR2A promoted the proliferation, migration and invasion of HCC. In terms of mechanism, we found that circACVR2A can directly interact with miR511-5p and act as a miRNA sponge to regulate the expression of related proteins in PI3K-Akt signaling pathway.In HCC, circACVR2A can mediate miR-511-5p/mRNA network to activate PI3K signal pathway. This shows that the molecular regulatory network with circACVR2A as the core is a new potential target for diagnosis and treatment of hepatocellular carcinoma., (© 2024. The Author(s).)

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease characterized by the development of arteriovenous malformations (AVMs) that can result in significant morbidity and mortality. HHT is caused primarily by mutations in bone morphogenetic protein receptors ACVRL1/ALK1, a signaling receptor, or endoglin (ENG), an accessory receptor. Because overexpression of Acvrl1 prevents AVM development in both Acvrl1 and Eng null mice, enhancing ACVRL1 expression may be a promising approach to development of targeted therapies for HHT. Therefore, we sought to understand the molecular mechanism of ACVRL1 regulation. We previously demonstrated in zebrafish embryos that acvrl1 is predominantly expressed in arterial endothelial cells and that expression requires blood flow. Here, we document that flow dependence exhibits regional heterogeneity and that acvrl1 expression is rapidly restored after reinitiation of flow. Furthermore, we find that acvrl1 expression is significantly decreased in mutants that lack the circulating Alk1 ligand, Bmp10, and that, in the absence of flow, intravascular injection of BMP10 or the related ligand, BMP9, restores acvrl1 expression in an Alk1-dependent manner. Using a transgenic acvrl1:egfp reporter line, we find that flow and Bmp10 regulate acvrl1 at the level of transcription. Finally, we observe similar ALK1 ligand-dependent increases in ACVRL1 in human endothelial cells subjected to shear stress. These data suggest that ligand-dependent Alk1 activity acts downstream of blood flow to maintain or enhance acvrl1 expression via a positive feedback mechanism, and that ALK1 activating therapeutics may have dual functionality by increasing both ALK1 signaling flux and ACVRL1 expression., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Telangiectases and arteriovenous malformations (AVMs) are the characteristic lesions of Hereditary Hemorrhagic Telangiectasia (HHT). Somatic second-hit loss-of-function variations in the HHT causative genes, ENG and ACVRL1 , have been described in dermal telangiectasias. It is unclear if somatic second-hit mutations also cause the formation of AVMs and nasal telangiectasias in HHT. To investigate the genetic mechanism of AVM formation in HHT, we evaluated multiple affected tissues from fourteen individuals. DNA was extracted from fresh/frozen tissue of 15 nasal telangiectasia, 4 dermal telangiectasia, and 9 normal control tissue biopsies, from nine unrelated individuals with HHT. DNA from six formalin-fixed paraffin-embedded (FFPE) AVM tissues (brain, lung, liver, and gallbladder) from five individuals was evaluated. A 736 vascular malformation and cancer gene next-generation sequencing (NGS) panel was used to evaluate these tissues down to 1% somatic mosaicism. Somatic second-hit mutations were identified in three in four AVM biopsies (75%) or half of the FFPE (50%) samples, including the loss of heterozygosity in ENG in one brain AVM sample, in which the germline mutation occurred in a different allele than a nearby somatic mutation (both are loss-of-function mutations). Eight of nine (88.9%) patients in whom telangiectasia tissues were evaluated had a somatic mutation ranging from 0.68 to 1.96% in the same gene with the germline mutation. Six of fifteen (40%) nasal and two of four (50%) dermal telangiectasia had a detectable somatic second hit. Additional low-level somatic mutations in other genes were identified in several telangiectasias. This is the first report that nasal telangiectasias and solid organ AVMs in HHT are caused by very-low-level somatic biallelic second-hit mutations.

Background: An imbalance of antiproliferative BMP (bone morphogenetic protein) signaling and proliferative TGF-β (transforming growth factor-β) signaling is implicated in the development of pulmonary arterial hypertension (PAH). The posttranslational modification (eg, phosphorylation and ubiquitination) of TGF-β family receptors, including BMPR2 (bone morphogenetic protein type 2 receptor)/ALK2 (activin receptor-like kinase-2) and TGF-βR2/R1, and receptor-regulated Smads significantly affects their activity and thus regulates the target cell fate. BRCC3 modifies the activity and stability of its substrate proteins through K63-dependent deubiquitination. By modulating the posttranslational modifications of the BMP/TGF-β-PPARγ pathway, BRCC3 may play a role in pulmonary vascular remodeling, hence the pathogenesis of PAH., Methods: Bioinformatic analyses were used to explore the mechanism by which BRCC3 deubiquitinates ALK2. Cultured pulmonary artery smooth muscle cells (PASMCs), mouse models, and specimens from patients with idiopathic PAH were used to investigate the rebalance between BMP and TGF-β signaling in regulating ALK2 phosphorylation and ubiquitination in the context of pulmonary hypertension., Results: BRCC3 was significantly downregulated in PASMCs from patients with PAH and animals with experimental pulmonary hypertension. BRCC3, by de-ubiquitinating ALK2 at Lys-472 and Lys-475, activated receptor-regulated Smad1/5/9, which resulted in transcriptional activation of BMP-regulated PPARγ, p53, and Id1. Overexpression of BRCC3 also attenuated TGF-β signaling by downregulating TGF-β expression and inhibiting phosphorylation of Smad3. Experiments in vitro indicated that overexpression of BRCC3 or the de-ubiquitin-mimetic ALK2-K472/475R attenuated PASMC proliferation and migration and enhanced PASMC apoptosis. In SM22α-BRCC3-Tg mice, pulmonary hypertension was ameliorated because of activation of the ALK2-Smad1/5-PPARγ axis in PASMCs. In contrast, Brcc3 -/- mice showed increased susceptibility of experimental pulmonary hypertension because of inhibition of the ALK2-Smad1/5 signaling., Conclusions: These results suggest a pivotal role of BRCC3 in sustaining pulmonary vascular homeostasis by maintaining the integrity of the BMP signaling (ie, the ALK2-Smad1/5-PPARγ axis) while suppressing TGF-β signaling in PASMCs. Such rebalance of BMP/TGF-β pathways is translationally important for PAH alleviation., Competing Interests: None.

Hemoglobinopathies are one of the most common single-gene genetic disorders globally, with approximately 1% to 5% of the global population carrying the mutated gene for thalassemia. Thalassemia are classified into transfusion-dependent thalassemia and non-transfusion-dependent thalassemia based on the need for blood transfusion. Traditional treatment modalities include blood transfusion, splenectomy, hydroxyurea therapy, and iron chelation therapy, which are now widely used for clinical treatment and constitute the main methods recommended in the β-thalassemia treatment guidelines. However, there are multiple barriers and limitations to the application of these approaches, and there is an urgent need to explore new therapeutic approaches. With the in-depth study of the pathophysiological process of β-thalassemia, a deeper understanding of the pathogenesis of the disease has been gained. It has been demonstrated that the pathogenesis of thalassemia is closely related to ineffective erythropoiesis (IE), imbalance in the ratio of α/β-globin protein chains and iron overload. New therapeutic approaches are emerging for different pathogenic mechanisms. Among them, new drugs for the treatment of IE mainly include activin receptor II trap ligands, Janus kinase 2 inhibitors, pyruvate kinase activators, and glycine transporter protein 1 inhibitors. Correcting the imbalance in the hemoglobin chain is mainly due to emerging technologies such as bone marrow transplantation and gene editing. Measures in reducing iron overload are associated with inhibiting the activity of transferrin and hepcidin. These new approaches provide new ideas and options for the treatment and management of β-thalassemia.

In the highly regulative embryo of the sea urchin Paracentrotus lividus, establishment of the dorsal-ventral (D/V) axis critically depends on the zygotic expression of the TGF-β nodal in the ventral ectoderm. nodal expression is first induced ubiquitously in the 32-cell embryo and becomes progressively restricted to the presumptive ventral ectoderm by the early blastula stage. This early spatial restriction of nodal expression is independent of Lefty, and instead relies on the activity of Panda, a maternally expressed TGF-β ligand related to Lefty and Inhibins, which is required maternally for D/V axis specification. However, the mechanism by which Panda restricts the early nodal expression has remained enigmatic and it is not known if Panda works like a BMP ligand by opposing Nodal and antagonizing Smad2/3 signaling, or if it works like Lefty by sequestering an essential component of the Nodal signaling pathway. In this study, we report that Panda functions as an antagonist of the TGF-β type II receptor ACVRII (Activin receptor type II), which is the only type II receptor for Nodal signaling in the sea urchin and is also a type II receptor for BMP ligands. Inhibiting translation of acvrII mRNA disrupted D/V patterning across all 3 germ layers and caused acvrII morphants to develop with a typical Nodal loss-of-function phenotype. In contrast, embryos overexpressing acvrII displayed strong ectopic Smad1/5/8 signaling at blastula stages and developed as dorsalized larvae, a phenotype very similar to that caused by over activation of BMP signaling. Remarkably, embryos co-injected with acvrII mRNA and panda mRNA did not show ectopic Smad1/5/8 signaling and developed with a largely normal dorsal-ventral polarity. Furthermore, using an axis induction assay, we found that Panda blocks the ability of ACVRII to orient the D/V axis when overexpressed locally. Using co-immunoprecipitation, we showed that Panda physically interacts with ACVRII, as well as with the Nodal co-receptor Cripto, and with TBR3 (Betaglycan), which is a non-signaling receptor for Inhibins in mammals. At the molecular level, we have traced back the antagonistic activity of Panda to the presence of a single proline residue, conserved with all the Lefty factors, in the ACVRII binding motif of Panda, instead of a serine as in most of TGF-β ligands. Conversion of this proline to a serine converted Panda from an antagonist that opposed Nodal signaling and promoted dorsalization to an agonist that promoted Nodal signaling and triggered ventralization when overexpressed. Finally, using phylogenomics, we analyzed the emergence of the agonist and antagonist form of Panda in the course of evolution. Our data are consistent with the idea that the presence of a serine at that position, like in most TGF-β, was the ancestral condition and that the initial function of Panda was possibly in promoting and not in antagonizing Nodal signaling. These results highlight the existence of key functional and structural elements conserved between Panda and Lefty, allow to draw an intriguing parallel between sea urchin Panda and mammalian Inhibin α and raise the unexpected possibility that the original function of Panda may have been in activation of the Nodal pathway rather than in its inhibition., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Viswanathan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

This study probes the utility of biomarkers for microsatellite instability (MSI) detection and elucidates the molecular dynamics propelling colorectal cancer (CRC) progression. We synthesized a primer panel targeting 725 MSI loci, informed by The Cancer Genome Atlas (TCGA) and ancillary databases, to construct an amplicon library for next-generation sequencing (NGS). K-means clustering facilitated the distillation of 8 prime MSI loci, including activin A receptor type 2A (ACVR2A). Subsequently, we explored ACVR2A's influence on CRC advancement through in vivo tumor experiments and hematoxylin-eosin (HE) staining. Transwell assays gauged ACVR2A's role in CRC cell migration and invasion, while colony formation assays appraised cell proliferation. Western blotting illuminated the impact of ACVR2A suppression on CRC's PI3K/AKT/mTOR pathway protein expressions under hypoxia. Additionally, ACVR2A's influence on CRC-induced angiogenesis was quantified via angiogenesis assays. K-means clustering of NGS data pinpointed 32 MSI loci specific to tumor and DNA mismatch repair deficiency (dMMR) tissues. ACVR2A emerged as a pivotal biomarker, discerning MSI-H tissues with 90.97% sensitivity. A curated 8-loci set demonstrated 100% sensitivity and specificity for MSI-H detection in CRC. In vitro analyses corroborated ACVR2A's critical role, revealing its suppression of CRC proliferation, migration, and invasion. Moreover, ACVR2A inhibition under CRC-induced hypoxia markedly escalated MMP3, CyclinA, CyclinD1, and HIF1α protein expressions, alongside angiogenesis, by triggering the PI3K/AKT/mTOR cascade. The 8-loci ensemble stands as the optimal marker for MSI-H identification in CRC. ACVR2A, a central element within this group, deters CRC progression, while its suppression amplifies PI3K/AKT/mTOR signaling and angiogenesis under hypoxic stress., (© 2024. The Author(s).)

The functional role of TGFβ type I receptor, activin-like kinase (ALK)-1 in post-myocardial infarction (MI) cardiac remodeling is unknown. We hypothesize that reduced ALK1 activity reduces survival and promotes cardiac fibrosis after MI. MI was induced in wild-type (WT), and ALK +/- mice by left coronary ligation. After 14 days ALK1 +/- mice had reduced survival with a higher rate of cardiac rupture compared to WT mice. ALK1 +/- left ventricles (LVs) had increased volumes at the end of systole and at the end of diastole. After MI ALK1 +/- LVs had increased profibrotic SMAD3 signaling, type 1 collagen, and fibrosis as well as increased levels of TGFβ1 co-receptor, endoglin, VEGF, and ALK1 ligands BMP9 and BMP10. ALK1 +/- LVs had decreased levels of stromal-derived factor 1α. These data identify the critical role of ALK1 in post-MI survival and cardiac remodeling and implicate ALK1 as a potential therapeutic target to improve survival after MI., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Myeloid immune cells are abundant in both ruptured and unruptured brain arteriovenous malformations (bAVMs). The role of central nervous system (CNS) resident and circulating monocyte-derived macrophages in bAVM pathogenesis has not been fully understood. We hypothesize that CNS resident macrophages enhance bAVM development and hemorrhage. RNA sequencing using cultured endothelial cells (ECs) and mouse bAVM samples revealed that downregulation of two bAVM causative genes, activin-like kinase 1 (ALK1) or endoglin, increased inflammation and innate immune signaling. To understand the role of CNS resident macrophages in bAVM development and hemorrhage, we administrated a colony-stimulating factor 1 receptor inhibitor to bAVM mice with brain focal Alk1 deletion. Transient depletion of CNS resident macrophages at an early stage of bAVM development mitigated the phenotype severity of bAVM, including a prolonged inhibition of angiogenesis, dysplastic vasculature formation, and infiltration of CNS resident and circulating monocyte-derived macrophages during bAVM development. Transient depletion of CNS resident macrophages increased EC tight junction protein expression, reduced the number of dysplasia vessels and severe hemorrhage in established bAVMs. Thus, EC AVM causative gene mutation can activate CNS resident macrophages promoting bAVM progression. CNS resident macrophage could be a therapeutic target to mitigate the development and severity of bAVMs., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Abstract: For monogenic diseases caused by pathogenic loss-of-function DNA variants, attention focuses on dysregulated gene-specific pathways, usually considering molecular subtypes together within causal genes. To better understand phenotypic variability in hereditary hemorrhagic telangiectasia (HHT), we subcategorized pathogenic DNA variants in ENG/endoglin, ACVRL1/ALK1, and SMAD4 if they generated premature termination codons (PTCs) subject to nonsense-mediated decay. In 3 patient cohorts, a PTC-based classification system explained some previously puzzling hemorrhage variability. In blood outgrowth endothelial cells (BOECs) derived from patients with ACVRL1+/PTC, ENG+/PTC, and SMAD4+/PTC genotypes, PTC-containing RNA transcripts persisted at low levels (8%-23% expected, varying between replicate cultures); genes differentially expressed to Bonferroni P < .05 in HHT+/PTC BOECs clustered significantly only to generic protein terms (isopeptide-bond/ubiquitin-like conjugation) and pulse-chase experiments detected subtle protein maturation differences but no evidence for PTC-truncated protein. BOECs displaying highest PTC persistence were discriminated in unsupervised hierarchical clustering of near-invariant housekeeper genes, with patterns compatible with higher cellular stress in BOECs with >11% PTC persistence. To test directionality, we used a HeLa reporter system to detect induction of activating transcription factor 4 (ATF4), which controls expression of stress-adaptive genes, and showed that ENG Q436X but not ENG R93X directly induced ATF4. AlphaFold accurately modeled relevant ENG domains, with AlphaMissense suggesting that readthrough substitutions would be benign for ENG R93X and other less rare ENG nonsense variants but more damaging for Q436X. We conclude that PTCs should be distinguished from other loss-of-function variants, PTC transcript levels increase in stressed cells, and readthrough proteins and mechanisms provide promising research avenues., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant form of vascular dysplasia. Genetic diagnosis is made by identifying loss-of-function variants in genes, such as ENG and ACVRL1. However, the causal mechanisms of various variants of unknown significance remains unclear. In this study, we analyzed 12 Japanese patients from 11 families who were clinically diagnosed with HHT. Sequencing analysis identified 11 distinct variants in ACVRL1 and ENG. Three of the 11 were truncating variants, leading to a definitive diagnosis, whereas the remaining eight were splice-site and missense variants that required functional analyses. In silico splicing analyses demonstrated that three variants, c.526-3C > G and c.598C > G in ACVRL1, and c.690-1G > A in ENG, caused aberrant splicing, as confirmed by a minigene assay. The five remaining missense variants were p.Arg67Gln, p.Ile256Asn, p.Leu285Pro, and p.Pro424Leu in ACVRL and p.Pro165His in ENG. Nanoluciferase-based bioluminescence analyses demonstrated that these ACVRL1 variants impaired cell membrane trafficking, resulting in the loss of bone morphogenetic protein 9 (BMP9) signal transduction. In contrast, the ENG mutation impaired BMP9 signaling despite normal cell membrane expression. The updated functional analysis methods performed in this study will facilitate effective genetic testing and appropriate medical care for patients with HHT., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Heterozygous activin receptor-like kinase 1 (ALK1) mutations are associated with two vascular diseases: hereditary hemorrhagic telangiectasia (HHT) and more rarely pulmonary arterial hypertension (PAH). Here, we aimed to understand the impact of ALK1 mutations on BMP9 and BMP10 transcriptomic responses in endothelial cells. Endothelial colony-forming cells (ECFCs) and microvascular endothelial cells (HMVECs) carrying loss of function ALK1 mutations were isolated from newborn HHT and adult PAH donors, respectively. RNA-sequencing was performed on each type of cells compared to controls following an 18 h stimulation with BMP9 or BMP10. In control ECFCs, BMP9 and BMP10 stimulations induced similar transcriptomic responses with around 800 differentially expressed genes (DEGs). ALK1-mutated ECFCs unexpectedly revealed highly similar transcriptomic profiles to controls, both at the baseline and upon stimulation, and normal activation of Smad1/5 that could not be explained by a compensation in cell-surface ALK1 level. Conversely, PAH HMVECs revealed strong transcriptional dysregulations compared to controls with > 1200 DEGs at the baseline. Consequently, because our study involved two variables, ALK1 genotype and BMP stimulation, we performed two-factor differential expression analysis and identified 44 BMP9-dysregulated genes in mutated HMVECs, but none in ECFCs. Yet, the impaired regulation of at least one hit, namely lunatic fringe (LFNG), was validated by RT-qPCR in three different ALK1-mutated endothelial models. In conclusion, ALK1 heterozygosity only modified the BMP9/BMP10 regulation of few genes, including LFNG involved in NOTCH signaling. Future studies will uncover whether dysregulations in such hits are enough to promote HHT/PAH pathogenesis, making them potential therapeutic targets, or if second hits are necessary., (© 2024. The Author(s).)

Hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber disease, is a dominant inherited vascular disorder. The clinical diagnosis is based on the Curaçao criteria and pathogenic variants in the ENG and ACVRL1 genes are responsible for most cases of HHT. Four families with a negative targeted gene panel and selected by a multidisciplinary team were selected and whole-genome sequencing was performed according to the recommendations of the French National Plan for Genomic Medicine. Structural variations were confirmed by standard molecular cytogenetic analysis (FISH). In two families with a definite diagnosis of HHT, we identified two different paracentric inversions of chromosome 9, both disrupting the ENG gene. These inversions are considered as pathogenic and causative for the HHT phenotype of the patients. This is the first time structural variations are reported to cause HHT. As such balanced events are often missed by exon-based sequencing (panel, exome), structural variations may be an under-recognized cause of HHT. Genome sequencing for the detection of these events could be suggested for patients with a definite diagnosis of HHT and in whom no causative pathogenic variant was identified., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease caused by a gain-of-function mutation in ACVR1, which is a bone morphogenetic protein (BMP) type I receptor. Moreover, it causes progressive heterotopic ossification (HO) in connective tissues. Using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and mouse models, we elucidated the underlying mechanisms of FOP pathogenesis and identified a candidate drug for FOP., Methods: In the current study, healthy mesenchymal stem/stromal cells derived from iPSCs (iMSCs) expressing ACVR2B-Fc (iMSC ACVR2B-Fc ), which is a neutralizing receptobody, were constructed. Furthermore, patient-derived iMSCs and FOP mouse model (ACVR1 R206H , female) were used to confirm the inhibitory function of ACVR2B-Fc fusion protein secreted by iMSC ACVR2B-Fc on BMP signaling pathways and HO development, respectively., Results: We found that secreted ACVR2B-Fc attenuated BMP signaling initiated by Activin-A and BMP-9 in both iMSCs and FOP-iMSCs in vitro. Transplantation of ACVR2B-Fc-expressing iMSCs reduced primary HO in a transgenic mouse model of FOP. Notably, a local injection of ACVR2B-Fc-expressing iMSCs and not an intraperitoneal injection improved the treadmill performance, suggesting compound effects of ACVR2B-Fc and iMSCs., Conclusions: These results offer a new perspective for treating FOP through stem cell therapy., (© 2024. The Author(s).)

Mutations in activin-like kinase 2 (ALK2), e.g., ALK2-R206H, induce aberrant signaling to SMAD1/5/8, leading to Fibrodysplasia Ossificans Progressiva (FOP). In spite of extensive studies, the underlying mechanism is still unclear. Here, we quantified the homomeric and heteromeric interactions of ACVR2A, ACVR2B, ALK2-WT, and ALK2-R206H by combining IgG-mediated immobilization of one receptor with fluorescence recovery after photobleaching (FRAP) measurements on the lateral diffusion of a co-expressed receptor. ACVR2B formed stable homomeric complexes that were enhanced by Activin A (ActA), while ACVR2A required ActA for homodimerization. ALK2-WT, but not ALK2-R206H, exhibited homomeric complexes unaffected by ActA. ACVR2B formed ActA-enhanced heterocomplexes with ALK2-R206H or ALK2-WT, while ACVR2A interacted mainly with ALK2-WT. The extent of the homomeric complex formation of ACVR2A or ACVR2B was reflected in their ability to induce the oligomerization of ALK2-R206H and ALK2-WT. Thus, ACVR2B, which forms dimers without ligand, induced ActA-independent ALK2-R206H clustering but required ActA for enhancing the oligomerization of the largely dimeric ALK2-WT. In contrast, ACVR2A, which undergoes homodimerization in response to ActA, required ActA to induce ALK2-R206H oligomerization. To investigate whether these interactions are translated into signaling, we studied signaling by the FOP-inducing hyperactive ALK2-R206H mutant, with ALK2-WT signaling as control. The activation of SMAD1/5/8 signaling in cells expressing ALK2-R206H alone or together with ACVR2A or ACVR2B was measured by blotting for pSMAD1/5/8 and by transcriptional activation assays using BRE-Luc reporter. In line with the biophysical studies, ACVR2B activated ALK2-R206H without ligand, while activation by ACVR2A was weaker and required ActA. We propose that the homodimerization of ACVR2B or ACVR2A dictates their ability to recruit ALK2-R206H into higher complexes, enabling the homomeric interactions of ALK2-R206H receptors and, subsequently, their activation.

Background: Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disease inherited in an autosomal dominant manner. Disease-causing variants in endoglin (ENG) and activin A receptor type II-like 1 (ACVRL1) genes are detected in around 90% of the patients; also 2% of patients harbor pathogenic variants at SMAD4 and GDF2. Importantly, the genetic cause of 8% of patients with clinical HHT remains unknown. Here, we present new putative genetic drivers of HHT., Methods: To identify new HHT genetic drivers, we performed exome sequencing of 19 HHT patients and relatives with unknown HHT genetic etiology. We applied a multistep filtration strategy to catalog deleterious variants and prioritize gene candidates based on their known relevance in endothelial cell biology. Additionally, we performed in vitro validation of one of the identified variants., Results: We identified variants in the INHA, HIF1A, JAK2, DNM2, POSTN, ANGPTL4, FOXO1 and SMAD6 genes as putative drivers in HHT. We have identified the SMAD6 p.(Glu407Lys) variant in one of the families; this is a loss-of-function variant leading to the activation of the BMP/TGFβ signaling in endothelial cells., Conclusions: Variants in these genes should be considered for genetic testing in patients with HHT phenotype and negative for ACVRL1/ENG mutations., Competing Interests: Declaration of Competing Interest Authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Brain arteriovenous malformation (BAVM) is a rare but serious cerebrovascular disease whose pathogenesis has not been fully elucidated. Studies have found that epigenetic regulation, genetic variation and their signaling pathways, immune inflammation, may be the cause of BAVM the main reason. This review comprehensively analyzes the key pathways and inflammatory factors related to BAVMs, and explores their interplay with epigenetic regulation and genetics. Studies have found that epigenetic regulation such as DNA methylation, non-coding RNAs and m6A RNA modification can regulate endothelial cell proliferation, apoptosis, migration and damage repair of vascular malformations through different target gene pathways. Gene defects such as KRAS, ACVRL1 and EPHB4 lead to a disordered vascular environment, which may promote abnormal proliferation of blood vessels through ERK, NOTCH, mTOR, Wnt and other pathways. PDGF-B and PDGFR-β were responsible for the recruitment of vascular adventitial cells and smooth muscle cells in the extracellular matrix environment of blood vessels, and played an important role in the pathological process of BAVM. Recent single-cell sequencing data revealed the diversity of various cell types within BAVM, as well as the heterogeneous expression of vascular-associated antigens, while neutrophils, macrophages and cytokines such as IL-6, IL-1, TNF-α, and IL-17A in BAVM tissue were significantly increased. Currently, there are no specific drugs targeting BAVMs, and biomarkers for BAVM formation, bleeding, and recurrence are lacking clinically. Therefore, further studies on molecular biological mechanisms will help to gain insight into the pathogenesis of BAVM and develop potential therapeutic strategies., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: To assess the utility of the Curaçao criteria by age over time in children with hereditary hemorrhagic telangiectasia (HHT)., Study Design: This was a single-center, retrospective analysis of patients attending the HHT clinic at the Hospital for Sick Children (Toronto, Canada) between 2000 and 2019. The evaluation of the Curaçao criteria was completed during initial and follow-up visits. Screening for pulmonary and brain arteriovenous malformations was completed at 5 yearly intervals., Results: A total of 116 patients with genetic confirmation of HHT were included in the analysis. At initial screening at a median (IQR) age of 8.4 (2.8, 12.9) years, 41% met criteria for a definite clinical diagnosis (≥3 criteria). In children <6 years at presentation, only 23% fulfilled at least 3 criteria initially. In longitudinal follow-up, 63% reached a definite clinical diagnosis, with a median (IQR) follow-up duration of 5.2 (3.2, 7.9) years (P = .005). Specifically, more patients met the epistaxis and telangiectasia criteria at last visit compared with initial (79% vs 60%; P = .006; 47% vs 30%; P = .02) but not for the arteriovenous malformation criterion (59% vs 57%; P = .65)., Conclusions: In the pediatric population, most patients do not meet definite clinical criteria of HHT at initial presentation. Although the number of diagnostic criteria met increased over time, mainly due to new onset of epistaxis and telangiectasia, accuracy remained low during follow-up visits. Relying solely on clinical criteria may lead to underdiagnosis of HHT in children., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

The transforming growth factor β (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 matrix metalloproteinase 9 (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., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology.)

To elucidate the pathogenesis of vein of Galen malformations (VOGMs), the most common and most severe of congenital brain arteriovenous malformations, we performed an integrated analysis of 310 VOGM proband-family exomes and 336,326 human cerebrovasculature single-cell transcriptomes. We found the Ras suppressor p120 RasGAP (RASA1) harbored a genome-wide significant burden of loss-of-function de novo variants (2042.5-fold, p = 4.79 x 10 -7 ). Rare, damaging transmitted variants were enriched in Ephrin receptor-B4 (EPHB4) (17.5-fold, p = 1.22 x 10 -5 ), which cooperates with p120 RasGAP to regulate vascular development. Additional probands had damaging variants in ACVRL1, NOTCH1, ITGB1, and PTPN11. ACVRL1 variants were also identified in a multi-generational VOGM pedigree. Integrative genomic analysis defined developing endothelial cells as a likely spatio-temporal locus of VOGM pathophysiology. Mice expressing a VOGM-specific EPHB4 kinase-domain missense variant (Phe867Leu) exhibited disrupted developmental angiogenesis and impaired hierarchical development of arterial-capillary-venous networks, but only in the presence of a "second-hit" allele. These results illuminate human arterio-venous development and VOGM pathobiology and have implications for patients and their families., (© 2023. The Author(s).)

Purpose: Pulmonary arterial hypertension (PAH) is a rare, progressive vasculopathy with significant cardiopulmonary morbidity and mortality. Genetic testing is currently recommended for adults diagnosed with heritable, idiopathic, anorexigen-, hereditary hemorrhagic telangiectasia-, and congenital heart disease-associated PAH, PAH with overt features of venous/capillary involvement, and all children diagnosed with PAH. Variants in at least 27 genes have putative evidence for PAH causality. Rigorous assessment of the evidence is needed to inform genetic testing., Methods: An international panel of experts in PAH applied a semi-quantitative scoring system developed by the NIH Clinical Genome Resource to classify the relative strength of evidence supporting PAH gene-disease relationships based on genetic and experimental evidence., Results: Twelve genes (BMPR2, ACVRL1, ATP13A3, CAV1, EIF2AK4, ENG, GDF2, KCNK3, KDR, SMAD9, SOX17, and TBX4) were classified as having definitive evidence and 3 genes (ABCC8, GGCX, and TET2) with moderate evidence. Six genes (AQP1, BMP10, FBLN2, KLF2, KLK1, and PDGFD) were classified as having limited evidence for causal effects of variants. TOPBP1 was classified as having no known PAH relationship. Five genes (BMPR1A, BMPR1B, NOTCH3, SMAD1, and SMAD4) were disputed because of a paucity of genetic evidence over time., Conclusion: We recommend that genetic testing includes all genes with definitive evidence and that caution be taken in the interpretation of variants identified in genes with moderate or limited evidence. Genes with no known evidence for PAH or disputed genes should not be included in genetic testing., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2023 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Background: Multitarget tyrosine kinase inhibitors (mTKIs) such as Regorafenib and Sorafenib have already been approved for the treatment of many solid tumours. However, the efficacy of mTKIs in colorectal cancer (CRC) is limited; the underlined mechanism remains largely elusive. Our study was aimed to find out the resistance mechanism of mTKIs in CRC., Methods: RNA sequencing was used to identify the expression of Activin A receptor-like type 1 (ACVRL1) under the treatment of mTKIs. Gain/loss-of-function experiments were performed to assess the biological function of ACVRL1 in resistance to mTKIs. The underlying mechanisms of ACVRL1-mediated mTKI resistance were investigated by using liquid chromatography-mass spectrometry assays (LC-MS), co-immunoprecipitation assays (Co-IP), chromatin immunoprecipitation assays, ubiquitination assays, dual luciferase reporter assays, etc. RESULTS: RNA sequencing identified the activation of ACVRL1 under the treatment of mTKIs in CRC cells. ACVRL1 knockdown and overexpression significantly affects the sensitivity of CRC cells to mTKIs both in vitro and vivo. Mechanistically, we found the β-catenin/TCF-1-KCNQ1OT1/miR-7-5p axis mediated the activation of ACVRL1. Furthermore, LC-MS assays indicated the interaction between ACVRL1 and glutathione peroxidase 2(GPX2) protein. IP assay defined ACVRL1 truncation (282-503aa) could be responsible for interacting with GPX2, and rescue experiments with ACVRL1 truncations confirmed the importance of this interaction in driving mTKI resistance. Co-IP assays confirmed that ACVRL1 associates with ubiquitin-specific peptidase 15(USP15) which directly deubiquinates GPX2 at the K187(K, lysine) site, leading to the accumulation of GPX2 protein. Rescue experiments performed with the lysine mutants in GPX2 CRISPR knockout cell model confirmed the importance of GPX2 K187 mutant. As a result, the increased ROS clearance and decreased cell apoptosis eventually lead to mTKI resistance in CRC., Conclusions: Our results demonstrate that the Wnt/β-catenin/KCNQ1OT1/miR-7-5p/ACVRL1/GPX2 biological axis plays a vital role in CRC, targeting which may be an effective approach for overcoming mTKI resistance., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Objective: To describe the clinical characteristics, diagnosis, genetic features and treatment of hereditary pulmonary hypertension complicated with suspected hereditary hemorrhagic telangiectasia (HHT). Methods: Firstly, we summarized and analyzed the clinical data of two cases of suspected HHT admitted to the Department of Pulmonary and Critical Care Medicine, the Second Xiangya Hospital, Central South University. Secondly, the genes of peripheral blood of patients and their families were completely sequenced and sanger sequencing was performed to verify the variation sites, and then the mRNA deletion caused by the variation was further verified. Thirdly, "HHT" "FPAH" and " BMPR2 gene variation" were used as keywords,and the related literatures of Wanfang database and PubMed database from January 2000 to November 2021 were searched and reviewed. Results: We found two patients in a family from Yiyang, Hunan province, who had symptoms of hemoptysis or pulmonary hypertension without epistaxis or other clinical features of HHT. However, both patients had pulmonary vascular abnormalities and pulmonary hypertension in their lungs. We found that BMPR2 gene variation (NM&#95;001204.7:c.1128+1G>T) was positive and ENG, ACVRL1 and SMAD4 genes were negative. Family analysis and Sanger verification were carried out on 16 individuals in 4 generations of the family (7 of whom were found to carry the mutant gene), and then transcriptional level mRNA sequencing further confirmed that the variation resulted in the deletion of exon 8 and exon 9, and amino acid sequence estimation revealed that the amino acids of the protein from 323 to 425 were deleted. We thought that the incomplete translation of BMPR2 gene could lead to BMPRⅡ dysfunction. Therefore, it was diagnosed as hereditary pulmonary hypertension with suspected HHT. Both patients were suggested to reduce the pulmonary artery pressure, and at the same time, the whole-body imaging examination should be performed to screen other arteriovenous malformations, and the annual cardiac color Doppler ultrasound should be reviewed to evaluate the changes of pulmonary artery pressure. Conclusions: Hereditary pulmonary hypertension (HPAH) is a group of diseases with increasing pulmonary vascular resistance caused by genetic factors, including familial PAH and simple PAH. Variation in the BMPR2 gene is an important pathogenic factor of HPAH. Therefore, we should pay attention to the inquiry of family history when we clinically encounter young patients with pulmonary hypertension. If the cause is unknown, genetic testing is recommended. HHT is a rare autosomal dominant genetic disease. The possibility of this disease should be considered in clinical manifestations such as familial pulmonary vascular abnormality, pulmonary hypertension and recurrent epistaxis. There is no effective specific treatment for HPAH and HHT, which are treated symptomatically (including blood pressure reduction and hemostasis, etc. ). It is suggested for these patients that pulmonary artery pressure should be dynamically monitored and have genetic counseling before giving birth.

Hereditary hemorrhagic telangiectasia (HHT) type 2 is an autosomal dominant disease in which one allele of the ACVRL1 gene is mutated. Patients exhibit disturbances in TGF-beta/BMP-dependent angiogenesis and, clinically, often present with severe nosebleeds as well as a reduced quality of life. The aim of our study was to use CRISPR/Cas9 to knockout ACVRL1 in normal induced pluripotent stem cells (iPSCs) and evaluate the effects on TGF-beta- and BMP-related gene expression as well as angiogenesis. The CRISPR/Cas9 knockout of the ACVRL1 gene was carried out in previously characterized wild-type ( ACVRL1 wt/wt ) iPSCs. An HHT type 2 iPS cell line was generated via a single-allele knockout ( ACVRL1 wt/mut ) in wild-type ( ACVRL1 wt/wt ) iPSCs, resulting in a heterozygous 17 bp frameshift deletion in the ACVRL1 gene [NG&#95;009549.1:g.13707&#95;13723del; NM&#95;000020.3:c.1137&#95;1153del]. After the generation of embryoid bodies (EBs), endothelial differentiation was induced via adding 4 ng/mL BMP4, 2% B27, and 10 ng/mL VEGF. Endothelial differentiation was monitored via immunocytochemistry. An analysis of 151 TGF-beta/BMP-related genes was performed via RT-qPCR through the use of mRNA derived from single iPS cell cultures as well as endothelial cells derived from EBs after endothelial differentiation. Differential TGF-beta/BMP gene expression was observed between ACVRL1 wt/wt and ACVRL1 wt/mut iPSCs as well as endothelial cells. EBs derived from CRISPR/Cas9-designed ACVRL1 mutant HHT type 2 iPSCs, together with their isogenic wild-type iPSC counterparts, can serve as valuable resources for HHT type 2 in vitro studies.

Background: The pathogenic mechanism of brain arteriovenous malformation (bAVM) is poorly understood. A growing body of evidence indicates that genetic factors play crucial roles in bAVM. This study examined genetic variants associated with bAVM through quantitative synthesis and qualitative description of literature., Methods: Five databases were searched to gather potentially relevant articles published up to January 2022. STATA 14.0 software was used for statistical analyses. Pooled odds ratios and 95% confidence intervals were calculated with random effect models, and heterogeneity was assessed using the Cochran Q test and quantified with the I 2 test. Sensitivity and publication bias were analyzed to test the robustness of the associations. Variants identified in only one study or with great heterogeneity were not suitable for pooling association analysis, and therefore a qualitative systematic review was performed., Results: In total, 30 papers were included in a systematic review involving 4709 cases and 7832 controls, where 17 papers were in a meta-analysis. A suggested association of bAVM was observed with ACVRL1 rs2071219 in the additive model and CDKN2B-AS1 rs1333040 in the recessive and additive models. Other variants of genes that could not be analyzed were summarized by qualitative description. These genes were mostly involved in bone morphogenic protein/transforming growth factor beta (BMP/TGF-β), vascular endothelial growth factor/vascular endothelial growth factor receptor (VEGF/VEGFR), and RAS-mitogen activated protein kinase (MAPK) signaling and inflammation., Conclusions: According to our meta-analysis, ACVRL1 rs2071219 and CDKN2B-AS1 rs1333040 were potentially associated with bAVM. Multiple pathological signaling pathways could affect disease development. Future studies should aim to determine the interaction of candidate genes with environmental risk factors and to elucidate detailed mechanisms of action of variants and genes.1., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Blood vessels can play dual roles in tissue growth by transporting gases and nutrients and by regulating tissue stem cell activity via signaling. Correlative evidence implicates skin endothelial cells (ECs) as signaling niches of hair follicle stem cells (HFSCs), but functional demonstration from gene depletion of signaling molecules in ECs is missing to date. Here, we show that depletion of the vasculature-factor Alk1 increases BMP4 secretion from ECs, which delays HFSC activation. Furthermore, while previous evidence suggests a lymphatic vessel role in adult HFSC activation possibly through tissue drainage, a blood vessel role has not yet been addressed. Genetic perturbation of the ALK1-BMP4 axis in all ECs or the lymphatic ECs specifically unveils inhibition of HFSC activation by blood vessels. Our work suggests a broader relevance of blood vessels, adding adult HFSCs to the EC functional repertoire as signaling niches for the adult stem cells., (© 2023 The Authors.)

Low-density lipoprotein (LDL) accumulation in the arterial wall contributes to atherosclerosis initiation and progression 1 . Activin A receptor-like type 1 (ACVRL1, called activin-like kinase receptor (ALK1)) is a recently identified receptor that mediates LDL entry and transcytosis in endothelial cells (ECs) 2,3 . However, the role of this pathway in vivo is not yet known. In the present study, we show that genetic deletion of ALK1 in arterial ECs of mice substantially limits LDL accumulation, macrophage infiltration and atherosclerosis without affecting cholesterol or triglyceride levels. Moreover, a selective monoclonal antibody binding ALK1 efficiently blocked LDL transcytosis, but not bone morphogenetic protein-9 (BMP9) signaling, dramatically reducing plaque formation in LDL receptor knockout mice fed a high-fat diet. Thus, our results demonstrate that blocking LDL transcytosis into the endothelium may be a promising therapeutic strategy that targets the initiating event of atherosclerotic cardiovascular disease., (© 2023. The Author(s).)

This review aimed to provide an overview of pulmonary arteriovenous malformations, including the major clinical and radiological presentations, investigation, and treatment algorithm of the condition. The primary etiology of pulmonary arteriovenous malformations is hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber syndrome, with mutations in the ENG gene on chromosome 9 (HHT type 1) or in the ACVRL1/ALK1 complex (HHT type 2). Epistaxis should always be evaluated when repeated, when associated with anemia, and in some cases of hypoxemia. In the investigation, contrast echocardiography and chest CT are essential for evaluating this condition. Embolization is the best treatment choice, especially for correction in cases of hypoxemia or to avoid systemic infections. Finally, disease management was addressed in special conditions such as pregnancy. CT follow-up should be performed every 3-5 years, depending on the size of the afferent and efferent vessels, and antibiotic prophylactic care should always be oriented. Ultimately, knowledge of the disease by health professionals is a crucial point for the early diagnosis of these patients in clinical practice, which can potentially modify the natural course of the disease.

Hereditary hemorrhagic telangiectasia (HHT) is a rare genetic disease caused by mutations affecting components of bone morphogenetic protein (BMP)/transforming growth factor-β (TGF-β) signaling in endothelial cells. This disorder is characterized by arteriovenous malformations that are prone to rupture, and the ensuing hemorrhages are responsible for iron-deficiency anemia. Along with activin receptor-like kinase (ALK1), mutations in endoglin are associated with the vast majority of HHT cases. In this study, we characterized the zebrafish endoglin locus and demonstrated that it produces two phylogenetically conserved protein isoforms. Functional analysis of a CRISPR/Cas9 zebrafish endoglin mutant revealed that Endoglin deficiency is lethal during the course from juvenile stage to adulthood. Endoglin-deficient zebrafish develop cardiomegaly, resulting in heart failure and hypochromic anemia, which both stem from chronic hypoxia. endoglin mutant zebrafish display structural alterations of the developing gills and underlying vascular network that coincide with hypoxia. Finally, phenylhydrazine treatment demonstrated that lowering hematocrit/blood viscosity alleviates heart failure and enhances the survival of Endoglin-deficient fish. Overall, our data link Endoglin deficiency to heart failure and establish zebrafish as a valuable HHT model., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Background: Noncompaction of ventricular myocardium(NVM) is a rare kind of cardiomyopathy associated with genetic mutations and nongenetic factors, among which the isolated right ventricular noncompaction (iRVNC) is the most rare type. ACVRL1 is the pathogenic gene of type 2 hereditary hemorrhagic telangiectasia (HHT2), and there's no NVM reported to be associated with ACVRL1 mutation., Case Presentation: This is a rare case diagnosed as iRVNC and pulmonary hypertention with ACVRL1 mutation detected., Conclusion: iRVNC in this case may be due to ACVRL1 mutation, secondary to pulmonary hypertention and right ventricular failure caused by ACVRL1 mutation, or they happened in the same case coincidently., (© 2023. The Author(s).)

Hereditary haemorrhagic telangiectasia (HHT) causes arteriovenous malformations (AVMs) in multiple organs to cause bleeding, neurological and other complications. HHT is caused by mutations in the BMP co-receptor endoglin. We characterised a range of vascular phenotypes in embryonic and adult endoglin mutant zebrafish and the effect of inhibiting different pathways downstream of Vegf signalling. Adult endoglin mutant zebrafish developed skin AVMs, retinal vascular abnormalities and cardiac enlargement. Embryonic endoglin mutants developed an enlarged basilar artery (similar to the previously described enlarged aorta and cardinal vein) and larger numbers of endothelial membrane cysts (kugeln) on cerebral vessels. Vegf inhibition prevented these embryonic phenotypes, leading us to investigate specific Vegf signalling pathways. Inhibiting mTOR or MEK pathways prevented abnormal trunk and cerebral vasculature phenotypes, whereas inhibiting Nos or Mapk pathways had no effect. Combined subtherapeutic mTOR and MEK inhibition prevented vascular abnormalities, confirming synergy between these pathways in HHT. These results indicate that the HHT-like phenotype in zebrafish endoglin mutants can be mitigated through modulation of Vegf signalling. Combined low-dose MEK and mTOR pathway inhibition could represent a novel therapeutic strategy in HHT., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Objective: Heritability of stroke is assumed not to be low, especially in the young stroke population. However, most genetic studies have been performed in highly selected patients with typical clinical or neuroimaging characteristics. We investigated the prevalence of 15 Mendelian stroke genes and explored the relationships between variants and the clinical and neuroimaging characteristics in a large, unselected, young stroke population., Methods: We enrolled patients aged ≤55 years with stroke or transient ischemic attack from a prospective, nationwide, multicenter stroke registry. We identified clinically relevant genetic variants (CRGVs) in 15 Mendelian stroke genes (GLA, NOTCH3, HTRA1, RNF213, ACVRL1, ENG, CBS, TREX1, ABCC6, COL4A1, FBN1, NF1, COL3A1, MT-TL1, and APP) using a customized, targeted next generation sequencing panel., Results: Among 1,033 patients, 131 (12.7%) had 28 CRGVs, most frequently in RNF213 (n = 59), followed by ABCC6 (n = 53) and NOTCH3 (n = 15). The frequency of CRGVs differed by ischemic stroke subtypes (p < 0.01): the highest in other determined etiology (20.1%), followed by large artery atherosclerosis (13.6%). It also differed between patients aged ≤35 years and those aged 51 to 55 years (17.1% vs 9.3%, p = 0.02). Only 27.1% and 26.7% of patients with RNF213 and NOTCH3 variants had typical neuroimaging features of the corresponding disorders, respectively. Variants of uncertain significance (VUSs) were found in 15.4% patients., Interpretation: CRGVs in 15 Mendelian stroke genes may not be uncommon in the young stroke population. The majority of patients with CRGVs did not have typical features of the corresponding monogenic disorders. Clinical implications of having CRGVs or VUSs should be explored. ANN NEUROL 2023;93:768-782., (© 2022 American Neurological Association.)

Circular RNAs (circRNAs) exert regulatory roles in cerebrovascular disease. Human brain microvascular endothelial cells (HBMECs) participated in brain vascular dysfunction in ischemic stroke. Herein, the functions of circ&#95;0000566 in oxygen-glucose deprivation and reoxygenation (OGD/R)-induced HBMECs were investigated. The expression of circ&#95;0000566, miR-18a-5p, and Activin receptor type 2B (ACVR2B) was measured via quantitative real-time PCR (qRT-PCR). Cell Counting Kit-8 (CCK-8) and flow cytometry assays were utilized to detect cell viability and cell apoptosis. Western blot assay was employed to measure the levels of apoptotic-related proteins and ACVR2B. The secretion of IL-1β, IL-6, and TNF-α was detected via corresponding kits. The relationship between miR-18a-5p and circ&#95;0000566 or ACVR2B was examined via dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Circ&#95;0000566 and ACVR2B were highly expressed, while miR-18a-5p was down-regulated in OGD/R-treated HBMECs. OGD/R treatment promoted HBMECs apoptosis and inflammation and suppressed cell viability, which could be attenuated by silencing of circ&#95;0000566. Circ&#95;0000566 acted as a miR-18a-5p sponge to contribute to OGD/R-induced HBMECs injury. ACVR2B served as a direct target of miR-18a-5p, and ACVR2B overexpression might abolish the inhibitory role of miR-18a-5p on OGD/R-treated HBMEC injury. Circ&#95;0000566 sponged miR-18a-5p to regulate OGD/R-induced HBMECs injury via regulating ACVR2B expression., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disease inherited in an autosomal dominant manner. Disease-causing variants in endoglin ( ENG ) and activin A receptor type II-like 1 ( ACVRL1 ) genes are detected in more than 90% of the patients undergoing molecular testing. The identification of variants of unknown significance is often seen as a challenge in clinical practice that makes family screening and genetic counseling difficult. Here, we show that the implementation of cDNA analysis to assess the effect of splice site variants on mRNA splicing is a powerful tool., Methods: Gene panel sequencing of genes associated with HHT and other arteriovenous malformation-related syndromes was performed. To evaluate the effect of the splice site variants, cDNA analysis of ENG and ACVRL1 genes was carried out., Results: three novel splice site variants were identified in ENG (c.68-2A > T and c.1311+4&#95;1311+8del) and ACVLR1 (c.526-6C > G) genes correspondingly in three individuals with HHT that met ≥ 3 Curaçao criteria. All three variants led to an aberrant splicing inducing exon skipping ( ENG :c.68-2A > T and ACVRL1 :c.526-6C > G) or intron retention ( ENG :c.1311+4&#95;1311+8del) allowing the confirmation of the predicted effect on splicing and the reclassification from unknown significance to pathogenic/likely pathogenic of two of them., Conclusions: RNA analysis should be performed to assess and/or confirm the impact of variants on splicing. The molecular diagnosis of HHT patients is crucial to allow family screening and accurate genetic counseling. A multidisciplinary approach including clinicians and geneticists is crucial when dealing with patients with rare diseases.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic vascular disorder characterized by the presence of arteriovenous malformation (AVM) in multiple organs. HHT is caused by mutations in genes encoding major constituents for transforming growth factor-β (TGF-β) family signaling: endoglin (ENG), activin receptor-like kinase 1 (ALK1), and SMAD4. The identity of physiological ligands for this ENG-ALK1 signaling pertinent to AVM formation has yet to be clearly determined. To investigate whether bone morphogenetic protein 9 (BMP9), BMP10, or both are physiological ligands of ENG-ALK1 signaling involved in arteriovenous network formation, we generated a novel Bmp10 conditional knockout mouse strain. We examined whether global Bmp10-inducible knockout (iKO) mice develop AVMs at neonatal and adult stages in comparison with control, Bmp9-KO, and Bmp9/10-double KO (dKO) mice. Bmp10-iKO and Bmp9/10-dKO mice showed AVMs in developing retina, postnatal brain, and adult wounded skin, while Bmp9-KO did not display any noticeable vascular defects. Bmp10 deficiency resulted in increased proliferation and size of endothelial cells in AVM vessels. The impaired neurovascular integrity in the brain and retina of Bmp10-iKO and Bmp9/10-dKO mice was detected. Bmp9/10-dKO mice exhibited the lethality and vascular malformation similar to Bmp10-iKO mice, but their phenotypes were more pronounced. Administration of BMP10 protein, but not BMP9 protein, prevented retinal AVM in Bmp9/10-dKO and endothelial-specific Eng-iKO mice. These data indicate that BMP10 is indispensable for the development of a proper arteriovenous network, whereas BMP9 has limited compensatory functions for the loss of BMP10. We suggest that BMP10 is the most relevant physiological ligand of the ENG-ALK1 signaling pathway pertinent to HHT pathogenesis., (© 2022. The Author(s).)

Objective: Genetic alterations are increasingly recognized as etiologic factors linked to the pathogenesis and development of cerebrovascular anomalies. Their identification allows for advanced screening and targeted therapeutic approaches. The authors aimed to describe the role of a collaborative approach to care and genetic testing in pediatric patients with neurovascular anomalies, with the objectives of identifying what genetic testing recommendations were made, the yield of genetic testing, and the implications for familial screening and management at present and in the future., Methods: The authors performed a descriptive retrospective cohort study examining pediatric patients genetically screened through the Pediatric Neurovascular Program of a single treatment center. Patients 18 years of age and younger with neurovascular anomalies, diagnosed radiographically or histopathologically, were evaluated for germline genetic testing. Patient demographic data and germline genetic testing and recommendation, clinical, treatment, and outcome data were collected and analyzed., Results: Sixty patients were included; 29 (47.5%) were female. The mean age at consultation was 11.0 ± 4.9 years. Diagnoses included cerebral arteriovenous malformations (AVMs) (n = 23), cerebral cavernous malformations (n = 19), non-neurofibromatosis/non-sickle cell moyamoya (n = 8), diffuse cerebral proliferative angiopathy, and megalencephaly-capillary malformation. Of the 56 patients recommended to have genetic testing, 40 completed it. Genetic alterations were found in 13 (23%) patients. Four patients with AVMs had RASA1, GDF2, and ACVRL1 mutations. Four patients with cavernous malformations had Krit1 mutations. One with moyamoya disease had an RNF213 mutation. Three patients with megalencephaly-capillary malformation had PIK3CA mutations, and 1 patient with a cavernous sinus lesion had an MED12 mutation. The majority of AVM patients were treated surgically. Patients with diffuse cerebral proliferative angiopathy were treated medically with sirolimus. At-risk relatives of 3 patients positive for genetic anomalies had also been tested., Conclusions: This study demonstrates a role for exploring genetic alterations in the identification and treatment of pediatric neurovascular disease pathogenesis. Germline genetic mutations were found in almost one-quarter of the patients screened in this study, results that helped to identify medically targeted treatment modalities for some pediatric neurovascular patients. Insight into the genetic etiology of vascular anomalies may provide broader clinical implications for risk assessment, family screening, follow-up surveillance, and medical management.

Cardiomyopathies are ascribed to a variety of etiologies, present with diverse clinical phenotypes, and lack disease-modifying treatments. Mounting evidence implicates dysregulated activin receptor signaling in heart disease and highlights inhibition of this pathway as a potential therapeutic target. Here, we explored the effects of activin ligand inhibition using ActRIIB:ALK4-Fc, a heterodimeric receptor fusion protein, in two mechanistically distinct murine models of cardiomyopathy. Treatment with ActRIIB:ALK4-Fc significantly improved systolic or diastolic function in cardiomyopathy induced by neuromuscular disease or diabetes mellitus. Moreover, ActRIIB:ALK4-Fc corrected Ca 2+ handling protein expression in diseased heart tissues, suggesting that activin signaling inhibition could alleviate cardiomyopathies in part by rebalancing aberrant intracellular Ca 2+ homeostasis-a common underlying pathomechanism in diverse heart diseases., (© 2022 Federation of European Biochemical Societies.)

Background: Fluid shear stress enhances endothelial SMAD1/5 signaling via the BMP9-bound ALK1 receptor complex supported by the co-receptor Endoglin. While moderate SMAD1/5 activation is required to maintain endothelial quiescence, excessive SMAD1/5 signaling promotes endothelial dysfunction. Increased BMP signaling participates in endothelial-to-mesenchymal transition and inflammation culminating in vascular diseases such as atherosclerosis. While the function of Endoglin has so far been described under picomolar concentrations of BMP9 and short-term shear application, we investigated Endoglin under physiological BMP9 and long-term pathophysiological shear conditions., Results: We report here that knock-down of Endoglin leads to exacerbated SMAD1/5 phosphorylation and atheroprone gene expression profile in HUVECs sheared for 24 h. Making use of the ligand-trap ALK1-Fc, we furthermore show that this increase is dependent on BMP9/10. Mechanistically, we reveal that long-term exposure of ECs to low laminar shear stress leads to enhanced Endoglin expression and endocytosis of Endoglin in Caveolin-1-positive early endosomes. In these endosomes, we could localize the ALK1-Endoglin complex, labeled BMP9 as well as SMAD1, highlighting Caveolin-1 vesicles as a SMAD signaling compartment in cells exposed to low atheroprone laminar shear stress., Conclusions: We identified Endoglin to be essential in preventing excessive activation of SMAD1/5 under physiological flow conditions and Caveolin-1-positive early endosomes as a new flow-regulated signaling compartment for BMP9-ALK1-Endoglin signaling axis in atheroprone flow conditions., (© 2022. The Author(s).)

Background: Circular RNAs (circRNAs) are attractive candidates to be used as biomarkers of human cancers, including lung adenocarcinoma (LUAD). Our study aimed to investigate the functions and regulatory mechanisms of hsa&#95;circ&#95;0129047 in the tumorigenesis of LUAD., Methods: Reverse transcription-quantitative polymerase chain reaction was performed to determine the circRNA, microRNA (miRNA), and mRNA expression levels in LUAD cell lines and tissues. Tumor xenografts were established in nude mice to evaluate whether hsa&#95;circ&#95;0129047 affected LUAD tumor development in vivo. Cell counting kit-8 and transwell assays were performed to assess the mechanisms by which hsa&#95;circ&#95;0129047 influenced the viability and migration of LUAD cells, respectively. Apoptosis was evaluated via determination of the levels of the apoptotic markers, B-cell lymphoma-2, and Bcl-2-associated X, via Western blotting. Dual-luciferase reporter assay, RNA immunoprecipitation assay, and Pearson's correlation analysis were performed to determine the relationships among miR-375 and hsa&#95;circ&#95;0129047 and activin A receptor-like type 1 (ACVRL1)., Results: Downregulation of hsa&#95;circ&#95;0129047 levels was observed in LUAD cell lines and tissues. Meanwhile, the upregulation of hsa&#95;circ&#95;0129047 levels repressed the proliferative, migratory, and survival capacities of LUAD cells in vitro. Hsa&#95;circ&#95;0129047 exerted antitumor effects during in vivo tumor development. Finally, we demonstrated that hsa&#95;circ&#95;0129047 sponged miR-375. This interaction facilitated the expression of the downstream target of miR-375, ACVRL1, whose upregulation inhibited the development and malignancy of LUAD., Conclusion: These findings demonstrate that hsa&#95;circ&#95;0129047 functions as a tumor inhibitor in LUAD by modulating the miR-375/ACVRL1 axis. Hence, hsa&#95;circ&#95;0129047 may be a promising biomarker and gene target for LUAD treatment., (© 2022 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

The effect of breast cancer heterogeneity on prognosis of patients is still unclear, especially the role of immune cells in prognosis of breast cancer. In this study, single cell transcriptome sequencing data of breast cancer were used to analyze the relationship between breast cancer heterogeneity and prognosis. In this study, 14 cell clusters were identified in two single-cell datasets (GSE75688 and G118389). Proportion analysis of immune cells showed that NK cells were significantly aggregated in triple negative breast cancer, and the proportion of macrophages was significantly increased in primary breast cancer, while B cells, T cells, and neutrophils may be involved in the metastasis of breast cancer. The results of ligand receptor interaction network revealed that macrophages and DC cells were the most frequently interacting cells with other cells in breast cancer. The results of WGCNA analysis suggested that the MEblue module is most relevant to the overall survival time of triple negative breast cancer. Twenty-four prognostic genes in the blue module were identified by univariate Cox regression analysis and KM survival analysis. Multivariate regression analysis combined with risk analysis was used to analyze 24 prognostic genes to construct a prognostic model. The verification result of our prognostic model showed that there were significant differences in the expression of PCDH12, SLIT3, ACVRL1, and DLL4 genes between the high-risk group and the low-risk group, which can be used as prognostic biomarkers., (© 2022. The Author(s).)

Background: Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder, affecting 1:5000 individuals worldwide. All the genes associated to the disease (ENG, ACVRL1, SMAD4, GDF2) belong to the TGF-β/BMPs signaling pathway. We found 19 HHT unrelated families, coming from a Northern Italy region and sharing the ACVRL1 in-frame deletion c.289&#95;294del (p.H97&#95;N98)., Methods: To test the hypothesis of a founder effect, we analyzed 88 subjects from 19 families (66 variant carriers, showing clinical signs of HHT, and 22 non-carriers, unaffected) using eight microsatellite markers within 3.7 Mb around the ACVRL1 locus. After the haplotype reconstruction, age estimation of the variant was carried out., Results: We observed a common disease haplotype in 16/19 families, while three families showed evidence of recombination around the ACVRL1 locus. The subsequent age estimation analyses suggested that the mutation occurred about 8 generations ago, corresponding to about 200 years ago. We also present novel in silico and modeling data supporting the variant pathogenicity: the deletion alters the protein stability and removes the unique extracellular glycosylation site., Conclusion: We have demonstrated, for the first time, a "founder effect" for a HHT pathogenic variant in Italy., (© 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)