Blood flow regulates acvrl1 transcription via ligand-dependent Alk1 activity.

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.

Impact of heterozygous ALK1 mutations on the transcriptomic response to BMP9 and BMP10 in endothelial cells from hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension donors.

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.

Endoglin mutants retained in the endoplasmic reticulum exacerbate loss of function in hereditary hemorrhagic telangiectasia type 1 (HHT1) by exerting dominant negative effects on the wild type allele.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder affecting 1 in 5000-8000 individuals. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is the most common HHT and manifests as diverse vascular malformations ranging from mild symptoms such as epistaxis and mucosal and cutaneous telangiectases to severe arteriovenous malformations (AVMs) in the lungs, brain or liver. HHT1 is caused by heterozygous mutations in the ENG gene, which encodes endoglin, the TGFß homodimeric co-receptor. It was previously shown that some endoglin HHT1-causing variants failed to traffic to the plasma membrane due to their retention in the endoplasmic reticulum (ER) and consequent degradation by ER-associated degradation (ERAD). Endoglin is a homodimer formed in the ER, and we therefore hypothesized that mixed heterodimers might form between ER-retained variants and WT protein, thus hampering its maturation and trafficking to the plasma membrane causing dominant negative effects. Indeed, HA-tagged ER-retained mutants formed heterodimers with Myc-tagged WT endoglin. Moreover, variants L32R, V105D, P165L, I271N and C363Y adversely affected the trafficking of WT endoglin by reducing its maturation and plasma membrane localization. These results strongly suggest dominant negative effects exerted by these ER-retained variants aggravating endoglin loss of function in patients expressing them in the heterozygous state with the WT allele. Moreover, this study may help explain some of the variability observed among HHT1 patients due to the additional loss of function exerted by the dominant negative effects in addition to that due to haploinsufficiency. These findings might also have implications for some of the many conditions impacted by ERAD.

Potential angiogenic biomarkers in hereditary hemorrhagic telangiectasia and other vascular diseases.

Biomarkers are new tools framed in precision and personalized medicine. Hereditary hemorrhagic telangiectasia (HHT) is a rare genetic vascular disease with disturbances in the angiogenic pathways. Descriptive evidence supports that some angiogenesis-related molecules are differently detected in HHT patients compared to healthy subjects. These molecules are also related to diagnosis, prognosis, complications and therapy monitoring in other common vascular diseases. Despite the need for improving knowledge before applying them in daily clinical practice, there are good candidates to be considered as potential biomarkers in HHT and other vascular diseases. In the present review, the authors aim to summarize and discuss current evidence regarding the main putative angiogenic biomarkers by describing the biological role of each biomarker, the evidence related to HHT and their potential use in this and other common vascular diseases from a clinical point-of-view.

BMP10 functions independently from BMP9 for the development of a proper arteriovenous network.

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-ß (TGF-ß) 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.

Vascular defects associated with hereditary hemorrhagic telangiectasia revealed in patient-derived isogenic iPSCs in 3D vessels on chip.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by weak blood vessels. HHT1 is caused by mutations in the ENDOGLIN (ENG) gene. Here, we generated induced pluripotent stem cells (hiPSCs) from a patient with rare mosaic HHT1 with tissues containing both mutant (ENGc.1678C>T) and normal cells, enabling derivation of isogenic diseased and healthy hiPSCs, respectively. We showed reduced ENG expression in HHT1 endothelial cells (HHT1-hiPSC-ECs), reflecting haploinsufficiency. HHT1c.1678C>T-hiPSC-ECs and the healthy isogenic control behaved similarly in two-dimensional (2D) culture, forming functionally indistinguishable vascular networks. However, when grown in 3D organ-on-chip devices under microfluidic flow, lumenized vessels formed in which defective vascular organization was evident: interaction between inner ECs and surrounding pericytes was decreased, and there was evidence for vascular leakage. Organs on chip thus revealed features of HHT in hiPSC-derived blood vessels that were not evident in conventional 2D assays.

Neuropilin-1 deficiency in vascular smooth muscle cells is associated with hereditary hemorrhagic telangiectasia arteriovenous malformations.

Patients with hereditary hemorrhagic telangiectasia (HHT) have arteriovenous malformations (AVMs) with genetic mutations involving the activin-A receptor like type 1 (ACVRL1 or ALK1) and endoglin (ENG). Recent studies have shown that Neuropilin-1 (NRP-1) inhibits ALK1. We investigated the expression of NRP-1 in livers of patients with HHT and found that there was a significant reduction in NRP-1 in perivascular smooth muscle cells (SMCs). We used Nrp1SM22KO mice (Nrp1 was ablated in SMCs) and found hemorrhage, increased immune cell infiltration with a decrease in SMCs, and pericyte lining in lungs and liver in adult mice. Histologic examination revealed lung arteriovenous fistulas (AVFs) with enlarged liver vessels. Evaluation of the retina vessels at P5 from Nrp1SM22KO mice demonstrated dilated capillaries with a reduction of pericytes. In inflow artery of surgical AVFs from the Nrp1SM22KO versus WT mice, there was a significant decrease in Tgfb1, Eng, and Alk1 expression and phosphorylated SMAD1/5/8 (pSMAD1/5/8), with an increase in apoptosis. TGF-ß1-stimulated aortic SMCs from Nrp1SM22KO versus WT mice have decreased pSMAD1/5/8 and increased apoptosis. Coimmunoprecipitation experiments revealed that NRP-1 interacts with ALK1 and ENG in SMCs. In summary, NRP-1 deletion in SMCs leads to reduced ALK1, ENG, and pSMAD1/5/8 signaling and reduced cell death associated with AVM formation.

Identification of Exosomal MicroRNA Signature by Liquid Biopsy in Hereditary Hemorrhagic Telangiectasia Patients.

Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal dominant vascular dysplasia characterized by epistaxis, mucocutaneous telangiectases, and arteriovenous malformations (AVM) in the visceral organs. The diagnosis of HHT is based on clinical Curaçao criteria, which show limited sensitivity in children and young patients. Here, we carried out a liquid biopsy by which we isolated total RNA from plasma exosome samples. A cohort of 15 HHT type 1 patients, 15 HHT type 2 patients, and 10 healthy relatives were analyzed. Upon gene expression data processing and normalization, a statistical analysis was performed to explore similarities in microRNA expression patterns among samples and detect differentially expressed microRNAs between HHT samples and the control group. We found a disease-associated molecular fingerprint of 35 miRNAs over-represented in HHT vs. controls, with eight being specific for HHT1 and 11 for HHT2; we also found 30 under-represented, including nine distinct for HHT1 and nine for HHT2. The analysis of the receiver operating characteristic (ROC) curves showed that eight miRNAs had good (AUC > 75%) or excellent (AUC > 90%) diagnosis value for HHT and even for type HHT1 and HHT2. In addition, we identified the cellular origin of these miRNAs among the cell types involved in the vascular malformations. Interestingly, we found that only some of them were incorporated into exosomes, which suggests a key functional role of these exosomal miRNAs in the pathophysiology of HHT.

BMP Receptor Inhibition Enhances Tissue Repair in Endoglin Heterozygous Mice.

Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a severe vascular disorder caused by mutations in the TGFß/BMP co-receptor endoglin. Endoglin haploinsufficiency results in vascular malformations and impaired neoangiogenesis. Furthermore, HHT1 patients display an impaired immune response. To date it is not fully understood how endoglin haploinsufficient immune cells contribute to HHT1 pathology. Therefore, we investigated the immune response during tissue repair in Eng+/- mice, a model for HHT1. Eng+/- mice exhibited prolonged infiltration of macrophages after experimentally induced myocardial infarction. Moreover, there was an increased number of inflammatory M1-like macrophages (Ly6Chigh/CD206-) at the expense of reparative M2-like macrophages (Ly6Clow/CD206+). Interestingly, HHT1 patients also showed an increased number of inflammatory macrophages. In vitro analysis revealed that TGFß-induced differentiation of Eng+/- monocytes into M2-like macrophages was blunted. Inhibiting BMP signaling by treating monocytes with LDN-193189 normalized their differentiation. Finally, LDN treatment improved heart function after MI and enhanced vascularization in both wild type and Eng+/- mice. The beneficial effect of LDN was also observed in the hind limb ischemia model. While blood flow recovery was hampered in vehicle-treated animals, LDN treatment improved tissue perfusion recovery in Eng+/- mice. In conclusion, BMPR kinase inhibition restored HHT1 macrophage imbalance in vitro and improved tissue repair after ischemic injury in Eng+/- mice.

Hereditary Haemorrhagic Telangiectasia, an Inherited Vascular Disorder in Need of Improved Evidence-Based Pharmaceutical Interventions.

Hereditary haemorrhagic telangiectasia (HHT) is characterised by arteriovenous malformations (AVMs). These vascular abnormalities form when arteries and veins directly connect, bypassing the local capillary system. Large AVMs may occur in the lungs, liver and brain, increasing the risk of morbidity and mortality. Smaller AVMs, known as telangiectases, are prevalent on the skin and mucosal lining of the nose, mouth and gastrointestinal tract and are prone to haemorrhage. HHT is primarily associated with a reduction in endoglin (ENG) or ACVRL1 activity due to loss-of-function mutations. ENG and ACVRL1 transmembrane receptors are expressed on endothelial cells (ECs) and bind to circulating ligands BMP9 and BMP10 with high affinity. Ligand binding to the receptor complex leads to activation of the SMAD1/5/8 signalling pathway to regulate downstream gene expression. Various genetic animal models demonstrate that disruption of this pathway in ECs results in AVMs. The vascular abnormalities underlying AVM formation result from abnormal EC responses to angiogenic and haemodynamic cues, and include increased proliferation, reduced migration against the direction of blood flow and an increased EC footprint. There is growing evidence that targeting VEGF signalling has beneficial outcomes in HHT patients and in animal models of this disease. The anti-VEGF inhibitor bevacizumab reduces epistaxis and has a normalising effect on high cardiac output in HHT patients with hepatic AVMs. Blocking VEGF signalling also reduces vascular malformations in mouse models of HHT1 and HHT2. However, VEGF signalling is complex and drives numerous downstream pathways, and it is not yet clear which pathway (or combination of pathways) is critical to target. This review will consider the recent evidence gained from HHT clinical and preclinical studies that are increasing our understanding of HHT pathobiology and informing therapeutic strategies.

Overexpression of Activin Receptor-Like Kinase 1 in Endothelial Cells Suppresses Development of Arteriovenous Malformations in Mouse Models of Hereditary Hemorrhagic Telangiectasia.

RATIONALE: Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease caused by mutations in ENG, ALK1, or SMAD4. Since proteins from all 3 HHT genes are components of signal transduction of TGF-ß (transforming growth factor ß) family members, it has been hypothesized that HHT is a disease caused by defects in the ENG-ALK1-SMAD4 linear signaling. However, in vivo evidence supporting this hypothesis is scarce. OBJECTIVE: We tested this hypothesis and investigated the therapeutic effects and potential risks of induced-ALK1 or -ENG overexpression (OE) for HHT. METHODS AND RESULTS: We generated a novel mouse allele (ROSA26Alk1) in which HA (human influenza hemagglutinin)-tagged ALK1 and bicistronic eGFP expression are induced by Cre activity. We examined whether ALK1-OE using the ROSA26Alk1 allele could suppress the development of arteriovenous malformations (AVMs) in wounded adult skin and developing retinas of Alk1- and Eng-inducible knockout (iKO) mice. We also used a similar approach to investigate whether ENG-OE could rescue AVMs. Biochemical and immunofluorescence analyses confirmed the Cre-dependent OE of the ALK1-HA transgene. We could not detect any pathological signs in ALK1-OE mice up to 3 months after induction. ALK1-OE prevented the development of retinal AVMs and wound-induced skin AVMs in Eng-iKO as well as Alk1-iKO mice. ALK1-OE normalized expression of SMAD and NOTCH target genes in ENG-deficient endothelial cells (ECs) and restored the effect of BMP9 (bone morphogenetic protein 9) on suppression of phosphor-AKT levels in these endothelial cells. On the other hand, ENG-OE could not inhibit the AVM development in Alk1-iKO models. CONCLUSIONS: These data support the notion that ENG and ALK1 form a linear signaling pathway for the formation of a proper arteriovenous network during angiogenesis. We suggest that ALK1 OE or activation can be an effective therapeutic strategy for HHT. Further research is required to study whether this therapy could be translated into treatment for humans.

Intranasal Efudix reduces epistaxis in hereditary hemorrhagic telangiectasia.

BACKGROUND: Local application of fluorouracil (Efudix, 5-FU) induces sclerosis in patients with sinonasal tumors and superficial basocellular skin carcinoma. As a 'back against the wall' treatment, we investigated the local effect of nasally applied 5-FU and whether this could decrease the burden of severe epistaxis in patients with hereditary hemorrhagic telangiectasia (HHT). METHODS: HHT patients with severe and frequent epistaxis, subsequent anemia and a necessity for blood and/or iron infusions were treated with a nasal tampon with 5-FU. This tampon was placed unilaterally in the nasal cavity on the side of the most severe epistaxis and replaced once weekly during 4 weeks. Outcome measures were safety and side effects, the aspect of the nasal mucosa measured with the mucosal HHT score, the epistaxis severity score (ESS), hemoglobin and ferritin plasma levels, and quality of life assessment pre-treatment, one and three months post-treatment. RESULTS: Six HHT patients participated. During treatment and follow-up, the nasal mucosa turned more pale and sclerotic and the number of telangiectases diminished. The mucosal HHT score improved and the ESS declined (p = 0.01). The decline of ESS persisted up to 3 months post-5-FU treatment. Moreover, mean hemoglobin levels increased from 6.0 pre-5-FU to 6.8 after one month post-5-FU. CONCLUSION: Unilateral application of 5-FU on a nasal tampon diminished the severity and frequency of epistaxis in all HHT patients. This effect sustained up to three months post-treatment, despite the fact that the contralateral side remained untreated. Subsequently, hemoglobin levels increased. Intranasal 5-FU is a promising entity for further research on epistaxis treatment in HHT patients.

Future treatments for hereditary hemorrhagic telangiectasia.

Hereditary Hemorrhagic Telangiectasia (HHT), also known as Rendu-Osler syndrome, is a genetic vascular disorder affecting 1 in 5000-8000 individuals worldwide. This rare disease is characterized by various vascular defects including epistaxis, blood vessel dilations (telangiectasia) and arteriovenous malformations (AVM) in several organs. About 90% of the cases are associated with heterozygous mutations of ACVRL1 or ENG genes, that respectively encode a bone morphogenetic protein receptor (activin receptor-like kinase 1, ALK1) and a co-receptor named endoglin. Less frequent mutations found in the remaining 10% of patients also affect the gene SMAD4 which is part of the transcriptional complex directly activated by this pathway. Presently, the therapeutic treatments for HHT are intended to reduce the symptoms of the disease. However, recent progress has been made using drugs that target VEGF (vascular endothelial growth factor) and the angiogenic pathway with the use of bevacizumab (anti-VEGF antibody). Furthermore, several exciting high-throughput screenings and preclinical studies have identified new molecular targets directly related to the signaling pathways affected in the disease. These include FKBP12, PI3-kinase and angiopoietin-2. This review aims at reporting these recent developments that should soon allow a better care of HHT patients.

Correcting Smad1/5/8, mTOR, and VEGFR2 treats pathology in hereditary hemorrhagic telangiectasia models.

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

Recent Advances in Basic Research for Brain Arteriovenous Malformation.

Arteriovenous malformations (AVMs) are abnormal connections of vessels that shunt blood directly from arteries into veins. Rupture of brain AVMs (bAVMs) can cause life-threatening intracranial bleeding. Even though the majority of bAVM cases are sporadic without a family history, some cases are familial. Most of the familial cases of bAVMs are associated with a genetic disorder called hereditary hemorrhagic telangiectasia (HHT). The mechanism of bAVM formation is not fully understood. The most important advances in bAVM basic science research is the identification of somatic mutations of genes in RAS-MAPK pathways. However, the mechanisms by which mutations of these genes lead to AVM formation are largely unknown. In this review, we summarized the latest advance in bAVM studies and discussed some pathways that play important roles in bAVM pathogenesis. We also discussed the therapeutic implications of these pathways.

PI3K (Phosphatidylinositol 3-Kinase) Activation and Endothelial Cell Proliferation in Patients with Hemorrhagic Hereditary Telangiectasia Type 1.

Hemorrhagic hereditary telangiectasia (HHT) type 2 patients have increased activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway in telangiectasia. The main objective is to evaluate the activation of the PI3K pathway in cutaneous telangiectasia of HHT1 patients. A cutaneous biopsy of a digital hand telangiectasia was performed in seven HHT1 and eight HHT2 patients and compared with six controls. The study was approved by the Clinical Research Ethics Committee of our center. A histopathological pattern with more dilated and superficial vessels that pushed up the epidermis was identified in HHT patients regardless of the type of mutation and was associated with older age, as opposed to the common telangiectasia pattern. The mean proliferation index (Ki-67) was statistically higher in endothelial cells (EC) from HHT1 than in controls. The percentage of positive EC for pNDRG1, pAKT, and pS6 in HHT1 patients versus controls resulted in higher values, statistically significant for pNDRG1 and pS6. In conclusion, we detected an increase in EC proliferation linked to overactivation of the PI3K pathway in cutaneous telangiectasia biopsies from HHT1 patients. Our results suggest that PI3K inhibitors could be used as novel therapeutic agents for HHT.

Endoglin is a conserved regulator of vasculogenesis in zebrafish - implications for hereditary haemorrhagic telangiectasia.

Hereditary haemorrhagic telangiectasia (HHT) is a progressive vascular disease with high mortality and prevalence. There is no effective treatment of HHT due to the lack of comprehensive knowledge of its underlying pathological mechanisms. The majority of HHT1 patients carry endoglin (ENG) mutations. Here, we used Danio rerio (zebrafish) as an in vivo model to investigate the effects of endoglin knockdown on vascular development. According to phylogenetic analyses and amino acid sequence similarity analyses, we confirmed that endoglin is conserved in vertebrates and descended from a single common ancestor. Endoglin is highly expressed in the vasculature beginning at the segmentation period in zebrafish. Upon endoglin knockdown by morpholinos, we observed disruption in the intersegmental vessels (ISVs) and decreased expression of several vascular markers. RNA sequencing (RNA-Seq) results implied that the BMP-binding endothelial regulator (bmper) is a gene affected by endoglin knockdown. Rescue experiments demonstrated that overexpression of bmper significantly increased the number of endothelial cells (ECs) and reduced the defects at ISVs in zebrafish. Moreover, there was enhanced tube formation in ENG mutant ECs derived from a HHT patient after human recombinant BMPER (hrBMPER) stimulation. Taken together, our results suggest that bmper, a potential downstream gene of ENG, could be targeted to improve vascular integrity in HHT.

Safety of thalidomide and bevacizumab in patients with hereditary hemorrhagic telangiectasia.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is a multisystemic inherited vascular dysplasia that leads to nosebleeds and visceral arteriovenous malformations (AVMs). Anti-angiogenic drugs thalidomide and bevacizumab have been increasingly used off-label with variable results. The HHT working group within the ERN for Rare Multisystemic Vascular Diseases (VASCERN), developed a questionnaire-based retrospective capture of adverse events (AEs) classified using the Common Terminology Criteria for Adverse Events. RESULTS: Sixty-nine HHT patients received bevacizumab, 37 (50.6%) for high output cardiac failure/hepatic AVMs, and 32 (49.4%) for bleeding; the 69 patients received bevacizumab for a mean of 11 months for a total of 63.8 person/years treatment. 67 received thalidomide, all for epistaxis and/or gastrointestinal bleeding; they received thalidomide for a mean of 13.4 months/patient for a total of 75 person/years treatment. AEs were reported in 58 patients, 33 with bevacizumab, 37 with thalidomide. 32 grade 1-3 AEs related to bevacizumab were reported with an average incidence rate of 50 per 100 person-years. 34 grade 1-3 AEs related to thalidomide were reported with an average incidence rate of 45.3 per 100 person-years. Bevacizumab AEs were more common in females (27 AEs in 46 women) than males (6 in 23, p < 0.001). Thalidomide AEs occurred at more similar rates in males (25 AEs in 41 men, 60.9%) and females (12 in 26 (46.2%), but were more common in ENG patients (17 in 17) than in ACVRL1 (14 in 34, p < 0.0001). For bevacizumab, the most common reports were of joint pains (7/69, 10%), headache (3/69, 4.4%) and proteinuria (2/69, 3%), and for thalidomide, peripheral neuropathy (12/67, 18%); drowsiness (8/67, 12%); and dizziness (6/67, 9%). Fatal adverse events were more common in males (p = 0.009), and in patients with ENG pathogenic variants (p = 0.012). One fatal AE was possibly related to bevacizumab (average incidence rate: 1.5 per 100 person-years); 3 fatal AEs were possibly related to thalidomide (average incidence rate: 4 per 100 person-years). CONCLUSIONS: With potential increase in use of Bevacizumab and Thalidomide in HHT patients, data presented support appropriate weighing of the toxicities which can arise in HHT settings and the practice recommendations for their prevention and management.

Pentraxin 3 level is elevated in hereditary hemorrhagic telangiectasia and reflects the severity of disease-associated epistaxis.

OBJECTIVES/HYPOTHESIS: We aimed to investigate if vascular endothelial growth factor (VEGF) and other angiogenic and inflammatory factors correlated with the clinical presentation in hereditary hemorrhagic telangiectasia (HHT) patients, particularly in regard to the severity of epistaxis. STUDY DESIGN: Prospective, comparative, single-center study. METHODS: One hundred nine samples were collected from 75 HHT patients attending the ear, nose, and throat department at Oslo University Hospital from February 2012 to August 2013. For comparison, samples were collected from 16 healthy controls. Angiogenic and inflammatory factors related to endothelial cell activation were analyzed by enzyme immunoassays. The grade of epistaxis was evaluated using the Epistaxis Severity Score and epistaxis Intensity, Frequency, and Need for Blood Transfusion score at the day of blood sampling. The presence of internal organ manifestations in the HHT group was recorded. RESULTS: Pentraxin 3 (PTX3) was the only factor that was significantly higher in the HHT patients than the controls and showed significant correlation to the epistaxis severity grade and the hemoglobin level. The VEGF level was higher in the HHT patients compared to controls but not to a significant degree. In addition, a significant correlation of the level of VEGF and the grade of epistaxis could not be observed. Also, no significant correlations were observed between the presence of internal organ manifestations and the level of angiogenic factors. CONCLUSIONS: PTX3, at least partly reflecting vascular inflammation, can be a potential biomarker for the severity of HHT associated epistaxis. The serum level of VEGF was not correlated with the severity of epistaxis in the HHT patients. LEVEL OF EVIDENCE: 2 Laryngoscope, 129:E44-E49, 2019.

Decreased Expression of Vascular Endothelial Growth Factor Receptor 1 Contributes to the Pathogenesis of Hereditary Hemorrhagic Telangiectasia Type 2.

BACKGROUND: Hereditary Hemorrhagic Telangiectasia type 2 (HHT2) is an inherited genetic disorder characterized by vascular malformations and hemorrhage. HHT2 results from ACVRL1 haploinsufficiency, the remaining wild-type allele being unable to contribute sufficient protein to sustain endothelial cell function. Blood vessels function normally but are prone to respond to angiogenic stimuli, leading to the development of telangiectasic lesions that can bleed. How ACVRL1 haploinsufficiency leads to pathological angiogenesis is unknown. METHODS: We took advantage of Acvrl1+/- mutant mice that exhibit HHT2 vascular lesions and focused on the neonatal retina and the airway system after Mycoplasma pulmonis infection, as physiological and pathological models of angiogenesis, respectively. We elucidated underlying disease mechanisms in vitro by generating Acvrl1+/- mouse embryonic stem cell lines that underwent sprouting angiogenesis and performed genetic complementation experiments. Finally, HHT2 plasma samples and skin biopsies were analyzed to determine whether the mechanisms evident in mice are conserved in humans. RESULTS: Acvrl1+/- retinas at postnatal day 7 showed excessive angiogenesis and numerous endothelial "tip cells" at the vascular front that displayed migratory defects. Vascular endothelial growth factor receptor 1 (VEGFR1; Flt-1) levels were reduced in Acvrl1+/- mice and HHT2 patients, suggesting similar mechanisms in humans. In sprouting angiogenesis, VEGFR1 is expressed in stalk cells to inhibit VEGFR2 (Flk-1, KDR) signaling and thus limit tip cell formation. Soluble VEGFR1 (sVEGFR1) is also secreted, creating a VEGF gradient that promotes orientated sprout migration. Acvrl1+/- embryonic stem cell lines recapitulated the vascular anomalies in Acvrl1+/- (HHT2) mice. Genetic insertion of either the membrane or soluble form of VEGFR1 into the ROSA26 locus of Acvrl1+/- embryonic stem cell lines prevented the vascular anomalies, suggesting that high VEGFR2 activity in Acvrl1+/- endothelial cells induces HHT2 vascular anomalies. To confirm our hypothesis, Acvrl1+/- mice were infected by Mycoplasma pulmonis to induce sustained airway inflammation. Infected Acvrl1+/- tracheas showed excessive angiogenesis with the formation of multiple telangiectases, vascular defects that were prevented by VEGFR2 blocking antibodies. CONCLUSIONS: Our findings demonstrate a key role of VEGFR1 in HHT2 pathogenesis and provide mechanisms explaining why HHT2 blood vessels respond abnormally to angiogenic signals. This supports the case for using anti-VEGF therapy in HHT2.