Virginia Albiñana

Estrogen therapy for hereditary haemorrhagic telangiectasia (HHT): Effects of raloxifene, on Endoglin and ALK1 expression in endothelial cells

Hereditary haemorrhagic telangiectasia (HHT), or Rendu-Osler-Weber syndrome, is an autosomal dominant vascular disease. The clinical manifestations are epistaxis, mucocutaneous and gastrointestinal telangiectases, and arteriovenous malformations. There are two predominant types of HHT caused by mutations in Endoglin (ENG) and activin receptor-like kinase 1 (ALK1) (ACVRL1) genes, HHT1 and HHT2, respectively. No cure for HHT has been found and there is a current need to find new effective drug treatments for the disease. Some patients show severe epistaxis which interferes with their quality of life. We report preliminary results obtained with Raloxifene to treat epistaxis in postmenopausal HHT women diagnosed with osteoporosis. We tried to unravel the molecular mechanisms involved in the therapeutic effects of raloxifene. ENG and ACVRL1 genes code for proteins involved in the transforming growth factor beta pathway and it is widely accepted that haploinsufficiency is the origin for the pathogenicity of HHT. Therefore, identification of drugs able to increase the expression of those genes is essential to propose new therapies for HHT. In vitro results show that raloxifene increases the protein and mRNA expression of ENG and ALK1 in cultured endothelial cells. Raloxifene also stimulates the promoter activity of these genes, suggesting a transcriptional regulation of ENG and ALK1. Furthermore, Raloxifene improved endothelial cell functions like tubulogenesis and migration in agreement with the reported functional roles of Endoglin and ALK1. Our pilot study provides a further hint that oral administration of raloxifene may be beneficial for epistaxis treatment in HHT menopausal women. The molecular mechanisms of raloxifene involve counteracting the haploinsufficiency of ENG and ALK1.

Propranolol as antiangiogenic candidate for the therapy of hereditary haemorrhagic telangiectasia

The β-blocker propranolol, originally designed for cardiological indications (angina, cardiac arrhythmias and high blood pressure), is nowadays, considered the most efficient drug for the treatment in infantile haemangiomas (IH), a vascular tumour that affects 5-10% of all infants. However, its potential therapeutic benefits in other vascular anomalies remain to be explored. In the present work we have assessed the impact of propranolol in endothelial cell cultures to test if this drug could be used in the vascular disease hereditary haemorrhagic telangiectasia (HHT). This rare disease is the result of abnormal angiogenesis with epistaxis, mucocutaneous and gastrointestinal telangiectases, as well as arteriovenous malformations in several organs, as clinical manifestations. Mutations in Endoglin (ENG) and ACVLR1 (ALK1) genes, lead to HHT1 and HHT2, respectively. Endoglin and ALK1 are involved in the TGF-β1 signalling pathway and play a critical role for the proper development of the blood vessels. As HHT is due to a deregulation of key angiogenic factors, inhibitors of angiogenesis have been used to normalise the nasal vasculature eliminating epistaxis derived from telangiectases. Thus, the antiangiogenic properties of propranolol were tested in endothelial cells. The drug was able to decrease cellular migration and tube formation, concomitantly with reduced RNA and protein levels of ENG and ALK1. Moreover, the drug showed apoptotic effects which could explain cell death in IH. Interestingly, propranolol showed some profibrinolytic activity, decreasing PAI-1 levels. These results suggest that local administration of propranolol in the nose mucosa to control epistaxis might be a potential therapeutic approach in HHT.

Immunosuppressor FK506 increases endoglin and activin receptor-like kinase 1 expression and modulates transforming growth factor-β1 signaling in endothelial cells.

Hereditary hemorrhagic telangiectasia (HHT), or Rendu-Osler-Weber syndrome, is an autosomal-dominant vascular disease. The clinical manifestations are epistaxis, mucocutaneous and gastrointestinal telangiectases, and arteriovenous malformations in internal organs. Patients show severe epistaxis, and/or gastrointestinal bleeding, both of which notably interfere with their quality of life. There are two predominant types of HHT caused by mutations in endoglin (ENG) and ACVRL1/activin receptor-like kinase 1 (ALK1) genes, named HHT1 and HHT2, respectively. ENG and ALK1 code for proteins involved in the transforming growth factor (TGF)-β1 signaling pathway, and it is widely accepted that HHT pathogenicity results from haploinsufficiency. No cure for HHT has been found, so identification of drugs able to increase the expression of these genes is essential when proposing new therapies. We report the efficacy of tacrolimus (FK506) in increasing ENG and ALK1 expression. The rationale comes from a case report of a patient with HHT who received a liver transplantation after hepatic failure due to a liver arteriovenous malformation. The liver was transplanted, and the immunosuppressor FK506 was used to prevent the rejection. After the first month of FK506 treatment, the internal and external telangiectases, epistaxes, and anemia disappeared. Here, we find that the immunosuppressor FK506 increases the protein and mRNA expression of ENG and ALK1 in cultured endothelial cells and enhances the TGF-β1/ALK1 signaling pathway and endothelial cell functions like tubulogenesis and migration. These results suggest that the mechanism of action of FK506 involves a partial correction of endoglin and ALK1 haploinsufficiency and may therefore be an interesting drug for use in patients with HHT who undergo transplantation.

Mutation study of Spanish patients with Hereditary Hemorrhagic Telangiectasia

BackgroundHereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant and age-dependent vascular disorder characterised mainly by mutations in the Endoglin (ENG) or activin receptor-like kinase-1 (ALK1, ACVRL1) genes.MethodsHere, we have identified 22 ALK1 mutations and 15 ENG mutations, many of which had not previously been reported, in independent Spanish families afflicted with HHT.ResultsWe identified mutations in thirty-seven unrelated families. A detailed analysis of clinical symptoms was recorded for each patient analyzed, with a higher significant presence of pulmonary arteriovenous malformations (PAVM) in HHT1 patients over HHT2. Twenty-two mutations in ALK1 and fifteen in ENG genes were identified. Many of them, almost half, represented new mutations in ALK1 and in ENG. Missense mutations in ENG and ALK1 were localized in a tridimensional protein structure model.ConclusionOverall, ALK1 mutations (HHT2) were predominant over ENG mutations (HHT1) in our Spanish population, in agreement with previous data from our country and other Mediterranean countries (France, Italy), but different to Northern Europe or North America. There was a significant increase of PAVM associated with HHT1 over HHT2 in these families.

Propranolol reduces viability and induces apoptosis in hemangioblastoma cells from von Hippel-Lindau patients

BackgroundVon Hippel-Lindau (VHL) disease is a rare oncological disease with an incidence of 1:36,000, and is characterized by the growth of different types of tumors: hemangioblastomas in the central nervous system (CNS) and retina, renal carcinoma, pheochromocytomas, pancreatic serous cystadenoma, and endolymphatic sac tumors. These tumors do not express VHL protein (pVHL). pVHL ubiquitinates hypoxia inducible factor (HIF) protein for degradation by the proteasome; in the absence of VHL, HIF translocates to the nucleus to activate the expression of its target genes. Targeting VHL-derived tumors with drugs that have reduced side effects is urgent to avoid repeat CNS surgeries. Recent reports have shown that propranolol, a β-blocker used for the treatment of hypertension and other cardiac and neurological diseases, is the best option for infantile hemangioma (IH). Propranolol could be an efficient treatment to control hemangioblastoma growth in VHL disease because of its antiangiogenic effects demonstrated in IH and the hypothetical impact on HIF levels.MethodsHeLa 9X (HRE) hypoxia responsive element cell line and primary hemangioblastoma-derived cells were subjected to propranolol treatment and cell viability and apoptosis were evaluated. HIF1-α and Hif-2α expression after propranolol treatment was analyzed by western blotting. Quantitative PCR was performed to study the mRNA expression of HIF target genes. Vascular endothelial growth factor (VEGF) was measured in culture supernatants by immunoassay.ResultsPropranolol downregulated HIF-dependent transcription in HeLa 9XHRE cells. Under hypoxic conditions, propranolol decreased the expression of HIF target genes in hemangioblastoma cells, which stopped proliferating and died following long-term treatment. These results suggests that propranolol treatment promoted reduced HIF protein expression and corresponding downregulation of HIF target genes, and inhibited cell proliferation in parallel with induction of cell death by apoptosis.ConclusionsOur results suggest that propranolol could reduce the growth of HIF-dependent tumors and may thus be a promising treatment to delay surgery in VHL patients.

Research on potential biomarkers in hereditary hemorrhagic telangiectasia

Hereditary hemorrhagic telangiectasia (HHT) is a genetically heterogeneous disorder, involving mutations in two predominant genes known as Endoglin (ENG; HHT1) and activin receptor-like kinase 1 (ACVRL1/ALK1; HHT2), as well as in some less frequent genes, such as MADH4/SMAD4 (JP-HHT) or BMP9/GDF2 (HHT5). The diagnosis of HHT patients currently remains at the clinical level, according to the “Curaçao criteria,” whereas the molecular diagnosis is used to confirm or rule out suspected HHT cases, especially when a well characterized index case is present in the family or in an isolated population. Unfortunately, many suspected patients do not present a clear HHT diagnosis or do not show pathogenic mutations in HHT genes, prompting the need to investigate additional biomarkers of the disease. Here, several HHT biomarkers and novel methodological approaches developed during the last years will be reviewed. On one hand, products detected in plasma or serum samples: soluble proteins (vascular endothelial growth factor, transforming growth factor β1, soluble endoglin, angiopoietin-2) and microRNA variants (miR-27a, miR-205, miR-210). On the other hand, differential HHT gene expression fingerprinting, next generation sequencing of a panel of genes involved in HHT, and infrared spectroscopy combined with artificial neural network patterns will also be reviewed. All these biomarkers might help to improve and refine HHT diagnosis by distinguishing from the non-HHT population.

Mutation affecting the proximal promoter of Endoglin as the origin of hereditary hemorrhagic telangiectasia type 1

BackgroundHereditary hemorrhagic telangiectasia (HHT) is a vascular multi-organ system disorder. Its diagnostic criteria include epistaxis, telangiectases in mucocutaneous sites, arteriovenous malformations (AVMs), and familial inheritance. HHT is transmitted as an autosomal dominant condition, caused in 85% of cases by mutations in either Endoglin (ENG) or Activin receptor-like kinase (ACVRL1/ACVRL1/ALK1) genes. Pathogenic mutations have been described in exons, splice junctions and, in a few cases with ENG mutations, in the proximal promoter, which creates a new ATG start site. However, no mutations affecting transcription regulation have been described to date in HHT, and this type of mutation is rarely identified in the literature on rare diseases.MethodsSequencing data from a family with HHT lead to single nucleotide change, c.-58G > A. The functionality and pathogenicity of this change was analyzed by in vitro mutagenesis, quantitative PCR and Gel shift assay. Student t test was used for statistical significance.ResultsA single nucleotide change, c.-58G > A, in the proximal ENG promoter co-segregated with HHT clinical features in an HHT family. This mutation was present in the proband and in 2 other symptomatic members, whereas 2 asymptomatic relatives did not harbor the mutation. Analysis of RNA from activated monocytes from the probands and the healthy brother revealed reduced ENG mRNA expression in the HHT patient (p = 0.005). Site-directed mutagenesis of the ENG promoter resulted in a three-fold decrease in luciferase activity of the mutant c.-58A allele compared to wild type (p = 0.005). Finally, gel shift assay identified a DNA-protein specific complex.ConclusionsThe novel ENG c.-58G > A substitution in the ENG promoter co-segregates with HHT symptoms in a family and appears to affect the transcriptional regulation of the gene, resulting in reduced ENG expression. ENG c.-58G > A may therefore be a pathogenic HHT mutation leading to haploinsufficiency of Endoglin and HHT symptoms. To the best of our knowledge, this is the first report of a pathogenic mutation in HHT involving the binding site for a transcription factor in the promoter of ENG.

Copy number variations in endoglin locus: mapping of large deletions in Spanish families with hereditary hemorrhagic telangiectasia type 1

BackgroundThe hereditary hemorrhagic telangiectasia syndrome (HHT), also known as the Rendu–Osler-Weber syndrome is a multiorganic vascular disorder inherited as an autosomal dominant trait. Diagnostic clinical criteria include: epistaxis, telangiectases in mucocutaneous and gastrointestinal sites, arteriovenous malformations (AVMs) most commonly found in pulmonary, hepatic and cerebral circulations, and familial inheritance. HHT is transmitted in 90% of the cases as an autosomal dominant condition due to mutations in either endoglin (ENG), or activin receptor-like kinase 1 (ACVRL1/ALK1) genes (HHT type 1 and 2, respectively).MethodsWe have carried out a genetic analysis of four independent Spanish families with HHT clinical criteria, which has permitted the identification of new large deletions in ENG. These mutations were first detected using the MLPA technique and subsequently, the deletion breakpoints were mapped using a customized copy number variation (CNV) microarray. The array was designed to cover the ENG gene and surrounding areas.ResultsAll tested families carried large deletions ranging from 3-kb to 100-kb, involving the ENG gene promoter, several ENG exons, and the two downstream genes FGSH and CDK9. Interestingly, common breakpoints coincident with Alu repetitive sequences were found among these families.ConclusionsThe systematic hybridization of DNA from HHT families, with deletions or duplications, to custom designed microarrays, could allow the mapping of breakpoints, coincident with repetitive Alu sequences that might act as “hot spots” in the development of chromosomal anomalies.

Propranolol Decreases the Viability and Triggers Apoptosis inHemangioblastoma Cells from Von Hippel-Lindau Patients

Abstract In our previous paper, propranolol is used in hemangioblastomas primary cultures from patients with von Hippel- Lindau disease. This is a rare inherited oncologic condition characterized by the growth of tumors affecting mainly the central nervous system, but also kidneys, pancreas, adrenal glands, retinas and endolymphatic sacs (inner ear). Up to date, the only treatment for these patients is surgery. The search for drugs able, at least, to stop the development of hemangioblastomas, has led our group to propose propranolol, a non-specific beta blocker, as a drug to test. According to the results of this work, propranolol would act by decreasing hypoxia signaling pathway, which is constitutively active in VHL patients, normalizing the hypoxia target genes involved in angiogenesis, survival and stemness in the hemangioblastoma cells. The results in vitro are promising and, in the absence of serious side effects, propranolol could be assayed as a therapy in the clinical practice for VHL patients.

Case Report: Propranolol increases the therapeutic response to temozolomide in a patient with metastatic paraganglioma

This case report presents the clinical evolution and management of a patient with a hereditary paraganglioma syndrome. This disease is characterized by rare tumors of neural crest origin that are symmetrically distributed along the paravertebral axis from the base of the skull and neck to the pelvis. In addition, these patients may develop renal cancer, gastrointestinal stromal tumors, pituitary adenomas, and bone metastasis in some cases. To date no successful therapeutic treatment has been reported. Total resection with postoperative radiotherapy and chemotherapy have been advocated, especially for the multiple metastasis. Here we show how the combination of high doses of the beta blocker propranolol (3 mg/Kg/day) and the DNA intercalating agent, temozolomide, has been successful in the treatment of a SDHA metastatic paraganglioma.