Repurposing propranolol as a drug for the treatment of retinal haemangioblastomas in von Hippel-Lindau disease.

BACKGROUND: Von 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 tumours. Haemangioblastomas in the central nervous system (CNS) and retina, renal carcinoma and pheochromocytomas are the most common tumours. The absence of treatment for VHL leads to the need of repeated surgeries as the only option for these patients. Targeting VHL-derived tumours with drugs with reduced side effects is urgent to avoid repeated CNS surgeries. Recent reports have demonstrated that propranolol, a ß-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 haemangioblastoma growth in VHL disease given its antiangiogenic effects that were recently demonstrated by us. The main objective of the present study was the assessment of the efficacy and safety of propranolol on retinal haemangioblastoma in von Hippel-Lindau disease (VHL). METHODS: 7 VHL patients, from different regions of Spain, affected from juxtapapillary or peripheral haemangioblastomas were administered 120 mg propranolol daily. Patients were evaluated every 3 months for 12 months, at Virgen de la Salud Hospital (Toledo). The patients had juxtapapillary or peripheral haemangioblastomas but had refused standard treatments. RESULTS: Propranolol was initiated with a progressive increase up to a final dose of 120 mg daily. All tumours remained stable, and no new tumours appeared. The reabsorption of retinal exudation was noted in the two patients having exudates. No adverse effects were recorded. VEGF and miRNA 210 levels were monitored in the plasma of patients as possible biomarkers of VHL. These levels decreased in all cases from the first month of treatment. CONCLUSIONS: Although more studies are necessary, the results of this work suggest that propranolol is a drug to be considered in the treatment of VHL patients with retinal haemangioblastomas. VEGF and miRNA 210 could be used as biomarkers of the VHL disease activity. TRIAL REGISTRATION: The study has a clinical trial design and was registered at EU Clinical Trials Register and Spanish Clinical Studies Registry, EudraCT Number: 2014-003671-30 . Registered 2 September 2014.

Chronic hypoxia-inducible transcription factor-2 activation stably transforms juxtaglomerular renin cells into fibroblast-like cells in vivo.

On the basis of previous observations that deletion of the von Hippel-Lindau protein (pVHL) in juxtaglomerular (JG) cells of the kidney suppresses renin and induces erythropoietin expression, this study aimed to characterize the events underlying this striking change of hormone expression. We found that renin cell-specific deletion of pVHL in mice leads to a phenotype switch in JG cells, from a cuboid and multiple vesicle-containing form into a flat and elongated form without vesicles. This shift of cell phenotype was accompanied by the disappearance of marker proteins for renin cells (e.g., aldo-keto reductase family 1, member 7 and connexin 40) and by the appearance of markers of fibroblast-like cells (e.g., collagen I, ecto-5'-nucleotidase, and PDGF receptor-ß). Furthermore, hypoxia-inducible transcription factor-2α (HIF-2α) protein constitutively accumulated in these transformed cells. Codeletion of pVHL and HIF-2α in JG cells completely prevented the phenotypic changes. Similar to renin expression in normal JG cells, angiotensin II negatively regulated erythropoietin expression in the transformed cells. In summary, chronic activation of HIF-2 in renal JG cells leads to a reprogramming of the cells into fibroblast-like cells resembling native erythropoietin-producing cells located in the tubulointerstitium.

Detection of large deletions in the VHL gene using a Real-Time PCR with SYBR Green.

Mutation in VHL gene causes the von Hippel-Lindau (VHL) disease, a dominantly inherited familial cancer syndrome. The VHL mutation pattern includes point mutations, small deletions and large deletions. While most mutations can be identified during sequencing, large deletions often remain unnoticed in initial mutational screening. We evaluated the utility of a previously described real-time quantitative PCR (RQ-PCR) using SYBR Green for detection of larger deletions in the VHL gene and normalized the data using two reference genes with a normal copy number i.e., ZNF80 (3q13.31) and GPR15 (3q12.1). DNA sequencing was also done on all cases included in the study. SJNB-6 cell line demonstrating distal 3p loss was used as a positive control for deletion. Out of 21 individual cases included of VHL disease, 2 cases were found with partial deletion by RQ-PCR, with an exon 1 deletion, while PCR-sequencing identified 5 cases with base pair substitution and 1 with splice site variant which were not picked up by RQ-PCR. RQ-PCR proved to be fast, accurate and sensitive for identifying large deletions and can be incorporated into the routine work-up for detection of large deletions in VHL disease.

Hemangioblastomas and neurogenic polyglobulia.

BACKGROUND: Neurogenic polyglobulia occurs with central nervous system hemangioblastomas. Among the suggested mechanisms are extramedullary hematopoiesis in the tumor tissue and germline mutations of the von Hippel-Lindau (VHL) tumor suppressor gene. OBJECTIVE: To determine the frequency and driving mechanisms of polyglobulia in central nervous system hemangioblastomas. METHODS: We performed a retrospective analysis of pre- and postoperative (at 3 and 12 months) hemoglobin levels in a consecutive series of patients with hemangioblastomas operated on in our institution from 1996 to 2009. We performed molecular genetic analyses for mutations of the VHL tumor suppressor gene. RESULTS: Preoperative hemoglobin levels were available from 164 patients. The average hemoglobin level (15.2 g/dL in males and 13.1 g/dL in females) was within normal range according to our standards. Of 22 patients with increased preoperative hemoglobin levels (>17 g/dL in males and >15 g/dL in females), 8 presented with pathological hemoglobin (>18.5 g/dL in males and >16.5 g/dL in females) according to World Health Organization criteria. Surgical removal of the hemangioblastoma resulted in a permanent cure of polyglobulia in all patients. Six of the 8 patients with pathological hemoglobin elevation carried a germline mutation of the VHL tumor suppressor gene. CONCLUSION: Neurogenic polyglobulia occurs in a subset of patients with hemangioblastomas. This phenomenon is mostly observed in VHL mutation carriers, but also occurs in patients with sporadic hemangioblastomas. Removal of the tumor results in the permanent cure of polyglobulia. Our observations suggest that polyglobulia is an effect by the tumor itself, either due to paraneoplasia or extramedullary hematopoiesis.

Cardiopulmonary function in two human disorders of the hypoxia-inducible factor (HIF) pathway: von Hippel-Lindau disease and HIF-2alpha gain-of-function mutation.

The hypoxia-inducible factors (HIFs; isoforms HIF-1α, HIF-2α, HIF-3α) mediate many responses to hypoxia. Their regulation is principally by oxygen-dependent degradation, which is initiated by hydroxylation of specific proline residues followed by binding of von Hippel-Lindau (VHL) protein. Chuvash polycythemia is a disorder with elevated HIF. It arises through germline homozygosity for hypomorphic VHL alleles and has a phenotype of hematological, cardiopulmonary, and metabolic abnormalities. This study explores the phenotype of two other HIF pathway diseases: classic VHL disease and HIF-2α gain-of-function mutation. No cardiopulmonary abnormalities were detected in classic VHL disease. HIF-2α gain-of-function mutations were associated with pulmonary hypertension, increased cardiac output, increased heart rate, and increased pulmonary ventilation relative to metabolism. Comparison of the HIF-2α gain-of-function responses with data from studies of Chuvash polycythemia suggested that other aspects of the Chuvash phenotype were diminished or absent. In classic VHL disease, patients are germline heterozygous for mutations in VHL, and the present results suggest that a single wild-type allele for VHL is sufficient to maintain normal cardiopulmonary function. The HIF-2α gain-of-function phenotype may be more limited than the Chuvash phenotype either because HIF-1α is not elevated in the former condition, or because other HIF-independent functions of VHL are perturbed in Chuvash polycythemia.

Plasma proteome profiling of von Hippel-Lindau disease after total and subtotal nephrectomy: a preliminary study.

OBJECTIVES: Common treatment of renal cell carcinoma associated with von Hippel-Lindau (VHL) disease is total (bilateral) or subtotal nephrectomy. Whereas total nephrectomy is associated with absolutely no residual renal function, subtotal nephrectomy frequently leads to chronic kidney disease (CKD) with some residual renal functions. However, molecular mechanisms underlying CKD remain unclear and the diagnosis of CKD is frequently accomplished at its late stage. DESIGN AND METHODS: We performed a plasma proteomics study to compare the plasma proteome profile of VHL patient who underwent total nephrectomy to the profiles of VHL patient with subtotal nephrectomy and healthy control. Totally 100 mug proteins from each sample was resolved by two-dimensional electrophoresis (2-DE) in triplicate and visualized with SYPRO Ruby fluorescence stain. RESULTS: The normal plasma proteome profile markedly differed from the profiles of VHL patients. Comparative analysis between total versus subtotal nephrectomized patients revealed significant differences in levels of 20 plasma proteins. Pathway analysis revealed two important networks involving in lipid metabolism, molecular transport, carbohydrate metabolism, cellular growth and proliferation, and small molecule biochemistry, in which these identified and other proteins interplayed. CONCLUSIONS: Our data identified potential biomarkers for CKD. Further characterization of these identified proteins might also lead to better understanding of molecular mechanisms underlying CKD.

[The detection of mutations in VHL gene from a single cell in a patient with von Hippel-Lindau disease].

OBJECTIVE: To explore a technology for diagnosing VHL mutations from a single cell and provide experimental evidences for the feasibility of applying technology in detecting genetic mutations from a single cell. METHODS: After whole genome amplification (WGA) based on multiple displacement amplication (MDA) for a single cell, we did regular PCR following sequencing and detected the genotypes using the real time PCR based on TaqMan probes. We detected VHL mutations by the different terminal fluorescent changing. RESULTS: The rate of amplification for single cell based on MDA was 90.91%. The rate of contamination was 0. After sequencing, the allele drop out (ADO) rate of heterozygotes was 26.67%(8/30); combined with the different terminal fluorescent changing, the rate of ADO of heterozygotes was 16.67%. CONCLUSION: WGA based on MDA for a single cell followed by regular PCR with sequencing and real time PCR can specially and accurately detect the VHL genotypes of single cells.

Bilateral testicular adrenal rests after bilateral adrenalectomies in a cushingoid patient with von Hippel-Lindau disease.

We report a case of bilateral testicular masses in a 25-year-old man with von Hippel-Lindau disease presenting with cushingoid symptoms. His medical history was significant for bilateral adrenalectomies secondary to pheochromocytomas, and he began steroid therapy at that time. After exhaustive endocrinologic, radiographic, and physical examinations, the testicular masses were postulated to be active adrenal rest tissue. Bilateral testicular venous sampling found elevated glucocorticoids that were responsive to dexamethasone suppression, which confirmed the testicular masses as testicular adrenal rests without the need for surgical intervention. Successful conservative management consisted of appropriate steroid manipulation and radiographic evaluation and resulted in the resolution of presenting symptoms, a decrease in size of the bilateral testicular masses, and testicular conservation in this young man.

Utility of plasma free metanephrines for detecting childhood pheochromocytoma.

Measurements of plasma free metanephrines, normetanephrine (NMN) and metanephrine (MN), provide a sensitive test for diagnosis of pheochromocytoma in adults but have not been evaluated in children. We therefore established reference ranges for plasma and urinary metanephrines and the catecholamines, norepinephrine (NE) and epinephrine (E), in 86 healthy children (age 5-17). A group of 158 healthy adults (age 18-72) served as a comparison group. Pediatric reference ranges were applied to examine the diagnostic utility of the various tests in 45 children evaluated for pheochromocytoma (age 8-17; 38 with von Hippel-Lindau syndrome), with tumors found on 12 occasions. Upper reference limits for E and MN were higher and those for NE and NMN lower in children than in adults. Boys had higher plasma levels of E and MN and higher urinary excretion of all four amines than girls. Plasma free metanephrines provided a diagnostic test with values for sensitivity (100%) and specificity (94%) that were equal to or higher than those of other tests. In two children screened for pheochromocytoma on multiple occasions, use of pediatric reference ranges for plasma free metanephrines indicated the tumor a year earlier than indicated using adult reference ranges. The findings indicate that plasma free metanephrines provide a sensitive tool for detection of pheochromocytoma in children. Age appropriate reference ranges should be used and gender differences should be considered.

Duodenal somatostatinoma and erythrocytosis in a patient with von Hippel-Lindau disease type 2A.

A female with von Hippel-Lindau (VHL) disease type 2A first presented with erythrocytosis at the age of 9 years. This patient revealed multiple paragangliomas at age 22. After the removal of tumors, a retinal hemangioblastoma developed. Our diagnosis of VHL disease type 2A was confirmed. Moreover, systemic examination showed a duodenal somatostatinoma. Frequent and long-term monitoring is important for patients with pheochromocytomas or paragangliomas, and a screening for VHL disease and other hereditary cancer syndromes is recommended. Recognition of neuroendocrine tumors as a manifestation of VHL disease permits earlier diagnosis and improves prognosis.

Elevated ocular levels of vascular endothelial growth factor in patients with von Hippel-Lindau disease.

BACKGROUND: Von Hippel Lindau disease (VHL) is a rare autosomal dominant inherited disorder characterized by highly vascularized tumors in various organs. The abundant presence of endothelial cells in VHL tumors strongly suggest a role of the VHL tumor suppressor gene in the regulation of angiogenesis. Recently, in vitro studies have shown that the VHL tumor suppressor gene regulates the expression of vascular endothelial growth factor (VEGF). We investigated whether VHL patiens have increased levels of VEGF in their body fluids. PATIENTS AND METHODS: The concentration of VEGF was measured in fluid of the anterior chamber of the eye, serum, urine, and fluid from renal cysts of VHL patients and unaffected individuals by ELISA. In addition, levels of basic fibroblast growth factor (bFGF), interleukin-8 (IL-8) and endothelin-1 (ET-1) were measured in urine and serum of VHL patients and control subjects. RESULTS: In 80% of the VHL patients VEGF was detectable in aqueous fluid of the anterior chamber of their eyes. A strong positive correlation (r = 0.90) was found between the age of VHL patients and ocular VEGF concentrations. At comparable age, VEGF levels in ocular fluid of VHL patients were significantly higher (P < 0.001) than in unaffected subjects. No correlation was found between VEGF concentration and the presence of retinal angiomas. A 10 and 16 fold increase of VEGF concentration was seen in fluid from two independent VHL-related cysts as compared with VEGF serum levels of the same patient. The mean concentration of VEGF in serum of VHL patients (n = 15) (319 +/- 84 pg/ml) was higher than in matched controls (238 +/- 68 pg/ml; P = NS). The mean concentration of VEGF in urine of VHL patients (128 +/- 36 pg/ml) was lower than in matched controls (183 +/- 25 pg/ml; P = NS). Concentrations of VEGF did not correlate with the presence of VHL-related tumors. No differences were observed between concentrations of bFGF, IL-8 and ET-1 in serum and urine of VHL patients and matched controls. CONCLUSIONS: These findings support a role for the VHL tumor suppressor gene in the in vivo regulation of VEGF.

Chromogranin A in familial pheochromocytoma: diagnostic screening value, prediction of tumor mass, and post-resection kinetics indicating two-compartment distribution.

PURPOSE: Chromogranin A, co-released with catecholamines from the adrenal medullary and sympathetic neuronal vesicles, is elevated in plasma from patients with pheochromocytoma. We assessed its diagnostic screening value, its plasma level in correlation with tumor mass, and its disposition kinetics in familial pheochromocytoma and sporadic pheochromocytoma. PATIENTS AND METHODS: The sensitivity and specificity of chromogranin A's diagnostic value for pheochromocytoma were established through one kindred with familial pheochromocytoma associated with von Hippel-Lindau syndrome (13 available members) and in seven subjects with sporadic pheochromocytoma. Serial postoperative plasma samples were also obtained (5 minutes to 4 days) from eight subjects with pheochromocytoma in order to study chromogranin A post-resection kinetics. Chromogranin A was measured by radioimmunoassay based on purified pheochromocytoma chromogranin A. RESULTS: In this kindred, elevations of chromogranin A (greater than 52 ng/mL) were sensitive (83%, five of six) and specific (100%, 10 of 10) in detecting familial pheochromocytoma; these diagnostic values comparable to those achieved by conventional evaluations for pheochromocytoma, such as urinary catecholamines, urinary catecholamine metabolites or imaging methods. Elevated levels of plasma chromogranin A specifically indicated pheochromocytoma, rather than von Hippel-Lindau syndrome gene carrier status. In 13 preoperative subjects with either familial or sporadic pheochromocytoma, plasma chromogranin A concentration predicted tumor size (r = 0.81, p less than 0.01). The change in chromogranin A plasma concentration after pheochromocytoma resection best fit a two-compartment model, with an initial rapid half-life time of 16 minutes, followed by a longer half-life time of 520 minutes. The model also predicted a 23.8:1 compartmental ratio of extravascular/intravascular chromogranin A, suggesting substantial tissue sequestration or binding of chromogranin A. CONCLUSIONS: (1) Plasma chromogranin A is a valuable (sensitive and specific) diagnostic tool in detecting both familial and sporadic pheochromocytoma. (2) The concentration of plasma chromogranin A predicts the size of the pheochromocytoma. (3) Chromogranin A post-resection kinetics suggest extravascular sequestration of chromogranin A.