Dissecting the mechanisms of bone loss in Gorham-Stout disease

Journal Pre-proof Dissecting the mechanisms of bone loss in Gorham-Stout disease

Michela Rossi, Paola Sabrina Buonuomo, Giulia Battafarano, Antonella Conforti, Eda Mariani, Mattia Algeri, Simone Pelle, Matteo D'Agostini, Marina Macchiaiolo, Rita De Vito, Michaela Veronika Gonfiantini, Alessandro Jenkner, Ippolita Rana, Andrea Bartuli, Andrea Del Fattore PII:

S8756-3282(19)30361-8

DOI:

https://doi.org/10.1016/j.bone.2019.115068

Reference:

BON 115068

To appear in:

Bone

Received date:

1 July 2019

Revised date:

11 September 2019

Accepted date:

11 September 2019

Please cite this article as: M. Rossi, P.S. Buonuomo, G. Battafarano, et al., Dissecting the mechanisms of bone loss in Gorham-Stout disease, Bone(2018), https://doi.org/10.1016/ j.bone.2019.115068

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© 2018 Published by Elsevier.

Journal Pre-proof Dissecting the mechanisms of bone loss in Gorham-Stout disease Michela Rossia, Paola Sabrina Buonuomob, Giulia Battafaranoa, Antonella Confortic, Eda Marianid, Mattia Algeric, Simone Pellee, Matteo D’Agostinif, Marina Macchiaiolob, Rita De Vitog, Michaela Veronika Gonfiantinib, Alessandro Jenknerh, Ippolita Ranab, Andrea Bartulib§ and Andrea Del Fattorea§* a

Bone Physiopathology Group, Multifactorial Disease and Complex Phenotype Research Area, Bambino

Gesù Children’s Hospital, Rome, Italy; bRare Diseases and Medical Genetic Unit, Bambino Gesù

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Children's Hospital, Rome, Italy; cDepartment of Pediatric Hematology and Oncology, Bambino Gesù

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Children's Hospital, Rome, Italy; dResearch Laboratories, Bambino Gesù Children's Hospital, Rome,

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Italy; e“Villa Aurora” Clinic, Rome, Italy; fClinical Laboratory, Bambino Gesù Children’s Hospital,

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Rome, Italy; gHistopathology, Bambino Gesù Children's Hospital, Rome, Italy; hDivision of Immunology

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and Infectious Diseases Department of Pediatrics, Bambino Gesù Children Hospital, Rome, Italy. Grants of support: This study was supported by Million Dollar Bike Ride Grant Program, Orphan Disease

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Center, University of Pennsylvania #MDBR-17-116-GLA/GSD59 to ADF and AB, and by grants from

Equal contributors

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§

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the Italian Ministry of Health (Ricerca Corrente) to ADF.

*Corresponding author:

Andrea Del Fattore, PhD Bone Physiopathology Group, Multifactorial Disease and Complex Phenotype Research Area, Bambino Gesù Children's Hospital, Viale San Paolo 15, 00146 Rome, Italy Phone: +39 06.6859.3740 Email: [email protected]

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Journal Pre-proof Disclosure Page

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The authors have no conflict of interest to declare

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Journal Pre-proof Abstract Gorham-Stout disease (GSD) is a rare disorder characterized by progressive osteolysis and angiomatous proliferation. Since the mechanisms leading to bone loss in GSD are not completely understood, we performed histological, serum, cellular and molecular analyses of 7 patients. Increased vessels, osteoclast number and osteocyte lacunar area were revealed in patients’ bone biopsies. Biochemical analysis of sera showed high levels of ICTP, Sclerostin, VEGF-A and IL-6. In vitro experiments revealed increased

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osteoclast differentiation and activity, and impaired mineralization ability of osteoblasts. To evaluate the

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involvement of systemic factors in GSD, control cells were treated with patients’ sera and displayed an

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increase of osteoclastogenesis, bone resorption activity and a reduction of osteoblast function.

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Interestingly, GSD sera stimulated the vessel formation by endothelial cells EA.hy926. These results

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suggest that bone cell autonomous alterations with the cooperation of systemic factors are involved in massive bone loss and angiomatous proliferation observed in GSD patients.

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Keywords: Gorham-Stout disease, Osteolysis, Osteoclast, Bone Histomorphometry, Osteoblast

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Journal Pre-proof Introduction Gorham-Stout disease is a very rare disorder characterized by extensive and progressive osteolysis and angiomatous proliferation, without new bone formation [1, 2]. Since the first description of a patient affected by GSD in the 1838, only ~300 patients were reported so far [3]. However, since some cases may be misdiagnosed or undiagnosed, there is consensus that there are more patients than those reported. GSD does not show sex predilection (1.6:1; male:female ratio) or racial and geographic distribution. The

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age of patients is from 1 month to 75 years old [4].

The disease may affect the appendicular or the axial skeleton. Although the shoulder and the pelvis are

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the common sites of involvement, skull, humerus, scapula, sternum, ribs, pelvis and femur can be affected

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[3, 4]. Hu et al. reported a case series, in which the bone lesions predominantly localize in femurs [5].

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At the onset of the disease, X rays analysis displays a patchy osteoporosis condition. GSD proceeds with skeletal deformity and progressive bone loss with concentric shrinkage of long bones [6, 7]. Eventually,

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a complete loss of bone occurs resulting in the appearance of the so-called “vanishing bone” disease. The

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quality of life is very poor in patients since they display most frequently pain, functional impairment and

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swelling of the affected regions. When ribs, scapula or thoracic vertebrae are affected, development of chylothorax from the extension of lymphangiectasia into pleural cavity or via invasion of the thoracic duct can occur. Moreover, bone infection and subsequent septic shock, spinal cord involvement and paraplegia due to vertebral lesions have been reported. Without surgical intervention, the morbidity and mortality rate is very high [8]. The diagnosis of GSD is challenging and it is usually performed by exclusion criteria requiring many examinations. Indeed, to define a proper diagnosis it needs to rule out neoplastic processes, infections, and metabolic and endocrine disorders [3, 7, 9]. Blood tests are often normal in GSD patients, with the possible exception of increased levels of alkaline phosphatase [4]. Additionally, a diagnostic role could 4

Journal Pre-proof be played by plain radiographs, bone scan, computed tomography and magnetic resonance imaging (MRI), even if the results from bone scan and MRI are variable [10-15]. All these procedures are useful, but the diagnosis must be confirmed by histopathological analysis of the lesions, that reveals evidence of progressive bone resorption, angiomatous tissue and absence of cellular atypia [16]. So far, the genetic alterations involved in the etiopathogenesis are not entirely known [3]. Preliminary work performed by Prof. Lorenzo’s group showed genetic imbalance in GSD patients [17], but the

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molecular mechanisms underlying GSD and the excessive bone resorption need to be still identified.

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Since in GSD patients a lymphatic and vascular proliferation within bone is also observed, it was

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suggested a relevant role of lymphatic and blood endothelial cells that stimulate osteoclast differentiation

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and function by TNFα and IL-6 secretion [18, 19]. Devlin et al. revealed high levels of IL-6 in serum

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from a patient [20]. Macrophages also produce VEGF-A, -C and -D that stimulate osteoclasts and lymphangiogenesis [21-24]. Moreover, TNFα secreted by endothelial cells and by macrophages can

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inhibit osteoblast function, blocking bone formation [25]. Indeed, a remarkable aspect of the osteolytic

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process is the absence of increased osteoblast activity along surfaces of remaining bone fragments in sections of affected tissues; the disappearing bone is replaced by fibrovascular tissue rather than newly

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formed woven repaired bone [26]. Due to its rarity, there are no set guidelines for the treatment and management of GSD [4]. The therapeutic options for the patients are based on drug treatments, radiation and surgery [4, 27-30]. Several pharmaceuticals have been used to treat GSD including anti-VEGF-A antibody, interferon alpha 2b, propranolol, steroids, vitamin D, calcitonin and bisphosphonates [4, 3137]. In the present study, histological, cellular and molecular aspects of the disease have been investigated in a group of seven patients with the aim to provide insights for diagnosis and for new therapeutic approaches for this rare disease. 5

Journal Pre-proof Materials and Methods Patients Seven patients (Table 1) were recruited by the Rare Diseases and Medical Genetics Unit of Bambino Gesù Children’s Hospital headed by Dr Andrea Bartuli, with the informed consent of their parents. The diagnosis of Gorham-Stout disease was based on radiological and bone biopsy analysis. Control subjects

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were matched for age and gender and tested for standard serum markers to exclude any inflammatory

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status.

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Histological/Histomorphometric analysis of bone biopsies

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Bone biopsies of 6 pediatric patients and 4 controls were fixed and processed for paraffin embedding

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with previous decalcification. For immunohistochemistry analysis, sections were labelled with antibodies against human CD68 (Abcam, Cambridge, UK), CD31 and Podoplanin (Agilent, Santa Clara, CA).

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Primary antibody incubations were carried out at room temperature for 1 h, followed by secondary

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incubations for 1 h at room temperature with the corresponding secondary antibody (Agilent, Santa Clara, CA). Histomorphometric measurements were carried out on 2 to 5 micron thick sections, with an

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interactive image analysis system (NIS-Elements BR 4.50.00). Nomenclature, symbols and units of histomorphometric bone parameters were those suggested by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (ASBMR) [38]. For the fibrotic area, the ratio between the fibrotic area in the bone marrow and the total bone marrow area was calculated. Serum markers Peripheral blood samples from 6 pediatric patients and 16 controls were centrifugated 1000 g x 10 min at room temperature and serum was collected, aliquoted and stored at -80°C. Pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen (ICTP) (Orion Diagnostica, Espoo, Finland), Tartrate6

Journal Pre-proof Resistant Acid Phosphatase (TRAcP) (IDS, United Kingdom), Procollagen type I N-terminal propeptide (P1NP) (Cusabio Technology LLC, Huston, TX), Sclerostin (Biomedica, Vienna, Austria), Dickkopf WNT signaling pathway inhibitor 1 (DKK1) (Biomedica, Vienna, Austria), Interleukin-6 (IL-6) (Diaclone SAS Besancon Cedex, France), Vascular Endothelial Growth Factor A (VEGF-A) (Diaclone SAS Besancon Cedex, France) and VEGF-C (IBL International, Hamburg, Germany) were measured by ELISA kits, according to the manufacturers’ instructions.

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Human osteoclast primary cultures

Osteoclasts were differentiated from Peripheral Blood Mononucelar Cells (PBMC) of 5 patients and 6

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controls as previously described [39]. Briefly, peripheral blood diluted in Phosphate Buffered Saline

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(PBS) solution was layered over Ficoll 1.077 g/ml (Lympholyte) centrifuged at 400 g for 30 min. ‘‘Buffy

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coat’’ was collected and washed twice with PBS. Cells were resuspended in DMEM medium containing 50 U/ml penicillin, 50 mg/ml streptomycin, 2 mM L-glutamine and 10% FBS (Fetal Bovin Serum). Then

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1x106 cells/cm2 were plated on cell culture dishes or on bovine bone slices (IDS, PANTEC) to evaluate

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bone resorption activity. After 3 h, cell cultures were rinsed to remove non-adherent cells. Cells were

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cultured in the presence of 20 ng/ml Macrophage Colony-Stimulating Factor (M-CSF, PeproTech, United Kingdom) and 30 ng/ml Receptor Activator of Nuclear factor kappa-Β Ligand (RANKL, PeproTech, United Kingdom) for 14 days to form mature osteoclasts. For the bone resorption assay, cells were cultured for further 4 days on bovine bone slices. Medium and factors were replaced every 3-4 days. Bone resorption activity Osteoclasts from controls and patients were differentiated on bovine bone slices. After 18 days of culture, cells were eventually fixed in paraformaldehyde and slices were stained for TRAcP to count osteoclasts; then they were cleaned free of cells by prolonged sonication, stained with 1% toluidine blue and observed 7

Journal Pre-proof by conventional light microscopy. Resorption pit area was measured by image analysis system (NIS Elements BR 4.50.00) and normalized for the osteoclast number. Mesenchymal stem/stromal cells cultures Mesenchymal stem/stromal cells (MSC) were isolated from 5 ml of bone marrow aspirate by density gradient centrifugation (Ficoll 1.077 g/ml; Lympholyte) and plated in non-coated 75 cm2 tissue culture

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flasks at a density of 160,000/cm2 in complete culture medium: DMEM supplemented with 10% FBS,

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50 U/ml penicillin and 50 mg/ml streptomycin. Cultures were maintained at 37°C in a humidified atmosphere, containing 5% CO2. After 48 h adhesion, non-adherent cells were removed and culture

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medium was replaced twice a week. MSC samples were phenotypically characterized by flow cytometry

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at passages 2-3. The osteogenic differentiation ability was evaluated by incubating cells with αMEM,

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10% FBS, 50 U/ml penicillin, 50 mg/ml streptomycin and 2 mM L-glutamine supplemented with 10-7 M dexamethasone and 50 µg/ml L-ascorbic acid; starting from day +7 of the culture, 5 mM β-

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glycerophosphate was added to the medium. After 3 weeks of culture, cells were washed with PBS, fixed

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Flow Cytometry Analysis

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with 10% formalin in PBS for 10 minutes or used for RNA isolation or mineralization assay.

All antibodies were purchased from BD. Mesenchymal stem cells were harvested from culture plates, centrifuged at 300 g for 5 min, and resuspended in PBS/FBS (2%). Single cell suspensions were incubated in the dark for 20 min at 4°C with directly conjugated monoclonal antibodies (BD, San Jose, CA) directed against the following human surface molecules: CD13 (1:40 APC Cy7 conjugated), CD34 (1:20 PE-conjugated), CD45 (1:20 FITC conjugated), CD73 (1:20 FITC conjugated), CD90 (1:40 PEconjugated; clone 2A3) and CD105 (1:20 PerCP-Cy5 conjugated). After labeling, cells were washed twice in PBS/FBS (2%), and data were acquired with a FACS LSRFortessa (BD). Flow cytometer 8

Journal Pre-proof profiles were analyzed using FACSDiva software (BD). A minimum of 20,000 events were collected per dataset. Mineralization assay Sorted ALP positive osteoblasts were cultured for 3 weeks in standard medium supplemented with 10 mM β-glycerophosphate and 50 μg/ml ascorbic acid before detection of mineralization by von Kossa

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staining (Bio Optica Milano Spa, Milan, Italy).

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Osteoblast isolation

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Bone fragments were obtained from healthy subjects who underwent femoral surgery for traumatic

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fractures. Bone fragments were subjected to sequential digestion with type IV collagenase (Sigma-

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Aldrich, USA) and trypsin (Gibco, USA). Cells obtained with this method were evaluated for ALP

Serum treatment

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activity (kit #86, Sigma-Aldrich, USA) and expression of osteoblast markers.

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A pool of sera from at least 6 patients and 6 sex- and age-matched controls were used to treat healthy

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donors cells. Healthy donors PBMC were cultured in DMEM and treated only with 10% of sera pool from controls and patients for 2 weeks. For the evaluation of bone resorption, control PBMC were differentiated on bovine bone slices for 2 weeks, and then treated for 4 days with sera from controls and patients. For the osteogenic differentiation protocol, MSC isolated from healthy donors were cultured for 4 weeks in αMEM supplemented with 10 mM β-glycerophosphate, 50 μg/ml ascorbic acid and 10% pool of sera. For the mineralization assay, control osteoblasts were cultured for 4 weeks in medium supplemented with 10 mM β-glycerophosphate, 50 μg/ml ascorbic acid and 10% pool of sera from controls and GSD patients. Regarding gene expression analysis, healthy donors osteoblasts were treated for 48 h with 10% pool of sera isolated from controls and patients. 9

Journal Pre-proof Vessel formation EA.hy926 cells (1×106/well) were grown on thin Matrigel™ (basement membrane matrix, BD Biosciences) layers pre-gelled in the wells of a 24-well cell culture plate (200 μL of pure matrigel solution, 1 h at 37 °C), cultured in DMEM and treated only with 10% pool of sera isolated from controls (N=6) and patients (N=6). Tubular networks were documented by light microscopy after 18 h incubation.

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Real-Time RT-PCR

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Total RNA was extracted using the TriPure isolation reagent (Sigma-Aldrich, USA) and 1 µg was reverse

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transcribed and the equivalent of 50 ng was used for PCR reactions using SensiFAST SYBR Hi-ROX

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(Bioline, UK). Primer sequences are listed in Table 2.

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Gene array

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RNA samples from osteoclasts and osteoblasts were hybridized on Human Clariom D (Thermo Fisher Scientific) gene chip and were analyzed using the Transcriptome Analysis Console 4.0 software (Applied

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Biosystem, Foster City, CA, USA by Thermo Fisher Scientific, Waltham, MA, USA). The significantly

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regulated transcripts with an expression level at least 1.5 fold different vs. control samples (p ≤ 0.05) were functionally classified according to the Gene Ontology (GO) annotations and submitted to the pathway analysis using the PANTHER expression analysis tools (http://pantherdb.org/) [40]. A pathways enrichment analysis was carried out to prioritize pathways that might have an association with disease phenotype. The enrichment of all pathways associated to the differentially expressed genes compared to the distribution of genes included on the Affymetrix microarray chip was therefore analyzed and p values ≤ 0.05, calculated by the binomial statistical test, were considered as significant enrichment. Statistics

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Journal Pre-proof Data were expressed as the mean ± s.d. of at least three independent experiments. Statistical analysis was performed by one-way analysis of variance, followed by the unpaired Student’s t test or the MannWhitney U test. A p value < 0.05 was considered statistically significant.

Results

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Patients recruitment

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Patients enrolled in this study were recruited by the Rare Disease Program of our Hospital. Clinical data

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of GSD patients were collected and described in Table 1. We collected data from 7 patients (6 males, 1

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female, median age 12,2 yrs, range 3-31). Progressive osteolysis and angiomatous proliferation are

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always the keys to the diagnosis. According to literature, clinical presentation of GSD is variable and depends on the site of involvement; as shown in Table 1, patients presented a wide phenotypic spectrum

skeleton

or

both

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usually

presents

with

cutaneous

swelling

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to

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appendicular

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varying from only one affected bone to severely affected conditions. The disease affects the axial or the

angiomatous/lymphatic lesions. Less frequently, the first symptom is chylothorax. This is a rare and

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severe complication of GSD and in our cohort was present in two patients.Histomorphometry Bone biopsy analysis

Histological analysis of bone biopsies isolated from patients revealed fibrous tissue in bone marrow stroma (Figure 1A, Supplementary Figure 1A) reaching about 60% of bone medullary total area (Figure 1B). Immunohistochemistry for the endothelial-specific antigen CD31 showed an increase of vascularization (Figure 1C, Supplementary Figure 1B). Indeed histomorphometric analysis revealed a trend of increase of vessel formation (Figure 1D) and about 4-fold increase of vessel area (Figure 1E) in patients’ biopsies. Immunohistochemistry for the lymphatic endothelial cells marker podoplanin revealed 11

Journal Pre-proof the lymphatic nature of the vessels in GSD patients (Figure 1F, Supplementary Figure 1C). CD68 staining analysis showed a great number of osteoclasts (Figure 1G, Supplementary Figure 1D) as confirmed by high levels of Osteoclast Surface/Bone Surface (Oc.S/BS) (Figure 1H) and Osteoclast Number/Bone Surface (Oc.N/BS) (Figure 1I). Since the osteolysis in Gorham-Stout patients is progressive and rapid we evaluated the possible involvement of osteocytes in bone erosion. Although histomorphometric analysis did not reveal any

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statistically significant differences in osteocyte number, osteocyte lacunae were significantly enlarged

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(Figure 1L-N), suggesting an involvement of osteocyte resorption in GSD.

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Serum markers

Evaluation of bone markers was performed in sera samples. Pyridinoline cross-linked carboxyterminal

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telopeptide of type I collagen (ICTP) values were elevated in patients, suggesting a generally increase of

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osteoclast bone resorption (Table 3). To analyze alterations of osteoclast differentiation and/or life span, TRAcP levels were measured and no significant differences were revealed between patients and healthy

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donors (Table 3). Interestingly, high levels of Sclerostin were found in patients with no alterations of

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other bone formation markers including P1NP and DKK1 (Table 3). Additionally, we revealed an increase of IL-6 and VEGF-A in GSD patients, with no changes of VEGF-C levels (Table 3). Characterization of GSD Osteoclasts As noted above, Gorham-Stout patients displayed an enhancement of osteoclast number in bone biopsies. This prompted us to evaluate a cell-autonomously altered differentiation of osteoclast precursors in GSD patients. Actually, PBMC isolated from patients showed a ~2-fold increased ability to differentiate into osteoclasts (Figure 2A-B), with higher number of nuclei per cell (Figure 2C-D). Indeed, ~20% of patient osteoclasts contained more than 10 nuclei (Figure 2D). About 75% of affected osteoclasts displayed 12

Journal Pre-proof lamellipodia, stress fibers and membrane ruffling, suggesting a more motile phenotype(Figure 2E-F). The activity of osteoclasts to resorb bone was evaluated plating cells on bovine bone slices. Interestingly, about 4.5-fold increase of bone resorption activity was observed in GSD osteoclasts (Figure 2G-H). To understand the molecular mechanisms involved in the alterations of osteoclasts obtained from patients, large scale microarray analysis was performed using the Human Clariom D array, which allows to interrogate more than 540,000 transcripts sourced from the largest number of public databases.

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Regarding the gene expression profiles, 106 modulated transcripts satisfied the Bonferroni-corrected p

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value criterion (p<0.05) and the fold change criterion (FC>I1.5I) were found. In particular, 40 and 66

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transcripts resulted to be under- and over-expressed, respectively (Tables 4-5). To validate our gene

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profiling results, comparative Real‐Time RT‐PCR was performed for a randomly selected subset of

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genes. We confirmed a decrease of solute carrier family 28 member 3 (SLC28A3) (Figure 3A), Midline2 (MID2) (Figure 3B) and Potassium Channel Tetramerization Domain Containing 9 (KCTD9) (Figure

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3C). Upregulation of other genes, such as LDL Receptor Related Protein 6 (LRP6) (Figure 3D) and

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Defensin beta 113 (DEFB113) (Figure 3E), was also confirmed by Real‐Time RT‐PCR.

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The Gene Ontology analysis showed that the modulated transcripts may be ascribed to molecular processes that can play a role in GSD osteoclasts including: binding, catalytic activity, channel regulator activity, receptor activity and signal transducer activity. Pathways analysis revealed an enrichment of the Angiotensin II-stimulated signaling through G proteins and beta-arrestin (P05911), PI3 kinase pathway (P00048) and EGF receptor signaling pathway (P00018) (Table 6). Analysis of GSD MSC and Osteoblasts Since in GSD patients the excessive bone resorption is not counterpoised by bone formation activity by osteoblasts, we isolated for the first time bone marrow MSC from a patient (patient #5). GSD-MSC 13

Journal Pre-proof displayed the characteristic spindle-shaped morphology (Figure 4A) and the same immunophenotype as healthy donors MSC; in particular, more than 95% of GSD-MSC were positive for CD13, CD73, CD90 and CD105, whereas they did not express hematopoietic markers (Figure 4B). After incubation with osteo-induction medium, GSD-MSC demonstrated no alterations of ALP activity as compared with control MSC (Figure 4C-D). To evaluate whether in GSD patients the reduced bone formation could be related to osteoblast activity

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defects, we sorted ALP+ osteoblasts from MSC differentiated cultures and we plated them for

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mineralization assay. As shown in Figure 4E-F, mineralized nodule formation was decreased in GSD

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osteoblasts.

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To investigate whether increased osteoclastogenesis could have also been due to the changes of

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RANKL/OPG (Osteoprotegerin) axis, the osteoclastogenic potential of GSD osteoblasts was evaluated.

(Figure 4G).

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Real-Time RT-PCR expression analysis revealed an increase of RANKL/OPG ratio in patient cells

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To investigate the mechanisms underlying the impairment of osteoblast function, gene expression

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analysis was performed. Our final analysis, using a cut of at least 1.5 and a statistical significance of p<0.05, revealed 72 genes that were modulated in Gorham-Stout osteoblasts. Indeed, 28 genes were downregulated and 44 were upregulated as listed in the tables 7-8. Modulation of genes such as Maestro Heat Like Repeat Family Member 2B (MROH2B) (Figure 5A), Epiphycan (EPYC) (Figure 5B), Doublecortin Domain Containing 1 (DCDC1) (Figure 5C), Relaxin/insulin-like Family Peptide receptor 1 (RFXP1) (Figure 5D) and the most upregulated Matrix metallopeptidase 13 (MMP13) (Figure 5E) was also confirmed by Real‐Time RT‐PCR.

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Journal Pre-proof Moreover, bioinformatic analysis suggested in GSD osteoblasts an enrichment of biological process involved in extracellular matrix and structure organization, endochondral ossification and bone morphogenesis, collagen metabolic process, negative regulation of ossification and regulation of ALP activity (Table 9). Effect of GSD sera on osteoclasts

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To address whether imbalance of systemic factors could be involved in the pathogenesis of GSD, we

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analyzed the effects of GSD sera in cell cultures. Cytokines’ imbalance suggested that osteoclast formation could be also affected when peripheral blood precursors are exposed to GSD sera. To address

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this hypothesis, we incubated control PBMC with 10% serum derived from patients and healthy donors.

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Interestingly, an increase of osteoclast formation was observed in cultures treated with patients’ sera as

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revealed by TRAcP staining (Figure 6A) and the quantification of multinucleated (>3 nuclei) TRAcP positive cells (Figure 6B); no difference was revealed in the number of nuclei per cell (Number of

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nuclei/osteoclast; Control: 4.86±0.25; GSD: 5.45±0.41; p=0.24). To evaluate the effects of systemic

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factors on bone resorption activity, we treated control osteoclasts plated on bovine bone slices with sera

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samples derived from patients and healthy donors. Interestingly, an increase of the bone resorption activity was observed in osteoclast cultures incubated with sera isolated from patients (Figure 6C-D). Effects of GSD sera on osteoblasts Regarding the effects of sera on osteoblasts differentiation and activity, MSC from healthy donors were treated with patients’ and heathy donors’s sera and osteogenic medium. ALP staining did not reveal any alterations of osteogenic ability (Densitrometric arbitrary unit; Control: 1.00±0.02; GSD: 0.88±0.07; p=0.19). However, control osteoblasts treated for 4 weeks with patients’ sera showed a reduced ability to form mineralized nodules (Figure 6E). To evaluate the effects of the treatment on osteoblast markers, 15

Journal Pre-proof osteoblasts isolated from bone fragments of healthy donors were treated for 48 h with patients’ and healthy donors’ sera. A reduction of the expression of Alkaline Phosphatase (Figure 6F) and Collagen 1a2 (Figure 6G) was revealed in GSD sera treated cells. Patients’ sera stimulated RANKL (Figure 6H) and downregulated OPG gene expression (Figure 6I); these modulations led to a significant increase of the RANKL/OPG ratio (Figure 6L).

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Effects of GSD sera on endothelial cells

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To evaluate whether systemic factors could influence the angiomatous proliferation, EA.hy926 endothelial hybrid cells were cultured on Matrigel and their ability to form tube formation upon sera

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treatment was evaluated. As shown in Figure 6, cells treated with sera isolated from patients are inclined

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to form more vessels with increased dimensions compared to cells treated with control sera (Figure 6M-

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Discussion

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To date, clinical studies on Gorham-Stout disease have been characterized by poor insight into the molecular and cellular mechanisms leading to progressive osteolysis. As a result, the diagnosis of GSD

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is based on exclusion criteria and bone biopsy analysis. The lack of specific biomarkers for this disease leads to the involvement of many diagnostic examinations and a strong delay to obtain correct diagnosis. In this study, some serum biomarkers have been highlighted as potentially relevant and specific diagnostic tools for this rare disease. The most promising of such markers appear to be ICTP, Sclerostin, VEGF-A and IL-6 that can cover all the cellular aspects of GSD that we also studied at cellular level. ICTP levels were increased in all the patients reflecting the great rate of bone resorption activity. High levels of bone formation inhibitor Sclerostin in GSD could be associated to defective bone regeneration following the osteolysis process. The increased level of VEGF-A reveals the angiomatous proliferation, 16

Journal Pre-proof pointing the attention to the second feature of this disease. Devlin et al. already documented high levels of IL-6 in GSD patients [20]; IL-6 can stimulate osteoclastogenesis [41] and angiogenesis by inducing VEGF expression [42]. The high number of osteoclasts observed in patients’ bone biopsy is due to primary alteration of cells and to effects of systemic factors that could stimulate mononuclear cells to differentiate into osteoclasts. Particularly, we showed that PBMC isolated from patients differentiate into osteoclasts with higher

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number of nuclei than control cells. GSD osteoclasts are also more active to resorb bone. This feature

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may be related to their motile phenotype since migration is essential for osteoclast bone resorption

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activity [43]. We performed transcriptomic analysis and an enrichment of the Angiotensin II-stimulated

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signaling through G proteins and beta-arrestin (P05911), PI3 kinase pathway (P00048) and EGF receptor

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signaling pathway (P00018) was found. All these pathways are involved in osteoclast differentiation and function and are correlated with PTEN (phosphatase and tensin homolog). It was demonstrated that

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PTEN overexpression suppressed RANKL-mediated osteoclast differentiation and osteopontin-

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stimulated cell migration [44]. The relevance of PTEN pathway in GSD was already evidenced by Hopman et al. who reported in a patient with Hamartoma Tumor Syndrome and Gorham-Stout

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Phenomenon a germline heterozygous mutation c.517 C>T (p.Arg173Cys) in exon 6 of PTEN [45]. Moreover, transcriptomic analysis revealed a reduction of MID2, SLC28A3, KCTD9 and an increase of LRP6 and DEFB113. Mid2 (Midline2) is an ubiquitin ligase that regulates microtubule dynamics and cell division [46]. Slc28a3 is a nucleoside transporter that is involved in many cellular processes, including neurotransmission, vascular tone and adenosine concentration. Slc28a3 shows broad specificity for pyrimidine and purine nucleosides [47]. Kctd9 belongs to the human potassium channel tetramerization domain (KCTD) family and is involved in NK cell development and functions [48].

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Journal Pre-proof Despite the regulation of these genes, to the best of our knowledge they have not been associated so far with bone metabolism, and we are now taking the challenge to investigate their role in this context. Conversely, the lipoprotein receptor-related protein 6 (LRP6) plays a critical role in skeletal development and homeostasis in adult since it is involved in Wnt pathway [49]. Although Wnt/β catenin signaling is essential for the bone formation, its role in osteoclasts is still under investigation. Weivoda et al. demonstrated that deletion of LRP5/6 in early osteoclast lineage reduced osteoclast numbers [50]. It is

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possible that the increased osteoclastogenesis in GSD patients could be also related to high levels of

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Lrp6. However, further investigation on GSD osteoclast precursors is important to understand the

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mechanisms by which these cells showed a great fusion index.

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Defensin β113 belongs to a family of innate host defense peptides with pleiotropic activities, generally

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secreted by various innate immune cells such as neutrophils and skin epithelial cells. The role of Defensin β113 in bone is poorly studied. However, it has been shown that human osteoblasts express beta defensins

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and particularly in response to stimulation by S. aureus [51]. The increase of Defensin β113 in GSD

ur

osteoclasts supports the notion that osteoclasts likely function as the innate immune cells of the bone,

Jo

thus are highly regulated to appropriately respond to stress and inflammatory changes in their microenvironment [52].

To date, osteoblast differentiation and activity in GSD patients have never been investigated. It was only reported that the increased bone resorption observed in GSD is not counterbalanced by bone formation activity and bone is replaced by fibrotic tissue [3, 26]. We isolated for the first time MSC from the bone marrow of a patient and differentiated them into osteoblasts. Although we did not observe any differences regarding their osteogenic ability, we assessed an impairment of their activity to form bone matrix as confirmed by mineralization assay.

18

Journal Pre-proof Our preliminary transcriptomic analysis revealed an increase of MMP13 gene expression in GSD osteoblasts. Hayami et al. revealed alterations of osteoblastic markers, including a reduction of Osterix and Osteopontin, in human periodontal ligament (PDL) cells overexpressing MMP-13 [53]. Interestingly, previous studies showed that MMP13-overexpressing cells promote capillary formation of endothelial cells [54] and that Matrix metalloproteinase-13 is required for osteocytic perilacunar remodeling [55]. Indeed, in bone biopsy of our patients we observed osteocytes with enlarged lacuna, that could be related

of

to osteocyte mediated-bone resorption activity [56, 57].

ro

Among the significantly upregulated genes in GSD osteoblasts, we also recognized RXFP1

-p

(Relaxin/insulin-like family peptide receptor 1). Rxfp1 is a G-protein coupled receptor containing

re

extracellular leucine-rich repeats for relaxin (RLN) binding; RLN is a polypeptide member of the insulin-

lP

like hormone family released by the ovarian corpora lutea and testis into the blood stream. Duarte et al. demonstrated higher ALP activity in MC3T3-E1 cells treated with small interfering-Rxfp [58]. Ahamad

ur

and -13 proteins [59].

na

et al. showed that in fibrocartilaginous cells the overexpression of Rxfp1 resulted in increased MMP-9

Jo

The exact function MROH2B and DCDC1 in bone are not entirely known. MROH2B encodes a protein belonging to the maestro heat-like repeat family. Dcdc1 contains a single doublecortin domain and is unable to bind microtubules and to regulate microtubule polymerization. However, DCDC1 is one the genes associated with bone mineral density or fracture risk in major genome-wide association studies [60]. EPYC gene encoded Dermatan sulfate proteoglycan 3; it is a member of the small leucine-rich repeat proteoglycan family, regulating fibrillogenesis by interacting with collagen fibrils and other extracellular matrix proteins [61]. Moreover, its downregulation was shown in osteoblasts isolated from patients with Idiopathic Scoliosis [62]. To confirm osteoblast cell autonomous defects in GSD and to evaluate whether the reduced mineralization ability of GSD cells could be associated to Epyc 19

Journal Pre-proof downregulation, further studies using MSC isolated from other patients should be performed. However, our paper is the first report that points the attention also to the involvement of osteoblasts and MSC in the complex scenario of GSD. Furthermore, we demonstrated alterations of systemic factors able to influence the differentiation and function of osteoclasts and osteoblasts. Indeed, it was reported that Sclerostin reduces alkaline phosphatase activity and mineralization in osteogenic cultures of murine MSC and human primary

of

osteoblasts [63-65]. According to these results, we observed in osteoblasts isolated form healthy donors

ro

and treated with GSD sera a decrease of ALP and COL1A2 gene expression and of mineralization activity.

-p

Sclerostin was shown to stimulate osteoclastogenesis upregulating the expression of RANKL and

re

downregulating OPG in osteoblast lineage cells [66, 67]. The increase of RANKL/OPG ratio observed

lP

in control cells treated with GSD sera could rely, to some extent, on this Sclerostin regulation. Devlin et al. showed that patient serum induces osteoclast formation in an IL-6-dependent manner [20]. IL-6

na

influences bone remodeling, acting also on osteoblasts as demonstrated in transgenic overexpressing IL-

ur

6 mice characterized by increased osteoclastogenesis and reduced osteoblast activity [68]. However, VEGF can cooperate with IL-6 to alter bone microenvironment in GSD patients. Indeed Yang et al.

Jo

demonstrated that VEGF treatment of purified murine bone marrow osteoclast precursors directly enhances their survival, differentiation into mature osteoclasts and resorption ability, activating both the PI3-kinase/Akt and MEK/ERK pathways [24]. Hu et al. (2016) showed that the deletion of Vegfr2 in osteoblastic cells increases osteoblast maturation and mineralization in bone repair experiments [5]. Interestingly, Oranger et al. recently demonstrated that VEGF expression and tubule formation are stimulated in human umbilical vein endothelial cells treated with Sclerostin, that may be considered as a bona fide angiogenic molecule [69]. The increase of VEGF-A and Sclerostin levels in patients could also explain the increased vascularization of EA.hy926 cells treated with patients’ sera. However, the

20

Journal Pre-proof relevance of endothelial growth factors in the pathogenesis of Gorham-Stout disease was recently reported. Homminick et al. generated a transgenic mouse expressing VEGF-C in bone under the control of Osterix promoter, showing osteoporosis phenotype and bone lymphatics [70]. Gorham-Stout disease lacks of a standard therapeutic approach. Hammer et al. reported a stabilization of clinical and radiological picture in a patient treated with 30 mg pamidronate i.v. every 3 months [71]. In an elegant review published in 2016, Liu et al. summarized the results obtained by bisphosphonates

of

treatments. After 3-month to 17-year follow-up, disease progress arrested in 8/10 patients; 1/10 reported

ro

no beneficial effects after treatment with alendronate and one had a moderate clinical improvement in

-p

combination with radiotherapy [37]. Moreover, a combined approach of bisphosphonates with

re

mammalian target of rapamycin (mTOR) inhibitor sirolimus to arrest angiogenesis was successfully

lP

tested in some patients [72, 73]. However, the use of anti-osteoporosis drugs as bisphosphonates has no action on bone formation and is often associated with side-effects, including the atrial fibrillation,

na

osteonecrosis of the jaw, and “frozen bone”, characterized by over-suppression of bone turnover [74];

ur

the pediatric administration is much more cumbersome, with unclear scheme of treatments and likelihood of adverse effects otherwise not seen in adults [75]. The increased levels of Sclerostin that we observed

Jo

in our patients could suggest a future approach based on Sclerostin inhibitors that are very promising to reduce fracture risk in osteoporotic patients stimulating bone formation and inhibiting bone resorption too [76]. In summary, our results suggest that a multi-targeted therapeutic plan should be tested to combine anti-osteoclast treatment, bone anabolic drug and angiogenesis inhibitors to face the complex features of this multifactorial disease.

21

Journal Pre-proof Acknowledgements This work was supported by the Million Dollar Bike Ride Grant Program, Orphan Disease Center, University of Pennsylvania grant ##MDBR-17-116-GLA/GSD59 to ADF and AB and by grants from the Italian Ministry of Health (“Ricerca corrente”) to ADF. Authors’ roles: MR and RDV performed histologic and histomorphometric analysis; MR and EM

of

performed osteoclast culture; GB and ADF contributed to gene expression analysis; SP isolated

ro

osteoblasts from healthy donors; PSB, MM, MVG, IR, and AJ recruited the patients and performed ELISA assays. AC, MA and MDA contributed to the hematologic tests and discussion of the results.

-p

ADF and AB designed and supervised the work and wrote the paper. All authors reviewed the manuscript

re

and approved the final version. ADF is the guarantor of this work and, as such, had full access to all of

lP

the data in the study and takes responsibility for the integrity of the data and the accuracy of the data

Jo

ur

na

analysis.

22

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Journal Pre-proof Figure Legends Figure 1. Bone biopsy analysis. A) Hematoxylin/eosin staining revealed a stroma rich in fibres (asterisks). Scale bar: 50 µm. B) Histomorphometric analysis of Fibrotic Area/Total Area. C) Immunohistochemical analysis of CD31. The asterisk indicates CD31+ vessels in control. Scale bar: 50 µm. D-E) Histomorphometric analysis of Vessel Number and Vessel Area/Total Area. F) Immunohistochemical analysis of podoplanin. Scale bar: 50 µm. G) Immunohistochemical staining with

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anti-CD68 antibody. Scale bar: 50 µm. H-I) Histomorphometric analysis of Oc.S/BS (Osteoclast

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Surface/Bone Surface) and Oc.N/BS (Osteoclast Number/Bone Surface). L) Hematoxylin/eosin staining

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showing dilated perilacunar area. Scale bar: 15 µm. M-N) Histomorphometric analysis of the osteocyte

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number and osteocyte lacunar mean area. Results are mean±SD. *p<0.05.

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Figure 2. Osteoclast cultures from peripheral blood mononuclear cells of patients and healthy donors as controls cultured in the presence of 20 ng/mL M-CSF and 30 ng/mL RANKL to differentiate into

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osteoclasts (OC). A) TRAcP staining. Scale bar: 75 µm. B) Number of TRAcP positive multinucleated

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(>3 nuclei) cells. C) TRAcP and Hoechst stainings of osteoclasts. Scale bar: 75 µm. D) Percentage of

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osteoclasts grouped by number of nuclei. E) TRITC-phalloidin staining. Scale bar: 50 µm. F) Percentage of osteoclasts with motile phenotype. G) Toluidine blue staining of bone slices revealing bone resorption lacunae. Scale bar: 100 µm. H) Quantification of resorbed area per osteoclast. Results are mean±SD. *p<0.05; ***p<0.001. Figure 3. Validation of osteoclast gene array. RNA was extracted from GSD and control osteoclast cultures and reverse transcribed, then cDNA was subjected to comparative Real-Time PCR using primer pairs and conditions specific for A) SLC28A3, B) MID2, C) KCTD9, D) LRP6 and E) DEFB113 genes. Values are expressed as mean±SD. *p<0.05; **p<0.01.

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Journal Pre-proof Figure 4. Mesenchymal stem cells and osteoblasts analysis isolated from patient #5 and four controls. A) Phase contrast microscopy showing the morphology of bone marrow-derived MSC (passage 2). Scale bar: 200 µm. B) FACS analysis of mesenchymal stem cells for CD13, CD34, CD45, CD73, CD90 and CD105 markers. MSC were positive for CD13, CD73, CD90 and CD105 surface antigens and negative for CD34 and CD45 molecules. C-D) Osteogenic differentiation ability of MSC treated for 3 weeks with osteogenic medium. C) Alkaline phosphatase (ALP) staining (Scale bar: 380 µm) and D) densitometric

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analysis. E) Sorted ALP+ cells were incubated for 3 weeks with osteogenic medium and von Kossa

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staining was performed. Scale bar: 150 µm. F) Densitometric analysis of mineralized area. G) RNA was

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extracted from osteoblasts and reverse transcribed, then cDNA was subjected to comparative Real-Time

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PCR using primer pairs and conditions specific for RANKL and OPG genes. RANKL/OPG ratio was

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evaluated. For the controls, results are expressed as mean±SD. Figure 5. Validation of osteoblast gene array. RNA was extracted from osteoblast cultures of patient #5

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and four controls and reverse transcribed, then cDNA was subjected to comparative Real-Time PCR

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using primer pairs and conditions specific for A) MROH2B, B) EPYC, C) DCDC1, D) RXFP1 and E)

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MMP13 genes. For the controls, results are expressed as mean±SD. Figure 6. Effects of sera isolated from patients on bone and endothelial cells. A) TRAcP staining of osteoclast cultures obtained from PBMC of healthy donors treated for 2 weeks with pool of sera isolated from patients and controls. Scale bar: 75 µm. B) Count of multinucleated (>3) TRAcP positive cells. C) Blue toluidine staining of resorption lacunae formed by control osteoclasts differentiated on bovine bone slices and then treated for 4 days with sera from patients and controls. Scale bar: 50 µm. D) Quantification of resorbed area. E) Densitometric analysis of mineralized nodules of control osteoblast cultures treated with sera isolated from patients and controls. F-L) RNA was extracted from control osteoblasts treated with patient and control sera and reverse transcribed; then cDNA was subjected to comparative Real33

Journal Pre-proof Time PCR using primer pairs and conditions specific for F) ALP, G) COL1A2, H) RANKL and I) OPG genes; L) Computation of the RANKL/OPG ratio. M) Representative pictures of vessel formation by endothelial cells EA.hy926 treated with patients’ and healthy donors’ sera. Scale bar: 100 µm. N) Quantification of number and O) area of vessels formed after treatment with sera. Results are mean±SD.

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*p<0.05; **p<0.01; ***p<0.001.

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Journal Pre-proof Supplementary Figure Legend Supplementary Figure 1. Representative pictures of bone biopsy analysis of other GSD patients. A) Hematoxylin/eosin staining (N=3 patients). Scale bar: 40 µm. B) Immunohistochemical analysis of CD31 (N=4 patients). Scale bar: 50 µm. C) Immunohistochemical analysis of podoplanin (N=3 patients). Scale bar: 50 µm. D) Immunohistochemical staining with anti-CD68 antibody (N=3 patients). Scale bar: 50

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