Dataset Information

Effect of Col1a2 mutation in the gene expression of immature and mature osteoblasts in a mouse model of osteognesis imperfecta (OI)

ABSTRACT: In Europe, approximately 85-90% of individuals with Osteogenesis Imperfecta (OI) have dominant pathogenic variants in the COL1A1 or COL1A2 genes whilst for Asian, especially Indian and Chinese cohorts, this ratio is much lower. This leads to decreased or abnormal Collagen type I production. Subsequently, bone formation is strongly reduced, causing bone fragility and liability to fractures throughout life. OI is clinically classified in 5 types with the severity ranging from mild to lethal depending on the gene and the type and location of the OI-causative variant and the subsequent effect on (pro) collagen type I synthesis. However, the specific effects on the phenotype and function of osteoblasts are not fully understood. To investigate this, the OI murine model was used, with the oim/oim (OIM) mice closest resembling severely deforming OI type 3 in humans. We showed that in OIM, COL1 mutation results in a multifactorial inhibition of the osteogenic differentiation and maturation as well as inhibition of osteoclastogenesis. The phenotype of differentiated OIM osteoblasts also differs from that of wild type mature osteoblasts, with upregulated oxidative cell stress and autophagy pathways, possibly in response to the intracellular accumulation of type I collagen mRNA. The extracellular accumulation of defective type I collagen fibres contributes to activation of the TGF- signalling pathway and activates the inflammatory pathway. These effects combine to destabilise the balance of bone turnover, increasing bone fragility. Together, these findings identify the complex mechanisms underlying OI bone fragility in the OIM model of severe OI and can potentially enable identification of clinically relevant endpoints to assess the efficacy of innovative pro-osteogenic treatment for patients with OI.

ORGANISM(S): Mus musculus

PROVIDER: GSE273779 | GEO | 2024/08/25

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Osteogenesis Imperfecta (OI) is a genetic, rare disease characterized by bone fragility, with a wide range in the severity of clinical manifestations. The majority of the cases are due to mutations in COL1A1 or COL1A2 genes which code for the type I collagen molecule. Mesenchymal stem cells (MSCs), as the progenitors of the osteoblasts, the main type I collagen secreting cell type in the bone, have been proposed and tested as an innovative therapy for OI with promising but transient outcomes. In this study, we performed a phase I clinical trial based on reiterative infusions of histocompatible MSCs in two pediatric patients affected by severe and moderate OI. The aim of this study was to assess the safety and effectiveness of this cell therapy in non-immunosuppressed OI patients. The host response to MSCs was also evaluated by collecting the sera from OI patients before, along and after the cell therapy. The results of this clinical trial are twofold: the sequential administration of MSCs is safe and improve the bone parameters and quality of life of OI patients. Moreover, MSCs therapy elicits a pro-osteogenic paracrine response in patients, especially noticeable in that affected by severe OI. These results indicate the feasibility and potential of reiterative MSCs infusion for OI and highlight the paracrine response shown by OI patients as a consequence of MSCs treatment. Study design and patients: The Mesenchymal Stem Cell Therapy for the Treatment of Osteogenesis Imperfecta (TERCELOI) study (www.clinical trials.gov: # NCT02172885, EudraCT number: 2012-002553-38) is an independent multi-center Phase I clinical trial to evaluate the feasibility, safety and potential efficacy of infused sibling HLA-matched MSCs in non-immunosuppressed children with OI. PBMCs isolation and serum collection: Venous blood samples were collected before the cell therapy (basal serum), along the cell therapy (1 week, 1 month and 4 months after each MSCs infusion) and during the follow-up period (1 and 2 years after the fifth and last MSCs infusion). MLR assay: PBMCs isolated from patients were resuspended in PBS+FBS (5%) and stained with carboxyfluoroscein succinate-ester (CFSE) (Molecular Probes, USA). Bone mineral density: Bone mineral density (BMD) at the lumbar spine (LS) from L1 to L4, was measured by dual energy x-ray absorptiometry (DXA) on a whole-body scanner within a pediatric platform (Hologic QDR densitometer). Raw measurements were converted to Z-scores for analysis using reference standards for age and pubertal status. OI-MSCs isolation and characterization and culture: OI-MSCs were derived from discarded and donated bone fragments of OI pediatric patients undergoing corrective surgery. All patients suffered Type III-IV OI, with mutations in either COL1A1 or COL1A2 genes. The donation was approved by the Basque Clinical Research Ethics Committee. Briefly, bone chips were mechanically flushed with PBS and then cut into small pieces to extrude cells without the use of enzymes. The cut bone pieces were let undisturbed during 14 days until OI-MSCs migrated to cell culture plate. OI-MSCs were assessed for the expression of CD105, CD90 and CD73 and the absence of CD14, CD34, CD45, CD19 and HLA-DR. MSCs were also tested for their potential to differentiate to osteoblasts and adipocytes using specific cell culture media. OI-MSCs were cultured under standard growth medium (DMEM low glucose with glutamax (Gibco), penicillin/streptomycin (Gibco) and fetal bovine serum at 10% (Sigma-Aldrich, USA). Osteogenesis induction medium (OIM) was composed of standard medium plus ascorbic acid 0.2 mM, beta-glycerophosphate 10 mM, and dexamethasone 10 nM (all from Sigma-Aldrich). When specified, FBS was replaced by P01 and P02 serum samples at 2.5%. RNAseq and Q-PCR validation: OI-MSCs from 10 OI pediatric patients were seeded in 96-well plates (1 000 cells/well) and the following day cultured in the presence of OIM. Next, total RNA was isolated with the AllPrep kit (QIAGEN, USA). cDNA library (TrueSeq stranded Total cDNA library, Illumina, USA) and sequencing at HiSeq 4000 (PE100nt, 50 million reads/sample).

Project description:Vascular Calcification (VC) is a life threatening cardiovascular complication that accounts for high death rates particularly in association with diabetes, atherosclerosis and Chronic Kidney Disease (CKD. during VC, Vascular Smooth Muscle Cells (VSMCs) undergo reprogramming into osteoblast-like cells in response to calcium and phosphate mineral imbalance in CKD patients. This osteogenic switch is driven by the expression of several bone markers such as Runt- related transcription factor 2 (Runx2), Alkaline Phosphatase (ALP), Osteopontin (OPN), Matrix-Gla Protein (MGP), Osteocalcin (OCN), Type I Collagen (COL1), and Bone Sialo Proteins (BSPs). Recent studies showed that VSMCs secrete heterogeneous populations of Extracellular Vesicles (EVs)11 that are enriched under physiological conditions by calcification inhibitors such as Fetuin-A and MGP12,13,14. Interestingly, under pathological conditions, secreted EVs have a pro-calcifying profile and thereby act as nucleating foci for hydroxyapatite crystallization and propagation11,15. Electron Microscopy based studies revealed that EVs were embedded between collagen and elastin fibrils of arterial walls suggesting that calcification can be initiated through the EVs’ direct physical interaction with them13,16. We previously showed that a specific oligogalacturonic acid with a degree of polymerization of 8 (DP8) was able to inhibit vascular calcification. This inhibition was partly due to a decrease of osteogenic markers’s expression but also to the masking of a consensus sequence found in COL1 i.e. GFOGER sequence, thus preventing EVs from binding to COL1. Although the use of DP8 in-vivo seems to be compromised due to probable enzymatic digestion, these results suggested that the inhibition of VSMCs’ osteogenic switch and the prevention of EVs-COL1 interaction could represent new potential therapeutic targets for inhibiting VC17. Since we have already showed that the triple-helical GFOGER consensus sequence forms a preferential binding site for EVs17, we thereby chemically synthesized a GFOGER peptide in order to determine its ability to inhibit VC on VSMCs and on an aortic ring ex-vivo model. We also aim to decipher the mechanisms of inhibition by investigating the osteogenic markers’ expression as well as the protein content of secreted EVs in the presence of the GFOGER peptide.

Dataset Information

Effect of Col1a2 mutation in the gene expression of immature and mature osteoblasts in a mouse model of osteognesis imperfecta (OI)

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets