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Abstract Number: 1627

HIF-1alpha Knockdown Down-Regulates Glycolytic Metabolism and Induces Rheumatoid Synovial Fibroblast Cell Death

Manuel J. Del Rey1, Alicia Usategui1, Álvaro Valín1, María Sánchez-Aragó2, José M. Cuezva2, Carmen M. García-Herrero1, María Galindo1, Juan D. Cañete3, Francisco J. Blanco4, Gabriel Criado1 and Jose L. Pablos1, 1Servicio de Reumatología, Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain, 2Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Investigación Hospital 12 de Octubre, Universidad Autónoma de Madrid, Madrid, Spain, 3Unitat d’Artritis, Servei de Reumatologia, Hospital Clínic de Barcelona and Institut d’Investigacions Biomèdiques August Pí i Sunyer, Barcelona, Spain, 4Laboratorio de Investigación Osteoarticular y del Envejecimiento, Instituto de Investigación Biomédica de A Coruña, INIBIC, A Coruña, Spain

Meeting: 2015 ACR/ARHP Annual Meeting

Date of first publication: September 29, 2015

Keywords: Fibroblasts, Metabolism, rheumatoid arthritis (RA) and synovial cells, synovial fluid

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Session Information

Date: Monday, November 9, 2015

Title: Rheumatoid Arthritis - Human Etiology and Pathogenesis Poster II

Session Type: ACR Poster Session B

Session Time: 9:00AM-11:00AM

Background/Purpose: Intense synovial fibroblast (SF) hyperplasia contributes to the chronic inflammation and osteoarticular destruction that characterizes rheumatoid arthritis (RA). Hypoxia-inducible factor 1α (HIF-1α) plays a pivotal role in the metabolic adaptation of SF in hypoxic inflamed joints. The purpose of this study was to analyze the impact of HIF-1α knockdown on metabolic regulation and cell survival of rheumatoid arthritis SF (RASF). 

Methods: HIF-1α was silenced using lentiviral vectors or siRNA duplexes transfer strategies. Cell viability upon HIF-1α silencing was analyzed by Alamar Blue assay in SF lines from RA, osteoarthritic or healthy synovium and LC5 lung fibroblasts. To analyze the effect of HIF-1α silencing on RASF in vivo, RASF were engrafted into an air pouch in NOD scid gamma (NSG) mice and siRNA duplexes (siHIF-1α or control, 9 mice per group) were administered locally. After 11 days mice were sacrificed, pouch membranes dissected and analyzed by immunoperoxidase labeling to quantify the number of human nuclei per pouch wall area. Proteomic analysis was performed in silenced HIF-1α RASF by iTRAQ labelling, and expression of identified glycolytic enzymes as GAPDH, TPI1 and ENO1 was confirmed by western blot. Energy metabolism was analyzed in silenced RASF and LC5 cells by measuring oxygen consumption rate (OCR) with a XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA), and lactate production by Lactate colorimetric assay kit II (Biovision, Milpitas, CA, USA).

Results: HIF-1α silencing induced cell death in cultured SF regardless of their origen but not in LC5 cells. The number of RASF engrafted into the air pouch was significantly reduced in HIF-1α compared to control siRNA injected mice (205±174 vs 393±237 nuclei/mm2, p=0.04 Mann-Whitney U-test). iTRAQ  analysis identified 321 proteins in HIF-1α silenced and control RASF, down-regulated proteins included several glycolytic enzymes but only GAPDH expression was significantly decreased in siHIF-1α transduced compared to non-silenced RASF (ratio GAPDH/β-actin 0.54±0.28 vs 0.85±0.18, p=0.004 Wilcoxon test). The oligomycin sensitive respiratory rate (OSR) as indicator of oxidative phosphorylation was significantly reduced in RASF compared to LC5 (14.1±3.5 vs 62.2±8.8 pmol O2/min/5000 cells, p<0.0001 Mann-Whitney U-test). Lactate production was significantly higher in RASF compared to LC5 (26.6±12.6 vs 12.2±1.9 nmol/mg/hour, p=0.003 Mann-Whitney U-test), and was reduced by HIF-1α silencing. Treatment of RASF with the GAPDH inhibitor 3-bromopyruvate (3BrP) also induced cell death in cultured RASF.

Conclusion: Our data show that RASF metabolism is mainly glycolytic and demonstrate that the regulatory role of HIF-1α in the glycolytic metabolism is critical for SF survival. In addition, local targeting of HIF-1α may provide a feasible strategy to reduce the pathogenic contribution of SF to chronic arthritis.


Disclosure: M. J. Del Rey, None; A. Usategui, None; Valín, None; M. Sánchez-Aragó, None; J. M. Cuezva, None; C. M. García-Herrero, None; M. Galindo, None; J. D. Cañete, None; F. J. Blanco, Pfizer, Bioiberica, and Gebro Pharma, 5; G. Criado, None; J. L. Pablos, None.

To cite this abstract in AMA style:

Del Rey MJ, Usategui A, Valín , Sánchez-Aragó M, Cuezva JM, García-Herrero CM, Galindo M, Cañete JD, Blanco FJ, Criado G, Pablos JL. HIF-1alpha Knockdown Down-Regulates Glycolytic Metabolism and Induces Rheumatoid Synovial Fibroblast Cell Death [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/hif-1alpha-knockdown-down-regulates-glycolytic-metabolism-and-induces-rheumatoid-synovial-fibroblast-cell-death/. Accessed .
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