RT info:eu-repo/semantics/doctoralThesis
T1 Metabolic rewiring is required for inflammation, calcification, and osteogenic differentiation of human aortic valve interstitial cells exposed to an inflammatory milieu, and mimics the metabolic phenotype in calcified aortic valves
A1 Sánchez-Bayuela Recio, Tania
A2 Universidad de Valladolid. Escuela de Doctorado
K1 Cardiología
K1 Metabolism, CAVD
K1 Metabolismo, CAVD
K1 3207.04 Patología Cardiovascular
AB Introduction and objectives: Inflammation has been linked to metabolic reprogramming in several diseases, including cardiovascular pathogenesis. Calcific aortic valve disease (CAVD) is an increasingly prevalent valvulopathy, yet surgical replacement is the only available therapy. CAVD is characterized by a damaged endothelium, inflammation, exaggerated matrix remodeling, calcification, and metabolic changes. At the cellular level, recent evidence disclose the interplay between innate immunity/inflammatory pathways, i.e., Toll-like receptors (TLR)3/4 and interferon-γ receptor signaling, on the differentiation, calcification, and inflammation of valve interstitial cells (VIC) via stabilization of hypoxia-inducible factor (HIF)-1α. Given that this transcription factor is associated with metabolic reprogramming in several diseases, inflammation and metabolism may work in an interconnected manner in CAVD. Therefore, the main goal of this study was to investigate the metabolic reprogramming of VIC under inflammatory settings, and its contribution to the processes relevant to CAVD pathogenesis.Material and methods: Human VIC from patients with no valve disease were used as a model. To mimic an inflammatory environment, cells were treated with pro-inflammatory cytokines and pathogen patterns recognized by TLRs. Metabolic analysis was performed by real-time metabolic analysis using Seahorse extracellular flux assays, and [U-13C]-glucose tracing, by liquid chromatography/mass spectrometry. Metabolic gene profiles and metabolite production were evaluated by qPCR, Western blot, and commercial kits. Inflammation, calcification, and apoptosis were studied using Western blot, ELISA, qPCR, immunofluorescence, flow cytometry, gene silencing, and in vitro calcification assays. Validation of findings in human VIC was performed in quiescent VIC dedifferentiated from human VIC, in porcine 3D VIC-valve endothelial cell co-cultures, as well  in valve leaflets and VIC explanted from patients with/without CAVD. Results: The main finding of the study is that inflammatory stimuli drive a metabolic reprogramming of VIC to a hyperglycolytic phenotype that mimics the metabolic phenotype in calcified valves. It is characterized by enhanced glycolysis and glycolytic ATP production, impaired pentose phosphate pathway (PPP), as well as damaged mitochondrial function with uncoupling of electron transport chain (ETC) and oxidative phosphorylation (OXPHOS). Furthermore, metabolic dysregulation is associated with reactive oxygen species (ROS) production, as well as increased reliance on glucose uptake for energy production and metabolite accumulation. Pharmacological approaches to metabolic routes demonstrate the role of glycolysis upregulation in processes relevant to CAVD, such as VIC differentiation, calcification, and inflammation, and have further highlight the contribution of PPP and oxidative stress in these processes. Our findings further reveal the involvement of the Janus kinase (JAK)-STAT/HIF-1α and nuclear factor (NF)-kB pathways in the metabolic reprogramming. Finally, the shift in VIC, also found in 3D VIC-VEC co-cultures exposed to inflammatory stimuli, replicates the hyperglycolytic profile of calcified cells and valve leaflets.Conclusion:  Inflammation drives a metabolic shift in human VIC, mirroring the glycolytic phenotype in calcified valves, which is characterized by hyperglycolysis that is necessary to support inflammation, calcification, and osteogenic differentiation of VIC. Additional reprogramming of complementary catabolic pathways, such as PPP, tricarboxylic acid cycle, and oxidative phosphorylation, generates redox homeostasis alterations that further contribute to pathological processes in VIC. Thus, inflammation-triggered changes in metabolic phenotypes may play a relevant  pathogenic role in the early stages of CAVD, and the identified metabolic routes may provide therapeutic clues for the disease.
YR 2024
FD 2024
LK https://uvadoc.uva.es/handle/10324/67180
UL https://uvadoc.uva.es/handle/10324/67180
LA eng
NO Escuela de Doctorado
DS UVaDOC
RD 17-may-2024