Abstract
Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability.
Highlights
Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels
ECs Remodel Their Metabolism upon Morphogenesis—To identify metabolic pathways potentially involved in controlling EC functions, we used integrative metabolic analysis tool (iMAT) to integrate time-resolved proteomic data of human umbilical vein endothelial cells (HUVECs, referred to as ECs throughout the Results section) grown on matrigel with genome-scale metabolic network model (GSMM) (Fig. 1A)
Pathway enrichment analysis based on fold change reaction flux between early and late matrigel highlighted fatty acid oxidation (FAO) in peroxisome, the organelle where very-long-chain fatty acids (FAs) can be oxidized into short-chain FAs and released into the cytosol, as the most up-regulated pathway when the network was fully assembled, and tricarboxylic acid cycle (TCAc) as the most down-regulated
Summary
Reagents, and Treatments—Human umbilical vein endothelial cells (HUVECs) isolated from 2–5 umbilical cords were pooled and cultured in EGM-2 (Lonza, Basel, Switzerland). Matrigel Assay—HUVECs were seeded and cultured on solidified Matrigel in EGM-2 medium with the indicated stimuli and harvested for MS analysis using Cell recovery solution according to manufacturer’s instructions and as previously described [19]. Proteome Matrigel—For each sample, ϳ2 g of digested peptides were eluted from reverse phase column with a flow of 200 nl/min in 190 min gradient, from 5% to 30% ACN in 0.5% acetic acid. Metabolomics Data Analysis—The peak areas (ϭ measured intensity) of different metabolites were determined using LCquan software (Thermo Fisher Scientific) where metabolites were identified by the exact mass of the singly charged ion and by known retention time on the HPLC column. The .raw MS files and search/identification files obtained with MaxQuant have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org/cgi/GetDataset) via the PRIDE partner repository with the dataset identifier PXD001186
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