Fatty acid metabolism promotes TRPV4 activity in lung microvascular endothelial cells in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a morbid disease characterized by significant lung endothelial cell (EC) dysfunction. Prior work has shown that microvascular endothelial cells (MVECs) isolated from animals with experimental PAH and patients with PAH exhibit significant abnormalities in metabolism and calcium signaling. With regards to metabolism, we and others have shown evidence of increased aerobic glycolysis and evidence of increased utilization of alternate fuel sources (such as fatty acids) in PAH EC. In the realm of calcium signaling, our prior work linked increased activity of the transient receptor potential vanilloid-4 (TRPV4) channel to increased proliferation of MVECs isolated from the Sugen/Hypoxia rat model of PAH (SuHx-MVECs). However, the relationship between metabolic shifts and calcium abnormalities was not clear. Specifically, whether shifts in metabolism were responsible for increasing TRPV4 channel activity in SuHx-MVECs was not known. In this study, using human data, serum samples from SuHx rats, and SuHx-MVECs, we describe the consequences of increased MVEC fatty acid oxidation in PAH. In human samples, we observed an increase in long-chain fatty acid levels that was associated with PAH severity. Next, using SuHx rats and SuHx-MVECs, we observed increased intracellular levels of lipids. We also show that increasing intracellular lipid content increases TRPV4 activity, whereas inhibiting fatty acid oxidation normalizes basal calcium levels in SuHx-MVECs. By exploring the fate of fatty acid-derived carbons, we observed that the metabolite linking increased intracellular lipids to TRPV4 activity was ß-hydroxybutyrate (BOHB), a product of fatty acid oxidation. Finally, we show that BOHB supplementation alone is sufficient to sensitize the TRPV4 channel in rat and mouse MVECs. Returning to humans, we observe a transpulmonary BOHB gradient in human patients with PAH. Thus, we establish a link between fatty acid oxidation, BOHB production, and TRPV4 activity in MVECs in PAH. These data provide new insight into metabolic regulation of calcium signaling in lung MVECs in PAH.NEW & NOTEWORTHY In this paper, we explore the link between metabolism and intracellular calcium levels in microvascular endothelial cells (MVECs) in pulmonary arterial hypertension (PAH). We show that fatty acid oxidation promotes sensitivity of the transient receptor potential vanilloid-4 (TRPV4) calcium channel in MVECs isolated from a rodent model of PAH.

MLKL polymerization-induced lysosomal membrane permeabilization promotes necroptosis.

Mixed lineage kinase-like protein (MLKL) forms amyloid-like polymers to promote necroptosis; however, the mechanism through which these polymers trigger cell death is not clear. We have determined that activated MLKL translocates to the lysosomal membrane during necroptosis induction. The subsequent polymerization of MLKL induces lysosome clustering and fusion and eventual lysosomal membrane permeabilization (LMP). This LMP leads to the rapid release of lysosomal contents into the cytosol, resulting in a massive surge in cathepsin levels, with Cathepsin B (CTSB) as a significant contributor to the ensuing cell death as it cleaves many proteins essential for cell survival. Importantly, chemical inhibition or knockdown of CTSB protects cells from necroptosis. Furthermore, induced polymerization of the MLKL N-terminal domain (NTD) also triggers LMP, leading to CTSB release and subsequent cell death. These findings clearly establish the critical role of MLKL polymerization induced lysosomal membrane permeabilization (MPI-LMP) in the process of necroptosis.