Biology subjects

Levitan, I.

Publications and source records attributed to Levitan, I..

4 recordsLinked to original sources

bioRxiv2025

A Single Mutation in TRPC6 Protects Mice from Acute Lung Injury by Regenerating Endothelium

Regenerating vascular endothelium under sepsis, trauma, and viral infections is vital for promoting the resolution of inflammatory diseases such as acute lung injury (ALI). Transient receptor potential canonical (TRPC) channels mediated Ca2+ entry compromises organ functions and survival from lung injury. Through decoding the domain in TRPC6 responsible for vascular injury, we unveiled the intricate molecular mechanisms underlying vascular regeneration in injured tissue. We found that the substitution of isoleucine111 within the Ist ankyrin domain of TRPC6 for its isomer 111leucine (I111L-TRPC6) altered channel localization at the membrane, blocked TRPC6-mediated Ca2+ entry and cation currents without affecting TRPC6 protein expression. Next, we delivered WT-TRPC6 and I111L-TRPC6 to the endothelial cells (ECs) of TRPC6 knockout mice using liposomes and found that while WT-TRPC6 induced lung vascular inflammatory injury and EC death these responses were blocked in lungs expressing I111L-TRPC6 mutant. Instead, the I111L-TRPC6 mutant promoted lung EC proliferation and prevented vascular injury. These responses were recapitulated in a preclinical mouse model of ALI after injection of engineered TRPC6-blocking peptide, suggesting a novel strategy for regenerating anti-inflammatory vascular niche and preventing ALI therapeutically.

cell biology↗

bioRxiv2024

The Glycolytic Metabolite Methylglyoxal Covalently Inactivates the NLRP3 Inflammasome.

The NLRP3 inflammasome promotes inflammation in disease, yet the full repertoire of mechanisms regulating its activity are not well delineated. Among established regulatory mechanisms, covalent modification of NLRP3 has emerged as a common route for pharmacological inactivation of this protein. Here, we show that inhibition of the glycolytic enzyme PGK1 results in the accumulation of methylglyoxal, a reactive metabolite whose increased levels decrease NLRP3 assembly and inflammatory signaling in cells. We find that methylglyoxal inactivates NLRP3 via a non-enzymatic, covalent crosslinking-based mechanism, promoting inter- and intra-protein MICA posttranslational linkages within NLRP3. This work establishes NLRP3 as capable of sensing a host of electrophilic chemicals, both exogenous small molecules and endogenous reactive metabolites, and suggests a mechanism by which glycolytic flux can moderate the activation status of a central inflammatory signaling pathway.

cell biology↗

bioRxiv2023

Endothelial stiffening induced by CD36-mediated lipid uptake leads to endothelial barrier disruption and contributes to atherosclerotic lesions

BackgroundTo determine the impact of endothelial stiffening induced by CD36-mediated lipid uptake in the disruption of aortic endothelial barrier and development of atherosclerosis in mouse models of obesity and hypercholesterolemia. Approach and ResultsEndothelial-specific inducible downregulation of CD36 results in abrogating the stiffening of aortic endothelium induced by a short-term (6-8 weeks) high-fat Western diet in intact freshly isolated mouse aortas of Cdh5.CreERT2CD36fl/fl mice, as assessed by atomic force microscopy. No effect was observed on the stiffness of aortic vascular wall assessed in the same groups of mice by echocardiography. Prevention of WD-induced endothelial stiffening by the downregulation of endothelial CD36 was associated with a protective effect against endothelial barrier disruption, assessed by morphological analysis of VE-cadherin junctions and penetration of Evans blue dye into the aortic wall. These protective effects were independent of the changes in the serum lipid profiles. Furthermore, endothelial specific downregulation of CD36 in hypercholesterolemic Cdh5.CreERT2CD36fl/flLDLR-/- mice also led to significant decrease in endothelial stiffening after 4-5 months of high fat diet and a significant decrease in the areas of atherosclerotic lesion. In both models, significant endothelial stiffening was observed specifically in male mice, while female mice exhibited less endothelial stiffening and less severe atherosclerosic phenotype, consistent with endothelial stiffening playing an important role in aortic vascular disease in a sex-dependent way. Mechanistically, we show in vitro that CD36-mdiated uptake of long chain saturated fatty acids, particularly palmitic acid, induces endothelial stiffening via activation of RhoA/ROCK pathway. Moreover, palmitic acid-induced endothelial stiffening critically depends on the expression of a RhoA inhibitory protein, Rho-GDI-1. ConclusionsWe conclude that stiffening of the aortic endothelium by CD36-mediated uptake of fatty acids contributes significantly to WD-induced vascular dysfunction and atherosclerosis. We further propose that fatty acids may activate RhoA by inducing its dissociation from Rho-GDI-1.

cell biology↗

bioRxiv2023

Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-beta (TGF-{beta}) is one of the most established drivers of fibrotic processes. TGF-{beta} promotes transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-{beta} robustly upregulates the expression of the calcium-activated chloride channel Anoctamin-1 (ANO1) in human lung fibroblasts (HLF) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle alpha-actin (SMA)-positive myofibroblasts. TGF-{beta}-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1 and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate pro-fibrotic TGF-{beta} signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-{beta} treatment of HLF results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-{beta}-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that (i) ANO1 is a TGF-{beta}-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-{beta}; and (ii) ANO1 mediates TGF-{beta}-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein, but independent of WNK1 kinase activity. NEW & NOTEWORTHYThis study describes a novel mechanism of differentiation of human lung fibroblasts (HLF) to myofibroblasts - the key process in the pathogenesis of pulmonary fibrosis. TGF-{beta} drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive With-No-Lysine (K) kinase (WNK1) to activate pro-fibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation.

cell biology↗