Galectin-1 Modulates Cell Adhesions, Caveolae, and Vascular Permeability in Kidney Endothelial Cells -- Insights from Proteomics, Phosphoproteomics, and Functional Studies

Abstract

Microvascular inflammation and endothelial injury, triggered by interferon-gamma (IFN{gamma}), are hallmarks of antibody-mediated rejection (ABMR), the leading cause of premature kidney allograft loss. Glomerular extracellular matrix (ECM) remodeling and endothelial caveolae formation are important aspects of chronic ABMR. We found galectin-1, an immunomodulatory protein that interacts with the ECM, to be increased in the glomeruli of patients with ABMR, while its gene (LGALS1) expression was decreased by IFN{gamma} stimulation in glomerular endothelial cells. Mechanisms underlying endothelial dysfunction in ABMR, its links to ECM remodeling, and the role of immunomodulatory proteins such as galectin-1 remain incompletely understood. Here we studied the effects of galectin-1 modulation in glomerular microvascular endothelial cells (GMECs) in vitro. We demonstrated that galectin-1 was mainly expressed by glomerular endothelial cells in ABMR kidneys. To model key aspects of endothelial injury in ABMR, we knocked down LGALS1 in GMECs, followed by stimulation with IFN{gamma} and performed label-free quantitative proteomic and phosphoproteomic profiling of GMECs. Proteomic analysis identified 5446 proteins (FDR<0.01), of which 236, 827, and 267 were differentially expressed in response to LGALS1 knockdown, IFN{gamma} treatment, and their interaction, respectively (FDR<0.05). Both LGALS1 knockdown and the interaction between treatments significantly altered expression of adhesion proteins (FDR<0.01), particularly integrin subunit {beta}5, which was validated. Phosphoproteomic profiling identified 2727 phosphopeptides (FDR<0.01), with 28 that were differentially expressed across LGALS1 knockdown, IFN{gamma} treatment, and their interaction (P<0.01). Phosphorylation of CAVN1 and co-localization with its partner CAV1, critical for caveolar formation, were decreased in GMECs upon LGALS1 knockdown, IFN{gamma} stimulation, or both. In a microfluidic model of the glomerular microvasculature, addition of recombinant galectin-1 increased both endothelial permeability and secretion of proinflammatory cytokines, in LGALS1-silenced GMECs. Thus, endothelial signaling pathways regulated by inflammatory cues and galectin-1 contribute to endothelial injury and caveolae formation, highlighting galectin-1 as a potential therapeutic target in ABMR. SynopsisGalectin-1 is expressed by kidney glomerular endothelium. This study reveals that modifying galectin-1 in endothelial cells, in the presence of IFN{gamma} perturbs cytoskeletal, adhesion and caveolar proteins resulting in altered endothelial permeability. O_LILGALS1 knockdown increased ECM proteins and decreased interferon-induced proteins. C_LIO_LILGALS1 knockdown and IFN{gamma} treatment perturbed cell adhesion proteins such as ITGB5. C_LIO_LICAVN1 phosphorylation and colocalization with CAV1 decreased upon LGALS1 knockdown. C_LIO_LIExtracellular galectin-1 increased microvascular permeability in response to IFN{gamma}. C_LI