Galectin‐1 promotes choroidal neovascularization and subretinal fibrosis mediatedviaepithelialmesenchymal transition

Abstract

VEGFA and TGF‐β are known major angiogenic and fibrogenic factors. Galectin‐1, encoded by lectin, galactoside‐binding, soluble (LGALS)1, has attracted growing attention for its facilitatory role in angiogenesis and fibrosis through its modification of VEGFA and TGF‐β receptor signaling pathways. We reveal galectin‐1 involvement in the mouse model of laser‐induced choroidal neovascularization (CNV) and subretinal fibrosis, both of which represent the pathogenesis of age‐related macular degeneration (AMD). Neither deletion nor overexpression of Lgals1 affected physiologic retinal development or visual function. Galectin‐1/Lgals1 was upregulated by CNV induction, whereas deletion of Lgals1 suppressed CNV together with downstream molecules of VEGF receptor (VEGFR)2. Loss of Lgals1 also attenuated subretinal fibrosis, expression of epithelial‐mesenchymal transition (EMT) markers including Snai1, and phosphorylation of SMAD family member 2. Supporting these in vivo findings, silencing of LGALS1 in human retinal pigment epithelial (RPE) cells inhibited TGF‐β1‐induced EMT‐related molecules and cell motilities. Conversely, overexpression of Lgals1 enhanced CNV and subretinal fibrosis. Specimens from patients with AMD demonstrated colocalization of galectin‐1 with VEGFR2 in neovascular endothelial cells and with phosphorylated SMAD2 in RPE cells. These results suggested a biologic significance of galectin‐1 as a key promotor for both angiogenesis and fibrosis in eyes with AMD.—Wu, D., Kanda, A., Liu, Y., Kase, S., Noda, K., Ishida, S. Galectin‐1 promotes choroidal neovascularization and subretinal fibrosis mediated via epithelialmesenchymal transition. FASEB J. 33, 2498–2513 (2019). http://www.fasebj.org

Glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced Müller glial galectin‐1 expression via HIF‐1α destabilization

“…Total RNA isolation was performed from cells using SuperPrep II Cell Lysis & RT Kit for qPCR (TOYOBO) and from tissue samples using TRI reagent (Molecular research centre), as previously described . The following primers were used: human LGALS1 (forward 5′‐CGC TAA GAG CTT CGT GCT GAA C‐3′, reverse 5′‐CAC ACC TCT GCA ACA CTT CCA G‐3′), human HIF1A (HIF‐1α; forward 5′‐TGC TCA TCA GTT GCC ACT TC‐3′, reverse 5′‐TCC TCA CAC GCA AAT AGC TG‐3′), human VEGFA (forward 5′‐CAG ATT ATG CGG ATC AAA CCT CA‐3′; reverse 5′‐CAA GGC CCA CAG GGA TTT TC‐3′), human TSC22D3 (forward 5′‐ATC TGC AAC CGC AAC ATC GAC C‐3′, reverse 5′‐GCA TAC ATC AGA TGA TTC TTC ACC‐3′), human DUSP1 (forward 5′‐CTG CCT TGA TCA ACG TCT CA‐3′, reverse 5′‐CTG TGC CTT GTG GTT GTC CT‐3′), human ACTB (β‐actin; forward 5′‐CTG GAA CGG TGA AGG TGA CA‐3′, reverse 5′‐ AAG GGA CTT CCT GTA ACA ATG CA‐3′), mouse Lgals1 (forward 5′‐GTC TCA GGA ATC TCT TCG CTT C‐3′, reverse 5′‐TCC CCG AAC TTT GAG ACA TTC‐3′, probe 5′‐TTC AAT CAT GGC CTG TGG TCT GGT‐3′), mouse Tsc22d3 (forward 5′‐TCA ATG AGG GCA TCT GCA ACC G‐3′, reverse 5′‐CAT CAG GTG GTT CTT CAC GAG G‐3′), and mouse Actb (forward 5′‐CAT CCG TAA AGA CCT CTA TGC CAA C‐3′, reverse 5′‐ATG GAG CCA CCG ATC CAC A‐3′).…”

“…Immunofluorescence analyses were performed as described previously . Serial sections were incubated with the following primary antibodies: goat anti‐galectin‐1 (1:100, R&D systems), rabbit anti‐glial fibrillary acidic protein (1:200, GFAP; Leica), mouse anti‐HIF‐1α (1:100, Novus Biologicals), rabbit anti‐HIF‐1α (1:100, Cell signaling technology) and mouse anti‐TSC22D3 (1:50, Santa Cruz Biotechnology) antibodies.…”

Glucocorticoid receptor inhibits Müller glial galectin‐1 expression via DUSP1‐dependent and ‐independent deactivation of AP‐1 signalling

“…Immunofluorescence analyses were performed as described previously . Serial sections were incubated with the following primary antibodies: mouse anti‐galectin‐1 (1:50, Santa Cruz Biotechnology), mouse and rabbit anti‐glial fibrillary acidic protein (GFAP) (1:200, Leica), rabbit anti‐phosphorylated ATF2 (1:100), rabbit anti‐phosphorylated c‐Fos (1:100), and rabbit anti‐phosphorylated c‐Jun (1:100, Cell Signaling Technology), rabbit anti‐DUSP1 (1:100, Millipore) and rabbit anti‐GR (1:100, Thermo Fisher Scientific) antibodies.…”

“…Immunofluorescence analyses were performed as described previously. 4,5,15 Serial sections were incubated with the following primary antibodies: mouse anti-galectin-1 (1:50, Santa Cruz Biotechnology), mouse and rabbit anti-glial fibrillary acidic protein (GFAP) (1:200, Leica), rabbit anti-phosphorylated ATF2 (1:100), rabbit anti-phospho-…”

“…Total RNA isolation and reverse transcription were performed from cells using SuperPrep Cell Lysis & RT Kit for qPCR (TOYOBO) and from tissues using PureLink RNA Mini Kit (Thermo Fisher Scientifc) and GoScrip Reverse Transcriptase (Promega), as previously described. 4,5,15 All primers are listed in Table S1. Real-time qPCR was performed using the GoTaq qPCR Master Mix (Promega) and…”