Journal: International Journal of Molecular Sciences

Article Title: Dysregulated Retinoic Acid Signaling in the Pathogenesis of Pseudoexfoliation Syndrome

doi: 10.3390/ijms23115977

Figure Lengend Snippet: Expression, activity, and regulation of nuclear receptors RARα and RXRα in ocular tissues of PEX and control eyes. ( A ) Western blot analysis of RARα expression in the ciliary body and iris tissue from PEX patients compared to normal donors (n = 3). Protein expression is normalized to the house-keeping gene β-actin and is expressed relative to expression in controls (set to 1). ( B ) Electrophoretic mobility shift assay using oligonucleotides containing retinoic acid response element (RARE) consensus binding sequences and nuclear extracts (4 µg) from human Tenon’s capsule fibroblasts derived from PEX and control eyes. Unlabeled oligonucleotides were used as competitors. Quantitative analysis of the protein-DNA complexes shows mean values ± SD (n = 5) relative to the control set to 100%. ( C ) Real-time PCR analysis of RARA and RXRA regulation by TGF-β1 without and with TGF-β1 inhibitor SB 431542 in cultured human Tenon’s capsule fibroblasts (hTCF, n=3) and human trabecular meshwork cells (hTMC, n = 3) (* p < 0.05, ** p < 0.01, *** p < 0.001; unpaired t -test).

Article Snippet: Primary human trabecular meshwork cells (hTMC) from four different donors (fetal to 24 years; 2 male, 2 female) were obtained from Provitro (Berlin, Germany) and grown in DMEM supplemented with 10% FBS and 1% PSA.

Techniques: Expressing, Activity Assay, Control, Western Blot, Electrophoretic Mobility Shift Assay, Binding Assay, Derivative Assay, Real-time Polymerase Chain Reaction, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Dysregulated Retinoic Acid Signaling in the Pathogenesis of Pseudoexfoliation Syndrome

doi: 10.3390/ijms23115977

Figure Lengend Snippet: Effects of RARA (retinoic acid receptor alpha) and RXRA (retinoid X receptor alpha) knockdown on matrix synthesis in PEX-relevant cell types. ( A ) Quantitative real-time PCR analysis of RARA , RXRA , LOXL1 (lysyl oxidase-like 1) , ELN (elastin), FBN1 (fibrillin-1), LTBP1 (latent transforming growth factor beta binding protein 1), LTBP2 , FBLN4 (fibulin-4), FBLN5 , MFAP2 (microfibril associated protein 2), TGFB1 (transforming growth factor beta 1), TGFBR2 (transforming growth factor beta receptor 2), CTGF (connective tissue growth factor), MMP2 (matrix metalloproteinase 2), TIMP2 (tissue inhibitor of metalloproteinases 2), COL1A1 (collagen type 1 alpha 1), COL3A1 , COL4A1 , and FN1 (fibronectin-1) mRNA in human Tenon’s capsule fibroblasts (hTCF) (n = 4) and trabecular meshwork cells (hTMC) (n = 4) transfected with RARA - or RXRA -specific siRNA or scrambled control siRNA. Expression levels were normalized to GAPDH and HPRT1 and expressed as means ± SD relative to controls set to 1 (red dashed line). ( B , C ) Western blot analysis of RARα ( B ) as well as LOXL1, elastin, and fibrillin-1 ( C ) protein after RARA siRNA-mediated gene silencing in hTCF and hTMC (n = 4) compared to cells transfected with scrambled non-targeting siRNA (Ctrl). Protein expression is normalized to the house-keeping gene β-actin and is expressed relative to expression in controls (set to 1); (* p < 0.05, ** p < 0.01, *** p < 0.001; unpaired t -test).

Article Snippet: Primary human trabecular meshwork cells (hTMC) from four different donors (fetal to 24 years; 2 male, 2 female) were obtained from Provitro (Berlin, Germany) and grown in DMEM supplemented with 10% FBS and 1% PSA.

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Binding Assay, Transfection, Control, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Dysregulated Retinoic Acid Signaling in the Pathogenesis of Pseudoexfoliation Syndrome

doi: 10.3390/ijms23115977

Figure Lengend Snippet: Effects of the RAR antagonist BMS 493 on matrix synthesis in PEX-relevant cell types. Quantitative real-time PCR analysis of RARA (retinoic acid receptor alpha), LOXL1 (lysyl oxidase-like 1) , ELN (elastin), FBN1 (fibrillin-1), LTBP1 (latent transforming growth factor beta binding protein 1), LTBP2 , FBLN4 (fibulin-4), FBLN5 , MFAP2 (microfibril associated protein 2), TGFB1 (transforming growth factor beta 1), TGFBR2 (transforming growth factor beta receptor 2), CTGF (connective tissue growth factor), MMP2 (matrix metalloproteinase 2), TIMP2 (tissue inhibitor of metalloproteinases 2), COL1A1 (collagen type 1 alpha 1), COL3A1 , COL4A1 , and FN1 (fibronectin-1) mRNA in human Tenon’s capsule fibroblasts (hTCF) (n = 4) and trabecular meshwork cells (hTMC) (n = 4) treated with the pan-RAR antagonist BMS 493 at a concentration of 5 µM. Expression levels were normalized to GAPDH and HPRT1 and expressed as means ± SD relative to untreated controls set to 1 (red dashed line); (* p < 0.05, ** p < 0.01, *** p < 0.001; unpaired t -test).

Article Snippet: Primary human trabecular meshwork cells (hTMC) from four different donors (fetal to 24 years; 2 male, 2 female) were obtained from Provitro (Berlin, Germany) and grown in DMEM supplemented with 10% FBS and 1% PSA.

Techniques: Real-time Polymerase Chain Reaction, Binding Assay, Concentration Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Dysregulated Retinoic Acid Signaling in the Pathogenesis of Pseudoexfoliation Syndrome

doi: 10.3390/ijms23115977

Figure Lengend Snippet: Effects of retinoic acid signaling activation on matrix synthesis in PEX-relevant cell types. ( A ) Quantitative real-time PCR analysis of RARA (retinoic acid receptor alpha), LOXL1 (lysyl oxidase-like 1) , ELN (elastin), FBN1 (fibrillin-1), LTBP1 (latent transforming growth factor beta binding protein 1), LTBP2 , FBLN4 (fibulin-4), FBLN5 , MFAP2 (microfibril associated protein 2), TGFB1 (transforming growth factor beta 1), TGFBR2 (transforming growth factor beta receptor 2), CTGF (connective tissue growth factor), MMP2 (matrix metalloproteinase 2), TIMP2 (tissue inhibitor of metalloproteinases 2), COL1A1 (collagen type 1 alpha 1), COL3A1 , COL4A1 , and FN1 (fibronectin-1) mRNA in human Tenon’s capsule fibroblasts (hTCF) (n = 4) and trabecular meshwork cells (hTMC) (n = 4) treated with 2 µM all- trans retinoic acid (ATRA). ( B ) Relative mRNA expression levels of LOXL1 , ELN , FBN1 , and LTBP1 in human non-pigmented ciliary epithelial (NPE), iris pigment epithelial (IPE) and lens epithelial (LEP) cells treated with 2 µM ATRA. ( C ) Relative mRNA expression levels of LOXL1 , ELN , FBN1 , and LTBP1 in hTCF treated with 2 µM ATRA or 10 µM of synthetic agonists selective for RARα (AM 80), RARβ (AC 261066), RARγ (CD 1530), RXRα (CD 3254) and pan-RXR (LGD 1069). Expression levels were normalized to GAPDH and HPRT1 and expressed as means ± SD relative to untreated controls set to 1; (* p < 0.05, ** p < 0.01, *** p < 0.001; unpaired t -test).

Article Snippet: Primary human trabecular meshwork cells (hTMC) from four different donors (fetal to 24 years; 2 male, 2 female) were obtained from Provitro (Berlin, Germany) and grown in DMEM supplemented with 10% FBS and 1% PSA.

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Binding Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Dysregulated Retinoic Acid Signaling in the Pathogenesis of Pseudoexfoliation Syndrome

doi: 10.3390/ijms23115977

Figure Lengend Snippet: Effects of retinoic acid signaling activation and inhibition on TGF-β1-induced matrix synthesis in PEX-relevant cell types. ( A ) Quantitative real-time PCR analysis of LOXL1 (lysyl oxidase-like 1) , ELN (elastin), FBN1 (fibrillin-1), and LTBP1 (latent transforming growth factor beta binding protein 1) mRNA in human Tenon’s capsule fibroblasts (hTCF) (n = 4) and trabecular meshwork cells (hTMC) (n = 4) treated with 2 µM all- trans retinoic acid (ATRA), 5 µM BMS 493 (BMS, pan-RAR antagonist), 5 ng/mL transforming growth factor-ß1 (TGF-β1), or 5 ng/mL TGF-β1 together with 2 µM ATRA. ( B ) Western blot analysis of LOXL1, elastin, and fibrillin-1 protein expression as well as total and phosphorylated Smad2 in hTCF and hTMC without stimulation (control, Ctrl) or in response to 2 µM ATRA, 5 µM BMS 493, 5 ng/mL TGF-β1, or 5 ng/mL TGF-β1 together with 2 µM ATRA (n = 4). Expression levels were normalized to GAPDH and HPRT1 (PCR) and β-actin (Western blot), respectively, and expressed as means ± SD relative to untreated controls set to 1 (* p < 0.05, ** p < 0.01, *** p < 0.001; unpaired t -test).

Article Snippet: Primary human trabecular meshwork cells (hTMC) from four different donors (fetal to 24 years; 2 male, 2 female) were obtained from Provitro (Berlin, Germany) and grown in DMEM supplemented with 10% FBS and 1% PSA.

Techniques: Activation Assay, Inhibition, Real-time Polymerase Chain Reaction, Binding Assay, Western Blot, Expressing, Control