Journal: bioRxiv

Article Title: A recombinant norrin-derived growth factor retains the complete multifunctionality of human norrin

doi: 10.1101/2025.10.15.682582

Figure Lengend Snippet: (A) Noregen FZD4 receptor binding activity compared to Norrin, EC 50 19 ng/mL & 34 ng/mL respectively. (B) Noregen dose response curve for TCF/LEF reporter activation in HEK-293 cells (EC 50 11 ng/mL). (C) Noregen induced Axin-2 gene expression in HRMECs. Values are means +/-SD; *P<0.05, **P<0.01, ***P<0.001 & ****P<0.0001.

Article Snippet: The cells were weaned to media containing no hydrocortisone hemisuccinate prior to stimulation with Noregen or rh norrin (R&D Systems, Minneapolis MN;3014-NR).

Techniques: Binding Assay, Activity Assay, Activation Assay, Gene Expression

Journal: bioRxiv

Article Title: A recombinant norrin-derived growth factor retains the complete multifunctionality of human norrin

doi: 10.1101/2025.10.15.682582

Figure Lengend Snippet: (A) Noregen significantly increases HRMEC proliferation as seen by a drop in capacitance when treated at 20 and 500 ng/mL. (B) Norrin increases proliferation to a similar extent as Noregen at the higher 500 ng/mL treatment level, but to a lesser extent at 20 ng/mL. (C) Comparison of Noregen and norrin treatments at 20 ng/mL. An earlier drop in capacitance indicates faster growth in Noregen treated cells.

Article Snippet: The cells were weaned to media containing no hydrocortisone hemisuccinate prior to stimulation with Noregen or rh norrin (R&D Systems, Minneapolis MN;3014-NR).

Techniques: Comparison

Journal: bioRxiv

Article Title: A recombinant norrin-derived growth factor retains the complete multifunctionality of human norrin

doi: 10.1101/2025.10.15.682582

Figure Lengend Snippet: (A) Reduction in normalized avascular area [AVA to Total Area ratio] in Noregen & norrin injected eyes. The [AVA to Total Area ratio] in injected eyes was normalized to fellow un-injected eyes. Noregen levels were 2, 20 or 200 ng. Values are means +/-SEM; *P<0.05, **P<0.01. (B) Dose dependent reduction in normalized avascular area seen when graphing in comparison to averaged Vehicle from combined cohorts. (C) Example retinal flatmount images with AVA highlighted in white. (D) Confocal images of superficial (S), intermediate (I) & deep (D) vascular beds in Noregen(200 ng/eye) and vehicle injected OIR eyes.

Article Snippet: The cells were weaned to media containing no hydrocortisone hemisuccinate prior to stimulation with Noregen or rh norrin (R&D Systems, Minneapolis MN;3014-NR).

Techniques: Injection, Comparison

Journal: bioRxiv

Article Title: A recombinant norrin-derived growth factor retains the complete multifunctionality of human norrin

doi: 10.1101/2025.10.15.682582

Figure Lengend Snippet: (A) Diagram illustrating TGF-ß1 and BMP-2 pathways. Norrin inhibits signaling via SMAD7 induction, thereby inhibiting pathway activation. (B) Noregen reduction in basal level and TGF-ß1 induced SMAD reporter activation. Values are means +/-SD; *P<0.05, **P<0.01, ***P<0.001. (C) ZO-1 immunostaining of HRMECs treated with TGF-ß1 or BMP-2 alone (translocation of ZO-1 to cytosol) or in combination with Noregen (restoration of ZO-1 at plasma membrane).

Article Snippet: The cells were weaned to media containing no hydrocortisone hemisuccinate prior to stimulation with Noregen or rh norrin (R&D Systems, Minneapolis MN;3014-NR).

Techniques: Activation Assay, Immunostaining, Translocation Assay, Clinical Proteomics, Membrane

Journal: Science Advances

Article Title: Wnt signaling activates MFSD2A to suppress vascular endothelial transcytosis and maintain blood-retinal barrier

doi: 10.1126/sciadv.aba7457

Figure Lengend Snippet: ( A and B ) Mfsd2a mRNA levels were increased in HRMEC treated with lithium chloride (LiCl), using control sodium chloride (NaCl). ( C and D ) Mfsd2a mRNA and protein levels were induced by Wnt3a-conditioned medium (Wnt3a-CM) and suppressed by the Wnt inhibitor XAV939. n-p-β-catenin, nonphosphorylated β-catenin. ( E ) Three promoter regions upstream of MFSD2A gene were identified containing putative Wnt-responsive TCF-binding motifs (TTCAAAG): P1 (−2841 to −2211 bp), P2 (−1900 to −1099 bp), and P3 (−1145 to −168 bp), followed by cloning and ligation with a luciferase reporter, and transfected with an active β-catenin plasmid in HEK293 cells. Luciferase activity was measured. ( F ) Mutation of TCF-binding site #1 (Mut-P1) (mutated to CCTGGGT) partially abolished the Wnt/β-catenin–responsive luciferase reporter activity compared with native P1. ( G ) Scheme of in vitro HRP transcytosis assay in ECs. ( H and I ) Transferred HRP in the lower chambers was measured to indicate the transcytosis levels through the EC monolayer. Activation of Wnt signaling activation was achieved by treatment with Wnt3a-CM (H) or human recombinant Norrin (I), and inhibition by XAV939 treatment. ( J and K ) siRNA targeting MFSD2A (si- M2A ) suppressed MFSD2A mRNA (J) and protein levels (K) in HRMEC compared with si-control (si-Ctrl). ( L ) HRP-based in vitro transcytosis was used to detect transcytosis levels in HRMEC with Wnt3a-CM in combination with si- M2A or si-control (si-Ctrl) treatment (K). Data are expressed as individual values plus means ± SD. n = 3 to 4 per group. Statistical differences between groups were analyzed using a one-way analysis of variance (ANOVA) statistical test with Dunnett’s multiple comparisons tests or two-tailed unpaired t tests. * P < 0.05, ** P < 0.01.

Article Snippet: Wnt3a-conditioned medium/control-conditioned medium (50% mixed with basic EBM2 medium), human recombinant Norrin (R&D Systems, Minneapolis, MN; 3014-NR), and XAV939 (10 μM, Selleckchem, Houston, TX; S1180) were added for 24-hour treatment.

Techniques: Control, Binding Assay, Cloning, Ligation, Luciferase, Transfection, Plasmid Preparation, Activity Assay, Mutagenesis, In Vitro, Activation Assay, Recombinant, Inhibition, Two Tailed Test

Journal: Science Advances

Article Title: Wnt signaling activates MFSD2A to suppress vascular endothelial transcytosis and maintain blood-retinal barrier

doi: 10.1126/sciadv.aba7457

Figure Lengend Snippet: ( A and B ) Long-chain polyunsaturated fatty acids in the retina or brain were measured. Both DHA and EPA were down-regulated in the retinas of Lrp5 −/− and Ndp y/− mice compared with their respective controls (A), but remained unchanged in the brains of the same mice (B). Data are expressed as individual values plus means ± SD. n = 4 per group. Statistical differences between groups were analyzed using two-tailed unpaired t tests. * P < 0.05, ** P < 0.01. n.s., not significant. ( C ) Scheme illustration on the role of Wnt signaling in controlling inner BRB integrity by limiting MFSD2A-mediated EC caveolar transcytosis. Canonical Wnt signaling is activated by binding of the ligand (Norrin or Wnts) to the receptor complex containing Frizzed4 (FZD4) and co-receptors (LRP5 or LRP6), leading to the prevention of β-catenin ubiquitination and degradation. Stabilized β-catenin then translocates to the nucleus and works with TCF to bind the TCF-responsive motif in the promoter region of MFSD2A, directly regulating its gene transcription. MFSD2A protein is located on cellular plasma membrane to suppress CAV-1 protein levels and block the formation of CAV-1–positive caveolae, thereby limiting EC transcytosis and maintaining inner BRB integrity.

Article Snippet: Wnt3a-conditioned medium/control-conditioned medium (50% mixed with basic EBM2 medium), human recombinant Norrin (R&D Systems, Minneapolis, MN; 3014-NR), and XAV939 (10 μM, Selleckchem, Houston, TX; S1180) were added for 24-hour treatment.

Techniques: Two Tailed Test, Binding Assay, Ubiquitin Proteomics, Clinical Proteomics, Membrane, Blocking Assay