Journal: The Journal of Experimental Medicine

Article Title: Endothelial LRP1 protects against neurodegeneration by blocking cyclophilin A

doi: 10.1084/jem.20202207

Figure Lengend Snippet: Activation of CypA-MMP9 pathway after Lrp1 endothelial loss. (A–D) ZO-1 (A, purple), occludin (B, purple), and claudin 5 (C, purple) colocalization with endothelial lectin (green) in the cortex of 2-mo-old Lrp1 lox/lox ; Tie2-Cre mice and Lrp1 lox/lox controls, and quantification of ZO-1, occludin, and claudin 5 length on lectin + endothelial profiles in these mice (D). Scale bar = 25 µm. Boxes in A–C are sites taken for higher magnification insets shown below single and merged images, scale bar = 10 µm. Mean ± SEM, n = 5 mice/group. (E and F) Immunoblotting for ZO-1, occludin, and claudin 5 in brain capillaries (E) and their relative abundance compared with β-actin (F) in Lrp1 lox/lox ; Tie2-Cre and Lrp1 lox/lox mice. Mean ± SEM, n = 3 mice/group. β-actin, loading control. (G) CypA, MMP9, and lectin + endothelium (left: purple, CypA and lectin; white, MMP9 and lectin); CypA, lectin + endothelium, and CD13 + pericytes (middle: purple, CypA and lectin), and MMP9, lectin + endothelium, and CD13 + pericytes (right: purple, MMP9 and lectin) in brain capillaries from Lrp1 lox/lox ; Tie2-Cre and Lrp1 lox/lox mice. Scale bar = 10 µm. (H) Ppia and Mmp9 mRNA relative abundance normalized to Gapdh (housekeeping gene) in brain endothelial cells and pericytes from Lrp1 lox/lox ; Tie2-Cre and Lrp1 lox/lox mice. Mean ± SEM, n = 5 isolates/group. (I) Gelatinase activity in brain endothelial cell medium from Lrp1 lox/lox and Lrp1 lox/lox ; Tie2-Cre mice with or without si Mmp9 or si Mmp2 . AU, arbitrary units. Mean ± SEM, n = 4 isolates/group. (J and K) Inhibition of Mmp9 mRNA (J) and activated MMP9 (K) by CypA silencing (si. Ppia ), the CypA inhibitor cyclosporine A (CsA; 42 nM), and the NF-κB inhibitor PDTC (20 µM) in brain endothelial cells from Lrp1 lox/lox ; Tie2-Cre mice. (L–N) Inhibition of Ppia mRNA (L), Mmp9 mRNA (M), and active MMP9 (N) in brain endothelial cells from Lrp1 lox/lox ; Tie2-Cre mice after adenoviral LRP1 (Ad.m LRP1 ) reexpression in the presence of astrocyte-derived murine apoE (40 nM). Ad GFP , control. Mean ± SEM, n = 3 isolates/group. All mice were 2 mo old. In D and F, significance was determined by Student’s t test. In H–N, significance was determined by one-way ANOVA followed by Bonferroni post hoc test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Membranes were blocked with 5% milk, incubated with the following primary antibodies: rabbit anti–ZO-1 (catalog number 40–2200, 1:1,000; Invitrogen), rabbit anti-occludin (catalog number16453, 1:500; BD Biosciences), rabbit anti–claudin-5 (catalog number ab15106, 1:1,000; Abcam), rabbit anti–GLUT-1 (CBL242, 1:500; Millipore), and rabbit anti-human MFSD2A (catalog number A18288, 1:1,000; ABclonal).

Techniques: Activation Assay, Western Blot, Activity Assay, Inhibition, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: Endothelial LRP1 protects against neurodegeneration by blocking cyclophilin A

doi: 10.1084/jem.20202207

Figure Lengend Snippet: Cyclophilin A inhibitor Debio-025 reverses vascular phenotype and protects from neurodegeneration in Lrp1 lox/lox ; Tie2-Cre mice. (A and B) MMP9 immunoreactivity in cortical endothelium (A) and quantification (B) in Lrp1 lox/lox ; Tie2-Cre mice after vehicle or Debio-025 treatment. Scale bar = 10 µm. Mean ± SEM, n = 10 mice/group. (C–E) IgG and fibrin deposits in the cortex (C) and quantification of their deposits in the cortex (Ctx) and hippocampus (Hp; D and E) in Lrp1 lox/lox and Lrp1 lox/lox ; Tie2-Cre mice after vehicle or Debio-025 treatment. Scale bar = 20 µm. Mean ± SEM, n = 14 mice/group. (F–I) ZO-1 (F, purple), occludin (G, purple), and claudin 5 (H, purple) colocalization with endothelial lectin (green) in the cortex of 4-mo-old Lrp1 lox/lox ; Tie2-Cre mice treated with vehicle or Debio-25, and quantification of ZO-1, occludin, and claudin 5 length on lectin + endothelial capillary profiles in these mice (I). Scale bar = 25 µm. Boxes in F–H are sites taken for higher magnification insets shown below single and merged images. Scale bar = 10 µm. White (merged), colocalization of ZO-1, occludin, or collagen with lectin. Mean ± SEM, n = 5 mice/group. (J and K) BBB K trans maps (J) and K trans values in the cortex and hippocampus (K) in Lrp1 lox/lox ; Tie2-Cre mice after vehicle or Debio-025 treatment, compared with Lrp1 lox/lox mice treated with vehicle. Scale bar = 0.5 mm. Mean ± SEM, n = 5–6 mice/group. (L–N) NeuN + neurons and SMI312 + neurites (L) and quantification of NeuN + neurons and SMI312 + neurites in the cortex and hippocampus (M and N) in 4-mo-old Lrp1 lox/lox and Lrp1 lox/lox ; Tie2-Cre mice after vehicle or Debio-025 treatment. Scale bar = 50 µm. Mean ± SEM, n = 14 mice/group. (O–R) Burrowing (O), nest construction (P), novel object location (Q), and recognition (R) in Lrp1 lox/lox and Lrp1 lox/lox ; Tie2-Cre mice after vehicle or Debio-025 treatment. Mean ± SEM, n = 14 mice/group. All mice were 4 mo old. Debio-025 (10 mg/kg/d) or vehicle were given for 30 d starting at 3 mo of age. In B, significance was determined by Student’s t test. In D, E, I, K, and M–R, significance was determined by one-way ANOVA followed by Bonferroni post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Membranes were blocked with 5% milk, incubated with the following primary antibodies: rabbit anti–ZO-1 (catalog number 40–2200, 1:1,000; Invitrogen), rabbit anti-occludin (catalog number16453, 1:500; BD Biosciences), rabbit anti–claudin-5 (catalog number ab15106, 1:1,000; Abcam), rabbit anti–GLUT-1 (CBL242, 1:500; Millipore), and rabbit anti-human MFSD2A (catalog number A18288, 1:1,000; ABclonal).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Usnea improves high-fat diet- and vitamin D3-induced atherosclerosis in rats by remodeling intestinal flora homeostasis

doi: 10.3389/fphar.2022.1064872

Figure Lengend Snippet: UEE affected the concentrations of occludin and ZO-1. (A) UEE increased the expression of ZO-1; (B) UEE increased the concentration of occludin; and (C) potential correlation analysis between the intestinal flora associated with AS and pharmacokinetic markers. Rectangles represent the intestinal flora related to AS; circles denote pharmacokinetic markers; red indicates a positive association, whereas blue indicates a negative correlation. Results are expressed as the mean ± SD ( n = 3 per group). * p < 0.05, ** p < 0.01.

Article Snippet: Anti-CYP7A1 rabbit monoclonal antibody (ID: 510615), anti-zonula occludens-1 (ZO-1) rabbit monoclonal antibody (ID: AF5145), anti-occludin rabbit monoclonal antibody (ID: 502601), anti-FMO3 rabbit monoclonal antibody (ID: 389347), anti-β-actin rat antibody (ID: DD1027), goat anti-rabbit IgG (H&L) (ID: JJ0903), and goat anti-rat secondary antibody (ID: II1120) were provided by Chengdu Zen Bioscience Co., Ltd. (Chengdu, China).

Techniques: Expressing, Concentration Assay