Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: A bioengineered lymphatic vessel model for studying lymphatic endothelial cell-cell junction and barrier function

doi: 10.1111/micc.12730

Figure Lengend Snippet: (A) VE-cadherin (VE-cad) and junctional adhesion molecule A (JAM-A) staining to visualize adherens junctions and tight junctions in LV-on-chip embedded in Collagen 1 or Collagen 1 + fibronectin matrix. (B) A loss of function experiment by using anti-integrin α5 neutralizing antibodies. In the Collagen 1 + fibronectin condition, 50 μg/ml of anti-integrin α5 antibodies or the vehicle were treated, and VE-cad, JAM-A, and F-actin were visualized. (C) Quantification of the relative junction area was performed, illustrating a significantly higher junction area in cells grown in the Collagen 1 + fibronectin condition compared to the cells grown in either the Collagen 1 or Collagen 1 + fibronectin + integrin α5 antibodies conditions. ** p = 0.0041 (Collagen 1 + fibronectin vs. Collagen 1); ** p = 0.0010 (Collagen 1 + fibronectin vs. Collagen 1 + fibronectin + integrin α5 antibodies); One-way ANOVA with Tukey’s HSD tests, n = 6 per group. Data are expressed as mean ± S.E.M. (D) Lymphatic vessel barrier function. 70 kDa dextran were perfused into the vessel lumens and dextran diffusion was observed in real time under microscopy. Superimposed red dashed lines represent the edges of the vessel lumens. (E) Quantification of the permeability of LEC-generated engineered LVs in different ECM and antibody treatment conditions. ** p = 0.0017 (Collagen 1 vs. Collagen 1 + fibronectin); * p = 0.0114 (Collagen 1 + fibronectin vs. Collagen 1 + fibronectin + integrin α5 antibodies); not-significant (ns) p = 0.4524 (Collagen 1 vs. Collagen 1 + fibronectin + integrin α5 antibodies). One-way ANOVA with Tukey’s HSD tests, n = 5 per group. Data are expressed as mean ± S.E.M.

Article Snippet: Primary antibodies detecting VE-cadherin (Santa Cruz, 1:100; or Abcam, 1:100), integrin α5 antibody (clone: SNAKA51) (Sigma, 1:200), JAM-A (Santa Cruz, 1:100), CD31 (DAKO, 1:200), Prox-1 (Abcam, 1:100), and CCL21 (MyBioSource, 1:100) were incubated in blocking buffer overnight at 4°C.

Techniques: Staining, Diffusion-based Assay, Microscopy, Permeability, Generated

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: A bioengineered lymphatic vessel model for studying lymphatic endothelial cell-cell junction and barrier function

doi: 10.1111/micc.12730

Figure Lengend Snippet: (A) Activated integrin α5 was visualized in both ECM composition conditions by using anti-integrin α5 antibody (clone: SNAKA51) that can only detect the activated form of the integrin α5. F-actin was also observed in these conditions. (B) LECs in Collagen 1 were pre-treated with anti-integrin α5 antibodies (clone: SNAKA51) antibodies to activate integrin α5 in LECs. The fixed samples were stained with anti-VE-cadherin antibodies, anti-JAM-A antibodies, and phalloidin to visualize adherens junctions and F-actin. (C) Quantification of the relative junction area was performed, illustrating a significantly higher junction area in integrin α5 activated cells compared to the control LECs. ** p = 0.0020; Two tailed unpaired Student t-test, n = 6 per group. Data are expressed as mean ± S.E.M. (D) Control LECs or LECs with activated integrin α5 were seeded in LV-on-chip and cultured for 3 days on the rocking platform. 70 kDa dextran was introduced to the lymphatic lumens. Dextran diffusion was observed at 0 and 1 minutes under microscopy. Superimposed red dashed lines represent the edges of the vessel lumens. (E) Quantification of the permeability of LEC-generated engineered LVs in collagen 1 with and without integrin α5 activation. ** p = 0.0021. Two tailed unpaired Student t-test, n = 5 per group. Data are expressed as mean ± S.E.M. (F) This table summarizes our findings regarding LEC permeability and integrin α5 activity. LVs grown in Collagen 1 without any activator treatment showed high LEC permeability and low integrin α5 activity. In contrast, LVs grown in either Collagen 1 + Fibronectin or LVs grown in only Collagen 1 with integrin α5 activator pre-treatment both showed low LEC permeability and high integrin α5 activity.

Article Snippet: Primary antibodies detecting VE-cadherin (Santa Cruz, 1:100; or Abcam, 1:100), integrin α5 antibody (clone: SNAKA51) (Sigma, 1:200), JAM-A (Santa Cruz, 1:100), CD31 (DAKO, 1:200), Prox-1 (Abcam, 1:100), and CCL21 (MyBioSource, 1:100) were incubated in blocking buffer overnight at 4°C.

Techniques: Staining, Two Tailed Test, Cell Culture, Diffusion-based Assay, Microscopy, Permeability, Generated, Activation Assay, Activity Assay

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: EZH2 modulates the DNA methylome and controls T cell adhesion through junctional adhesion molecule-A in lupus patients

doi: 10.1002/art.40338

Figure Lengend Snippet: Overexpression of EZH2 in stimulated CD4+ T cells affects JAM-A expression and enzymes and proteins involved in DNA methylation. (A) Overexpression of EZH2 in control CD4+ T cells was confirmed by qPCR (0.016 ± 0.008 vs. 0.525 ± 1.225 for control vs. EZH2 vector, n=14, p=0.0001). (B) EZH2 overexpression led to increase in JAM-A and H3K27me3 expression in CD4+ T cells. Representative figure of n=3. (C) JAM-A was overexpressed in naïve CD4+ T cells from lupus patients compared to control after overnight stimulation (densitometry analysis: Controls 0.0026 ± 0.0002 vs. lupus 0.0038 ± 0.0009; p=0.047, n=4). (D–F) EZH2 overexpression reduced the expression of MeCP2 (n=14, 0.003 ± 0.002 vs. 0.002 ± 0.001 control vs. EZH2 vector, p=0.04) and DNMT3A (n=14, 0.003 ± 0.001 vs. 0.002 ± 0.001 control vs. EZH2 vector, p=0.02), but had no effect on DNMT1 (n=14, 0.006 ± 0.002 vs. 0.006 ± 0.002 control vs. EZH2 vector, p=0.42). Results are expressed as mean +/− SD and p<0.05 was considered significant.

Article Snippet: Stimulated EZH2-overexpressing CD4+ T cells (50,000 cells/well in 100 μl RPMI medium) were labeled with Calcein AM (cell-permeant dye, 5 μM; Invitrogen) in the presence or absence of neutralizing antibodies to JAM-A (50 μg/ml, BioLegend) or isotype control (Mouse IgG1, κ, BioLegend).

Techniques: Over Expression, Expressing, DNA Methylation Assay, Plasmid Preparation

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: EZH2 modulates the DNA methylome and controls T cell adhesion through junctional adhesion molecule-A in lupus patients

doi: 10.1002/art.40338

Figure Lengend Snippet: Effect of EZH2 on lymphocyte adhesion. (A) EZH2-overexpressing stimulated CD4+ T cells showed increased adhesion to HMVECs compared to cells transfected with an empty vector (n=8, control vs. EZH2 vector 1.0 ± 0.0 vs. 1.94 ± 0.74, p=0.02). (B) The increased adhesion of EZH2-overexpressing CD4+ T cells was blocked by neutralizing antibodies to JAM-A (One-way ANOVA p=0.002; n=6, control vector: IgG vs. JAM-A 1.00 ± 0.09 vs. 0.92 ± 0.15, post-hoc p=0.99; EZH2 vector: IgG vs. JAM-A 1.77 ± 0.57 vs. 1.04 ± 0.38, post-hoc p=0.01. Control vector IgG group vs. EZH2 vector IgG group post-hoc p=0.008). (C) Naïve CD4+ T cells from lupus patients stimulated overnight adhere to HMVECs significantly more than cells from healthy controls (n=6, control vs. SLE 1.0 ± 0.0 vs. 2.43 ± 2.31, p=0.002). (D) Blocking JAM-A reduced the ability of lupus CD4+ T cells to adhere to HMVECs (One-way ANOVA p=0.007; n=6, control: IgG vs. JAM-A 1.00 ± 0.09 vs. 1.05 ± 0.38, p=1.00; SLE: IgG vs. JAM-A 2.19 ± 1.08 vs. 1.12 ± 0.31, p=0.03. Control IgG group vs. SLE IgG group p=0.01). (E) Treating stimulated lupus CD4+ T cells with EZH2 inhibitor DZNep reduced EZH2 mRNA levels significantly (n=5, PBS vs. DZNep 0.013 ± 0.005 vs. 0.005 ± 0.002, p=0.02]. (F) At the protein levels, DZNep decreased EZH2, JAM-A, H3K27me3 levels in stimulated lupus CD4+ T cells. (G) Pre-treating stimulated CD4+ T cells from lupus patients with DZNep for 3 days significantly reduced their adhesion to HMVECs compared to cells treated with PBS (n=6, PBS vs. DZNep: 1.0 ± 0.0 vs. 0.42 ± 0.21, p=0.002). (H) Representative pictures of the adhesion assay: stimulated CD4+ T cells (in green) isolated from a lupus patients showed increased ability to adhere to HMVECs (in red) compared to a healthy control, and DZNep significantly reduced adhesion of CD4+ T cells from the same lupus patient. (I) Pre-treating stimulated CD4+ T cells isolated from lupus patients with GSK126 for 3 days led to significant reduction in cell adhesion compared to cells treated with DMSO (n=6, DMSO vs. GSK126: 1.0 ± 0.0 vs. 0.47 ± 0.32, p=0.048). Results are expressed as mean +/− SD and p<0.05 was considered significant.

Article Snippet: Stimulated EZH2-overexpressing CD4+ T cells (50,000 cells/well in 100 μl RPMI medium) were labeled with Calcein AM (cell-permeant dye, 5 μM; Invitrogen) in the presence or absence of neutralizing antibodies to JAM-A (50 μg/ml, BioLegend) or isotype control (Mouse IgG1, κ, BioLegend).

Techniques: Transfection, Plasmid Preparation, Blocking Assay, Cell Adhesion Assay, Isolation