EMBO/soda-nel huggingface dataset:
The retinal pigment epithelium as a gateway for monocyte trafficking into the eye (2016)
Figure 2

Figure 2. ONC elicits an early and transient elevation in the expression of leukocyte trafficking molecules in the RPE.

(A, C-E) Changes in mRNA transcript levels of an array of adhesion molecules (A), chemokines (C, D) and cytokine receptors (E) in retinas from ONC eyes compared with contralateral noninjured retinas, 8 hours, and 1, 3 and 7 days after ONC. n = 6-13 per group. Data shown are representative of three independent experiments. (B) Representative micrographs showing the expression of ICAM-1 and VCAM-1 (red) by the RPE (green) 12 hours after ONC. Scale bar, 20 µm. Bar graphs throughout the figure show mean ± SE of each group. Asterisks above bars indicate significant differences compared with noninjured tissue; significant differences between different time points are indicated by asterisks between bars. *, P < 0.05; **, P < 0.01; *** P < 0.001. noninj – noninjured control; ONC – optic nerve crush.
The retinal pigment epithelium as a gateway for monocyte trafficking into the eye (2016)
Figure 3

Figure 3. Monocyte/macrophage dynamics in the RPE and retina after optic nerve crush.

(A, B) Quantitative flow cytometric analysis of myeloid cells in the RPE complex (A) and retinas (B) from injured (filled bars) and contralateral noninjured eyes (empty bars), on days 1, 3 and 7 after ONC. Cells were pre-gated on total leukocytes (CD45+), and analyzed for the expression of CD11b. CD11b+ cells were further discriminated based on their expression levels of Ly6C. n = 6-7 per group. Bar graphs throughout the figure show mean ± SE of each group. Asterisks above bars indicate significant differences compared with noninjured tissue; significant differences between different time points are indicated by asterisks between bars. *, P < 0.05; **, P < 0.01; *** P < 0.001. noninj – noninjured control; ONC – optic nerve crush.
The retinal pigment epithelium as a gateway for monocyte trafficking into the eye (2016)
Figure 4

Figure 4. Blocking VCAM-1 after ONC affects retinal monocyte infiltration and the local cytokine bias

Mice were subjected to ONC and i.v.-injected with either an anti-VCAM-1 blocking antibody or an isotype control antibody. Eyes were collected after 2 or 3 days for flow cytometric or Real-Time PCR analysis, respectively. (A) Flow cytometric quantification of myeloid cells in retinas from mice that were treated with anti-VCAM-1 antibody, as compared to isotype-treated controls. n = 3 per group. Asterisks above bars indicate significant differences compared with noninjured tissue; significant differences between different antibody treatments are indicated by asterisks between bars. (B) Expression ratios of Tnf, Il12a and Il1b, compared to Tgfb2, showing that blocking VCAM-1 results in skewing the retina towards a pro-inflammatory milieu after ONC. n = 11-13 per group. Data shown are representative of two independent experiments. Bar graphs throughout the figure show mean ± SE of each group. *, P < 0.05; **, P < 0.01; *** P < 0.001. noninj – noninjured control; ONC – optic nerve crush.
The retinal pigment epithelium as a gateway for monocyte trafficking into the eye (2016)
Figure 5

Figure 5. Live imaging of infiltrating monocytes in the eye.

(A) Live imaging of noninjured and ONC eyes, showing the presence of DiR+ monocyte clusters in injured eyes only. Some of the cell clusters are co-localized with GFP, indicating monocytes that have crossed the RPE and infiltrated the retina. n = 2-3 per group. Data shown are representative of two independent experiments. (B) Representative micrographs of eye sections collected from mice 1 day after ONC, or 29 days after the induction of EAU, stained for β-dystroglycan (red, arrows) and laminin (green), showing a comparable structure of blood vessels in the inner retina of ONC eyes and noninjured controls, which is different from the appearance of blood vessels in EAU retina. Scale bar, 50 µm. (C-E) [Actbegfp/+ WT] BM chimeric mice were generated and subjected to ONC. (C) Representative micrographs showing the abundance of GFP+ cells (green) in the RPE (RPE65, red) and retina of the ONC eye compared to noninjured contralateral eye. Retinal layers can be distinguished by Hoechst staining (blue; scale bar, 100 µm). (D) Higher magnification micrographs of ONC eyes, showing the association of blood-derived GFP+ cells (green, arrows) with the RPE (red); Note processes extending from the cells to the RPE (scale bar, 20 µm). (E) Representative micrograph of ONC eye showing GFP+ cells (green) in proximity to the GCL, the site of dying retinal ganglion cells (scale bar, 20 µm). n = 4 per group. EAU – experimental autoimmune uveoretinitis; GCL – ganglion cell layer; noninj – noninjured control; ONC – optic nerve crush; ONH – optic nerve head; RPE – retinal pigment epithelium.
The retinal pigment epithelium as a gateway for monocyte trafficking into the eye (2016)
Figure 6

Figure 6. The expression of leukocyte trafficking molecules by the RPE is upregulated following ocular glutamate intoxication.

(A-D) Changes in mRNA transcript levels of a series of adhesion molecules (A), chemokines (B, C) and cytokine receptors (D) in injured retinas compared with contralateral noninjured retinas, assessed 1 day after GT intoxication. n = 6 per group. (E) Representative micrographs showing the expression of ICAM-1 and VCAM-1 (red) by the RPE (green) 1 day after GT intoxication. Bar graphs throughout the figure show mean ± SE of each group. *, P < 0.05; **, P < 0.01; *** P < 0.001. GT – glutamate; noninj – noninjured control.
The retinal pigment epithelium as a gateway for monocyte trafficking into the eye (2016)
Figure 7

Figure 7. Neuroprotective monocytes localize to the RPE/SRS after glutamate intoxication.

(A) Representative micrograph of an eye from day 7 after glutamate intoxication, from a mouse that was i.v.-injected with monocytes, showing monocyte localization to the SRS after this mode of application, and indicating a potential homing route for the cells (IL-10, red; CX3CR1-GFP, green). (B-E) Mice were injected i.v.t. with either monocytes or PBS 1 day after GT intoxication, and ocular sections from 7 days after the injury were analyzed. (B, C) Representative micrographs of noninjured and injured retinas stained for the RGC marker, Brn3a (red; scale bar, 100 µm), and quantification of RGC survival. Arrows indicate Brn3a+ cells. Bar graph shows mean ± SE of each group. *, P < 0.05. (D, E) Representative micrographs of GT-intoxicated eyes that were i.v.t.-injected with monocytes, showing the expression of an array of cytokines and neurotrophic factors (red) by the injected monocytes (CX3CR1-GFP, green). The injected cells were found in the vitreous and in proximity to injured RGCs (left panels), as well as in the SRS, adjacent to the RPE (right panels). Insets show higher magnification of representative cells (scale bar, 100 µm; inset scale, 20 µm). n = 4-5 per group. GCL – ganglion cell layer; GT – glutamate; mon – monocytes; RGC – retinal ganglion cell; RPE – retinal pigment epithelium; SRS – subretinal space; vit – vitreous.
Shear stress control of vascular leaks and atheromas through Tie2 activation by VE-PTP sequestration (2023)
Figure 1

Figure 1. Downstream polarization of VE-PTP in endothelial cells.

(A) Regions of mouse aortic arch and thoracic aorta studied.

(B) Downstream polarization of VE-PTP (red) in endothelial cells in the thoracic aorta. Blood flow: left to right. VE-cadherin, green.

(C) Higher magnification views of a region similar to B, showing variability of VE-PTP polarization ranging from conspicuous (upper) to subtle (lower). Nuclei, blue (lamin A/C).

(D) Dot plot of proportions of VE-PTP staining in upstream and downstream halves of endothelial cells in thoracic aorta of wild-type mouse. Each dot is one endothelial cell. Mean ± SEM, n = 15 cells.

(E) VE-PTP staining (red) in aortic arch endothelial cells contrasts distinct polarization in outer curvature and diffuse staining in inner curvature. Endothelial cells are elongated in outer curvature, but polygonal in inner curvature with jagged cell borders. VE-PTP, red. VE-cadherin, green.

(F) Contrasting directions of blood flow in the two regions.

(G) VE-PTP staining (red) in HUVEC under static conditions and after laminar flow (15 dyn/cm2; 5 min - 24 h). Nuclei, blue (Hoechst 33342). Faint diffuse VE-PTP staining in the static control contrasts with downstream polarization after flow and change from diffuse downstream staining at 5 min to dot-like staining at 30 min. Intracellular VE-PTP is more dispersed at 24 h. See related images in Appendix Figure S2.

(H) HUVEC were grown under static conditions or for 24 h under 15 dyn/cm2 flow and cell lysates were immunoblotted for VE-PTP and tubulin (as indicated).

Data information: ***P < 1 ×10-15, by Student's t-test in D. Scale bars: 40 µm in B, 20 µm in C and G, 10 µm in E.
Shear stress control of vascular leaks and atheromas through Tie2 activation by VE-PTP sequestration (2023)
Figure 2

Figure 2. VE-PTP internalization into endosomes under flow conditions.

(A) Confocal microscopic images of VE-PTP (red) and early endosome marker EEA1 (green) showing colocalization in some endosomes near downstream tip of endothelial cells (arrowheads) in thoracic aorta of wild-type mouse. Blood flow left to right. Patches of VE-PTP and many scattered endosomes without colocalization are also present.

(B) Similar field showing VE-PTP and early endosome marker Rab5 (green) with colocalization in some dot-like endosomes (arrowheads).

(C) VE-PTP internalization in HUVEC into EEA1 endosomes (arrowheads) under static conditions and laminar flow of 15 dyn/cm2 of shear force for 5, 10, and 30 min.

(D) Quantification of VE-PTP staining signals of images shown in C, which co-localize with EEA1. Mean ± SEM, n= 10 pooled in 3 independent experiments

(E) HUVEC exposed to same conditions as in C, followed by fixation with (upper row) or without (lower row) permeabilization with 0.3% Triton X-100, and staining for VE-PTP (red) and nuclei (blue, Hoechst 33342).

(F) VE-PTP on cell surface measured by cell-based ELISA assay of HUVEC after exposure as in C and D. Red and black lines show measurements with anti-VE-PTP antibody and isotype control antibody. Mean ± SEM, n = 6 independent experiments/group.

Data information: **P < 0.01, ***P < 0.001 by one-way ANOVA followed by Dunnett's test in D and F. P < 1 ×10-15 (0 min vs. 10 min and 30 min) in D. P = 0.0080 (0 min vs. 5 min) and 0.0056 (0 min vs. 10 min) and 0.0010 (0 min vs. 30 min) in F. Scale bars: 10 µm in A-C, E.
Shear stress control of vascular leaks and atheromas through Tie2 activation by VE-PTP sequestration (2023)
Figure 3

Figure 3. Tie2 phosphorylation, VE-PTP, and VE-cadherin distribution in endothelial cells of aorta.

(A) Strong staining for Tie2-pY992 (green) and polarized VE-PTP (red) in endothelial cells of thoracic aorta of wild-type mouse.

(B) Enlargement of region marked by white box in A, comparing the distributions of Tie2-pY992 at endothelial cell borders and focal adhesions (arrowheads) with VE-PTP polarized to the downstream cell tip of endothelial cells.

(C-D) Strong Tie2-pY992 (green) staining at cell borders and VE-PTP (red) downstream polarization in outer curvature in C in contrast to weak, dispersed staining for Tie2-pY992 and VE-PTP in inner curvature in D of aortic arch of wild-type mouse.

(E-F) Tie2-pY992 (green) at endothelial junctions marked by VE-cadherin (red). (E) Contrasting patterns and intensity of Tie2-pY992 in outer and inner curvatures. (F) Tie2-pY992 mean fluorescence intensity at junctions (normalized to outer curvature = 1.0). Mean ± SEM, n = 6 mice/group.

Data information: ***P = 0.000049, by Welch's t-test in F. Scale bars: 25 µm in A, 20 µm in B-E.
Shear stress control of vascular leaks and atheromas through Tie2 activation by VE-PTP sequestration (2023)
Figure 4

Figure 4. Endothelial cell junctions and plasma leakage in the aorta.

(A) VE-cadherin (green) of adherens junctions is continuous at endothelial cell borders in both the outer and inner curvature, but junctions in inner curvature are jagged and have tiny finger-like processes, and endothelial cell shape is markedly different.

(B) FGD5, a GTPase exchange factor required for the anti-leakage effect of Tie2 activation, is continuous in outer curvature, but is discontinuous in the inner curvature.

(C) Little leakage of anti-fibrinogen antibody (red) in outer curvature compared to extensive leakage in inner curvature. VE-cadherin (green).

(D) Gradient of anti-fibrinogen antibody leakage (red) at interface of outer (upper) and inner curvatures (lower). VE-cadherin (green).

(E) Comparison of distribution of fluorescence intensities of extravasated antibody showing significantly stronger fluorescence due to greater leakage in inner curvature.

(F) Mean fluorescence intensity value for each mouse in the two groups shown in E. Mean ± SEM, n = 7 mice/group.

(G) Localized distribution of intense leakage (red) around ostium of an intercostal artery (arrow). VE-cadherin (green).

(H) Opposite distributions of extravasation and VE-PTP polarization in endothelial cells in thoracic aorta. Left and right panels are the same image with colors switched to highlight leakage (red) and polarized VE-PTP (green, arrowheads) on the left, and polarized VE-PTP (red, arrowheads), VE-cadherin (green), and leakage (blue) on the right.

Data information: *P = 0.0219, by Kolmogorov-Smirnov two-sample test in E and ***P = 0.00000025 by Student's t-test in F. Scale bars: 20 µm in A-D, G and H.
Shear stress control of vascular leaks and atheromas through Tie2 activation by VE-PTP sequestration (2023)
Figure 5

Figure 5. Shear stress promotion of VE-PTP-dependent Tie2 activation.

(A) Polarized staining for VE-PTP (red) and strong Tie2-pY992 (green) in the aortic outer curvature but not inner curvature of VE-PTPfl/fl mice compared to the absence of VE-PTP and equally strong Tie2-pY992 staining in both curvatures of VE-PTPiECKO mice. Blood flow left to right.

(B) Measurements of Tie2-pY992 staining intensity at intercellular junctions in aortic arch endothelial cells showing lower values in the inner curvature than in the upper curvature of VE-PTPfl/fl mice but equally high values in both regions of VE-PTPiECKO mice. Normalized to mean value for outer curvature of VE-PTPfl/fl mice scaled to 1.0. Mean ± SEM, n = 6 mice/group.

(C) HUVEC transfected with control siRNA or VE-PTP siRNA and exposed to static or laminar shear force at 15 dyn/cm2 for 30 min, followed by immunoprecipitation of Tie2. Left. Blot showing immunoprecipitates (top) or cell lysates (bottom) immunoblotted for pan-phosphotyrosine (antibody 4G10), Tie2, or VE-PTP. Ratio of staining for phosphorylated Tie2 and total Tie2 (Tie2-pY to Tie2, n = 4 experiments). Right. Relative Tie2-pY levels in HUVEC after control siRNA or VE-PTP siRNA. Normalized to static control = 1. Mean ± SEM

(D) Effect of shear stress on the phosphorylation levels of Tie2 Y992 at cellular junctions. HUVECs treated with either control siRNA (left) or Tie2 siRNA (right), were exposed to 15 dyn/cm2 shear stress for 5 min. The resulting cells were fixed and stained for Tie2-pY992 (red) and VE-cadherin (green).

(E) Quantification of junctional Tie2-pY992 levels. Mean ± SEM, n = 4 experiments.

Data information: *P < 0.05, **P < 0.01, by two-way ANOVA followed by Tukey test in B, C and E. N.S. not significant. P = 0.0455 (outer curvature, VE-PTPfl/fl vs, inner curvature, VE-PTPfl/fl), 0.048 (inner curvature, VE-PTPfl/fl vs. inner curvature, VE-PTPiECKO) and 0.99 (outer curvature, VE-PTPiECKO vs. inner curvature, VE-PTPiECKO) in B. P = 0.023 (Static, Control siRNA vs. Flow, Control siRNA), 0,013 (Static, Control siRNA vs. Static, VE-PTP siRNA) and 0.99 (Static, VE-PTP siRNA vs. Flow, VE-PTP siRNA) in C. P = 0.0058 (Control siRNA) and 0.99 (Tie2 siRNA) in E. Scale bar: 20 µm in A and D.
Shear stress control of vascular leaks and atheromas through Tie2 activation by VE-PTP sequestration (2023)
Figure 6

Figure 6. Relevance of VE-PTP and Tie2 for flow-induced endothelial junction tightening.

(A-B) Red fluorescence of extravasated anti-fibrinogen antibody in aortic arch inner curvature of VE-PTPfl/fl mouse (control) and VE-PTPiECKO mouse at 30 min after i.v. injection of the antibody.

(C-D) Amounts of extravasated antibody in the inner curvature reflected by the distribution of fluorescence intensities in C and box and whisker plots in D, where boxes show 1st, 2nd (median), and 3rd quartiles and whiskers show maximum and minimum fluorescence intensity (x marks the mean). Both plots document significantly less leakage in the inner curvature of VE-PTPiECKO mice (n = 9) than of VE-PTPfl/fl mice (n = 7).

(E) HUVECs cultured on biotinylated gelatin were treated either with control siRNA, VE-PTP siRNA, Tie2 siRNA or VE-cadherin siRNA, then exposed to 15 dyn/cm2 shear stress for 30 minutes followed by 3 min incubation with Streptavidin-Alexa647 (red), washing, fixation and staining for VE-cadherin (green).

(F and G) Quantification of Streptavidin leakage area per image. Mean ± SEM, n = 19 pooled in ten independent experiments (F), and n = 6 pooled in three independent experiments (G).

Data information: *P < 0.05, **P < 0.01, ***P < 0.001, by Kolmogorov-Smirnov test in C (maximum difference in cumulative frequency curves = 39%), by Student's t-test in D, and by Mann-Whitney U-test with Bonferroni correction in F and G. P = 0.00051 in C. P = 0.0214 in D. P = 0.030 (Control siRNA), 0.17 (VE-PTP siRNA) and 0.99 (Tie2 siRNA) in F. P = 0.0043 (Control siRNA and 0.0043 (VE-cadherin siRNA) in G. Scale bars: 100 µm in A and B, 50 µm in E.
Shear stress control of vascular leaks and atheromas through Tie2 activation by VE-PTP sequestration (2023)
Figure 7

Figure 7. Reduction of atheromas and vascular leakage in ApoE-/- mice with VE-PTP gene deletion on high-fat diet.

(A) Oil Red O staining of atheromas in ApoE-/-/VE-PTPfl/fl mice and ApoE-/-/VE-PTPiECKO mice on high-fat diet for 10 wk.

(B) Fractional area occupied by Oil Red O-stained atheromas in aortic arch, brachiocephalic artery, at intercostal artery ostia, and away from ostia. Each dot is value for one mouse. Mean ± SEM, n = 13-18 mice/group.

(C) Sites of leakage revealed by extravasated anti-fibrinogen antibody in aortas of ApoE-/-/VE-PTPfl/fl mouse (left) and ApoE-/-/VE-PTPiECKO mouse (right) on high-fat diet for 7 wk. Arrowheads mark intercostal artery ostia; black asterisks mark strong leakage at aortic root.

(D-E) Fluorescence intensity distributions in D and mean intensities in E of extravasated anti-fibrinogen antibody in inner curvature of ApoE-/-/VE-PTPfl/fl mice and ApoE-/-/VE-PTPiECKO mice on high-fat diet for 7 wk. Mean ± SEM, n = 7 mice/group.

(F-G) Atheromas in ApoE-/- mice marked by extravasated anti-fibrinogen antibody (red to yellow) in inner curvature at low magnification and enlarged (white box) in F and in thoracic aorta in G. VE-cadherin (green).

Data information: *P < 0.05, **P < 0.01, ***P < 0.001, by two-way ANOVA followed by Bonferroni test in B, Kolmogorov-Smirnov test in D, and Student's t-test in E. N.S. not significant. P = 0.00069 (aortic arch), 0.00017 (brachiocephalic artery), 0.0010 (Ostia in descending aorta) and 0.99 (far from ostia in descending sorta) in B. P = 0.0010 in D. P = 0.018 in E. Scale bars: 1 mm in A upper row and C, 300 µm in A lower row, 400 µm in F left, 100 µm in F right, 50 µm in G.
Shear stress control of vascular leaks and atheromas through Tie2 activation by VE-PTP sequestration (2023)
Figure 8

Figure 8. VE-PTP inhibitor AKB-9785 amplification of Tie2 phosphorylation and reduction in atherogenesis.

(A-B) Tie2-pY992 staining in aortic endothelial cells is shown with VE-cadherin in A and with VE-PTP in B. Images in A show the thoracic aorta 1 hr after the second of two doses of AKB-9785 (6 hr after first dose). Images in B show the aortic arch 1 hr after a single dose of AKB-9785. Tie2-pY992 staining at cell borders is stronger after AKB-9785 in the thoracic aorta and in the inner curvature but not outer curvature.

(C) Measurements of Tie2-pY992 intensity at intercellular junctions in aortic arch of mice shown in B. Mean ± SEM, n = 8 mice/group.

(D) Atheromas stained with Oil Red O in aortas of ApoE-/- mice treated with AKB-9785 daily for 11 wk while on high-fat diet for the final 10 wk. Regions of thoracic aorta outlined by white boxes are shown at higher magnification in lower row.

(E) Fractional area occupied by Oil Red O-stained atheromas in aortic arch, brachiocephalic artery, at intercostal artery ostia, and away from ostia. Each dot is value for one mouse. Mean ± SEM, n = 17 mice/group.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001, by two-way ANOVA followed by Tukey test in C and by Bonferroni test in E. N.S. not significant. P = 0.0005 (outer curvature, vehicle vs. inner curvature, vehicle), 0.042 (inner curvature, vehicle vs. inner curvature, AKB-9778) and 0.52 (outer curvature, AKB-9785 vs. inner curvature, AKB-9785) in C. P = 0.00033 (aortic arch), 0.0216 (brachiocephalic artery), 0.0020 (ostia in descending aorta) and 0.99 (far from ostia in descending aorta) in E. Scale bars: 20 µm in A and B, 1 mm in D upper row, 300 µm in D lower row.
PARP-1 regulates DNA repair factor availability (2018)
Figure 1

Figure 1. PARP-1 enzymatic activity is increased as a function of disease progression and is associated with poor outcome. (A) Tissue microarrays (TMAs) from primary PCa (n=132) and CRPC (n=148) were stained via immunohistochemistry for poly(ADP-ribose) (PAR), and scored by a clinical pathologist (T. Parsons) for intensity (0-3) and percentage (0-3). (B) PAR score was generated via the equation: (intensity x 1) + (percentage x 2). PAR scores were compared between primary and CRPC. ****=p value<0.0001 by Chi-square test. (C) Manual PAR scores were divided in to quartiles and then were compared to progression-free survival in the CRPC TMAs. *=p<0.05, ns=not statistically significant by Log-rank (Mantel-Cox). 1st quartile vs. 2nd quartile, p=0.1482; 1st quartile vs. 3rd quartile, p=0.5794; 1st quartile vs. 4th quartile, p= 0.0160; 2nd quartile vs. 3rd quartile, p=0.3869; 2nd quartile vs. 4th quartile, p=0.2110; 3rd vs. 4th quartile, p=0.0201. (D) Top Left: Representative image of one TMA core after multiplex fluorescent IHC for γH2AX (green), PAR (red), PARP-1 (purple), with DNA (blue). Top Right: Insets of parent image at left. Numbers above inset columns coincide with numbers on image at left that were chosen for further magnification and representation (boxed areas). Bottom Left: Percent positive staining for PAR for the entirety of each TMA cohort. Bottom Middle: Percent positive staining for PARP-1. Bottom right: γH2AX for the entirety of each TMA cohort. Data was considered after a median intensity cutoff, and analyzed for statistical significance using two-tailed Student's t-test for PAR, PARP-1, and γH2AX, respectively. Exact p values are indicated. (E) Two-tailed Spearman correlation test between PAR and γH2AX (% positive with a median intensity cut-off). Exact p values are indicated when available.