Journal: The American Journal of Pathology

Article Title: Preferential Lymphatic Growth in Bronchus-Associated Lymphoid Tissue in Sustained Lung Inflammation

doi: 10.1016/j.ajpath.2014.01.021

Figure Lengend Snippet: Number and distribution of lymphatics in lung of pathogen-free mice. Lymphatics are stained for Prox1-EGFP immunoreactivity (green). Smooth muscle cells stained for αSMA (red) delineate the wall of airways and blood vessels. A: Overview of the distribution of lymphatics in the midportion of the left lung. Lymphatics encircle major bronchi and blood vessels and follow branches toward the lung perimeter. No lymphatics are present in the visceral pleura. Boxed regions in A are shown enlarged in B (A, middle box), C (A, top box), and E (A, bottom box) B: Smooth surfaced lymphatics on bronchus (Br) near the hilum. Typical of bronchi, bands of smooth muscle cells (αSMA, red) are oriented perpendicular to the airway axis. Also visible are small branches of the pulmonary artery (PA; arrows) and pulmonary vein (PV; arrowhead). C: Smooth surfaced lymphatics on a small branch of PV (arrowhead). Small branches of PA that lack lymphatics are also visible (arrows). D: Enlargement of boxed region in C showing the relationship of lymphatics (arrowhead) to smooth muscle of small PV (arrows). E: Lymphatics in distal lung on small branches of PV. F: Relative abundance of lymphatics around major bronchi, branches of PA and vein, and distal lung parenchyma in cross section (200 μm thick) of the mid region of the left lung. Dots show the mean area density of lymphatics marked by VEGFR-3 staining in each mouse. Values for Br and pulmonary vessels are not significantly different from one another. ∗P < 0.05 between Br and pulmonary vessels (N = 13 to 15 mice per group). Scale bars: 200 μm (A–C and E); 50 μm (D).

Article Snippet: Inhibition of VEGF-2 and/or VEGFR-3 Signaling Function-blocking rat monoclonal antibodies were used to block VEGFR-2 (clone DC101) and/or VEGFR-3 (clone mF4-31C1) (ImClone Systems, New York, NY).

Techniques: Staining

Journal: The American Journal of Pathology

Article Title: Preferential Lymphatic Growth in Bronchus-Associated Lymphoid Tissue in Sustained Lung Inflammation

doi: 10.1016/j.ajpath.2014.01.021

Figure Lengend Snippet: Expansion of lymphatic network in mouse lung after M. pulmonis infection. A: Overview of lymphatics in the midportion of the left lung near the hilum after infection for 28 days. Abundant lymphatics (Prox1-EGFP, green) found around major bronchus (Br), pulmonary artery (PA), and pulmonary vein (PV), shown by smooth muscle (αSMA, red) in the wall, but few lymphatics in the distal lung. Boxed regions are shown enlarged in C (A, lower box) and Supplemental Figure S2A (A, upper box). Comparison of few small lymphatics (Prox1-EGFP, green) on a pulmonary vein branch in lung of pathogen-free mouse (B) with abundant larger lymphatics on a pulmonary vein after infection for 28 days (C). Comparison of smooth surfaced lymphatic in lung of pathogen-free mouse (D) and lymphatics with sprouts (arrowheads) in lung after infection for 7 days (E). Lymphatics shown by VEGFR-3 immunoreactivity (green). Comparison of few lymphatics (Prox1-EGFP, green) in the narrow space around Br and PA of lung of pathogen-free mouse (F) with abundant lymphatics in cuff of lymphoid tissue around pulmonary artery and bronchus after infection for 28 days (G). Region of lymphatics in lymphoid tissue is sharply demarcated from surrounding lung parenchyma with type 1 alveolar epithelial cells shown by aquaporin-5 immunoreactivity (red). H: Time course of expansion of lymphatic network in left lung from pathogen-free state (0) to 7, 14, and 28 days after infection. Each red dot represents the average value for lymphatics near bronchi, pulmonary arteries, and pulmonary veins in each mouse at one time point. Mean values after infection are significantly different from baseline (∗P < 0.05). Blue dots are corresponding values for lung parenchyma, which are significantly less than red values but not different from one another (N = 4 to 15 mice per group). I: Cells with VEGF-C immunoreactivity (red, arrows) near lymphatics (Prox1-EGFP, green) in BALT around PA after infection for 7 days. J: Higher-magnification view of I showing the distribution of cells in which staining for VEGF-C (red) and CD45 (green) are colocalized (orange, arrows). Scale bars: 200 μm (A, F, and G); 50 μm (B–E and I); 20 μm (J). Br, bronchi; PA, pulmonary artery.

Article Snippet: Inhibition of VEGF-2 and/or VEGFR-3 Signaling Function-blocking rat monoclonal antibodies were used to block VEGFR-2 (clone DC101) and/or VEGFR-3 (clone mF4-31C1) (ImClone Systems, New York, NY).

Techniques: Infection, Staining

Journal: The American Journal of Pathology

Article Title: Preferential Lymphatic Growth in Bronchus-Associated Lymphoid Tissue in Sustained Lung Inflammation

doi: 10.1016/j.ajpath.2014.01.021

Figure Lengend Snippet: Effect of treatment of mice with control IgG or function-blocking antibodies to VEGFR-2 (DC101) and/or VEGFR-3 (mF4-31C1) on lymphangiogenesis and angiogenesis in lung during M. pulmonis infection for 14 days. Pathogen-free controls had no treatment. A–E: Lymphatics around bronchus (Br), pulmonary artery (PA), or pulmonary vein (PV) near hilum. Lung sections stained for VEGFR-3 (red) and αSMA (green). A: Sparse lymphatics in pathogen-free mouse. B: Abundant lymphatics in infected mouse treated with control IgG. C: Abundant lymphatics in infected mouse treated with anti–VEGFR-2. D: Sparse lymphatics in infected mouse treated with anti–VEGFR-3. E: Sparse lymphatics in infected mouse treated with anti–VEGFR-2 and anti–VEGFR-3. F: Relative abundance of lymphatics in lung of pathogen-free mice and infected mice treated with VEGFR blocking antibodies. Red dots represent average value for lymphatics around bronchus, pulmonary artery, and pulmonary vein in each mouse. Blue dots represent corresponding values for lung parenchyma. ∗P < 0.05 compared to baseline; †P < 0.05 compared to infected mice treated with control IgG. No significant differences among lung parenchyma groups (N = 5 to 11 mice per group). G: H&E-stained section of left lung of infected mouse treated with anti–VEGFR-2 and anti–VEGFR-3 showing BALT with lymphoid follicle (arrow) around Br and PA. H: Amount of BALT, expressed as percentage of total lung area, measured in H&E-stained lung sections of pathogen-free mice and infected mice treated with VEGFR blocking antibodies. BALT was not present in lungs of pathogen-free mice. Significantly different from pathogen-free group (∗P < 0.05), but no significant difference was found among the treatment groups (N = 5 to 8 mice per group). I: Bronchial lymph node wet weight in pathogen-free mice and infected mice treated with VEGFR blocking antibodies. Significantly different from pathogen-free group (∗P < 0.05) or other groups of infected mice (†P < 0.05) (N = 5 to 11 mice per group). J and K: Blood vessels (PECAM-1, green) and leukocytes (CD45, red) in BALT near bronchus. Infected mouse treated with control IgG. J: BALT contains abundant leukocytes, blood capillaries (arrowheads), and HEVs (arrow). Infected mouse treated with anti–VEGFR-2 and anti–VEGFR-3. K: BALT has fewer blood capillaries (arrowhead), but larger blood vessels (HEVs, arrow) are still present.

Article Snippet: Inhibition of VEGF-2 and/or VEGFR-3 Signaling Function-blocking rat monoclonal antibodies were used to block VEGFR-2 (clone DC101) and/or VEGFR-3 (clone mF4-31C1) (ImClone Systems, New York, NY).

Techniques: Blocking Assay, Infection, Staining

Journal: The American Journal of Pathology

Article Title: Preferential Lymphatic Growth in Bronchus-Associated Lymphoid Tissue in Sustained Lung Inflammation

doi: 10.1016/j.ajpath.2014.01.021

Figure Lengend Snippet: Reversibility of BALT formation, but not lymphangiogenesis, after M. pulmonis infection. H&E-stained sections of mouse left lung comparing pathogen-free state (A), infection for 14 days (B), infection for 28 days with vehicle treatment during final 14 days (C), and infection for 28 days with antibiotic (oxytetracycline) during final 14 days (D). BALT is abundant around bronchus (Br) and pulmonary artery (PA) in infected mice, except in antibiotic-treated group. Confocal microscopic images of lungs under same conditions as in A to D comparing the sparse lymphatics (VEGFR-3, red) around major Br and PA in pathogen-free lung (E) with the abundant lymphatics after infection, where lymphatics are similarly numerous in all infected groups (F–H). I: Percentage of lung sectional area occupied by BALT under same conditions as in A to D. J: Area density of lung lymphatics shown by VEGFR-3 immunoreactivity around major bronchi and pulmonary vessels under same conditions as in A to D. Values are greater after infection and not reduced by antibiotic (Oxy) during final 14 days of 28-day infection. K: Bronchial lymph node weight under same conditions as in A to D. N = 4 to 5 mice per group. ∗P < 0.05 from pathogen-free group, †P < 0.05 from 28-day infection with vehicle (Veh) treatment.

Article Snippet: Inhibition of VEGF-2 and/or VEGFR-3 Signaling Function-blocking rat monoclonal antibodies were used to block VEGFR-2 (clone DC101) and/or VEGFR-3 (clone mF4-31C1) (ImClone Systems, New York, NY).

Techniques: Infection, Staining

Journal: The Journal of Cell Biology

Article Title: VEGF is a chemoattractant for FGF-2–stimulated neural progenitors

doi: 10.1083/jcb.200308040

Figure Lengend Snippet: VEGF stimulates chemotaxis of neural progenitors through VEGFR2. (A) Scatter plots showing the migation patterns of neural progenitors under control conditions or in the presence of VEGFR blockers. Cells treated with the VEGFR2-blocking Ab (DC101) lost the chemotactic response to VEGF. In contrast, the VEGFR1-blocking Ab (MF1) did not affect progenitor migration. (B) Speed and FMI under different migration conditions. Data are shown as the mean ± SEM from three independent experiments. *, P < 0.01 by two-tailed unpaired t test, significantly different from DC101-treated cells. (C and D) Migration tracks of representative cells (four for each condition) exposed to a VEGF concentration gradient, in the presence of either VEGFR2-blocking Ab (C) or control (polysialic acid blocking) Ab (D). The starting point for each cell is the intersection between the X and Y axes (0,0), and the source of VEGF is at the top in the gradient condition.

Article Snippet: MF1, a VEGFR1 blocking Ab, DC101, and a VEGFR2 blocking Ab (both added at 20 μg/ml; provided by D. Hicklin, ImClone Systems Inc., New York, NY) were used to block the function of the corresponding VEGFR.

Techniques: Chemotaxis Assay, Control, Blocking Assay, Migration, Two Tailed Test, Concentration Assay

Journal: Cancer research

Article Title: Anti-tumor effects of anti-Semaphorin 4D antibody unravel a novel pro-invasive mechanism of vascular targeting agents

doi: 10.1158/0008-5472.CAN-18-3436

Figure Lengend Snippet: A) Double immunofluorescence of Sema4D and F4/80 in IgG1 and anti-Sema4D (α-S4D) treated samples (2 wks treatment). White arrows reveal the expression of Sema4D by some TAMs. B-D) Quantification of the number of intratumoral total TAMs, Sema4D negative TAMs or Sema4D positive TAMs per field and the percentage of intratumoral Sema4D positive TAMs per total number of TAMs. IgG1 treated mice were used as a control. Mann-Whitney test (n≥20). E) Quantification of the number of migrated RAW 264 cells per field in untreated, IgG1, anti-Sema4D and recombinant Sema4D (rS4D) treatment conditions. Results are presented as number of migrated cells per field normalized by the untreated control. Mann-Whitney test (n≥45). F) Quantification of the number of migrated RAW 264 cells per field in parental and sh Sema4D, sh CD72, sh PlexinB2 and sh NS RAW 264 cells. Results are presented as number of migrated cells per field normalized by the parental control. Mann-Whitney test (n≥30). G) Quantification of the number of migrated RAW 264 cells per field in untreated and anti-Sema4D treatment conditions in sh Sema4D ans sh NS (non-silencing control) RAW 264 cells. Mann-Whitney test (n≥45).

Article Snippet: Four week-long treatments in RIP1-Tag2 mice started at 12 weeks of age with: 1) Anti-Semaphorin 4D Mab67 function blocking murine IgG1 antibody (anti-Sema4D) kindly provided by Vaccinex Inc, Rochester, NY ( 20 ), 2) anti-VEGFR2 blocking antibody (DC101) purified in our laboratory or 3) ChromPure Mouse IgG1 whole molecule as isotype control (Jackson Immuno Research Laboratories, Inc).

Techniques: Immunofluorescence, Expressing, Control, MANN-WHITNEY, Recombinant

Journal: Cancer research

Article Title: Anti-tumor effects of anti-Semaphorin 4D antibody unravel a novel pro-invasive mechanism of vascular targeting agents

doi: 10.1158/0008-5472.CAN-18-3436

Figure Lengend Snippet: A) Triple immunofluorescence co-staining for Insulin, F4/80 and Sema4D in tumor fronts of IgG1 and anti-Sema4D treated animals. B-D) Quantification of the number of peritumoral total TAMs, Sema4D negative TAMs or Sema4D positive TAMS normalized by the perimeter of the base tumor protrusions (μm) in the invasive fronts and the percentage of Sema4D positive TAMs per total of TAMs. Mann-Whitney test (n≥20). IgG1 treated mice were used as a control. E) Quantification of invasive βTC4 cells per field in the presence of the conditioned media of untreated, IgG1 added or treated and anti-Sema4D added or treated RAW 264 cells used as chemoattractant in Matrigel® transwell assay. IgG1 treatment is used as an isotype control. Results are presented as number of invasive cells per field normalized by the untreated control. Representative experiment of n=3. Mann-Whitney test (n>19 fields). F) Quantification of invasive βTC4 cells per field in the presence of the conditioned media of parental, sh Sema4D, sh CD72, sh PlexinB2 and sh NS (non-silencing control) RAW 264 cells used as chemoattractant in Matrigel® transwell assay. Results are presented as number of invasive cells per field normalized by the parental control. Representative experiment of n=3. Mann-Whitney test (n>20 fields).

Article Snippet: Four week-long treatments in RIP1-Tag2 mice started at 12 weeks of age with: 1) Anti-Semaphorin 4D Mab67 function blocking murine IgG1 antibody (anti-Sema4D) kindly provided by Vaccinex Inc, Rochester, NY ( 20 ), 2) anti-VEGFR2 blocking antibody (DC101) purified in our laboratory or 3) ChromPure Mouse IgG1 whole molecule as isotype control (Jackson Immuno Research Laboratories, Inc).

Techniques: Immunofluorescence, Staining, MANN-WHITNEY, Control, Transwell Assay

Journal: Cancer research

Article Title: Anti-tumor effects of anti-Semaphorin 4D antibody unravel a novel pro-invasive mechanism of vascular targeting agents

doi: 10.1158/0008-5472.CAN-18-3436

Figure Lengend Snippet: A) Levels of stromal cell-derived factor 1 (SDF1) in supernatants of RAW 264 cells treated with IgG1 or anti-Sema4D. Anti-Sema4D added medium was used as a control. T-test (n=4). B) Quantification of SDF1 protein release by ELISA analysis of conditioned media from control and anti-Sema4D treated RAW parental and sh Sema4D cells. Results are presented as ng of SDF1 per total protein μg for each condition normalized by the untreated controls. Mann-Whitney test (n=3). C) Quantification of in vitro matrigel invasion assay of βTC4 cells in presence of basal medium, medium containing SDF1, AMD3100 or both. Results are presented as number of invasive cells per field normalized by the basal control. Representative experiment of n=3. Mann-Whitney test (n≥ 30 fields). D) Quantification of in vitro matrigel invasion assay in which βTC4 cells were incubated with conditioned media from RAW 264 cells untreated or treated with IgG1 or anti-Sema4D in presence of SDF1 or its AMD3100. Results are presented as number of invasive cells per field normalized by the untreated control. Representative experiment of n=3. Mann-Whitney test (n≥ 30 fields). E) Immunohistochemistry (left) and quantification (right) of the incidence of CXR4 expressing tumors in control and anti-Sema4D treated mice. Chi-square test (n>17 tumors). F) Immunohistochemistry (left) and quantification (right) of the number of SDF1 positive round intratumoral cells per field in control and anti-Sema4D treated mice. Mann-Whitney test (n>85 tumors). G) Incidence of SDF1 expressing tumors according to the invasive capacity of the tumor fronts and the treatment regime. H) Quantification of the number of SDF1 positive Sema4D positive cells per total number of Sema4D positive cells per tumor field of control and anti-Sema4D treated mice. Mann-Whitney test (n>17 tumors).

Article Snippet: Four week-long treatments in RIP1-Tag2 mice started at 12 weeks of age with: 1) Anti-Semaphorin 4D Mab67 function blocking murine IgG1 antibody (anti-Sema4D) kindly provided by Vaccinex Inc, Rochester, NY ( 20 ), 2) anti-VEGFR2 blocking antibody (DC101) purified in our laboratory or 3) ChromPure Mouse IgG1 whole molecule as isotype control (Jackson Immuno Research Laboratories, Inc).

Techniques: Derivative Assay, Control, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, In Vitro, Invasion Assay, Incubation, Immunohistochemistry, Expressing