Journal: PLoS ONE

Article Title: Phosphorylation of Akt and ERK1/2 Is Required for VEGF-A/VEGFR2-Induced Proliferation and Migration of Lymphatic Endothelium

doi: 10.1371/journal.pone.0028947

Figure Lengend Snippet: A–C : PROX1 ( A ) is localized in the nucleus ( B ) in primary human LECs ( C ). D : Cell viability/proliferation was measured with Cell Titer Blue reagent after culturing LECs for 48 hours in EGM-2MV media (positive control), reduced-serum media (negative control), or with VEGF-A (100 ng/ml) in the presence or absence of r84 (500 molar excess), a functional blocking antibody against VEGFR1 (500 molar excess), or control IgG (500 molar excess). r84 blocked VEGF-A-induced proliferation/viability of LECs whereas the other antibodies had no effect. E : LECs were seeded in the upper chamber of a transwell insert and allowed to migrate overnight toward EGM-2MV (positive control), reduced-serum media (negative control), or VEGF-A (100 ng/ml) in the presence or absence of r84 (500 molar excess), a functional blocking antibody against VEGFR1 (500 molar excess), or control IgG (500 molar excess). The number of LECs that migrated to the lower chamber was counted and normalized to the positive control. r84 blocked VEGF-A-induced migration whereas the other antibodies had no effect. For panels C and D, significance tested by ANOVA. Asterisk P<0.05 compared to VEGF-A. ns = not significant compared to VEGF-A.

Article Snippet: The VEGFR1 functional blocking antibody (6.12) was from ImClone.

Techniques: Positive Control, Negative Control, Functional Assay, Blocking Assay, Control, Migration

Journal: Respiratory Research

Article Title: Role of IGF-1 pathway in lung fibroblast activation

doi: 10.1186/1465-9921-14-102

Figure Lengend Snippet: Decreased αSMA promoter activity and αSMA protein expression in bleomycin-injured mice treated with IGF-1 receptor blocking antibody (A12). (A) Representative H&E sections of αSMA-GFP mice at day 21 after bleomycin injury. Fibrotic regions of lung parenchyma are indicated by ( ) and normal lung parenchyma are indicated by ( n ). (B) αSMA immunostaining of bleomycin-injured lung in an αSMA-GFP mouse. Note the overlap of αSMA staining (red) with αSMA-GFP expression (green) in the peribronchiolar fibrotic region indicated by ( ). Scale bars represent 100 μm. (C) (Left) Representative fluorescent images of αSMA-GFP mice treated with A12 (b and d) showed less αSMA promoter activity as indicated by GFP (green) positive cells, compared to control mice (a and c) at d21 after bleomycin instillation. (Right) Percentage of αSMA-GFP + cells/total number of DAPI + cells, quantification by NIH ImageJ (n = 4 mice/group, mean ± SEM). (D) (Left) Representative images of αSMA staining by immunofluorescent microscopy of the same A12-treated mice (b and d) compared to control mice (a and c) at d21 after bleomycin instillation. Large airways and vasculature staining for αSMA, indicated by an asterisk (*), were masked in the analysis. Interstitial staining, indicated by an arrow ( ), was included in the analysis. (Right) Ratio of αSMA staining area per DAPI + area (n = 4 mice/group, mean ± SEM).

Article Snippet: Function-blocking antibody to the human IGF-1 receptor (A12) and keyhole limpet hemocyanin (KLH) isotype control antibody were a generous gift from Dale Ludwig (ImClone Systems) [ , ].

Techniques: Activity Assay, Expressing, Blocking Assay, Immunostaining, Staining, Control, Microscopy

Journal: Respiratory Research

Article Title: Role of IGF-1 pathway in lung fibroblast activation

doi: 10.1186/1465-9921-14-102

Figure Lengend Snippet: Effect of matrix stiffness on response to IGF-1 treatment. (A) MLF on tissue culture plate or collagen I-coated tissue culture plate (stiff substrates) were treated with IGF-1 (100 ng/ml), TGFβ (1 ng/ml) or IGF/TGFβ (100 ng/ml and 1 ng/ml, respectively), or serum-free media (negative control) for 24 hr. (B) MLF on collagen I (1 mg/ml) hydrogel (soft substrate) were treated with IGF-1 (100 ng/ml), IGF-1 (100 ng/ml) with A12 (40 μg/ml) or PI3 kinase inhibitor LY294002 (Ly, 50 μM;) for 24 h. (C) MLF isolated from bleomycin-injured C57Bl6 mice were treated with the indicated cytokine. Real time PCR analyses of myofibroblast markers Acta2 , Col1a1 , and Col3a1 were performed. Data were normalized to HPRT expression. Y-axis represents fold increase compared to serum-free control (n = 3, mean ± SEM, * p < 0.05 compared to serum-free control).

Article Snippet: Function-blocking antibody to the human IGF-1 receptor (A12) and keyhole limpet hemocyanin (KLH) isotype control antibody were a generous gift from Dale Ludwig (ImClone Systems) [ , ].

Techniques: Negative Control, Isolation, Real-time Polymerase Chain Reaction, Expressing, Control

Journal: Respiratory Research

Article Title: Role of IGF-1 pathway in lung fibroblast activation

doi: 10.1186/1465-9921-14-102

Figure Lengend Snippet: IGF-1 treatment increases αSMA stress fibers. A . MLF treated with IGF-1 (100 ng/ml), TGF-β1 (10 ng/ml) or IGF-1/TGF-β1 (100 ng/ml and 10 ng/ml, respectively) with or without A12 (40 μg/ml) for 24 hr. Negative control is serum free media. Cells were co-stained for F-actin (red) and αSMA (green). B . The mean ratio (±SEM) of αSMA stress fiber (+) fibroblasts over all F-actin (+) fibroblasts is presented in the bar graph. * p < 0.05 compared to serum-free control. ** p < <0.01 compared to serum-free control.

Article Snippet: Function-blocking antibody to the human IGF-1 receptor (A12) and keyhole limpet hemocyanin (KLH) isotype control antibody were a generous gift from Dale Ludwig (ImClone Systems) [ , ].

Techniques: Negative Control, Staining, Control

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 American Journal of Pathology

Article Title: Plasticity of Button-Like Junctions in the Endothelium of Airway Lymphatics in Development and Inflammation

doi: 10.1016/j.ajpath.2012.02.019

Figure Lengend Snippet: Reversal of button-to-zipper conversion in inflamed airways. A and B: Lymphatic area density in mucosa overlying cartilage rings (A) and normalized bronchial lymph node and lung weights (B). *P < 0.05 versus pathogen-free mice; †P < 0.05 versus the 14-day infected baseline group. Dex, dexamethasone. C–E: Zippers in lymphatic endothelium shown after M. pulmonis infection and vehicle treatment by staining for VE-cadherin (red) and LYVE-1 (green). Enlargement of boxed regions in C shows zippers (arrows) in both new (D) and existing (E) lymphatics. F–H: Buttons in nonregressed lymphatic endothelium shown after M. pulmonis infection and Dex treatment. Enlargement of boxed regions in F shows buttons (arrows) in the oak leaf–shaped endothelial cells of new (G) and existing (H) lymphatics. I–K: Zippers (arrows) in lymphatic endothelium shown after M. pulmonis infection and anti-VEGFR-3 antibody treatment. Enlargement of boxed regions in I shows zippers (arrows) in both new (J) and existing (K) lymphatics. Scale bars: 50 μm (C, F, and I); 20 μm (D, E, G, H, J, and K).

Article Snippet: Function-blocking, rat monoclonal anti-VEGFR-3 antibody (clone mF4-31C1; ImClone Systems, Inc., New York, NY) was injected i.p. into 8-week-old, pathogen-free or infected C57BL/6 mice at an initial dose of 2 mg per mouse and then 0.8 mg per mouse every other day for 14 days.

Techniques: Infection, Staining