Journal: Experimental and Therapeutic Medicine

Article Title: Expression levels of VEGF-C and VEGFR-3 in renal cell carcinoma and their association with lymph node metastasis

doi: 10.3892/etm.2021.9986

Figure Lengend Snippet: Immunohistochemical staining of VEGFR-3 expression. VEGFR-3 immunohistochemical staining in (A) normal renal tissues and (B) RCC tissues. (C) Lymphatic endothelial cells in RCC tissues were VEGFR-3 + (indicated by arrows). Scale bars, 100 µm. (D) Correlation between VEGF-C and VEGFR-3 expression in RCC tissues. Statistical significance was determined by Spearman's correlation test. RCC, renal cell carcinoma; VEGF-C, vascular endothelial growth factor-C; VEGFR-3, VEGF receptor-3.

Article Snippet: For antigen retrieval, the tissue sections were incubated with 0.01 M sodium citrate (pH 6) in a microwave oven at 95˚C for 10 min, followed by blocking with 5% normal goat serum (cat. no. ZLI-9021; OriGene Technologies, Inc.) for 10 min at room temperature, the tissue sections were incubated with rabbit anti-human VEGF-C monoclonal antibody (cat. no. BA0548; 1:200; Wuhan Boster Biological Technology Co., Ltd.), rabbit anti-human VEGFR-3 monoclonal antibody (cat. no. A01276-3; 1:200; Wuhan Boster Biological Technology Co., Ltd.) and mouse anti-human D2-40 monoclonal antibody (cat. no. ZM-0465; undiluted; OriGene Technologies, Inc.) for 12 h at 4˚C.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Developmental Cell

Article Title: A spatial vascular transcriptomic, proteomic, and phosphoproteomic atlas unveils an angiocrine Tie–Wnt signaling axis in the liver

doi: 10.1016/j.devcel.2021.05.001

Figure Lengend Snippet:

Article Snippet: VEGFR3 (Flt4) (Mm01292604_m1) TaqMan probe , Thermo Fisher Scientific , Cat#4331182.

Techniques: Recombinant, Fluorescence, Staining, Protease Inhibitor, Lysis, Extraction, Isolation, DNA Extraction, Purification, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, RNA HS Assay, Labeling, Sequencing, Control, Software

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: List of the analyzed ECM components.

Article Snippet: FLT4/VEGF3 , FLT4-Hs00176607_m1.

Techniques:

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: Invasion spectrum (the mRNA expression pattern of invasion-associated extracellular matrix components) differs in patients with ‘worse’ and ‘better’ prognoses. mRNA expression measurements were performed twice for each gene to confirm the data. A longer bar on the logarithmic scale indicates reduced expression. *P<0.05 vs. group A (Mann-Whitney U test). Group A, OS <24 months; group B, OS >24 months; BCAN, brevican; CD44, cluster of differentiation 44; CSPG5, chondroitin sulfate proteoglycan 5; EGFR, epidermal growth factor receptor; FLT4, Fms-related tyrosine kinase 4; HMMR, hyaluronan-mediated motility receptor; IDH1, isocitrate dehydrogenase 1; ITGAV, integrin-αV; MDM2, murine double minute 2; MMP-2, matrix metallopeptidase 2; NCAN, neurocan; PDGFA, platelet-derived growth factor α; TNC, tenascin C; VCAN, versican; OS, overall survival.

Article Snippet: FLT4/VEGF3 , FLT4-Hs00176607_m1.

Techniques: Expressing, MANN-WHITNEY, Derivative Assay

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: A total of 3 invasion-associated extracellular matrix components demonstrated significantly different expression levels in the samples from different prognostic groups. All 3 molecules had increased expression in patients in group A, indicating that the level of these molecules was associated with tumor invasiveness and patient survival. Group A, OS <24 months; group B, OS >24 month.; FLT4, Fms-related tyrosine kinase 4; MDM2, murine double minute 2; MMP-2, matrix metallopeptidase 2; rel., relative; OS, overall survival.

Article Snippet: FLT4/VEGF3 , FLT4-Hs00176607_m1.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: Immunohistochemical images of glioblastoma stained for FLT4/VEGF-3. (A) Overexpression of FLT4/VEGF-3 in patients with glioblastoma with a worse prognosis. (B) Moderate positivity in patients with a better prognosis (staining is notably reduced compared with that in patients with a worse prognosis). (C) Negative control. All images depicted are at ×20 magnification. FLT4, Fms-related tyrosine kinase 4; VEGF, vascular endothelial growth factor.

Article Snippet: FLT4/VEGF3 , FLT4-Hs00176607_m1.

Techniques: Immunohistochemical staining, Staining, Over Expression, Negative Control

Journal: Nature Communications

Article Title: Brain-to-cervical lymph node signaling after stroke

doi: 10.1038/s41467-019-13324-w

Figure Lengend Snippet: Cervical lymph node activation in rats after stroke: a Evans Blue dye (2%, 10 µL) was injected into lateral ventricles in normal male Sprague Dawley (SD) rats. Within 30 min, Evans Blue fluorescence was detected in deep and superficial cervical lymph nodes (CLNs) but not in axillary lymph nodes (ALNs) or inguinal lymph nodes (ILNs). Scale: 500 nm. b – d Male SD rats were subjected to 100 min of transient focal cerebral ischemia. Deep and superficial CLNs were collected and analyzed by immunohistochemistry. We did not count LYVE-1 or Ki67 single-positive cells to exclude potential signal from the macrophage population. b Proliferation of lymphatic endothelial cells were also observed in deep CLNs, but it was not statistically significant. c LYVE-1 positive lymphatic endothelial cells in the area of subcapsular sinus in superficial CLNs rapidly proliferated as early as 3 h until 24 h after focal ischemia ( n = 4 biologically independent animals). d At the same time, ILNs were collected as a negative control. Note: Some of Ki67 could be co-expressed with immune cells including macrophages and T cells . * P < 0.05, ** P < 0.01 vs Sham, one-way ANOVA followed by Fisher’s LSD test. Scale: 100 nm. e , f Western blot, following immunoprecipitation, demonstrated that VEGFR3 was phosphorylated in superficial CLNs at 24 h after transient focal ischemia ( n = 3). * P < 0.05, unpaired t -test. g PBS (5 µL) or MAZ51 (50 ng/5 µL) was injected into lateral ventricles immediately after reperfusion and FACS analysis was performed at 24 h. h FACS analysis indicated that focal ischemia promoted the proliferation of VEGFR3 positive cells in the superficial CLNs. Intracerebroventricular injection of MAZ51 significantly suppressed the proliferation of VEGFR3-positive cells in CLNs (Sham; n = 3, MCAO; n = 11, MCAO + MAZ51; n = 7 biologically independent animals). * P < 0.05, ** P < 0.01, one-way ANOVA followed by Fisher’s LSD test. i Immunohistochemistry demonstrated that MAZ51 treatment decreased the proliferation of LYVE-1 positive lymphatic endothelium that was observed after ischemia ( n = 4). Scale: 100 nm. ** P < 0.01, unpaired t -test. All values are mean +/− SD.

Article Snippet: Anti-β-actin (1:1,000, A5441, Sigma-aldrich), anti-p-Tyr antibody (1:500, sc-7020, Santa Cruz), anti-VEGF-C antibody (1:500, sc-374628, Santa Cruz), anti-VEGFR3 antibody (1:500, sc-365748, Santa Cruz), anti-iNOS antibody (1:500, ab3523, Abcam), anti-IL-1β antibody (1:500, ab9722, Abcam), anti-TNF-α antibody (1:200, GTX110520, GeneTex), anti-TGF-β antibody (1:200, ab64715, Abcam), anti-CCL28 antibody (1:500, MAB717, R&D systems), anti-Podoplanin antibody (1:200, sc-166906, Santa Cruz), anti-CD31 antibody (1:200, 550274, BD biosciences), anti-LYVE-1 antibody (1:500, NB100–725B, NOVUS biologicals), anti-vWF antibody (1:100, A0082, Agilent), FITC anti-CD4 antibody (1:100, 561828, BD Biosciences), FITC Neutrophil antibody (1:100, ab53453, Abcam), PE anti-CD34 antibody (1:100, 551387, BD Biosciences), APC anti-F4/80 antibody (1:200, 123116, BioLegend), FITC anti-CD16/32 antibody (1:200, 101306, BioLegend), PE anti-CD11b antibody (1:200, 557397, BD biosciences), anti-CD45 antibody (1:400, 20103–1-AP, Proteintech).

Techniques: Activation Assay, Injection, Fluorescence, Immunohistochemistry, Negative Control, Western Blot, Immunoprecipitation

Journal: Nature Communications

Article Title: Brain-to-cervical lymph node signaling after stroke

doi: 10.1038/s41467-019-13324-w

Figure Lengend Snippet: Cervical lymphatic inflammation through VEGFR3 tyrosine kinase in mice after stroke: a Male C57BL6 mice were subjected to transient focal ischemia and, immediately after reperfusion the vehicle (PBS 10 µL) or MAZ51 (3 ng/10 µL) were injected into the nasal cavity. b MAZ51 treatment suppressed tyrosine phosphorylation in superficial CLN lymphatic endothelium at 72 h after focal ischemia (Sham; n = 4, MCAO; n = 9, MCAO + MAZ51; n = 8 biologically independent animals). * P < 0.05, one-way ANOVA followed by Fisher’s LSD test. c FACS analysis demonstrated that MAZ51 treatment significantly decreased pro-inflammatory TNF-α positive macrophages in superficial CLNs; no clear changes were noticed in TGF-β positive macrophages. * P < 0.05, one-way ANOVA followed by Fisher’s LSD test. d – f . Immunostaining revealed that CD169 positive macrophages increased TNF-α, while MAZ51 treatment decreased pro-inflammatory macrophages. Scale: 100 nm. g – h Confocal microscopy analysis demonstrated that TNF-α was highly co-localized with CD169 positive macrophages in the subcapsular sinus of superficial CLNs. MAZ51 treatment decreased the co-localization. TNF-α expression was not observed in ILN macrophages. Scale: 100 nm. i – l Western blot confirmed that TNF-α, IL-1β and CCL28 were significantly increased in post-stroke superficial CLNs. MAZ51 treatment reduced cytokine/chemokine expression ( n = 4 biologically independent animals). ILNs weakly responded to focal cerebral ischemia as to TNF-α, IL-1β or CCL28 expression ( n = 3 biologically independent animals). * P < 0.05, ** P < 0.01, one-way ANOVA followed by Fisher’s LSD test. All values are mean +/− SD.

Article Snippet: Anti-β-actin (1:1,000, A5441, Sigma-aldrich), anti-p-Tyr antibody (1:500, sc-7020, Santa Cruz), anti-VEGF-C antibody (1:500, sc-374628, Santa Cruz), anti-VEGFR3 antibody (1:500, sc-365748, Santa Cruz), anti-iNOS antibody (1:500, ab3523, Abcam), anti-IL-1β antibody (1:500, ab9722, Abcam), anti-TNF-α antibody (1:200, GTX110520, GeneTex), anti-TGF-β antibody (1:200, ab64715, Abcam), anti-CCL28 antibody (1:500, MAB717, R&D systems), anti-Podoplanin antibody (1:200, sc-166906, Santa Cruz), anti-CD31 antibody (1:200, 550274, BD biosciences), anti-LYVE-1 antibody (1:500, NB100–725B, NOVUS biologicals), anti-vWF antibody (1:100, A0082, Agilent), FITC anti-CD4 antibody (1:100, 561828, BD Biosciences), FITC Neutrophil antibody (1:100, ab53453, Abcam), PE anti-CD34 antibody (1:100, 551387, BD Biosciences), APC anti-F4/80 antibody (1:200, 123116, BioLegend), FITC anti-CD16/32 antibody (1:200, 101306, BioLegend), PE anti-CD11b antibody (1:200, 557397, BD biosciences), anti-CD45 antibody (1:400, 20103–1-AP, Proteintech).

Techniques: Injection, Phospho-proteomics, Immunostaining, Confocal Microscopy, Expressing, Western Blot

Journal: Nature Communications

Article Title: Brain-to-cervical lymph node signaling after stroke

doi: 10.1038/s41467-019-13324-w

Figure Lengend Snippet: Activated lymphatic endothelium upregulates pro-inflammatory macrophages in vitro: a LYVE-1 antibody-conjugated magnetic beads were used for lymphatic endothelial isolation using CLNs isolated from 4 mice. b VEGFR3 was activated by adding VEGF-C (10 ng/ml) to mouse CLN lymphatic endothelial cells for 24 h, then cells were co-cultured with mouse peritoneal macrophages for another 24 h and analyzed by immunostaining. c Immunostaining showed that the expression of iNOS (M1-like) was upregulated, but CD206 (M2-like) expression did not change. Scale: 50 nm. d – f Western blot confirmed that iNOS and IL-1β were upregulated, but TGF-β did not change. * P < 0.05, unpaired t-test. g – i VEGF-C (10 ng/mL) was directly added to macrophages for 24 h. Western blot confirmed that VEGF-C itself did not change macrophage iNOS, IL-1β, and TGF-β expressions ( n = 6 biologically independent cells, n = 3 biologically independent experiments), unpaired t -test. All values are mean +/− SD.

Article Snippet: Anti-β-actin (1:1,000, A5441, Sigma-aldrich), anti-p-Tyr antibody (1:500, sc-7020, Santa Cruz), anti-VEGF-C antibody (1:500, sc-374628, Santa Cruz), anti-VEGFR3 antibody (1:500, sc-365748, Santa Cruz), anti-iNOS antibody (1:500, ab3523, Abcam), anti-IL-1β antibody (1:500, ab9722, Abcam), anti-TNF-α antibody (1:200, GTX110520, GeneTex), anti-TGF-β antibody (1:200, ab64715, Abcam), anti-CCL28 antibody (1:500, MAB717, R&D systems), anti-Podoplanin antibody (1:200, sc-166906, Santa Cruz), anti-CD31 antibody (1:200, 550274, BD biosciences), anti-LYVE-1 antibody (1:500, NB100–725B, NOVUS biologicals), anti-vWF antibody (1:100, A0082, Agilent), FITC anti-CD4 antibody (1:100, 561828, BD Biosciences), FITC Neutrophil antibody (1:100, ab53453, Abcam), PE anti-CD34 antibody (1:100, 551387, BD Biosciences), APC anti-F4/80 antibody (1:200, 123116, BioLegend), FITC anti-CD16/32 antibody (1:200, 101306, BioLegend), PE anti-CD11b antibody (1:200, 557397, BD biosciences), anti-CD45 antibody (1:400, 20103–1-AP, Proteintech).

Techniques: In Vitro, Magnetic Beads, Isolation, Cell Culture, Immunostaining, Expressing, Western Blot

Journal: Nature Communications

Article Title: Brain-to-cervical lymph node signaling after stroke

doi: 10.1038/s41467-019-13324-w

Figure Lengend Snippet: Superficial cervical node lymphadenectomy reduces brain damage after stroke: a Superficial CLNs were surgically removed from C57BL6 mice, then mice were subjected to transient 60 min focal cerebral ischemia. b , c Flow cytometry analysis demonstrated that accumulations of neutrophils and F4/80 positive monocytes/macrophages in both systemic circulation ( b ) and injured brain ( c ) were significantly reduced by superficial CLN removal (Control; n = 3, CLN removal; n = 4 biologically independent animals). * P < 0.05, ** P < 0.01, one-way ANOVA followed by Fisher’s LSD test. d . Percentage of IgG leaked area in the ipsilateral hemisphere was assessed. CLN removal did not significantly influence IgG leakage after focal ischemia ( n = 4 biologically independent animals). unpaired t -test. e CLN lymphadenectomy significantly decreased pro-inflammatory macrophages in ipsilateral cortex and striatum ( n = 4 biologically independent animals), unpaired t -test. Scale: 100 nm. f CLN lymphadenectomy significantly reduced infarct volume at 72 h post-stroke (Control; n = 10, CLN removal; n = 11 biologically independent animals). Control: 99.9 + /− 22.3 mm 3 , CLN removal: 73.4 + /− 22.7 mm 3 . * P < 0.05, unpaired t -test. g Inguinal lymph nodes (ILN) lymphadenectomy did not decrease infarct volume at 72 h post-stroke (Control; n = 7, ILN removal; n = 6 biologically independent animals). Control: 90.2 +/− 19.1 mm 3 , ILN removal: 89.9 +/− 9.6 mm 3 , unpaired t -test. All values are mean +/− SD. h Schematic of the proposed brain - to-cervical lymph node pathway in the regulation of inflammatory response via VEGFR3 signaling after focal cerebral ischemia.

Article Snippet: Anti-β-actin (1:1,000, A5441, Sigma-aldrich), anti-p-Tyr antibody (1:500, sc-7020, Santa Cruz), anti-VEGF-C antibody (1:500, sc-374628, Santa Cruz), anti-VEGFR3 antibody (1:500, sc-365748, Santa Cruz), anti-iNOS antibody (1:500, ab3523, Abcam), anti-IL-1β antibody (1:500, ab9722, Abcam), anti-TNF-α antibody (1:200, GTX110520, GeneTex), anti-TGF-β antibody (1:200, ab64715, Abcam), anti-CCL28 antibody (1:500, MAB717, R&D systems), anti-Podoplanin antibody (1:200, sc-166906, Santa Cruz), anti-CD31 antibody (1:200, 550274, BD biosciences), anti-LYVE-1 antibody (1:500, NB100–725B, NOVUS biologicals), anti-vWF antibody (1:100, A0082, Agilent), FITC anti-CD4 antibody (1:100, 561828, BD Biosciences), FITC Neutrophil antibody (1:100, ab53453, Abcam), PE anti-CD34 antibody (1:100, 551387, BD Biosciences), APC anti-F4/80 antibody (1:200, 123116, BioLegend), FITC anti-CD16/32 antibody (1:200, 101306, BioLegend), PE anti-CD11b antibody (1:200, 557397, BD biosciences), anti-CD45 antibody (1:400, 20103–1-AP, Proteintech).

Techniques: Flow Cytometry, Control