Journal: Experimental Biology and Medicine

Article Title: Engineering ADSCs by manipulating YAP for lymphedema treatment in a mouse tail model

doi: 10.3389/ebm.2024.10295

Figure Lengend Snippet: VEGFC successfully induced lymphatic endothelial transdifferentiation of ADSCs. (A–C) PCR tests showed the upregulation of VEGFR-3, Prox-1, Lyve-1 during the lymphatic endothelial transdifferentiation of ADSCs. (D) VEGFR-3 as a typical LEC marker was detected by immunofluorescence staining after VEGFC-induction at the indicated times. Scale bar = 50 μm. (E) Immunofluorescence intensity analysis of VEGFR-3. (F, H) Western blot showed the increased VEGFR-3 expression during the lymphatic endothelial transdifferentiation of ADSCs. (G) ADSCs were seeded on Matrigel after 7-day induction, and tube formation was evaluated at 12 h postseeding. VEGFC group generated tube-like structure while control group did not exhibit tubes. Scale bar = 100 μm. Bars: means ± standard deviation. n = 3 in each group, ns: no significant, ** P < 0.01, *** P < 0.001.

Article Snippet: For the lymphatic transdifferentiation of ADSCs, VEGFC (100 ng/mL, HY-P77864, MedChem Express, Monmouth Junction, NJ, United States) was used for 7 days.

Techniques: Marker, Immunofluorescence, Staining, Western Blot, Expressing, Generated, Control, Standard Deviation

Journal: Experimental Biology and Medicine

Article Title: Engineering ADSCs by manipulating YAP for lymphedema treatment in a mouse tail model

doi: 10.3389/ebm.2024.10295

Figure Lengend Snippet: Effect of lymphatic endothelial transdifferentiation and verteporfin on the expression of YAP. (A) Immunostaining of YAP in the control group and VEGFC group. Scale bar = 50 μm. (B) Immunofluorescence intensity analysis of YAP. (C–E) Western blot showed the decreased nuclear YAP expression and increased cytosolic YAP expression in ADSCs after lymphatic endothelial transdifferentiation. (F) PCR test showed that verteporfin suppressed the expression of YAP in ADSCs at the concentration of 20 μM. (G , H) Western blot showed the continuously inhibitory effect of verteporfin on YAP expression in ADSCs. Bars: means ± standard deviation. n = 3 in each group; ns, no significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For the lymphatic transdifferentiation of ADSCs, VEGFC (100 ng/mL, HY-P77864, MedChem Express, Monmouth Junction, NJ, United States) was used for 7 days.

Techniques: Expressing, Immunostaining, Control, Immunofluorescence, Western Blot, Concentration Assay, Standard Deviation

Journal: Experimental Biology and Medicine

Article Title: Engineering ADSCs by manipulating YAP for lymphedema treatment in a mouse tail model

doi: 10.3389/ebm.2024.10295

Figure Lengend Snippet: The downregulation of YAP enhanced the lymphatic endothelial transdifferentiation of ADSCs in vitro . 20 μM verteporfin preconditioning for 48 h downregulated the expression of YAP in ADSCs. Under this inhibitory effect, higher expression levels of VEGFR-3 can be detected after differentiation of ADSCs, and larger density of tube formation can be observed. (A) Lymphatic endothelial transdifferentiation of ADSCs was conducted after 20 μM-verteporfin preconditioning for 48 h, and immunofluorescence staining indicated higher level of VEGFR-3. Scale bar = 50 μm. (B) Immunofluorescence intensity analysis of VEGFR-3. (C, D) Higher expression level of VEGFR-3 was confirmed by western blot. (E, F) The tube formation assay showed that VEGFC (+) verteporfin (+) group generated more tube-like structure than VEGFC (+) verteporfin (−) group. And quantification of master segments was analysed. Scale bar = 200 μm. Bars: means ± standard deviation. n = 3 in each group, ns: no significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For the lymphatic transdifferentiation of ADSCs, VEGFC (100 ng/mL, HY-P77864, MedChem Express, Monmouth Junction, NJ, United States) was used for 7 days.

Techniques: In Vitro, Expressing, Immunofluorescence, Staining, Western Blot, Tube Formation Assay, Generated, Standard Deviation

Journal: Applied Sciences

Article Title: Systemic Antibiotic and Nonsteroidal Anti-Inflammatory Drug Treatment Decreases the Level of Endogenous Angiogenic Vascular Endothelial Growth Factor in Inflamed Human Periapical Tissues

doi: 10.3390/app11114976

Figure Lengend Snippet: Figure 2. Example of electrophoretic separation of PCR products for VEGF-B, MWM-molecular weight marker (50–500 bp); 1–6-samples (84 bp); 7-positive control; 8-negative control.

Article Snippet: In all the groups, VEGF levels were measured using a RayBio Human VEGF ELISA kit (RayBiotech Inc.) according to the manufacturer’s specifications.

Techniques: Molecular Weight, Marker, Positive Control, Negative Control

Journal: Applied Sciences

Article Title: Systemic Antibiotic and Nonsteroidal Anti-Inflammatory Drug Treatment Decreases the Level of Endogenous Angiogenic Vascular Endothelial Growth Factor in Inflamed Human Periapical Tissues

doi: 10.3390/app11114976

Figure Lengend Snippet: Figure 3. VEGF concentration in all groups of patients: group I (NSAIDs), group II (antibiotics), group II (NSAID and antibiotics) and control group (no drug treatment).

Article Snippet: In all the groups, VEGF levels were measured using a RayBio Human VEGF ELISA kit (RayBiotech Inc.) according to the manufacturer’s specifications.

Techniques: Concentration Assay, Control

Journal: Cancer Medicine

Article Title: The MIR181A2HG / miR ‐5680/ VCAN ‐ CD44 Axis Regulates Gastric Cancer Lymph Node Metastasis by Promoting M2 Macrophage Polarization

doi: 10.1002/cam4.70600

Figure Lengend Snippet: GC cell CM induces M2‐like polarization of macrophages, promoting lymphangiogenesis. (A) Immunofluorescence shows changes in the M2‐type macrophage marker (CD163) after treatment with conditioned medium from different treatment groups: Si‐NC (control group) and si‐#1 (si‐MIR181A2HG‐1) (scale bar, 50 μm). (B) Quantification of the Proportion of M2 Macrophages (CD163) Among Total Macrophages (CD68). (C) Schematic of the co‐culture model of GC cells and macrophages promoting HLECs lymphangiogenesis. (D) Typical images of macrophage morphological changes after treatment with PMA and GC cell CM (scale bar, 20 μm). (E–H) qRT‐PCR detection of typical M2 markers (CD163, CD206, and IL‐10) and M1 markers (iNOS, IL‐6, and TNFα) in PMA‐treated THP‐1 cells cultured with CM from different treatment groups (si‐NC, si‐#1 and si‐#2) of GC cells. (I) ELISA experiment detects the content of VEGF‐C in the CM after culturing macrophages with CM from different treatment groups (si‐NC, si‐#1 and si‐#2) of GC cells. (J) Immunofluorescence shows changes in the macrophage marker (CD163) after treatment with CM from different treatment groups: Si‐NC, si‐#1 and si‐#2 (scale bar, 50 μm). (K) Tube formation and transwell experiments detect the effects of tube formation and migration invasion ability of HLECS by macrophage CM from different treatment groups: Si‐NC, si‐#1 and si‐#1 + recombinant VEGF‐C protein. Statistical significance was assessed using two‐tailed t ‐tests. * p < 0.05, ** p < 0.01, *** p < 0.001 (scale bar, 50 μm).

Article Snippet: On the other hand, HLECs are cultured in Endothelial Cell Medium (ECM) supplemented with 10% serum and 0.1% endothelial cell growth factor. recombinant VEGF‐C and VCAN protein were purchased from MedChemExpress (New Jersey, USA), Calcein AM were purchased from Beyotime (Shanghai, China), and Phorbol‐12‐myristate‐13‐acetate (PMA) was sourced from SIGMA (Missouri, USA).

Techniques: Immunofluorescence, Marker, Control, Co-Culture Assay, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Migration, Recombinant, Two Tailed Test

Journal: Cancer Medicine

Article Title: The MIR181A2HG / miR ‐5680/ VCAN ‐ CD44 Axis Regulates Gastric Cancer Lymph Node Metastasis by Promoting M2 Macrophage Polarization

doi: 10.1002/cam4.70600

Figure Lengend Snippet: The MIR181A2HG/miR‐5680/VCAN axis affects lymphangiogenesis by influencing the polarization of M2‐type macrophages. (A) Elisa detection of VCAN protein expression levels in different treatment groups of GC cells: NC, si‐#1 (si‐MIR181A2HG‐1), si‐#1 + miR‐5680 inhibitor, si‐#1 + miR‐5680 inhibitor +si‐VCAN. (B–E) qRT‐PCR detection of typical M2 markers (CD163, CD206, and IL‐10) and M1 markers (iNOS, IL‐6, and TNFα) in PMA‐treated THP‐1 cells cultured with CM from different treatment groups (NC, si‐#1 (si‐MIR181A2HG‐1), si‐#1 + miR‐5680 inhibitor, si‐#1 + miR‐5680 inhibitor +si‐VCAN) of GC cells. (F) ELISA experiment detects the content of VEGF‐C in the supernatant after culturing macrophages with CM from different treatment groups (NC, si‐#1 (si‐MIR181A2HG‐1), si‐#1 + miR‐5680 inhibitor, si‐#1 + miR‐5680 inhibitor +si‐VCAN) of GC cells. (G–K) Tube formation and transwell experiments detect the effects and quantification of tube formation and migration invasion ability of HLECS by macrophage CM from different treatment groups: NC, si‐#1 (si‐MIR181A2HG‐1), si‐#1 + miR‐5680 inhibitor, si‐#1 + miR‐5680 inhibitor +si‐VCAN. Statistical significance was assessed using two‐tailed t ‐tests. * p < 0.05, ** p < 0.01, *** p < 0.001 (scale bar, 50 μm).

Article Snippet: On the other hand, HLECs are cultured in Endothelial Cell Medium (ECM) supplemented with 10% serum and 0.1% endothelial cell growth factor. recombinant VEGF‐C and VCAN protein were purchased from MedChemExpress (New Jersey, USA), Calcein AM were purchased from Beyotime (Shanghai, China), and Phorbol‐12‐myristate‐13‐acetate (PMA) was sourced from SIGMA (Missouri, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Cell Culture, Migration, Two Tailed Test

Journal: Cancer Medicine

Article Title: The MIR181A2HG / miR ‐5680/ VCAN ‐ CD44 Axis Regulates Gastric Cancer Lymph Node Metastasis by Promoting M2 Macrophage Polarization

doi: 10.1002/cam4.70600

Figure Lengend Snippet: VCAN Induces M2 Macrophage Activation via Binding to CD44. (A) Prediction of VCAN interacting proteins based on online databases String, GeneMANIA, Hitpredict, Biogrid, Hint. (B) String database predicts CD44 interacts with VCAN. Purple line: Experimentally determined. Green line: Text mining. Black line: Co‐expression. (C) Co‐IP experiment detects the mutual binding of CD44 and VCAN in polarized macrophages. (D) Immunofluorescence detects the co‐localization of CD44 and VCAN in polarized macrophages (scale bar, 50 μm). (E–H) qRT‐PCR detection of typical M2 markers (CD163, CD206, and IL‐10) and M1 markers (iNOS, IL‐6, and TNFα) in PMA‐treated THP‐1 cells cultured with CM from different treatment groups (NC, VCAN recombinant protein, VCAN recombinant protein + anti‐CD44) of GC cells. (I) Immunofluorescence shows changes in the macrophage marker (CD163) after treatment with CM from different treatment groups: NC, VCAN recombinant protein, VCAN recombinant protein + anti‐CD44 (scale bar, 50 μm). (J) ELISA experiment detects the content of VEGF‐C in the supernatant after culturing macrophages with CM from different treatment groups (NC, VCAN recombinant protein, VCAN recombinant protein + anti‐CD44) of GC cells. (K) Tube formation and transwell experiments detect the effects of macrophage CM from different treatment groups (NC, VCAN recombinant protein, VCAN recombinant protein + anti‐CD44) on the tube formation and migration invasion ability of HLECS (scale bar, 50 μm). (L) Co‐IP experiment detects the mutual binding of CD44 and VCAN in GC cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: On the other hand, HLECs are cultured in Endothelial Cell Medium (ECM) supplemented with 10% serum and 0.1% endothelial cell growth factor. recombinant VEGF‐C and VCAN protein were purchased from MedChemExpress (New Jersey, USA), Calcein AM were purchased from Beyotime (Shanghai, China), and Phorbol‐12‐myristate‐13‐acetate (PMA) was sourced from SIGMA (Missouri, USA).

Techniques: Activation Assay, Binding Assay, Expressing, Co-Immunoprecipitation Assay, Immunofluorescence, Quantitative RT-PCR, Cell Culture, Recombinant, Marker, Enzyme-linked Immunosorbent Assay, Migration

Journal: Life Science Alliance

Article Title: BACH family members regulate angiogenesis and lymphangiogenesis by modulating VEGFC expression

doi: 10.26508/lsa.202000666

Figure Lengend Snippet: (A) Conservation of BACH sites in human, mouse, and zebrafish. The distal site is completely conserved. The proximal site is fully conserved between mouse and human, whereas there are three BACH sites at very close proximity in zebrafish. All proximal sites differ by one nucleotide from the consensus sequence. (B) Chromatin immunoprecipitation assay, followed by PCR measurements, was performed using primer mapping to the above human BACH proximal and distal regulatory sites and DNA precipitated with nonspecific IgG, HA-tag, or BACH1 antibodies. (C) Schematic representation of the wild-type human VEGFC promoter-driven luciferase (Luc) reporters (blue) (pVEGFCwt-Luc) and of three constructs deleted either from proximal (nt. −623 to −603, pVEGFCΔPro-Luc) or distal (nt. −2074 to −2054, pVEGFCΔDis-Luc) BACH-binding sites or a combination thereof (nt −623 to −603 and −2074 to −2054, pVEGFCΔProDis-Luc). Numbers refer to the nucleotide positions relative to ATG (translation initiation). (D) Quantification of dual–luciferase activity in human ES2 cells driven from pVEGFCwt-Luc, pVEGFCΔPro-Luc, pVEGFCΔDis-Luc, and pVEGFCΔProDis-Luc constructs. Relative luciferase activity is shown as a percentage of the pVEGFCwt-Luc value (mean ± SEM, n = 3). * P < 0.0001, Kruskal–Wallis test. (E) Immunofluorescence staining of human ES2 cells stably expressing either an empty pIRES vector (Control) or N-terminally HA-tagged BACH 1 ( BACH1 ) with antibodies directed against the HA tag (red, left panel) or against VEGFC (red, right panel). Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. TSS, transcription start site.

Article Snippet: The 5′ regulatory region of human VEGFC gene ( NG_034216.1 ) encompassing 2,274 nucleotides (−1 to −2,274 in relation to ATG, ) was synthesized by GenScript.

Techniques: Sequencing, Chromatin Immunoprecipitation, Luciferase, Construct, Binding Assay, Activity Assay, Immunofluorescence, Staining, Stable Transfection, Expressing, Plasmid Preparation, Control