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: 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: YAP-downregulation ADSCs reduced the degree of swelling and improved fibrosis mouse tail lymphedema models. (A) Representative images of the mouse tail at 1, 2, 3, and 4 weeks after surgery. (B, C) Tail diameter was measured before and after surgery at the site of 5 mm and 10 mm distal from the incision. (D) Masson staining of the subcutaneous tissue of mouse tail at 2 and 4 weeks after surgery. Scale bar = 500 μm. (E) Statistical analysis of subcutaneous fibrosis ratio. (F) Representative results for Picro-Sirius red staining of mouse tail at 2 and 4 weeks after surgery. Red-yellow fibers represented collagen type I and green fibers with weak birefringence represented collagen type III. Scale bar = 20 μm. (G) Collagen type I/III ratio showed that more Collagen type III was produced in ADSCs (VEGFC+ verteporfin) group. n = 5 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: Staining, Produced

Journal: The Journal of pathology

Article Title: Effect of VEGFC on lymph flow and inflammation-induced alveolar bone loss

doi: 10.1002/path.5456

Figure Lengend Snippet: AAV-VEGFC increases local lymphatic drainage and reduces alveolar bone loss in RA-associated periodontitis. Four-month-old TNF-Tg mice received local injection of AAV-GFP control virus (left maxillary) or AAV-VEGFC virus (right maxillary) into periodontal tissues of the left and right maxillary first molar, respectively. Two months after viral injection, mice were sacrificed and analyzed. (A) Expression of virus-encoded human VEGFC in periodontal tissues of TNF-Tg mice. (B) Paraffin sections of maxillae were subjected to IF for PDPN (red) and αSMA (green). Representative images of periodontal tissue showing the distribution of PDPN+/αSMA− lymphatic capillaries (red), PDPN+/αSMA+ collecting LVs (yellow), and PDPN−/αSMA+ blood vessels (green). Quantification of the PDPN+αSMA− area or PDPN+αSMA+ area to tissue area (%) was determined. The number of PDPN+αSMA+ collecting LVs per mm2 tissue areas (#/mm2) was calculated. (C) An example of in vivo washout of ICG in periodontal tissues reflecting lymph flow through all time points. ICG clearance (%) was quantified. Values shown are mean ± SEM. *p < 0.05 versus AAV-GFP control virus at the same time point. (D) Representative 3D scanned sections (upper panels) and reconstructed sections (lower panels) along the longitudinal direction of the maxillae. Bone mineral density (BMD, g/cm2) was analyzed. (E) Representative images of H&E-stained paraffin sections. Measurement of the bone levels by comparing the distance from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) in μm was determined. (F) Representative images of TRAP-stained paraffin sections. The percentage of alveolar bone surface covered by osteoclasts (Oc.S/B.S) was determined. (G) Representative images of ALP-stained paraffin sections. The percentage of alveolar bone surface covered by osteoblasts (Ob.S/B.S) was determined. *p < 0.05 versus AAV-GFP control virus. N = 6–8. A two-tailed unpaired Student’s t-test was performed.

Article Snippet: A recombinant adeno-associated virus (AAV) encoding a full-length human VEGFC cDNA (NM_005429) was purchased from Genechem Biotech Inc (Shanghai, PR China).

Techniques: Injection, Virus, Expressing, In Vivo, Staining, Two Tailed Test

Journal: The Journal of pathology

Article Title: Effect of VEGFC on lymph flow and inflammation-induced alveolar bone loss

doi: 10.1002/path.5456

Figure Lengend Snippet: AAV-VEGFC increases local lymphatic drainage and reduces alveolar bone loss in ligature-induced periodontitis. Two-month-old WT mice were randomly divided into two groups (six mice per group). One group received local injection of AAV-GFP control virus in periodontal tissues of both the left and right maxillary first molar. Another group received AAV-VEGFC virus injection. Two weeks later, a 5–0 silk ligature was tied around the right maxillary first molar and the contralateral tooth was left unligated to serve as the baseline control. All the mice were sacrificed and analyzed 2 weeks after placement of the ligature. (A) Paraffin sections of maxillae were subjected to IF for PDPN (red) and αSMA (green). Representative images of periodontal tissue showing the distribution of PDPN+/αSMA− lymphatic capillaries (red), PDPN+/αSMA+ collecting LVs (yellow), and PDPN−/αSMA+ blood vessels (green). Quantification of the PDPN+αSMA− area or PDPN+αSMA+ area to tissue area (%) was determined. The number of PDPN+αSMA+ collecting LVs per mm2 tissue areas (#/mm2) was calculated. (B) An example of in vivo washout of ICG in periodontal tissues reflecting lymph flow through all time points. ICG clearance (%) was quantified. Values shown are mean ± SEM. *p < 0.05 versus control + AAV-GFP, #p < 0.05 versus periodontitis + AAV-VEGFC, at the same time point. (C) Representative 3D scanned sections (upper panels) and reconstructed sections (lower panels) along the longitudinal direction of the maxillae. Bone mineral density (BMD, g/cm2) was analyzed. (D) Representative images of H&E-stained paraffin sections. Measurement of the bone levels by comparing the distance from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) in μm was determined. (E) Representative images of TRAP-stained paraffin sections. The percentage of alveolar bone surface covered by osteoclasts (Oc.S/B.S) was determined. (F) Representative images of ALP-stained paraffin sections. The percentage of alveolar bone surface covered by osteoblasts (Ob.S/B.S) was determined. *p < 0.05 in the indicated groups. N = 6. One-way ANOVA followed by Dunnett’s post hoc multiple comparisons test was performed.

Article Snippet: A recombinant adeno-associated virus (AAV) encoding a full-length human VEGFC cDNA (NM_005429) was purchased from Genechem Biotech Inc (Shanghai, PR China).

Techniques: Injection, Virus, In Vivo, Staining

Journal: Cancer Communications

Article Title: NAT10‐mediated ac 4 C‐modified ANKZF1 promotes tumor progression and lymphangiogenesis in clear‐cell renal cell carcinoma by attenuating YWHAE‐driven cytoplasmic retention of YAP1

doi: 10.1002/cac2.12523

Figure Lengend Snippet: NAT10 promoted tumor lymphangiogenesis in ccRCC. (A) GSEA showed the associations between the VEGF signaling and the NAT10 mRNA levels in ccRCC. FDR q < 25% was considered statistically significant. Patients were categorized into low and high subgroups using median expression (50%) as the cut‐off. (B) Cell proliferation curves of HLECs treated with CM from the NAT10‐overexpressed ccRCC cells and the control cells ( n = 4) ( t ‐test for statistics). (C) Cell proliferation curves of HLECs treated with CM from the NAT10‐knockdown ccRCC cells and the control cells ( n = 4) ( t ‐test for statistics). (D) Transwell assays for HLECs treated with CM from the NAT10‐overexpressed ccRCC cells and the control cells ( n = 3) ( t ‐test for statistics). (E) Transwell assays for HLECs treated with CM from the NAT10‐knockdown ccRCC cells and the control cells ( n = 3) ( t ‐test for statistics). (F) Tube formation assays for HLECs treated with CM from the NAT10‐overexpressed ccRCC cells and the control cells ( n = 3) ( t ‐test for statistics). (G) Tube formation assays for HLECs treated with CM from the NAT10‐knockdown ccRCC cells and the control cells ( n = 3) ( t ‐test for statistics). (H) ELISAs were used to detect the VEGFD concentrations in CM from ccRCC cells with NAT10 overexpression or knockdown ( n = 3) ( t ‐test for statistics). (I) IHC of NAT10, VEGFC/D, and LYVE1 in subcutaneous tumors from the NAT10‐knockdown group and the control group ( n = 5) (Mann‐Whitney U for statistics). Results represented at least three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001). Abbreviations: NAT10, N‐acetyltransferase 10; ccRCC, clear‐cell renal cell carcinoma; GSEA, gene set enrichment analysis; FDR, false discovery rate; NES, normalized enrichment score; VEGF, vascular endothelial growth factor; HLEC, human lymphatic endothelial cell; CM, conditioned medium; ELISA, Enzyme‐linked immunosorbent assay; VEGFC/D, vascular endothelial growth factor‐C/D; LYVE1, lymphatic vessel endothelial hyaluronan receptor 1; KEGG, kyoto encyclopedia of genes and genomes.

Article Snippet: The VEGFC/D concentrations in CM from ccRCC cells were detected using human VEGFC/D ELISA Kits (#E‐EL‐H1600c and #E‐EL‐H1601c, Elabscience, Wuhan, Hubei, China) following the manufacturer's protocol.

Techniques: Expressing, Control, Knockdown, Over Expression, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay