Journal: International Journal of Molecular Sciences

Article Title: Potassium Dehydroandrograpolide Succinate Targets NRP1 Mediated VEGFR2/VE-Cadherin Signaling Pathway to Promote Endothelial Barrier Repair

doi: 10.3390/ijms24043096

Figure Lengend Snippet: PDA activates the VEGF signaling pathway after vascular injury. ( A ) HUVEC-Cs were treated with PDA (10 μM) for 24 h. The transcription levels of the VEGF family were evaluated with real-time PCR ( n = 6). ( B ) Western blotting was performed to determine the expression of VEGFR2 in HUVECs after PDA (10 μM, 24 h) treatment at different doses, and the quantification data are evaluated in ( C ) ( n = 8). ( D ) IF staining was performed to detect the expression of VEGFR2 in the HUVECs after PDA (10 μM) treatment for 24 h, and the quantification data are evaluated in ( E ) ( n = 8). ( F ) IHC staining was performed against CD31 and VEGFR2 antibodies to evaluate the expression of VEGFR2 in a left carotid artery partial ligation model following PDA treatment for 28 days. Yellow arrows indicate regions containing CD31–positive and VEGFR2-positive vascular endothelial cells. The quantification data are evaluated in ( G ) ( n = 8). ( H ) IF staining was used to evaluate the expression of VEGFR2 in blood vessels within the anterior tibial muscle of mice that were intramuscularly injected with cardiotoxin and treated with PDA (20 mg/kg) for 7 consecutive days, and the quantification data are evaluated in ( I ) ( n = 8). Analytical data are expressed as means ± SEM * p < 0.05.

Article Snippet: The antibodies used in this study included CD31 (Cell Signaling Technology, Danvers, MA, USA), NRP1 (Proteintech, Chicago, IL, USA), VE-Cad (Cell Signaling Technology), VEGFR2 (Cell Signaling Technology), Mac2 (Sigma, St. Louis, MO, USA) and BRDU (Invitrogen, Waltham, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Staining, Immunohistochemistry, Ligation, Injection

Journal: International Journal of Molecular Sciences

Article Title: Potassium Dehydroandrograpolide Succinate Targets NRP1 Mediated VEGFR2/VE-Cadherin Signaling Pathway to Promote Endothelial Barrier Repair

doi: 10.3390/ijms24043096

Figure Lengend Snippet: PDA regulates interactions between NRP1, VEGFR and VE-Cad within vascular endothelial cells. ( A ) Molecular docking simulation performed to evaluate the binding energy of PDA with NRP1, VEGFR2 and VE-Cad using Autodock Vina 1.5.6 software, which was developed by Olson’s research group. PDA worked as a receptor, and NRP1, VEGFR2 and VE-Cad were used as ligands to detect the docking sites between receptors and ligands. The three-dimensional structures of VEGFR2 and NRP1 were obtained from the RCSBPDB database ( http://www.rcsb.org/ , accessed on 16 August 2022) and that of VE-Cad was obtained from the SWISS-Model database ( http://swissmodel.expasy.org/ , accessed on 20 August 2022). When values for the binding energy were less than zero, those proteins were considered to spontaneously bind and interact with each other. ( B ) The binding energies of PDA with NRP1, VEGFR2 and VE-Cad were determined based on a molecular docking simulation. ( C ) IF staining of NRP1 and VEGFR2 was performed in the skeletal muscle injury model to determine their co-localization expression in vascular endothelial cells. ( D ) Immunofluorescence staining of NRP1 and VE-Cad was performed in the skeletal muscle injury model to determine their co-localization expression in vascular endothelial cells. ( E ) The interaction between NRP1, VEGFR2 and VE-Cad was validated using the CO-immunoprecipitation assay in HUVECs.

Article Snippet: The antibodies used in this study included CD31 (Cell Signaling Technology, Danvers, MA, USA), NRP1 (Proteintech, Chicago, IL, USA), VE-Cad (Cell Signaling Technology), VEGFR2 (Cell Signaling Technology), Mac2 (Sigma, St. Louis, MO, USA) and BRDU (Invitrogen, Waltham, MA, USA).

Techniques: Binding Assay, Software, Staining, Expressing, Immunofluorescence, Co-Immunoprecipitation Assay

Journal: International Journal of Molecular Sciences

Article Title: Potassium Dehydroandrograpolide Succinate Targets NRP1 Mediated VEGFR2/VE-Cadherin Signaling Pathway to Promote Endothelial Barrier Repair

doi: 10.3390/ijms24043096

Figure Lengend Snippet: PDA regulates vascular endothelial barrier function through the NRP1/VEGFR2/VE-Cad signaling pathway. ( A ) After knockdown of NRP1 in HUVECs, real-time PCR was performed to evaluate transcription levels of NRP1-, VEGFR2- and VE-Cad-related genes ( n = 6). ( B ) After knockdown of NRP1, HUVECs were treated with PDA (10 μM) for 24 h and real-time PCR was performed to evaluate transcription levels of NRP1-, VEGFR2- and VE-Cad-related genes ( n = 6). ( C ) After knockdown of NRP1, IF staining was performed to detect the expression of VE-Cad in the HUVECs following PDA (10 μM) treatment for 24 h, and the quantification data are evaluated in ( D ) ( n = 8). ( E ) After knockdown of NRP1, HUVECs were treated with PDA (10 μM) for 24 h and real-time PCR was performed to evaluate transcription levels of inflammation-related genes ( n = 6). ( F ) After knockdown of NRP1, HUVEC-C and THP-1 were co-cultured and treated with PDA (10 μM) for 12 h, and the quantification data are evaluated in ( G ). ( H ) Schematic diagram indicating that PDA promoted endothelial barrier repair in pathological vascular remodeling. Analytical data are expressed as means ± SEM * p < 0.05.

Article Snippet: The antibodies used in this study included CD31 (Cell Signaling Technology, Danvers, MA, USA), NRP1 (Proteintech, Chicago, IL, USA), VE-Cad (Cell Signaling Technology), VEGFR2 (Cell Signaling Technology), Mac2 (Sigma, St. Louis, MO, USA) and BRDU (Invitrogen, Waltham, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Staining, Expressing, Cell Culture

Journal: Theranostics

Article Title: Cancer-associated fibroblasts facilitate premetastatic niche formation through lncRNA SNHG5-mediated angiogenesis and vascular permeability in breast cancer

doi: 10.7150/thno.74753

Figure Lengend Snippet: Breast CAF-derived CCL2 and CCL5 activate P38 MAPK signaling in endothelial cells. (A-B) Western blotting was used to analyze p-P38, P38, p-VEGFR2, VEGFR2, ZO-1, and Occludin levels in HUVECs incubated with CM from NF/Ctrl, CCL2, CCL5, CCL2/CCL5 or CAF/shNC, shCCL2, shCCL5, shCCL2/shCCL5. The pixel intensity of p-P38 was normalized to that of β-Actin for the quantification of p-P38 levels (B). (C-D) Western blotting was used to analyze the levels of p-P38, P38, p-VEGFR2, VEGFR2, ZO-1 and Occludin in HUVECs incubated with FBS-free medium with DMSO, rCCL2, rCCL5, rCCL2 and rCCL5 or rCCL2 and rCCL5 combined with axitinib, RS102895, maraviroc, or cenicriviroc. Levels of p-P38 (D) were quantified by normalizing its relative pixel intensity to β-Actin. (E-F) Effects of CM from NF/Ctrl, CCL2, CCL5, CCL2/CCL5 or CAF/shNC, shCCL2, shCCL5, shCCL2/shCCL5 on tube formation (E) and permeability (F) of HUVECs. (G-H) Effects of rCCL2, rCCL5, rCCL2 and rCCL5 or rCCL2 and rCCL5 combined with axitinib, RS102895, maraviroc, or cenicriviroc on tube formation (G) and permeability (H) of HUVECs. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: A total of 50-80 μg protein was separated using 8-12% SDS-PAGE gel and electrically transferred to 0.22 μM PVDF membrane (Bio-Rad, USA), which was blocked with 5% nonfat milk at room temperature for 3 h and then incubated at 4 °C overnight with the special primary antibodies: Occludin (Abcam, ab216327, 1:1000), ZO-1 (Cell Signaling, #8193, 1:1000), ZNF281 (SAB, #43566, 1:1000), IGF2BP2 (Proteintech, 11601-1-AP, 1:2000), VEGFA (Abcam, ab1316, 1:1000), CCL2 (SAB, #45065, 1:500), CCL5 (SAB, #32935, 1:500), p-AKT (CST, #12694, 1:1000), AKT (CST, #9272, 1:1000), p-STAT3 (CST, #9131, 1:500), STAT3 (CST, #4904, 1:500), p-P38 (CST, #4631, 1:500), P38 (CST, #9212, 1:1000), p-VEGFR2 (Sangon, D155165, 1:800), VEGFR2 (Sangon, D151118, 1:800), and β-Actin (Biosharp, 1:1000).

Techniques: Derivative Assay, Western Blot, Incubation, Permeability