Journal: Renal Failure

Article Title: miR-29a-3p/ Vegfa axis modulates high phosphate-induced vascular smooth muscle cell calcification

doi: 10.1080/0886022x.2025.2489712

Figure Lengend Snippet: Figure 1. Identification of Vegfa as a direct target of miR-29a-3p in VSMCs using bioinformatics and dual-luciferase assay. (A) Venn diagram depicting the overlap of miR-29a-3p target genes predicted by four miRNA target prediction tools: miRTarBase, miRWalk, TargetScan, and miRDB. The box highlights the seven genes identified as common targets across all tools. (B) Sequence alignment of miR-29a-3p with the 3′-UTR region of wild-type and mutated Vegfa mRNA. The predicted binding site and seed region are marked in red. (C) Dual-luciferase reporter assay results for VSMCs co-transfected with a control vector, a vector containing the wild type (WT) Vegfa 3′-UTR sequence, or the MUT Vegfa sequence, along with miR-29a-3p mimics or a negative control miRNA (NT). Relative luciferase activity was measured, and data are presented as mean ± SD from three independent experiments.

Article Snippet: VSMCs recovered by centrifugation were homogenized and separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (China National Pharmaceutical Group Corporation Chemical reagent Co., Ltd., China; Cat. #30166428). the proteins were then transferred to polyvinylidene fluoride membranes (Millipore Co., uSa; Cat. #ISEQ00010). the blots were stained with rabbit polyclonal antibodies against VEGFa (1:10,000; Proteintech, uSa; Cat. #19003-1-aP), and mouse monoclonal antibodies against β-actin (1:10,000, Servicebio, China; Cat. #Gb15001), followed by anti-rabbit IgG (1:10,000, SIMubIotECH, China; Cat. #S2001), and anti-mouse IgG horseradish peroxidase conjugates (1:10,000, abclonal, China; Cat. #aS003).

Techniques: Luciferase, Sequencing, Binding Assay, Reporter Assay, Transfection, Control, Plasmid Preparation, Negative Control, Activity Assay

Journal: Renal Failure

Article Title: miR-29a-3p/ Vegfa axis modulates high phosphate-induced vascular smooth muscle cell calcification

doi: 10.1080/0886022x.2025.2489712

Figure Lengend Snippet: Figure 2. miR-29a-3p overexpression suppresses Vegfa expression in VSMCs. (A) Representative Western blot analysis of VEGFA protein levels in VSMCs under standard culture conditions (control), high phosphate conditions (Pi), and high phosphate conditions following miR-29a-3p overexpression (Pi + miR-29a-3p). β-actin served as a loading control. Quantitative analysis of VEGFA protein levels is shown as relative fold change normalized to β-actin. (B) Vegfa mRNA levels quantified using RT-qPCR in the same experimental groups. GAPDH was used as an internal control for normalization. Data represent the mean ± SD of five independent biological replicates.

Article Snippet: VSMCs recovered by centrifugation were homogenized and separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (China National Pharmaceutical Group Corporation Chemical reagent Co., Ltd., China; Cat. #30166428). the proteins were then transferred to polyvinylidene fluoride membranes (Millipore Co., uSa; Cat. #ISEQ00010). the blots were stained with rabbit polyclonal antibodies against VEGFa (1:10,000; Proteintech, uSa; Cat. #19003-1-aP), and mouse monoclonal antibodies against β-actin (1:10,000, Servicebio, China; Cat. #Gb15001), followed by anti-rabbit IgG (1:10,000, SIMubIotECH, China; Cat. #S2001), and anti-mouse IgG horseradish peroxidase conjugates (1:10,000, abclonal, China; Cat. #aS003).

Techniques: Over Expression, Expressing, Western Blot, Control, Quantitative RT-PCR

Journal: Renal Failure

Article Title: miR-29a-3p/ Vegfa axis modulates high phosphate-induced vascular smooth muscle cell calcification

doi: 10.1080/0886022x.2025.2489712

Figure Lengend Snippet: Figure 3. Vegfa knockdown and miR-29a-3p overexpression mitigate high phosphate-induced VSMCs calcification. (A–C) Analysis of Vegfa knockdown effects in VSMCs cultured in normal media (control) or high phosphate conditions (Pi). (A) Vegfa mRNA levels were quantified via RT-qPCR. (B) ARS with corresponding spectrophotometric quantification to assess cellular mineralization. (C) Quantification of intracellular Ca2+ content. Cells were treated with small interfering RNA targeting Vegfa (siVegfa) or a NT control. (D–F) Investigation of the role of miR-29a-3p in VSMCs cultured under high phosphate conditions. (D) Vegfa mRNA expression measured using RT-qPCR, (E) mineralization assessed using ARS and spectrophotometric quantification, and (F) intracellular Ca2+ levels were quantified. Cells were transfected with either an empty vector (VE) or a Vegfa-overexpressing construct (OE), combined with a NT miRNA mimic or a miR-29a-3p mimic. All experiments were performed in five replicates, and error bars represent the SD. GAPDH was used as an internal control in RT-qPCR experiments. Scale bar = 100 µm.

Article Snippet: VSMCs recovered by centrifugation were homogenized and separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (China National Pharmaceutical Group Corporation Chemical reagent Co., Ltd., China; Cat. #30166428). the proteins were then transferred to polyvinylidene fluoride membranes (Millipore Co., uSa; Cat. #ISEQ00010). the blots were stained with rabbit polyclonal antibodies against VEGFa (1:10,000; Proteintech, uSa; Cat. #19003-1-aP), and mouse monoclonal antibodies against β-actin (1:10,000, Servicebio, China; Cat. #Gb15001), followed by anti-rabbit IgG (1:10,000, SIMubIotECH, China; Cat. #S2001), and anti-mouse IgG horseradish peroxidase conjugates (1:10,000, abclonal, China; Cat. #aS003).

Techniques: Knockdown, Over Expression, Cell Culture, Control, Quantitative RT-PCR, Small Interfering RNA, Expressing, Transfection, Plasmid Preparation, Construct

Journal: Scientific Reports

Article Title: Role of Kindlin 2 in prostate cancer

doi: 10.1038/s41598-024-70202-2

Figure Lengend Snippet: K2KO reduces growth of PC3 cells in vivo and reduces tumor angiogenesis and VEGF-R2 phosphorylation. ( A ). K2KO markedly suppressed tumor growth compared to WT PC3 cells as measured by IVIS. ( B ) Representative immunofluorescence confocal micrographs of sections of tumors from mice implanted with WT PC3 cells or K2KO PC3 cells stained with anti-vWF (red) to detect tumor-associated blood vessels. ( C ) Representative immunofluorescence confocal micrographs of sections of tumors, stained for CA9. ( D ) Quantification of CA9 staining (n = 5, mice per group). ( E ) Representative immunofluorescence confocal micrographs of sections of tumors, stained for total VEGF-R2. ( F ) Quantification of VEGF-R2 staining (n = 5, mice per group). ( G ) Representative immunofluorescence confocal micrographs of sections of tumors, stained for phospho VEGF-R2. ( H ) Quantification of phospho VEGF-R2 staining (n = 5, mice per group. Cell nuclei are counterstained with DAPI (blue). Bar, 200 μm.

Article Snippet: Mouse mAb against K2 (clone 3A3) was from Millipore Sigma, rabbit mAb against actin (D18C11) was from Cell Signaling Technology, mouse mAb against vinculin was from Millipore Sigma; mouse anti human aVb3-AF488 (clone LM609) was from Milipore Sigma; mouse anti CA9 mAb was from Abcam; mouse anti EGFP mAb (B2) was from Santa Cruz Biotechnology; rabbit anti VEGF-R mAb was from Cell Signalling Technology; anti-human mouse mAb against β1-integrin PE-conjugated was from BD Pharmingen; rabbit anti- human AR-AF488 was from Millipore Sigma; Alexa-coupled secondary antibodies, and Alexa-coupled phalloidins were from Thermo Fisher Scientific; rabbit polyclonal antibodies against phospho-VEGF R2 (Tyr1054, Tyr1059) were from Invitrogen; rabbit polyclonal antibodies against vWf were from Agilent; horseradish peroxidase–conjugated secondary antibodies were from Bio-Rad; bovine fibronectin was from Milipore Sigma, human fibrinogen was from Milipore Sigma, testosterone was from Milipore Sigma; ECL reagent was from Roche, RPMI, DMEM/F12, penicillin/streptomycin, and l -glutamine were from the Media Lab (Cleveland Clinic).

Techniques: In Vivo, Phospho-proteomics, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: The Role of Vascular Actors in Two Dimensional Dialogue of Human Bone Marrow Stromal Cell and Endothelial Cell for Inducing Self-Assembled Network

doi: 10.1371/journal.pone.0016767

Figure Lengend Snippet: (A) Gene expression VEGF 165 in HBMSC, HUVEC, co-HBMSC and co-HUVEC after being cultured for different time. Data of gene expression was quantified relative to the same gene expression in HBMSC after 6 hours of culture (same in the following gene expression analysis). a and b indicated the difference p≤0.05 or p≤0.01, respectively. Interestingly, we found that VEGF 165 was only expressed by HBMSC and co-HBMSC and VEGF 165 expression in co-HBMSC is much higher than that in HBMSC during 6 hours–18 hours. However, this upregulation in co-HBMSC diminished at 24 hours. (B) Gene expression of KDR in HBMSC, HUVEC, co-HBMSC and co-HUVEC after being cultured for different time. KDR was mainly expressed by endothelial cells and it was much higher in co-HUVEC than in HUVEC from 14–24 hours. At 6 hours, no significant difference has been observed. (C) Gene expression of uPA in HBMSC, HUVEC, co-HBMSC and co-HUVEC after being cultured for different time. uPA has a similar expression style as to the expression of KDR. (D) Gene expression of uPAR in HBMSC, HUVEC, co-HBMSC and co-HUVEC after being cultured for different time. uPAR expressed by all cells and it is higher expressed by co-cultured cells as compared to mono-cultured cell. (E) Immunofluorescence staining of KDR in cells in green. Nuclear was stained in blue with DAPI. Scare bars represent 50 µm. It can be clearly seen that the expression of KDR is much higher in co-cultured cells than in mono-cultured HUVEC. No much KDR could be detected in HBMSC by immunofluorescence.

Article Snippet: Mouse monoclonal antibody against human VE-cad (Hycult Biotechnology) was used at 15 µg/ml, rabbit polyclonal antibody against human VEGF 165 (Millpore) was used at 10 µg/ml and rabbit polyclonal antibody against human uPAR (American Diagostica Inc.) was used at 5 µg/ml.

Techniques: Expressing, Cell Culture, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: The Role of Vascular Actors in Two Dimensional Dialogue of Human Bone Marrow Stromal Cell and Endothelial Cell for Inducing Self-Assembled Network

doi: 10.1371/journal.pone.0016767

Figure Lengend Snippet: (A) HBMSC and HUVEC were co-cultured with neutralizing antibodies against VE-cad, VEGF 165 and uPAR for 24 hours. We found that the neutralization of VE-cad, VEGF 165 and uPAR suppressed the formation of self-assembled network in co-culture system without impairing the viability of cells. Addition of isotype control mouse antibody or normal IgG rabbit antibody had no effect on self-assembled network formation. Scare bars represent 200 µm. (B) The migration velocity of co-HUVEC were analyzed from the images taken by time-lapse videomicroscopy observing co-cultures with or without neutralizing antibody. Interestingly, co-HUVEC in co-culture incubated with VE-cad neutralizing antibody was still be able to migrate. The migration speed is very close to the co-HUVEC in co-culture without any neutralizing antibody. However, the neutralization of VEGF 165 and uPAR totally blocked the migration of co-HUVEC in co-culture system.

Article Snippet: Mouse monoclonal antibody against human VE-cad (Hycult Biotechnology) was used at 15 µg/ml, rabbit polyclonal antibody against human VEGF 165 (Millpore) was used at 10 µg/ml and rabbit polyclonal antibody against human uPAR (American Diagostica Inc.) was used at 5 µg/ml.

Techniques: Cell Culture, Neutralization, Co-Culture Assay, Migration, Incubation

Journal: PLoS ONE

Article Title: The Role of Vascular Actors in Two Dimensional Dialogue of Human Bone Marrow Stromal Cell and Endothelial Cell for Inducing Self-Assembled Network

doi: 10.1371/journal.pone.0016767

Figure Lengend Snippet: (A) Gene expressions of VEGF 165 in HBMSC, HUVEC, Co-HBMSC and co-HUVEC cultured with (bars with shadow) or without VE-cad neutralizing antibody. Neutralization of VE-cad has no strong effects on expression of VEGF 165 . (B) Gene expressions of uPA in HBMSC, HUVEC, Co-HBMSC and co-HUVEC cultured with (bars with shadow) or without VE-cad neutralizing antibody. Neutralization of VE-cad significantly downregulated the expression of uPA in co-HUVEC at 14 hours and 16 hours and this effect decreased at 24 hours. However, the expression of uPA in co-HUVEC still maintained a high level. (C) Gene expressions of uPAR in HBMSC, HUVEC, Co-HBMSC and co-HUVEC cultured with (bars with shadow) or without VE-cad neutralizing antibody. uPAR was transiently affected by neutralization of VE-cad. Its expression statistically decreased in co-HBMSC at 6 hours and 14 hours. (D) Gene expression of uPA in HBMSC, HUVEC, Co-HBMSC and co-HUVEC with (bars with shadow) or without VEGF 165 neutralizing antibody. Neutralization of VEGF 165 strongly suppressed the expression of uPA in co-HUVEC. (E) Gene expression of uPAR in HBMSC, HUVEC, Co-HBMSC and co-HUVEC with (bars with shadow) or without VEGF 165 neutralizing antibody. Gene expression of uPAR in co-cultured cells was downregulated all the time after the addition of VEGF 165 neutralizing antibody. a and b indicated the difference p≤0.05 or p≤0.01, respectively.

Article Snippet: Mouse monoclonal antibody against human VE-cad (Hycult Biotechnology) was used at 15 µg/ml, rabbit polyclonal antibody against human VEGF 165 (Millpore) was used at 10 µg/ml and rabbit polyclonal antibody against human uPAR (American Diagostica Inc.) was used at 5 µg/ml.

Techniques: Cell Culture, Neutralization, Expressing

Journal: PLoS ONE

Article Title: The Role of Vascular Actors in Two Dimensional Dialogue of Human Bone Marrow Stromal Cell and Endothelial Cell for Inducing Self-Assembled Network

doi: 10.1371/journal.pone.0016767

Figure Lengend Snippet: Primer sequences used in Q-PCR.

Article Snippet: Mouse monoclonal antibody against human VE-cad (Hycult Biotechnology) was used at 15 µg/ml, rabbit polyclonal antibody against human VEGF 165 (Millpore) was used at 10 µg/ml and rabbit polyclonal antibody against human uPAR (American Diagostica Inc.) was used at 5 µg/ml.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Dihydroartemisinin Inhibits Laser-Induced Choroidal Neovascularization in a Mouse Model of Neovascular AMD

doi: 10.3389/fphar.2022.838263

Figure Lengend Snippet: VEGFR2 and NF-κB p65 expression at the site of choroidal neovascularization (CNV) lesions analyzed by immunostaining images. The CNV lesion was indicated by the red ellipse. (A) The sections were fluorescently labeled with VEGFR2 antibody (green) and isolectin B4 (red). VEGFR2 had a little positive staining in retinal capillary endothelial cells in both the vehicle and dihydroartemisinin (DHA) groups. However, in CNV formation spots, the positive staining area of VEGFR2 in the DHA group was smaller than that in the vehicle group (white arrow). (B) The sections were fluorescently labeled with NF-κB p65 antibody (green) and isolectin B4 (red). The positive staining area of NF-κB p65 in the DHA group was significantly reduced compared to that in the vehicle group, which was mainly seen in CNV areas (yellow arrow). GCL, Ganglion Cell Layer; INL, Inner Nuclear Layer; ONL, Outer Nuclear Layer. Scale bar, 50 μm.

Article Snippet: The sections were stained with monoclonal antibodies against NF-κB p65 (CST, 8,242, Beverly, MA, United States) or with polyclonal antibodies against VEGFR2 (CST, 2,478, Beverly, MA, United States) at 4 °C overnight and then incubated with corresponding secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen, United States) for 1 h at room temperature in the dark, while the nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI; Sigma Aldrich).

Techniques: Expressing, Immunostaining, Labeling, Staining

Journal: Frontiers in Pharmacology

Article Title: Dihydroartemisinin Inhibits Laser-Induced Choroidal Neovascularization in a Mouse Model of Neovascular AMD

doi: 10.3389/fphar.2022.838263

Figure Lengend Snippet: The impact of DHA on the protein expression levels of the NF-κB family, VEGF, and VEGFR2 in the RPE-choroid complex of a laser-induced CNV mouse model. Mice were intragastrically administered vehicle or DHA once a day for 12 days. (A) The typical Western blot results showed changes in NF-κB family, VEGF, and VEGFR2 protein expression. (B) The quantitative analysis results demonstrated that the expression levels of NF-κB p65, NF-κB1 p50, Rel B, VEGF, and VEGFR2 were markedly increased in the vehicle group compared to the normal control group. DHA significantly inhibited the expression of NF-κB p65, NF-κB1 p50, VEGF, and VEGFR2. n = 3 experiments; * p < .05 vs Nor; # p < .05 vs Laser + Vehicle; one-way ANOVA followed by Tukey’s test. CNV: Choroidal Neovascularization; DHA: Dihydroartemisinin.

Article Snippet: The sections were stained with monoclonal antibodies against NF-κB p65 (CST, 8,242, Beverly, MA, United States) or with polyclonal antibodies against VEGFR2 (CST, 2,478, Beverly, MA, United States) at 4 °C overnight and then incubated with corresponding secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen, United States) for 1 h at room temperature in the dark, while the nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI; Sigma Aldrich).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Dihydroartemisinin Inhibits Laser-Induced Choroidal Neovascularization in a Mouse Model of Neovascular AMD

doi: 10.3389/fphar.2022.838263

Figure Lengend Snippet: Schematic illustration of the mechanisms of the anti-CNV effects of DHA. In addition to the downregulation of VEGF expression, DHA inhibits VEGFR2 expression and angiogenesis through suppression of the NF-κB pathway.

Article Snippet: The sections were stained with monoclonal antibodies against NF-κB p65 (CST, 8,242, Beverly, MA, United States) or with polyclonal antibodies against VEGFR2 (CST, 2,478, Beverly, MA, United States) at 4 °C overnight and then incubated with corresponding secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen, United States) for 1 h at room temperature in the dark, while the nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI; Sigma Aldrich).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Pentagalloyl Glucose, a Major Compound in Mango Seed Kernel, Exhibits Distinct Gastroprotective Effects in Indomethacin-Induced Gastropathy in Rats via Modulating the NO/eNOS/iNOS Signaling Pathway

doi: 10.3389/fphar.2022.800986

Figure Lengend Snippet: Upper panel represents the effect of pretreatment with either pentagalloyl glucose (PGG) (100 or 200 mg/kg) or famotidine on gastric VEGF level of indomethacin-induced gastric injury. The lower panel represents densitometry analysis of the ratio of VEGF protein over β-actin protein. Data are expressed as mean ± SEM, n = 3. * and @ Statistically significant from control or indomethacin groups of rats, respectively, at p < 0.05 by one-way ANOVA was used for statistical analysis followed by Tukey’s post hoc test.

Article Snippet: Ten percent SDS-PAGE was used to separate tissue proteins, which were then transferred to nitrocellulose membranes using the Trans-Blot ® semi-dry transfer cell (Bio- Rad, Hercules, CA, United States) at 20 v for 15 min. After blocking with 1 × Tris-buffered saline/0.1% Tween 20 (TBST) with 5% non-fat dry milk for 1 h, rabbit polyclonal antibodies against VEGF (Catalog no: CAB12303; Assay genie, Dublin, Ireland; dilution, 1:500); primary rabbit polyclonal antibodies against PECAM-1 (Catalog no: PA5-96055; Invitrogen, Thermo Fisher Scientific corporation, Massachusetts, United States; dilution, 1:1,000); and primary rabbit monoclonal antibodies against β-actin (Catalog no: M01263; Boster Biological Technology, Pleasanton, CA, United States; diluted at 1:1,000) were all identified by the antibodies used (Sigma-Aldrich, United States).

Techniques: Control