anti mouse vegfr2 polyclonal antibody  (R&D Systems)

Journal: Advanced Science

Article Title: Selective Targeting of Tip Endothelial Cells as a Therapeutic Strategy for Tumor Angiogenesis

doi: 10.1002/advs.202512975

Figure Lengend Snippet: PRND is selectively expressed in tip endothelial cells ( Tip EC) and promotes tip cell–like behavior. (A) UMAP projection of tumor‐associated endothelial cells (ECs), colored by inferred EC subtypes: arterial, venous, stalk‐like, tip‐like, and transition. (B) Overlay of PRND expression on the UMAP shows selective enrichment in Tip EC. (C) Violin plot showing PRND expression across EC subtypes, with highest level detected in Tip EC. (D) Volcano plot comparing gene expression between tip‐like and stalk‐like ECs. PRND is among the most significantly upregulated genes in tip‐like ECs. Genes with adjusted p < 0.05 and Log2 fold change > 0.25 were considered differenially expressed. (E) Quantification of double (CD34 + Dll4 + ) and triple (CD34 + Dll4 + CXCR4 + ) gated Tip EC after 24 h treatment with recombinant Doppel (rDpl, 1 µg mL −1 ) in HDMECs. Dots represent independent experiments, n = 3 per condition. Two‐tailed Student's t ‐test; p * < 0.05. (F) Representative images of VEGF‐induced endothelial sprouting from HUVEC spheroids treated with increasing concentrations of rDpl. White asterisks indicate HUVEC sprouts. Scale bar, 100 µm. Right panel: Quantification of sprouts per spheroid. n = 5 experimental replicates; 40–50 spheroids were counted per experiment. See Also Figure . Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple‐comparison test; p ** < 0.01, nonsignificant comparisions not shown. (G) Heatmap of the top 20 differentially expressed genes (DEGs) between PRND + and PRND − ECs. A total of 1500 randomly selected cells per group were analyzed. Rows represent genes; columns represent individual cells. Expression values were log‐normalized and Z‐scored. Color scale: red = high expression; blue = low expression. (H) WikiPathways enrichment analysis of DEGs in PRND + versus PRND − ECs, highlighting VEGF/VEGFR2 signaling as the top enriched pathway. Dot size indicates gene count; color represents adjusted p ‐value. All data are presented as mean ± standard deviation (SD).

Article Snippet: For VEGFR2 staining, goat anti‐mouse VEGFR2 polyclonal antibody (1:200, AF644, R&D Systems) followed by Alexa 488‐linked donkey anti‐goat antibodies were used.

Techniques: Expressing, Gene Expression, Recombinant, Two Tailed Test, Comparison, Standard Deviation

Journal: Advanced Science

Article Title: Selective Targeting of Tip Endothelial Cells as a Therapeutic Strategy for Tumor Angiogenesis

doi: 10.1002/advs.202512975

Figure Lengend Snippet: Doppel promotes tip cell morphology and cytoskeletal remodeling. (A) Volcano plot showing differentially expressed genes (DEGs) between PRND + versus PRND − within the Tip EC population. Genes with adjusted p < 0.05 and Log2 fold change > 0.25 were considered significant. (B) Gene Ontology enrichment analysis of DEGs in PRND + versus PRND − Tip ECs. Dot size indicates gene count; color represents adjusted p‐ value. Pathways related to actin cytoskeleton and cell motility are highlighted. (C) Quantification of EC length in HDMECs transfected with either mock‐ or Doppel‐constructs. Doppel‐transfected cells exhibit elongated morphology and filopodia‐like protrusions., n = 6 experimental replicates. Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple‐comparison test; p * < 0.05, nonsignificant comparisions not shown. Corresponding images are shown in Figure . White arrows indicate filopodia. Scale bar, 100 µm. (D) Representative confocal images of HDMECs transfected with Doppel cDNA or mock vector. Doppel‐PLA, red dots; phalloidin, green; DAPI, blue. Scale bar, 10 µm. (E) Representative confocal image showing subcellular distribution of VEGFR2 in Doppel‐ and mock‐transfected HDMECs. In Doppel‐expressing cells, VEGFR2 localizes toward the leading edge. VEGFR2‐PLA, red dots; Phalloidin, green; DAPI, blue. Scale bar, 10 µm. Right panel: Number of VEGFR2‐PLA dots either in cytoplasm or leading‐edge. n = 5 experimental replicates; 50–100 cells were counted per experiment. Statistical analysis was performed using two‐tailed Student's t ‐test; p **** < 0.0001, p ** < 0.01. (F) RTK phospho‐array analysis of Hu Dpl cells transfected with scramble or Doppel siRNA. Right panel: Quantification by pixel density. n = 3 experimental replicates. (G) Western blot analysis for phosphorylated and total Src in ECs isolated from SCC7 and CT26 tumors following transfection with either Doppel or scramble siRNA. Doppel protein levels confirm knockdown efficiency. (H) Western blot analysis for active and total CDC42 and RhoA in Hu Dpl cells following transfection with either Doppel or scramble siRNA under VEGF stimulation. All data are presented as mean ± standard deviation (SD).

Article Snippet: For VEGFR2 staining, goat anti‐mouse VEGFR2 polyclonal antibody (1:200, AF644, R&D Systems) followed by Alexa 488‐linked donkey anti‐goat antibodies were used.

Techniques: Transfection, Construct, Comparison, Plasmid Preparation, Expressing, Two Tailed Test, Western Blot, Isolation, Knockdown, Standard Deviation

Journal: Advanced Science

Article Title: Selective Targeting of Tip Endothelial Cells as a Therapeutic Strategy for Tumor Angiogenesis

doi: 10.1002/advs.202512975

Figure Lengend Snippet: Doppel facilitates Tip EC mobility and stability through VEGFR2‐mediated signaling. (A) Schematic of a bioengineering platform with alternating VEGF‐rich (V + ) and VEGF‐depleted (V − ) stripes for directional endothelial cell migration. (B) Brightfield and confocal microscopy images showing VEGFR2 localization relative to VEGF‐A gradients in HUVEC and Hu Dpl cells. n = 9∼10 experimental replicates. VEGFR2, red; Phalloidin, green; DAPI, blue. Scale bar, 10 µm. Right panel: Ratio of VEGFR2‐proximal PLA dots in V + and V − areas for both HUVEC and Hu Dpl cells. (C) Schematic of a bioengineered angiogenesis platform incorporating ECs to visualize Tip EC migration and representative fluorescent image showing the sprouting of Hu Dpl cells along a V + stripe. CD31, red; VEGFR2, green; DAPI, blue. Scale bar, 100 µm (D) Confocal image of Tip EC cells expressing activated integrin in Hu Dpl sprouts. Activated αVβ3 integrein, red; VEGFR2, green; DAPI, blue. Scale bar, 100 µm (E) Quantification of sprouting length in Hu Dpl versus HUVEC spheroids per V + stripe. n = 10 sprouts. (F) Quantification of Tip EC number in Hu Dpl versus HUVEC spheroids per V + stripe. n = 20 individual fields. (G) Representative confocal image of Dll4 expression in Tip EC from Hu Dpl versus HUVEC. Dll4, purple (left)/ grayscale (right); VEGFR2, green; DAPI, blue. Scale bar, 25 µm. Right panel: Quantification of Dll4‐positive signals per nucleus in Hu Dpl versus HUVEC sprouts in the V + stripes. n = 20 individual fields. (H) Schematic model illustrating Doppel's role in Tip EC differentiation, filopodia extension and directed migration through VEGFR2 signaling. Doppel enhances VEGFR2 localization to the leading edge and activates Dll4 and downstream Src signaling pathways that stabilize tip cell identity and motility. All data are presented as mean ± standard deviation (SD). Statistical analysis was performed using two‐tailed Student's t ‐test unless otherwise stated; p **** < 0.0001, p *** < 0.001, p ** < 0.01.

Article Snippet: For VEGFR2 staining, goat anti‐mouse VEGFR2 polyclonal antibody (1:200, AF644, R&D Systems) followed by Alexa 488‐linked donkey anti‐goat antibodies were used.

Techniques: Migration, Confocal Microscopy, Expressing, Protein-Protein interactions, Standard Deviation, Two Tailed Test

Journal: Advanced Science

Article Title: Selective Targeting of Tip Endothelial Cells as a Therapeutic Strategy for Tumor Angiogenesis

doi: 10.1002/advs.202512975

Figure Lengend Snippet: Selective depletion of Tip ECs via Doppel blocking antibodies. (A) Tumor growth curves following treatment with anti‐Dll4 (navy), anti‐VEGFR2 (yellow), anti‐Doppel (red) antibodies, as well as control (gray). Mice were treated at 10 mg kg −1 once every 4 days, n = 7. Two‐way ANOVA followed by Tukey's multiple‐comparison test on day 12. (B,C) Flow cytometric quantification of endothelial subtypes in tumors: CD31 + CXCR4 + Tip ECs (B) and CD31 + SELP + stalk ECs (C). (D,E) Proportion of VEGFR2 + cells in Tip EC (D) and stalk EC (E) subsets. (F) Schematic representation of Tip EC states based on relative VEGFR2 expression. Top: Highly differentiated Tip ECs exhibiting a high tip/stalk VEGFR2 + ratio. Bottom: Competing or unstable Tip ECs characterized by a low tip/stalk VEGFR2 + ratio, indicating phenotypic plasticity and competition with neighbouring Stalk EC. (G) Quantification of VEGFR2 + Tip EC/ Stalk EC ratio in tumors from each group. (H) Representative confocal images of tumor vasculature stained for endothelial and Tip EC markers. White asterisks indicate CD31 + CXCR4 + Tip ECs. CXCR4, green; CD31, red; DAPI, blue. Scale bar, 20 µm. (I) Quantification of Tip ECs per vessel. Dots indicate individual vessels. (J) Representative immunofluorescence images of liver tissue sections of mice treated with anti‐VEGFR2 antibody, anti‐Dll4 antibody, and anti‐Doppel antibody, as well as control. red; CD31, green; DAPI, blue. (K) Tumor growth of 4T1 orthotopic tumors treated with control IgG (gray), anti‐Doppel antibody (red), and anti‐VEGFR2 (yellow). Mice were treated at 10 mg kg −1 once every 4 days, n =7. Two‐way ANOVA followed by Tukey's multiple‐comparison test. (L) Flow‐cytometric quantification of tip endothelial cells ( Tip EC: CD31 + CXCR4 + ). All data are presented as mean ± standard deviation (SD). Arrows indicate the treatment initiation and tumor size at that time. Statistical analysis was performed using one/two‐way ANOVA followed by Tukey's multiple‐comparison test unless otherwise stated; p **** < 0.0001, p *** < 0.001, p ** < 0.01, p * < 0.05, nonsignificant comparisons not shown.

Article Snippet: For VEGFR2 staining, goat anti‐mouse VEGFR2 polyclonal antibody (1:200, AF644, R&D Systems) followed by Alexa 488‐linked donkey anti‐goat antibodies were used.

Techniques: Blocking Assay, Control, Comparison, Expressing, Staining, Immunofluorescence, Standard Deviation

Journal: Science Advances

Article Title: Low-flow intussusception and metastable VEGFR2 signaling launch angiogenesis in ischemic muscle

doi: 10.1126/sciadv.abg9509

Figure Lengend Snippet: ( A ) Confocal micrographs of projected optical sections of native EDL muscle (top row, 20-μm-thick projection) and EDL muscle 5 days after ischemic injury (bottom row, 16 μm thick), immunostained for CD31 (green), VEGFR2 (grayscale), p-VEGFR2 (red), and DAPI (blue). Arrows depict the borders of a mother vessel with several microdivisions/early splitting zones (asterisks). Strong p-VEGFR2 immunoreactivity is present in the primordial vessels but not capillaries. ( B ) Epifluorescence micrographs of transverse sections of EDL muscle 5 days after ischemic injury and 16 hours after receiving vehicle (left) or apatinib (right), immunostained for CD31, p-VEGFR2, and DAPI. p-VEGFR2 signals are diminished in CD31-positive microvessels and nonvascular cells in mice receiving apatinib. ( C ) RBC transit maps of microvascular networks in regenerating (5-day) EDL muscle 16 hours after receiving apatinib, cabozantinib or ZM323881 (small-molecule inhibitors of VEGFR2) or DC101 (VEGFR2-blocking antibody), and respective controls. Pillars have been circled yellow (arrows) and microdivisions/splits are outlined in dashed blue lines. Graphs depicting the density of microvascular networks are shown on the right [means ± SE; n = 26/13, 17/12, 17/19, and 21/18 distinct muscle territories (0.63 mm 2 ) in the EDL subjected to control or designated blocking agent]. The effects of cabozantinib and ZM323881 were studied using the same vehicle controls [dimethyl sulfoxide/polyethylene glycol (PEG)/saline], and P values are Bonferroni corrected. ( D and E ) Graphs depicting the density of pillars (D) and splits (E) in EDL muscle 5 days after ischemic injury. Data are expressed relative to the respective controls, depicted by the dashed line (means ± SE, n = 13, 12, 19, and 18).

Article Snippet: Triple immunostaining was performed using rat monoclonal anti-CD31 antibody (1:20; Clone SZ31, Dianova), rabbit polyclonal anti–p-VEGFR2 Y1054/1059 antibody, and goat polyclonal anti-VEGFR2 antibody (1:100; AF644, R&D Systems).

Techniques: Blocking Assay, Control, Saline

Journal: Science Advances

Article Title: Low-flow intussusception and metastable VEGFR2 signaling launch angiogenesis in ischemic muscle

doi: 10.1126/sciadv.abg9509

Figure Lengend Snippet: ( A ) Confocal micrographs of planar-projected optical sections of a primordial vessel in EDL muscle 5 days after ischemic injury immunostained for CD31 (green) and p-(activated) VEGFR2 (red), with TO-PRO-3–stained nuclei. A CD31-expressing pillar is present (arrow). There is abundant p-VEGFR2 signal in endothelial cells of the wall but not the pillar. ( B ) Graph depicting p-VEGFR2 signals in endothelial pillars versus wall-lining endothelium of the same vessel. N = 8 pillars from different primordial vessels. Wall and pillar endothelial cell data from a given primordial vessel are connected. A.U., arbitrary units. ( C ) Confocal planar projection of a primordial vessel showing a CD31-expressing pillar endothelial cell (arrow) with modest p-VEGFR2 signal lateralized to one face, orthogonal to the vessel long axis (small arrows). ( D ) Confocal orthogonal (XZ, YZ) reconstructions of a day 5 primordial vessel showing an endothelial cell pillar structure inside the lumen. p-VEGFR2 signal is abundant in the lining endothelium, but on the pillar p-VEGFR2 appears as a discrete lateralized signal. ( E ) Distribution of p-VEGFR2–positive pillars with diffuse versus lateralized p-VEGFR2 signal. ( F ) Confocal planar projections through a pillar within a primordial vessel in day 5–regenerated muscle showing VEGFR2 activity that is focal, lateralized, and enriched at the midzone of the pillar (arrows). The two planar projections are 3 μm apart. A schematic of the micrograph is depicted (right). ( G ) Distribution of p-VEGFR2 locations in lumen-crossing pillars.

Article Snippet: Triple immunostaining was performed using rat monoclonal anti-CD31 antibody (1:20; Clone SZ31, Dianova), rabbit polyclonal anti–p-VEGFR2 Y1054/1059 antibody, and goat polyclonal anti-VEGFR2 antibody (1:100; AF644, R&D Systems).

Techniques: Staining, Expressing, Activity Assay

Journal: Science Advances

Article Title: Low-flow intussusception and metastable VEGFR2 signaling launch angiogenesis in ischemic muscle

doi: 10.1126/sciadv.abg9509

Figure Lengend Snippet: ( A ) Graph depicting the aspect ratio of control and VEGFR2 knockdown endothelial cells lining a PDMS microfluidic device subjected to ultralow shear stress (0.3 dyne/cm 2 ). A total of 394 cells with control siRNA and 379 with VEGFR2 siRNA from four different devices were quantified. Median values and all data points are shown. ( B ) Pillar content, defined as the proportion of endothelial cells at microchannel shoulders that formed a pillar, for differentially labeled control and VEGFR2 knockdown endothelial cells. Data are from four independent experiments and depicted as mean and SE. ( C to E ) Confocal microscopy volume projections of differentially labeled control and VEGFR2 knockdown endothelial cells lining a microfluidic device and subjected to ultralow shear stress. In (C) and (D), control-siRNA cells express GFP, and VEGFR2-siRNA cells express RFP. In (E), the labeling is reversed. Nuclei were stained with DAPI. In (C) and (E), pillars are composed of VEGFR2-siRNA cell projections that span across the lumen and connect with a control-siRNA cell on the opposite wall. In (D), a VEGFR2-siRNA cell body crosses the lumen and is connected on either side with control siRNA cells lining the device walls. ( F ) Pillar content of microfluidic channels lined by HUVECs subjected to ultralow shear stress (0.3 dyne/cm 2 ) and infused with L-NAME (10 μM) or water vehicle. ( G ) Pillar content of endothelial cell–lined microchannels under ultralow shear conditions infused with DETA NONOate (1 μM), ZM323881 (10 nM), or the combined interventions. Data are means and SE.

Article Snippet: Triple immunostaining was performed using rat monoclonal anti-CD31 antibody (1:20; Clone SZ31, Dianova), rabbit polyclonal anti–p-VEGFR2 Y1054/1059 antibody, and goat polyclonal anti-VEGFR2 antibody (1:100; AF644, R&D Systems).

Techniques: Control, Knockdown, Shear, Labeling, Confocal Microscopy, Staining