Journal: bioRxiv

Article Title: OBESITY-INDUCED ENDOTHELIAL FENESTRATION AND CAPILLARY LEAKAGE CONTRIBUTE TO INCREASED PAIN SENSATION

doi: 10.64898/2026.03.13.711502

Figure Lengend Snippet: (A) Schematic diagram of skin vasculature illustrates the organization of blood vessels in the skin layers. In the deep dermis, there are large-diameter blood vessels, including arteries and veins. The intermediate dermis contains arterioles and venules that branch from these arteries and veins, forming an intricate network. Lymphatic vessels are also located in the intermediate dermis. In the superficial dermis, capillaries form a highly branched network. (B) A representative maximum projection image from a whole-mount immunohistochemical analysis of mouse ear skin is presented. This analysis uses the pan-endothelial cell (EC) marker PECAM-1 to visualize skin vasculature from the deep to the superficial dermis. Different colors in the image indicate varying depths (Z-depth) within the dermis. Scale bar: 100 μm. (C) Representative whole-mount images of PECAM-1 + vasculature in the superficial, intermediate, and deep dermis of mouse ear skin are shown. Scale bars: 100 μm. (D) Experimental outline for generating diet-induced obesity (DIO) mice. Mice were fed either regular diet (10 Kcal % fat) or high-fat diet (60 Kcal % fat) from 6 weeks-of-age to 22 weeks-of-age. (E) Representative whole-mount images of the superficial dermal vasculature in the ear skin of control and DIO mice at 22 weeks-of-age, labeled with antibodies for the vascular smooth muscle cell marker αSMA (green or white), the vascular permeability marker PLVAP (MECA-32, red or white), along with PECAM-1 (blue or white) are presented. Scale bars: 100 μm. (F) Quantification of PLVAP + /PECAM-1 + capillaries in the superficial dermal vasculature from control and DIO mice is shown. The sample size is N = 6 in each group. (G) Representative transmission electron microscopy images of capillary ECs in the superficial dermis from control and DIO mice are presented. The dotted box regions in the left panels are magnified in the right panels. Fenestrae were observed only in DIO capillary ECs (arrowheads). Scale bars: 200 nm. (H) Quantification of endothelial fenestration in control and DIO capillary ECs is provided, showing both the number and percentage of non-fenestrated and fenestrated ECs. The sample sizes are as follows: N = 18 in control, N = 27 in DIO. Results are shown as the mean ± SEM. *p<0.05. P values were determined by the parametric two-tailed t test. The schematic diagrams and graphic summary were partially created with BioRender.com .

Article Snippet: A neutralizing anti-PLVAP antibody (clone MECA-32, BioXCell, BE0200) or a rat IgG2a isotype control (BioXCell, BE0089) was diluted to a concentration of 2.11 mg/ml in a 0.9% sodium chloride solution (Millipore Sigma, S8776).

Techniques: Immunohistochemical staining, Marker, Control, Labeling, Permeability, Transmission Assay, Electron Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: OBESITY-INDUCED ENDOTHELIAL FENESTRATION AND CAPILLARY LEAKAGE CONTRIBUTE TO INCREASED PAIN SENSATION

doi: 10.64898/2026.03.13.711502

Figure Lengend Snippet: (A) Schematic diagram illustrates capillary ECs in the superficial dermal vasculature with or without a neutralizing anti-PLVAP antibody. In DIO capillary ECs, fenestrae form, which facilitates molecular leakage from blood to tissue (left). The anti-PLVAP antibody binds to the PLVAP protein, possibly obstructing the fenestrae and inhibiting molecular leakage (right). (B) Schematic diagram illustrating the administration of the anti-PLVAP antibody into DIO mice from 20 weeks-of-age to 22 weeks-of-age using an osmotic pump. (C) Illustration shows intravital imaging of mouse ear skin, along with representative images of dextran extravasation from superficial capillaries. Lectin (green) labels capillaries, and dextran (40 kDa, red) is visible inside the capillaries immediately after injection (t=0), gradually extravasating thereafter (t=10). Scale bars: 100 μm. (D) Representative time-course images show the extravasation kinetics of dextran (40 kDa) in control skin, DIO skin treated with saline, and DIO skin treated with the neutralizing anti-PLVAP antibody. Lectin labels capillaries (green) in the superficial dermis. Time-course rainbow color images show the intensity of dextran. The amount of extravasated dextran is quantified based on the intensity of the dextran signal outside the lectin + capillaries. Scale bars: 100 μm. (E) Changes in dextran (40 kDa) extravasation are shown for control skin (blue), DIO skin treated with saline (red), and DIO skin treated with the neutralizing anti-PLVAP antibody (green). (F) Quantitative measurements of dextran (40 kDa) extravasation at the 4-minute mark are shown. The sample sizes are as follows: N = 13 in control, N = 11 in DIO + Saline, N = 9 in DIO + PLVAP ab. Results are shown as the mean ± SEM. *p<0.05, **p<0.01. P values were determined by the parametric two-tailed t test. The schematic diagrams and graphic summary were partially created with BioRender.com .

Article Snippet: A neutralizing anti-PLVAP antibody (clone MECA-32, BioXCell, BE0200) or a rat IgG2a isotype control (BioXCell, BE0089) was diluted to a concentration of 2.11 mg/ml in a 0.9% sodium chloride solution (Millipore Sigma, S8776).

Techniques: Imaging, Injection, Control, Saline, Two Tailed Test

Journal: bioRxiv

Article Title: OBESITY-INDUCED ENDOTHELIAL FENESTRATION AND CAPILLARY LEAKAGE CONTRIBUTE TO INCREASED PAIN SENSATION

doi: 10.64898/2026.03.13.711502

Figure Lengend Snippet: (A) Schematic diagram illustrates the changes in vascular structure and sensory functions in the skin between control and DIO mice. In the skin of DIO mice, capillary ECs become fenestrated, leading to increased vascular permeability. Additionally, DIO mice exhibit enhanced pain behavior and sensory hypersensitivity . (B) Illustration shows the implantation of an osmotic pump in sensory neuron-specific Pirt-GCaMP3 calcium reporter mice. This pump is used to administer saline, the IgG control, or the neutralizing anti-PLVAP antibody. The sample sizes are as follows: N = 6 in Pirt-GCaMP3 mice on a control diet (control), N = 8 in Pirt-GCaMP3 mice with DIO receiving saline (DIO + Saline), N = 5 in Pirt-GCaMP3 mice with DIO receiving IgG control (DIO + IgG control), N = 8 in Pirt-GCaMP3 mice with DIO receiving the anti-PLVAP antibody (DIO + PLVAP Ab). (C) Illustrations depict the capsaicin-mediated acute pain behavior assay (left) and ex vivo Ca 2+ imaging of peripheral terminals of nociceptive neurons located in the epidermis of the ear skin (right). (D) Total forelimb wiping responses following capsaicin application are shown for control, DIO + Saline, DIO + IgG control, and DIO + PLVAP Ab. (E) Quantification of Ca 2+ responses within the ear skin of control mice, DIO mice with saline, DIO mice with the IgG, and DIO mice with the neutralizing anti-PLVAP antibody is shown. The Ca 2+ transients were normalized by the baseline Ca 2+ transient (ΔF/F 0 ). (F) The integrated Ca 2+ transient (ΔF/F0) was calculated as the area under the curve (AUC). Results are shown as the mean ± SEM. *p<0.05, ***p<0.001. P values were determined by the parametric two-tailed t test. The schematic diagrams and graphic summary were partially created with BioRender.com .

Article Snippet: A neutralizing anti-PLVAP antibody (clone MECA-32, BioXCell, BE0200) or a rat IgG2a isotype control (BioXCell, BE0089) was diluted to a concentration of 2.11 mg/ml in a 0.9% sodium chloride solution (Millipore Sigma, S8776).

Techniques: Control, Permeability, Saline, Behavioral Assay, Ex Vivo, Imaging, Two Tailed Test

Journal: bioRxiv

Article Title: OBESITY-INDUCED ENDOTHELIAL FENESTRATION AND CAPILLARY LEAKAGE CONTRIBUTE TO INCREASED PAIN SENSATION

doi: 10.64898/2026.03.13.711502

Figure Lengend Snippet: (A) Representative section immunohistochemical images of ear skin from control mice, DIO mice treated with saline, and DIO mice treated with the neutralizing anti-PLVAP antibody are presented. This assay uses the antibodies for FOXO1 (green), the keratinocyte marker K14 (red), along with the nuclear marker TOPRO3 (blue). Each inset displays the pattern of FOXO1 expression in a single keratinocyte. Dashed lines indicate the boundary between the epidermis and the dermis. “Epi” indicates the epidermis; “D” indicates the dermis. Scale bars: 20 μm. (B) Quantification of nuclear FOXO1 expression in keratinocytes is provided. The percentages of nuclear FOXO1 expression within the total FOXO1 expression in keratinocytes are presented. The sample sizes are as follows: N = 5 in control, N = 5 in DIO + Saline, N = 7 in DIO + PLVAP Ab. (C) Representative X-gal staining images of ear skin from NGF-LacZ control mice, DIO mice with saline, and DIO mice with the neutralizing anti-PLVAP antibody (blue) are presented. Dashed lines indicate the boundary between the epidermis and the dermis. Scale bars: 50 μm. (D) Quantification of the LacZ-positive area in the epidermis is provided. The sample sizes are as follows: N = 9 in control, N = 15 in DIO + Saline, N = 9 in DIO + PLVAP Ab. (E) Graphical summary illustrates how vascular hyperpermeability leads to sensory hypersensitivity in DIO skin. Increased permeability in the superficial dermal capillaries facilitates the diffusion of insulin into the epidermis, activating insulin signaling in epidermal keratinocytes. This activation leads to NGF upregulation in these keratinocytes, which in turn promotes sensory hypersensitivity in DIO skin. A neutralizing anti-PLVAP antibody reduces the diffusion of insulin, thereby decreasing NGF expression in the epidermal keratinocytes and alleviating sensory hypersensitivity. Results are shown as the mean ± SEM. *p<0.05, **p<0.01. P values were determined by the parametric two-tailed t test. The schematic diagrams and graphic summary were partially created with BioRender.com .

Article Snippet: A neutralizing anti-PLVAP antibody (clone MECA-32, BioXCell, BE0200) or a rat IgG2a isotype control (BioXCell, BE0089) was diluted to a concentration of 2.11 mg/ml in a 0.9% sodium chloride solution (Millipore Sigma, S8776).

Techniques: Immunohistochemical staining, Control, Saline, Marker, Expressing, Staining, Permeability, Diffusion-based Assay, Activation Assay, Two Tailed Test

Journal:

Article Title: Notch signaling is essential for vascular morphogenesis in mice

doi:

Figure Lengend Snippet: Yolk sac and placental defects in mutant embryos. (A–D) Morphology of embryos in their yolk sacs at E9.5. Large vitelline blood vessels (arrowheads) are observed in the Notch1+/− and Notch4−/− yolk sacs, but not in the Notch1−/− and Notch1−/− Notch4−/− mutants. (E–G) PECAM-1-stained yolk sacs. The yolk sacs of the Notch1−/− and Notch1−/− Notch4−/− mutant embryos are at the primitive vascular plexus stage and have not undergone vascular remodeling to form the large and small blood vessels of the mature yolk sac. (H,I) Histological sections of PECAM-1-stained yolk sacs. The Notch1+/− yolk sac (H) has differentiated both small capillaries (arrowheads) and large vitelline collecting vessels (arrow). The Notch1−/− yolk sac (I) exhibits a disorganized, confluent vascular plexus. (J,K) Histological sections of placentas at E9.5. In the Notch1+/− Notch4−/− control embryo (J), embryonic blood vessels containing nucleated erythrocytes (arrowheads) have invaded the labyrinthine layer of the placenta. In the Notch1−/− Notch4−/− mutant embryo (K), embryonic blood vessels are present at the edge of the placenta but have not invaded the labyrinthine layer.

Article Snippet: Embryos for immunohistochemistry were fixed in 4% paraformaldehyde in PBS and stained with a monoclonal anti-PECAM-1 antibody (clone MEC 13.3; Pharmingen).

Techniques: Mutagenesis, Staining

Journal:

Article Title: Notch signaling is essential for vascular morphogenesis in mice

doi:

Figure Lengend Snippet: Defects in vascular remodeling in Notch1−/− and Notch1−/− Notch4−/− mutant embryos. (A–G) PECAM-1-stained whole mount embryos. (A–D) Defective morphogenesis of the main trunk of the anterior cardinal vein (arrowhead) in Notch1−/− (B) and Notch1−/− Notch4−/− mutant embryos (C,D). The double mutant embryo in D is more severely affected than the embryo in C. (E–G) In the Notch1+/− Notch4−/− control embryo (E), intersomitic vessels (arrowheads) differentiate, whereas these vessels are not observed in the Notch1−/− (F) and Notch1−/− Notch4−/− (G) mutant embryos. (H–J) Histological sections of PECAM-1-stained embryos at the level of the otic vesicle. In the N1+/− control embryo (H), both the dorsal aortae (arrowheads) and the anterior cardinal veins (arrows) have open lumens and normal morphology. In the Notch1−/− Notch4−/− mutant embryo (J), endothelial cells have differentiated but both the dorsal aortae and the anterior cardinal veins have an abnormal, collapsed morphology. In the less-severely affected Notch1−/− mutant embryo (I), the anterior cardinal veins still have an open lumen but the dorsal aortae are collapsed.

Article Snippet: Embryos for immunohistochemistry were fixed in 4% paraformaldehyde in PBS and stained with a monoclonal anti-PECAM-1 antibody (clone MEC 13.3; Pharmingen).

Techniques: Mutagenesis, Staining

Journal: The Journal of Comparative Neurology

Article Title: Sympathetic innervation of inguinal white adipose tissue in the mouse

doi: 10.1002/cne.25031

Figure Lengend Snippet: List of used resources

Article Snippet: Then, tissues were incubated with primary antibodies (chicken anti‐GFP [1:400], Abcam, Cambridge, MA; rabbit anti‐TH [1:400], Millipore, Burlington, MA; CD31/PECAM‐1 [1:300], Novus Biologicals, Littleton, CO) in primary incubation solution (92 ml PTwH (1 ml of 10 mg/ml heparin stock solution, 2ml Tween‐20, quantum satis to 1 liter with PBS), 5 ml DMSO, 3 ml NDS) on a shaking incubator at 37°C for at least 7 days.

Techniques: Sequencing, Positive Control, Concentration Assay, Recombinant, Transgenic Assay, Expressing, Cell Culture, Transfection, Affinity Purification, Staining, Membrane, Mutagenesis, Virus, Software