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: The Journal of Clinical Investigation

Article Title: Claudin-2 deficiency associates with hypercalciuria in mice and human kidney stone disease

doi: 10.1172/JCI127750

Figure Lengend Snippet: (A–C) Confocal microscopy of papillary sections from Cldn2–/y mice shows von Kossa staining of deposits (pseudocolored white) colabeled with the following tubule markers: (A) Inner medullary collecting duct (AQP2) and thin descending limbs (AQP1). (B) Thin descending limbs (AQP1) and thin ascending limbs (CLC-K). (C) Inner medullary collecting ducts (AQP2) and vasa recta (MECA32). Association of calcium deposits with tubule markers is infrequent, but instances of colocalization with AQP1- or CLC-K–positive thin limbs are occasionally seen (arrows in A and B). Scale bars: 50 μm. (D and E) Electron microscopy of papillae from 5-month-old Cldn2–/y mice shows small mineral deposits within type 4 thin descending limb cells with frequent tight junctions (arrowheads) (D) as well as type 3 thin ascending limb cells lacking these features (E). Scale bars: 2 μm.

Article Snippet: The primary antibodies used were CLC-K (1:200; Alomone Labs ACL-004), AQP1 (1:500; Abcam ab9566), AQP2 (1:500; a gift from Mark Knepper, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA), MECA32 (1:100; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa, USA).

Techniques: Confocal Microscopy, Staining, Electron Microscopy

Journal: PLoS ONE

Article Title: Caveolae, Fenestrae and Transendothelial Channels Retain PV1 on the Surface of Endothelial Cells

doi: 10.1371/journal.pone.0032655

Figure Lengend Snippet: A–B) Protein levels of PV1, Cav1 and CD31 in the lung (A) and kidney (B) total membranes of Cav1−/−, Cav1+/− and WT mice were detected by immunoblotting. C) Protein levels of PV1, cavin-1 and CD31 in the lung total membranes of cavin-1−/− and WT mice were detected by immunoblotting. D) PV1 mRNA levels in the lung ( left panel ) and kidney ( right panel ) of WT, Cav1−/− and cavin-1−/− mice.

Article Snippet: Prior to the experiment the MLEC-wt and MLEC-Cav1KO cells were serum-starved (2 h, 37°C) in serum-free endothelial basal medium 2 (EBM2) (Lonza), followed by labeling (30 min, 10°C) with fluorophore coupled rat anti-mouse PV1 MECA-32 mAb (1.5 μg/ml) in EBM2 supplemented with 2% BSA.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Caveolae, Fenestrae and Transendothelial Channels Retain PV1 on the Surface of Endothelial Cells

doi: 10.1371/journal.pone.0032655

Figure Lengend Snippet: A) Protein levels of PV1 in MLEC-wt( WT ), MLEC-Cav1KO ( Cav1KO ) and MLEC-Cav1-ECRC ( ECRC ) cells detected by immunoblotting with anti-PV1 antibodies. M - Corresponds to membrane proteins, C – cytosolic proteins. Equal amount of membrane protein was loaded whereas the cytosolic proteins were normalized to membrane extract volume. The membrane and cytosolic proteins were also partially deglycosylated with PNGase F ( + ), which resulted in the drop in PV1 molecular weight. Note very low PV1 level in Cav1KO cells and increased PV1 protein level in cells reconstituted with Cav1 (Cav1-ECRC). The top and bottom panels are different exposures of the same blot. B) PV1 is predominantly associated with caveolae on the surface of lung endothelial cells. PV1 ( red ) colocalizes with Cav1-EGFP ( green ) at the plasma membrane of live MLEC, as shown by TIRFM. Insets demonstrate the extensive colocalization of the two labels ( white arrowheads ). Scale bars −20 µm.

Article Snippet: Prior to the experiment the MLEC-wt and MLEC-Cav1KO cells were serum-starved (2 h, 37°C) in serum-free endothelial basal medium 2 (EBM2) (Lonza), followed by labeling (30 min, 10°C) with fluorophore coupled rat anti-mouse PV1 MECA-32 mAb (1.5 μg/ml) in EBM2 supplemented with 2% BSA.

Techniques: Western Blot, Membrane, Molecular Weight, Clinical Proteomics

Journal: PLoS ONE

Article Title: Caveolae, Fenestrae and Transendothelial Channels Retain PV1 on the Surface of Endothelial Cells

doi: 10.1371/journal.pone.0032655

Figure Lengend Snippet: A) PV1 mRNA levels in MLEC-wt ( WT ) and MLEC-Cav1KO ( Cav1KO ) cells measured by real time quantitative PCR. The data was obtained from quadruplicate samples and normalized to b-actin mRNA levels (ΔΔCt). Bars – SEM. B) Pulse 35 S metabolic labeling of MLEC-WT ( top panel ) and MLEC-Cav1KO ( bottom panel ) cells followed by PV1 immunoprecipitation at the indicated time points and 35 S fluorography. Duplicate samples are shown for each time point assessed. PV1 has four active N-glycosylation sites and therefore shows five bands, the lowest representing the non-glycosylated form and the four higher bands representing various degrees of N-glycosylation. C) Densitometric quantitation of the amount of PV1 translated after 10 min pulse with 35 S-methionine and cysteine in MLEC-WT and MLEC-Cav1KO cells. Error bars correspond to SEM (n = 3).

Article Snippet: Prior to the experiment the MLEC-wt and MLEC-Cav1KO cells were serum-starved (2 h, 37°C) in serum-free endothelial basal medium 2 (EBM2) (Lonza), followed by labeling (30 min, 10°C) with fluorophore coupled rat anti-mouse PV1 MECA-32 mAb (1.5 μg/ml) in EBM2 supplemented with 2% BSA.

Techniques: Real-time Polymerase Chain Reaction, Labeling, Immunoprecipitation, Glycoproteomics, Quantitation Assay

Journal: PLoS ONE

Article Title: Caveolae, Fenestrae and Transendothelial Channels Retain PV1 on the Surface of Endothelial Cells

doi: 10.1371/journal.pone.0032655

Figure Lengend Snippet: A) Schematic of the timeline ( upper right ) and the principal steps of PV1 internalization flow cytometric assay ( right ). An example of data gating and fluorescence intensity histogram is given in the lower left panels. B) Amount of PV1 on the surface of MLEC-wt ( WT ) and MLEC-Cav1KO ( Cav1 KO) at t 0 expressed as median fluorescence intensity per cell from fluorophore-labeled anti-PV1 ( PV1 ). Labeling of cells with isotype control non-immune antibodies showed the level of unspecific binding ( control ) (error bars correspond to stdev, n = 4, *p<0.01). C) Amount of internalized PV1 at different time points in MLEC-WT at 37°C ( solid line ) and 4°C ( dashed line ) expressed as median fluorescence intensity per cell from fluorophore-labeled anti-PV1 ( PV1 ) (stdev, n = 6, *p<0.01). D) PV1 internalization in MLEC-WT ( WT, top panels ) and MLEC-Cav1KO ( Cav1 KO, bottom panels) cells at different time points, as detected by confocal microscopy. Images are maximum projections of confocal stacks in green channel (PV1, lower panels ) or green merged with blue (nuclei labeled with DAPI, upper panels ). E) Internalization rate of PV1 in MLEC-WT ( solid line, solid circles ) and Cav1KO ( dashed line, open circles ) cells, expressed as a percentage from the total amount of PV1 on the cell surface. (stdev, n = 4 per time point, *p<0.01). F) Degradation curves of 35 S labeled PV1 in MLEC-Cav1KO (Cav1KO, dashed line , open circles ) and MLEC-WT (WT, solid circles ), isolated from Cav1−/− and wild type mice, respectively. Data is representative of three experiments carried out in duplicate. G, H) PV1 degradation rates were measured in MLEC-WT (WT) and MLEC-Cav1KO (Cav1KO) treated with lysosomal or proteasomal inhibitors. G) Western blots used for densitometric quantifications of PV1 protein level. H) Quantitation of protein levels of PV1.

Article Snippet: Prior to the experiment the MLEC-wt and MLEC-Cav1KO cells were serum-starved (2 h, 37°C) in serum-free endothelial basal medium 2 (EBM2) (Lonza), followed by labeling (30 min, 10°C) with fluorophore coupled rat anti-mouse PV1 MECA-32 mAb (1.5 μg/ml) in EBM2 supplemented with 2% BSA.

Techniques: Flow Cytometry, Fluorescence, Labeling, Control, Binding Assay, Confocal Microscopy, Isolation, Western Blot, Quantitation Assay

Journal: PLoS ONE

Article Title: Caveolae, Fenestrae and Transendothelial Channels Retain PV1 on the Surface of Endothelial Cells

doi: 10.1371/journal.pone.0032655

Figure Lengend Snippet: A) PV1 does not colocalize with clathrin-GFP on the cell surface. Confocal micrographs of MLEC-WT transfected with clathrin-GFP ( Clathrin, green ) and labeled with fluorescent anti-PV1 antibodies ( PV1, red ). The insets represent a low power field with two transfected cells. The areas in shaded in grey are magnified in lowed panels. B–G) PV1 and transferrin internalization rates in MLECs were quantified by flow cytometry. Error bars correspond to StDev. B–D) Percentage of fluorescent antibody labeled PV1 internalized from the cell surface. B) PV1 internalization at 15 and 60 min in presence and absence of the clathrin pathway inhibitor PitStop2 ( PS2 ) or the inactive PitStop2 negative control ( NC ) (n = 4, p >0.05). C,D) PV1 internalization at 15 and 60 min in presence of dynamin inhibitors Dyngo-4a (C) (n = 4, p >0.05) or Dynasore ( D ) (n = 4, p>0.05). E) Median fluorescent intensity of transferrin-Alexa647 internalized within 10 min in the presence and absence of PitStop2, Dynasore or Dyngo4a (n = 4, * p <0.01). D–G) Internalization of PV1 (F) and transferrin (G) at 15 min in untransfected MLECs (mock) and MLECs transfected with eGFP-encoding plasmid (GFP), dynamin 2-eGFP fusion (Dyn2 wt) or dominant-negative form of dynamin 2 fused to eGFP (Dyn2 K44A MLECs (n = 4, * p <0.01). H) Schematic of PV1 ( green ) trafficking in ECs. De novo formed PV1 enters the secretory pathway and arrives at the cell surface by exocytosis ( green arrow ) using secretory vesicles (Step 1). On the plasma membrane PV1 is targeted to caveolae, fenestrae or TEC (Step 2) where it forms diaphragms. PV1 is internalized via clathrin- and dynamin-independent endocytic mechanism ( Step 3 and 4 ) followed by degradation in the lysosomes ( Step 5 ).

Article Snippet: Prior to the experiment the MLEC-wt and MLEC-Cav1KO cells were serum-starved (2 h, 37°C) in serum-free endothelial basal medium 2 (EBM2) (Lonza), followed by labeling (30 min, 10°C) with fluorophore coupled rat anti-mouse PV1 MECA-32 mAb (1.5 μg/ml) in EBM2 supplemented with 2% BSA.

Techniques: Transfection, Labeling, Flow Cytometry, Negative Control, Plasmid Preparation, Dominant Negative Mutation, Clinical Proteomics, Membrane