Claudin5 protects the peripheral endothelial barrier in an organ and vessel-type-specific manner
- Department of Immunology, Genetics and Pathology, Uppsala University, Sweden
- Beijer Gene- and Neuro Laboratory and Science for Life Laboratories, Uppsala University, Sweden
- Oulu Center for Cell-Matrix Research, Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, University of Oulu, Finland
- Department of Medicine Huddinge, Karolinska Institutet, Campus Flemingsberg, Neo, Sweden
Figures
Figure 1 with 5 supplements
Patterning of the EC barrier at the single-cell level.
(A) Principal component analysis of the distances within 400 trajectories calculated with integrated data of murine datasets of ear skin, trachea, skeletal muscle, and heart blood endothelial cells …
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Figure 1—source data 1
Spreadsheets detailing the results of the differential gene expression analysis conducted between mouse BEC subtypes in ear skin.
- https://cdn.elifesciences.org/articles/78517/elife-78517-fig1-data1-v2.xlsx
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Figure 1—source data 2
Spreadsheets detailing the results of the differential gene expression analysis conducted between mouse BEC subtypes in trachea.
- https://cdn.elifesciences.org/articles/78517/elife-78517-fig1-data2-v2.xlsx
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Figure 1—source data 3
Spreadsheets detailing the results of the differential gene expression analysis conducted between mouse BEC subtypes in skeletal muscle.
- https://cdn.elifesciences.org/articles/78517/elife-78517-fig1-data3-v2.xlsx
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Figure 1—source data 4
Spreadsheets detailing the results of the differential gene expression analysis conducted between mouse BEC subtypes in heart.
- https://cdn.elifesciences.org/articles/78517/elife-78517-fig1-data4-v2.xlsx
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Figure 1—source data 5
Spreadsheet detailing the results of the differential gene expression analysis conducted between human dermal BEC subtypes.
- https://cdn.elifesciences.org/articles/78517/elife-78517-fig1-data5-v2.xlsx
Figure 1—figure supplement 1
Gating strategy for the FACS isolation of single blood vessel BECs from the mouse ear skin.
Figure 1—figure supplement 2
BEC subset allocation and clustering of individual mouse organs.
(A) Ear skin, (B) trachea, (C) skeletal muscle, and (D) heart mouse BECs colored by subset as defined by analysis of integrated data. The number of cells in each subtype are specified in each …
Figure 1—figure supplement 3
Expression of mouse endothelial junctional components.
Violin plots of gene expression for mouse endothelial junctional components.Gene expression was normalized to account for differences in sample library size and imputed to account for dropouts in …
Figure 1—figure supplement 4
Analysis of human skin BECs.
(A) Uniform manifold approximation and projection (UMAP) of human dermal BECs showing the distance of an isolated trajectory calculated with tSpace. (B) Equidistant binning of the trajectory shown …
Figure 1—figure supplement 5
Expression of human dermal endothelial junctional components.
Violin plots of gene expression for human dermal endothelial junctional components. Gene expression was normalized to account for differences in library size and imputed to account for dropouts in …
Figure 2 with 1 supplement
Organotypic integrity of the EC barrier.
(A) Leakage patterning in Cldn5(BAC)-GFP mouse ear skin in response to intradermal histamine. Left, overlay of Cldn5(BAC)-GFP-positive and -negative vessels (visualised through circulating TRITC …
Figure 2—figure supplement 1
Histamine leakage in skeletal muscle and heart vasculature.
Leakage and Cldn5(BAC)-GFP expression patterning in skeletal muscle and heart in response to the systemic administration of histamine. Magnified images of dashed boxes are shown to the right of the …
Figure 3 with 1 supplement
Claudin5 exhibits organotypic protection of the EC barrier.
(A) Schematic illustration of systemic tamoxifen regime. (B) Representative western blot of Claudin5 protein expression in control and Cldn5 iECKO mice. (C) Quantification of Claudin5 protein …
Figure 3—figure supplement 1
Cldn5 targeting and histamine-induced leakage quantification.
(A) Schematic diagram showing the targeting strategy of Cldn5 floxed mice. (B) Quantification of histamine-induced (10 mg/kg) leakage of 2000 kDa dextran from the heart, skeletal muscle and ear skin …
Figure 4 with 1 supplement
Loss of Claudin5 differentially affects vessel subtypes in the ear dermis.
(A) Representative images of histamine-induced 2000 kDa dextran leakage in the ear skin of control (left) and Cldn5 iECKO (right) mice. (B) Representative time-lapse images of 2000 kDa dextran …
Figure 4—figure supplement 1
4-hydroxytamoxifen-mediated Claudin5 loss, histamine receptor expression and vessel-specific leakage in Cldn5 iECKO mice.
(A) Schematic illustration of topical 4-hydroxytamoxifen regime. (B) Claudin5 protein expression in the ear skin of control and Cldn5 iECKO mice following topical tamoxifen treatment. Left, …
Figure 5 with 1 supplement
Claudin5 regulates junction protein expression.
(A–C) Area (A) width (B) and intensity (C) of electron dense regions in the ear skin of control and Cldn5 iECKO mice after visualisation by TEM. Right, representative TEM images of junctions in the …
Figure 5—figure supplement 1
Endothelial junction protein expression in Cldn5 iECKO mice.
(A) Representative western blots of ESAM, Cingulin, and JAM-A expression in control and Cldn5 iECKO mouse lung lysates. (B) Scatter graphs showing the correlation between Claudin5 expression levels …
Videos
Video 1
Histamine-mediated leakage in Cldn5(BAC)-GFP mice.
Extravasation of circulating 2000 kDa TRITC Dextran (pseudocolor) after intradermal injection of histamine in the ear dermis of Cldn5(BAC)-GFP mice. The first 30 frames show a still at t=0 to show Cl…
Video 2
Histamine-mediated leakage in control and Cldn5 iECKO mice.
Extravasation of circulating 2000 kDa FITC Dextran (pseudocolor) in control (left) and Cldn5 iECKO (right) mice after intradermal injection of histamine in the ear dermis.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | C57BL/6 J | Taconic | B6-F/M | |
| Genetic reagent (Mus musculus) | Cldn5(BAC)-GFP | Laviña et al., 2018 | N/A | |
| Genetic reagent (Mus musculus) | Cldn5 iECKO | This paper | N/A | See Figure 3—figure supplement 1 and Methods-Animals |
| Genetic reagent (Mus musculus) | Cldn5fl/fl; Rosa26lox-STOP-lox-YFP; Cdh5CreERT2 | This paper | N/A | See Methods-Animals |
| Antibody | Mouse monoclonal anti-GAPDH | Millipore | MAB374 | 1:1,000 |
| Antibody | Rat monoclonal anti-CD31 | BD Biosciences | 553370 | 1:100 |
| Antibody | Goat polyclonal anti-CD31 | R&D Systems | AF3628 | 1:100 |
| Antibody | Goat polyclonal anti-VE-Cadherin | R&D Systems | AF1002 | 1:100, 1:1000 |
| Antibody | Chicken polyclonal anti-GFP | Abcam | Ab13970 | 1:100 |
| Antibody | Rabbit polyclonal anti-Claudin5 | ThermoFischer Scientific | 341600 | 1:100, 1:1000 |
| Antibody | Rabbit polyclonal anti-ZO-1 | ThermoFischer Scientific | 617300 | 1:100, 1:1000 |
| Antibody | Rabbit polyclonal anti-Occludin | ThermoFischer Scientific | 711500 | 1:1,000 |
| Antibody | Rabbit monoclonal anti-JAM-A | Martìn-Padura et al., 1998 | N/A | 1:1000 |
| Antibody | Rabbit polyclonal anti-Cingulin | Cardellini et al., 1996 | N/A | 1:1000 |
| Antibody | Goat polyclonal anti-ESAM | R&D Systems | AF2827 | 1:1000 |
| Antibody | Goat polyclonal anti-collagen IV | Merck Millipore | AB789 | 1:100 |
| Antibody | Mouse monoclonal anti-αSMA FITC | Sigma Aldrich | F3777 | 1:100 |
| Antibody | Mouse monoclonal anti- αSMA Cy3 | Sigma Aldrich | C6198 | 1:100 |
| Antibody | Rat monoclonal Anti-CD16/32 | ThermoFischer Scientific | 14-0161-85 | 1:100 |
| Antibody | Rat monoclonal Anti-CD31 FITC | BD Biosciences | 553372 | 1:50 |
| Antibody | Rat monoclonal Anti-CD45 APC | BioLegend | 103112 | 1:50 |
| Antibody | Rat monoclonal Anti-Lyve1 eFluor 660 | ThermoFischer Scientific | 50–0443082 | 1:50 |
| Antibody | Donkey polyclonal anti-rat alexa 488 | ThermoFischer Scientific | A21208 | 1:400 |
| Antibody | Donkey polyclonal anti-rat alexa 594 | ThermoFischer Scientific | A21209 | 1:400 |
| Antibody | Donkey polyclonal anti-rabbit alexa 488 | ThermoFischer Scientific | A21206 | 1:400 |
| Antibody | Donkey polyclonal anti-rabbit alexa 568 | ThermoFischer Scientific | A10042 | 1:400 |
| Antibody | Donkey polyclonal anti-goat alexa 647 | ImmunoResearch Laboratories | 705-605-147 | 1:400 |
| Antibody | Donkey polyclonal anti-chicken alexa 488 | ImmunoResearch Laboratories | 703-545-155 | 1:400 |
| Antibody | Sheep polyclonal anti-mouse HRP | Cytiva | NA931 | 1:10,000 |
| Antibody | Sheep polyclonal anti-rabbit HRP | Cytiva | NA934 | 1:10,000 |
| Sequence-based reagent | Cldn5 probe | ACD Bio | 491611-C2 | |
| Sequence-based reagent | 3-plex negative control probes | ACD Bio | 320871 | |
| Sequence-based reagent | 3-plex positive control probes | ACD Bio | 320811 | |
| Sequence-based reagent | GAPDH | ThermoFischer Scientific | Mm99999915_g1 | |
| Sequence-based reagent | Cldn5 | ThermoFischer Scientific | Mm00727012_s1 | |
| Sequence-based reagent | Cdh5 | ThermoFischer Scientific | Mm00486938_m1 | |
| Sequence-based reagent | Tjp1 | ThermoFischer Scientific | Mm01320638_m1 | |
| Sequence-based reagent | Ocln | ThermoFischer Scientific | Mm00500912_m1 | |
| Sequence-based reagent | F11r | ThermoFischer Scientific | Mm00554113_m1 | |
| Sequence-based reagent | Cgn | ThermoFischer Scientific | Mm01263534_m1 | |
| Sequence-based reagent | Esam | ThermoFischer Scientific | Mm00518378_m1 | |
| Peptide, recombinant protein | Collagenase IV | Worthington | LS004183 | |
| Peptide, recombinant protein | DNase I | Worthington | LS006333 | |
| Peptide, recombinant protein | HRP | SigmaAldrich | 77332 | |
| Commercial assay or kit | RNAscope Fluorescent Multiplex Assay | ACD Bio | 322340, 320851 | |
| Commercial assay or kit | RNeasy Plus kit | Qiagen | 74034 | |
| Commercial assay or kit | iScript Adv cDNA Kit for RT-qPCR | Bio-Rad | 1725038 | |
| Chemical compound, drug | Tamoxifen | SigmaAldrich | T5648 | |
| Chemical compound, drug | 4-hydroxytamoxifen | SigmaAldrich | H7904 | |
| Chemical compound, drug | Oxazolone | SigmaAldrich | E0753 | |
| Chemical compound, drug | Histamine | SigmaAldrich | H7125 | |
| Other | Live/Dead near IR cell stain | ThermoFischer Scientific | L10119 | See Materials and methods-Ear dermal single cell isolation |
| Other | Phosphatase inhibitor cocktail | Roche | 04906837001 | See Materials and methods-Western blot analysis |
| Other | LDS sample buffer | Invitrogen | NP0007 | See Materials and methods-Western blot analysis |
| Other | Sample reducing agent | Invitrogen | NP0009 | See Materials and methods-Western blot analysis |
| Other | MOPS SDS running buffer | Invitrogen | NP0001 | See Materials and methods-Western blot analysis |
| Other | PVDF membrane | Thermofischer Scientific | 88518 | See Materials and methods-Western blot analysis |
| Other | NuPAGE transfer buffer | Novex | NP006 | See Materials and methods-Western blot analysis |
| Other | RNAlater | ThermoFischer Scientific | AM7024 | See Materials and methods-quantitative PCR |
| Other | 2000 kDa FITC Dextran | SigmaAldrich | FD2000S | See Materials and methods-permeability analysis |
| Other | 2000 kDa TRITC Dextran Lysine Fixable | ThermoFischer Scientific | D7139 | See Materials and methods-permeability analysis |
| Other | 10 kDa TRITC Dextran | ThermoFischer Scientific | D1817 | See Materials and methods-permeability analysis |
| Other | 4 kDa TRITC Dextran | Tdb labs | TD4 | See Materials and methods-permeability analysis |
| Other | 10 kDa FITC Dextran | Tdb labs | FD10 | See Materials and methods-permeability analysis |
| Other | 70 kDa TRITC Dextran | Tdb labs | TD70 | See Materials and methods-permeability analysis |
| Other | 2000 kDa FITC Dextran lysine fixable | Tdb labs | FLD2000 | See Materials and methods-permeability analysis |
| Other | Anti-Isolectin GS-IB4 | Molecular Probes | I32450 | See Materials and methods-Immunohistochemistry |
