Conditional deletion of the <i>Podxl</i> locus in endothelial cells.

<p>(<b>A</b>) Schematic representation of the <i>Podxl</i> transgenic allele, floxed allele (Podxl^F/F) and deleted allele (<i>Podxl</i>^Δ<i>F/F</i>). Exons are depicted as vertical lines. Inserted loxP and frt sites are depicted with black and grey arrowheads, respectively. The Neo^R cassette is represented by a box. (<b>B</b>) Capillary gel electrophoresis of genomic DNA isolated and amplified (PCR) from primary lung endothelial cells prepared from mice harboring wild type (WT, 122 bp), floxed (Flox, 171 bp) or functionally deleted (via <i>Cdh5</i>-Cre) <i>Podxl</i> alleles (ΔFlox, 285 bp) (<b>C</b>) qRT-PCR evaluation of podocalyxin mRNA in highly vascularized adult tissues harvested from <i>Podxl</i>^F/F (black bars) and <i>Podxl</i>^ΔEC (white bars) mice (n = 3–6). Expression levels were quantified relative to <i>Gapdh</i> and then normalized to the mean <i>Podxl</i> expression in the <i>Podxl^F/F</i> tissues. *Significantly different compared to <i>Podxl^F/F</i> mouse tissue where P<0.05 by Student's <i>t</i> test. Error bars represent the SEM.</p

Podocalyxin expression on lung endothelial cells is required for efficient spreading on laminin and collagen I matrices.

<p>(<b>A</b>) Static adhesion of lung mECs isolated from <i>Podxl^F/F</i> (black bars) and <i>Podxl</i>^ΔEC (white bars) mice to surfaces coated with fibronectin, laminin, gelatin, collagen I, and collagen IV. Primary mEC were plated on matrix-coated plates for 90 min, washed and adhesion quantified by crystal violet absorbance. The average absorbance for the uncoated wells was normalized to 1 and the relative absorbance (proportional to adhesion) is shown. (<b>B</b>) Spreading of lung mECs isolated from <i>Podxl^F/F</i> (black bars) and <i>Podxl</i>^ΔEC (white bars) mice. Primary mECs were plated on matrix-coated transwells (0.4 µm pore size) and cultured for 48 h. The % monolayer coverage (spreading) was assessed by the threshold area of the cell monolayer via ImageJ on at least 3 independent cultures per genotype. The mean adhesion reported here was pooled from two independent experiments. (<b>C</b>) Representative bright field micrographs of spreading assay described in (E). Error bars  =  SEM, *Significantly different with P<0.05 by Student's <i>t</i> test or one-sample.</p

Podocalyxin deletion results in increased vascular permeability without altered endothelial cell frequency or immunophenotype.

<p>(<b>A</b>) Vascular permeability in lungs assessed by a modified Miles assay. Female mice were treated with PBS (naïve) or LPS (2 mg/kg) intra-tracheally 24 h before sacrifice <i>Podxl^F/F</i> (black bars), and <i>Podxl</i>^ΔEC (white bars) (n = 5–7 mice per genotype). One hour before sacrifice, mice received 20 mg/kg Evan's blue dye via the lateral tail vein. *Significantly different with P<0.05 by when compared to Podxl<i>^F/F</i> naïve mice. #Significantly different (P<0.05) than LPS treated Podxl<i>^F/F</i> mice by one-way ANOVA. (<b>B</b>) Lung edema presented as a ratio of wet/dry lung weight. Lungs were excised and weighed immediately for wet weight and subsequently dried at 60°C for 36 h and weighed again to determine the dry weight (n = 6 mice). (<b>C</b>) Vessel density was determined by the ratio of von Willebrand factor-positive (vWF⁺) staining to total lung tissue. Data represent 4 images per mouse and 6 mice per group. (<b>D</b>) Lung tissue displays normal expression of endothelial cell markers by flow cytometry. Shown are representative flow cytometry histograms. The frequency of CD31⁺ endothelial cells from each genotype is shown in the first profile. All subsequent profiles were gated on CD31⁺ cells in order to focus exclusively on marker expression by endothelia. (<b>E</b>) Expression of junctional proteins in lung. Lungs from <i>Podxl^F/F</i> and <i>Podxl</i>^ΔEC mice were homogenized in RIPA buffer and proteins were resolved on 8% SDS-PAGE gel followed by immunoblotting with the antibodies indicated. Actin was used as an internal loading control. The immunoblots shown are from one experiment and are representative examples of 4 independent mice/genotype. (<b>F</b>) Localization of junctional proteins in lung. Sections from inflated lungs of <i>Podxl^F/F</i> and <i>Podxl</i>^ΔEC mice were stained for ZO-1 (green), claudin-5 (red), and nuclei (DAPI, blue). In endothelial cells, ZO-1 and claudin-5 co-localize (yellow), and ZO-1 is also detected in epithelial cell junctions (green). The immunofluorescence micrographs are representative of 3 images per mouse and 3 mice per genotype.</p

<i>Cdh5</i>-Cre fails to efficiently delete podocalyxin in a subset of organ vascular beds.

<p>(<b>A</b>) In the wildtype heart podocalyxin is expressed by all endothelial cells. In the <i>Podxl</i>^ΔEC mice, podocalyxin is efficiently deleted in large vessels such as the pulmonary artery (arrow, 20x mag. panels) but deletion in the smaller trabecular vessels of the heart muscle is variable (see 10x mag. panels). (<b>B</b>) In the liver of <i>Podxl</i>^ΔEC mice, podocalyxin expression is ablated in the major vessels (portal and central veins, hepatic artery) but not in sinusoidal endothelial cells. (<b>C</b>) In the brain, podocalyxin is normally expressed in the ventricles and endothelial cells, including the microvasculature. Sections of the brain from <i>Podxl</i>^ΔEC mice display similar staining to control mice indicating poor recombination of loxP sites by <i>Cdh5</i>-Cre in brain. Adjacent H&E sections demonstrate normal gross morphology podocalyxin-deficient tissues.</p

Deletion of vascular podocalyxin contributes to structural and functional changes in the lung.

<p>(<b>A</b>) H&E stained sections of lungs inflated to 25 cm H₂O with open thoracic cavity from <i>Podxl^F/F</i> and <i>Podxl</i>^ΔEC mice obtained at 4 weeks and 10 weeks post-natal. Loss of podocalyxin results in increased mean linear intercept (MLI) at 10 weeks of age. The MLI values were determined by computer-assisted image analysis (n = 6 mice per genotype). (<b>B</b>) Mean lung volumes of <i>Podxl^F/F</i> and <i>Podxl</i>^ΔEC mice at 2, 4 and 10 weeks (n = 4–6 mice per genotype). Representative images of inflated lungs at 4 and 10 weeks are shown below the graphs. (<b>C</b>) H&E stained sections of lungs inflated with constant volume with closed chest wall from adult <i>Podxl^F/F</i> and <i>Podxl</i>^ΔEC mice (n = 6 mice per genotype). (<b>D</b>) Resistance measurements from primewave-8 (Rn) and snapshot (R) perturbation. *Significantly different with P<0.05 by Student's <i>t</i> test; **Significantly different with P<0.01 by Student's <i>t</i> test at each time point. The error bars represent the SEM.</p

<i>Cdh5</i>-Cre drives efficient deletion of podocalyxin in the lung.

<p>(<b>A</b>) Podocalyxin expression is completely abrogated in the lung of <i>Podxl</i>^ΔEC mice. (<b>B</b>, <b>C</b>) Within the aorta and vessels of the small intestine (arrows), podocalyxin is deleted in all but a few isolated cells in <i>Podxl</i>^ΔEC mice. (<b>D</b>) Within the kidney, podocalyxin (brown staining) is efficiently deleted in the glomerular endothelial cells (arrows, 40x mag. panels) and larger vessels (arrowheads, 10x mag. panels). Positive staining in the glomerulus of <i>Podxl</i>^ΔEC kidney are likely podocyte epithelial cells. Adjacent H&E sections included demonstrating normal morphology with loss of podocalyxin expression in these tissues.</p

Podocalyxin deletion results in altered integrin and laminin expression in primary lung endothelial cells.

<p>(<b>A</b>) Integrins gene expression in <i>Podxl^F/F</i> (black bars) and <i>Podxl</i>^ΔEC (white bars) cultured lung mEC (results are the mean of 3 independent mEC primary cultures per genotype). (<b>B</b>) Cell surface expression of integrins on lung mECs isolated from <i>Podxl</i>^F/F (blue line) and <i>Podxl</i>^ΔEC (green line) mice. Shown are representative histograms from flow cytometric assays from one experiment. (<b>C</b>) Surface expression levels of integrins were determined by flow cytometry using the mean fluorescence intensity (MFI) of the integrin staining in primary mEC cultures. The mean change in the MFI of <i>Podxl</i>^ΔEC mECs (white bars) compared to <i>Podxl</i>^F/F mEC (black bars, normalized to 1) are from 4 independently derived mEC cultures. Error bars  =  SEM, *Significantly different with P<0.05 or ***significantly different with P<0.005 using one-sample <i>t</i> test with hypothetical value set to 1 (normalized control).</p

Real time qPCR primer list.

<p>Real time qPCR primer list.</p