Loss of vascular CD34 results in increased sensitivity to lung injury

Survival during lung injury requires a coordinated program of damage limitation and rapid repair. CD34 is a cell surface sialomucin expressed by epithelial, vascular and stromal cells that promotes cell adhesion, coordinates inflammatory cell recruitment, and drives angiogenesis. To test whether CD34 also orchestrates pulmonary damage and repair, we induced acute lung injury in wild type (WT) and Cd34-/- mice by bleomycin (BLM) administration. We found that Cd34-/- mice displayed severe weight loss and early mortality compared to WT controls. Despite equivalent early airway inflammation to WT mice, CD34-deficient animals developed interstitial edema and endothelial delamination, suggesting impaired endothelial function. Chimeric Cd34-/- mice reconstituted with WT hematopoietic cells exhibited early mortality compared to WT mice reconstituted with Cd34-/- cells, supporting an endothelial defect. CD34-deficient mice were also more sensitive to lung damage caused by influenza infection, showing greater weight loss and more extensive pulmonary remodeling. Together our data suggest that CD34 plays an essential role in maintaining vascular integrity in the lung in response to chemical- and infection-induced, tissue damage

UNBOUND

Unbound showcases the graduating class from the fashion design school at Fanshawe College. Unbound describes the creative spirit and achievements of our twenty-seven emerging Canadian fashion designers. Unbound 2014 is a professional collaboration between Fanshawe College, Community and Professionals in the Fashion Industry. As you turn the pages, admire their accomplishments - the results of three years of passion, hard work, and dedication.https://first.fanshawec.ca/famd_design_fashiondesign_unbound/1003/thumbnail.jp

Podocalyxin promotes vascular barrier function

The CD34-family sialomucin, podocalyxin (Podxl), is broadly expressed on the luminal face of blood vessels in adult mammals; however, its biological function on vascular endothelial cells (vEC) is not well-defined. Here, we reveal specific functions for podocalyxin in maintaining endothelial barriers using HUVEC monolayers as a model in vitro. Detailed analysis of barrier HUVEC characteristics using electrical cell-substrate impedance sensing (ECIS) and live cell imaging revealed essential roles for podocalyxin in maintaining cell-cell and cell-matrix interactions. Thus, podocalyxin-deficient HUVEC fail to form a functional barrier when plated on several extracellular matrix (ECM) substrates. Regardless of ECM substrate, these monolayers lack adherens junctions and focal adhesions; and display a disorganized cortical actin cytoskeleton. To explore an in vivo function of podocalyxin, we conditionally deleted Podxl in vEC using the Tie2Cre strain (PodxlΔTie2Cre). Although we did not detect altered permeability in naïve mice at steady state, systemic priming with lipopolysaccharides (LPS) disrupted the blood-brain barrier (BBB) in PodxlΔTie2Cre but not WT mice. To study the potential consequence of this BBB breach, we used a selective agonist of PAR-1, a thrombin receptor expressed by neurons and glial cells. As a polar peptide, the PAR-1 agonist (TFLLRN), is normally excluded from CNS parenchyma by the BBB. In response to systemic administration of TFLLRN, LPS-primed PodxlΔTie2Cre mice experienced a dramatic behavioral change marked by a severely dampened neurological electrical activity. We conclude that podocalyxin expression by CNS vECs is required to maintain BBB integrity under inflammatory conditions. Supplementary materials available at: http://hdl.handle.net/2429/68998Medicine, Faculty ofExperimental Medicine, Division ofMedicine, Department ofGraduat

Conventional laboratory housing increases morbidity and mortality in research rodents: results of a meta-analysis

Background Over 120 million mice and rats are used annually in research, conventionally housed in shoebox-sized cages that restrict natural behaviours (e.g. nesting and burrowing). This can reduce physical fitness, impair thermoregulation and reduce welfare (e.g. inducing abnormal stereotypic behaviours). In humans, chronic stress has biological costs, increasing disease risks and potentially shortening life. Using a pre-registered protocol ( https://atrium.lib.uoguelph.ca/xmlui/handle/10214/17955 ), this meta-analysis therefore tested the hypothesis that, compared to rodents in ‘enriched’ housing that better meets their needs, conventional housing increases stress-related morbidity and all-cause mortality. Results Comprehensive searches (via Ovid, CABI, Web of Science, Proquest and SCOPUS on May 24 2020) yielded 10,094 publications. Screening for inclusion criteria (published in English, using mice or rats and providing ‘enrichments’ in long-term housing) yielded 214 studies (within 165 articles, using 6495 animals: 59.1% mice; 68.2% male; 31.8% isolation-housed), and data on all-cause mortality plus five experimentally induced stress-sensitive diseases: anxiety, cancer, cardiovascular disease, depression and stroke. The Systematic Review Center for Laboratory animal Experimentation (SYRCLE) tool assessed individual studies’ risks of bias. Random-effects meta-analyses supported the hypothesis: conventional housing significantly exacerbated disease severity with medium to large effect sizes: cancer (SMD = 0.71, 95% CI = 0.54–0.88); cardiovascular disease (SMD = 0.72, 95% CI = 0.35–1.09); stroke (SMD = 0.87, 95% CI = 0.59–1.15); signs of anxiety (SMD = 0.91, 95% CI = 0.56–1.25); signs of depression (SMD = 1.24, 95% CI = 0.98–1.49). It also increased mortality rates (hazard ratio = 1.48, 95% CI = 1.25–1.74; relative median survival = 0.91, 95% CI = 0.89–0.94). Meta-regressions indicated that such housing effects were ubiquitous across species and sexes, but could not identify the most impactful improvements to conventional housing. Data variability (assessed via coefficient of variation) was also not increased by ‘enriched’ housing. Conclusions Conventional housing appears sufficiently distressing to compromise rodent health, raising ethical concerns. Results also add to previous work to show that research rodents are typically CRAMPED (cold, rotund, abnormal, male-biased, poorly surviving, enclosed and distressed), raising questions about the validity and generalisability of the data they generate. This research was funded by NSERC, Canada.Applied Science, Faculty ofMedicine, Faculty ofBiomedical Engineering, School ofReviewedFacult

Podocalyxin regulates murine lung vascular permeability by altering endothelial cell adhesion.

Despite the widespread use of CD34-family sialomucins (CD34, podocalyxin and endoglycan) as vascular endothelial cell markers, there is remarkably little known of their vascular function. Podocalyxin (gene name Podxl), in particular, has been difficult to study in adult vasculature as germ-line deletion of podocalyxin in mice leads to kidney malformations and perinatal death. We generated mice that conditionally delete podocalyxin in vascular endothelial cells (Podxl(ΔEC) mice) to study the homeostatic role of podocalyxin in adult mouse vessels. Although Podxl(ΔEC) adult mice are viable, their lungs display increased lung volume and changes to the matrix composition. Intriguingly, this was associated with increased basal and inflammation-induced pulmonary vascular permeability. To further investigate the etiology of these defects, we isolated mouse pulmonary endothelial cells. Podxl(ΔEC) endothelial cells display mildly enhanced static adhesion to fibronectin but spread normally when plated on fibronectin-coated transwells. In contrast, Podxl(ΔEC) endothelial cells exhibit a severely impaired ability to spread on laminin and, to a lesser extent, collagen I coated transwells. The data suggest that, in endothelial cells, podocalyxin plays a previously unrecognized role in maintaining vascular integrity, likely through orchestrating interactions with extracellular matrix components and basement membranes, and that this influences downstream epithelial architecture

Correction: CD34 Promotes Pathological Epi-Retinal Neovascularization in a Mouse Model of Oxygen-Induced Retinopathy.

[This corrects the article DOI: 10.1371/journal.pone.0157902.]

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

<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