Podocalyxin in the Diagnosis and Treatment of Cancer

Kelly M. McNagny, Michael R. Hughes, Marcia L. Graves, Erin J. DeBruin, Kimberly Snyder, Jane Cipollone, Michelle Turvey, Poh C. Tan, Shaun McColl and Calvin D. Roskelle

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

Depressed mood in pregnancy: Prevalence and correlates in two Cape Town peri-urban settlements

<p>Abstract</p> <p>Background</p> <p>The disability associated with depression and its impact on maternal and child health has important implications for public health policy. While the prevalence of postnatal depression is high, there are no prevalence data on antenatal depression in South Africa. The purpose of this study was to determine the prevalence and correlates of depressed mood in pregnancy in Cape Town peri-urban settlements.</p> <p>Methods</p> <p>This study reports on baseline data collected from the Philani Mentor Mothers Project (PMMP), a community-based, cluster-randomized controlled trial on the outskirts of Cape Town, South Africa. The PMMP aims to evaluate the effectiveness of a home-based intervention for preventing and managing illnesses related to HIV, TB, alcohol use and malnutrition in pregnant mothers and their infants. Participants were 1062 pregnant women from Khayelitsha and Mfuleni, Cape Town. Measures included the Edinburgh Postnatal Depression Scale (EPDS), the Derived AUDIT-C, indices for social support with regards to partner and parents, and questions concerning socio-demographics, intimate partner violence, and the current pregnancy. Data were analysed using bivariate analyses followed by logistic regression.</p> <p>Results</p> <p>Depressed mood in pregnancy was reported by 39% of mothers. The strongest predictors of depressed mood were lack of partner support, intimate partner violence, having a household income below R2000 per month, and younger age.</p> <p>Conclusions</p> <p>The high prevalence of depressed mood in pregnancy necessitates early screening and intervention in primary health care and antenatal settings for depression. The effectiveness and scalability of community-based interventions for maternal depression must be developed for pregnant women in peri-urban settlements.</p> <p>Trial registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT00972699">NCT00972699</a>.</p

The structural and functional importance of the CD34 family in lung function and vascular cell biology

Despite the widespread use of CD34-family sialomucins (CD34, podocalyxin (Podxl) and endoglycan) as vascular endothelial cell (EC) markers, there is remarkably little known of their role in vascular development and functions with the exception of vessel lumen formation in the developing mouse embryo (Podxl) and vessel patency during tumor angiogenesis and inflammation (CD34). Because germ-line deletion of Podxl in mice causes perinatal death, we generated mice that conditionally delete Podxl in vascular endothelial cells (PodxlΔEC mice) to study the role of podocalyxin in adult mouse vessels. Although PodxlΔEC adult mice are viable and thrive, we discovered increased basal and inflammationinduced pulmonary vascular permeability. Furthermore, PodxlΔEC mouse adult lungs display airspace enlargement with increased collagen deposition and exhibit a gene expression profile similar to regenerating lung. To study whether endothelial cell morphology influences the defective lung architecture and the vascular permeability phenotype in PodxlΔEC mice, we isolated primary vascular ECs from lung tissue. PodxlΔEC ECs display enhanced adhesion to fibronectin (FN) in a static adhesion assay. When plated on matrix-coated transwells, PodxlΔEC EC spread normally on FN but display defective spreading on laminin and collagen I. Thus, expression of Podxl in EC is required for normal lung architecture and function in adult mice and adhesion of EC to extracellular matrix components. Although its expression has not been well characterized, in humans, endoglycan expression has been reported in vascularized tissues. In mouse blood vessels, we showed that vascular smooth muscle cells (vSMC), but not EC, express the highest levels of endoglycan. Using a mouse aortic smooth muscle line (MOVAS-1) we found that forced expression of endoglycan enhances basal but not platelet derived growth factor (PDGF)ββ-dependent vSMC migration in vitro. Further studies to understand the role of endoglycan in primary vSMC showed that endoglycan is upregulated with differentiation to a contractile phenotype, but is not influenced by inflammatory stimuli or mitogenic factors. The findings of this thesis suggest that the CD34 family regulates vascular development and function with a role for podocalyxin in EC-matrix adhesion relevant to normal lung function and a role for endoglycan in SMC differentiation and migration.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

IL-7Rα and L-selectin, but not CD103 or CD34, are required for murine peanut-induced anaphylaxis

Background: Allergy to peanuts results in severe anaphylactic responses in affected individuals, and has dramatic effects on society and public policy. Despite the health impacts of peanut-induced anaphylaxis (PIA), relatively little is known about immune mechanisms underlying the disease. Using a mouse model of PIA, we evaluated mice with deletions in four distinct immune molecules (IL7Rα, L-selectin, CD34, CD103), for perturbed responses. Methods: PIA was induced by intragastric sensitization with peanut antigen and cholera toxin adjuvant, followed by intraperitoneal challenge with crude peanut extract (CPE). Disease outcome was assessed by monitoring body temperature, clinical symptoms, and serum histamine levels. Resistant mice were evaluated for total and antigen specific serum IgE, as well as susceptibility to passive systemic anaphylaxis. Results: PIA responses were dramatically reduced in IL7Rα−/− and L-selectin−/− mice, despite normal peanut-specific IgE production and susceptibility to passive systemic anaphylaxis. In contrast, CD34−/− and CD103−/− mice exhibited robust PIA responses, indistinguishable from wild type controls. Conclusions: Loss of L-selectin or IL7Rα function is sufficient to impair PIA, while CD34 or CD103 ablation has no effect on disease severity. More broadly, our findings suggest that future food allergy interventions should focus on disrupting sensitization to food allergens and limiting antigen-specific late-phase responses. Conversely, therapies targeting immune cell migration following antigen challenge are unlikely to have significant benefits, particularly considering the rapid kinetics of PIA.Medicine, Faculty ofNon UBCReviewedFacult

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

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

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