Losartan metabolite EXP3179 is a unique blood pressure-lowering AT1R antagonist with direct, rapid endothelium-dependent vasoactive properties.

BACKGROUND AND PURPOSE: Losartan is an anti-hypertensive angiotensin II (ANGII) type 1 receptor (AT1R) blocker (ARB) with many unexpected therapeutic properties, even in non-blood pressure (BP)-related diseases. Administered as a prodrug, losartan undergoes serial metabolism into EXP3179, a metabolite alleged to lack AT1R-blocking properties, and EXP3174, the dominant AT1R antagonist. Having observed that losartan can decrease vascular tone in mice with low AT1R expression and inhibit Marfan aortic widening at very high doses, we investigated whether EXP3179 may have unique, AT1R-independent effects on vascular tone and endothelial function. EXPERIMENTAL APPROACH: We compared the AT1R blocking capabilities of EXP3179 and EXP3174 using AT1R-expressing cell lines. Their BP lowering and vasoactive properties were studied in normal, hypertensive and transgenic rodents, and ex vivo wire myography. KEY RESULTS: We observed that both EXP3179 and EXP3174 can fully block (100%) AT1R signaling in vitro and significantly decrease BP in normotensive and spontaneously hypertensive rats. Only EXP3179 prevented PE-induced contraction by up to 65% (p < 0.01) in L-NAME and endothelium removal-sensitive fashion. Use of transgenic mice revealed that these effects involve the eNOS/caveolin-1 axis and the endothelium-dependent hyperpolarization factor (EDHF). CONCLUSION AND IMPLICATIONS: We provide direct structure-activity evidence that EXP3179 is a BP-lowering AT1R blocker with unique endothelial function-enhancing properties not shared with losartan or EXP3174. The major pharmacological effects of losartan in patients are therefore likely more complex than simple blockade of AT1R by EXP3174, which helps rationalize its therapeutic and prophylactic properties, especially at very high doses. Reports relying on EXP3179 as an AT1R-independent losartan analogue may require careful re-evaluation.

Prenatal antibiotic exposure, asthma, and the atopic march: A systematic review and meta-analysis.

Antibiotic use during pregnancy may increase the risk for asthma in children. We performed a meta-analysis assessing prenatal antibiotic exposure and the risk for childhood wheeze or asthma, as well as for diseases associated with the atopic march. A systematic literature search protocol (PROSPERO-ID: CRD42020191940) was registered and searches were completed using Medline, Proquest, Embase, and the Cochrane central register of controlled trials. Screening for inclusion criteria: published in English, German, French, Dutch, or Arabic, intervention (use of any antibiotic at any time point during pregnancy), and disease (reporting atopic disease incidence in children with a primary outcome of asthma or wheeze), and exclusion criteria: reviews, preclinical data, and descriptive studies, yielded 27 studies. Study quality was assessed using the Newcastle-Ottawa Assessment Scale. Quality of the evidence was assessed using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. Our meta-analysis demonstrates that antibiotic use during pregnancy is associated with an increased relative risk (RR) of developing wheeze RR 1.51 (95% CI: 1.17-1.94) or asthma RR 1.28 (95% CI 1.22-1.34) during childhood. Assessment of the atopic march in association with asthma or wheeze revealed that antibiotic use during pregnancy also increases the risk for eczema/dermatitis RR 1.28 (95% CI: 1.06-1.53) and allergic rhinitis RR 1.13 (95% CI: 1.02-1.25). One study found an increase in food allergy RR 1.81 (95% CI: 1.11-2.95). Maternal antibiotic use during pregnancy is associated with an increased risk for wheeze or asthma development in children, as well as for diseases involved in the atopic march. There was high heterogeneity in the data, and the certainty of the evidence was determined to be low quality, highlighting the need for more high-quality studies on this topic. These results have importance for antibiotic stewardship throughout the prenatal period. This work was supported by the Deutsche Forschungsgemeinschaft and the Konrad Adenauer Foundation.

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.

Antibiotic Treatment in an Animal Model of Inflammatory Lung Disease.

Allergic disease is on the rise and yet the underlying cause and risk factors are not fully understood. While lifesaving in many circumstances, the use of antibiotics and the subsequent disruption of the microbiome are positively correlated with the development of allergies. Here, we describe the use of the antibiotic vancomycin in combination with the papain-induced mouse model of allergic disease that allows for the assessment of microbiome perturbations and the impact on allergy development.

Abortive γδTCR rearrangements suggest ILC2s are derived from T-cell precursors.

Innate lymphoid cells (ILCs) are a recently identified subset of leukocytes that play a central role in pathogen surveillance and resistance, modulation of immune response, and tissue repair. They are remarkably similar to CD4+ T-helper subsets in terms of function and transcription factors required for their development but are distinguished by their lack of antigen-specific receptors. Despite their similarities, the absence of a surface T-cell receptor (TCR) and presence of ILCs and precursors in adult bone marrow has led to speculation that ILCs and T cells develop separately from lineages that branch at the point of precursors within the bone marrow. Considering the common lineage markers and effector cytokine profiles shared between ILCs and T cells, it is surprising that the status of the TCR loci in ILCs was not fully explored at the time of their discovery. Here, we demonstrate that a high proportion of peripheral tissue ILC2s have TCRγ chain gene rearrangements and TCRδ locus deletions. Detailed analyses of these loci show abundant frameshifts and premature stop codons that would encode nonfunctional TCR proteins. Collectively, these data argue that ILC2 can develop from T cells that fail to appropriately rearrange TCR genes, potentially within the thymus.

A sticky wicket: Defining molecular functions for CD34 in hematopoietic cells.

The CD34 cell surface antigen is widely expressed in tissues on cells with progenitor-like properties and on mature vascular endothelia. In adult human bone marrow, CD34 marks hematopoietic stem and progenitor cells (HSPCs) starting from the bulk of hematopoietic stem cells with long-term repopulating potential (LT-HSCs) throughout expansion and differentiation of oligopotent and unipotent progenitors. CD34 protein surface expression is typically lost as cells mature into terminal effectors. Because of this expression pattern of HSPCs, CD34 has had a central role in the evaluation or selection of donor graft tissue in HSC transplant (HSCT). Given its clinical importance, it is surprising that the biological functions of CD34 are still poorly understood. This enigma is due, in part, to CD34's context-specific role as both a pro-adhesive and anti-adhesive molecule and its potential functional redundancy with other sialomucins. Moreover, there are also critical differences in the regulation of CD34 expression on HSPCs in humans and experimental mice. In this review, we highlight some of the more well-defined functions of CD34 in HSPCs with a focus on proposed functions most relevant to HSCT biology.

Reduced genetic potential for butyrate fermentation in the gut microbiome of infants who develop allergic sensitization.

BACKGROUND: Allergic disease is the most frequent chronic health issue in children and has been linked to early-life gut microbiome dysbiosis. Many lines of evidence suggest that microbially derived short-chain fatty acids, and particularly butyrate, can promote immune tolerance. OBJECTIVE: We sought to determine whether bacterial butyrate production in the gut during early infancy is protective against the development of atopic disease in children. METHODS: We used shotgun metagenomic analysis to determine whether dysbiosis in butyrate fermentation could be identified in human infants, before their developing allergic disease. RESULTS: We found that the microbiome of infants who went on to develop allergic sensitization later in childhood lacked genes encoding key enzymes for carbohydrate breakdown and butyrate production. CONCLUSIONS: Our findings support the importance of microbial carbohydrate metabolism during early infancy in protecting against the development of allergies.

Podocalyxin is required for maintaining blood-brain barrier function during acute inflammation.

Podocalyxin (Podxl) is broadly expressed on the luminal face of most blood vessels in adult vertebrates, yet its function on these cells is poorly defined. In the present study, we identified specific functions for Podxl in maintaining endothelial barrier function. Using electrical cell substrate impedance sensing and live imaging, we found that, in the absence of Podxl, human umbilical vein endothelial cells fail to form an efficient barrier when plated on several extracellular matrix substrates. In addition, these monolayers lack adherens junctions and focal adhesions and display a disorganized cortical actin cytoskeleton. Thus, Podxl has a key role in promoting the appropriate endothelial morphogenesis required to form functional barriers. This conclusion is further supported by analyses of mutant mice in which we conditionally deleted a floxed allele of Podxl in vascular endothelial cells (vECs) using Tie2Cre mice (PodxlΔTie2Cre). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood-brain barrier (BBB) in PodxlΔTie2Cre mice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH2) of the protease-activated receptor-1 (PAR-1), a thrombin receptor expressed by vECs, neuronal cells, and glial cells. In response to systemic administration of TFLLR-NH2, LPS-primed PodxlΔTie2Cre mice become completely immobilized for a 5-min period, coinciding with severely dampened neuroelectric activity. We conclude that Podxl expression by CNS tissue vECs is essential for BBB maintenance under inflammatory conditions.

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 administration. We found that Cd34-/- mice displayed severe weight loss and early mortality compared with 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 with 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.

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.].

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

The sialomucins CD34 and podocalyxin (PODXL) are anti-adhesive molecules expressed at the luminal membrane of endothelial cells of small blood vessels and facilitate vascular lumen formation in the developing mouse aorta. CD34 transcript and protein levels are increased during human angiogenesis, its expression is particularly enriched on endothelial tip cell filopodia and CD34 is a marker for tip cells in vitro. Here, we investigated whether CD34 merely marks endothelial tip cells or has a functional role in tip cells and angiogenesis. We assessed that silencing CD34 in human microvascular endothelial cells has little effect on endothelial cell migration or invasion, but has a significant effect on vascular-endothelial growth factor-induced angiogenic sprouting activity in vitro. In vivo, the absence of CD34 reduced the density of filopodia on retinal endothelial tip cells in neonatal mice, but did not influence the overall architecture of the retinal vascular network. In oxygen-induced retinopathy, Cd34-/- mice showed normal intra-retinal regenerative angiogenesis but the number of pathological epi-retinal neovascular tufts were reduced. We conclude that CD34 is not essential for developmental vascularization in the retina, but its expression promotes the formation of pathological, invasive vessels during neovascularization.

DMSO Represses Inflammatory Cytokine Production from Human Blood Cells and Reduces Autoimmune Arthritis.

Dimethyl sulfoxide (DMSO) is currently used as an alternative treatment for various inflammatory conditions as well as for cancer. Despite its widespread use, there is a paucity of data regarding its safety and efficacy as well as its mechanism of action in human cells. Herein, we demonstrate that DMSO has ex-vivo anti-inflammatory activity using Escherichia coli- (E. coli) and herpes simplex virus-1 (HSV-1)-stimulated whole human blood. Specifically, we found that between 0.5%-2%, DMSO significantly suppressed the expression of many pro-inflammatory cytokines/chemokines and prostaglandin E2 (PGE2). However, a significant reduction in monocyte viability was also observed at 2% DMSO, suggesting a narrow window of efficacy. Anti-inflammatory concentrations of DMSO suppressed E. coli-induced ERK1/2, p38, JNK and Akt phosphorylation, suggesting DMSO acts on these signaling pathways to suppress inflammatory cytokine/chemokine production. Although DMSO induces the differentiation of B16/F10 melanoma cells in vitro, topical administration of DMSO to mice subcutaneously implanted with B16 melanoma cells was ineffective at reducing tumor growth, DMSO was also found to block mouse macrophages from polarizing to either an M1- or an M2-phenotype, which may contribute to its inability to slow tumor growth. Topical administration of DMSO, however, significantly mitigated K/BxN serum-induced arthritis in mice, and this was associated with reduced levels of pro-inflammatory cytokines in the joints and white blood cell levels in the blood. Thus, while we cannot confirm the efficacy of DMSO as an anti-cancer agent, the use of DMSO in arthritis warrants further investigation to ascertain its therapeutic potential.

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.