Pericyte, but not astrocyte, hypoxia inducible factor-1 (HIF-1) drives hypoxia-induced vascular permeability in vivo

Background Ways to prevent disease-induced vascular modifications that accelerate brain damage remain largely elusive. Improved understanding of perivascular cell signalling could provide unparalleled insight as these cells impact vascular stability and functionality of the neurovascular unit as a whole. Identifying key drivers of astrocyte and pericyte responses that modify cell–cell interactions and crosstalk during injury is key. At the cellular level, injury-induced outcomes are closely entwined with activation of the hypoxia-inducible factor-1 (HIF-1) pathway. Studies clearly suggest that endothelial HIF-1 signalling increases blood–brain barrier permeability but the influence of perivascular HIF-1 induction on outcome is unknown. Using novel mouse lines with astrocyte and pericyte targeted HIF-1 loss of function, we herein show that vascular stability in vivo is differentially impacted by perivascular hypoxia-induced HIF-1 stabilization. Methods To facilitate HIF-1 deletion in adult mice without developmental complications, novel Cre-inducible astrocyte-targeted (GFAP-CreERT2; HIF-1αfl/fl and GLAST-CreERT2; HIF-1αfl/fl) and pericyte-targeted (SMMHC-CreERT2; HIF-1αfl/fl) transgenic animals were generated. Mice in their home cages were exposed to either normoxia (21% O2) or hypoxia (8% O2) for 96 h in an oxygen-controlled humidified glove box. All lines were similarly responsive to hypoxic challenge and post-Cre activation showed significantly reduced HIF-1 target gene levels in the individual cells as predicted. Results Unexpectedly, hypoxia-induced vascular remodelling was unaffected by HIF-1 loss of function in the two astrocyte lines but effectively blocked in the pericyte line. In correlation, hypoxia-induced barrier permeability and water accumulation were abrogated only in pericyte targeted HIF-1 loss of function mice. In contrast to expectation, brain and serum levels of hypoxia-induced VEGF, TGF-β and MMPs (genes known to mediate vascular remodelling) were unaffected by HIF-1 deletion in all lines. However, in agreement with the permeability data, immunofluorescence and electron microscopy showed clear prevention of hypoxia-induced tight junction disruption in the pericyte loss of function line. Conclusion This study shows that pericyte but not astrocyte HIF-1 stabilization modulates endothelial tight junction functionality and thereby plays a pivotal role in hypoxia-induced vascular dysfunction. Whether the cells respond similarly or differentially to other injury stimuli will be of significant relevance.

miR-27a-3p regulates expression of intercellular junctions at the brain endothelium and controls the endothelial barrier permeability

Background The brain endothelial barrier permeability is governed by tight and adherens junction protein complexes that restrict paracellular permeability at the blood-brain barrier (BBB). Dysfunction of the inter-endothelial junctions has been implicated in neurological disorders such as multiple sclerosis, stroke and Alzheimer’s disease. The molecular mechanisms underlying junctional dysfunction during BBB impairment remain elusive. MicroRNAs (miRNAs) have emerged as versatile regulators of the BBB function under physiological and pathological conditions, and altered levels of BBB-associated microRNAs were demonstrated in a number of brain pathologies including neurodegeneration and neuroinflammatory diseases. Among the altered micro-RNAs, miR-27a-3p was found to be downregulated in a number of neurological diseases characterized by loss of inter-endothelial junctions and disruption of the barrier integrity. However, the relationship between miR-27a-3p and tight and adherens junctions at the brain endothelium remains unexplored. Whether miR-27a-3p is involved in regulation of the junctions at the brain endothelium remains to be determined. Methods Using a gain-and-loss of function approach, we modulated levels of miR-27a-3p in an in-vitro model of the brain endothelium, key component of the BBB, and examined the resultant effect on the barrier paracellular permeability and on the expression of essential tight and adherens junctions. The mechanisms governing the regulation of junctional proteins by miR-27a-3p were also explored. Results Our results showed that miR-27a-3p inhibitor increases the barrier permeability and causes reduction of claudin-5 and occludin, two proteins highly enriched at the tight junction, while miR-27a-3p mimic reduced the paracellular leakage and increased claudin-5 and occludin protein levels. Interestingly, we found that miR-27-3p induces expression of claudin-5 and occludin by downregulating Glycogen Synthase Kinase 3 beta (GSK3ß) and activating Wnt/ß-catenin signaling, a key pathway required for the BBB maintenance. Conclusion For the first time, we showed that miR-27a-3p is a positive regulator of key tight junction proteins, claudin-5 and occludin, at the brain endothelium through targeting GSK3ß gene and activating Wnt/ß-catenin signaling. Thus, miR-27a-3p may constitute a novel therapeutic target that could be exploited to prevent BBB dysfunction and preserves its integrity in neurological disorders characterized by impairment of the barrier’s function.

Effect of Probiotics on the Performance and Intestinal Health of Broiler Chickens Infected with Eimeria tenella

Coccidiosis is an important parasitic disease of poultry with great economic importance. Due to drug resistance issues, the study was conducted to investigate how probiotics (Lactobacillus plantarum or L. plantarum) affected oocysts per gram of feces (OPG), fecal scores, feed conversion ratio (FCR), immunomodulatory effect in terms of the cell-mediated and humoral immune response. Serum chemistry (ALT, AST, LDH, and creatinine) was measured in different treated chicken groups. mRNA expression levels of antioxidant enzymes (SOD 1 and CAT), peptide transporter 1 (PepT 1), and tight junction proteins (ZO and CLDN 1) were also examined in chicken groups infected with Eimeria tenella (E. tenella). Chickens supplemented with L. plantarum 1 × 108 CFU (colony-forming unit) showed an improved cell-mediated and humoral immune response, compared with the control group (p < 0.05). Probiotics also enhanced the performance of antioxidant enzymes, PepT 1, and tight junction proteins, and improved serum chemistry (AST, ALT, and LDH), compared with control-infected, non-medicated chickens. However, no significant difference (p > 0.05) was observed in CLDN 1 expression level and creatinine in all treated chicken groups. These findings demonstrated that probiotics supplementation in the feed can protect the birds against E. tenella infection.

Identification of Different Proteins Binding to Na, K-ATPase α1 in LPS-Induced ARDS Cell Model by Proteomic Analysis

Background: Acute respiratory distress syndrome (ARDS) is characterized by refractory hypoxemia caused by accumulation of pulmonary fluid, which is related to inflammatory cell infiltration, impaired tight junction of pulmonary epithelium and impaired Na, K-ATPase function, especially Na, K-ATPase α1 subunit. Up until now, the pathogenic mechanism at the level of protein during lipopolysaccharide- (LPS-) induced ARDS remains unclear.Methods: Using an unbiased, discovery and quantitative proteomic approach, we discovered the differentially expressed proteins binding to Na, K-ATPase α1 between LPS-A549 cells and Control-A549 cells. These Na, K-ATPase α1 interacting proteins were screened by co-immunoprecipitation (Co-IP) technology. Among them, some of the differentially expressed proteins with significant performance were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The protein interaction network was constructed by the related Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Several differentially expressed proteins were validated by Western blot.Results: Of identified 1598 proteins, 89 were differentially expressed proteins between LPS-A549 cells and Control-A549 cells. Intriguingly, protein-protein interaction network showed that there were 244 significantly enriched co-expression among 60 proteins in the group control-A549. while the group LPS-A549 showed 43 significant enriched interactions among 29 proteins. The related GO and KEGG analysis found evident phenomena of ubiquitination and deubiquitination, as well as the pathways related to autophagy. Among proteins with rich abundance, there were several intriguing ones, including the deubiquitinase (OTUB1), the tight junction protein zonula occludens-1 (ZO-1), the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complexes (CUL4B) and the autophagy-related protein sequestosome-1 (SQSTM1).Conclusions: In conclusion, our proteomic approach revealed targets related to the occurrence and development of ARDS, being the first study to investigate significant differences in Na, K-ATPase α1 interacting proteins between LPS-induced ARDS cell model and control-A549 cell. These proteins may help the clinical diagnosis and facilitate the personalized treatment of ARDS.

Direct Inhibition of the First PDZ Domain of ZO-1 by Glycyrrhizin is a Possible Mechanism of Tight Junction Opening of Caco-2 Cells.

Glycyrrhizin (GL) is known to exhibit a variety of useful pharmacological activities, including anti-inflammation, anti-hepatotoxicity, and enhancement of intestinal drug absorption. GL has been reported to modify the assembly of...

Tetrapeptides Modelled to the Androgen Binding Site of ZIP9 Stimulate Expression of Tight Junction Proteins and Tight Junction Formation in Sertoli Cells

Androgens stimulate the expression of tight junction (TJ) proteins and the formation of the blood–testis barrier (BTB). Interactions of testosterone with the zinc transporter ZIP9 stimulate the expression of TJ-forming proteins and promote TJ formation in Sertoli cells. In order to investigate androgenic effects mediated by ZIP9 but not by the nuclear androgen receptor (AR), the effects of three tetrapeptides fitting the androgen binding site of ZIP9 were compared with those induced by testosterone in a Sertoli cell line expressing ZIP9 but not the AR. Three tetrapeptides and testosterone displaced testosterone-BSA-FITC from the surface of 93RS2 cells and stimulated the non-classical testosterone signaling pathway that includes the activation of Erk1/2 kinases and transcription factors CREB and ATF-1. The expression of the TJ-associated proteins ZO-1 and claudin-5 was triggered as was the re-distribution of claudin-1 from the cytosol to the membrane and nucleus. Furthermore, TJ formation was stimulated, indicated by increased transepithelial electrical resistance. Silencing ZIP9 expression by siRNA prevented all of these responses. These results are consistent with an alternative pathway for testosterone action at the BTB that does not involve the nuclear AR and highlight the significant role of ZIP9 as a cell-surface androgen receptor that stimulates TJ formation.