Decreased expression of VE-cadherin and claudin-5 and increased phosphorylation of VE-cadherin in vascular endothelium in nasal polyps

VE-cadherin and claudin-5 are major components of adherens and tight junctions of vascular endothelial cells and a decrease in their expression and an increase in the tyrosine-phosphorylation of VE-cadherin are associated with an increase in endothelial paracellular permeability. To clarify the mechanism underlying the development of edema in nasal polyps, we studied these molecules in polyp microvessels. Normal inferior turbinate mucosal tissues and nasal polyps from patients treated with or without glucocorticoid were stained for VE-cadherin or claudin-5 and CD31 by a double-immunofluorescence method and the immunofluorescence intensities were graded 1–3 with increasing intensity. To correct for differences in fluorescence intensity attributable to a different endothelial area being exposed in a section or to the thickness of a section, the relative immunofluorescence intensity was estimated by dividing the grade of VE-cadherin or claudin-5 by that of CD31 in each microvessel. Tyrosine-phosphorylation of VE-cadherin was examined by Western blot analysis. The relative intensities of VE-cadherin and claudin-5 in the CD31-positive microvessels significantly decreased in the following order; inferior turbinate mucosa, treated polyps and untreated polyps. The ratio of tyrosine-phosphorylated VE-cadherin to VE-cadherin was significantly higher in untreated polyps than in the inferior turbinate mucosa and treated polyps, between which no significant difference in the ratio was seen. Thus, in nasal polyps, the barrier function of endothelial adherens and tight junctions is weakened, although glucocorticoid treatment improves this weakened barrier function.     

Unfolded protein response regulates P53 expression in the pulmonary endothelium

Lung endothelial barrier dysfunction leads to severe pathologies, including the lethal Acute Respiratory Distress Syndrome. P53 has been associated with anti‐inflammatory activities. The current study employs a variety of unfolded protein response (UPR) activators and inhibitors to investigate the regulation of P53 by UPR in lung cells. The bovine cells that were exposed to the UPR inductors brefeldin A, dithiothreitol, and thapsigargin; demonstrated elevated expression levels of P53 compared to the vehicle‐treated cells. On the contrary, the UPR inhibitors N‐acetyl cysteine, kifunensine, and ATP‐competitive IRE1α kinase‐inhibiting RNase attenuator; produced the opposite effects. The outcomes of the present study reveal a positive regulation between UPR and P53. Since it has been shown that a mild induction of the unfolded protein response opposes inflammation, we suggest that P53 is involved in those protective activities in the lung.     

Rac1 regulates cardiovascular development and postnatal function of endothelium

Rac1 is a member of the small Rho GTPase family, which controls actin cytoskeleton and focal adhesion dynamics in cellular protrusions. While Rac1 therefore contributes to regulation of endothelial cell-cell and cell-matrix interactions, a detailed understanding of its role in endothelium function is lacking. Recently, the role of Rac1 in development and postnatal regulation of the cardiovascular system has been investigated in murine models lacking Rac1 specifically in endothelium. Homozygous endothelial deletion was lethal, primarily due to defects in angiogenesis. Rac1-deficient endothelial cells were unable to form cellular protrusions/lamellipodia, leading to impaired cell-cell and cell-matrix interactions, and resulting in dysfunctional adhesion, motility, permeability and capillary morphogenesis. Development was normal in the heterozygous model, however a hypertensive phenotype was observed as a result of reduced nitric oxide signalling. Nitric oxide synthase activity was regulated by Rac1 at multiple levels; expression, mRNA stability and uptake of the nitric oxide synthase substrate L-arginine. Therefore, Rac1 activity is essential in regulating developmental and postnatal angiogenesis and cardiovascular function, by controlling nitric oxide production, and formation of endothelial cell protrusions.     

Rac1 regulates cardiovascular development and postnatal function of endothelium

Rac1 is a member of the small Rho GTPase family, which controls actin cytoskeleton and focal adhesion dynamics in cellular protrusions. While Rac1 therefore contributes to regulation of endothelial cell-cell and cell-matrix interactions, a detailed understanding of its role in endothelium function is lacking. Recently, the role of Rac1 in development and postnatal regulation of the cardiovascular system has been investigated in murine models lacking Rac1 specifically in endothelium. Homozygous endothelial deletion was lethal, primarily due to defects in angiogenesis. Rac1-deficient endothelial cells were unable to form cellular protrusions/lamellipodia, leading to impaired cell-cell and cell-matrix interactions, and resulting in dysfunctional adhesion, motility, permeability and capillary morphogenesis. Development was normal in the heterozygous model, however a hypertensive phenotype was observed as a result of reduced nitric oxide signalling. Nitric oxide synthase activity was regulated by Rac1 at multiple levels; expression, mRNA stability and uptake of the nitric oxide synthase substrate L-arginine. Therefore, Rac1 activity is essential in regulating developmental and postnatal angiogenesis and cardiovascular function, by controlling nitric oxide production, and formation of endothelial cell protrusions.Key words: Rac1, angiogenesis, endothelial, motility, lamellipodia, nitric oxide, nitric oxide synthase     

Cell cycle dynamics and complement expression distinguishes mature haematopoietic subsets arising from hemogenic endothelium

The emergence of haematopoietic stem and progenitor cells (HSPCs) from hemogenic endothelium results in the formation of sizeable HSPC clusters attached to the vascular wall. We evaluate the cell cycle and proliferation of HSPCs involved in cluster formation, as well as the molecular signatures from their initial appearance to the point when cluster cells are capable of adult engraftment (definitive HSCs). We uncover a non-clonal origin of HSPC clusters with differing cell cycle, migration, and cell signaling attributes. In addition, we find that the complement cascade is highly enriched in mature HSPC clusters, possibly delineating a new role for this pathway in engraftment.     

NO regulates LPS-stimulated cyclooxygenase gene expression and activity in pulmonary artery endothelium

We examined whether nitric oxide (NO) inhibits prostanoid synthesis through actions on cyclooxygenase (COX) gene expression and activity. Bovine pulmonary artery endothelial cells were pretreated for 30 min with the NO donors 1 mM S-nitroso-N-acetylpenicillamine (SNAP), 0.5 mM sodium nitroprusside (SNP), or 0.2 microM spermine NONOate; controls included cells pretreated with either 1 mM N-acetyl-D-penicillamine or the NO synthase (NOS) inhibitor 1 mM N(G)-nitro-L-arginine methyl ester with and without addition of lipopolysaccharide (LPS; 0.1 microg/ml) for 8 h. COX-1 and COX-2 gene and protein expression were examined by RT-PCR and Western analysis, respectively; prostanoid measurements were made by gas chromatography-mass spectrometry, and COX activity was studied after a 30-min incubation with 30 microM arachidonic acid. LPS induced COX-2 gene and protein expression and caused an increase in COX activity and an eightfold increase in 6-keto-PGF(1alpha) release. LPS-stimulated COX-2 gene expression was decreased by approximately 50% by the NO donors. In contrast, LPS caused a significant reduction in COX-1 gene expression and treatment with NO donors had little effect. SNAP, SNP, and NONOate significantly suppressed LPS-stimulated COX activity and 6-keto-PGF(1alpha) release. Our data indicate that increased generation of NO attenuates LPS-stimulated COX-2 gene expression and activity, whereas inhibition of endogenous NOS has little effect.     

SOX9 regulates prostaglandin D synthase gene transcription in vivo to ensure testis development

In mammals, male sex is determined by the Y-chromosomal gene Sry (sex-determining region of Y chromosome). The expression of Sry and subsequently Sox9 (SRY box containing gene 9) in precursors of the supporting cell lineage results in the differentiation of these cells into Sertoli cells. Sertoli cells in turn orchestrate the development of all other male-specific cell types. To ensure that Sertoli cells differentiate in sufficient numbers to induce normal testis development, the early testis produces prostaglandin D(2) (PGD(2)), which recruits cells of the supporting cell lineage to a Sertoli cell fate. Here we show that the gene encoding prostaglandin D synthase (Pgds), the enzyme that produces PGD(2), is expressed in Sertoli cells immediately after the onset of Sox9 expression. Promoter analysis in silico and in vitro identified a paired SOX/SRY binding site. Interestingly, only SOX9, and not SRY, was able to bind as a dimer to this site and transactivate the Pgds promoter. In line with this, a transgenic mouse model showed that Pgds expression is not affected by ectopic Sry expression. Finally, chromatin immunoprecipitation proved that SOX9 but not SRY binds to the Pgds promoter in vivo.     

Wnt7b regulates placental development in mice.

Secreted Wnt proteins regulate many developmental processes in multicellular organisms. We have generated a targeted mutation in the mouse Wnt7b gene. Homozygous Wnt7b mutant mice die at midgestation stages as a result of placental abnormalities. Wnt7b expression in the chorion is required for fusion of the chorion and allantois during placental development. The alpha4 integrin protein, required for chorioallantoic fusion, is not expressed by cells in the mutant chorion. Wnt7b also is required for normal organization of cells in the chorionic plate. Thus, Wnt7b signaling is central to the early stages of placental development in mammals.     

Differentiation-dependent expression of proteins in brain endothelium during development of the blood-brain barrier

The blood-brain barrier is a specific property of differentiated brain endothelium. To study the differentiation of blood vessels in the brain, we have correlated the expression of a number of proteins in brain endothelial cells with the development of the blood-brain barrier in mouse, quail, and chick embryos. Using histochemical methods, alkaline phosphatase activity was found to be present in all species and appeared around embryonic Days 17 (mouse), 14 (quail), and 12 (chick). Butyrylcholinesterase activity was found in the mouse and quail but not the chick brain vasculature, and appeared around Days 17 (mouse) and 15 (quail). gamma-Glutamyltranspeptidase activity was demonstrated histochemically in mouse but not in chick and quail brain capillaries, beginning at Day 15. Transferrin receptor was localized on brain endothelium in all species by immunofluorescence methods using monoclonal antibodies. It appeared at Days 15 and 11 in mouse and chick embryonic brain, respectively. The staining of all markers in embryonic brain was compared with adult brain endothelium and the leptomeningeal blood vessels. The expression of these proteins was correlated with the development of the blood-brain barrier by studying the permeability of brain endothelium for the protein horseradish peroxidase during mouse embryogenesis. Vessels in the telencephalon were found to become impermeable around Day 16 of development. Taken together the results of previous investigations and those presented here, we conclude that a number of proteins are sequentially expressed in brain endothelial cells correlating in time with the formation of the blood-brain barrier in different species.     

Role of metalloproteinases at the onset of liver development

At the onset of liver development, the hepatic precursor cells, namely, the hepatoblasts, derive from the ventral foregut endoderm and form a bud surrounded by a basement membrane (BM). To initiate liver growth, the hepatoblasts migrate across the BM and invade the neighboring septum transversum mesenchyme. In the present study, carried out in the mouse embryo, we searched for effectors involved in this process and we examined the role of matrix metalloproteinases (MMPs). We found expression of a broad range of MMPs, among which MMP-2 was predominantly expressed in the septum transversum and MMP-14 in the hepatoblasts. Using a new liver explant culture system we showed that inhibition of MMP activity represses migration of the hepatoblasts. We conclude that MMPs are required to initiate expansion of the liver during development and that our culture system provides a new model to study hepatoblast migration.