The Bcl I single nucleotide polymorphism of the human glucocorticoid receptor gene h-GR/NR3C1 promoter in patients with bronchial asthma: pilot study

Bcl I in the promoter polymorphism observed within h-GR/NR3C1 gene may play an important role in the development of bronchial asthma and resistance to GCs in the severe bronchial asthma. The aim of the investigation was to study the correlation between this h-GR/NR3C1 gene polymorphism and occurrence of asthma in the population of Polish asthmatics. Peripheral blood was obtained from 70 healthy volunteers and 59 asthma patients. Structuralized anamnesis, spirometry and allergy skin prick tests were performed in all participants. Genotyping was carried out with PCR–RFLP method. In healthy, non-atopic population variants of Bcl I: GG, GC, CC were found with frequency 0.129/0.471/0.400, respectively. In asthma patients Bcl I: GG, GC, CC occurred with respective frequencies of 0.410/0.462/0.128. Chi-square analysis revealed a significantly different (P < 0.05) distribution between cases and controls for the Bcl I polymorphism. The Bcl I polymorphism of h-GR/NR3C1 gene is significantly associated with bronchial asthma, susceptibility to the development of severe form and resistance to GCs in Polish population

VEGF and Angiopoietin-1 Exert Opposing Effects on Cell Junctions by Regulating the Rho GEF Syx

Vascular endothelial growth factor (VEGF) and Ang1 (Angiopoietin-1) have opposing effects on vascular permeability, but the molecular basis of these effects is not fully known. We report in this paper that VEGF and Ang1 regulate endothelial cell (EC) junctions by determining the localization of the RhoA-specific guanine nucleotide exchange factor Syx. Syx was recruited to junctions by members of the Crumbs polarity complex and promoted junction integrity by activating Diaphanous. VEGF caused translocation of Syx from cell junctions, promoting junction disassembly, whereas Ang1 maintained Syx at the junctions, inducing junction stabilization. The VEGF-induced translocation of Syx from EC junctions was caused by PKD1 (protein kinase D1)-mediated phosphorylation of Syx at Ser806, which reduced Syx association to its junctional anchors. In support of the pivotal role of Syx in regulating EC junctions, syx−/− mice had defective junctions, resulting in vascular leakiness, edema, and impaired heart function

Targeting Src family kinases inhibits bevacizumab-induced glioma cell invasion.

Anti-VEGF antibody therapy with bevacizumab provides significant clinical benefit in patients with recurrent glioblastoma multiforme (GBM). Unfortunately, progression on bevacizumab therapy is often associated with a diffuse disease recurrence pattern, which limits subsequent therapeutic options. Therefore, there is an urgent need to understand bevacizumab's influence on glioma biology and block it's actions towards cell invasion. To explore the mechanism(s) of GBM cell invasion we have examined a panel of serially transplanted human GBM lines grown either in short-term culture, as xenografts in mouse flank, or injected orthotopically in mouse brain. Using an orthotopic xenograft model that exhibits increased invasiveness upon bevacizumab treatment, we also tested the effect of dasatinib, a broad spectrum SFK inhibitor, on bevacizumab-induced invasion.We show that 1) activation of Src family kinases (SFKs) is common in GBM, 2) the relative invasiveness of 17 serially transplanted GBM xenografts correlates strongly with p120 catenin phosphorylation at Y228, a Src kinase site, and 3) SFK activation assessed immunohistochemically in orthotopic xenografts, as well as the phosphorylation of downstream substrates occurs specifically at the invasive tumor edge. Further, we show that SFK signaling is markedly elevated at the invasive tumor front upon bevacizumab administration, and that dasatinib treatment effectively blocked the increased invasion induced by bevacizumab.Our data are consistent with the hypothesis that the increased invasiveness associated with anti-VEGF therapy is due to increased SFK signaling, and support testing the combination of dasatinib with bevacizumab in the clinic

Solution fibre spinning technique for the fabrication of tuneable decellularised matrix-laden fibres and fibrous micromembranes

Recreating tissue-specific microenvironments of the extracellular matrix (ECM) in vitro is of broad interest for the fields of tissue engineering and organ-on-a-chip. Here, we present biofunctional ECM protein fibres and suspended membranes, with tuneable biochemical, mechanical and topographical properties. This soft and entirely biologic membrane scaffold, formed by micro-nano-fibres using low voltage electrospinning, displays three unique characteristics for potential cell culture applications: high-content of key ECM proteins, single-layered mesh membrane, and flexibility for in situ integration into a range of device setups. Extracellular matrix (ECM) powder derived from urinary bladder, was used to fabricate the ECM-laden fibres and membranes. The highest ECM concentration in the dry protein fibre was 50 wt%, with the rest consisting of gelatin. Key ECM proteins, including collagen IV, laminin, and fibronectin, were shown to be preserved post the biofabrication process. The single fibre tensile Young's modulus can be tuned for over two orders of magnitude between ∼600 kPa and 50 MPa depending on the ECM content. Combining the fibre mesh printing with 3D printed or microfabricated structures, culture devices were constructed for endothelial layer formation, and a trans-membrane co-culture formed by glomerular cell types of podocytes and glomerular endothelial cells, demonstrating feasibility of the membrane culture. Our cell culture observation points to the importance of membrane mechanical property and re-modelling ability as a factor for soft membrane-based cell cultures. The ECM-laden fibres and membranes presented here would see potential applications in in vitro assays, and tailoring structure and biological functions of tissue engineering scaffolds. Statement of Significance: Recreating tissue-specific microenvironments of the extracellular matrix (ECM) is of broad interest for the fields of tissue engineering and organ-on-a-chip. Both the biochemical and biophysical signatures of the engineered ECM interplay to affect cell response. Currently, there are limited biomaterials processing methods which allow to design ECM membrane properties flexibly and rapidly. Solvents and additives used in many existing processes also induced unwanted ECM protein degradation and toxic residues. This paper presents a solution fibre spinning technique, where careful selection of the solution combination led to well-preserved ECM proteins with tuneable composition. This technique also provides a highly versatile approach to fabricate ECM fibres and membranes, leading to designable fibre Young's modulus for over two orders of magnitude

Dasatinib inhibits activation of SFKs and downstream targets in glioma.

<p><b>A.</b> Migration of SF767 cells in the absence or presence of 10 µM dasatinib or 10 µM PP2 was determined using a trans-well migration assay. Inhibition of SFKs with dasatinib or PP2 resulted in fewer SF767 cells migrating toward the chemoattractant. The differences vs. control (DMSO) treatment are statistically significant (n = 4; one-way ANOVA with Dunnett's Multiple Comparisons post test; *** indicates p<0.0001). <b>B.</b> Immunofluorescent staining of actin (white) in SF767 cells after control (DMSO) treatment or treatment with dasatinib (10 µM for 24 hours). For each treatment, the top image is the x-z orientation and bottom is the x-y orientation. Blue staining is DAPI; scale bar is 10 µm. <b>C.</b> Whole cell lysates of SF767 cells treated for 24 hours with 10 µM dasatinib (+) or DMSO (−) were western blotted for total Src, Y416-phosphorylated Src, total p120-catenin, Y228-phosphorylated p120, Y172-phosphorylated Vav2, actin (as a loading control), or active or total Rac1.</p

Activation of Src and p120 phosphorylation correlates with increased glioma invasiveness.

<p><b>A.</b> Seventeen glioma cell lines propagated as xenografts in mouse flank were examined by western blot for expression of active (Y416-phosphorylated) and total Src and Y228-phosphorylated and total p120 catenin proteins. Actin is a loading control. The positive control lysate is from MDA231 cells. Xenograft lines were previously classified based on their relative invasiveness as highly or moderately invasive (data boxed in gray) or minimally or non-invasive (not boxed). <b>B.</b> The level of Y228-phosphorylated p120 catenin expression (relative to actin expression) as determined by Western blot for each cell line was plotted vs. relative glioma invasiveness. The lines through the data indicate the median for each invasiveness category; n = 8 highly/moderately invasive lines and n = 9 minimally/non-invasive lines; **indicates a statistical difference (one-tailed, unpaired t test) between the two categories of invasiveness at p<0.005. <b>C.</b> Human prostate tumor, breast tumor, astrocytoma, and GBM samples were examined by immunohistochemistry for total p120 catenin expression (using the 15D2 antibody), and Y228-phosphorylated p120 catenin expression. Bar: 100 µm.</p

The invasive front of human GBM is a site of SFK activation.

<p>The spatial distribution of active SFK (as assessed by Y416-phosphorylated Src), Y228-phosphorylated p120 catenin, Y410-phosphorylated p130cas and Y172-phosphorylated Vav2 was assessed by immunohistochemistry of orthotopic GBM39 xenograft samples. Core indicates an image from the non-invading tumor core; rim indicates an image from the tumor's leading edge where the tumor interfaces with and can invade into the surrounding normal brain. Bar: 100 µm.</p