Reorganization of the endothelial cell (EC) cytoskeleton and cell adhesive complexes provides a structural basis for increased vascular permeability implicated in the pathogenesis of many diseases, ...including asthma, sepsis, and acute respiratory distress syndrome (ARDS). We have recently described the barrier-protective effects of hepatocyte growth factor (HGF) on the human pulmonary EC. In the present study, we explored the involvement of Rac-GTPase and Rac-specific nucleotide exchange factor Tiam1 in the mechanisms of EC barrier protection by HGF. HGF protected EC monolayers from thrombin-induced hyperpermeability, disruption of intercellular junctions, and formation of stress fibers and paracellular gaps by inhibiting thrombin-induced activation of Rho GTPase, Rho association with nucleotide exchange factor p115-RhoGEF, and myosin light chain phosphorylation, which was opposed by stimulation of Rac-dependent signaling. The pharmacological Rac inhibitor or silencing RNA (siRNA) based depletion of either Rac or Tiam1 significantly attenuated HGF-induced peripheral translocation of Rac effector cortactin, cortical actin ring formation, and EC barrier enhancement. Moreover, Tiam1 knockdown using the siRNA approach, attenuated the protective effect of HGF against thrombin-induced activation of Rho signaling, monolayer disruption, and EC hyperpermeability. This study demonstrates the Tiam1/Rac-dependent mechanism of HGF-induced EC barrier protection and provides novel mechanistic insights into regulation of EC permeability via dynamic interactions between Rho- and Tiam1/Rac-mediated pathways. --Birukova, A., Alekseeva, E., Mikaelyan, A., and Birukov, K. G. HGF attenuates thrombin-induced endothelial permeability by Tiam1-mediated activation of the Rac pathway and by Tiam1/Rac-dependent inhibition of the Rho pathway.
Abstract
Elevated plasma levels of hyaluronic acid (HA) is a disease marker in liver pathology and other inflammatory disorders. Inhibition of HA synthesis with coumarin 4-methylumbelliferone (4MU) ...has a beneficial effect in animal models of fibrosis, inflammation, cancer and metabolic syndrome. 4MU is an active compound of approved choleretic drug hymecromone with low bioavailability and a broad spectrum of action. New, more specific and efficient inhibitors of hyaluronan synthases (HAS) are required. We have tested several newly synthesized coumarin compounds and commercial chitin synthesis inhibitors to inhibit HA production in cell culture assay. Coumarin derivative compound VII (10′-methyl-6′-phenyl-3′H-spiropiperidine-4,2′-pyrano3,2-gchromene-4′,8′-dione) demonstrated inhibition of HA secretion by NIH3T3 cells with the half-maximal inhibitory concentration (IC50) = 1.69 ± 0.75 μΜ superior to 4MU (IC50 = 8.68 ± 1.6 μΜ). Inhibitors of chitin synthesis, etoxazole, buprofezin, triflumuron, reduced HA deposition with IC50 of 4.21 ± 3.82 μΜ, 1.24 ± 0.87 μΜ and 1.48 ± 1.44 μΜ, respectively. Etoxazole reduced HA production and prevented collagen fibre formation in the CCl4 liver fibrosis model in mice similar to 4MU. Bioinformatics analysis revealed homology between chitin synthases and HAS enzymes, particularly in the pore-forming domain, containing the proposed site for etoxazole binding.
Abstract
Background: Insulin-like growth factor 1 (IGF1) exerts a broad anti-apoptotic function, and IGF1/IGF1-receptor (IGF-1R) signaling pathway plays a crucial role in human cancer development and ...progression. Stromal cells associated with cancer cells are considered an important component of tumor microenvironment, including pancreatic cancers. Previously we have shown an increased expression of IGF1 in pancreatic ductal adenocarcinoma stroma. The goal of our study was to investigate the effect of IGF1 on expression of important for tumor progression genes that regulate epithelial-mesenchymal transition (EMT) in cancer and stromal cells of pancreatic tumors. Methods: The effect of IGF1 was tested on five pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-2, MiaPaCa-2 and Panc1), all cultivated on cell medium with reduced levels of serum. Also, we used primary cultures of pancreatic ductal adenocarcinoma stromal cells on early passages. Gene expression was analyzed via Western Blotting and qRT-PCR. Results: In reduced serum medium IGF1 has been shown to stimulate proliferation in all cell lines and primary cultures. In cancer cell lines MiaPaCa-2 and Panc1 incubation with IGF1 lead to an increase in levels of SNAIL and Zeb1 proteins after 120 hours of incubation. There was an elevation in expression of Twist protein in all cancer cell lines after 120 hours of incubation. However, we have not detected a decrease in levels of epithelial differentiation marker E-cadherin, nor a notable increase in levels of mesenchymal differentiation markers N-cadherin and Vimentin in IGF1-treated cancer cell lines. Only a slight decrease in E-cadherin expression was shown in IGF1-treated AsPC-1 and Panc1 cells. Treatment of primary stromal cells of pancreatic ductal adenocarcinoma with IGF1 results in a slight elevation of N-cadherin and Vimentin expression, as well as a significant increase in anti-apoptotic protein survivin levels. Incubation of stromal cells with specific low-molecular weight inhibitor of IGF-1R was shown to significantly increase levels of SNAI1 gene expression and increase SNAIL protein levels. Conclusions: Analysis of EMT markers and transcription factors in pancreatic cancer cell lines does not allow us to draw conclusions about the ability of IGF1 to cause EMT. Despite increased levels of SNAIL, Zeb1 and Twist proteins, epithelial cancer cells retained stable expression of E-cadherin and did not show an increase in mesenchymal marker expression. In pancreatic tumor stromal cultures IGF1 increases mesenchymal cell phenotype and, evidently, increases cell survival rate by stimulating survivin expression. The effect of IGF-1R inhibitor on the activation of SNAIL expression, as we assume, was caused by interactions between signaling pathways of IGF1 and SHH in stromal cell cultures.
The work is supported by grant of Russian Foundation of Basic Research 13-04-40171-KOMFI
Note: This abstract was not presented at the meeting.
Citation Format: Marina R. Kopantseva, Eugenia Usova, Arsen Mikaelyan, Maria Kostina, Olga Melekhina, Vyacheslav Egorov, Eugene P. Kopantzev. Effect of IGF1 on cancer and stromal cells of human pancreatic tumors. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1540. doi:10.1158/1538-7445.AM2015-1540
Reorganization of the endothelial cell (EC) cytoskeleton and cell adhesive complexes provides a structural basis for increased vascular permeability implicated in the pathogenesis of many diseases, ...including asthma, sepsis, and acute respiratory distress syndrome (ARDS). We have recently described the barrier‐protective effects of hepatocyte growth factor (HGF) on the human pulmonary EC. In the present study, we explored the involvement of Rac‐GTPase and Rac‐specific nucleotide exchange factor Tiam1 in the mechanisms of EC barrier protection by HGF. HGF protected EC monolayers from thrombin‐induced hy‐perpermeability, disruption of intercellular junctions, and formation of stress fibers and paracellular gaps by inhibiting thrombin‐induced activation of Rho GTPase, Rho association with nucleotide exchange factor p115‐RhoGEF, and myosin light chain phosphorylation, which was opposed by stimulation of Rac‐dependent signaling. The pharmacological Rac inhibitor or silencing RNA (siRNA) based depletion of either Rac or Tiam1 significantly attenuated HGF‐induced peripheral translocation of Rac effector cortactin, cortical actin ring formation, and EC barrier enhancement. Moreover, Tiam1 knockdown using the siRNA approach, attenuated the protective effect of HGF against throm‐bin‐induced activation of Rho signaling, monolayer disruption, and EC hyperpermeability. This study demonstrates the Tiam1/Rac‐dependent mechanism of HGF‐induced EC barrier protection and provides novel mechanistic insights into regulation of EC permeabilityvia dynamic interactions between Rho‐ and Tiam1/Rac‐medi‐ated pathways.—Birukova, A., Alekseeva, E., Mikaelyan, A., and Birukov, K. G. HGF attenuates thrombin‐induced endothelial permeability by Tiam1‐mediated activation of the Rac pathway and by Tiam1/Rac‐dependent inhibition of the Rho pathway. FASEB J. 21, 2776–2786 (2007)
Purpose: Neural crest stem cells derived from the boundary cap (bNCSCs), markedly promote survival, proliferation and function of insulin producing β-cells in vitro and in vivo after ...coculture/transplantation with pancreatic islets
1, 2
. Recently, we have shown that beneficial effects on β-cells require cadherin contacts between bNCSCs and β-cells
3, 4
. Here we investigated whether hair follicle (HF) NCSCs, a potential source for human allogeneic transplantation, exert similar positive effects on β-cells. Materials and Methods: We established cocultures of HF-NCSCs or bNCSCs from mice expressing enhanced green fluorescent protein together with pancreatic islets from DxRed expressing mice or NMRI mice and compared their migration towards islet cells and effect on proliferation of β-cells as well as intracellular relations between NCSCs and islets using qRT-PCR analysis and immunohistochemistry. Results: Whereas both types of NCSCs migrated extensively in the presence of islets, only bNCSCs demonstrated directed migration toward islets, induced β-cell proliferation and increased the presence of cadherin at the junctions between bNCSCs and β-cells. Even in direct contact between β-cells and HF-NCSCs, no cadherin expression was detected. Conclusions: These observations indicate that HF-NCSCs do not confer the same positive effect on β-cells as demonstrated for bNCSCs. Furthermore, these data suggest that induction of cadherin expression by HF-NCSCs may be useful for their ability to support β-cells in coculture and after transplantation.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Single-stranded long oligonucleotide-based (50- to 70-mer) microarrays offer several advantages over conventional cDNA microarrays. These include the easy preparation of the probes, low cost of array ...production, and low cross-contamination during probe handling. However, the application of oligonucleotide microarrays for the analysis of global gene expression with small amounts of total RNA using the conventional oligo(dT)-T7 promoter-based amplification is hampered by the single-stranded nature (sense strand) of oligonucleotide probes in microarrays. In this report, we describe modified RNA amplification methods generating antisense-labeled cDNA targets and a successful application for oligonucleotide microarray gene expression analysis. In the first round, mRNA was amplified linearly with oligo(dT)
T7-primed reverse transcription and in vitro transcription by T7 RNA polymerase. In the second round, random 9-mer T3 primers and T3 RNA polymerase were used to generate sense-strand amplified RNA (aRNA). Fluorescently labeled cDNA targets were generated from the aRNA and hybridized to the oligonucleotide microarrays. Our data show that the amplification provides highly reproducible results, as evidenced by a significant correlation between the amplified and nonamplified samples. We also demonstrate that amplification of RNA derived from laser-microdissected tumor samples reproduced the gene expression profiles that were obtained from total RNA isolated from the same samples.
Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exhibits potent barrier protective effects on pulmonary endothelium, which are mediated by small GTPases Rac and Cdc42. ...However, upstream mechanisms of OxPAPC-induced small GTPase activation are not known. We studied involvement of Rac/Cdc42-specific guanine nucleotide exchange factors (GEFs) Tiam1 and betaPIX in OxPAPC-induced Rac activation, cytoskeletal remodeling, and barrier protective responses in the human pulmonary endothelial cells (EC). OxPAPC induced membrane translocation of Tiam1, betaPIX, Cdc42, and Rac, but did not affect intracellular distribution of Rho and Rho-specific GEF p115-RhoGEF. Protein depletion of Tiam1 and betaPIX using siRNA approach abolished OxPAPC-induced activation of Rac and its effector PAK1. EC transfection with Tiam1-, betaPIX-, or PAK1-specific siRNA dramatically attenuated OxPAPC-induced barrier enhancement, peripheral actin cytoskeletal enhancement, and translocation of actin-binding proteins cortactin and Arp3. These results show for the first time that Tiam1 and betaPIX mediate OxPAPC-induced Rac activation, cytoskeletal remodeling, and barrier protective response in pulmonary endothelium.
The design and the prototype of a platform for video capturing and real-time processing are presented. Compared to other platforms, support of different input/output interfaces is achieved. Frame ...double synchronization is implemented by caching data in DDR2 memory leading to video transmission stability. Standard interfaces have been used between the main blocks to simplify the integration of image processing algorithms. The maximum input video resolution and usage of an external decoder for analog video inputs are the main limitations of the platform.
PDF
