The Role and Mechanism of SIRT6 in Regulating Phenotype Transformation of Vascular Smooth Muscle Cells in Abdominal Aortic Aneurysm

Background. Data mining of current gene expression databases has not been previously performed to determine whether sirtuin 6 (SIRT6) expression participates in the pathological process of abdominal aortic aneurysm (AAA). The present study was aimed at investigating the role and mechanism of SIRT6 in regulating phenotype transformation of vascular smooth muscle cells (VSMC) in AAA. Methods. Three gene expression microarray datasets of AAA patients in the Gene Expression Omnibus (GEO) database and one dataset of SIRT6-knockout (KO) mice were selected, and the differentially expressed genes (DEGs) were identified using GEO2R. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of both the AAA-related DEGs and the SIRT6-related DEGs were conducted. Results. GEO2R analysis showed that the expression of SIRT6 was downregulated for three groups and upregulated for one group in the three datasets, and none of them satisfied statistical significance. There were top 5 DEGs (KYNU, NPTX2, SCRG1, GRK5, and RGS5) in both of the human AAA group and SIRT6-KO mouse group. Top 25 ontology of the SIRT6-KO-related DEGs showed that several pathways including tryptophan catabolic process to kynurenine and negative regulation of cell growth were enriched in the tissues of thickness aortic wall biopsies of AAA patients. Conclusions. Although SIRT6 mRNA level itself did not change among AAA patients, SIRT6 may play an important role in regulating several signaling pathways with significant association with AAA, suggesting that SIRT6 mRNA upregulation is a protective factor for VSMC against AAA.

Lysozyme Improves the Inhibitory Effects of Panax notoginseng Saponins on Phenotype Transformation of Vascular Smooth Muscle Cells by Binding to Ginsenoside Re

Panax notoginseng saponins (PNS) have been used to treat cardiovascular diseases for hundreds of years in China. Lysozyme can bind to exogenous compounds and promote their activity. Nevertheless, knowledge of whether there is a synergistic role between lysozyme and PNS is far from sufficient. In this study, we show that the mixture of PNS and lysozyme synergistically inhibited platelet derived growth factor BB (PDGF-BB)-induced vascular smooth muscle cell (VSMC) viability, and in the five main components of PNS, GS-Re, but not GS-Rb1, NG-R1, GS-Rg1, or GS-Rd, reduced VSMC viability by combined application with lysozyme. Next, the supramolecular complexes formed by GS-Re and lysozyme were detected by mass spectrometry, and the binding ability increased with the concentration ratio of GS-Re to lysozyme from 4:1 to 12:1. In the supramolecular complexes, the relative contents of α-helix of lysozyme were increased, which was beneficial for stabilizing the structure of lysozyme. The 12:1 mixture of GS-Re and lysozyme (12.8 μmol/L GS-Re+1.067 μmol/L lysozyme) repressed PDGF-BB-induced VSMC viability, proliferation, and migration, which were associated with the upregulated differentiated markers and downregulated dedifferentiated markers. Finally, in CaCl2-induced rodent abdominal aortic aneurysm (AAA) models, we found that the 12:1 mixture of GS-Re and lysozyme slowed down AAA progression and reversed phenotype transformation of VSMCs. Thus, Gs-Re combined with a small amount of lysozyme may provide a novel therapeutic strategy for vascular remodeling-associated cardiovascular diseases.

A newly-evolved chimeric lysin motif receptor-like kinase in Medicago truncatula spp. tricycla R108 extends its Rhizobia symbiotic partnership

Rhizobial lipochitooligosaccharidic Nod factors, specified by nod genes, are the primary determinants of host specificity in the legume-Rhizobia symbiosis. A Sinorhizobium meliloti nodF/nodL mutant produces Nod factors that differ from wild-type ones in lacking an O-acetate, and with a different acyl chain on the terminal non-reducing sugar. This mutant is defective in nodulation with various Medicago hosts. We examined the nodulation ability of M. truncatula cv Jemalong A17 and M. truncatula ssp. tricycla R108 with the nodF/nodL mutant. We then applied genetic and functional approaches to study the genetic basis and mechanism of nodulation of R108 by this mutant. We show that the nodF/nodL mutant can nodulate R108 but not A17. Using genomics and reverse genetics, we identified a newly-evolved gene in R108, LYK2bis, which is responsible for the phenotype. Transformation with LYK2bis allows A17 to gain nodulation with the nodF/nodL mutant. We found that LYK2bis is involved in specific NF signalling and interacts with the key receptor protein NFP. Our findings reveal that a newly-evolved gene in R108, LYK2bis, extends nodulation specificity to strains producing non-O-acetylated NFs. Interaction between LYK2bis and NFP provides a means of integrating the nodulation signalling pathways.

Microfluidic Lab-on-a-Chip Based on UHF-Dielectrophoresis for Stemness Phenotype Characterization and Discrimination among Glioblastoma Cells

Glioblastoma (GBM) is one of the most aggressive solid tumors, particularly due to the presence of cancer stem cells (CSCs). Nowadays, the characterization of this cell type with an efficient, fast and low-cost method remains an issue. Hence, we have developed a microfluidic lab-on-a-chip based on dielectrophoresis (DEP) single cell electro-manipulation to measure the two crossover frequencies: fx01 in the low-frequency range (below 500 kHz) and fx02 in the ultra-high-frequency range (UHF, above 50 MHz). First, in vitro conditions were investigated. An U87-MG cell line was cultured in different conditions in order to induce an undifferentiated phenotype. Then, ex vivo GBM cells from patients’ primary cell culture were passed through the developed microfluidic system and characterized in order to reflect clinical conditions. This article demonstrates that the usual exploitation of low-frequency range DEP does not allow the discrimination of the undifferentiated GBM cells from the differentiated one. However, the presented study highlights the use of UHF-DEP as a relevant discriminant parameter. The proposed microfluidic lab-on-a-chip is able to follow the kinetics of U87-MG phenotype transformation in a CSC enrichment medium and the cancer stem cells phenotype acquirement.

Microfluidic Lab-On-a-Chip based on UHF-Dielectrophoresis for Stemness Phenotype Characterization and Discrimination among Glioblastoma Cells

Glioblastoma (GBM) is one of the most aggressive solid tumors, particularly due to the presence of cancer stem cells (CSCs). Today the characterization of this type of cells with an efficient, fast and low-cost method remains an issue. Hence, we have developed a microfluidic lab-on-a-chip based on dielectrophoresis (DEP) single cell electro-manipulation to measure the two crossover frequencies: fx01 in low frequency range (below 500 kHz) and fx02 in Ultra High Frequency range (UHF, above 50 MHz). First, in vitro conditions were investigated. U87-MG cell lines were cultured in different conditions in order to induce an undifferentiated phenotype. Then, ex vivo GBM cells from patients’ primary cell culture, were passed through the developed microfluidic system and characterized in order to reflect clinical conditions. This article demonstrates that the usual exploitation of low frequency range DEP does not allow the discrimination of the undifferentiated from the differentiated phenotypes of GBM cells. However, the presented study highlights the use of UHF-DEP as a relevant discriminant parameter. The proposed microfluidic lab-on-a-chip is able to follow the kinetic of U87-MG phenotype transformation in a CSC enrichment medium and their cancer stem cells phenotype acquirement.

Melatonin Ameliorates Axonal Hypomyelination of Periventricular White Matter by Transforming A1 to A2 Astrocyte via JAK2/STAT3 Pathway in Neonatal Rats Induced by Lipopolysaccharide Injection

Background: Astrocyte A1/A2 phenotypes may play differential role in the pathogenesis of periventricular white matter (PWM) damage in septic postnatal rats. In this study, we sought to determine whether melatonin(MEL) would improve the axonal hypomyelination and neurological dysfunction, and, if so, to ascertain whether this may be related to transformation of astrocyte A1 to A2 phenotype.Methods: One-day-old Sprague–Dawley rats were divided into control, LPS, and LPS+MEL groups. Immunofluorescence was performed to detect IBA1, GFAP, MAG, C3 and S100A10 in the PWM of neonatal rats. C1q, IL-1α and TNF-α expression were assessed by immunofluorescence and ELISA. Electron microscopy was conducted to observe alterations of axonal myelin sheath in the PWM, and the number of PLP and MBP positive oligdendrocytes was caculated using in situ hybridization. The effects of MEL on locomotor ability, spatial learning and memory were evaluated by behavioral testing. In vitro, A1 astrocyte was induced by IL-1α, C1q and TNF-α, the effect of MEL on C3 and S100A10 expression was determined by Western blot and immunofluorescence. JAK2/STAT3 signaling pathway was investigated to determine whether it was involved in modulation of A1/A2 phenotype transformation.Results: At 1 and 3 days after LPS injection, IBA1+ microglia in the PWM were significantly increased in cell numbers which generated excess amounts of IL-1α, TNF-α, and C1q. The number of A1 astrocytes was significantly increased at 7-28d after LPS injection. In rats given MEL treatment, the number of A1 astrocytes was significantly decreaed, but that of A2 astrocytes, PLP+, MBP+ and MAG+ cells was increased. By electron microscopy, ultrastructural features of axonal hypomyelination were attenuated by MEL. Furthermore, MEL improved neurological dysfunction as evaluated by different neurological tests. In vitro, MEL decreased the C3 significantly, and upregulated expression of S100A10 in primary astrocytes subjected to IL-1α, TNF-α and C1q treatment. Additionally, JAK2/STAT3 signaling pathway was found to be involved in modulation of A1/A2 phenotype transformation. Conclusions: MEL effectively alleviates PWMD of septic neonatal rats, and that it is most likely through modulating astrocyte phenotypic transformation from A1 to A2 via the MT1/JAK2/STAT3 pathway.

Proliferative Vascular Smooth Muscle Cells Stimulate Extracellular Matrix Production via Osteopontin/p38 MAPK Signaling Pathway

<b><i>Introduction:</i></b> Extracellular matrix disorder and cellular phenotype transformation are the major histopathological features associated with ascending aortic aneurysms. Rare studies have investigated the relationship between cellular phenotype transformation and the abnormalities of the matrix constituents. In this study, we investigated whether the cellular phenotype transformation resulted in the extracellular matrix disorder. <b><i>Methods:</i></b> Aortic samples were obtained from 20 patients undergoing operations for ascending aortic aneurysms. Control aortic samples were obtained from 15 patients who underwent coronary artery bypass graft. The protein levels of osteopontin (OPN), collagen, and elastin were examined using Western blot, and quantitative reverse transcriptase-PCR was used to analyze the mRNA expression of collagen and elastin. In vitro experiment, vascular smooth muscle cells (VSMCs) were treated with recombinant human OPN (rh-OPN) or p38 MAPK inhibitor (SB203580) to investigate whether OPN and p38 MAPK regulated the expression of collagen and elastin. <b><i>Results:</i></b> The protein level of OPN and collagen III increased in ascending aortic aneurysm samples, compared with controls (<i>p</i> &#x3c; 0.05). There was no difference in the protein level of elastin between aneurysm tissues and the controls. VSMCs treated with rh-OPN increased the collagen III and elastin protein level and mRNA expression (<i>p</i> &#x3c; 0.05). Cells treated with SB203580 decreased the collagen III and elastin protein level and mRNA expression (<i>p</i> &#x3c; 0.05). Furthermore, VSMCs incubated with SB203580 reduced the rh-OPN-induced production of collagen III and elastin (<i>p</i> &#x3c; 0.05). <b><i>Conclusion:</i></b> OPN, the proliferative VSMCs maker, increased the expression of extracellular matrix. OPN/p38 MAPK signaling pathways may protect against ascending aortic aneurysm progression.