NADPH oxidase 4 mediates insulin-stimulated HIF-1α and VEGF expression, and angiogenesis in vitro

Acute intensive insulin therapy causes a transient worsening of diabetic retinopathy in type 1 diabetes patients and is related to VEGF expression. Reactive oxygen species (ROS) have been shown to be involved in HIF-1α and VEGF expression induced by insulin, but the role of specific ROS sources has not been fully elucidated. In this study we examined the role of NADPH oxidase subunit 4 (Nox4) in insulin-stimulated HIF-1α and VEGF expression, and angiogenic responses in human microvascular endothelial cells (HMVECs). Here we demonstrate that knockdown of Nox4 by siRNA reduced insulin-stimulated ROS generation, the tyrosine phosphorylation of IR-β and IRS-1, but did not change the serine phosphorylation of IRS-1. Nox4 gene silencing had a much greater inhibitory effect on insulin-induced AKT activation than ERK1/2 activation, whereas it had little effect on the expression of the phosphatases such as MKP-1 and SHIP. Inhibition of Nox4 expression inhibited the transcriptional activity of VEGF through HIF-1. Overexpression of wild-type Nox4 was sufficient to increase VEGF transcriptional activity, and further enhanced insulin-stimulated the activation of VEGF. Downregulation of Nox4 expression decreased insulin-stimulated mRNA and protein expression of HIF-1α, but did not change the rate of HIF-1α degradation. Inhibition of Nox4 impaired insulin-stimulated VEGF expression, cell migration, cell proliferation, and tube formation in HMVECs. Our data indicate that Nox4-derived ROS are essential for HIF-1α-dependent VEGF expression, and angiogenesis in vitro induced by insulin. Nox4 may be an attractive therapeutic target for diabetic retinopathy caused by intensive insulin treatment

Escape-zone-based optimal evasion guidance against multiple orbital pursuers

The orbital evasion problem is getting increasing attention because of the increase of space maneuvering objects. In this paper, an escape-zone-based optimal orbital evasion guidance law for an evading spacecraft on near circular reference orbit is proposed against multiple pursuing spacecraft with impulsive thrust. The relative reachable domain is introduced first and approximated as an ellipsoid propagating along the nominal trajectory under the short-term assumption. The escape zone for the impulsive evasion problem is presented herein as a geometric description of the set of terminal positions for all the impulsive evasion trajectories that are not threatened by the maneuvers of pursuers at the maneuver moment. A general method is developed next to calculate the defined escape zone through finding the intersection of two relative reachable domain approximate ellipsoids at arbitrary intersection moment. Then, the two-sided optimal strategies for the orbital evasion problem are analyzed according to whether the escape zone exists, based on which the escape value is defined and used as the basis of the proposed orbital evasion guidance scheme. Finally, numerical examples demonstrate the usefulness of the presented method for calculating escape zone and the effectiveness of the proposed evasion guidance scheme against multiple pursuing spacecraft

Full-length transcriptome analysis and identification of genes involved in asarinin and aristolochic acid biosynthesis in medicinal plant Asarum sieboldii

Asarum sieboldii a well-known traditional Chinese medicinal herb, is used for curing inflammation and ache. It contains both bioactive ingredient asarinin and the toxic compound aristolochic acid. To address further breeding demand, genes involved in the biosynthetic pathways of asarinin and aristolochic acid should be explored. Therefore, we sequenced the full-length transcriptome of A. sieboldii using PacBio Iso-Seq to determine the candidate transcripts that encode the biosynthetic enzymes of asarinin and aristolochic acid. In our study, in total of 63,023 full-length transcripts were generated with an average length of 1,371 bp from roots, stems and leaves tissues, of which 49,593 transcripts (78.69%) were annotated against public databases. Furthermore, 555 alternative splicing (AS), 10,869 lncRNAs as well as their 11,291 target genes and 17,909 SSRs were identified. Our data also revealed that 97 candidate transcripts related to asarinin metabolism, of which 6 novel genes that encoded enzymes involved in asarinin biosynthesis were initially reported. 56 transcripts related to aristolochic acid biosynthesis were also identified, especially CYP81B. In summary, our transcriptome data provide a useful resource to study gene function and genetic engineering in A. sieboldii.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

NADPH Oxidase 4 Mediates Insulin-Stimulated HIF-1α and VEGF Expression, and Angiogenesis <em>In Vitro</em>

<div><p>Acute intensive insulin therapy causes a transient worsening of diabetic retinopathy in type 1 diabetes patients and is related to VEGF expression. Reactive oxygen species (ROS) have been shown to be involved in HIF-1α and VEGF expression induced by insulin, but the role of specific ROS sources has not been fully elucidated. In this study we examined the role of NADPH oxidase subunit 4 (Nox4) in insulin-stimulated HIF-1α and VEGF expression, and angiogenic responses in human microvascular endothelial cells (HMVECs). Here we demonstrate that knockdown of Nox4 by siRNA reduced insulin-stimulated ROS generation, the tyrosine phosphorylation of IR-β and IRS-1, but did not change the serine phosphorylation of IRS-1. Nox4 gene silencing had a much greater inhibitory effect on insulin-induced AKT activation than ERK1/2 activation, whereas it had little effect on the expression of the phosphatases such as MKP-1 and SHIP. Inhibition of Nox4 expression inhibited the transcriptional activity of VEGF through HIF-1. Overexpression of wild-type Nox4 was sufficient to increase VEGF transcriptional activity, and further enhanced insulin-stimulated the activation of VEGF. Downregulation of Nox4 expression decreased insulin-stimulated mRNA and protein expression of HIF-1α, but did not change the rate of HIF-1α degradation. Inhibition of Nox4 impaired insulin-stimulated VEGF expression, cell migration, cell proliferation, and tube formation in HMVECs. Our data indicate that Nox4-derived ROS are essential for HIF-1α-dependent VEGF expression, and angiogenesis <em>in vitro</em> induced by insulin. Nox4 may be an attractive therapeutic target for diabetic retinopathy caused by intensive insulin treatment.</p> </div

Human induced pluripotent stem cells derived endothelial cells mimicking vascular inflammatory response under flow

Endothelial cells (ECs) have great potential in vascular diseases research and regenerative medicine. Autologous human ECs are difficult to acquire in sufficient numbers in vitro, and human induced pluripotent stem cells (iPSCs) offer unique opportunity to generate ECs for these purposes. In this work, we present a new and efficient method to simply differentiate human iPSCs into functional ECs, which can respond to physiological level of flow and inflammatory stimulation on a fabricated microdevice. The endothelial-like cells were differentiated from human iPSCs within only one week, according to the inducing development principle. The expression of endothelial progenitor and endothelial marker genes (GATA2, RUNX1, CD34, and CD31) increased on the second and fourth days after the initial inducing process. The differentiated ECs exhibited strong expression of cells-specific markers (CD31 and von Willebrand factor antibody), similar to that present in human umbilical vein endothelial cells. In addition, the hiPSC derived ECs were able to form tubular structure and respond to vascular-like flow generated on a microdevice. Furthermore, the human induced pluripotent stem cell-endothelial cells (hiPSC-ECs) pretreated with tumor necrosis factor (TNF-alpha) were susceptible to adhesion to human monocyte line U937 under flow condition, indicating the feasibility of this hiPSCs derived microsystem for mimicking the inflammatory response of endothelial cells under physiological and pathological process. (C) 2016 AIP Publishing LLC

Nox4 mediates insulin-induced VEGF mRNA and protein expression.

<p>HMVECs were transfected with Nox4 siRNA or the control siRNA for 24 h. The cells were growth arrested and then treated with 100 nM insulin. (A) In 12 hours, VEGF 165 mRNA level was analyzed by real time-PCR. (B) In 24 hours, the VEGF protein levels in the supernatants were determined by ELISA assay. *<i>P</i><0.05, **<i>P</i><0.01 vs. the control siRNA group; #<i>P</i><0.05, ##<i>P</i><0.01 vs. the insulin+control siRNA group.</p

Nox4 mediates insulin-induced HMVEC migration, proliferation, and angiogenesis <i>in vitro</i>.

<p>HMVECs were transfected with the control or Nox4 siRNA for 24 hours. (A) The cells were growth arrested followed by stimulation with insulin (100 nM) for 10 hours. Cell migration was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048393#s2" target="_blank">Methods</a>. The data are expressed as the fold change relative to the control. (B) Growth-arrested cells were treated with insulin (100 nM) for 24 hours. Cell proliferation was measured using CCK-8 assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048393#s2" target="_blank">Methods</a>. (C) and (D) HMVECs transfected with the control or Nox4 siRNA were seeded onto Matrigel and incubated with or without insulin (100 nM), treated or not with H₂O₂ (0.1 µM) for 12 hours. The relative tube lengths were normalized by the negative control. Bar = 1000 µm. **<i>P</i><0.01 vs. the control siRNA group; #<i>P</i><0.05, ##<i>P</i><0.01 vs. the insulin+control siRNA group.</p