scholarly journals Hypoxia down-regulates sFlt-1 (sVEGFR-1) expression in human microvascular endothelial cells by a mechanism involving mRNA alternative processing

2011 ◽  
Vol 436 (2) ◽  
pp. 399-407 ◽  
Author(s):  
Takayuki Ikeda ◽  
Li Sun ◽  
Naoki Tsuruoka ◽  
Yasuhito Ishigaki ◽  
Yasuo Yoshitomi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Mrna Processing ◽  
Soluble Form ◽  
Microvascular Endothelial Cells ◽  
Cis Element ◽  
Alternative Processing ◽  
O2 Concentration ◽  
Membrane Bound ◽  
Microvascular Endothelial ◽  
Endothelial Growth Factor

sFlt-1 (soluble Flt-1) potently inhibits angiogenesis by binding extracellularly to VEGF (vascular endothelial growth factor). In the present paper, we report that hypoxia down-regulates sFlt-1 expression in HMVECs (human microvascular endothelial cells), a constituent of microvessels where angiogenesis occurs. Hypoxia (5–1% O2) increased VEGF expression in HMVECs. In contrast, the levels of sFlt-1 mRNA and protein in HMVECs decreased significantly as the O2 concentration fell, whereas mFlt-1 (membrane-bound Flt-1) mRNA and protein remained unchanged. This suggested that hypoxia selectively regulates alternative 3′-end processing of sFlt-1 pre-mRNA. We have also demonstrated that sFlt-1 overexpression in lentiviral-construct-infected HMVECs counteracted VEGF-induced endothelial cell growth. We next identified cis-elements involved in sFlt-1 mRNA processing in HMVECs using a human Flt-1 minigene and found that two non-contiguous AUUAAA sequences function as the poly(A) signal. Furthermore, we identified a cis-element in intron 13 that regulates sFlt-1 mRNA processing. Mutagenesis of the U-rich region in intron 13 caused a significant decrease in the soluble-form/membrane-form RNA ratio in the minigene-transfected HMVECs. These results suggest that decreased sFlt-1 expression due to hypoxia contributes to hypoxia-induced angiogenesis and reveals a novel mechanism regulating angiogenesis by alternative mRNA 3′-end processing.

Abstract 474: Effects of Cigarette Smoke on CD146 Expression and Function in Pulmonary Endothelial Cells

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Adelheid Kratzer ◽  
Jonas Salys ◽  
Benjy Gonzalez ◽  
Hong Wei Chu ◽  
Martin Zamora ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cigarette Smoke ◽  
Immunofluorescent Staining ◽  
Microvascular Endothelial Cells ◽  
Pulmonary Microvascular Endothelial Cells ◽  
Pulmonary Endothelial Cells ◽  
Membrane Bound ◽  
Microvascular Endothelial ◽  
Cd146 Expression

Background and Objectives: Cell adhesion molecule CD146 is a transmembrane glycoprotein constitutively expressed in all types of endothelial cells (EC). It exists in two forms: a membrane-anchored form (CD146) and a soluble, extracellular and cleaved form (sCD146). The plasma concentration of sCD146 is modulated in inflammatory diseases that involve endothelial alterations. We investigated the role of endothelial CD146 in cigarette smoke-induced emphysema in vivo and in pulmonary endothelial cells (EC) in vitro . Methods: Sprague Dawley rats exposed to cigarette smoke for 2 months developed significant emphysematous changes (measured by mean linear intercept). Levels of sCD146 were subsequently measured in the circulation as well as in the bronchoalveolar lavage fluid (BALf) via ELISA. In vitro studies were carried out in rat pulmonary microvascular endothelial cells using CSE. Results: CD146 is highly expressed in rat pulmonary microvascular endothelial cells (RPMVEC) and to a much lower extent, in pulmonary macrovascular endothelial cells (RPAEC). Treatment of RPMVEC with cigarette smoke extract (CSE) in vitro resulted in decreased expression of membrane-bound CD146 as well as a reduced gene expression and increased sCD146 levels in the culture medium after 12 hours. Moreover, CSE-induced downregulation of CD146 expression resulted in increased vascular permeability of RPMVEC, as measured by EVANs Blue assay and migration of CFSE-labeled rat alveolar macrophage. Immunofluorescent staining revealed that CSE treatment resulted in translocation of membrane-bound CD146 into the nucleus. Subsequent western blot analysis showed changes in ERK and AKT activation and signaling. Similar results were found upon siRNA silencing of CD146, implicating a role for CD146 in tissue inflammation and integrity. Circulating levels of sCD146 were also elevated in plasma and BALf of patients with COPD and correlated, in part, with the presence of anti-endothelial autoantibodies. Additionally, we found decreased expression of membrane-bound CD146 in lung tissues of COPD patients. Conclusions: Our data suggest that CD146 plays an important role in pulmonary vascular EC function. Moreover, levels of circulating soluble CD146 can be a predictor of vascular endothelial cell injury.

scholarly journals Heme relieves the inhibition of proliferation, migration and angiogenesis of HMEC-1 under hyperoxia by inhibiting BACH1

2020 ◽  
Author(s):  
Lan Jian ◽  
Yang Mei ◽  
Yuan Rongdi
Keyword(s):  
Endothelial Cells ◽  
Phase I ◽  
Scratch Test ◽  
Cell Counting ◽  
Endothelial Cell Proliferation ◽  
Microvascular Endothelial Cells ◽  
Inhibition Of Proliferation ◽  
Retinal Angiogenesis ◽  
Microvascular Endothelial ◽  
Endothelial Growth Factor

Abstract Background The relatively hyperoxia inhibited vascular endothelial growth factor (VEGF) in retina is the main cause of angiogenesis retardation in phase I retinopathy of prematurity (ROP). Human retinal angiogenesis is related to the proliferation, migration and angiogenesis of microvascular endothelial cells. Previous studies have confirmed that BTB and CNC homology l (BACH1) can inhibit VEGF and angiogenesis, while heme can specifically degrade BACH1. However, the effect of heme on endothelial cells and ROP remains unknown. Methods In this report, we established a model of human microvascular endothelial cells (HMEC-1) induced by 40% hyperoxia to simulate the relatively hyperoxia of phase I ROP. Meanwhile, heme was added to investigate the effects on the growth and viability of HMEC-1. Cell counting kit 8 (CCK8) and 5-ethynyl-2′-deox-yuridine (EDU) methods were used to detect the proliferation ability. Cell scratch test and matrigel matrix glue were used to detect the migration or angiogenesis ability. Western blot and immunofluorescence methods were used to detect the relative protein expression of BACH1 and VEGF. Results The proliferation, migration and angiogenesis of HMEC-1 were inhibited under hyperoxia. Moderate heme can promote endothelial cell proliferation, while excessive can inhibit, 20 µM heme could inhibit the expression of BACH1, promote the expression of VEGF, and relieve the inhibition of proliferation, migration and angiogenesis induced by hyperoxia in HMEC-1. Conclusions 20 µM heme can relieve the inhibitory effects induced by hyperoxia, via the mechanism of promoting VEGF by inhibiting BACH1 in HMEC-1, and maybe a potential medicine for retinal angiogenesis retardation induced by relatively hyperoxia in phase I ROP.

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