QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 13 May 2003
doi: 10.1242/jcs.00470

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, C. Articles by Kumar, S. Search for Related Content PubMed PubMed Citation Articles by Li, C. Articles by Kumar, S.

CD105 prevents apoptosis in hypoxic endothelial cells

¹ Department of Pathology, Medical School, University of Manchester and Christie Hospital, Manchester M13 9PT, UK
² Department of Biological Sciences, Manchester Metropolitan University, Manchester, UK
³ University of Manchester Academic Unit of Obstetrics and Gynaecology, St Mary's Hospital, Manchester M13 0JH, UK
⁴ Institute Reina Sofia de Investigacion Nefrologica, Department of Physiology and Pharmacology, Salamanca University, Salamanca, Spain
⁵ Department of Immunology, Centro de Investigaciones Biologicas, CSIC, 28006 Madrid, Spain

View larger version (29K): [in a new window]   Fig. 1. Hypoxia induces CD105 protein expression. HDMECs were cultured under hypoxic conditions for up to 24 hours, and CD105 expression was quantified by flow cytometry (A), immunoblotting (B) and ELISA (C) as described in Materials and Methods. (A) Hypoxic culture resulted in a maximal cell surface expression of CD105 at 16 hours (86.3% increase). The data represent five samples at each time point collected from five separate experiments (*P<0.05 and **P<0.01 compared with 0 hours as analysed by one-way ANOVA followed by the Duncan test). (B) Cell extracts were resolved on 4-7.5% SDS-PAGE under non-reducing conditions and electrophoretically transferred onto membranes. The blot was probed using mAb E9 and a rabbit anti--actin antibody. The maximal expression (as quantified on a densitometer) of CD105 was seen at 16 hours of hypoxic culture. Similar results were observed when the blots were reprobed using mAb 44G4 (data not shown). The bar chart shows the CD105 signal intensity relative to actin, pooled from three experiments. (C) Conditioned medium was collected, and soluble CD105 levels were quantified using a chemiluminescence ELISA system. CD105 levels peaked at 16 hours of hypoxic culture. The data represent six replicates at each time point pooled from three separate experiments (*P<0.05 and **P<0.01 compared with 0 hours as analyzed by one-way ANOVA followed by the Duncan test). Vertical bars indicate the standard error of the means.

View larger version (27K): [in a new window]   Fig. 2. Effect of hypoxia on CD105 transcription. (A) Northern blot analysis. Total RNA was extracted from HDMECs and fractionated on a 1% denaturing agarose gel. After blotting onto a nitrocellulose membrane, the fractionated RNA was probed using 32P-labelled cDNAs for CD105 or GAPDH, visualised on a phosphorimager and quantified using a densitometer. Maximal expression of CD105 mRNA was observed at 3 hours with a threefold increase over normoxic culture. The bar chart represents CD105 mRNA relative to GAPDH mRNA signal intensity collected from three experiments. (B) Hypoxia activates the CD105 promoter. HDMECs (the same batch of cells as for panel A) co-transfected with plasmid pXP2/pCD105/luc and CMVßgal were grown under hypoxic condition for up to 24 hours. Luciferase activity was determined and normalised to ß-galactosidase activity. CD105 promoter activity peaked between 3 hours and 6 hours of culture (*P<0.05 and **P<0.01 compared with 0 hours as analysed by one-way ANOVA followed by the Duncan test). Data represent six replicates at each time point collected from three separate experiments. Vertical bars indicate standard error of the means.

View larger version (65K): [in a new window]   Fig. 3. Cell cycle analysis and TUNEL staining of cells cultured under hypoxic conditions. HDMECs were cultured under hypoxic conditions for 0, 6 or 24 hours as indicated. (A) For cell cycle analysis, DNA was stained using propidium iodide and analysed by flow cytometry. Hypoxic culture resulted in an increased number of cells arrested at the G0/G1 phases and an increased level of DNA fragmentation (blue profile). (B) TUNEL staining was performed on the same batch of cells used for cell cycle analysis. Apple green staining of nuclei corresponds to apoptotic cells. Hypoxic culture led to an increased number of apoptotic cells (original magnification: x250). All experiments were performed at least three times and produced similar results.

View larger version (21K): [in a new window]   Fig. 4. Analysis of anti- and pro-apoptotic proteins under hypoxic conditions. Cells collected after 24 hours of culture under normoxic or hypoxic conditions were subjected to immunostaining using specific antibodies and FACS analysis. The ratio of Bcl-2 to Bax dropped from 1.36 in normoxia to 0.79 in hypoxia. Data were expressed as means±s.e.m. pooled from duplicate samples of three experiments (*P<0.05 and **P<0.01, student's t-test).

View larger version (42K): [in a new window]   Fig. 5. CD105 mRNA and protein expression in ODN-treated and untreated cells. HDMECs were cultured for 72 hours in normoxia in the presence or absence of ODN plus DMRIE-C, followed by exposure to hypoxia for 24 hours. CD105 protein levels were assessed by immunoblotting and FACS, and mRNA levels were assessed by northern blotting. (A) Immunoblotting revealed that CD105 protein was reduced by 58% in AS-ODN-treated cells (lane 2) compared with SC-ODN-treated (lane 3) and untreated cells (lane 1). The bar chart indicates the ratio of CD105 relative to CD31 signal intensity collected from three experiments (means±s.e.m.). (B) FACS analysis of cell surface CD105 indicated a 60% reduction in AS-ODN-treated (blue profile) compared with SC-ODN-treated (green profile) or untreated (black profile) cells. The red profile represents a negative control, where mAb E9 was substituted with pre-immunised mouse serum. The plot is a representative of five similar experiments. (C) Northern blotting of CD105 and GAPDH mRNA showed that CD105 mRNA was markedly degraded by AS ODN (lane 2) but not by SC ODN (lane 3). Lane 1 is mRNA extracted from untreated cells, and it has no evident alteration compared with lane 3. The bar chart shows CD105 mRNA, relative to GAPDH mRNA signal intensity, pooled from three experiments (means±s.e.m.).

View larger version (27K): [in a new window]   Fig. 6. Effect of CD105 antisense ODN on cell apoptosis. Three groups of cells, AS- or SC-ODN-treated or untreated HDMECs, were exposed to hypoxia and/or TGF-ß1 for 24 hours, TUNEL-stained and analysed by FACS. The fluorescence intensity (A) and percentage of apoptotic cells (B) were determined. The apoptotic effect of TGFß1 was observed to be concentration dependent, with the maximal effect of TGFß1 at 10 ng/ml. Data were expressed as means±s.e.m. collected from six samples of three experiments. (*P<0.05 and **P<0.01, one-way ANOVA followed by the Duncan test).