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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 20, 2003 as doi:10.1096/fj.03-0097fje. |
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Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK;
* Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA;
UK MRC HGMP Resource Centre, Hinxton, Cambridge CB10 1SB, UK;
Medical Research Council Biostatistics Unit, Cambridge CB2 2SR, UK; and
Department of Biochemistry, University of Sydney, NSW 2006, Australia
3Correspondence: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK. E-mail: cgp22{at}cam.ac.uk
SPECIFIC AIMS
To investigate changes in the transcriptome and glycome of endothelial cells (ECs) that may contribute to the apoptotic process.
PRINCIPAL FINDINGS
1. Partial survival factor deprivation (SFD) leads to morphological changes in EC consistent with apoptosis
To induce apoptosis, we cultured human umbilical vein endothelial cells (HUVEC) in optimal media to passage 5 and partially deprived replicate cultures of survival factors by replacement of proprietary supplement with 2% charcoal-stripped fetal calf serum. Cells began to show signs of stress within 1 h of SFD. At 28 h, progression through the cell cycle had ceased; only 60% of cells remained adherent to the substrate and 
10–15% of adherent cells appeared to be undergoing apoptosis. Time lapse microscopy and immunocytochemical staining confirmed the sequence of morphological changes were consistent with apoptosis. After 48 h, 30% of cells remained but apoptotic incidence remained constant. We analyzed transcriptome changes at 28 and 48 h to allow cells sufficient time to accumulate late SFD-induced responses likely to determine cell fate.
2. Statistical analysis of gene arrays revealed the transcriptome was altered in survival factor-deprived endothelial cells
We extracted RNA from healthy (control) and SFD endothelial cell (EC) cultures derived from five individuals, and prepared and hybridized complex cRNA probes to Affymetrix U95A gene chips. Analysis of normalized data suggested that the vast majority of 12,600 transcripts were not significantly regulated by SFD (Fig. 1
a). We used Bayesian t tests to identify transcripts regulated consistently in all five experiments (P
0.01). 171 of these transcripts were up- and 495 down-regulated at least twofold and only 10 transcripts were regulated 
2-fold between 28 h and 48 h SFD, largely representing a partial reversal of the regulation that had occurred in the initial 28 h. Independent component analysis identified two significant "components" (patterns of related transcript changes) associated with 28 h SFD (Fig. 1b
) comprised largely of transcripts with potential coregulation. Hierarchical clustering separated SFD from non-SFD cultures but failed to identify biologically significant grouping of the transcript data (Fig. 1c
).
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3. Transcript changes induced by SFD encode proteins likely to promote EC death, down-regulate survival signals, and promote phagocytosis of apoptotic bodies
The majority of SFD-dependent transcript changes represented those associated with apoptosis. Several transcripts encoding components of the apoptotic machinery were up-regulated. Down-regulated transcripts included those encoding anti-apoptotic proteins, EC growth and survival factors, survival factor receptors, and intracellular signaling molecules involved in the transduction of survival signals in EC, particularly those associated with G-protein signaling, known to play an essential role in determining EC fate. The chemokine monocyte chemoattractant protein-1 was up-regulated greatly after SFD, and this may enhance recruitment of macrophages to regions of cell death. Figure 2
summarizes some genes found to be significantly up- or down-regulated in this study.
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We used Affymetrix gene arrays to assess the response of SFD EC to the survival factor vascular endothelial growth factor-A (VEGF-A). Only 5 transcripts were regulated by VEGF-A in SFD cells vs. 92 transcripts in healthy EC, suggesting SFD cells have a reduced capacity to respond to survival factors.
4. Quantitative PCR and immunohistochemical analysis validate a subset of results at the transcript and protein level
We repeated the analysis of transcripts within our RNAs using quantitative PCR (TaqMan). The fold changes detected by Affymetrix and TaqMan concurred closely. We found MCP-1 to be highly up-regulated in SFD conditions, and used immunocytochemical analysis of healthy and SFD HUVEC to confirm that the protein this transcript encodes was also up-regulated. Coimmunohistochemistry using a marker for cells undergoing apoptosis (anti-active caspase 3 antibody) suggested that MCP-1 was up-regulated in every SFD cell prior to caspase 3 activation, but remained expressed in cells undergoing apoptosis.
5. Sulfation of heparan sulfate glycosaminoglycans (HSGAGs) is reduced in SFD EC
To determine whether SFD-induced EC apoptosis was associated with saccharide changes, we analyzed the composition of cell surface HSGAGs. Exhaustive digestion of heparan sulfates, followed by capillary electrophoresis, revealed that the relative sulfation of saccharide building blocks was reduced after 48 h SFD (Fig. 3
).
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CONCLUSIONS
We cultured ECs under conditions of SFD to study stress-related changes that may induce apoptosis at the level of the transcriptome. We used five replicate arrays, each time using biologically independent samples, resulting in data we believe to be highly reproducible and reliable. By treating healthy and SFD cells with VEGF-A, we showed that the transcriptome response to this survival factor was relatively reduced in SFD. This may be due in part to reduced levels of transcripts encoding proteins associated with survival such as receptors and components of the intracellular signaling machinery. The apoptosis-associated reduction in HSGAG sulfation observed for the first time in this study may further reduce the ability of stressed or apoptotic cells to respond to survival factors. This may result in part from the reduced abundance of enzymes that promote HSGAG sulfation or increased abundance of enzymes that remove sulfates from HSGAGs.
In this study, we have combined genomics with glycomic analysis to identify an apoptosis-related program of EC transcriptome and glycome regulation. Based on our results, we propose that the cell biology of stress-induced apoptosis is underpinned by synergy between previously described protein-based changes and the changes to the transcriptome and glycome presented here.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0097fje 
2 C.G.P. and S.K.S. contributed equally to this work.
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