Western array analysis of human atherosclerotic plaques: downregulation of apoptosis-linked gene 2.
OBJECTIVE
In recent years, microarray techniques have been used to characterize differences in mRNA populations between atherosclerotic plaques and normal arterial tissue. Because proteomics provide an attractive complementary approach to genomics, we used Western array technology as a global protein profiling method to identify differentially expressed proteins with potential pathobiological relevance in human atherosclerotic plaques.


METHODS
Cell lysates from human carotid endarterectomy specimens and non-atherosclerotic mammary arteries were screened with monoclonal antibodies (823 in total) that were combined into unique cocktails. Hits were verified with traditional Western blotting.


RESULTS
Seven proteins with a >5-fold relative expression difference were identified. One of the most apparent changes in human plaques was the downregulation of apoptosis-linked gene 2 (ALG-2), a positive mediator of apoptotic cell death. Differential expression of ALG-2 in human plaques relative to mammary arteries was not confirmed by real-time quantitative RT-PCR, suggesting post-transcriptional regulation. Uptake of aggregated LDL (agLDL) downregulated ALG-2 protein expression in THP-1 macrophages, but not in smooth muscle cells (SMCs). Transfection of THP-1 cells with ALG-2-specific small interfering RNA (siRNA) caused ALG-2 depletion and inhibited the execution phase of apoptosis (DNA fragmentation) but did not affect caspase-3 activation, annexin-V labeling and necrotic cell death.


CONCLUSIONS
Western array screening of carotid endarterectomy specimens revealed a strong downregulation of ALG-2 protein. Because ALG-2 has pro-apoptotic potential, our results point to a novel survival mechanism against cell death in human atherosclerotic plaques.
Martinet
Wim
W
Division of Pharmacology, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium. wim.martinet@ua.ac.be
Schrijvers
Dorien M
DM
De Meyer
Guido R Y
GR
Herman
Arnold G
AG
Kockx
Mark M
MM
D000368:Aged; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001161:Arteriosclerosis / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002339:Carotid Arteries / Q000378:metabolism; D002460:Cell Line; D015870:Gene Expression; D020868:Gene Silencing; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D008297:Male; D008323:Mammary Arteries / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; C097753:PDCD6 protein, human; D034741:RNA, Small Interfering