(Circulation. 1996;93:1095-1106.)
© 1996 American Heart Association, Inc.
Articles |
Platelet-Derived Growth Factor–A mRNA Expression in Fetal, Normal Adult, and Atherosclerotic Human Aortas
Analysis by Competitive Polymerase Chain Reaction
From the Department of Pathology, University of Washington, School of Medicine (Seattle).
Background To understand which growth factors are important for growth of atherosclerotic plaques, it is necessary to know the factor's relative abundance and how its gene is regulated in relation to cell proliferation. We tested whether platelet-derived growth factor–A (PDGF-A) mRNA levels correlated with cell proliferation in developing aorta, normal adult aorta, and atherosclerotic plaques.
Methods and Results We developed a competitive reverse transcription–polymerase chain reaction (RT-PCR) assay to measure human PDGF-A mRNA levels in small tissue samples. A mutated PDGF-A synthetic RNA was used as an internal standard to compete with endogenous PDGF-A mRNA for amplification. The assay is highly sensitive and much more precise than routine RT-PCR. Correction for heteroduplex pairing between the endogenous and mutant PCR products correlates precisely with synthetic RNA standards and quantitative Northern blotting. Immunostaining with the proliferation marker (proliferating cell nuclear antigen) showed the following rank order of proliferation: fetal aorta>>atherosclerotic plaque>normal aortic media. PDGF-A mRNA levels, however, did not correlate with proliferation. Normal adult aorta contained the most PDGF-A mRNA (34.0±7.6 amol/µg total RNA). Fetal aortas were intermediate (10.2±1.6 amol/µg total RNA); advanced atherosclerotic plaques contained the least PDGF-A mRNA (0.3±0.1 amol/µg total RNA). PDGF-A protein was readily detectable in normal media by immunostaining. Advanced plaques generally had less cell-associated PDGF-A protein, although A-chain was also detected in plaque matrix.
Conclusions PDGF-A mRNA and protein do not correlate with proliferation among these three groups. The significance of high levels of PDGF-A mRNA in the "quiescent" aortic media is unknown, but it clearly does not promote cell replication.
Key Words: atherosclerosis • growth substances • polymerase chain reaction • cells
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