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Quantitation of platelet-derived growth factor receptors in human arterial smooth muscle cells in vitro
A Krettek1, G Fager, P Jernberg
1Wallenberg Laboratory for Cardiovascular Research, Göteborg University, Sweden. alexandra.krettek@wlab.wall.gu.se
Abstract:
Platelet-derived growth factor (PDGF) is suggested to play an important role in the development of atherosclerosis as a migratory and mitogenic stimulus to arterial smooth muscle cells (ASMCs). Stimulated and unstimulated ASMCs were studied with respect to PDGF receptor (PDGF-R) mRNA and protein expression. Quantitative RT-PCR was developed for simultaneous evaluation of both PDGF-R alpha and -R beta mRNA expression and a quantitative ELISA for estimation of corresponding PDGF-R subunits. On the mRNA level, the overall PDGF-R beta expression was approximately 100 times lower than that of PDGF-R alpha. Furthermore, although PDGF-R alpha mRNA levels were high irrespective of hASMC phenotype, PDGF-R beta mRNA was influenced by serum stimulation with lower copy numbers in proliferating and confluent cells compared with quiescent cells. On the protein level, quiescent hASMCs expressed 10 times more PDGF-R beta than PDGF-R alpha. Serum stimulation decreased cell surface PDGF-Rs, with most prominent loss of PDGF-R alpha (ELISA and immunohistochemistry). Our results suggest a differential regulatory pattern for PDGF-R alpha and -R beta and are compatible with the usage of alternative promoters for regulation of -R alpha expression. Further, it seems that the number of available receptor subunits is not the only determinant of variations in cell stimulation with different PDGF isoforms.
Insights
Platelet-derived growth factor receptors (PDGF-R) alpha and beta show differential regulation in arterial smooth muscle cells (ASMCs). Their expression levels vary with cell phenotype and serum stimulation, impacting atherosclerosis development.
Area of Science:
- Cardiovascular Biology
- Molecular Cell Biology
- Atherosclerosis Research
Background:
- Platelet-derived growth factor (PDGF) is implicated in atherosclerosis development.
- PDGF acts as a stimulus for smooth muscle cell migration and proliferation.
- Understanding PDGF receptor (PDGF-R) regulation in arterial smooth muscle cells (ASMCs) is crucial.
Purpose of the Study:
- To investigate the differential expression of PDGF-R alpha and PDGF-R beta mRNA and protein in ASMCs.
- To explore the regulation of PDGF-R expression in response to cell phenotype and serum stimulation.
- To elucidate the role of PDGF-R expression in ASMC behavior relevant to atherosclerosis.
Main Methods:
- Quantitative reverse transcription-PCR (RT-PCR) for simultaneous evaluation of PDGF-R alpha and -R beta mRNA.
- Quantitative enzyme-linked immunosorbent assay (ELISA) for PDGF-R alpha and -R beta protein subunits.
- Immunohistochemistry to assess cell surface receptor expression.
Main Results:
- PDGF-R beta mRNA levels were approximately 100 times lower than PDGF-R alpha mRNA.
- PDGF-R beta mRNA expression decreased in proliferating and confluent cells compared to quiescent cells.
- Quiescent ASMCs expressed 10 times more PDGF-R beta protein than PDGF-R alpha protein.
- Serum stimulation led to a decrease in cell surface PDGF-Rs, particularly PDGF-R alpha.
Conclusions:
- PDGF-R alpha and -R beta exhibit distinct regulatory patterns in ASMCs.
- Alternative promoter usage may regulate PDGF-R alpha expression.
- Receptor subunit availability is not the sole factor determining cellular response to PDGF isoforms.