A quantitative method for determination of endothelial mRNA expression in vivo: induction of platelet-derived growth factor by endotoxin.
Quantitation of mRNA expression by endothelial cells in vivo has been limited to larger animals from which sufficient amounts of RNA could be isolated for Northern blot analysis. In the present study, we established a technique to isolate endothelial RNA from rat aortas using en face preparations. This RNA was not contaminated with RNA from smooth muscle cells as demonstrated by the absence of smooth muscle alpha-actin RNA. Following lipopolysaccharide (LPS) administration to rats, quantitation of platelet-derived growth factor (PDGF) ligand and receptor mRNA expression was carried out by competitive reverse transcriptase-polymerase chain reaction and normalized to glyceraldehyde-3 phosphate dehydrogenase. The results of the competitive reverse transcriptase-polymerase chain reaction were compared with those obtained by en face in situ hybridization. Aortic endothelium showed a 140-fold increase in PDGF-A mRNA expression 4 hours after LPS injection. Expression levels of this growth factor declined to near base line levels within 36 hours of the LPS injection. A 52-fold increase in PDGF-B mRNA was seen at 12 hours after LPS injection but expression levels were approximately 300-fold lower than for PDGF-A. These data indicate that changes in PDGF expression by endothelium in vivo can greatly exceed those observed in cultured cells. This method should permit study of endothelial gene regulation in a variety of pathological conditions in vivo.
Taguchi
J
J
First Department of Internal Medicine, University of Tokyo, Japan.
Murry
C E
CE
Herren
B I
BI
Pech
M
M
Schwartz
S M
SM
Lindner
V
V
D000199:Actins / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000378:metabolism; D001483:Base Sequence; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D017403:In Situ Hybridization; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012044:Regression Analysis; D013997:Time Factors; D014158:Transcription, Genetic
D000199:Actins; D008070:Lipopolysaccharides; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger; C075278:platelet-derived growth factor A; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases