ESTABLISHMENT OF A QUANTITATIVE RT-PCR FOR DETECTION OF VASCULAR CELL-ADHESION MOLECULE-1 TRANSCRIPTS IN ENDOTHELIAL-CELLS AFTER STIMULATION WITH ADVANCED GLYCATION ENDPRODUCTS

Advanced glycation endproducts (AGE) are supposed to increase endothel ial expression of adhesion molecules like vascular cell adhesion molec ule-1 (VCAM-1) by inducing an intracellular stress with subsequent act ivation of nuclear transcription factor NF-kappa-B. Quantitative analy sis of VCAM-1-transcription has not been demonstrated concerning this topic. Thus, the aim of this study was to establish quantitative rever se transcription polymerase chain reaction (RT-PCR) assays using a spa cer gene in order to measure the amounts of specific mRNA for VCAM-1 i n human umbilical vein endothelial cells (HUVEC) which were stimulated with AGE-albumin (AGE-BSA). A recombinant RNA-standard was synthesize d and used as internal RT-PCR standard. The amount of VCAM-1-mRNA in u nstimulated HUVEC was found to be 2.2+/- 2.7 copies per cell. After st imulation with AGE-BSA, mRNA-levels were elevated to 38.9+/-10.9 copie s per cell. Positive controls (stimulated with lipopolysaccharide) rev ealed mRNA-levels of 78.7+/-27.5 copies per cell. We conclude that qua ntitative RT-PCR using the spacer gene technique is a valid and reliab le method for the measurement of small amounts of specific mRNA.