Measurement by quantitative reverse transcription-polymerase chain reaction of the levels of tumor necrosis factor alpha mRNA in atherosclerotic arteries in Watanabe heritable hyperlipidemic rabbits.

Tumor necrosis factor-alpha (TNF-alpha), produced by activated monocytes and other cells, has been proposed as a mediator of importance in atherosclerosis. In this study, we use a newly developed technique, quantitative reverse transcription-polymerase chain reaction (RT-PCR), to measure the mRNA levels of TNF-alpha in arteries of Watanabe Heritable Hyperlipidemic (WHHL) rabbits in relation to the progression of atherosclerosis. Co-amplification of known amounts of TNF-alpha RNA and TNF-alpha internal control RNA indicated that the quantitative RT-PCR method was quite reliable, with a < 5% difference between TNF-alpha mRNA levels deduced from the standard curve and actually loaded TNF-alpha mRNA. As another control, TNF-alpha mRNA levels in lipopolysaccharide-induced (LPS-induced) and uninduced monocytes were measured, and a 9.3-fold increase in the TNF-alpha mRNA levels was observed in LPS-induced monocytes. TNF-alpha mRNA levels in the aortic arches of healthy New Zealand White (NZW) rabbits was 0.0112 -/+ 0.0016 (SD) pg per ng tissue RNA. TNF-alpha mRNA levels in the aortic arches and descending thoracic aortas of 6-month-old WHHL rabbits were 0.0273 -/+ 0.0066 pg and 0.0176 -/+ 0.0013 pg per ng tissue RNA, respectively. TNF-alpha mRNA levels in the aortic arches and descending thoracic aortas of 18-month-old WHHL rabbits were much higher than in those of 6-month-old WHHL rabbits, with values of 0.2107 -/+ 0.0205 pg and 0.1043 -/+ 0.0196 pg per ng tissue RNA, respectively. These findings indicate that: a) Quantitative RT-PCR can be used to measure levels of small abundance RNA in normal and diseased tissues accurately; b) no significant increase in TNF-alpha mRNA levels was observed in the aortic arches and descending thoracic aortas of 6-month-old WHHL rabbits compared with those of NZW rabbits, although they had multiple raised intimal lesions; and c) a significantly elevated total tissue level of TNF-alpha mRNA is demonstrable late in the course of atherosclerosis in 18-month-old WHHL rabbits (Bonferroni method). These findings suggest that TNF-alpha might play an important role in the progression of advanced atherosclerosis, although total tissue levels of TNF-alpha mRNA are not unequivocally elevated earlier in the course of the disease.