We used two different VSMCs cell lines
We used two different VSMCs cell lines, KSB and CSD (isolated from two different thoracic aortas). Rat aortic VSMCs were isolated from medial segments from thoracic rate 25 sta synthesis from Wistar-Kyoto rats (six to eight weeks old, Charles River Wiga GmbH) and cultured over several passages as described . Quiescent VSMCs were stimulated with 50 ng/ml PDGF-BB or 100 nM Ang II for different time periods. When quiescent unstimulated cells were compared with PDGF-BB or Ang II-stimulated cells most bands were the same except for one differentially expressed band, denoted as the P-band . The P-band was seen in Ang II- or PDGF-BB-stimulated cells at 30 minutes (lanes 2 KSB, lane 2 CSD or lanes 4 KSB, 4 CSD), respectively, compared to quiescent unstimulated cells (lane 1 KSB and 1 CSD). The P-band was not visible in Ang II- or PDGF-BB-stimulated cells at 12 hours (lanes 3 KSB, 3 CSD or 5 KSB, 5 CSD, respectively). shows a single amplified cDNA fragment (lane 1) with a molecular size between 603 and 310 bp (lane 2, III digest DNA marker). The cDNA fragment was completely sequenced. The nucleotide sequence is shown in . The results show that the cDNA fragment was flanked by the sequence corresponding to arbitrary decanucleotide primer. In contrast, the cDNA fragment was not flanked by the sequences corresponding to the DNA squences of the oligo(dT) primer. Apparently, the oligo(dT) primer hybridized to an internal site and not to 3′-end. This phenomenon was also observed by others . The nucleotide sequence of the P-band cDNA fragment was analysed by searching for homologies in the nucleotide databases at EMBL using the search engines FASTA and BLAST. The nucleotide sequence of P-band cDNA fragment was found to be >99% identical to rat gp130mRNA (accession number, Z47987). Thus, we designated the P-band cDNA as gp130-related cDNA. To confirm regulation of the gp130mRNA in response to Ang II and PDGF-BB stimulation, Northern blotting analysis was performed using the [α-P]dCTP-labeled gp130-related cDNA fragment as DNA probe. As demonstrated in , stimulation of the VSMCs with PDGF-BB resulted in a time-dependent decrease in the gp130-related mRNA. A pronounced decrease in the mRNA level occurred six hours after stimulation and the level remained markedly low within 12 hours after stimulation, compared with unstimulated cells. Densitometric analysis of the band intensities revealed that stimulation of the VSMCs with PDGF-BB for six, eight and 12 hours caused 70, 95 and 95% reductions of the gp130-related mRNA level in unstimulated cells, respectively. The findings obtained by the Northern blotting approach were confirmed by RT-PCR using the specific gp130primer for detection of 524 bp gp130cDNA. As shown in , stimulation of VSMCs with PDGF-BB resulted in a marked decline of the 524 bp gp130cDNA occurring after six hours of stimulation. The decline remained stable within 12 hours after stimulation of VSMCs with PDGF-BB. Stimulation of the VSMCs for 15 minutes, 30 minutes, one hour and two hours did not influence the 524 bp gp130cDNA level, compared wth unstimulated cells. The 384 bp GAPDH cDNA level was nearly the same within two hours after stimulation. Although 384 bp GAPDH cDNA level was higher after six and 12 hours of stimulation with PDGF-BB, the 524 bp gp130cDNA level at six and 12 hours was markedly lower, compared with the level at the other stimulation time periods. Stimulation of the VSMC with Ang II also resulted in a time-dependent decrease in gp130-related mRNA but the maximal decrease occurred four hours after stimulation and level remained markedly low within 12 hours after stimulation . Again, our results show that stimulation of the VSMCs with Ang II four, six and 12 hours resulted in a 90% reduction in the gp130-related mRNA. The findings obtained by the Northern blotting approach were confirmed by RT-PCR using the specific gp130primer for detection of 524 bp gp130cDNA. Again, stimulation time periods, the 524 bp gp 130cDNA levels at 6 and 12 hours were markedly lower. Since changes in gp130cDNA by RT-PCR reflect semiquantitative changes in gp130mRNA is is obvious that both growth factors remarkably attenuate the gp130mRNA six hours after stimulation of the VSMCs.