Transforming growth factor beta signaling in hypertensive remodeling of porcine aorta

A porcine aortic coarctation model was used to examine regulation of gene expression in early hypertensive vascular remodeling. Aortic segments were collected proximal (high pressure) and distal (low pressure) to the coarctation after 2 weeks of sustained hypertension (mean arterial pressure>150mmHg). Porcine 10K oligoarrays used for gene expression profiling of the two regions of aorta revealed downregulation of cytoskeletal and upregulation of extracellular region genes relative to the whole genome. A genomic database search for transforming growth factor-β (TGFβ) control elements (TCE) showed that 19% of the genes that changed expression due to hypertension contained putative TCEs. Real time PCR and microarray analysis showed no change in expression of TGFβ1, TGFβ2, TGFβ3, or bone morphogenetc proteins (BMP)2 and 4, yet immunohistochemical staining for phosphorylated SMAD2, an indicator of TGFβ signaling, and for phosphorylated SMAD1/5/8, an indicator of signaling through the BMPs, showed the highest percentage of positively stained cells in the proximal aortic segments of occluded animals. For TGFβ signaling, this increase was significantly different from sham-operated controls. Western blot analysis showed no difference in total TGFβ1 protein levels with respect to treatment or aortic segment. Immunohistochemistry showed that the protein levels of latency associated peptide, was decreased in proximal segments of occluded animals. Collectively, these results suggest that activation of TGFβ, but not altered expression, may be a major mechanism regulating early hypertensive vascular remodeling. Dye-coupled RNA samples isolated from the same animal were paired according to the balanced block experimental design with a complete dye swap. Quality control was done by combining 3 flagging systems: F635-B635>10; 40% of pixels>B635 +2SD; F635/B635>2.5. If the gene failed only one of these criteria, it was flagged and was not included in further analysis. Same criteria were used to flag measurements in 532 channel. Filtering: A gene had to have absent flags in at least 4 out of 6 samples in order to be included in the analysis. Genes with at least 1.5 times upregulated or 1.5 downregulated gene expression and T-test p<0.05 were considered to be significant.