Fig. 4.

Receptor for advanced glycation end products (RAGE) interacts with inositol monophosphatase 1 (IMPA1) in pulmonary arterial hypertension (PAH). A: mass spectrometry analysis of proteins coimmunoprecipitated (co-IP) with RAGE from lungs identified IMPA1 as a novel RAGE interacting partner. RAGE-IMPA1 complex was discovered in both samples from PAH animals (week 5) but not in control (peptide probability P > 0; 4 peptides with P > 89%). B: time course of RAGE-IMPA1 interaction was investigated by co-IP. There was no interaction between RAGE and IMPA1 in controls from week 1 RAGE/IMPA1 were efficiently co-IP (n = 4 for all groups). Results are expressed as box and whisker plots (boxes: 25th to 75th percentile of the data; whiskers: minimum to maximum; line represents the median value). *P < 0.05 vs. control. Statistical analysis was performed by Newman-Keuls multiple-comparisons test. C: the formation of the RAGE-IMPA1 complex in the pulmonary vascular wall was visualized using the proximity ligation assay (PLA) method. Images are representative of 6 pulmonary arteries/at n = 4 rats/group. Gray images were taken using the light microscopy (×20) to visualize the tissue structure; red fluorescent signal indicates RAGE-IMPA1 interaction; blue fluorescent signal indicates nuclei stained by DAPI. Yellow square represents the area of magnification (×100). The white marker corresponds to 25 μm. D: the amplified signal from the RAGE-IMPA1 complex (red) was registered in the media of hypertrophied pulmonary arteries, as seen on enlarged images from weeks 1 (Wk1) and 5 (Wk5) (D). The yellow dotted line is traced following the external lamina of the vessel. Gray arrowheads point on the endothelial cells that have no red PLA signal; white arrows point on the cells in the pulmonary artery media and adventitia cells that have bright red PLA signal. IB, immunoblot.