Analysis of Changes in Endothelial Cell Protein Expression and Phosphorylation Induced by Cleaved High Molecular Weight Kininogen

High molecular weight kininogen (HK) is an abundant plasma protein that functions as a critical cofactor in the kallikrein-kinin system. HK normally circulates in the single chain form, but is cleaved by plasma kallikrein to release the nonapeptide bradykinin and form cleaved high molecular weight kininogen (HKa) that consists of a heavy and light chain linked by a single disulfide bond. Conformational changes occurring in cleaved kininogen result in increased exposure of histidine and glycine-rich regions within kininogen domain 5 that impart HKa with unique properties, including the ability to inhibit angiogenesis by causing selective apoptosis of proliferating endothelial cells. However, neither the receptors that mediate the antiangiogenic activity of HKa nor the signaling pathways that lead to apoptosis have been rigorously defined. In this study we attempted to define specific signaling pathways activated following exposure of proliferating endothelial cells to HKa using a high-throughput, unbiased, microarray approach (Kinexus, Vancouver BC).

Endothelial cells were cultured at low density and stimulated to proliferate using 20 ng/ml bFGF in the absence or presence of HKa (15 nM). At various time points (20, 60 and 300 minutes) total cell extracts were prepared and analyzed using the Kinexus antibody microarray that includes 530 pan-specific and 270 phospho-site specific antibodies. In cells exposed to HKa, the analysis revealed increased expression of 109, 141 and 162 proteins, and decreased expression of 117, 68 and 59 proteins at the 20 min, 60 min, and 300 minute time points, respectively. In cells exposed to HKa, the number of newly-phosphorylated proteins increased from 30 at 20 minutes to 61 at 300 minutes after HKa treatment. Segregation of proteins whose expression level and/or phosphorylation state changed following exposure of cells to HKa into families demonstrated that HKa primarily targets protein kinases (61–70% of all proteins affected at the various time points), transcription factors (8–11%), and phosphatases (4–5%). Increased expression of several proteins involved in apoptosis, such as caspases 4, 6 and 7 and DNA fragmentation factors 35 and 45, and increased phosphorylation of stress regulated activating transcription factor 2 (ATF2) and apoptosis signal regulating protein kinase1 (ASK1) were evident within 20 minutes of exposure of cells to HKa. Metacore and Ingenuity pathway analysis of proteins that exhibited rapid changes in expression or phosphorylation revealed activation of several major signaling pathways including apoptosis, DNA damage response, angiogenesis, inflammation, and tissue remodeling and wound repair.

Exposure of proliferating endothelial cells to HKa led to rapid changes in protein expression and phosphorylation. Most remarkable was the increased expression of several caspases within 20 minutes of addition of HKa to cells. Patterns of protein expression were consistent with activation of several pathways related to apoptosis, inflammation and tissue remodeling. These findings support suspected physiological functions of HK/HKa in vivo, and suggest specific proteins that may be targeted to further dissect effects of HKa on discrete cellular functions.

No relevant conflicts of interest to declare.