Analysis of Changes in Endothelial Cell Protein Expression and Phosphorylation Induced by Cleaved High Molecular Weight Kininogen
Notice bibliographique
Résumé
Abstract Abstract 5195 Background and objective: 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). Results: 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. Conclusion: 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. Disclosures: No relevant conflicts of interest to declare.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,002 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».