Transcriptional Regulation of Thrombin-Induced Endothelial VEGF Induction and Proangiogenic Response

Abstract

Thrombin, the ligand of the protease-activated receptor 1 (PAR1), is a well-known stimulator of proangiogenic responses in vascular endothelial cells (ECs), which are mediated through the induction of vascular endothelial growth factor (VEGF). However, the transcriptional events underlying this thrombin-induced VEGF induction and angiogenic response are less well understood at present. As reported here, we conducted detailed promotor activation and signal transduction pathway studies in human microvascular ECs, to decipher the transcription factors and the intracellular signaling events underlying the thrombin and PAR-1-induced endothelial VEGF induction. We found that c-FOS is a key transcription factor controlling thrombin-induced EC VEGF synthesis and angiogenesis. Upon the binding and internalization of its G-protein-coupled PAR-1 receptor, thrombin triggers ERK1/2 signaling and activation of the nuclear AP-1/c-FOS transcription factor complex, which then leads to VEGF transcription, extracellular secretion, and concomitant proangiogenic responses of ECs. In conclusion, exposure of human microvascular ECs to thrombin triggers signaling through the PAR-1–ERK1/2–AP-1/c-FOS axis to control VEGF gene transcription and VEGF-induced angiogenesis. These observations offer a greater understanding of endothelial responses to thromboinflammation, which may help to interpret the results of clinical trials tackling the conditions associated with endothelial injury and thrombosis.

Autoantibodies from Patients with Scleroderma Renal Crisis Promote PAR-1 Receptor Activation and IL-6 Production in Endothelial Cells

“…First, the effect of SSc-IgG on PAR-1 activation can be blocked by the thrombin inhibitor refludan. Second, the signalling pathway evoked by SSc-IgG resembles closely that induced by thrombin on endothelial VEGF expression [ 35 ].…”

“…Human dermal microvascular endothelial cells (HMECs, catalogue no. CRL-3243) were purchased from ATCC ® (Manassas, VA, USA) and cultured as described previously [ 35 ].…”

Expanded Hemodialysis Therapy Ameliorates Uremia-Induced Systemic Microinflammation and Endothelial Dysfunction by Modulating VEGF, TNF-α and AP-1 Signaling

“…The samples were centrifuged at 2000 g for 15 min, followed by cryostorage at −80°C to be used for the EC incubation assay and multiplex analysis presented in Figures 2 , 3 . Quantification of soluble markers in patient serum was conducted on citrated plasma using the Milliplex Human Cytokine assay (Millipore) and Luminex-based immunoassays for soluble receptor proteins and specific ELISAs ( 28 , 37 ). For mechanistic experiments ( Figures 4 – 6 ), samples of 20 patients were pooled at equal volumes to obtain a uremic serum pool (USP) and non-uremic serum from 14 healthy donors (Age 36 ± 9.2 years; nine males, five females) was collected at our department, to generate a healthy serum pool (HSP) ( Table S1 ) ( 17 ).…”

“…Luciferase activity was assessed using the dual-luciferase reporter assay system (Promega, Mannheim, Germany) according to the manufacturer's protocol. Luciferase activity was (37). The TNF-a protein concentrations were measured using human TNF-a Antibody Pair Kits according to the manufacturer's instructions (ThermoFisher) and explained elsewhere (34).…”

“…Altered VEGF production is associated with an increased morbidity and mortality in HD patients (21)(22)(23)32). VEGF is also a major growth and survival factor for endothelial cells (ECs) and essential for angiogenesis (35)(36)(37). Considering proinflammatory inducers/regulators of maladaptive uremic signaling, in particular tumor necrosis factor (TNF) family members, e.g.…”