Insulin is known to act as an anti-inflammatory agent and protect vascular endothelial cells during ischaemic damage in vivo. Although it is known that insulin signals in part through phosphatidylinositol 3-kinases (PI3Ks) and Akt, its effects on endothelial junctions and actin cytoskeleton are unknown. This study aimed to characterise endothelial responses to insulin, identify endothelial insulin-‐induced changes in protein phosphorylation and determine the roles of these changes in regulating endothelial functions. Insulin stimulation induced dose-dependent Akt activation in both primary human umbilical vein endothelial cells (HUVECs) and an endothelial cell line, human bone-marrow endothelial cells (HBMECs). Insulin decreased basal HUVEC permeability, increased angiogenic loop formation in vitro and increased cell migration in a wound-healing model, compared to untreated cells. Insulin-stimulated changes in protein phosphorylation were identified using a 14-3-3 affinity purification proteomic screen, as 14-3-3 proteins interact specifically with phosphorylated Ser/Thr residues within 14-3-3-binding motifs. A total of 390 14-3-3-binding proteins were identified from insulin-stimulated HBMECs, from which 12 proteins were selected based on predicted roles in endothelial cytoskeleton regulation. Validation of these hits, performed by Far-Western overlay analysis, identified 4 IGF-I-regulated 14-3-3-binding proteins: Parg1 (ARHGAP29), RICH-1 (ARHGAP17), LMO7 and Epsin2. Parg1 depletion in HUVECs induced stress fibre formation, increased endothelial permeability, severely decreased angiogenic loop formation and decreased cell migration, compared to siRNA control-treated cells. This suggests that Parg1 regulates contractility and hence could affect endothelial cell-cell junctional stability. Depletion of RICH-1 and LMO7 in HUVECs resulted in mislocalisation of the tight junction protein ZO-1. However, this did not affect endothelial permeability, suggesting that these proteins are important for maintaining tight junction integrity. LMO7 and Epsin2 depletion each resulted in an increase in angiogenic loop formation, but did not detectably affect cell migration. Insulin stimulation of Epsin2 might increase lamellipodium formation, although further studies are required to establish the mechanisms involved. In conclusion, this thesis describes a 14-3-3-based proteomic screen that identified novel regulators of endothelial function. These proteins could contribute to the anti-inflammatory roles of insulin.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:677057 |
| Date | January 2014 |
| Creators | Philippeos, Christina |
| Contributors | Ridley, Anne |
| Publisher | King's College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://kclpure.kcl.ac.uk/portal/en/theses/insulin-signalling-in-endothelial-cells(8e35db48-dc9c-41be-b1aa-1fbe241fc356).html |
Page generated in 0.0026 seconds