The Roles of the Collagen Binding Integrins in the Regulation of Endothelial Cell Function
type: CreativeWork
description: Abstract The aim of this project was to first investigate the binding preferences of collagen binding integrins (α1β1, α2β1, α10β1 and α11β1), and to then assess their role in the regulation of endothelial cell (EC) behaviour using human umbilical vein endothelial cells (HUVECs). First, quantitative real-time polymerase chain reaction (qPCR) was performed to quantify the expression levels of the four integrins. Functional roles of these integrins were then investigated using commercially available inhibitors (TC-I-15, Obtustatin and 6F1) to modulate integrin function, and small interfering RNA (siRNA) to reduce expression levels. qPCR demonstrates that α2 is the most abundant receptor subunit transcript in HUVECs followed by α10 and α1; α11 was barely detectable. After integrin inhibition or knockdown, functional assays measuring changes in proliferation, apoptosis, adhesion, migration and angiogenesis were carried out. Synthetic triple helical collagenous peptides (THPs), based on the canonical GFOGER amino acid sequence, were used to probe the binding preferences of the integrins. Initially recombinant integrin αI-domains were studied, before utilising C2C12 cell lines stably expressing the full-length integrin receptors. The four integrins showed very similar, overlapping, binding profiles, with α1β1 and α10β1 binding strongly to GLOGEN motifs and moderately to GFOGER, and α2β1 and α11β1 binding strongly to GFOGER and moderately to GLOGEN. The specificities of the inhibitors TC-I-15, Obtustatin, and 6F1, for different integrins were characterised using static adhesion assays and real-time xCELLigence adhesion assays. TC-I-15 showed cross-reactivity between α1β1 and α2β1 and inhibited both receptors, Obtustatin was specific to α1β1 only and 6F1 was specific to α2β1. No cellular toxicity was observed for any of these inhibitors. siRNA was optimised to efficiently and selectively target each receptor and 90% knockdowns of α1β1, α2β1 and α10β1 mRNA were achieved in H…
abstract: Abstract The aim of this project was to first investigate the binding preferences of collagen binding integrins (α1β1, α2β1, α10β1 and α11β1), and to then assess their role in the regulation of endothelial cell (EC) behaviour using human umbilical vein endothelial cells (HUVECs). First, quantitative real-time polymerase chain reaction (qPCR) was performed to quantify the expression levels of the four integrins. Functional roles of these integrins were then investigated using commercially available inhibitors (TC-I-15, Obtustatin and 6F1) to modulate integrin function, and small interfering RNA (siRNA) to reduce expression levels. qPCR demonstrates that α2 is the most abundant receptor subunit transcript in HUVECs followed by α10 and α1; α11 was barely detectable. After integrin inhibition or knockdown, functional assays measuring changes in proliferation, apoptosis, adhesion, migration and angiogenesis were carried out. Synthetic triple helical collagenous peptides (THPs), based on the canonical GFOGER amino acid sequence, were used to probe the binding preferences of the integrins. Initially recombinant integrin αI-domains were studied, before utilising C2C12 cell lines stably expressing the full-length integrin receptors. The four integrins showed very similar, overlapping, binding profiles, with α1β1 and α10β1 binding strongly to GLOGEN motifs and moderately to GFOGER, and α2β1 and α11β1 binding strongly to GFOGER and moderately to GLOGEN. The specificities of the inhibitors TC-I-15, Obtustatin, and 6F1, for different integrins were characterised using static adhesion assays and real-time xCELLigence adhesion assays. TC-I-15 showed cross-reactivity between α1β1 and α2β1 and inhibited both receptors, Obtustatin was specific to α1β1 only and 6F1 was specific to α2β1. No cellular toxicity was observed for any of these inhibitors. siRNA was optimised to efficiently and selectively target each receptor and 90% knockdowns of α1β1, α2β1 and α10β1 mRNA were achieved in H…
author: Hunter, Emma Joanne
language: en
keywords: collagen, integrin, endothelial, HUVEC, cardiovascular, extracellular matrix, angiogenesis, migration, proliferation, siRNA, TC-I-15, Obtustatin, 6F1
license: https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2373c8fa-269f-48b3-a708-62e6ede8d026/download
license: https://www.rioxx.net/licenses/all-rights-reserved/
date published: 2020-04-25
date modified: 2026-07-22T22:24:01Z
provider: Cambridge University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Cambridge

PropertyValue
property ID: DOI
value: 10.17863/CAM.50699