The Roles of the Collagen Binding Integrins in the Regulation of Endothelial Cell Function
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 HUVECs. Functional assays were carried out downstream of integrin inhibition or siRNA treatment. HUVEC proliferation, measured by cell number quantification and 5-ethynyl-2’-deoxyuridine (EdU) incorporation analysis, was unaffected by inhibition or siRNA knockdown of any integrin receptor. Knockdown or inhibition of α2β1 severely hindered HUVEC attachment and spreading to collagen and THPs, measured by static and real-time adhesion assays. Cell migration, in time-lapse microscopy random movement assays, was also impaired after either knockdown or inhibition of α2β1. In addition, inhibition of α2β1 using TC-I-15 and 6F1 impeded angiogenesis in tube formation assays on a Geltrex substrate. However, siRNA knockdown of α2β1 had no effect on tube formation. This observation highlights the differences in signalling events between inhibiting a receptor that is present and simply lacking the receptor altogether. No compensatory upregulation of other α-subunits was observed. In conclusion, the inhibition or knockdown of integrin α2β1 has a significant effect on the behaviour of HUVECs. In contrast, inhibition or knockdown of α1β1 and α10β1 does not significantly affect HUVEC cellular response. Additionally, the results presented here suggest that inhibitors and antibodies targeting collagen-binding integrins must be rigorously tested for cross-reactivity before use. Finally, these findings show that TC-I-15 could have an interesting therapeutic value in blocking angiogenesis and the migration of endothelial cells.
collagen, integrin, endothelial, HUVEC, cardiovascular, extracellular matrix, angiogenesis, migration, proliferation, siRNA, TC-I-15, Obtustatin, 6F1
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