Preventing the anti-angiogenic escape through the endothelial adhesome

Gontarczyk, Aleksander (2019) Preventing the anti-angiogenic escape through the endothelial adhesome. Doctoral thesis, University of East Anglia.

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Abstract

αVβ3 integrin is a promising target for therapy due to its upregulation in angiogenic vasculature. However, Cilengitide, an αVβ3 integrin inhibitor (RGD mimetic), has failed late stage clinical trials due to lack of overall survival benefit. We have previously shown that depleting β3-integrin acutely in the mouse endothelium leads to smaller tumours in vivo, whereas constitutive depletion of endothelial expression of this molecule causes an increase in tumour angiogenesis. In the context of cancer therapy, long-term depletion or inhibition of β3-integrin is subject to treatment escape via unknown mechanisms of resistance. At the molecular level, much of the regulation of endothelial cell migration and adhesion, and therefore angiogenesis, occurs via the ‘adhesome’ – the sub-proteome present in focal adhesion complexes. We isolated and compared WT, β3-HET, β3-KO and αVβ3-inhibitor-treated adhesomes by mass spectrometry. Among approximately 300 proteins whose abundance significantly changed, three intermediate filament proteins were upregulated in the β3-integrin depleted adhesome, namely: nestin, plectin and vimentin. All three molecules are known to regulate integrins and play a role in angiogenesis.

Given this upregulation of intermediate filaments in the adhesome, I set out to determine whether they are playing a differential role in angiogenesis depending on β3-integrin depletion. At first, I took an siRNA approach to determine the functional consequence of knocking down each target, but, overall, results from this approach were inconclusive. However, I observed that Withaferin A, an inhibitor of vimentin, impaired proliferation and migration of β3-HET, but not WT, endothelial cells. I then decided to look at the effects of Withaferin A on subcutaneous tumour growth in vivo, in combination with long-term endothelial-specific depletion of β3-integrin (via the β3-floxed Tie1.Cre model). The drug did not significantly inhibit tumour growth, but tumour angiogenesis was significantly reduced in the Cre +ve, and not in the Cre -ve background. Similarly, Withaferin A inhibited microvascular sprouting of β3-floxed Tie1.Cre +ve aortic rings, but not of Cre -ve rings. I also utilised pSico technology as an shRNA delivery system and observed impaired microvascular sprouting when depleting nestin. Additionally, I generated tdTomato-labelled nestin constructs which could be utilised to visualise nestin in vitro. Overall, the findings described in this thesis suggest co-targeting β3-integrin and intermediate filaments, such as those made up of vimentin, as an improved anti-angiogenic strategy.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Jennifer Whitaker
Date Deposited:	11 Jun 2019 09:30
Last Modified:	11 Jun 2019 09:30
URI:	https://ueaeprints.uea.ac.uk/id/eprint/71308
DOI:

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