Regulation of RhoGTPase signalling at endothelial junctions and blood vessel integrity

Lead Research Organisation: University College London
Department Name: Institute of Ophthalmology

Abstract

Endothelial cells form a single layer of cells, lining the walls of blood and lymphatic vessels. Individual endothelial cells interact with each other via molecular complexes that mediate adhesion but also function as sensors that transmit information about the environment, such as the presence or absence of neighbouring cells, to the cell interior. Development and function of blood vessel requires endothelia to form functional cell-cell junctions that mediate adhesion and restrict trafficking of cells and molecules along the space in between neighbouring cells. These junctions are essential for blood and lymph vessel integrity, and are highly regulated to enable dynamic developmental processes, such as the growth of new vessels, or during physiological and pathological conditions that can affect vessel wall integrity. Deregulation of endothelial cell junctions occurs in different diseases, such as diabetes or inflammatory conditions, and can lead to vessel leakage or rupture (e.g., haemorrhagic stroke).

This proposal focuses on the identification of molecular mechanisms that regulate endothelial dynamic behaviour via cell-cell junctions and the importance of such mechanisms for blood vessel growth and vascular stability. Our unpublished data indicate that a protein that we previously identified in another cell type is also expressed in endothelial cells and regulates formation of cell-cell junctions and expression of functionally important endothelial genes. We have evidence that this protein is crucial for normal mammalian development and a functional cardiovascular system. Deficiency of this protein leads to internal bleeding and as well as defects in abdominal closure that are reminiscent of a human inherited disease.

The overall aim of this proposal is to understand the role of p114RhoGEF in endothelial cells during normal development and for the function of the vascular system. We also propose experiments designed to determine how this pathway that stabilises cell-cell junctions is regulated with the aim to target this signalling mechanism in the future for possible therapeutic intervention for diseases that lead to vascular leakage.

Knowledge of how cells sense their neighbours and transmit such information to the cell interior is not only important for the understanding of fundamental processes as they occur during the development of the vasculature, organs and tissues, but also to comprehend how different diseases interfere with the normal functioning of our cells. Hence, identifying new mechanisms that guide cell behaviour in response to contact with neighbours will help us to think of new ways to counteract vascular leakage and to treat wide-spread diseases such as cardiovascular conditions, age-related diseases such as diabetes and cancer.

Technical Summary

Endothelial cells line the walls of blood and lymphatic vessels. Angiogenesis, vessel integrity and endothelial homoeostasis require endothelial cells to form functional cell-cell junctions. Tight junctions are one of those cell-cell junctions and can be intertwined with adherens junctions in endothelia. This proposal focuses on the role of endothelial tight junctions in blood vessel stability and endothelial homeostasis. We have identified a Rho signalling pathway that is centred on a guanine nucleotide exchange factor that drives junctional actomyosin activation. Our preliminary data indicate that this pathway regulates endothelial junction assembly in vitro and expression of functionally important endothelial genes, and is required for normal development in mouse, a developmental defect that involves vessel stability defects leading to haemorrhages. Our hypothesis is that this Rho signalling pathway regulates junctional actomyosin, endothelial differentiation, vasoprotective responses, and, thereby, epithelial homeostasis and vessel integrity. Aim 1 is to determine the role of this signalling mechanism in endothelial processes and cell-cell tension important for endothelial function and angiogenesis, and employ in vitro angiogenesis models. We also aim to determine how this pathway is regulated and how it affects expression of identified candidate genes and endothelial differentiation. Aim 2 will be to test the in vivo relevance of these findings using loss of function mouse models. Hence, we expect these studies to disclose the role of this Rho signalling mechanism in development and its importance and function in cardiovascular development and vessel integrity. The expected results will be important for the understanding and the development of therapeutic applications for diseases that lead or involve blood vessel leakage, such as diabetes, age-related diseases and inflammation, and haemorrhagic stroke.

Planned Impact

Who will benefit from this research?

The immediate beneficiaries will be scientists working in allied fields ranging from developmental biology, tissue engineering, and cardiovascular biology. However, the project is likely to impact medical researchers and translational scientists working on therapeutic applications for various diseases including age-related epithelial degenerations, congenital defects associated with ventral folding morphogenesis, cardiovascular conditions, or diabetes-induced epithelial and endothelial malfunctions. Diseases such as diabetes and age-related conditions represent an increasing burden for the NHS and our society. Hence, insights into the biology and function of endothelial cell junctions will benefit medical scientists, clinicians and, ultimately, patients as well as the NHS and the general public.


How will they benefit from this research?

The research will benefit allied scientists by providing them with the molecular details of a new mechanism that links cell-cell adhesion to the regulation of endothelial cell and tissue dynamics that they can then test in their respective model systems. Translational scientists will benefit in a similar way. In our own research environment, these results can be used to understand many pathological processes that lead to or involve vascular dysfunction, leakage and ruptures. Examples are diabetes, inflammation and haemorrhagic stroke as well as vascular dysplasia and fragility in cerebral cavernous malformation and Clarkson disease (systemic capillary leakage syndrome), a lethal blood vessel disease that has been recently linked to polymorphisms in the p114RhoGEF. This project will also benefit scientist that develop new approaches for tissue engineering and transplantation. Clinicians and patients will then benefit in the longer term by the availability of new approaches to treat common diseases.

The project will also involve training of a postdoctoral fellow that can benefit the private sector as well as public services through the NHS.

The expected results are likely to start to benefit other scientists within the lifetime of this grant.
 
Description ARHGEF18, a guanine nucleotide exchange factor that activates RHOA, a small GTPase protein is involved during mouse placenta development and function. Furthermore, the retinal structure in individuals with ARHGEF18 mutations resembled that seen in subjects with CRB1 mutations. Five mutations were found on six alleles in the three individuals: c.808A>G (p.Thr270Ala), c.1617+5G>A (p.Asp540Glyfs*63), c.1996C>T (p.Arg666*), c.2632G>T (p.Glu878*), and c.2738_2761del (p.Arg913_Glu920del). Functional tests suggest that each disease genotype might retain some ARHGEF18 activity, such that the phenotype described here is not the consequence of nullizygosity. In particular, the p.Thr270Ala missense variant affects a highly conserved residue in the DBL homology domain, which is required for the interaction and activation of RHOA.
We also have a manuscript under revision:
Abstract
Coordination of cell-cell adhesion and the actin cytoskeleton is key to tissue
development and function. Morphogenesis of organs such as the placenta
requires complex cellular processes ranging from branching morphogenesis to
cell-cell fusion; however, the molecular mechanisms that control cell adhesion
and actomyosin dynamics to power such processes are not well understood.
Here we show that deletion of p114RhoGEF/ARHGEF18, a tight junctionassociated
guanine nucleotide exchange factor for RhoA that forms complexes
with myosin II and Rock II, is required to drive placenta development. In vitro and in vivo experiments indicate that
p114RhoGEF guides trophoblast differentiation and behaviour by coordinating
actomyosin remodelling and gene expression. In cultured
trophoblast models, actomyosin remodelling and
hormonogenesis are inhibited upon p114RhoGEF depletion. Our
data thus indicate that p114RhoGEF links cell-cell junctions and actomyosin
dynamics to the regulation of placenta morphogenesis.
Exploitation Route Further funding, collaborations, publications, novel therapeutic approaches
Sectors Education,Pharmaceuticals and Medical Biotechnology,Other

URL https://www.biorxiv.org/content/10.1101/2020.07.12.199141v1
 
Description Coordination of cell-cell adhesion and the actin cytoskeleton is key to tissue development and function. Morphogenesis of organs such as the placenta requires complex cellular processes ranging from branching morphogenesis to cell-cell fusion; however, the molecular mechanisms that control cell adhesion and actomyosin dynamics to power such processes are not well understood. Here we show that deletion of p114RhoGEF/ARHGEF18, a tight junctionassociated guanine nucleotide exchange factor for RhoA that forms complexes with myosin II and Rock II, is required to drive placenta development. In vitro and in vivo experiments indicate that p114RhoGEF guides trophoblast differentiation and behaviour by coordinating actomyosin remodelling and gene expression. In cultured trophoblast models, actomyosin remodelling and hormonogenesis are inhibited upon p114RhoGEF depletion. Our data thus indicate that p114RhoGEF links cell-cell junctions and actomyosin dynamics to the regulation of placenta morphogenesis.
First Year Of Impact 2018
Sector Healthcare,Other
Impact Types Societal,Economic

 
Description Fight for Sight, PhD studentship, Cell-cell junctions in retina degeneration disease: The role of p114RhoGEF, a junction-associated Rho GEF
Amount £100,000 (GBP)
Funding ID Ref number 1609 
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2018 
End 09/2021
 
Description MarvelD3 signalling and retinal tissue stress
Amount £122,242 (GBP)
Funding ID GR001000 
Organisation Moorfields Eye Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2020 
End 12/2021
 
Description Polarity signalling in retinal degeneration
Amount £8,670 (GBP)
Funding ID BRC3_039 
Organisation Moorfields Eye Hospital NHS Foundation Trust 
Department NIHR Moorfields Biomedical Research Centre
Sector Academic/University
Country United Kingdom
Start 10/2017 
End 03/2018
 
Description Regulation of RhoGTPase signalling at endothelial junctions and blood vessel integrity.
Amount £519,340 (GBP)
Funding ID BB/N001133/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2016 
End 03/2019
 
Title Analysis of the role of tight junction proteins in vivo during mouse development 
Description Analysis of the role of tight junction proteins in cell-based models and in vivo during mouse development in endothelial cells 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2016 
Provided To Others? Yes  
Impact Analysis of the role of tight junction proteins in cell-based models and in vivo during mouse development in endothelial cells 
 
Title p114Rho-GEF controls endothelial adherens junctions, cell-cell tension, angiogenesis and barrier formation 
Description Characterisation of the molecular mechanism by which p114Rho-GEF controls endothelial adherens junctions, cell-cell tension, angiogenesis and barrier formation 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2016 
Provided To Others? No  
Impact Endothelial cell junctions have an important role in the function of the endothelium. They provide adhesive contacts between neighbouring cells and provide communication between cells. Proteins at the endothelial junctions are capable of detecting stretch, stress and damage and providing signals to intercellular effector pathways. p114Rho-GEF contributes to the regulation of endothelial cytoskeletal arrangement, tight junction protein localisation and inflammation. Further characterization of the molecular mechanisms involved in these p114Rho-GEF-dependent pathways may therefore highlight novel therapeutic interventions for vascular diseases, such as atherosclerosis. 
 
Description Dimitris K. Grammatopoulos, Translational and Experimental Medicine, Warwick Medical School and Institute of Precision Biomarkers, Dept. of Pathology, University Hospital Coventry and Warwickshire NHS Trust, Coventry, United Kingdom 
Organisation University Hospitals Coventry and Warwickshire NHS Trust
Department Paediatrics University Hospitals Coventry and Warwickshire
Country United Kingdom 
Sector Public 
PI Contribution In vitro experiments to test how p114RhoGEF cooperates with PKA signalling, to guide trophoblast differentiation and behaviour by coordinating actomyosin remodelling and gene expression. In cultured trophoblast models, actomyosin remodelling, cell-cell fusion and hormonogenesis are inhibited upon p114RhoGEF depletion. Our data thus indicate that p114RhoGEF links cell-cell junctions, actomyosin dynamics to the regulation of PKA signalling during placenta morphogenesis.
Collaborator Contribution BeWo cells and Human chorionic gonadotropin (hCG) secretion assay
Impact A manuscript under revision
Start Year 2018
 
Description Dr. Marcus Fruttiger 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Regulation of RhoGTPase signalling at endothelial junctions and blood vessel integrity during mouse development
Collaborator Contribution Advice on mouse techniques
Impact - 04.16 - 03.19: Biotechnology and Biological Sciences Research Council. Principal applicant (co-applicants: Karl Matter and Marcus Fruttiger) Regulation of RhoGTPase signalling at endothelial junctions and blood vessel integrity. Amount: £519,340. Ref BB/N001133/1
Start Year 2011
 
Description Distinguished Lecture UCL Medicine 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Discussion of future work
Year(s) Of Engagement Activity 2017
 
Description Institute of Ageing and Chronic Disease, Liverpool, UK, invited speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Institute of Ageing and Chronic Disease, Liverpool, UK, invited speaker
Year(s) Of Engagement Activity 2019
 
Description PhD students Berlin 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Lecture for students of a PhD programme in Germany and discussions about their own research projects
Year(s) Of Engagement Activity 2017
 
Description RB, Poster presentation - UCL Institute of Ophthalmology Annual Symposium, 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact RB, Poster presentation - UCL Institute of Ophthalmology Annual Symposium, around 100 students, postdocs and group leaders attended for the annual research symposium, questions and discussion at the poster during coffee and lunch breaks
Year(s) Of Engagement Activity 2019