retina vasculature sprouting front

ATTRACT: Arterial flow as attractor for endothelial cell migration

Fondation Leducq Transatlantic Network of Excellence

The project

What is Leducq ATTRACT about?

The cells that form the inner lining of arteries are called endothelial cells. It was once thought that endothelial cells were an immobile interface between the blood flowing past them and the tissues which are perfused with oxygen. However, recent discoveries by our network’s members have shown that endothelial cells can change position within vessels, and that they actually have been observed to migrate in the opposite direction of blood flow. This finding has led us to examine whether this endothelial cell migration plays a role in the normal development of blood vessels. In addition, we hope to understand how endothelial cell migration may play a role in disease. In vascular disease such as stroke and coronary artery disease, the atherosclerotic process disrupts the endothelial layer and changes the pattern of cell migration. Our network will investigate the principles of normal vessel function and how endothelial cell movement and repair are maladaptive after a stroke. We hope to learn how to harness the beneficial movement of the endothelial cells so that patients with neurovascular disease have better outcomes, and abnormal vascular connections are avoided.

 

Participating labs

 

Holger Gerhardt Lab

Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin (Germany)

Website

The Integrative Vascular Biology Laboratory aims to unravel the fundamental principles and molecular/genetic regulation of functional vascular network formation in development and disease.

 

 

Doug Marchuk Lab

Duke University Medical Center, Durham, NC (USA)

Website

Doug's laboratory studies the genetics of cardiovascular disease using both the human and the mouse as a model system.

 

 

Claudio Franco Lab

Instittuto de Medicina Molecular, Lisbon (Portugal)

Website

The Vascular Morphogenesis Lab has the aim to understand the molecular mechanisms regulating coordinated endothelial cell behaviour during sprouting and remodelling phases of the angiogenic process.

 

 

Anne Eichmann Lab

Yale University School of Medicine, New Haven (USA)

Website

Anne's lab studies vascular and lymphatic development, with particular emphasis on mechanisms that direct patterning and guidance.

 

 

Lena Claesson-Welsh Lab

Uppsala University (Sweden)

Website

Lena's lab studies how blood vessels are formed and which factors control the process, positively or negatively. Major topics of her research are how vascular endothelial growth factor (VEGF) regulates angiogenesis and the signal transduction pathways which regulate blood vessel permeability.

 

 

Miguel Bernabeu Lab

The University of Edinburg (UK)

Website

Miguel is a Computational Biologist specialised in microvascular Biomechanics and Mechanobiology. His research interests concern the study of vascular remodelling during angiogenesis in order to identify molecular targets for its regulation. In addition, he explores the translational potential of these findings for the treatment diabetic retinopathy.

 

 

S. Paul Oh Lab

Barrow Neurological Institute, Phoenix (USA)

Website

Paul Oh’s research interests include identifying cellular mechanisms responsible for cerebrovascular disorders and assessing novel therapies to treat these disorders.

Publications

Here you can find the most recent publications from the network groups, relevant for the Leducq project.

Cerebral Cavernous Malformations Develop Through Clonal Expansion of Mutant Endothelial Cells

September 2018, in Circulation Research

Matthew R. Detter, Daniel A. Snellings, and Douglas A. Marchuk

Rationale:

Vascular malformations arise in vessels throughout the entire body. Causative genetic mutations have been identified for many of these diseases; however, little is known about the mutant cell lineage within these malformations.

Objective:

We utilize an inducible mouse model of cerebral cavernous malformations (CCMs) coupled with a multicolor fluorescent reporter to visualize the contribution of mutant endothelial cells (ECs) to the malformation.

Methods and Results:

We combined a Ccm3 mouse model with the confetti fluorescent reporter to simultaneously delete Ccm3 and label the mutant EC with 1 of 4 possible colors. We acquired Z-series confocal images from serial brain sections and created 3-dimensional reconstructions of entire CCMs to visualize mutant ECs during CCM development. We observed a pronounced pattern of CCMs lined with mutant ECs labeled with a single confetti color (n=42). The close 3-dimensional distribution, as determined by the nearest neighbor analysis, of the clonally dominant ECs within the CCM was statistically different than the background confetti labeling of ECs in non-CCM control brain slices as well as a computer simulation (P<0.001). Many of the small (<100 μm diameter) CCMs consisted, almost exclusively, of the clonally dominant mutant ECs labeled with the same confetti color, whereas the large (>100 μm diameter) CCMs contained both the clonally dominant mutant cells and wild-type ECs. We propose of model of CCM development in which an EC acquires a second somatic mutation, undergoes clonal expansion to initiate CCM formation, and then incorporates neighboring wild-type ECs to increase the size of the malformation.

Conclusions:

This is the first study to visualize, with single-cell resolution, the clonal expansion of mutant ECs within CCMs. The incorporation of wild-type ECs into the growing malformation presents another series of cellular events whose elucidation would enhance our understanding of CCMs and may provide novel therapeutic opportunities.

Link to article

 

NCK-dependent pericyte migration promotes pathological neovascularization in ischemic retinopathy

August 2018 in Nature communications

Alexandre Dubrac, Steffen E. Künzel, Sandrine H. Künzel, Jinyu Li, Rachana Radhamani Chandran, Kathleen Martin, Daniel M. Greif, Ralf H. Adams & Anne Eichmann 

Pericytes are mural cells that surround capillaries and control angiogenesis and capillary barrier function. During sprouting angiogenesis, endothelial cell-derived platelet-derived growth factor-B (PDGF-B) regulates pericyte proliferation and migration via the platelet-derived growth factor receptor-β (PDGFRβ). PDGF-B overexpression has been associated with proliferative retinopathy, but the underlying mechanisms remain poorly understood. Here we show that abnormal, α-SMA-expressing pericytes cover angiogenic sprouts and pathological neovascular tufts (NVTs) in a mouse model of oxygen-induced retinopathy. Genetic lineage tracing demonstrates that pericytes acquire α-SMA expression during NVT formation. Pericyte depletion through inducible endothelial-specific knockout of Pdgf-b decreases NVT formation and impairs revascularization. Inactivation of the NCK1 and NCK2 adaptor proteins inhibits pericyte migration by preventing PDGF-B-induced phosphorylation of PDGFRβ at Y1009 and PAK activation. Loss of Nck1 and Nck2 in mural cells prevents NVT formation and vascular leakage and promotes revascularization, suggesting PDGFRβ-Y1009/NCK signaling as a potential target for the treatment of retinopathies.

https://www.nature.com/articles/s41467-018-05926-7

 

A Fully Discrete Open Source Framework for the Simulation of Vascular Remodelling.

July 2018, Conf Proc IEEE Eng Med Biol Soc

James M. Osbotne and Miguel O. Bernabeu

 

In this paper we present a novel computational framework for the theoretical study of the interaction between haemodynamics and vessel biology, with particular applications to the study of vascular remodelling. We introduce the mathematical formulation, validate the numerical method against an analytical solution derived for a simplified case, and present a case study of tissue remodelling in response to flow.

Link to article

SMAD4 Prevents Flow Induced Arterial-Venous Malformations by Inhibiting Casein Kinase 2

July 2018, in Circulation

Roxana Ola, Sandrine H. Künzel, Feng Zhang, Gael Genet, Raja Chakraborty, Laurence Pibouin-Fragner, Kathleen Martin, William Sessa, Alexandre Dubrac, and Anne Eichmann

Background—Hereditary Hemorrhagic Telangiectasia (HHT) is an inherited vascular disorder that causes arterial-venous malformations (AVMs). Mutations in the genes encoding Endoglin (ENG) and Activin-receptor-like kinase 1 (AVCRL1 encoding ALK1) cause HHT type 1 and 2, respectively. Mutations in the SMAD4 gene are present in families with Juvenile Polyposis/HHT syndrome that involves AVMs. SMAD4 is a downstream effector of Transforming growth factor-β (TGFβ)/Bone morphogenetic protein (BMP) family ligands that signal via Activin like kinase receptors (ALKs). Ligand-neutralizing antibodies or inducible, endothelial-specific Alk1 deletion induce AVMs in mouse models as a result of increased PI3K/AKT signaling. Here we addressed if SMAD4 was required for BMP9-ALK1 effects on PI3K/AKT pathway activation.

Methods: We generated a tamoxifen-inducible, postnatal endothelial-specific Smad4 mutant mice (Smad4iΔEC).

Results: We found that loss of endothelial Smad4 resulted in AVM formation and lethality. AVMs formed in regions with high blood flow in developing retinas and other tissues. Mechanistically, BMP9 signaling antagonized flow-induced AKT activation in an ALK1 and SMAD4 dependent manner. Smad4iΔEC endothelial cells in AVMs displayed increased PI3K/AKT signaling, and pharmacological PI3K inhibitors or endothelial Akt1 deletion both rescued AVM formation in Smad4iΔEC mice. BMP9-induced SMAD4 inhibited Casein Kinase 2 (CK2) transcription, in turn limiting PTEN phosphorylation and AKT activation. Consequently, CK2 inhibition prevented AVM formation in Smad4iΔEC mice.

Conclusions: Our study reveals SMAD4 as an essential effector of BMP9-10/ALK1 signaling that affects AVM pathogenesis via regulation of CK2 expression and PI3K/AKT1 activation.

Link to article

Intravital imaging-based analysis tools for vessel identification and assessment of concurrent dynamic vascular events

July 2018, in Nature Communications

Naoki Honkura, Mark Richards, Bàrbara Laviña, Miguel Sáinz-Jaspeado, Christer Betsholtz, and Lena Claesson-Welsh

The vasculature undergoes changes in diameter, permeability and blood flow in response to specific stimuli. The dynamics and interdependence of these responses in different vessels are largely unknown. Here we report a non-invasive technique to study dynamic events in different vessel categories by multi-photon microscopy and an image analysis tool, RVDM (relative velocity, direction, and morphology) allowing the identification of vessel categories by their red blood cell (RBC) parameters. Moreover, Claudin5 promoter-driven green fluorescent protein (GFP) expression is used to distinguish capillary subtypes. Intradermal injection of vascular endothelial growth factor A (VEGFA) is shown to induce leakage of circulating dextran, with vessel-type-dependent kinetics, from capillaries and venules devoid of GFP expression. VEGFA-induced leakage in capillaries coincides with vessel dilation and reduced flow velocity. Thus, intravital imaging of non-invasive stimulation combined with RVDM analysis allows for recording and quantification of very rapid events in the vasculature.

Link to article

PolNet: A Tool to Quantify Network-Level Cell Polarity and Blood Flow in Vascular Remodeling

May 2018, in Biophysical Journal

Miguel O. Bernabeu, Martin L. Jones, Rupert W. Nash, Anna Pezzarossa, Peter V. Coveney, Holger Gerhardt, and Claudio A. Franco

In this article, we present PolNet, an open-source software tool for the study of blood flow and cell-level biological activity during vessel morphogenesis. We provide an image acquisition, segmentation, and analysis protocol to quantify endothelial cell polarity in entire in vivo vascular networks. In combination, we use computational fluid dynamics to characterize the hemodynamics of the vascular networks under study. The tool enables, to our knowledge for the first time, a network-level analysis of polarity and flow for individual endothelial cells. To date, PolNet has proven invaluable for the study of endothelial cell polarization and migration during vascular patterning, as demonstrated by two recent publications. Additionally, the tool can be easily extended to correlate blood flow with other experimental observations at the cellular/molecular level. We release the source code of our tool under the Lesser General Public License.

Link to article

 

News

March 2019

We are thrilled to announce that Anne Eichmann has been awarded the 2019 Judah Folkman Award in Vascular Biology from NAVBO! What a great and well deserved honour! Congratulations to Anne and her team!

 

http://www.navbo.org/awards/judah-folkman-award/eichmann

 


 

February 2019

Yi Jin, a postdoc in the lab of Lena Claesson-Welsh, is currently visiting the lab of Claudio Franco in Portugal. Yi is working on Src family kinases in vascular biology and went to Portugal to learn the models of flow and scratch wound assay, the cell polarity analysis for the in vitro models as well as how to use the Polnet software. We wish Yi a productive and interesting stay at the iMM in Lisbon!

 


 

January 2019

Gordon Research Seminar (GRS) organized by Gina Zarkada

Molecular and Cellular Dynamics of Endothelial Cells in Health and Disease

August 3 - 4, 2019 in Newport, RI, US

The Gordon Research Seminar (GRS) on Angiogenesis provides an excellent platform for graduate students, post-docs, and other young scientists to meet and exchange data and new ideas. The GRS provides an exceptional and vibrant environment for networking, career guidance and opportunity awareness, therefore presenting a unique learning experience for participants.

The 2019 GRS on Angiogenesis will introduce and highlight key developments in the field. We will discuss the basics of cardiovascular biology and disease with an emphasis on novel approaches and innovative technologies to study these processes. We will also focus on the heterogeneity of the endothelium, and the implications of functional genomics in the future directions of our research. The special format of the GRS allows up-and-coming vascular biologists to discuss their science in a relaxed environment, and interact informally in an open discussion forum. The seminar will conclude with a panel discussion on academic and professional development with established investigators and senior researchers.

Deadline for applications: May 3, 2019. 

 


 

December 2018

lisbon tower at dusk

Save the date: Portugal meeting 2019

Host: Claudio Franco ---------------

------------------- Dates: April 2-4th

More info coming soon! ------------

 


 

November 2018

                      Schedule for the video conference meetings in 2019

 
January 14th AE: Hyojin Park PO: Yong Hwan Kim
February 4th LCW: Suvi Jauhiainen DM: Dan Snellings
March 4th MB: Qi Zhou CF: Catarina Fonseca
April

Lisbon meeting (no VC)

 
May 13th LCW: Mark Richards PO: Chul Han
June 3rd HG: Ines Fechner LCW: Elin Sjöberg
July 8th LCW: Lakshmi Venkatraman DM: Matt Detter
August summer break  
September 9th HG: Andre Rosa CF: Isabela Fortunato
October 7th DM: Sarah Wetzel Strong AE: Kevin Boye
November 4th LCW: Tor Persson Skare HG: Olya Oppenheim
December 9th PO: Hyunwoo Choi LCW: Ross Smith

 

Download VC meeting schedule 2019


 

October 2018

Second Leducq Attract meeting in Durham, North Carolina.


 

May 2018

From now on, our network will gather once monthly for a video conference meeting. Every meeting will consist of two scientific talks by PhD students and postdocs. Here is the schedule for 2018:

May 7th CF Isabela Fortunato AE: Alex Dubrac
June 11th LCW: Miguel Sainz HG: Andre Rosa
July 2nd AE: Kevin Boyle PO: Hyunwoo Choi
August 6th: Summer break    
September 17th CF: Maria Dominguez DM: Han Kyu Lee
October 1st AE: Gina Zarkada HG Tijana Perovic
November 5th HG: Anna Szymborska MB: Lowell Edgar
December 3rd LCW: Yi Jin CF: Marie Ouarne

 

April 2018

Our first Leducq ATTRACT meeting took place in Paris on April 10th-11th and was hosted by Anne Eichmann and her team at the Paris Cardiovascular Research Center (PARCC)

full retina

Funding:

Fondation Leducq Transatlantic

Network of Excellence

 

European Coordinator:

Holger GERHARDT, Germany

 

North American Coordinator:

Douglas MARCHUK, USA

 

Members:

Miguel BERNABEU, UK

Lena CLAESSON-WELSH, Sweden

Anne EICHMANN, USA

Claudio FRANCO, Portugal

Paul OH, USA

 

Contact:

Petya Georgieva

Network Administrative Officer

Petya.georgieva@mdc-berlin.de