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.

Vasohibin 1 selectively regulates secondary sprouting and lymphangiogenesis in the zebrafish trunk

Feb 2021, Development

Marta Bastos de Oliveira, Katja Meier, Simone Jung, Eireen Bartels-Klein, Baptiste Coxam, Ilse Geudens, Anna Szymborska, Renae Skoczylas, Ines Fechner, Katarzyna Koltowska and Holger Gerhardt

Previous studies have shown that Vasohibin 1 (Vash1) is stimulated by VEGFs in endothelial cells and that its overexpression interferes with angiogenesis in vivo. Recently, Vash1 was found to mediate tubulin detyrosination, a post-translational modification that is implicated in many cell functions, such as cell division. Here, we used the zebrafish embryo to investigate the cellular and subcellular mechanisms of Vash1 on endothelial microtubules during formation of the trunk vasculature. We show that microtubules within venous- derived secondary sprouts are strongly and selectively detyrosinated in comparison with other endothelial cells, and that this difference is lost upon vash1 knockdown. Vash1 depletion in zebrafish specifically affected secondary sprouting from the posterior cardinal vein, increasing endothelial cell divisions and cell number in the sprouts. We show that altering secondary sprout numbers and structure upon Vash1 depletion leads to defective lymphatic vessel formation and ectopic lymphatic progenitor specification in the zebrafish trunk.

KEY WORDS: Lymphangiogenesis, Vash1, Microtubules, Tubulin detyrosination, Sprouting angiogenesis

https://dev.biologists.org/content/148/4/dev194993

On the preservation of vessel bifurcations during flow-mediated angiogenic remodelling

Feb 2021, PLOS Computational biology

Lowell T. Edgar, Claudio A. Franco, Holger Gerhardt, Miguel O. Bernabeu

During developmental angiogenesis, endothelial cells respond to shear stress by migrating and remodelling the initially hyperbranched plexus, removing certain vessels whilst maintaining others. In this study, we argue that the key regulator of vessel preservation is cell decision behaviour at bifurcations. At flow-convergent bifurcations where migration paths diverge, cells must finely tune migration along both possible paths if the bifurcation is to persist. Experiments have demonstrated that disrupting the cells’ ability to sense shear or the junction forces transmitted between cells impacts the preservation of bifurcations during the remodelling process. However, how these migratory cues integrate during cell decision making remains poorly understood. Therefore, we present the first agent-based model of endothelial cell flow-mediated migration suitable for interrogating the mechanisms behind bifurcation stability. The model simulates flow in a bifurcated vessel network composed of agents representing endothelial cells arranged into a lumen which migrate against flow. Upon approaching a bifurcation where more than one migration path exists, agents refer to a stochastic bifurcation rule which models the decision cells make as a combination of flow-based and collective-based migratory cues. With this rule, cells favour branches with relatively larger shear stress or cell number. We found that cells must integrate both cues nearly equally to maximise bifurcation stability. In simulations with stable bifurcations, we found competitive oscillations between flow and collective cues, and simulations that lost the bifurcation were unable to maintain these oscillations. The competition between these two cues is haemodynamic in origin, and demonstrates that a natural defence against bifurcation loss during remodelling exists: as vessel lumens narrow due to cell efflux, resistance to flow and shear stress increases, attracting new cells to enter and rescue the vessel from regression. Our work provides theoretical insight into the role of junction force transmission has in stabilising vasculature during remodelling and as an emergent mechanism to avoid functional shunting.

https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1007715

 

Permeability of the Endothelial Barrier: Identifying and Reconciling Controversies

Dec 2020, Trends of Molecular Medicine

Lena Claesson-WelshElisabetta DejanaDonald M McDonald 

Leakage from blood vessels into tissues is governed by mechanisms that control endothelial barrier function to maintain homeostasis. Dysregulated endothelial permeability contributes to many conditions and can influence disease morbidity and treatment. Diverse approaches used to study endothelial permeability have yielded a wealth of valuable insights. Yet, ongoing questions, technical challenges, and unresolved controversies relating to the mechanisms and relative contributions of barrier regulation, transendothelial sieving, and transport of fluid, solutes, and particulates complicate interpretations in the context of vascular physiology and pathophysiology. Here, we describe recent in vivo findings and other advances in understanding endothelial barrier function with the goal of identifying and reconciling controversies over cellular and molecular processes that regulate the vascular barrier in health and disease.

https://www.sciencedirect.com/science/article/pii/S1471491420302938?via%3Dihub

Abnormal morphology biases hematocrit distribution in tumor vasculature and contributes to heterogeneity in tissue oxygenation

October 2020, Proceedings of the National Academy of Sciences of the U.S.A.

Miguel O Bernabeu, Jakub Köry, James A Grogan, Bostjan Markelc, Albert Beardo, Mayeul d'Avezac, Romain Enjalbert, Jakob Kaeppler, Nicholas Daly, James Hetherington, Timm Krüger, Philip K Maini, Joe M Pitt-Francis, Ruth J Muschel, Tomás Alarcón, Helen M Byrne 

Oxygen heterogeneity in solid tumors is recognized as a limiting factor for therapeutic efficacy. This heterogeneity arises from the abnormal vascular structure of the tumor, but the precise mechanisms linking abnormal structure and compromised oxygen transport are only partially understood. In this paper, we investigate the role that red blood cell (RBC) transport plays in establishing oxygen heterogeneity in tumor tissue. We focus on heterogeneity driven by network effects, which are challenging to observe experimentally due to the reduced fields of view typically considered. Motivated by our findings of abnormal vascular patterns linked to deviations from current RBC transport theory, we calculated average vessel lengths [Formula: see text] and diameters [Formula: see text] from tumor allografts of three cancer cell lines and observed a substantial reduction in the ratio [Formula: see text] compared to physiological conditions. Mathematical modeling reveals that small values of the ratio λ (i.e., [Formula: see text]) can bias hematocrit distribution in tumor vascular networks and drive heterogeneous oxygenation of tumor tissue. Finally, we show an increase in the value of λ in tumor vascular networks following treatment with the antiangiogenic cancer agent DC101. Based on our findings, we propose λ as an effective way of monitoring the efficacy of antiangiogenic agents and as a proxy measure of perfusion and oxygenation in tumor tissue undergoing antiangiogenic treatment.

https://www.pnas.org/content/early/2020/10/26/2007770117.long

Overexpression of Activin Receptor-Like Kinase 1 in Endothelial Cells Suppresses Development of Arteriovenous Malformations in Mouse Models of Hereditary Hemorrhagic Telangiectasia

July 2020, Circulation research

Yong Hwan Kim, Phuong-Nhung Vu, Se-woon Choe, Chang-Jin Jeon, Helen M. Arthur, Calvin P.H. Vary, Young Jae Lee, S. Paul Oh.

Rationale:

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease caused by mutations in ENG, ALK1, or SMAD4. Since proteins from all 3 HHT genes are components of signal transduction of TGF-β (transforming growth factor β) family members, it has been hypothesized that HHT is a disease caused by defects in the ENG-ALK1-SMAD4 linear signaling. However, in vivo evidence supporting this hypothesis is scarce.

Objective:

We tested this hypothesis and investigated the therapeutic effects and potential risks of induced-ALK1 or -ENG overexpression (OE) for HHT.

Methods and Results:

We generated a novel mouse allele (ROSA26Alk1) in which HA (human influenza hemagglutinin)-tagged ALK1 and bicistronic eGFP expression are induced by Cre activity. We examined whether ALK1-OE using the ROSA26Alk1 allele could suppress the development of arteriovenous malformations (AVMs) in wounded adult skin and developing retinas of Alk1- and Eng-inducible knockout (iKO) mice. We also used a similar approach to investigate whether ENG-OE could rescue AVMs. Biochemical and immunofluorescence analyses confirmed the Cre-dependent OE of the ALK1-HA transgene. We could not detect any pathological signs in ALK1-OE mice up to 3 months after induction. ALK1-OE prevented the development of retinal AVMs and wound-induced skin AVMs in Eng-iKO as well as Alk1-iKO mice. ALK1-OE normalized expression of SMAD and NOTCH target genes in ENG-deficient endothelial cells (ECs) and restored the effect of BMP9 (bone morphogenetic protein 9) on suppression of phosphor-AKT levels in these endothelial cells. On the other hand, ENG-OE could not inhibit the AVM development in Alk1-iKO models.

Conclusions:

These data support the notion that ENG and ALK1 form a linear signaling pathway for the formation of a proper arteriovenous network during angiogenesis. We suggest that ALK1 OE or activation can be an effective therapeutic strategy for HHT. Further research is required to study whether this therapy could be translated into treatment for humans.

https://www.ahajournals.org/doi/10.1161/CIRCRESAHA.119.316267?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed

 

Endothelial cells on the move: dynamics in vascular morphogenesis and disease

July 2020, Vascular Biology

Catarina G Fonseca, Pedro Barbacena, and Claudio A Franco

The vascular system is a hierarchically organized network of blood vessels that play crucial roles in embryogenesis, homeostasis and disease. Blood vessels are built by endothelial cells – the cells lining the interior of blood vessels – through a process named vascular morphogenesis. Endothelial cells react to different biomechanical signals in their environment by adjusting their behavior to: (1) invade, proliferate and fuse to form new vessels (angiogenesis); (2) remodel, regress and establish a hierarchy in the network (patterning); and (3) maintain network stability (quiescence). Each step involves the coordination of endothelial cell differentiation, proliferation, polarity, migration, rearrangements and shape changes to ensure network integrity and an efficient barrier between blood and tissues. In this review, we highlighted the relevance and the mechanisms involving endothelial cell migration during different steps of vascular morphogenesis. We further present evidence on how impaired endothelial cell dynamics can contribute to pathology.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7487603/?report=reader

Novel Murine Models of Cerebral Cavernous Malformations

July 2020, Angiogenesis

Matthew R Detter, Robert Shenkar, Christian R Benavides, Catherine A Neilson, Thomas Moore, Rhonda Lightle, Nicholas Hobson, Le Shen, Ying Cao, Romuald Girard, Dongdong Zhang, Erin Griffin, Carol J Gallione, Issam A Awad, Douglas A Marchuk

Cerebral cavernous malformations (CCMs) are ectatic capillary-venous malformations that develop in approximately 0.5% of the population. Patients with CCMs may develop headaches, focal neurologic deficits, seizures, and hemorrhages. While symptomatic CCMs, depending upon the anatomic location, can be surgically removed, there is currently no pharmaceutical therapy to treat CCMs. Several mouse models have been developed to better understand CCM pathogenesis and test therapeutics. The most common mouse models induce a large CCM burden that is anatomically restricted to the cerebellum and contributes to lethality in the early days of life. These inducible models thus have a relatively short period for drug administration. We developed an inducible CCM3 mouse model that develops CCMs after weaning and provides a longer period for potential therapeutic intervention. Using this new model, three recently proposed CCM therapies, fasudil, tempol, vitamin D3, and a combination of the three drugs, failed to substantially reduce CCM formation when treatment was administered for 5 weeks, from postnatal day 21 (P21) to P56. We next restricted Ccm3 deletion to the brain vasculature and provided greater time (121 days) for CCMs to develop chronic hemorrhage, recapitulating the human lesions. We also developed the first model of acute CCM hemorrhage by injecting mice harboring CCMs with lipopolysaccharide. These efficient models will enable future drug studies to more precisely target clinically relevant features of CCM disease: CCM formation, chronic hemorrhage, and acute hemorrhage.

https://link.springer.com/article/10.1007%2Fs10456-020-09736-8

Lymphatic Endothelial Cell Junctions: Molecular Regulation in Physiology and Diseases

May 2020, Frontiers in Physiology

Feng Zhang, Georgia Zarkada, Sanjun Yi and Anne Eichmann

Lymphatic endothelial cells (LECs) lining lymphatic vessels develop specialized cell-cell junctions that are crucial for the maintenance of vessel integrity and proper lymphatic vascular functions. Successful lymphatic drainage requires a division of labor between lymphatic capillaries that take up lymph via open “button-like” junctions, and collectors that transport lymph to veins, which have tight “zipper-like” junctions that prevent lymph leakage. In recent years, progress has been made in the understanding of these specialized junctions, as a result of the application of state-of-the-art imaging tools and novel transgenic animal models. In this review, we discuss lymphatic development and mechanisms governing junction remodeling between button and zipper-like states in LECs. Understanding lymphatic junction remodeling is important in order to unravel lymphatic drainage regulation in obesity and inflammatory diseases and may pave the way towards future novel therapeutic interventions.

https://www.frontiersin.org/articles/10.3389/fphys.2020.00509/full#ack1

PI3Kβ-Regulated Pericyte Maturation Governs Vascular Remodeling

May 2020, Circulation

Ana M. Figueiredo, Pilar Villacampa, Rodrigo Diéguez-Hurtado, Juan José Lozano, Piotr Kobialka, Ana Rosa Cortazar, Anabel Martinez-Romero, Ana Angulo-Urarte, Claudio A. Franco, Marc Claret, Ana María Aransay, Ralf H. Adams, Arkaitz Carracedo, and Mariona Graupera

Background: Pericytes regulate vessel stabilization and function and their loss is associated with diseases such as diabetic retinopathy or cancer. Despite their physiological importance, pericyte function and molecular regulation during angiogenesis remain poorly understood.

Methods: To decipher the transcriptomic programs of pericytes during angiogenesis, we crossed the Pdgfrb(BAC)-CreERT2 into the RiboTagflox/flox mice. Pericyte morphological changes were assessed in mural cell-specific R26-mTmG reporter mice, in which low doses of tamoxifen allowed labeling of single cell pericytes at high resolution. To study the role of phosphoinositide 3-kinase (PI3K) signaling in pericyte biology during angiogenesis, we used genetic mouse models which allow selective inactivation of PI3Kα and PI3Kβ isoforms and their negative regulator PTEN (phosphate and tensin homologue deleted on chromosome ten, PTEN) in mural cells.

Results: At the onset of angiogenesis, pericytes exhibit molecular traits of cell proliferation and activated PI3K signaling, whereas during vascular remodeling pericytes upregulate genes involved in mature pericyte cell function, together with a remarkable decrease in PI3K signaling. Immature pericytes showed stellate shape and high proliferation, and mature pericytes were quiescent and elongated. Unexpectedly, we demonstrate that the PI3Kβ, but not PI3Kα, regulates pericyte proliferation and maturation during vessel formation. Genetic PI3Kβ inactivation in pericytes triggered early pericyte maturation. Conversely, unleashing PI3K signaling by means of PTEN deletion delayed pericyte maturation. Pericyte maturation was necessary to undergo vessel remodeling during angiogenesis.

Conclusions: Our results identify new molecular and morphological traits associated to pericyte maturation and uncover PI3Kβ activity as a checkpoint to ensure appropriate vessel formation. In turn, our results may open new therapeutic opportunities to regulate angiogenesis in pathological processes through the manipulation of pericyte PI3Kβ activity.

https://www.ahajournals.org/doi/abs/10.1161/CIRCULATIONAHA.119.042354

Association between erythrocyte dynamics and vessel remodelling in developmental vascular networks

May 2020, bioRxiv

Qi Zhou, Tijana Perovic, Ines Fechner, Lowell T. Edgar, Peter R. Hoskins, Holger Gerhardt, Timm Krüger, Miguel O. Bernabeu

Sprouting angiogenesis is an essential vascularisation mechanism and consists of two phases: sprouting and remodelling. The remodelling phase is driven by rearrangements of endothelial cells (ECs) within the primitive vascular plexus. Prior work has uncovered how ECs polarise and migrate in response to flow-induced wall shear stress (WSS). However, the question of how the presence of red blood cells (RBCs), and their profound impact on microvascular haemodynamics, affect vascular remodelling has not been addressed. Here, we extend our computational framework to model blood flow in developmental mouse retina as a suspension of RBCs. Our results demonstrate a previously unreported highly heterogeneous distribution of RBCs in the post-sprouting vascular network. Furthermore, we report a strong association between vessel regression and RBC depletion, and identify plasma skimming as the driving mechanism. Live imaging in a developmental zebrafish model confirms this association. Taken together, our results indicate that RBC dynamics are fundamental for establishing the regional WSS differences driving vascular remodelling via their ability to modulate effective viscosity.

https://www.biorxiv.org/content/10.1101/2020.05.21.106914v1

 

Vascular permeability in retinopathy is regulated by VEGFR2 Y949 signaling to VE-cadherin.

April 2020, eLife

Ross O Smith, Takeshi Ninchoji, Emma Gordon, Helder André, Elisabetta Dejana, Dietmar Vestweber, Anders Kvanta, Lena Claesson-Welsh

Edema stemming from leaky blood vessels is common in eye diseases such as age-related macular degeneration and diabetic retinopathy. Whereas therapies targeting vascular endothelial growth factor A (VEGFA) can suppress leakage, side-effects include vascular rarefaction and geographic atrophy. By challenging mouse models representing different steps in VEGFA/VEGF receptor 2 (VEGFR2)-induced vascular permeability, we show that targeting signaling downstream of VEGFR2 pY949 limits vascular permeability in retinopathy induced by high oxygen or by laser-wounding. Although suppressed permeability is accompanied by reduced pathological neoangiogenesis in oxygen-induced retinopathy, similarly sized lesions leak less in mutant mice, separating regulation of permeability from angiogenesis. Strikingly, vascular endothelial (VE)-cadherin phosphorylation at the Y685, but not Y658, residue is reduced when VEGFR2 pY949 signaling is impaired. These findings support a mechanism whereby VE-cadherin Y685 phosphorylation is selectively associated with excessive vascular leakage. Therapeutically, targeting VEGFR2-regulated VE-cadherin phosphorylation could suppress edema while leaving other VEGFR2-dependent functions intact.

https://elifesciences.org/articles/54056

TMEM100 is a key factor for specification of lymphatic endothelial progenitors

February 2020, Angiogenesis

Eun-Hye Moon, Yong Hwan Kim, Phuong-Nhung Vu, Hyunjin Yoo, Kwonho Hong, Young Jae Lee & S. Paul Oh

Background: TMEM100 is identified as a downstream gene of bone morphogenetic protein 9 (BMP9) signaling via activin receptor-like kinase 1 (ALK1), which is known to participate in lymphangiogenesis as well as angiogenesis. TMEM100 has been shown to be important for blood vessel formation and maintenance, but its role in the development of lymphatic vasculature remains unknown. The objective is to investigate the role of TMEM100 in development of the lymphatic system.

Methods and results: Global Tmem100 gene deletion was induced by tamoxifen on 10.5 days post-coitus. Tmem100-inducible knockout (iKO) embryos in embryonic days (E)14.5–16.5 exhibited edema and blood-filled enlarged lymphatics with misconnections between veins and lymphatic vessels. For a reciprocal approach, we have generated a novel mouse line in which TMEM100 overexpression (OE) can be induced in endothelial cells by intercrossing with Tie2-Cre driver. TMEM100-OE embryos at E12.5–14.5 exhibited edema with small size and number of lymphatic vessels, the exact opposite phenotypes of Tmem100-iKOs. In Tmem100-iKO embryos, the number of progenitors of lymphatic endothelial cells (LECs) in the cardinal vein was increased, while it was decreased in TMEM100-OE embryos. The activity of NOTCH signaling, which limits the number of progenitors of LECs in the cardinal vein, was decreased in Tmem100-iKO embryos, whereas it was increased in TMEM100-OE embryos.

Conclusion: TMEM100 plays an important role in the specification of LECs in the cardinal veins, at least in part, by regulating the NOTCH signaling.

https://link.springer.com/article/10.1007/s10456-020-09713-1

Correcting Smad1/5/8, mTOR, and VEGFR2 treats pathology in hereditary hemorrhagic telangiectasia models

February 2020, The Journal of Clinical Investigation

Santiago Ruiz, Haitian Zhao, Pallavi Chandakkar, Julien Papoin, Hyunwoo Choi, Aya Nomura-Kitabayashi, Radhika Patel, Matthew Gillen, Li Diao, Prodyot K. Chatterjee, Mingzhu He, Yousef Al-Abed, Ping Wang, Christine N. Metz, S. Paul Oh, Lionel Blanc, Fabien Campagne, and Philippe Marambaud

Hereditary hemorrhagic telangiectasia (HHT), a genetic bleeding disorder leading to systemic arteriovenous malformations (AVMs), is caused by loss-of-function mutations in the ALK1/ENG/Smad1/5/8 pathway. Evidence suggests that HHT pathogenesis strongly relies on overactivated PI3K/Akt/mTOR and VEGFR2 pathways in endothelial cells (ECs). In the BMP9/10-immunoblocked (BMP9/10ib) neonatal mouse model of HHT, we report here that the mTOR inhibitor, sirolimus, and the receptor tyrosine kinase inhibitor, nintedanib, could synergistically fully block, but also reversed, retinal AVMs to avert retinal bleeding and anemia. Sirolimus plus nintedanib prevented vascular pathology in the oral mucosa, lungs, and liver of the BMP9/10ib mice, as well as significantly reduced gastrointestinal bleeding and anemia in inducible ALK1-deficient adult mice. Mechanistically, in vivo in BMP9/10ib mouse ECs, sirolimus and nintedanib blocked the overactivation of mTOR and VEGFR2, respectively. Furthermore, we found that sirolimus activated ALK2-mediated Smad1/5/8 signaling in primary ECs — including in HHT patient blood outgrowth ECs — and partially rescued Smad1/5/8 activity in vivo in BMP9/10ib mouse ECs. These data demonstrate that the combined correction of endothelial Smad1/5/8, mTOR, and VEGFR2 pathways opposes HHT pathogenesis. Repurposing of sirolimus plus nintedanib might provide therapeutic benefit in patients with HHT.

https://www.jci.org/articles/view/127425/pdf

 

Mouse retinal cell behaviour in space and time using light sheet fluorescence microscopy

February 2020, eLIFE

Claudia Prahst, Parham Ashrafzadeh, Thomas Mead, Ana Figueiredo, Karen Chang, Douglas Richardson, Lakshmi Venkaraman, Mark Richards, Ana Martins Russo, Kyle Harrington, Marie Ouarné, Andreia Pena, Dong Feng Chen, Lena Claesson-Welsh, Kin-Sang Cho, Claudio A Franco, Katie Bentley

As the general population ages, more people are affected by eye diseases, such as retinopathies. It is therefore critical to improve imaging of eye disease mouse models. Here, we demonstrate that 1) rapid, quantitative 3D and 4D (time lapse) imaging of cellular and subcellular processes in the mouse eye is feasible, with and without tissue clearing, using light-sheet fluorescent microscopy (LSFM); 2) flat-mounting retinas for confocal microscopy significantly distorts tissue morphology, confirmed by quantitative correlative LSFM-Confocal imaging of vessels; 3) LSFM readily reveals new features of even well-studied eye disease mouse models, such as the oxygen-induced retinopathy (OIR) model, including a previously unappreciated 'knotted' morphology to pathological vascular tufts, abnormal cell motility and altered filopodia dynamics when live-imaged. We conclude that quantitative 3D/4D LSFM imaging and analysis has the potential to advance our understanding of the eye, in particular pathological, neuro-vascular, degenerative processes.

https://elifesciences.org/articles/49779

c-Src controls stability of sprouting blood vessels in the developing retina independently of cell-cell adhesion through focal adhesion assembly

February 2020, Development

Lilian Schimmel, Daisuke Fukuhara, Mark Richards, Yi Jin, Patricia Essebier, Emmanuelle Frampton, Marie Hedlund, Elisabetta Dejana, Lena Claesson-Welsh, Emma Gordon

Endothelial cell adhesion is implicated in blood vessel sprout formation, yet how adhesion controls angiogenesis, and whether it occurs via rapid remodeling of adherens junctions, focal adhesion assembly, or both, remains poorly understood. Furthermore, how endothelial cell adhesion is controlled in particular tissues and under different conditions remains unexplored. Here, we identified an unexpected role for spatiotemporal c-Src activity in sprouting angiogenesis in the retina, which is in contrast to the dominant focus on c-Src's role in maintenance of vascular integrity. Thus, mice specifically deficient in endothelial c-Src displayed significantly reduced blood vessel sprouting and loss in actin-rich filopodial protrusions at the vascular front of the developing retina. In contrast to what has been observed during vascular leakage, endothelial cell-cell adhesion was unaffected by loss of c-Src. Instead, decreased angiogenic sprouting was due to loss of focal adhesion assembly and cell-matrix adhesion, resulting in loss of sprout stability. These results demonstrate c-Src signaling at specified endothelial cell membrane compartments (adherens junctions or focal adhesions) control vascular processes in a tissue and context dependent manner.

https://dev.biologists.org/content/early/2020/02/26/dev.185405.long

Anatomy and function of the vertebral column lymphatic network in mice

October 2019, Nature Communications

Laurent Jacob, Ligia Simoes Braga Boisserand, Luiz Henrique Medeiros Geraldo, Jose de Brito Neto, Thomas Mathivet, Salli Antila, Besma Barka, Yunling Xu, Jean-Mickael Thomas, Juliette Pestel, Marie-Stéphane Aigrot, Eric Song, Harri Nurmi, Seyoung Lee, Kari Alitalo, Nicolas Renier, Anne Eichmann & Jean-Leon Thomas

Cranial lymphatic vessels (LVs) are involved in the transport of fluids, macromolecules and central nervous system (CNS) immune responses. Little information about spinal LVs is available, because these delicate structures are embedded within vertebral tissues and difficult to visualize using traditional histology. Here we show an extended vertebral column LV network using three-dimensional imaging of decalcified iDISCO+-clarified spine segments. Vertebral LVs connect to peripheral sensory and sympathetic ganglia and form metameric vertebral circuits connecting to lymph nodes and the thoracic duct. They drain the epidural space and the dura mater around the spinal cord and associate with leukocytes. Vertebral LVs remodel extensively after spinal cord injury and VEGF-C-induced vertebral lymphangiogenesis exacerbates the inflammatory responses, T cell infiltration and demyelination following focal spinal cord lesion. Therefore, vertebral LVs add to skull meningeal LVs as gatekeepers of CNS immunity and may be potential targets to improve the maintenance and repair of spinal tissues.

https://doi.org/10.1038/s41467-019-12568-w

Somatic Mutations in Vascular Malformations of Hereditary Hemorrhagic Telangiectasia Result in Bi-allelic Loss of ENG or ACVRL1

October 2019, The American Journal of Human Genetics

Daniel A. Snellings, Carol J. Gallione, Dewi S. Clark, Nicholas T. Vozoris, Marie E. Faughnan, Douglas A. Marchuk

Hereditary hemorrhagic telangiectasia (HHT) is a Mendelian disease characterized by vascular malformations (VMs) including visceral arteriovenous malformations and mucosal telangiectasia. HHT is caused by loss-of-function (LoF) mutations in one of three genes, ENG, ACVRL1, or SMAD4, and is inherited as an autosomal-dominant condition. Intriguingly, the constitutional mutation causing HHT is present throughout the body, yet the multiple VMs in individuals with HHT occur focally, rather than manifesting as a systemic vascular defect. This disconnect between genotype and phenotype suggests that a local event is necessary for the development of VMs. We investigated the hypothesis that local somatic mutations seed the formation HHT-related telangiectasia in a genetic two-hit mechanism. We identified low-frequency somatic mutations in 9/19 telangiectasia through the use of next-generation sequencing. We established phase for seven of nine samples, which confirms that the germline and somatic mutations in all seven samples exist in trans configuration; this is consistent with a genetic two-hit mechanism. These combined data suggest that bi-allelic loss of ENG or ACVRL1 may be a required event in the development of telangiectasia, and that rather than haploinsufficiency, VMs in HHT are caused by a Knudsonian two-hit mechanism.

https://doi.org/10.1016/j.ajhg.2019.09.010

Endothelial PKA activity regulates angiogenesis by limiting autophagy through phosphorylation of ATG16L1

October 2019, eLIFE

Xiaocheng Zhao, Pavel Nedvetsky, Fabio Stanchi, Anne-Clemence Vion, Oliver Popp, Kerstin Zühlke, Gunnar Dittmar, Enno Klussmann, Holger Gerhardt

The cAMP-dependent protein kinase A (PKA) regulates various cellular functions in health and disease. In endothelial cells PKA activity promotes vessel maturation and limits tip cell formation. Here, we used a chemical genetic screen to identify endothelial-specific direct substrates of PKA in human umbilical vein endothelial cells (HUVEC) that may mediate these effects. Amongst several candidates, we identified ATG16L1, a regulator of autophagy, as novel target of PKA. Biochemical validation, mass spectrometry and peptide spot arrays revealed that PKA phosphorylates ATG16L1α at Ser268 and ATG16L1β at Ser269, driving phosphorylation-dependent degradation of ATG16L1 protein. Reducing PKA activity increased ATG16L1 protein levels and endothelial autophagy. Mouse in vivo genetics and pharmacological experiments demonstrated that autophagy inhibition partially rescues vascular hypersprouting caused by PKA deficiency. Together these results indicate that endothelial PKA activity mediates a critical switch from active sprouting to quiescence in part through phosphorylation of ATG16L1, which in turn reduces endothelial autophagy.

https://doi.org/10.7554/eLife.46380.001

Myc‐dependent endothelial proliferation is controlled by phosphotyrosine 1212 in VEGF receptor‐2

September 2019, EMBO Reports

Chiara Testini, Ross O Smith, Yi Jin, Pernilla Martinsson, Ying Sun, Marie Hedlund, Miguel Sáinz-Jaspeado, Masabumi Shibuya, Mats Hellström & Lena Claesson-Welsh

Exaggerated signaling by vascular endothelial growth factor (VEGF)‐A and its receptor, VEGFR2, in pathologies results in poor vessel function. Still, pharmacological suppression of VEGFA/VEGFR2 may aggravate disease. Delineating VEGFR2 signaling in vivo provides strategies for suppression of specific VEGFR2‐induced pathways. Three VEGFR2 tyrosine residues (Y949, Y1212, and Y1173) induce downstream signaling. Here, we show that knock‐in of phenylalanine to create VEGFR2 Y1212F in C57Bl/6 and FVB mouse strains leads to loss of growth factor receptor‐bound protein 2‐ and phosphoinositide 3′‐kinase (PI3K)p85 signaling. C57Bl/6 Vegfr2Y1212F/Y1212F show reduced embryonic endothelial cell (EC) proliferation and partial lethality. FVB Vegfr2Y1212F/Y1212F show reduced postnatal EC proliferation. Reduced EC proliferation in Vegfr2Y1212F/Y1212F explants is rescued by c‐Myc overexpression. We conclude that VEGFR2 Y1212 signaling induces activation of extracellular‐signal‐regulated kinase (ERK)1/2 and Akt pathways required for c‐Myc‐dependent gene regulation, endothelial proliferation, and vessel stability.

https://doi.org/10.15252/embr.201947845

Novel Neuroprotective Loci Modulating Ischemic Stroke Volume in Wild-Derived Inbred Mouse Strains

September 2019, GENETICS

Han Kyu Lee, Samuel J. Widmayer, Min-Nung Huang, David L. Aylor and Douglas A. Marchuk

To identify genes involved in cerebral infarction we have employed a forward genetic approach in inbred mouse strains, using quantitative trait locus (QTL) mapping for cerebral infarct volume after middle cerebral artery occlusion. We had previously observed that infarct volume is inversely correlated with cerebral collateral vessel density in most strains. In this study, we expanded the pool of allelic variation among classical inbred mouse strains by utilizing the eight founder strains of the Collaborative Cross and found a wild-derived strain, WSB/EiJ, that breaks this general rule that collateral vessel density inversely correlates with infarct volume. WSB/EiJ and another wild-derived strain, CAST/EiJ, show the highest collateral vessel densities of any inbred strain, but infarct volume of WSB/EiJ mice is 8.7-fold larger than that of CAST/EiJ mice. QTL mapping between these strains identified four new neuroprotective loci modulating cerebral infarct volume while not affecting collateral vessel phenotypes. To identify causative variants in genes we surveyed non-synonymous coding SNPs between CAST/EiJ and WSB/EiJ and found 96 genes harboring coding SNPs predicted to be damaging and mapping within one of the four intervals. In addition, we performed RNA sequencing for brain tissue of CAST/EiJ and WSB/EiJ mice and identified 79 candidate genes mapping in one of the four intervals showing strain-specific differences in expression. The identification of the genes underlying these neuroprotective loci will provide new understanding of genetic risk factors of ischemic stroke which may provide novel targets for future therapeutic intervention of human ischemic stroke.

https://doi.org/10.1534/genetics.119.302555

Artery-vein specification in the zebrafish trunk is pre-patterned by heterogeneous Notch activity and balanced by flow-mediated fine-tuning

August 2019, Development

Ilse Geudens, Baptiste Coxam, Silvanus Alt, Véronique Gebala, Anne-Clémence Vion, Katja Meier, Andre Rosa, Holger Gerhardt

How developing vascular networks acquire the right balance of arteries, veins and lymphatic vessels to efficiently supply and drain tissues is poorly understood. In zebrafish embryos, the robust and regular 50:50 global balance of intersegmental veins and arteries that form along the trunk prompts the intriguing question of how does the organism keep ‘count’? Previous studies have suggested that the ultimate fate of an intersegmental vessel (ISV) is determined by the identity of the approaching secondary sprout emerging from the posterior cardinal vein. Here, we show that the formation of a balanced trunk vasculature involves an early heterogeneity in endothelial cell behaviour and Notch signalling activity in the seemingly identical primary ISVs that is independent of secondary sprouting and flow. We show that Notch signalling mediates the local patterning of ISVs, and an adaptive flow-mediated mechanism subsequently fine-tunes the global balance of arteries and veins along the trunk. We propose that this dual mechanism provides the adaptability required to establish a balanced network of arteries, veins and lymphatic vessels.

https://dev.biologists.org/content/146/16/dev181024.long

ATTRACT: Arterial Flow as Attractor for Endothelial Cell Migration

July 2019, Circulation research

Petya B. Georgieva, Douglas A. Marchuk, Holger Gerhardt and Leducq ATTRACT Consortium

Non-canonical Wnt signaling regulates junctional mechanocoupling during angiogenic collective cell migration

June 2019, Elife

Joana R CarvalhoIsabela C FortunatoCatarina G FonsecaAnna PezzarossaPedro BarbacenaMaria A Dominguez-CejudoFrancisca F VasconcelosNuno C SantosFilomena A CarvalhoClaudio A Franco

Morphogenesis of hierarchical vascular networks depends on the integration of multiple biomechanical signals by endothelial cells, the cells lining the interior of blood vessels. Expansion of vascular networks arises through sprouting angiogenesis, a process involving extensive cell rearrangements and collective cell migration. Yet, the mechanisms controlling angiogenic collective behavior remain poorly understood. Here, we show this collective cell behavior is regulated by non-canonical Wnt signaling. We identify that Wnt5a specifically activates Cdc42 at cell junctions downstream of ROR2 to reinforce coupling between adherens junctions and the actin cytoskeleton. We show that Wnt5a signaling stabilizes vinculin binding to alpha-catenin, and abrogation of vinculin in vivo and in vitro leads to uncoordinated polarity and deficient sprouting angiogenesis in Mus musculus. Our findings highlight how non-canonical Wnt signaling coordinates collective cell behavior during vascular morphogenesis by fine-tuning junctional mechanocoupling between endothelial cells.

https://elifesciences.org/articles/45853

 

Endophilin-A2 dependent VEGFR2 endocytosis promotes sprouting angiogenesis

May 2019,  Nat Communications

Gael Genet, Kevin Boyé, Thomas Mathivet, Roxana Ola, Feng Zhang, Alexandre Dubrac, Jinyu Li, Nafiisha Genet, Luiz Henrique Geraldo, Lorena Benedetti, Steffen Künzel, Laurence Pibouin-Fragner, Jean-Leon Thomas & Anne Eichmann

Endothelial cell migration, proliferation and survival are triggered by VEGF-A activation of VEGFR2. However, how these cell behaviors are regulated individually is still unknown. Here we identify Endophilin-A2 (ENDOA2), a BAR-domain protein that orchestrates CLATHRIN-independent internalization, as a critical mediator of endothelial cell migration and sprouting angiogenesis. We show that EndoA2 knockout mice exhibit postnatal angiogenesis defects and impaired front-rear polarization of sprouting tip cells. ENDOA2 deficiency reduces VEGFR2 internalization and inhibits downstream activation of the signaling effector PAK but not ERK, thereby affecting front-rear polarity and migration but not proliferation or survival. Mechanistically, VEGFR2 is directed towards ENDOA2-mediated endocytosis by the SLIT2-ROBO pathway via SLIT-ROBO-GAP1 bridging of ENDOA2 and ROBO1. Blocking ENDOA2-mediated endothelial cell migration attenuates pathological angiogenesis in oxygen-induced retinopathy models. This work identifies a specific endocytic pathway controlling a subset of VEGFR2 mediated responses that could be targeted to prevent excessive sprouting angiogenesis in pathological conditions.

https://www.nature.com/articles/s41467-019-10359-x

 

GNrep mouse: A reporter mouse for front-rear cell polarity.

April 2019, in Genesis

Pedro Barbacena, Marie Ouarné, Jody J. Haigh, Francisca F. Vasconcelos, Anna Pezzarossa, and Claudio A. Franco

Cell migration is essential during development, regeneration, homeostasis, and disease. Depending on the microenvironment, cells use different mechanisms to migrate. Yet, all modes of migration require the establishment of an intracellular front-rear polarity axis for directional movement. Although front-rear polarity can be easily identified in in vitro conditions, its assessment in vivo by live-imaging is challenging due to tissue complexity and lack of reliable markers. Here, we describe a novel and unique double fluorescent reporter mouse line to study front-rear cell polarity in living tissues, called GNrep. This mouse line simultaneously labels Golgi complexes and nuclei allowing the assignment of a nucleus-to-Golgi axis to each cell, which functions as a readout for cell front-rear polarity. As a proof-of-principle, we validated the efficiency of the GNrep line using an endothelial-specific Cre mouse line. We show that the GNrep labels the nucleus and the Golgi apparatus of endothelial cells with very high efficiency and high specificity. Importantly, the features of fluorescent intensity and localization for both mCherry and eGFP fluorescent intensity and localization allow automated segmentation and assignment of polarity vectors in complex tissues, making GNrep a great tool to study cell behavior in large-scale automated analyses. Altogether, the GNrep mouse line, in combination with different Cre recombinase lines, is a novel and unique tool to study of front-rear polarity in mice, both in fixed tissues or in intravital live imaging. This new line will be instrumental to understand cell migration and polarity in development, homeostasis, and disease.

https://onlinelibrary.wiley.com/doi/full/10.1002/dvg.23299

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

January 2021

Happy New Year, may it bring scientific success and good health to all of us and our loved ones!

From January onwards, Petya Jordan takes back her role as Network Administrative Officer. Many thanks to Tanja Florin for her outstanding work for the network during last year!

 


November 2020


October 2020


September 2020

The winners of the first Leducq ATTRACT network Young Investigator Award have been selected!

Congratulations to Elin Sjöberg and Andre Rosa! Their project proposals have been developed during the 2020 Online Summer Symposium and evaluated by all network PIs in a competitive process. The funded projects are collaborative studies of topics that are related to, and will benefit the ATTRACT network.

The YIA have been installed as a career development opportunity for the network trainees. Due to its the large success it will be repeated during the next all-members meeting in summer 2021.


June 2020

Despite the unsual circumstances and moving the in-person meeting from Uppsala to an entirely online symposium, this year's all-members symposium was a big success.


 

April 2020

Unusual circumstances require unusual measurements. 

The decision has not been easy but due to the Covid19 pandemic we have decided to postpone our 2020 all-member Summer Symposium in Uppsala by one year. Importantly, we do not want this to weaken our network's strong connections and excellent research commitments. Therefore, this year, we will have a virtual online symposium instead. This will allow all our trainees to present their research to our network, discuss new findings, get valuable feedback and spark new ideas. But above all, it will keep our community united in these difficult times. 

More information to come...

Stay safe and healthy!


 

December 2019

Happy Holidays!

It's been another exciting year of our network with many direct collaborations, great achievements such as top notch publications, super interesting video conference-style seminars and a variety of lab visits between the members of the network.

Of course there are many events already planned for 2020. One part is the continuation of our monthly video conference seminars for which you can find the schedule below.

Stay tuned for further announcement regarding upcoming events, achievements and fun pictures.


 

November 2019 

Lowell Edgar, a postdoc in Miguel Bernabeu’s lab at the University of Edinburgh, visited Holger Gerhardt’s group at MDC Berlin where he is collaborating with Andre Rosa and Wolfgang Giese to develop his model of cell migration. His fruitful week-long visit was celebrated with a joyful dinner in Berlin.

Members from the Gerhardt group celebrating Lowell's visit to Berlin at Friday dinner. From left to right: Baptiste Coxam, Lowell Edgar, Holger Gerhardt, Wolfgang Giese, Tanja Florin, Petya Jordan, Jennifer Paech, Olya Oppenheim and Andre Rosa.


 

October 2019 

Second group leader meeting on October 24 at the Cardiovascular Research Center at Yale University, New Haven, CT.

Some of the group leaders at the October meeting in Yale. From left to right: Claudio Franco, host Anne Eichmann, Holger Gerhardt, Paul Oh, and Doug Marchuk.

Due to travel difficulties, Holger Gerhardt joined remotely, while Lena Claesson-Welsh and Miguel Bernabeu had to run to catch the shuttle back to the airport. A very short, but productive meeting.


 

October 2019

New NAO of our Network

Hi! I’m Tanja Florin and I’m excited to join the ATTRACT network as Network Administrative Officer (NAO) for the next year, replacing Petya during her maternity leave. Even though I personally won’t be participating in any scientific research for the network, I’m thrilled to be working with world-class scientists and creative minds. A little bit about myself: I recently obtained my Ph.D. in Biomolecular Sciences from the University of Illinois at Chicago where I focused my studies on mechanisms of protein synthesis inhibition in bacteria. After returning to my home country Germany this summer, I switched gears and decided to become more active in science management and coordination. Besides, I’m also following my passion, pursuing different science outreach & engagement projects locally and globally. I’m looking forward to getting to know the members of the network and to many fruitful interactions.

 

 


 

September 2019

Lena Claesson-Welsh talked to EMBO about quality of peer review. Find the full article here.

 

On being a reviewer:

"I review papers for several reasons: I need my own submissions reviewed, I learn about where the field is moving, new technologies, reagents and so on, and I also find out more about the journal, its principles and ambition.

When I review, I first of all want to see beyond the experimental details to focus on the larger picture and the concepts the authors wish to address and how they relate to where the field is moving. If there are conceptual problems, these belong to my major criticisms. I want to be fair and not force the authors to try to solve something that is essentially impossible to do, especially within a deadline. It is bad when authors feel compelled to scramble up shaky results in no time, leading to unfounded conclusions. I try to suggest how my comments can be addressed. I don’t want to list page up and down with technical details. If there are too many technical issues and consistent errors, they should be mentioned but then it’s better to recommend rejection."

 

On quality in peer review:

"Peer review is important because it improves the quality of the papers and, in the long run, promotes more sound science. For me, quality in peer review means that the reviewer knows the field and can appreciate the authors’ goal and efforts and provide constructive criticism. A reviewer should also be open to new ideas and concepts if they are well underbuilt. Moreover, quality means that the editor forms an independent impression and can take a decision and withstand unreasonable, unfair criticism.

A good review is when the reviewer’s intention is to help you improve the work. That’s normally clear from the type and style of comments. Criticism should be worded in positive manner and one should avoid diminishing the authors’ efforts. Giving suggestions for how the criticism can be addressed is useful. And one should always take the authors’ perspective into consideration."

 

On transparency in peer review:

"I consider the transparent peer review process valuable since it most likely motivates reviewers to give their comments in a more constructive manner. It is also important that EMBO Press provides all material used for the editorial decision-making. Especially for younger PIs this is very valuable.

The transparency takes some of the reviewer anonymity away, which forces the reviewers to be more careful in their wording. The anonymity cannot be entirely removed as it would make it harder to give stern criticism and reject papers that must be rejected."

 

We are grateful to EMBO for permission to publish the text of the interview here.

 

 


 

 

July 2019

Our invited "News & views" article just came out in Circulation research! In this paper, we tell the story about the ATTRACT network: how the idea first came up, how the proposal was written and funded, first results we have achieved and much more. Just like with the research work, many network members joined forces for this article and contributed with ideas, images and feedback. Big thank you to everyone!

You can find the article here:

News & views article "ATTRACT"

 

 


 

 

June 2019

Wolfgang Giese, a postdoc who recently joined the lab of Holger Gerhardt in Berlin, visited the lab of Miguel Bernabeu in Edinburgh for two days. The aim of the visit was to coordinate the modelling approaches applied in both labs and starting a collaborative project with Lowell Edgar. The team of Miguel together with Wolfgang joined forces not only in research, but also in a pub quiz and won the main prize: a pineapple and a voucher! Certainly a very successful collaboration start!

 

 

 

 


 

 

April 2019

Our third network meeting took place in sunny Portugal, April 2nd-4th at Castello Palmela with 23 participants from all member labs! The program was packed with scientific presentations but we also went for a visit to the Bacalhoa castle and winery. Everyone appreciated the fantastic location but most of all, the opportunities for lively and productive discussions and interactions. In an anonymous survey, participants gave the retreat a 4.87 out of 5 stars! All in all, a very successful meeting - looking forward to the next one!

 

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)

2020 Summer Symposium

This year, our summer symposium will be held online. You will find all information here. For access to the video conference room, check with Tanja Florin.

2020 Leducq ATTRACT network Summer symposium

June 16-18, online via Zoom

Day 1

June 16th

Time EDT Time CEST Duration
09:15 15:15 Opening reception/set up time (optional) 00:15
09:30 15:30 Opening words, welcome, logistics 00:10
09:40 15:40 Main achievements, goals 00:15
09:55 15:55 Catarina Fonseca (Franco lab)
Unveiling new regulators of flow dependent ECs polarization
00:25
10:20 16:20 Anna Szymborska (Gerhardt lab)
Dynamics of endothelial junctions in angiogenesis: from high-resolution imaging to proteomics
00:25
10:45 16:45 Coffee break 00:20
11:05 17:05 Grant development Workshop pitches 00:45
11:50 17:50 Lunch/Dinner break 00:40
12:30 18:30 Yi Jin (Claesson-Welsh lab)
Src family kinase Yes1 controls the phosphorylation of VE-Cadherin and regulates collective endothelial cell migration.
00:25
12:55 18:55 Lowell Edgar (Bernabeu lab)
Novel force driven agent based models of flow-mediated angiogenic remodelling
00:25
13:20 19:20 Conclusion Day 1, Outlook Day 2 00:15
13:35 19:35 End

 

Day 2

June 17th

Time EDT Time CEST Duration
09:15 15:15 Coffee/set up time (optional) 00:15
09:30 15:30 Workshop details 00:10
09:40 15:40 Grant development Workshop, breakout rooms 01:30
11:10 17:10 Coffee break 00:20
11:30 17:30 Ines Fechner (Gerhardt lab)
Investigating the role of Rabep2 in vascular development
00:25
11:55 17:55 Han Kyu Lee (Marchuk lab)
A role of Rabep2 in modulating ischemic stroke infarction
00:25
12:20 18:20 Lunch/Dinner break 00:40
13:00 19:00 Kevin Boye (Eichmann lab)
Unc5B regulates Blood-Brain Barrier tight-junctions
00:25
13:25 19:25 Round up Day 2, Outlook Day 3 00:15
13:40 17:10 Coffee break 00:20
14:00 20:00 Social Activity: Trivia/Pub quiz in teams 1:30
15:30 21:30 End

 

Day 3

June 18th

Time EDT Time CEST Duration
09:15 15:15 Coffee/set up time (optional) 00:15
09:30 15:30 Grant development Workshop, breakout rooms 00:45
10:15 16:15 Coffee break 00:20
10:35 16:35 Charles Zhou (Bernabeu lab)
Association between erythrocyte dynamics and vessel remodelling in developmental vascular networks.
00:15
10:50 16:50 Ana Villaplana-Velasco (Bernabeu lab)
The genetics underlying human retinal vascular phenotypes
00:15
11:05 17:05 Coffee break 00:20
11:25 17:25 Technical Workshops 1 & 2 in parallel breakout sessions 00:50
12:15 18:15 Lunch/Dinner break 00:40
12:55 18:55 Chul Han (Oh lab)
Scl-CreER;Eng model – Determining the critical developmental stage for the development of brain AVM
00:25
13:20 19:20 Hyunwoo Choi (Oh lab)
BMP10 has a unique function for the development of proper arteriovenous network
00:25
13:45 19:45 Closing Remarks 00:15
14:00 20:00 End

 

Documents for download

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:

Network Administrative Officer

Paulina Wakula

paulina.wakula@mdc-berlin.de

 

Former NAO: Petya Jordan, Tanja Florin