Periodic Reporting for period 1 - STRomA (Novel Matrix Stiffness-regulated Genes in Lymphangiogenesis and Angiogenesis)
Reporting period: 2019-10-01 to 2021-09-30
From October 2019 until September 2021, the Marie Skłodowska Curie Action ‘Novel Matrix Stiffness-regulated Genes in Lymphangiogenesis and Angiogenesis’ (Acronym: STRomA) has been analyzing the role of endothelial proteins that are regulated via the surrounding tissue environment of endothelial cells (ECs).
ECs form the innermost cell layer of all blood and lymphatic vessel types in the body and function as a dynamic barrier between the circulating blood or lymph on their luminal side (the inside of the vessel) and the surrounding tissue on their abluminal side. ECs recognize and respond to mechanical forces from the luminal and abluminal microenvironment through their cell-cell and cell-matrix adhesions and translate mechanical stimuli into biological responses in a process called mechanotransduction. The composition and mechanical properties of the surrounding tissue environment, the so called extracellular matrix (ECM), that enwraps the ECs, differ across the vascular tree and in development and disease.
It is important to note that most vascular diseases, such as cardiovascular disease, atherosclerosis and lymphedema, are frequently associated with pathological ECM changes. However, our knowledge on how, in particular, changes of ECM stiffness regulate vascular development and contribute to blood and lymphatic vessels dysfunction is still limited. Findings of the STRomA action have enabled a better understanding of ECM stiffness-regulated endothelial signaling mechanisms and have the far-reaching potential to provide novel therapeutic targets to treat vascular diseases associated with pathological ECM changes.
Using human primary EC cultures and genetically modified mouse models in combination with advanced microscopy and live cell imaging, objectives of STRomA have been (i) to elucidate the function of an actin-regulating protein family in angiogenesis, (ii) to identify the role of a cGMP-regulating protein in lymphangiogenesis and (iii) to develop novel fluorescent stiffness sensor tools to live-visualize stiffness changes in ECs. A parallel goal of the STRomA action has been to foster the development of the individual researcher (IR) and support her to become an independent research group leader.
ECs form the innermost cell layer of all blood and lymphatic vessel types in the body and function as a dynamic barrier between the circulating blood or lymph on their luminal side (the inside of the vessel) and the surrounding tissue on their abluminal side. ECs recognize and respond to mechanical forces from the luminal and abluminal microenvironment through their cell-cell and cell-matrix adhesions and translate mechanical stimuli into biological responses in a process called mechanotransduction. The composition and mechanical properties of the surrounding tissue environment, the so called extracellular matrix (ECM), that enwraps the ECs, differ across the vascular tree and in development and disease.
It is important to note that most vascular diseases, such as cardiovascular disease, atherosclerosis and lymphedema, are frequently associated with pathological ECM changes. However, our knowledge on how, in particular, changes of ECM stiffness regulate vascular development and contribute to blood and lymphatic vessels dysfunction is still limited. Findings of the STRomA action have enabled a better understanding of ECM stiffness-regulated endothelial signaling mechanisms and have the far-reaching potential to provide novel therapeutic targets to treat vascular diseases associated with pathological ECM changes.
Using human primary EC cultures and genetically modified mouse models in combination with advanced microscopy and live cell imaging, objectives of STRomA have been (i) to elucidate the function of an actin-regulating protein family in angiogenesis, (ii) to identify the role of a cGMP-regulating protein in lymphangiogenesis and (iii) to develop novel fluorescent stiffness sensor tools to live-visualize stiffness changes in ECs. A parallel goal of the STRomA action has been to foster the development of the individual researcher (IR) and support her to become an independent research group leader.
The STRomA action was divided into three scientific work packages (WPs):
As part of WP1, a pivotal role of the Ena/VASP protein EVL has been described in retinal angiogenesis. Global genetic deletion of EVL (but not VASP) and endothelial-specific EVL deletion compromised the radial outgrowth of the vascular plexus in mice. Furthermore, gene sets involved in blood vessel development and angiogenesis were found to be down-regulated in EVL-deficient retinal ECs. Consistently, EVL deletion impaired vascular endothelial growth factor (VEGF)-induced EC proliferation and sprouting, and reduced the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. The study was published in EMBO Reports with the IR as shared first and co-corresponding author. Due to the IR’s expertise on vascular signaling and imaging, during the initial phase of the COVID-19 pandemic, the STRomA action contributed to an in-depth analysis of angiogenic and pathological changes of the pulmonary blood vasculature to identify underlying mechanisms of pulmonary blood vessel failure in severe COVID-19 cases. The study was published in EBioMedicine with the IR as senior and corresponding author.
In WP2, the STRomA action has identified a novel role of a molecule which is involved in intracellular cGMP signaling and is predominantly regulated in lymphatic ECs. The identified molecule specifically regulates embryonic lymphangiogenesis. The results of WP have already been presented in the renowned Kloster Seeon Angiogenesis conference in September 2021 (with the IR as invited speaker) and the manuscript is currently prepared for publication. Furthermore, as part of WP2, the STRomA action has contributed to identify a role of additional lymphatic molecules that specifically regulates lymphatic vessel maintenance. The transmembrane ligand EphrinB2 and its receptor EphB4 have been identified as critical homeostatic regulators of collecting lymphatic vessel integrity. Conditional gene deletion in mice revealed that EphrinB2/EphB4 signaling is dispensable for blood endothelial barrier function, but required for stabilization of lymphatic EC junctions in different organs of juvenile and adult mice. Studies in primary human lymphatic ECs further showed that basal EphrinB2/EphB4 signaling controls junctional localization of the tight junction protein CLDN5 and junction stability via Rac1/Rho-mediated regulation of cytoskeletal contractility. EphrinB2/EphB4 signaling therefore provides a potential therapeutic target to selectively modulate lymphatic vessel permeability and function. The study has been presented in the Gordon Research Conference on Lymphatics in 2020 (with the IR as invited speaker) and has been published with the IR as first author and co-corresponding author in eLife.
Within the WP 3, the STRomA action has successfully developed endothelial stiffness sensors. The sensors are currently employed as fundamental tools within the research group and will be published in the near future.
The data collected during the STRomA action will continuously inform and enhance several publications in the coming years, in addition to the ones produced and published during the fellowship itself. To increase visibility of the core topics of the STRomA action, the IR has furthermore edited a special issue on ‘Lymphangiogenesis in Development and Disease - Taking a Fresh Look at the Forming Lymphatics’ in the International Journal of Molecular Sciences and published a systematic review on ‘The Importance of Mechanical Forces for in vitro Endothelial Cell Biology’ in Frontiers of Physiology. At the same time, STRomA action updates and continuous result reports have been disseminated to the public via the laboratory twitter account (@VascSensing_Lab) and press releases of the University Medical Center Hamburg (UKE). Career development, that further included regular meetings with the supervisor, and dissemination and communication of the STRomA action have been managed under WP4 and WP5.
As part of WP1, a pivotal role of the Ena/VASP protein EVL has been described in retinal angiogenesis. Global genetic deletion of EVL (but not VASP) and endothelial-specific EVL deletion compromised the radial outgrowth of the vascular plexus in mice. Furthermore, gene sets involved in blood vessel development and angiogenesis were found to be down-regulated in EVL-deficient retinal ECs. Consistently, EVL deletion impaired vascular endothelial growth factor (VEGF)-induced EC proliferation and sprouting, and reduced the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. The study was published in EMBO Reports with the IR as shared first and co-corresponding author. Due to the IR’s expertise on vascular signaling and imaging, during the initial phase of the COVID-19 pandemic, the STRomA action contributed to an in-depth analysis of angiogenic and pathological changes of the pulmonary blood vasculature to identify underlying mechanisms of pulmonary blood vessel failure in severe COVID-19 cases. The study was published in EBioMedicine with the IR as senior and corresponding author.
In WP2, the STRomA action has identified a novel role of a molecule which is involved in intracellular cGMP signaling and is predominantly regulated in lymphatic ECs. The identified molecule specifically regulates embryonic lymphangiogenesis. The results of WP have already been presented in the renowned Kloster Seeon Angiogenesis conference in September 2021 (with the IR as invited speaker) and the manuscript is currently prepared for publication. Furthermore, as part of WP2, the STRomA action has contributed to identify a role of additional lymphatic molecules that specifically regulates lymphatic vessel maintenance. The transmembrane ligand EphrinB2 and its receptor EphB4 have been identified as critical homeostatic regulators of collecting lymphatic vessel integrity. Conditional gene deletion in mice revealed that EphrinB2/EphB4 signaling is dispensable for blood endothelial barrier function, but required for stabilization of lymphatic EC junctions in different organs of juvenile and adult mice. Studies in primary human lymphatic ECs further showed that basal EphrinB2/EphB4 signaling controls junctional localization of the tight junction protein CLDN5 and junction stability via Rac1/Rho-mediated regulation of cytoskeletal contractility. EphrinB2/EphB4 signaling therefore provides a potential therapeutic target to selectively modulate lymphatic vessel permeability and function. The study has been presented in the Gordon Research Conference on Lymphatics in 2020 (with the IR as invited speaker) and has been published with the IR as first author and co-corresponding author in eLife.
Within the WP 3, the STRomA action has successfully developed endothelial stiffness sensors. The sensors are currently employed as fundamental tools within the research group and will be published in the near future.
The data collected during the STRomA action will continuously inform and enhance several publications in the coming years, in addition to the ones produced and published during the fellowship itself. To increase visibility of the core topics of the STRomA action, the IR has furthermore edited a special issue on ‘Lymphangiogenesis in Development and Disease - Taking a Fresh Look at the Forming Lymphatics’ in the International Journal of Molecular Sciences and published a systematic review on ‘The Importance of Mechanical Forces for in vitro Endothelial Cell Biology’ in Frontiers of Physiology. At the same time, STRomA action updates and continuous result reports have been disseminated to the public via the laboratory twitter account (@VascSensing_Lab) and press releases of the University Medical Center Hamburg (UKE). Career development, that further included regular meetings with the supervisor, and dissemination and communication of the STRomA action have been managed under WP4 and WP5.
The IR’s research has successfully advanced European excellence in the interdisciplinary research field that combines vascular developmental biology and dysfunctional (lymph)angiogenesis with matrix biology. The STRomA action has built a solid foundation for the IR’s independent research group but also has stimulated many new research ideas: In the translational and clinical research environment at the UKE, the IR has become more and more curious about vascular diseases, specifically the role of ECs in disease development and progression. In the future, the IR aims to explore in more detail the interplay of ECM stiffness changes and endothelial function in vascular diseases that are accompanied by pathological changes of tissue stiffness.