Frank L. Bos

ccbe1 is required for embryonic lymphangiogenesis and venous sprouting

Lymphatic vessels have important roles in fluid homeostasis, fat absorption, inflammation and cancer metastasis and develop in a dynamic process (called lymphangiogenesis) involving budding, migration and proliferation of lymphangioblasts. Using a genetic screen in zebrafish we identify ccbe1 (collagen and calcium-binding EGF domain-1) as indispensible for embryonic lymphangiogenesis. Ccbe1 acts at the same stage of development as Vegfc and is required for lymphangioblast budding and angiogenic sprouting from venous endothelium.

CCBE1 Is Essential for Mammalian Lymphatic Vascular Development and Enhances the Lymphangiogenic Effect of Vascular Endothelial Growth Factor-C In Vivo

Rationale: Collagen- and calcium-binding EGF domains 1 (CCBE1) has been associated with Hennekam syndrome, in which patients have lymphedema, lymphangiectasias, and other cardiovascular anomalies. Insight into the molecular role of CCBE1 is completely lacking, and mouse models for the disease do not exist. Objective: CCBE1 deficient mice were generated to understand the function of CCBE1 in cardiovascular development, and CCBE1 recombinant protein was used in both in vivo and in vitro settings to gain insight into the molecular function of CCBE1. Methods and Results: Phenotypic analysis of murine Ccbe1 mutant embryos showed a complete lack of definitive lymphatic structures, even though Prox1+ lymphatic endothelial cells get specified within the cardinal vein. Mutant mice die prenatally. Proximity ligation assays indicate that vascular endothelial growth factor receptor 3 activation appears unaltered in mutants. Human CCBE1 protein binds to components of the extracellular matrix in vitro, and CCBE1 protein strongly enhances vascular endothelial growth factor-C–mediated lymphangiogenesis in a corneal micropocket assay. Conclusions: Our data identify CCBE1 as a factor critically required for budding and migration of Prox-1+ lymphatic endothelial cells from the cardinal vein. CCBE1 probably exerts these effects through binding to components of the extracellular matrix. CCBE1 has little lymphangiogenic effect on its own but dramatically enhances the lymphangiogenic effect of vascular endothelial growth factor-C in vivo. Thus, our data suggest CCBE1 to be essential but not sufficient for lymphangiogenesis.

Arteries provide essential guidance cues for lymphatic endothelial cells in the zebrafish trunk

The endothelial cells of the vertebrate lymphatic system assemble into complex networks, but local cues that guide the migration of this distinct set of cells are currently unknown. As a model for lymphatic patterning, we have studied the simple vascular network of the zebrafish trunk consisting of three types of lymphatic vessels that develop in close connection with the blood vasculature. We have generated transgenic lines that allow us to distinguish between arterial, venous and lymphatic endothelial cells (LECs) within a single zebrafish embryo. We found that LECs migrate exclusively along arteries in a manner that suggests that arterial endothelial cells serve as the LEC migratory substrate. In the absence of intersegmental arteries, LEC migration in the trunk is blocked. Our data therefore demonstrate a crucial role for arteries in LEC guidance.

Endothelial Cell–Specific FGD5 Involvement in Vascular Pruning Defines Neovessel Fate in Mice

Background— New vessel formation contributes to organ development during embryogenesis and tissue repair in response to mechanical damage, inflammation, and ischemia in adult organisms. Early angiogenesis includes formation of an excessive primitive network that needs to be reorganized into a secondary vascular network with higher hierarchical structure. Vascular pruning, the removal of aberrant neovessels by apoptosis, is a vital step in this process. Although multiple molecular pathways for early angiogenesis have been identified, little is known about the genetic regulators of secondary network development. Methods and Results— Using a transcriptomics approach, we identified a new endothelial specific gene named FYVE, RhoGEF, and PH domain–containing 5 (FGD5) that plays a crucial role in vascular pruning. Loss- and gain-of-function studies demonstrate that FGD5 inhibits neovascularization, indicated by in vitro tube-formation, aortic-ring, and coated-bead assays and by in vivo coated-bead plug assays and studies in the murine retina model. FGD5 promotes apoptosis-induced vaso-obliteration via induction of the hey1-p53 pathway by direct binding and activation of cdc42. Indeed, FGD5 correlates with apoptosis in endothelial cells during vascular remodeling and was linked to rising p21CIP1 levels in aging mice. Conclusion— We have identified FGD5 as a novel genetic regulator of vascular pruning by activation of endothelial cell–targeted apoptosis.

Ets2 Determines the Inflammatory State of Endothelial Cells in Advanced Atherosclerotic Lesions

Rationale: Neovascularization is required for embryonic development and plays a central role in diseases in adults. In atherosclerosis, the role of neovascularization remains to be elucidated. In a genome-wide microarray-screen of Flk1+ angioblasts during murine embryogenesis, the v-ets erythroblastosis virus E26 oncogene homolog 2 (Ets2) transcription factor was identified as a potential angiogenic factor. Objectives: We assessed the role of Ets2 in endothelial cells during atherosclerotic lesion progression toward plaque instability. Methods and Results: In 91 patients treated for carotid artery disease, Ets2 levels showed modest correlations with capillary growth, thrombogenicity, and rising levels of tumor necrosis factor-&agr; (TNF&agr;), monocyte chemoattractant protein 1, and interleukin-6 in the atherosclerotic lesions. Experiments in ApoE−/− mice, using a vulnerable plaque model, showed that Ets2 expression was increased under atherogenic conditions and was augmented specifically in the vulnerable versus stable lesions. In endothelial cell cultures, Ets2 expression and activation was responsive to the atherogenic cytokine TNF&agr;. In the murine vulnerable plaque model, overexpression of Ets2 promoted lesion growth with neovessel formation, hemorrhaging, and plaque destabilization. In contrast, Ets2 silencing, using a lentiviral shRNA construct, promoted lesion stabilization. In vitro studies showed that Ets2 was crucial for TNF&agr;-induced expression of monocyte chemoattractant protein 1, interleukin-6, and vascular cell adhesion molecule 1 in endothelial cells. In addition, Ets2 promoted tube formation and amplified TNF&agr;-induced loss of vascular endothelial integrity. Evaluation in a murine retina model further validated the role of Ets2 in regulating vessel inflammation and endothelial leakage. Conclusions: We provide the first evidence for the plaque-destabilizing role of Ets2 in atherosclerosis development by induction of an intraplaque proinflammatory phenotype in endothelial cells.

Functional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain

RATIONALE Collagen- and calcium-binding EGF domain-containing protein 1 (CCBE1) is essential for lymphangiogenesis in vertebrates and has been associated with Hennekam syndrome. Recently, CCBE1 has emerged as a crucial regulator of vascular endothelial growth factor-C (VEGFC) signaling. OBJECTIVE CCBE1 is a secreted protein characterized by 2 EGF domains and 2 collagen repeats. The functional role of the different CCBE1 protein domains is completely unknown. Here, we analyzed the functional role of the different CCBE1 domains in vivo and in vitro. METHODS AND RESULTS We analyzed the functionality of several CCBE1 deletion mutants by generating knock-in mice expressing these mutants, by analyzing their ability to enhance Vegfc signaling in vivo in zebrafish, and by testing their ability to induce VEGFC processing in vitro. We found that deleting the collagen domains of CCBE1 has a much stronger effect on CCBE1 activity than deleting the EGF domains. First, although CCBE1ΔCollagen mice fully phenocopy CCBE1 knock-out mice, CCBE1ΔEGF knock-in embryos still form rudimentary lymphatics. Second, Ccbe1ΔEGF, but not Ccbe1ΔCollagen, could partially substitute for Ccbe1 to enhance Vegfc signaling in zebrafish. Third, CCBE1ΔEGF, similarly to CCBE1, but not CCBE1ΔCollagen could activate VEGFC processing in vitro. Furthermore, a Hennekam syndrome mutation within the collagen domain has a stronger effect than a Hennekam syndrome mutation within the EGF domain. CONCLUSIONS We propose that the collagen domains of CCBE1 are crucial for the activation of VEGFC in vitro and in vivo. The EGF domains of CCBE1 are dispensable for regulation of VEGFC processing in vitro, however, they are necessary for full lymphangiogenic activity of CCBE1 in vivo.Rationale: Collagen- and calcium-binding EGF domain–containing protein 1 (CCBE1) is essential for lymphangiogenesis in vertebrates and has been associated with Hennekam syndrome. Recently, CCBE1 has emerged as a crucial regulator of vascular endothelial growth factor-C (VEGFC) signaling. Objective: CCBE1 is a secreted protein characterized by 2 EGF domains and 2 collagen repeats. The functional role of the different CCBE1 protein domains is completely unknown. Here, we analyzed the functional role of the different CCBE1 domains in vivo and in vitro. Methods and Results: We analyzed the functionality of several CCBE1 deletion mutants by generating knock-in mice expressing these mutants, by analyzing their ability to enhance Vegfc signaling in vivo in zebrafish, and by testing their ability to induce VEGFC processing in vitro. We found that deleting the collagen domains of CCBE1 has a much stronger effect on CCBE1 activity than deleting the EGF domains. First, although CCBE1ΔCollagen mice fully phenocopy CCBE1 knock-out mice, CCBE1ΔEGF knock-in embryos still form rudimentary lymphatics. Second, Ccbe1ΔEGF, but not Ccbe1ΔCollagen, could partially substitute for Ccbe1 to enhance Vegfc signaling in zebrafish. Third, CCBE1ΔEGF, similarly to CCBE1, but not CCBE1ΔCollagen could activate VEGFC processing in vitro. Furthermore, a Hennekam syndrome mutation within the collagen domain has a stronger effect than a Hennekam syndrome mutation within the EGF domain. Conclusions: We propose that the collagen domains of CCBE1 are crucial for the activation of VEGFC in vitro and in vivo. The EGF domains of CCBE1 are dispensable for regulation of VEGFC processing in vitro, however, they are necessary for full lymphangiogenic activity of CCBE1 in vivo. # Novelty and Significance {#article-title-22}

THSD1 preserves vascular integrity and protects against intraplaque haemorrhaging in ApoE−/− mice

AIMS Impairment of the endothelial barrier leads to microvascular breakdown in cardiovascular disease and is involved in intraplaque haemorrhaging and the progression of advanced atherosclerotic lesions that are vulnerable to rupture. The exact mechanism that regulates vascular integrity requires further definition. Using a microarray screen for angiogenesis-associated genes during murine embryogenesis, we identified thrombospondin type I domain 1 (THSD1) as a new putative angiopotent factor with unknown biological function. We sought to characterize the role of THSD1 in endothelial cells during vascular development and cardiovascular disease. METHODS AND RESULTS Functional knockdown of Thsd1 in zebrafish embryos and in a murine retina vascularization model induced severe haemorrhaging without affecting neovascular growth. In human carotid endarterectomy specimens, THSD1 expression by endothelial cells was detected in advanced atherosclerotic lesions with intraplaque haemorrhaging, but was absent in stable lesions, implying involvement of THSD1 in neovascular bleeding. In vitro, stimulation with pro-atherogenic factors (3% O2 and TNFα) decreased THSD1 expression in human endothelial cells, whereas stimulation with an anti-atherogenic factor (IL10) showed opposite effect. Therapeutic evaluation in a murine advanced atherosclerosis model showed that Thsd1 overexpression decreased plaque vulnerability by attenuating intraplaque vascular leakage, subsequently reducing macrophage accumulation and necrotic core size. Mechanistic studies in human endothelial cells demonstrated that THSD1 activates FAK-PI3K, leading to Rac1-mediated actin cytoskeleton regulation of adherens junctions and focal adhesion assembly. CONCLUSION THSD1 is a new regulator of endothelial barrier function during vascular development and protects intraplaque microvessels against haemorrhaging in advanced atherosclerotic lesions.

Directing Nanoparticle Biodistribution through Evasion and Exploitation of Stab2-Dependent Nanoparticle Uptake

Up to 99% of systemically administered nanoparticles are cleared through the liver. Within the liver, most nanoparticles are thought to be sequestered by macrophages (Kupffer cells), although significant nanoparticle interactions with other hepatic cells have also been observed. To achieve effective cell-specific targeting of drugs through nanoparticle encapsulation, improved mechanistic understanding of nanoparticle-liver interactions is required. Here, we show the caudal vein of the embryonic zebrafish ( Danio rerio) can be used as a model for assessing nanoparticle interactions with mammalian liver sinusoidal (or scavenger) endothelial cells (SECs) and macrophages. We observe that anionic nanoparticles are primarily taken up by SECs and identify an essential requirement for the scavenger receptor, stabilin-2 ( stab2) in this process. Importantly, nanoparticle-SEC interactions can be blocked by dextran sulfate, a competitive inhibitor of stab2 and other scavenger receptors. Finally, we exploit nanoparticle-SEC interactions to demonstrate targeted intracellular drug delivery resulting in the selective deletion of a single blood vessel in the zebrafish embryo. Together, we propose stab2 inhibition or targeting as a general approach for modifying nanoparticle-liver interactions of a wide range of nanomedicines.

A blood capillary plexus-derived population of progenitor cells contributes to genesis of the dermal lymphatic vasculature during embryonic development

ABSTRACT Despite the essential role of the lymphatic vasculature in tissue homeostasis and disease, knowledge of the organ-specific origins of lymphatic endothelial progenitor cells remains limited. The assumption that most murine embryonic lymphatic endothelial cells (LECs) are venous derived has recently been challenged. Here, we show that the embryonic dermal blood capillary plexus constitutes an additional, local source of LECs that contributes to the formation of the dermal lymphatic vascular network. We describe a novel mechanism whereby rare PROX1-positive endothelial cells exit the capillary plexus in a Ccbe1-dependent manner to establish discrete LEC clusters. As development proceeds, these clusters expand and further contribute to the growing lymphatic system. Lineage tracing and analyses of Gata2-deficient mice confirmed that these clusters are endothelial in origin. Furthermore, ectopic expression of Vegfc in the vasculature increased the number of PROX1-positive progenitors within the capillary bed. Our work reveals a novel source of lymphatic endothelial progenitors employed during construction of the dermal lymphatic vasculature and demonstrates that the blood vasculature is likely to remain an ongoing source of LECs during organogenesis, raising the question of whether a similar mechanism operates during pathological lymphangiogenesis. Highlighted Article: Genetic lineage tracing and targeted gene disruption and gain of function identify a novel source of lymphatic endothelial progenitors that contribute to the formation of lymphatic vasculature in mouse skin.

Functional Dissection of the CCBE1 Protein

RATIONALE Collagen- and calcium-binding EGF domain-containing protein 1 (CCBE1) is essential for lymphangiogenesis in vertebrates and has been associated with Hennekam syndrome. Recently, CCBE1 has emerged as a crucial regulator of vascular endothelial growth factor-C (VEGFC) signaling. OBJECTIVE CCBE1 is a secreted protein characterized by 2 EGF domains and 2 collagen repeats. The functional role of the different CCBE1 protein domains is completely unknown. Here, we analyzed the functional role of the different CCBE1 domains in vivo and in vitro. METHODS AND RESULTS We analyzed the functionality of several CCBE1 deletion mutants by generating knock-in mice expressing these mutants, by analyzing their ability to enhance Vegfc signaling in vivo in zebrafish, and by testing their ability to induce VEGFC processing in vitro. We found that deleting the collagen domains of CCBE1 has a much stronger effect on CCBE1 activity than deleting the EGF domains. First, although CCBE1ΔCollagen mice fully phenocopy CCBE1 knock-out mice, CCBE1ΔEGF knock-in embryos still form rudimentary lymphatics. Second, Ccbe1ΔEGF, but not Ccbe1ΔCollagen, could partially substitute for Ccbe1 to enhance Vegfc signaling in zebrafish. Third, CCBE1ΔEGF, similarly to CCBE1, but not CCBE1ΔCollagen could activate VEGFC processing in vitro. Furthermore, a Hennekam syndrome mutation within the collagen domain has a stronger effect than a Hennekam syndrome mutation within the EGF domain. CONCLUSIONS We propose that the collagen domains of CCBE1 are crucial for the activation of VEGFC in vitro and in vivo. The EGF domains of CCBE1 are dispensable for regulation of VEGFC processing in vitro, however, they are necessary for full lymphangiogenic activity of CCBE1 in vivo.Rationale: Collagen- and calcium-binding EGF domain–containing protein 1 (CCBE1) is essential for lymphangiogenesis in vertebrates and has been associated with Hennekam syndrome. Recently, CCBE1 has emerged as a crucial regulator of vascular endothelial growth factor-C (VEGFC) signaling. Objective: CCBE1 is a secreted protein characterized by 2 EGF domains and 2 collagen repeats. The functional role of the different CCBE1 protein domains is completely unknown. Here, we analyzed the functional role of the different CCBE1 domains in vivo and in vitro. Methods and Results: We analyzed the functionality of several CCBE1 deletion mutants by generating knock-in mice expressing these mutants, by analyzing their ability to enhance Vegfc signaling in vivo in zebrafish, and by testing their ability to induce VEGFC processing in vitro. We found that deleting the collagen domains of CCBE1 has a much stronger effect on CCBE1 activity than deleting the EGF domains. First, although CCBE1ΔCollagen mice fully phenocopy CCBE1 knock-out mice, CCBE1ΔEGF knock-in embryos still form rudimentary lymphatics. Second, Ccbe1ΔEGF, but not Ccbe1ΔCollagen, could partially substitute for Ccbe1 to enhance Vegfc signaling in zebrafish. Third, CCBE1ΔEGF, similarly to CCBE1, but not CCBE1ΔCollagen could activate VEGFC processing in vitro. Furthermore, a Hennekam syndrome mutation within the collagen domain has a stronger effect than a Hennekam syndrome mutation within the EGF domain. Conclusions: We propose that the collagen domains of CCBE1 are crucial for the activation of VEGFC in vitro and in vivo. The EGF domains of CCBE1 are dispensable for regulation of VEGFC processing in vitro, however, they are necessary for full lymphangiogenic activity of CCBE1 in vivo. # Novelty and Significance {#article-title-22}