Wouter Vandevelde

Role of VEGF-D and VEGFR-3 in developmental lymphangiogenesis, a chemicogenetic study in Xenopus tadpoles

The importance of the lymphangiogenic factor VEGF-D and its receptor VEGFR-3 in early lymphatic development remains largely unresolved. We therefore investigated their role in Xenopus laevis tadpoles, a small animal model allowing chemicogenetic dissection of developmental lymphangiogenesis. Single morpholino antisense oligo knockdown of xVEGF-D did not affect lymphatic commitment, but transiently impaired lymphatic endothelial cell (LEC) migration. Notably, combined knockdown of xVEGF-D with xVEGF-C at suboptimal morpholino concentrations resulted in more severe migration defects and lymphedema formation than the corresponding single knockdowns. Knockdown of VEGFR-3 or treatment with the VEGFR-3 inhibitor MAZ51 similarly impaired lymph vessel formation and function and caused pronounced edema. VEGFR-3 silencing by morpholino knockdown, MAZ51 treatment, or xVEGF-C/D double knockdown also resulted in dilation and dysfunction of the lymph heart. These findings document a critical role of VEGFR-3 in embryonic lymphatic development and function, and reveal a previously unrecognized modifier role of VEGF-D in the regulation of embryonic lymphangiogenesis in frog embryos.

Transcription factor COUP-TFII is indispensable for venous and lymphatic development in zebrafish and Xenopus laevis

Transcription factors play a central role in cell fate determination. Gene targeting in mice revealed that Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII, also known as Nuclear Receptor 2F2 or NR2F2) induces a venous phenotype in endothelial cells (ECs). More recently, NR2F2 was shown to be required for initiating the expression of Prox1, responsible for lymphatic commitment of venous ECs. Small animal models like zebrafish embryos and Xenopus laevis tadpoles have been very useful to elucidate mechanisms of (lymph) vascular development. Therefore, the role of NR2F2 in (lymph) vascular development was studied by eliminating its expression in these models. Like in mice, absence of NR2F2 in zebrafish resulted in distinct vascular defects including loss of venous marker expression, major trunk vessel fusion and vascular leakage. Both in zebrafish and Xenopus the development of the main lymphatic structures was severely hampered. NR2F2 knockdown significantly decreased prox1 expression in zebrafish ECs and the same manipulation affected lymphatic (L)EC commitment, migration and function in Xenopus tadpoles. Therefore, the role of NR2F2 in EC fate determination is evolutionary conserved.

Role of synectin in lymphatic development in zebrafish and frogs

The molecular basis of lymphangiogenesis remains incompletely characterized. Here, we document a novel role for the PDZ domain-containing scaffold protein synectin in lymphangiogenesis using genetic studies in zebrafish and tadpoles. In zebrafish, the thoracic duct arises from parachordal lymphangioblast cells, which in turn derive from secondary lymphangiogenic sprouts from the posterior cardinal vein. Morpholino knockdown of synectin in zebrafish impaired formation of the thoracic duct, due to selective defects in lymphangiogenic but not angiogenic sprouting. Synectin genetically interacted with Vegfr3 and neuropilin-2a in regulating lymphangiogenesis. Silencing of synectin in tadpoles caused lymphatic defects due to an underdevelopment and impaired migration of Prox-1(+) lymphatic endothelial cells. Molecular analysis further revealed that synectin regulated Sox18-induced expression of Prox-1 and vascular endothelial growth factor C-induced migration of lymphatic endothelial cells in vitro. These findings reveal a novel role for synectin in lymphatic development.

Liprin β1 is highly expressed in lymphatic vasculature and is important for lymphatic vessel integrity

The lymphatic vasculature is important for the regulation of tissue fluid homeostasis, immune response, and lipid absorption, and the development of in vitro models should allow for a better understanding of the mechanisms regulating lymphatic vascular growth, repair, and function. Here we report isolation and characterization of lymphatic endothelial cells from human intestine and show that intestinal lymphatic endothelial cells have a related but distinct gene expression profile from human dermal lymphatic endothelial cells. Furthermore, we identify liprin beta1, a member of the family of LAR transmembrane tyrosine phosphatase-interacting proteins, as highly expressed in intestinal lymphatic endothelial cells in vitro and lymphatic vasculature in vivo, and show that it plays an important role in the maintenance of lymphatic vessel integrity in Xenopus tadpoles.

Claudin-like protein 24 interacts with the VEGFR-2 and VEGFR-3 pathways and regulates lymphatic vessel development

The Claudin-like protein of 24 kDa (CLP24) is a hypoxia-regulated transmembrane protein of unknown function. We show here that clp24 knockdown in Danio rerio and Xenopus laevis results in defective lymphatic development. Targeted disruption of Clp24 in mice led to enlarged lymphatic vessels having an abnormal smooth muscle cell coating. We also show that the Clp24(-/-) phenotype was further aggravated in the Vegfr2(+/LacZ) or Vegfr3(+/LacZ) backgrounds and that CLP24 interacts with vascular endothelial growth factor receptor-2 (VEGFR-2) and VEGFR-3 and attenuates the transcription factor CREB phosphorylation via these receptors. Our results indicate that CLP24 is a novel regulator of VEGFR-2 and VEGFR-3 signaling pathways and of normal lymphatic vessel structure.

A transgenic Xenopus laevis reporter model to study lymphangiogenesis

Summary The importance of the blood- and lymph vessels in the transport of essential fluids, gases, macromolecules and cells in vertebrates warrants optimal insight into the regulatory mechanisms underlying their development. Mouse and zebrafish models of lymphatic development are instrumental for gene discovery and gene characterization but are challenging for certain aspects, e.g. no direct accessibility of embryonic stages, or non-straightforward visualization of early lymphatic sprouting, respectively. We previously demonstrated that the Xenopus tadpole is a valuable model to study the processes of lymphatic development. However, a fluorescent Xenopus reporter directly visualizing the lymph vessels was lacking. Here, we created transgenic Tg(Flk1:eGFP) Xenopus laevis reporter lines expressing green fluorescent protein (GFP) in blood- and lymph vessels driven by the Flk1 (VEGFR-2) promoter. We also established a high-resolution fluorescent dye labeling technique selectively and persistently visualizing lymphatic endothelial cells, even in conditions of impaired lymph vessel formation or drainage function upon silencing of lymphangiogenic factors. Next, we applied the model to dynamically document blood and lymphatic sprouting and patterning of the initially avascular tadpole fin. Furthermore, quantifiable models of spontaneous or induced lymphatic sprouting into the tadpole fin were developed for dynamic analysis of loss-of-function and gain-of-function phenotypes using pharmacologic or genetic manipulation. Together with angiography and lymphangiography to assess functionality, Tg(Flk1:eGFP) reporter tadpoles readily allowed detailed lymphatic phenotyping of live tadpoles by fluorescence microscopy. The Tg(Flk1:eGFP) tadpoles represent a versatile model for functional lymph/angiogenomics and drug screening.

Cardiovascular Research turns the spotlight onto the right ventricle

This month’s issue of Cardiovascular Research is dedicated to the right ventricle. As introduced by the guest editors, this has long been considered the ‘lesser’ ventricle. A PubMed search for ‘left ventricle’ (LV) shows 25 217 publications since 2010, whereas a search for ‘right ventricle’ (RV) results in 21 684 publications in the same time period. However, most documents include both terms, so if we compare the publications that exclusively cover the ‘left ventricle’ we get 6 270 documents, and in contrast there are only 2 737 publications specifically on the ‘right ventricle’. Even if this is a crude approximation, it aligns with a message that is present in several of the review articles of this Spotlight Issue, namely that our knowledge of the RV is less than that of the LV. At the same time, available knowledge clearly indicates the particularities of the RV and underscores that we cannot simply ‘copy-paste’ insights from the LV to the RV. In the past years, we also noticed that the RV was less often the topic of submitted manuscripts but seemed to gain attention. We have browsed the recent issues of Cardiovascular Research, bringing together relevant publications in this virtual issue including selected articles from Europace and European Heart Journal, to complement the series of reviews. Filling of the RV and diastolic functions were studied by Pérez Del Villar et al., focusing on the restoring forces and the role of suction. The interaction between LV and RV function, focusing on diastolic dysfunction, was recently underscored in a study on HFpEF patients demonstrating impaired RV functional reserve. The causes and mechanisms underlying arrhythmogenic RV disease are still being unravelled, and diagnostic testing presents challenges. The power of advanced and next generation sequencing can support differential diagnosis and positioning within the group of cardiomyopathies. Mechanisms of remodeling and targets for treatment are mostly studied in models of RV hypertrophy and failure consequent on pulmonary hypertension (PHT). Within the RV, apoptosis and loss of myocytes may temporarily be compensated by the remaining myocytes. Targeting pathways that reduce PHT and vascular remodelling are associated with improved RV remodelling. While this could be due to the reduced haemodynamic load, some of these approaches may involve some direct effects on the RV, as e.g. the neuregulin pathway. Other mechanistic studies emphasize the tight link between the vascular remodelling and RV remodelling, as for miR-223-IGF-IR. Thrombospondin-1 may be important in signalling within the lungs and pulmonary vascular smooth muscle cells in response to hypoxia, but interrupting this pathway benefits cardiac function as well. Conflict of interest: none declared.

Hot topics and networks in Cardiovascular Research.

‘Publish or perish’ refers to the essence of journals as instruments for sharing novel and reliable information to advance scientific knowledge. Editors are expected to present high-quality, novel, and relevant studies to the community by a process of review and selection from submitted manuscripts. Peer review is an essential part of this selection process providing expert input on all three aspects for the final editorial decision. In addition, regular evaluation of the outcome of the editorial work is essential to guide the process. ISI provides metrics of which the impact factor is best-known, but there are many others. All of these give single numerical value to the aggregate of the articles but do not inform about the content of the journal. Here, after 3 years in office, we wanted to take stock of the research published in Cardiovascular Research in 2013 and 2014. Are we on track for the impact and visibility we aim to achieve? What topics are emerging? Have publication policies provided more visibility to these publications? Taking a global view of the results of the first 2 years, the topics of the papers published and their visibility in the research community, is a useful step in analysing the decisions made in the past. Overall, 379 original research articles and 80 reviews were published in Cardiovascular Research in 2013 and 2014. One of the main indicators of visibility of these publications is the number of citations obtained from Web of Science online on 21 September 2015, totalling 3199 citations. Publications in Cardiovascular Research enjoy a high visibility, with an average citation rate of 7 citations per publication, a low percentage of only 7% that have …

Cardiovascular Research as a forum for publications from China: present, past and future

There has been a rapid growth of publications from China over the last 20 years, with China becoming the second largest producer of scientific papers across all disciplines in the year 2007.1 Focused investment in building scientific capacity in China has significantly contributed to the growth in publication output. In 1998, the launch of the ‘985’ project increased Chinas commitment to improve the global standing and publication activities of a select group of 24 universities.2 Research performance has also gained from the commitment of the Chinese government to increase the Gross Domestic Expenditure on Research and Development (GERD). A common indicator used is the ratio of GERD to Gross Domestic Product (GDP) and in China, this ratio has grown steadily from 1.07% in 2002 to reach 1.84% in 2011.3 More specifically, the funding provided through the National Natural Science Foundation of China (NSFC; http://www.nsfc.gov.cn/publish/portal1/tab293/) and the National Basic Research Program of China (NBRPC) have had a significant impact in fuelling basic research, for example with almost 40 million Euros [312 million Chinese Renminbi (RMB)] being assigned by the NSFC to experimental cardiovascular studies in 2013 alone.4 These policy and funding commitments in China have affected the submissions to Cardiovascular Research over the past 40 years. The number of publications from Chinese authors in Cardiovascular Research has grown progressively from one article being published in the 1970s, three in the 1980s, and seven being published from 1990 to 1994, before finally seeing more than one publication per year starting from 1998 onwards. Cardiovascular Research has experienced a steady increase in the submissions resulting from research based in China, which has raised the interest of …

Editorial highlights from Cardiovascular Research

In the past years, the editorial office has regularly taken stock of what the journal published and how papers fared. We have been particularly interested in the impact of the editorial choices and the evolution in the field. Following up on our 2015 editorial report, we have now examined the papers published in 2015 and 2016, identifying topics and zooming in on papers highlighted as editors’ choice, mostly papers accompanied by an editorial. We then compared the outcome to the previous analysis.