Cardiovascular Research (2009) 81, 362–369 doi:10.1093/cvr/cvn294 A crucial role of caldesmon in vascular development in vivo Ping-Pin Zheng1, Lies-Anne Severijnen2, Marcel van der Weiden1, Rob Willemsen2†, and Johan M. Kros1*† 1Department of Pathology, Erasmus Medical Center, JNI Room 230-c, Dr Molewaterplein 50, PO Box 1738, 3000 DR Rotterdam, The Netherlands; and 2Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands Downloaded from https://academic.oup.com/cardiovascres/article/81/2/362/286189 by guest on 24 September 2021 Received 24 July 2008; revised 28 October 2008; accepted 29 October 2008; online publish-ahead-of-print 3 November 2008 Time for primary review: 13 days KEYWORDS Aims We explored the in vivo effects of knockdown of caldesmon on vascular development in zebrafish. Caldesmon; Methods and results We investigated the effects of caldesmon knockdown on the vascular development Vascular development; in a zebrafish model with special attention for the trunk and head vessels including the aortic arches. We Zebrafish model; examined the developing fishes at various time points. The vascular abnormalities observed in the cal- Vasculogenesis; desmon morphants were morphologically and functionally characterized in detail in fixed and living Angiogenesis embryos. The knockdown of caldesmon caused serious defects in vasculogenesis and angiogenesis in zebrafish morphants, and the vascular integrity and blood circulation were concomitantly impaired. Conclusion The data provide the first functional assessment of the role of caldesmon in vascular development in vivo, indicating that this molecule plays a crucial role in vasculogenesis and angio- genesis in vivo. Interfering with caldesmon opens new therapeutic avenues for anti-angiogenesis in cancer and ischaemic cardiovascular disease. 1. Introduction primordial hindbrain channel, the anterior cardinal vein, and the basilar artery in the head.8 The formation of most Caldesmon (CaD) is evolutionally conserved among ver- 1 of the subsequent vessels in the embryo occurs by sprouting tebrates. The zebrafish homologue is similar to mammalian from pre-existing vessels in a process known as angiogenesis. low-molecular-weight caldesmon (l-CaD). Previously, we Many (presumably angiogenic) blood vessels that sub- reported the specific upregulation of this protein (l-CaD) in sequently develop in the zebrafish have orthologues in glioma neovasculature and its association with migration other vertebrates and among these are the central cranial and proliferation of endothelial cells (ECs) and endothelial 2–7 arteries and the massive network of microvessels in the progenitor cells (EPCs) in human tumours. The findings head, the dorsal longitudinal anastomotic vessels (DLAVs), triggered us to explore the effects of this protein on the the intersegmental vessels (ISVs), the subintestinal veins development of blood vessels in vivo for the design of new (SIVs), the caudal vessel plexus (CVP), the parachordal therapeutic strategies. Here we explored the effects of vessels, the vertebral vessels in the trunk, and more.9 The knockdown of CaD on the vascular development in zebrafish embryology of the aortic arch (AA) system in zebrafish is embryos. Zebrafish embryos can survive several days very similar to that of birds and mammals.10 Six pairs of without a functioning circulatory system, allowing detailed vessels, connecting the ventral aorta to the lateral DA, analysis of the animals with severe cardiovascular defects. emerge in a cranial-to-caudal sequence, each of which is The development of the vascular system in vertebrates embedded in its respective pharyngeal arch which is collec- occurs by two distinct processes: vasculogenesis and angio- tively known as the branchial AAs.10 The AA primordia arise genesis. The same primary vasculogenic vessels that estab- by vasculogenesis and extend via angiogenesis.10 At the mol- lish the initial circulatory circuits in other vertebrate ecular level, several important genes, including VEGF,Flk-1/ embryos are also present in the zebrafish. These vessels KDR, Fli-1, Flt-1, Tie-1, and Tie-2, have been cloned in include the dorsal aorta (DA) and posterior cardinal vein zebrafish and show expression patterns similar to those (PCV) in the trunk and the internal carotid artery, the in mammals.11,12 The striking conservation of vascular anatomy and the expression pattern of the associated genes across the vertebrate phyla indicate similar vasculo- * Corresponding author. Tel: þ31 10 7043905; fax: þ31 10 7043905. E-mail address: [email protected] genic and angiogenic signalling pathways for blood vessel † These authors contributed equally to this work. formation and patterning. Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2008. For permissions please email: [email protected]. Caldesmon in vascular development 363 2. Methods (3 Â 10 min), followed by incubating with horseradish peroxidase- conjugated secondary antibody (1:1000) for 2 h at RT and washed 2.1 Morpholino injections and verification (3 Â 10 min). The target protein spots were visualized by enhanced of the specificity chemiluminescence (Amersham Biosciences Corp., Piscataway, NJ, USA). The films were scanned for analysis and imaging. The caldesmon antisense morpholino oligonucleotides (MOs) and 5-base mismatch controls were purchased from Gene Tools (Philo- math, OR, USA). The caldesmon antisense MO1 5-AGTAAAGTCTCTTA 2.3 Morpholino rescue experiment TTCTTCAACGC-3 and MO2 5-TAAGAGTTCATCCTGTAGAGTGATG-3 were designed to inhibit translation of the caldesmon RNA (gene: The cDNA of human CaD served as a template containing a T7 RNA ENSDARG00000032052; transcript: ENSDART00000067366; trans- polymerase promoter. RNA was in vitro synthesized using the mMES- lation: ENSDARP00000067365) and a 5-base mismatch control 5- AGA SAGE mMACHINE kit (Ambion, Austin, TX, USA) according to the AAACTCTCTTATTGTTGAAGGC-3 was used. First, in a titration exper- manufacturer’s protocol and co-injected with the morpholinos. iment, the MOs were injected into the yolk sac of zebrafish embryos Downloaded from https://academic.oup.com/cardiovascres/article/81/2/362/286189 by guest on 24 September 2021 between one- and two-cell stages at different concentrations (2, 4, 2.4 Immunostaining of whole mount and sections and 8 ng/embryo), and the embryos were raised at 28.58C until analysis under standard laboratory conditions. The concentration Briefly, embryos were fixed by 4% PFA at RT for minimal 3 h following (4 ng/embryo) was used in all the subsequent experiments, standard procedures. Embryos were treated with 1 M NH4Cl at RT for because the survival of the embryos was satisfactory (.86%). The 3 h to quench autofluorescence, permeabilized, blocked by 5% goat use of zebrafish embryos was approved by the Institutional Review serum in PBST for 1 h at RT, and incubated with the selected primary Board for experimental animals. antibodies: VEGFR2/Flk1 (Lab Vision), VEGFR1/Flt1 (Lab Vision), There are several options to determine whether a phenotype is endothelial nitric oxide synthase (eNOS) (Lab Vision), CD105 (Lab the result of knocking down a gene-of-interest by blocking trans- Vision), Glut-1 (Dako), occludin (Zymed), ZO-1 (Zymed), and Tie-2 lation with morpholinos (MO): (a) quantification of the target (R&D System) at dilution 1:50 to 100 for two to three overnights protein by an antibody (check if the translation is blocked); (b) at 48C. After post-incubation washing, embryos were incubated RNA rescue experiments; (c) a control morpholino with 5 bp mis- with FITC- or rhodamine-conjugated goat-anti-rabbit or goat-anti- match (discussed earlier); and (d) application of a second MO with mouse (Jackson ImmunoResearch Laboratories, Inc.) at a dilution similar blocking effects (discussed earlier). We verified the speci- of 1:100 for two overnights at 48C. After thoroughly washing, fluor- ficity of the MO phenotype in our experiments by combining the escence images were recorded by a fluorescence microscope and/or above-mentioned methods. confocal laser scanning microscopy (CLSM). Embryos at identical developmental stages, processed without primary antibody, were used as controls for each experiment. 40,6-diamidino-2-phenylindole 2.2 Quantification of the homologue protein (DAPI) was used for nuclei counterstaining. of caldesmon Immunohistochemical analysis of sections was performed by stan- dard methods. Briefly, embryos were fixed, dehydrated, embedded Quantification of the homologue protein was performed by vision in paraffin, and sectioned (5 mm). Sections were deparaffinized, assay (see whole-mount staining and section immunohistochemis- blocked, antigen-retrieved, incubated with the selected primary try), dot blot, and whole-mount enzyme-linked immunosorbent antibodies: EP050852 (Eurogentec) at 1:100 and Glut-1 (Dako) at assay (ELISA) by using the antibody EP050852 (Eurogentec, 1:150, washed, and stained by AP-conjugated secondary antibody. Belgium) against the protein. Western blotting was impossible AP-based substrate was used for visualization. because the antibody was not working in a reducing status. The cross-species reactivity of the antibodies used was confirmed In whole-mount ELISA, zebrafish embryos were fixed in 4% paraf- by immunohistochemistry, western blotting, and dot blot, unless ormaldehyde (PFA) at least 3 h, washed and permeabilized by TBST already tested by others,13–15 or the antibody was specifically (0.05% Triton X-100 in TBS), and treated