Silvia Carra

Aβ peptides accelerate the senescence of endothelial cells in vitro and in vivo, impairing angiogenesis

Cerebral amyloid angiopathy (CAA) caused by amyloid β (Aß) deposition around brain microvessels results in vascular degenerative changes. Antiangiogenic Aß properties are known to contribute to the compromised cerebrovascular architecture. Here we hypothesize that Aß peptides impair angiogenesis by causing endothelial cells to enter senescence at an early stage of vascular development. Wild‐type (WT) Aß and its mutated variant E22Q peptide, endowed with marked vascular tropism, were used in this study. In vivo, in zebrafish embryos, the WT or E22Q peptides reduced embryo survival with an IC50 of 6.1 and 4.7 μM, respectively. The 2.5 μM concentration, showing minimal toxicity, was chosen. Alkaline phosphatase staining revealed disorganized vessel patterning, narrowing, and reduced branching of vessels. ß‐Galactosidase staining and the cyclindependent kinase inhibitor p21 expression, indicative of senescence, were increased. In vitro, WT and E22Q reduced endothelial cell survival with an IC50 of 12.3 and 8.8 μM, respectively. The 5 μM concentration, devoid of acute effects on the endothelium, was applied chronically to long‐term cultured human umbilical vein endothelial cells (HUVECs). We observed reduced cumulative population doubling, which coincided with ß‐galactosidase accumulation, down‐regulation of telomerase reversetranscriptase mRNA expression, decreased telomerase activity, and p21 activation. Senescent HUVECs showed marked angiogenesis impairment, as Aß treatment reduced tube sprouting. The endothelial injuries caused by the E22Q peptide were much more aggressive than those induced by the WT peptide. Premature Aß‐induced senescence of the endothelium, producing progressive alterations of microvessel morphology and functions, may represent one of the underlying mechanisms for sporadic or heritable CAA.—Donnini, S., Solito, R., Cetti, E., Corti, F., Giachetti, A., Carra, S., Beltrame, M., Cotelli, F., Ziche, M. Aß peptides accelerate the senescence of endothelial cells in vitro and in vivo, impairing angiogenesis. FASEBJ. 24, 2385–2395 (2010). www.fasebj.org

Crucial role of zebrafish prox1

BackgroundProx1, the vertebrate homolog of prospero in Drosophila melanogaster, is a divergent homeogene that regulates cell proliferation, fate determination and differentiation during vertebrate embryonic development.ResultsHere we report that, in zebrafish, prox1 is widely expressed in several districts of the Central Nervous System (CNS). Specifically, we evidenced prox1 expression in a group of neurons, already positive for otp1, located in the hypothalamus at the level of the posterior tuberculum (PT). Prox1 knock-down determines the severe loss of hypothalamic catecholaminergic (CA) neurons, identified by tyrosine hydroxylase (TH) expression, and the synergistic prox1/otp1 overexpression induces the appearance of hypothalamic supernumerary TH-positive neurons and ectopic TH-positive cells on the yolk epitelium.ConclusionOur findings indicate that prox1 activity is crucial for the proper development of the otp1-positive hypothalamic neuronal precursors to their terminal CA phenotype.

Sox18 Genetically Interacts With VegfC to Regulate Lymphangiogenesis in Zebrafish

Objective—Lymphangiogenesis is regulated by transcription factors and by growth factor pathways, but their interplay has not been extensively studied so far. We addressed this issue in zebrafish. Approach and Results—Mutations in the transcription factor–coding gene SOX18 and in VEGFR3 cause lymphedema, and the VEGFR3/Flt4 ligand VEGFC plays an evolutionarily conserved role in lymphangiogenesis. Here, we report a strong genetic interaction between Sox18 and VegfC in the early phases of lymphatic development in zebrafish. Knockdown of sox18 selectively impaired lymphatic sprouting from the cardinal vein and resulted in defective lymphatic thoracic duct formation. Sox18 and the related protein Sox7 play redundant roles in arteriovenous differentiation. We used a novel transgenic line that enables inducible expression of a dominant-negative mutant form of mouse Sox18 protein. Our data led us to conclude that Sox18 is crucially involved in lymphangiogenesis after arteriovenous differentiation. Combined partial knockdown of sox18 and vegfc, using subcritical doses of specific morpholinos, revealed a synergistic interaction in both venous and lymphatic sprouting from the cardinal vein and greatly impaired thoracic duct formation. Conclusions—This interaction suggests a previously unappreciated crosstalk between the growth factor and transcription factor pathways that regulate lymphangiogenesis in development and disease.

Crucial role of zebrafish prox1 in hypothalamic catecholaminergic neurons development

BackgroundProx1, the vertebrate homolog of prospero in Drosophila melanogaster, is a divergent homeogene that regulates cell proliferation, fate determination and differentiation during vertebrate embryonic development.ResultsHere we report that, in zebrafish, prox1 is widely expressed in several districts of the Central Nervous System (CNS). Specifically, we evidenced prox1 expression in a group of neurons, already positive for otp1, located in the hypothalamus at the level of the posterior tuberculum (PT). Prox1 knock-down determines the severe loss of hypothalamic catecholaminergic (CA) neurons, identified by tyrosine hydroxylase (TH) expression, and the synergistic prox1/otp1 overexpression induces the appearance of hypothalamic supernumerary TH-positive neurons and ectopic TH-positive cells on the yolk epitelium.ConclusionOur findings indicate that prox1 activity is crucial for the proper development of the otp1-positive hypothalamic neuronal precursors to their terminal CA phenotype.

Zebrafish Collagen Type I: Molecular and Biochemical Characterization of the Major Structural Protein in Bone and Skin.

Over the last years the zebrafish imposed itself as a powerful model to study skeletal diseases, but a limit to its use is the poor characterization of collagen type I, the most abundant protein in bone and skin. In tetrapods collagen type I is a trimer mainly composed of two α1 chains and one α2 chain, encoded by COL1A1 and COL1A2 genes, respectively. In contrast, in zebrafish three type I collagen genes exist, col1a1a, col1a1b and col1a2 coding for α1(I), α3(I) and α2(I) chains. During embryonic and larval development the three collagen type I genes showed a similar spatio-temporal expression pattern, indicating their co-regulation and interdependence at these stages. In both embryonic and adult tissues, the presence of the three α(I) chains was demonstrated, although in embryos α1(I) was present in two distinct glycosylated states, suggesting a developmental-specific collagen composition. Even though in adult bone, skin and scales equal amounts of α1(I), α3(I) and α2(I) chains are present, the presented data suggest a tissue-specific stoichiometry and/or post-translational modification status for collagen type I. In conclusion, this data will be useful to properly interpret results and insights gained from zebrafish models of skeletal diseases.

Ve-ptp modulates vascular integrity by promoting adherens junction maturation.

Background Endothelial cell junctions control blood vessel permeability. Altered permeability can be associated with vascular fragility that leads to vessel weakness and haemorrhage formation. In vivo studies on the function of genes involved in the maintenance of vascular integrity are essential to better understand the molecular basis of diseases linked to permeability defects. Ve-ptp (Vascular Endothelial-Protein Tyrosine Phosphatase) is a transmembrane protein present at endothelial adherens junctions (AJs). Methodology/Principal Findings We investigated the role of Ve-ptp in AJ maturation/stability and in the modulation of endothelial permeability using zebrafish (Danio rerio). Whole-mount in situ hybridizations revealed zve-ptp expression exclusively in the developing vascular system. Generation of altered zve-ptp transcripts, induced separately by two different splicing morpholinos, resulted in permeability defects closely linked to vascular wall fragility. The ultrastructural analysis revealed a statistically significant reduction of junction complexes and the presence of immature AJs in zve-ptp morphants but not in control embryos. Conclusions/Significance Here we show the first in vivo evidence of a potentially critical role played by Ve-ptp in AJ maturation, an important event for permeability modulation and for the development of a functional vascular system.

ADAP2 in heart development: a candidate gene for the occurrence of cardiovascular malformations in NF1 microdeletion syndrome

Background Cardiovascular malformations have a higher incidence in patients with NF1 microdeletion syndrome compared to NF1 patients with intragenic mutation, presumably owing to haploinsufficiency of one or more genes included in the deletion interval and involved in heart development. In order to identify which genes could be responsible for cardiovascular malformations in the deleted patients, we carried out expression studies in mouse embryos and functional studies in zebrafish. Methods and results The expression analysis of three candidate genes included in the NF1 deletion interval, ADAP2, SUZ12 and UTP6, performed by in situ hybridisation, showed the expression of ADAP2 murine ortholog in heart during fundamental phases of cardiac morphogenesis. In order to investigate the role of ADAP2 in cardiac development, we performed loss-of-function experiments of zebrafish ADAP2 ortholog, adap2, by injecting two different morpholino oligos (adap2-MO and UTR-adap2-MO). adap2-MOs-injected embryos (morphants) displayed in vivo circulatory and heart shape defects. The molecular characterisation of morphants with cardiac specific markers showed that the injection of adap2-MOs causes defects in heart jogging and looping. Additionally, morphological and molecular analysis of adap2 morphants demonstrated that the loss of adap2 function leads to defective valvulogenesis, suggesting a correlation between ADAP2 haploinsufficiency and the occurrence of valve defects in NF1-microdeleted patients. Conclusions Overall, our findings indicate that ADAP2 has a role in heart development, and might be a reliable candidate gene for the occurrence of cardiovascular malformations in patients with NF1 microdeletion and, more generally, for the occurrence of a subset of congenital heart defects.

In vivo Functional Consequences of Human THRA Variants Expressed in the Zebrafish.

BACKGROUND Heterozygous mutations in the thyroid hormone receptor alpha (THRA) gene cause resistance to thyroid hormone alpha (RTHα), a disease characterized by variable manifestations reminiscent of untreated congenital hypothyroidism but a raised triiodothyronine/thyroxine ratio and normal thyrotropin levels. It was recently described that zebrafish embryos expressing a dominant negative (DN) form of thraa recapitulate the key features of RTHα, and that zebrafish and human receptors are functionally interchangeable. METHODS This study expressed several human thyroid hormone receptor alpha (hTRα) variants in zebrafish embryos and analyzed the resulting phenotypes. RESULTS All hTRα-injected embryos showed variable defects, including cerebral and cardiac edema likely caused by an aberrant looping during heart development, anemia, and an incomplete formation of the vascular network. Moreover, the hTRα-injected embryos presented severe defects of motorneurons and craniofacial development, thus affecting their autonomous feeding and swimming behaviors. Surprisingly, expression of all hTRα mutants had no detectable effect on thyrotropin beta and thyrotropin-releasing hormone transcripts, indicating that their DN action is limited on the thyroid hormone reception beta 2 targets at the hypothalamic/pituitary level in vivo. As previously described in vitro, treatment with high triiodothyronine doses can efficiently revert the observed defects only in embryos injected with missense hTRα variants. CONCLUSION Injection of human THRA variants in zebrafish embryos causes tissue-specific defects recapitulating most of the RTHα clinical and biochemical manifestations. The described manipulation of zebrafish embryos represents a novel in vivo model to screen the functional consequences of THRA variants and the rescue potential of new therapeutic compounds.

The Coiled-Coil domain containing 80 (ccdc80) gene regulates gadd45β2 expression in the developing somites of zebrafish as a new player of the hedgehog pathway

The Coiled‐Coil Domain Containing 80 (CCDC80) gene has been identified as strongly induced in rat thyroid PC CL3 cells immortalized by the adenoviral E1A gene. In human, CCDC80 is a potential oncosoppressor due to its down‐regulation in several tumor cell lines and tissues and it is expressed in almost all tissues. CCDC80 has homologous in mouse, chicken, and zebrafish. We cloned the zebrafish ccdc80 and analyzed its expression and function during embryonic development. The in‐silico translated zebrafish protein shares high similarity with its mammalian homologous, with nuclear localization signals and a signal peptide. Gene expression analysis demonstrates that zebrafish ccdc80 is maternally and zygotically expressed throughout the development. In particular, ccdc80 is strongly expressed in the notochord and it is under the regulation of the Hedgehog pathway. In this work we investigated the functional effects of ccdc80‐loss‐of‐function during embryonic development and verified its interaction with gadd45β2 in somitogenesis. J. Cell. Physiol. 230: 821–830, 2015.

Spatiotemporal image correlation analysis of blood flow in branched vessel networks of zebrafish embryos

Abstract. Ramification of blood circulation is relevant in a number of physiological and pathological conditions. The oxygen exchange occurs largely in the capillary bed, and the cancer progression is closely linked to the angiogenesis around the tumor mass. Optical microscopy has made impressive improvements in in vivo imaging and dynamic studies based on correlation analysis of time stacks of images. Here, we develop and test advanced methods that allow mapping the flow fields in branched vessel networks at the resolution of 10 to 20  μm. The methods, based on the application of spatiotemporal image correlation spectroscopy and its extension to cross-correlation analysis, are applied here to the case of early stage embryos of zebrafish.