Final Report Summary - NETRIN-ANGIO (Role of netrin signaling in blood vessel pathfinding)
The central aim of this project was to identify candidate molecules involved in the guidance of vascular sprouts by using zebrafish and mouse as animal models and taking advantage of the striking similarities between the vascular and nervous systems. Indeed, a remarkable recent finding is that well-known axon guidance cues have been involved in vessel pathfinding as well. Molecules belonging to the netrin/DCC/UNC, Slit/Robo, semaphorin/NRP/plexin and Eph/ephrin families were originally described as axon guidance cues and can act on endothelial cells (ECs) [1, 2]. However, a role for netrin molecules in developmental vessel navigation or pathological angiogenesis is on debate, since it has been reported that they exhibit pro- and anti-angiogenic activities [3-5]. Although this controversial response has been linked to cell survival [6], it is not clear whether other mechanisms could be also involved.
In the nervous system, netrins induce attraction, repulsion, adhesion, proliferation and survival. These different cellular effects of netrin signaling depends on (i) the subset of receptors that is expressed by the target cell and (ii) the intracellular levels of second messengers. However, these two aspects of netrin-mediated signaling have not been studied in detail in ECs. Upon netrin stimulation, intracellular signaling involves tyrosine phosphorylation, phosphatidylinositol signaling and regulation of small Rho GTPases. In addition, downstream signaling regulates gene transcription and protein translation, although these effects remained poorly characterized, both in neurons and ECs.
The aim of this project was to analyze the effects triggered by netrins on ECs at the molecular and in vivo functional level.
Therefore, we studied modifications on gene expression patterns in cultured ECs after stimulation with two different netrin ligands: Netrin1 and Netrin4. We have performed mRNA microarrays to determine the gene expression profile of primary cultures of human venous and arterial ECs, upon Netrin1 or Netrin4 stimulation. In venous ECs, we found that the expression of 165 genes was regulated by Netrin1, 115 genes by Netrin4, while 98 genes were regulated in a opposed manner by both ligands (p-val<0.001). In arterial ECs, we found that 165 genes were regulated by Netrin1, 386 genes by Netrin4 and 121 genes were differentially regulated by both ligands (p-val<0.001). This differential regulation was further confirmed by quantitative RT-PCR for some of the candidates. Taking together, these results indicate that netrins induce a considerable shift in EC expression pattern, in both arteries and veins. Moreover, this genetic program is not common for both types of ECs, as only 7-13% of the regulated genes are shared by arterial and venous ECs.
To further characterize the role of these candidate genes during angiogenesis, we performed an unbiased selection of 34 candidates. These candidates have been implicated in different biological processes, including proliferation, cell signaling, gene regulation, adhesion, mRNA processing, or they had an unknown function. To determine the in vivo relevance of these candidates in the developmental formation of blood vessels, we used the larvae of Zebrafish as animal model. To determine the expression of the zebrafish orthologous genes of the candidates, we synthesized complementary DNA from a pool of different stages of zebrafish embryos and cloned probes to perform in situ hybridization (ISH) in zebrafish embryos. Analysis of the vasculature on zebrafish embryos revealed that 65% of these candidates (22/34) were expressed in the blood vessels (see figure 1 for expression pattern of 14 of the positive candidate genes). This list includes both known and previously unknown genes.
To study more in detail the role of these candidate genes during developmental angiogenesis, we microinjected morpholino antisense oligomers (morpholinos) into 1-2 cell stage of zebrafish embryos to specifically knockdown the expression of the candidates. The study particularly focused on the sprouting of stereotyped intersomitic vessels (ISVs) from the dorsal aorta.
In the nervous system, netrins induce attraction, repulsion, adhesion, proliferation and survival. These different cellular effects of netrin signaling depends on (i) the subset of receptors that is expressed by the target cell and (ii) the intracellular levels of second messengers. However, these two aspects of netrin-mediated signaling have not been studied in detail in ECs. Upon netrin stimulation, intracellular signaling involves tyrosine phosphorylation, phosphatidylinositol signaling and regulation of small Rho GTPases. In addition, downstream signaling regulates gene transcription and protein translation, although these effects remained poorly characterized, both in neurons and ECs.
The aim of this project was to analyze the effects triggered by netrins on ECs at the molecular and in vivo functional level.
Therefore, we studied modifications on gene expression patterns in cultured ECs after stimulation with two different netrin ligands: Netrin1 and Netrin4. We have performed mRNA microarrays to determine the gene expression profile of primary cultures of human venous and arterial ECs, upon Netrin1 or Netrin4 stimulation. In venous ECs, we found that the expression of 165 genes was regulated by Netrin1, 115 genes by Netrin4, while 98 genes were regulated in a opposed manner by both ligands (p-val<0.001). In arterial ECs, we found that 165 genes were regulated by Netrin1, 386 genes by Netrin4 and 121 genes were differentially regulated by both ligands (p-val<0.001). This differential regulation was further confirmed by quantitative RT-PCR for some of the candidates. Taking together, these results indicate that netrins induce a considerable shift in EC expression pattern, in both arteries and veins. Moreover, this genetic program is not common for both types of ECs, as only 7-13% of the regulated genes are shared by arterial and venous ECs.
To further characterize the role of these candidate genes during angiogenesis, we performed an unbiased selection of 34 candidates. These candidates have been implicated in different biological processes, including proliferation, cell signaling, gene regulation, adhesion, mRNA processing, or they had an unknown function. To determine the in vivo relevance of these candidates in the developmental formation of blood vessels, we used the larvae of Zebrafish as animal model. To determine the expression of the zebrafish orthologous genes of the candidates, we synthesized complementary DNA from a pool of different stages of zebrafish embryos and cloned probes to perform in situ hybridization (ISH) in zebrafish embryos. Analysis of the vasculature on zebrafish embryos revealed that 65% of these candidates (22/34) were expressed in the blood vessels (see figure 1 for expression pattern of 14 of the positive candidate genes). This list includes both known and previously unknown genes.
To study more in detail the role of these candidate genes during developmental angiogenesis, we microinjected morpholino antisense oligomers (morpholinos) into 1-2 cell stage of zebrafish embryos to specifically knockdown the expression of the candidates. The study particularly focused on the sprouting of stereotyped intersomitic vessels (ISVs) from the dorsal aorta.
