Images were taken at 48 hpf. metabolic product of HMGCR, reverses the inhibitory effect of aplexone on venous angiogenesis. In addition, aplexone treatment inhibits protein prenylation and blocking the activity of geranylgeranyl transferase induces a venous angiogenesis phenotype resembling that observed in aplexone-treated embryos. Furthermore, endothelial cells of venous origin have higher levels of proteins requiring geranylgeranylation than arterial endothelial cells and inhibiting the activity of Rac or Rho kinase effectively reduces the migration of venous, but not arterial, endothelial cells. Taken together, our findings indicate that angiogenesis is differentially regulated by the HMGCR pathway via an arteriovenous-dependent requirement for protein prenylation in zebrafish and human endothelial cells. and upon stimulation by VEGF, whereas ECs of the posterior cardinal vein (PCV) express a distinct set of genes such as and (Lawson et al., 2001; Zhong et al., 2001; Lawson et al., 2002). Similarly, in mouse embryos, capillaries of arterial origin express ephrin B2 and those of venous origin express (Wang et al., 1998). Disrupting this arteriovenous lineage-specific expression pattern blocks circulation, highlighting the essential role for arteriovenous identity in establishing blood circulation (Gerety et al., 1999; Gerety and Anderson, 2002). In addition to the diversity in their transcriptional profiles, ECs exhibit different cellular behaviors according to their arteriovenous origins. In zebrafish, angioblasts migrate from their lateral MNS position to the midline in two waves to form the vascular cord. It has been hypothesized that angioblasts destined to form the DA migrate first, whereas the angioblasts destined to form the PCV migrate at a later stage (Torres-Vazquez et al., 2003; Jin et al., 2005; Williams et al., 2010). A pathway involving signaling molecules such as VEGF, Notch, PI3K and Eph/ephrin then directs a dorsal migration of ECs to form DA and a ventral migration to form PCV (Herbert et al., 2009). The diversity in lineage-dependent cellular behavior is further evident in the differential timing of angiogenesis during the formation of the dorsoventrally positioned intersegmental vessels (ISVs) in the trunk. Two waves of ISV sprouting were noted in zebrafish depending on the origin of ECs (Isogai et al., 2003; Hogan et al., 2009; Ellertsdottir et al., 2010). The first wave occurs at around 20 hours post fertilization (hpf) when ECs of the DA migrate dorsally in response to signals, including VEGF and Notch to form the primary, aorta-derived vascular network. The second wave occurs about 16 hours later (36 hpf) when a new set of vascular sprouts emerges exclusively from the PCV. Some of these secondary sprouts connect with the primary ISVs, linking the posterior cardinal vein to the primary vascular network (Isogai et al., 2003; Hogan et al., 2009; Ellertsdottir et al., 2010). The distinct timings of the arterial-derived primary sprouts and the venous-derived secondary sprouts indicate that arterial and venous angiogenesis are differentially regulated during development. How the distinct molecular identities of arteries and veins influence lineage-specific angiogenesis is currently not known. The optical clarity and rapid development of zebrafish embryos, along with the fact that they are fertilized externally, offer an excellent opportunity to MNS conduct in vivo screens for compounds that modulate biological processes of interest (Zon and Peterson, 2005; Walsh and Chang, 2006). Traditionally, ISV formation has served as a model for angiogenesis in zebrafish. Here, we show that ECs of the caudal vein also undergo active angiogenesis, providing an additional model for venous angiogenesis. We screened a collection of small molecules for compounds that preferentially suppress angiogenesis by endothelial cells of either arterial or venous origin, using caudal vein morphogenesis and ISV formation as indicators. In this screen, we identified a novel compound, aplexone, that can effectively block angiogenesis from the vein, but has limited impact on arterial angiogenesis. We offer several lines of evidence demonstrating that aplexone targets the HMG-CoA reductase pathway, disrupts protein geranylgeranylation and effectively inhibits venous EC migration both in zebrafish embryos and cultured human.(F) The percentage of embryos with ectopic primordial germ cells in control (embryos by 24 hpf (G), but fail to migrate to midline in embryos treated with 30 M aplexone (H). levels. Injecting mevalonate, a metabolic product of HMGCR, reverses the inhibitory effect of aplexone on venous angiogenesis. In addition, aplexone treatment inhibits protein prenylation and blocking the activity of geranylgeranyl transferase induces a venous angiogenesis phenotype resembling that observed in aplexone-treated embryos. Furthermore, endothelial cells of venous origin have higher levels of proteins requiring geranylgeranylation than arterial endothelial cells and inhibiting the activity of Rac or Rho kinase effectively reduces the migration of venous, but not arterial, endothelial cells. Taken together, our findings indicate that angiogenesis is differentially regulated by the HMGCR pathway via an arteriovenous-dependent requirement for CLIP1 protein prenylation in zebrafish and human endothelial cells. and upon stimulation by VEGF, whereas ECs of the posterior cardinal vein (PCV) express a distinct set of genes such as and (Lawson et al., 2001; Zhong et al., 2001; Lawson et al., 2002). Similarly, in mouse embryos, capillaries of arterial origin express ephrin B2 and those of venous origin express (Wang et al., 1998). Disrupting this arteriovenous lineage-specific expression pattern blocks circulation, highlighting the essential role for arteriovenous identity in establishing blood circulation (Gerety et al., 1999; Gerety and Anderson, MNS 2002). In addition to the diversity in their transcriptional profiles, ECs exhibit different cellular behaviors according to their arteriovenous origins. In zebrafish, angioblasts migrate from their lateral position to the midline in two waves to form the vascular cord. It has been hypothesized that angioblasts destined to form the DA migrate first, whereas the angioblasts destined to form the PCV migrate at a later stage (Torres-Vazquez et al., 2003; Jin et al., 2005; Williams et al., 2010). A pathway involving signaling molecules such as VEGF, Notch, PI3K and Eph/ephrin then directs a dorsal migration of ECs to form DA and a ventral migration to form PCV (Herbert et al., 2009). The diversity in lineage-dependent cellular behavior is further evident in the differential timing of angiogenesis during the formation of the dorsoventrally positioned intersegmental vessels (ISVs) in the trunk. Two waves of ISV sprouting were noted in zebrafish depending on the origin of ECs (Isogai et al., 2003; Hogan et al., 2009; Ellertsdottir et al., 2010). The first wave occurs at around 20 hours post fertilization (hpf) when ECs of the DA migrate dorsally in response to signals, including VEGF and Notch to form the primary, aorta-derived vascular network. The second wave occurs about 16 hours later (36 hpf) when a new set of vascular sprouts emerges exclusively from the PCV. Some of these secondary sprouts connect with the primary ISVs, linking the posterior cardinal vein to the primary vascular network (Isogai et al., 2003; Hogan et al., 2009; Ellertsdottir et al., 2010). The distinct timings of the arterial-derived primary sprouts and the venous-derived secondary sprouts indicate that arterial and venous angiogenesis are differentially regulated during development. How the distinct molecular identities of arteries and veins influence lineage-specific angiogenesis is currently not known. The optical clarity and rapid development of zebrafish embryos, along with the fact that they are fertilized externally, offer an excellent opportunity to conduct in vivo screens for compounds that modulate biological processes of interest (Zon and Peterson, 2005; Walsh and Chang, 2006). Traditionally, ISV formation has served as a model for angiogenesis in zebrafish. Here, we show that ECs of the caudal vein also undergo active angiogenesis, providing an additional model for venous angiogenesis. We screened a collection of small molecules for compounds that preferentially suppress angiogenesis by endothelial cells of either arterial or venous origin, using caudal vein morphogenesis and ISV formation as indicators. In this screen, we identified a novel compound, aplexone, that can effectively block angiogenesis from your vein, but offers limited impact on arterial angiogenesis. We offer several lines of evidence demonstrating that aplexone focuses on the HMG-CoA reductase pathway, disrupts protein geranylgeranylation and efficiently inhibits venous EC migration both in zebrafish embryos and cultured human being ECs. In addition, we find that venous ECs have higher levels of proteins requiring geranylgeranylation than arterial ECs, and their migration is definitely more sensitive to changes in protein prenylation. Taken together, our findings show that angiogenesis is definitely differentially regulated based on the arteriovenous source of ECs and that the HMGCR biochemical pathway has a crucial part in.
