Nalogues have been synthesized [5,6]. In addition to its antibiotic action [1,4,7], aeroplysinin-1 has been shown to have a wide spectrum of anti-tumoral action [8?11]. Aeroplysinin-1 has been shown to display a strong anti-tumor effect on EGF-dependent tumor cell lines through its claimed inhibitory effect on the intrinsic protein tyrosine kinase activity of EGF-receptor kinase complex [9]. We have previously characterized aeroplysinin-1 as a potent anti-angiogenic compound in vitro and in vivo [12]. Most of the in vitro assays in that article were carried out with primary cultures of MedChemExpress Itacitinib bovine aortic endothelial cells (BAEC). However, although BAEC are widely used as model cell cultures for angiogenesis research, some concerns have been raised due to the facts that theydo not come from microvessels and they do not come from humans or model animals [13,14]. Therefore, a first objective of the present study was to test whether the results obtained in different in vitro angiogenesis-related assays are dependent on the origins of the endothelial cells. To fulfil this objective, we have made use of three types of human endothelial cells, namely, EVLC-2 (endothelial venous line cells), RF-24 (an immortalized line of HUVEC, human umbilical vein endothelial cells) and HMEC (immortalized human microvascular endothelial cells). Once demonstrated that our results are consistently reproduced in the three types of tested human endothelial cells, we used primary cultures of HUVEC to evaluate short-term effects of aeroplysinin1 on angiogenesis-related genes expressed by human umbilical vein endothelial cells (HUVEC), by using commercial angiogenesis gene arrays and alternative validation procedures. Since results point to modulation of genes related with inflammation, we proceeded further by using a commercial cytokine array and alternative validation procedures. Furthermore, several key experiments were also carried out with the THP-1 humanAeroplysinin-1 Inhibits Pro-Inflammatory Moleculesmonocyte cell line. In this case, results confirmed that monocyte cell proliferation was inhibited and the expression levels of cyclooxygenase-2 protein by these cells was decreased upon treatment with aeroplysinin-1. Altogether, the results shown here support a description of aeroplysinin-1 as an inhibitor of angiogenesis in human endothelial cells and as a new potent inhibitor of pro-inflammatory biomolecules.determined by the invasion assay described in Material and Methods.Aeroplysinin-1 Treatment Induces Partial Inhibition of Two Angiogenesis Genes Related to Inflammation in HUVECIn order to evaluate short-term effects of aeroplysinin-1 on angiogenesis-related genes expressed by proliferating HUVEC, we used a human angiogenesis gene array. A typical result is shown in Figure 2A. Due to intrinsic variability of biological samples and the experimental procedure, we only 24786787 took into account those changes in gene expression consistently repeated in five independent experiments. This stringent requirement was fulfilled by few genes. From them, two genes had the clearest changes upon aeroplysin-1 treatment: thrombospondin 1 (TSP-1) and monocyte 166518-60-1 chemoattractant protein-1 (MCP-1). Aeroplysinin-1 (10 mM for 6 h) decreased the expression levels of TSP-1 protein and MCP1 mRNA to 6568 and 34613 of their respective control values. In this study, we confirmed our gene array results by using semiquantitative RT-PCR for the MCP-1 mRNA expression levels (Figure 2B) and Western b.Nalogues have been synthesized [5,6]. In addition to its antibiotic action [1,4,7], aeroplysinin-1 has been shown to have a wide spectrum of anti-tumoral action [8?11]. Aeroplysinin-1 has been shown to display a strong anti-tumor effect on EGF-dependent tumor cell lines through its claimed inhibitory effect on the intrinsic protein tyrosine kinase activity of EGF-receptor kinase complex [9]. We have previously characterized aeroplysinin-1 as a potent anti-angiogenic compound in vitro and in vivo [12]. Most of the in vitro assays in that article were carried out with primary cultures of bovine aortic endothelial cells (BAEC). However, although BAEC are widely used as model cell cultures for angiogenesis research, some concerns have been raised due to the facts that theydo not come from microvessels and they do not come from humans or model animals [13,14]. Therefore, a first objective of the present study was to test whether the results obtained in different in vitro angiogenesis-related assays are dependent on the origins of the endothelial cells. To fulfil this objective, we have made use of three types of human endothelial cells, namely, EVLC-2 (endothelial venous line cells), RF-24 (an immortalized line of HUVEC, human umbilical vein endothelial cells) and HMEC (immortalized human microvascular endothelial cells). Once demonstrated that our results are consistently reproduced in the three types of tested human endothelial cells, we used primary cultures of HUVEC to evaluate short-term effects of aeroplysinin1 on angiogenesis-related genes expressed by human umbilical vein endothelial cells (HUVEC), by using commercial angiogenesis gene arrays and alternative validation procedures. Since results point to modulation of genes related with inflammation, we proceeded further by using a commercial cytokine array and alternative validation procedures. Furthermore, several key experiments were also carried out with the THP-1 humanAeroplysinin-1 Inhibits Pro-Inflammatory Moleculesmonocyte cell line. In this case, results confirmed that monocyte cell proliferation was inhibited and the expression levels of cyclooxygenase-2 protein by these cells was decreased upon treatment with aeroplysinin-1. Altogether, the results shown here support a description of aeroplysinin-1 as an inhibitor of angiogenesis in human endothelial cells and as a new potent inhibitor of pro-inflammatory biomolecules.determined by the invasion assay described in Material and Methods.Aeroplysinin-1 Treatment Induces Partial Inhibition of Two Angiogenesis Genes Related to Inflammation in HUVECIn order to evaluate short-term effects of aeroplysinin-1 on angiogenesis-related genes expressed by proliferating HUVEC, we used a human angiogenesis gene array. A typical result is shown in Figure 2A. Due to intrinsic variability of biological samples and the experimental procedure, we only 24786787 took into account those changes in gene expression consistently repeated in five independent experiments. This stringent requirement was fulfilled by few genes. From them, two genes had the clearest changes upon aeroplysin-1 treatment: thrombospondin 1 (TSP-1) and monocyte chemoattractant protein-1 (MCP-1). Aeroplysinin-1 (10 mM for 6 h) decreased the expression levels of TSP-1 protein and MCP1 mRNA to 6568 and 34613 of their respective control values. In this study, we confirmed our gene array results by using semiquantitative RT-PCR for the MCP-1 mRNA expression levels (Figure 2B) and Western b.