Lotting for TSP-1 protein levels (Figure 2D). We could not detect a signal for MCP-1 protein in HUVEC by Western blotting due to a lack of sensitivity, but we could observe the inhibitory effect in the level of secreted MCP-1 to the medium by using a high sensitive ELISA assay: in samples of conditioned media from control HUVEC, there was 4.3260.11 fg MCP-1/cell, whereas in conditioned media from HUVEC treated with aeroplysinin-1 (10 mM for 6 h) there was 2.3060.41 fg MCP1/cell (Figure 2C). That means that aeroplysinin-1 ASP015K treatment decreased the levels of secreted MCP-1 by more than 45 . These additional results undoubtedly show that both MCP-1 mRNA and secreted protein and TSP-1 protein levels are decreased in samples from HUVEC treated with aeroplysinin-1 (10 mM for 6 h), as compared to control, untreated HUVEC.Results Aeroplysinin-1 Treatment Inhibits Key Steps of Angiogenesis in Human Endothelial CellsIn a previous report, we identified and characterized aeroplysinin-1 as a potent anti-angiogenic compound affecting several key steps of the process in BAEC [12]. Since that study was carried out using endothelial cells isolated from a great vessel (aorta) from no human source (cow), we began the present study trying to confirm the anti-angiogenic effects of aeroplysinin-1 in human endothelial cells from medium size vessels and microvessels. Table 1 shows the IC50 values determined in proliferation assays using MTT as described in Material and Methods. These values were in the low micromolar range, as it was the case of the published effect on BAEC proliferation [12]. The ML 264 formation of a three-dimensional network of newly formed vessels is the final key event of the angiogenic process. In vitro, endothelial cells plated on Matrigel align themselves forming cords (Figure 1A, controls). Figure 1A (treatments) shows that treatment for 6 h with low micromolar concentrations of aeroplysinin-1 resulted in complete inhibition of endothelial cell alignment and cord formation for the three immortalized human endothelial cell lines tested. At lower concentrations of aeroplysinin-1 than those inducing complete inhibition of cord formation, partial inhibition could be observed in a dose-response manner, as shown in Figure 1A (histogram, below). Matrix metalloproteinase-2 (MMP-2) is a key extracellular enzyme involved in basal membrane remodeling, a required step to allow for endothelial cell migration during active angiogenesis. Figure 1B shows that 2.5 mM aeroplysinin-1 induced a partial inhibition of MMP-2 in conditioned media from RF-24 and HMEC and an almost total inhibition of MMP-2 in conditioned media from EVLC-2. For the rest of the study, we used either immortalized (RF-24) or primary cultures of HUVEC. Figure 1C (histogram, above) shows that 20 mM aeroplysinin-1 significantly inhibits RF-24 cell migratory potential after 7 h of treatment, as determined in a “wound healing” assay, under conditions not affecting cell viability (data not shown). However, this effect on migration was not detectable when we made use of a migration assay based on a transwell (Figure 1C, time-course curve, below). Figure 1D shows that 3?0 mM aeroplysinin-1 did not significantly affect the RF-24 cell invasive potential through a layer of Matrigel as Table 1. IC50 values for aeroplysinin-1 treatment on human endothelial cells determined by the MTT assay.Aeroplysinin-1 Treatment Induces Partial Inhibition of the Secretion of Pro-inflammatory Cytokines by HUVEC to.Lotting for TSP-1 protein levels (Figure 2D). We could not detect a signal for MCP-1 protein in HUVEC by Western blotting due to a lack of sensitivity, but we could observe the inhibitory effect in the level of secreted MCP-1 to the medium by using a high sensitive ELISA assay: in samples of conditioned media from control HUVEC, there was 4.3260.11 fg MCP-1/cell, whereas in conditioned media from HUVEC treated with aeroplysinin-1 (10 mM for 6 h) there was 2.3060.41 fg MCP1/cell (Figure 2C). That means that aeroplysinin-1 treatment decreased the levels of secreted MCP-1 by more than 45 . These additional results undoubtedly show that both MCP-1 mRNA and secreted protein and TSP-1 protein levels are decreased in samples from HUVEC treated with aeroplysinin-1 (10 mM for 6 h), as compared to control, untreated HUVEC.Results Aeroplysinin-1 Treatment Inhibits Key Steps of Angiogenesis in Human Endothelial CellsIn a previous report, we identified and characterized aeroplysinin-1 as a potent anti-angiogenic compound affecting several key steps of the process in BAEC [12]. Since that study was carried out using endothelial cells isolated from a great vessel (aorta) from no human source (cow), we began the present study trying to confirm the anti-angiogenic effects of aeroplysinin-1 in human endothelial cells from medium size vessels and microvessels. Table 1 shows the IC50 values determined in proliferation assays using MTT as described in Material and Methods. These values were in the low micromolar range, as it was the case of the published effect on BAEC proliferation [12]. The formation of a three-dimensional network of newly formed vessels is the final key event of the angiogenic process. In vitro, endothelial cells plated on Matrigel align themselves forming cords (Figure 1A, controls). Figure 1A (treatments) shows that treatment for 6 h with low micromolar concentrations of aeroplysinin-1 resulted in complete inhibition of endothelial cell alignment and cord formation for the three immortalized human endothelial cell lines tested. At lower concentrations of aeroplysinin-1 than those inducing complete inhibition of cord formation, partial inhibition could be observed in a dose-response manner, as shown in Figure 1A (histogram, below). Matrix metalloproteinase-2 (MMP-2) is a key extracellular enzyme involved in basal membrane remodeling, a required step to allow for endothelial cell migration during active angiogenesis. Figure 1B shows that 2.5 mM aeroplysinin-1 induced a partial inhibition of MMP-2 in conditioned media from RF-24 and HMEC and an almost total inhibition of MMP-2 in conditioned media from EVLC-2. For the rest of the study, we used either immortalized (RF-24) or primary cultures of HUVEC. Figure 1C (histogram, above) shows that 20 mM aeroplysinin-1 significantly inhibits RF-24 cell migratory potential after 7 h of treatment, as determined in a “wound healing” assay, under conditions not affecting cell viability (data not shown). However, this effect on migration was not detectable when we made use of a migration assay based on a transwell (Figure 1C, time-course curve, below). Figure 1D shows that 3?0 mM aeroplysinin-1 did not significantly affect the RF-24 cell invasive potential through a layer of Matrigel as Table 1. IC50 values for aeroplysinin-1 treatment on human endothelial cells determined by the MTT assay.Aeroplysinin-1 Treatment Induces Partial Inhibition of the Secretion of Pro-inflammatory Cytokines by HUVEC to.
