Decreased Tube Formation Research Articles

Anti-angiogenic activity of torilin, a sesquiterpene compound isolated fromTorilis japonica

Torilin is a sesquiterpene compound purified from fruits of Torilis japonica (Umbelliferae). In this study, we demonstrated the anti-angiogenic activity of torilin using in vivo and in vitro assay systems. Torilin decreased both neovascularization of chick embryos in the chorioallantoic membrane assay and basic fibroblast growth factor-induced vessel formation in the mouse Matrigel plug assay. Torilin also reduced the proliferation and tube formation of human umbilical vein endothelial cells. In addition, the concentrated conditioned media obtained from torilin-treated HepG2 human hepatoblastoma cells blocked the angiogenic activation of torilin-untreated concentrated conditioned media, indicating that torilin may have an inhibitory effect on tumor-induced angiogenesis. To determine what molecules were involved in the anti-angiogenic activity, we examined the expression of hypoxia-inducible angiogenic factors in torilin-treated HepG2 cells. Torilin significantly down-regulated the expression of hypoxia-inducible vascular endothelial growth factor and insulin-like growth factor-II. Taken together, our data suggest that torilin may be a strong angiogenic inhibitor with the ability to decrease tube formation of vascular endothelial cells and to reduce expression of angiogenic factors of tumor cells.

Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways

Endothelial cell differentiation into capillary structures is a complex process that requires the concerted effects of several extracellular matrix proteases, including plasminogen activators. Here, the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) was evaluated in an in vitro model of endothelial morphogenesis involving organization of human umbilical vein endothelial cells into tubular structures when they are cultured on the basement membrane preparation, Matrigel. Both uPA and tPA were detected in HUVEC cultures on Matrigel, and inhibitors of plasminogen activators or of serine proteases decreased the extent of the tube network formed by the cells. The decrease resulting from serine protease inhibitors was additive to that from matrix metalloproteinase inhibitors which have previously been shown to decrease tube formation in this model, suggesting that the two classes of proteases modulate tube formation by distinct mechanisms. Plasminogen activator inhibitor (PAI)-1 decreased tube formation by 50% when added up to 4.5 h after the initiation of an 18 h assay and caused 25% inhibition when added 9.5 h after culture initiation, indicating that the effects of plasminogen activators are not limited to an early event in the differentiation process. Steady-state expression of mRNA for uPA increased during the first several hours of culture on Matrigel, further supporting a role for PA activity throughout the process of tube formation. These findings suggested that PAs may affect multiple events during tube-forming activity. A fucosylated peptide comprising the amino-terminal domain of uPA that binds to the uPA receptor (uPAR) but lacking proteolytic activity enhanced tube formation. In contrast, a defucosylated form of the same peptide had no effect. Since fucosylation of this fragment has been shown to be essential in other models of cell stimulation by uPA-uPAR interaction, these data support the hypothesis that uPA enhances endothelial morphogenesis both through proteolytic activity and via uPAR occupancy. Plasminogen activators could facilitate angiogenesis in vivo.

Type IV collagenase(s) and TIMPs modulate endothelial cell morphogenesis in vitro.

It has been proposed that proteases are important in endothelial cell behavior. We examined the contribution of the gelatinase/type IV collagenase system in an in vitro model of endothelial differentiation. Human umbilical vein endothelial cells rapidly align and form networks of tubes when cultured on a basement membrane preparation, Matrigel. Zymograms of culture supernates demonstrate a 72-kD and a 92-kD gelatinase activity; the cells produce most of the 72-kD gelatinase, whereas the 92-kD activity is derived entirely from the Matrigel. Addition of antibodies against type IV gelatinase/collagenase decreases the area of the tube network. Both tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, similarly decrease tube formation when added to cultures. Conversely, exogenous recombinant 72-kD gelatinase increases tube-forming activity. The effects of the anti-gelatinase antibodies and the TIMPs are not additive. Inhibition by either antibodies or TIMPs is greatest when they are added at culture initiation, suggesting that the protease activity is important in the early steps of morphogenesis. However, culture of the cells on Matrigel does not increase early expression of mRNA for the 72-kD gelatinase. Expression of message for the enzyme actually decreases during the course of the assay, while transcription of mRNAs for TIMPs increases, further supporting the concept that collagenases facilitate an early event in tube formation. These data demonstrate that gelatinase/type IV collagenase activity is important in endothelial cell morphogenesis on Matrigel, and suggest a role for collagenases in formation of new capillaries in vivo.