NF-KB activation in endothelial cells is critical for the activity of anαiostatic agents

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Angiostatic agents are increasingly important in the clinical management of cancer. Many angiostatic agents interfere with the angiogenic process by blocking the proliferation/ survival, the migration, or the maturation of endothelial cells. The molecular targets of these agents have only been partially discovered (3, 31, 32). A thorough understanding of the mechanism of action of angiostatic agents, mainly at the level of proximal signaling events, is still required for the development of efficient antitumor therapies. The current report describes that angiostatic agents with a direct activity on endothelial cells, such as 16K hPRL, anginex, endostatin, and angiostatin, activate NF-κB in endothelial cells.

NF-κB activation was observed in endothelial cells already after 30 min of stimulation with angiostatic agents. This time-course activation is comparable with what has been described previously for TNF-α in endothelial cells (33). These results suggest that the NF-κB pathway is an early event in response to binding of angiostatic agents to their receptors. The transgenic mice harboring a NF-κB-responsive promoter element driving a lacZ reporter gene provided the opportunity to evaluate NF-κB activation in vivo after treatment with angiostatic agents in tumor endothelial cells both spatially and quantitatively.

Twenty-four hours after treatment with anginex or endostatin, NF-κB was activated in tumor endothelial cells as measured by β-galactosidase activity and expression of lacZ and IkBα mRNA. To our knowledge, this is the first report illustrating activation of NF-κB in tumor endothelial cells after treatment with angiostatic agents. In this context, it is interesting to note that NF-κB is known to be activated by proinflammatory cytokines such as TNF-α, interleukin-1, and IFN-γ. These cytokines have antiangiogenesis activity as well. This is most well known for TNF-α (34) and IFN-γ(35), which, depending on the dose, can even induce apoptosis in endothelial cells. Interleukin-1 is mostly known as an angiogenesis stimulator, but some reports identified inhibitory activities as well (36).

The role of NF-κB in endothelial cells is poorly understood (37). We found previously that, in bovine endothelial cells, 16K hPRL induces caspase-dependent apoptosis by a mechanism that requires activation of NF-κB (30). Interestingly, endostatin and anginex have also been described to induce apoptosis in endothelial cells (12). We here showed that NF-κB is induced in human and mouse endothelial cells after exposure to any of these inhibitors. However, specific inhibition of NF-κB does not counteract anginex-induced inhibition of proliferation/viability but abrogate anginex-induced inhibition of sprouting. These results suggest that even if NF-κB activation is a common pathway activated by angiostatic agents, the role of NF-κB is agent-specific and/or cell-specific.

Even if BAYll 70-82 has been shown previously to target specifically the NF-κB pathway at concentrations <20 µmol/L, we cannot rule out the possibility that this compound may have off-target effects in endothelial cells. Therefore, p65 silencing would be an optimal strategy to confirm our results. However, transfection of primary endothelial cells such as HUVEC is a challenging option. In our hands, optimized transfection protocols gave an efficiency rate of ~30%, indicating that the NF-κB pathway could be blocked in a maximum of 30% of the cells and therefore making the data interpretation difficult. In addition, the toxicity we observed following transfection was not negligible, as is not unusual with primary endothelial cells, suggesting that many stress-related pathways would be activated by the transfection process and confuse the results about the role of NF-κB in angiogenesis.

Very recently, NF-κB signaling has been found to regulate endothelial cell integrity and vascular homeostasis in vivo. Treatment of zebrafish embryos with NF-κB inhibitors provoked vascular leakage and altered vessel morphology (38). Tie2 promoter/enhancer-IκB^S32A_S36A transgenic mice, in which the endothelial-specific Tie2 promoter drives the expression of a trans-dominant-negative mutant of NF-κB and therefore present an inhibition of endothelial NF-κB signaling, developed normally and displayed, in particular, a normal pattern of vascular development (39). However, inoculated tumors grew faster in these mice, and histologic analysis revealed a striking increase in tumor vascularization. The molecular mechanisms accounting for such increase are still unknown. Our data are in accordance with these observations and furthermore suggest that NF-κB activation is mainly involved in the spatial organization of endothelial cells such as sprouting.

The activation of NF-κB can also be connected with an indirect antitumor activity through reversal of endothelial unresponsiveness to inflammatory signals, a process called endothelial cell anergy. The latter is defined by us as the absence of leukocyte-binding adhesion molecules on endothelial cells and the unresponsiveness to inflammatory cytokines (40). This phenomenon occurs due to exposure of endothelial cells to angiogenic growth factors. NF-κB is best known for its regulation of proinflammatory genes. We have reported previously that angiogenic factors (e.g., bFGF) down-regulate endothelial adhesion molecules such as ICAM-1, vascular cell adhesion molecule-1, and E-selectin through NF-κB inhibition (41). In addition, tumor endothelial cells displayed a reduced expression of adhesion molecules compared with that of normal endothelial cells, accounting for the observation of a reduced number of infiltrated leukocytes within the tumor (42). Accordingly, we found in the current study that NF-κB activation by angiostatic agents is correlated with ICAM-1 up-regulation at the endothelial cell surface. This expression further results in an enhanced NF-κB-dependent leukocyte-endothelial cell interaction. Together, these data show that anginex, and possibly other angiogenesis inhibitors as well, overcome endothelial cell anergy by activation of the NF-κB pathway. This activity may be shared with the abovementioned proinflammatory cytokines that can have intrinsic antiangiogenesis activities as well (34-36). Therefore, activation of NF-κB in endothelial cells resulting in stimulation of antitumor immunity can clearly result in an antitumor outcome.

The fact that different biochemically and biologically unrelated angiostatic agents activate the NF-κB pathway is intriguing. Recently, galectin-1 has been identified as the anginex receptor at the surface of endothelial cells (43). The importance of gal-1 in angiogenesis has been further illustrated in the zebrafish model, where expression knockdown results in impaired vascular guidance and growth of dysfunctional vessels. However, thus far, no direct evidence has linked galectin-1 and the NF-κB pathway. Endo-statin may exert its antiangiogenic effect through its binding to α₅β₁ integrin or to binding to glypican (44, 45). However, it is not obvious which endostatin receptor conveys the effective antiangiogenic signals. Our data showing that NF-κB pathway is activated by different angiostatic agents already after 1 h of treatment suggest that a common receptor/ coreceptor exist at the surface of endothelial cells. Further studies will be required to decipher the pathway upstream NF-κB activation up to the receptor.

We report for the first time that activation of NF-κB is a common pathway used by multiple and biochemically unrelated angiostatic agents. NF-κB activation has been observed both in vitro and in vivo in tumor endothelial cells. Furthermore, we showed that NF-κB inhibition impairs the ability of angiostatic agents to block sprouting of endothelial cells and to overcome endothelial cell anergy. This is of special interest because, in tumor cells, NF-κB activation has been associated to inhibition of apoptosis. Therefore, currently novel treatment strategies are being developed based on inhibition of NF-κB. Although this would cause an antitumor effect in tumor cells, it is suggested to coincide with proangiogenic activity. Based on our results, the antitumor activity of angiostatic agents is linked to activation of NF-κB in endothelial cells. This activation could result in stimulation of antitumor immunity and in inhibition of angiogenesis. It is attractive to speculate that optimal therapeutic results could be achieved by either (or both) targeting inhibitors of NF-κB to tumor cells and/or by specific activation of NF-κB in endothelial cells.