CD40-dependent Activation of Phosphatidylinositol 3-Kinase/Akt Pathway Mediates Endothelial Cell Survival and in Vitro Angiogenesis

Abstract

CD40 has been involved in tumor and inflammatory neoangiogenesis. In this study we determined that stimulation of endothelial CD40 with sCD154 induced resistance to apoptosis and in vitro vessel-like formation by human microvascular endothelial cells (HMEC). These effects were determined to be mediated by CD40-dependent signaling because they were inhibited by a soluble CD40-muIg fusion protein. Moreover, apoptosis of HMEC was associated with an impairment of Akt phosphorylation, which was restored by stimulation with sCD154. The anti-apoptotic effect as well as in vitro vessel-like formation and Akt phosphorylation were inhibited by treatment of HMEC with two unrelated pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002. CD40 stimulation induced a rapid increase in Akt enzymatic activity that was not prevented by cycloheximide, an inhibitor of protein synthesis. The enhanced Akt activity induced by stimulation of endothelial CD40 was temporarily correlated with the association of CD40 with TRAF6, c-Cbl, and the p85 subunit of PI3K. Expression of negativedominant Akt inhibited the activation of endogenous Akt through CD40 stimulation, despite the observation that association of CD40 with TRAF6, c-Cbl, and PI3K was intact. The defective activation of Akt abrogated not only the anti-apoptotic effect of CD40 stimulation but also the proliferative response, the enhanced motility, and the in vitro formation of vessel-like tubular structures by CD40-stimulated HMEC. In conclusion, these results suggest that endothelial CD40, through activation of the PI3K/Akt signaling pathway, regulates cell survival, proliferation, migration, and vessel-like structure formation, all steps considered critical for angiogenesis.CD40 is a member of the tumor necrosis factor (TNF) 1 receptor superfamily, which provides activation signals in antigen-presenting cells such as B cells, macrophages, and dendritic cells (1, 2). Among the molecular mechanisms that link immunity to inflammation, the interaction between CD40 and its counterreceptor CD154 has rapidly emerged as a key system in the regulation of vascular pathophysiological processes such as atherogenesis (3, 4), tumor neoangiogenesis (5, 6), and inflammation (2, 8). Under physiological conditions, CD40 is expressed at low levels on endothelial cells but is up-regulated in areas of inflammation (9). Ligation of endothelial CD40 by CD154, either expressed on activated monocytes or T cells (2) or disgorged by platelets upon activation (10), induces production of various inflammatory cytokines and chemokines, procoagulant activity, adhesion molecules, metalloproteinases, and inflammatory mediators (11-14). These mediators have been implicated in the development and progression of atherosclerosis. In situ analysis of human atherosclerotic lesions revealed the co-expression of CD154 and CD40 on vascular endothelium and smooth muscle cells (15). Blockade of CD40-CD154 interaction in atherosclerosis was found not only to diminish the formation and pr...

Prostaglandin E2 stimulates angiogenesis by activating the nitric oxide/cGMP pathway in human umbilical vein endothelial cells

“…It has been demonstrated that cAMP causes PI3K-dependent Akt activation in hepatocytes and hepatic WIF-B9 cells, probably in a PKA-dependent manner (Li et al, 2000;Kagawa et al, 2002). The PI3K/Akt signaling pathway can be induced by VEGF (Kosmidou et al, 2001;Laughner et al, 2001) and sphingosine 1-phosphate (Morales-Ruiz et al, 2001) and plays a central role in angiogenesis by promoting endothelial sprouting, proliferation, migration, and vessel-like structure formation (Deregibus et al, 2003;Licht et al, 2003). These evidences suggest that many angiogenic factors activate the PI3K/Akt signaling pathway, which is a central mediator of angiogenesis (Zachary, 2001).…”

Role of Pax2 in Apoptosis Resistance and Proinvasive Phenotype of Kaposi's Sarcoma Cells

“…Endothelial cells were characterized as endothelial cells by morphology and positive staining for von Willebrand factor antigen and for CD31, CD105, and the fucosylated receptors for plant lectins (Ulex europeus I and Bandeira simplicifolia). Cytoplasmic staining was positive for vimentin and negative for cytokeratin and desmin, as previously described (17). A previously characterized renal carcinoma cell line (K1) was used as positive control for Pax2 expression (18).…”

“…Migration tracks were generated by marking the positions of the nuclei of individual cells on each image. The net migratory speed (velocity straight line) was calculated by the MicroImage software based on the straight line distance between the starting and ending points divided by the time of observation (17). Migration of at least 30 cells was analyzed for each experimental condition.…”

Potentiation of irinotecan sensitivity by Se-methylselenocysteine in an in vivo tumor model is associated with downregulation of cyclooxygenase-2, inducible nitric oxide synthase, and hypoxia-inducible factor 1α expression, resulting in reduced angiogenesis

“…These results suggest the possibility that the inhibition of neoangiogenesis in vivo may be associated with the antitumor synergy of irinotecan and MSC. Endothelial CD40 affects several steps important for angiogenesis, including cell proliferation, motility, and formation of vessel-like structures, through the PI3K/Akt signaling pathway Deregibus et al, 2003), suggesting that Akt mediates angiogenesis. To confirm whether the inhibition of SN-38-induced angiogenesis involves Akt inactivation, we examined the effect of MSC on SN-38-induced Akt expression and phosphorylation.…”