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VEGF-A and _Vß₃ integrin synergistically rescue angiogenesis via N-Ras and PI3-K signaling in human microvascular endothelial cells¹

ZHAO-JUN LIU^*,, RUTHANNE SNYDER^*,, AKINOBU SOMA, TAKASHI SHIRAKAWA, BARRY L. ZIOBER, RONALD M. FAIRMAN^*, MEENHARD HERLYN and OMAIDA C. VELAZQUEZ^*,,2

^* Department of Surgery, School of Medicine, University of Pennsylvania;
The Wistar Institute; and
Department of Otorhinolaryngology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, USA

²Correspondence: Department of Surgery, School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA. E-mail: omaida.velazquez{at}uphs.upenn.edu

SPECIFIC AIMS

We recently reported that normal fibroblasts mediate capillary-like network formation of human microvascular endothelial cells (HMVEC) in a three-dimensional (3-D) angiogenesis model. The present study investigated the molecular mechanisms involved in this process. We show the existence of N-Ras- and PI3-K-dependent signaling mechanisms for the synergistic effect of VEGF-A and _Vß₃ integrin on fibroblast-mediated microvascular network formation.

PRINCIPAL FINDINGS

1. Synergistic effect of VEGF-A and _Vß₃ integrinon microvascular network formation
Angiogenesis depends on inputs from growth factors and vascular cell adhesion signals. We first examined the role for _Vß₃ integrin in a capillary-like network formation induced by fibroblasts. Overexpression of a dominant-negative mutant ß₃ integrin (DN-ß₃) in HMVEC mediated by adenoviral vector significantly suppressed network formation. A similar effect was achieved by adding anti-_Vß₃ blocking antibody LM609, demonstrating a critical role for _Vß₃ integrin in modulating fibroblast-induced capillary network formation of HMVEC in vitro.

Our previous findings indicated that VEGF-A plays a critical role in network formation. To determine whether VEGF-A exerts its function completely through the activation of integrins or via an alternative collaborative mechanism, we assessed the effect of transduced fibroblasts overexpressing VEGF-A on capillary-like network formation by various HMVEC transductants grown under reduced serum conditions. In our 3-D model, cultured HMVEC monolayers are able to differentiate into capillary-like networks by supplying the medium with 5% FBS. Under reduced serum conditions, however, capillary-like network formation was completely inhibited, but the inhibition was partially rescued by overexpression of VEGF-A. Addition of LM609 suppressed this effect, implicating the involvement of _Vß₃ in the VEGF-A-mediated rescue. Although overexpression of wild-type ß₃ integrin (WT-ß₃) on HMVEC alone failed to restore capillary-like network formation, simultaneous expression of VEGF-A in fibroblasts and WT-ß₃ integrin on HMVEC significantly restored network formation. VEGF-A and ß₃ integrin work synergistically, suggesting a collaborative effect between the angiogenic factor VEGF-A and _Vß₃ integrin on vascular network formation in our 3-D model.

2. Differential roles of N-Ras and Rho/Rac/Cdc42 in modulating microvascular network formation
To investigate the signal pathways that mediate the synergistic effect of VEGF-A and _Vß₃ integrin on network formation, we examined the roles of small GTPases because they can be activated by VEGFR or _Vß₃ integrin. We introduced dominant-negative mutants of N-Ras^N17, RhoA^N19, Rac^N17, and Cdc42^N17 into WT-ß₃-HMVEC, which were engineered to express WT-ß₃ integrin simultaneously. We subsequently used these cells in our 3-D model overlaid by fibroblasts that were transduced with recombinant adenovirus (VEGF₁₂₁/Ad5) and cultured in reduced serum conditions in order to examine whether any of these mutants could interfere with VEGF-A-induced and _Vß₃ integrin-mediated network formation. WT forms of N-Ras, RhoA, Rac, and Cdc42 were transduced into WT-ß₃-HMVEC and compared with LacZ/Ad5-transduced WT-ß₃-HMVEC. We found that formation of capillary-like networks was significantly inhibited in HMVEC expressing N-Ras^N17, whereas expression of RhoA^N19/Rac^N17/Cdc42^N17, N-Ras, or WT-RhoA/Rac/Cdc42 did not affect vascular network formation. Our data indicate the presence of a N-Ras-dependent but RhoA/Rac/Cdc42-independent mechanism that mediates the synergistic effect of VEGF-A and _Vß₃ integrin on fibroblast-induced angiogenesis.

3. Role of PI3-K, but not Akt, in modulating formation of microvascular networks
We also investigated the contribution of the PI3-K pathway, which involves PI3-K and its downstream effector, Akt, in the collaborative effect of VEGF-A and ß₃ integrin on network formation. Constitutively active forms of PI3-K (Myr-p110) and Akt (Myr-Akt) were tested for their ability to rescue the DN-ß₃-induced defect in vascular network formation. The mutant was transduced into DN-ß₃-HMVEC, engineered to express DN-ß₃ integrin simultaneously. The cells were then tested in our 3-D model under the previous conditions. The network formation of DN-ß₃-HMVEC could be rescued by Myr-p110 but not by Myr-Akt (Fig. 1 ). Consistent with these findings, expression of Myr-p110 but not Myr-Akt further increased the number of vascular networks formed by WT-ß₃-HMVEC. Immunostaining of the monolayers of HMVEC showed that the morphology of Myr-Akt-transduced HMVEC has been changed, suggesting that Myr-Akt might cause a phenotype alteration that would not favor network formation. Thus, our data implicate a differential role for PI3-K vs. Akt in the regulation of VEGF-A- and _Vß₃ integrin-mediated angiogenesis.

View larger version (51K): [in this window] [in a new window]   Figure 1. Differential effect of PI3-K and Akt on vascular network formation. a) Effect of ECM components on PI3-K activity. HMVEC were coated on type I collagen or vitronectin for 1 h. Cells were harvested and PI3-kinase activity was assessed. The PIP3 product, separated by thin-layer chromatography, and the origins are indicated. b) Expression of exogenous Myr-p110 resulted in activation of endogenous Akt activity. Myr-p110/Ad5-transduced HMVEC were harvested 48 h post-transduction and whole-cell lysates were analyzed by Western blot to detect phosphorylation of endogenous Akt. The total Akt level is also visualized. c) Expression of exogenous Myr-Akt. Myr-Akt/Ad5-transduced HMVEC were harvested 48 h post-transduction and whole-cell lysates were analyzed by Western blot. ß-Actin level is shown to demonstrate an equal loading condition. d) Phosphorylation of Akt. The membrane used in panel c was subsequently stripped and blotted with Ab recognizing phosphorylated Akt. e) Specific effect of PI3-K on rescue of vascular network formation in DN-ß3/Ad5-transduced HMVEC under reduced serum conditions. HMVEC transduced with Myr-p110/Ad5 or Myr-Akt/Ad5 were tested in a 3-D model and compared with that of nontransduced or lacZ/Ad5-transduced cells. Morphology of vascular networks, x10. Statistic data are means ± SD of 4 separate experiments with a total of 16 samples for each condition. *P < 0.05, Student’s t test. f) Effect of PI3-K and Akt on vascular network formation in WT-ß3/Ad5-transduced HMVEC under reduced serum conditions. Morphology of vascular networks, x10. Statistic data are means ± SD of 4 separate experiments with a total of 16 samples for each condition. *P < 0.05, Student’s t test. g) Percentage of Myr-p110/Ad5- and Myr-Akt/Ad5-transduced HMVEC. Immunostaining of cell monolayer was performed. Total cell number is shown as 100% by open bars. Filled bars represent percentage of cells overexpressing mutant proteins. x20. Statistic data are means ± SD of 3 separate experiments.

PI3-K functions as a downstream target of N-Ras in HMVEC
We further investigated the potential linkage between PI3-K and N-Ras in HMVEC. Coimmunoprecipitation experiment demonstrated that N-Ras could be coimmunoprecipitated with p85 in N-Ras-transduced HMVEC, implicating an association between PI3-K and N-Ras. Moreover, Myr-p110 could functionally rescue the effects of the dominant negative mutant N-Ras^N17 on network formation, strongly implying that PI3-K is a downstream signal transducer of Ras in HMVEC.

CONCLUSIONS AND SIGNIFICANCE

Using our unique in vitro 3-D angiogenesis model, we observed synergistic angiogenic effects exerted by VEGF-A and _Vß₃ integrin on fibroblast-mediated angiogenesis in HMVEC, and that these effects depend on both N-Ras and PI3-K but not on Rho/Rac/Cdc42 or Akt. PI3-K functions as a downstream target of N-Ras (Fig. 2 ). Findings derived from approaches of this type might have important implications for understanding the signaling mechanisms involved in modulating vasculogenesis and angiogenesis. Moreover, such studies might serve in identifying novel targets with clinical implications for regulating normal and/or neoplastic neovascularization.

View larger version (25K): [in this window] [in a new window]   Figure 2. Schematic illustration of VEGF-A-induced and ß3 integrin-mediated signaling pathways in HMVEC. The two signaling systems synergistically modulate vascular network formation in which PI3-K- and N-Ras-dependent, but Akt- and Rho family-independent, mechanisms are involved. PI3-K functions downstream of N-Ras.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1171fje; doi: 10.1096/fj.02-1171fje

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