HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/2/338 03-0271fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ERBER, R. Articles by VAJKOCZY, P. Search for Related Content PubMed PubMed Citation Articles by ERBER, R. Articles by VAJKOCZY, P.

Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms¹

RALF ERBER, ANDREAS THURNHER, ALICE D. KATSEN^*, GESINE GROTH, HEINZ KERGER, HANS-PETER HAMMES, MICHAEL D. MENGER^*, AXEL ULLRICH and PETER VAJKOCZY²

Department of Neurosurgery, Medical Faculty of the University of Heidelberg, Mannheim, Germany;
^* Institute for Clinical and Experimental Surgery, University of Saarland, Homburg/Saar, Germany;
Department of Anesthesiology and Intensive Care Medicine, Medical Faculty of the University of Heidelberg, Mannheim, Germany;
Vth Medicine Clinic, Medical Faculty of the University of Heidelberg, Mannheim, Germany; and
Department of Molecular Biology, Max-Planck-Institute for Biochemistry, Martinsried, Germany

² Correspondence: Department of Neurosurgery, Klinikum Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, D-68167 Mannheim, Germany. E-mail: peter.vajkoczy{at}nch.ma.uni-heidelberg.de

SPECIFIC AIMS

Vascular endothelial growth factor (VEGF) acts not only as a mitogenic but also as a survival factor for angiogenic endothelial cells; therefore, selective targeting of VEGF signaling may provide a therapeutic means to enforce tumor blood vessel regression. The specific aims were to study 1) the anti-vascular efficacy of and potential resistance mechanisms to a selective targeting of VEGF signaling, 2) whether tumor blood vessel regression can be enhanced by a simultaneous targeting of endothelial cells and pericytes, and 3) to define the microcirculatory consequences of the consecutive tumor vessel destruction.

PRINCIPAL FINDINGS

1. Selective targeting of VEGF signaling fails to induce regression of tumor vessels
To determine whether targeting of VEGF signaling enforces tumor blood vessel regression we treated established C6 tumors with SU5416, a selective inhibitor of the VEGF receptor VEGFR-2, and assessed the tumor microvasculature by intravital fluorescence videomicroscopy. Although SU5416 suppressed the formation of new tumor blood vessels, it had no effect on the density, diameter, or architecture of already established tumor blood vessels.

2. Pericytes protect tumor blood vessels from enforced regression
To address the mechanisms underlying this resistance of tumor blood vessels to selective VEGFR-2 inhibition, we performed morphological and molecular analyses, with special reference to pericytes since their contact to endothelial cells may stabilize new blood vessels and promote endothelial survival. Eighty percent of established tumor blood vessels were associated with pericytes; as a result of selective VEGFR-2 targeting, additional pericytes were recruited to tumor blood vessels and provided an intimate vascular coverage. In parallel, selective VEGFR-2 inhibition resulted in pericytic up-regulation of the endothelial cell survival factor angiopoietin-1 (Ang-1), suggesting that pericytes not only stabilized tumor blood vessels by cell-to-cell contact but also protected them by providing endothelial survival signals.

3. Targeting of endothelial cells and pericytes induces tumor blood vessel regression and hypoxia
To test whether simultaneous targeting of endothelial cells and pericytes would be more effective in enforcing tumor blood vessel regression, we treated tumors with SU6668, an inhibitor that targets not only VEGFR-2 (like SU5416), but also platelet-derived growth factor receptor-ß (PDGFR-ß) expressed by pericytes. Under these conditions, tumor blood vessels rapidly disintegrated and regressed (Fig. 1 ). To further address the microcirculatory consequences of this blood vessel regression, we studied tumor oxygenation by the phosphorescence quenching technique, which revealed an increase in tumor hypoxia (P<0.05), reflecting a substantial perfusion and nutritional supply deficit.

View larger version (86K): [in this window] [in a new window]   Figure 1. Targeting of VEGFR-2 plus PDGFR-ß induces enforced regression of tumor blood vessels. A–C) Overview of rapid regression of C6 tumor blood vessels within 24 h after initiation of SU6668 treatment. Bars = 100 µm. D) Vascular sprouts were most susceptible to SU6668 treatment, disrupting within 6 h after initiation of SU6668 treatment. Arrow indicates small hemorrhage from disrupted sprout. Bar indicates 50 µm. E) Destabilized tumor blood vessel 18 h after initiation of SU6668 treatment. Note extravasation of high molecular weight fluorescent marker and hemorrhage (indicated by arrow). Bar indicates 50 µm. F, G\) Host blood vessels within the adjacent skin muscle (F) and subcutaneous tissue (G) remained largely unaffected by 18 h of SU6668 treatment, revealing tumor specificity of vessel regression. F) Arrows indicate perfused and still functional skin muscle capillaries. G) A capillary and postcapillary venule within the subcutaneous tissue. Bar indicates 50 µm. H) Quantitative analysis of vessel density, vessel diameter, and tumor size. Animals were treated with DMSO (n=4; 50 µL DMSO i.p.) or SU6668 (n=5 animals; 75 mg·kg-1·day-1 in 50 µL DMSO i.p.). All parameters were analyzed off-line using a computer-assisted image analysis system. The mean ± SD values are represented. Statistical analysis was performed using ANOVA, followed by unpaired Student’s t test. *P < 0.05.

4. Regression of tumor blood vessels involves endothelial cell apoptosis and detachment
To study the mechanisms underlying enforced regression of tumor blood vessels, we performed TUNEL stainings and electron microscopy 24 h after treatment. SU6668-treated tumor blood vessels were characterized by endothelial cell apoptosis and detachment from the vessel wall into the vascular lumen. To confirm these results in vivo, SU6668-treated animals received the active caspase inhibitor ZVAD-fmk, which abolished endothelial cell apoptosis and tumor blood vessel regression.

5. Inhibition of VEGFR plus PDGFR-ß interferes with pericyte-mediated endothelial cell survival mechanisms
To understand the distinct effects of selective VEGFR-2 inhibition vs. VEGFR-2 plus PDGFR-ß inhibition, we next studied pericyte–endothelial cell interaction in SU6668-treated tumors. The amount of blood vessels that colocalized with pericytes and the extent of pericytic Ang-1 were comparable among SU5416-treated and SU6668-treated tumors. However, electron microscopy demonstrated that although pericytes were still present, their association with endothelial cells in SU6668-treated tumors was less intimate than in the SU5416 group, showing that inhibition of VEGFR-2 plus PDGFR-ß negatively influences the pericyte–endothelial cell interaction, interfering with pericyte-mediated endothelial cell survival mechanisms.

CONCLUSIONS

Our findings demonstrate that selective inhibition of VEGF signaling fails to enforce regression of tumor blood vessels. This was unexpected because VEGF not only mediates endothelial cell proliferation, but also endothelial cell survival, and VEGF withdrawal has been shown to ablate immature tumor vessels (i.e., vessels lacking pericyte association). The resistance to vessel regression in our study may be explained in part by the high maturation index of C6 tumor blood vessels (80%). However, our study has revealed at least two other mechanisms of how pericytes may confer resistance to targeted tumor blood vessels in vivo (Fig. 2 ). First, pericytes may be recruited to immature vessels and form an intimate association with endothelial cells, thereby stabilizing these vessels through an enhanced cell-to-cell contact. Second, our analysis has suggested that the proapoptotic effect of selective VEGFR-2 inhibition may be overwhelmed by the increased activity of endothelial cell survival factors other than VEGF, such as Ang-1. These findings are striking since it had recently been suggested that endothelial cells, in contrast to tumor cells, are resistant to the development of an acquired resistance. This resistance may be overcome by inhibition of VEGFR-2 plus PDGFR-ß, which simultaneously targets endothelial cells and pericytes and acts as a potent anti-vascular strategy, inducing tumor endothelial cell apoptosis, tumor vessel regression, and tumor hypoxia (Fig. 2) . Due to this central role of pericytes in tumor angiogenesis and blood vessel maturation, an extended anti-angiogenic strategy (i.e., targeting of endothelial cells and pericytes) represents an attractive approach in treating patients that present with tumors at an advanced stage. Future studies will have to address whether these pericytic resistance mechanisms to VEGFR-2 targeting can be generalized to other tumor entities. Experimental studies will also have to address the cellular and molecular mechanism underlying pericyte/endothelial cell interaction, e.g., how Ang-1/Tie2 and PDGF-B/PDGFR-ß interact in mediating blood vessel maturation.

View larger version (31K): [in this window] [in a new window]   Figure 2. Summary of the consequences of selective VEGFR targeting and VEGFR plus PDGFR-ß targeting for established tumor blood vessels. After selective VEGFR targeting, pericytes were recruited and conferred resistance to vessel regression by an intimate vascular coverage and expression of the endothelial survival factor Ang-1. In contrast, VEGFR plus PDGFR-ß targeting, which simultaneously targets endothelial cells and pericytes, acts as a potent anti-vascular strategy, inducing endothelial cell apoptosis and tumor blood vessel regression.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0271fje

This article has been cited by other articles:

				M. E. Lacouture, L. M. Reilly, P. Gerami, and J. Guitart Hand foot skin reaction in cancer patients treated with the multikinase inhibitors sorafenib and sunitinib Ann. Onc., June 10, 2008; (2008) mdn389v1. [Abstract] [Full Text] [PDF]

				K. Reddy, Z. Zhou, K. Schadler, S.-F. Jia, and E. S. Kleinerman Bone Marrow Subsets Differentiate into Endothelial Cells and Pericytes Contributing to Ewing's Tumor Vessels Mol. Cancer Res., June 1, 2008; 6(6): 929 - 936. [Abstract] [Full Text] [PDF]

				S. Lamy, E. Beaulieu, D. Labbe, V. Bedard, A. Moghrabi, S. Barrette, D. Gingras, and R. Beliveau Delphinidin, a dietary anthocyanidin, inhibits platelet-derived growth factor ligand/receptor (PDGF/PDGFR) signaling Carcinogenesis, May 1, 2008; 29(5): 1033 - 1041. [Abstract] [Full Text] [PDF]

				C. Timke, H. Zieher, A. Roth, K. Hauser, K. E. Lipson, K. J. Weber, J. Debus, A. Abdollahi, and P. E. Huber Combination of Vascular Endothelial Growth Factor Receptor/Platelet-Derived Growth Factor Receptor Inhibition Markedly Improves Radiation Tumor Therapy Clin. Cancer Res., April 1, 2008; 14(7): 2210 - 2219. [Abstract] [Full Text] [PDF]

				A. Kadenhe-Chiweshe, J. Papa, K. W. McCrudden, J. Frischer, J.-O Bae, J. Huang, J. Fisher, J. H. Lefkowitch, N. Feirt, J. Rudge, et al. Sustained VEGF Blockade Results in Microenvironmental Sequestration of VEGF by Tumors and Persistent VEGF Receptor-2 Activation Mol. Cancer Res., January 1, 2008; 6(1): 1 - 9. [Abstract] [Full Text] [PDF]

				L. B. Saltz, L. S. Rosen, J. L. Marshall, R. J. Belt, H. I. Hurwitz, S. G. Eckhardt, E. K. Bergsland, D. G. Haller, A. C. Lockhart, C. M. Rocha Lima, et al. Phase II Trial of Sunitinib in Patients With Metastatic Colorectal Cancer After Failure of Standard Therapy J. Clin. Oncol., October 20, 2007; 25(30): 4793 - 4799. [Abstract] [Full Text] [PDF]

				S. de Bouard, P. Herlin, J. G. Christensen, E. Lemoisson, P. Gauduchon, E. Raymond, and J.-S. Guillamo Antiangiogenic and anti-invasive effects of sunitinib on experimental human glioblastoma Neuro-oncol, October 1, 2007; 9(4): 412 - 423. [Abstract] [Full Text] [PDF]

				C. Oehler-Janne, W. Jochum, O. Riesterer, A. Broggini-Tenzer, G. Caravatti, V. Vuong, and M. Pruschy Hypoxia modulation and radiosensitization by the novel dual EGFR and VEGFR inhibitor AEE788 in spontaneous and related allograft tumor models Mol. Cancer Ther., September 1, 2007; 6(9): 2496 - 2504. [Abstract] [Full Text] [PDF]

				B. Sennino, B. L. Falcon, D. McCauley, T. Le, T. McCauley, J. C. Kurz, A. Haskell, D. M. Epstein, and D. M. McDonald Sequential Loss of Tumor Vessel Pericytes and Endothelial Cells after Inhibition of Platelet-Derived Growth Factor B by Selective Aptamer AX102 Cancer Res., August 1, 2007; 67(15): 7358 - 7367. [Abstract] [Full Text] [PDF]

				M.-E. Jockovich, M. L. Bajenaru, Y. Pina, F. Suarez, W. Feuer, M. E. Fini, and T. G. Murray Retinoblastoma Tumor Vessel Maturation Impacts Efficacy of Vessel Targeting in the LHBETATAG Mouse Model Invest. Ophthalmol. Vis. Sci., June 1, 2007; 48(6): 2476 - 2482. [Abstract] [Full Text] [PDF]

				E. Zetterberg, A. M. Vannucchi, A. R. Migliaccio, W. Vainchenker, M. Tulliez, R. Dickie, H. Hasselbalch, R. Rogers, and J. Palmblad Pericyte coverage of abnormal blood vessels in myelofibrotic bone marrows Haematologica, May 1, 2007; 92(5): 597 - 604. [Abstract] [Full Text] [PDF]

				K.-Y. Tsai, C.-H. Yang, T.-t. Kuo, H.-S. Hong, and J. W.C. Chang Hand-Foot Syndrome and Seborrheic Dermatitis-Like Rash Induced by Sunitinib in a Patient With Advanced Renal Cell Carcinoma J. Clin. Oncol., December 20, 2006; 24(36): 5786 - 5788. [Full Text] [PDF]

				Y. Kitadai, T. Sasaki, T. Kuwai, T. Nakamura, C. D. Bucana, and I. J. Fidler Targeting the Expression of Platelet-Derived Growth Factor Receptor by Reactive Stroma Inhibits Growth and Metastasis of Human Colon Carcinoma Am. J. Pathol., December 1, 2006; 169(6): 2054 - 2065. [Abstract] [Full Text] [PDF]

				A. P. Hall Review of the Pericyte during Angiogenesis and its Role in Cancer and Diabetic Retinopathy Toxicol Pathol, October 1, 2006; 34(6): 763 - 775. [PDF]

				M. F. McCarty and K. I. Block Preadministration of High-Dose Salicylates, Suppressors of NF-{kappa}B Activation, May Increase the Chemosensitivity of Many Cancers: An Example of Proapoptotic Signal Modulation Therapy. Integr Cancer Ther, September 1, 2006; 5(3): 252 - 268. [Abstract] [PDF]

				L. Zhang, J. A.F. Hannay, J. Liu, P. Das, M. Zhan, T. Nguyen, D. J. Hicklin, D. Yu, R. E. Pollock, and D. Lev Vascular Endothelial Growth Factor Overexpression by Soft Tissue Sarcoma Cells: Implications for Tumor Growth, Metastasis, and Chemoresistance. Cancer Res., September 1, 2006; 66(17): 8770 - 8778. [Abstract] [Full Text] [PDF]

				M. F. McCarty and K. I. Block Toward a core nutraceutical program for cancer management. Integr Cancer Ther, June 1, 2006; 5(2): 150 - 171. [Abstract] [PDF]

				N. Jo, C. Mailhos, M. Ju, E. Cheung, J. Bradley, K. Nishijima, G. S. Robinson, A. P. Adamis, and D. T. Shima Inhibition of Platelet-Derived Growth Factor B Signaling Enhances the Efficacy of Anti-Vascular Endothelial Growth Factor Therapy in Multiple Models of Ocular Neovascularization Am. J. Pathol., June 1, 2006; 168(6): 2036 - 2053. [Abstract] [Full Text] [PDF]

				A. Lee, J. Frischer, A. Serur, J. Huang, J.-O Bae, Z. N. Kornfield, L. Eljuga, C. J. Shawber, N. Feirt, M. Mansukhani, et al. Inhibition of cyclooxygenase-2 disrupts tumor vascular mural cell recruitment and survival signaling. Cancer Res., April 15, 2006; 66(8): 4378 - 4384. [Abstract] [Full Text] [PDF]

				E. Naumova, P. Ubezio, A. Garofalo, P. Borsotti, L. Cassis, E. Riccardi, E. Scanziani, S. A. Eccles, M. R. Bani, and R. Giavazzi The Vascular Targeting Property of Paclitaxel Is Enhanced by SU6668, a Receptor Tyrosine Kinase Inhibitor, Causing Apoptosis of Endothelial Cells and Inhibition of Angiogenesis. Clin. Cancer Res., March 15, 2006; 12(6): 1839 - 1849. [Abstract] [Full Text] [PDF]

				V. J. Yao, M. G. Ozawa, A. S. Varner, I. M. Kasman, Y. H. Chanthery, R. Pasqualini, W. Arap, and D. M. McDonald Antiangiogenic therapy decreases integrin expression in normalized tumor blood vessels. Cancer Res., March 1, 2006; 66(5): 2639 - 2649. [Abstract] [Full Text] [PDF]

				A. M. Jubb, H. I. Hurwitz, W. Bai, E. B. Holmgren, P. Tobin, A. S. Guerrero, F. Kabbinavar, S. N. Holden, W. F. Novotny, G. D. Frantz, et al. Impact of Vascular Endothelial Growth Factor-A Expression, Thrombospondin-2 Expression, and Microvessel Density on the Treatment Effect of Bevacizumab in Metastatic Colorectal Cancer J. Clin. Oncol., January 10, 2006; 24(2): 217 - 227. [Abstract] [Full Text] [PDF]

				K. Yokoi, T. Sasaki, C. D. Bucana, D. Fan, C. H. Baker, Y. Kitadai, T. Kuwai, J. L. Abbruzzese, and I. J. Fidler Simultaneous Inhibition of EGFR, VEGFR, and Platelet-Derived Growth Factor Receptor Signaling Combined with Gemcitabine Produces Therapy of Human Pancreatic Carcinoma and Prolongs Survival in an Orthotopic Nude Mouse Model Cancer Res., November 15, 2005; 65(22): 10371 - 10380. [Abstract] [Full Text] [PDF]

				R. G. Bagley, W. Weber, C. Rouleau, and B. A. Teicher Pericytes and Endothelial Precursor Cells: Cellular Interactions and Contributions to Malignancy Cancer Res., November 1, 2005; 65(21): 9741 - 9750. [Abstract] [Full Text] [PDF]

				J. Baranowska-Kortylewicz, M. Abe, K. Pietras, Z. P. Kortylewicz, T. Kurizaki, J. Nearman, J. Paulsson, R. L. Mosley, C. A. Enke, and A. Ostman Effect of Platelet-Derived Growth Factor Receptor-{beta} Inhibition with STI571 on Radioimmunotherapy Cancer Res., September 1, 2005; 65(17): 7824 - 7831. [Abstract] [Full Text] [PDF]

				S. Jodele, C. F. Chantrain, L. Blavier, C. Lutzko, G. M. Crooks, H. Shimada, L. M. Coussens, and Y. A. DeClerck The Contribution of Bone Marrow-Derived Cells to the Tumor Vasculature in Neuroblastoma Is Matrix Metalloproteinase-9 Dependent Cancer Res., April 15, 2005; 65(8): 3200 - 3208. [Abstract] [Full Text] [PDF]

				K. Pietras and D. Hanahan A Multitargeted, Metronomic, and Maximum-Tolerated Dose "Chemo-Switch" Regimen is Antiangiogenic, Producing Objective Responses and Survival Benefit in a Mouse Model of Cancer J. Clin. Oncol., February 10, 2005; 23(5): 939 - 952. [Abstract] [Full Text] [PDF]

				V. J. Yao, M. G. Ozawa, M. Trepel, W. Arap, D. M. McDonald, and R. Pasqualini Targeting Pancreatic Islets with Phage Display Assisted by Laser Pressure Catapult Microdissection Am. J. Pathol., February 1, 2005; 166(2): 625 - 636. [Abstract] [Full Text] [PDF]

				M. Zareie, E. D. Keuning, P. M. ter Wee, R. H. J. Beelen, and J. van den Born Peritoneal dialysis fluid-induced changes of the peritoneal membrane are reversible after peritoneal rest in rats Nephrol. Dial. Transplant., January 1, 2005; 20(1): 189 - 193. [Abstract] [Full Text] [PDF]

				M. Furuhashi, T. Sjoblom, A. Abramsson, J. Ellingsen, P. Micke, H. Li, E. Bergsten-Folestad, U. Eriksson, R. Heuchel, C. Betsholtz, et al. Platelet-Derived Growth Factor Production by B16 Melanoma Cells Leads to Increased Pericyte Abundance in Tumors and an Associated Increase in Tumor Growth Rate Cancer Res., April 15, 2004; 64(8): 2725 - 2733. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 18/2/338 03-0271fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ERBER, R. Articles by VAJKOCZY, P. Search for Related Content PubMed PubMed Citation Articles by ERBER, R. Articles by VAJKOCZY, P.