Ann. N.Y. Acad. Sci. ISSN 0077-8923 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Issue: Targeting VEGF-Mediated Tumor Angiogenesis in Cancer Therapy Angiopoietin-2: a multifaceted cytokine that functions in both angiogenesis and inflammation Alexander Scholz,1 Karl H. Plate,2,3,4,5 and Yvonne Reiss2,3,4,5 1 Department of Pathology, Laboratory of Immunology and Vascular Biology, Stanford University School of Medicine, Stanford, California. 2 Edinger Institute/Institute of Neurology, Frankfurt University Medical School, Frankfurt, Germany. 3 German Center for Cardiovascular Research (DZHK), Frankfurt, Germany. 4 German Cancer Consortium (DKTK), Frankfurt, Germany. 5 German Cancer Research Center (DKFZ), Heidelberg, Germany Address for correspondence: Alexander Scholz, Ph.D., Department of Pathology, Laboratory of Immunology and Vascular Biology, Stanford University School of Medicine, Stanford, California 94304. alexander.scholz@stanford.edu Angiogenesis and inflammation are two highly linked processes. In the last decade, several factors with dual function in both of these major pathways have been identified. This review focuses on angiopoietin-2 (Ang-2), an important proangiogenic factor that has more recently been implicated in mediating inflammatory processes as well. Ang-2 is upregulated in multiple inflammatory diseases and has been implicated in the direct control of inflammation-related signaling pathways. As a consequence of its multiple roles, designs for therapeutic targeting of Ang-2 should consider the dual function of this factor in regulating angiogenesis and inflammation. Keywords: angiopoietin; Ang-2; inflammation; myeloid cell; innate immune cell Introduction The angiopoietins (Ang) are a family of secreted factors comprising Ang-1,1 Ang-2,2 and Ang-3 (Ang-4 in humans).3 While Ang-1 has been widely accepted as an agonist for the Tyr kinase with Ig and epidermal growth factor homology domains (Tie2), Ang-2 has been described as a context-dependent antagonist interfering with Ang-1–induced Tie2 phosphorylation.4 Interestingly, both ligands bind with similar kinetics to the receptor’s second Ig domain.5,6 Genetic mouse models demonstrated that the phenotypes of Ang-1-7 and Tie2-deficient mice8,9 and mice with endothelial-specific overexpression of Ang-22 are highly similar. Thus, the concept arose that Ang-1–induced paracrine Tie2 phosphorylation is an important stimulus for vascular integrity and homeostasis and is constantly active in healthy adult vasculature.1,10 In contrast, Ang-2 expression is tightly regulated, and its expression is mostly restricted to the vascular endothelium.11–14 Upon secretion from Weibel–Palade bodies,15 Ang- 2 acts on Tie2 in an autocrine manner and competes with Ang-1 for receptor binding, leading to disturbances in endothelial junctional integrity,16 pericyte drop-off,2,17 and an overall priming of the vascular bed for angiogenic sprouting. The latter is highly dependent on the local cytokine milieu. In conjunction with vascular endothelial growth factor (VEGF), Ang-2 has been reported to induce vascular sprouting, whereas in the absence of VEGF, Ang-2 induces endothelial cell (EC) apoptosis.18 Because of its proangiogenic properties, Ang-2 has become an interesting target in antiangiogenic tumor therapy, with several inhibitors in current clinical trials.19 The role of Ang-2 in the induction of angiogenic sprouting has been reviewed elsewhere.20–22 This review will therefore focus on the proinflammatory effects of Ang-2 and the potential therapeutic implications arising from those. Ang-2 at the interface between leukocytes and the endothelium The link between the angiopoietins and inflammation was hypothesized soon after their discovery. At first, the anti-inflammatory properties of Ang-1 were assessed and mainly attributed to their effect on vascular integrity and the reduction in plasma leakage.23–25 Later, the molecular mechanisms doi: 10.1111/nyas.12726 C 2015 New York Academy of Sciences. Ann. N.Y. Acad. Sci. xxxx (2015) 1–7  1 Angiopoietin-2 and inflammation Scholz et al. Figure 1. Comparison of autocrine and paracrine Ang-2 signaling during inflammatory responses. Angiogenic and inflammatory stimuli trigger the release of Ang-2 from endothelial Weibel–Palade bodies. Ang-2 can act either in an autocrine manner on endothelial cells or in a paracrine manner on leukocytes, in particular cells of the myeloid lineage. While Ang-2–induced autocrine signaling effects are well understood and entail the induction of adhesion molecules and vascular leakage, paracrine Ang-2 signaling is still enigmatic. Paracrine Ang-2 signaling has been described in monocytes/macrophages and neutrophils and is either Tie2 dependent (blue receptor) or the responsible receptor has not yet been identified (black receptor). Upon stimulation, innate immune cells can respond with increased adhesion to the vessel wall, triggered in a ␤2-integrin–dependent manner. Furthermore, upregulation of IL-10, downregulation of TNF-␣ and IL-6, and induction of CD206 expression has been described in monocytes/macrophages, indicative of an M2-polarized phenotype. In addition, IL-8 induction in neutrophils has been linked to Ang-2 stimulation. underlying the stabilizing functions of Ang-1 were further investigated. Besides an increase in pericyte coverage, Ang-1 additionally tightens the EC junctions by sequestering small kinases to prevent VEGF-induced VE-cadherin phosphorylation.26 The role of Ang-2 in inflammation was long projected on the basis of the assumption that it counteracts the anti-inflammatory effects of Ang-124,27 and on the finding that it is upregulated in several inflammatory diseases.28–37 The first mechanistic evidence arose in 2006, when it was shown that Ang-2 sensitized Tie2+ ECs toward inflammatory stimuli such as tumor necrosis factor ␣ (TNF-␣).38 Following this sensitization, ECs strongly upregulated adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) or vascular cell adhesion molecule 1 (VCAM-1), thus inducing leukocyte adhesion (Fig. 1). In fact, using intravital fluorescence videomicroscopy, the authors showed that, in 2 mice lacking Ang-2 (Ang2 knockout (KO)), leukocytes failed to arrest. Consequently, the number of infiltrating cells in thioglycollate-induced peritonitis was strongly reduced in Ang2 KO mice, but could be rescued by administration of recombinant Ang-2.38 Supporting the autocrine proinflammatory effects of Ang-2, it has been shown that Ang-2 is required for the transformation of capillaries into venules, which consequently mediates leukocyte trafficking in a mouse airway inflammation model.39 Beyond multiple studies that focused on the autocrine effects of endothelial secreted Ang-2 on the EC-expressed Tie2 receptor, recent evidence in the literature suggests an additional direct paracrine action of Ang-2 on immune cells (Fig. 1). It has been shown that Ang-2 acts as a chemoattractant, mediating the in vitro migration of a specific subset of human monocytes that is characterized by the expression of the Tie2 receptor (Tie2 expressing monocytes (TEMs)).40,41 Blockade of Tie2 using a neutralizing antibody was able to abolish Ang-2– mediated migration.40 Another group has described the expression of Tie2 on human neutrophils. Tie2expressing neutrophils show increased adhesion upon Ang-2 stimulation42 and, similar to monocytes, directed migration toward Ang-2.43 Interestingly, in these studies, Ang-1 and Ang-2 triggered additive effects.42,43 Further evidence for a paracrine function of Ang-2 is based on the observation that human neutrophils react to Ang-2 stimulation with the upregulation of IL-8RB and IL-10RB, whereas Ang-1 stimulation induced IL-1␤ and IL1-Ra.44 The induction of specific cytokines by angiopoietins has also been shown in IL-10–polarized macrophages,45 indicative of a more general effect of angiopoietins on innate immune cells. In line with these observations, Ang-2 triggered the infiltration of neutrophils and monocytes into a murine sponge/matrigel model.46 In addition, we were able to demonstrate that Ang-2 generally attracts innate immune cells in a paracrine, integrin-dependent manner.47 Mice with inducible, EC-specific overexpression of Ang-2 showed massive influx of myeloid cells into multiple organs in a time-dependent manner. Using an in vitro flow-chamber model, we demonstrated that Ang-2 is very potent in inducing human monocyte adhesion on ICAM-1 and VCAM-1, while it had no effect on lymphocyte adhesion. Furthermore, Ang-2 was specifically upregulated in the vasculature C 2015 New York Academy of Sciences. Ann. N.Y. Acad. Sci. xxxx (2015) 1–7  Scholz et al. Angiopoietin-2 and inflammation of human myeloid cell–dominated inflammation, whereas it is absent in lymphoid-dominated inflammatory diseases.47 This specificity in recruiting cells of the myeloid lineage is of particular interest in targeted tumor therapy, as discussed next. Ang-2 in cancer inflammation The role of cancer-related inflammation, especially the infiltration of innate immune cells, has been extensively reviewed.48–51 In most solid cancers, larger numbers of infiltrating tumor-associated macrophages (TAMs) correlate with worse patient survival. This could mainly be attributed to the alternative polarization of TAMs, rendering them proangiogenic and immunosuppressive. However, the molecular mechanisms underlying the recruitment and polarization of innate immune cells in solid tumors are poorly understood. Ang-2 is highly upregulated in the vasculature of mouse and human neoplasms and, given its effects on EC activation and direct myeloid cell recruitment, it represents an interesting candidate to mediate tumor immune cell homing. We showed in a subcutaneous Lewis lung carcinoma model that tumors grown in mice with EC-specific Ang-2 overexpression show increased infiltration with innate immune cells, in particular TEMs.52 Circulating TEMs are already innately proangiogenic; however, stimulation with Ang-2 could further increase their expression of angiogenic and immunosuppressive genes.52 This is of particular interest, as the recruitment of proangiogenic myeloid cells has been discussed as a potential mechanism for resistance to antiangiogenic therapy.53 More recently, Mazzieri et al. showed that blockade of Ang-2 in two different spontaneous mouse tumor models did not impair the recruitment but rather the close association of TEM with the tumor vasculature.54 Blocking the EC–TEM interaction impaired the restoration of tumor neovascularization and thus enhanced antiangiogenic therapy. In another study, the inhibition of Ang-2 reduced the amount of infiltrating TEM in a Colo-205 xenograft model.55 Interestingly, in some tumors, Ang-2 was upregulated and counteracted antiangiogenic therapy targeting VEGF. Double inhibition of both VEGF and Ang-2 thus greatly enhanced the efficacy of antiangiogenic therapy in those tumors.56 This type of compensatory mechanism has been described for Ang-2 as well. In a mouse model of liver metastasis, the lack of Ang-2 strongly Figure 2. Contribution of autocrine and paracrine Ang-2 signaling in different inflammatory diseases. On the basis of the current literature, the contribution of direct effects of Ang-2 on leukocytes (paracrine) and on endothelial cells (autocrine) in different inflammation-related diseases is shown. Activating or inhibiting arrows indicate how Ang-2 affects the course of the particular disease, whereas their position in relation to autocrine or paracrine signaling indicates the expected contribution of these pathways to the inflammatory response. For example, Ang-2 has been described to worsen disease outcome in sepsis, and this has been attributed to direct, destabilizing effects on the endothelium. In contrast, cancer-related inflammation equally entails endothelial stimulation and direct effects on tumor-infiltrating innate immune cells. IBD, inflammatory bowel disease. upregulated granulocyte colony-stimulating factor, leading to the recruitment of CD11b+ Tie2+ immune cells, which restored neoangiogenesis and tumor growth.57 Nevertheless, Ang-2 is a promising candidate for combined anticancer therapy, as it targets two major protumorigenic processes, angiogenesis and inflammation (Fig. 2). Ang-2 in autoimmune diseases In addition to neoplastic diseases, Ang-2 expression is also upregulated in human autoimmune diseases, such as psoriasis32 and arthritis.33–35,58 Both types of disease are characterized by extensive angiogenesis and inflammatory cell recruitment. In this context, Ang-2 expression has been shown to correlate with arthritis severity in patients.58 More recently, the functional role of Ang-2 in arthritis has been addressed using a fully humanized blocking antibody directed against Ang-2.59 The authors showed that anti-Ang-2 therapy significantly improved the clinical score of animals with psoriatic and rheumatoid arthritis. The rationale for the use of Ang-2 blockade was based on the observation that Ang-2 and TNF-␣ act cooperatively on synovial macrophages to induce a proinflammatory phenotype.58 In line with these C 2015 New York Academy of Sciences. Ann. N.Y. Acad. Sci. xxxx (2015) 1–7  3 Angiopoietin-2 and inflammation Scholz et al. findings, dual inhibition of TNF-␣ and Ang-2 by a bispecific antibody was superior to targeting TNF-␣ alone.60 Interestingly, sequestering Ang-2 in combination with VEGF using double antiangiogenic protein in collagen-induced arthritis reduced the disease severity and also decreased F4/80+ macrophage infiltrate, indicative of similar but distinct roles of these angiogenic factors in arthritis.61 Ang-2 in inflammatory bowel disease Inflammatory bowel disease (IBD) encompasses ulcerative colitis and Crohn’s disease (CD) and is characterized by uncontrolled inflammation of the intestinal mucosa. Excessive recruitment of neutrophils to the gut wall and inadequate control of their proinflammatory activation are thought to be the critical underlying mechanisms in IBD. However, the role of the Ang/Tie system during IBD is only poorly understood. On the one hand, studies show that patient serum levels of Ang-2 are elevated during IBD, while Ang-1 levels are decreased.28,29 Conversely, during colonrestricted CD, Ang-2 levels were significantly lower in patients as compared to healthy controls,62 indicative of distinct roles of Ang-2 at different IBD sites. Interestingly, in a mouse model of dextran sodium sulfate (DSS)-induced colitis, genetic ablation of Angpt2 decreased the influx of inflammatory cells to the gut wall but failed to improve clinical symptoms. On the contrary, the clinical outcome in Angpt2 KO mice in experimental colitis was worse compared to wild-type littermates.63 Ang-2 in sepsis Sepsis is a systemic inflammatory response to infection. The high mortality in septic patients is owed to multiorgan dysfunction caused by a dramatic drop in blood pressure. ECs are supposed to be the main mediators of septic shock, reacting to a massive cytokine storm with the loss of vascular integrity and increased plasma leakage. A number of studies have described Ang-2 to be highly upregulated in the serum of septic patients, and its expression strongly correlates with severity of the disease.30,31,64 It has been shown that intravenously administered lipopolysaccharide (LPS) strongly upregulated Ang-2 expression in several organs, which was accompanied by reduced Tie2 phosphorylation.65 Currently, evidence in the literature suggests that 4 the vessel-destabilizing functions of Ang-2 are the main contributors to the septic phenotype (Fig. 2). Ziegler et al. showed that mice with endothelialspecific, but not cardiomyocyte-specific, Ang-2 overexpression develop sepsis-like hemodynamic alterations.66 These effects were antagonized by injecting mice with an Angpt1- or Pdgfb-encoding adeno-associated virus, suggesting a strong involvement of Ang-2–mediated pericyte detachment during septic shock. Furthermore, targeting Ang-2 with a neutralizing antibody decreased mortality in LPS-induced endotoxic shock.66 Another study used the synthetic Tie2-activating ligand vasculotide in a cecal ligation–puncture model and demonstrated a significant enhancement of survival.67 Similar results were obtained when injecting Mat-Ang-1, a stable Ang-1 variant, into mice with LPS-induced septic shock.68 These studies indicate that the tight balance between vascular-destabilizing Ang-2 and stabilizing Ang-1 could predict the outcome during septic shock. This is supported by the fact that even the loss of one Ang2 allele improved survival in different sepsis models.64 This finding was accompanied by reduced VCAM-1 expression in Ang2+/– mice and induced inflammatory cell infiltration.64 Although the vast majority of studies link Ang-2 to a worse disease outcome, Tzepi et al. showed that injection of recombinant Ang-2 was protective against septic shock induced in mice by inactivated Pseudomonas aeruginosa or Escherichia coli. In contrast, Ang-2 was not able to protect against LPSinduced septic shock, indicating crucial differences in the septic models that have been used.69 Whether the detrimental role of Ang-2 during septic shock is primarily attributable to the destabilization of the endothelium or additionally dependent on the recruitment of proinflammatory immune cells remains to be investigated. Concluding remarks In the past decade, numerous studies convincingly demonstrated a link between Ang-2 signaling and the recruitment of innate immune cells, both in a Tie2-dependent and Tie2-independent manner (Fig. 1). Thus, Ang-2 has been widely termed a proinflammatory molecule. Yet, it is questionable whether Ang-2 is indeed proinflammatory or might even operate as an anti-inflammatory factor. For example, we observed massive recruitment of C 2015 New York Academy of Sciences. Ann. N.Y. Acad. Sci. xxxx (2015) 1–7  Scholz et al. myeloid cells into organs of Ang-2–overexpressing mice without signs of active tissue damage.47 This is supported by the finding that during DSSinduced colitis, Ang-2 deficiency impaired neutrophil recruitment but did not improve disease progression.63 In addition, monocytes recruited by Ang-2 into tumors are also highly immunosuppressive, a characteristic further enhanced by Ang-2 stimulation.52 Furthermore, Ang-2–stimulated TAMs are strong producers of the anti-inflammatory cytokine IL-10 and promote the expansion of FoxP3+ regulatory T cells, a lymphoid subset that is highly immunosuppressive and known to correlate with a worse prognosis.70 In a more recent study, Ang-2 induced a M2-like phenotype in monocytes while Ang-1 induced a proinflammatory signaling cascade in monocytes via p38 and Erk1/2, leading to increased TNF-␣ release71 (Fig. 1). However, whether this concept holds true in vivo needs to be investigated. Accordingly, Ang-2 deficiency does not protect against LPS-induced acute kidney injury, but leads to even higher TNF-␣ and IL-6 levels.72 Thus, it remains to be determined whether Ang-2 triggers classic inflammatory responses or rather attracts myeloid cells without additional obvious signs of inflammation. Resolution of this question will have strong implications for the use of Ang-2 as a therapeutic target: depending on the type of disease, it has to be taken into consideration if the antiinflammatory (i.e., during tumor therapy) or proinflammatory (i.e., during IBD) actions of myeloid cells are the objective of therapeutic targeting. Acknowledgments We would like to thank Eugene C. Butcher for insightful contributions and Cathrin J. Czupalla and Rebecca Kunder for proofreading and improvements of the manuscript. This work was supported by the Collaborative Research Center “Vascular differentiation and remodeling” (CRC/Transregio23, project C1) to Y.R. and K.H.P., a start-up grant from the Cluster of Excellence 147 “Cardiopulmonary system” (ECCPS), and a postdoctoral research fellowship (SCHO 1555/1-1) to A.S. by the German Research Council (DFG). Conflicts of interest The authors declare no conflicts of interest. Angiopoietin-2 and inflammation References 1. Davis, S. et al. 1996. Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell 87: 1161–1169. 2. Maisonpierre, P.C. 1997. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277: 55–60. 3. Valenzuela, D.M. et al. 1999. Angiopoietins 3 and 4: diverging gene counterparts in mice and humans. Proc. Natl. Acad. Sci. USA 96: 1904–1909. 4. Reiss, Y. et al. 2007. Angiopoietin-2 impairs revascularization after limb ischemia. Circ. Res. 101: 88–96. 5. 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