Angiogenic and antiangiogenic chemokines.

Marone G, Granata F (eds): Angiogenesis, Lymphangiogenesis and Clinical Implications. Chem Immunol Allergy. Basel, Karger, 2014, vol 99, pp 89–104 (DOI: 10.1159/000353317)

Angiogenic and Antiangiogenic Chemokines Daniela Bosisio a ⋅ Valentina Salvi a ⋅ Vincenzo Gagliostro a ⋅ Silvano Sozzani a, b

a

Department of Molecular and Translational Medicine, University of Brescia, Brescia, and Clinical and Research Center, Rozzano, Italy

b Humanitas

Abstract Chemokines are a family of vertebrate-specific, small-secreted molecules that were originally identified as mediators of leukocyte migration and tissue positioning during the immune response. Subsequently, chemokines were discovered to control movement also of endothelial cells and other cell types in many different contexts. The human chemokine system comprises about 50 chemokines and more than 20 receptors belonging to the seven-transmembrane receptor family. In the present chapter, we review the literature supporting a role for chemokines in angiogenesis and lymphangiogenesis. We highlight that chemokines exert both pro- and antiangiogenic roles either by acting directly on endothelial cells or by recruiting leukocytes that, in turn, secrete angiogenic mediators. This latter mode of action is possibly the most relevant in tumor angiogenesis. Finally, we explore the angiogenic properties of nonchemokine chemoattractant molecules. Copyright © 2014 S. Karger AG, Basel

Chemokines (contraction for ‘chemotactic cytokines’) are a family of functionally related small-secreted molecules, named after their leukocyte chemoattractant and cytokine-like activities. Their hallmark function is directing receptor-bearing cells to specific locations in the body, along either soluble or matrix-bound concentration gradients. Despite originally identified as mediators of leukocyte migration and tissue positioning during the immune response, chemokines were soon discovered to control movement also of endothelial cells and other cell types. The human chemokine system comprises about 50 chemokines and more than 20 receptors (fig. 1). A typical chemokine molecule contains four cysteine residues re-

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Chemokine System

ELR+

NH2

C

COOH CXCL6 CXCL8

CXC C

ELR–

CXCL4 CXCL9 CXCL10 CXCL11 CXCL12 CXCL13 CXCL16 CXCL12

CXCR1 CXCR2 CXCR3

CC NH2

C

COOH

CC C

C NH2

COOH

C

CCL3 CCL5 CCL7 CCL8 CCL13 CCL14 CCL15 CCL16 CCL23

CCL2 CCL7 CCL8 CCL13

CCL5 CCL7 CCL8 CCL11 CCL13 CCL15 CCL24 CCL26

CCR1

CCR2

CCR3

XCL1 XCL2

C XCR1

CXCR4

CXCR5

CCL17 CCL22

CCL3 CCL4 CCL5 CCL8

CCL20

CCL19 CCL21

CCL1

CCL27 CCL25 CCL28

CCL2 CCL8 CCL13 CCL19 CCL21 CCL25

CCR4

CCR5

CCR6

CCR7

CCR8

CCR9

CCR11

CX3C NH2

C CX3C C

CXCR6

CXCR7

COOH

CCR10

CX3CL1 CX3CR1

Fig. 1. Chemokine subfamilies. Subfamilies of chemokines are classified according to amount of cysteine residues deposited at the N-terminal domain of the molecules. These subfamilies are the CXC, CC, C and CX3C chemokines. The CXC class further comprises chemokines that do or do not contain a glu-leu-arg (ELR) amino acid sequence at their N-terminus, and are thus indicated as ELR– and ELR+ chemokines, respectively. The first part of the chemokine names identifies the subfamily and L stands for ‘ligand’ followed by a progressive number. Chemokines exert their biological effects through cell-surface receptors that belong to the superfamily of seven-transmembrane-domain Gprotein-coupled receptors. The chemokine-receptor nomenclature follows that of chemokines, and the receptors are thus designated CXCR, CCR, XCR and CX3CR followed by a progressive number. Please note that the vast majority of chemokine receptors bind more than one chemokine, but also some ligands can bind to several receptors. From a functional point of view, chemokines can be classified as homeostatic/constitutive or inflammatory/inducible. Homeostatic chemokines (shown in italics, grey) are produced in a tonic fashion in lymphoid and/or nonlymphoid organs and probably direct the normal traffic of leukocytes under physiological conditions. Inflammatory chemokines (shown in plain, black) are produced in response to microbial, inflammatory or immune signals and account for the increased recruitment of leukocytes under these conditions. The distinction between constitutive and inducible chemokines is useful but schematic because the two realms overlap in terms of pathology and molecules. Chemokines to date known as both constitutive and inflammatory are shown in italics, black. This figure shows all known ligand/receptor pairs of each subfamily and conveys information about the inflammatory (black), constitutive (grey italics) or both inflammatory and constitutive (black italics) natures of all ligands.

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Bosisio · Salvi · Gagliostro · Sozzani Marone G, Granata F (eds): Angiogenesis, Lymphangiogenesis and Clinical Implications. Chem Immunol Allergy. Basel, Karger, 2014, vol 99, pp 89–104 (DOI: 10.1159/000353317)


CXC

CXCL1 CXCL2 CXCL3 CXCL5 CXCL6 CXCL7 CXCL8

Chemokines in Angiogenesis Marone G, Granata F (eds): Angiogenesis, Lymphangiogenesis and Clinical Implications. Chem Immunol Allergy. Basel, Karger, 2014, vol 99, pp 89–104 (DOI: 10.1159/000353317)

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sponsible for the formation of intramolecular disulphide bonds. Based on the presence and position of conserved cysteine residues, chemokines can be divided into four subgroups [1]: CXC and CX3C chemokines are characterized by the presence of a single or three amino acids between the first two N-terminal cysteines, respectively, while members of the CC class lack this amino acid. In addition, the so called C chemokines lack the first and third cysteine residues. Only one CX3C and two C chemokines have been described so far. The CXC class further comprises chemokines that contain or not a glu-leu-arg (ELR) amino acid sequence at their N-terminus, thus indicated as ELR– and ELR+ chemokines, respectively (fig. 1). Many of the genes encoding chemokines cluster at specific loci: in humans for instance, CC chemokine genes are grouped at 17q11.2–12, CXC chemokine genes at 4q13 and the two C chemokines on chromosome 1. Thus, chemokines are thought to have arisen by duplication and divergence from a primordial chemokine gene [1]. In order to induce chemotaxis, chemokines bind to seven-transmembrane G-protein-coupled receptors (GPCRs) belonging to the family of class A rhodopsin-like GPCRs. Chemokine receptor nomenclature follows that of the ligands, e.g. CC and CXC chemokines bind CC and CXC receptors (or CCRs and CXCRs), respectively [1]. Ten CCRs, seven CXCRs, one CX3CR and one XCR have been described so far. As shown in figure 1, the vast majority of chemokine receptors bind more than one chemokine, but also some ligands can bind to several receptors [2]. This makes the chemokine system redundant by definition: however, it is becoming more and more evident that the chemokine system is also tightly regulated and that specificity to receptor-ligand interactions may often be conferred by refined spatial and temporal control mechanisms [3]. Additional system complexity comes from the fact that chemokine receptors can homo- and heterodimerize and by the existence of atypical receptors which sequester chemokines without activating cell response [4], thus functioning to shape gradients and dampening responses (see below). From a functional point of view, chemokines are usually categorized as homeostatic or inflammatory (fig. 1). Homeostatic chemokines (shown in grey italics in fig. 1), which are constitutively secreted, play key roles in organogenesis by directing and maintaining stem cells to the site of organ development and by inducing angiogenesis [5]. In addition, homeostatic chemokines oversee the organization and grant the functions of the immune system by recruiting precursors and maintaining the trafficking of the leukocyte subtypes. Important examples of these chemokines are CCL25 that drives T cell progenitors to the thymus and α4β7+ T cells to the intestine, and CCL28 that recruits IgA+ plasmablasts to the mammary gland. Inflammatory chemokines are instead induced during inflammation and control the recruitment of effector leukocytes in infection, inflammation, tissue injury and tumors. CXC chemokines are active on neutrophils (PMN) and lymphocytes while CC chemokines exert their action on multiple leukocyte subtypes, including monocytes, basophils, eosinophils, T lymphocytes, dendritic cells and NK cells, but they are generally inactive on PMN. CCL11 displays the most restricted spectrum of action being selectively active

on eosinophils, basophils and Th2 mast cells. C and CX3C chemokines are active on lymphoid cells (T lymphocytes and NK cells) and CX3CL1 is also active on monocytes. The distinction between constitutive and inducible chemokines is useful but schematic because the two realms overlap in terms of pathology and molecules. In neoplasia, for instance, inducible chemokines such as CCL2 are made constitutively. Moreover, certain constitutive chemokines expressed in lymphoid organs in the absence of deliberate stimulation, such as CCL22, are also produced in an inducible way (shown in black italics in fig. 1).

The binding of a chemokine to its receptor activates complex signal transduction pathways inducing integrin activation, polarization of the actin cytoskeleton and internalization of the receptor [6]. Intracellular signaling through GPCRs strictly depends on the activation of associated heterotrimeric G-protein (Gα, Gβ and Gγ). Typically, chemokine receptors associate with G proteins containing Gαi subunits that are blocked by pertussis toxin. Thus, this drug has been extensively used to address the role of G proteins in chemokine receptor signaling. Recently, however, some chemokine receptors (e.g. CXCR4) have been shown to utilize pertussis toxin-resistant subunits, such as Gαq [7]. G-protein activation leads to the activation of several downstream signaling cascades involving MAP (mitogen-activated protein) kinases, RAS and Rho GTPases and phosphoinositide 3-kinase (PI3K), which directly influence cell migration and proliferation. Of note, chemokine receptor signaling also promotes cell survival via the activation of protein kinase B (PKB/AKT) and by upregulating the expression of antiapoptotic genes [8]. More recently, it has been shown that chemokine receptors can transactivate other signaling pathways, such as the Jak-STATs, and receptors, such as tyrosine kinase receptors. The chemokine system also comprises receptors that bind chemokines without eliciting conventional signal transduction. These receptors, labeled as ‘atypical’, work as decoy receptors that shape local gradient by sequestering chemokines, but may also activate nonclassical signaling pathways. Atypical receptors include D6, DARC, CCRL1 and CXCR7 [4, 9]. Transient signaling is a common characteristic of most chemokine receptors, but one which requires rapid inactivation. This is achieved through receptor phosphorylation (which alters the three-dimensional conformation thus impairing the interaction with G proteins), desensitization and internalization (for an exhaustive review, see [10]). Of note, a single receptor may undergo internalization or not depending on the ligand, as described for CCR5 that is internalized by CCL3 and CCL5 but not by CCL4. The molecular mechanisms underlying this effect remain to be explained, although it may imply alternative usage of different intracellular mediators. Endocyto-

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Cell Activation by Chemokine Receptors

sis can follow classic clathrin- and dynamin-dependent pathways (as described for CXCR1, CXCR2, CXCR4 and CCR7) or caveolin-dependent pathways (DARC and CCRL1), or both routes (CCR2, CCR4 and CCR5). After internalization, receptors can be recycled or degraded. Often, the fate of a receptor depends on the ligand that its binding and one specific ligand can induce different fates for different receptors. For example, CCL5 induces recycling of CCR5 and degradation of CCR1 and CCR3. All in all, these reports underscore the importance of ligand-dependent fine-tuning of the amount of receptor to be exposed on the cell surface. Thus, the extreme complexity of chemokine receptor signaling, together with receptor promiscuity and ligand variety account for the refined regulation of cell activation by chemokines.

Chemokines of the CXC family are key mediators of angiogenesis in both physiological and pathological conditions. Within this family, all ELR+ chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7 and CXCL8) are proangiogenic while ELR– members (CXCL4, CXCL9, CXCL10, CXCL11, CXCL14) are antiangiogenic [11]. The only remarkable exception to this rule is the ELR- chemokine CXCL12, which is currently considered to be proangiogenic (see further). In mice, all angiogenic ELR+ chemokines signal via CXCR2, while in humans CXCL6 and CXCL8 also bind to CXCR1 [2]. Both receptors are expressed by endothelial cells and were demonstrated to be able to induce chemotaxis. However, since some endothelial cell population that express CXCR2 only can migrate in response to angiogenic chemokines [12], CXCR2 is generally assumed as the major angiogenic receptors also in humans. Of note, CXCR2 is also present on immune cells and is particularly important for neutrophil recruitment. Consistently, in a model of wound healing in CXCR2-deficient mice, the impairment of neovascularization paralleled with a modification of the relative composition of the inflammatory infiltrate [13]. This evidence raise the interesting possibility that CXCR2 may not stimulate angiogenesis only through endothelial cell chemotaxis, but also by recruiting other growthfactor producing cells that may in turn activate phosphotyrosine receptors and angiogenic sprouting (fig. 2). Endothelial specific CXCR2 knockout mice are expected in order to answer this question. Despite the proangiogenic role of ELR+ chemokines is unquestioned, CXCR2 seems dispensable for embryonic angiogenesis since CXCR2 knockout mice develop normally [14]. Thus, a different set of chemokines must be used by the embryo, or, less probably, that the role of chemokines is redundant. The dissection of embryonic angiogenesis in these mice is awaited to solve this question. A partial answer comes, however, from the analysis of the proangiogenic functions of CXCL12 and of its two receptors, CXCR4 and CXCR7 [15].


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Chemokines in Angiogenesis

Blood vessel

Angiogenic sprouting

Chemokine Chemokine receptor Endothelial cell Recruited leukocyte

Growth factor Phosphotyrosine receptor Chemokine-producing cell

CXCL12 is currently regarded as the only ELR– proangiogenic chemokine, yet in the past its function was considered uncertain since it can antagonize the proangiogenic activity of ELR+ chemokines and vascular endothelial growth factor (VEGF). This chemokine is the first to be expressed during embryogenesis and mice lacking CXCL12 or CXCR4 show defective embryonic blood vessel development in the intestine and kidney [16]. However, the vast majority of vessels in other organs form normally, thus suggesting the existence of compensatory mechanisms and/or that the function of CXCL12/CXCR4 is region-specific. In addition, while mice lacking other angiogenic signaling pathways such as VEGF-A or Notch die very early during embryonic development, half of these mutants come to light [16], further supporting the existence of CXCL12/CXCR4-independent mechanisms and the possibility that this

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Fig. 2. Direct and indirect roles of chemokines during angiogenesis. Chemokines, secreted by stromal cells, endothelial cells or leukocytes (grey arrows), may induce direct migration and angiogenic sprouting of chemokine receptor-expressing endothelial cells. In parallel, chemokines attract chemokine receptor-expressing leukocytes that in turn secrete proangiogenic growth factors such as VEGF.


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chemokine may be important mainly for the fine-tuning of vasculature development. In the adult, surface expression of both receptors can be induced upon activation of endothelial cells with growth factors or inflammatory cytokines [17], although the mRNA for CXCR7 can be found in many cell types indicating the existence of posttranscriptional regulatory mechanisms [17]. To date, the role of each receptor is not completely clear, nor is it known whether they can both activate cells. As discussed earlier, CXCR4 is a canonical G-coupled chemokine receptor, while CXCR7 is an ‘atypical’ receptor that activates alternative pathways and is possibly more involved in enhancing survival and adhesion than chemotaxis [17]. CXCR4 and CXCR7 have been shown to heterodimerize [18] and in this configuration CXCR7 may alter the signaling of CXCR4 or even act as a decoy that sequesters CXCL12, thus preventing its binding to CXCR4 [19]. Another atypical receptor, DARC, by binding to CXCL1, CXCL5 and CXCL8 may work to sequester excess angiogenic chemokines [9]. When overexpressed in endothelial cells DARC decreased the proangiogenic properties of ELR+ CXC chemokines [20], while DARC deficient mice showed increased levels of these chemokines as well as increased angiogenesis in a model of prostate adenocarcinoma [21]. Among chemokines that may have a direct role in angiogenesis, also some CC members need to be mentioned [22]. For instance, endothelial cells can express CCR2 and migrate towards its ligand CCL2. Moreover, CCL2 can promote blood vessel formation in angiogenic assays in vivo. A number of studies also support the pro-angiogenic properties of CCL5, although its role remains controversial. Angiostatic, ELR– chemokines include CXCL4, CXCL9, CXCL10, CXCL11 and CXCL14. Many of these chemokines are inducible by IFNs, including CXCL9, 10 and 11 [23–26] and bind to CXCR3. Their angiostatic functions would depend on the capability of this receptor to inhibit the proliferation of endothelial cells [27]. In humans, CXCR3 exists in three different splicing variants [27, 28]: CXCR3A, expressed by lymphocytes, CXCR3B, expressed by endothelial cells, and CXCR3-alt, whose function in angiogenesis remains undefined. In mice, however, CXCR3B does not exist and CXCL10 exert antiproliferative effects independently of CXCR3 [29], indicating species-specificity of the angiostatic effects of these chemokines. IFN-inducible chemokines may also exert indirect antiangiogenic effects by recruiting CXCR3-expressing Th1 lymphocytes and NK cells, a concept known as ‘immunoangiostasis’ [30]. These cells, in fact, induce cell-mediated immunity and release more IFN and IFN-inducible cytokines leading to inhibition of angiogenesis. CXCL4, one of the major platelet-derived chemokine, was the first angiostatic chemokine to be described [28]. In humans, two nonallelic variants of CXCL4 exist and they are known as CXCL4 (binding to CXCR3B [27]) and CXCL4L1 (binding to both CXCR3A and CXCR3B [28]). Of the two, CXCL4L1 was found to be more potent in inhibiting both bFGF and CXCL8/IL-8 driven angiogenesis [28]. The mechanisms through which these chemokines inhibit angiogenesis are not completely understood. It’s been shown that they cause random endothelial cell locomotion, disturb directed

migration towards angiogenic chemokines and may also inhibit endothelial cells proliferation. These effects depend in part by their capability to block the binding of VEGF and bFGF to their receptors, but also mechanism that does not involve direct binding to either VEGF ligands or receptors must exist since CXCL4 mutants lacking heparin-binding capacity still inhibit angiogenesis. Also CXCL14 was found to inhibit endothelial cell chemotaxis to CXCL8, VEGF and bFGF in vitro and to potently inhibit angiogenesis in vivo [31], but its receptor as well as its mechanisms of action remains to be enlightened.

Chemokines in Tumor Angiogenesis

Tumor-Associated Leukocytes and Angiogenesis In 1863, Virchow noted that euplastic tissues contain a ‘lymphoreticular infiltrate’, suggesting the connection between cancer and inflammation [32]. Many tumors of epithelial origin contain a leukocyte infiltrate consisting predominantly of macrophages and T lymphocytes. Leukocytes infiltrate tumors in response to tumor-derived chemokines produced by tumor cells, resident and infiltrating stromal cells [33]. CCL2 and CCL5, shown to be upregulated in a number of carcinomas [23], are major attractors of macrophage precursors. Macrophages are mononuclear phagocytic cells that can come in a number of flavors displaying disparate activities. A successful classification of these cells is based on their mode of activation and biological activity [34]: classically activated macrophages (also known as M1) are activated by Th1 cells and possess potent proinflammatory activity, while alternatively activated macrophages (or M2) differentiate during Th2-driven responses, are considered noninflammatory and support

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Tumor growth is strictly dependent on angiogenesis; once a tumor reaches a few millimeters in diameter, further tumor expansion requires neovascularization. Once this is achieved, tumor growth is usually rapid and allows the potential for metastasis. Much of the original work on tumor angiogenesis has been focused on the family of VEGF. However, it is now clear that chemokines play a fundamental role in the angiogenic switch of malignancies in organs such as skin, pancreas, intestines, lung, brain, reproductive organs, kidney and others. Chemokine-dependent angiogenesis may be either direct (i.e. tumor cells produce chemokines that act on endothelial cells) or indirect (when chemokines produced within the tumor attract leukocytes which in turn release proangiogenic mediators), the latter being probably the most relevant mode of action of chemokines in tumor angiogenesis (fig. 3). In vivo, however, these two modes of action can hardly be distinguished because of the potent activity of chemokines on leukocytes and of the simultaneous upregulation of multiple factors during tumor progression. Chemokines also contribute to tumor progression by enhancing tumor cell survival and metastatic spread (fig. 3), but these aspects will not be further analyzed hereby (for a review, see [8]).

Growth/survival metastasis invasion

Recruitment Differentiation (M2, Th2) Angiogenesis Inflammation

Angiogenesis Chemokine Chemokine receptor Endothelial cell

Tumour cell Infiltrating leukocyte

wound healing and angiogenesis via the production of VEGF and prostaglandin E2. Th2 cells, generally regarded as tumor-promoting, are abundant in human cancer where they are recruited by gradients of locally produced CCL17 and CCL22. These cells, together with CCL2/17/22 themselves and with other anti-inflammatory mediators such as prostaglandin E2, promote M2 polarization of TAM. In fact, TAM associated with actively growing tumors appears to be almost exclusively polarized toward a M2 phenotype [35]. A similar classification has been proposed for dendritic cells and alternatively activated dendritic cells have shown proangiogenic activity [36]. The tumor-fostering and proangiogenic role of TAM is supported by a number of studies, and generally high macrophage counts are a poor prognostic sign [35]. For instance, in PyMT mice (susceptible to mammary carcinoma) deficient in CSF-1, the progression to malignancy and metastasis was delayed due to paucity of TAM and in breast cancer macrophage infiltration correlated with vascularity. However, macrophages are extremely plastic and can shift phenotype according to the microenvironmental condi-


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Fig. 3. Role of chemokines in malignancy. Chemokines play a fundamental role in the angiogenic switch of malignancies both directly, when tumor cells produce chemokines that act on endothelial cells, and indirectly, i.e. by attracting leukocytes which in turn release more chemokines and proangiogenic mediators. The latter mode of action is probably the most relevant in this setting, despite the two being hardly distinguishable in vivo because of the potent activity of chemokines on leukocytes and the simultaneous upregulation of multiple factors during tumor progression. Please note that chemokines also influence the protumorigenic polarization of infiltrating leukocytes and contribute to tumor progression by enhancing tumor cell survival and metastatic spread.

Direct Induction of Angiogenesis by Tumor Cells Tumor cells may also induce angiogenesis by producing proangiogenic factors, including VEGF and chemokines that act directly on endothelial cells [8]. Although with some exception, most endothelial cell types express receptors for ELR+ angiogenic chemokine as well as CXCR4 and CCR1/2. An increase in the levels of ELR+ CXC chemokines has been reported in a number of tumor settings including ovarian cancer, non-small-cell lung cancer (NSCLC), prostate cancer, and renal cell carcinoma and in many cases their tissue levels directly correlate with the extent of tumor angiogenesis and inversely correlate with survival [23]. In patients with NSCLC, high CXCL8 levels also correlated with extensive TAM infiltration. The role of CXC chemokines in tumor angiogenesis is confirmed by in vitro and in vivo experimental models. Transfection of melanocytes with CXCL1, 2 and 3 enabled them to form tumors in nude mice, and those tumors were highly vascularized. Work by Yoneda et al. [40] showed a role for CXCL8 in the angiogenesis and hence progression of human ovarian cancer xenografts in nude mice. CXCL8 contributed to angiogenesis in models of NSCLC in vivo and its inhibition attenuated tumor development [41]. Consistently, ablation of the atypical receptor DARC in a prostate cancer model resulted in increased vascularization and malignancy of the tumors [21]. Also CC chemokines binding to CCR1 and CCR2, such as CCL2, CCL4, CCL11 and CCL16, can induce direct tumor angiogenesis [22]. However, these chemokines always correlate with massive infiltration of proangiogenic macrophages that often makes difficult or impossible to discriminate the real contribution of direct and indirect angiogenesis. Conversely, angiostatic ELR– chemokines may have therapeutic benefit in cancer. The expression of CXCL9 and 10 in Burkitt’s lymphoma cell lines in nude mice was

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tions. For instance, in regressive tumors TAM can display M1 polarization and tumoricidal activity. Consistently, patients with pancreatic carcinoma with high levels of circulating CCL2 have been shown to have a significantly higher survival rate, while Nesbit et al. [37] demonstrated that CCL2 overexpression in melanoma cells led to tumor destruction in nude mice, due to a massive monocyte/macrophage infiltrate. Thus, manipulation of the local chemokine milieu may represent an interesting therapeutic target in terms of M2 TAM reprogramming, blocking of their recruitment or enhancement of the antitumor immune response. A number of chemokines including CCL1, CCL2, CCL5, CCL20, CXCL10 and XCL1 have been overexpressed in murine tumor models and have all led to an enhanced immune response and tumor rejection. Tumors generated by a CCL21-transduced cell line showed a less aggressive phenotype that correlated with an increased infiltrate including immature dendritic cells and CD8+ T cells [38]. In a study of esophageal carcinoma by Schumacher et al. [39], the intratumoral CD8+ T cell infiltrate showed proliferative activity and IFN-γ secretion and this infiltrate (rather than the peritumoral T cell infiltrate) was correlated with a good prognosis in both squamous cell and adenocarcinomas.

higher in tumors that spontaneously regressed and was correlated with impaired angiogenesis [23]. Production of CXCL10 from adenocarcinoma or squamous cell carcinoma cell lines inoculated in SCID mice was inversely correlated with tumor growth [42]. According to the previously described concept of immunoangiostasis, this effect is not only due to direct inhibition of angiogenesis but also to increased extravasation of Th1 cells and induction of cell-mediated immunity. Since the balance between proand antiangiogenic chemokines can regulate angiogenesis which indirectly affects tumour growth, interfering with this balance may have therapeutic benefits.

Chemokines in Lymphangiogenesis

In addition to their effects on vascular endothelial cells, chemokines also play a role in the formation of the lymphatic system [37]. Apart for VEGF-C, most of the local signals that guide the positioning of the first lymphatic progenitors in mammals are still poorly defined. Once the lymphatic sacs are established, at the site of future lymph node formation there begin to appear cells known as lymphoid tissue inducer cells (LTIs). LTIs are probably of hematopoietic origin and are characterized by the expression of CD45, CD4, IL-7 receptor-α and CXCR5, but they do not express CD3 and T cell receptor. Since mice deficient for both CXCL13 and IL-7 receptor-α do not develop lymph nodes, it is likely that CXCR5 is crucial to promote the recruitment and accumulation of LTIs to the sites of emerging lymph nodes. Further lymph node development is coordinated by a set of complex interactions between LTIs and local stromal cells that, for this reason, are also known as organizer cells. Once activated by LTβ, organizer cells secrete CXCL13, CCL21 and CCL19, thus inducing the recruitment of more LTIs and initiating the segregation of B and T cell areas. CCL21 and CCL19 are crucial for the development of facial, cervical, brachial, and axillary lymph nodes and their absence (or the absence of the corresponding receptor CCR7) in CXCL13–/– (or CXCR5–/–) mice results in failure to form any peripheral lymph node except for mesenteric ones. This phenotype suggests a highly cooperative function of CXCL13, CCL19 and CCL21 during lymph node formation. Similar mechanisms also support the formation of tertiary lymphoid organs at sites of chronic inflammation.

Although chemokines by themselves provide an extensive network of chemotactic signals, also nonchemokine molecules such as lipid mediators (e.g. LTB4 and S1P), pathogen-derived products (e.g. the pathogen-associated molecular profile fMLP), antimicrobial peptides (e.g. chemerin), complement products and other normal constituents of our body may be chemotactic and regulate trafficking of leukocytes, and of other cell types, both in physiological and pathological conditions [43]. Similarly,


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Nonchemokine Chemoattractants in Angiogenesis

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to chemokines, all of these chemoattractants bind to and activate G protein-coupled seven transmembrane receptors. The finding that endothelial cells express S1P1, one of the receptors for the pleiotropic lysophospholipid mediator sphingosine-1-phosphate (S1P), and that S1P can induce endothelial cell migration and formation of capillary-like tube structures in vitro dates back to the late 1990s. In vivo, deletion of the S1pr1 gene resulted in embryonic lethality due to failure of vascular maturation while S1P1 silencing by RNA interference inhibited tumor angiogenesis (for a review, see [44]). More recently, S1P was shown to play a key role also in endothelial cell senescence by binding to another receptor, S1P2, which impairs chemotaxis and morphogenic responses of endothelial cells through the activation of the lipid phosphatase PTEN [45]. Thus, S1P can exert different and even contrasting functions in angiogenesis depending on the balance of receptors expressed by a given cell type. Very recently, endothelial cells were also shown to express BLT2 that when stimulated with its specific ligands, namely 12(S)-HETE and LTB4, caused significant angiogenic activity [46]. Of interest, BLT2 ligands were reported to be increased at sites of tumor angiogenesis. Despite the precise mechanisms by which BLT2 stimulates that angiogenesis remain unknown, the authors inferred that this receptor may mediate VEGF-induced angiogenesis. In fact, its expression was significantly upregulated by VEGF and, conversely, its knockdown attenuated VEGF-induced angiogenesis in vitro and in vivo. Of note, BLT2 exerts also indirect proangiogenic effects since BLT2expressing mast cell were shown to contribute to tumor angiogenesis via the production of IL-1β-induced CXCL8 [47]. The anaphylatoxins C3a and C5a are key component of the complement cascade that exert chemotactic functions by binding to class A rhodopsin-like G-coupled seven transmembrane receptors [48]. These receptors are expressed by leukocytes that are recruited to the site of complement activation during infections, but also by endothelial cells. A potential role for complement in the regulation of angiogenesis was originally shown in age-related macular degeneration. In this model, C3a and C5a stimulated the secretion of VEGF, while neovascularization and VEGF levels were decreased in C3aR–/– and C5aR–/– mice and wild-type mice treated with C3aR and C5aR antagonists or neutralizing antibodies against C3a and C5a [49]. Similar results were recently obtained using an ovarian tumor model, where C5a activated endothelial cells and the pharmacological inhibition of C5aR inhibited the release of VEGF [50]. By contrast, in a model of retinopathy of prematurity the C5a-C5aR axis was found to potently inhibit angiogenesis [51]. In this study, antibody-mediated blockade of C5a, treatment with C5aR antagonist, or C5aR deficiency resulted in enhanced pathological angiogenesis. The antiangiogenic activity of complement did not depend on direct inhibition of endothelial cell functions. Instead, it was mediated by macrophages, polarized by C5a towards an angiogenesis-inhibitory phenotype, since macrophage depletion in vivo reversed the increased neovascularization associated with C5aR deficiency. These apparently contrasting results indicate that the specific path-

ological milieu is able to determine how complement influences both direct and indirect angiogenesis. Chemerin is an adipokine and chemoattractant protein that serves as a ligand for the seven-transmembrane spanning G protein-coupled receptors CMKLR1 (chemokine-like receptor 1, also known as ChemR23) and the atypical chemokine receptor CCRL2. While activation of CMKLR1 triggers calcium mobilization, internalization and cell migration, CCRL2 does not appear to be a signaling receptor and is thought to play a role in the regulation of chemerin bioavailability. Another recently reported high affinity chemerin receptor is GPR1 (G protein-coupled receptor 1), which triggers calcium efflux in the absence of chemerin-dependent cell migration. The exact role of the three receptors in regulating chemerin activities in immune responses and other cellular processes is still not completely clear (for a review, see [52]). In addition, these molecules serve as receptors also for other ligands (e.g. ChemR23 also binds resolvin E1) and activate different intracellular mediators [53]. Two independent studies have demonstrated that chemerin acts on human umbilical vein endothelial cells (HUVECs) as a proangiogenic factor by promoting migration, capillary tube formation and activation of gelatinases; in one of them, this effect was found to be mediated by the expression of functional ChemR23 [54, 55]. By contrast, in a very recent paper Monnier et al. [56] analyzed the expression of chemerin receptors in several human endothelial cells, including HUVECs, and could never detect ChemR23 or GPR1, while CCRL2 was always present. Similarly, absolute quantitative PCR analysis experiments performed in our lab called ChemR23 and GPR1 as absent in HUVECs [Salvi, unpubl. data]. Despite these discrepancies possibly depend on different culture conditions that could affect gene regulation, the expression and balance of chemerin receptors on endothelial cells remains a matter of debate thus making it difficult to address the molecular mechanisms that activate chemerin-dependent angiogenesis. Also, the role of chemerin-recruited infiltrating cells in the regulation of angiogenesis still awaits to be dissected. For instance, ChemR23 is expressed and functional in dendritic cells, which may display pro- or antiangiogenic properties according to their activation state [36]. Interestingly, the lipid mediator Resolvin E1, which also signals through ChemR23, reduced inflammatory angiogenesis in a model of corneal neovascularization by stimulating resolution mechanisms of the innate immune responses [57]. Thus, progresses in understanding how the three receptors regulate activities of chemerin and/or other ligands are awaited to help dissecting their role in angiogenesis in different pathophysiological settings.

Since the original observations on the proangiogenic effects of CXCL8, a wealth of literature has provided evidence on the extensive role of chemokines and other chemotactic factors in the regulation of angiogenesis and lymphangiogenesis. Chemo-


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Concluding Remarks

kines can be classified for their ability to promote or to inhibit the angiogenic process and be considered crucial mediators of the whole inflammatory process. A lot of evidence is also available to support a role of chemokine in pathogenesis, especially in tumor growth, through their ability to regulate the angiogenic process. However, the correct understanding of the direct role of chemokines on endothelial cells rather than their indirect role through the recruitment of angiogenic factor-producing leukocytes still needs to be fully elucidated.

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Prof. Silvano Sozzani Dipartimento di Medicina Molecolare e Traslazionale Università degli Studi di Brescia, Viale Europa 11 IT–25123 Brescia (Italy) E-Mail sozzani @ med.unibs.it

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CXC and CC chemokines as angiogenic modulators in nonhaematological tumors.

Angiogenic and Antiangiogenic Factors in Preterm Neonates Born to Mothers with and without Preeclampsia.

Balance of antiangiogenic and angiogenic factors in the context of the etiology of preeclampsia.

Quantitative insight in utilizing circulating angiogenic factors as biomarkers for antiangiogenic therapy: systems pharmacology approach.

The use of angiogenic and antiangiogenic factors in the differential diagnosis of pre-eclampsia, antiphospholipid syndrome nephropathy and lupus nephritis.

Lysophosphatidic acid alters the expression profiles of angiogenic factors, cytokines, and chemokines in mouse liver sinusoidal endothelial cells.

Chemokines and alcoholic hepatitis: are chemokines good therapeutic targets?

Antiangiogenic agents targeting different angiogenic pathways have opposite effects on tumor hypoxia in R-18 human melanoma xenografts.

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Chemokines and immunity.

Semi-synthesis of chemokines.

Chemokines and microRNAs in atherosclerosis.

Teleost Chemokines and Their Receptors.

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Corneal neovascularization and contemporary antiangiogenic therapeutics.

Sunitinib: the antiangiogenic effects and beyond.

Antiangiogenic treatment of meningiomas.

Antiangiogenic therapies for glioblastoma.

Angiogenesis and antiangiogenic agents in cervical cancer.

Chemogenesis of an antiangiogenic glycosaminoglycan.

Fucoidans: pro- or antiangiogenic agents?

Chemokines in cancer.

Chemokines in cellular positioning and human disease.

Chemokines and chemokine receptors in multiple sclerosis.