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Integrin Structure

Integrins comprise a family of cell surface heterodimeric complexes formed by the noncovalent association of two subunits, α and β (Takada et al. 2007). There are 18 α and 8 β subunits capable of forming 24 different functional heterodimers. The αβ composition of the heterodimer largely determines the ligand specificity, although some ligands can bind directly to individual subunits, such as collagens, to the I-domain on the α subunit. Each individual subunit consists of a large extracellular domain (about 750 amino acids for the β subunits and around to 1000 amino acids for the α subunits), a single transmembrane domain (22–24 amino acids), and a short cytoplasmic tail (15–58 amino acids, except for the β4 subunit, which contains over 1000 intracellular residues). The cytoplasmic domain is essential for the regulation of integrin activity and function: on the one side it controls extracellular ligand-binding activity of the complex (“inside-out” signaling), while on the other, it initiates cellular responses upon ligand binding (“outside-in” signaling) (Ginsberg et al. 2005). In resting, nonligated integrins, the β subunit cytoplasmic domain interacts with the α subunit cytoplasmic domain, thereby maintaining the receptor in its inactive state (Luo et al. 2007). Binding of the cytoplasmic protein talin to the β subunit cytoplasmic domain disrupts this interaction, resulting in a conformational change of the extracellular domain leading to a high affinity ligand-binding state (affinity maturation). The “released” β cytoplasmic tail interacts with additional intracellular structural (e.g., paxillin, vinculin), adaptor (e.g., Shc, Cas), and signaling (e.g., FAK, ILK) proteins, thereby initiating cytoskeletal rearrangement and cell signaling events. Ligated integrins can cluster to form small focal contacts at the cell periphery, large focal adhesions retracted from the cell border, or fibrillar adhesions located underneath the cell body along actin stress fibers (Romer et al. 2006).

Integrin Functions

Cell Adhesion

Integrins are the main cell adhesion receptors for ECM proteins for virtually every cell, including endothelial cells (Hynes 2007). A particular feature of integrins is their ability to recognize short amino acid sequences on exposed loops of their cognate ligands, the tripeptide RGD being the best known and studied. In addition, integrins also bind matricellular proteins, such as thrombospondins, and cell surface molecules, such as ICAMs (for a comprehensive detailed list of ligands, see (Takada et al. 2007)). Ligand binding specificity is promiscuous and redundant: that is, one integrin can bind several different ligands, and many different integrins can bind to the same ligand. Redundancy may be an advantage when the cellular response needed in a particular context (e.g., survival or migration during matrix remodeling) is more important than the nature of the ECM protein eliciting. For example integrin αVβ3 binds to many ECM proteins present at sites of inflammation, coagulation, and tissue remodeling. Promiscuity may reflect the need to initiate different signaling events and cellular responses from the same ECM. For example, integrin α5β1 and αVβ6 bind to fibronectin, but α5β1 suppresses cell migration, while αVβ6 stimulates it (Coutifaris et al. 2005; Scott et al. 2004). More recently, integrins have been reported to bind to a multitude of noncanonical ligands, which themselves are known modulators of vascular functions, including VEGF (Vlahakis et al. 2007), FGF (Murakami et al. 2008), angiopoietins (Camenisch et al. 2002), or matrix-bound VEGFR-1 (Orecchia et al. 2003). These observations open the intriguing possibility that angiogenic growth factors, when associated to the ECM, may modulate endothelial cell functions by signaling through integrins in complement to their activities mediated by their canonical receptors.

Cell Signaling

Integrin ligation also initiates signaling cascades, modulating complex cell functions like spreading, migration, survival, proliferation, or differentiation (Alghisi and Ruegg 2006; Stupack 2007) (Fig. 6.1). As integrins do not have intrinsic enzymatic activity, they need to recruit cytoplasmic structural (e.g., α-actinin, talin, vinculin) and signaling (e.g., FAK, paxillin, and Src family kinases) proteins at adhesion complexes to initiate signal transduction (Luo et al. 2007; Romer et al. 2006). Many signaling pathways activated by integrins are also activated by growth factor receptors, and maximal signal transduction is achieved when integrins and growth factor receptors are concomitantly engaged. Signaling pathways activated by integrins, including in angiogenesis, comprise: MAPK, Akt/PKB, Rho family GTPases, and NF-κB (Mahabeleshwar et al. 2006).

Fig. 6.1
figure 1_6

Integrin functions and how to inhibit them. Integrins act as cell adhesion and motility mediators (green arrows) or as signal transducers (red arrows). These functions can be modulated by growth factors and their receptors (green and red dashed arrows). On one hand, the inhibition or integrin function can be achieved extracellularly by the action of peptidomimetics (a), peptides, most frequently RGD-based, but also as noncanonical peptides (b) or antibodies (c). These three classes of inhibitors could also be used as imaging tools if they are labeled with a detectable tracers. On the other hand, peptides disrupting or blocking the interaction between the β integrin cytoplasmic tail with cytoplasmic adaptor or signaling proteins inhibit integrin function and may be developed in the future as therapeutic tools (d)

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A pathway activated by integrin, particularly relevant to vascular biology and angiogenesis, is the COX-2/prostaglandin pathway. Integrin-mediated adhesion and binding of soluble ligands induce COX-2 mRNA expression and stabilize COX-2 protein in endothelial cells resulting in enhanced prostaglandin production (Zaric and Ruegg 2005). In turn, prostaglandins activate the adenylcyclase via prostane receptor signaling, resulting in PKA activation, accelerated αVβ3-dependent cell adhesion, spreading, and migration in a Rac1-dependent manner (Dormond and Ruegg 2003). Consistent with these findings, COX-2 inhibitors inhibit αVβ3-dependent endothelial cell spreading and migration in vitro and angiogenesis in vivo.

Integrins in Tumor Angiogenesis

  • Integrin αVβ3 was the first integrin associated with angiogenesis (Brooks et al. 1994). αVβ3 is highly expressed in angiogenic endothelial cells in granulation tissue and in malignant tumors, but is virtually absent from quiescent endothelial cells (Hynes 2007). Inhibition of αVβ3 with a function-blocking monoclonal antibody, or RGD-based peptides, or peptidomimetics suppressed corneal neovascularization (Klotz et al. 2000), hypoxia-induced retinal neovascularization (Hammes et al. 1996), tumor angiogenesis, and tumor progression in various in vivo models (MacDonald et al. 2001; Reinmuth et al. 2003), and endothelial cell sprouting and angiogenesis in an in vitro 3D model of angiogenesis (Nisato et al. 2004). Importantly, quiescent and pre-existing vessels were not perturbed by these treatments. The results obtained with pharmacological antagonists of integrins αVβ3/αVβ5 contrast with results obtained through genetic approaches. Mice deficient in αV integrins and lacking αVβ1, αVβ3, αVβ5, αVβ6, and αVβ8 expression, were still able to undergo extensive developmental vasculogenesis and angiogenesis, although they died in utero or shortly after birth (Bader et al. 1998). Analysis of the phenotype of individual β integrin knock-out mice showed that the β8 knock-out was the only one to reproduce the αV knock-out phenotype (Zhu et al. 2002), thereby revealing a role for αVβ8 in the association between cerebral microvessels and brain parenchymal cells. Deletion of the β3 subunit did not significantly disrupt vascular development, although some embryos died in utero due to placenta defects, while others died postnatally due to bleeding and anemia (Hodivala-Dilke et al. 1999). β3-deficiency reproduce the inherited human bleeding disorder known as Glanzmann thrombasthenia due to the concomitant lack of αIIbβ3 (Tomiyama 2000). Paradoxically, mice lacking αVβ3 integrins had enhanced pathological angiogenesis, including tumor angiogenesis (Reynolds et al. 2002), associated with enhanced VEGFR-2 signaling (Reynolds et al. 2004). The reason for the divergence of the results obtained with pharmacological inhibition vs. genetic deletion of αVβ3 are not yet fully clear (Hynes 2002).

  • More recently, β1 integrins (i.e., α1β1, α2β1, α3β1, α4β1, α5β1, αVβ1), αVβ8, and α6β4 have also been shown to promote angiogenesis (Alghisi and Ruegg 2006; Serini et al. 2006). β1 integrin expression on vascular endothelial cells is dispensable for vasculogenesis, but crucial for embryonic angiogenesis (Tanjore et al. 2007). Deletion of the α5 gene is embryonically lethal and is associated with vascular and cardiac defects (Francis et al. 2002). α5β1 is up regulated in angiogenesis and blocking anti-α5 antibodies suppressed VEGF-induced tumor angiogenesis in both chick embryo and murine models (Collo and Pepper 1999; Kim et al. 2000). An α5β1 antagonist in combination with chemotherapy reduced metastasis and suppressed angiogenesis at metastatic lesions (Stoeltzing et al. 2003).

  • Integrins α1β1 and α2β1 are highly upregulated by VEGF in cultured endothelial cells, resulting in enhanced cell spreading on collagen I, while anti-α1β1 and anti-α2β1 antibodies inhibited VEGF-driven angiogenesis in vivo. Combined administration of anti-α1β1 and anti-α2β1 antibodies to mice bearing squamous cell carcinoma xenografts, resulted in reduced tumor angiogenesis and tumor growth (Hong et al. 2004; Perruzzi et al. 2003; Senger et al. 2002). The role of α2β1 for the regulation of murine wound angiogenesis was confirmed in a genetic approach (Zweers et al. 2007).

  • α6β4 promotes an invasive endothelial cell phenotype at the early phase of angiogenesis in response to growth factors (FGF-2, VEGF) (Nikolopoulos et al. 2004). Genetic studies have revealed that α6β4 signaling promotes both angiogenesis and tumorigenesis. Importantly, α6β4 combines with multiple receptor tyrosine kinases, including ErbB2, EGF-R and c-Met, and enhances their signaling function (Giancotti 2007).

Integrin Antagonists with Antiangiogenic Activities

Four different types of integrin antagonists have been developed: antibodies, endogenous inhibitors, peptides, and nonpeptidic antagonists. We describe here the main representative drugs within each class that have shown antiangiogenic activity in preclinical models, with particular emphasis on drugs that entered clinical testing. These and additional inhibitors are summarized in Table 6.1.

Table 6.1 Integrin antagonists in clinical trials
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Antibodies

  • LM609/MEDI-522/Vitaxin. The anti-αVβ3 monoclonal antibody LM609 blocked endothelial cell adhesion, migration, and sprouting in vitro and angiogenesis in vivo in the CAM assay (Brooks et al. 1994). Subsequently, LM609 was humanized and affinity maturated allowing the isolation of an antibody with a 90-fold improved affinity (MEDI-522 or Vitaxin) (Wu et al. 1998). Phase I studies demonstrated that treatment was well tolerated with little or no toxicity. The most common side effect was infusion-related fevers. Doses of 1 mg/kg/week or more produced plasma concentrations sufficient to saturate αVβ3 in vitro. Vitaxin demonstrated a half-life longer than five days with no tendency to accumulation. One partial response and several stable diseases were observed (Posey et al. 2001). Combination of Vitaxin with chemotherapy was well tolerated. There was possible effect on tumor perfusion detected by dynamic computed tomography imaging, but no objective antitumor responses (McNeel et al. 2005). In treated patients, there was evidence of reduced FAK phosphorylation in skin wound vessels, consistent with inhibition of αVβ3 signaling (Zhang et al. 2007). Vitaxin has entered Phase II trials mostly on hormone-refractory prostate cancers or metastatic melanoma (www.clinicaltrials.gov).

  • CNTO 95. CNTO 95 is a pan anti-αV fully humanized antibody. In a human melanoma xenograft model, wherein CNTO 95 recognized αVβ3 and αVβ5 on human tumor cells but not mouse cells, CNTO 95 treatment inhibited tumor growth by 80%. In a nude rat, human skin xenograft tumor model where CNTO 95 blocks αVβ3 and αVβ5 on both human tumor cells and human skin endothelial cells, treatment reduced final tumor weight by >99% (Trikha et al. 2004). The antibody did not show any adverse effects in monkeys (Martin et al. 2005) and entered Phase I clinical trials in various solid tumors, including ovarian, colorectal, melanoma, and renal cell carcinoma. Results on these patients showed that CNTO 95 was well tolerated up to weekly doses of 10 mg/kg (www.asco.org). CNTO 95 is now in Phase I/II in combination with other chemotherapeutic drugs in Stage IV melanoma or metastatic HRPC.

  • M200/Volociximab. M200 is an affinity maturated humanized chimeric monoclonal antibody blocking α5β1 integrin. It inhibited tumor angiogenesis and tumor growth in a rabbit syngenic tumor model (M200 does not bind to rodent α5β1) despite a 20-fold lower affinity for rabbit integrin, relative to human (Bhaskar et al. 2007a). A function blocking rat-anti-mouse α5β1 antibody with features similar to M200 was shown to inhibit angiogenesis and suppress tumor growth and metastasis in mice (Bhaskar et al. 2007b; Ramakrishnan et al. 2006). Based on this activity profile, Volociximab was tested in Phase I trials in various refractory solid tumors including renal cell carcinoma and metastatic melanoma. The study data showed that adverse events were generally mild to moderate in intensity and there were no dose limiting toxicities. Volociximab is currently evaluated in Phase II trials as a single agent (Kuwada 2007). Combination trials with chemotherapy are planned.

  • c7E3/Abciximab/ReoPro. c7E3 is a humanized monoclonal antibody Fab fragment approved for use as adjunct therapy to prevent cardiac ischemic complications in patients undergoing coronary angioplasty (Cohen et al. 2000). c7E3 also interacts with integrins αVβ3 and Mac-1 (αMβ2). In animal models, c7E3 inhibited tumor growth and angiogenesis (Nakada et al. 2006).

Endogenous Antagonists

  • Endostatin is a carboxyl-terminal fragment of Collagen XVIII inhibiting endothelial cell proliferation in vitro and angiogenesis and tumor growth in vivo (O’Reilly et al. 1997). The generation of endostatin from Collagen XII is mediated by various proteases (e.g., cathepsin L and MMPs). The antiangiogenic activity of endostatin is due, at least in part, to binding to integrin α5β1 and caveolin-1 on endothelial cells, causing downregulation of RhoA activity and Src family kinase-dependent disassembly of focal adhesions and actin stress fibers, resulting in decreased matrix deposition and migration (Wickstrom et al. 2005). Recombinant human endostatin entered clinical testing and was found to be safe and well tolerated (Hansma et al. 2005; Herbst et al. 2002).

  • Tumstatin consists of the carboxyl-terminal noncollagenous 1 (NC1) domain of the α3 chain of Collagen IV. It inhibited in vivo neovascularization in Matrigel plug assays, suppressed tumor growth in xenograft models, and induced endothelial cell apoptosis (Hamano and Kalluri 2005). Tumstatin binds to αVβ3 integrin in endothelial cells, and selectively inhibits protein synthesis by suppressing mTOR (Maeshima et al. 2002).

Peptides

  • EMD121974/Cilengitide. The discovery that many integrins recognize their ligands through short amino acid sequences, most notably RGD, led to the development of small peptides that competitively blocked ligand–receptor interaction. Cyclized peptides were up to 100-fold more selective than linear counterparts, and cyclic pentapeptides that possessed two hydrophobic amino acids next to the recognition sequence proved to be highly active and selective for αVβ3/αVβ5. The cyclic pentapeptide cyclo(-Arg-Gly-Asp-D-Phe-Val-) (EMD66203 Merck KGaA) showed nanomolar inhibition of vitronectin binding to the αVβ3 integrin without interfering with αIIbβ3 integrin (Haubner et al. 1996). Modification of the amino acids flanking the RGD sequence led to the synthesis of EMD121974 (Cilengitide) inhibiting αVβ3 integrin binding to vitronectin with an IC50 of 0.6 nM versus 900 nM for the αIIbβ3 integrin (Smith 2003). Cilengitide showed antitumor effects in brain, melanoma, head and neck, and brain tumors (MacDonald et al. 2001; Mitjans et al. 2000; Raguse et al. 2004; Taga et al. 2002). In Phase I, studies of cilengitide were well tolerated with no dose-limiting toxicities, and showed evidence of activity in recurrent malignant gliomas (Nabors et al. 2007). Cilengitide is now in Phase II clinical trials, alone and in combination with radio and chemotherapies, in solid tumors, leukemia, and lymphoma (Stupp and Ruegg 2007).

  • ATN-161 is a peptide derived from the α5β1-binding sequence PHSRN present in fibronectin (Livant et al. 2000). Chemical modifications of this sequence led to the synthesis of ATN-161 (Ac-PHSCN-NH2) that, in contrast to other integrin antagonist peptides, is not a RGD-based peptide. ATN-161 possesses antitumorigenic antiangiogenic activities in mice in the absence of toxicities (Livant et al. 2000). ATN-161 was tested in a Phase I study in patients with advanced solid tumors for up to 14 cycles of 4 weeks and was well tolerated at all dose levels. Approximately, one-third of the treated patients manifested prolonged stable disease (Cianfrocca et al. 2006).

Non-peptidic Inhibitors

Peptidomimetics are compounds containing non-peptidic structural elements mimicking the action(s) of a natural parent peptide. Peptidomimetics can be administered orally, are insensitive to protease-mediated degradation, and have longer stability (Cacciari and Spalluto 2005).

  • SCH221153 was obtained by screening and further modifying an RGD-based peptidomimetic library. It targets αVβ3 and αVβ5 integrins with IC50 of 3.2 and 1.7 nM, respectively. SCH221153 inhibited endothelial cell adhesion to vitronectin and suppressed angiogenesis in a CAM assay (Kumar et al. 2001).

  • BCH-14661 and BCH-15046 are integrin antagonists that induce cell detachment and apoptosis of angiogenic endothelial grown on RGD-based matrices (i.e., vitronectin and fibronectin). BCH-14661 is specific for αVβ3, while BCH-15046 antagonizes αVβ3, αVβ5 and α5β1 (Meerovitch et al. 2003). BCH-15046 was also capable to induce endothelial cell apoptosis independently of cell detachment.

  • Thiolutin is a non-peptidic antagonist of cell adhesion interfering with integrin-post-receptor events (Minamiguchi et al. 2001). The antiadhesive effect of thiolutin is due to decreased paxillin protein expression, disruption of focal adhesions, and cell detachment.

  • SJ749, which structure mimics RGD-based sequences, is a potent inhibitor of α5β1 integrin (IC50 around 0.8 nM), and it inhibited angiogenesis in the CAM assay (Kim et al. 2000). The structure of this non-peptidic compound bound to the head of the α5β1 integrin has been resolved, thereby opening new perspectives in rational design to improve its specificity and binding constant (Marinelli et al. 2005).

  • JSM6427 is another α5β1-specific peptidomimetic inhibitor with antiangiogenic activities (Umeda et al. 2006). Interestingly, JSM6427 inhibited inflammatory lymphangiogenesis (Dietrich et al. 2007) suggesting the possibility of combined targeting angiogenesis and lymphangiogenesis by targeting one integrin.

Open Questions and Current Developments

Preclinical studies suggest that vascular integrins are valuable targets for antiangiogenic treatments. Results obtained in Phase I clinical trials have shown that the integrin antagonists tested so far are well tolerated and hint some antitumor activity. Phase II trials aimed at demonstrating antiangiogenic/antitumor activities are ongoing (Stupp and Ruegg 2007). Important basic questions on the role of integrins in angiogenesis and their therapeutic targeting, however, have remained unanswered and new ones have emerged. In this section we review some of the open outstanding questions.

Most Relevant Targets

Endothelial cells can express up to 12 integrins (αVβ3, αVβ5, α5β1, α1β1, α2β1, α6β4, α3β1, α4β1, α6β1, α8β1, αVβ1, and αVβ8) (Alghisi and Ruegg 2006). At this point, we do not know which one of these integrins is the best therapeutic target for antiangiogenic treatments, and if angiogenic vessels in different tumors or at different stages of development may use different integrins. More preclinical work is needed to test and compare the suitability of individual integrins as therapeutic target and to evaluate the possibility of combined targeting.

Combination Therapies

Combined treatment with integrin antagonists and chemotherapy or radiotherapy has shown enhanced therapeutic efficacy in preclinical models (Ruegg and Mutter 2007). Since combination therapy appears to be a general rule for antiangiogenic treatments in humans, the critical issue is to define the best combination in term of drug association, timing, and schedule.

  • Radiotherapy. Integrin antagonists enhance efficacy of radiotherapy. Radiation was found to upregulate αVβ3 expression in endothelial cells and to induce activation of Akt/PKB, possibly as a mechanism for the tumor vasculature to escape or recover from radiation-induced injury. Inhibitors of αVβ3 integrin suppressed radiation-induced Akt/PKB phosphorylation, increased cell death and enhanced antiangiogenic and antitumor effects in xenograft models (Abdollahi et al. 2005; Ning et al. 2007). Using a different model, cilengitide sensitized tumors to radioimmunotherapy (Burke et al. 2002). These results reinforce the rationale of combining vascular integrin antagonists with radiotherapy.

  • Chemotherapy. Combination of ATN-161 (α5β1 antagonist) with 5-fluoroacil synergized the reduction of the number of liver metastases and tumor burden of CT26 colon cancer cells in mice (Stoeltzing et al. 2003). Liver tumors in the ATN-161 and ATN-161/5-FU groups had significantly fewer microvessels than tumors in the control or 5-FU-treated groups.

  • Tumor Necrosis Factor (TNF). TNF is used in combination with high dose chemotherapy in an isolation limb perfusion setting to treat advanced cancers of the limbs (Lejeune et al. 2006). The mechanism by which TNF exerts its antitumor activity involves detachment and death of angiogenic endothelial cells expressing αVβ3 (Ruegg et al. 1998). Integrin-mediated adhesion is required for TNF-induced Akt/PKB activation, an event essential for the survival of TNF-stimulated endothelial cells (Bieler et al. 2007). Consistent with these results, cilengitide sensitizes endothelial cells to TNF-induced death in vitro. Thus, combined administration of cilengitide may open new perspectives to the therapeutic use of TNF as anticancer agent.

  • Tyrosine Kinase Inhibitors (TKI). Since integrins facilitate signaling from several receptor tyrosine kinases, including ErbB2, VEGFR-2, EGF-R, and Met, it is reasonable to hypothesize that integrin inhibition may sensitize endothelial cells to currently available TKI antiangiogenic drugs (e.g., bevacizumab, sorafenib, sunitinib, temsirolimus) or to other TKI with antiangiogenic activities, such as EGFR antagonists (e.g., cetuximab or gefitinib), or PDGFRs inhibitors (e.g., Imatinib). Indeed, combined administration of cilengitide and SU5416, a VEGR-2 TKI reduced tumoral vessel density and intratumoral blood flow compared to single drug treatments (Strieth et al. 2006). α6β4 might be an interesting integrin to target in combination with ErbB2, EGFR, and Met inhibitors, since, in addition to antiangiogenic effects, it may also have direct antitumor activity, as α6β4 and ErbB2, EGF-R and Met are expressed on many carcinoma cells (Giancotti 2007). The endogenous antiangiogenic peptide tumstatin was shown to exert direct antitumoral effects in αVβ3, expressing glioma cells in vitro and in vivo by suppressing αVβ3-dependent Akt and mTOR signaling (Kawaguchi et al. 2006), suggesting the possibility that a combination strategy may be chosen in a way to target angiogenic endothelial cells and tumor cells.

Drug Targeting

Vascular integrins expressed on tumoral vessels, such as αVβ3 have been used to target drugs to tumors. Cationic nanoparticles coupled with an integrin αVβ3-targeting ligand were used to deliver a dominant-negative mutant Raf gene to angiogenic blood vessels in tumor-bearing mice, resulting in apoptosis of the tumor vessels and regression of established primary and metastatic tumors (Hood et al. 2002). Paclitaxel (Taxol), an antitumor drug commonly used for the treatment of advanced metastatic breast cancer, conjugated with an RGD-based peptide had a better uptake kinetic in vivo compared to free paclitaxel (i.e., 4 h for free PTX vs. 2 h for the PTX-RGD conjugate), although it did not show enhanced potency at the cellular level(Chen et al. 2005). These experiments demonstrate the feasibility of integrin-based targeted drug delivery to tumors. More recently, several studies reported that conjugation of αVβ3-targeting RGD peptides or peptidomimetics to carrier proteins (e.g., antibody), synthetic scaffold structures, or micelles (e.g., PEG-polyLys-associated with plasmid DNA) resulted in improved pharmacokinetics, retention in the tumor tissue, and cellular uptake (Mitra et al. 2006; Oba et al. 2007; Shin et al. 2007). αVβ3-integrin-targeted nanoparticles rapidly taken up by αVβ3-positive angiogenic vessels and tumors were developed for delivery and imaging purposes (Xie et al. 2007).

Tumor Imaging

Vascular integrins upregulated in angiogenic vessels have also been explored for noninvasive tumor imaging purposes. Most approaches have targeted αVβ3 in combination with positron emission tomography (PET) and Magnetic Resonance Imaging (MRI) imaging techniques (Choe and Lee 2007). The proof of concept experiment was reported already in 1998, where gadolinium-labeled LM609 was used to detect angiogenesis in a rabbit tumor model.

Before this approach be successfully translated into the clinic, however, substantial gains in sensitivity brought about by improved coils, pulse sequences, and contrast agents were needed (Barrett et al. 2007). Thanks to its high sensitivity, PET technology has been preferred and used in animal models and in humans to detect αVβ3 using 18F-labeled monomeric or multimeric RGD peptides. The level of expression αVβ3 detected by PET, correlated with the level of αVβ3 determined by immunohistochemistry, suggesting that this approach may be used for the noninvasive measurement of αVβ3, and monitoring antiangiogenic therapy in patients (Beer et al. 2006). 64Cu-DOTA-labeled Vitaxin (Abegrin) were used in animal models and showed high levels of late tumor activity accumulation (i.e., 71 h) post injection (Cai et al. 2006). Similarly, 99mTc-labeled RGD peptides were used to image tumors and angiogenic vascular beds by gamma camera (Decristoforo et al. 2006) or single photon emission computed tomography (SPECT) in experimental models (Liu et al. 2007). Recently, near-infrared fluorescence imaging coupled with 3D optical imaging systems have been used to image αVβ3-positive tumor vessels and tumor cells in mice using Cy5.5-RGD peptides (Hsu et al. 2006). Taken together, these results illustrate the potential of employing integrin-targeted molecular probes to image tumor vasculature and monitoring response to therapy.

Future Directions

New Generation of Extracellular Antagonists

While most efforts have been focused on the generation of small molecular inhibitors based on the RGD sequence or the ligand-binding pocket, recent studies have reported inhibitors acting in a RGD-independent manner, such as tumstatin (Maeshima et al. 2000) or ATN-161 (Livant et al. 2000). The resolution of the 3D structure of cilengitide-αVβ3 complex (Xiong et al. 2002) and of SJ749-α5β1 complex (Marinelli et al. 2005), allows for the exploration of additional regions of the receptor for binding of novel inhibitory molecules, “in silico” design and virtual screenings of improved or fully novel antagonists (Zhou et al. 2006). Furthermore, “broad spectrum” inhibitors blocking several angiogenic integrins (e.g., β1 and β3) may be developed, as suggested by the recent report of BCH-15046, a αVβ3, αVβ5 and α5β1 antagonist (Meerovitch et al. 2003).

Targeting the Integrin Intracellular Domains

Interaction of the cytoplasmic tail of the β subunit is essential for integrin function (Travis et al. 2003). Expression of isolated β integrin subunit cytoplasmic and transmembrane domains in adherent endothelial cells caused cell detachment and death in vitro and in vivo (Hasmim et al. 2005; Oguey et al. 2000), consistent with the notion that overexpression of isolated β-cytoplasmic domain competes for binding of essential cytoplasmic adaptor proteins (e.g., talin), resulting in “mechanical uncoupling”of the integrins from focal adhesions and cytoskeletal structures. These results also suggest the possibility of targeting the cytoplasmic domain for therapeutic purposes. Two main problems need to be addressed: the first one concerns integrin specificity (current constructs that do not differentiate between β1 and β3 integrins). The second problem concerns intracellular delivery: to allow penetration into the cell, an inhibitory peptide has to be fused to cytoplasmic transduction peptides (Kim et al. 2006). Alternatively, non-peptidic drugs may be developed to disrupt β3-tail interaction with structural or signaling cytoplasmic proteins.

Targeting Angiogenic Precursor Cells and Inflammatory Cells

Bone marrow cells are mobilized during tumor growth and recruited at tumor sites to promote tumor angiogenesis. While some of the cells include true endothelial precursors giving rise to mature endothelial cells, most of them are of monocyte/macrophage lineage (De Palma and Naldini 2006). Monocyte/macrophage is very sensitive to hypoxia and produces angiogenic factors and chemokines that stimulate tumor angiogenesis, progression, and metastasis (Condeelis and Pollard 2006). Since leukocytes and inflammatory cells use integrins to extravasate and migrate through the stroma, such as αLβ2, α4β1, αMβ2, αVβ3, or α5β1, it may be reasonable to inhibit their recruitment to tumor sites by targeting their integrins (Ulbrich et al. 2003). For example, antagonists of integrin α4β1 blocked extravasation of monocytes into tumor tissue and prevented monocyte macrophage colonization of tumors and tumor angiogenesis (Jin et al. 2006). Since it is also possible that some of the antiangiogenic effects observed with αVβ3 or α5β1 antagonists may be due to the inhibition of leukocyte recruitment, in future experimental and clinical studies it will be important to monitor the effect of integrin inhibitors on the recruitment of inflammatory cells to tumor sites. Of note is the observation that Cilengitide inhibited proliferation and differentiation of human endothelial progenitor cells in vitro (Loges et al. 2007).

Conclusions

Preclinical evidence indicates that integrins expressed in angiogenic endothelial cells are potentially relevant targets for antiangiogenic therapies in cancer. Early clinical trials have provided initial evidence of activity in human cancers, and ongoing clinical trials tell us whether they may bring benefits to human cancer treatment. If this is the case, besides further clinical trials, there will be a race to generate novel, more potent, and orally bioavailable “second generation” antagonists as well as to define the best combination strategy. The possibility of coupling the use of integrin antagonists for therapeutic purposes with non-invasive imaging of vascular integrins would open the possibility to select patients expressing high levels of the target, and therefore those who are most likely to benefit from the treatment.