key: cord-0749068-udcw1mx5
authors: Lu, Patrick Y.; Xie, Frank Y.; Woodle, Martin C.
title: Modulation of angiogenesis with siRNA inhibitors for novel therapeutics
date: 2005-03-31
journal: Trends in Molecular Medicine
DOI: 10.1016/j.molmed.2005.01.005
sha: 2fea166ad56fbacf1da0677c3b1a89e450083aa3
doc_id: 749068
cord_uid: udcw1mx5

Cancer and many other serious diseases are characterized by the uncontrolled growth of new blood vessels. Recently, RNA interference (RNAi) has reinvigorated the therapeutic prospects for inhibiting gene expression and promises many advantages over binding inhibitors, including high specificity, which is essential for targeted therapeutics. This article describes the latest developments using small-interfering RNA (siRNA) inhibitors to downregulate various angiogenic and tumor-associated factors, both in cell-culture assays and in animal disease models. The majority of research efforts are currently focused on understanding gene function, as well as proof-of-concept for siRNA-mediated anti-angiogenesis. The prospects for siRNA therapeutics, both advantages and looming hurdles, are evaluated.

Angiogenesis is the process of generating new capillary blood vessels from pre-existing blood vessels which involves multiple gene products expressed by various cell types and an integrated sequence of events. This uncontrolled process of new blood vessel growth from the preexisting circulation network is an important pathogenic cause of tumor growth, many blinding ocular conditions and inflammatory diseases [1] . Angiogenesis can be characterized distinctly as hemangiogenesis (HA; blood neovascularization) and lymphangiogenesis (LA; lymphatic neovascularization), the latter being an important initial step in tumor metastasis and transplant sensitization [2] . During recent years, much has been learned about the stimulators and inhibitors of HA and LA, and members of the vascular endothelial growth factor (VEGF) family have emerged as prime mediators of both processes [3] . Therefore, identifying and evaluating the specific inhibitors of pro-angiogenesis factors has been the focus of anti-angiogenesis research with a goal for therapeutic development. The emergence of RNA interference (RNAi; Box 1), a natural mechanism for posttranscriptional gene silencing (PTGS) [4] , offers a promising approach to develop a powerful class of inhibitors applicable to angiogenesis, with either a chemically synthesized small-interfering RNA (siRNA) oligonucleotide or a gene expression vector producing short-hairpin RNA (shRNA) as the therapeutic agent ( Figure 1 ) [5] . Here, the latest developments for using RNAi agents to regulate angiogenesis are reviewed, including studies to identify the genes involved in controlling the angiogenesis process and efforts to develop novel anti-angiogenic therapeutics for the treatment of cancer, ocular neovascularization and rheumatoid arthritis.

Hypoxia (inadequate oxygen), which is one of the key early initiators of angiogenesis, is followed by the production of nitric-oxide synthetases that are responsible for governing vascular tone and regulating growth factors, such as VEGF, angiopoietins, fibroblast growth factors (FGFs) and their receptors. Genes involved in matrix metabolism, including matrix metalloproteinases (MMPs), plasminogenactivator receptors and inhibitors and collagen prolyl hydroxylase, have also been reported as crucial in angiogenesis. The functional validation of angiogenic factors for their specific role has been greatly facilitated by the use of RNAi inhibitors, revealing a network involving the early activation of the VEGF pathway and interactions among MMPs and adhesion molecules, leading to the regulation of signal transduction pathways.

The VEGF pathway Several pathologies are associated with the upregulation of the VEGF pathway. The VEGF family consists of five growth factors that bind to and activate three distinct receptors. VEGF-A binds to VEGFR1 and VEGFR2, whereas placental growth factor (PIGF) and VEGF-B bind only to VEGFR1. VEGF-C and VEGF-D bind to VEGFR2 and VEGFR3.

Active intermediates of the endogenous RNA-interference process, small-interfering RNA oligos, or siRNAs, have enabled an easy-tomake and easy-to-use gene inhibitor that can be used intracellularly by an RNA-induced silencing complex (RISC) to degrade homologous mRNA with high specificity and potency ( Figure 1 ) [4] . Using siRNA to inhibit genes in vitro and in vivo has improved studies on the mechanism of action for many disease genes, including those involved in the angiogenesis process [5] . The capability of using siRNA in vivo to validate angiogenesis factors as drug targets is uniquely important, because its pathological impact can only be characterized accurately in animal disease models. With the emergence of clinically viable delivery vehicles, anti-angiogenesis RNAi agents appear to have a promising and unprecedented role for the treatment of many serious human diseases that result from excessive angiogenesis.

VEGF has received considerable attention. The transcription factor hypoxia inducible factor (HIF)-1 is a key determinant of hypoxia-regulated gene expression, including VEGF. The inhibition of HIF-1a by siRNA markedly attenuated the induction of VEGF and several other key genes, including heme oxygenase I (HO-1) and phosphoglycerate kinase (PGK) [6] , indicating a role for VEGF in oxygen-dependent cell-cycle regulation. Progesterone receptor (PR) B also preferentially regulates VEGF expression in breast cancer cells, identified using siRNA [7] . In cell-culture-based assays, the expression of VEGF 165 was specifically inhibited using siRNA in HeLa cells, ovarian carcinoma cells and melanoma cells [8] . VEGF 165 is the predominant protein among the major splice variants of VEGF-A, which include: VEGF 121 , VEGF 165 , VEGF 189 and VEGF 206 amino acids, each one comprising a specific exon addition. In a different study, siRNAs selectively blocked a splice variant of VEGF (VEGF 165b ) showing its inhibitory function; antibodies against VEGF cannot distinguish this variant from other isoforms [9] , giving an example of siRNA selectivity. The existence of this splicing to switch VEGF into an inhibitor potentially represents an interesting point for therapeutic intervention; that is, using VEGF 165b -specific siRNA to reduce the levels of VEGF protein. The inhibition of breast tumor growth by the intratumoral delivery of siRNA targeting VEGF has been demonstrated in an MDA-435 xenograft model [10] . In parallel studies of VEGF-siRNA intratumoral administration, the suppression of tumor angiogenesis and growth was also observed in a PtdCho-3 xenograft model [11] . In eye disease, VEGF-siRNA was used as an inhibitor of choroidal neovascularization (CNV) induced by laser photocoagulation in a murine retina model [12] . Using a ligand-directed nanoparticle that was amenable to systemic administration, VEGF-siRNA mediated anti-angiogenesis activity and suppressed ocular neovascularization induced by herpes simplex virus (HSV) infection in mice [13] .

Other studies have used siRNA to inhibit factors upstream of VEGF. When Src homology 2 domain adaptor protein (Shb) was inhibited with siRNA, VEGF-dependent cellular migration was reduced, resulting from a loss of stimulated phosphatidylinositol 3-kinase, the phosphorylation of focal adhesion kinase, the generation of focal adhesions and stress fiber formation [14] . Another protein, IQGAP1 (IQ-motif-containing GTPase-activating protein 1), which is expressed in endothelial cells (ECs), was found, through siRNA-mediated IQGAP1 knockdown, to be involved in VEGF-stimulated reactive oxygen species production, Akt phosphorylation, endothelial migration and proliferation [15] .

Studies of VEGF receptors have also benefited from siRNA technology. Using a neuroblastoma syngenic tumor model [16] and HSV-induced ocular neovascularization [13] , siRNAs specific to murine VEGFR2 inhibited angiogenesis in both tissues, reducing tumor growth and ocular neovasculature, respectively. Similarly, siRNAs specific to murine VEGFR1 also demonstrated antiangiogenic effects in the mouse ocular model [13] .

Although most vascular basement membrane (BM) components sustain the growth and survival of the vascular endothelium, the MMPs and vascular integrins have emerged as key regulators of angiogenesis. MMP9 and MMP2 are important for the mobilization of sequestered VEGF and initiation of tumor angiogenesis [17] , whereas specific integrins mediate interactions between endothelial cells and the BM by activating the integrin receptor signaling that controls many key functions, such as proliferation [18] .

The knockdown of MMP-9 with siRNA [19] concurrently resulted in increased levels of surface E-cadherin and a redistribution of b-catenin at the plasma membrane, in addition to its physical association with E-cadherin. In a cell-culture assay, the reduction of bovine aortic smooth muscle cell (BASMC) migration caused by the incubation with conditioned media can be completely reversed by siRNA knockdown of MMP-2, but not MMP-9, expression [20] . To indirectly block the induction of MMP activity, a novel MMP regulator, RECK (reversion-inducing cysteine-rich protein, with Kazal motifs), was downregulated using siRNA [21] , causing diminished activation of MMP-2. In a separate study, siRNA knockdown of a ubiquitous Mint isoform protein, Mint-3, inhibited membrane type 5 (MT5)-MMP activity [22] , indicating that Mint proteins might be the adaptor proteins that regulate the trafficking of MT-MMPs.

The cleavage of CD44, which is a cell-surface glycoprotein that is involved in cell adhesion and migration and contributes to the migration and invasion of tumor cells, is regulated by MMPs. It can be suppressed by the metalloproteinase inhibitor KB-R7785 and tissue inhibitor of metalloproteinases-1 (TIMP-1) or enhanced by the metalloproteinase disintegrin ADAM10, as demonstrated by siRNA-mediated knockdown [23] . A different cell-surface molecule, CD13/aminopeptide N (CD13/APN), has been identified as a potent regulator of angiogenesis and its transcription in endothelial cells is induced by VEGF through the RAS-MAPK (mitogen-activated protein kinase) pathway. The role of Ets-2 in the induction of CD13/APN was revealed when Ets-2 mRNA and protein were downregulated using Ets-2-specific siRNA [24] . Another metalloprotease disintegrin, ADAM12, was characterized by siRNA knockdown in C2C12 myoblast cells, suggesting that ADAM12-mediated adhesion and/or signaling might have a role in the determination of the pool of reserve cells during myoblast differentiation [25] . This protein has also been recognized as a key enzyme implicated in ectodomain shedding of membraneanchored heparin-binding proHB-EGF-dependent epidermal growth factor receptor (EGFR) transactivation [26] . When an siRNA for Pacsin 3, which is a protein bound to ADAM12, was used in HT1080 cells, the inhibition of this gene attenuated the shedding of proHB-EGF induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) and angiotensin II. FAK has a crucial role in the adhesion to collagens. Using FAK siRNAs to inhibit its expression resulted in a strong inhibition of adhesion not only to collagen I but also to collagen IV and fibronectin [27] .

Integrins initiate signal transduction through intracellular molecules and collaborate with other membrane receptor-mediated signal pathways, including the transforming growth factor (TGF)b1 pathway. When the effects of cell adhesion status on the TGFb1-mediated Erk1/2 regulation was evaluated in a gastric carcinoma cell variant [28] , the role of Smad protein in enhancing TGFb1-mediated Erk1/2 activation was revealed with a Smad2 siRNA. Smad2 protein is part of a signal transduction pathway leading to the transcriptional regulation of multiple cellular processes, such as cell proliferation, apoptosis and differentiation. The effects of carcinoembryonic antigen-related cell-adhesion molecule 6 (CEACAM6) crosslinking on pancreatic adenocarcinoma cellular interaction with extracellular matrix (ECM) components, vitronectin (avb3 integrin) and fibronectin (a5b1 integrin) were characterized using siRNA knockdown of CEACAM6, leading to increased ECM component adhesion [29] . Integrin-linked kinase (ILK) stimulated the expression of VEGF through the stimulation of HIF1a expression in a PKB-Akt-and mTOR-FRAP-dependent manner. With siRNA inhibition of ILK, many significant effects were observed, including the inhibition of HIF-1a and VEGF expression, in addition to reduced VEGFmediated endothelial cell migration and capillary formation in vitro, culminating in reduced angiogenesis in vivo [30] . These data demonstrated an essential role for ILK in two key aspects of tumor angiogenesis: VEGF expression by tumor cells and VEGF-stimulated blood vessel formation. Interestingly, a widely expressed focal adhesion protein, PINCH-1, was found to form a ternary complex with ILK and a-parvin [31] . Using siRNAs, PINCH-1 and ILK, but not a-parvin, were found to be crucial for promoting cell spreading and motility.

The cooperative roles of platelet-derived growth factor receptor (PDGFr) and avb3 integrin in the glioblastoma cell migration have been shown by PDGF stimulation of vitronectin-adherent cells, promoting the recruitment of avb3 integrin [32] . The Lck/yes-related novel protein (Lyn) was preferentially associated with avb3 integrin in the victronectin-adherent cells, both in the presence and absence of PDGF stimulation. The downregulation of Lyn expression using siRNA inhibits the cell migration that is mediated by avb3 integrin in PDGF-stimulated cells. In fibrotic liver, the fate of stellate cells was influenced by the ECM through an intermediary of avb3 integrin, shown using siRNA to silence the a(v) subunit [33] . In a different study, microfilaments were found to associate with avb3-integrin-positive focal contacts in endothelial cells. When vimentin expression was inhibited by siRNA, the cells assembled focal contacts smaller than normal and showed decreased adhesion [34] . A laminin adhesion receptor, a6b4 integrin, was demonstrated, using an siRNA targeting either subunit of a6b4, to have a key role in the invasive phenotype of MDA-MB-231 breast carcinoma cells when surface expression was inhibited [35] .

In addition to receptors for the VEGF family, which includes PDGF, the inhibition of other receptors, such as EGFR (erbB1), is a promising anti-angiogenesis approach. When erbB1 expression was blocked (90% inhibition) in A431 human epidermal carcinoma cells using siRNA, EGF induction of tyrosine phosphorylation was lost along with cell proliferation, and apoptosis was induced [36] . Similarly, using retrovirus-mediated transfer of Her-2/neu siRNA, breast and ovarian tumor cells exhibited slower proliferation, increased apoptosis, increased G 0 -G 1 arrest and decreased tumor growth [37] . Her-2/neu siRNA also increased the expression of the antiangiogenic factor thrombospondin-1 and decreased the expression of VEGF, suggesting that Her-2/neu stimulates tumor growth in part by regulating angiogenesis. When a pSUPER plasmid expressing shRNA for S100A10, a key plasminogen receptor on the extracellular cell surface, was transfected into colorectal (CCL-222) cancer cells, the RNAi-mediated downregulation of S100A10 resulted in a 45% loss of plasminogen binding and a complete loss in plasminogen-dependent cellular invasiveness [38] . An inducible RNAi mediated the inhibition of endogenous CXCR4, a receptor with pleiotropic roles in angiogenesis, host immune response, homing and tumor metastasis, significantly inhibited a breast cancer cell migration in vitro [39] . Furthermore, a pro-apoptotic protein, Bim, was identified, using siRNAs, as a crucial mediator of anoikis in epithelial cells [40] . The communication between G-protein-coupled receptor (GPCR) and EGFR signaling systems that are involved in the cell-surface proteolysis of EGF-like precursors was revealed using siRNA, which demonstrated the role of amphiregulin (AR) and prevented GPCR-induced EGFR tyrosine phosphorylation, downstream mitogenic signaling events, cell proliferation, migration and activation of the survival mediator Akt/PKB [41] . In the same study, siRNA for the metalloprotease-disintegrin TNFa-converting enzyme (TACE) suppressed GPCR-stimulated AR release, EGFR activation and downstream events.

Factors in signal transduction pathways VEGF promotes angiogenesis through the activation of signaling pathways that include the activation of a series of relay proteins by the binding of VEGF to its appropriate receptor, which transmits a signal to the nucleus of the endothelial cells; the nuclear signal ultimately prompts a group of genes to make products needed for new endothelial cell growth. VEGF stimulation of sphingosine kinase (SPK) affects not only endothelial cell signaling but also tumor cells expressing VEGF receptors [42] . In T24 bladder tumor cells, VEGF inhibition reduced cellular sphingosine levels and raised those of sphingosine-1-phosphate. siRNA targeting SPK1, but not SPK2, blocked VEGF-induced accumulation of Ras-GTP and phospho-ERK, but not EGF-induced accumulation of phospho-ERK1/2. The involvement, revealed by siRNA, of diacylglycerol kinase a (Dgka) in hepatocyte growth factor (HGF)-stimulated cell migration, which impaired angiogenesis in vitro, indicated its essential role in both proliferative and migratory response to VEGF, and suggested that it is a novel therapeutic target for controlling angiogenesis [43] . In a different study, the inhibition of Mcl-1 by siRNA decreased proliferation and induced apoptosis, supporting the notion that VEGFinduced multiple myeloma cell proliferation and survival is mediated by Mcl-1, and provided the preclinical framework for novel therapeutics targeting both Mcl-1 and/or VEGF to improve patient outcome in multiple myeloma [44] . Growth-arrest-specific protein 1 (Gas1), which is upregulated by the junction membrane protein vascular endothelial cadherin (VEC) in 293 cells, was inhibited by siRNA in cell and allantois organ cultures, which resulted in endothelial cells that could not be protected from apoptosis by VEGF, once again providing evidence for another potential drug target [45] .

One of the key factors in the downstream signal transduction pathway of VEGF receptors is B-Raf kinase, which constitutively activates the MEK-ERK pathway. Cell proliferation of melanoma cells with increased B-Raf levels was strongly inhibited by siRNA-mediated depletion of the mutant B-Raf protein [46] . When FAK was silenced using siRNA in primary human colon cancer cells or SW620 colonocytes, pressure-stimulated adhesion was prevented and pressure-activated FAK397, Src and FAK576 phosphorylation were also ablated [47] . Integrinlinked kinase-associated serine-threonine phosphatase 2C (ILKAP) selectively associates with ILK to modulate cell adhesion and growth factor signaling. When ILKAP is suppressed with siRNA, cell entry into S phase increased, consistent with its antagonism of ILK [48] . The role of diphosphoinositol polyphosphates (DIP) was also characterized by siRNA-mediated downregulation, indicating that DIP inactivates Rho and activates Rac following EGF stimulation [49] . Using siRNA to inhibit Disabled-2 (DAB2) protein expression in K562 cells modulated both cell-cell adhesion and the phosphorylation of MAPK [50] . Knocking down 3-phosphoinositide-dependent protein kinase-1 (PDK1) with siRNA demonstrated that PDK1 maintains steady-state phosphorylated MEK levels and cell growth [51] . Interestingly, the downregulation of PDK1 reduced MEK and MAPK activities but could not prolong MAPK signaling. The interaction of (thyroid receptor interacting protein 6) TRIP6/ZRP-1 (zyxinrelated protein 1) with lysophosphatidic acid (LPA) receptor was induced by LPA and associated with the activation of actin rearrangement, focal adhesion assembly and cell migration [52] , as revealed by TRIP6 siRNA in SKOV3 ovarian cancer cells.

RNAi is an endogenous mechanism for the potent and specific inhibition of gene expression, which can be diverted to act on cellular genes by introducing siRNA agents. Their success as a powerful research tool, illustrated by the many advances described above, is fueling enthusiasm for siRNA as a novel modality for antiangiogenesis therapeutics, a large unmet clinical need (Box 2).

Anti-VEGF siRNA to treat cancer There are two distinct approaches to achieve antiangiogenesis activity for cancer treatments: (i) activating endogenous or exogenously delivering anti-angiogenesis factors; or (ii) delivering inhibitors to reduce the activities of endogenous pro-angiogenesis factors. siRNA is a particularly useful inhibitor with both potency and sequence-specific selectivity that has been demonstrated to induce a phenotypic response in cell-culture studies of anti-angiogenesis (Tables 1-4) . However, their therapeutic potential will only be realized when the in vivo anti-angiogenesis efficacy of the siRNA agents can be achieved with clinically feasible delivery systems [53] .

Using intratumoral delivery of VEGF-targeted siRNA, the inhibition of tumor growth was observed in MDA MB-435 and MCF-7 human xenograft breast cancer models [10] . In tumors expressing both thrombospondin-1 (TSP1) and VEGF, the effect of TSP1 to reduce vascularization and tumor growth were restored using the Box 2. siRNA, antisense, ribozyme and DNAzyme

RNAi is a newly discovered cellular pathway for the silencing of sequence-specific genes at the mRNA level by the introduction of cognate double-stranded RNA. Similarly, gene-silencing nucleic acids, such as ribozymes, DNA enzymes (DNAzymes), antisense oligonucleotides (ODNs) and siRNAs, depend on hybridization to accessible sites within target mRNA for activity. However, the accurate prediction of accessible sites as a means to facilitate reagent design is problematic. In a study attempting to evaluate the use of scanning arrays for the effective design of ribozyme, DNAzyme and siRNA sequences targeting the EGFR mRNA [67] , all three types complementary to the same site were effective in a dosedependent manner, using Lipofectin-mediated transfection. In fact, the effects achieved correlated in all cases with concomitant dosedependent reduction in EGFR protein expression. However, the ribozyme and DNAzyme exhibited similar but low potencies, with IC 50 values of w750 nM, whereas siRNA was significantly more potent, with an IC 50 of w100 nM. In a separate comparison, the combination of plasmid-expressed antisense (AS)-and sense (S)-RNA sequences was more effective than expressed AS-RNA alone for target gene knockdown. Expressed S-RNA alone had no effect [68] . Furthermore, the potency of 22-nucleotide siRNAs targeting the reporter gene luciferase was one order of magnitude stronger than that of a phosphorothioate AS-DNA. Overall, many more studies comparing the efficiencies of gene silencing by siRNA with other nucleic acid inhibitors find, in most cases, better siRNA potency [5] . systemic administration of aqueous VEGF siRNA but failed to show signs of target inhibition [54] . In the same report, local delivery of the siRNA into the tumors had no effect on VEGF expression. In a different study, intratumoral delivery of VEGF siRNA with a cationic carrier resulted in dramatically suppressed angiogenesis and growth [11] . These preclinical studies illustrate the growing interest in siRNA-based anti-angiogenesis therapeutics for the treatment of cancers.

It has been recognized that the local delivery of antitumor agents is limited to only a few tumor types with clinical relevance. Therefore, systemic administration of siRNA will provide the greatest clinical benefit as a treatment, especially for disseminated metastatic cancer. To that end, VEGF-pathway siRNA agents with in vivo validated activity were further evaluated by systemic administration to mice bearing neuroblastoma tumors using a ligand-directed nanoparticle carrier, and exhibited the inhibition of target expression, angiogenesis and tumor growth after repeated dosing [16] . The observed efficacy was achieved with a potency amenable to clinical application, further strengthening the promise of siRNA as a therapeutic modality.

Anti-VEGF siRNA to treat ocular neovascularization diseases New and uncontrolled blood vessel development in eyes is a pivotal process in the pathogenesis of several ocular neovascularization (NV) diseases, including age-related macular degeneration (AMD), diabetic retinopathy (RA) and herpes simplex virus (HSV)-induced stromal keratitis (SK). These ocular diseases often lead to blindness; unfortunately, managing them therapeutically is challenging. The systemic administration of ligand-directed nanoparticles carrying anti-VEGF siRNA in a mouse SK model was applied to measure its anti-angiogenesis therapeutic potential [13] . In the study, potent siRNAs targeting VEGFA, VEGFR1 and VEGFR2 were validated first in vitro, and mixed with a polymer conjugate to form nanoparticles 100-120 nm in diameter (Box 3). The tri-functional polymer was composed of branched polyethylenimine at one end, polyethylene glycol (PEG) in the middle and an RGD peptide motif at other end. This polymer was found to self assemble with negatively charged siRNA into a particle with the RGD peptide exposed on its surface [16] . The RGD peptide is a specific ligand for the avb3 and a5b1 integrins of activated endothelial cells of the neovasculature in the diseased tissue, and PEG helps to prevent nonspecific binding to other tissues, such that the VEGF siRNA can be delivered in a ligand-directed endocytosis. By comparison, subconjuctival administration of siRNA resulted in weaker and shorter anti-angiogenesis efficacy. In addition, the combination of siRNAs targeting three pro-angiogenesis genes, VEGF, VEGFR1 and VEGFR2, achieved stronger efficacy than that of siRNA only targeting one of the three proangiogenesis genes. Using a different model, the feasibility of siRNAmediated anti-angiogenesis in retinal cells in vitro and in the murine retina in vivo was evaluated [12] . siRNAs specific to murine and human VEGF were tested in vitro using chemically induced hypoxia to modulate endogenous VEGF expression. In vivo studies evaluating the effects of naked siRNA on levels of VEGF were performed first by co-injecting recombinant adenoviruses carrying enhanced green-fluorescent protein (eGFP) or hVEGF cDNAs, then by using laser-induced choroidal neovascularization (CNV) along with subretinal delivery of the appropriate siRNAs in mice. The siRNAs effectively and specifically inhibited hypoxia-induced hVEGF levels in human cell lines or with adenoviral-expressed hVEGF in vivo. Subretinal delivery of mVEGF-siRNA significantly inhibited CNV after laser photocoagulation in mice.

Anti-angiogenesis siRNA to treat rheumatoid arthritis Rheumatoid arthritis (RA) is one of the most severe articular diseases, characterized by synovial hyperplasia followed by impaired joint function with a severe impact on quality of life. Although the mechanisms that regulate synovial cell outgrowth are not fully understood, there is evidence that VEGF has an important role in the development of RA [55] [56] [57] [58] . In patients with RA, both serum basic fibroblast growth factor (bFGF) and VEGF concentrations were increased, and when those patients were treated with steroids, their synovial fluid levels of bFGF and VEGF were reduced [56] . The investigation of polymorphisms of the VEGF gene in patients with RA and their relationship with clinical features and the radiographic progression of joint disease indicated that the frequencies of two haplotypes (CGCT and AAGT) were significantly increased in patients with RA compared with controls [55] . The VEGF receptor inhibitor PTK787/ ZK222584 caused a dose-dependent reduction in the vascularity of a mouse RA model and it inhibited knee swelling by 40% in antigen-induced arthritis and inhibited both severity scores (by 51%) and global histological scores in the mouse model [57] . Furthermore, treatment using infliximab, an anti-TNFa antibody resulted in the reduction of soluble adhesion molecules and VEGF and achieved a rapid suppression of disease activity [58] . This indicates that the pathology of RA is closely related to the overexpression of VEGF. One signature in RA joints is the overexpression of TNF which, with other cytokines, induced angiogenesis in inflammation tissue. Therefore, using siRNA inhibitors to modulate the relevant pathways might provide therapeutic potentials for the treatment of RA.

After primary RA synovial fibroblasts (RASFs) were treated by TNFa, several genes were selected, using microarray analysis, based upon their differential expression status [59] . Using siRNA to downregulate one of these genes, GG2-1, which is a TNFa-inducible FLICE-like inhibitory protein (FLIP)-like gene, significantly enhanced apoptosis, decreased proliferation and decreased the production of MMP-1 in TNFa-stimulated RASFs. Akt protein kinase B had an important role in TNFa-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in rheumatoid synovial cells. Using siRNA to inactivate Akt significantly increased TRAIL-mediated apoptosis in synovial cells [60] . Mutations in the human CIAS1 gene, which codes for Cryopyrin, have been identified in a continuum of inflammatory disorders. Cryopyrin mutants induce potent NF-kB activity that is dependent on the expression of ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), an adaptor protein previously suggested to mediate Cryopyrin signaling. The induction of Cryopyrin activity resulted in ASC binding and the secretion of IL-1b, an effect that was abolished by the inhibition of ASC expression with siRNA [61] . Even though no efficacy data have been reported using siRNA to reverse RA pathology in animal disease models, the potential application is very promising; the downregulation of RA-causing cytokines and their receptors and of VEGF and its signaling factors, Box 3. A cocktail of siRNAs targeting multiple genes Many human diseases result from the overexpression of multiple endogenous and exogenous pathogenic genes. Angiogenesisrelated diseases represent this typical characteristic caused by the abnormal overexpression of multiple genes. Moreover, disease progression and the development of drug resistance can also circumvent the effect of single-drug treatments. One strategy to overcome those hurdles is using a combination of multiple drugs. siRNAs should provide a unique advantage for combination therapy by combining multiple siRNA oligos targeting multiple diseasecausing genes without dramatic differences in the drug chemical properties. The combination of siRNAs targeting VEGFA, VEGFR1 and VEGFR2 has demonstrated an improved anti-angiogenesis potency compared with siRNAs targeting only one factor [13] . The combination appears to have achieved a synergistic effect by the knockdown of all three targets simultaneously, resulting in a potent anti-angiogenesis efficacy. Anti-angiogenic molecules can fall into five main categories according to their mode of action: (i) inhibitors of pro-angiogenic growth factors and their corresponding receptors, such as VEGF, VEGFR1 and VEGFR2, bFGF and FGF receptors and PDGF; (ii) protease inhibitors that prevent the breakdown of the surrounding matrix, which is needed for blood-vessel growth; (iii) endogenous inhibitors of angiogenesis, such as endostatin; (iv) inhibitors of cellular adhesion molecules; and (v) molecules with undefined mechanisms. Owing to the complications of angiogenesis pathways, combining multiple siRNAs targeting angiogenic factors in each category might enable the identification of potent antiangiogenic agents for potential therapeutic applications. The same strategy was applied during an attempt to achieve stronger anti-SARS coronavirus activity with remarkable success [69] . either individually or with siRNAs in combination, represents a novel approach for the treatment of RA.

Concluding remarks: siRNA, a powerful antiangiogenesis agent The modulation of angiogenesis pathways using siRNA inhibitors of gene expression has proven to be a powerful approach for validating gene functions of the relevant factors in vitro and in vivo. The rapid development of experimental evidence improving our understanding of angiogenesis and approaches for intervention has clearly demonstrated the power of RNAi, which has unique advantages over previous methods for gene-function studies [62] , including nucleic acid inhibitors (antisense, ribozyme and DNAzyme). The information obtained for each individual factor not only contributes to the global understanding of angiogenesis but also provides strong support for efforts to develop novel therapeutics for the treatment of various angiogenesis diseases, including cancer, ocular neovascularization diseases and rheumatoid arthritis. Nonetheless, although the in vivo studies described in this article provide the groundwork for potential therapeutic applications of RNAi technology, thorough preclinical studies, such as pharmacology and toxicology, of specific siRNA therapeutic candidates remain. Some reports have revealed the occurrence of incomplete selectivity of siRNA oligos, including 'off-target effects' [63] and the activation of interferons [64, 65] , which raises concerns for safety and unwanted side effects. It is fair to say that all those studies were based upon cellculture experiments without support from the in vivo study. A recent study using systemic delivery (intraperitoneal and intravenous) of naked siRNA duplexes into mice indicated a lack of interferon response compared with that of polyinosinic acid: polycytidylic acid [poly(I:C)], an analog of the long-dsRNA ligand for Toll-like receptor 3 [66] . With clinically viable delivery emerging, siRNA inhibitors have achieved therapeutic efficacy in several animal disease models of angiogenesis. The dual-targeted therapeutics (Box 4) and the combination of siRNAs targeting multiple pro-angiogenesis factors have further demonstrated the advantages of this Box 4. Dual-targeted anti-angiogenesis therapeutics Inhibiting the expression of drug-target genes with siRNAs is quite different from the inhibitory actions of conventional drugs such as monoclonal antibodies and small-molecule inhibitors, which bind to and inhibit existing proteins. This represents a novel and distinct class of therapeutic inhibitors that will probably have a different range of applicable drug targets, albeit with substantial overlap. Most importantly, the selectivity conveyed by the gene sequence, which could potentially be as stringent as a single nucleotide difference between two genes, promises to aid in therapeutic approaches by providing much better target selectivity. In addition, using a tissue-targeting nanoparticle delivery with ligand-directed tissue selectivity enables the potential for a dual-targeted therapeutic approach, and one that is uniquely useful for enhancing the extracellular stability of the siRNA agent, avoiding effects on the targeted gene in normal tissues and increasing the activity of siRNA in disease tissue ( Figure I ). Using this systemic delivery of siRNAs targeting VEGF pathway factors at sites of neovascularization, anti-angiogenesis efficacies were achieved in a neuroblastoma tumor model [16] and in an HSV-induced mouse ocular SK model [13] . Identifying ligands binding to adhesion molecules and integrin receptors for tissue-targeted delivery of siRNA agents will provide synergistic effects to further improve antiangiogenesis efficacy. A layered, self-assembled siRNA nanoparticle system [16] is used to deliver siRNAs specific to genes involved in the VEGF pathway to achieve a dual-targeted antiangiogenesis therapeutic effect. The first targeted effect is achieved by using the RGD peptide as the surface ligand of the nanoparticle to target integrin expression on tumor neovasculature and thus avoid effects on the targeted gene in normal tissues. The second target effect is achieved by using siRNA specifically against genes involved in VEGF pathway, such as VEGF, VEGFR1, and VEGFR2, to inhibit the angiogenesis activity in the targeted tumor tissue and thus generate anti-tumor efficacy.

novel therapeutic modality. As with any emerging technologies, many issues related to siRNA therapeutics must be addressed diligently before the success of this novel approach can be achieved in clinics.

Clinical translation of angiogenesis inhibitors

Lymphangiogenesis and cancer metastasis

VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment

Gene silencing in mammals by small interfering RNAs

siRNA-mediated antitumorigenesis for drug target validation and therapeutics

RNA interference for HIF-1a inhibits its downstream signalling and affects cellular proliferation

Progestin-dependent induction of vascular endothelial growth factor in human breast cancer cells: preferential regulation by progesterone receptor B

siRNA agents inhibit oncogene expression and attenuate human tumor cell growth

Differentiated human podocytes endogenously express an inhibitory isoform of vascular endothelial growth factor (VEGF 165b ) mRNA and protein

Delivering siRNA to animal disease models for validation of novel drug targets in vivo

A small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics

Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model

Inhibition of ocular angiogenesis by siRNA targeting vascular endothelial growth factor-pathway genes; therapeutic strategy for herpetic stromal keratitis

The adaptor protein shb binds to tyrosine 1175 in vascular endothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration

QGAP1, a novel vascular endothelial growth factor receptor binding protein, is involved in reactive oxygen species-dependent endothelial migration and proliferation

Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically-stablilized nanoparticle

Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis

Basement membranes: structure, assembly and role in tumor angiogenesis

Matrix metalloproteinase-9 silencing by RNA interference triggers the migratory-adhesive switch in Ewing's sarcoma cells

Cyclic strain-induced endothelial MMP-2: role in vascular smooth muscle cell migration

Histone deacetylase inhibitor up-regulates RECK to inhibit MMP-2 activation and cancer cell invasion

Mint-3 regulates the retrieval of the internalized membrane-type matrix metalloproteinase, MT5-MMP, to the plasma membrane by binding to its carboxyl end motif EWV

Engagement of CD44 promotes Rac activation and CD44 cleavage during tumor cell migration

CD13/APN transcription is induced by RAS/MAPK-mediated phosphorylation of Ets-2 in activated endothelial cells

Role of metalloprotease disintegrin ADAM12 in determination of quiescent reserve cells during myogenic differentiation in vitro

PACSIN3 binds ADAM12/meltrin a and upregulates ectodomain shedding of heparin-binding epidermal growth factor-like growth factor

Absence of adhesion triggers differential FAK and SAPKp38 signals in SW620 human colon cancer cells that may inhibit adhesiveness and lead to cell death

Smad2 mediates Erk1/2 activation by TGF-b1 in suspended, but not in adherent, gastric carcinoma cells

c-Src-dependent cross-talk between CEACAM6 and avb3 integrin enhances pancreatic adenocarcinoma cell adhesion to extracellular matrix components

Regulation of tumor angiogenesis by integrinlinked kinase (ILK)

PINCH-1 is an obligate partner of integrinlinked kinase (ILK) functioning in cell shape modulation, motility, and survival

The pattern of enhancement of Src kinase activity on platelet-derived growth factor stimulation of glioblastoma cells is affected by the integrin engaged

Engagement of avb3 integrin regulates proliferation and apoptosis of hepatic stellate cells

The vimentin cytoskeleton regulates focal contact size and adhesion of endothelial cells subjected to shear stress

Use of RNA interference to inhibit integrin (a6b4)-mediated invasion and migration of breast carcinoma cells

Small interfering RNAs suppress the expression of endogenous and GFP-fused epidermal growth factor receptor (erbB1) and induce apoptosis in erbB1-overexpressing cells

Inhibition of breast and ovarian tumor growth through multiple signaling pathways by using retrovirus-mediated small interfering RNA against Her-2/neu gene expression

RNA interference-mediated silencing of the S100A10 gene attenuates plasmin generation and invasiveness of Colo 222 colorectal cancer cells

Down-regulation of CXCR4 by inducible small interfering RNA inhibits breast cancer cell invasion in vitro

Integrins and EGFR coordinately regulate the pro-apoptotic protein Bim to prevent anoikis

TACE cleavage of proamphiregulin regulates GPCR-induced proliferation and motility of cancer cells

Sphingosine kinase mediates vascular endothelial growth factor-induced activation of ras and mitogen-activated protein kinases

Activation of diacylglycerol kinase a is required for VEGF-induced angiogenic signaling in vitro

VEGF induces MCL-1 upregulation and protects multiple myeloma cells against apoptosis

Gas1 is induced by VE-cadherin and vascular endothelial growth factor and inhibits endothelial cell apoptosis

Mutation of B-Raf in human choroidal melanoma cells mediates cell proliferation and transformation through the MEK/ERK pathway

Pressure activates colon cancer cell adhesion by inside-out focal adhesion complex and actin cytoskeletal signaling

ILKAP regulates ILK signaling and inhibits anchorage-independent growth

DIP (mDia interacting protein) is a key molecule regulating Rho and Rac in a Src-dependent manner

Disabled-2 small interfering RNA modulates cellular adhesive function and MAPK activity during megakaryocytic differentiation of K562 cells

Involvement of 3-phosphoinositide-dependent protein kinases-1 in the MEK/MAPK signal-transduction pathway

TRIP6 enhances lysophosphatidic acid-induced cell migration by interacting with the lysophosphatidic acid 2 receptor

Delivering siRNA in vivo For functional genomics can novel therapeutics

siRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth

VEGF gene polymorphisms and susceptibility to rheumatoid arthritis

Synovial concentrations of the angiogenic peptides bFGF and VEGF do not discriminate rheumatoid arthritis from other forms of inflammatory arthritis. Scand

Angiogenesis inhibition by the novel VEGF receptor tyrosine kinase inhibitor, PTK787/ZK222584, causes significant anti-arthritic effects in models of rheumatoid arthritis

Reduction of soluble adhesion molecules (sICAM-1, sVCAM-1, and sE-selectin) and vascular endothelial growth factor levels in serum of rheumatoid arthritis patients following multiple intravenous infusions of infliximab

Novel tumor necrosis factor a-regulated genes in rheumatoid arthritis

Akt is an endogenous inhibitor toward tumor necrosis factor-related apoptosis inducing ligand-mediated apoptosis in rheumatoid synovial cells

Cryopyrin-induced interleukin 1b secretion in monocytic cells: enhanced activity of disease-associated mutants and requirement for ASC

Pharmaceutical prospects for RNA interference

Expression profiling reveals off-target gene regulation by RNAi

Activation of the interferon system by shortinterfering RNAs

Small interfering RNAs mediate sequenceindependent gene suppression and induce immune activation by signaling through toll-like receptor 3

Lack of interferon response in animals to naked siRNAs

Gene silencing nucleic acids designed by scanning arrays: anti-EGFR activity of siRNA, ribozyme and DNA enzymes targeting a single hybridization-accessible region using the same delivery system

RNA interference may be more potent than antisense RNA in human cancer cell lines

Prophylactic and therapeutic effects of small interfering RNA targeting SARS-coronavirus