Human IgG1 antibodies suppress angiogenesis in a target-independent manner.

Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world's population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in mice. Here we show bevacizumab suppressed angiogenesis in three mouse models not via Vegfa blockade but rather Fc-mediated signaling through FcγRI (CD64) and c-Cbl, impairing macrophage migration. Other approved humanized or human IgG1 antibodies without mouse targets (adalimumab, alemtuzumab, ofatumumab, omalizumab, palivizumab and tocilizumab), mouse IgG2a, and overexpression of human IgG1-Fc or mouse IgG2a-Fc, also inhibited angiogenesis in wild-type and FcγR humanized mice. This anti-angiogenic effect was abolished by Fcgr1 ablation or knockdown, Fc cleavage, IgG-Fc inhibition, disruption of Fc-FcγR interaction, or elimination of FcRγ-initated signaling. Furthermore, bevacizumab's Fc region potentiated its anti-angiogenic activity in humanized VEGFA mice. Finally, mice deficient in FcγRI exhibited increased developmental and pathological angiogenesis. These findings reveal an unexpected anti-angiogenic function for FcγRI and a potentially concerning off-target effect of hIgG1 therapies.

ERK1/2 activation is a therapeutic target in age-related macular degeneration.

Deficient expression of the RNase III DICER1, which leads to the accumulation of cytotoxic Alu RNA, has been implicated in degeneration of the retinal pigmented epithelium (RPE) in geographic atrophy (GA), a late stage of age-related macular degeneration that causes blindness in millions of people worldwide. Here we show increased extracellular-signal-regulated kinase (ERK) 1/2 phosphorylation in the RPE of human eyes with GA and that RPE degeneration in mouse eyes and in human cell culture induced by DICER1 depletion or Alu RNA exposure is mediated via ERK1/2 signaling. Alu RNA overexpression or DICER1 knockdown increases ERK1/2 phosphorylation in the RPE in mice and in human cell culture. Alu RNA-induced RPE degeneration in mice is rescued by intravitreous administration of PD98059, an inhibitor of the ERK1/2-activating kinase MEK1, but not by inhibitors of other MAP kinases such as p38 or JNK. These findings reveal a previously unrecognized function of ERK1/2 in the pathogenesis of GA and provide a mechanistic basis for evaluation of ERK1/2 inhibition in treatment of this disease.

Evaluation of extended release brimonidine intravitreal device in normotensive rabbit eyes.

PURPOSE: To evaluate the safety profile of a brimonidine extended release intravitreal implant, in normotensive rabbit eyes. METHODS: Devices were made from hollow poly-l-lactic acid (PLA) tubes and contained hundred micrograms of brimonidine pamoate. Device was injected intravitreally in one eye of 12 New Zealand pigmented rabbits, whereas other eye was injected with a sham implant in masked fashion. Ocular examination was conducted at baseline and months 1, 3 and 6 including dilated fundus examination and electro-retinogram (ERG). Four rabbits were sacrificed at each time-point for retinal histology. ERG data were compared between groups and time-points using anova. RESULTS: No complications were reported from either eye of any rabbits over a 6-month period. Photopic A wave was reduced in the control eye at 1 month compared with baseline (p < 0.01). There was no significant difference in other ERG parameters between the groups at different time-points. Gross retinal histology was normal at all time-points. CONCLUSION: Extended release intravitreal brimonidine device was found to be safe and in normotensive rabbit eyes.

DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88.

Alu RNA accumulation due to DICER1 deficiency in the retinal pigmented epithelium (RPE) is implicated in geographic atrophy (GA), an advanced form of age-related macular degeneration that causes blindness in millions of individuals. The mechanism of Alu RNA-induced cytotoxicity is unknown. Here we show that DICER1 deficit or Alu RNA exposure activates the NLRP3 inflammasome and triggers TLR-independent MyD88 signaling via IL18 in the RPE. Genetic or pharmacological inhibition of inflammasome components (NLRP3, Pycard, Caspase-1), MyD88, or IL18 prevents RPE degeneration induced by DICER1 loss or Alu RNA exposure. These findings, coupled with our observation that human GA RPE contains elevated amounts of NLRP3, PYCARD, and IL18 and evidence of increased Caspase-1 and MyD88 activation, provide a rationale for targeting this pathway in GA. Our findings also reveal a function of the inflammasome outside the immune system and an immunomodulatory action of mobile elements.

Short-interfering RNAs induce retinal degeneration via TLR3 and IRF3.

The discovery of sequence-specific gene silencing by endogenous double-stranded RNAs (dsRNA) has propelled synthetic short-interfering RNAs (siRNAs) to the forefront of targeted pharmaceutical engineering. The first clinical trials utilized 21-nucleotide (nt) siRNAs for the treatment of neovascular age-related macular degeneration (AMD). Surprisingly, these compounds were not formulated for cell permeation, which is required for bona fide RNA interference (RNAi). We showed that these "naked" siRNAs suppress neovascularization in mice not via RNAi but via sequence-independent activation of cell surface Toll-like receptor-3 (TLR3). Here, we demonstrate that noninternalized siRNAs induce retinal degeneration in mice by activating surface TLR3 on retinal pigmented epithelial cells. Cholesterol conjugated siRNAs capable of cell permeation and triggering RNAi also induce the same phenotype. Retinal degeneration was not observed after treatment with siRNAs shorter than 21-nts. Other cytosolic dsRNA sensors are not critical to this response. TLR3 activation triggers caspase-3-mediated apoptotic death of the retinal pigment epithelium (RPE) via nuclear translocation of interferon regulatory factor-3. While this unexpected adverse effect of siRNAs has implications for future clinical trials, these findings also introduce a new preclinical model of geographic atrophy (GA), a late stage of dry AMD that causes blindness in millions worldwide.

DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration.

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

The multifactorial nature of retinal vascular disease.

Retinal vascular disease is the most common cause of macular edema (ME). While there are several etiologies of vascular compromise and subsequent macular leakage, diabetic retinopathy is the most prevalent and continues to challenge ophthalmologists and frustrate patients due to its refractory nature. In response to this epidemic, diabetic ME (DME) along with cystoid ME (CME) have been areas of active investigation both in the clinic and the laboratory. Several decades of basic science research have revealed a growing and complex array of cytokine growth factors and proinflammatory mediators which are capable of inciting the cellular changes that result in accumulation of fluid within the retina. Much of this new molecular foundation provides the current and fundamental scaffold for understanding the pathologic process of ME while simultaneously identifying potential therapeutic targets. Whereas CME has classically been treated with corticosteroids and nonsteroidal antiinflammatory drugs, recent clinical studies have demonstrated improved visual outcomes for DME treatment with light focal/grid laser, corticosteroids and anti-vascular endothelial growth factor antibodies. Yet, each of these treatments has differential effects on the multifactorial mechanisms of ME. This article reviews the anatomical, cellular and molecular derangements associated with ME and highlights specific pathways targeted by current treatments.

Alternatively spliced vascular endothelial growth factor receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth.

Disruption of the precise balance of positive and negative molecular regulators of blood and lymphatic vessel growth can lead to myriad diseases. Although dozens of natural inhibitors of hemangiogenesis have been identified, an endogenous selective inhibitor of lymphatic vessel growth has not to our knowledge been previously described. We report the existence of a splice variant of the gene encoding vascular endothelial growth factor receptor-2 (Vegfr-2) that encodes a secreted form of the protein, designated soluble Vegfr-2 (sVegfr-2), that inhibits developmental and reparative lymphangiogenesis by blocking Vegf-c function. Tissue-specific loss of sVegfr-2 in mice induced, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without affecting blood vasculature. Administration of sVegfr-2 inhibited lymphangiogenesis but not hemangiogenesis induced by corneal suture injury or transplantation, enhanced corneal allograft survival and suppressed lymphangioma cellular proliferation. Naturally occurring sVegfr-2 thus acts as a molecular uncoupler of blood and lymphatic vessels; modulation of sVegfr-2 might have therapeutic effects in treating lymphatic vascular malformations, transplantation rejection and, potentially, tumor lymphangiogenesis and lymphedema (pages 993-994).

CCR3 is a target for age-related macular degeneration diagnosis and therapy.

Age-related macular degeneration (AMD), a leading cause of blindness worldwide, is as prevalent as cancer in industrialized nations. Most blindness in AMD results from invasion of the retina by choroidal neovascularisation (CNV). Here we show that the eosinophil/mast cell chemokine receptor CCR3 is specifically expressed in choroidal neovascular endothelial cells in humans with AMD, and that despite the expression of its ligands eotaxin-1, -2 and -3, neither eosinophils nor mast cells are present in human CNV. Genetic or pharmacological targeting of CCR3 or eotaxins inhibited injury-induced CNV in mice. CNV suppression by CCR3 blockade was due to direct inhibition of endothelial cell proliferation, and was uncoupled from inflammation because it occurred in mice lacking eosinophils or mast cells, and was independent of macrophage and neutrophil recruitment. CCR3 blockade was more effective at reducing CNV than vascular endothelial growth factor A (VEGF-A) neutralization, which is in clinical use at present, and, unlike VEGF-A blockade, is not toxic to the mouse retina. In vivo imaging with CCR3-targeting quantum dots located spontaneous CNV invisible to standard fluorescein angiography in mice before retinal invasion. CCR3 targeting might reduce vision loss due to AMD through early detection and therapeutic angioinhibition.

Small interfering RNA-induced TLR3 activation inhibits blood and lymphatic vessel growth.

Neovascularization in response to tissue injury consists of the dual invasion of blood (hemangiogenesis) and lymphatic (lymphangiogenesis) vessels. We reported recently that 21-nt or longer small interfering RNAs (siRNAs) can suppress hemangiogenesis in mouse models of choroidal neovascularization and dermal wound healing independently of RNA interference by directly activating Toll-like receptor 3 (TLR3), a double-stranded RNA immune receptor, on the cell surface of blood endothelial cells. Here, we show that a 21-nt nontargeted siRNA suppresses both hemangiogenesis and lymphangiogenesis in mouse models of neovascularization induced by corneal sutures or hindlimb ischemia as efficiently as a 21-nt siRNA targeting vascular endothelial growth factor-A. In contrast, a 7-nt nontargeted siRNA, which is too short to activate TLR3, does not block hemangiogenesis or lymphangiogenesis in these models. Exposure to 21-nt siRNA, which we demonstrate is not internalized unless cell-permeating moieties are used, triggers phosphorylation of cell surface TLR3 on lymphatic endothelial cells and induces apoptosis. These findings introduce TLR3 activation as a method of jointly suppressing blood and lymphatic neovascularization and simultaneously raise new concerns about the undesirable effects of siRNAs on both circulatory systems.

Sequence- and target-independent angiogenesis suppression by siRNA via TLR3.

Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro- and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon-alpha/beta activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon-gamma and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3-RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world's population, and that siRNAs might induce unanticipated vascular or immune effects.

Corneal avascularity is due to soluble VEGF receptor-1.

Corneal avascularity-the absence of blood vessels in the cornea-is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

Loss of SPARC-mediated VEGFR-1 suppression after injury reveals a novel antiangiogenic activity of VEGF-A.

VEGF-A promotes angiogenesis in many tissues. Here we report that choroidal neovascularization (CNV) incited by injury was increased by excess VEGF-A before injury but was suppressed by VEGF-A after injury. This unorthodox antiangiogenic effect was mediated via VEGFR-1 activation and VEGFR-2 deactivation, the latter via Src homology domain 2-containing (SH2-containing) tyrosine phosphatase-1 (SHP-1). The VEGFR-1-specific ligand placental growth factor-1 (PlGF-1), but not VEGF-E, which selectively binds VEGFR-2, mimicked these responses. Excess VEGF-A increased CNV before injury because VEGFR-1 activation was silenced by secreted protein, acidic and rich in cysteine (SPARC). The transient decline of SPARC after injury revealed a temporal window in which VEGF-A signaling was routed principally through VEGFR-1. These observations indicate that therapeutic design of VEGF-A inhibition should include consideration of the level and activity of SPARC.

Drusen complement components C3a and C5a promote choroidal neovascularization.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrialized nations, affecting 30-50 million people worldwide. The earliest clinical hallmark of AMD is the presence of drusen, extracellular deposits that accumulate beneath the retinal pigmented epithelium. Although drusen nearly always precede and increase the risk of choroidal neovascularization (CNV), the late vision-threatening stage of AMD, it is unknown whether drusen contribute to the development of CNV. Both in patients with AMD and in a recently described mouse model of AMD, early subretinal pigmented epithelium deposition of complement components C3 and C5 occurs, suggesting a contributing role for these inflammatory proteins in the development of AMD. Here we provide evidence that bioactive fragments of these complement components (C3a and C5a) are present in drusen of patients with AMD, and that C3a and C5a induce VEGF expression in vitro and in vivo. Further, we demonstrate that C3a and C5a are generated early in the course of laser-induced CNV, an accelerated model of neovascular AMD driven by VEGF and recruitment of leukocytes into the choroid. We also show that genetic ablation of receptors for C3a or C5a reduces VEGF expression, leukocyte recruitment, and CNV formation after laser injury, and that antibody-mediated neutralization of C3a or C5a or pharmacological blockade of their receptors also reduces CNV. Collectively, these findings establish a mechanistic basis for the clinical observation that drusen predispose to CNV, revealing a role for immunological phenomena in angiogenesis and providing therapeutic targets for AMD.

Recruitment of marrow-derived endothelial cells to experimental choroidal neovascularization by local expression of vascular endothelial growth factor.

PURPOSE: The question of whether adult animals maintain a reservoir of endothelial progenitor cells (EPCs) in the bone marrow that is involved in neovascularization is under investigation. The following study was undertaken to examine the potential contribution of EPCs to the development of choroidal neovascularization (CNV) in adult mice and to examine the role of local expression of vascular endothelial growth factor (VEGF) in this process. METHODS: Lethally irradiated, adult female nude mice were engrafted with whole bone marrow isolated from male transgenic mice expressing LacZ driven by the endothelial specific Tie-2 promoter. Two months, following bone marrow reconstitution, confirmed by quantitative Taqman PCR, an E1-deleted adenoviral vector expressing vascular endothelial growth factor (165) (Ad.VEGF(165)) was injected subretinally to induce CNV, confirmed by collagen IV immunohistochemistry. Bone marrow-derived endothelial cells were detected using either X-gal staining or Y chromosome in situ hybridization. Y chromosome positive cells within the CNV were confirmed to be endothelial cells by lectin staining. RESULTS: Subretinal Ad.VEGF(165) was capable of inducing CNV. Four-week old lesions were found to contain LacZ expressing cells within the CNV in bone marrow transplanted animals but not in negative control animals. Eighteen percent of all Y chromosome positive cells within the CNV were found to be lectin positive while 27% of all endothelial cells within the CNV were Y chromosome positive. CONCLUSION: Engrafted bone marrow-derived EPCs were shown to differentiate into endothelial cells at the site of subretinal VEGF-induced CNV in mice. These results suggest that EPCs contribute to the formation of neovascularization and that subretinal expression of VEGF might play an important role in recruitment of these cells to the site of CNV.