Valeria Tarallo

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.

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.

Vascular Endothelial Growth Factor Receptor-1 Contributes to Resistance to Anti–Epidermal Growth Factor Receptor Drugs in Human Cancer Cells

Purpose: The resistance to selective EGFR inhibitors involves the activation of alternative signaling pathways, and Akt activation and VEGF induction have been described in EGFR inhibitor–resistant tumors. Combined inhibition of EGFR and other signaling proteins has become a successful therapeutic approach, stimulating the search for further determinants of resistance as basis for novel therapeutic strategies. Experimental Design: We established human cancer cell lines with various degrees of EGFR expression and sensitivity to EGFR inhibitors and analyzed signal transducers under the control of EGFR-dependent and EGFR-independent pathways. Results: Multitargeted inhibitor vandetanib (ZD6474) inhibited the growth and the phosphorylation of Akt and its effector p70S6 kinase in both wild-type and EGFR inhibitor–resistant human colon, prostate, and breast cancer cells. We found that the resistant cell lines exhibit, as common feature, VEGFR-1/Flt-1 overexpression, increased secretion of VEGF and placental growth factor, and augmented migration capabilities and that vandetanib is able to antagonize them. Accordingly, a new kinase assay revealed that in addition to VEGF receptor (VEGFR)-2, RET, and EGFR, vandetanib efficiently inhibits also VEGFR-1. The contribution of VEGFR-1 to the resistant phenotype was further supported by the demonstration that VEGFR-1 silencing in resistant cells restored sensitivity to anti-EGFR drugs and impaired migration capabilities, whereas exogenous VEGFR-1 overexpression in wild-type cells conferred resistance to these agents. Conclusions: This study shows that VEGFR-1 contributes to anti-EGFR drug resistance in different human cancer cells. Moreover, vandetanib inhibits VEGFR-1 activation, cell proliferation, and migration, suggesting its potential utility in patients resistant to EGFR inhibitors.

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.

TLR-Independent and P2X7-Dependent Signaling Mediate Alu RNA-Induced NLRP3 Inflammasome Activation in Geographic Atrophy

PURPOSE Accumulation of Alu RNA transcripts due to DICER1 deficiency in the retinal pigmented epithelium (RPE) promotes geographic atrophy. Recently we showed that Alu RNA activated the NLRP3 inflammasome, leading to RPE cell death via interleukin-18 (IL-18)-mediated MyD88 signaling. However, the molecular basis for NLRP3 inflammasome activation by Alu RNA is not well understood. We sought to decipher the key signaling events triggered by Alu RNA that lead to priming and activation of the NLRP3 inflammasome and, ultimately, to RPE degeneration by investigating the roles of the purinoreceptor P2X7, the transcription factor NF-κB, and the Toll-like receptors (TLRs) in these processes. METHODS Human and mouse RPE cells were transfected with a plasmid encoding an Alu element (pAlu) or an in vitro-transcribed Alu RNA. Inflammasome priming was assessed by measuring NLRP3 and IL18 mRNA levels by real-time quantitative PCR. Using immunoblotting, we assessed NF-κB activation by monitoring phosphorylation of its p65 subunit, and inflammasome activation by monitoring caspase-1 cleavage into its active form. RPE degeneration was induced in mice by subretinal transfection of pAlu or Alu RNA. The NF-κB inhibitor BAY 11-7082, the P2X7 receptor antagonist A-740003, and the NLRP3 inflammasome inhibitor glyburide were delivered by intravitreous injections. We studied wild-type (WT) C57Bl/6J, P2rx7(-/-), Nfkb1(-/-), and Tlr23479(-/-) mice. RPE degeneration was assessed by fundus photography and zonula occludens-1 (ZO-1) staining of mouse RPE. RESULTS Alu RNA-induced NF-κB activation, independent of TLR-1, -2, -3, -4, -6, -7, and -9 signaling, was required for priming the NLRP3 inflammasome. Nfkb1(-/-) and P2rx7(-/-) mice and WT mice treated with the pharmacological inhibitors of NF-κB, P2X7, or NLRP3, were protected against Alu RNA-induced RPE degeneration. CONCLUSIONS NF-κB and P2X7 are critical signaling intermediates in Alu RNA-induced inflammasome priming and RPE degeneration. These molecules are novel targets for rational drug development for geographic atrophy.

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.

DICER1/Alu RNA dysmetabolism induces Caspase-8–mediated cell death in age-related macular degeneration

Significance Geographic atrophy is a late stage of age-related macular degeneration (AMD) that causes blindness in millions worldwide characterized by death of the retinal pigmented epithelium (RPE). We previously reported that RPE death is due to a deficiency in the enzyme DICER1, which leads to accumulation of toxic Alu RNA. We also demonstrated that Alu RNA causes RPE death by activating an immune platform called the NLRP3 inflammasome. However, the precise mechanisms of RPE death in this disease remained unresolved. The present study indicates that Alu RNA induces RPE death by activating the enzyme Caspase-8 downstream of inflammasome activation and that blocking Caspase-8 rescues RPE degeneration. This implicates apoptosis as the cell death pathway responsible for Alu RNA cytotoxicity, and these findings provide new potential therapeutic targets for this disease. Geographic atrophy, an advanced form of age-related macular degeneration (AMD) characterized by death of the retinal pigmented epithelium (RPE), causes untreatable blindness in millions worldwide. The RPE of human eyes with geographic atrophy accumulates toxic Alu RNA in response to a deficit in the enzyme DICER1, which in turn leads to activation of the NLRP3 inflammasome and elaboration of IL-18. Despite these recent insights, it is still unclear how RPE cells die during the course of the disease. In this study, we implicate the involvement of Caspase-8 as a critical mediator of RPE degeneration. Here we show that DICER1 deficiency, Alu RNA accumulation, and IL-18 up-regulation lead to RPE cell death via activation of Caspase-8 through a Fas ligand-dependent mechanism. Coupled with our observation of increased Caspase-8 expression in the RPE of human eyes with geographic atrophy, our findings provide a rationale for targeting this apoptotic pathway in this disease.

Modulation of Angiogenesis by a Tetrameric Tripeptide That Antagonizes Vascular Endothelial Growth Factor Receptor 1

Vascular endothelial growth factor receptor-1 (VEGFR-1, also known as Flt-1) is involved in complex biological processes often associated to severe pathological conditions like cancer, inflammation, and metastasis formation. Consequently, the search for antagonists of Flt-1 has recently gained a growing interest. Here we report the identification of a tetrameric tripeptide from a combinatorial peptide library built using non-natural amino acids, which binds Flt-1 and inhibits in vitro its interaction with placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) A and B (IC50 ∼ 10 μm). The peptide is stable in serum for 7 days and prevents both Flt-1 phosphorylation and the capillary-like tube formation of human primary endothelial cells stimulated by PlGF or VEGF-A. Conversely, the identified peptide does not interfere in VEGF-induced VEGFR-2 activation. In vivo, this peptide inhibits VEGF-A- and PlGF-induced neoangiogenesis in the chicken embryo chorioallantoic membrane assay. In contrast, in the cornea, where avascularity is maintained by high levels of expression of the soluble form of Flt-1 receptor (sFlt-1) that prevents the VEGF-A activity, the peptide is able to stimulate corneal mouse neovascularization in physiological condition, as reported previously for others neutralizing anti-Flt-1 molecules. This tetrameric tripeptide represents a new, promising compound for therapeutic approaches in pathologies where Flt-1 activation plays a crucial role.

Anti–neuropilin-1 peptide inhibition of synoviocyte survival, angiogenesis, and experimental arthritis

OBJECTIVE To delineate the role of neuropilin-1 (NP-1), a vascular endothelial growth factor receptor (VEGFR), in rheumatoid inflammation and to determine whether blockade of NP-1 could suppress synoviocyte survival and angiogenesis. METHODS VEGF(111-165) peptide, which encompasses the NP-1 binding domain of VEGF(165), was generated by cleaving VEGF(165) with plasmin. The effect of this peptide on the interaction between VEGF(165) and its receptor was determined by (125)I-VEGFR binding assay. Assays to determine synoviocyte apoptosis, adhesion, and migration were performed in the presence of VEGF(165) and/or the peptide. VEGF(165)-induced angiogenesis was assessed by measuring the proliferation, tube formation, and wounding migration of endothelial cells (ECs). Mice were immunized with type II collagen to induce experimental arthritis. RESULTS VEGF(111-165) peptide specifically inhibited the binding of (125)I-VEGF(165) to NP-1 on rheumatoid synoviocytes and ECs. The peptide eliminated the VEGF(165)-mediated increase in synoviocyte survival and activation of p-ERK and Bcl-2. The peptide also completely inhibited a VEGF(165)-induced increase in synoviocyte adhesion and migration. In addition, the anti-NP-1 peptide blocked VEGF(165)-stimulated proliferation, capillary tube formation, and wounding migration of ECs in vitro. VEGF(165)-induced neovascularization in a Matrigel plug in mice was also blocked by treatment with the peptide. Finally, subcutaneous injection of anti-NP-1 peptide suppressed arthritis severity and autoantibody formation in mice with experimental arthritis and inhibited synoviocyte hyperplasia and angiogenesis in arthritic joints. CONCLUSION Anti-NP-1 peptide suppressed VEGF(165)-induced increases in synoviocyte survival and angiogenesis, and thereby blocked experimental arthritis. Our findings suggest that anti-NP-1 peptide could be useful in alleviating chronic arthritis.

IL-18 is not therapeutic for neovascular age-related macular degeneration

Up to 50 million people worldwide are afflicted with the devastating blinding disease age-related macular degeneration (AMD)1–3. The vast majority of patients have the currently untreatable “dry” or atrophic form of AMD, characterized by NLRP3 inflammasome-driven degeneration of the retinal pigment epithelium (RPE) supportive cell layer4,5. Blockade of the NLRP3 inflammasome is a next-generation therapeutic target in dry AMD; however, it was recently reported that inflammasome-mediated production of IL18 potentially safeguards the retina against the other, often more visually devastating form of AMD, for which dry AMD patients are at greatly increased risk of developing, known as choroidal neovascularization (CNV)6. Therefore, it is essential, prior to initiating inflammasome-targeting clinical trials, to directly and rigorously assess whether modulating IL18 or the NLRP3 inflammasome affects CNV and RPE cell health.