Effects of metformin on retinoblastoma growth in vitro and in vivo

Recent studies suggest that the anti-diabetic drug metformin may reduce the risk of cancer and have anti-proliferative effects for some but not all cancers. In this study, we examined the effects of metformin on human retinoblastoma cell proliferation in vitro and in vivo. Two different human retinoblastoma cell lines (Y79, WERI) were treated with metformin in vitro and xenografts of Y79 cells were established in nu/nu immune-deficient mice and used to assess the effects of pharmacological levels of metformin in vivo. Metformin inhibited proliferation of the retinoblastoma cells in vitro. Similar to other studies, high concentrations of metformin (mM) blocked the cell cycle in G0–G1, indicated by a strong decrease of G1 cyclins, especially cyclin D, cyclin-dependent kinases (4 and 6), and flow cytometry assessment of the cell cycle. This was associated with activation of AMPK, inhibition of the mTOR pathways and autophagy marker LC3B. However, metformin failed to suppress growth of xenografted tumors of Y79 human retinoblastoma cells in nu/nu mice, even when treated with a maximally tolerated dose level achieved in human patients. In conclusion, suprapharmacological levels (mM) of metformin, well above those tolerated in vivo, inhibited the proliferation of retinoblastoma cells in vitro. However, physiological levels of metformin, such as seen in the clinical setting, did not affect the growth of retinoblastoma cells in vitro or in vivo. This suggests that the potential beneficial effects of metformin seen in epidemiological studies may be limited to specific tumor types or be related to indirect effects/mechanisms not observed under acute laboratory conditions

VEGF\u3csub\u3e164\u3c/sub\u3e-Mediated Inflammation is Required for Pathological, but Not Physiological, Ischemia-Induced Retinal Neovascularization

Hypoxia-induced VEGF governs both physiological retinal vascular development and pathological retinal neovascularization. In the current paper, the mechanisms of physiological and pathological neovascularization are compared and contrasted. During pathological neovascularization, both the absolute and relative expression levels for VEGF164 increased to a greater degree than during physiological neovascularization. Furthermore, extensive leukocyte adhesion was observed at the leading edge of pathological, but not physiological, neovascularization. When a VEGF164-specific neutralizing aptamer was administered, it potently suppressed the leukocyte adhesion and pathological neovascularization, whereas it had little or no effect on physiological neovascularization. In parallel experiments, genetically altered VEGF164-deficient (VEGF120/188) mice exhibited no difference in physiological neovascularization when compared with wild-type (VEGF+/+) controls. In contrast, administration of a VEGFR-1/Fc fusion protein, which blocks all VEGF isoforms, led to significant suppression of both pathological and physiological neovascularization. In addition, the targeted inactivation of monocyte lineage cells with clodronate-liposomes led to the suppression of pathological neovascularization. Conversely, the blockade of T lymphocyte–mediated immune responses with an anti-CD2 antibody exacerbated pathological neovascularization. These data highlight important molecular and cellular differences between physiological and pathological retinal neovascularization. During pathological neovascularization, VEGF164 selectively induces inflammation and cellular immunity. These processes provide positive and negative angiogenic regulation, respectively. Together, new therapeutic approaches for selectively targeting pathological, but not physiological, retinal neovascularization are outlined

Increased Choroidal Neovascularization following Laser Induction in Mice Lacking Lysyl Oxidase-like 1

PURPOSE. Age-related degradation of the elastic lamina in Bruch's membrane may have a permissive effect on the growth of choroidal neovascularization (CNV). This study investigated the influence of defective elastic fiber maintenance in the development of laser-induced CNV. METHODS. A mouse lacking lysyl oxidase-like (LOXL)-1, an enzyme essential for elastin polymerization, was studied. The morphologic characteristics of the elastic lamina within Bruch's membrane were examined in mutant and wild-type (WT) eyes. Laser-induced CNV was evaluated by fluorescein angiography and choroidal flat mounts. Immunohistochemistry for elastin was performed on the CNV lesions, and vascular endothelial growth factor (VEGF) levels were determined by ELISA. Soluble elastin and matrix metalloproteinase (MMPs) levels were also analyzed by immunoblotting. RESULTS. The elastic lamina of Bruch's membrane in the LOXL1-deficient mice was fragmented and less continuous than in the WT controls. The mutant mice showed increased levels of soluble elastin peptides and reduced elastin polymer deposition in neovascular membranes. Significantly larger CNV with greater leakage on fluorescein angiography developed in mutant mice. VEGF levels in the RPE/choroid were higher in the knockout mice on days 7 and 14 after laser (P Ͻ 0.05). MT1-MMP (MMP14) was also elevated after laser in the LOXL1 mutant eyes compared to the WT controls. CONCLUSIONS. These results show that a systemic defect in elastic fiber deposition affects Bruch's membrane integrity and leads to more aggressive CNV growth. The latter may be partially mediated by abnormal signaling from the accumulation of soluble elastin peptides. (Invest Ophthalmol Vis Sci. 2008;49:2599 -2605) DOI:10.1167/iovs.07-1508 C horoidal neovascularization (CNV) is the predominant cause of severe visual loss in age-related macular degeneration (AMD) and other macular diseases. Bruch's membrane integrity is known to be compromised in many conditions characterized by choroidal neovascularization, such as traumatic choroidal rupture, angioid streaks, and myopia. Additionally, laser rupture of Bruch's membrane is a well-established means of inducing experimental choroidal neovascularization. Such evidence indicates that Bruch's membrane serves as an important barrier to CNV formation, but the structural characteristics important in this barrier function are still under investigation. Bruch's membrane lies between the neural retina and the choriocapillaris. There are five layers to this structure: the basement membrane of the retinal pigment epithelium, an inner collagenous layer, the central elastic lamina composed of elastic fibers, the outer collagenous layer, and the basement membrane of the choriocapillaris endothelium. Numerous morphologic changes in Bruch's membrane have been described with aging, particularly the development of drusen and basal laminar deposits, but also calcification, changes in thickness, and decreases in hydraulic conductivity. 1 Loss of elastic fibers is a hallmark of connective tissue aging, and the central elastic lamina of Bruch's membrane has long been postulated to have a barrier function against CNV. 2,3 Recently, Chong et al. 5 Elastic fibers are amorphous polymers composed of the protein elastin, known as tropoelastin in its monomeric form. Oxidative deamination of lysine residues, an initial step of elastin polymerization, requires lysyl oxidases. 6 Lysyl oxidaselike (LOXL) protein 1 has been shown to guide the spatially defined deposition of elastin and is essential for the maintenance of elastic fibers. 7 LOXL1-deficient mice develop multiple systemic defects including pelvic organ prolapse, emphysematous changes in the lungs, and vascular abnormalities attributable to a failure in elastic fiber maintenance

VEGF164-mediated Inflammation Is Required for Pathological, but Not Physiological, Ischemia-induced Retinal Neovascularization

Hypoxia-induced VEGF governs both physiological retinal vascular development and pathological retinal neovascularization. In the current paper, the mechanisms of physiological and pathological neovascularization are compared and contrasted. During pathological neovascularization, both the absolute and relative expression levels for VEGF(164) increased to a greater degree than during physiological neovascularization. Furthermore, extensive leukocyte adhesion was observed at the leading edge of pathological, but not physiological, neovascularization. When a VEGF(164)-specific neutralizing aptamer was administered, it potently suppressed the leukocyte adhesion and pathological neovascularization, whereas it had little or no effect on physiological neovascularization. In parallel experiments, genetically altered VEGF(164)-deficient (VEGF(120/188)) mice exhibited no difference in physiological neovascularization when compared with wild-type (VEGF(+/+)) controls. In contrast, administration of a VEGFk-1/Fc fusion protein, which blocks all VEGF isoforms, led to significant suppression of both pathological and physiological neovascularization. In addition, the targeted inactivation of monocyte lineage cells with clodronate-liposomes led to the suppression of pathological neovascularization. Conversely, the blockade of T lymphocyte-mediated immune responses with an anti-CD2 antibody exacerbated pathological neovascularization. These data highlight important molecular and cellular differences between physiological and pathological retinal neovascularization. During pathological neovascularization, VEGF(164) selectively induces inflammation and cellular immunity. These processes provide positive and negative angiogenic regulation, respectively. Together, new therapeutic approaches for selectively targeting pathological, but not physiological, retinal neovascularization are outlined

Utilizing Targeted Gene Therapy with Nanoparticles Binding Alpha v Beta 3 for Imaging and Treating Choroidal Neovascularization

Purpose: The integrin αvβ3 is differentially expressed on neovascular endothelial cells. We investigated whether a novel intravenously injectable αvβ3 integrin-ligand coupled nanoparticle (NP) can target choroidal neovascular membranes (CNV) for imaging and targeted gene therapy. Methods: CNV lesions were induced in rats using laser photocoagulation. The utility of NP for in vivo imaging and gene delivery was evaluated by coupling the NP with a green fluorescing protein plasmid (NP-GFPg). Rhodamine labeling (Rd-NP) was used to localize NP in choroidal flatmounts. Rd-NP-GFPg particles were injected intravenously on weeks 1, 2, or 3. In the treatment arm, rats received NP containing a dominant negative Raf mutant gene (NP-ATPμ-Raf) on days 1, 3, and 5. The change in CNV size and leakage, and TUNEL positive cells were quantified. Results: GFP plasmid expression was seen in vivo up to 3 days after injection of Rd-NP-GFPg. Choroidal flatmounts confirmed the localization of the NP and the expression of GFP plasmid in the CNV. Treating the CNV with NP-ATPμ-Raf decreased the CNV size by 42% (P<0.001). OCT analysis revealed that the reduction of CNV size started on day 5 and reached statistical significance by day 7. Fluorescein angiography grading showed significantly less leakage in the treated CNV (P<0.001). There were significantly more apoptotic (TUNEL-positive) nuclei in the treated CNV. Conclusion: Systemic administration of αvβ3 targeted NP can be used to label the abnormal blood vessels of CNV for imaging. Targeted gene delivery with NP-ATPμ-Raf leads to a reduction in size and leakage of the CNV by induction of apoptosis in the CNV

Germline BAP1 Inactivation Is Preferentially Associated with Metastatic Ocular Melanoma and Cutaneous-Ocular Melanoma Families

Background: BAP1 has been shown to be a target of both somatic alteration in high-risk ocular melanomas (OM) and germline inactivation in a few individuals from cancer-prone families. These findings suggest that constitutional BAP1 changes may predispose individuals to metastatic OM and that familial permeation of deleterious alleles could delineate a new cancer syndrome. Design: To characterize BAP1’s contribution to melanoma risk, we sequenced BAP1 in a set of 100 patients with OM, including 50 metastatic OM cases and 50 matched non-metastatic OM controls, and 200 individuals with cutaneous melanoma (CM) including 7 CM patients from CM-OM families and 193 CM patients from CM-non-OM kindreds. Results: Germline BAP1 mutations were detected in 4/50 patients with metastatic OM and 0/50 cases of non-metastatic OM (8 % vs. 0%, p = 0.059). Since 2/4 of the BAP1 carriers reported a family history of CM, we analyzed 200 additional hereditary CM patients and found mutations in 2/7 CM probands from CM-OM families and 1/193 probands from CM-non-OM kindreds (29 % vs. 0.52%, p =.003). Germline mutations co-segregated with both CM and OM phenotypes and were associated with the presence of unique nevoid melanomas and highly atypical nevoid melanoma-like melanocytic proliferations (NEMMPs). Interestingly, 7/14 germline variants identified to date reside in C-terminus suggesting that the BRCA1 binding domain i

Issues with the Specificity of Immunological Reagents for NLRP3: Implications for Age-related Macular Degeneration

Contradictory data have been presented regarding the implication of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in age-related macular degeneration (AMD), the leading cause of vision loss in the Western world. Recognizing that antibody specificity may explain this discrepancy and in line with recent National Institutes of Health (NIH) guidelines requiring authentication of key biological resources, the specificity of anti-NLRP3 antibodies was assessed to elucidate whether non-immune RPE cells express NLRP3. Using validated resources, NLRP3 was not detected in human primary or human established RPE cell lines under multiple inflammasome-priming conditions, including purported NLRP3 stimuli in RPE such as DICER1 deletion and Alu RNA transfection. Furthermore, NLRP3 was below detection limits in ex vivo macular RPE from AMD patients, as well as in human induced pluripotent stem cell (hiPSC)-derived RPE from patients with overactive NLRP3 syndrome (Chronic infantile neurologic cutaneous and articulate, CINCA syndrome). Evidence presented in this study provides new data regarding the interpretation of published results reporting NLRP3 expression and upregulation in RPE and addresses the role that this inflammasome plays in AMD pathogenesis

Systematic genomic and translational efficiency studies of uveal melanoma

To further our understanding of the somatic genetic basis of uveal melanoma, we sequenced the protein-coding regions of 52 primary tumors and 3 liver metastases together with paired normal DNA. Known recurrent mutations were identified in GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. The role of mutated EIF1AX was tested using loss of function approaches including viability and translational efficiency assays. Knockdown of both wild type and mutant EIF1AX was lethal to uveal melanoma cells. We probed the function of N-terminal tail EIF1AX mutations by performing RNA sequencing of polysome-associated transcripts in cells expressing endogenous wild type or mutant EIF1AX. Ribosome occupancy of the global translational apparatus was sensitive to suppression of wild type but not mutant EIF1AX. Together, these studies suggest that cells expressing mutant EIF1AX may exhibit aberrant translational regulation, which may provide clonal selective advantage in the subset of uveal melanoma that harbors this mutation