Sulfated CXCR3 Peptide Trap Use as a Promising Therapeutic Approach for Age-Related Macular Degeneration.

BACKGROUND AND OBJECTIVES: Chemokines have various biological functions and potential roles in the development or progression of neuroinflammatory diseases. However, the specific pathogenic roles of chemokines in the major cause for vision loss among the elderly, the leading cause of blindness in older individuals, remain elusive. Chemokines interact with their receptors expressed in the endothelium and on leukocytes. The sulfation of tyrosine residues in chemokine receptors increases the strength of ligand-receptor interaction and modulates signaling. Therefore, in the present study, we aimed to construct a human recombinant sulfated CXCR3 peptide trap (hCXCR3-S2) and mouse recombinant sulfated CXCR3 peptide trap (mCXCR3-S2) to demonstrate in vivo effects in preventing choroidal neovascularization (CNV) and chemotaxis. MATERIALS AND METHODS: We generated expression vectors for mCXCR3-S2 and hCXCR3-S2 with GST domains and their respective cDNA sequences. Following overexpression in E. coli BL21 (DE3), we purified the fusion proteins from cell lysates using affinity chromatography. First, the impact of hCXCR3-S2 was validated in vitro. Subsequently, the in vivo efficacy of mCXCR3-S2 was investigated using a laser-induced CNV mouse model, a mouse model of neovascular age-related macular degeneration (AMD). RESULTS: hCXCR3-S2 inhibited the migration and invasion of two human cancer cell lines. Intravitreal injection of mCXCR3-S2 attenuated CNV and macrophage recruitment in neovascular lesions of mouse models. These in vitro and in vivo effects were significantly stronger with CXCR3-S2 than with wild-type CXCR3 peptides. CONCLUSION: These findings demonstrate that the sulfated form of the CXCR3 peptide trap is a valuable tool that could be supplemented with antivascular endothelial growth factors in AMD treatment.

Supramolecular Senolytics via Intracellular Oligomerization of Peptides in Response to Elevated Reactive Oxygen Species Levels in Aging Cells.

Senolytics, which eliminate senescent cells from tissues, represent an emerging therapeutic strategy for various age-related diseases. Most senolytics target antiapoptotic proteins, which are overexpressed in senescent cells, limiting specificity and inducing severe side effects. To overcome these limitations, we constructed self-assembling senolytics targeting senescent cells with an intracellular oligomerization system. Intracellular aryl-dithiol-containing peptide oligomerization occurred only inside the mitochondria of senescent cells due to selective localization of the peptides by RGD-mediated cellular uptake into integrin αvß3-overexpressed senescent cells and elevated levels of reactive oxygen species, which can be used as a chemical fuel for disulfide formation. This oligomerization results in an artificial protein-like nanoassembly with a stable α-helix secondary structure, which can disrupt the mitochondrial membrane via multivalent interactions because the mitochondrial membrane of senescent cells has weaker integrity than that of normal cells. These three specificities (integrin αvß3, high ROS, and weak mitochondrial membrane integrity) of senescent cells work in combination; therefore, this intramitochondrial oligomerization system can selectively induce apoptosis of senescent cells without side effects on normal cells. Significant reductions in key senescence markers and amelioration of retinal degeneration were observed after elimination of the senescent retinal pigment epithelium by this peptide senolytic in an age-related macular degeneration mouse model and in aged mice, and this effect was accompanied by improved visual function. This system provides a strategy for the treatment of age-related diseases using supramolecular senolytics.

Accelerated aging phenotypes in the retinal pigment epithelium of Zmpste24-deficient mice.

Age-related macular degeneration (AMD) is a chronic and progressive disease characterized by degeneration of the retinal pigment epithelium (RPE) and retina that ultimately leads to loss of vision. The pathological mechanisms of AMD are not fully known. Cellular senescence, which is a state of cell cycle arrest induced by DNA-damage or aging, is hypothesized to critically affect the pathogenesis of AMD. In this study, we examined the relationship between cellular senescence and RPE/retinal degeneration in mouse models of natural aging and accelerated aging. We performed a bulk RNA sequencing of the RPE cells from adult (8 months old) and naturally-aged old (24 months old) mice and found that common signatures of senescence and AMD pathology - inflammation, apoptosis, and blood vessel formation - are upregulated in the RPE of old mice. Next, we investigated markers of senescence and the degree of RPE/retinal degeneration in Zmpste24-deficient (Zmpste24-/-) mice, which is a model for progeria and accelerated aging. We found that Zmpste24-/- mice display markedly greater level of senescence-related markers in RPE and significant RPE/retinal degeneration compared to wild-type mice, in a manner consistent with natural aging. Overall, these results provide support for the association between cellular senescence of RPE and the pathogenesis of AMD, and suggest the use of Zmpste24-/- mice as a novel senescent RPE model of AMD.

Single-cell transcriptome of the mouse retinal pigment epithelium in response to a low-dose of doxorubicin.

Cellular senescence of the retinal pigment epithelium (RPE) is thought to play an important role in vision-threatening retinal degenerative diseases, such as age-related macular degeneration (AMD). However, the single-cell RNA profiles of control RPE tissue and RPE tissue exhibiting cellular senescence are not well known. We have analyzed the single-cell transcriptomes of control mice and mice with low-dose doxorubicin (Dox)-induced RPE senescence (Dox-RPE). Our results have identified 4 main subpopulations in the control RPE that exhibit heterogeneous biological activities and play roles in ATP synthesis, cell mobility/differentiation, mRNA processing, and catalytic activity. In Dox-RPE mice, cellular senescence mainly occurs in the specific cluster, which has been characterized by catalytic activity in the control RPE. Furthermore, in the Dox-RPE mice, 6 genes that have not previously been associated with senescence also show altered expression in 4 clusters. Our results might serve as a useful reference for the study of control and senescent RPE.

Targeting senescent retinal pigment epithelial cells facilitates retinal regeneration in mouse models of age-related macular degeneration.

Although age-related macular degeneration (AMD) is a multifactorial disorder with angiogenic, immune, and inflammatory components, the most common clinical treatment strategies are antiangiogenic therapies. However, these strategies are only applicable to neovascular AMD, which accounts for less than 20% of all AMD cases, and there are no FDA-approved drugs for the treatment of dry AMD, which accounts for ~ 80% of AMD cases. Here, we report that the elimination of senescent cells is a potential novel therapeutic approach for the treatment of all types of AMD. We identified senescent retinal pigment epithelium (RPE) cells in animal models of AMD and determined their contributions to retinal degeneration. We further confirmed that the clearance of senescent RPE cells with the MDM2-p53 inhibitor Nutlin-3a ameliorated retinal degeneration. These findings provide new insights into the use of senescent cells as a therapeutic target for the treatment of AMD.

Mitochondrial Membrane Disrupting Molecules for Selective Killing of Senescent Cells.

Cellular senescence, a stable form of cell cycle arrest, facilitates protection from tumorigenesis and aids in tissue repair as they accumulate in the body at an early age. However, long-term retention of senescent cells causes inflammation, aging of the tissue, and progression of deadly diseases such as obesity, diabetes, and atherosclerosis. Various attempts have been made to achieve selective elimination of senescent cells from the body, yet little has been explored in designing the mitochondria-targeted senolytic agent. Many characteristics of senescence are associated with mitochondria. Here we have designed a library of alkyl-monoquaternary ammonium-triphenyl phosphine (TPP) and alkyl-diquaternary ammonium-TPP of varying alkyl chain lengths, which target the mitochondria; we also studied their senolytic properties. It was observed that the alkyl-diquaternary ammonium-TPP with the longest chain length induced apoptosis in senescent cells selectively via an increase of reactive oxygen species (ROS) and mitochondrial membrane disruption. This study demonstrates that mitochondria could be a potential target for designing new small molecules as senolytic agents for the treatment of a variety of dysfunctions associated with pathological aging.

Recombinant sulfated CCR2 peptide trap reduces retinal degeneration in mice.

Chemokine receptors are generally sulfated at tyrosine residues of the N-terminal region. Tyrosine sulfation of the C-C chemokine receptor type 2 (CCR2) enhances its interaction with the chemokine ligand CCL2. Here, we generated a recombinant sulfated CCR2 peptide trap (mCCR2-S2) and investigated its effects on retinal degeneration in mice. Treatment with mCCR2-S2 reduced choroidal neovascularization (CNV) in a laser-induced CNV mouse model. In NaIO3-injected mice, treatment with mCCR2-S2 increased the outer nuclear layer thickness and rhodopsin expression in the retinas compared to that in mice treated with mCCR2-wild-type or glutathione S-transferase controls. Furthermore, glial fibrillary acidic protein (GFAP) expression and macrophage infiltration were decreased in mCCR2-S2-treated retinas. Recombinant mCCR2-S2 suppressed CNV development and retinal degeneration, possibly by regulating macrophage infiltration. Thus, the sulfated form of the CCR2 peptide trap may be a useful tool for treating patients with retinal degeneration, such as those with age-related macular degeneration and intraocular inflammatory disorders.

Quantitative proteomic analysis of aqueous humor from patients with drusen and reticular pseudodrusen in age-related macular degeneration.

BACKGROUND: To identify novel biomarkers related to the pathogenesis of dry age-related macular degeneration (AMD), we adopted a human retinal pigment epithelial (RPE) cell culture model that mimics some features of dry AMD including the accumulation of intra- and sub-RPE deposits. Then, we investigated the aqueous humor (AH) proteome using a data-independent acquisition method (sequential window acquisition of all theoretical fragment ion mass spectrometry) for dry AMD patients and controls. METHODS: After uniformly pigmented polarized monolayers of human fetal primary RPE (hfRPE) cells were established, the cells were exposed to 4-hydroxy-2-nonenal (4-HNE), followed by Western blotting, immunofluorescence analysis and ELISA of cells or conditioned media for several proteins of interest. Data-dependent acquisition for identification of the AH proteome and SWATH-based mass spectrometry were performed for 11 dry AMD patients according to their phenotypes (including soft drusen and reticular pseudodrusen [RPD]) and 2 controls (3 groups). RESULTS: Increased intra- and sub-RPE deposits were observed in 4-HNE-treated hfRPE cells compared with control cultures based on APOA1, cathepsin D, and clusterin immunoreactivity. Additionally, the differential abundance of proteins in apical and basal chambers with or without 4-HNE treatment confirmed the polarized secretion of proteins from hfRPE cells. A total of 119 proteins were quantified in dry AMD patients and controls by SWATH-MS. Sixty-five proteins exhibited significantly altered abundance among the three groups. A two-dimensional principal component analysis plot was generated to identify typical proteins related to the pathogenesis of dry AMD. Among the identified proteins, eight proteins, including APOA1, CFHR2, and CLUS, were previously considered major components or regulators of drusen. Three proteins (SERPINA4, LUM, and KERA proteins) have not been previously described as components of drusen or as being related to dry AMD. Interestingly, the LUM and KERA proteins, which are related to extracellular matrix organization, were upregulated in both RPD and soft drusen. CONCLUSIONS: Differential protein expression in the AH between patients with drusen and RPD was quantified using SWATH-MS in the present study. Detailed proteomic analyses of dry AMD patients might provide insights into the in vivo biology of drusen and RPD.

Effects of Ranibizumab, Bevacizumab, and Aflibercept on Senescent Retinal Pigment Epithelial Cells.

PURPOSE: Anti-vascular endothelial growth factor (VEGF) agents have been used for the last 10 years, but their safety profile, including cytotoxicity against various ocular cells such as retinal pigment epithelial (RPE) cells, remains a serious concern. Safety studies of VEGF agents conducted to date have primarily relied on healthy RPE cells. In this study, we assessed the safety of three anti-VEGF agents, namely, ranibizumab, bevacizumab, and aflibercept, on senescent RPE cells. METHODS: Senescent human induced pluripotent stem cell-derived RPE cells were generated by continuous replication and confirmed with senescence biomarkers. The viability, proliferation, protein expression, and phagocytosis of the senescent RPE cells were characterized 3 days after anti-VEGF treatment with clinical doses of ranibizumab, bevacizumab, or aflibercept. RESULTS: Clinical doses of ranibizumab, bevacizumab, or aflibercept did not decrease the viability or alter proliferation of senescent RPE cells. In addition, the anti-VEGF agents did not induce additional senescence, impair the protein expression of zonula occludens-1 and RPE65, or reduce the phagocytosis capacity of senescent RPE cells. CONCLUSIONS: Clinical dosages of ranibizumab, bevacizumab, or aflibercept do not induce significant cytotoxicity in senescent RPE cells.

Ambient fine particulate matters induce cell death and inflammatory response by influencing mitochondria function in human corneal epithelial cells.

Ambient fine particulate matter (AFP) is a main risk factor for the cornea as ultraviolet light. However, the mechanism of corneal damage following exposure to AFP has been poorly understood. In this study, we first confirmed that AFP can penetrate the cornea of mice, considering that two-dimensional cell culture systems are limited in reflecting the situation in vivo. Then, we investigated the toxic mechanism using human corneal epithelial (HCET) cells. At 24h after exposure, AFP located within the autophagosome-like vacuoles, and cell proliferation was clearly inhibited in all the tested concentration. Production of ROS and NO and secretion of pro-inflammatory cytokines were elevated in a dose-dependent manner. Additionally, conversion of LC3B from I-type to II-type and activation of caspase cascade which show autophagic- and apoptotic cell death, respectively, were observed in cells exposed to AFP. Furthermore, AFP decreased mitochondrial volume, inhibited ATP production, and altered the expression of metabolism-related genes. Taken together, we suggest that AFP induces cell death and inflammatory response by influencing mitochondrial function in HCET cells. In addition, we recommend that stringent air quality regulations are needed for eye health.

Nano-sized iron particles may induce multiple pathways of cell death following generation of mistranscripted RNA in human corneal epithelial cells.

Iron is closely associated with an ambient particulate matters-induced inflammatory response, and the cornea that covers the front of the eye, is among tissues exposed directly to ambient particulate matters. Prior to this study, we confirmed that nano-sized iron particles (FeNPs) can penetrate the cornea. Thus, we identified the toxic mechanism of FeNPs using human corneal epithelial cells. At 24h after exposure, FeNPs located inside autophagosome-like vacuoles or freely within human corneal epithelial cells. Level of inflammatory mediators including nitric oxide, cytokines, and a chemokine was notably elevated accompanied by the increased generation of reactive oxygen species. Additionally, cell proliferation dose-dependently decreased, and level of multiple pathways of cell death-related indicators was clearly altered following exposure to FeNPs. Furthermore, expression of gene encoding DNA binding protein inhibitor (1, 2, and 3), which are correlated to inhibition of the binding of mistranscripted RNA, was significantly down-regulated. More importantly, expression of p-Akt and caspase-3 and conversion to LC3B-II from LC3B-I was enhanced by pretreatment with a caspase-1 inhibitor. Taken together, we suggest that FeNPs may induce multiple pathways of cell death via generation of mistranscripted RNA, and these cell death pathways may influence by cross-talk. Furthermore, we propose the need of further study for the possibility of tumorigenesis following exposure to FeNPs.