IL-10-induced modulation of macrophage polarization suppresses outer-blood-retinal barrier disruption in the streptozotocin-induced early diabetic retinopathy mouse model.

Diabetic retinopathy (DR) is associated with ocular inflammation leading to retinal barrier breakdown, vascular leakage, macular edema, and vision loss. DR is not only a microvascular disease but also involves retinal neurodegeneration, demonstrating that pathological changes associated with neuroinflammation precede microvascular injury in early DR. Macrophage activation plays a central role in neuroinflammation. During DR, the inflammatory response depends on the polarization of retinal macrophages, triggering pro-inflammatory (M1) or anti-inflammatory (M2) activity. This study aimed to determine the role of macrophages in vascular leakage through the tight junction complexes of retinal pigment epithelium, which is the outer blood-retinal barrier (BRB). Furthermore, we aimed to assess whether interleukin-10 (IL-10), a representative M2-inducer, can decrease inflammatory macrophages and alleviate outer-BRB disruption. We found that modulation of macrophage polarization affects the structural and functional integrity of ARPE-19 cells in a co-culture system under high-glucose conditions. Furthermore, we demonstrated that intravitreal IL-10 injection induces an increase in the ratio of anti-inflammatory macrophages and effectively suppresses outer-BRB disruption and vascular leakage in a mouse model of early-stage streptozotocin-induced diabetes. Our results suggest that modulation of macrophage polarization by IL-10 administration during early-stage DR has a promising protective effect against outer-BRB disruption and vascular leakage. This finding provides valuable insights for early intervention in DR.

Mitochondrial transplantation attenuates oligomeric amyloid-beta-induced mitochondrial dysfunction and tight junction protein destruction in retinal pigment epithelium.

Transplantation of mitochondria derived from mesenchymal stem cells (MSCs) has emerged as a new treatment method to improve mitochondrial dysfunction and alleviate cell impairment. Interest in using extrinsic mitochondrial transplantation as a therapeutic approach has been increasing because it has been confirmed to be effective in treating various diseases related to mitochondrial dysfunction, including ischemia, cardiovascular disease, and toxic damage. To support this application, we conducted an experiment to deliver external mitochondria to retinal pigment epithelial cells treated with oligomeric amyloid-beta (oAß). Externally delivered amyloid-beta internalizes into cells and interacts with mitochondria, resulting in mitochondrial dysfunction and intracellular damage, including increased reactive oxygen species and destruction of tight junction proteins. Externally delivered mitochondria were confirmed to alleviate mitochondrial dysfunction and tight junction protein disruption as well as improve internalized oAß clearance. These results were also confirmed in a mouse model in vivo. Overall, these findings indicate that the transfer of external mitochondria isolated from MSCs has potential as a new treatment method for age-related macular degeneration, which involves oAß-induced changes to the retinal pigment epithelium.

Inhibition of cellular senescence hallmarks by mitochondrial transplantation in senescence-induced ARPE-19 cells.

Retinal pigment epithelium (RPE) damage is a major factor in age-related macular degeneration (AMD). The RPE in AMD shows mitochondrial dysfunction suggesting an association of AMD with mitochondrial function. Therefore, exogenous mitochondrial transplantation for restoring and replacing dysfunctional mitochondria may be an effective therapeutic strategy for AMD. Here, we investigated the effects of extrinsic mitochondrial transplantation on senescence-induced ARPE-19 cells. We demonstrated mitochondrial dysfunction in replicative senescence-induced ARPE-19 cells after repeated passage. Imbalanced mitophagy and mitochondrial dynamics resulted in increased mitochondrial numbers and elevated levels of mitochondrial and intracellular reactive oxygen species. Exogenous mitochondrial transplantation improved mitochondrial dysfunction and alleviated cellular senescence hallmarks, such as increased cell size, increased senescence-associated ß-galactosidase activity, augmented NF-κB activity, increased inflammatory cytokines, and upregulated the cyclin-dependent kinase inhibitors p21 and p16. Further, cellular senescence properties were improved by exogenous mitochondrial transplantation in oxidative stress-induced senescent ARPE-19 cells. These results indicate that exogenous mitochondrial transplantation modulates cellular senescence and may be considered a novel therapeutic strategy for AMD.

Cell-type-specific Modulation of Non-homologous End Joining of Gamma Ray-induced DNA Double-strand Breaks by cAMP Signaling in Human Cancer Cells.

BACKGROUND: Cyclic AMP (cAMP) signaling is activated by various hormones and neurotransmitters and regulates numerous physiological phenomena, including energy metabolism, gene expression, and proliferation. cAMP signaling plays a role in the repair of DNA damage, but its specific function is inconsistent in the literature. The present study aimed to investigate the mechanism of the different roles of cAMP signaling in DNA repair by analyzing the cell-type differences in the modulation of DNA repair by cAMP signaling following γ-ray irradiation. METHODS: cAMP signaling was activated in human malignant melanoma cells (SK-MEL-2 and SK-MEL-28), human uterine cervical cancer cells (HeLa and SiHa) and human non-small cell lung cancer cells (H1299 and A549) by expressing a constitutively active mutant of the long-form stimulatory α subunit of GTP-binding protein or by treating with isoproterenol and prostaglandin E2 before γ-ray irradiation. DNA damage was quantitated by western blot analysis of γ-H2AX, and non-homologous end joining (NHEJ) was assessed by fluorescent reporter plasmid repair assay and immunofluorescence of microscopic foci of XRCC4 and DNA-ligase IV. RESULTS: cAMP signaling modulated DNA damage, apoptosis and the NHEJ repair following γ-ray irradiation differently depending upon the cell type. cAMP signaling regulated the phosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at Ser2056 and Thr2609 in cell-type-specific manners following γ-ray irradiation, an activity that was mediated by protein kinase A. CONCLUSION: cAMP signaling modulates the NHEJ repair of γ-ray-induced DNA damage in melanoma cells, uterine cervical cancer cells and lung cancer cells in a cell-type-specific manner, and the modulation is likely mediated by protein kinase A-dependent phosphorylation of DNA-PKcs. This study suggests that cell- and tissue-specific modulation of DNA damage repair by cAMP signaling may contribute to improve the therapeutic efficiency of radiation therapy.

Inhibition of non-homologous end joining of gamma ray-induced DNA double-strand breaks by cAMP signaling in lung cancer cells.

DNA double-strand breaks (DSB) are formed by various exogenous and endogenous factors and are repaired by homologous recombination and non-homologous end joining (NHEJ). DNA-dependent protein kinase (DNA-PK) is the principal enzyme for NHEJ. We explored the role and the underlying mechanism of cAMP signaling in the NHEJ repair of DSBs resulted from gamma ray irradiation to non-small cell lung cancer (NSLC) cells. Activated cAMP signaling by expression of an activated stimulatory GTP-binding protein or by pretreatment with isoproterenol and prostaglandin E2, delayed the repair of DSBs resulted from gamma ray irradiation, and the delaying effects depended on protein kinase A (PKA). Activated cAMP signaling suppressed XRCC4 and DNA ligase IV recruitment into DSB foci, and reduced phosphorylation at T2609 in DNA-PK catalytic subunit (DNA-PKcs) with a concomitant increase in phosphorylation at S2056 in PKA-dependent ways following gamma ray irradiation. cAMP signaling decreased phosphorylation of T2609 by protein phosphatase 2A-dependent inhibition of ATM. We conclude that cAMP signaling delays the repair of gamma ray-induced DNA DSBs in NSLC cells by inhibiting NHEJ via PKA-dependent pathways, and that cAMP signaling differentially modulates DNA-PKcs phosphorylation at S2056 and T2609, which might contribute to the inhibition of NHEJ in NSLC cells.