MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by post-transcriptional inhibition of mRNA translation. Dysregulation of miRNAs, including circulating miRNAs, has been reported to play an important role in the development of various diseases, including fibrotic diseases. Aberrant expression of miRNAs in the vitreous humor of vitreoretinal diseased eyes has been reported. However, the expression pattern of miRNAs present in the vitreous humor of proliferative vitreoretinal disease (PVD) patients, including proliferative diabetic retinopathy (PDR), and proliferative vitreoretinopathy (PVR), remains unknown. To investigate the factors important for the development of PVD, we characterized the miRNAs present in the vitreous humor of PVD patients and analyzed the expression profiles of 377 miRNAs using quantitative polymerase chain reaction-based miRNA arrays. The expression of a specific subset of miRNAs, previously reported to be associated with the development of angiogenesis and fibrosis, was significantly altered in the vitreous of PVD patients. Among these miRNAs, we identified miR-21 as a candidate fibrotic miRNA with an important role in the pathogenesis of PVD. Increased miR-21 levels in the vitreous were associated with retinal fibrosis, including PVR and PDR. Because epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPECs) plays a critical role in retinal fibrosis, the expression of miR-21 in human RPECs was determined. Its expression in RPECs was induced by transforming growth factor-β, a key growth factor involved in fibrogenesis, and was enhanced by high glucose culture conditions, suggesting that miR-21 expression positively correlates with disease progression. Gain- and loss-of-function studies revealed that miR-21 promoted cell proliferation and migration of ARPE-19 cells without affecting EMT-related gene expression. Together, our studies have identified miR-21 as a potential disease-modifying miRNA in the vitreous humor that is involved in the development of retinal fibrosis and may be a novel marker of PVD.

Significance: Retinal ischemia causes hypoxia-induced neovascularization that stimulates production of proangiogenic cytokines such as VEGF by the hypoxia-inducible transcription factors HIF-1 and HIF-2. Current therapies for ischemic retinal diseases target circulating VEGF but do not modulate the activity of the transcription factors that control its production. We demonstrate that inhibiting HIF-mediated transcription with a peptide derived from the intrinsically disordered protein CITED2, a negative feedback regulator of HIF-1α, reduces pathological angiogenesis in a mouse model of ischemic retinopathy. Combining the CITED2 peptide with the VEGF-Trap aflibercept causes potent suppression of neovascularization and promotes revascularization of the ischemic retina, an outcome not observed for anti-VEGF agents alone, suggesting that dual targeting of the HIF transcription factors and VEGF may be advantageous.

Abnormal subretinal neovascularization is a characteristic of vision-threatening retinal diseases, including macular telangiectasia (MacTel) and retinal angiomatous proliferation (RAP). Subretinal neovascular tufts and photoreceptor dysfunction are observed in very-low-density lipoprotein receptor ( Vldlr ^–/– ) mutant mice. These changes mirror those observed in patients with MacTel and RAP, but the pathogenesis is largely unknown. In this study, we show that retinal microglia were closely associated with retinal neovascular tufts in Vldlr ^–/– mice and retinal tissue from patients with MacTel; ablation of microglia/macrophages dramatically prevented formation of retinal neovascular tufts and improved neuronal function, as assessed by electroretinography. Vldlr ^–/– mice with retinal pigmented epithelium–specific (RPE-specific) Vegfa had greatly reduced subretinal infiltration of microglia/macrophages, subsequently reducing neovascular tufts. These findings highlight the contribution of microglia/macrophages to the pathogenesis of neovascularization, provide valuable clues regarding potential causative cellular mechanisms for subretinal neovascularization in patients with MacTel and RAP and suggest that targeting microglia activation may be a therapeutic option in these diseases.

In the retina, microglia are resident immune cells that are essential for development and function. Retinal microglia play a central role in mediating pathological degeneration in diseases such as glaucoma, retinitis pigmentosa, age‐related neurodegeneration, ischemic retinopathy, and diabetic retinopathy. Current models of mature human retinal organoids (ROs) derived from iPS cell (hiPSC) do not contain resident microglia integrated into retinal layers. Increasing cellular diversity in ROs by including resident microglia would more accurately represent the native retina and better model diseases in which microglia play a key role. In this study, we develop a new 3D in vitro tissue model of microglia‐containing retinal organoids by co‐culturing ROs and hiPSC‐derived macrophage precursor cells (MPCs). We optimized the parameters for successful integration of MPCs into retinal organoids. We show that while in the ROs, MPCs migrate to the equivalent of the outer plexiform layer where retinal microglia cells reside in healthy retinal tissue. While there, they develop a mature morphology characterized by small cell bodies and long branching processes which is only observed in vivo. During this maturation process these MPCs cycle through an activated phase followed by a stable mature microglial phase as seen by the down regulation of pro‐inflammatory cytokines and upregulation of anti‐inflammatory cytokines. Finally, we characterized mature ROs with integrated MPCs using RNAseq showing an enrichment of cell‐type specific microglia markers. We propose that this co‐culture system may be useful for understanding the pathogenesis of retinal diseases involving retinal microglia and for drug discovery directly in human tissue.

Diabetic macular edema (DME) is a common cause of visual impairment in patients with diabetes. Although intravitreal anti-vascular endothelial growth factor (VEGF) injections were efficacious in clinical trials, several patients exhibited a poor response. This study aimed to compare clinical features between patients who were susceptible to intravitreal anti-VEGF injections for DME and those who were not. A single-center, retrospective study of 102 such patients was conducted (123 eyes; mean ± standard deviation age, 63.4 ± 10.8 years; 57.8% males). Systemic and ocular data, assessed at baseline and after a month, were compared between good (>20% decrease in central macular thickness (CMT)) and poor (≤20% decrease in CMT) responders using the Mann–Whitney U test/Fisher’s exact test. Eighty-one eyes (65.9%) were good responders. The glycosylated hemoglobin level was higher (p = 0.011) in poor (7.5% ± 0.94%) than in good (7.04% ± 1.19%) responders. The foveal avascular zone was larger (p = 0.0003) in poor (0.67 ± 0.33 μm²) than in good (0.47 ± 0.23 μm²) responders. The number of microaneurysms in the pericapillary network was higher (p = 0.0007) in poor (2.7 ± 2.2) than in good (1.4 ± 2.0) responders. Baseline glycemic control and macular ischemia may be associated with the short-term response to intravitreal anti-VEGF injections.