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Our previous investigations have shown that bone marrow-derived cells (BMCs), including mesenchymal stem cells (MSCs), contribute to the development of choroidal neovascularization (CNV) as sources of cells and angiogenic factors. Two main steps for circulating BMCs to integrate into CNV lesions are extracellular matrix remodeling and consequential cell migration. MicroRNAs (miRNAs) were found to be involved in CNV development; however, little is known about whether miRNAs regulate the contribution of BMCs to CNV. In the present study, we found that the expression of miR-188-5p was decreased in cultured hypoxic MSCs and BMCs within laser-induced CNV in mice. Matrix metalloproteinase 2 (MMP-2) and MMP-13 were both discovered as targets of miR-188-5p by bioinformatics predictions and dual-luciferase reporter system. Accordingly, increased expression of MMP-2/13 was found in hypoxic MSCs and BMCs in CNV lesions. Furthermore, miR-188-5p mimic transfection caused downregulation of MMP-2/13 in hypoxic MSCs and decreased tube formation of co-cultured vascular endothelial cells. Intravitreal injections of a miR-188-5p agomir attenuated the severity of CNV and inhibited the migration of BMCs into CNV lesions in mice. Our study suggests that miR-188-5p regulates the contribution of BMCs to CNV development by targeting MMP-2/13-mediated extracellular matrix degeneration, and miR-188-5p serves as a therapeutic target to treat CNV-related diseases.

Choroidal neovascularization (CNV) is the common pathological basis of irreversible visual impairment encountered in a variety of chorioretinal diseases; the pathogenesis of its development is complicated and still imperfectly understood. Recent studies indicated that delta-like ligand 4 (Dll4), one of the Notch family ligands might participate in the HIF-1α-VEGF pathway to regulate CNV angiogenesis. But little is known about the influence and potential mechanism of Dll4/Notch signals on CNV angiogenesis. Real-time RT-PCR, Western blotting were used to analyze the expression alteration of Dll4, VEGF and HIF-1α in hypoxic RF/6A cells. Immunofluorescence staining, a laser-induced rat CNV model and intravitreal injection techniques were used to confirm the relationships among these molecules in vitro and in vivo. RPE-RF/6A cell co-culture systems were used to investigate the effects of Dll4/Notch signals on CNV angiogenesis. We found that the Dll4 was involved in hypoxia signaling in CNV angiogenesis. Results from the co-culture system showed that the enhancement of Dll4 expression in RF/6A cells led to the significantly faster proliferation and stronger tube forming ability, but inhibited cells migration and invasion across a monolayer of RPE cells in hypoxic environment, while siRNA-mediated Dll4 silencing caused the opposite effects. Pharmacological disruption of Notch signaling using gamma-secretase inhibitor (GSI) produced similar, but not identical effects, to that caused by the Dll4 siRNA. In addition, the expression of several key molecules involved in the angiogenesis of CNV was altered in RF/6A cells showing constitutively active Dll4 expression. These results suggest that Dll4 play an important role in CNV angiogenesis, which appears to be regulated by HIF-1α and VEGF during the progression of CNV under hypoxic conditions. Targeting Dll4/Notch signaling may facilitate further understanding of the mechanisms that underlie CNV angiogenesis.

Bone marrow (BM)-derived endothelial progenitor cells (EPC) have therapeutic potentials in promoting tissue regeneration, but how these cells are modulated in vivo has been elusive. Here, we report that RBP-J, the critical transcription factor mediating Notch signaling, modulates EPC through CXCR4. In a mouse partial hepatectomy (PHx) model, RBP-J deficient EPC showed attenuated capacities of homing and facilitating liver regeneration. In resting mice, the conditional deletion of RBP-J led to a decrease of BM EPC, with a concomitant increase of EPC in the peripheral blood. This was accompanied by a down-regulation of CXCR4 on EPC in BM, although CXCR4 expression on EPC in the circulation was up-regulated in the absence of RBP-J. PHx in RBP-J deficient mice induced stronger EPC mobilization. In vitro, RBP-J deficient EPC showed lowered capacities of adhering, migrating, and forming vessel-like structures in three-dimensional cultures. Over-expression of CXCR4 could at least rescue the defects in vessel formation by the RBP-J deficient EPC. These data suggested that the RBP-J-mediated Notch signaling regulated EPC mobilization and function, at least partially through dynamic modulation of CXCR4 expression. Our findings not only provide new insights into the regulation of EPC, but also have implications for clinical therapies using EPC in diseases.