Microglial Galectin3 enhances endothelial metabolism and promotes pathological angiogenesis via Notch inhibition by competitively binding to Jag1.

Microglia were considered as immune cells in inflammation until their angiogenic role was widely understood. Although the pro-inflammatory role of microglia in retinal angiogenesis has been explored, little is known about its role in pro-angiogenesis and the microglia-endothelia interaction. Here, we report that galectin-3 (Gal3) released by activated microglia functions as a communicator between microglia and endothelia and competitively binds to Jag1, thus inhibiting the Notch signaling pathway and enhancing endothelial angiogenic metabolism to promote angiogenesis. These results suggest that Gal3 may be a novel and effective target in the treatment of retinal angiogenesis.

Notch activation promotes endothelial quiescence by repressing MYC expression via miR-218.

After angiogenesis-activated embryonic and early postnatal vascularization, endothelial cells (ECs) in most tissues enter a quiescent state necessary for proper tissue perfusion and EC functions. Notch signaling is essential for maintaining EC quiescence, but the mechanisms of action remain elusive. Here, we show that microRNA-218 (miR-218) is a downstream effector of Notch in quiescent ECs. Notch activation upregulated, while Notch blockade downregulated, miR-218 and its host gene Slit2, likely via transactivation of the Slit2 promoter. Overexpressing miR-218 in human umbilical vein ECs (HUVECs) significantly repressed cell proliferation and sprouting in vitro. Transcriptomics showed that miR-218 overexpression attenuated the MYC proto-oncogene, bHLH transcription factor (MYC, also known as c-myc) signature. MYC overexpression rescued miR-218-mediated proliferation and sprouting defects in HUVECs. MYC was repressed by miR-218 via multiple mechanisms, including reduction of MYC mRNA, repression of MYC translation by targeting heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), and promoting MYC degradation by targeting EYA3. Inhibition of miR-218 partially reversed Notch-induced repression of HUVEC proliferation and sprouting. In vivo, intravitreal injection of miR-218 reduced retinal EC proliferation accompanied by MYC repression, attenuated pathological choroidal neovascularization, and rescued retinal EC hyper-sprouting induced by Notch blockade. In summary, miR-218 mediates the effect of Notch activation of EC quiescence via MYC and is a potential treatment for angiogenesis-related diseases.

Integrin α5ß1 promotes BMCs mobilization and differentiation to exacerbate choroidal neovascularization.

Choroidal neovascularization (CNV) is an acknowledged pathogenic mechanism of various ocular diseases, and in situ cells and mobilized bone marrow-derived cells (BMCs) are thought to participate in this process. We aimed to evaluate the roles of integrin α5 in BMCs and vascular endothelial cells (VECs) in the CNV process mediated by SDF-1/CXCR4 signaling. Adult wild-type mice were engrafted with whole BMCs obtained from GFP transgenic mice and then laser injured to induce CNV. BMCs and RF/6A cells were cultured to discover the mechanism of CNV in vitro. BMCs were mobilized to CNV areas, which expressed elevated SDF-1 and CXCR4. When SDF-1 was intravitreally injected, the number of BMCs was profoundly increased. In the SDF-1-treated group, the levels of integrin α5 expressed on BMCs and VECs were significantly higher than those on the cells in the control group. SDF-1 significantly increased the expression and positive ratio of integrin α5, which was involved in the recruitment and differentiation of BMCs into BMC-derived VECs, and these effects were suppressed by the CXCR4 inhibitor AMD3100. The PI3K/AKT pathway rather than the ERK pathway mediated SDF-1/CXCR4 induction of integrin α5. Integrin α5 suppression efficiently prevented the production of TGF-ß and bFGF but not VEGF. Inhibiting the SDF-1/CXCR4-PI3K/AKT-integrin α5 axis reduced CNV severity. Integrin α5 participates in BMC recruitment and differentiation in SDF-1/CXCR4-induced CNV and inhibition of this pathway may be a new approach to inhibit CNV.

SNAI1, an endothelial-mesenchymal transition transcription factor, promotes the early phase of ocular neovascularization.

Ocular neovascularization is a comprehensive process involved in retinal vascular development and several blinding diseases such as age-related macular degeneration and retinopathy of prematurity, with vascular endothelial growth factor (VEGF) regarded as the master regulator. However, the qualified effect of anti-VEGF therapy reveals that the underlying mechanisms are still not clearly identified. To initialize angiogenesis, endothelial cells undergo a phenotype switching to generate highly migratory and invasive cells. This process shares certain similar characters observed in endothelial-mesenchymal transition (EndMT). Here, we found that SNAI1, an EndMT transcription factor, was expressed by endothelial cells in both physiological and pathological ocular neovascularization. SNAI1 overexpression triggered cell morphological change and enhanced cell motility, while loss of SNAI1 attenuated migration, invasion and sprouting. RNA sequence analysis further revealed that SNAI1 knockdown decreased the expression of genes related to cytoskeleton rearrangement and ECM remodeling. Moreover, intravitreal injection of small interfering RNA of SNAI1 suppressed new vessel formation in developing retina as well as mice model of choroidal neovascularization and oxygen-induced retinopathy. Therefore, we propose that the EndMT transcription factor SNAI1 promotes the early phase of ocular neovascularization and may provide a potential therapeutic target.

The Notch ligand delta-like 3 promotes tumor growth and inhibits Notch signaling in lung cancer cells in mice.

Although it has been suggested that Dll3, one of the Notch ligands, promotes the proliferation and inhibits the apoptosis of cancer cells, the role of Dll3 in cancers remains unclear. In this study, we found that in the murine Lewis lung carcinoma (LLC) cells, the level of Dll3 mRNA changed upon tumor microenvironment (TME) stimulation, namely, decreased under hypoxia or stimulated with tumor necrosis factor (TNF)-α. Dll3 was also expressed at higher level in human lung carcinoma tissues than in the para-carcinoma tissues. Overexpression of Dll3 in LLC cells promoted cell proliferation and reduced apoptosis in vitro, and enhanced tumor growth when inoculated in vivo in mice. The Dll3-mediated proliferation could be due to increased Akt phosphorylation in LLC cells, because an Akt inhibitor counteracted Dll3-induced proliferation. Moreover, Dll3 overexpression promoted PI3K/Akt signaling through inhibiting Notch signaling.

Myeloid-Specific Blockade of Notch Signaling Attenuates Choroidal Neovascularization through Compromised Macrophage Infiltration and Polarization in Mice.

Macrophages have been recognized as an important inflammatory component in choroidal neovascularization (CNV). However, it is unclear how these cells are activated and polarized, how they affect angiogenesis and what the underlining mechanisms are during CNV. Notch signaling has been implicated in macrophage activation. Previously we have shown that inducible disruption of RBP-J, the critical transcription factor of Notch signaling, in adult mice results in enhanced CNV, but it is unclear what is the role of macrophage-specific Notch signaling in the development of CNV. In the current study, by using the myeloid specific RBP-J knockout mouse model combined with the laser-induced CNV model, we show that disruption of Notch signaling in macrophages displayed attenuated CNV growth, reduced macrophage infiltration and activation, and alleviated angiogenic response after laser induction. The inhibition of CNV occurred with reduced expression of VEGF and TNF-α in infiltrating inflammatory macrophages in myeloid specific RBP-J knockout mice. These changes might result in direct inhibition of EC lumen formation, as shown in an in vitro study. Therefore, clinical intervention of Notch signaling in CNV needs to pinpoint myeloid lineage to avoid the counteractive effects of global inhibition.

miR-342-5p Is a Notch Downstream Molecule and Regulates Multiple Angiogenic Pathways Including Notch, Vascular Endothelial Growth Factor and Transforming Growth Factor ß Signaling.

BACKGROUND: Endothelial cells (ECs) form blood vessels through angiogenesis that is regulated by coordination of vascular endothelial growth factor (VEGF), Notch, transforming growth factor ß, and other signals, but the detailed molecular mechanisms remain unclear. METHODS AND RESULTS: Small RNA sequencing initially identified miR-342-5p as a novel downstream molecule of Notch signaling in ECs. Reporter assay, quantitative reverse transcription polymerase chain reaction and Western blot analysis indicated that miR-342-5p targeted endoglin and modulated transforming growth factor ß signaling by repressing SMAD1/5 phosphorylation in ECs. Transfection of miR-342-5p inhibited EC proliferation and lumen formation and reduced angiogenesis in vitro and in vivo, as assayed by using a fibrin beads-based sprouting assay, mouse aortic ring culture, and intravitreal injection of miR-342-5p agomir in P3 pups. Moreover, miR-342-5p promoted the migration of ECs, accompanied by reduced endothelial markers and increased mesenchymal markers, indicative of increased endothelial-mesenchymal transition. Transfection of endoglin at least partially reversed endothelial-mesenchymal transition induced by miR-342-5p. The expression of miR-342-5p was upregulated by transforming growth factor ß, and inhibition of miR-342-5p attenuated the inhibitory effects of transforming growth factor ß on lumen formation and sprouting by ECs. In addition, VEGF repressed miR-342-5p expression, and transfection of miR-342-5p repressed VEGFR2 and VEGFR3 expression and VEGF-triggered Akt phosphorylation in ECs. miR-342-5p repressed angiogenesis in a laser-induced choroidal neovascularization model in mice, highlighting its clinical potential. CONCLUSIONS: miR-342-5p acts as a multifunctional angiogenic repressor mediating the effects and interaction among angiogenic pathways.