Microglia-Derived Spp1 Promotes Pathological Retinal Neovascularization via Activating Endothelial Kit/Akt/mTOR Signaling

Abstract

Pathological retinal neovascularization (RNV) is the main character of ischemic ocular diseases, which causes severe visual impairments. Though retinal microglia are well acknowledged to play important roles in both physiological and pathological angiogenesis, the molecular mechanisms by which microglia communicates with endothelial cells (EC) remain unknown. In this study, using single-cell RNA sequencing, we revealed that the pro-inflammatory secreted protein Spp1 was the most upregulated gene in microglia in the mouse model of oxygen-induced retinopathy (OIR). Bioinformatic analysis showed that the expression of Spp1 in microglia was respectively regulated via nuclear factor-kappa B (NF-κB) and hypoxia-inducible factor 1α (HIF-1α) pathways, which was further confirmed through in vitro assays using BV2 microglia cell line. To mimic microglia-EC communication, the bEnd.3 endothelial cell line was cultured with conditional medium (CM) from BV2. We found that adding recombinant Spp1 to bEnd.3 as well as treating with hypoxic BV2 CM significantly enhanced EC proliferation and migration, while Spp1 neutralizing blocked those CM-induced effects. Moreover, RNA sequencing of BV2 CM-treated bEnd.3 revealed a significant downregulation of Kit, one of the type III tyrosine kinase receptors that plays a critical role in cell growth and activation. We further revealed that Spp1 increased phosphorylation and expression level of Akt/mTOR signaling cascade, which might account for its pro-angiogenic effects. Finally, we showed that intravitreal injection of Spp1 neutralizing antibody attenuated pathological RNV and improved visual function. Taken together, our work suggests that Spp1 mediates microglia-EC communication in RNV via activating endothelial Kit/Akt/mTOR signaling and is a potential target to treat ischemic ocular diseases.

Single‐cell RNA sequencing in exploring the pathogenesis of diabetic retinopathy

“…Besides, SPP1 was observed significantly enriched in this activated microglial cluster compared with other cell types, in accordance with the findings from animal models by Bai et al. 56 However, in a study by Corano‐Scheri et al., 37 a cluster of macrophages showing high expression of pro‐angiogenetic chemokines, such as CXCL1, CXCL2 and CCL2, was identified, instead of microglia. The authors claimed that they were unable to replicate the conclusion of Hu et al.…”

“…Pseudo-time analysis further revealed that this profibrotic cluster was the major activation state of microglia involved in the formation of FVM and differentiated from retina-resident microglia rather than from monocyte-derived macrophages. Besides, SPP1 was observed significantly enriched in this activated microglial cluster compared with other cell types, in accordance with the findings from animal models by Bai et al 56 However, in a study by Corano-Scheri et al, 37 a cluster of macrophages showing high expression of pro-angiogenetic chemokines, such as CXCL1, CXCL2 and CCL2, was identified, instead of microglia. The authors claimed that they were unable to replicate the conclusion of Hu et al 34 as they regarded the majority of immune cells in FVM as macrophages rather than microglia.…”

“… 55 and Bai et al. 56 both performed scRNA‐seq in mice with oxygen‐induced retinopathy (OIR), a commonly used model for proliferative retinopathy. Igf1 and Spp1, respectively, were observed to be upregulated in OIR microglia compared to control microglia.…”

“…pro-inflammatory cytokines at the early stage of DR and identified microglia as the major source of interleukin (IL)-1β and tumour necrosis factor (TNF). To address the function of microglia in late DR, Liu et al 55 and Bai et al 56 both performed scRNA-seq in mice with oxygeninduced retinopathy (OIR), a commonly used model for proliferative retinopathy. Igf1 and Spp1, respectively, were observed to be upregulated in OIR microglia compared to control microglia.…”

“…As shown in Table 1 , the application of scRNA‐seq to the study of DR has seen significant advancements. 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 …”