Yolk sac progenitor cells in gastrula ultimately evolve into retinal microglia through three developmental waves from differentiation to primitive bone marrow cells. At mouse E7.0–7.5, the yolk sac progenitor cells in the blood land specifically express the RUNX family transcription factor 1 (Runx1) and differentiate to C-kit⁺ early bone marrow erythroid progenitor cells. At mouse E8.0, bone marrow erythroid progenitor cells enter the first wave of growth and precisely express CD41 and CD45. At E8.25, CD41⁺ and CD45⁺ cells access the second wave of development and further distinguish to the precursor microglia under the regulation of transcription factors such as SPi-1 proto-oncogene (PU.1), interferon regulatory factor (Irf8) and Runx1. At mouse E9.5, the precursor microglia enter the central nervous system (CNS) supported by Sodium/Calcium exchanger protein (NCX-1) and then colonize in the retina (Ginhoux et al., 2010; Prinz et al., 2021). At mouse E10.5, these cells eventually differentiate into the naive microglia under the stimulus factor of transforming growth factor beta (Tgfβ), colony stimulating factor 1 receptor (CSF1R), and interleukin 34 (IL34). EMR1/ADGRE1 (F4/80⁺) microglial population can be identified in the retina at E11.5 (Ajami et al., 2007; Tay et al., 2017). At mouse E12.5, the number of F4/80⁺ cell populations steadily increases and then proliferates and migrates from the central area to the retinal periphery (Santos et al., 2008). At mouse E14.5, microglia possess the function of synaptic pruning and shaping neural circuits (Figure 1A).

Microglia progressively migrate to the whole retina during development and are finally located in ganglion cell layer (GCL), inner plexiform layer (IPL), outer plexiform layer (OPL), and nerve fiber layer (NFL) layers (Lee et al., 2008). Microglial CSF1R is one of the basic receptors to colonize the retina (Ginhoux et al., 2010). CSF1R, as a tyrosine kinase transmembrane receptor, has two activating ligands (Wei et al., 2010); (1) CSF-1, known as macrophage colony stimulating factor (M-CSF), plays an essential role in regulating the proliferation, differentiation, and survival of macrophages (Sherr et al., 1985); (2) IL34, mainly produced by glial cells and neurons, considers a substitute ligand for CSF-1 (Wei et al., 2010). When CSF-1 and IL34 bind to CSF1R, they encourage non-covalent dimerization of the receptor chains and transphosphorylation of tyrosine residues (Yu et al., 2008). And CSF-1 and IL34 have different affinities for D2 and D3 protein domains from CSF1R (Stanley and Chitu, 2014). Furthermore, unlikely microglia in OPL, microglia depend on IL-34 secreted by other neurons to localize in IPL and have the feature of assisting cone and rod in transferring chemical signals (Greter et al., 2012).

The neuronal regeneration capacity is limited in the damaged adult mammalian retina (Jorstad et al., 2017). Nevertheless, microglia repopulate the retina via individual mitosis or migration of the microglia/macrophage outside the retina to guarantee population quantity (Zhang et al., 2018). Huang et al. used PLX5622 to eliminate nearly all endogenous microglia in the retina of Cx3cr1^+/GFP transgenic mice and discovered two microglial refilling ways: The epibiotic microglia of the optic nerve enter the retinal optic disc and then refill from the retinal center to retinal periphery; another microglial refill way derived from corpus ciliare/iris is in the opposite direction with above (Huang et al., 2018a). In addition, the newly recolonized microglia compulsively own similar phenotypes to the endogenous microglia in function, morphology and proliferation under the influence of the retina-tissue environment (Jin et al., 2017; McPherson et al., 2019).

Retinal neuronal activity can affect microglial phenotypes (Anderson et al., 2022). The polarization phenotypes of retinal microglia present two extreme states: pro-inflammatory (M1) and anti-inflammatory (M2; Figure 1B). M1-microglia stay in “Classical activation” which secretes high levels of TNF-α, IL-1β, ROS, etc. Retinal IL-1β is primarily expressed by microglia and adding small doses of exogenous IL-1β increases neuronal survival ability in the excitotoxic condition (Todd et al., 2019). Appropriate activation of M1 microglia also facilitates axonal regeneration in trauma sites (Kigerl et al., 2009). However, continued activation of M1 microglia leads to irreversible neuronal damage (Tang and Le, 2016). In RD10, activated infiltrating microglia continuously secreted IL-1β which further accelerated the degeneration of the rod (Zhao et al., 2015).

The microglial phenotypes are in dynamic alternation during the disease process. M2 microglia can be subdivided into “alternative activation” and “acquired deactivation,” hinging on the activation environment and stimulation factors (Walker and Lue, 2015; Siddiqui et al., 2016). Alternative activated M2 microglia are heavily linked with functions such as anti-inflammatory repair and extracellular matrix reconstitution. In contrast, acquired deactivated M2 microglia convert their phenotype in response to anti-inflammatory factors (e.g., TGFβ1, IL-4) from the environment (Caruso et al., 2020; Chen et al., 2022). M2 microglia increase phagocytosis of erythrocytes and tissue debris, which facilitates hematoma regression (Lan et al., 2017). M1 transformation to M2 microglia alleviates the degeneration of photoreceptors in the mouse model RD1 (Zhou et al., 2018; Figure 1B). The conversion of the M1/ M2 phenotype in the appropriate period of acute or chronic retinopathy may provide better therapeutic benefits.

However, with the discovery of the biomarker, retinal microglia likewise generated different subsets of species, and their classification should not be limited to M1 or M2 in the fully polarized state (Wieghofer et al., 2021). In the meanwhile, Liu et al., had pointed out that a proliferative retinopathy-associated subset of microglia presented to perivascular newborn tufts in the oxygen-induced model (Liu Z. et al., 2022). Therefore, a rational classification of these microglia subsets and naming them with specialized nomenclature is necessary.

Retina, as a functional unit of the CNS, is mainly composed of six types of neurons (rod, cone, amacrine, bipolar, horizontal and ganglion) which convert light from the external environment into neural chemical signal and then transfer it to the brain through the optic nerve. And there are three species of glia cells; thereinto, astrocyte and Müller provide retina nutrition and supporting function. Microglia, as the third type of glia in the retina, supervises the homeostasis of the retinal environment in real time and resists foreign microorganism invasion. In structure, the retina is divided into: (1) ONL: cytons of cone and rod; (2) OPL: synapses of cones, rods and horizontal; (3) INL: cytons of bipolar, horizontal and amacrine and Müller; (4) IPL: synapses of bipolar, amacrine cells, retinal ganglion cells and Müller; (5) GCL: cytons of retinal ganglion cells (Figure 2).

The retina of humans, mice and rats have formed three kinds of vasculature systems in the long course of evolution. And the sequence of their development is choroidal vasculature - hyaloid vasculature-retinal vasculature (Lutty and McLeod, 2018). Retina, as a high oxygen consumption and metabolic demand tissue in the CNS (Kooragayala et al., 2015), has a dual blood supply vascular system: (1) Retinal vasculature: Central artery and vein radially extend along retinal exterior and then form arterioles and venules. Arterioles expand to the IPL and OPL layers and then continue to nurture capillaries. Blood backflow to venules along the capillaries eventually returns to the central vein and leaves the visual system through the optic nerve, and completes the blood cycle of vision. At 14 weeks of gestation in humans, the retinal vasculature starts to develop and enters the epilogue at 23 weeks (Hasegawa et al., 2008). Unlike humans, the retinal vasculature of the mouse comes to maturity about 1–2 weeks after postnatal development, and the time of maturation varies between strains (Stahl et al., 2010). After the development of the retinal vascular system, three layers of vascular plexus are differentiated in the GCL, IPL, and OPL, respectively called superficial vascular plexus, intermediate vascular plexus, and deep vascular plexus. And the growth rate of the deep is preferred to the middle (Fruttiger, 2007). (2) Choroid vasculature also consists of three blood plexuses. The outermost plexus is called Haller’s layer, the middle one is the Sattler layer, and the innermost anterior is the capillaries (Lutty and McLeod, 2018). The anterior capillaries allow plasma to congregate the surface of Bruch’s membrane, and vesicles in RPE deliver nutrients and oxygen from plasma to the rod and cone.

Pathological angiogenesis is the leading irreversible cause of blindness among potentially blinding eye diseases. Angiogenesis is a process defined as forming new blood vessels on basis of existing capillaries under the combined action of angiogenic factors and endothelial cells. Endothelial cells consolidate their proliferation and transferability under the action of vascular endothelial growth factor (VEGF), insulin like growth factor 1 (IGF1) and Notch family receptors and their ligands; some endothelial cells grow the filopodia and become endothelial tip cells (Gerhardt et al., 2003; Cao et al., 2017). Mature neovascularization connects with the initial vessels to participate in blood circulation and become a section of the vascular system (Zoya Tahergorabil, 2012). In multiple retinal diseases, such as RAP, the neovascularization extending to the avascular area aggravated vision loss in patients (Spaide, 2013). And the activated microglia are involved in angiogenesis during disease onset.

BRB is vital to retinal function. Sharing resemblances with the blood–brain barrier in the brain, BRB protects the retina and isolates pathogens and microorganisms. The BRB consists of the inner blood-retinal barrier (iBRB) and the outer blood-retinal barrier (oBRB). Endothelial cells in the iBRB safeguard the integrity of the iBRB under the action of the neurovascular unit formed by microglia, Müller cells, astrocytes and pericytes (O'Leary and Campbell, 2021). And the oBRB is composed of choroid, Bruch’s membrane and RPE (Figure 2B).

In angiodynamic researches, fluorescence angiography (VFA) is commonly utilized to examine the half-rise, half-fall and offset time of blood flow filling in retinal arteries, veins and capillaries after intravenous injection of drugs to check the damage of the blood-retinal barrier, vascular leakage and angiogenesis (Hui et al., 2014).

Microglia settle in the retina through the optic nerve, vitreous body, and ciliary body before retinal vasculature matures (Diaz-Araya et al., 1995). Their ramifications contact endothelial stalk cells and filopodia on tip cells in the period of mouse retinal vasculature development (Checchin et al., 2006). And microglia may lead tip cells to clarify the direction of neovascularization which is ultimately limited in retinal GCL, IPL and OPL layers (Haupt et al., 2019). Sharing resemblances with the brain, after treatment with lipopolysaccharide, the primary microglia up-regulate the VEGF-A and PDGF-BB expression level of RMEC which promotes the ability of tubes formation, migration and proliferation (Li et al., 2014; Ding et al., 2018). After using PLX5622 to deplete microglia, retinal choroidal vessels begin to atrophy, and RPE present dysfunction (Yang et al., 2020). Microglia and retinal vessels are positively correlated in terms of quantity during development (Checchin et al., 2006; Zeng et al., 2022). The diminished number of retinal microglia result in sparse vascular density during the second and fourth postnatal days in CSF^−/− deficient mice (Rymo et al., 2011). Exogenous microglia within intravitreal injection promotes the decreasing density and area of vascular triggered by resident microglia depletion during the retina development process (Checchin et al., 2006). Activated pro-inflammatory microglia are involved in the remodeling of retinal vasculature in the PKD model (Chen et al., 2021). This evidence is sufficient to demonstrate the indispensable role of microglia in adjacent retinal vasculature (Figures 3A–D).

Altered endothelial cell permeability is the central mechanism of BRB dysfunction during retinopathy (Sun et al., 2021). The defective BRB accelerates the death of the cone leading to swift vision degeneration (Ivanova et al., 2019). Targeting activated microglia is a crucial factor in repairing damaged BRB. They recruit neutrophil infiltration, aggravating the damage to the blood-retina barrier and optic nerve cell death in the retinal vein occlusion model (Jovanovic et al., 2020). Microglia can be activated by hyperglycemia in the retinal environment (Zhang et al., 2019). And the activated microglia engulf endothelial cells which exacerbates the phenomenon of acellular capillaries and albumin leakage in the diabetic model (Xie et al., 2021). Inflammatory factors secreted by activated microglia aggravate the damage of BRB (Usui-Ouchi et al., 2020). Pro-inflammatory microglia activate the TLR4/MyD88/NF-κB p65 signaling axis and the NF-κB p65 nuclear translocation promotes the inflammatory factors resulting in the BRB breakdown (Fang et al., 2021). In the co-culture system, unstimulated microglia promote the expression of endothelial tight junction protein (TJP), Zonula Ocluden-1 (ZO-1) and Occludin (Mehrabadi et al., 2017). However, activated microglia secrete IL-1β which stimulates VEGF releasing; subsequently, VEGF can down-regulate the expression of ZO-1 and Claudin-5 in endothelial cells in hypoxia conditions (Inada et al., 2021). And the ablation of activated microglia alleviates innate immunity stimulated by LPS and protects retinal BRB integrity through up-regulating Occludin, ZO1 and Claudin-5 (Kokona et al., 2018; Figures 3E–H).

In conclusion, activated microglia produce inflammatory factors and participate in the damage of BRB. In the hypoxia condition, microglia assist neovascularization and guide them incorrect positioning which causes the secondary lesions and accelerates the disorder progression in patients. Therefore, profoundly investigating the characteristics of microglia under pathological conditions may become a key to treating retinal diseases. After this, we choose one system and two targets we are more interested in. They contribute to retinal vasoconstriction, neovascularization and microglial cytoactive.

The human retina has locally subsisting RAAS systems (Wagner et al., 1996). RAAS controls vasoconstriction, regulates the release of vascular endothelial growth factor (VEGF) to participate in angiogenesis and increase vascular permeability. Moreover, AT1R, as an essential receptor with cardiovascular homeostasis, facilitates the efficacy of Ang II. And Ang II participates in aldosterone release under the action of aldosterone synthase (Maning et al., 2017). Aldosterone further binds to mineralocorticoid receptor (MR) regulating internal water-electrolyte balance and influencing cardiovascular diseases (Waanders et al., 2011). In addition, the aldosterone with the intravitreal injection can mimic clinical symptoms of central serous chorioretinopathy (CSC; Yu et al., 2022).

Retinal microglia have RAAS. In healthy conditions, Microglial CX3CR1 receives neuronal CX3CL1 signal which may govern the velocity of retina blood flow through RAAS (Mills et al., 2021; Figure 4B). When the exogenous Ang II binds to AT1R on the surface of N9(a microglia cell line) and primary microglia, the ROCK activation stimulates the NOX activation via P38 (Rodriguez-Perez et al., 2015). NOX is one of ROS manufacturers which facilitates the process of retinopathy (Ahmad et al., 2021). In addition, the binding of Ang II to AT1R motivates the translocation process of NF-кβ and STAT3 which accelerates the TNF-α releasing (Abadir et al., 2011). Adding AT1R antagonist effectively reverses microglial inflammatory phenotype and reduces the release of inflammatory factors stimulated by exogenous Ang II (Phipps et al., 2018). AT1R up-regulates at 12 h after ischemia, the candesartan, as an AT1R antagonist, effectively reduces the inflammation (Fukuda et al., 2010). The AT1R antagonist diminishes NF-Kβ nuclear translocation and STAT3 phosphorylation which declines the release of TNF-α, IL-10, ROS and nitrite accumulation in BV2 microglia treated with LPS (Bhat et al., 2016).

Microglia also express MR and aldosterone synthase. Microglial density significantly up-regulates in both REN-2 transgenic rats (rats overexpress renin and Ang II) and the Oxygen-Induced Retinopathy(OIR) model; Valsartan (an antagonist of AT1R) and Spironolactone (an antagonist of MR) effectively diminish the VEGF, CCL5 and IFN-γ secreted by the primary microglia in vitro hypoxia condition (Rana et al., 2020). Activated microglia can promote retinal neovascularization in some cases (Hatzopoulos et al., 2014). Using FAD286 (an aldosterone synthase inhibitor) reduces the density of Iba1⁺ microglia and inhibits approximately 89% of neovascularization and 67% of neovascular tufts in the OIR model (Deliyanti et al., 2012).

According to the clues above, we can summarize microglia may regulate the velocity of retina blood flow through RAAS in healthy conditions. However, in chronic diseases, RAAS converts microglia to the pro-inflammatory phenotype, which releases inflammatory factors and injures the integrity of BRB. Reactive microglia promote angiogenesis when the survival environment situates in relative hypoxia.

The ACE/Ang II/AT2R signaling pathway is associated with anti-angiogenesis, anti-inflammatory and partly balances the influence of RAAS (Tao et al., 2016; Carey, 2017). The expression of AT2R decreases with age and is lower than AT1R in the adult mouse retinas (Yoon et al., 2016; Verma et al., 2019). The up-regulation of AT2R effectively prevents damage to the optic nerve and the polymorphism of G/A at the 1,675 site may be related to arteriolar diameter (Kurihara et al., 2006; Liu et al., 2011). Moreover, the activation of the AngII-AT2R signaling pathway can reverse the microglial inflammatory phenotype (Gao W. et al., 2022).

Utilizing exogenous Ang II activates the GSK3β through upregulating the phosphorylation of Y216 and downregulating the phosphorylation of S9 which degrade the NRF2 expression and inhibit microglial antioxidant capacity; the escalation of Ang II concurrently leads to mitochondrial dysfunction through stimulating phosphorylation of PKCα/β and P-PKCδ, activating phosphorylation of NOX-2^p47phox and generating ROS accumulation (Bhat et al., 2019). AT2R binding to AngII stimulates the activation of PP2A (Guimond and Gallo-Payet, 2012). PP2A guarantees neuronal survival and silences PKC/ERK/NF-кB signaling pathway to prevent inflammatory cascade reaction stimulated by LPS (Egger et al., 2003; Bononi et al., 2011). CGP42112A, as an AT2R agonist, induces PP2A activation by declining the p-Y307-PP2A expression and inhibits NOX-2 activation by declining the P-S345-P47^phox expression; in addition, CGP42112A reduces the ROS production induced by Ang II and converts microglial phenotype from pro-inflammatory to anti-inflammatory (Bhat et al., 2019). Delaying administration of C21 (an agonist of AT2R) after 3 days post-stroke aggrandizes the number of anti-inflammatory microglia and effectively improves the rate of survivability, sensorimotor and cognitive deficits (Jackson et al., 2020; Sumners et al., 2020). Adding CGP42112 boosts the proliferation ability of microglial; the newborn cells predominantly present the ramified structure in peripheral infarct and the area of the infarct is diminished after 3 days of stoke; however, using PD4123319 (an AT2R antagonist) reverses the protective function of CGP42112 (McCarthy et al., 2012). We speculate that CGP42112 may promote anti-inflammatory microglia which maintain neuronal survivability. And in the central infarct core, the continuous release of inflammatory factors stimulates microglia to form different phenotypes with microglia in the peripheral infarct area. Whereafter, McCarthy et al. find that under the C21 stimulation, microglia releases brain derived neurotrophic factor (BDNF) which protects neuron survival and improves vasodilation (McCarthy et al., 2014). In addition, the upregulation of VEGF stimulated by PD123319 promotes the proliferation and migration ability of endothelial cells; on the contrary, CGP42112A selectively inhibits vascularization driven by VEGF in the OIR model (Carbajo-Lozoya et al., 2012).

Therefore, we speculate there may be a similar mechanism to brain-derived microglia in retinal microglia. Retinal microglia may preserve neuronal survival and blood vessels through the ACE/Ang II/AT2R signaling pathway which is expected to treat neoangiogenic retinopathy.

ACE2/Ang-(1–7)/MasR promotes vasodilation and has demonstrated anti-inflammatory properties (Liu et al., 2016). ACE2, a homolog of ACE, hydrolyzes Ang I and Ang II to Ang1-7; Ang1-7 binds to Mas receptor (MasR) which is encoded by MAS1 proto-oncogene (Mas1; Jiang et al., 2013). In oxidative stress injury induced by Ang II, the Ang1-7 activation protects the survival of dopaminergic neurons, alleviates microglia-induced inflammatory responses and enhances the survival rate of hypertensive stroke rats model (Regenhardt et al., 2014; Liu et al., 2016; Costa-Besada et al., 2018).

Retinal microglia express MasR which gradually decreases with age (Tuhina Prasad and Li, 2014). In primary retinal microglia, the expression of MasR is up-regulated following the hypoxia condition which is a necessary element to stimulate angiogenesis in retinal development process (Gariano and Gardner, 2005; Foulquier et al., 2019). In MAS1 deficient mice model, the rate of angiogenesis is slower than in controls, and the number of perivascular microglia and filopodia of endothelial tip cells is significantly decreased; under the stimulating of MAS agonist AVE0991, the mRNA expression level of IL-10, Notch1, Dll4, and Jag1 are increased (Foulquier et al., 2019). The contact points between microglial ramifications and endothelial cells activate the Notch1 signal which facilitates tip cells to recruit microglia during retinal development in newborn mice (Outtz et al., 2011). Microglia can stimulate vessel sprouting and branching of aortic ring explants through soluble factors and angiogenic factors; in turn, aortic ring explants recruit microglia to migrate toward them (Rymo et al., 2011). Therefore, we have reasonable doubt to suspect that microglia interact with endothelial cells and guide the tip cells in the neovascular to migrate toward the retinal periphery through the MAS signaling pathway during retinal development (Figure 4C).

The primary microglia of the brain also express MasR (Rivas-Santisteban et al., 2021). Nevertheless, renin-angiotensin system becomes unbalanced under the long-term stimulation of LPS in the brain; when ACE2/ Ang(1–7)/MasR signal axis is stimulated by AVE0991, microglia convert their phenotype from pro-inflammatory to anti-inflammatory (Dang et al., 2021). Utilizing DIZE, an ACE2 activator, diminishes inflammation of activated microglia, up-regulates cell survival proteins and down-regulates neuronal apoptotic proteins through stimulating the ACE2/ Ang (1–7)/MasR signal axis in the 6-OHDA induced Parkinson’s model; nevertheless, the A-779, a MasR antagonist, balances the protective affection of DIZE (Gupta et al., 2022). MasR can form a dimer with AT1R or AT2R. The expression of these dimers in striatal microglia is higher than those in cerebral neurons; when microglia are treated with IFN-γ and LPS, the expression of AT1R-MasR is up-regulated and the expression of AT2R-MasR is down-regulated; Rivas et al. propose the possibility of forming heterotrimer among AT1R, AT2R and MasR (Rivas-Santisteban et al., 2021).

Based on the above clues, we consider the dysfunction of the microglial renin-angiotensin system loses the ability to control retinal vasoconstriction in retinal vascular malformation diseases which reduces the adequate oxygen supply to retinal neurons leading to the deterioration of vision. The molecular relationship among AT1R, AT2R, and MasR needs to be further verified in retinal microglia (Figure 4).