1. Introduction

Microglia, which are derived from the yolk sac, are the primary resident mononuclear macrophages in the retina and are regarded as the first line of active immune defenders. They express many pattern recognition receptors and immune receptors, such as AT1R, AT2R, and TβRII, empowering their function of guiding blood vessel formation, guaranteeing the survival of neurons, monitoring neuronal activity, pruning synapses of neurons, sustaining density of dendritic spines and clearing apoptotic cells or protein aggregates (Takahashi et al., 2005; Parkhurst et al., 2013; Cramer et al., 2022; He et al., 2022; Figure 1). Retinal microglia mainly present high branch structures to guarantee retinal homeostasis. When the balance of the retinal microenvironment is broken, microglia change their phenotypes, such as amoeboid mode, and stay in an activated state. Activated microglia are one of the essential neurotoxic mediators of neuroinflammation (Deng et al., 2009; Zhao et al., 2015). They rapidly move to damage sites and secrete pro-inflammatory factors (e.g., IL-1β, IL-6, TNF-α), chemokines (e.g., CCL2, CCL4, CXCL10) resulting in secondary damage to neuronal death, thereby accelerating the disease progression of patients (Wu et al., 2021; Puthenparampil et al., 2022).

Figure 1. (A) A stepwise microglia developmental program in the retina. Mature microglia maintain a stable population derived from self-renewal. E-embryo. (B) Ramified microglia can be activated to two polarized phenotypes: M1 and M2. The polarization of M1 microglia increased the expression level of proinflammatory factors. Moreover, the polarization of M2 microglia expressed a high level of anti-inflammatory factors. (C) The functions of microglia in healthy retina tissue: Phagocytosis of apoptotic cells and debris; releasing neurotrophic factors to neurons; maintaining neural circuits; ensuring the survival of neurons; pruning neuronal synapses; promoting angiogenesis in retinal development; controlling the retinal vascular blood flow rate.

The retina, a highly active tissue member of the central nervous system, requires high oxygen consumption and metabolic demands. The retinal vascular systems transport nutrients to the retina and break down metabolites. In retinal diseases, such as proliferative diabetic retinopathy and retinopathy of prematurity, neovascularization exacerbate the loss of sight. Microglia participate in neovascularization and present co-localization with the neovascular plexus. According to recently published papers, researchers classified microglia into pro-inflammatory and anti-inflammatory based on the classification method of macrophages (Cano-Cano et al., 2021). The conversation from the pro-inflammatory microglia to the anti-inflammatory-microglia effectively reduces the formation of neovascularization (Zhou et al., 2021). In addition, endothelial cells cooperate with other types of cells and form the blood-retinal barrier (BRB) which strictly limits the exchange of various molecules and segregates the plasma and the tissue. Activated microglia promote the dysfunction of BRB which further aggravates plasma leakage and the death of cells. And it is possible that interventions targeting microglia could alleviate neovascularization and the damage to the blood-retinal barrier, thereby helping patients preserve their sight.

This review narrates the correlation of microglia with retinal neovascularization and the blood-retinal barrier. Targeting microglial activation may represent an attractive therapeutic method in retinopathy. Microglia involve in retinal vascularization during normal retinal development. Under some retinopathy, microglia participate in pathological angiogenesis, particularly in hypoxia conditions. The large number of inflammatory factors released by continuously activated microglia can accelerate the loss of vision and lead to the death of retinal endothelial cells, pericytes, retinal pigment epithelium (RPE), and neurons. Pro-inflammatory microglial death is fatal to the retinal disease process. Hence, we recapitulate three microglial signaling pathways we are more interested in. They may monitor retinal vascular function and decipher how microglia migrate near blood vessels through intercellular signaling crosstalk. This evidence demonstrates the vital role of microglia in the retinal vasculature and BRB. Finally, we summarize the research progress of microglia induced by iPSC (iMG) which is a reliable method to predict the molecular mechanisms of retinal microglia in vitro. And the unique properties of microglia may contribute to vascularizing human retinal organoids.

2. Retina microglia, retina vessel, and blood-retinal barrier 2.1. Microglia in retina 2.1.1. Derivation and localization of retinal microglia

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).

2.1.2. Microglial colonization and recolonization after ablation

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).

2.1.3. The classification of retinal microglia

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.

2.2. Microglia correlation with retinal vasculature and BRB in retina

Microglial branches contact retinal blood vessels, secrete nutritional factors and angiogenic factors, control the apoptosis of pericytes and endothelial cells, and timely eliminate redundancy vessel debris which plat an outstanding significance to maintaining retinal function (Ritter et al., 2006; Jolivel et al., 2015; Harsing et al., 2021). They regulate vessel diameter and blood flow velocity through neurovascular coupling (Császár et al., 2021; Mills et al., 2021). Astrocyte and Müller cells are also members of neurovascular coupling. Astrocytes mainly contact the superficial vascular plexus and the regulation ability of Müller cells is principally limited in the intermediate vascular plexus in stable conditions (Biesecker et al., 2016).

IPL and OPL are the main oxygen-consuming layers (Yu and Cringle, 2001). In these layers, microglial branches co-locate with blood vessels which is sufficient to justify the importance of microglia to the retinal blood vessel and the BRB. Therefore, in this section, we briefly introduce the structure of retina and then summarize the relevance of microglia to the retinal neovascularization and BRB.

2.2.1. Structure of retina

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).

Figure 2. (A) Genesis of retinal cells during the development of the human and mouse. FWK-fetal week; ME-mouse embryo; MP-mouse postnatally. (B) Schematic picture of the retina, the iBRB and oBRB. The main types of tight junction protiens are different in iBRB and oBRB.

2.2.2. Retinal vasculature

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.

2.2.3. BRB

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).

2.2.4. Microglial relationship with retinal vasculature

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).

Figure 3. (A) Microglia monitor the retinal pericytes and endothelial cells and regulate the rate of retinal blood flow in healthy retina. B-D. The microglial responses in different retinal dysfunction environments. (B) Activated microglia promote apoptosis of neuronal cells, endothelial cells, and pericytes. Persistent activation of microglia alters the permeability of endothelial cells and promotes the appearance of vascular leakage. (C) Microglia guide the formation of neovascularization when the retina stays in a comparatively hypoxic environment. (D) In an inflammatory environment, activated microglia phagocytose the neovascular plexus or engulf apoptotic endothelial cells. (E) The major types of tight juctional molecules between endothelial cells of the iBRB in healthy retina. (F) Inflammatory factors secreted by activated microglia facilitate the downregulation of tight junctional molecules between endothelial cells, and vascular leakage occurs. (G) The significant types of tight junctional molecules between RPE cells of the oBRB in healthy retina. (H) Inflammatory factors secreted by activated microglia accelerate the downregulation of tight junctional molecules between RPE cells, capillary leakage, and RPE cells apoptosis. Choroidal neovascularization invasion to subretinal space in a relatively hypoxic environment.

2.2.5. Microglial relationship with BRB

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.

3. Potential targets of microglia in retinopathy 3.1. The microglial renin-angiotensin system contributes to retinal vasoconstriction and angiogenesis

Renin-angiotensin system was initially considered a humoral system that governed blood pressure and water-sodium homeostasis. In recent studies, researchers find that renin-angiotensin system has the function of regulating ocular circulation and balancing intraocular pressure. The upregulation of the renin-angiotensin system contributes to the formation of blood vessels in the developing retina (Sarlos and Wilkinson-Berka, 2005). Dysregulation of the renin-angiotensin system is commonly seen in retinopathy, such as ROP. Blocking the renin-angiotensin system could postpone neovascularization forming (Misrak Tadesse et al., 2001).

In the renin-angiotensin system, renin, a rate-limiting enzyme which the precursor is pro-renin, disassembles angiotensinogen (Agt) encoded by the AGT gene to angiotensin I (Ang I). Ang I subsequently hydrolyze to effector molecule angiotensin II (Ang II) under angiotensin-converting enzyme (ACE). In the model of proliferative Diabetic Retinopathy (DR) and Age-Related Macular Degeneration (AMD), Ang II upregulates the secretion of angiogenic factors and growth factors, provokes microglial activation and leads to hypertension (Moravski et al., 2000; Shi et al., 2010; Hatzopoulos et al., 2014). And up-regulating the expression of retinal Ang II promotes the expression of TNF-α secreted by the activated glial cells which contributes to the death of RGC (Jeon et al., 2022). Activated microglia secrete Agt and Ang II resulting in angiogenesis during tissue injury or disease (Mills et al., 2021).

However, the functions are dissimilar when Ang II binds with various receptors. The common receptors of Ang II are angiotensin subtype-1 receptor (AT1R), Ang II type 2 (AT2R) and MAS-R (Figure 4A).

Figure 4. Schematic diagram of RAS signal pathways in microglia. (A) During disease onset, the RAS in microglia is imbalanced. Ang II is biased towards binding to AT1R and MR which promote microglia to pro-inflammatory functions. Among these receptors, the presence and function of AT2R in retinal microglia remain to be demonstrated. (B) The crosstalk among three glia cells controls blood flow rate through RAS. (C) Microglial MasR controls blood vessel formation during retinal development.

3.1.1. Renin-angiotensin-aldosterone (RAAS) and AT1R

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.

3.1.2. Ace/Ang II/AT2R

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-2p47phox 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-P47phox 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.

3.1.3. ACE2/Ang1-7/MasR

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).

3.2. Microglial CD39 may provide anti-inflammatory treatment and protect BBB integrity during retinopathy

In the mammalian retina, ATP and adenosine (ADO) are crucial molecules that participate in vascular remodeling and form the neurovascular coupling (Zeiner et al., 2019; Losenkova et al., 2022). Abnormally elevated expression of ATP is a common phenomenon in many retinopathies. There are an abundant supply of adenosine triphosphate (ATP) ecto-nucleotidases near retinal blood vessels which are classified into three main types: nucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39), NTPDase2 and ecto-5′-nucleotidase (CD73). Thereinto, CD39 is an extracellular enzyme encoded by ENTPD1 which hydrolyzes ATP to adenosine diphosphate (ADP) and adenosine (ADO) by cooperating with CD73. And retinal CD39 are mainly produced by microglia and endothelial cells. At the early stage of retinopathy, the CD39 expression is significantly up-regulated to maintain the balance between ATP and ADO; when retinal disease develops in the advanced stage, the number of CD39+ microglia decrease prominently and the balance between ATP and ADO is broken; The overladen ATP in the retinal microenvironment mediate inflammatory reaction that leads to neuronal death and M1 microglial activation (Lu et al., 2015; Hu et al., 2017; Rodrigues-Neves et al., 2018).

In addition to eliminating the redundant ATP in the internal environment, CD39 can remodel the anti-inflammatory properties of macrophages (Villacampa et al., 2015; Jakovljevic et al., 2019). CD39 and CD73 synergistically inhibit the multiplication capacity of T cells and decrease the production of inflammatory factors in the EAU mouse model (Chen et al., 2016). Similarly, in type I diabetic hypothalamus, the expression of CD39 in microglia and blood vessels is diminished by 30%, and the integrity of the blood–brain barrier is impaired; Minocycline can effectively promote the expression of CD39 and the pro-inflammatory microglial activation is decreased (Bulavina et al., 2013; Hu et al., 2015).

The ADO, the hydrolysate of ATP, participates in the microglial contracting branches process and recruits them to migrate toward the lesion area (Matyash et al., 2017; Lim et al., 2018). The microglia migration capacity decreases significantly in CD39-deficient mice; after adding exogenous ecto-nucleotidases or ADO, microglial migration ability recovered (Farber et al., 2008).Microglial CD39 mitigates neuronal overactivation through the ATP/AMP/ADO/A1R signaling axis (Badimon et al., 2020). ADO can bind to different receptors and perform different functions. Adenosine A1 receptor (A1R) and A2aR are involved in regulating angiogenesis, blood flow rate and inflammatory response. For example, in the diabetic retina, the ADO releasing and formation stimulated by Triamcinolone promotes the A1R activation which prevents cells from osmotic swelling and results in ion efflux through potassium and chloride channels (Wurm et al., 2008). And the A2aR activation can inhibit phagocytic activity and migration capacity of primary microglia (Ingwersen et al., 2016). The downstream response mechanism of retinal microglia receiving ADO signals is still unclear and needs further exploration (Figure 5A).

Figure 5. Schematic diagram of partly signal pathways in retinal microglia. (A) Microglia express CD39 and CD73 that progressively hydrolyze ATP to ADO together. (B) In the OIR model, microglial necrosis promotes the leakage of FGF, which stimulates retinal neoangiogenesis. Microglia express proangiogenic factors to promote retinal vascular proliferation, and the binding of (C) In the IR model, a competing endogenous RNA network (ceRNET) stimulates microglial pyroptosis. The maturation of pro-caspase1 in inflammasome and the activation and assembly of GSDMD protiens lead to the leak age of inflammatory factors, which ultimately result in the apoptosis of retinal neurons and damage to the BRB. The switching status of microglial signal pathways in different retinal environments and the target genes in upstream and downstream need to be further explored.

Interestingly, microglia create a spatially arranged network in the retinal parenchyma and form a local “purinergic junctions” system with CD39low/CD73− neuronal cell bodies and CD39high/CD73− retinal blood vessels through their CD39high/CD73low branches (Losenkova et al., 2022). Further exploring the mechanisms through which a broad spectrum of soluble and membrane-binding enzymes synergistically regulates purine levels in the retina may serve as potential therapeutic targets for the treatment of retinopathy (Zeiner et al., 2019). Increasing the number of CD39+ microglia subset may contribute to the restoration of the retinal barrier and alleviate inflammation and reduce retinal vascularization.

3.3. Microglial Tgfβ1 and its receptor TβRII involve in vascular remodeling and cellular phenotype conversion

Transforming growth factor beta (Tgfβ), as a multifunctional cytokine which is associated with AMD susceptibility, is vital to sustain the specificity of microglia, ensure the stability of retinal vascular endothelial cells and BBB (Walshe et al., 2009; Braunger et al., 2013; Bohlen et al., 2017; Zarkada et al., 2021). TGFβ can up-regulate SNAIL through AKT and polarize macrophages to anti-inflammatory phenotype; when blocking TGFβ/SNAIL signal transduction, they promote the output of inflammatory factors and convert their phenotype to pro-inflammatory (Zhang et al., 2016). The Tgfβ up-regulation inhibits pro-inflammatory microglial activation and reduces the TNF-α and IL-6 releasing in microglia during retinopathy (Kim et al., 2004; Taylor et al., 2017; Yang et al., 2021).

In mammals, three members of the Tgfβ isoform family encoded by independent genes are identified: Tgfβ1, Tgfβ2 and Tgfβ3 (Anderson et al., 1995). Tgfβ1, as a potential therapeutic target, reduces microglia-mediated neuroinflammation and improves outcomes of intracerebral hemorrhage after acute injury (Taylor et al., 2017). Although the expression of Tgfβ1 is the lowest one of the Tgfβ family in the adult retina, the ability of Tgfβ1 has been proven to induce microglial conversion from the pro-inflammatory phenotype to anti-inflammatory phenotype which is related to neuroprotection and the anti-inflammatory treatment (Tosi et al., 2018; Caruso et al., 2020). In Rho−/− and PDE6βmut/mut mouse model of retinitis pigmentosa, AAV8-mediated supplementation of Tgfβ1 effectively prolongs the cone degeneration time through up-regulating Spp1 and down-regulating Gas6 of microglia, thereby indicating the therapeutic potential of precisely polarizing pro-inflammatory to anti-inflammatory microglia (Wang et al., 2020). And Tgfβ1 can also promote the up-regulation of CD73, inhibit the proliferation of activated T cells and reduce inflammation of the internal environment in the EAU model (Chen et al., 2016). Surprisingly, Tgfβ1 expressed by microglia frequently associates with vascular remodeling. In the microglial autocrine Tgfβ1 signaling pathway, Kindlin3, as an intracellular adapter molecule, is associated with microglial polarization; when Kindlin3 is knockout, high myosin contractility contributes to ERK phosphorylation and further promotes the overexpression of Tgfβ1 which results in angiogenesis (Dudiki et al., 2020).

The Tgfβ receptor consists of two structurally similar sub-families (TβRI and TβRII) and a transmembrane proteoglycan (beta-glycan/TβRIII). Thereinto, TβRII belongs to the serine and threonine transferase receptor, which is highly expressed in retinal microglia and endothelial cells (Ma et al., 2019). After Tgfβ ligation to TβRII, SMAD2 and SMAD3 are phosphorylated and form complexes with SMAD4 which transfer to the nucleus and further promote the transcription of target genes (Javelaud and Mauviel, 2004). In TβRII–deficient condition, the retina presents a critically pathological change in structure and function, such as the shortage of pericyte differentiation and retinal capillaries, which leads to microaneurysms, hemorrhages, microglial activation and proliferative retinopathy (Braunger et al., 2015). According to the collaborative genome-wide association study, TβRII, as a receptor for Tgfβ signaling pathway, was also associated with AMD susceptibility (Fritsche et al., 2013).

Nevertheless, because of the postnatal lethality of TGFβ1-deficient mice or lethal embryonic phenotypes of TβRII-deficient mice, was limited to investigate how TGFβ1 regulates microglial activation through TβRII in adults (Shull et al., 1992; Oshima et al., 1996). Therefore, Zoller et al. constructed Cx3cr1CreERT2:Tgfbr2fl/fl mouse model to persuade the conditional deletion of TβRII in adult microglia and found that TβRII-deficient microglia change the original morphology, up-regulate microglial activation and increase phosphorylation of TAK1 (Zoller et al., 2018). The ablation of TβRII in retinal microglia induces the secondary apoptosis of Müller and reduces mRNA expression level of microglial ‘sensome’ transcripts, such as Siglech (Hickman et al., 2013; Ma et al., 2019). Although TβRII-deficient microglia do not induce obvious changes in the retinal structure of adults, they promote pathological angiogenesis after laser injury (Ma et al., 2019). Similarly, in OIR, TβRII-deficient microglia secrete chemokines and up-regulate Igf1 expression which exacerbates retinal neovascularization plexus forming (Usui-Ouchi et al., 2022; Figure 5B).

According to these shreds of evidence, we draw partly microglial signaling pathways we are more interested in (Figure 5). Then we speculate that retinal TGFβ1 promotes the up-regulation of CD73; CD39 in microglia or endothelial cells cooperates with CD73 to hydrolyze ATP to ADP and ADO; and the hydrolysis production ADO can recruit microglia. In retinal vascular diseases, such as diabetic retinopathy and retinopathy of prematurity, the expression of ATP and ADO are upregulated. This may partly explain why microglia appear in adjacent vessel and injury sites. Intracellular molecular mechanisms in microglia responding to ADO need to be explored in depth. In addition, the Tgfβ1/TβRII signaling pathway in microglia effectively decreases microglial activation and exhibits a protective effect on neurons in retinopathy. And the disruption of the Tgfβ1/TβRII signaling pathway in microglia accelerates pathological angiogenesis during retinal injury and hypoxic processing. The relevance of Tgfβ1 to microglia CD39 and the renin-angiotensin system is currently unknown.

4. Effect of microglia on retinal vasculature and BRB in various retinopathy models 4.1. Microglia in diabetic retinopathy

DR, characterized by the apoptosis of pericytes and endothelial cells as an early clinical feature, results in the damage of BRB integrity and leakage of plasma. Although microglia may be a member of the BRB and act as a secondary protective barrier for extravasated proteins, we cannot ignore the inflammatory response induced by activated microglia. Microglia is the main source of retinal TNF-α after retinal injury. At 3 h of TNF-α intravitreal injections, endothelial cells appeared necrotic and revascularization began at 24 h injection (Claudio et al., 1994). The massive IFN-γ and IL-6 in DR activate microglial STAT3 phosphorylation which stimulates TNF-α secretion, suppresses the kinase activity in AKT/p70S6 signal axis and leads to the apoptosis of pericytes. Under IL-6 stimulation, the activated microglia recruit to the vicinity of RPE and secrete TNF-α to downregulate the expression of ZO-1 and Occludin in the RPE which disrupt the integrity of oBRB; whereas using STAT3 inhibitors modify the influence of microglia to RPE (Jo et al., 2019). With a similar mechanism, the activated microglia increase the permeability of endothelial cells in iBRB (Yun et al., 2017).

In addition, the normal retina responds to light stimulation through neurovascular coupling which expands the blood flow per unit time; whereas in the early stages of type I and II diabetes mellitus, patients normally present a deferred reaction under the stimulation of flickering (Lim et al., 2014). Microglial branches contact the pericytes, endothelial cells, and neuronal synapses in the retinal vasculature and regulate retinal vasoconstriction through the renin-angiotensin system; nevertheless, microglia lost this ability in the early STZ-induced mouse model (Mills et al., 2021). Asiatic acid upregulates the protein arginine (Arg-1) expression and reduces NF-κB p65 nuclear translocation in microglia; the microglial phenotype is reversed and alleviates the disease process of DR (Fang et al., 2021). Erythropoietin protects the BRB by increasing phosphorylation of the Src/Akt/cofilin signaling axis to inhibit microglia engulfing endothelial cells (Xie et al., 2021). CD5-2, as a novel oligonucleotide-based drug, enlarges the VE-cadherin transcription which is silenced by miR-27a in endothelial cells and defends the integrity of BRB and preserves the coverage of pericytes; the activation state of microglia is suppressed after treatment with CD5-2 (Ting et al., 2019). Melatonin can inhibit microglia activation through the PI3K/Akt/Stat3/NF-κB signal axis and protect pericytes from apoptosis (Tang L. et al., 2022). And MicroRNA-93-5p can restrain M1 microglia activation by silencing STAT3 (Wang et al., 2021).

4.2. Microglia in age-related macular degeneration

AMD is a neurodegenerative disease of retinal macular degeneration which is associated with age and can mainly divide into three types: (1) Geographic atrophy; (2) choroidal neovascularization; (3) retinal angiomatous proliferation (RAP). In healthy human retinas, the macular region has no blood vessels on account of the abundantly antiangiogenic factors expressed by cells (Lutty and McLeod, 2018). And the high density of cones in this area indicates more susceptibility to oxidative stress.

Quite unlike humans, the retina of mice does not have macular structures. Hence, external assistance is needed to establish disease models and explore the mechanisms involved in macular degeneration. For example, the NRL-deficient transgenic model assists us in researching retinal degenerative diseases related to cones and decreases the interference of rods (Samardzija et al., 2014). Barben et al., based on this model and knocked out the von Hippel Lindau protein to mimic the degenerative death mechanism of cone under chronic hypoxic conditions in AMD and found that microglia infiltrate to the fundus and the upregulation of H1F-1A, VEGF, and Fgf2 related to neovascularization during early retinal development (Barben et al., 2018). In contrast, the sterile 1% NaIO3 can mimic geographic atrophy by injecting the tail vein (Enzbrenner et al., 2021). And in the 5XFAD mouse model, the presence of deposits on the fundus can mimic types 2 and 3 AMD while using the low-dose efavirenz reduces microglial activation, neovascular formation, and accumulations of amyloid β plaques in focus (El-Darzi et al., 2022).

The microglial number in perivascular and subretinal increases with age. The increased number of microglia and lipofuscin deposition synergistically contribute to the risk of the AMD process (Xu et al., 2008). And the structurally altered HTRA1 contributes to the Tgfβ signaling in autocrine microglia which downregulates phosphorylation of SMAD2 and similarly increases the risk of AMD (Friedrich et al., 2015). Moreover, the high-fat diet activates the immune response of retinal microglia; IL-1β released by the activated microglia provokes the cellular iron sequestration reaction which sparks the toxic accumulation of iron in RPE cells; subsequently, the RPE presents oxidative stress and electrophysiological dysfunction facilitating the process of AMD (Sterling et al., 2022).

4.3. Microglia in other types of retinal diseases

Autoimmune uveitis (Au) is a chronic inflammatory intraocular disease mediated by the autoimmune system. In AU, the abnormal activation of microglia increase the production of inducible nitric oxide synthase (iNOS) which accelerates retinal degeneration and the loss of iBRB integrity, the leakage and abnormal proliferation of capillaries. In the EAU mouse model, the addition of Icariin upregulates PRDX3 which transfers the microglia phenotype from pro-inflammatory to anti-inflammatory and alleviates the state of illness (Wang et al., 2022). Thereinto, PRDX3, a primary isoform of six peroxidases in mitochondria, swiftly scavenges abnormal accumulation of H2O2 to decrease apoptosis and damage caused by oxidative stress (Rebelo et al., 2021).

In addition, CSC is characterized by the dilation and leakage of choroidal vasculature leading to the accumulation of subretinal fluid and serous detachment of the neurosensory retina which can mimic by using aldosterone with the intravitreal injection (Zhao et al., 2012; Yu et al., 2022). CSC may be associated with inappropriate activation of MR (Zhao et al., 2017). The melatonin through intraperitoneal injection rescues microglial infiltration mediated by activation of the IL-17A/NF-kb signaling pathway and significantly reduces CX3CR1 and cyclooxygenase 2 secreted by the activated microglia in the CSC mouse model (Yu et al., 2022).

4.4. Microglia in oxygen-induced retinopathy (OIR)

Microglia are involved in neoangiogenesis and are activated prior to neovascularization in the central avascular region during OIR (Ritter et al., 2006; Fischer et al., 2011). At the beginning of the OIR model construction, mice are exposed to an oxygen-rich environment and the capillaries appear to atrophy; when the survival environment returns to the normoxia, neovascularization occurs in the retina. At postnatal 12 days, the NF-κβ/STAT3 signaling pathway is activated in pro-inflammatory microglia which are presented in the central and peripheral neovascular plexus of the retina; and at postnatal 17 days, anti-inflammatory microglia gradually emerge (Li et al., 2021). During this period, the decreased number of microglia can exacerbate retinal vascular degeneration (Liu J. et al., 2022). Microglia activate the RIP1/ RIP3 signaling pathway to promote the phosphorylation of MLKL which translocate to the cytoplasmic membrane and regulates the activation of ion channels leading to necroptotic of microglia; the necrotic microglia release FGF2 and HIF-1A which stimulate retinal neoangiogenesis; and the knockdown of RIP3 effectively alleviate angiogenesis (Kubota et al., 2009).

The deletion of CCN1, a gene that encodes the extracellular matrix-associated integrin-binding protein, exacerbates the pro-inflammatory responses of microglia and leads to the malformation of retinal vasculature in the OIR model (Yan et al., 2015). And the administration of Celastrol reverses the activation of the miR-17-5p/HIF-1α/VEGF signaling pathway which reduces retinal neovascularization by inhibiting the microglial activation and inflammation; the proliferation, migration and tube formation ability of human retinal microvascular endothelial cells are also inhibited in vitro culture (Zhao et al., 2022). In addition, KC7F2, a novel molecular compound, reduces retinal angiogenesis by reducing the co-localization ratio between microglia and neovascularization and inhibits the activation of the HIF1α/VEGF signaling pathway in human umbilical vein endothelial cells (Tang X. et al., 2022).

4.5. Microglia in ischemia–reperfusion (IR) model

The retinal IR model is another common disease model used in experiments to mimic glaucoma, DR and retinal arterial obstruction (Osborne