Glial cells are essential regulators of retinal angiogenesis, secreting pro- and anti-angiogenic factors necessary for this process. In the mammalian retina, vascular endothelial cells are always accompanied by astrocytes. Areas of the retina with low vascular density have few astrocytes, whereas avascular areas lack these glial cells (Schnitzer, 1988a, 1987). In humans and guinea pigs, the foveal zone is completely avascular and devoid of astrocytes (Ikui et al., 1976; Small et al., 1991), whereas, in rabbits, astrocytes are restricted to a small, unique vascularized zone called the medullary rays (Schnitzer, 1988a, 1988b).

An essential factor that regulates retinal angiogenesis is the VEGF (Gerhardt et al., 2003; Stone et al., 1995). The concentration of this protein increases in the retina during embryonic development and gradually decreases in the postnatal stages (Famiglietti et al., 2003). Knockout mouse models have shown that deficiency of this growth factor leads to abnormal development of blood vessels and a lethal phenotype (Carmeliet et al., 1996; Ferrara et al., 1996). VEGF is produced by several cell types of the mouse NR, mainly astrocytes and MGC. Using cell-specific deletions of the Vegfa gene by the Cre/LoxP system, it was shown that the absence of this growth factor in astrocytes altered the growth of the SVP. In contrast, deleting Vegfa in neurons and MGC impaired DVP growth without altering SVP growth (Biswas et al., 2020; Rattner et al., 2019). These data indicate that VEGFA derived from glial sources contributes to developing and maintaining various vascular plexuses in the retina (Biswas et al., 2020; Stone et al., 1995).

In the retina, the Norrin-Frizzled-4 (Fzd4)-low-density lipoprotein receptor-related protein 5 (Lrp5)-tetraspanin 12 (Tspan12) signaling module is closely associated with angiogenesis. MGC mainly secretes Norrin in the retina and binds to its high-affinity receptor Fzd4, which is expressed in neurons and endothelial cells (Ye et al., 2009). Disruption of the Norrin pathway in the retina, either genetically or by neutralizing antibodies, results in loss of retinal vascular integrity (Paes et al., 2011; Wang et al., 2012), delay of SVP growth, and loss of DVP (Chen et al., 2012; Luhmann et al., 2005; Ye et al., 2009). These studies suggest that MGC-derived factors, such as VEGF and Norrin, are crucial for retinal angiogenesis and play a predominant role in DVP formation and a partial role in SVP during angiogenic vascular development stages.

As mentioned earlier, glial cells are essential regulators in retinal angiogenesis, and MGC, in particular, are involved in the structural organization of the BRB (García and Vecino, 2003). Blood capillaries are lined by the processes of MGC, which serve as a communicating system for metabolic exchange between the vasculature and neurons (García and Vecino, 2003). One of the characteristic proteins in these barriers is Aquaporin-4 (AQP4). In the retina, this protein is expressed in the end-feet of astrocytes surrounding the SVP and in MGCs in contact with the DVP (Nicchia et al., 2016). Studies using AQP4 knockout mice show vascular leakage only in the DVP, suggesting that expression of this protein in MGC but not astrocytes is critical for maintaining BRB integrity (Nicchia et al., 2016).

An interesting study showed that implanting MGC in the anterior chamber of rats’ eyes resulted in the formation of new blood vessels, indicating a close relationship between vessel formation and MGC’s ability to regulate their functions (Tout et al., 1993). On the other hand, the relationship between endothelial cells and MGC has been the subject of studies in recent years, which will be discussed in the following sections.

The interactions between MGC and other cell members of iBRB, such as endothelial cells, have been studied mainly using in vitro models, in which these cells are co-cultured to understand their involvement and linkage in physiology and pathology. In these studies, MGC has been hypothesized to maintain an “anti-angiogenic background” under physiological conditions (Yafai et al., 2007), significantly limiting events such as angiogenesis.

The anti-angiogenic effect is evident when endothelial cells are co-cultured with MGC in vitro. MIO-M1 cells (Moorfields/Institute of Ophthalmology-Müller 1), a human immortalized MGC line, induce an anti-angiogenic state in bovine retinal endothelial cells (BRECs), resulting in an increase in proliferation and a decrease in apoptosis, and cell migration rates (Yafai et al., 2004), and cell permeability (Tretiach et al., 2005, 2004). Moreover, when BREC cells were exposed to a conditioned medium of MIO-M1 cells, cell proliferation and ERK1/2 activity decreased, independent of their activation by VEGF (Yafai et al., 2007, 2004). These studies demonstrate the ability of MGC, which, together with endothelial cells, to maintain control over retinal angiogenesis, partly mediated by the release of angiogenic and anti-angiogenic factors.

In the neural retina, MGC has been described as the cellular entity responsible for releasing angiogenic mediators that play an essential role in neuro-retinal vascular homeostasis. Accordingly, VEGF plays a central role in vascular development and homeostasis. This factor is a polypeptide secreted by various tissues in response to hypoxia and activates its receptors on endothelial cells to promote survival, proliferation, permeability, and cell migration (Chung and Ferrara, 2011). Molecular and environmental mediators positively regulate its expression and release, including hypoxia, inflammation, excitotoxicity, oxidative stress, and hyperglycemia (Behl and Kotwani, 2015). Under these conditions, Vegf gene transcription is controlled by hypoxia-inducible factor (HIF) proteins and α-β heterodimeric transcription factors that are stabilized by tissue hypoxia and act as intracellular oxygen sensors (Semenza, 2014).

In the neural retina, MGCs are the predominant source of VEGF (Fu et al., 2015; Saint-Geniez et al., 2008; Wang et al., 2015). The angiogenic effects of VEGF are mediated by VEGFR2, which is expressed in all retinal cells but is more prominent in MGC and retinal endothelial cells (Saint-Geniez et al., 2008; Simmons et al., 2018). Both cells increase VEGFR2 expression in response to VEGF (Rossino et al., 2020). However, the type and duration of VEGF stimulus predict the neuroprotective or neurodegenerative behavior of GMCs (Bai et al., 2009; Jiang et al., 2014; Subirada et al., 2018). In addition, MGC-derived VEGF may exert an autocrine loop that promotes the expression of more retinal VEGF (Rossino et al., 2020). The angiogenic effects of VEGF are mediated by VEGFR2, which is expressed in all retinal cells but is more prominent in MGC and retinal endothelial cells (Kim et al., 1999; Saint-Geniez et al., 2008; Simmons et al., 2018). Both cells increase VEGFR2 expression in response to VEGF (Rossino et al., 2020). Temporal expression of endogenous VEGF promotes axonal growth and post-ischemia and aids in reorganizing glial processes within retinal tissue (Hayakawa et al., 2011; Hermann and Zechariah, 2009; Storkebaum and Carmeliet, 2004). However, the type and duration of VEGF stimulus predict the neuroprotective or neurodegenerative behavior of GMCs (Bai et al., 2009; Jiang et al., 2014; Subirada et al., 2018).

Moreover, hypoxia upregulates numerous factors, including EGF, which may contribute to the induction of abnormal angiogenesis and damage the structural arrangement of BRB. Stimulation of EGF receptors (EGF-R) in MGC activates proliferative and migratory behavior, often leading to progressive and permanent vision loss. In vitro experiments have demonstrated a link between VEGF and EGF in MGC. Exposure to exogenous VEGF increases the expression of EGF-R in rat MGC lines (rMC-1) to an even greater extent than the expression induced by its cognate ligand, EGF (a 20-fold increase compared with a 3-fold increase) (Peña and Vazquez, 2020). In the retina, paracrine signaling of VEGF has been linked to the upregulation of heparin-binding EGF ligand (HB-EGF) after injury, activating through EGF-R (Peña and Vazquez, 2020). Upregulation of EGF-R via HB-EGF stimulates MGC to initiate dedifferentiation responses via wnt/β-catenin (Wan et al., 2012).

In vasoproliferative retinopathies, rupture of the BRB and occlusion of the micro-capillaries are common events. In response to this event, MGC increases the secretion of pro-angiogenic factors, such as VEGF and fibroblast growth factor (FGF) (Peña and Vazquez, 2020). bFGF can induce proliferation, migration, and extracellular matrix proteolysis in endothelial cells (Yafai et al., 2013), whereas FGF2 activates proliferation and gliotic responses in MGC (Hollborn et al., 2004), and induces expression of proinflammatory genes in MIO-M1, and seems to play a significant role in activation of MGC during DR (Rezzola et al., 2021). In this disease, FGF2 has been detected in epiretinal membranes formed by gliotic events of MGC (Hueber et al., 1997).

In addition to VEGF, HIF upregulates factors such as ANGPTL4 in MGC in vitro under hypoxic conditions and in vivo in the ischemic inner retina (Xin et al., 2013). Secretion of ANGPTL4 has been shown to modulate the disposition of circulating triglycerides by inhibiting lipoprotein lipase (Yin et al., 2009). ANGPTL4 may promote disruption of vascular integrity by sequentially interacting with integrin α5β1, vascular endothelial-cadherin, and claudin-5 (Huang et al., 2011).

The anti-angiogenic factors antagonize the angiogenic process in different tissues; the PEDF is the primary inhibitor of angiogenesis in the retina (Dawson et al., 1999). PEDF is a glycoprotein first identified in the culture medium of RPE cells (Tombran-Tink and Johnson, 1989) and is now known to block the migration and proliferation of endothelial cells (Duh et al., 2002). High levels of PEDF have been detected in ocular compartments such as the cornea and vitreous, indicating its critical role in maintaining local avascularity (Karakousis et al., 2001). In the neural retina, MGCs are essential producers of PEDF (Eichler et al., 2004a). The anti-angiogenic mechanism of PEDF involves blocking the angiogenic signaling pathway activated by VEGF, specifically nuclear translocation of HIF-1, and inhibiting the phosphorylation of ERK1/2, a pathway involved in retinal angiogenesis. This mechanism has been described in retinal capillary endothelial (RCEC) cells (Zhang et al., 2006).

In contrast, in microvascular diseases like diabetic retinopathy, PEDF levels in the vitreous and aqueous humor decrease markedly. This seems to be closely related to the progression of the retinopathy (Boehm et al., 2003; Ogata et al., 2002, 2001). Exogenous administration of PEDF significantly inhibited neovascularization in animal models of ischemic retinopathies (Duh et al., 2002; Stellmach et al., 2001), possibly by inducing apoptosis in the endothelial cells of the new vessels (Chen et al., 2006; Volpert et al., 2002). Experimental hypoxia models induce overexpression of VEGF and reduction of PEDF in MGC (Eichler et al., 2004a; Yafai et al., 2007). In addition, PEDF has neurotrophic and neuroprotective functions in the retina (Cao et al., 2001, 1999; Ogata et al., 2001; Takita et al., 2003). The anti-angiogenic mechanism of PEDF involves blocking the angiogenic signaling pathway activated by VEGF, specifically nuclear translocation of HIF-1, and inhibiting the phosphorylation of ERK1/2, a pathway involved in retinal angiogenesis. This mechanism has been described in BREC cells (Bullard et al., 2003; Hutchings et al., 2002; Zhang et al., 2006). In NR, the MGC is the primary source of both VEGF and PEDF (Fu et al., 2015; Saint-Geniez et al., 2008; Unterlauft et al., 2014; Wang et al., 2010). This balance between pro- and anti-angiogenic factors, which exist under healthy physiological conditions, is disrupted during pathological events.

The mammalian TGF-β family includes TGF-β1, TGF-β2, and TGF-β3. TGF-β2 is a dimeric polypeptide that regulates cellular growth, proliferation, apoptosis, differentiation, extracellular matrix synthesis, and immunosuppression (Fuchshofer et al., 2005; Lu et al., 2004; Pardali et al., 2010; Roberts and Sporn, 1993). MGC produces substantial amounts of TGF-β2, inhibiting the proliferation of retinal microvascular endothelial cells (Eichler et al., 2004b, 2001). This factor binds to the serine/threonine kinase type II TGF-β2 receptor located in BRECs, which activates and phosphorylates Smad2 and Smad3, attenuating the phosphorylation state of ERK1/2. Thus, TGF-β2 released by MGC has anti-angiogenic activity by inhibiting the proliferation of retinal endothelial cells (Yafai et al., 2014). It has also been described that TGF-β2 can exert anti- or pro-angiogenic effects depending on the cellular conditions, with the anti-angiogenic being maintained under a physiological environment (Yafai et al., 2014).

TSP-1, a sizable multi-domain glycoprotein of the extracellular matrix, can inhibit angiogenesis by inducing apoptosis in endothelial cells (Jiménez et al., 2000); therefore, TSP-1 is involved in the anti-angiogenic background effect in the healthy retina. Furthermore, TSP-1 may render endothelial cells refractory to important angiogenic inducers such as VEGF. The mechanism of TSP-1 depends on its binding to the CD36 receptor, which results from the interaction of upregulated FasL with CD95/Fas and its subsequent apoptosis induction in endothelial cells (Jiménez et al., 2000). TSP-1 also competes with FGF for binding to the endothelial cell surface (Eichler et al., 2004b).

Although the identities and functions of many factors and cytokines that modulate angiogenesis are still unknown, glial cells such as MGC are evidently involved in regulating vascular homeostasis and related processes such as inflammation by releasing protein factors that regulate the activity and behavior of various cells, such as retinal endothelial cells (Figure 3).

Diabetic retinopathy (DR) is the leading cause of adult blindness in the working-age population in industrialized countries (Biswas et al., 2020). DR is characterized in its early stages by microvascular damage, capillary occlusion, and angiogenesis in the proliferative diabetic retinopathy stage (Yafai et al., 2004). An increase in VEGF has been reported as an essential marker of disease progression and the occurrence of events related to angiogenesis and BRB disruption (Aiello et al., 1994; Malecaze et al., 1994). DR is considered a multifactorial disease, with processes such as hyperglycemia, oxidative stress, inflammation, hypoxia, and dyslipidemia contributing to its development (Al-Kharashi, 2018).

Transcriptomic studies performed on the retinas of patients with DR and healthy individuals have identified upregulated genes, particularly those related to inflammatory responses and angiogenesis (Tang and Kern, 2011). The highly neovascular phenotype in DR reflects an imbalance between angiogenic and anti-angiogenic factors. In the vitreous of patients with DR, there is a decrease in PEDF and an increase in VEGF (Ogata et al., 2002, 2001). This increase in VEGF is observed in vitro in MGC cultures exposed to hyperglycemic or hypoxic conditions (Fletcher et al., 2007). Moreover, treatment with intravitreal injections of PEDF for 4 weeks in streptozotocin-induced diabetic rats ameliorates the diabetes-induced changes such as a decrease in a- and b-wave amplitude in electroretinogram, overexpression of glial fibrillary acidic protein (GFAP) in MGC, increase in VEGF, and disruption of BRB (Yoshida et al., 2009).

On the other hand, the pathological changes in the retina during PDR, activated MGC may progress to reactive gliosis, a nonspecific reactive response of glial cells to damage characterized by uncontrolled proliferation, migration, and increased expression of gliosis markers (Bringmann et al., 2006). In addition, MGC can undergo fibrotic transdifferentiation, which contributes to the formation of fibrovascular epiretinal membranes (ERM) that can exert tractional forces on the retinal surface and cause retinal detachment (Oberstein et al., 2011; Roy et al., 2016). In diabetic mouse retinas, ERM formation was stimulated by TGF-β1 through the PI3K/Akt signaling pathway (Zhang and Kong, 2020). These glial-derived structures have low PEDF levels (Lange et al., 2008), which are inversely correlated with the degree of retinal neovascularization (Gao et al., 2001), and present positivity for molecular markers related to glial-mesenchymal transition, including SNAIL proteins, smooth muscle proteins, VEGFA and TGF-β receptors (Wu et al., 2020).

Placental Growth Factor (PlGF), a member of the VEGF family, is also detected at high levels in vitreous PDR patients (Mitamura et al., 2002). PlGF is significantly released from the MGC and plays a physiological role in cellular survival. Its dysregulation may correlate with the development of DR (Sensenbrenner, 1993). In contrast, a deficiency of PIGF in diabetic mice prevents BRB breakdown and retinal cell death (Huang et al., 2015).

Most studies have focused on animal models of type 1 DM, although the highest prevalence of diabetes worldwide is type 2. The Goto-Kakizaki (GK) rat is a genetic model of non-obese type 2 diabetes that develops glucose intolerance corresponding to insulin resistance (Hachana et al., 2020). In this animal model, adult rats have abundant lipid deposits in Bruch’s membrane (Kimura et al., 1982). This is consistent with humans with type 2 DM exhibiting increased serum concentrations of various fatty acids associated with disease progression (Korani et al., 2012). RD is activated by cellular damage in the retina stimulated by the diabetic environment, including increased levels of intraocular free fatty acids. Free fatty acids may serve as triggers for releasing inflammatory cytokines from MGC. The resulting cytokines may be potent stimulators of retinal endothelial pathologies, such as leukostasis, vascular permeability, and basement membrane thinning (Capozzi et al., 2020). In humans, MGC exposed to fatty acids such as linoleic acid and oleic acid, production of VEGF, interleukin-6 (IL-6), and IL-8 was observed. This response was not recapitulated in human astrocytes or retinal pigment epithelial cell lines (ARPE-19). Other fatty acids, such as palmitic acid, have also shown a robust angiogenic response in the mouse retina, blocked by inhibition of the PPARβ/δ receptor (Capozzi et al., 2020). These reports suggest specific mechanisms of MGC for producing pro-angiogenic factors in response to lipid molecules, which are vital for the progression of DR (Capozzi et al., 2016).

In current treatments of DR, VEGFA activity is neutralized by intravitreal administration of anti-VEGF antibodies such as ranibizumab or aflibercept (Cai et al., 2021). A significant increase in apoptosis and autophagy has been observed in MGC treated with these drugs (Le, 2017; Saint-Geniez et al., 2008). This complements recent evidence that the VEGF released by MGC protects itself and retinal neurons (Le, 2017; Matsuda et al., 2017) and acts as a neuroprotective factor when adverse conditions threaten neuronal survival (Amato et al., 2016). Moreover, disruption of VEGFR2 in rat MGC and mouse retina under diabetic conditions leads to apoptosis of these cell types (Fu et al., 2015; Saint-Geniez et al., 2008). This effect might be different in the MIO-M1 cell line (Matsuda et al., 2017).

In recent years, vitreous humor from patients with PDR in retinal cell cultures has been used as an in vitro model of DR. In MIO-M1 cells, applying vitreous obtained from PDR patients increased cell proliferation, migration, and expression of proinflammatory cytokines and chemokines (Rezzola et al., 2021). Surprisingly, this inflammatory phenotype did not appear to be mediated by stimulation with VEGF or bFGF. Anti-VEGF treatments such as ranibizumab had no protective effect. However, using FGFR receptor antagonists (BGJ398) and anti-FGF traps (NSC12) inhibited the proinflammatory responses induced by the vitreous PDR patients (Rezzola et al., 2021). Exosomes expressing the microRNA miR-9-3p, derived from MGC, and whose described mechanism involves binding to coding sequences of the sphingosine-1-phosphate receptor S1P1 in endothelial cells. miR-9-3p were identified in the vitreous of PDR patients, which subsequently activates VEGFR2 phosphorylation and internalization in the presence or absence of exogenous VEGFA, inducing proliferation, migration, and tube-forming ability in primary human retinal endothelial cells (hREC) (Liu et al., 2022). Other studies have found increased levels of PCED1B Antisense RNA 1 (PCED1B-AS1) in the vitreous of DR patients (Wang et al., 2022). In in vitro cultures of MGC exposed to hypoxic or hyperglycemic conditions induced by CoCl₂ or high glucose, respectively, PCED1B-AS1 significantly attenuated the upregulation of VEGF and MCP-1. It also attenuated VEGF-induced proliferation and migration of HRMECs (Wang et al., 2022). Information suggests that both are markedly released in MGC and play a promising role in treating DR (Liu et al., 2021; Patel et al., 1994).

Hypoxic conditions promote a significant increase in pro-angiogenic factors in MGC, including VEGF and ANGPTL4, leading to angiogenesis and increased vascular permeability of endothelial vascular cells (Ou et al., 2019). Both in vitro and in vivo, the upregulation of both proteins in MGC is regulated by HIF-1 (Babapoor-Farrokhran et al., 2015). ANGPTL4 expression was also increased in the vitreous and aqueous humor of PDR patients and localized mainly in areas of retinal angiogenesis. ANGPTL4 has been associated with promoting vascular permeability and macular edema in diabetic patients. Inhibition of ANGPTL4 expression reduced the angiogenic potential of hypoxic MGC, and this effect was additive with inhibition of VEGF expression (Babapoor-Farrokhran et al., 2015). Another factor associated with these hypoxic conditions is bFGF. Ischemia–reperfusion or post-ischemia conditions in rat retinas increased bFGF levels in MGC or isolated MGC in vitro. In PDR patients, bFGF immunoreactivity colocalizes with GFAP-positive cells in surgically removed retinal tissues. The cells synthesize mRNA and the bFGF protein and release it, which is increased under hypoxia or oxidative stress and stimulated by growth factors and cytokines, including pro-inflammatory factors. The conditioned culture medium of cells exposed to hypoxia for 72 h had a stimulatory effect on retinal endothelial cell proliferation and ERK1/2 phosphorylation. Treatment with neutralizing bFGF antibodies suppressed both effects similarly to anti-VEGF antibodies (Yafai et al., 2013).

Many of these pathologic effects appear to be a side effect of the increased expression of pro-angiogenic and pro-inflammatory cytokines and growth factors in the diabetic retina, mainly promoted by the active response of MGC.

The retina is among the last organs to undergo vascularization during fetal development, initiating around 16 weeks’ gestation. At this stage, vasculogenesis starts in the posterior region of the superficial retina, precisely the optic disk, and extends towards the periphery. By 25 weeks gestation, angiogenesis begins from the optic disk and progresses peripherally, developing a deeper vascular network. This process is driven by the hypoxic uterine environment, which induces pro-angiogenic growth factors that promote retinal blood vessel growth to meet the increasing oxygen demands of the developing retina. Complete retinal vascularization is achieved by approximately 36–40 weeks’ gestation. The relatively hyperoxic postnatal environment then prevents further vasoproliferation (Koc and Bas, 2024; Tsang et al., 2019).

Retinopathy of prematurity (ROP) is the most common cause of visual impairment in infancy worldwide and causes 170,000 infants to go blind annually around the world (Hartnett and Penn, 2012; Hoppe et al., 2020). It occurs primarily in premature infants because of high oxygen concentrations in incubators (Park et al., 2010). ROP is a two-stage ocular disease, and the first stage is vaso-obliteration caused by hyperoxia due to excessive oxygen consumption to support respiratory function (Mutlu and Sarici, 2007). Delayed growth of retinal vessels and degeneration of preexisting vessels are hallmarks of this stage. The second stage is vasoproliferative, triggered by relative hypoxia caused by the retinal vessels not carrying enough oxygen (Smith, 2003). The second stage is abnormal retina neovascularization at the interface between avascular and vascularized areas (Cai et al., 2021). The severity of retinopathy of prematurity (ROP) is inversely related to gestational age. Since retinal vascularization is typically completed around birth, premature infants often exhibit a larger area of retinal avascularity. Postnatally, exposure to a hyperoxic environment exacerbates vasoattenuation of the sparse retinal vasculature. When these infants are subsequently returned to a normoxic room air environment, retinal hypoxia ensues, leading to the development of ROP (Penn, 2008).

MGC produces TGF-β2 under physiological and post-ischemic conditions. In MGC cultures with BRECs, TGF-β2 is secreted by MGC bound to TGF-β receptors in BRECs and activated anti-proliferative signaling pathways (Yafai et al., 2014). In the immortalized human MGC line, MIO-M1, and guinea pig MGC, hypoxia resulted in decreased release of TGF-β2 and PEDF but increased release of TSP-1. All three factors inhibited the proliferation of retinal endothelial cells. It is also possible that hypoxia-induced VEGF stimulates the production of TSP-1 in MGC, inhibiting endothelial cell growth via a negative feedback mechanism (Eichler et al., 2004b).

In the retinas of a rat ROP model, the oxygen-induced retinopathy (OIR) model, VEGFA mRNA was found mainly in the INL corresponding to CRALBP-positive MGC (Wang et al., 2013). In this model, specific knockdown of MGC-derived VEGF164 by short hairpin RNAs maintained long-term inhibition of intravitreal neovascularization and limited cell death (Jiang et al., 2014) without affecting retinal vascular development and reducing VEGFR2 phosphorylation in retinal endothelial cells (Wang et al., 2013). An in vitro iBRB model co-culture mouse microvascular endothelial cells and MGC exposed to hypoxia showed that the response to the oxygen deprivation is more harmful in endothelial cells, compared with the iBRB model without MGC, resulting in more impaired barrier function in the iBRB in vitro model (Inada et al., 2021). This suggests that communication between MGC and endothelial cells requires highly orchestrated mechanisms in which MGC can act as sensors of retinal damage.

Angiogenesis is a complex process for vascular development and pathologic conditions such as age-related macular degeneration (AMD). Intraretinal and subretinal neovascularization originates from the inner retinal vessels in a subtype of AMD called retinal angiomatous proliferation (RAP) (Dorrell et al., 2009). RAP is one of the most common causes of blindness worldwide (Miller, 2013). It arises from superficial vascular plexus (SVP) capillaries and develops initially intraretinal (stage I), subretinal (stage II), and finally to choroidal neovascularization and retinal-choroidal anastomosis (stage III) (Wei et al., 2019). Pathologically, RAP is an intraretinal angiomatous complex in the outer retina (Monson et al., 2008; Wei et al., 2019). Few animal models simulate RAP. In a mouse model of RAP, which has characteristics like human patients and involves deletion of the very low-density lipoprotein receptor (VLDLR), MGC was activated near the lesion and showed increased expression of VEGF and bFGF. In the retinas of this model, there was also an increase in phosphorylation of Akt and MAPK and translocation of NFkB (Li et al., 2007). Recently, a model involving intravitreal administration of DL-2-aminoadipic acid (DL-AAA), an inhibitor of glutamine synthetase (GS) toxic to MGC, was described. After 8 to 12 weeks, administration of DL-AAA in rabbits resulted in intraretinal detachment with retinal degeneration, retinal and choroidal inflammation, retinal vascular leakage, retinal hemorrhage, dilated retinal vessels, and retinal angiogenesis with epiretinal vessels growing toward the peripheral retina and central area. The mechanism by which DL-AAA produces these effects has yet to be fully understood. However, it has been hypothesized that DL-AAA alters the connections between BRB, causing retinal metabolic dysfunction, retinal ischemia, and inflammation, resulting in high concentrations of VEGF that induce a chronic state of neovascularization (Kumar et al., 2022). Interestingly, in this animal model, the phenotype RAP is achieved by direct changes in MGC and their physiological function by promoting its secretory phenotype of proangiogenic factors that stimulate retinal angiogenesis (Kumar et al., 2022; Mao and Liu, 2013; Sapieha et al., 2010).

VEGF has been described as one of the main pro-angiogenic factors; however, several studies over the past few decades have identified several pro-angiogenic and anti-angiogenic molecules, many of which are released by MGC. These include ANGPLT4, PEDF, BDNF, and Angiopoietin-2, among others (Babapoor-Farrokhran et al., 2015; Calado et al., 2016; Gong et al., 2012; Seki et al., 2004; Striglia et al., 2020). Novel therapies for treating vasoproliferative retinopathies could involve modulating the natural release of cytokines or growth factors from MGC to shift pro-angiogenic and neurodegenerative retinal microenvironments toward anti-angiogenic, neuroprotective, and pro-survival states. At this moment, it is crucial to understand in detail the molecular mechanisms of anti-angiogenic proteins or growth factors, not only in MGC but also in other retinal cell players. Understanding the specific expression of the release system and receptors that modulate the downstream release of growth factors and cytokines will enable pharmacological or genetic interventions to block excessive release without compromising the function of other glial or non-glial cells. In this regard, the extended localization of MGC, adjacent to the vitreous and subretinal space, makes it one of the first cell types to respond to intravitreal or subretinal injections (Devoldere et al., 2019), thus facilitating the delivery of specific drugs that modulate the synthesis and release of angiogenic proteins. Moreover, numerous studies show that neural retinal damage occurs before detectable vascular damage (Sinclair and Schwartz, 2019; Verma et al., 2011), suggesting that modulating the pro- and anti-angiogenic environment of MGC could influence neuronal, glial, and retinal endothelial survival, damage, or death, not only in proliferative stages of DR but also at an early stage.

EVs are a less-explored molecular tool in Müller cells and the retina. They are nano-sized vesicles secreted into the extracellular environment by all forms of living cells. They are essential elements in cell-to-cell communication and carry bioactive molecules such as amino acids, proteins, RNA, and lipids. Substantial evidence suggests that EVs can modulate angiogenesis in organs and have potential therapeutic applications (Todorova et al., 2017). However, compared to other organs and tissues, the available information regarding angiogenic mechanisms, biogenesis, and cargo of EVs in the retina is considerably limited.

Nevertheless, there is evidence that Müller cells secrete EVs capable of modulating angiogenesis in endothelial cells (Liu et al., 2022). There is also evidence that EVs loaded with anti-VEGF molecules can reduce the frequency of intravitreal injections for the treatment of DR in an animal model (Reddy et al., 2023). Further research is needed to determine their protein and non-protein cargo and how these molecules might modulate angiogenesis in retinal tissues. Understanding these mechanisms will enhance our ability to modulate them in various vasoproliferative pathologies through EV-based therapies.

In the last decade, heterogeneity within the MGC population has begun to be explored (Roesch et al., 2008; Syrbe et al., 2018; Voigt et al., 2019). This heterogeneity has been reported regarding MGC localization within the retina, which also influences the cellular morphology, physiology, and expression of several proteins, such as the enzyme GS and glutamate transporter (GLAST), structural cytoskeletal proteins like GFAP, calcium channels, and several transcription factors (Bringmann et al., 2018; Distler and Dreher, 1996; Matet et al., 2015; Reichenbach and Bringmann, 2013, 2020; Nishikawa and Tamai, 2001; Roesch et al., 2008; Vecino et al., 2016). Recently, MGC heterogeneity has also begun to be explored in their cellular response in animal models of OIR and DR (Deng et al., 2024; Yao et al., 2024). However, this heterogeneity has not yet been studied in terms of the MGC’s ability to secrete specific cytokines or growth factors and, in turn, MGC’s ability to induce retinal angiogenesis in the early stage of the disease. This heterogeneity also affects the points above, potentially determining specialization in the upstream and downstream mechanisms involved in the release of pro- and anti-angiogenic proteins, either in soluble form or within EVs, depending on retinal location and the expression of the proteins involved in synthesis and release within these glial cells.

Although this review focuses on cytokines and growth factors (protein-based molecules), it is well known that a broad set of RNA molecules (e.g., miRNAs, ncRNAs, etc.) directly modulate angiogenesis in various tissues, including the retina (Biswas et al., 2021; Gandhi et al., 2024; Vishwakarma and Kaur, 2023). Furthermore, recent ophthalmological research has increasingly focused on understanding the involvement of fatty acids and lipophilic molecules in retinal angiogenesis, which is directly linked to the release of protein molecules that control this process (Elmasry et al., 2019; Fleming, 2019; Gabrielle, 2022). Understanding these processes at the molecular and cellular levels and within the context of the entire tissue will enable us to design targeted therapies aimed at key cellular players involved in the development of vasoproliferative retinopathies.