The retinal microvascular system is an indispensable constituent of the normal retina (21). The distribution of the microvasculature in the retina is specific, not spread through all layers of the retina. Currently, the microvasculature is found in four layers of the retina: (1) the deep part of the INL, (2) the border of the shallow INL and deep IPL, (3) the shallow part of the IPL and the RGCL, and (4) the NFL, and the photoreceptor layer is devoid of retinal blood flow (22, 23). Vascular endothelial cells and pericytes are important components of the retinal microvasculature, sharing a common basement membrane (24). The structure and function of the microvasculature rely on interactions between pericytes and endothelial cells, which are disturbed in some retinal vascular diseases, such as DR, retinal vascular occlusion and retinopathy of prematurity (21). Additionally, pericytes and endothelial cells are important components of the retinal NVU, and interactions between the two types of cells are necessary for the complete structure and normal function of the retinal NVU (21). In addition, the pericyte-endothelial interaction is an essential component of the internal blood retina-barrier (BRB), which is a highly selective barrier protecting the retina from the blood circulation (25, 26).

Retinal neurons are sensitive cells of various types in the retinal NVU. Retinal neurons include five major cell types: photoreceptors, horizontal cells, bipolar cells, amacrine cells and ganglion cells (27). Photoreceptors are light-sensing cells that are categorized into cone and rod cell types (28). The ONL contains the cone and rod photoreceptor cell bodies, the INL contains the cell bodies of amacrine, bipolar and horizontal cells, and the GCL mainly consists of ganglion cell bodies (29). Photoreceptor cells are in contact with secondary neurons (bipolar and horizontal cells), which in turn are in contact with ganglion cells in the IPL. In the retina, ganglion cells are the output neurons integrating information (30). Axons of ganglion cells comprise the NFL that sends the visual signal to the visual cortex through the optic nerve (31, 32).

Müller cells and astrocytes are two types of macroglia in the retina (33). Müller cells are the most abundant, accounting for 90% (34). In addition to their larger number, the distribution of Müller cells is also wide. Müller cells penetrate almost all layers of the retina and contact a variety of retinal cells (35). Due to their unique position, the normal function of Müller cells is necessary to maintain retinal homeostasis. Müller cells participate in structural support and metabolic nutrition in a healthy retina. For instance, Müller cells participate in the regulation of nutrition metabolism and protection of neurons (35). In comparison, retinal astrocytes are found only in the NFL and GCL (36). Astrocytes play a pivotal role in the metabolism and mechanical support of the neurons and serve as an essential component in the internal BRB (34, 36, 37).

In the brain and retina, microglia are resident immune cells that monitor their surroundings (38). Under normal circumstances, microglia spread over the NFL, GCL, IPL, INL, and OPL of the retina (34). According to recent research, microglia are present in the ONL only under pathological conditions (38). As immune cells of the central nervous system (CNS) (39), the functions of microglia are subdivided into six major categories: (1) Phagocytosis: Microglia predominantly clear cellular waste from the retina (40). (2) Immune Functions: Microglia are thought to participate in antigen presentation, inflammatory reactions and complement activation during defense against infectious substances and to facilitate tissue repair and immune regulation in the retina (38). (3) Microglia participate in regulating progenitor cell proliferation, differentiation, and neuronal survival (41). (4) Microglia are necessary to maintain synaptic transmission based on the synaptic structure and normal visual function in the adult retina (42). (5) Microglia have an essential role in angiogenesis. (6) Microglia are necessary to maintain retinal homeostasis (43).

Generally, the retinal NVU participates in retinal nutrition and metabolism and provides an appropriate environment for neural signal transmission (12, 44). Furthermore, RPE cells and the retinal NVU are the core components of the BRB, an important protective barrier that is comprised of two parts: the internal BRB and the outer BRB. Most of the components of the retinal NVU are involved in the composition of the internal BRB ( Figure 2 ) (45). The outer BRB is mainly comprised of tight junctions of RPE cells (46). Functionally, the internal BRB is essential in maintaining the microenvironmental homeostasis of the inner retina layers, and the outer BRB mainly regulates the transfer of solutes and nutrients from the blood to the photoreceptors (47–49).

According to previous studies, many components of the retinal NVU are affected in individuals with diabetes (50). The proper function of every element of this retinal NVU is critical for normal retinal function. In individuals with DR, damage to various cells in the retinal NVU leads to dysfunction of every component associated with the development of DR (51). The diabetic environment damages the retinal NVU through various pathways, such as oxidative stress, endoplasmic reticulum stress and inflammation (52).

As living standards improve, the number of people with metabolic diseases is increasing annually (82). Obesity and DM are common metabolic diseases worldwide. As mentioned above, NLRP3 inflammasomes activate caspase-1 to trigger pyroptosis. Eventually, IL-1β and IL-18 are secreted by pyroptotic cells. Many studies have found that NLRP3 inflammasomes correlated with pyroptosis participate in the pathogenic mechanism of some metabolic diseases, including obesity and type 2 diabetes mellitus (83).

Obesity is a metabolic disorder with multifactorial pathogenesis. In addition, obesity is also a risk factor for T2DM. Macrophages of monocyte origin infiltrate tissues as one of the pathological changes of obesity. Monocytes in the peripheral blood of obese patients exhibit high inflammatory caspase activity. In monocytes, saturated fatty acids activate caspase-4/5 to induce the production and release of IL-1β and IL-18, eventually leading to obesity-related inflammation (84). Moreover, higher expression of NLRP3 is detected in adipose tissue from obese individuals than in adipose tissue from metabolically healthy individuals (85). Of note, NLRP3 knockout prevented mice fed a high-fat diet from becoming obese (86). Adipocytes not only store energy but also secrete adipokines to regulate metabolism (87). Adipocyte dysfunction (e.g., decreased levels of insulin-sensitive adipokines and increased levels of proinflammatory cytokines) are related to insulin resistance and T2DM (88). Pancreatic β-cell dysfunction and insulin resistance are the main characteristics of T2DM. Previous studies have suggested that the NLRP3 inflammasome is closely associated with the pathogenesis of T2DM (89). HG, free fatty acids and a high-fat diet promote the activation of the NLRP3 inflammasome in patients with T2DM (90). Activation of NLRP3 inflammasomes induces excessive secretion of IL-1β and IL-18. Increased IL-1β levels cause the dysregulation of blood sugar levels by impairing pancreatic β-cells and inducing insulin resistance (83). The high expression of IL-1β receptors on pancreatic β-cells not only contributes to increase production of IL-1β, but also facilitates the spread of inflammatory signals through the NF-κB pathway, eventually leading to pancreatic β-cell dysfunction (91). IL-1β promotes insulin resistance by reducing the tyrosine phosphorylation and mRNA expression of insulin receptor substrate-1, and inducing the expression of tumor necrosis factor α (92). Additionally, IL-1β and IL-18 decrease the insulin sensitivity of target organs by inducing lymphocytes to accumulate in adipose tissue (93). In conclusion, NLRP3 inflammasomes are critical for the development and progression of obesity and T2DM, especially IL-1β, a product of NLRP3 inflammasome activation. Importantly, pyroptosis is associated with the occurrence and development of DM but also with the development of its complications, such as diabetic cardiomyopathy (94), diabetic nephropathy (95) and DR (96).

One of the earliest hallmarks of DR is microvascular changes, accompanied by the loss of pericytes, basement membrane thickening and the destruction of tight junctions between endothelial cells, together with hyperpermeability, capillary nonperfusion, microaneurysms, and the subsequent loss of endothelial cells (125, 126). Pericyte and endothelial cell death is a fatal blow to the retinal microvasculature. Multiple forms of cell death have been observed in diabetes-induced pericytes and endothelial cell death. Based on previous evidence, pericytes may die due to apoptosis and necrosis, and endothelial cells predominantly undergo apoptosis during the development of DR (127). However, some studies have found that pyroptosis might participate in the death of vascular cells in the retina and the pathological changes in DR.

Recently, HG was revealed to significantly induce the release of inflammatory cytokines and pore formation HRPs, resulting in pericyte lysis (102). Based on these findings, HG induces inflammation and pyroptosis in HRPs. Furthermore, HG induces retinal pericyte pyroptosis through the NLRP3-caspase-1 pathway (102). Coincidentally, HRPs undergo caspase-1-dependent pyroptosis after treatment with advanced glycation end product modified bovine serum albumin (AGE-BSA), which often appears in the diabetic environment (128). LncRNA myocardial infarction-associated transcript (MIAT) regulates caspase-1 expression by sponging miR-342–3p, ultimately resulting in the pyroptosis of HRPs treated with AGE-BSA (128). Notably, the authors used immunofluorescence staining and observed that AGE-BSA-induced HRPs exhibited phenotypic features of pyroptosis, including pyknosis, cell swelling, and hyperpermeability in the plasma membrane (128). Chen et al. documented that the protein expression of caspase-1, NLRP3, ASC, IL-1β, and IL-18 was significantly upregulated in retinal tissues of streptozotocin-induced diabetic rats (129). Furthermore, HG activated the NLRP3 inflammasome in HG-exposed HRMECs by the ROS-TXNIP pathway (129). Other studies have also shown that HRMECs undergo pyroptotic cell death under diabetic-like conditions (107). Platania et al. suggested that some miRNAs, such as miR-20a-5p, miR-20b and miR-106a-5p, are dysregulated in the retina and blood circulation of diabetic mice. These miRNAs can modulate the expression of DR-related factors, such as vascular endothelial growth factor (VEGF), participating in the progression of DR (130). Our group has reported that HRMECs cultured under HG conditions suffer from pyroptosis. Notably, miR-590-3p targets NLRP1 and inactivates the NOX4 signaling pathway to inhibit pyroptosis in HRMECs (101). In addition, prostaglandin E2 (an inflammatory mediator) participates in the activation of NLRP3 inflammasomes in HRMECs (131). Mcc950 selectively inhibits NLRP3 inflammasomes, thereby inhibiting human retinal endothelial cell (HREC) dysfunction under HG conditions (132).

The presence of gasdermin pores on the cell membrane is one of the characteristics of pyroptotic death. These pores destroy the osmotic potential, causing the cells to swell and eventually lyse (133). Diabetic environments such as HG and AGE-BSA promote retinal microvascular cell loss through pyroptosis (107). The loss of these two types of cells results in decreased pericyte-endothelial interactions and their miscommunication and contributes to microvascular instability. In addition, the loss of pericytes contributes to the formation of acellular capillaries, capillary occlusions, microaneurysms and hemorrhage (134). These significant pathological changes occur in DR. Vascular occlusion may lead to perfusion failure and retinal ischemic-hypoxic injury. The latter increases the expression of VEGF in glial cells and endothelial cells. Moreover, vascular occlusion may lead to retinal neuron dysfunction and even neuronal death. Furthermore, vascular cell death causes the destruction of the BRB (135), which increases vascular permeability and the possibility of inflammatory cells entering the retinal microenvironment. These processes are also the main features of DR (25). Alterations in BRB integrity lead to diabetic macular edema, eventually resulting in a severe visual impairment without timely intervention (136). Hyperglycemia-induced pyroptosis in the retinal microvasculature not only causes the death of pericytes and endothelial cells but also increases the number of inflammatory mediators, including IL-18 and IL-1β (102, 107). Researchers found increased levels of proinflammatory mediators in the serum or aqueous humor in patients with DR compared with normal controls (20). These inflammatory cytokines participate in triggering an even more excessive inflammatory reaction, promoting the development of DR (66, 137).

Neurons in the retinal NVU are the major cells that transmit light signals and form vision. Extensive studies have shown the presence of retinal neuronal degeneration in the early stage of DR, even earlier than visible vasculopathy (138, 139). Two features of neurodegeneration in the retina are neuronal death and reactive gliosis (9). Many researchers have provided a description of neuronal apoptosis in DR. Pyroptosis, a form of programmed cell death involved in inflammation, can occur in retinal ganglion cells (140). DM causes hypoxia in retinal tissue and leads to an imbalance in retinal immune responses (141). Hypoxia-induced factor-1 (HIF-1) is continuously produced and degraded under hypoxic conditions. Then, HIF-1α functions as a transcription factor to activate the genes encoding the proangiogenic growth factors, IL-6 and IL-8 (141). Moreover, loss of pericytes potentially leads to acellular capillary formation, which is associated with vascular occlusion and leads to nonperfusion and retinal ischemic-hypoxic injury. Ischemia-hypoxia upregulates the expression of HIF-1 (25). Pyroptosis participates in retinal ischemic damage and promotes retinal ganglion cell death in acute glaucoma (142). The caspase-8-HIF-1α-NLRP12/NLRP3/NLRC4 pathway initiates neuroinflammation and pyroptosis (142). Moreover, pyroptosis is an alternative pathway through which photoreceptors degenerate after retinal detachment (143). Additionally, caspase-1/3/4/5 activities were found to be increased in a streptozotocin-induced diabetes mouse model (140). A previous study reported that NLRP3, ASC, and caspase-1 were specifically located in the GCL and the INL and ONL in the retinas of diabetic rats according to immunohistochemical results (99). Simultaneously, other authors have also documented that the expression levels of NLRP3, ASC, and caspase-1 were increased in retinal cells of diabetic rats (99).

Müller cells and astrocytes of the retinal NVU participate in retinal structural support and maintain retinal homeostasis. Studies have found that approximately 15% of Müller cells die after 7 months in retinas of the diabetic mice (127). After exposure to HG, caspase-1 activity and IL-1β production in Müller cells increase and subsequently induce cell death (144). Furthermore, inhibition of the caspase-1/IL-1β pathway prevents the loss of Müller cells under diabetic conditions (127). The aforementioned evidence revealed that pyroptotic death may be responsible for Müller cell death under diabetic conditions. Consistently, HG-induced nuclear accumulation of GAPDH in Müller cells relies on activating the caspase-1/IL-1β pathway. More interestingly, the accumulation of GAPDH in the nucleus is associated with the induction of cell death (145). Due to their important locations and functions, Müller cell loss will lead to an incomplete retinal structure. For example, Müller cell loss promotes the destruction of the internal BRB integrity and increases the vascular permeability and the loss of neuroprotective effects, affecting both neurons and blood cells (35). Specific removal of Müller cells from the retina leads to retinoschisis, showing that Müller cells hold the neural layers together to protect the neural tissue from ripping apart (146). Previous studies have reported that selective removal of Müller cells results in photoreceptor apoptosis, BRB breakdown, and vascular telangiectasis (147). In addition, destruction of the cell-to-cell communication between Müller cells and retinal pericytes promotes pericyte death (148). The loss of Müller cells in DM is also related to the formation of aneurysms, a clinical feature of DR (35). Furthermore, Müller cells are the major source of IL-1β (149). Long-term production of IL-1β from Müller cells affects the viability of endothelial cells in a paracrine manner (150). Due to the high sensitivity of endothelial cells to IL-1β, endothelial cells die after responding to this inflammatory cytokine (150). In addition, the death of endothelial cells promotes the formation of acellular capillaries, which are the main hallmark of DR pathology (151).

Microglia are specific innate immune cells of the retina that monitor the environment and remove metabolic waste. As more in-depth research on pyroptosis has been conducted, pyroptosis has also been identified in microglia. Microglial pyroptosis occurs in various disease states, such as after spinal cord injury (152), ischemic brain injury (153), retinal ischemia and reperfusion injury (154), and DR. Retinal ischemia and reperfusion injury (I/R) is the basis of multiple retinal diseases, including DR, glaucoma, and retinal artery occlusion (155). Accumulating studies have shown that I/R promotes retinal microglial pyroptotic death, which is associated with lncRNA H19 (154). In addition, S100A12 is closely related to the incidence and severity of DR. S100A12 represents a proinflammatory trigger for retinal microglial activation by activating the NLRP3 inflammasome in a diabetic environment (156). Moreover, HG was recently shown to induce retinal microglial pyroptosis through NLPR3 inflammasome signaling (157). HG upregulated the protein expression of NLPR3, caspase-1, GSDMD, and IL-1β in retinal microglia (157). In another study, HG-induced retinal cells produced more IL-1β, and the IL-1β induced microglial proliferation (158). Moreover, IL-1β is mainly produced by microglia under diabetic conditions (159). Overactive microglia produce various proinflammatory and cytotoxic factors, including IL-1β, TNF-α, and ROS, which lead to chronic inflammation and contribute to the destruction of hemostasis in the NVU, BRB breakdown and worsening of the pathology of DR (160).

Accumulating research suggests that IL-1β and IL-18 may partly come from the pyroptosis-mediated cell death of retinal cells in diabetic rats and mice (99). IL-1β and IL-18 are the two key cytokines that undergo maturation through cleavage by active caspase-1 and are released through pyroptosis. In addition, IL-1β is the most studied IL-1 family member in retinopathy, such as DR (161). These proinflammatory mediators lead to persistent low-grade inflammation, affecting the hemostasis of the retinal NVU. Under HG conditions, IL-1β induces pericyte apoptosis by activating NF-κB, thereby increasing vascular permeability (162). Additionally, IL-1β affects glial cells (microglia and macroglia) and ultimately results in neural changes (158). Moreover, the increased levels of IL-1β coincide with increasing retinal neovascularization (163). Coincidentally, IL-18 may promote retinal angiogenesis in active PDR together with VEGF or through VEGF (164). Accumulated studies indicate that glucocorticoids show anti-inflammatory efficacy by inhibiting key proinflammatory mediators such as IL-1β (165). Thus, glucocorticoids may have potential use in modulating pyroptosis. Collectively, IL-1β and IL-18 produced during pyroptosis participate in inflammation, promoting the progression of DR.