Understanding the Molecular Basis of Pathological Retinal Angiogenesis: Roles of Lipids, Non-Coding RNAs, and Cellular Crosstalk

Authors: Anamika Sharma, Summer Kizy, Nikhlesh K. Singh

Abstract

Pathological retinal neovascularization is a prevalent cause of blindness, impacting millions of individuals across all age groups, from infants to older adults. It is a key clinical feature of several retinal diseases, such as diabetic retinopathy (DR), retinopathy of prematurity (ROP), age-related macular degeneration (AMD), and Coats disease. Researchers have extensively investigated the involvement of vascular endothelial growth factor (VEGF) signaling, hypoxia inducible factor (HIF) signaling, and inflammation in the regulation of pathological retinal neovascularization. Recent breakthroughs have demonstrated the influence of several substances and cells in the retina on the regulation of pathological retinal neovascularization. This review emphasizes the rising significance of substances such as lipid mediators, non-coding ribonucleic acids, as well as the involvement of several retinal cell types, including endothelial cells (ECs), pericytes, glial cells, and the retinal pigment epithelium (RPE) in the regulation of pathological neovascularization. Here, we emphasize the interactions among these cell types in maintaining retinal homeostasis and their role in abnormal neovascularization in diseases like DR, AMD, ROP, etc. This review aims to highlight the importance of molecules and cells that go beyond the VEGF-centric therapeutic focus to treat pathological neovascularization.

Aging populations and the rising prevalence of diabetes have made acquired retinal diseases, such as proliferative retinopathies, a primary cause of global blindness.^1^ Therefore, understanding the physiology of the retina, its supportive components, and the processes that lead to retinal pathologies, including proliferative retinopathies, has become increasingly important. The retina lines the posterior aspect of the eye and has a thickness of approximately 0.5 mm.^2^ To maintain homeostasis and facilitate complex visual processing, the cells of the vertebrate retina are organized into multiple layers (Fig. 1). The visual function of the neural retina relies on the retinal microvasculature to deliver nutrients and oxygen to the retinal cells while removing waste. The blood-retinal barrier (BRB) prevents large molecules in the blood from entering the neural tissue. As part of the central nervous system, a neurovascular unit is formed through the coordinated interactions among vascular cells, neurons, and glial cells, including Muller cells and astrocytes.^3^ The retinal neurovascular unit plays a crucial role in maintaining environmental homeostasis. However, several diseases and conditions, such as inflammation, ischemia, and diabetic retinopathy (DR), can lead to BRB breakdown, disrupt interactions among neurovascular unit cells, and alter the architecture of the retinal microvasculature. These changes can further worsen inflammation, ischemic damage, and pathological processes.^4^^–^^6^

Angiogenesis is a physiological process in which new blood vessels are generated from pre-existing vessels, playing a vital role in growth and development in multicellular organisms.^7^ Angiogenesis in the retina is crucial for maintaining vascular homeostasis under normal physiological settings, facilitated by a delicate equilibrium between proangiogenic and antiangiogenic factors. However, this balance can be disrupted by factors such as retinal hypoxia, ischemia, or inflammation, potentially leading to pathological neovascularization in conditions like DR, retinopathy of prematurity (ROP), and age-related macular degeneration (AMD).^8^

ROP is a disease characterized by abnormal neoangiogenesis in the retinas of premature infants. In the initial phase of ROP, hyperoxia hinders retinal vascularization in preterm infants. As the retina matures and its metabolic demands increase, a relative hypoxic condition arises, resulting in abnormal angiogenesis or retinal neovascularization. In severe cases of ROP, fibrotic proliferation can lead to retinal detachment, which may result in permanent vision loss.^9^^,^^10^ DR is the primary eye problem associated with diabetes mellitus. The global prevalence of DR was estimated to be 103 million, constituting 22.27% of the total diabetes burden in 2020, with projections suggesting that this number could increase to 160.5 million by 2045.^11^ DR is classified into two main categories based on the presence of non-proliferative DR (NPDR) and proliferative DR (PDR). NPDR is characterized by structural changes in the retinal blood vessels, which can lead to hemorrhages. As NPDR advances to PDR, new and fragile blood vessels form in the retina. These delicate vessels are prone to damage, leading to significant bleeding and, ultimately, the risk of blindness.^12^^,^^13^

AMD is a leading contributor to vision impairment in older adults, with its prevalence expected to increase as the global older population grows.^10^ The pathophysiology of this complex condition involves multiple components, including dysregulation of the complement system, fatty acid metabolism, angiogenesis, inflammation, and extracellular matrix (ECM) pathways.^14^ Although AMD is a complex, multifactorial disease, it can be categorized into two main “dry or geographic AMD” and “wet or neovascular AMD.” In the early stages, AMD typically presents as “dry” AMD with yellowish deposits referred to as drusen in the subretinal or retinal pigment epithelium (RPE) areas.^14^ In contrast, “wet or neovascular” AMD is characterized by choroidal neovascularization (CNV), where the underlying choroid develops new blood vessels. This process can result in complications such as hemorrhage, edema, and ultimately the death of photoreceptor cells.^15^ Coats disease is a rare neovascular retinal disease that primarily affects male patients and is often diagnosed within the first 2 decades of life, with most cases being unilateral. The defining characteristic of Coats disease is retinal telangiectasia (disrupted, spider-like vessels). This condition is often accompanied by capillary dropout, vascular aneurysm, exudation, and accumulation of subretinal or intraretinal fluid. Additional common manifestations include visual acuity, strabismus, nystagmus, retinal detachment, hemorrhage, fibrosis, macular edema, and pain.^16^^–^^18^

All these proliferative retinopathies are characterized by abnormal blood vessel growth, resulting in BRB breakdown, increased leukocyte adhesion, and edema, ultimately leading to retinal neovascular tuft formation and vision impairment (Fig. 2). This unregulated neovascularization can also result in hemorrhage, fibrosis, and irreversible damage to the retinal tissue.^8^ Pathological angiogenesis begins with an increased expression of proangiogenic molecules, including vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), and other growth factors. In the presence of proangiogenic factors, first the ECM is degraded by MMPs, which stimulate the endothelial cells (ECs) to proliferate and migrate. Subsequently, these ECs develop into primary sprouts and immature tubes, resulting in the reconstruction of capillaries and maturation of blood vessels.^19^ The treatment options for pathological retinal neovascularization include laser photocoagulation, cryotherapy, vitreoretinal surgery, and anti-VEGF therapies.^16^^–^^18^^,^^20^ The disorders mentioned, along with the severity of their advanced stages, highlight the risks associated with retinal neovascularization and demonstrate the value of understanding the cells involved in this process.

Retinal Cell Population Affecting Retinal Neovascularization

Vascular ECs

Vascular ECs play a crucial role in the pathophysiology of proliferative retinopathies, making them a potential target for therapeutic intervention. Under normal physiological conditions, ECs form a monolayer that lines the inner surfaces of capillaries, facilitating the transport of oxygen and nutrients to the retina. ECs swiftly transit from a dormant state to a proliferative and migratory state in response to proangiogenic stimuli such as hypoxia, which initiate the development of new blood vessels from pre-existing vessels, known as neoangiogenesis. Neoangiogenesis involves multiple processes, including the proliferation and migration of ECs, the reorganization of the ECM, and tubulogenesis.^21^ Hypoxia acts as a crucial trigger for retinal angiogenesis by initiating a shift to anaerobic metabolism, characterized by glycolysis and lactate production. Additionally, hypoxia increases the expression of angiogenic growth factors such as VEGF and erythropoietin (EPO), which facilitate EC proliferation and the remodeling of blood vessels.^22^^,^^23^ A complex interplay of angiogenic and angiostatic factors determines the activation of the “angiogenic switch,” leading to angiogenesis. This switch is confined to a specific subset of ECs, which subsequently migrate, proliferate, form lumens, and develop vessel walls while preserving the integrity of the existing vascular network.^24^^,^^25^ Interactions with ECMs are crucial for the survival of ECs by influencing the expression of Fas and c-Flip. Fas is a receptor involved in mediating apoptosis. The interaction between ECM and ECs enhances c-Flip expression, which inhibits Fas and prevents apoptosis. Consequently, adherence to the ECM protects ECs from cell death.^26^

It has been shown that pathological retinal neovascularization correlates with an increased number of tip cells, which are located at the growing edge of newly formed blood vessels. This phenomenon, along with excessive finger-like projections known as filopodial protrusions, lead to disorganized microvascular networks that are distinct from physiological angiogenesis.^27^ The underlying cellular processes in pathological retinal conditions are mediated by metabolic pathways. Various studies performed in our laboratory have highlighted the role of EC signaling in ischemia-induced retinal neovascularization. We have shown that VEGFA, through its receptors VEGFR1 and VEGFR2, is a key regulator of angiogenic processes in human retinal microvascular endothelial cells (HRMVECs). Using a mouse model of oxygen-induced retinopathy (OIR), we have shown that both VEGFA and VEGFB regulate hypoxia-induced activation of the Src-PLD1-PKCg-cPLA2 pathway, which leads to retinal neovascularization.^28^ Additionally, we have established that the VEGFC-induced CREB-DLL4-NOTCH1 signaling pathway plays a crucial role in regulating the formation of retinal EC tip cells and retinal neovascularization.^29^ Furthermore, our findings indicate that VAGFA-induced Pak1-p38MAPKβ-cofilin signaling is essential for the proliferation of HRMVECs and formation of tip cells,^30^ whereas Pyk2-mediated STAT3-cJun signaling is involved in regulating the migration and tube formation of human retinal ECs.^31^

Our research studies on the role of inflammation and inflammatory molecules in a mouse model of proliferative retinopathies has revealed that interleukin-33 (IL-33) regulates NF-kappaB-Jagged1-mediated Notch1 activation and retinal EC sprouting angiogenesis.^32^ Additionally, we have also demonstrated that IL-33-induced PKCµ/PRKD1-p38 MAPK-a-catenin signaling affects endothelial permeability and the integrity of inner BRB integrity.^33^ Our analysis of EC adhesion molecules shows that ischemia-induced VCAM-1-JunB-CXCL1 signaling influences retinal EC sprouting and contributes to retinal neovascularization.^34^ Furthermore, we have recently shown that blocking or suppressing the cleavage and activation of IL-33 by neutrophil proteases may reduce neovascularization in cases of proliferative retinopathy.^35^ Additionally, we have recently shown that EC-specific ADAM10 modulates pathological retinal angiogenesis in the ischemic retina, underscoring its potential as a therapeutic target for proliferative retinopathies.^36^

Recent findings highlight the crucial role of endothelial metabolism in regulating angiogenesis, demonstrating that ECs rely on fatty acid oxidation and glucose metabolism for energy production and various functions. The genetic or pharmacological inhibition of carnitine palmitoyl transferase I (CPT1), results in functional abnormalities in differentiation, proliferation, and barrier function in ECs.^37^ Furthermore, fatty acid-binding protein (FABP) and fatty acid transport protein have been identified as regulators of EC function in response to VEGF stimulation. The absence of FABP4 in ECs leads to a significant reduction in proliferation, migration, and sprouting.^38^ Similarly, the lack of EC-specific GLUT1 results in diminished glycolysis, activation of AMP-activated protein kinase (AMPK), and a decrease in cell proliferation. The conditional ablation of Glut1 in murine ECs compromises angiogenesis in the retina and brain, primarily due to a reduction in tip cells in vivo.^39^ Due to the metabolic requirements of angiogenic ECs, targeting endothelial metabolism has emerged as a therapeutic strategy for retinal vascular disorders.^40^ Recent studies indicate that the activation of peroxisome proliferator-activated receptor-alpha (PPARa), particularly through treatment with fenofibrate (a medication for hypercholesterolemia) restores mitochondrial function, enhances the viability of endothelial colony-forming cells (ECFCs), and reduces pathological angiogenesis in models of DR.^41^ Metabolic-targeted therapies, especially those that inhibit pathological tip cells, provide a precise and effective approach to suppress aberrant angiogenesis while preserving physiological vascular homeostasis.^27^ Thus, a thorough understanding of signaling and metabolism of ECs that drive angiogenic events and retinal neovascularization is essential for developing innovative therapeutic strategies for retinal vascular disorders. This understanding will ultimately enhance the clinical outcomes for patients with vision-threatening conditions.

Pericytes

Having established the crucial role of ECs in the retina, it is important to highlight pericytes, which serve multiple functions, including the maintenance of EC integrity.^42^^–^^45^ Carl Eberth from Germany and Charles-Marie Rouget from France independently described pericytes in 1871 and 1873, referring to them as “Rouget cells.” In 1923, Karl Zimmermann, a German anatomist, renamed them “pericytes” to emphasize their perivascular location.^43^^,^^45^ Pericytes are specialized mural cells situated at the perivascular site on the abluminal side of micro vessels. By extending the processes around and along precapillary arterioles and capillaries within the basement membrane, pericytes encircle numerous ECs and their tight junctions. The detachment of pericytes from the basement membrane alters their function.^43^^,^^45^^–^^47^

Retinal pericytes and ECs interact closely due to the thin basement membrane of the retina. The depletion of pericytes disrupts the vascular architecture because these cells provide antiapoptotic and antiproliferative support to ECs.^43^^,^^45^ Pericyte coverage in the retina is greater than that observed in any other tissue. The pericyte-to-EC ratio in the retina is 1, compared to 2.5 in the kidneys, 5 in the brain cortex, 10 in the lungs, and 100 in the skeletal muscle. The high abundance of pericytes in the retina highlights their structural and functional significance.^46^^,^^48^^–^^50^ Furthermore, pericyte coverage is considered essential for the survival of ECs, particularly in stressful conditions such as hyperglycemia associated with diabetes mellitus.^42^^–^^45^

The relationship between ECs and pericytes is interdependent. Consequently, the balance between pericytes and ECs is carefully regulated through a series of signaling pathways that operate in both autocrine and paracrine manners. The two most commonly used molecular markers for retinal pericytes are neural/glial antigen 2 (NG2) and platelet-derived growth factor receptor beta (PDGFRb).^43^^,^^45^^,^^46^ However, the expression of pericyte markers varies depending on the tissue type, developmental stage, and activation state.^47^^,^^51^

In DR, activated pericytes contribute to the worsening of retinal inflammation. In response to various inflammatory cytokines, pericytes secrete proinflammatory mediators, including IL-1b, TNF-a, IL-6, reactive oxygen species (ROS), nitric oxide, low-density lipoprotein receptor (LDLR)-related protein-1, and MMP2/MMP9, which play a role in activating ECs, microglia, and astrocytes.^45^^,^^52^ Additionally, pericytes secrete IL-10 and regulate immune cell trafficking through the secretion of CXCL10, CXCL8, and CCL5.^53^ Furthermore, when stimulated by IFN-g, pericytes produce MHC class I and II molecules, indicating their potential role as antigen-presenting cells.^45^

Pericytes play a crucial role in maintaining vascular stability and EC barrier function through both direct interaction and paracrine signaling. Pericytes are responsible for secreting and activating signaling molecules, such as TGF-beta and ANGPT1, which facilitate communication between pericytes and ECs, thereby influencing EC function.^54^ A mouse model for pericyte depletion was created using diphtheria toxin receptor (DTR), specifically the Pdgfrb-CreERT2 ROSA-DTR (DTR^iPC^) double transgenic mice.^55^ In these mice, the pericyte reduction was more evident around actively forming capillaries, resulting in a reduced radial outgrowth of retinal vessels.^55^ By disrupting the VEGF(A)-VEGFR2 signaling pathway, researchers demonstrated that pericytes regulate EC proliferation and tip cell formation during retinal angiogenesis. Furthermore, the pericyte-specific inactivation of VEGFR1 in a mouse line (Flt1^iPC^) replicated the defects in endothelial sprouting and filopodia formation observed in pericyte-depleted mice. Thus, it was concluded that pericytes regulate VEGF-dependent EC sprouting through VEGFR1 signaling.^55^

A separate study investigated the role of pericytes in the formation, maturation, and preservation of the BRB by selectively depleting PDGFB in ECs, which resulted in reduced pericyte coverage. PDGFB is a signaling molecule that binds to pericytes and promotes BRB formation. The authors observed that insufficient pericyte coverage leads to impairment of the BRB and increased leakage in developing the retinal vasculature, negatively impacting visual function. Additionally, they found that inactivating FOXO1 and ANGPT2 mitigated pathological events, as these factors contribute to BRB impairment and destabilization of the blood vessels when overactive.^56^ ANGPT1 and Tie2 are essential for the formation of retinal vasculature and maturation of the BRB. Research indicates that ANGPT1 is expressed by the retinal neurons rather than pericytes, which contradicts earlier findings that pericytes primarily express ANGPT1 and play a role in stabilizing blood vessels.^57^ Additionally, studies have indicated that the removal of pericytes through the specific knockout of PDGF-beta in ECs leads to a reduction in neovascular tuft formation and overall neovascularization.^58^ Furthermore, it has been observed that pericytes that express a-smooth muscle actin are present on neovascular tufts in a mouse model of OIR. The adaptor proteins NCK1 and NCK2 are also implicated in regulating pericyte migration and neovascular tuft formation (Fig. 3).

Disturbances in the number and function of pericytes have been associated with impairments in the retinal microvasculature, leading to retinal ischemia and neovascularization. The essential interactions between pericytes and ECs, their varied responses to angiogenic cytokines and growth factors, and their role in the BRB stress in proliferative retinal diseases present a promising area for research focused on the identification of new therapeutic targets for proliferative retinopathies.

Glial Cells

In normal physiological conditions, glial cells play a crucial role in regulating and supporting the retinal environment. The retina consists of three types of glial Muller cells, microglia, and astrocytes.

Muller Cells

Muller cells are the primary type of glial cells found in the retina.^59^ They span the entire thickness of the retina, forming end feet and microvilli by extending their glial processes toward both the internal and outer limiting membranes.^60^ These processes encircle the blood vessels and make direct contact with vascular ECs and retinal neurons. Positioned between the retinal blood vessels and ganglion cells, Muller cells facilitate the transport of molecules between blood vessels and neurons.^61^^,^^62^ For energy metabolism, glucose from the bloodstream is transported into Muller cells, where lactate dehydrogenases facilitate glycogen synthesis and storage. In Muller cells, the glycolytic pathway primarily produces ATP while consuming minimal oxygen. As a result, Muller cells demonstrate a tolerance to sustained hypoxia and hypoglycemia, effectively conserving oxygen and providing energy to neurons. At the same time, significant amounts of lactate are generated during glycolysis, which serves as a metabolic substrate for neurons.^19^^,^^63^ Furthermore, Muller cells regulate the transport of total retinal glutamate and maintain low extracellular glutamate levels to prevent toxicity induced due to glutamate. Glutamate, secreted by neurons, is reabsorbed by the Muller cells via the glutamate-aspartate transporter.^64^ Muller cells have high levels of glutamine synthetase, which converts the recovered glutamate to glutamine,^60^ which is again absorbed by neurons.^65^

Under pathological conditions, the Muller cells activate several intracellular signaling pathways to enhance the expression of proangiogenic factors while suppressing antiangiogenic factors.^19^ The proliferation and dedifferentiation of Muller cells amplify these effects.^66^ Muller cell gliosis, marked by GFAP upregulation, is linked to nearly all retinal diseases and signifies that Muller cells have become “activated” or “reactive” in response to pathological changes in the retina.^60^ Mice exposed with OIR exhibit a higher number of gliotic (stressed) Muller cells in the ischemic central retina compared to the perfused peripheral retina.^67^ Ischemic Muller cells acquire a proangiogenic phenotype through the overexpression and activation of the HIF-1a and HIF-2a signaling pathways.^19^ Additionally, the downregulation of HIF-1a and HIF-2a expression in the Muller cells diminishes the overexpression of VEGF.^68^ VEGF produced by Muller cells play a critical role in ischemia-induced retinal neovascularization and vascular leakage. The conditional deletion of VEGF in Muller cells in mice subjected to OIR significantly inhibits retinal neovascularization and disruption of BRB.^69^ Studies indicate that HIF-1a promotes the expression of angiopoietin-like 4 (ANGPTL4) in hypoxic Muller cells. ANGPTL4 is a crucial proangiogenic factor that functions independently of VEGF.^67^^,^^70^ Additionally, the inhibition of ANGPTL4 further reduces the angiogenic potential of hypoxic Muller cells beyond the effects of VEGF inhbition.^70^ Therefore, targeting Muller cells through their metabolic or angiogenic influences may present a promising treatment strategy for proliferative retinopathies.

Astrocytes

Astrocytes are primarily found in the retinal nerve fiber and ganglion cell layer, where they are closely associated with neurons and major blood vessels. They play a crucial role in the development and functioning of the retinal vascular system, including blood circulation and BRB formation.^71^^,^^72^ Additionally, astrocytes extend their terminals to the walls of retinal blood vessels, encircling them to influence barrier function.^73^

Astrocytes play a vital role in physiological retinal neurovascular interactions and are present in the immature, avascular retina before the onset of vascular development.^74^ When astrocytes experience ischemia or hypoxia, they release angiogenic molecules that promote the radial migration of ECs into the hypoxic regions toward the peripheral retina.^75^ Additionally, the exposure of astrocytes to hyperoxia enhances their differentiation in the developing retina.^76^

The VEGF produced from astrocytes directly influences the tip cells and enhances filopodial expansion in the developing retina.^77^ In the OIR model, the damage to the retinal astrocytic template disrupts proper vessel formation, leading to pathological neovascularization.^78^ Furthermore, the targeted deletion of PDGFRa in the astrocytes significantly disrupts astrocyte organization and retinal angiogenesis.^79^ The intravitreal delivery of astrocytes or astrocyte-conditioned medium effectively salvages endogenous astrocytes from degeneration in the vaso-obliterated region, speeds up the revascularization of normal retinal plexuses, and reduces pathological retinal neovascularization. The observed effects may be partially attributed to the presence of basic fibroblast growth factor and VEGF in the astrocyte-conditioned media.^80^ Astrocytes were identified using antibodies that target the intermediate filament, GFAP, showcasing their net-like arrangement and star-like morphology in the retina. Oxygen-induced retinal damage led to a decrease in the number of astrocytes, along with changes in their shape and distribution. Additionally, the findings also suggest that administering melatonin may help restore the astrocyte network, thereby promoting the growth of tip cells and reducing the size of the vascular tufts.^81^

Astrocytes facilitate both physiological blood vessel development and the progression of pathological retinal neovascularization. Therefore, it can be emphasized that astrocytes can be used as a key therapeutic target in the prevention of aberrant retinal neovascularization.

Microglia

Microglia make up 5% to 20% of all glial cells and serve as the resident macrophages of the retina. They originate from the progenitor cells found in the yolk sac during early embryonic development. Once they mature, they act as resident immune cells that continuously monitor their surrounding tissue.^82^^–^^85^ The activation of microglia is meticulously regulated by various inhibitory pathways, and they serve as the primary cellular element of the retina’s innate immune system.^86^^,^^87^ Cytokines released by microglia play a crucial role in regulating cell death, cell survival, and retinal inflammation.^88^ In a diabetic state, hyperglycemia and glycated albumin are the key factors that activate the retinal microglia.^89^ Once activated, retinal microglia release mediators that promote neuronal apoptosis.^90^ The sustained activation of microglia in the diabetic retina is predominantly proinflammatory, resulting in a chronic inflammatory state that may contribute to retinal damage.^91^^,^^92^

The number of activated microglia increases in the ischemic retina, and this morphological change indicates that they have switched into an inflammatory state. In this state, they release proinflammatory cytokines, including IL-1, TNF-a, and IL-6. This inflammatory response contributes to pathological retinal neovascularization.^93^^–^^95^ The activated microglia are associated with OIR-induced neovascular tufts, suggesting that these cells may play a role in retinal neovascularization.^81^ The activated microglia produce IL-1β, which sustains their activation and induces microvascular damage by secreting the proapoptotic/repulsive factor, semaphorin 3A from the neighboring neurons during the initial hyperoxic phases of OIR.^96^ The activated microglia in the retinas exposed to OIR have been demonstrated to secrete interleukin-17A (IL-17A), which interacts with IL-17 receptors on Muller cells and RGC, thereby facilitating VEGF secretion and contributing to vasculopathy.^97^ Contrastingly, microglia-derived exosomes provide protective benefits against OIR by reducing photoreceptor damage and retinal neovascularization. This protective effect may be associated with the activation of endosomes in hypoxic conditions, facilitating the delivery of microRNA (miRNA)-24-3p. This miRNA inhibits inositol-requiring enzyme 1a (IRE1a), a stress response protein, within the photoreceptors.^98^ Consequently, whereas microglia are crucial for retinal immune surveillance and vascular development, their dysregulation due to chronic activation or excessive cytokine release, significantly contributes to the pathogenesis of neovascular retinal diseases.

Retinal Pigment Epithelium Cells

RPE cells form a cuboidal monolayer comprised of hexagonal pigmented cells, located between the choriocapillaris and outer segments of photoreceptors.^99^^,^^100^ The apical surface of the RPE features microvilli and faces the outer segments of the photoreceptors, whereas the basolateral surface is directed toward Bruch’s membrane, distinguishing it from the choriocapillaris.^100^^,^^101^ The primary biological functions of the RPE in the retina are depicted in Figure 4. The RPE maintains its polarity through a complex network of tight junctions situated on the apical side, which creates a barrier to paracellular diffusion.^102^ These tight junctions consist of claudins, occludins, cell adhesion molecules, tricellulin, and zonula occludens.^103^ Occludins and claudins play a critical role in maintaining the integrity of tight junctions.^101^ The outer BRB is established at the RPE through tight junctions between the RPE cells, which regulate the transport of fluids and molecules to and from the neural retina.^104^ The RPE creates a distinct microenvironment for the retina, characterized by its polarity and the asymmetrical distribution of organelles, proteins, and functional specializations.^105^ The structural components of the RPE, including tight junctions and polarity, work together to perform essential functions. For instance, the RPE is necessary to sustain photoreceptors through various mechanisms and is instrumental in ensuring both the structural and functional integrity of these cells.^100^

RPE is crucial for maintaining a healthy retina, as it regulates the flow of nutrients to the photoreceptors and is highly pigmented to reduce light dispersion.^106^ However, in pathological conditions such as AMD and diabetes, the function of the RPE is significantly impaired. Studies conducted in a controlled environment have shown that RPE cells cultured in a diabetic setting produce more ROS compared with those cultured in a nondiabetic environment.^107^ Studies have shown that inhibiting RPE65 in mice using retinylamine reduces a diabetes-induced increase in the superoxide levels within the retina.^108^ This suggests that the visual cycle in diabetic RPE cells play a role in elevating the oxidative stress, particularly in photoreceptor cells. Additionally, high glucose levels in diabetes have been found to cause RPE cells to downregulate GLUT-1 and lower the antioxidant levels, which may lead to damage in the retinal tissue.^109^ Research involving diabetic and ischemic mice indicates that vascular leakage occurs when the RPE’s tight junctions break down, underscoring the essential barrier function of RPE cells.^110^ RPE dysfunction is also observed in various pathological conditions, such as AMD and Alzheimer’s disease. The accumulation of amyloid-beta (Ab) in the RPE, mediated by RAGE/p38 MAPK-related endocytosis, can weaken and disorganize tight junctions and, in some cases, cause their disintegration.^111^^,^^112^ In wet AMD, the choriocapillaris complex is thought to be the main site of BRB dysfunction; however, subsequent damage to the RPE significantly contributes to visual impairment.^113^

The RPE is essential for retinal health by acting as a metabolic gatekeeper, structural barrier, and regulator of oxidative stress. RPE dysfunction is widely recognized as a significant factor in the etiology of retinal degenerative diseases, highlighting its potential as a target for therapeutic intervention.

Bioactive Lipids and Retinal Angiogenesis

Lipid mediators derived from membrane lipids are prevalent in various tissues, including in the retina. Lipids and their derivatives not only serve as the essential components of cell membranes but also act as signaling molecules, which is why they are referred to as “bioactive lipids.” They play a vital role in numerous biological functions by modulating various signaling pathways. Bioactive lipids are critical in processes such as promoting angiogenesis, regulating inflammation, and maintaining homeostasis. Bioactive lipids include a diverse array of compounds, with the most common forms originating from polyunsaturated fatty acids (PUFAs).^8^

PUFAs, sphingolipids, and glycerophospholipids demonstrate significant proangiogenic or antiangiogenic properties, as shown by various preclinical and clinical studies.^114^ Bioactive lipids, particularly PUFAs and their derivatives, have been linked to the development of DR, ROP, and AMD. Recent research studies suggest that a higher intake of ω-3 PUFAs may prevent the progression of retinal neovascularization. Both ω-3 and ω-6 PUFAs, along with their derivatives such as resolvins, lipoxins, and protectins possess anti-inflammatory properties. These compounds can limit the expression of VEGF and TNF-a while inhibiting EC proliferation, ultimately leading to reduced pathological retinal neovascularization.^115^

The dietary intake of ω-3 PUFAs has been reported to significantly reduce retinal angiogenesis in ROP and is associated with decreased ER stress in the retina.^116^ Furthermore, ω‐3 PUFAs, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), offer protection to photoreceptors from oxidative stress, emphasizing their protective role against the development of AMD and pathological retinal neovascularization.^117^^–^^119^ A prospective PREDIMED trial found that the participants with type 2 diabetes mellitus who consumed at least 500 mg of ω-3 PUFAs per day experienced a 48% reduction in their risk of developing sight-threatening DR over a 6-year period.^120^ In addition, a 2023 study involving 1356 patients with type 2 diabetes found that higher blood levels of DHA were inversely correlated with the occurrence and severity of DR.^121^ Similarly, a study on patients with type 1 diabetes found that elevated blood ω-3 PUFA levels were correlated with reduced severity of DR and improved retinal perfusion.^122^ In contrast to these findings, the ASCEND-Eye trial has shown that consumption of 1 gram of ω‐3 PUFAs by patients with type 2 diabetes over 6.5 years has no significant effect on the incidence of DR and AMD compared with that of placebo controls.^123^ The lack of effect of omega-3 PUFAs on DR and AMD in the ASCEND-Eye trial, compared to the PREDIMED study, may stem from differences in the types of fish supplements utilized or the substitution of fish with less healthy food options.

The proposed protective effects of ω‐3 PUFAs are also attributed to the 5‐lipoxygenase (LO)‐dependent oxidation of DHA to 4‐hydroxy‐decosahexaenoic acid. This process, which involves a PPARg‐dependent mechanism, inhibits endothelial proliferation and angiogenesis.^124^ However, this mechanism is not applicable to AMD, as oxidized phospholipids have been found to be elevated in the eyes of patients with AMD, leading to CNV in mice.^125^ Diets rich in ω‐3 PUFAs, such as DHA or EPA, are proposed as a potential therapeutic strategy to prevent DR due to their anti-inflammatory properties and their ability to protect against pathological neovascularization in the retina.^118^^,^^126^^,^^127^ Conversely, 5-LO (ALOX5) and 12/15-LO (ALOX15), enzymes that oxidize PUFAs, may play a role in the pathogenesis of DR. The diabetic mice lacking 5-LO (5‐LO^−/−^), there was a reduction in the retinal capillary degeneration after 9 months of diabetes, unlike the 12/15‐LO^−/−^ mice, which did not exhibit similar protective effects against this degeneration. Furthermore, the retinas of both diabetic 5‐LO^−/−^ and 12/15‐LO^−/−^ mice showed lower levels of leukocyte adhesion at 3 months of age.^128^ This observation implies that the absence of 5‐LO and 12/15‐LO may correlate with a decreased risk of disease progression, as leukocyte adhesion can trigger harmful inflammatory responses. Additionally, there was a significant increase in bioactive lipids derived from 12/15 LO in the vitreous and epiretinal membranes of patients with proliferative DR, suggesting a potential link between these lipids and disease.^129^^,^^130^ Genetic deletion or pharmacological inhibition of 12/15‐LO has been shown to significantly reduce retinal neovascularization in a murine model of OIR and restore the barrier integrity in DR.^130^^,^^131^ Research indicates that the dysfunction of retinal ECs (RECs) induced by hyperglycemia is associated with endothelial 12/15-LO, rather than leukocytic 12/15-LO.^132^ When Muller cells were treated with hydroxy eicosatetraenoic acids (HETEs), which are metabolites of 12/15-LO, there was an overexpression of proangiogenic and proinflammatory molecules, including IL-6, VEGF, and TNF-a, alongside a concurrent inhibition of the angiostatic and neurotrophic factor, PEDF.^130^^,^^133^ Furthermore, inhibiting 12/15-LO or cytochrome P450 significantly reduced the retinal neovascularization in a murine OIR model, highlighting their role in pathological retinal neovascularization.^130^^,^^134^ Collectively, these findings support the theory that 12/15-LO is implicated in the pathogenicity of DR, suggesting an indirect effect of bioactive lipids such as PUFAs.

Recently, fenofibrate has been commonly used to treat high serum cholesterol and triglyceride levels. Studies such as FIELD trials have shown that fenofibrate can reduce the requirement of laser treatment in patients with DR by 1.5% over 5 years.^135^ Similarly, the ACCORD-Eye Study has shown that the addition of fenofibrate to statin therapy significantly reduces DR progression over a period of 4 years compared with that of therapies with statin alone.^135^ In 2024, the findings of the LENS study further validated that a daily intake of fenofibrate (145 mg) reduces the onset of macular edema and advancement of early DR in both patients with type 1 and type 2 diabetes.^136^ Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that plays a crucial role in vessel morphogenesis, vascular leakage, and lethality by interacting with S1P receptors 1 through 5 (S1PR1-5).^137^ S1P2R is significantly upregulated in ECs during hypoxic conditions. Additionally, the genetic deletion of S1P2 in mice markedly decreased OIR-induced pathological neovascularization and the infiltration of inflammatory cells in the vitreous chamber.^138^ Arachidonic acid and its metabolites, including prostaglandins and leukotrienes, are shown to influence pathological retinal neovascularization.^139^

In summary, bioactive lipids play a crucial role in regulating retinal neovascularization, demonstrating both proangiogenic and antiangiogenic effects, as detailed in the Table. Understanding the functions of bioactive lipids, their metabolites, and the metabolic pathways they influence could aid in the development of new therapeutics for proliferative retinopathies.

Ribonucleic Acids and Retinal Neovascularization

A significant portion of the human genome that lacks protein-coding genes was once deemed “junk” DNA. However, advancements in the sequencing techniques over the past decade have enabled researchers to explore the non-coding regions of the genome more thoroughly.^140^ Although protein-coding genes make up only a small fraction of the human genome, it is important to note that nearly the entire genome is transcribed.^141^ The non-coding regions of the genome are categorized into long non-coding RNAs (lncRNAs), miRNAs, and circular RNAs. Recent studies have highlighted the involvement of lncRNAs and miRNAs in retinal neovascularization. Figure 5 depicts the biosynthesis and biological functions of miRNA and lncRNA, whereas the Table summarizes their proangiogenic and antiangiogenic effects.

Long Non-Coding RNAs

Non-coding RNAs that are longer than 200 nucleotides are referred to as lncRNAs. Although they possess 5′ capped ends and undergo splicing of similar to mRNAs, lncRNAs do not contain open reading frames, indicating they lack the ability to code proteins. Additionally, the expression of lncRNA is less stable than that of mRNA and can vary based on the species, tissue, or cell type.^142^ Once expressed, lncRNAs are essential for various cellular processes, including transcription regulation, post-transcriptional modifications such as splicing, the organization of organelles and cellular structures, and maintenance of genome integrity.^143^ The functions of lncRNAs may differ depending on their specific cellular locations. Approximately 15% of lncRNAs are located in the cytoplasm, whereas the majority are found in the nucleus.^144^^,^^145^ Those located in the nucleus can influence gene expression, whereas those in the cytoplasm may act as sponges for miRNA.^146^

LncRNAs are classified as regulatory non-coding RNAs due to their structural characteristics.^147^ In the eye, lncRNAs demonstrate tissue specificity and play a vital role in the pathophysiology of various ocular diseases.^148^^,^^149^ A considerable number of lncRNAs have been studied for their involvement in ROP. Specifically, lncRNA MALAT1 contributes to the progression of ROP by promoting proangiogenic processes, including the activation of the VEGF/Akt pathway and expression of inflammatory cytokines such as IL-6, IL-1, and TNF-a. Additionally, MALAT1, a gene expressed maternally, regulates early growth responses by functioning as a sponge for miR-124-3p miRNA, which regulates EGR1 expression.^150^ EGR1 encodes for angiogenic and inflammatory factors.^151^ Together, MALAT1 and EGR1 form a novel regulatory axis under hypoxic conditions associated with ROP and OIR.^150^^,^^151^ The lncRNA TUG1 acts as a sponge for miR-299-3p, a known suppressor of VEGFA. The depletion of lncRNA TUG1 reduces OIR-induced inflammation, apoptosis, VEGFA expression, and retinal neovascularization.^152^ In a study involving OIR mice, researchers discovered that the lentivirus-mediated overexpression of MEG3 in the retina inhibited retinal neovascularization through the VEGF/PI3K/Akt signaling pathway.^153^

MIAT, MALAT1, and RNCR3 lncRNA play a detrimental role in the molecular processes associated with DR. MIAT lncRNA modulates miRNA-29, which influences cellular apoptosis.^154^ In the animal models of DR, the MIAT pathway modulates EC function by creating a feedback loop that includes VEGF and miR-150-5p.^155^ Elevated levels of lncRNA MALAT1 have been observed in these models, and inhibiting MALAT1 has been shown to ameliorate streptozotocin-induced DR in rats. This lncRNA modulates EC activity by regulating the expression of cell cycle inhibitory genes (p21 and p27Kip1) and S-phase cyclins (cyclin A2, cyclin B1, and cyclin B2).^156^^,^^157^ Elevated glucose levels lead to the upregulation of RNCR3, and depleting this lncRNA restores vascular integrity while reducing leakage and inflammation. LncRNA RNCR3 regulates the KLF2/miR-185-5p pathway, and its depletion results in decreased retinal EC migration and proliferation, thereby improving retinal vascular integrity.^158^ Alongside those lncRNAs that exacerbate DR, there are protective lncRNAs whose suppression or loss may contribute to the progression of DR. For instance, lncRNA H19 inhibits the production of TGF-1 signaling proteins through miR-200b, which helps prevent endothelial-mesenchymal transition in DR.^159^ In addition, maternally expressed gene 3 (MEG3) acts as a tumor suppressor lncRNA. Research has shown that the depletion of lncRNA-MEG3 increases PI3K and Akt activity, suggesting its role in angiogenesis.^160^^,^^161^

LncRNAs play a role in the development of AMD. Patients with AMD show an increased expression of Vax2os1 and Vax2os2 lncRNAs. During retinal development, the overexpression of Vax2os1 disrupts cell cycle progression and hinders the differentiation of retinal photoreceptor progenitors.^162^ The lncRNA ZNF503-AS1 is upregulated in the cytoplasm of RPE cells during differentiation, whereas its expression is downregulated in the RPE-choroid of individuals with atrophic AMD. NF-kB has been identified as a potential transcription factor for ZNF503-AS1. The dedifferentiation of RPE cells plays a significant role in the pathophysiology of dry AMD, making ZNF503-AS1 a compelling biomarker and potential target for treatment.^163^ LINC00167 lncRNA functions as a sponge for miR-203a-3p, enhancing the transcription of SOCS3, which inhibits the JAK/STAT signaling pathway. The regulation of the LINC00167/miR-203a-3p/SOCS3 axis contributes to the attenuation of AMD progression by preserving the differentiation of RPE cells.^164^ In contrast, lncRNAs MIAT and MALAT1 do not provide protective effects; instead, they promote retinal neoangiogenesis in AMD. MIAT lncRNA regulates vascular permeability via the miR-150-5p/VEGF pathway in both the retina and cornea, with its expression levels changing during instances of neurovascular dysfunction. Elevated VEGF levels lead to abnormal vessel proliferation. Similarly, the inhibition of MALAT1 impacts the proliferation, migration, and angiogenesis of retinal ECs by targeting miR-125b and disrupting the integrity of the VE-cadherin/-catenin complex. These findings indicate that MALAT1 may represent a therapeutic target for disorders related to retinal neoangiogenesis, such as AMD.^165^^,^^166^

Numerous studies indicate that lncRNA play a regulatory role in gene expression processes, EC activity, and miRNA interactions. This regulation can either protect against or stimulate neoangiogenesis, making lncRNA potential targets for treatment.

MiRNAs

The miRNAs are a class of post-transcriptional gene regulators that go beyond interactions with lncRNA and play a role in controlling gene expression in various contexts.^167^ These miRNAs are short non-coding RNAs, typically ranging from 19 to 25 nucleotides in length. The miRNAs regulate gene expression by targeting mRNA transcripts, which leads to reduced protein levels.^168^^–^^170^ The RNA-induced silencing complex (RISC) is a protein complex that incorporates miRNA and uses it to identify complementary mRNA, resulting in gene silencing.^171^^,^^172^

The miRNAs play a crucial role in suppressing proliferative retinopathy, suggesting that their overexpression could serve as a potential therapeutic strategy. Utilizing a murine model of OIR, it has been shown that miR-329, miR-31, miR-150, and miR-184 exhibit anti-neovascular effects. A microarray analysis demonstrated a significant downregulation of miR-31, -150, and -184 in the retinas of OIR mice relative to normoxic controls. Furthermore, the overexpression of miR-31, -150, or -184 in the retina significantly reduced ischemia-induced retinal neovascularization.^172^^,^^173^ Additionally, a recent study identified miR-150 as a key suppressor of pathological angiogenesis, showing inhibitory effects on both CNV and retinal neovascularization.^174^ The miR-329 regulates retinal neovascularization by targeting CD146. The CD146 levels were significantly increased in the OIR retinas, whereas miR-329 levels decreased during the retinal neovascularization phase.^175^ The study indicates that the inhibition of CD146 with miR-329 is crucial for reducing retinal neovascularization by 40%.^175^ However, this inhibition does not affect the vaso-obliteration in ischemic retinas.^175^ These findings suggest that whereas miR-329 is effective in reducing vasculature-related abnormalities, it does not promote vessel regeneration. In addition, miR-329 has been shown to downregulate CD146 expression, thereby inhibiting VEGF-induced Src-p38 MAPK-NF-kB activation.^175^ Based on these studies, a potential therapeutic approach for retinal neovascularization may involve the intravitreal administration of miRNA mimics or the upregulation of pre-miRNA. Furthermore, miR-221 and miR-222, which belong to the same family, have been shown to inhibit angiogenic events in ECs by targeting c-kit and indirectly modulating the expression of endothelial nitric oxide synthase.^176^^,^^177^ It has been demonstrated that miR-222 can inhibit retinal neovascularization by blocking STAT5A signaling in a mouse model of oxygen-induced retinopathy.^178^ Additionally, miR-221/222 show promigratory, antiapoptotic, and pro-proliferative properties in vascular smooth muscle cells. These findings suggest that miR-221/222 have biological functions that are specific to different cell types within vascular walls.^179^

However, studies have shown that miR-155 and miR-21 enhance retinal neovascularization in the OIR model. The expression levels of miR-155 were significantly elevated in the OIR retinas compared with normoxic retinas. The inhibition of miR-155 via the lentiviral technique or genetic knockout resulted in a decrease in neovascular tufts and avascular regions within the OIR retinas.^180^^,^^181^ Similarly, miR-21 is a crucial microRNA associated with tumor growth, angiogenesis, and metastasis, and is highly expressed in bovine retinal microvascular ECs.^182^ Additionally, it was observed that inhibiting miR-21 significantly hampers the proliferation, migration, and tube formation of bovine retinal ECs.^182^ A separate study showed that the overexpression of miR-21 in HUVECs markedly reduced EC proliferation, migration, and tube formation. In contrast, the suppression of endogenous miR-21 produced opposite effects. Mechanistic investigations suggest that miR-21 targets RhoB, which disrupts the formation of actin stress fibers, thereby inhibiting angiogenesis by impairing EC migration and tube formation.^183^ The observed discrepancies in these results may stem from the differing cell culture and assay systems used in the experiments, suggesting a potential species-specific effect.

Overall, miRNAs play a significant role in retinal neovascularization, with various subsets functioning as the inhibitors or promoters of angiogenesis. The observed downregulation of antiangiogenic miRNAs and upregulation of proangiogenic miRNAs in OIR retinas indicate that miRNA-based therapies, including miRNA mimics or inhibitors, may present a promising strategy for addressing pathological retinal neovascularization. However, it is critical to keep in mind the differences in miRNA function across species and cell types when developing therapeutic approaches.

Emerging Directions

Since the approval of the first angiogenic inhibitor, bevacizumab, for the treatment of proliferative retinopathies, most therapeutic targets have concentrated on anti-VEGF medications. Recently, there has been significant research emphasis on identifying additional angiogenic inhibitors and immune checkpoint inhibitors beyond anti-VEGF therapies. Recent research utilizing single-cell transcriptomics has revealed the antiangiogenic role of mitochondria-localized glutamic acid-rich protein (Mgarp) in DR. The inhibition of Mgarp significantly reduced the proliferation, migration, and tube-forming abilities of retinal ECs.^184^ Single-cell transcriptomics have similarly identified microglial subtypes associated with proliferative retinopathy. Among these subtypes, the proliferative retinopathy-associated microglia (PRAM) are localized within and around neovascular tufts. These cells are hypermetabolic and express elevated levels of proangiogenic genes and glycolytic enzymes, indicating their potential to promote retinal angiogenesis.^185^ The analysis of single-cell transcriptomics in fibrovascular membranes linked to proliferative DR identifies adipocyte enhancer-binding protein 1 (AEBP1) as a key regulator of fibrogenesis. AEBP1 signaling influences the transformation of pericytes into myofibroblasts, suggesting that targeting AEBP1 could potentially reduce scar tissue formation in advanced DR.^186^ In addition, single-cell transcriptomic profiling has been utilized to clarify the cellular heterogeneity and intercellular communication in retinal diseases, particularly in the CNV model. This profiling has been instrumental in linking and understanding the roles of ECs, fibroblasts, and macrophage cell clusters in the progression of neovascular AMD. Through a CellChat analysis, a complex cell-cell communication network was established to illustrate the interactions between EC clusters and fibroblasts as well as macrophages in neovascular AMD. These interactions have been demonstrated to influence the angiogenic effects, fibrotic responses, and inflammatory processes.^187^

In addition to single-cell transcriptomics, researchers are utilizing a lipidomic analysis to assess the progression of DR and neovascular AMD. In the vitreous humor of patients with PDR, differentially expressed lipids, including phosphatidylcholine, phosphatidylserine, phosphatidylinositol, cholesteryl ester, and sphingomyelin, have been identified as potential metabolic biomarkers for the clinical diagnosis and prognosis of DR.^188^ Mass spectrometry-based metabolomic and lipidomic profiling effectively categorizes the stages of DR, potentially facilitating early diagnosis and targeted interventions for the disease. Notably, sphingolipid and purine metabolism were significantly changed in both the proliferative and non-proliferative stages of DR. In cases of PDR, glycine-serine-threonine metabolism was particularly prominent, whereas tyrosine metabolism was disrupted in the non-proliferative DR.^189^ Therefore, a lipidomic analysis can be used not only to assess the stages of proliferative retinopathies, but also to identify the therapeutic targets for various stages of retinal diseases.

Another significant particle that has garnered interest in the diagnosis, pathogenesis, and treatment of proliferative retinopathies is the extracellular vesicle (EV). EVs are nanoscale membrane-bound vesicles of varying sizes. They act as natural messengers between cells, carrying a diverse range of cargo, including RNAs, lipids, proteins, and other molecules. They play a crucial role in intercellular communication, immune modulation, and the regulation of both physiological and pathological processes. Due to their low immunogenicity and exceptional biocompatibility, these vesicles are utilized as therapeutics and as targeted drug delivery nanocarriers.^190^ The serum levels of extracellular vesicle-derived miR-26b-5p and miRNA-3976 are elevated in patients with DR. Additionally, miRNA-3976 is involved in the regulation of NF-kB-related pathways and may function as a prospective biomarker for DR.^191^^,^^192^ Numerous studies have demonstrated that EVs derived from human umbilical cord mesenchymal stem cells (hucMSCs), which contain specific miRNAs such as miR-17-3p,^193^ miR-18b,^194^ miR-30c-5p,^195^ and miR-22-3p,^196^ may help reduce inflammatory responses and oxidative damage in mouse models of DR by targeting key angiogenic and inflammatory signaling pathways. Additionally, the intravitreal delivery of these hucMSCs-derived EVs has been found to reduce neurodegeneration in animal models of DR.^197^

In view of the above-mentioned emerging approaches, incorporating single-cell transcriptomics and lipidomics into future research could enhance our understanding of the pathogenesis and improve our ability to diagnose proliferative retinopathies in a more precise manner. The research on EV-based delivery systems for their therapeutic potential against proliferative retinopathies may lead to the development of new targeted treatment strategies.

Conclusions

Vascular homeostasis is disturbed in pathological conditions such as DR, AMD, and ROP, leading to the growth of abnormal blood vessels and ultimately resulting in vision loss. Over the past 2 decades, anti-VEGF therapies have been used to treat these vascular pathologies. However, several common issues associated with anti-VEGF treatments need to be addressed, including the burden of frequent, lifelong therapies, and the potential systemic side effects. These limitations drive researchers to explore the cellular and molecular mechanisms involved in pathological retinal neovascularization to develop effective therapeutic strategies. New evidence shows that lipid mediators, lncRNAs, and miRNAs all play a role in the cellular and molecular processes of the retina. When these are combined with new delivery vehicles, it might lead to the creation of therapeutic regimens that reduce the global burden of retinal proliferative diseases. Furthermore, the studies presented here shed light on the role of retinal cellular interactions in pathological retinal neovascularization and their influence on angiogenic and inflammatory effects, potentially paving the way for new targeted treatment strategies.