Molecular and Cellular Mechanisms Involved in the Pathophysiology of Retinal Vascular Disease—Interplay Between Inflammation and Oxidative Stress

1.1. A Brief Overview of Retinal Physiology and Function

The retina, located in the posterior segment of the eye, is a highly developed nervous tissue consisting of ten different layers [2], six types of neurons (rod, cone, amacrine, bipolar, horizontal, and ganglion), and three types of glial cells (astrocytes, Müller cells, and microglia) (Figure 1) [3,4]. The blood–retinal barrier (BRB) controls the entry of fluid and electrolytes into the extracellular space and maintains homeostasis [5]. The inner component of the BRB is formed by the retinal vascular endothelium (RVE), while the outer BRB consists of the retinal pigment epithelium (RPE), both of which have tight junctions [5]. Anatomically, the RPE serves as a structural barrier separating the retina from the choroid [3]. The morphological structure of the retina, as well as the vascularization of the retina, including the fenestrated capillaries of the choroid located below the retina, are shown in Figure 1.

Light stimulation initiates the transmission of visual signals from photoreceptors to ganglion cells. Ganglion cell axons form the retinal nerve fiber layer (RNFL), which together form the optic nerve. There are two important structures in the central area of the retina: the macula and the optic disk [6]. The fovea, which is located within the macula, contains the highest concentration of photoreceptors and is responsible for central and precise vision [6]. The optic disk, in addition, corresponds to the optic nerve head and serves as the entry point for blood vessels into the eye [7].

Two primary vascular systems that are crucial for maintaining adequate retinal blood flow are retinal and choroidal blood vessels. The retinal vascular system is responsible for 20–30% of blood flow, while the choroidal circulation provides 65–80% of blood flow [8,9]. The vascular network of the inner retina is composed of retinal arterioles and venules that originate from the central retinal artery and drain into the central retinal vein as they cross the inner retinal surface [10], while the outer retina, composed of photoreceptors, remains avascular [11]. The central retinal artery originates from the internal carotid artery via the ophthalmic artery, and the central retinal vein drains directly or indirectly via the superior ophthalmic vein into the cavernous sinus [10]. These retinal vessels run through the center of the optic nerve. The metabolic needs of photoreceptors are primarily met by the choroidal vessels. The choriocapillaris, a dense network of fenestrated capillaries that work closely with the RPE, is located in the choroid [12]. Equipped with specialized tight junctions and transport mechanisms, the RPE facilitates the exchange of nutrients and waste products between the choroidal circulation and the outer retina [12]. The retinal circulation has a high oxygen extraction fraction, which contrasts with the choroidal circulation and is characterized by a low oxygen extraction fraction [10]. In the healthy adult retina, the vasculature is usually mature and remains in a quiescent, nonangiogenic state maintained by a balance between proangiogenic factors, including VEGF, insulin-like growth factor 1 (IGF-1), and erythropoietin (EPO), as well as antiangiogenic factors, such as pigment epithelium-derived factor (PEDF) [13].

1.2. Retinal Vascular Diseases—Diabetic Retinopathy

Diabetic retinopathy (DR) is one of the most severe complications of type 1 and type 2 diabetes mellitus (DM) and is the most common RVD leading to visual impairment and blindness [14]. By the year 2030, the global incidence of DR is estimated to reach approximately 454 million people, primarily due to increasing obesity, increased exposure to obesogenic environments, increased life expectancy, and improved diagnostic capabilities for detecting the disease [15]. Factors outside the eye that contribute to the onset and progression of DR include inadequate glycemic control, hypertension, dyslipidemia, prolonged duration of diabetes mellitus, pregnancy, and genetic predisposition [14].

One of the characteristic manifestations of DR, first documented by ophthalmoscopy in the 19th century, is the initial appearance of microaneurysms. Over time, these microaneurysms progress to exudative changes, macular edema, ischemia, collateralization, and dilatation of the venules, eventually culminating in proliferative changes [16]. Retinal microvascular changes in DR patients include thickening of the basement membrane; loss of endothelial tight junctions and pericytes; increased vascular permeability; capillary occlusion; microaneurysms; and, in later stages, loss of endothelial cells (ECs) and their transition to mesenchymal cells (Figure 2) [17]. The progressive nature of DR can culminate in retinal ischemia, increased vascular permeability, neovascularization, and macular edema [18]. Ischemia of the retina, manifested as acellular capillaries, is an early sign of DR and has been observed in various experimental models of DR and humans [19]. As a microvascular complication of DM, DR damages not only the retinal microvasculature but also neurons and glial cells [20]. This disease is considered a progressive neurovascular complication characterized by neuronal dysfunction followed by microvascular damage [20].

DR progression typically includes a non-proliferative stage of DR (NPDR) characterized by progressive intraretinal microvascular changes and a proliferative stage (PDR) characterized by the growth of newly formed vessels on the retina or optic nerve head [21]. Common features of NPDR include visible microaneurysms and flame or blot hemorrhages, while PDR is characterized by the growth of new vessels and retinal tissue edema [10]. The most common pathological features of NPDR and PDR are presented in Figure 2. The use of noninvasive imaging techniques, particularly optical coherence tomography (OCT), in conjunction with the introduction of intravitreal anti-vascular endothelial growth factor (anti-VEGF) therapies has significantly improved the early detection and treatment of diabetic macular edema (DME) and led to an improvement in the visual performance of those affected [22]. DME is a specific complication of DR that can occur at all degrees of severity in both NPDR and PDR patients [23]. DME, which occurs in approximately 25% of diabetic patients, is a major factor contributing to visual impairment. It is characterized by central thickening of the fovea, as observed via OCT, and fluorescein leakage, as detected via angiography [16].

DR manifests through a complex interplay among biochemical processes that are particularly influenced by persistent hyperglycemia. These processes include increased levels of advanced glycation end products/receptors (AGE/RAGE), activation of the polyol pathway, activation of protein kinase C (PKC), and activation of the hexosamine pathway, which increases the production of reactive oxygen species (ROS) and, consequently, oxidative damage [24]. Increased OS and inflammatory stimuli result in inflammation, which is a widely found pathogenetic factor in the DR. Increased production of proinflammatory cytokines, including tumor necrosis factor α (TNF-a), interleukin 1 (IL-1), IL-17, and IL-6, contributes to maintaining circulus vitiosus and persistent inflammation and OS [24]. Proteomic studies have indicated changes in the plasma levels of several proteins. Retinol-binding protein 1 (RBP1), diphosphoinositol polyphosphohydrolase 3 alpha, and neuroglobin (NGB) were downregulated, while subunit gamma 2 (HBG2) and CD160 antigen18 were upregulated [25]. Vascular endothelial growth factor (VEGF), the production of which is closely related to inflammation and OS, plays a key role in the pathophysiology of DR and DME [24]. An increase in angiogenic platelet-derived growth factor (PDGF) and a decrease in antiangiogenic PEDF in the vitreous fluid of individuals with DR represent common chemokine changes [26].

1.3. Retinal Vascular Diseases—Retinopathy of Prematurity

Retinopathy of prematurity (ROP), originally described in 1942 as retrolental fibroplasia, is characterized by abnormal neovascularization of the retina and affects 30% to 50% of newborns with very low birth weight, especially those born preterm [27]. In high-income countries, the incidence of ROP decreases with increasing gestational age at birth, and ROP predominantly, although not exclusively, affects extremely preterm infants (born before the 28th week of gestation) [28]. ROP can lead to severe visual impairment and blindness, while other complications include the development of glaucoma, strabismus, myopia, and amblyopia [29]. The pathophysiology of ROP is multifactorial, with short gestation, low birth weight, and hyperoxia being the most common predisposing factors [30]. The hallmark of ROP pathogenesis is a biphasic pattern characterized by an initial impairment of retinal vascular development and subsequent excessive vascular proliferation that spreads from the retina into the vitreous [28]. Early detection and timely intervention are critical for preventing vision loss in infants with ROP. A key milestone in understanding the pathophysiology of ROP was the discovery of the role of VEGF in the process of retinal neovascularization [31]. Identification of this pathological mechanism plays a crucial role in the development of novel interventions involving intravitreal injections of anti-VEGF antibodies [32]. The anti-VEGF agents ranibizumab, bevacizumab, and aflibercept are frequently used to limit pathological neovascularization of the vitreous in preterm infants [28]. The main pathological features of ROP are presented in Figure 3 and include excessive growth of the retinal blood vessels, formation of the fibrovascular ridge, and, in the final stages, detached retina.

1.4. Retinal Vascular Diseases—Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is one of the main causes of blindness due to the deterioration of high-resolution central vision in older people [33]. Numerous modifiable and nonmodifiable risk factors have been associated with the development and progression of AMD [33]. The most common nonmodifiable risk factors include older age (≥65 years), a northern European region, and a family history [34]. AMD affects men and women to a similar extent, although a strong genetic component is also responsible for the occurrence of the disease [33]. The majority of AMD patients (85–90%) suffer from the currently untreatable, atrophic (dry) form of the disease, while the remaining patients (10–15%) develop an exudative (wet) form of AMD [35].

The pathogenesis of AMD is complex and includes genetic susceptibility, age-related disruption of retinal homeostasis, impaired lipid metabolism, immune activation leading to chronic inflammation, OS and ECM dysfunction, all of which contribute to the disease [36]. Pathology changes in patients with AMD affect the inner retinal layers of the macula and adjacent vessels, resulting in impaired central vision [37]. In dry AMD, there is gradual degeneration of the RPE and the outer retinal layers [38]. The wet form is characterized by abnormal choroidal neovascularization that invades both the RPE and the retina in a disorderly manner [38]. In the early clinical stages, both the dry and wet forms of AMD are characterized by the accumulation of lysosomal lipofuscin and extracellular drusen deposits and by degeneration of the RPE [35]. The accumulation of deposits on the retina, known as drusen, is a hallmark clinical feature of AMD and indicates the “dry” form of the disease [37]. Drusen are primarily composed of lipids, proteins, and carbohydrates and can be visualized as small white or yellowish deposits on the macula [33]. Approximately 10–15% of AMD patients develop advanced exudative disease. Differences in the dry and wet form of the AMD are presented in Figure 4.

There are a variety of diagnostic tools for the detection of AMD, including fundus examination, color fundus photography, fundus autofluorescence (FAF), optical coherence tomography (OCT), infrared reflectance (IR), and fluorescein/indocyanine green angiography (FGA) or OCT angiography [38]. The involvement of VEGF in the pathogenesis of neovascular AMD was a breakthrough finding that led to the introduction of anti-VEGF agents as standard treatment for these patients [39]. However, there are still no suitable treatments for the dry form of AMD [35]. Approaches under investigation to slow the progression of AMD include drugs with antioxidant properties, inhibitors targeting the complement cascade, neuroprotective agents, visual cycle inhibitors, gene therapy, and cell-based therapies [39].

1.5. Retinal Vascular Occlusions

Acute retinal vascular occlusions, which include both retinal artery occlusions (RAOs) and retinal vein occlusions (RVOs), are the leading cause of visual impairment and loss. These conditions are widely associated with older age and cardiovascular risk factors; however, they differ significantly in pathophysiology and treatment [40]. Appropriate therapeutic strategies are needed to address the distinct features of RAOs and improve visual morbidity and clinical outcomes in these patients.

1.6. Carotid Artery Stenosis and Ocular Ischemic Syndrome

Carotid artery stenosis is a common risk factor for ocular ischemic complications such as amaurosis fugax, central retinal artery occlusion, and ocular ischemic syndrome, while the ophthalmic artery as a major branch of the internal carotid artery gives rise to the central retinal artery [63]. Reduced blood flow in the carotid artery after stenosis can lead to microcirculatory blockages in both the retina and choroid [64]. Lawrence and colleagues reported that the incidence of ocular symptoms in carotid artery stenosis is greater than 50% [65]. Consequently, without early intervention the metabolism of the retina is disturbed, reducing blood flow to the choroid and causing microstructural changes [66].

Carotid stenosis or carotid occlusion is a significant risk factor for retinal ischemic disease [67]. Lahme and colleagues reported reduced retinal flow density in patients with carotid artery stenosis [68]. Narrowing or blockage of the carotid artery, leading to reduced blood flow in the retina, causes conditions such as ocular ischemic syndrome (OIS) and retinal artery occlusion [67]. The mentioned characteristics are shown in Figure 6. In addition, retinal degeneration is one of the pathologic changes occurring during arterial or venous occlusion or diabetic retinopathy [69]. Chronic ocular ischemic disease can have an insidious onset and a variety of symptoms that, if left untreated, can lead to severe vision loss or serious cerebrovascular events [70]. Several risk factors, including hypertension and carotid occlusion, strongly affect retinal blood flow. Significant improvements have been observed after the use of hypolipidemic medications and carotid surgery [67]. Therefore, early detection of any impairment in ocular blood flow and mild retinal ischemia is essential to prevent ocular ischemic complications.

OIS is characterized by vision loss, orbital pain, visual field changes, and various signs in both the anterior and posterior segments of the eye. Approximately 7.5 cases per million people are diagnosed with OIS each year [71]. This condition is usually undiagnosed, with a poor prognosis, and accompanied by severe ischemic cardiovascular and cerebrovascular diseases. The 5-year survival rate of these patients is 60% [72]. The main cause of OIS is severe carotid stenosis caused by atherosclerosis leading to reduced perfusion pressure of the central retinal artery [71]. This ischemic insult increases the production of vascular endothelial growth factor (VEGF), resulting in neovascularization and increased vascular permeability [73]. OIS is diagnosed in the presence of amaurosis fugax with or without loss of vision, eye pain, conjunctival or episcleral injection, corneal edema, and ocular signs in the anterior and posterior chamber [74]. Fundus fluorescein angiography is commonly used to detect pathological changes, such as non-perfusion of the retina with or without neovascularization and internal carotid artery stenosis [71].

To better understand the pathophysiology of OIS, several experimental models have been developed in rats and mice [75]. In these models, the common or internal carotid artery is surgically occluded or constricted [75] either bilaterally or unilaterally. Researchers commonly perform bilateral occlusions of the common carotid arteries in rats to induce chronic cerebral hypoperfusion, which helps them study neurodegenerative disorders and diseases linked to reduced blood flow [76]. Generally, mice are more prone to ischemic injuries compared to rats, researchers have employed the method of unilateral occlusion of the common carotid artery in mice [75]. These studies have shown that ocular hypoperfusion can lead to similar pathological outcomes, including neovascularization, oxidative damage, and inflammation [75]. Du and colleagues reported increased oxidative and nitrosative stress in an animal model of OIS induced by bilateral common carotid artery, which was related to increased activity of RhoA/MERK1/ERK1/2/iNOS pathways throughout the retina [77].

Early diagnosis and intervention are critical for the treatment of OIS, the resolution of symptoms, and the prevention of permanent vision loss [74]. Treatment options for OIS management are very limited, involving both ocular and systemic treatment [75].

The therapeutic options for described RVDs are shown in Table 1.

2.1. Angiogenesis in Retinal Vascular Diseases

Retinal angiogenesis is a fundamental process involved in both normal vascular development and the pathogenesis of retinal neovascularization in ischemic retinal vasculopathy [95]. The inner BRB is involved in the protection of the inner retina and consists of ECs, the basement membrane, and mural cells [96]. Under physiological conditions, mural cells protect ECs by inhibiting angiogenesis, whereas in retinal diseases such as DR, the loss of mural cells triggers various deleterious processes, including EC death [96]. An imbalance between angiogenic (HIF-1α, PIGF, TGF-beta, FGF, and EDN1) and antiangiogenic (THBS1 and TIMPs) factors is a major factor in the development of retinal neovascularization and can lead to retinal detachment [97]. This phenomenon is common in diseases such as DR, RVOs, and ROP [98].

2.2. Inflammatory Pathways in Retinal Vascular Diseases

Ischemic conditions are followed by an inflammatory response called sterile inflammation [132]. Although both the retina and CNS are considered immune-privileged organs, as neither is capable of eliciting an immunological response [91], recent research has challenged the concept of retinal immune privilege as evidence of contrary events [85]. The role of inflammation in ocular vascular diseases is widely recognized, but its specific cellular origin and underlying signaling pathways are still controversial [133].

Immune privilege is an evolutionary mechanism for protecting components of the immune system; however, once compromised, the entire ocular tissue becomes highly susceptible to damage from inflammation [133]. However, an appropriate inflammatory response requires coordinated interactions between immune cells, endothelial cells, and neurons that initiate signaling cascades mediated by inflammatory factors [127,133].

2.3. Oxidative Stress and Vascular Dysfunction

The term OS describes a condition caused by an imbalance between the production of reactive oxygen species (ROS) and the capacity of the antioxidant defense system, resulting in excessive ROS accumulation and tissue damage [150]. Oxidative metabolism occurs mainly in the mitochondria, particularly in the electron transport chain (ETC) and in the endoplasmic reticulum. Even under optimal conditions, electron loss occurs, which contributes to the formation of ROS [150].

Both the anterior and posterior parts of the eye are susceptible to OS, and numerous diseases, including RVDs, are associated with increased levels of free radicals [151]. ROS are generated by the incomplete reduction of molecular oxygen and include hydrogen peroxide (H₂O₂), superoxide anion (O₂⁻), hydroxyl radical (OH⁻), peroxyl radical (ROO⁻), and singlet oxygen (O₂) [152]. In a physiological state, the cells of the retina are constantly exposed to increased OS caused by strong exposure to light and the function of visual signal transduction pathways, leading to the production of significant amounts of ROS. Factors such as the high oxygen consumption of the outer retina-RPE complex, exposure to strong light, abundance of photosensitizers in both the neurosensory retina and the RPE, and presence of easily oxidizable polyunsaturated fatty acids in the membranes of the outer photoreceptor segment collectively contribute to the initiation of cytotoxic responses [153]. The main sources of ROS in diabetes are oxidative phosphorylation in the mitochondria and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system (Nox) [154]. The Nox family includes Nox1-Nox5, dual oxidase 1 (Duox1), and dual oxidase 2 (Duox2), enzymes that can generate either superoxide or hydrogen peroxide, depending on the isoform [155]. Because reactive nitrogen species (RNS) are normally produced in the human body, it is important to mention that physiological concentrations of nitric oxide (NO) are essential for normal vision, while excessive NO concentrations can be harmful [151]. The enzyme responsible for NO synthesis is NOS synthase (NOS), which catalyzes this process in the presence of oxygen and NADPH. There are three isoforms of NOS, neuronal (nNOS), endothelial (eNOS), and inducible (iNOS), which are induced under pathological conditions [156,157].

In addition to other organisms that utilize oxygen, the human body has developed various antioxidant defense mechanisms. These mechanisms include low-molecular-weight and nonenzymatic antioxidants such as vitamins A, C, and E, as well as intracellular antioxidants and antioxidative enzymes, such as catalase, different isoforms of superoxide dismutase (Cu/Zn SOD or SOD1, Mn-SOD, extracellular SOD), reduced glutathione (GSH), and enzymes of the glutathione cycle, such as glutathione peroxidase (GPx) and glutathione reductase (GR) [155].

OS has been identified in several studies as an important factor in the pathophysiology of DR [158]. An increase in O₂⁻ is most often associated with diabetes and DR and is triggered mainly by hyperglycemia, hypoxia, or proinflammatory cytokines [158]. In addition, reduced levels of antioxidants are also present in the diabetic retina [159]. Müller glial cells play an important role in the antioxidant defense system and the pathogenesis of DR by interacting with cells in all retinal layers [160]. Under hyperglycemic conditions, Müller glial cells exhibit increased expression of heme oxygenase-1 and increased ROS production and consequently respond to OS by producing GSH, which prevents excitotoxicity in the retina [151]. However, decreased GSH levels were observed in the retinas of rats exposed to three weeks of hyperglycemia, which contributes to oxidative damage in DR [161]. An important phenomenon to note is metabolic memory, which represents microvascular changes that persist despite good glycemic control and lead to regression of retinopathy resistance [162]. This imprinting of vascular cells highlights the lasting effects of past periods of poor glycemic control on vascular health [130]. The progression of DR to the PDR stage is particularly associated with OS and reduced antioxidant capacity [163]. In addition, several studies have shown a strong correlation between plasma NO concentrations and the severity of DR. In particular, higher plasma NO concentrations correlate with more severe stages of DR [164]. This relationship suggested that NO plays an important role in the development and progression of DR and may contribute to the vascular dysfunction and retinal damage associated with this disease. The effects of OS on different cell types in the retina are shown in Figure 9.

OS is strongly implicated in the development of ROP. Shortly after birth, premature infants experience a rapid shift in oxygen levels as they transition from a low-oxygen environment in utero to a higher oxygen concentration outside the uterus [165]. Both hyperoxia and hypoxia are directly involved in ROP, while an increase in nitro-OS leads to the formation of nitrite, nitrate, and peroxynitrite (ONOO⁻), which contribute to subsequent microvascular damage in the retina [151]. In the pathogenesis of ROP, studies have highlighted the NO₂⁻-mediated peroxidation process as significant, focusing on nitrosative stress mediators such as trans-arachidonic acid isomers resulting from NO₂⁻-mediated arachidonic acid isomerization [151]. Specifically, trans-arachidonic acid isomers lead to the upregulation of thrombospondin-1 in retinal ECs, which may further inhibit angiogenesis by inducing the apoptosis of microvascular ECs [166]. In AMD, OS, which occurs mainly in RPE cells, is considered to be one of the main causes of this disease [167] (Figure 9). During periods of high metabolic stress, increased levels of ROS occur in RPE cells, leading to further accumulation of lipofuscin, which contributes to RPE cell dysfunction [151]. Enzymes such as NADPH oxidase and peroxidases are also considered to be additional sources of ROS in AMD [168]. A close link between OS and AMD has been confirmed by elevated levels of OS markers, such as malondialdehyde (MDA) and 8-hydroxy-2′-deoxyguanosine (8-OHdG), in the blood serum of individuals with AMD compared to individuals without AMD [169]. The effects of OS on the development of DR and AMD are shown in Figure 10.

OS is widely recognized as a major factor in the etiology and pathogenesis of various forms of retinopathies. However, despite extensive research, there are still many unresolved questions in understanding the exact mechanisms by which OS contributes to retinopathies [151]. Therefore, ongoing and future studies are crucial for deepening our understanding and unraveling the complexity of OS in the context of RVDs.