Diabetic Retinopathy Pathophysiology
Diabetic retinopathy (DR) is a microvascular disorder caused by vision-threatening damage to the retina, a long-term sequela of diabetes mellitus.
DR is the most common microvascular complication in diabetic patients and the leading global cause of vision loss in working middle-aged adults.  The propensity of developing DR is directly proportional to the age of the patient and duration of diabetes as well as with poor glycemic control and hypertension.
For more information on the disease entity, etiology, risk factors, diagnosis, and management, see Diabetic Retinopathy. This article provides detail on the mechanisms and pathological processes involved in DR.
DR can be classified clinically into non-proliferative (NPDR) and proliferative (PDR) forms, according to the presence or absence of retinal neovascularization, and it can present with or without macular edema (DME).
Mechanisms of Diabetic Retinopathy Subtypes
NPDR represents the early stage of DR, with increased vascular permeability and capillary occlusion being the two main observations in retinal vasculature. Based on the severity of retinal vascular lesions, NPDR is categorized into mild, moderate, and severe forms. Lesions vary from microaneurysms, dot and blot hemorrhages, hard exudates, and cotton wool spots to venous beading and intra-retinal microvascular abnormalities (IRMAs).  PDR represents a more advanced stage of DR characterized by the presence of neovascularization. The new abnormal vessels may bleed into the vitreous or cause a tractional retinal detachment, severely impairing vision. DME is characterized by thickening of the macula due to the accumulation of fluid within 500 µm of the center of the macula and it can occur at any stage of DR.  In patients with type I diabetes, PDR is the most prevalent vision-threatening condition. However, the most common cause of vision loss in type II diabetes patients is DME.
Hyperglycemia results in damage to retinal capillaries through the formation of advanced glycation endproducts (AGEs). The resulting endothelial damage compromises capillary walls and results in microaneurysms. Microaneurysms consequently rupture to form hemorrhages deep in the retina, appearing as "dots" on retinal examination, more commonly known as dot and blot hemorrhages. The fundamental pathologic process involved in capillary occlusion is believed to be the result of an activated leukocyte adhering to and damaging the retinal capillary wall, which results in eventual capillary occlusion. This obstruction can cause infarction of the nerve fiber layer, resulting in cotton-wool spots.
Inflammatory cytokines are significantly up-regulated in diabetes, and as a result, chronic inflammation and endothelial damage lead to increased vascular permeability of blood vessels. The pathologic process involved in DME is the resultant fluid leaking into the retina and depositing under the macula. Sediment left behind from this edema leads to waxy, yellow lipid byproducts referred to as hard exudates. Macular edema can occur in NPDR, but it is more common in more severe cases of DR where the increased vascular permeability is more advanced.
Continued ischemia stimulates retinal cells to release pro-angiogenic factors such as VEGF. Such factors stimulate neovascularization to bypass damaged retinal blood vessels. The formation of new blood vessels occurs from existing capillaries as a result of angiogenesis. These blood vessels usually arise in the interface between perfused and non-perfused areas of the retina in retinal neovascularization. They can also originate from the optic disk or iris (neovascularization of the disk/iris). These new vessels are extremely immature, fragile, permeable and bleed very easily, originating severe complications such as vitreous hemorrhage or tractional retinal detachment.
Several pathways are involved in the aforementioned processes.
Hyperglycemia and the regulation of metabolic pathways
Chronic hyperglycemia is the key promotor for the development and progression of DR due to its tissue-damaging effects, as described in the UKPDS and DCCT trials. However, genetic factors may play a role in individual susceptibility to those effects and other clinical factors like hypertension, dyslipidemia and pregnancy have also been implicated. 
Hyperglycemia leads to the activation of alternative pathways of glucose metabolism such as the polyol pathway, advanced glycation endproducts (AGEs) formation, protein kinase C (PKC) activation, hexosamine pathway flux and Poly(ADP-ribose) polymerase activation. The end result of these pathways is the activation of cytokines and growth factors, leading to vascular endothelial dysfunction, increased vascular permeability, and eventual microvascular occlusion. Microvascular occlusion then leads to retinal ischemia, which promotes neovascularization and the formation of IRMAs.
The Polyol Pathway
Excess glucose is metabolized via the polyol pathway to sorbitol. Sorbitol is impermeable to cellular membranes, accumulating inside the cell and inducing osmotic damage.  It can also be metabolized to fructose and subsequently to fructose-3-phosphate and deoxyglucosone, both of which are strong glycolyzing agents and lead to the deposition of AGEs. In addition, upregulation of the polyol pathway results in a reduced availability of NADPH, thereby enhancing the sensitivity of affected cells to oxidative stress.
Due to the high availability of glucose, AGEs formation is markedly increased in diabetic patients. AGEs have the capacity to cross-link proteins which alters their structure and function, affecting basement membranes, cellular receptors, and blood vessel wall components. Moreover, AGEs receptors activation induces prooxidant and pro-inflammatory cascades, thus exacerbating oxidative stress and leukocyte adhesion. The accumulation of AGEs has also been correlated to pericyte loss.
An increase in glycolysis activity also occurs during hyperglycemic episodes, elevating the synthesis of diacylglycerol (DAG) which in turn activates the PKC pathway. PKC activates the mitogen-activated protein kinase (MAPK) factors, leading to enhanced expression of stress-related proteins and mediators of vascular function such as c-Jun kinases and heat shock proteins. In particular, the PKC-β isoform increases VEGF expression. PKC activation also drives over-expression of NADPH oxidase and NFκB in vascular cells, exacerbating oxidative stress and inflammation.
Hexosamine Pathway Flux
In the hexosamine pathway, fructose-6-phosphate (F6P) is converted into uridine-5-diphospho-N-acetylgalactosamine (UDP-GlcNAc). O-GlcNAc transferase (OGT) catalyzes the addition of GlcNAc to serine and threonine residues at phosphorylation sites on SP1, upregulating its transcriptional activity and consequently the expression of transforming growth factor beta (TGFβ) and plasminogen activator inhibitor-1 (PAI-1) in vascular cells. The glycosylation of RNA polymerase-II transcription factors by OGT and UDP-GlcNAc affects the expression of multiple factors involved in DR pathophysiology, representing a key regulatory mechanism of glucose-responsive gene transcription.
Poly(ADP-Ribose) Polymerase Activation
Hyperglycemia-induced oxidative stress correlates to increased poly(ADP-ribose) polymerase (PARP) activation. The formation of ROS leads to NAD+ depletion and inhibition of glyceraldehyde phosphate dehydrogenase (GAPDH) through the depletion of the enzyme’s catalytic cofactor and PARP-mediated ribosylation. In conjunction, these molecular mechanisms contribute to DNA damage and endothelial cell dysfunction in diabetic blood vessels.
Several signaling pathways can be altered by having hyperglycemia in different tissues, which produces oxidative stress. Hyperglycemia activates a particular pathway involving diacylglycerol (DAG), the activation of protein kinase C (PKC), and the NADPH-oxidase system. This particular signaling pathway is involved in the control of angiogenesis, oxidative stress, and cell death.
Increasing evidence points to inflammation as a key factor in the pathogenesis of DR, although the exact molecular mechanisms are not well understood. The simultaneous course of multiple metabolic pathways, such as oxidative stress, AGEs, and increased VEGF expression all likely contribute to the inflammatory response. Chronic low-grade inflammation is a key driver of capillary occlusion and hypoxia that reinforces VEGF expression and concomitant hallmark vascular abnormalities of DR.
Inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), IL-8 and IL-1 were significantly up-regulated in diabetic patients, and their expression level is correlated with the severity of DR.  Leukostasis has been associated with occlusion of retinal microvasculature and is correlated with endothelium damage and BRB impairment in diabetic rats, contributing to endothelial cell loss and breakdown of BRB. 
Retinal glial cell dysfunction is also presumed to be involved in inflammation in DR. Under hyperglycemic stress, microglia activation increases secretion of TNF-α, IL-6, MCP-1 and VEGF. Numerous studies show that inflammation inhibition by using anti-inflammatory drugs such as intravitreal triamcinolone acetonide and NSAIDs like nepafenac reduces VEGF expression and vascular permeability, inhibits retinal cell death, diminishes leukostasis, and ultimately improves visual acuity.  
Vascular abnormalities and angiogenesis pathways
Hyperglycemia causes pericyte loss, apoptosis of endothelial cells and thickening of the basement membrane, which collectively contribute to the impairment of the BRB. Since pericytes are responsible for providing structural support for capillaries, their loss leads to microaneurysm formation. Furthermore, pronounced loss of pericytes and endothelial cells results in capillary occlusion and ischemia. Retinal ischemia/hypoxia leads to upregulation of VEGF through activation of hypoxia-inducible factor 1 (HIF-1).
Neural retina cells are also affected in DR pathophysiology. In fact, retinal neurodegeneration is an early event during the progression of DR that may even precede vascular apoptosis. Upregulation of pro-apoptotic molecules has been detected in retinal neurons in diabetic animals and humans.   Oxidative stress seems to be involved in the activation of these pathways. Pro-apoptotic mitochondrial proteins such as cytochrome c and apoptosis-inducing factor (AIF) were also found to be significantly increased, implicating mitochondrial dysfunction in retinal degeneration In diabetic patients, inner retinal thinning was detected with no DR or minimal DR. This highlights the sensitivity of neuronal cell types to apoptotic stimuli such as oxidative stress and mitochondrial dysfunction. Therefore, neuroprotective agents may play a role in preventing retinal neurodegeneration in early stages of DR. Neuronal and vascular cells interact with each other to regulate blood flow in the retina via an autonomic independent mechanism. New evidence shows that this interaction is uncoupled in DR .
The pathophysiology of DR is fascinating and complex, with many mechanisms that need further study. DR treatment is an economic burden due to the number of patients affected and the cost of anti-VEGF therapies. Therefore, filling the gaps in the landscape of DR pathophysiology is of the utmost importance for a better understanding of the disease.
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