Ординатура / Офтальмология / Английские материалы / Retinal Vascular Disease_Joussen, Gardner, Kirchhof_2007

7.5Retinal Hypoxia and Diabetic Retinopathy

Essentials

Diabetic retinopathy (DR) is classified into two groups based on the presence of abnormal neovessels: non-proliferative and proliferative

Hypoxia plays a significant role in the pathogenesis of DR since early hyperoxia reverses aspects of vision loss

Growth factors such as VEGF, TNF-, MIF and endothelin and interleukins upregulate adhesion molecules in the endothelium and leukocytes, resulting in leukostasis, reduction in blood flow and direct leukocytemediated endothelial apoptosis involving the Fas/FasL pathway

AGEs and protein glycation downregulate free radical scavengers and upregulate nitric oxide and angiogenic growth factors contributing to cellular demise

Hypoxia and hyperglycemia activate the PCK Akt and MAPK pathways that in turn upregulate transcription factors such as NF-kB and HIF-1 that are central in the upregulation of growth factors and adhesion molecules that are central in the pathogenesis of DR

7.5.1 Introduction

Diabetes mellitus (DM) is the leading cause of blindness in people between the ages of 20 and 74 in the United States, and DR will eventually affect most Type I diabetics. Blindness is 25 times more common in patients with DM than in controls. DR is classified into two main groups: non-proliferative (mild, moderate, moderately severe and severe) and proliferative (mild, moderate and high risk) diabetic retinopathy. Non-proliferative diabetic retinopathy (NPDR) is characterized by increased vascular permeability, dilation and tortuosity of the retinal veins, abnormal vascular communications between arterioles and venules, microaneurysms, intraretinal hemorrhages and cotton wool spots (areas of infarction in the nerve layer). Microvascular angiopathy results in exudation of plasma from breakdown of the bloodretinal barrier. The reabsorption of the exuded fluid results in the deposition of protein and lipid exudates (“hard exudates”). Proliferative diabetic retinopathy (PDR) is marked by the formation of neovessels in the area of the optic disk (NVD) or elsewhere (NVE). Fifty percent of Type I and 10 % of Type II diabetics

who had DR for 15 years will have PDR, whereas the prevalence is higher in Type II patients who require I 7 insulin [61, 62].

7.5.2Pathophysiology of Diabetic Retinopathy

Although tissue hypoxia has been for many years suggested to play a critical role in the progression of diabetic retinopathy, the exact time when hypoxia begins has so far remained unknown. Many animal models fail to demonstrate the existence of retinal hypoxia early in the course of diabetic retinopathy, whereas we know that by the time capillary non-per- fusion is evident tissue hypoxia has already occurred [133]. The diabetic retina is hypoxic even when few microaneurysms are present and before capillary dropout is present [74]. It was also demonstrated that early hyperoxia reverses the contrast sensitivity deficits and oscillatory potential reductions, stressing the importance of hypoxia in the pathogenesis of the cardinal manifestations of DR [133].

It is now known that reduction in the retinal blood flow is one of the earlier signs of diabetic retinopathy. Leukostasis, the adhesion of leukocytes in the vascular endothelium, also occurs early and is at least partially responsible for the diminution of the blood flow. Leukocytes are found in higher numbers in diabetic individuals, they possess higher amounts of adhesion molecules (ICAM-1, VCAM-1) and they are less deformable than in normal individuals. The hyperglycemic and hyperosmotic environment in combination with the upregulated expression of multiple growth factors and cytokines such as VEGF, TNF- and interleukins contributes to the enhanced expression of the adhesion molecules in the vascular endothelium and the leukocytes [54, 63]. It is also suggested that large periods of dark adaptation aggravate tissue hypoxia by depriving the inner retina of the small amount of oxygen diffusing from the choroid during light adaptation [133].

Leukocyte adhesion to the vascular endothelium results in the endothelial cell injury and death that leads to vascular leakage, acellular capillaries and ultimately capillary dropout and retinal hypoxia [54]. This is a continuing vicious cycle since retinal hypoxia enhances even further the adhesion of the leukocytes to the endothelium and the endothelial damage. The endothelial cell death involves a Fas/ FasL mediated apoptotic cell death and is prevented by the administration of neutralizing FasL antibodies in a rat model of diabetic retinopathy [56]. Both Fas and FasL are upregulated in the endothelial cells and leukocytes respectively, during the course of diabetes from various cytokines such as interfer- on-, IL-4, TNF- and IL-1 [54]. The crosslinking

130 I Pathogenesis of Retinal Vascular Disease

7 I

HYPERGLYCEMIA

↑ leucostasis

Neovascularization and macular edema

Fig. 7.3. Pathophysiology of hyperglycemia-induced macular edema and neovascularization. High glucose levels upregulate growth factors such as VEGF through the activation of kinases such as Akt and transcription factors such as HIF-1 . Upregulated VEGF promotes neovascularization directly and macular edema indirectly through the endothelial and pericyte dysfunction, and the breakdown of the blood-retina barrier

of Fas from FasL initiates an apoptotic process involving the activation of apical caspases such as caspase-8, the activation of mitochondria that funnels into the upregulation and activation of executional caspases such as caspase-3 that cleaves nucleic acids and important cellular proteins resulting in the cellular demise.

Chronic changes in the retinal metabolic pathways are central in DR and add to the hypoxic damage. Hyperglycemia results in increased intracellular glucose, accumulation of sorbitol (an intermediate metabolite), an increased lactate/pyruvate ratio and disturbance of the redox balance that results in cell damage. Protein glycation and AGEs as well as the downregulation of genes that code for free-radical scavengers contribute to the oxidative stress that results in the cellular demise, and microand macroangiopathy, further aggravating the cycle of hypoxia

and the upregulation of growth factors such as VEGF and TNF- [138]. Hypoxia also activates local vascular loops that attempt to reverse the decreased vascular flow such as the stimulation of the adenosine receptor. This promotes the accumulation of superoxide radicals from the adenosine catabolism, which upregulates the nitric oxide synthase activity and ultimately affects gene expression of transcripts such as VEGF that reduce the retinal blood flow and contribute to the cellular damage [129].

Hypoxia and hyperglycemia activate the main players of multiple transduction pathways such as PKC and MAPK that contribute to the endothelial and pericyte dysfunction, the breakdown of the blood-retinal barrier and cellular death [139]. We have found that multiple activators of PKC such as diacylglycerol and PKC-binding proteins are upregulated early in diabetes whereas PKC inhibitors such

as 14 – 3-3 are downregulated, contributing to the upregulation of VEGF [53]. In parallel, members of the ras family are activated, mediating the MAPKinduced VEGF upregulation. Activation of hsp proteins suchas hsp27 and hsp60 is also a consequence of the oxidative stress induced by hypoxia, although hsp70 is downregulated, likely contributing to the leukocyte-mediated endothelial damage observed in the diabetic retina [53]. Hypoxia and the subsequent generation of free radicals also results in the upregulation of eNOS and the generation of NO that mediates through akt the enhanced expression of adhesion molecules like ICAM-1 and growth factors such as VEGF [55]. It is possible that growth factors, cytokines, and reactive oxygen intermediates produced by inflammatory cells upregulate the proapoptotic genes as poorly in the endothelial cells and pericytes and collaborate with ischemia and oxidative stress to induce apoptosis.

Central to the pathogenesis of diabetic retinopathy is the upregulation of growth factors such as VEGF that play a major role in all its cardinal manifestations. The transcription of the VEGF gene is very sensitive to oxygen changes and it is strongly responsive to the hypoxic environment through the upregulation of the transcription factor HIF-1. Insulin and IGF-I also upregulate VEGF during the course of diabetes via the orchestrated activation of HIF-1 through akt and NF-kB through MAPK that bind to adjacent responsive elements at the VEGF promoter [96]. Multiple proinflammatory cytokines are upregulated in the hypoxic environment of the diabetic retina. A few that will be mentioned are MIF, a lymphokine that enhances the adhesion of leukocytes in the vasculature, and endothelin B receptor [53]. Endothelin (ET), its ligand, is a potent vasoconstrictor and a permeability factor that works via a PKC-mediated mechanism and regulates extracellular matrix protein gene expression in target organs [22].

Hyperglycemia and hypoxia during diabetes result in the formation of vascular microaneurysms, venular dilatation, thickening of the retinal basement membrane, and microvascular contractile cell (pericyte) death, leading to acellular capillaries, which tend to undergo occlusion, causing retinal ischemia. “Cotton-wool” spots and soft exudates represent ischemic areas of the nerve-fiber retina layer. Platelet microthrombi can form, leading to capillary occlusion [13]. Hemorrhages and/or extravasation of fluid and retinal edema promote more hypoxia. Growth of new blood vessels in the retina in response to retinal hypoxia is the hallmark of proliferative DR.

Central and branch retinal vein occlusion (CRVO and BRVO, respectively) result in retinal ischemia, hypoxia and neovascularization. Scatter photocoagulation can be performed therapeutically to suppress fluid accumulation and limit preretinal and iris neovascularization [36]. Vitrectomy and retinal detachment procedures are occasionally required in patients with uncontrolled vitreous hemorrhage and retinal membrane formation, which threatens the integrity of the macula [99]. Scatter photocoagulation of these ischemic hypoxic areas restores the local retinal pO2 to normal values within 2 weeks [99]. It has been proposed that photocoagulation decreases oxygen consumption in the outer retina by destroying photoreceptors. The glial scar that develops in their place allows oxygen to diffuse from the choriocapillaris into the retina without being consumed in the mitochondria of the photoreceptors, thus increasing inner retina O2 availability. As a consequence of reversing hypoxia, VEGF production and neovascularization are suppressed. Vasoconstriction of existing vessels occurs, as well, due to elevated pO2, thus increasing arteriolar resistance, decreasing hydrostatic pressure in capillaries and venules and suppressing fluid extravasation and edema formation, which in turn facilitates O2 diffusion in the extracellular fluid and promotes O2 delivery to cells, further downregulating VEGF production [120]. Hence, the reported inhibitory effect of photocoagulation on the development of retinal neovascularization could be due to a reversal effect on tissue hypoxia [99]. Vitrectomy also improves retinal oxygenation by allowing oxygen and other nutrients to be transported in water currents in the vitreous cavity from well-perfused, well-oxygenated to ischemic areas of the retina [120].

VEGF is upregulated in the eyes of animal models of CRVO, as well as in humans with CRVO [94]. Aqueous VEGF concentrations correlate with the onset, persistence, and regression of neovascularization; extent of retinal capillary non-perfusion; and vascular permeability. Following treatment with laser ablation of the hypoxic retina, neovascularization regressed only if VEGF concentrations were successfully reduced. Thus, a high aqueous VEGF level may suggest the need for initial or repeat treatment [16]. Retinal vein occlusion in the rat retina induced upregulation of VEGF and basic fibroblast growth factor (bFGF) expression, overall protein tyrosinephosphorylation, as well as specific tyrosine-phos- phorylation of PLC, PI3K, and MAPK within the distribution of the occluded vein [45]. BRVO increases the concentration of another HIF-depen-

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I Pathogenesis of Retinal Vascular Disease

dent factor, erythropoietin, in the vitreous fluid, which may act as an endogenous neuroprotective factor against ischemic retinal disorders [48]. On the other hand, pigment epithelium-derived factor (PEDF), a major antiangiogenic growth factor in the eye, is downregulated by hypoxia [27]. The PEDF concentrations in ocular fluids from patients with extensive non-diabetic retinal neovascularization caused by retinal vein occlusion were lower than in control patients. Levels of PEDF were restored in patients with retinal scatter photocoagulation compared with patients without previous photocoagulation [118]. These findings suggest that substitution of angiogenesis inhibitors may be an effective approach in the treatment of PDR.

Inhibition of the VEGF pathway, by either an antisense oligodeoxynucleotide against VEGF or an antiVEGF antibody, delivered to the retina via intravitreal injection, has been demonstrated to reduce ocular angiogenesis in a non-human primate model of retinal vein occlusion [11, 1], suggesting that anti-VEGF therapy at an early stage of ischemic retinal vein occlusion may be therapeutically beneficial. Similarly, adenovirus-mediated VHL intraocular gene transfer inhibited the development of angiogenesis in a monkey model of multiple BRVO [2], confirming that the HIF/VHL pathway is crucial in the upregulation of VEGF and induction of ocular neovascularization in this condition and suggesting that gene therapy based on VHL gene delivery has potential in the treatment of human ocular neovascularization.

7.7 Other Diseases

7.7.1 Cystoid Macular Edema

Essentials

Oxygen supplementation reduces foveal thickness in macular edema, likely reducing hypoxic damage

Oxygen plays a pathogenetic role in other diseases also. It was recently shown that supplemental oxygen with a nasal cannula reduced foveal thickness by an average of 42.5 % in patients with cystoid macular edema [89]. Although the pathophysiology behind this finding is unclear, it is known that oxygen can exert its beneficial actions independently of the atmospheric pressure. It is possible that improved oxygenation breaks the cycle of hypoxia and edema in the diabetic retina, a hypothesis that is supported by the worsening of the edema after the discontinuation of the oxygen laser in all the eyes except the ones that received additional focal laser therapy. A combi-

nation of laser treatment with supplemental oxygenation can be argued for the refractory cases of cystoid macular edema. In these cases supplemental oxygenation can reduce macular thickness below a critical point so that the benefits of focal laser photocoagulation may be realized.

7.7.2 Retinal Degeneration

Essentials

Rod loss that characterizes retinal degeneration increases oxygen concentration in the outer retina likely resulting in caspase independent oxidative cone loss that activates the mitochondria in later stages and is not associated with DNA fragmentation

Increased inner retina oxygen concentration results in increased oxygen diffusion in the outer retina with subsequent capillary loss and cellular damage

Reduction of oxygen flow in animal models of retinal degeneration prevents hypoxic damage

Oxygen is also central to the pathogenesis of the retinal vasculature obliteration that accompanies photoreceptor loss in retinal degenerative diseases. It is believed that when the photoreceptors die oxygen diffuses freely from the choroid to the inner retina causing vasoconstriction of the major vessels and capillary loss. Reduction of the oxygen influx in animal models of retinal degenerative diseases when they are maintained under hypoxic conditions prevents this capillary loss [95].

Because choroidal levels do not have autoregulatory mechanisms controlled by oxygen levels, oxygen levels in the outer retina vary according to oxygen consumption. Therefore one could expect that during the course of retinitis pigmentosa (RP) as the rods die and the oxygen consumption in a particular retinal area decreases, the oxygen levels increase. It was indeed shown in transgenic rat models of RP that the oxygen levels in the outer retina increase significantly as the rods degenerate and decrease in number [141].

Since it is known that high levels of oxygen are damaging to the photoreceptors, it was hypothesized that cone death in retinal degenerations could represent a form of oxidative damage [122, 126]. It was recently shown in a transgenic rhodopsin pig model of RP that after the rods degenerate the cones show evidence of oxidative damage by expressing high amounts of biomarkers for oxidative damage to lipids proteins and DNA. From the above we can naturally

assume that retinal areas that have the highest concentration of rod photoreceptors such as the periphery would be affected first in retinal degenerations, whereas areas with high cone concentrations such as the macula would be spared; that is indeed the case.

Oxidative stress results in the generation of reactive oxygen species (ROS) that leads to the oxidative inactivation of the caspases [59, 134]. It seems that the oxidative damage has the key elements of an apoptotic cell death but is not dependent on caspase activation. In vitro and in animal models of RP this form of oxidative retinal cell apoptosis is not accompanied by caspase activation and is not inhibited by caspase inhibitors [30, 142]. One of the earliest events during ROS-induced photoreceptor death is the exposure of phosphatidylserine (PS) in the outer surface of the cellular membrane. Although PS exposure is traditionally a caspase dependent process, in this case it seems that it follows alternative regulatory mechanisms mainly controlled by ROS. Although in traditional forms of apoptosis the mitochondria are activated early and act as a positive loop in the activation of caspases, in this caspase independent oxidative death it seems that mitochondrial depolarization is a late step. This apoptotic death is not associated with low molecular weight DNA fragmentation at least in vitro that is a caspase dependent process and could involve other endonucleases. It is interesting to hypothesize that wide variability in the clinical phenotype of these diseases can be attributed to polymorphisms in key antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and metallothionines.

7.7.3 Glaucoma

Essentials

Hyperbaric oxygen treatment improves visual field outcomes in glaucoma without affecting the intraocular pressure (IOP)

Glaucoma is associated with vascular abnormalities such as vasospasm and hypotension that could be associated with altered microcirculation in the central retinal artery and hypoxic injury to the ganglion or supporting cells

Glutamate, reactive oxygen species, nitric oxide, calcium and TNF- are among the proposed mediators of the hypoxic damage HIF-1 is upregulated in animal models of glaucoma with differential localization and expression among the retinal cell subtypes that could be translated to differential sensitivity to glaucomatous damage

Growing evidence supports a vascular hypothesis in

the pathogenesis of glaucoma. Patients with glauco- I 7 ma treated with hyperbaric oxygen, a treatment that

does not interfere with IOP regulation, demonstrated improved visual field outcomes in relation to controls [15]. Retinal photocoagulation in an animal model of glaucoma with subsequent increase in the inner retinal oxygen distribution results in increased ganglion cell survival [91]. Vascular abnormalities that occur in glaucoma patients such as vasospasm, hypotension, and various vascular perfusion defects proven by angiographic studies lead investigators to believe that reduced vascular perfusion in the optic nerve can be one of the pathogenetic mechanisms in glaucoma. The altered microcirculation in the central retinal artery can be secondary to the IOP or secondary to abnormal blood pressure, vasospasm, hemorrhage, autoregulation, increased viscosity or altered rheological characteristics of the blood. The hypoxic/oligemic insult results then in decreased oxygen supply that can contribute to the ganglion cell death in patients with glaucoma. The hypoxic insult can also result in the death of astrocytes and supporting microglia and ganglion cell axons that can account for the cupping associated with glaucoma before the ganglion cell loss. It was hypothesized that the oligemic/hypoxic insult results in astrocytic injury that is propagated among various cell types (Müller, microglia) in the form of depolarization that is called spreading depression where various ions such as calcium and potassium and glutamate play a role [73]. The dysfunctioning astrocytes can then secrete various substances such as nitric oxide, prostaglandins, and D-serine that could exacerbate the injury to the ganglion cells and the supporting glial cells. Additionally the lamina cribrosa of the optic nerve is considered a transitional zone between an efficient (myelinated) and inefficient (non-myelinat- ed) system of action potential transmission and therefore is more susceptible to hypoxic injuries. That would lead to glutamate “leakage” from the ganglion cells and eventually increase its extracellular levels when the Müller cells’ capacity to remove it from the extracellular space is saturated. The increased levels of glutamate would differentially injure the ganglion cells according to their excitatory receptor profile [31].

Many mediators and molecular pathways have been proposed to mediate the hypoxic damage such as reactive oxygen species, glutamate excitotoxicity, nitric oxide mediated oxidative stress [88], calcium toxicity [65], and tumor necrosis factor-mediated apoptosis [124]. In murine models of glaucoma, HIF-1 was shown to be expressed in higher levels compared to normal controls and exhibit a spatial relationship with the functional damage that is

found in these eyes. HIF-1 immunoreactivity is 7 I higher in the inner retinal layers, giving rise to the hypothesis that ischemic axons in the optic nerve head initiate a retrograde signal to their cell bodies, upregulating this transcriptional factor [125]. Although the most prominent increase occurs in the inner retinal layer, one cannot exclude the possibility that all layers are hypoxic and this response is the result of a differential regulation of HIF-1. It is also intriguing that HIF-1 immunolocalization is cytoplasmic in the retinal ganglion cells and nuclear in the glial cells probably due to a differential activity of the regulatory signaling cascades in the different subtypes of the retinal cells. Differential HIF-1 expression could explain the differential sensitivity to the glaucomatous damage. It is still unclear what are the molecular pathways leading to the HIF-1 upregulation although nitric oxide, cytokines such as TNF- and reactive oxygen species have been implicated. One of the apoptotic genes that HIF-1 activates and is implicated in the glaucomatous and excitotoxic damage is p53 [34]. It is possible that HIF-1 initiates a cell death program in glaucomatous eyes through p53, which could be a transcription-

al activator of neuronal apoptosis.

7.7.4 Retinal Detachment

Essentials

Hyperoxia has a protective effect in animal models of retinal detachment by restoring the influx of oxygen from the choroids that is separated from the outer retina

Hypoxia may also be a contributing factor in vision loss in retinal detachment. It has been recently hypothesized that hyperoxia may be beneficial in preventing photoreceptor damage in retinal detachment [80, 106]. Detachment separates the inner segments from their O2 supply, and although no consuming tissue is found under the retina, the increased distance reduces the flux of O2 from the choroid to the photoreceptor inner segments. Hyperoxia should restore this flux, at least for detachments of moderate height, because increased choroidal pO2 can compensate for the increased distance. For large detachments, the photoreceptors may benefit more from increased amounts of O2 in the retinal circulation than in the choroidal circulation. The protective effect of hyperoxia on detached photoreceptors has been shown experimentally in cats, in which hyperoxia was able to save photoreceptors and prevent the activation of retinal glia normally caused by detachment.

7.8 Conclusions

Retinal hypoxia occurs in a diverse spectrum of ophthalmological disorders and is a potent stimulus for neovascularization. As visual acuity depends on the transparency of the eye structures, abnormal neovascularization is vision threatening. The VHL/HIF pathway is a pivotal regulator of the response to hypoxia and VEGF is its main effector cytokine. Pharmacological suppression of the VHL/HIF/VEGF could be an important advance for the treatment of such neovascularization and the preservation of vision.

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