Ординатура / Офтальмология / Английские материалы / Diabetes and Ocular Disease Past, Present, and Future Therapies 2nd edition_Scott, Flynn, Smiddy_2009

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Figure 20.4. Correlation of Vascular endothelial growth factor (VEGF) expression and ischemic retinal neovascularization in the mouse. When neonatal mice are exposed to alterations in oxygen concentration for several days, the normal vascularization pattern of the retina (A) is altered resulting in areas of nonperfusion (B, dark central areas) and retinal neovascularization (arrows) that closely resemble those observed in diabetic retinopathy. The production of VEGF is low under normal conditions (C) and markedly increased just prior to the onset of retinal neovascularization (D). (A and B) are retinal flat mounts from neonatal mice whose vasculature has been perfused with a fluorescein-conjugated dextran for visualization purposes. (C and D) are cross sectional in situ hybridization photomicrographs showing location of VEGF production. (Source: Adapted from Pierce et al. [65], with permission.)

concentrations of VEGF well below those found in eyes with active PDR (Fig. 20.7) [55]. Repetitive intravitreal injections of recombinant human VEGF are sufficient to produce iris neovascularization in a nonhuman primate leading to ectropion uveae and neovascular glaucoma (Fig. 20.8) [79]. Similarly, transgenic mice that overexpress VEGF in the photoreceptors develop extensive intraretinal neovascularization as confirmed by light, confocal and standard fluorescent microscopy (Fig. 20.9) [80,81]. Interestingly, the vessels originate from the retinal vasculature and grow toward the VEGF-producing photoreceptor layer, a morphology that is inverted compared to that observed in diabetic retinopathy.

Although increased permeability can occur in the absence of neovascularization as is often observed with diabetic macular edema (Fig. 20.1D), a universal characteristic of retinal proliferation is a corresponding increase in vascular permeability. VEGF is a very effective inducer of permeability, being 50,000 times more potent in the dermal microvasculature than is histamine in this regard [82]. In the eye, extravasated albumin and VEGF immunoreactivity co-localize [83,84]. Repeated injections of high concentrations of VEGF result in leakage of fluorescein dye from

Figure 20.9. Transgenic expression of Vascular endothelial growth factor (VEGF) in the photoreceptors produces intraretinal neovascularization. Transgenic mice over-expressing VEGF in the photoreceptors demonstrated marked intraretinal neovascularization (arrows) that appeared to be proliferating toward the site of VEGF expression in the outer retina. (Source: Adapted from Okamoto et al. [81], with permission.)

tissue isolation can have significant effects. In contrast, an immunohistochemical evaluation of postmortem human eyes with NPDR, but without extensive retinal nonperfusion, demonstrated increased VEGF expression as compared with nondiabetic controls (Fig. 20.11) [90,91]. Repeated injections of high concentrations of VEGF into the normal nonhuman primate eye produce retinal changes resembling NPDR including vascular tortuosity, capillary abnormalities resembling microaneurysms, and leakage of fluorescein (Fig. 20.12) [85]. Intravitreal injections of physiologic concentrations of VEGF in rats alter retinal blood flow and venous caliber in the same manner as observed in diabetic patients with increasingly severe diabetic retinopathy [92]. Furthermore, diabetes accentuates the retina’s response to VEGF as compared with nondiabetic animals. As shown in Figure 20.13, these data suggest that, even early in the course of diabetes, the retina may have both increased expression as well as an accentuated response to VEGF. Such expression and response could theoretically result in a positive feedback loop that might eventually induce enough retinal ischemia and VEGF expression to stimulate intraocular neovascularization. This hypothesis raises the intriguing possibility that inhibitors of VEGF action might prove beneficial not only for the neovascular and permeability complications of diabetes, but also as a prevention of retinopathy progression in the nonproliferative stages.

VEGF AS CAUSAL MEDIATOR OF ISCHEMIA-INDUCED

RETINAL NEOVASCULARIZATION

Direct evidence that VEGF expression is necessary for ischemia-induced retinal and iris neovascularization in animals has been obtained using multiple different agents that inhibit VEGF. These originally included VEGF receptor chimeric proteins [93], neutralizing antibodies [94], and antisense phosphorothioate

Figure 20.10. Vascular endothelial growth factor (VEGF) induces retinal vascular permeability. The ability of intravitreal injections of VEGF to induce retinal vascular permeability in rats was evaluated utilizing vitreous fluorophotometry (A). A dose-dependent five-fold increase in retinal vascular permeability was evident with physiologic concentrations of VEGF that were consistent with those observed in patients with active proliferative diabetic retinopathy (Fig. 20.5). The retinal vasculature was also perfused with a fluorescein-conjugated dextran approximately the size of albumin that is retained within the lumen of normal vessels. The normal retinal vessel architecture of an animal that received a control intravitreal injection is shown in

(B). Note that the fluorescence is primarily retained within the vasculature. However, as shown in (C), intravitreal VEGF injection induced a readily apparent increase in vessel permeability to the fluorescent compound. (Source: Adapted from Aiello et al. [86], with permission.)

oligodeoxynucleotides [95]. These VEGF inhibitors suppressed ischemia-induced intraocular neovascularization by up to 77% in up to 100% of animals studied. The average magnitude of inhibition was approximately 50%. Similar results were obtained for iris neovascularization in primates [94] (Fig. 20.14A and 20.14B) and retinal neovascularization in mice (Fig. 20.14B and 20.14C) [93]. No toxicity

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Figure 20.11. Vascular endothelial growth factor (VEGF) expression is increased in patients with nonproliferative diabetic retinopathy and minimal retinal ischemia. Immunohistochemical evaluation of VEGF protein was performed in patients with nonproliferative diabetic retinopathy without extensive areas of retinal nonprofusion. Increased VEGF expression (arrows) was observed in the periphery (A) and the macula (B). (Source: Adapted from Amin et al. [91], with permission.)

was evident by light microscopic evaluation in these relatively short duration studies. As discussed earlier, VEGF over-expression in the photoreceptors of a transgenic mouse was sufficient to produce extensive retinal neovascularization (Fig. 20.9) [80,81]. These data demonstrate that, although the neovascular response is undoubtedly modulated by a wide variety of factors, VEGF appears necessary and sufficient to induce retinal and iris angiogenesis, particularly as a sequelae of the retinal ischemia characteristic of diabetic retinopathy. In addition, these findings strongly suggest that any agent that blocks VEGF action may result in a significant, although perhaps not a complete, reduction in intraocular neovascularization. However, early clinical studies as discussed below show remarkable sensitivity of PDR to anti-VEGF molecules with near-total resolution of neovascularization within 1 week of treatment [96–98]. In contrast, clinical impression is that the response of macular edema to anti-VEGF treatment may not be as sensitive or as complete.

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Figure 20.12. Intravitreal injection of Vascular endothelial growth factor (VEGF) into the nonhuman primate induces retinal changes resembling nonproliferative diabetic retinopathy. Repetitive intravitreal injections of VEGF into the normal primate eye resulted in vascular tortuosity and capillary abnormalities resembling microaneurysms (A). Increased VEGF dose resulted in capillary nonperfusion and retinal vascular leakage of fluorescein (B). (Source: Adapted from Tolentino et al. [85], with permission.)

BASIC MECHANISMS AND TARGETS IN DIABETIC RETINOPATHY

The detailed biochemical mechanisms that underlie the intracellular processes permitting VEGF expression and signaling are becoming better understood. One important area, from a potential therapeutic standpoint, is the mechanism by which hypoxia increases VEGF expression. The endogenous nucleoside adenosine appears to serve an important role in this regard (Fig. 20.15) [99–103]. As demonstrated in Figure 20.15, hypoxia increases adenosine concentrations severalfold [99,101,102] by inhibiting an enzyme (adenosine kinase) that usually converts adenosine to adenosine monophospate (AMP) [104]. In retinal endothelial cells, the specific adenosine receptors that mediate the induction of VEGF expression are known. In addition, several of the molecules involved in the intracellular signaling of the adenosine stimulus have been identified and include adenyl cyclase and protein kinase A [103]. Adenosine receptors also work in concert with the VEGF receptor to increase endothelial cell migration and vessel formation [102].

Retinal blood flow

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Figure 20.13. Theoretical mechanism by which Vascular endothelial growth factor (VEGF) may mediate the progression of nonproliferative diabetic retinopathy. In early diabetes, molecules such as ET-1 and NO reduce retinal blood flow. This combined with oxidative stress may produce an initial hypoxic stimulus for VEGF expression. The diabetic state further enhances retinal VEGF sensitivity, inducing the vascular abnormalities characteristic of nonproliferative diabetic retinopathy. A positive feedback loop occurs as the development of ischemic areas creates localized hypoxia and further stimulates VEGF production. The down-regulation of ET-1 and nitric oxide by VEGF further increases retinal blood flow as retinopathy advances. Once vascular damage results in extensive retinal ischemia, VEGF concentrations become high enough to induce intraocular neovascularization. ET-1, endothelin 1; NO, nitric oxide; RBF, retinal blood flow. (Source: Adapted from Clermont et al. [92], with permission.)

Thus, inhibition of adenosine or its receptors would be expected to suppress VEGF expression under hypoxic conditions and decrease subsequent vasculogenesis. Inhibitors of adenosine receptors do indeed have this action in cell culture suggesting that they might prove useful in the treatment of diabetic retinopathy [100–103]; however, more study is required to determine the actual clinical applicability of these agents.

Basic FGF (bFGF) was studied extensively as a probable mediator of angiogenesis in diabetic retinopathy until the transgenic mouse data discussed earlier made it unlikely that the induction of neovascularization is its primary role [31]. It should be noted, however, that the mitogenic actions of bFGF and VEGF are potently synergistic both in vivo [32] and in vitro [33,34]. The mechanism of this synergy has been partially elucidated. As shown in Figure 20.16, bFGF increases VEGF [105,106] and VEGF receptor 2 expression (kinase domain receptor [KDR], VEGFR2) [107]. VEGF activity is closely correlated with cellular KDR expression. Even under conditions where KDR expression is low and VEGF’s stimulatory activity is minimal, bFGF dramatically increases KDR expression subsequently

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Figure 20.14. Inhibition of Vascular endothelial growth factor (VEGF) suppresses retinal ischemia-induced iris and retinal neovascularization. Retinal ischemia in the primate characteristically produces iris neovascularization while similar ischemia in the neonatal mouse produces retinal neovascularization. VEGF neutralizing antibodies injected into the vitreous of primates with retinal ischemia produced by laser-induced retinal vein occlusion resulted in marked suppression of the iris neovascularization (A, normal yellow iris color) that is normally observed in eyes not receiving the inhibitor (B, abnormal red iris color). Similarly, a VEGF chimeric receptor protein, which binds to VEGF and inhibits its action, was injected intravitreally into neonatal mice with retinal ischemia. These animals universally develop retinal neovascularization in the absence of VEGF inhibition (C, arrows). However, intravitreal injection of the VEGF receptor chimeric protein reduced retinal neovascularization as shown here in the contralateral eye of the same animal (D). (Source: A and B adapted from Adamis et al. [94], C and D adapted from Aiello et al. [93]; with permission.)

allowing VEGF to efficiently induce both mitogenesis and further KDR expression. Basic FGF’s induction of VEGF receptor expression requires activation of PKC and MAP kinase. VEGF also increases both thrombin [108] and plasminogen activator expression [109], which can release bioactive bFGF from the extracellular matrix and further potentiate the response [110]. These data demonstrate that the VEGF receptor KDR is a critical regulating component of the VEGF pathway and suggest that compounds that inhibit its function or reduce its expression are likely to be effective inhibitors of neovascularization associated with diabetes. Indeed, this approach has already been proven successful in animals by suppressing angiogenesis, endothelial cell proliferation, tumor growth, tumor metastasis and cancerassociated mortality [95,111–117].