Endogenous insulin signaling in the RPE contributes to the maintenance of rod photoreceptor function in diabetes.

In diabetes, there are two major physiological aberrations: (i) Loss of insulin signaling due to absence of insulin (type 1 diabetes) or insulin resistance (type 2 diabetes) and (ii) increased blood glucose levels. The retina has a high proclivity to damage following diabetes, and much of the pathology seen in diabetic retinopathy has been ascribed to hyperglycemia and downstream cascades activated by increased blood glucose. However, less attention has been focused on the direct role of insulin on retinal physiology, likely due to the fact that uptake of glucose in retinal cells is not insulin-dependent. The retinal pigment epithelium (RPE) is instrumental in maintaining the structural and functional integrity of the retina. Recent studies have suggested that RPE dysfunction is a precursor of, and contributes to, the development of diabetic retinopathy. To evaluate the role of insulin on RPE cell function directly, we generated a RPE specific insulin receptor (IR) knockout (RPEIRKO) mouse using the Cre-loxP system. Using this mouse, we sought to determine the impact of insulin-mediated signaling in the RPE on retinal function under physiological control conditions as well as in streptozotocin (STZ)-induced diabetes. We demonstrate that loss of RPE-specific IR expression resulted in lower a- and b-wave electroretinogram amplitudes in diabetic mice as compared to diabetic mice that expressed IR on the RPE. Interestingly, RPEIRKO mice did not exhibit significant differences in the amplitude of the RPE-dependent electroretinogram c-wave as compared to diabetic controls. However, loss of IR-mediated signaling in the RPE reduced levels of reactive oxygen species and the expression of pro-inflammatory cytokines in the retina of diabetic mice. These results imply that IR-mediated signaling in the RPE regulates photoreceptor function and may play a role in the generation of oxidative stress and inflammation in the retina in diabetes.

Inhibition of EGF signaling protects the diabetic retina from insulin-induced vascular leakage.

Diabetes mellitus is a disease with considerable morbidity and mortality worldwide. Breakdown of the blood-retinal barrier and leakage from the retinal vasculature leads to diabetic macular edema, an important cause of vision loss in patients with diabetes. Although epidemiologic studies and randomized clinical trials suggest that glycemic control plays a major role in the development of vascular complications of diabetes, insulin therapies for control of glucose metabolism cannot prevent long-term retinal complications. The phenomenon of temporary paradoxical worsening of diabetic macular edema after insulin treatment has been observed in a number of studies. In prospective studies on non-insulin-dependent (type 2) diabetes mellitus patients, a change in treatment from oral drugs to insulin was often associated with a significant increased risk of retinopathy progression and visual impairment. Although insulin therapies are critical for regulation of the metabolic disease, their role in the retina is controversial. In this study with diabetic mice, insulin treatment resulted in increased vascular leakage apparently mediated by betacellulin and signaling via the epidermal growth factor (EGF) receptor. In addition, treatment with EGF receptor inhibitors reduced retinal vascular leakage in diabetic mice on insulin. These findings provide unique insight into the role of insulin signaling in mediating retinal effects in diabetes and open new avenues for therapeutics to treat the retinal complications of diabetes mellitus.

Cross-talk between vascular endothelial growth factor and matrix metalloproteinases in the induction of neovascularization in vivo.

Matrix metalloproteinases (MMPs), a specialized group of enzymes capable of proteolytically degrading extracellular matrix proteins, have been postulated to play an important role in angiogenesis. It has been suggested that MMPs can regulate neovascularization using mechanisms other than simple remodeling of the capillary basement membrane. To determine the interplay between vascular endothelial growth factor (VEGF) and MMPs, we investigated the induction of angiogenesis by recombinant active MMPs and VEGF in vivo. Using a rat corneal micropocket in vivo angiogenesis assay, we observed that the active form of MMP-9 could induce neovascularization in vivo when compared with the pro- form of the enzyme as a control. This angiogenic response could be inhibited by neutralizing VEGF antibody, which suggests that MMPs acts upstream of VEGF. Additional in vitro studies using extracellular matrix loaded with radiolabeled VEGF determined that active MMPs can enzymatically release sequestered VEGF. Interestingly, in vivo angiogenesis induced by VEGF could be inhibited by MMP inhibitors, indicating that MMPs also act downstream of VEGF. In addition, inflammation plays an important role in the induction of angiogenesis mediated by both VEGF and MMPs. Our results suggest that MMPs act both upstream and downstream of VEGF and imply that potential combination therapies of VEGF and MMP inhibitors may be a useful therapeutic approach in diseases of pathological neovascularization.

Impaired function of circulating CD34(+) CD45(-) cells in patients with proliferative diabetic retinopathy.

Proliferative diabetic retinopathy is a consequence of retinal ischemia due to capillary occlusion resulting from damage to the retinal microvascular endothelium. Recent evidence suggests that high levels of bone-marrow derived circulating endothelial progenitor cells (EPCs) contribute to the pathological neovascularization of ischemic tissues and are a critical risk factor for the development of these complications. In the absence of a consensus definition of a circulating EPC and its surface markers in humans we evaluated the functional properties of CD34(+) CD45(-) endothelial colony forming cells (ECFCs) in patients with proliferative diabetic retinopathy (PDR). Higher levels of circulating CD34(+) CD45(-) cells were observed in patients with PDR compared to controls. However, ECFCs from patients with PDR were impaired in their ability to migrate towards SDF-1 and human serum, incorporate into and form vascular tubes with human retinal endothelial cells. The results from these pilot studies suggest that ECFCs from patients with PDR are mobilized into the circulation but may be unable to migrate and repair damaged capillary endothelium. This suggests that ECFCs may be a potential therapeutic target in the prevention and treatment of diabetic vascular complications.

Role of FGF and Hyaluronan in Choroidal Neovascularization in Sorsby Fundus Dystrophy.

Sorsby's fundus dystrophy (SFD) is an inherited blinding disorder caused by mutations in the tissue inhibitor of metalloproteinase-3 (TIMP3) gene. The SFD pathology of macular degeneration with subretinal deposits and choroidal neovascularization (CNV) closely resembles that of the more common age-related macular degeneration (AMD). The objective of this study was to gain further insight into the molecular mechanism(s) by which mutant TIMP3 induces CNV. In this study we demonstrate that hyaluronan (HA), a large glycosaminoglycan, is elevated in the plasma and retinal pigment epithelium (RPE)/choroid of patients with AMD. Mice carrying the S179C-TIMP3 mutation also showed increased plasma levels of HA as well as accumulation of HA around the RPE in the retina. Human RPE cells expressing the S179C-TIMP3 mutation accumulated HA apically, intracellularly and basally when cultured long-term compared with cells expressing wildtype TIMP3. We recently reported that RPE cells carrying the S179C-TIMP3 mutation have the propensity to induce angiogenesis via basic fibroblast growth factor (FGF-2). We now demonstrate that FGF-2 induces accumulation of HA in RPE cells. These results suggest that the TIMP3-MMP-FGF-2-HA axis may have an important role in the pathogenesis of CNV in SFD and possibly AMD.

Changes in VEGF-related factors are associated with presence of inflammatory factors in carbohydrate metabolism disorders during pregnancy.

The aim of this study was to determine the action of molecules in carbohydrate metabolism disorders during pregnancy. The concentration of different types of cytokines and vascular endothelial growth factor (VEGF) in the plasma were measured in 4 groups of women: Group I, normal pregnancy (n = 10); Group II, patients with gestational DM (n = 12); Group III, pregnant patients with preexisting DM (n = 16); and Group IV, diabetic non-pregnant women (n = 22). The plasma VEGF concentration was significantly higher in the women in Group IV than in other groups (P <0.01). The concentration of the soluble form of the VEGF receptor-1 (sVEGFR-1) was significantly higher in Group I than in other groups (P <0.01). The concentration of soluble form of the VEGF receptor-2 (sVEGFR-2) was significantly lower in Groups I than in other groups (P <0.05). The concentrations of monocyte chemotactic protein-1 (MCP-1) and eotaxin were significantly lower in Group I than in Groups III and IV. The levels of interleukin (IL)-8, IL-6, and tumor necrosis factor-α (TNF-α) were significantly higher in Group I than in Group IV. Both the VEGF-related molecules and the Inflammatory cytokines are altered in pregnant women with the carbohydrate metabolism disorders.

Sorsby Fundus Dystrophy Mutation in Tissue Inhibitor of Metalloproteinase 3 (TIMP3) promotes Choroidal Neovascularization via a Fibroblast Growth Factor-dependent Mechanism.

Choroidal neovascularization (CNV) leads to loss of vision in patients with Sorsby Fundus Dystrophy (SFD), an inherited, macular degenerative disorder, caused by mutations in the Tissue Inhibitor of Metalloproteinase-3 (TIMP3) gene. SFD closely resembles age-related macular degeneration (AMD), which is the leading cause of blindness in the elderly population of the Western hemisphere. Variants in TIMP3 gene have recently been identified in patients with AMD. A majority of patients with AMD also lose vision as a consequence of choroidal neovascularization (CNV). Thus, understanding the molecular mechanisms that contribute to CNV as a consequence of TIMP-3 mutations will provide insight into the pathophysiology in SFD and likely the neovascular component of the more commonly seen AMD. While the role of VEGF in CNV has been studied extensively, it is becoming increasingly clear that other factors likely play a significant role. The objective of this study was to test the hypothesis that basic Fibroblast Growth Factor (bFGF) regulates SFD-related CNV. In this study we demonstrate that mice expressing mutant TIMP3 (Timp3S179C/S179C) showed reduced MMP inhibitory activity with an increase in MMP2 activity and bFGF levels, as well as accentuated CNV leakage when subjected to laser injury. S179C mutant-TIMP3 in retinal pigment epithelial (RPE) cells showed increased secretion of bFGF and conditioned medium from these cells induced increased angiogenesis in endothelial cells. These studies suggest that S179C-TIMP3 may promote angiogenesis and CNV via a FGFR-1-dependent pathway by increasing bFGF release and activity.

Tissue inhibitor of metalloproteinases-3 peptides inhibit angiogenesis and choroidal neovascularization in mice.

Tissue inhibitors of metalloproteinases (TIMPs) while originally characterized as inhibitors of matrix metalloproteinases (MMPs) have recently been shown to have a wide range of functions that are independent of their MMP inhibitory properties. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a potent inhibitor of VEGF-mediated angiogenesis and neovascularization through its ability to block the binding of VEGF to its receptor VEGFR-2. To identify and characterize the anti-angiogenic domain of TIMP-3, structure function analyses and synthetic peptide studies were performed using VEGF-mediated receptor binding, signaling, migration and proliferation. In addition, the ability of TIMP-3 peptides to inhibit CNV in a mouse model was evaluated. We demonstrate that the anti-angiogenic property resides in the COOH-terminal domain of TIMP-3 protein which can block the binding of VEGF specifically to its receptor VEGFR-2, but not to VEGFR-1 similar to the full-length wild-type protein. Synthetic peptides corresponding to putative loop 6 and tail region of TIMP-3 have anti-angiogenic properties as determined by inhibition of VEGF binding to VEGFR-2, VEGF-induced phosphorylation of VEGFR-2 and downstream signaling pathways as well as endothelial cell proliferation and migration in response to VEGF. In addition, we show that intravitreal administration of TIMP-3 peptide could inhibit the size of laser-induced choroidal neovascularization lesions in mice. Thus, we have identified TIMP-3 peptides to be efficient inhibitors of angiogenesis and have a potential to be used therapeutically in diseases with increased neovascularization.

Increased neovascularization in mice lacking tissue inhibitor of metalloproteinases-3.

PURPOSE: Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a matrix-bound inhibitor of matrix metalloproteinases (MMPs). The authors have previously determined a novel function of TIMP-3 to inhibit vascular endothelial growth factor (VEGF)-mediated angiogenesis. Here, the authors examined the in vivo angiogenic phenotype of ocular vessels in mice deficient in TIMP-3. METHODS: VEGF-mediated corneal neovascularization and laser-induced choroidal neovascularization (CNV) were examined in TIMP-3-null mice. The effects of the absence of TIMP-3 on the phosphorylation status of the VEGF-receptor-2 (VEGFR-2) and the downstream signaling pathways were evaluated biochemically. In addition, the activation state of MMPs in the retina of TIMP-3-deficient mice was examined by in situ zymography. RESULTS: The results of these studies determine an accentuation of pathologic VEGF-mediated angiogenesis in the cornea and laser-induced CNV in mice lacking TIMP-3. In the absence of the MMP inhibitor, pathophysiological changes were observed in the choroidal vasculature concomitantly with an increase in gelatinolytic activity. These results suggest that an imbalance of extracellular matrix homeostasis, together with a loss of an angiogenesis inhibitor, can prime vascular beds to be more responsive to an angiogenic stimulus. CONCLUSIONS: In light of the recent studies suggesting that genetic variants near TIMP-3 influence susceptibility to age-related macular degeneration, these results imply that TIMP-3 may regulate the development of the choroidal vasculature and is a likely contributor to increased susceptibility to choroidal neovascularization.

Betacellulin induces increased retinal vascular permeability in mice.

BACKGROUND: Diabetic maculopathy, the leading cause of vision loss in patients with type 2 diabetes, is characterized by hyper-permeability of retinal blood vessels with subsequent formation of macular edema and hard exudates. The degree of hyperglycemia and duration of diabetes have been suggested to be good predictors of retinal complications. Intervention studies have determined that while intensive treatment of diabetes reduced the development of proliferative diabetic retinopathy it was associated with a two to three-fold increased risk of severe hypoglycemia. Thus we hypothesized the need to identify downstream glycemic targets, which induce retinal vascular permeability that could be targeted therapeutically without the additional risks associated with intensive treatment of the hyperglycemia. Betacellulin is a 32 kD member of the epidermal growth factor family with mitogenic properties for the retinal pigment epithelial cells. This led us to hypothesize a role for betacellulin in the retinal vascular complications associated with diabetes. METHODS AND FINDINGS: In this study, using a mouse model of diabetes, we demonstrate that diabetic mice have accentuated retinal vascular permeability with a concomitant increased expression of a cleaved soluble form of betacellulin (s-Btc) in the retina. Intravitreal injection of soluble betacellulin induced retinal vascular permeability in normoglycemic and hyperglycemic mice. Western blot analysis of retinas from patients with diabetic retinopathy showed an increase in the active soluble form of betacellulin. In addition, an increase in the levels of A disintegrin and metalloproteinase (ADAM)-10 which plays a role in the cleavage of betacellulin was seen in the retinas of diabetic mice and humans. CONCLUSIONS: These results suggest that excessive amounts of betacellulin in the retina may contribute to the pathogenesis of diabetic macular edema.