Inhibition of EGF Signaling Protects the Diabetic Retina from Insulin-Induced Vascular Leakage

Abstract

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. 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. Diabetic maculopathy, an important cause of vision loss in patients with type 2 diabetes, is characterized by hyperpermeability of retinal blood vessels and subsequent formation of macular edema and hard exudates. Although the increase in retinal vascular permeability occurs both diffusely and in focal regions, the basic physiological defect that causes retinal vascular leakage is unknown. The blood–retinal barrier (BRB) isolates the retina from the bloodstream, establishing a favorable environmental milieu with the regulation of ionic balance, nutrient availability, and blockage of potentially toxic molecules that allows for optimal retinal function. The BRB consists of an inner BRB, formed by endothelial cells lining the retinal blood vessels and the outer BRB formed by the retinal pigment epithelium (RPE), a layer of epithelial cells between the retina and the non-neuronal choroid.1Erickson K.K. Sundstrom J.M. Antonetti D.A. Vascular permeability in ocular disease and the role of tight junctions.Angiogenesis. 2007; 10: 103-117Crossref PubMed Scopus (148) Google Scholar, 2Neuwelt E. Abbott N.J. Abrey L. Banks W.A. Blakley B. Davis T. Engelhardt B. Grammas P. Nedergaard M. Nutt J. Pardridge W. Rosenberg G.A. Smith Q. Drewes L.R. Strategies to advance translational research into brain barriers.Lancet Neurol. 2008; 7: 84-96Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar Disruption of the BRB is an important feature of diabetic retinopathy. Based on data from the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR), a prospective population-based cohort study of patients with type 1 and 2 diabetes mellitus, the prevalence of clinically significant macular edema is 5.9% for type 1 and 7.5% for type 2 diabetes.3Hirai F.E. Knudtson M.D. Klein B.E.K. Klein R. Clinically significant macular edema and survival in type 1 and type 2 diabetes.Am J Ophthalmol. 2008; 145: 700-706Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Although epidemiologic studies and randomized clinical trials suggest that glycemic control plays a major role in the development of vascular complications of diabetes,4The Diabetes Control and Complications Trial Research GroupImplications of the Diabetes Control and Complications Trial.Diabetes. 1993; 42: 1555-1558Crossref PubMed Scopus (76) Google Scholar insulin therapies for control of glucose metabolism may not prevent long-term complications.5Keenan H.A. Costacou T. Sun J.K. Doria A. Cavellerano J. Coney J. Orchard T.J. Aiello L.P. King G.L. Clinical factors associated with resistance to microvascular complications in diabetic patients of extreme disease duration: the 50-year medalist study.Diabetes Care. 2007; 30: 1995-1997Crossref PubMed Scopus (140) Google Scholar, 6Wong T.Y. Liew G. Tapp R.J. Schmidt M.I. Wang J.J. Mitchell P. Klein R. Klein B.E.K. Zimmet P. Shaw J. Relation between fasting glucose and retinopathy for diagnosis of diabetes: three population-based cross-sectional studies.Lancet. 2008; 371 ([Erratum appeared in Lancet 2008, 371:1838]): 736-743Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar Even though both laser photocoagulation and anti-VEGF therapies have shown significant promise in the treatment of proliferating vessels in proliferative diabetic retinopathy, diabetic macular edema (DME) appears to be more resistant to these treatment approaches, suggesting that other factors might contribute to this complication. We have recently reported the potential role of betacellulin (Btc) in inducing retinal vascular permeability in diabetes.7Anand-Apte B. Ebrahem Q. Cutler A. Farage E. Sugimoto M. Hollyfield J. Folkman J. Betacellulin induces increased retinal vascular permeability in mice.PLoS One. 2010; 5: e13444Crossref PubMed Scopus (23) Google Scholar Clinical trials and other studies have determined that initiation of acute intensive insulin therapy in patients with long-standing poor glycemic control results in a transient worsening of diabetic retinopathy.8The Kroc Collaborative Study GroupBlood glucose control and the evolution of diabetic retinopathy and albuminuria–a preliminary multicenter trial.N Engl J Med. 1984; 311: 365-372Crossref PubMed Scopus (499) Google Scholar, 9The Kroc Collaborative Study GroupDiabetic retinopathy after two years of intensified insulin treatment. Follow-up of the Kroc Collaborative Study.JAMA. 1988; 260: 37-41Crossref PubMed Scopus (144) Google Scholar, 10Keen H. Normoglycaemic re-entry and diabetic complications.Diabet Med. 1984; 1: 85-87Crossref PubMed Scopus (36) Google Scholar, 11Lauritzen T. Frost-Larsen K. Larsen H.W. Deckert T. Effect of 1 year of near-normal blood glucose levels on retinopathy in insulin-dependent diabetics.Lancet. 1983; 1: 200-204Abstract PubMed Scopus (321) Google Scholar, 12Lawson P.M. Champion M.C. Canny C. Kingsley R. White M.C. Dupré J. Kohner E.M. Continuous subcutaneous insulin infusion (CSII) does not prevent progression of proliferative and preproliferative retinopathy.Br J Ophthalmol. 1982; 66: 762-766Crossref PubMed Scopus (64) Google Scholar, 13Chantelau E. Kohner E.M. Why some cases of retinopathy worsen when diabetic control improves.BMJ. 1997; 315: 1105-1106Crossref PubMed Scopus (67) Google Scholar A change in treatment from oral drugs to insulin in patients with non–insulin-dependent (type 2) diabetes mellitus was associated with a significantly increased risk of retinopathy progression and visual impairment.14Henricsson M. Berntorp K. Berntorp E. Fernlund P. Sundkvist G. Progression of retinopathy after improved metabolic control in type 2 diabetic patients. Relation to IGF-1 and hemostatic variables.Diabetes Care. 1999; 22: 1944-1949Crossref PubMed Scopus (41) Google Scholar, 15Henricsson M. Janzon L. Groop L. Progression of retinopathy after change of treatment from oral antihyperglycemic agents to insulin in patients with NIDDM.Diabetes Care. 1995; 18: 1571-1576Crossref PubMed Scopus (49) Google Scholar, 16Henricsson M. Nilsson A. Janzon L. Groop L. The effect of glycaemic control and the introduction of insulin therapy on retinopathy in non-insulin-dependent diabetes mellitus.Diabet Med. 1997; 14: 123-131Crossref PubMed Scopus (87) Google Scholar In addition, it has been reported that patients who undergo total pancreatectomy for cancer develop severe diabetes because of the complete absence of insulin but rarely if ever develop proliferative diabetic retinopathy,17Tiengo A. Segato T. Briani G. Setti A. Del Prato S. Devide A. Padovan D. Virgili F. Crepaldi G. The presence of retinopathy in patients with secondary diabetes following pancreatectomy or chronic pancreatitis.Diabetes Care. 1983; 6: 570-574Crossref PubMed Scopus (29) Google Scholar even when they survive for more than one or two decades. These reports led us to model and evaluate the pathophysiological effects of insulin on the retinal vasculature and the potential crosstalk between insulin and Btc in the regulation of retinal vascular permeability. Recombinant human vascular endothelial growth factor (VEGF) was a kind gift from Genentech (South San Francisco, CA). Recombinant mouse Btc was from R&D Systems (Minneapolis. MN). Goat polyclonal anti-Btc antibody (sc-5802), goat polyclonal anti-actin antibody (sc-1615), rabbit polyclonal anti–zonula occludens-1 (anti–ZO-1) antibody (sc-10804), horseradish peroxidase–conjugated anti-goat IgG (sc-2353), normal rabbit IgG (sc-2027), and normal goat IgG (sc-2028) were from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase–conjugated anti-rabbit IgG antibody (NA934V) was from GE Healthcare (Chalfont St Giles, polyclonal antibody was from rabbit polyclonal antibody was from anti-goat IgG and anti-rabbit IgG were from CA). inhibitors were from and from studies were by the and of the and to for the and of in The were for in with the for Research in and for the of in and Research mice, to were from the of diabetes, mice three of in Control mice three of after the blood glucose levels were with a and CA). with blood glucose levels than were The were to the treatment were after the was a for two with the after the were and 2 after the of insulin was W. P. J. intensive insulin therapy diabetic barrier via and PubMed Scopus Google Scholar In mice were with and one insulin was in the subcutaneous of their and mouse retina were on for in of and were by for and to and a or were after with a horseradish peroxidase–conjugated anti-goat or anti-rabbit antibody a secondary and GE to The were with and were to the was by CA). retinal pigment epithelium cells were in with and in a with retinal cells were in complete complete Research with and in a with cells were in with and in a with cells were in for the of the were the and cells a of 2 on were in for with for goat polyclonal anti-Btc antibody was the and anti-goat IgG was the secondary cells were with rabbit polyclonal antibody a anti-rabbit IgG was a secondary of were in and an was by in for were with goat polyclonal anti-Btc antibody by anti-goat were a with a or a were were three were by or of by for was are data on the of from the in patients with type diabetes on insulin was with in with type 2 diabetes not R. Klein B.E.K. Davis M.D. The Wisconsin Epidemiologic Study of Diabetic The of macular Full Text PDF PubMed Scopus Google Scholar Although the in the of in patients not insulin was to the of patients a of diabetes or severe results from the study the of disease an increase in in patients insulin of disease insulin is a significant of macular edema, the type 2 diabetes cohort and with or more years of diabetes and two who not insulin of diagnosis and were not insulin and who been insulin for or more R. Klein B.E.K. Davis M.D. The Wisconsin Epidemiologic Study of Diabetic The of macular Full Text PDF PubMed Scopus Google Scholar with prevalence of macular edema the and for of diabetes, blood and determined that with type 2 diabetes insulin were significantly more to have macular edema model the effect of insulin on the BRB in a mouse model of diabetes and an B. Ebrahem Q. Cutler A. Farage E. Sugimoto M. Hollyfield J. Folkman J. Betacellulin induces increased retinal vascular permeability in mice.PLoS One. 2010; 5: e13444Crossref PubMed Scopus (23) Google Scholar, W. P. J. intensive insulin therapy diabetic barrier via and PubMed Scopus Google Scholar We of control mice control mice diabetic mice not with insulin blood glucose and diabetic mice with insulin after blood glucose and vascular permeability was after of diabetes and was to be increased from retinal 1 in mice to retinal 1 in diabetic mice Diabetic mice with insulin a increase in retinal vascular from to retinal These results suggest that increased retinal vascular permeability may not be to and that intensive insulin therapy in the presence of may in significant increase in BRB that this may be a model for the worsening of diabetic retinopathy observed after acute intensive insulin therapy in type 1 diabetes Diabetes Control and Complications Trial Research effect of intensive diabetes treatment on the progression of diabetic retinopathy in insulin-dependent diabetes Ophthalmol. 1995; PubMed Scopus Google Scholar, B. E. progression of severe retinopathy in type insulin-dependent diabetes mellitus patients after improved metabolic Diabetes 6: Full Text PDF PubMed Scopus Google Scholar, K. L. glucose and progression of diabetic the year results of the PubMed Scopus (144) Google Scholar We have reported that the increased retinal vascular permeability in diabetic mice is by Btc and and B. Ebrahem Q. Cutler A. Farage E. Sugimoto M. Hollyfield J. Folkman J. Betacellulin induces increased retinal vascular permeability in mice.PLoS One. 2010; 5: e13444Crossref PubMed Scopus (23) Google Scholar insulin treatment in diabetic mice induces increased of and from diabetic mice that were or with and Btc were increased in retinal from diabetic mice on with diabetic mice not with insulin evaluate Btc tight in retinal endothelial and pigment epithelial determined cells in the retina with Btc on from and human cells and cells and Btc with polyclonal Btc normal mouse retina of Btc in the and with control IgG We have that Btc induces retinal vascular leakage in in B. Ebrahem Q. Cutler A. Farage E. Sugimoto M. Hollyfield J. Folkman J. Betacellulin induces increased retinal vascular permeability in mice.PLoS One. 2010; 5: e13444Crossref PubMed Scopus (23) Google Scholar In the the effects of Btc on the tight of and endothelial cells Btc was to cells or of 1 or and the effects on tight were with of of were with in control cells and to Btc tight in to a in to the by Although it does that are more to Btc than are cells in this to be in insulin tight in the of in cells after treatment with insulin. The insulin treatment resulted in a of in with control cells of Btc and in cells an increase in both Btc and was increased with of insulin suggesting that insulin might in Btc to evaluate the increase in Btc is for the of tight treatment of cells with Btc the of tight We cells with insulin to Btc and of in cells with Btc In addition, of from control cells and cells a in Btc in cells with Btc but effect on that is of Btc has the effect in of tight to cells that been with insulin and The of resulted in an on the of the cells EGF to the of and and A. the 7: PubMed Scopus Google Scholar of a or by of and secondary Btc has been shown to and via EGF M. R. S. Betacellulin and of and signaling in vascular PubMed Scopus (60) Google Scholar Although the role of T. The factor a survival in 2007; PubMed Scopus Google Scholar, J. S. E. M. 1 induces via of the epidermal growth factor PubMed Scopus Google Scholar, C. A. A role for A and betacellulin in human and PubMed Scopus Google Scholar, J. T. J. D. T. signaling of cells in PubMed Scopus Google Scholar, M. R. M. J. R. T. S. S. H. T. M. and growth effect of betacellulin in human cancer J Google Scholar, H. M. K. P. H. M. L. therapy induces in the and diabetes in Med. PubMed Scopus Google Scholar, L. M. A and effect on of in PubMed Scopus Google Scholar, H. H. K. T. J. T. M. H. Betacellulin and A cells into PubMed Scopus Google Scholar, T. M. a factor for glucose in PubMed Scopus Google Scholar, H. J. T. K. T. K. R. induces insulin and in cells in the presence of PubMed Google Scholar and EGF receptor P. P. E. M. T. EGF receptor in 2008; PubMed Scopus (49) Google Scholar in has been in the retina has not been in We observed that insulin cells with Btc or a in that treatment with an might and retinal vascular We cells to insulin with a that is for after treatment with insulin and a in with cells not to We observed that the by with cells to insulin but not with We the of of to retinal vascular leakage in diabetic mice on insulin. inhibitors prevent the retinal vascular leakage in mice, of diabetic mice insulin and diabetic mice with insulin but diabetic mice insulin but with and diabetic mice with both insulin and treatment. were a with and retinal vascular leakage was after of diabetes. vascular leakage was a of vascular leakage observed in diabetic The increase in retinal vascular leakage observed in diabetic mice on insulin was reduced when diabetic mice were with edema, an important cause of vision in patients with diabetes, is a of increased retinal vascular permeability and of the Based on the results of randomized trials the Diabetic Diabetes Study Research control with or insulin with treatment and risk of complications in patients with type 2 diabetes ([Erratum appeared in Lancet Full Text Full Text PDF PubMed Scopus Google Scholar and the Diabetes Control and Complications Diabetes Control and Complications Trial Research effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes Engl J Med. 1993; PubMed Scopus Google Scholar intensive therapies for and the of intensive glycemic control in the risk of microvascular the Diabetes a of for for in Diabetes of for the clinical Care. Google Scholar in the Trial a significant increase in mortality in the intensive glycemic control to of this of the F. T. J. J. R. S. B. D. L. A. P. R. D. H. A. D. Trial Effect of intensive treatment of on microvascular in type 2 diabetes: an of the 2010; ([Erratum appeared in Lancet Full Text Full Text PDF PubMed Scopus Google Scholar the the study reported that the intensive glycemic control therapy significantly reduced the risk of progression of diabetic retinopathy but not the risk of vision Davis M.D. S. L. S. Study Study of therapies on retinopathy progression in type 2 Engl J Med. 2010; ([Erratum appeared in Engl J and in Engl J PubMed Scopus Google Scholar data that of insulin are to an increased of macular R. Klein B.E.K. The Wisconsin Epidemiologic Study of Diabetic The of to the and progression of diabetic 1995; PubMed Google Scholar The of Diabetes and Complications study determined a risk for development of complications of in patients on insulin therapy even and years after to intensive insulin normal Diabetes Control and Complications of Diabetes and Complications Research and in patients with type 1 diabetes years after a of intensive Engl J Med. ([Erratum appeared in Engl J PubMed Scopus Google Scholar, Sun W. Davis M.D. J.M. effect of intensive therapy on the risk of retinopathy complications in patients with type 1 diabetes years after the Diabetes Control and Complications Ophthalmol. 2008; PubMed Scopus Google Scholar findings from the study the study and for levels suggest that insulin may be with the of diabetic macular edema of blood Although data are the findings into the role of blood control in the regulation of retinal vascular to this is the that patients with of retinopathy often a paradoxical worsening of their retinopathy with the of levels of associated with the of insulin therapy to control their Kroc Collaborative Study GroupBlood glucose control and the evolution of diabetic retinopathy and albuminuria–a preliminary multicenter trial.N Engl J Med. 1984; 311: 365-372Crossref PubMed Scopus (499) Google Scholar, 9The Kroc Collaborative Study GroupDiabetic retinopathy after two years of intensified insulin treatment. Follow-up of the Kroc Collaborative Study.JAMA. 1988; 260: 37-41Crossref PubMed Scopus (144) Google Scholar, 10Keen H. Normoglycaemic re-entry and diabetic complications.Diabet Med. 1984; 1: 85-87Crossref PubMed Scopus (36) Google Scholar, 11Lauritzen T. Frost-Larsen K. Larsen H.W. Deckert T. Effect of 1 year of near-normal blood glucose levels on retinopathy in insulin-dependent diabetics.Lancet. 1983; 1: 200-204Abstract PubMed Scopus (321) Google Scholar, 12Lawson P.M. Champion M.C. Canny C. Kingsley R. White M.C. Dupré J. Kohner E.M. Continuous subcutaneous insulin infusion (CSII) does not prevent progression of proliferative and preproliferative retinopathy.Br J Ophthalmol. 1982; 66: 762-766Crossref PubMed Scopus (64) Google Scholar, 13Chantelau E. Kohner E.M. Why some cases of retinopathy worsen when diabetic control improves.BMJ. 1997; 315: 1105-1106Crossref PubMed Scopus (67) Google Scholar, K. L. of blood glucose control leads to transient of retinopathy in insulin diabetes the J PubMed Scopus Google Scholar worsening of diabetic retinopathy has been reported to be to of growth factor M. Berntorp K. Berntorp E. Fernlund P. Sundkvist G. Progression of retinopathy after improved metabolic control in type 2 diabetic patients. Relation to IGF-1 and hemostatic variables.Diabetes Care. 1999; 22: 1944-1949Crossref PubMed Scopus (41) Google Scholar, E. that of IGF-1 of diabetic retinopathy.Br J Ophthalmol. PubMed Scopus Google Scholar, E. R. K. of IGF-1 for treatment of worsening of diabetic a long-term of two 2010; PubMed Scopus Google Scholar, M. Berntorp K. Fernlund P. Sundkvist G. Progression of retinopathy in type 2 diabetic Care. PubMed Scopus Google Scholar the that contribute to the have of these the of therapies to prevent of worsening of retinopathy insulin in that acute intensive insulin therapy diabetic BRB via and W. P. J. intensive insulin therapy diabetic barrier via and PubMed Scopus Google the an mouse model to that in the The results suggest that the of macular edema with insulin may be a of the effect of Btc and EGF receptor signaling in the that the outer is that a of patients may be more to these effects that in between the insulin receptor and EGF receptor signaling has been reported to a role in growth and in J.M. R. J. signaling via growth and in One. 6: PubMed Scopus Google Scholar The that by the insulin EGF growth and the that may be other factors to the between insulin and EGF signaling that may be on type and or physiological has been shown to increase the of and the and of the of S. E. the and of the of by and 2007; PubMed Scopus Google Scholar EGF and Btc are important of G. K. S. J. D. P. for and in of PubMed Scopus Google Scholar the have an and signaling via the EGF receptor for the transient BRB that is a of intensive insulin In addition, have that the increased retinal vascular leakage after insulin the absence of inhibitors to the that be in the role of other of EGF receptor that to this phenomenon cannot be insulin or growth factor are resistant to retinal T. D. K. M. King G.L. M. of insulin and IGF-1 on vascular endothelial cells retinal PubMed Scopus Google Scholar edema was by A. A of insulin Scopus Google Scholar in to the development of severe acute edema in diabetic K. B. J. PubMed Scopus Google Scholar A that insulin the permeability of J. of insulin in the control of vascular J PubMed Scopus Google Scholar that insulin treatment have effects on vascular permeability. the have that insulin tight in cells and and the subsequent the of Btc plays a role in the of tight was by the of Btc to prevent of tight In diabetic mice, insulin signaling in retinal endothelial cells is to insulin signaling in T. insulin signaling in retinal in diabetic PubMed Scopus Google Scholar In diabetic mice and mouse insulin receptor and insulin receptor and were increased by T. insulin signaling in retinal in diabetic PubMed Scopus Google Scholar These findings suggest in the diabetic may be an increase in the of insulin receptor in the results in of of Btc to of endothelial tight retinal vascular and and diabetic retinopathy. retinal may be with insulin therapy to worsening of of insulin to the receptor the in the induces on and a of on that insulin receptor and associated and these in other and and the and to and a Although insulin signaling has been characterized in and to in the retina has to be in the of insulin and insulin receptor signaling in for diabetic 22: PubMed Scopus Google Scholar insulin receptor is to that of the it does not with the and is in a of Although studies a for in of the has been in retinal endothelial and the the in the to insulin signaling is T. insulin signaling in retinal in diabetic PubMed Scopus Google Scholar or L. M. Antonetti D.A. Diabetes retinal insulin receptor with and PubMed Scopus Google Scholar in diabetes In addition, it is that insulin might in on metabolic Btc signaling is to retinal the retina with or inhibitors a potentially to the retinal vascular leakage in some patients with diabetes. treatment of diabetes results in increased retinal vascular permeability mediated via the of EGF signaling may be a in with insulin to prevent retinal vascular