March 2009
Volume 50, Issue 3
Free
Retina  |   March 2009
Tyrosine Phosphorylation of Vitreous Inflammatory and Angiogenic Peptides and Proteins in Diabetic Retinopathy
Author Affiliations
  • Jordi L. Reverter
    From the Endocrinology and Nutrition Department, Germans Trias i Pujol University Hospital, Universitat Autònoma de Barcelona, Badalona, Spain; the
  • Jeroni Nadal
    Department of Vitreous and Retina Surgery, Centro de Oftalmología Barraquer, Universitat Autònoma de Barcelona, Barcelona, Spain; the
  • Josep María Fernández-Novell
    Department of Biochemistry and Molecular Biology and IRB (Institute for Research in Biomedicine), Universitat de Barcelona, Barcelona, Spain; and the
  • Joan Ballester
    Department of Medicine and Surgery, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain.
  • Laura Ramió-Lluch
    Department of Medicine and Surgery, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain.
  • María Montserrat Rivera
    Department of Medicine and Surgery, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain.
  • Javier Elizalde
    Department of Vitreous and Retina Surgery, Centro de Oftalmología Barraquer, Universitat Autònoma de Barcelona, Barcelona, Spain; the
  • Santiago Abengoechea
    Department of Vitreous and Retina Surgery, Centro de Oftalmología Barraquer, Universitat Autònoma de Barcelona, Barcelona, Spain; the
  • Joan J. Guinovart
    Department of Biochemistry and Molecular Biology and IRB (Institute for Research in Biomedicine), Universitat de Barcelona, Barcelona, Spain; and the
  • Joan Enric Rodríguez-Gil
    Department of Medicine and Surgery, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Investigative Ophthalmology & Visual Science March 2009, Vol.50, 1378-1382. doi:10.1167/iovs.08-2736
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      Jordi L. Reverter, Jeroni Nadal, Josep María Fernández-Novell, Joan Ballester, Laura Ramió-Lluch, María Montserrat Rivera, Javier Elizalde, Santiago Abengoechea, Joan J. Guinovart, Joan Enric Rodríguez-Gil; Tyrosine Phosphorylation of Vitreous Inflammatory and Angiogenic Peptides and Proteins in Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2009;50(3):1378-1382. doi: 10.1167/iovs.08-2736.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To evaluate the degree of phosphorylation of vitreous proteins in patients with type 2 diabetes mellitus and diabetic retinopathy compared with a group of control subjects without diabetes and of similar age and sex.

methods. In samples obtained after vitrectomy for diabetic retinopathy in patients and for macular hole in control subjects, immunoblot techniques were applied to a mini-array system for quantification of a wide range of chemokines and vasoactive peptides and proteins. Antiphosphotyrosine antibody was used for tyrosine phosphorylation evaluation and results were expressed as the percentage of variation compared with that in control subjects.

results. Samples from eight patients with type 2 diabetes and from eight control subjects were analyzed. The total quantity of proteins analyzed was similar in both patients and control subjects. Tyrosine phosphorylation was very significantly decreased (<20%, P < 0.05) in diabetic patients with respect to the control group in growth-related oncogene, human cytokine I-309, interleukin-13, monocyte colony-stimulating factor, macrophage-derived chemokine, stem cell factor, transforming growth factor-β1, angiogenin, and oncostatin M. A significant decrease in phosphorylation (between 20% and 40%, P < 0.05) was observed in epithelial neutrophil-activating peptide 78; granulocyte colony-stimulating factor; granulocyte-monocyte–stimulating colony factor; IL-5, -6, -7, -8, -10, and -12p40p70; monokine induced by interferon-γ; macrophage inflammatory protein 1-γ; and normal T expressed and secreted cytokine (RANTES) in comparison with that in the control subjects. The greatest decrease in phosphorylation status was found in IL-1-α and -1β.

conclusions. Diabetic retinopathy is associated with a decrease in tyrosine phosphorylation of many vitreous proteins which may indicate an alteration in protein functionality or action even before significant quantitative variations.

Diabetic retinopathy is the most common cause of visual loss in the working population and one of the primary reasons for blindness worldwide. 1 Chronic hyperglycemia is the main etiological factor in the development of diabetic retinopathy, but other diabetes-related factors such as blood pressure, lipid profile, or smoking may influence its development. 2 Several biochemical mechanisms have been identified in the pathogenesis of hyperglycemia-induced vascular damage including glucotoxin formation via the aldose reductase pathway, induction of alterations in cellular signaling by activation of protein kinase C, accelerated formation of advanced glycation end products, and increased oxidative stress. 3 The key etiologic components of diabetic retinopathy are hypoxia-induced inflammation and angiogenesis. Previous studies have investigated both vitreous and serum levels of angiogenic and inflammatory factors, 4 and inflammatory processes have been found to play an important role in the degeneration of retinal capillaries in diabetic patients. 5 Increases in levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and other inflammatory mediators have been reported in vitreous of diabetic patients. 6 In addition, inflammatory chemokines substantially contribute to inducing retinal damage by infiltration of ocular tissue by leukocytes. 7  
The recently developed proteome techniques applied to the vitreous of diabetic patients and nondiabetic control subjects have contributed to the identification of some novel pathogenic proteins and clinical biomarkers 8 9 associated with different biological pathways, such as lipid transport, kallikrein-kinin, coagulation, and complement systems. 10 However, most of previous studies have been focused on the concentration of targeted proteins, particularly molecules involved in angiogenesis and cellular proliferation, but not in protein functionality. 
Essential biological functions are often regulated via posttranslational modifications, especially phosphorylation. Thus, the detection of phosphoproteins and phosphorylation sites is important in comprehensively exploring human biological function. 11 Protein phosphorylation is brought about by protein tyrosine kinases, enzymes that add phosphate to specific tyrosines in target proteins. Phosphate is removed from phosphorylated tyrosines by protein tyrosine phosphatases enzymes. The reversible phosphorylation of tyrosines in proteins plays a key role in regulating many different processes in eukaryotic organisms, such as growth control, cell cycle control, differentiation, cell shape and movement, gene transcription, synaptic transmission, and insulin action. Phosphorylated tyrosines are recognized by specialized binding domains on other proteins, and such interactions are used to initiate intracellular signaling pathways. 12 Thus, evaluation of the degree of protein phosphorylation could provide significant information to the simple determination of protein quantity, although there is a great lack of knowledge about the effects of tyrosine phosphorylation in peptides within the milieu of the eye. 
The purpose of the present study was to evaluate tyrosine phosphorylation of a wide array of chemokines and vasoactive peptides by immunoblot analyses techniques applied to a mini-array system designed to quantify these proteins in vitreous samples obtained from patients with diabetic retinopathy and nondiabetic control subjects. 
Methods
Patients
We selected diabetic patients with diabetic retinopathy defined by fundus oculi performed by a specialized ophthalmologist and referred for vitrectomy, according to accepted clinical criteria. 13 Those with active or recent vitreous hemorrhage, previous ocular surgery, inflammatory ocular disease or trauma were excluded. The diagnosis of type 2 diabetes mellitus was made according to the criteria of the American Diabetes Association. 14 Demographic and clinical data, including age, sex, and history of clinical macrovascular disease and microvascular diabetic complications were recorded in all subjects. Blood samples were drawn by venipuncture between 0700 and 0800 hours after an overnight fast. Plasma glucose, total cholesterol, HDL cholesterol, and triglycerides were measured by routine clinical chemistry immediately after extraction. HbA1c was measured in blood samples with EDTA by high-pressure liquid chromatography using a fully automated analyzer (Adams Menarini HI-AUTO A1c 8160; Arkray, Kyoto, Japan) with an interassay coefficient of variation of 1.8% and 1.5% at HbA1c levels of 4.8% and 9.0%, respectively (reference range: 4%–5.8%). 
The control group included vitreous obtained from otherwise healthy subjects with idiopathic macular hole without history of any ocular diseases or minor pathologic conditions. 
The study was approved by the local Ethics Committee, in accordance with the Declaration of Helsinki and all participants gave their written informed consent before inclusion. 
Vitreous Collection
Vitreous fluid was obtained from individuals undergoing pars plana vitrectomy in accordance with approved Human Discarded Specimen Research Protocols from the institutional review board. Undiluted samples were collected at the time of surgery, immediately placed on ice, spun at 15.000g for 1 minute to remove insoluble material, and stored at −80°C. Harvested vitreous samples were collected in tubes (0.8–1.0 mL) by using a surgical system (Accurus; Alcon, Fort Worth, TX). To maintain intraocular pressure, vitreous, was removed slowly with air interchange. 
Processing Vitreous Samples for Array Analysis
One hundred fifty microliters of each vitreous humor sample was mixed immediately after thawing with 150 μL of the lysis buffer (1× cell lysis buffer) contained in a commercial human array antibody kit (RayBio Human Cytokine Antibody Array; RayBiotech, Inc., Norcross, GA). Cell lysis buffer was added (1×) with 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM benzamidine, 10 μg/mL leupeptin, and 1 mM Na2VO4 before mixing with the vitreous humor samples. The mixture of vitreous humor/lysis buffer was then homogenized by ultrasonication. Afterward, samples were centrifuged for 5 minutes at 5000g at 5°C and the resultant pellet was discarded. The total protein content of the obtained supernatants was measured to guarantee the presence of a minimum of 50 μg of total protein content per sample. Once the total protein content of the samples was determined, the samples were immediately used for the array analysis. Total protein content was analyzed by the Bradford method 15 with a commercial kit (Bio-Rad Laboratories, Hercules, CA). 
Array Analysis Technique
The detection of the total content of each of the proteins analyzed was performed after rigorous adherence to the protocol provided by the kit used. Thus, they were incubated with the biotin-conjugated anti-cytokine antibody provided in the kit. This direct protocol was followed in 16 separate samples: 8 from healthy individuals and 8 from diabetic patients. At the same time, these 16 samples were simultaneously used for array analysis of tyrosine phosphorylation levels to make a direct comparison between the protein content and the tyrosine phosphorylation status. Moreover, another 16 separate samples—8 from healthy individuals and 8 from diabetic patients—were processed to determine only the tyrosine phosphorylation status. The proteins determined in the commercial array used were the following: epithelial neutrophil-activating peptide 78 (ENA 78); granulocyte colony-stimulating factor (GCSF); granulocyte-monocyte–stimulating colony factor (GM-SCF); growth-related oncogene (GRO); GRO-α; human cytokine I-309 (I-309); IL-1α, -1β, -2, -3, -4, -5, -6, -7, -8, -10, -12p40p70, -13, and -15; IFN-γ; monocyte chemotactic protein (MCP)-1 -2, and -3; monocyte colony-stimulating factor (MCSF); macrophage-derived chemokine (MDC); monokine induced by interferon (MIG)-γ; macrophage inflammatory protein (MIP)-1γ; regulated on activation, normal T expressed and secreted cytokine (RANTES); stem cell factor (SCF); stromal-derived factor (SDF)-1; thymus- and activation-regulated chemokine (TARC); transforming growth factor (TGF)-β1; tumor necrosis factor (TNF)-α and -β; epidermal growth factor (EGF); insulin-like growth factor (IGF)-1; angiogenin; oncostatin M; thrombopoietin; vascular endothelial growth factor (VEGF); platelet-derived growth factor (PDGF)-88; and leptin. 
Tyrosine phosphorylation analysis was performed by modification of the original protocol included in the commercial kit. For this purpose, the incubation of the array membranes with the samples used and further washing of the incubated membranes was performed according to the protocol included in the kit. After this, samples were incubated for 1 hour at 15°C temperature with an anti-phosphotyrosine antibody (PY-20; BD Transduction Laboratory, Temecula, CA) diluted at 1/20,000 (vol/vol) in the blocking buffer provided with the commercial kit. Thereafter, samples were washed three times, 5 minutes each, with the 1× wash buffer I included in the commercial kit, and 2 additional times for 5 minutes each, with 1× wash buffer II, also included in the commercial kit. Samples were then incubated for 1 hour at 15°C with a goat anti-mouse IgG-HRP secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a dilution of 1/2000 (vol/vol) in the blocking buffer provided in the commercial kit. Afterward, samples were washed according to the manufacturer’s protocol. Finally, the membranes were incubated for 5 minutes with a chemiluminescence substrate (ECL Plus; GE Healthcare, Buckinghamshire, UK). They were then exposed to autoradiography films for 2 minutes on average. In all cases, the quantification of the positive spots obtained against a homogeneous background was performed with a commercial system (RayBio Analysis Tool; RayBiotech, Inc.) that was specifically designed for the analysis of the arrays used. 
All chemical reagents were of analytical grade and were obtained from Sigma-Aldrich (St. Louis, MO), Merck (Darmstadt, Germany), Bio-Rad Laboratories (Hercules, CA), and EMS (Fort Washington, PA). 
Statistical Analyses
Data were analyzed with commercial software (Statistical Analysis System for Windows; SAS Institute, Cary, NC). Data were expressed as the mean ± SD or the median and interquartile range, as appropriate. The comparison between biochemical data was performed with nonparametric tests. Determination of putative differences between healthy individuals and diabetic patients was performed by applying the GLM procedure included in the package. For an optimal application of the statistical procedures, data were previously normalized through an arcsin [√x/100] transformation, x being the transformed data. Differences were considered as significant with P < 0.05. 
Results
We included eight vitreous samples from eight male patients with type 2 diabetes and eight samples from eight male control subjects of a similar age (57 ± 8 vs. 57 ± 9 years; P = NS). The mean duration of diabetes was 15 ± 8.4 years. Diabetic patients presented proliferative diabetic retinopathy with significant macular edema in two. With respect to diabetic complications, 16% presented diabetic nephropathy, 11% cardiovascular and/or peripheral vascular disease, and 5% polyneuropathy. All the patients were treated with two doses of premixed insulin without oral hypoglycemic drugs and received similar doses of statin, aspirin, and ramipril. Blood glucose and HbA1 were increased in diabetic patients with respect to the control, as expected (187 ± 69 mg/dL vs. 91 ± 7 mg/dL, P < 0.05; 8.4 ± 1.8 vs. 4.6 ± 0.3%, P < 0.05; respectively). No significant differences were observed in cholesterol (201 ± 30 mg/dL vs. 191 ± 48 mg/dL; P = NS), HDL-cholesterol (54 ± 12 mg/dL vs. 53 ± 11 mg/dL; P = NS), or triglycerides (132 [81–155] mg/dL vs. 122 [75–140] mg/dL; P = NS]), between patients and control subjects. 
Presence of Interleukins and Other Cytokines in the Vitreous Humor of Healthy Individuals and Diabetic Patients
Simple array analysis showed the presence of a wide array of interleukins and other cytokines in the vitreous humors of healthy men. Notwithstanding, there was a great variation in the amount of each of these proteins. Thus, we performed an arbitrary classification of these proteins according to their mean intensity marks on the arrays when evaluated against the background. This classification was performed after the analysis of eight arrays from eight healthy individuals. After this classification, the proteins were classified as follows:
  1.  
    Proteins without any significant presence: IL-1α, IL-β, and leptin did not show any significant mark in any of the arrays used.
  2.  
    Proteins with low presence: ENA-78; GCSF; GM-SCF; GRO; GRO-α; I-309; IL-3, -6, -7, -10, -12p40p70, -15; IFN-γ; MCP-2, -3; MCSF; MDC; MIG; RANTES; SCF; SDF-1; TARC; TGF-β; TNF-α and -β; angiogenin; oncostatin M; thrombopoietin; VEGF; and PDGF-88 showed an intensity mark below 10% over the background.
  3.  
    Proteins with medium presence: IL-5, -8, and IL-13; MCP-1; MIP-1γ; and IGF-1 showed an intensity mark between 10% and 30% over the background.
  4.  
    Proteins with high presence: IL-2 and -4 and EGF showed a mean intensity mark above 30% over the background.
Diabetic patients showed no significant differences with respect to the control group in the expression and presence of any of the proteins tested in the vitreous humor as shown in the analysis of eight arrays from separate diabetic patients compared with eight arrays from eight healthy individuals. 
Tyrosine Phosphorylation Status of Interleukins and Other Immunomodulating Proteins in the Vitreous Humor of Healthy Individuals and Diabetic Patients
Analysis of the tyrosine phosphorylation from the proteins included in the arrays used showed the presence of the many phosphorylated interleukins and other cytokines in the vitreous humors of healthy men. On the other hand, and similar to that observed regarding protein content, there was a great variation in the amount of tyrosine phosphorylation of each of these proteins. Accordingly, we again performed an arbitrary classification of these phosphorylated proteins related to their mean intensity marks on the arrays when evaluated against the background. This classification was performed after the analysis of eight arrays from eight healthy individuals. After this classification, proteins were classified as follows:
  1.  
    Proteins with no significant phosphorylation: These proteins did not show any significant mark in any of the arrays used. Only leptin did not show any signal of phosphorylation.
  2.  
    Proteins with low tyrosine phosphorylation levels: GRO-α; IL-2, -4, and IL-15; IFN-γ; MCP-2; SDF-1; TARC; TNF-β; EGF; thrombopoietin; VEGF; and PDGF 88 showed an intensity mark below 30% over the background.
  3.  
    Proteins with medium tyrosine phosphorylation levels: GRO; I-309; IL-3, -5, -6, and -13; MCP-1; MCP-3; MCSF; MDC; MIG; SCF; TNF-α; IGF-1; angiogenin; and oncostatin M showed an intensity mark between 30% and 60% over the background.
  4.  
    Proteins with high tyrosine phosphorylation levels: IL-1α, -1β, -7, -8, -10, and -12p40p70; MIP-1γ; RANTES; and TGF-β1 showed an intensity mark above 60% over the background.
Vitreous from diabetic patients presented striking changes in the tyrosine phosphorylation patterns of the proteins studied. As shown in Table 1 , diabetic patients showed a significant (P < 0.05) decrease in the intensity of tyrosine phosphorylation of many of the proteins analyzed with variations in the intensity of this decrease. Thus, proteins such as GRO, I-309, IL-13, MCSF, MDC, SCF, TGF-β1, angiogenin, and oncostatin M demonstrated a significant (P < 0.05) decrease below 20% when compared with arrays form healthy individuals. Moreover, ENA-78; GCSF; GM-SCF; IL-5, -6, -7, -8, -10, and -12p40p70; MIG; MIP-1γ; and RANTES showed a significant (P < 0.05) decrease of between 20% to 40% in phosphorylation when compared with healthy individuals. Finally, both IL-1α and -1β showed the greatest decrease in phosphorylation status, reaching values of 54.8% ± 3.3% (mean ± SEM) for of IL-1α and 49.9% ± 3.3% (mean ± SEM) for IL-1β when compared with arrays from healthy individuals. In all the cases, tyrosine phosphorylation values of their respective control proteins from healthy individuals were adjusted to an arbitrary value of 100.0. There were no significant changes in diabetic patients in tyrosine phosphorylation of GRO-α; IL-2, -3, -4, and IL-15; IFN-γ; MCP-1; MCP-2 and -3; SDF-1; TARC; TNF-α and -β; EGF; IGF-1; thrombopoietin; VEGF; and PDGF-88. These results were also obtained in samples in which a simultaneous analysis of the total content for each protein was performed, thus indicating that the observed decrease in tyrosine phosphorylation was not due to a concomitant decrease in the total content of the proteins evaluated. 
Discussion
In our study, diabetic retinopathy was associated with a general decrease in the tyrosine phosphorylation status of angiogenic and inflammatory elements present in the vitreous fluid. In recent years, the development of proteome technology has opened a new approach to study microvascular diabetic complications, 10 and many peptides and proteins have been emerged as putative mediators of vascular damage. 16 However, the alterations in protein expression or quantity observed in these studies do not implicate a mechanistic role and may not be a cause but rather a consequence of the endothelial lesion. With the same caveats, our study could provide one more step in the comprehension of diabetic retinopathy, since the tyrosine phosphorylation status of many peptides and proteins is directly linked to its function. In this way, tyrosine phosphorylation is a widespread mechanism underlying the action of a broad range of different membrane receptors, including most of the peptides analyzed in this study, such as interleukins, 17 18 TNF, 19 IGF II, 20 or VEGF. 21 However, to our knowledge, there is a lack of literature regarding tyrosine phosphorylation of the peptides and proteins studied, despite that their structure, such as that of the interleukin family, showing different tyrosine residues that are susceptible to phosphorylation. 22 23 Only in some of the peptides analyzed such as angiogenin, has the presence of phosphorylated tyrosine residues been previously described, 24 and this change in phosphorylation status could lead to changes in the specific activity of angiogenin. At present, we can only speculate about the role of tyrosine phosphorylation in modulating, for example, interleukin action. However, the diabetes-linked changes observed in the phosphorylation status of the peptides analyzed in our study may be important from a clinical and research point of view, regardless of their specific biological significance. 
It is noteworthy that the decrease observed in the overall tyrosine phosphorylation of the peptides studied was not associated with significant changes in the total protein content of most of the proteins evaluated. However, results published by other authors indicate that diabetic retinopathy can be associated with changes in the total content of many of the peptides analyzed in vitreous humor, such as interferon-induced protein (IP)-10; MCP-1; VEGF; TNFα; IL-1β, -6, and -8; and even leptin. 25 26 27 28 29 30 31 32 33 A possible explanation for these differences could be related to the fact that our study was performed using laboratory technology that is unable to detect smaller changes in total protein content than those used by the previously reported works and by the statistical power of the number of samples. However, it should be pointed out that, despite these shortcomings, an overall decrease in tyrosine phosphorylation status was detected. From a practical point of view, these results demonstrate that changes in the tyrosine phosphorylation status of these peptides can be detected even in conditions in which no changes in the total protein content were observed, thus, yielding a new approach to detect functional alterations in the vitreous humor components that can be detected in conditions in which variations of total protein content are not appreciated. These results indicate that analysis of changes in tyrosine phosphorylation vitreous humor peptides could be a sensitive biomarker of the presence of ocular alterations related to diabetic retinopathy. 
Our results lead to a discussion of the probable cause(s) by which the diverse analyzed peptides demonstrate a decrease in their tyrosine phosphorylation status. In view of our data, there are at least two hypothetical explanations. The first would be a diabetes-related malfunction in the phosphodephosphorylation mechanisms in cells that synthesize and secrete these peptides. Phosphodephosphorylation of tyrosine residues is a posttranscriptional phenomenon, which is always performed through a whole battery of separate protein kinases and phosphatases under an intracellular basis. 12 The presence of changes in the tyrosine phosphorylation status of peptides present in an extracellular milieu, such as the vitreous humor, would indicate that some alteration in the functioning of these protein kinases and phosphatases occurs in the cells producing and secreting the peptides analyzed. In fact, a relationship has already been described between diabetes and alteration of phosphodephosphorylation mechanisms in metabolic pathways. Thus, it is well known that diabetes alters not only the basal activity but also the insulin-stimulated activation of several important tyrosine kinases such as Akt and glycogen synthase kinase (GSK)-3. 34 These alterations induce concomitant modifications in the normal functioning of most of the metabolic pathways controlled by these kinases. 
Another possible explanation for the decrease observed in tyrosine phosphorylation status is a reduction in phosphorylated residues by nitration, related to the well-documented excess of peroxynitrites in the milieu of the eye in diabetes. 35 In this regard, it has been described that diabetes induces retinal degeneration through a peroxynitrite-mediated inhibition in the signaling of the nerve growth factor (NGF). 35 This effect is mediated by a decreased phosphorylation of the NGF tyrosine kinase receptor, which prevents the normal signaling of NGF in retinal cells. 35 36 Thus, the overall decrease on tyrosine phosphorylation observed could be the consequence of the presence of high levels of superoxide radicals and peroxynitrites, affecting the final signaling of these phosphorylated peptides in their target cells. Our study was not designed to investigate the physiopathological mechanisms of protein phosphodephosphorylation, because we did not know the phosphorylation status of vitreous proteins in diabetes mellitus in advance. Future experiments are being designed to address this question. 
In conclusion, our results indicate that diabetic retinopathy involves changes in the tyrosine phosphorylation status of many angiogenic and inflammatory elements present in the vitreous fluid. Further research on this topic could provide additional understanding of the pathologic mechanisms of diabetic retinopathy. 
 
Table 1.
 
Comparison between Tyrosine Phosphorylation Intensity Values of Peptides and Proteins in Type 2 Diabetes Vitreous Fluid Samples versus Control Samples
Table 1.
 
Comparison between Tyrosine Phosphorylation Intensity Values of Peptides and Proteins in Type 2 Diabetes Vitreous Fluid Samples versus Control Samples
Proteins Healthy Individuals (n = 8) Diabetic Patients (n = 8)
ENA-78 100.0 ± 3.0 61.3 ± 2.0*
GCSF 100.0 ± 2.4 68.2 ± 2.1*
GM-SCF 100.0 ± 3.9 63.4 ± 2.4*
GRO 100.0 ± 2.3 81.2 ± 3.4*
GRO-α 100.0 ± 3.8 103.8 ± 3.1
I-309 100.0 ± 4.0 86.3 ± 4.0*
IL-1α 100.0 ± 3.2 54.8 ± 3.3*
IL-1β 100.0 ± 4.1 49.9 ± 3.3*
IL-2 100.0 ± 3.1 97.3 ± 2.5
IL-3 100.0 ± 4.2 90.6 ± 3.7
IL-4 100.0 ± 4.3 88.9 ± 4.2
IL-5 100.0 ± 3.1 75.3 ± 2.8*
IL-6 100.0 ± 2.3 68.0 ± 2.4*
IL-7 100.0 ± 1.6 62.1 ± 2.1*
IL-8 100.0 ± 1.7 72.8 ± 2.8*
IL-10 100.0 ± 1.4 76.2 ± 3.2*
IL-12p40p70 100.0 ± 1.9 77.8 ± 3.5*
IL-13 100.0 ± 4.0 84.9 ± 3.6*
IL-15 100.0 ± 4.1 99.8 ± 4.6
IFN-γ 100.0 ± 3.3 107.8 ± 5.9
MCP-1 100.0 ± 2.1 105.3 ± 5.8
MCP-2 100.0 ± 2.6 99.2 ± 4.2
MCP-3 100.0 ± 1.5 96.9 ± 4.2
MCSF 100.0 ± 1.3 83.8 ± 3.6*
MDC 100.0 ± 1.4 81.3 ± 4.6*
MIG 100.0 ± 3.2 74.0 ± 2.9*
MIP-1γ 100.0 ± 2.9 71.7 ± 3.6*
RANTES 100.0 ± 2.1 77.6 ± 3.6*
SCF 100.0 ± 2.4 81.6 ± 3.6*
SDF-1 100.0 ± 2.9 94.5 ± 3.9
TARC 100.0 ± 4.3 96.3 ± 3.8
TGF-β1 100.0 ± 3.9 84.9 ± 4.8*
TNF-α 100.0 ± 4.1 90.1 ± 4.7
TNF-β 100.0 ± 4.5 109.0 ± 4.2
EGF 100.0 ± 3.2 91.6 ± 4.5
IGF-1 100.0 ± 3.7 90-3 ± 5.4
Angiogenin 100.0 ± 2.1 86.8 ± 3.4*
Oncostatin M 100.0 ± 2.6 82.5 ± 3.5*
Thrombopoietin 100.0 ± 4.8 98.5 ± 4.1
VEGF 100.0 ± 3.7 98.6 ± 3.9
PDGF-88 100.0 ± 3.6 103.8 ± 4.0
AielloLP, GardnerTW, KingGL, et al. Diabetic retinopathy. Diabetes Care. 1998;21:143–156. [CrossRef] [PubMed]
FrankRN. Diabetic retinopathy. N Engl J Med. 2004;350:48–58. [CrossRef] [PubMed]
AronsonD. Hyperglycemia and the pathobiology of diabetic complications. Adv Cardiol. 2008;45:1–16. [PubMed]
JoN, WuGS, RaoNA. Upregulation of chemokine expression in the retinal vasculature in ischemia-reperfusion injury. Invest Ophthalmol Vis Sci. 2003;44:4054–4060. [CrossRef] [PubMed]
MitamuraY, HaradaC, HaradaT. Role of cytokines and trophic factors in the pathogenesis of diabetic retinopathy. Curr Diabetes Rev. 2005;1:73–81. [CrossRef] [PubMed]
YuukiT, KandaT, KimuraY, et al. Inflammatory cytokines in vitreous fluid and serum of patients with diabetic vitreoretinopathy. J Diabetes Complications. 2001;15:257–259. [CrossRef] [PubMed]
JoN, WuGS, RaoNA. Upregulation of chemokine expression in the retinal vasculature in ischemia-reperfusion injury. Invest Ophthalmol Vis Sci. 2003;44:4054–4560. [CrossRef] [PubMed]
KimT, KimSJ, KimK, et al. Profiling of vitreous proteomes from proliferative diabetic retinopathy and nondiabetic patients. Proteomics. 2007;7:4203–4215. [CrossRef] [PubMed]
García-RamírezM, CanalsF, HernándezC, et al. Proteomic analysis of human vitreous fluid by fluorescence-based difference gel electrophoresis (DIGE): a new strategy for identifying potential candidates in the pathogenesis of proliferative diabetic retinopathy. Diabetologia. 2007;50:1294–1303. [CrossRef] [PubMed]
GaoBB, ChenX, TimothyN, AielloLP, FeenerEP. Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy. J Proteome Res. 2008;7:2516–2525. [CrossRef] [PubMed]
CohenP. Protein phosphorylation and hormone action. Proc R Soc Lond B Biol Sci. 1988;22(234)115–144.
SchlessingerJ. Cell signalling by receptor tyrosine kinases. Cell. 2000;103:211–225. [CrossRef] [PubMed]
MohamedQ, GilliesMC, WongTY. Management of diabetic retinopathy: a systematic review. JAMA. 2007;298:902–916. [CrossRef] [PubMed]
Standards of medical care in diabetes-2007; American Diabetes Association. Diabetes Care. 2007;30:S42–S47. [CrossRef] [PubMed]
BradfordMM. A rapid and sensitive for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. [CrossRef] [PubMed]
MerchantML, KleinJB. Proteomics and diabetic nephropathy. Semin Nephrol. 2007;27:627–636. [CrossRef] [PubMed]
BagleyCJ, WoodcockJM, GuthridgeMA, StomskiFC, LópezAF. Structural and functional hot spots in cytokine receptors. Int J Hematol. 2001;73:299–307. [CrossRef] [PubMed]
HeinrichPC, BehrmannI, HaanS, HermannsHM, Müller-NewenG, SchaperF. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J. 2003;374:1–20. [CrossRef] [PubMed]
DarnayBG, AggarwalBB. Inhibition of protein tyrosine phosphatases causes phosphorylation of tyrosine-331 in the p60 TNF receptor and inactivates the receptor-associated kinase. FEBS Lett. 1997;410:361–367. [CrossRef] [PubMed]
CorveraS, WiteheadRE, MottolaC, CzechMP. The insulin-like growth factor II receptor is phosphorylated by a tyrosine kinase in adipocyte plasma membranes. J Biol Chem. 1986;261:7675–7679. [PubMed]
VeikkolaT, KarkkainenM, Claesson-WelshL, AlitaloK. Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res. 2000;60:203–212. [PubMed]
RobbRJ, KutnyRM, PanicoM, MorrisHR, ChowdhryV. Amino acid sequence and post-translational modification of human interleukin 2. Proc Natl Acad Sci USA. 1984;81:6486–6490. [CrossRef] [PubMed]
FiskerstrandC, SarganD. Nucleotide sequence of ovine interleukin-1 beta. Nucleic Acids Res. 1990;18:7165. [CrossRef] [PubMed]
TanigawaK, FujiharaM, SakamotoR, YanahiraS, OhtsukiK. Characterization of bovine angiogenin-1 and lactogenin-like protein as glycyrrhizin-binding proteins and their in vitro phosphorylation by C-kinase. Biol Pharm Bull. 2001;24:443–447. [CrossRef] [PubMed]
CicikE, TekinH, AkarS, et al. Interleukin-8, Nitric oxide and glutathione status in proliferative vitreoretinopathy and proliferative diabetic retinopathy. Ophthalmic Res. 2003;35:251–255. [CrossRef] [PubMed]
FunatsuH, YamashitaH, IkedaT, MimuraT, EguchiS, HoriS. Vitreous levels of interleukin-6 and vascular endothelial growth factor are related to diabetic macular edema. Ophthalmology. 2003;110:1690–1696. [CrossRef] [PubMed]
ErH, DoğanayS, OzerolE, YürekliM. Adrenomedullin and leptin levels in diabetic retinopathy and retinal diseases. Ophthalmologica. 2005;219:107–111. [CrossRef] [PubMed]
FunatsuH, YamashitaH, NomaH, et al. Aqueous humor levels of cytokines are related to vitreous levels and progression of diabetic retinopathy in diabetic patients. Graefes Arch Clin Exp Ophthalmol. 2005;243:3–8. [CrossRef] [PubMed]
DemircanN, SafranBG, SoyluM, OzcanAA, SizmazS. Determination of vitreous interleukin-1 (IL-1) and tumour necrosis factor (TNF) levels in proliferative diabetic retinopathy. Eye. 2006;20:1366–1369. [CrossRef] [PubMed]
MaierR, WegerM, Haller-SchoberEM, et al. Multiplex bead analysis of vitreous and serum concentrations of inflammatory and proangiogenic factors in diabetic patients. Mol Vis. 2008;14:637–643. [PubMed]
NomaH, FunatsuH, YamasakiM, et al. Aqueous humour levels of cytokines are correlated to vitreous levels and severity of macular oedema in branch retinal vein occlusion. Eye. 2008;22:42–48. [CrossRef] [PubMed]
KrookA, RothRA, JiangXJ, ZierathJR, Wallberg-HenrikssonH. Insulin-stimulated Akt kinase activity is reduced in skeletal muscle from NIDDM subjects. Diabetes. 1998;47:1281–1286. [CrossRef] [PubMed]
Eldar-FinkelmanH, ShreyerSA, ShinoharaMM, LeBoeufRC, KrebsEG. Increased glycogen synthase kinase-3 activity in diabetes- and obesity-prone C57BL/6J mice. Diabetes. 1999;48:1662–1666. [CrossRef] [PubMed]
ShaoJ, YamashitaH, QiaoL, FriedmanJE. Decreased Akt kinase activity and insulin resistance in C57BL/KsJ-Leprdb/db mice. J Endocrinol. 2000;167:107–115. [CrossRef] [PubMed]
AliTK, MatragoonS, PillaiBA, LiouGI, El-RemessyAB. Peroxynitrite mediates retinal neurodegeneration by inhibiting nerve growth factor survival signaling in experimental and human diabetes. Diabetes. 2008;57:889–898. [CrossRef] [PubMed]
JonnalaRR, BuccafuscoJJ. Inhibition of nerve growth factor signaling by peroxynitrite. J Neurosci Res. 2001;63:27–34. [CrossRef] [PubMed]
Table 1.
 
Comparison between Tyrosine Phosphorylation Intensity Values of Peptides and Proteins in Type 2 Diabetes Vitreous Fluid Samples versus Control Samples
Table 1.
 
Comparison between Tyrosine Phosphorylation Intensity Values of Peptides and Proteins in Type 2 Diabetes Vitreous Fluid Samples versus Control Samples
Proteins Healthy Individuals (n = 8) Diabetic Patients (n = 8)
ENA-78 100.0 ± 3.0 61.3 ± 2.0*
GCSF 100.0 ± 2.4 68.2 ± 2.1*
GM-SCF 100.0 ± 3.9 63.4 ± 2.4*
GRO 100.0 ± 2.3 81.2 ± 3.4*
GRO-α 100.0 ± 3.8 103.8 ± 3.1
I-309 100.0 ± 4.0 86.3 ± 4.0*
IL-1α 100.0 ± 3.2 54.8 ± 3.3*
IL-1β 100.0 ± 4.1 49.9 ± 3.3*
IL-2 100.0 ± 3.1 97.3 ± 2.5
IL-3 100.0 ± 4.2 90.6 ± 3.7
IL-4 100.0 ± 4.3 88.9 ± 4.2
IL-5 100.0 ± 3.1 75.3 ± 2.8*
IL-6 100.0 ± 2.3 68.0 ± 2.4*
IL-7 100.0 ± 1.6 62.1 ± 2.1*
IL-8 100.0 ± 1.7 72.8 ± 2.8*
IL-10 100.0 ± 1.4 76.2 ± 3.2*
IL-12p40p70 100.0 ± 1.9 77.8 ± 3.5*
IL-13 100.0 ± 4.0 84.9 ± 3.6*
IL-15 100.0 ± 4.1 99.8 ± 4.6
IFN-γ 100.0 ± 3.3 107.8 ± 5.9
MCP-1 100.0 ± 2.1 105.3 ± 5.8
MCP-2 100.0 ± 2.6 99.2 ± 4.2
MCP-3 100.0 ± 1.5 96.9 ± 4.2
MCSF 100.0 ± 1.3 83.8 ± 3.6*
MDC 100.0 ± 1.4 81.3 ± 4.6*
MIG 100.0 ± 3.2 74.0 ± 2.9*
MIP-1γ 100.0 ± 2.9 71.7 ± 3.6*
RANTES 100.0 ± 2.1 77.6 ± 3.6*
SCF 100.0 ± 2.4 81.6 ± 3.6*
SDF-1 100.0 ± 2.9 94.5 ± 3.9
TARC 100.0 ± 4.3 96.3 ± 3.8
TGF-β1 100.0 ± 3.9 84.9 ± 4.8*
TNF-α 100.0 ± 4.1 90.1 ± 4.7
TNF-β 100.0 ± 4.5 109.0 ± 4.2
EGF 100.0 ± 3.2 91.6 ± 4.5
IGF-1 100.0 ± 3.7 90-3 ± 5.4
Angiogenin 100.0 ± 2.1 86.8 ± 3.4*
Oncostatin M 100.0 ± 2.6 82.5 ± 3.5*
Thrombopoietin 100.0 ± 4.8 98.5 ± 4.1
VEGF 100.0 ± 3.7 98.6 ± 3.9
PDGF-88 100.0 ± 3.6 103.8 ± 4.0
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