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1.  The Angio-Fibrotic Switch of VEGF and CTGF in Proliferative Diabetic Retinopathy 
PLoS ONE  2008;3(7):e2675.
Background
In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) cause blindness by neovascularization and subsequent fibrosis, but their relative contribution to both processes is unknown. We hypothesize that the balance between levels of pro-angiogenic VEGF and pro-fibrotic CTGF regulates angiogenesis, the angio-fibrotic switch, and the resulting fibrosis and scarring.
Methods/Principal Findings
VEGF and CTGF were measured by ELISA in 68 vitreous samples of patients with proliferative DR (PDR, N = 32), macular hole (N = 13) or macular pucker (N = 23) and were related to clinical data, including degree of intra-ocular neovascularization and fibrosis. In addition, clinical cases of PDR (n = 4) were studied before and after pan-retinal photocoagulation and intra-vitreal injections with bevacizumab, an antibody against VEGF. Neovascularization and fibrosis in various degrees occurred almost exclusively in PDR patients. In PDR patients, vitreous CTGF levels were significantly associated with degree of fibrosis and with VEGF levels, but not with neovascularization, whereas VEGF levels were associated only with neovascularization. The ratio of CTGF and VEGF was the strongest predictor of degree of fibrosis. As predicted by these findings, patients with PDR demonstrated a temporary increase in intra-ocular fibrosis after anti-VEGF treatment or laser treatment.
Conclusions/Significance
CTGF is primarily a pro-fibrotic factor in the eye, and a shift in the balance between CTGF and VEGF is associated with the switch from angiogenesis to fibrosis in proliferative retinopathy.
doi:10.1371/journal.pone.0002675
PMCID: PMC2443281  PMID: 18628999
2.  Angiofibrotic response to VEGF inhibition in diabetic retinal detachment. Report No 1 
Archives of ophthalmology  2012;130(9):1127-1134.
Purpose
To assess the effect of bevacizumab on connective tissue growth factor (CTGF) and VEGF in ocular fluids of patients with diabetic traction retinal detachment. To determine whether intra- and post-operative complications are decreased in eyes given adjunctive pre-operative bevacizumab.
Methods
Twenty eyes of 19 patients were randomized to receive intravitreal bevacizumab or sham 3–7 days prior to vitrectomy for severe proliferative diabetic retinopathy. Aqueous samples prior to injection and at time of surgery were collected. Undiluted vitreous samples were extracted.
Results
Five eyes had decreased vascularization of membranes from pre-injection to time of surgery (all in treatment arm). Median visual acuity was 20/400 in controls at baseline and 3 months post-op; counts fingers in the treated group at baseline and 20/100 at 3 months (p=0.30 between controls and treated at 3 months). All retinas were attached at post-operative month 3.
Vitreous levels of VEGF were significantly lower in the bevacizumab group than the control group (p-value=0.03). Vitreous levels of CTGF were slightly lower in the bevacizumab group compared to controls but this was not statistically significant (p-value=0.38). CTGF levels in the aqueous were strongly correlated to those in the vitreous of controls (Spearman correlation coefficient 0.95, p-value<0.001).
Conclusions
Intravitreal bevacizumab reduces vitreous levels of VEGF and produces a clinically observable alteration in diabetic fibrovascular membranes. Ocular fluid levels of CTGF are not significantly affected within the week following VEGF inhibition. Retinal reattachment rates and visual acuity are not significantly altered by pre-operative intravitreal bevacizumab at post-operative month 3.
doi:10.1001/archophthalmol.2012.1611
PMCID: PMC3984910  PMID: 22965588
3.  Vitreous TIMP-1 levels associate with neovascularization and TGF-β2 levels but not with fibrosis in the clinical course of proliferative diabetic retinopathy 
In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and CCN2 (connective tissue growth factor; CTGF) cause blindness by neovascularization and subsequent fibrosis. This angio-fibrotic switch is associated with a shift in the balance between vitreous levels of CCN2 and VEGF in the eye. Here, we investigated the possible involvement of other important mediators of fibrosis, tissue inhibitor of metalloproteinases (TIMP)-1 and transforming growth factor (TGF)-β2, and of the matrix metalloproteinases (MMP)-2 and MMP-9, in the natural course of PDR. TIMP-1, activated TGF-β2, CCN2 and VEGF levels were measured by ELISA in 78 vitreous samples of patients with PDR (n = 28), diabetic patients without PDR (n = 24), and patients with the diabetes-unrelated retinal conditions macular hole (n = 10) or macular pucker (n = 16), and were related to MMP-2 and MMP-9 activity on zymograms and to clinical data, including degree of intra-ocular neovascularization and fibrosis. TIMP-1, CCN2 and VEGF levels, but not activated TGF-β2 levels, were significantly increased in the vitreous of diabetic patients, with the highest levels in PDR patients. CCN2 and the CCN2/VEGF ratio were the strongest predictors of degree of fibrosis. In diabetic patients with or without PDR, activated TGF-β2 levels correlated with TIMP-1 levels, whereas in PDR patients, TIMP-1 levels, MMP-2 and proMMP-9 were associated with degree of neovascularization, like VEGF levels, but not with fibrosis. We confirm here our previous findings that retinal fibrosis in PDR patients is significantly correlated with vitreous CCN2 levels and the CCN2/VEGF ratio. In contrast, TIMP-1, MMP-2 and MMP-9 appear to have a role in the angiogenic phase rather than in the fibrotic phase of PDR.
doi:10.1007/s12079-012-0178-y
PMCID: PMC3590360  PMID: 23054594
Diabetic retinopathy; CCN2; VEGF; TGF-β; TIMP-1; MMP-2; MMP-9; Neovascularization; Fibrosis
4.  Changes in vitreous VEGF, bFGF and fibrosis in proliferative diabetic retinopathy after intravitreal bevacizumab 
AIM
To evaluate the relationship between intravitreal bevacizumab (IVB) treatment and the levels of vitreous vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and vitreous-retina surface fibrosis in patients with proliferative diabetic retinopathy (PDR).
METHODS
This study was a prospective, open-label, controlled, randomized clinical trial. Sixty-eight eyes of PDR patients (n=53) and macular hole patients (n=15) were enrolled in this study. Thirty-four eyes of the PDR patients received IVB before vitrectomy. Twenty-three of the 34 PDR patients received IVB treatment 5d before vitrectomy (subgroup a), and 11 of the 34 PDR patients received IVB treatment greater than 2wk prior to vitrectomy (subgroup b). Nineteen of the PDR patients did not receive IVB treatment at any time prior to vitrectomy. The levels of bFGF and VEGF in vitreous samples were measured using enzyme-linked immunosorbent assay (ELISA) and the degree of vitreoretinal fibrosis was characterized using clinical data and data obtained intra-operatively.
RESULTS
In PDR patients, VEGF and bFGF levels were significantly increased compared to non-PDR (control) subject's eyes (P<0.01). In PDR patients, vitreous VEGF levels were significantly decreased following IVB treatment compared to PDR patients that did not receive IVB treatment (P<0.01). The degree of vitreoretinal fibrosis was significantly increased in subgroup b compared to subgroup a(P<0.05) and to patients that did not receive IVB (P<0.05). Vitreous bFGF levels were significantly greater in subgroup b than subgroup a (P<0.01) or in patients who did not receive IVB treatment (P<0.05). A Spearman's rank correlation test indicated that higher levels of vitreous bFGF, but not VEGF, correlated with the degree of vitreoretinal fibrosis.
CONCLUSION
We found that bFGF levels increase in PDR patient's vitreous after IVB treatment longer than two weeks prior to vitrectomy and correlated with the degree of fibrosis after IVB treatment. These findings suggest vitreous fibrosis is increased in PDR patients after IVB treatment may be due to increased levels of bFGF.
doi:10.3980/j.issn.2222-3959.2015.06.22
PMCID: PMC4651889  PMID: 26682173
proliferative diabetic retinopathy; vascular endothelial growth factor; basic fibroblast growth factor; fibrosis; bevacizumab
5.  Effect of Intravitreal Bevacizumab on Vascular Endothelial Growth Factor Expression in Patients with Proliferative Diabetic Retinopathy 
Yonsei Medical Journal  2010;52(1):151-157.
Purpose
To investigate the effect of bevacizumab (Avastin; Genentech, San Francisco, CA, USA) on vascular endothelial growth factor (VEGF) expression and inflammation in fibrovascular membranes in patients with proliferative diabetic retinopathy (PDR).
Materials and Methods
Fibrovascular membranes from 19 eyes of 18 patients with PDR were studied using immunohistochemistry and analyzed in the following 3 groups; group 1: 4 inactive PDR eyes, group 2: 10 active PDR eyes treated preoperatively with adjunctive intravitreal bevacizumab, group 3: five active PDR eyes not treated preoperatively with bevacizumab. Immunohistochemical staining for VEGF, CD31 and CD68 were done.
Results
The immunoreactivity to VEGF and CD 31-positive blood vessels was significantly higher in membranes from group 3 than group 1 (p = 0.007 for VEGF, 0.013 for CD 31-positive vessels). Intravitreal bevacizumab caused a reduction in VEGF expression and vascular densities in 4 out of 10 (40%) excised membranes from eyes with PDR. However, six membranes (60%) in group 2 still demonstrated relatively strong VEGF expression and high vascular density. Infiltration of macrophages was observed in 16 out of the 19 membranes, and the density of macrophages was increased in group 2 compared with group 1 (p = 0.043).
Conclusion
Intravitreal bevacizumab injections caused some reduction in VEGF expression and vascular densities in a limited number of active PDR patients. A single intravitreal bevacizumab injection may not be enough to induce complete blockage of VEGF and pathologic neovascularization in active PDR patients. Repeated injections, panretinal photocoagulation and/or PPV may be necessary following intravitreal bevacizumab to reinforce the anti-VEGF effect of the drug.
doi:10.3349/ymj.2011.52.1.151
PMCID: PMC3017691  PMID: 21155048
Intravitreal bevacizumab; proliferative diabetic retinopathy; vascular endothelial growth factor
6.  Osteopontin and Other Regulators of Angiogenesis and Fibrogenesis in the Vitreous from Patients with Proliferative Vitreoretinal Disorders 
Mediators of Inflammation  2012;2012:493043.
The aim of this study was to determine the levels of the angiogenic and fibrogenic factors osteopontin (OPN), high-mobility group box-1 (HMGB1), and connective tissue growth factor (CTGF) and the antiangiogenic and antifibrogenic pigment epithelium-derived factor (PEDF) in the vitreous fluid from patients with proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and rhegmatogenous retinal detachment with no PVR (RD). Vitreous samples from 48 PDR, 17 PVR and 30 RD patients were studied by enzyme-linked immunosorbent assay. OPN, HMGB1, CTGF, and PEDF levels were significantly higher in PDR patients than in RD patients (P < 0.001; 0.002; <0.001; <0.001, resp.). CTGF and PEDF levels were significantly higher in PVR patients than in RD patients (P < 0.001; 0.004, resp.). Exploratory logistic regression analysis identified significant associations between PDR and high levels of HMGB1, CTGF and PEDF, between PDR with active neovascularization and high levels of CTGF and PEDF, and between PDR with traction retinal detachment and high levels of HMGB1. In patients with PDR, there were significant correlations between the levels of PEDF and the levels of OPN (r = 0.544, P = 0.001), HMGB1 (r = 0.719, P < 0.001), and CTGF (r = 0.715, P < 0.001). In patients with PVR, there were significant correlations between the levels of OPN and the levels of HMGB1 (r = 0.484, P = 0.049) and PEDF (r = 0.559, P = 0.02). Our findings suggest that OPN, HMGB1, and CTGF contribute to the pathogenesis of proliferative vitreoretinal disorders and that increased levels of PEDF may be a response to counterbalance the activity of angiogenic and fibrogenic factors in PDR and PVR.
doi:10.1155/2012/493043
PMCID: PMC3465918  PMID: 23055574
7.  Differential expression of connective tissue growth factor in microglia and pericytes in the human diabetic retina 
The British Journal of Ophthalmology  2004;88(8):1082-1087.
Background/aim: Connective tissue growth factor (CTGF) stimulates extracellular matrix formation, fibrosis, and angiogenesis. It has a role in the pathogenesis of diabetic nephropathy and possibly in diabetic retinopathy (DR): in cultured retinal vascular cells CTGF is induced by VEGF-A. To further characterise this role the authors investigated CTGF expression in normal and diabetic human retina.
Methods: CTGF expression patterns were studied by immunohistochemistry in the retina of eyes of 36 diabetic persons and 18 non-diabetic controls and compared with markers of endothelial cells (CD31, PAL-E), pericytes (NG2), astrocytes (GFAP), and microglia (CD45).
Results: In the retina, distinct and specific staining of CTGF was observed in microglia, situated around or in close vicinity of retinal capillaries. In the control cases, sporadic staining of pericytes was also observed within the vascular wall. In contrast, in the retina of people with diabetes, CTGF staining in microglia was decreased and staining in pericytes was increased. This pattern of predominantly pericyte staining was observed in 20 out of 36 diabetic cases and in one out of 18 controls. The altered CTGF staining patterns in the diabetic cases did not correlate to staining of PAL-E, a marker of retinal vascular leakage associated with DR.
Conclusions: The study shows that CTGF is expressed in microglia in the normal retina whereas in a large subset of diabetic persons, CTGF expression shifts to microvascular pericytes. This altered CTGF expression pattern appears unrelated to manifest DR and may therefore represent a preclinical retinal change caused by diabetes. The results suggest a distinct, but as yet unidentified, role of CTGF in the pathogenesis of diabetic retinopathy.
doi:10.1136/bjo.2003.032045
PMCID: PMC1772287  PMID: 15258030
connective tissue growth factor; diabetic retinopathy; human; immunohistochemistry
8.  Intravitreal Injection of Ranibizumab and CTGF shRNA Improves Retinal Gene Expression and Microvessel Ultrastructure in a Rodent Model of Diabetes 
Therapeutic modalities targeting vascular endothelial growth factor (VEGF) have been used to treat neovascularization and macular edema. However, anti-VEGF treatment alone may cause up-regulation of connective tissue growth factor (CTGF) in the retina, increasing the risk of fibrosis and tractional retinal detachment. Therefore, in this study, we employ a novel dual-target intervention that involves intravitreal injection of the VEGF inhibitor ranibizumab and a transfection reagent-treated non-viral vector carrying anti-CTGF short hairpin RNA (shRNA) driven by human RNA polymerase III promoter U6. The effects of the dual-target intervention on the expression of VEGF and CTGF and on microvessel ultrastructure were examined in retina of streptozocin-induced diabetic rats. CTGF was significantly up-regulated at week 8 after diabetic induction, whereas VEGF was not up-regulated until week 10. The high expression of both genes was maintained at week 12. Transmission electron microscopy also revealed progressive exacerbation of microvessel ultrastructure during the same period. In addition, ranibizumab significantly lowered VEGF but elevated CTGF mRNA, whereas CTGF shRNA significantly reduced the mRNA levels of both CTGF and VEGF in diabetic retinas. Importantly, dual-target intervention normalized the transcript levels of both target genes and ameliorated retinal microvessel ultrastructural damage better than either single-target intervention. These results suggest the advantages of dual-target over single-target interventions in diabetic retina and reveal a novel therapeutic modality for diabetic retinopathy.
doi:10.3390/ijms15011606
PMCID: PMC3907889  PMID: 24451141
vascular endothelial growth factor; connective tissue growth factor; diabetic retinopathy; ranibizumab; shRNA
9.  Effects of bevacizumab on the neovascular membrane of proliferative diabetic retinopathy: reduction of endothelial cells and expressions of VEGF and HIF-1α 
Molecular Vision  2012;18:1-9.
Purpose
Anti-vascular endothelial growth factor (VEGF) agents have recently been used intravitreally during the perioperative period for proliferative diabetic retinopathy (PDR). However, the mechanism of theraputic effects of the agents remains unclear. This study aimed to investigate the effects of intravitreal bevacizumab (IVB) on retinal vascular endothelial cells and expressions of VEGF and hypoxia inducible factor-1α (HIF-1α) in PDR.
Methods
Twenty-four patients with PDR were enrolled and randomized to two groups. Twelve eyes of 12 patients of each group received either an intravitreal injection of 1.25 mg bevacizumab or a sham injection 6 days before vitrectomy. Neovascular membranes (NVMs) were collected during pars plana vitrectomy. The numbers of vascular endothelial cells in the NVMs were counted after staining with hematoxylin and eosin and von Willebrand. The expressions of VEGF and HIF-1α in the NVMs were detected through immunohistochemistry. Ten epiretinal membrane specimens from patients with proliferative vitreoretinopathy (PVR) without IVB treatment were set as an additional control.
Results
The number of vascular endothelial cells in NVMs of the IVB pretreated group was significantly lower than that of the sham group (21.5±3.94 versus 41.33±7.44, p=0.003). The IVB pretreated group also showed significantly lower levels of VEGF and HIF-1α in NVMs than those of the sham group (PHIF-1α=0.02, PVEGF<0.001). A stepwise regression analysis showed that IVB was a significant negative predictor for the numbers of vascular endothelial cells (β=–0.89, p<0.001) and the expressions of VEGF (β=–0.85, p<0.001) and HIF-1α (β=–0.64, p=0.001) in PDR patients. Epiretinal membranes of the PVR group showed negative staining of VEGF and HIF-1α.
Conclusions
Pretreatment with IVB in patients with PDR significantly decreased vascular endothelial cells and expressions of VEGF and HIF-1α, which further supports preoperative use of IVB in such patients.
PMCID: PMC3253067  PMID: 22232563
10.  Elevated LRP6 levels correlate with vascular endothelial growth factor in the vitreous of proliferative diabetic retinopathy 
Molecular Vision  2015;21:665-672.
Purpose
To measure intravitreal low-density lipoprotein receptor-related protein 6 (LRP6) and vascular endothelial growth factor (VEGF) levels in the eyes of patients with proliferative diabetic retinopathy (PDR) and to observe their correlation with PDR activity.
Methods
Fifty-five eyes of 55 patients were enrolled consecutively. Vitreous samples from 30 eyes with PDR and 25 eyes with nondiabetic macular disease were collected. Active PDR was present in 16 patients and quiescent PDR in 14 patients according to retinal neovascularization. LRP6 and VEGF concentrations in samples were determined using enzyme-linked immunosorbent assay (ELISA).
Results
ELISA revealed significant increases in the vitreous levels of VEGF in eyes affected with PDR compared to the controls (p<0.001). The mean concentrations of LRP6 were also higher in the vitreous samples from patients with PDR compared to the nondiabetic controls: 39.85 ng/ml and 15.48 ng/ml, respectively (p=0.002). In addition, the vitreous levels of LRP6 and VEGF were significantly higher in active PDR than in quiescent PDR (p=0.022 and p=0.015, respectively). Furthermore, a significant positive correlation was found between intravitreal levels of LRP6 and VEGF in patients with PDR (r=0.567, p=0.001). However, comparison of patients with PDR with controls revealed that the plasma levels of LRP6 were not significantly different between the two groups (p=0.636).
Conclusions
LRP6 and VEGF levels in the vitreous body from patients with PDR were increased and correlated mutually. LRP6 may be a good diagnostic biomarker and a new therapeutic target for PDR.
PMCID: PMC4462953  PMID: 26120271
11.  Angiotensin II and vascular endothelial growth factor in the vitreous fluid of patients with proliferative diabetic retinopathy 
Aims: To investigate the correlation between the level of angiotensin II and vascular endothelial growth factor (VEGF) in the vitreous fluid and the severity of proliferative diabetic retinopathy (PDR).
Methods: During vitreoretinal surgery at the Tokyo Women's Medical University, vitreous fluid samples were obtained from 51 eyes of diabetic patients with PDR, six eyes of diabetic patients without retinopathy, and 16 eyes of non-diabetic patients with ocular disease (controls). The VEGF levels in vitreous fluid and plasma were determined by enzyme linked immunosorbent assay, while angiotensin II levels were measured by radioimmunoassay.
Results: The vitreous fluid levels of VEGF and angiotensin II were significantly higher in patients with PDR than in non-diabetic patients or diabetic patients without retinopathy (all p<0.0001). The vitreous fluid level of angiotensin II was significantly correlated with that of VEGF (p<0.0001), and the vitreous concentrations of both VEGF and angiotensin II were significantly higher in patients with active PDR than in those with quiescent PDR (p<0.0001 and p=0.0005, respectively).
Conclusion: The authors found that both angiotensin II and VEGF levels were significantly higher in the vitreous fluid of patients with PDR than in that of non-diabetic patients or diabetic patients without retinopathy, and that the levels of both angiotensin II and VEGF were elevated in the active stage of PDR. These findings suggest that angiotensin II contributes to the development and progression of PDR in combination with VEGF.
PMCID: PMC1771054  PMID: 11864890
proliferative diabetic retinopathy; angiotensin II; vascular endothelial growth factor; angiogenesis
12.  Extracellular SOD and VEGF are increased in vitreous bodies from proliferative diabetic retinopathy patients 
Molecular Vision  2009;15:2663-2672.
Purpose
To evaluate the relationship between vascular endothelial growth factor (VEGF) and extracellular superoxide dismutase (EC-SOD) in vitreous body and serum in patients with proliferative diabetic retinopathy (PDR), and investigate the role of EC-SOD in PDR by evaluating its angiostatic effect, using an in vitro angiogenesis model. To investigate the role of EC-SOD in PDR by evaluating its angiostatic effect, using an in vitro angiogenesis model.
Methods
EC-SOD and VEGF concentrations in vitreous and serum samples from PDR and macular hole (MH) were measured by ELISA. The effects of EC-SOD on VEGF-induced proliferation, migration, and tube formation were evaluated using human umbilical vein endothelial cells (HUVECs). Moreover, the effects of EC-SOD on VEGF-induced proliferation and migration were evaluated in HUVECs and primary normal human retinal microvascular endothelial cells.
Results
Intravitreal concentrations of EC-SOD were significantly higher (p<0.01) in PDR (58.0±23.8 ng/ml, mean±SD) than in MH (29.3±6.6 ng/ml). Intravitreal concentrations of VEGF were dramatically higher (p<0.01) in PDR (798.2±882.7 pg/ml) than in MH (17.7±15.5 pg/ml). The serum concentrations of EC-SOD and VEGF did not differ between the two patient groups. The vitreous concentrations of VEGF correlated with those of EC-SOD in all patients (rs=0.61, p<0.001). In HUVECs, EC-SOD at 100 ng/ml significantly suppressed VEGF-induced proliferation and tube formation, but not VEGF-induced migration.
Conclusions
EC-SOD was increased together with VEGF in the vitreous body from PDR patients, suggesting that EC-SOD may play a pivotal role in the pathogenesis of angiogenesis.
PMCID: PMC2791042  PMID: 20011081
13.  Apelin in epiretinal membranes of patients with proliferative diabetic retinopathy 
Molecular Vision  2014;20:1122-1131.
Purpose
Formation of epiretinal membranes (ERMs) in the posterior fundus results in visual impairment. ERMs have been associated with numerous clinical conditions, including proliferative diabetic retinopathy (PDR), a neovascular disease. Apelin has been identified as a novel angiogenesis contributor. The aim of this study was to investigate the correlation between apelin and ERMs after PDR.
Methods
ERM samples were obtained by vitrectomy from 12 subjects with PDR (aged 57±6 years; duration of diabetes 16±7 years), and 12 subjects with idiopathic ERM (aged 68±5 years). The samples were processed for immunohistochemistry and reverse transcription–PCR (RT–PCR). We also analyzed samples from patients with PDR who received an intravitreal injection of bevacizumab (IVB) before vitrectomy.
Results
The mRNA expression of apelin was significantly higher in the PDR ERMs than in the idiopathic ERMs. Accordingly, immunohistochemical analysis revealed strong expression of apelin in all eight PDR ERMs without IVB, and was double-labeled with glial fibrillary acidic protein antibody (GFAP), platelet endothelial cell adhesion molecule-1 (CD31), cytokeratin (CK) and vascular endothelial growth factor (VEGF) but not with fibronectin. They were mainly located in the adventitia. In contrast, the expression of apelin was lower in the PDR ERMs after IVB and the idiopathic ERMs.
Conclusions
The results showed that apelin was involved in the formation of ERMs and promoted the formation of adventitia, including glial, endothelial, and RPE cells. Bevacizumab blocked the expression of apelin and regressed gliosis and angiogenesis.
PMCID: PMC4119233  PMID: 25324682
14.  Effects of laser photocoagulation on serum angiopoietin-1, angiopoietin-2, angiopoietin-1/angiopoietin-2 ratio, and soluble angiopoietin receptor Tie-2 levels in type 2 diabetic patients with proliferative diabetic retinopathy 
AIM
To determine the effects of laser photocoagulation on serum levels of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), soluble angiopoietin receptor Tie-2 (Tie-2), Ang-1/Ang-2 ratio and vascular endothelial growth factor (VEGF) in patients with type 2 diabetes mellitus (T2DM) and proliferative diabetic retinopathy (PDR). We also explored the role of the Ang/Tie system in PDR.
METHODS
160 patients with T2DM, including 50 patients with non-diabetic retinopathy (NDR), 58 patients with non-proliferative diabetic retinopathy (NPDR), and 52 patients with PDR were enrolled in this study. Serum Ang-1, Ang-2, Tie-2 receptor and VEGF levels were measured using enzyme-linked immunosorbent assays for all patients and were repeated in 26 patients who underwent laser photocoagulation two months after the procedure.
RESULTS
The median levels of Ang-2 and VEGF in serum were significantly higher in the NPDR group (4.23 ng/mL and 303.2 pg/mL, respectively) compared to the NDR group (2.67 ng/mL and 159.8 pg/mL, respectively, P<0.01), with the highest level in the PDR group (6.26 ng/mL and 531.2 pg/mL, respectively, P<0.01). The median level of Ang-1 was significantly higher in the NPDR group (10.77 ng/mL) compared to the NDR group (9.31 ng/mL) and the PDR groups (9.54 ng/mL) (P<0.05), while no difference was observed between the PDR and NDR groups. Ang-1/Ang-2 ratio of PDR group was lowest in three groups (1.49 vs 2.69 and 2.90, both P<0.01). The median level of Tie-2 was not significantly different among three groups (P>0.05). Ang-2 was positively correlated with VEGF and Tie-2 in the PDR and NPDR groups (both P<0.05). Among the 26 patients who underwent laser photocoagulation, serum Ang-2 and VEGF levels significantly decreased (both P<0.05), whereas serum Ang-1 level and Ang-1/Ang-2 ratio were weakly increased (P>0.05). The median levels of Ang-2 and VEGF in serum were highest in PDR group, however, Ang-1/Ang-2 ratio of PDR group was lowest in three groups.
CONCLUSION
Laser photocoagulation can reduce serum Ang-2 and VEGF levels. The Ang/Tie system and VEGF play an important role in the development and progression of T2DM patients with PDR.
doi:10.3980/j.issn.2222-3959.2014.04.11
PMCID: PMC4137200  PMID: 25161936
angiopoietin; receptor protein tyrosine kinase; diabetes mellitus; type 2; retinopathy; laser photocoagulation
15.  Inhibition of connective tissue growth factor by small interfering ribonucleic acid prevents increase in extracellular matrix molecules in a rodent model of diabetic retinopathy 
Molecular Vision  2012;18:874-886.
Purpose
Connective tissue growth factor (CTGF) is a profibrotic factor that induces extracellular matrix (ECM) production and angiogenesis, two processes involved in diabetic retinopathy (DR). In this study, we examined whether insulin therapy or a CTGF-specific small interfering RNA (siRNA) administered to diabetic rats decreased the levels of CTGF and of selected putative downstream genes in the retina.
Methods
Rats with streptozotocin-induced diabetes were used. Animals received either no treatment for 12 weeks or were administered constant insulin therapy. MRNA and protein levels of CTGF and select ECM genes were determined using real-time PCR and western blotting of the retina. Localization of CTGF in the retina was visualized using immunohistochemistry. A group of diabetic rats received intravitreal injection of CTGF siRNA, and the retinas were examined three days later.
Results
CTGF mRNA and protein significantly increased in the retinas of diabetic rats. Immunohistochemistry indicated that retinal Müller cells of diabetic rats expressed CTGF. Hyperglycemia upregulated mRNA levels of fibronectin, laminin β1, collagen IVα3, and vascular endothelial growth factor (VEGF), and this increase was prevented by insulin therapy. Treatment of diabetic rats with CTGF siRNA decreased laminin β1, collagen IVα3 mRNA, and CTGF mRNA and protein but did not affect fibronectin or vascular endothelial growth factor mRNA levels.
Conclusions
These results indicate that CTGF and ECM genes can be regulated using insulin. Importantly, these results also suggest that CTGF regulates changes in ECM molecules in DR.
PMCID: PMC3327439  PMID: 22511849
16.  Risk evaluation of outcome of vitreous surgery for proliferative diabetic retinopathy based on vitreous level of vascular endothelial growth factor and angiotensin II 
The British Journal of Ophthalmology  2004;88(8):1064-1068.
Aims: To ascertain whether measurement of the vitreous fluid levels of vascular endothelial growth factor (VEGF) or angiotensin II (Ang II) could predict the outcome of vitreous surgery in patients with proliferative diabetic retinopathy (PDR).
Methods: A prospective observational case study was performed in 61 consecutive patients (61 eyes) with PDR who underwent vitreoretinal surgery. Vitreous fluid samples were obtained during surgery. The VEGF level in vitreous fluid and plasma was determined by enzyme linked immunosorbent assay, while the Ang II level was measured by radioimmunoassay. Patients were prospectively followed for 6 months and the postoperative outcome was analysed by logistic regression analysis.
Results: No improvement and/or progression of PDR was seen in 15 (25%) of the 61 eyes. Vitreous levels of VEGF and Ang II were significantly higher in eyes with progression of PDR than in eyes with regression of PDR (p = 0.0044, and p = 0.0178, respectively). Multivariate logistic regression analysis showed that the vitreous VEGF level increased along with the progression of PDR after vitreous surgery (odds ratio 2.48, p = 0.0008).
Conclusion: A high vitreous fluid VEGF level is associated with a significant risk of postoperative progression of PDR. The vitreous level of VEGF at the time of surgery may be a useful predictor of the outcome.
doi:10.1136/bjo.2003.032656
PMCID: PMC1772284  PMID: 15258026
proliferative diabetic retinopathy; vitreous surgery; risk factor; vascular endothelial growth factor
17.  S100A4 is upregulated in proliferative diabetic retinopathy and correlates with markers of angiogenesis and fibrogenesis 
Molecular Vision  2014;20:1209-1224.
Purpose
The calcium-binding protein S100A4 is implicated in cancer cell invasion and metastasis, the stimulation of angiogenesis, the progression of fibrosis, and inflammatory disorders. We investigated the expression of S100A4 and correlated it with clinical disease activity as well as with the levels of osteopontin (OPN), soluble syndecan-1, and vascular endothelial growth factor (VEGF) in proliferative diabetic retinopathy (PDR). To reinforce the findings at the functional level, we examined the expressions of S100A4 and OPN in the retinas of diabetic rats and in human retinal microvascular endothelial cells (HRMECs) following exposure to VEGF and the proinflammatory cytokine tumor necrosis factor-α (TNF-α).
Methods
Vitreous samples from 30 PDR and 30 nondiabetic patients, epiretinal membranes from 14 patients with PDR, the retinas of rats, and HRMECs were studied by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, western blot analysis, and co-immunoprecipitation.
Results
ELISA revealed a significant increase in the expressions of S100A4, OPN, soluble syndecan-1, and VEGF in vitreous samples from PDR patients compared to nondiabetic controls (p = 0.001; <0.001; <0.001; <0.001, respectively). Significant positive correlations were found between the levels of S100A4, OPN (r = 0.52, p = <0.001), soluble syndecan-1 (r = 0.37, p = 0.012), and VEGF (r = 0.29, p = 0.044). In epiretinal membranes, S100A4 was expressed in the vascular endothelial cells and stromal CD45-expressing leukocytes. A significant positive correlation was detected between the number of blood vessels expressing CD31 and the number of stromal cells expressing S100A4 (r = 0.77, p = 0.001). Western blot analysis revealed a significant increase in the expressions of S100A4 and both intact and cleaved OPN in vitreous samples from PDR patients compared to nondiabetic controls, as well as in the retinas of diabetic rats. Co-immunoprecipitation studies revealed a positive interaction between S100A4 and the receptor for advanced glycation end products (RAGE) in the retinas of diabetic rats. TNF-α—but not VEGF—induced the upregulations of S100A4 and both intact and cleaved OPN in HRMECs.
Conclusions
S100A4 represents a valuable vitreous marker molecule in the pathogenesis of PDR and might become a new target for the treatment of PDR.
PMCID: PMC4160443  PMID: 25253987
18.  Local and genetic determinants of vascular endothelial growth factor expression in advanced proliferative diabetic retinopathy 
Molecular Vision  2008;14:1382-1387.
Purpose
In proliferative diabetic retinopathy (PDR) and other angiogenesis-associated diseases, increased levels of cytokines, inflammatory cells, growth factors, and angiogenic factors are present. Vascular endothelial growth factor (VEGF) appears to play a central role in mediating microvascular pathology in PDR. The purpose of the present study was to search for the association between the –634 C/G polymorphism of the VEGF gene and PDR. Moreover, it was hoped to determine whether serum and vitreous levels of VEGF are affected by genetic factors.
Methods
This cross-sectional case-control study enrolled 349 unrelated Slovene subjects (Caucasians) with type 2 diabetes mellitus. The case group consisted of 206 patients with an advanced form of PDR and for whom vitrectomy was performed, and the control group had 143 patients who had no clinical signs of diabetic retinopathy but did have type 2 diabetes of more than 10 years duration. To analyze the genotype distribution we had to compare the genotype frequencies in diabetics with PDR (cases, n=206) and diabetics without diabetic retinopathy (control group, n=143). Additionally, to evaluate the effect of diabetes on the VEGF serum levels 2 groups, diabetics and non diabetics, were compared. First group were diabetics (diabetics with PDR, n=104), and second group were 29 subjects without diabetes.
Results
The –634 C/G VEGF polymorphism was not associated with PDR. Mean serum and vitreous levels of VEGF were statistically significantly higher in PDR in comparison to the control group. Moreover, significantly higher serum and vitreous levels of VEGF were demonstrated in diabetics with the CC genotype compared to those with the other (CG + GG) genotypes.
Conclusions
VEGF is an important cytokine in PDR. Despite the effect of the –634 C/G VEGF polymorphism on serum and vitreous levels of VEGF in PDR, it failed to contribute to the genetic susceptibility to PDR.
PMCID: PMC2493030  PMID: 18682813
19.  Aflibercept Traps Galectin-1, an Angiogenic Factor Associated with Diabetic Retinopathy 
Scientific Reports  2015;5:17946.
Vascular endothelial growth factor (VEGF)-A-driven angiogenesis contributes to various disorders including cancer and proliferative diabetic retinopathy (PDR). Among several VEGF-A blockers clinically used is aflibercept, a chimeric VEGFR1/VEGFR2-based decoy receptor fused to the Fc fragment of IgG1 (i.e., VEGFR1/VEGFR2-Fc). Here, we revealed a novel anti-angiogenic function for aflibercept beyond its antagonism against VEGF family members. Immunoprecipitation and mass spectrometry analyses identified galectin-1 as an aflibercept-interacting protein. Biolayer interferometry revealed aflibercept binding to galectin-1 with higher affinity than VEGFR1-Fc and VEGFR2-Fc, which was abolished by deglycosylation of aflibercept with peptide:N-glycosidase F. Retinal LGALS1/Galectin-1 mRNA expression was enhanced in vitro by hypoxic stimulation and in vivo by induction of diseases including diabetes. Galectin-1 immunoreactivity co-localized with VEGFR2 in neovascular tissues surgically excised from human eyes with PDR. Compared with non-diabetic controls, intravitreal galectin-1 protein levels were elevated in PDR eyes, showing no correlation with increased VEGF-A levels. Preoperative injection of bevacizumab, a monoclonal antibody to VEGF-A, reduced the VEGF-A, but not galectin-1, levels. Galectin-1 application to human retinal microvascular endothelial cells up-regulated VEGFR2 phosphorylation, which was eliminated by aflibercept. Our present findings demonstrated the neutralizing efficacy of aflibercept against galectin-1, an angiogenic factor associated with PDR independently of VEGF-A.
doi:10.1038/srep17946
PMCID: PMC4673700  PMID: 26648523
20.  Vitreous and Plasma VEGF Levels as Predictive Factors in the Progression of Proliferative Diabetic Retinopathy after Vitrectomy 
PLoS ONE  2014;9(10):e110531.
Purpose
To investigate the vitreous and plasma levels of vascular endothelial growth factor (VEGF) in patients with proliferative diabetic retinopathy (PDR) and to determine whether they predict a disease prognosis after primary vitrectomy.
Methods
Fifty patients (50 eyes) with PDR who underwent pars plana vitrectomy (PPV) and 56 healthy controls (56 eyes) were enrolled in this retrospective study. Clinical data were collected and analyzed. Vitreous and plasma VEGF concentrations were measured using enzyme-linked immunosorbent assays. VEGF levels and clinical data were compared and analyzed to see if they provide a prognosis of PDR progression after primary vitrectomy at more than 6 months follow-up. Correlation of VEGF concentrations between vitreous fluid and plasma was analyzed.
Results
The average BCVA was significantly improved after surgery (P<0.001). Vitreous and plasma VEGF levels were significantly elevated in PDR patients than those in healthy controls (Pvitreous<0.001; Pplasma<0.001). Both vitreous and plasma VEGF levels were significantly higher in PDR progression group than in stable group (Pvitreous<0.001; Pplasma = 0.004). Multivariate logistic regression analyses showed that the increased vitreous VEGF level was associated with the progression of PDR after primary PPV (OR = 1.539; P = 0.036). Vitreous VEGF level was positively associated with plasma VEGF level in PDR patients (P<0.001).
Conclusion
The increased VEGF level in vitreous fluid may be identified as a significant predictive factor for the outcome of vitrectomy in patients with PDR.
doi:10.1371/journal.pone.0110531
PMCID: PMC4199758  PMID: 25329921
21.  Upregulated expression of vascular endothelial growth factor in proliferative diabetic retinopathy. 
AIMS/BACKGROUND: Vascular endothelial growth factor (VEGF) is a hypoxia induced angiogenic factor. Recent studies have shown that high levels of VEGF accumulate in the vitreous of patients with proliferative diabetic retinopathy (PDR). The purpose of the present study was to identify the retinal cells that upregulate VEGF expression in human PDR patients representing progressive stages of retina deterioration. METHODS: Thirteen formalin fixed and paraffin embedded enucleated eyes with PDR were used (eyes were enucleated because of being blind and painful as a result of neovascular glaucoma). Thin retina sections were hybridised in situ with a VEGF specific probe, to identify cells producing VEGF mRNA. RESULTS: All eyes with PDR showed upregulated expression of VEGF mRNA, specifically in the cells of the neurosensory retina. VEGF expression was upregulated in all three nuclear layers--namely, the ganglion cell layer, the inner nuclear layer, and the outer nuclear layer. However, in each patient, VEGF producing cells were mostly distributed in a different layer, or even confined to a specific region in that layer. For example, expression by the outer nuclear layer was mostly detected in detached (presumably hypoxic) regions of the retina. CONCLUSIONS: Progression of PDR is distinguished by a sustained, upregulated expression of VEGF by the neurosensory retina. Cells in all retina layers can potentially contribute to augmented VEGF production. The restricted population of VEGF producing cells in each case is likely to represent cells residing in ischaemic regions of the retina. Thus, VEGF may function as a linking factor between retinal ischaemia and PDR associated neovascularisation.
Images
PMCID: PMC505435  PMID: 8703862
22.  Clinical efficacy and mechanistic evaluation of aflibercept for proliferative diabetic retinopathy (acronym CLARITY): a multicentre phase IIb randomised active-controlled clinical trial 
BMJ Open  2015;5(9):e008405.
Introduction
Proliferative diabetic retinopathy (PDR) is the main cause of severe visual loss in people with diabetes mellitus. The standard treatment for this condition is panretinal photocoagulation (PRP). This laser treatment is inherently destructive, with predictable adverse effects on visual function, and a safer alternative is required. Intravitreal injection of vascular endothelial growth factor (VEGF) inhibitors can induce short-term regression of retinal neovascularisation. The aim of this randomised controlled trial is to determine the efficacy, safety and cost-effectiveness of intravitreal aflibercept, an inhibitor of VEGF-A, VEGF-B and placental growth factor (PLGF), in PDR, and to investigate the impact on local oxygenation.
Methods and analysis
This is a phase IIb randomised controlled single-masked multicentre clinical trial to determine the impact of repeated intravitreal aflibercept injections in the treatment and prevention of PDR. 220 participants with treatment-naïve or treated but active retinal neovascularisation in at least one eye will be randomly allocated 1:1 to intravitreal aflibercept injections or PRP for a period of 52 weeks. The primary outcome is the change in best-corrected visual acuity in the study eye at 52 weeks. Secondary outcomes include changes from baseline in other visual functions, anatomical changes and cost-effectiveness. Ocular and non-ocular adverse events will also be reported over 52 weeks.
Ethics and dissemination
The study has been approved by the National Research Ethics Service (NRES) committee with respect to scientific content and compliance with applicable research and human subjects’ regulations. Findings will be reported through scientific publications and research conferences. The results of this study will provide clinical evidence for the feasibility, efficacy safety and cost-effectiveness of intravitreal aflibercept for PDR.
Trial registration number
ISRCTN 32207582.
doi:10.1136/bmjopen-2015-008405
PMCID: PMC4577925  PMID: 26369798
23.  A review of anti-VEGF agents for proliferative diabetic retinopathy 
Eye  2014;28(5):510-520.
Previous research has implicated vascular endothelial growth factor (VEGF) in the pathogenesis of diabetic retinopathy (DR). Although many studies reviewed the use of anti-VEGF for diabetic macular oedema, little has been written about the use of anti-VEGF for proliferative diabetic retinopathy (PDR). This study is a review of relevant publications dealing with the use of anti-VEGF for the treatment of PDR. The articles were identified through systematic searches of PUBMED and the Cochrane Central Register of Controlled Trials. At the end of each section, we summarized the level of evidence of the scientific literature. Off-label use of anti-VEGF agents was found to be beneficial in PDR, especially in cases with neovascular glaucoma, persistent vitreous haemorrhage, and before vitrectomy. The disadvantages of the use of anti-VEGF are its short-effect duration, causing tractional retinal detachment in cases with pre-existing pre-retinal fibrosis and endophthalmitis in rare cases. There is no conclusive evidence from large randomized trials regarding the efficacy of anti-VEGF treatment in PDR. However, numerous case series, sound biochemical mechanism of action, and increasing experience with using anti-VEGF drugs can be used to support the ongoing use of this treatment modality in selected patients.
doi:10.1038/eye.2014.13
PMCID: PMC4017101  PMID: 24525867
diabetic retinopathy; proliferative diabetic retinopathy; panretinal photocoagulation; ranibizumab; Bevacizumab; vascular endothelial growth factor
24.  Levels of vascular endothelial growth factor are elevated in the vitreous of patients with subretinal neovascularisation. 
BACKGROUND: Vascular endothelial growth factor (VEGF) has been shown to play a major role in intraocular neovascularisation in ischaemic retinal diseases. Subretinal neovascularisation is an important cause of central visual loss, but little is known about the role of this growth factor in its pathogenesis. The aim of this study was to investigate the possible role of VEGF in the development of subretinal neovascularisation. METHODS: Undiluted vitreous samples were obtained from patients undergoing vitrectomy for removal of non-age-related subfoveal neovascular membranes (SFNM). For comparison vitreous from patients undergoing vitrectomy for idiopathic full thickness macular holes (FTMH) and proliferative diabetic retinopathy (PDR) was used. Indirect enzyme linked immunosorbent assay (ELISA), with an antibody directed against the conserved N-terminal region of human VEGF165, was used to determine vitreous levels of VEGF. The growth factor was also localised in the vitreous of patients with SFNM by western blot analysis. RESULTS: The mean (SE) VEGF concentration in the vitreous of patients with SFNM was 27.78 (2.22) ng/ml (n = 8), FTMH was 16.62 (0.9) ng/ml (n = 18), and PDR was 37.77 (3.28) ng/ml (n = 16). The differences between the PDR group and SFNM group versus the FTMH group were both significant (p = 0.0001 and p = 0.0015) as analysed by the Wilcoxon rank sum test). CONCLUSIONS: Vitreous levels of VEGF are significantly elevated in eyes with non-age-related subretinal neovascularisation compared with eyes with FTMH but not as elevated as in PDR. This suggests that VEGF is involved in subretinal angiogenesis.
Images
PMCID: PMC505466  PMID: 8703891
25.  Proliferative diabetic retinopathy and relations among antioxidant activity, oxidative stress, and VEGF in the vitreous body 
Molecular Vision  2010;16:130-136.
Purpose
To investigate the relationships among antioxidant activities, oxidative stress, and vascular endothelial growth factor (VEGF) in the vitreous body and serum from proliferative diabetic retinopathy (PDR) patients.
Methods
In 21 patients with PDR and 21 controls with macular hole (MH), the VEGF and lipid peroxide (Nε-hexanoyl-lysine [HEL]) levels in the vitreous and serum were measured by enzyme-linked immunosorbent assay, while antioxidant capacity (potential antioxidant [PAO]) was measured by chemical reduction of Cu2+.
Results
Both the PAO and HEL levels in the vitreous and serum were significantly higher in PDR patients than in those with MH (both p<0.01). The VEGF concentrations in the vitreous were higher in PDR patients than in those with MH (p<0.01); however, the VEGF concentrations in the serum were not different between the two groups (p=0.95). Positive correlations were found between the PAO and VEGF concentrations and between the HEL and VEGF concentrations in the vitreous of both the PDR and the MH patients.
Conclusions
Our study revealed that the PAO, HEL, and VEGF concentrations in the vitreous were increased in PDR versus MH patients and that there were positive correlations among these factors. This is consistent with VEGF and lipid peroxide levels in the vitreous playing some role in the pathogenesis of PDR.
PMCID: PMC2817014  PMID: 20142849

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