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.
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.
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.
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.
Diabetic retinopathy; CCN2; VEGF; TGF-β; TIMP-1; MMP-2; MMP-9; Neovascularization; Fibrosis
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.
proliferative diabetic retinopathy; angiotensin II; vascular endothelial growth factor; angiogenesis
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.
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.
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.
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.
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.
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).
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.
Intravitreal bevacizumab; proliferative diabetic retinopathy; vascular endothelial growth factor
To determine the intravitreal levels of vascular endothelial growth factor (VEGF) in eyes with anterior hyaloidal fibrovascular proliferation (AHFVP).
Three eyes of three patients who underwent vitrectomy for proliferative diabetic retinopathy (PDR) and subsequently developed an AHFVP (AHFVP group) were studied. We measured the level of VEGF in vitreous samples collected at the primary and following operations by enzyme-linked immunosorbent assay. The vitreous levels of VEGF in 25 eyes of 22 patients with PDR were also studied as controls (PDR group).
The averaged VEGF level in the samples collected at the primary surgery was 1.98 ± 2.23 ng/mL in the PDR group, and it was 9.07, 1.94, and 8.07 ng/mL in the AHFVP cases. After the primary surgery, the VEGF level rose up to 49.50, 15.60, and 50.60 ng/mL at the subsequent surgeries for respective cases of the AHFVP group. These levels of VEGF were more than five times higher than the baseline at the primary surgery.
The subsequent increase of the VEGF level after the primary surgery in eyes with an AHFVP suggests that the vitreous levels of VEGF are associated with the development of the AHFVP although only three eyes were studied.
VEGF; AHFVP; diabetic retinopathy
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.
connective tissue growth factor; diabetic retinopathy; human; immunohistochemistry
To investigate the significance of netrin-1 and vascular endothelial growth factor (VEGF) in the pathogenesis of retinal angiogenesis, the levels of netrin-1 and VEGF in the vitreous fluid and serum of the proliferative diabetic retinopathy (PDR) and non-proliferative diabetic retinopathy (non-PDR) patients were measured. We then determined the netrin-1 and VEGF expression in the oxygen induced retinopathy (OIR) mice retina.
A total of 18 eyes from 18 patients were included in our study and 10 of them were collected from PDR patients and 8 from non-PDR patients. Undiluted vitreous fluid samples were collected during pars plana vitrectomy. Appropriate blood samples were collected if possible. Netrin-1 and VEGF levels in the vitreous fluid and plasma were determined by Enzyme-linked Immunosorbent Assays. OIR mice models were established, and netrin-1 and VEGF levels were determined by immunohistochemistry analysis.
The levels of netrin-1 and VEGF in the vitreous of PDR patients were significantly higher than those in the controls (Mediannetrin-1=509.94 vs. 85.91 pg/ml, P<0.001 and MedianVEGF=762.60 vs. 77.52 pg/ml, P<0.001). Netrin-1 was mainly expressed in GCL and INL of the retina in mice. Both netrin-1 and VEGF were up-regulated in OIR mice.
Netrin-1 and VEGF levels were elevated in vitreous fluid of the PDR patients and the OIR mice retina. Therefore, netrin-1 may play an important role in pathological retinal angiogenesis.
Retinal angiogenesis; Netrin-1; VEGF
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.
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.
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.
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.
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.
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.
proliferative diabetic retinopathy; vitreous surgery; risk factor; vascular endothelial growth factor
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.
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.
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.
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.
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.
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.
A major issue concerning clinical trials is the availability of standardized assays to evaluate drug efficacy. Ideally, such assays should test the effect of a putative drug on the expression of a biomarker in biological fluids. In a recent study by Kuiper et al. (PLOS One, 3(7): e2675). The relative levels of vascular endothelial growth factor (VEGF) and CCN2 (connective tissue growth factor [CTGF]) were examined in proliferative diabetic retinopathy (PDR). This paper is the subject of this commentary.
This study was conducted to determine levels of angiogenic and endothelial progenitor cell mobilizing (vasculogenic) factors in vitreous fluid from proliferative diabetic retinopathy (PDR) patients and correlate their levels with clinical disease activity. Vascular endothelial growth factor (VEGF), soluble vascular endothelial growth factor receptor-2 (sVEGFR-2), stem cell factor (SCF), soluble c-kit (s-kit), endothelial nitric oxide synthase (eNOS), and prostaglandin E2 (PGE2) levels were measured by ELISA in vitreous samples from 34 PDR and 15 nondiabetic patients. eNOS was not detected. VEGF, sVEGFR-2, SCF, and s-kit levels were significantly higher in PDR with active neovascularization compared with quiescent PDR and nondiabetic patients (P < 0.001; 0.007; 0.001; <0.001, resp.). In contrast, PGE2 levels were significantly higher in nondiabetic patients compared with PDR patients (P < 0.001). There were significant correlations between levels of sVEGFR-2 versus SCF (r = 0.950, P < 0.001), sVEGFR-2 versus s-kit (r = 0.941, P < 0.001), and SCF versus s-kit (r = 0.970, P < 0.001). Our findings suggest that upregulation of VEGF, sVEGFR-2, SCF, and s-kit supports the contributions of angiogenesis and vasculogenesis in pathogenesis of PDR.
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.
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+.
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.
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.
Background/aim: Angiopoietin 1 and 2 interact with vascular endothelial growth factor (VEGF) to promote angiogenesis in animal and in vitro models. Although VEGF concentrations are elevated, there is little information regarding angiopoietin concentration in the vitreous of patients with diabetic retinopathy.
Methods: Angiopoietin concentrations were measured by luminescence immunoassay in vitreous samples from 17 patients with non-proliferative diabetic retinopathy (NPDR) and clinically significant diabetic macular oedema (CSMO), 10 patients with proliferative diabetic retinopathy (PDR), and five patients with macular hole (controls) obtained at pars plana vitrectomy.
Results: Angiopoietin 1 concentrations were low in patients with macular hole (median 17 pg/ml) while in NPDR with CSMO they were 2002 pg/ml (range 289–5820 pg/ml) and in PDR 186 pg/ml (range 26–2292 pg/ml). Angiopoietin 2 concentrations in NPDR with CSMO were a median of 4000 pg/ml (range 1341–14 329 pg/ml). For both macular hole and PDR patients angiopoietin 2 was below the limit of detection.
Conclusions: Angiopoietin 2 concentration was twice that of angiopoietin 1 in NPDR with CSMO. Angiopoietin 2 is the natural antagonist of angiopoietin 1 which is thought to act as an anti-permeability agent. The predominance of angiopoietin 2 may allow VEGF induced retinal vascular permeability in patients with CSMO. The relatively low concentration of both angiopoietin 1 and 2 in patients with proliferative diabetic retinopathy may reflect the established nature of the neovascularisation in cases proceeding to vitrectomy.
diabetic retinopathy; angiopoietin
The aim of this study was to measure the levels of high-mobility group box-1 (HMGB1) in the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) and to correlate its levels with clinical disease activity and the levels of vascular endothelial growth factor (VEGF), the angiogenic cytokine granulocyte-colony-stimulating factor (G-CSF), the endothelial cell angiogenic markers soluble vascular endothelial-cadherin (sVE-cadherin), and soluble endoglin (sEng). Vitreous samples from 36 PDR and 21 nondiabetic patients were studied by enzyme-linked immunosorbent assay. HMGB1, VEGF, sVE-cadherin, and sEng levels were significantly higher in PDR patients than in nondiabetics (P = 0.008; <0.001; <0.001; 0.003, resp.). G-CSF was detected in only 3 PDR samples. In the whole study group, there was significant positive correlation between the levels of HMGB1, and sVE-cadherin (r = 0.378, P = 0.007). In PDR patients, there was significant negative correlation between the levels of sVE-cadherin and sEng (r = −0.517, P = 0.0005). Exploratory regression analysis identified significant associations between active PDR and high levels of VEGF (odds ratio = 76.4; 95% confidence interval = 6.32–923) and high levels of sEng (odds ratio = 6.01; 95% confidence interval = 1.25–29.0). Our findings suggest that HMGB1, VEGF, sVE-cadherin and sEng regulate the angiogenesis in PDR.
Background/aims: Many cytokines are involved in the pathogenesis of retinal proliferative diseases, but none has been shown to be related to a specific disorder. The aim of this study was to provide a selective marker of diabetes induced proliferative retinopathies.
Methods: 10 vitreous samples from 10 subjects affected by quiescent proliferative diabetic retinopathy (PDR), 20 vitreous samples from 20 subjects affected by active PDR, and 15 samples from 15 patients with proliferative vitreoretinopathy (PVR) were studied. Samples from 18 patients with a macular hole (n = 8) or pucker (n = 10) served as controls. Vitreous samples were obtained via pars plana vitrectomy. The polyamines spermidine, putrescine, and spermine, vascular endothelial growth factor (VEGF), interleukin 8 (IL-8), and transforming growth factor 1β (TGF-1β) were measured by high performance liquid chromatography (HPLC) and enzyme linked immunosorbent assay (ELISA), and the correlation coefficients between the vitreous polyamine content and VEGF, IL-8, and TGF-1β levels were determined.
Results: Spermidine and putrescine were expressed in normal vitreous, but spermine was not detectable. In all the test groups spermidine was 3–4 times higher than in control vitreous and putrescine was similarly lower. The spermine content was up to 15 times higher only in vitreous from patients affected by PDR. Correlation coefficients showed that the spermidine and putrescine level variations correlated with the VEGF and IL-8 content in the active PDR and PVR groups, but not in those with quiescent PDR patients, while spermine was correlated to these cytokines in PDR, but not in PVR groups.
Conclusions: These data suggest a significant role for spermidine and putrescine as markers of proliferative diseases of the retina. The increase in spermine, restricted to diabetic states, may indicate that this polyamine is a unique and specific index of PDR.
polyamines; VEGF; proliferative diabetic retinopathy; proliferative vitreoretinopathy; vitrectomy; laboratory assay
To evaluate serum concentrations of angiogenesis-related cytokines in proliferative diabetic retinopathy (PDR) before and after vitrectomy.
Serum samples were collected from 30 PDR patients with varying severity before and after vitrectomy. Serum concentrations of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), interleukin-8 (IL-8) and interferon-inducible protein-10 (IP-10) were determined by enzyme-linked immunosorbent assays (ELISA).
Serum concentrations of VEGF, PEDF, IL-8 and IP-10 were significantly higher in PDR patients than that in controls, respectively (P<0.05). VEGF concentration decreased significantly in postoperative samples than that in preoperative samples (P<0.05). The concentrations of PEDF, IL-8 and IP-10 did not exhibit significant changes after vitrectomy.
Elevated cytokines levels in serum may be diagnostically useful in PDR. Angiogenesis-related cytokines play important roles in the development of PDR, and would instruct the risk assessment of pathogenetic condition in PDR patients.
proliferative diabetic retinopathy; cytokine; vitrectomy; enzyme-linked immunosorbent assay; angiogenesis
This study aims to investigate the levels of aqueous vascular endothelial growth factor (VEGF) in diabetic patient groups in comparison to normal subjects, and to correlate elevated VEGF with the severity of diabetic retinopathy (DR).
Materials and Methods:
Aqueous samples were obtained from 78 eyes of 74 patients undergoing intraocular surgery and they were examined by the enzyme-linked immunosorbent assay. Color photographs, optical coherence tomography scans, and fluorescein angiography were used to evaluate patients preoperatively.
A strong statistical correlation was found to exist between the level of aqueous VEGF and the severity of DR (P < 0.001), whereas, the VEGF levels in a control group and a diabetic group without DR were not significantly different (P = 0.985). Aqueous VEGF levels were significantly elevated in patients with proliferative DR (PDR) as compared to the control group (P < 0.001), to diabetic patients without retinopathy (NDR) (P < 0.001), and to diabetic patients with nonproliferative DR (NPDR) (P < 0.001). The aqueous VEGF levels were significantly higher in patients with active PDR than in those with quiescent PDR (P = 0.001). On the other hand, a statistically insignificant (P = 0.065) correlation was found between elevated aqueous VEGF and the presence of macular edema in the NPDR group.
VEGF was elevated in the aqueous humor of patients with DR compared to that in normal eyes. The aqueous VEGF level had a strong correlation with the severity of retinopathy along with a statistically insignificant difference in macular edema.
Diabetic retinopathy; macular edema; retinal ischemia; vascular endothelial growth factor
Connective tissue growth factor (CTGF) is a major fibrogenic factor. Increased retinal CTGF levels have been implicated to play a role in diabetic retinopathy. SERPINA3K is a serine proteinase inhibitor, and its levels were decreased in retinas with diabetic retinopathy. The purpose of this study was to investigate the role of SERPINA3K in the regulation of CTGF and fibrogenesis and its mechanism of action.
RESEARCH DESIGN AND METHODS
Adenovirus expressing SERPINA3K was injected intravitreally into streptozotocin-induced diabetic rats. CTGF expression was measured using Western blot analysis and real-time RT-PCR. Fibrosis was evaluated by quantifying retinal fibronectin using enzyme-linked immunosorbent assay. Wnt pathway activation was determined by phosphorylation of LDL receptor–related protein 6, a coreceptor of Wnt ligands, and stabilization of β-catenin, an essential effector of the canonical Wnt pathway.
Ad-SERPINA3K attenuated the CTGF and fibronectin overexpression in retinas of diabetic rats. In cultured retinal cells, SERPINA3K blocked the overproduction of CTGF induced by high glucose. Dickkopf-1, a specific Wnt antagonist, also attenuated the high-glucose–induced CTGF overexpression, indicating a role of Wnt signaling in CTGF overexpression in diabetes. Similarly, increased SERPINA3K blocked Wnt pathway activation in diabetic retinas and in cells treated with high glucose. Further, SERPINA3K also attenuated the Wnt3a-induced activation of the canonical Wnt pathway and the overexpression of CTGF.
SERPINA3K is an antifibrogenic factor, and its antifibrogenic activity is through blocking the Wnt pathway. Decreased SERPINA3K levels may contribute to the fibrosis in diabetic retinopathy.
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.
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.
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.
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.
The purpose of this study was to evaluate cell cycle changes in choroidal endothelial cells treated with varying doses of bevacizumab in the presence of a range of concentrations of vascular endothelial growth factor (VEGF). Bevacizumab, a drug widely used in the treatment of neovascular age-related macular degeneration, choroidal neovascularization, and proliferative diabetic retinopathy, neutralizes all isoforms of VEGF. However, the effect of intravitreal administration of bevacizumab on the choroidal endothelial cell cycle has not been established.
Monkey choroidal endothelial (RF/6A) cells were treated with VEGF 50 ng/mL and escalating doses of bevacizumab 0.1–2 mg/mL for 72 hours. Cell cycle changes in response to bevacizumab were analyzed by flow cytometry and propidium iodide staining. Cell proliferation was measured using the WST-1 assay. Morphological changes were recorded by bright field cell microscopy.
Bevacizumab inhibited proliferation of choroidal endothelial cells by stabilization of the cell cycle in G0/G1 phase. Cell cycle analysis of VEGF-enriched choroidal endothelial cells revealed a predominant increase in the G2/M population (21.84%, P, 0.01) and a decrease in the G0/G1 phase population (55.08%, P, 0.01). Addition of escalating doses of bevacizumab stabilized VEGF-enriched cells in the G0/G1 phase (55.08%, 54.49%, 56.3%, and 64% [P, 0.01]) and arrested proliferation by inhibiting the G2/M phase (21.84%, 21.46%, 20.59%, 20.94%, and 16.1% [P, 0.01]). The increase in G0/G1 subpopulation in VEGF-enriched and bevacizumab-treated cells compared with VEGF-enriched cells alone was dose-dependent.
Bevacizumab arrests proliferation of VEGF-enriched choroidal endothelial cells by stabilizing the cell cycle in the G0/G1 phase and inhibiting the G2/M phase in a dose-dependent fashion.
bevacizumab; age-related macular degeneration; vascular endothelial growth factor