Angiogenic potential of vitreous from proliferative diabetic retinopathy and eales' disease patients

Proliferative Diabetic Retinopathy (PDR) and Eales' Disease (ED) have different aetiologies although they share certain common clinical symptoms including pre-retinal neovascularization. Since there is a need to understand if the shared end-stage angiogenic pathology of PDR and ED is driven by common stimulating factors, we have studied the cytokines contained in vitreous from both patient groups and analyzed the angiogenic potential of these samples in vitro.Vitreous samples from patients with PDR (n = 13) and ED (n = 5) were quantified for various cytokines using a cytokine biochip array and sandwich ELISA. An additional group of patients (n = 5) with macular hole (MH) was also studied for comparison. To determine the angiogenic potential of these vitreous samples, they were analyzed for their ability to induce tubulogenesis in human microvascular endothelial cells. Further, the effect of anti-VEGF (Ranibizumab) and anti-IL-6 antibodies were studied on vitreous-mediated vascular tube formation.Elevated levels of IL-6, IL-8, MCP-1 and VEGF were observed in vitreous of both PDR and ED when compared to MH. PDR and ED vitreous induced greater levels of endothelial cell tube formation compared to controls without vitreous (P<0.05). When VEGF in vitreous was neutralized by clinically-relevant concentrations of Ranibizumab, tube length was reduced significantly in 5 of 6 PDR and 3 of 5 ED samples. Moreover, when treated with IL-6 neutralizing antibody, apparent reduction (71.4%) was observed in PDR vitreous samples.We have demonstrated that vitreous specimens from PDR and ED patients share common elevations of pro-inflammatory and pro-angiogenic cytokines. This suggests that common cytokine profiles link these two conditions

Matrix metalloproteinases (MMP-8, MMP-9) and the tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) in patients with fungal keratitis

Purpose: To study the infiltrating cells and quantify the levels of matrix metalloproteinases (MMP-8, MMP-9) and tissue inhibitor of metalloproteinases (TIMP-1, TIMP-2) in the cornea, tear, and serum of patients with fungal keratitis. Methods: Experimental study. Infected corneal tissue from 4 patients with fungal keratitis (group 1) scheduled to undergo therapeutic keratoplasty accounted for the histopathologic and cytospin smear analysis. Ten patients with fungal keratitis from group 2 served for the quantification of MMPs and TIMPs. Five patients with keratoconus undergoing penetrating keratoplasty and 5 cadaver corneas were chosen as controls for group 2. Corneal buttons obtained during keratoplasty, 15 to 20 &#956;L of tears collected using the capillary flow method, and 3 mL of blood was obtained from patients with fungal keratitis and patients with keratoconus. Corneal button sections from group 1 were stained with hematoxylin and eosin and Grocott methenamine silver nitrate for the histopathologic studies and Giemsa staining for the cytospin smear analysis. Enzyme-linked immunosorbent assay was used for the quantification of total MMP-8, MMP-9, TIMP-1, and TIMP-2 in the corneal homogenates, tear, and serum samples of group 2. Results: Corneal sections from group 1 revealed dense fungal filaments and a large proportion (91.4% &#177; 38%) of polymorphonuclear leukocytes (PMNs). Significant elevation in the levels of MMP-8 and MMP-9 (P &lt; 0.05) in the fungal keratitis corneas was observed in group 2 compared with the cadaver and keratoconus corneas. The ratio of MMP/TIMP was also higher in the fungal keratitis corneas. Conclusions: Infiltrating PMNs in the cornea of patients with fungal keratitis contributed to the increased activities of MMP-8 and MMP-9, thereby enhancing tissue destruction and derangement

Proinflammatory cytokines and angiogenic and anti-angiogenic factors in vitreous of patients with proliferative diabetic retinopathy and eales' disease

Purpose: To investigate the mechanism of angiogenesis in proliferative diabetic retinopathy (PDR) and Eales' disease (ED) on the basis of the levels of proinflammatory cytokines, angiogenic growth factor, and antiangiogenic factor in the vitreous humor. Methods: Twenty-five patients with PDR, 10 patients with ED, and 25 with macular hole (MH) as control subjects were studied. The concentration of the proinflammatory cytokines interleukin-6 (IL-6), IL-8, IL-1&#946;; chemokine-monocyte chemoattractant protein-1 (MCP-1); angiogenic factor-vascular endothelial growth factor (VEGF); and antiangiogenic factor-pigment epithelium derived factor (PEDF) in the vitreous fluid obtained from the eyes during vitrectomy were measured by sandwich enzyme linked immunosorbent assay (ELISA). Results: IL-6, IL-8, MCP-1, and VEGF levels in the vitreous were significantly higher in PDR (P &lt; 0.0001) and ED (P &lt; 0.0001) than in MH patients. Conversely, the vitreous level of PEDF was significantly reduced in PDR (P &lt; 0.0001) but not in ED. A significant correlation was observed between VEGF and IL-6 in ED patients. Conclusion: The authors demonstrate the importance of VEGF in retinal neovascularization of ED which is an idiopathic inflammatory venous occlusion. Further study is required to understand the interrelationship between VEGF and inflammatory cytokines in PDR and ED

Representative phase images of tube formation induced by vitreous from PDR/ED/MH patients in human dermal microvascular endothelial cell (HMEC).

<p>2×10⁵ HMECs in triplicate were exposed to vitreous alone or with RZB (0.125 mg/ml). Tube formation was observed and images were captured after 48 hours incubation. Each panel shows a part of the representative well. The tube length was quantified by NIS-Elements software (Nikon). Scale bar  = 100 µm. A. Control (without vitreous); B and C - PDR vitreous-induced tube formation, which had high levels of VEGF/IL-6/MCP-1; D-PDR vitreous with trace levels of cytokines showing a very few tube formation. E- G - ED vitreous-induced tube formation as in PDR. H - MH vitreous. I and J are images of vascular tubes in the presence of PDR/ED vitreous and anti-VEGF antibody, showing reduction in tube length compared to C and E respectively. Number in the images denotes the patient ID as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107551#pone-0107551-t001" target="_blank">table 1</a>. PDR - Proliferative diabetic retinopathy; ED- Eales' disease; MH- Macular Hole. RZB – Ranibizumab.</p

Effects of anti-IL-6 on vitreous-induced tube formation.

<p>HMECs were mixed with PDR or ED vitreous with or without anti-IL6 (0.1 µg/ml). Tube formation was observed and images were captured after 48 hours of incubation (in duplicate cultures). A and B are PDR or ED vitreous-induced tube formation. C and D are images of vascular tubes of corresponding vitreous with anti-IL6. E and F – shows the effect of anti-IL6 on vascular tube length for 6 PDR (E) and 2 ED (F) vitreous samples. The tube length was quantified by NIS-Elements software (Nikon). Number in the image denotes the patient ID as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107551#pone-0107551-t001" target="_blank">table 1</a>. PDR-Proliferative diabetic retinopathy; ED- Eales' disease. Scale bar  = 100 µm.</p

Resveratrol reverses the adverse effects of bevacizumab on cultured ARPE-19 cells

Abstract Age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR) are one of the major causes of blindness caused by neo-vascular changes in the retina. Intravitreal anti-VEGF injections are widely used in the treatment of wet-AMD and PDR. A significant percentage of treated patients have complications of repeated injections. Resveratrol (RES) is a polyphenol phytoalexin with anti-oxidative, anti-inflammatory and anti-proliferative properties. Hence, we hypothesized that if RES is used in combination with bevacizumab (BEV, anti-VEGF), it could reverse the adverse effects that precipitate fibrotic changes, drusen formation, tractional retinal detachment and so on. Human retinal pigment epithelial cells were treated with various combinations of BEV and RES. There was partial reduction in secreted VEGF levels compared to untreated controls. Epithelial-mesenchymal transition was lower in BEV + RES treated cultures compared to BEV treated cultures. The proliferation status was similar in BEV + RES as well as BEV treated cultures both groups. Phagocytosis was enhanced in the presence of BEV + RES compared to BEV. Furthermore, we observed that notch signaling was involved in reversing the adverse effects of BEV. This study paves way for a combinatorial strategy to treat as well as prevent adverse effects of therapy in patients with wet AMD and PDR

Angiogenic potential of vitreous in capillary tube formation.

<p>Experimental details are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107551#pone-0107551-g002" target="_blank">Fig. 2</a>. (A) PDR vitreous, (B) ED vitreous, (C) MH vitreous. Bar graph shows the mean concentration of the triplicate of each sample. PDR-proliferative diabetic retinopathy; ED- Eales' disease; MH- Macular Hole. Number in X-axis denotes the patient number as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107551#pone-0107551-t001" target="_blank">table 1</a>. **P<0.001; *P<0.05.</p