Additional file 1 of Vitrectomy with sulfur hexafluoride versus air tamponade for idiopathic macular hole: a retrospective study

Supplementary Material

The Effects of Pleiotrophin in Proliferative Diabetic Retinopathy

<div><p>Pleiotrophin (PTN), a secreted, multifunctional cytokine, is involved in angiogenic, fibrotic and neurodegenerative diseases. However, little is known about its effects on diabetic retinopathy, a neurovascular disease. To investigate the role of PTN in proliferative diabetic retinopathy (PDR), PTN concentration in the vitreous was evaluated in PDR patients and non-diabetic controls. PTN expression was observed in epiretinal membranes from patients. PTN knockdown was performed using small interfering (si)RNA, and the effects on retinal pigment epithelium (RPE) cells and human umbilical vascular endothelia cells (HUVECs) were observed <i>in vitro</i> under hyperglycemic and hypoxic conditions. Cell attachment, proliferation, migration, tube formation, cell cycle, apoptosis, extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation, and VEGF levels were studied. The vitreous PTN concentration in PDR patients was higher than that in non-diabetic controls, and PTN was highly expressed in the fibrovascular membranes of PDR patients. Under hyperglycemic and hypoxic conditions, PTN knockdown reduced cell attachment, proliferation, migration, and tube formation and induced cell cycle arrest and apoptosis <i>in vitro</i>. Mechanically, PTN depletion decreased ERK 1/2 phosphorylation. Recombinant PTN up regulated the concentration of VEGF <i>in vitro</i>, which can be attenuated by the ERK 1/2 inhibitor. Taken together, our results implied that elevated PTN in PDR patients might participate in the critical processes of the development of PDR, most likely playing roles in angiogenesis and proliferation, possibly by activating the ERK 1/2 pathway and regulating VEGF secretion. These findings provide new insight into the roles of PTN in PDR and suggest that PTN may become a new target for therapeutic intervention in PDR.</p></div

Effect of PTN-siRNA on RPE cells and HUVECs.

<p>PTN expression in human RPE cells and HUVECs was significantly knocked down in PTN-siRNA treated groups at the mRNA level, as measured by real-time RT-PCR 48 h after transfection. There’s no difference in NC and NS groups. The expression of NC was set to 100%. Data are expressed as the means ± SD of results from at least three independent experiments (**P < 0.01).</p

Effects of PTN on HUVEC tube formation.

<p>Microphotographs are representative of tube-like structure generation after a 4-h incubation on Matrigel. The length of tube branches per view field was measured. PTN-siRNA-treated HUVECs (panel C) showed an impaired capacity to form a regular vascular network. The length of the angiogenesis network was statistically significantly decreased compared to the control groups. Each experiment was repeated at least three separate times. Data are presented as the means ± SD. NC was set to 100%. **P < 0.01.</p

Detection of PTN on epiretinal membranes from PDR patients.

<p>Micrographs show immunofluorescence double staining of epiretinal PDR membrane sections (left column) and EMM sections (right column) for PTN (A, B) and CD31 (C, D). Nuclei were stained with DAPI (E, F). Images in the fourth row are merged (G, H). Double staining revealed PDR membranes expressing PTN and CD31. PTN expression was detected in CD31-labeled cells (panel G, short arrow) and in fibrous-like tissue (panel G, long arrow). However, neither PTN nor CD31 was detected in EMMs. Bar, 50 μm.</p

Effects of PTN on ERK 1/2 phosphorylation.

<p>Immunoblot imaging (A) and statistical analysis (B) for p-ERK 1/2 signaling pathways. The statistical results show that PTN-siRNA-treated group significantly inhibited p-ERK 1/2 levels. Western blot analyses were repeated three times, and qualitatively similar results were obtained. Data are presented as the means ± SD. **P < 0.01.</p

Effects of PTN on cell apoptosis and the cell cycle.

<p>(A) The statistical results of PTN on cell apoptosis. The percentage of early apoptotic cells plus late apoptotic cells in the PTN-siRNA-treated group was significantly higher than the controls, indicating that PTN depletion induces cell apoptosis in RPE cells and HUVECs. (B) The statistical data for the cell cycle distributions of NS-siRNA and PTN-siRNA treated groups in RPE cells and HUVECs. PTN-siRNA treatment induces cell cycle arrest in the G0/G1 phase and results in reduced cell numbers in the S phase. Data are presented as the means ± SD and presented in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115523#pone.0115523.t002" target="_blank">Table 2</a></b>. Each experiment was repeated three times. **P < 0.01.</p

Immunocytochemical assays for PTN in RPE cells and HUVECs.

<p>The fluorescence (green), representing PTN expression in cells, was very strong in NC (A, B) and NS (C, D) cells but was barely detectable in the PTN-siRNA–treated cells (E, F), further confirming the effectiveness of PTN-siRNA transfection. Bar, 100 μm.</p

Effects of recombinant PTN (rPTN) on VEGF₁₆₅ expression.

<p>The statistical results of secreted VEGF₁₆₅ concentrations, as measured with an ELISA assay at different rPTN concentrations at 6-h incubation timepoints in RPE cells (A) and HUVECs (B). The effects of co-incubation of U0126(10 μM, inhibitor of ERK 1/2) and rPTN on VEGF₁₆₅ expression were also observed. Each experiment was repeated in 6 wells and was duplicated at least three times. Data are presented as the means ± SD and presented in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115523#pone.0115523.t003" target="_blank">Table 3</a></b>. *P < 0.05 and ***P< 0.001.</p

Demographic, clinical, and laboratory data of subjects included in the study.

<p>Demographic, clinical, and laboratory data of subjects included in the study.</p