PGF expression in diabetic retina.

<p><b>(a–b) Comparison of PGF staining in non diabetic (a) and diabetic (b) retinas.</b> (<b>a</b>) Sections of eyes from adult control non diabetic rat showed co-expression of PGF and GFAP in glial Muller cells from the gcl (arrowheads) to the inl, and PGF expression in glial Müller cells which are not immuno-reactive for GFAP (white star). Scale bar = 50 µm. (<b>b</b>) A similar pattern was observed on retinal sections of eyes from three-month-old diabetic rats, with a strong immuno-reactivity for PGF at the gcl level. <b>(c) PGF detection by Western-blot in diabetic and non-diabetic retinas, from 1, 2, 5 and 12 month-old rats</b>. For each lane in which 40 µg of proteins were deposited, the blood sugar level of the represented rats is indicated between parentheses. <b>(d–e) Immunostaining for PGF and GFAP in sections from pVAX2-rPGF-1 ET- treated diabetic rat eyes, one month after ET.</b> Sections show PGF-expressing infiltrating cells in the sub retinal space (<b>d</b>, arrows) and confirmed PGF expression by RMG cells (<b>d</b>, arrowheads). GFAP staining showed gliosis induced by RMG cells (<b>d, e</b>). <b>ch</b>, choroid; <b>gcl</b>, ganglion cell layer; <b>inl,</b> inner nuclear layer; <b>ipl</b>, inner plexiform layer; <b>onl</b>, outer nuclear layer; <b>rpe</b>, retinal pigmented epithelium; Scale bar = 100 µm.</p

Morphological analysis of flat-mounted retina labeled with FITC-conjugated lectin from <i>Bandeira simplicifolia</i>.

<p><b>(a) Illustration of the procedure followed with “ImageJ” software.</b> (<b>a₁</b>) Selection of seven areas per group, in mid (700x500 px) and extreme (400x300 px) peripheries on mosaics (3007x2904 px) made with microscopic images at low magnification (x4). (<b>a₂</b>) Application of steerable filters for ridge detection in the selected image, with the plug-in “Feature Detector” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017462#pone.0017462-Jacob1" target="_blank">[57]</a>. (<b>a₃</b>) Transformation of the filtered image into a binary image allowing selection of the retinal vessels to calculate the vascularized area (area covered by vessels out of total area, <b>Av/At</b>). (<b>a₄</b>) Derivation of the vascular skeleton from the binary image, using the procedure “skeletonizes”. This second binary picture was used to calculate the tortuosity index corresponding to the ratio: real vessel length (<b>L2</b>, in red) out of the length of an imaginary straight line on the measured vessel (<b>L1</b>, in blue) as shown in d₁ and d₂. <b>(b–c) Quantitative analysis of the retinal vasculature from control (CTL) and PGF ET-treated eyes. (b)</b> Vascularized area calculated from the binary images which are illustrated in c₁ and c₂. <b>All data</b>: CTL, 18.8%±0.5%; PGF-ET, 23.7%±1.1%; ** p = 0.0034. <b>Mid periphery</b>: CTL, 19.4%±0.7%; PGF-ET, treated eyes 25.1%±0.8%; Bonferroni, ** p<0.01. <b>Extreme periphery</b>: CTL, 18.2%±0.7%; PGF-ET treated eyes 21.2%±1.7%; Bonferroni, ns. (<b>c</b>) Tortuosity index of capillaries calculated from the second binary images. CTL, controls 1.053±0.005; PGF-ET, treated eyes 1.180±0.014; *** p<0.0001; PGF ET/CTL = 1.12.</p

Analysis of flat-mounted diabetic retinas labeled with FITC-conjugated lectin from <i>Bandeira simplicifolia</i>, one month after ET.

<p><b>(a) Quantitative analysis of the retinal vasculature from control and PGF ET-treated diabetic rat eyes. (b) Microscopy of flat-mounted control (b₁, b₃) and PGF ET-treated (b₂, b₄) diabetic retinas.</b> (<b>b₁–b₂</b>) Optic microscopy on mosaics made with microscopic images at low magnification (x4) showing no evident difference of the retinal vascularization between control (b₁) and pVAX-2-rPGF-1 ET-treated eyes (b₂), except a weak retinal venous dilation in treated retinas (between arrowheads). (<b>b₃, b₄</b>) Confocal microscopy showing lectin-labeled cell infiltration (arrow) and micro-aneurysmal-like structures observed in both group (insets).</p

Fluorescein angiograms of Brown-Norway rat eyes.

<p><b>(a) Observations with a classic angiograph (Pro III Fundus camera, Kowa), 4 and 6 weeks after pVAX2-rPGF ET</b>. Angiograms were established with a scan angle of 30°. Vascular abnormalities were scored from 0 to 5 in accordance to the following grading: <b>Grade 0</b>, normal retinal vasculature, as observed in control fundus at week 6 (<b>ONH</b>, Optic Nerve Head); <b>+1 point</b> for each of the following changes - dilated (between white arrowheads) or tortuous (white arrows) vessels, microaneurysmal-like hyperfluorescent dots (black arrowheads) <10 or hyper-fluorescence around the ONH; <b>+2 points</b> for microaneurysmal-like hyper-fluorescent dots >10. <b>(b) Observations with a confocal scanning Laser Ophthalmoscope (cSLO, Heidelberg Retina Angiograph I), 2, 3 and 5 weeks after pVAX2-rPGF ET</b>. The same grading was used to score vascular abnormalities. The higher resolution of the cSLO allowed the observation of early fluorescein leakage (<b>Grade 1</b>), and the detection of strong vascular abnormalities at later stage (<b>+ 1 point</b>).</p

Immunostaining for PGF (a–e), GFAP (b, c) and von Willebrand factor (d, e) in membrane excised surgically from a 60-year-old woman with proliferative diabetic retinopathy.

<p>(<b>a</b>) PGF was immuno-detected on the epi-retinal membrane. It was localized in GFAP (<b>b–c</b>, arrows) and von Willebrand factor (vW) (<b>d–e</b>, arrows) immuno-positive cells. Red stars indicate cells expression only GFAP (c) or vW (d), and green stars show non-endothelial cells expressing PGF.</p

Effect of PGF over expression on retinal histology and on RPE cells.

<p><b>(a–b) Histological sections of retinas from eyes embedded in historesin and stained by Toluidine blue, three months after saline (a₁, b₁) and pVAX2-rPGF-1 ET (a₂–a₄, b₂–b₄).</b> (<b>a₁</b>) Normal histological section of the retina after pVAX2 ET. <b>ch</b>, choroid; <b>gcl</b>, ganglion cell layer; <b>inl</b>, inner nuclear layer; <b>onl</b>, outer nuclear layer; <b>rpe</b>, retinal pigmented epithelium; <b>Sc</b>, Sclera. Scale bar (a1–4)  = 100 µm. (<b>a₂</b>) Histological section of pVAX2-rPGF-1 ET-treated retinas, showing vascular retinal abnormalities in the inner part of the peripheral retina. (<b>a₃</b>) Sections showing retinal detachment (star) associated with pre-retinal proliferation (arrow) and edema at the ONL level (magnified in b3 inset). (<b>a₄</b>) Sections showing retinal detachment (star) associated with RPE barrier breakdown. (<b>b₁</b>) Section at high magnification showing normal RPE cells after PBS ET. Scale bar (a1–4)  = 10 µm. (<b>b₂–b₄</b>) Magnified RPE cells of the retinal sections (a₂–a₄) demonstrating morphological changes of RPE cells which appears swollen, and dilation of the chorio-capillaries (b₂, b₄ black arrowheads). Background has been subtracted in all pictures. <b>(c) Sustained blood-retinal barrier breakdown induced by rPGF-1 over expression.</b> Tight junctions were observed by occludin immuno-histochemistry on whole flat-mounted RPE cells. (<b>c1–3</b>) Normal tight junction-associated occludin was observed in PBS-ET treated eyes. Scale bar = 20 µm. (<b>c2–4</b>) Two months after pVAX2-rPGF-1 ET, junctions remained opened between some RPE cells (white arrows). At this opened junction level, infiltrating cells (stars) are also detected by the DAPI staining and recognized by the small size of their nuclei as compared to those of RPE cells.</p

Control of rPGF-1 expression.

<p><b>(a) PGF and VEGF expression analysis at the mRNA level, two months after pVAX2-rPGF ET.</b> Mean relative band intensity ±SEM determined after detection of rPGF-1, rVEGF and GAPDH mRNA expression in control eyes (in white, n = 5) or in pVAX2-rPGF-1 ET treated eyes (in black, n = 5); **, p = 0.0079. Representative RT-PCR products visualized by ethidium bromide staining are shown. <b>(b) Localization of rPGF-1 expression on sections of the ciliary muscle, one month after saline ET (Control) or pVAX2-rPGF ET (PGF ET).</b> Alpha-smooth muscle actin (αSMA) - Texas-Red labeling allows localization of the ciliary muscle (<b>cm</b>). Compared to control eye, white arrowheads indicate that after pVAX2-rPGF-1 ET, the protein of interest which is labeled in green, is localized in muscle fibers and in the epithelium of ciliary bodies (<b>cb</b>). No staining was observed when the primary antibody was omitted. Scale bars: 100 µm.</p

Angiogenic response on retinal vascular cells.

<p><b>(a-d) Confocal microscopy of vascular abnormalities within the neural retina, one month after pVAX2-rPGF-1 ET.</b> Confocal microscopy of flat-mounted neuroretinas from eyes in which rPGF-1 was over-expressed shows lectin-positive blood vessels and cells at different depth levels, from the superficial (1) to the deep vascular plexus (4). Scale bars = 50 µm. (<b>a, b</b>) The abnormalities noticed <i>in vivo</i> on SLO angiograms from two eyes were found on retinal flat-mounts by comparison of the vascular architectures. (<b>c, d</b>) For illustration, retinal flat-mounts have been turned into grey-scale pictures. Red asterisks indicate the presence of infiltrating cells around vascular abnormalization. <b>(e) Optic microscopy of vascular abnormalities within flat-mounted retinas, two months after ET.</b> Vascular abnormalities (<b>e₁</b>), sprouts (<b>e₂</b>) and microaneurysmal-like structures (<b>e₃</b>) were observed between the inner and the middle vascular beds. <b>(f) Q PCR analysis of VEGF, IL-1beta, TNF-alpha and IL-6, two months after ET,</b> in retina of control (in white, n = 3 in duplicate) and pVAX2-rPGF-1 ET treated (in black, n = 5 in duplicate) retina from BN rats. ***, p<0.005 <i>versus</i> wildtype mRNA expression. Data represent mean ± SEM.</p