Defects in fully developed retinal vasculature of Pls-deficient mice.

<p><b>A.</b> Quantification of retinal capillaries on confocal microscopy pictures of whole-mounted and FITC-dextran-perfused retinas from control and Pls-deficient mice. Three concentric circles (yellow dashed circles) centred on the optic nerve head were drawn to delimit central, mid-peripheric and far peripheric areas. The number of capillaries crossing a ring situated in the middle of each area (red dashed circles) was counted over 360°. <b>B.</b> Quantitative evaluation of retinal capillaries in adult control and Pls-deficient mice. The capillary density was significantly increased in central, mid-peripheric and far-peripheric areas of the retina of Pls-deficient mice (n = 6/group). <b>C.</b> Representative fluorescence microscopy pictures of isolectin-B4-labelled (ILB4) endothelial cells on retinal wholemounts of control and Pls-deficient mice. Retinal vasculature of adult Pls-deficient mice was characterized by tortuous large vessels (arrows), dilated arteries and veins (stars) and vascular lesions (circle). <b>D.</b> Representative electroretinographic response of Pls-deficient and control mice. The ERG traces of Pls-deficient mice (red trace) exhibited a specific alteration of the positive b-wave (arrow) that is typical of retinal hypoxia. Scale bar = 75 µm.</p

Pericyte recruitment and vessel stabilization in retinas of control, Pls-deficient and iPLA2-inhibited mice.

<p><b>A.</b> Relative expression of <i>PDGF</i> and <i>PDGFR</i> in control animals, Pls-deficient mice and iPLA2-treated mice examined by RT-qPCR (n = 4–6 per group) at PN7, PN14 and PN21. The relative expression of the genes was normalized to <i>gusb, hprt</i> and <i>b2m</i> genes and compared to the control level (set at 1). The expression of <i>pdgfb</i> and <i>pdgfrb</i> genes was significantly reduced in mice with iPLA2 inhibition at PN7 and in Pls-deficient and iPLA2-inhibited mice at PN21. <b>*</b>: Statistically significant difference when compared to control group (Student’s t-test, <i>P</i><0.05); <b>**</b>: statistically significant difference when compared to control group (Student’s t-test, <i>P</i><0.01); <b>***</b>: statistically significant difference when compared to control group (Student’s t-test, <i>P</i><0.001). <b>B., C. and D.</b> Confocal microscopy pictures of anti-NG2- (green, labelling pericytes) and isolectin-B4- (ILB4, red, labelling endothelial cells) labelled retinal whole mounts of control and Pls-deficient mice and mice at PN7 (<b>B.</b>), PN14 (<b>C.</b>) and PN21 (<b>D.</b>). No abnormality was observed in retinas of Pls-deficient and iPLA2-inhibited mice, suggesting normal vessel stabilization by pericytes. Scale bar = 75 µm.</p

Temporarily delayed outgrowth and increased retinal angiogenic activity in Pls-deficient and iPLA2-inhibited animals.

<p><b>A.</b> Representative fluorescence microscopy pictures of isolectin-B4-labelled (ILB4) endothelial cells on retinal whole mounts of control, Pls-deficient and iPLA2-treated mice at PN7. Dashed and solid arrows indicate the outgrowth distance and the retinal radius, respectively. <b>B.</b> Quantitative analysis of retinal outgrowth represented by the ratio (%) of outgrowth distance (l) to retinal radius length (L) from the optic nerve to the retinal periphery (R = l/L×100). A delay in retinal vascular outgrowth was observed in Pls-deficient mice and iPLA2-inhibited mice at PN7 (n = 6–18 per group) but not at PN14 (n = 6–10 per group). *: statistically significant difference when compared to control group (Kruskal-Wallis test, <i>P</i><0.05); <b>**</b>: statistically significant difference when compared to control group (Kruskal-Wallis test, <i>P</i><0.01). <b>C. and D.</b> Transcriptional analysis of angiogenic factors in retinas of control, Pls-deficient and iPLA2-inhibited mice (n = 6–8 per group) at PN7 (<b>C.</b>) and PN14 (<b>D.</b>). The relative expression of angiogenic genes was normalized to <i>gusb</i>, <i>hprt</i> and <i>b2m</i> genes and compared to control levels (set as 1). Pls-deficient and iPLA2-treated mice displayed fluctuations in the expression of genes encoding for pro- and anti-angiogenic proteins that were related to vascular phenotype. <b>*</b>: Statistically significant difference when compared to control group (Student’s t-test, <i>P</i><0.05); <b>**</b>: statistically significant difference when compared to control group (Student’s t-test, <i>P</i><0.01); <b>***</b>: statistically significant difference when compared to control group (Student’s t-test, <i>P</i><0.001). <b>E.</b> Representative pictures of ILB4-labelled retinal wholemounts showing angiogenic sprouts (arrowheads) in control, Pls-deficient and iPLA2-inhibited mice at PN14. <b>F.</b> Quantitative evaluation of angiogenic sprouts in retinas of control, Pls-deficient, and iPLA2-inhibited mice at PN14 (n = 10–12 per group). The number of angiogenic sprouts was significantly increased in mice deficient in Pls and in animals displaying a chemical inhibition of iPLA2 when compared to control mice (set as 100), suggesting greater sprouting activity. <b>***</b>: Statistically significant difference when compared to control group (Kruskal-Wallis test, <i>P</i><0.001).</p

Influence of astrocyte template on vessel architecture of adult mice.

<p><b>A.</b> Fluorescence microscopy pictures of anti-GFAP- (green) and isolectin-B4- (ILB4, red) labelled retinal whole mounts of control and Pls-deficient mice and mice at adult age. ILB4-positive cells (endothelial cells) and GFAP-positive cells (astrocytes) were co-localized, suggesting that vessel tortuosity is a secondary consequence of an abnormal arrangement of the astrocytic bed. <b>B.</b> Confocal microscopy pictures of anti-GFAP- (green, labelling astrocytes) and isolectin-B4- (ILB4, red, labelling endothelial cells) labelled retinal whole mounts of control, Pls-deficient and iPLA2-inhibited mice at PN14 and PN21. The retinal vasculatures of Pls-deficient and iPLA2-inhibited mice were characterized by vascular lesions that co-localized with activated astrocytes. These activated-astrocyte areas were sharply outlined at PN14, whereas they were less immuno-reactive to GFAP and had a fibrous aspect at PN21. <b>C.</b> Relative expression of GFAP, a marker of astroglial activity in control animals, Pls-deficient mice and iPLA2-treated mice examined by RT-qPCR (n = 4–6 per group) at PN14 and PN21. The relative expression of the <i>gfap</i> gene was normalized to <i>gusb</i>, <i>hprt</i> and <i>b2m</i> genes and compared to the control level (set at 1). The expression of the <i>gfap</i> gene was significantly increased in Pls-deficient and iPLA2-treated mice at PN14, suggesting increased astroglial activity at this age. <b>*</b>: Statistically significant difference when compared to control group (Student’s t-test, <i>P</i><0.05). Scale bar = 75 µm.</p

Greater retinal astroglial activity in Pls-deficient and iPLA2-inhibited animals at PN7.

<p><b>A.</b> Relative expression of genes encoding for two markers of astroglial activity in control animals (n = 4–5), Pls-deficient mice (n = 5) and iPLA2-treated mice (n = 6) examined by RT-qPCR. The relative expression of <i>gfap</i> and <i>fn1</i> genes encoding for GFAP and fibronectin, respectively, was normalized to <i>gusb, hprt</i> and <i>b2m</i> genes and compared to the control level (set at 1). The expression of <i>gfap</i> and <i>fn1</i> genes was significantly increased in Pls-deficient and iPLA2-treated mice at PN7, suggesting increased astroglial activity. <b>*</b>: Statistically significant difference when compared to control group (Student’s t-test, <i>P</i><0.05); <b>**</b>: statistically significant difference when compared to control group (Student’s t-test, <i>P</i><0.01). <b>B.</b> Confocal microscopy of anti-fibronectin (green) and ILB4 (red) labelled retinal whole mounts of control, Pls-deficient and iPLA2-inhibited mice at PN7 (n = 3–6 per group). The secretion of fibronectin protein by retinal astrocytes at the front of vascular outgrowth was more pronounced in Pls-deficient and iPLA2-inhibited animals when compared to controls, confirming greater astroglial activity. Scale bar = 150 µm.</p

Defects in hyaloid vasculature regression in mice with iPLA2 inhibition.

<p>A. Quantification of hyaloid arteries (HA) and <i>vasa hyaloidea propria</i> (VHP) vessels on depth-scan images from confocal cSLO angiography. VHP vessels (stars) were visualized and quantified at the level of the posterior lens, whereas HAs (arrowheads) were counted in the posterior eye in control and iPLA2-inhibited animals (n = 11 per group). <b>B.</b> Quantitative evaluation of hyaloid arteries (HA) and <i>vasa hyaloidea propria</i> (VHP) vessels in control and iPLA2-inhibited mice at PN21 and PN27. The numbers of HAs and VHPs were significantly higher in iPLA2-inhibited mice at PN21 and PN27 when compared to controls, thus confirming that the control of hyaloid vessel regression by Pls involves the iPLA2 enzyme. <b>*</b>: Statistically significant difference when compared to control group (Kruskal-Wallis test, <i>P</i><0.05); *<b>*</b>: statistically significant difference when compared to control group (Kruskal-Wallis test, <i>P</i><0.01); <b>***</b>: statistically significant difference when compared to control group (Kruskal-Wallis test, <i>P</i><0.001).</p

Symbol, name, assay ID, and GenBank reference of assayed genes.

<p>Symbol, name, assay ID, and GenBank reference of assayed genes.</p

Structure of conventional phospholipids and plasmalogens.

<p>Conventional phospholipids such as phosphatidyl-ethanolamine contain ester bonds to link R1 and R2 acyl-moieties at the <i>sn-</i>1 and <i>sn-</i>2 positions of glycerol, respectively. Ethanolamine-plasmalogens (also termed plasmenyl-ethanolamine) are characterized by the presence of a vinyl-ether bond at the <i>sn-</i>1 position of the glycerol backbone to link alkenyl-moieties and an ester bond at the <i>sn-</i>2 position to link acyl-residues.</p

Concentration of individual species of phosphatidyl-choline (PC) and plasmenyl-choline (PlsC) in erythrocytes from controls and diabetic patients without or with mild, moderate, severe or proliferative diabetic retinopathy (results are expressed as µg of mg phospholipids).

<p>Abbreviations of individual PC and PlsC species are as follows: position on the glycerol backbone as shown as sn-1/sn-2 of the fatty acid and fatty alcohol radicals (abbreviated as number of carbons: number of double bonds).</p>a<p>Based on Kruskall-Wallis test, significantly different when compared to controls (<i>P</i><0.05).</p><p>Concentration of individual species of phosphatidyl-choline (PC) and plasmenyl-choline (PlsC) in erythrocytes from controls and diabetic patients without or with mild, moderate, severe or proliferative diabetic retinopathy (results are expressed as µg of mg phospholipids).</p

Red blood cell membrane levels of docosahexaenoic acid (DHA), total n-3 PUFA, n-6 PUFA, and total n-6 PUFA/total n-3 PUFA in control subjects and diabetic patients with or without DR.

<p>Results are expressed as means ± SEM. * based on Kruskall-Wallis test (<i>P</i><0.05).</p