Choroidal flatmounts showing accumulation of rhodamine labeled NP and expression of GFP plasmid in the CNV.

<p>The CNV lesions are delineated by arrowheads in bright field images with false blue color (A and E). FITC-filtered images highlight the GFP expression one day after systemic injection of Rd-NP-GFPg (B) whereas non-targeted NP (Rd-ntNP-GFPg) does not induce GFP expression in CNV (F). Cy3-filtered images highlight that rhodamine-labeled NP (Rd-NP-GFPg) accumulates in the CNV (C), while rhodamine-labeled non-targeted NP (Rd-ntNP-GFPg) does not (G). Some particles can be visualized circulating in the choroidal vessels. Overlay of images A–C is presented in panel D and overlay of E–G is shown in H.</p

Bioimaging with NP-angiography showing GFP expression using the Topcon camera with fluorescein angiography filter settings.

<p>Late phase FAs (A and D) show the CNV lesions prior to injection of NP. Autofluorescent images taken prior to injection of NP reveal minimal background fluorescence of the CNV lesions (B and E). Injection of targeted NP carrying a GFP plasmid (NP-GFPg) causes increased fluorescence of the CNV lesions from GFP expression (C) whereas non-targeted NP carrying a GFP plasmid (ntNP-GFPg) does not cause any increase in the intensity of fluorescence of the CNV over background autofluorescence (F).</p

Late phase fluorescein angiography (FA) and choroidal flatmounts (<i>x10</i>) two weeks after laser photocoagulation.

<p>Representative lesions are from the control group (A–D) and the NP-ATPμ-Raf treated group (E and F). Group (A) received no treatment; (B) received intravenous injection of non-targeted NP containing ATPμ-Raf on days 1, 3, and 5 after laser CNV creation; (C) received intravenous injection of α_νβ₃ targeted-NP without ATPμ-Raf gene on days 1,3, and 5; (D) received injection of ATPμ-Raf gene without NP on days 1, 3, and 5; (E) received injection of α_νβ₃ targeted-NP containing ATPμ-Raf (NP-ATPμ-Raf) on days 1, 3, and 5; and (F) received injection of NP-ATPμ-Raf on days 3, 5, and 7. NP-ATPμ-Raf treated groups (E and F) had significantly lower grade CNV lesions on FA grading and smaller CNV size compared to the control group (A–D). No statistically significant difference in size was noted between the control groups A–D. Quantification of the CNV size on choroidal flat mounts is shown in (G). *P<0.01. Data are expressed as the mean ± SE.</p

Evaluation of endothelial cell apoptosis with TUNEL staining in frozen sections.

<p>Quantification of TUNEL positive cells showed significantly more TUNEL(+) cells/lesion (A) and TUNEL (+) cells/mm² (B) with treatment of NP-ATPμ-Raf compared to the control group on day 3 and 5 after laser injury. There was a statistically significant reduction of CNV size noted on day 7(C). Double-immunofluorescent staining of frozen sections (<i>x20</i>) obtained at 3, 5 and 7 days after laser photocoagulation for the endothelial cell marker CD31 and TUNEL stain (D). *P<0.01. Data are expressed as the mean ± SE.</p

<i>In vivo</i> evaluation of CNV utilizing SD-OCT.

<p>Quantification of CNV size using SD-OCT (A) reveals a decrease in CNV size, reaching statistical significance on day 7 (Mann- Whitney U test, p = 0.001) in the NP-ATPμ-Raf treated group compared to the control group. A hyper-reflective subretinal lesion is seen as delineated by the red dotted line (B). This lesion corresponds to the hyporeflective area on fundus reconstruction (red dotted circle, C). *P<0.01. Data are expressed as the mean ± SE.</p

Increased macrophage infiltration at the site of treated CNV.

<p>Macrophage infiltration was highest on day 3 with gradual decrease on days 5 and 7. Significantly higher number of macrophages were observed with the NP-ATPμ-Raf treated group compared to the control group on days 5 and 7 (A and B). There was a statistically significant reduction of CNV size noted on day 7(C). Immunofluorescent staining of representative frozen sections (<i>x20</i>) obtained at 3, 5, and 7 days after laser photocoagulation for ED 1, a marker for macrophage (D). *P<0.01. Data are expressed as the mean ± SE.</p

Representative ¹H NMR spectra of control plasma.

<p>500 MHz ¹H NMR spectra of blood plasma from a control subject: a) standard 1D spectrum; b) CPMG spectrum; c) diffusion-edited spectrum. Signal assignment: 1-lactate; 2-alanine; 3 -glutamine; 4-glucose; 5-isoleucine; 6-leucine; 7-valine; 8-lysine; 9-acetate; 10-pyruvate; 11-citrate; 12-creatine; 13-creatinine; 14-dimethyl sulfone; 15-TMAO, trimethylamine-<i>N</i>-Oxide; 16,proline; 17-methanol; 18-glycine; 19-tyrosine; 20-histidine; 21- phenylalanine; 22-formate; 23-C18H cholesterol; 24-CH₃ lipids; 25-(CH₂)_n lipids; 26-C<u>H</u>₂CH₂CO lipids; 27-C<u>H</u>₂CH₂C = C lipids; 28-C<u>H</u>₂C = C lipids; 29-C<u>H</u>₂CO lipids; 30-C = CC<u>H</u>₂CH = C lipids; 31-albumin lysil groups; 32-N(CH₃)₃ choline; 33-glyceryl C1,3H; 34-glyceryl C1,3H’; 35-glyceryl C2H; 36-HC = CH lipids; 37-NH protein region.</p

Boxplot graphs for metabolites varying in Coimbra cohort.

<p>Coimbra cohort: boxplot representations of the metabolite variations found statistically relevant (* indicates <i>p-</i>value < 0.05) in at least one pairwise PLS-DA model. Compound names in rectangles correspond to compounds differentiating between controls and early AMD patients. C: controls, E: early AMD, I: intermediate AMD, L: late AMD. F.A.: fatty acids.</p

Characterization of the study population.

<p>Characterization of the study populations (Coimbra and Boston cohorts), with corresponding number of subjects (n), age (years), female (F)/male (M) ratio, body mass index (BMI) (kg.m^-2) and smoking history.</p

Examples of PLS-DA score plots.

<p>PLS-DA scores scatter plots and MCCV quality parameters (pairwise model Q², Q²_median (obtained through MCCV), % CR, % sens. and % spec.) obtained for variable selected CPMG NMR spectra of late AMD patients <i>vs</i> controls, in the a) Coimbra cohort: late AMD patients (□, n = 32), controls (∎, n = 42) and b) Boston cohort: late AMD patients (◇, n = 38), controls (♦, n = 40).</p