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

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

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

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

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

Retinal nerve fiber layer (RNFL) thinning and glaucomatous optic neuropathy in sGCα₁^−/− mice. A

<p>: Quantitative analysis, assessed by SD-OCT (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060156#pone.0060156.s001" target="_blank">fig. S1</a>), of total retinal thickness (<b>left panel</b>) and RNFL thickness, in young (6-week-old, <b>middle panel</b>) and old (70-week-old, <b>right panel</b>) wild-type (WT, <i>n</i> = 19 and 13, respectively) and soluble guanylate cyclase α₁-deficient (sGCα₁^−/−) mice (<i>n</i> = 15 and 14, respectively; *<i>P</i> = 1.2×10⁻²). <b>B</b>: Representative whole-mount retinas from age-matched young (20-week-old) and old (56-week-old) WT and sGCα₁^−/− mice, reacted with antibodies directed against SMI32, staining retinal nerve fibers yellow. Scale bars: 500 μm. <b>C</b>: Representative confocal images, taken at a similar distance from the optic nerve, of flat-mounted retinas isolated from age-matched 52-week-old WT and sGCα₁^−/− mice that were reacted with antibodies directed against βIII Tubulin, and quantitative analysis of the number of RGCs/high-powered field (<i>n</i> = 8 and 7, respectively; *<i>P</i> = 3.6×10⁻²). A retinal ganglion cell (red) is indicated by an arrow. Scale bars: 20 μm. <b>D</b>: Representative cross sections through the optic nerve of 52-week-old WT and sGCα₁^−/− mice stained with paraphenylenediamine, and quantitative analysis of the calculated number of axons/optic nerve (ON). The arrow indicates an injured area in the optic nerve, characterized by the absence of well-formed myelinated axons (<i>n</i> = 7 and 6, respectively; *<i>P</i> = 4.9×10⁻²). Scale bars: 25 μm.</p

Retinal vascular dysfunction in sGCα₁^−/− mice. A

<p>: Representative trace depicting the diameter in one segment of a retinal arteriole in a wild-type (WT) mouse before, during (arrow), and after injection of the NO-donor compound sodium nitroprusside. Dashed lines indicate the diameter before and after sodium nitroprusside injection. <b>B</b>: Quantitative analysis of the change in diameter (double arrow in fig. 6A) induced by injection of 0.8 mg/kg sodium nitroprusside in WT and sGCα₁^−/− mice. <i>n</i> = 5 mice (3–4 arterioles per mouse were assessed, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060156#pone.0060156.s003" target="_blank">fig. S3</a>). *<i>P</i> = 4.1×10⁻³.</p

Intraocular pressure (IOP) increases with age in sGCα₁^−/− mice.

<p>IOP, measured serially at 2 time points (19±1 and 37±3 weeks) in eyes from age-matched wild-type (WT, <b>left panel</b>) and soluble guanylate cyclase α₁-deficient (sGCα₁^−/−) mice (<b>right panel</b>). While IOP remained stable in WT mice as they aged from 19 to 37 weeks (14±2 to 14±2 mmHg; <i>n</i> = 25; <i>P</i> = 0.67), IOP increased in sGCα₁^−/− mice (14±2 to 18±3 mmHg; <i>n</i> = 37; *<i>P</i> = 1.9×10⁻⁸).</p

Localization of sGC α₁ and β₁ subunits in the human and murine eye.

<p>Panels <b>A–C</b> depict tissue sections from human eyes. Panels <b>D–F</b> depict tissue sections from mouse eyes. <b>A</b>: Ciliary muscle (CM), stained for α-smooth muscle actin (red), sGCα₁ (green, <b>upper panel</b>), or sGCβ₁ (green, <b>lower panel</b>). Both sGCα₁ and sGCβ₁ co-localized with α-smooth muscle actin in CM (yellow in merged images). Scale bars: 100 μm. <b>B</b>: An arteriole in the iris (IA) and an arteriole in the retina (RA) were stained for α-smooth muscle actin (red), sGCα₁ (green, <b>upper panels</b>), or sGCβ₁ (green, <b>lower panels</b>). Both sGCα₁ and sGCβ₁ co-localized with α-smooth muscle actin in the smooth muscle cell layer of arterioles in the iris and retina (yellow in merged images). ONL: outer nuclear layer, INL: inner nuclear layer. Scale bars: 20 μm. <b>C</b>: sGCα₁ (<b>left panel</b>) and sGCβ₁ (<b>right panel</b>) expression was detected histologically in the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL, white arrow) of the retina. sGCα₁ and sGCβ₁ are visualized by green fluorescence. Scale bars: 20 μm. <b>D</b>: Adjacent sections of a wild-type (WT) murine eye were stained for α-smooth muscle actin (green, <b>left panel</b>) or sGCα₁ (red, <b>right panel</b>). sGCα₁ co-localized with α-smooth muscle actin in ciliary muscle (CM) and in arterioles in the ciliary body (CA). The iridocorneal angle is indicated. Scale bars: 50 μm. <b>E</b>: Adjacent sections of a WT murine eye were stained for α-smooth muscle actin (green, <b>left panel</b>) or sGCα₁ (red, <b>right panel</b>). sGCα₁ co-localized with α-smooth muscle actin in retinal arterioles (RA). Scale bars: 50 μm. <b>F</b>: sGCα₁ (<b>left panel</b>) and sGCβ₁ (<b>right panel</b>) expression was detected histologically in the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL, white arrow) of the mouse retina. sGCα₁ is visualized by brown peroxidase stain and sGCβ₁ is visualized by green fluorescence. Scale bars: 20 μm.</p