sCD59 Increases Retinal Müller Cell Activation in Diabetic Mice.

<p>(<b>a</b>). Representative retinal sections stained for GFAP from un-injected diabetic eyes (n=7) and un-injected non-diabetic eyes (n=6) showing similar staining of astrocytes (white arrowheads) in both groups. Scale bars = 29μm. Insets: Higher magnification of stained astrocytes. (<b>b</b>). Quantification of GFAP staining in un-injected diabetic eyes (n=7) and un-injected non-diabetic eyes (n=6) showing no difference in the staining of the astrocytes between the two groups. (<b>c</b>). Representative retinal sections stained for GFAP showing increased staining of astrocytes and Müller cells in AAV2/8-sCD59 injected diabetic eyes (n=7), when compared to AAV2/8-pA injected diabetic eyes (n=6). White arrowheads indicate Müller cell processes. Scale bar = 29μm. Insets: Higher magnification. (<b>d</b>). Quantification of GFAP staining showing a 180% increase in staining of the glial cells in AAV2/8-sCD59 injected diabetic eyes (n=7) when compared to AAV2/8-pA injected diabetic control eyes (n=6) [p=0.006]. n represents the number of eyes. Note: GFAP staining was assessed and quantified from both central as well as peripheral retinal sections in all groups.</p

AdCAGsCD59 protects against human MAC deposition in the larger (non-capillary) blood vessels of the liver.

<p>(A) Representative images indicating human MAC staining on the endothelial cell layer of a large blood vessel of murine liver perfused with mPECAM-1 antibody and NHS 7 days post-injection with AdCAGsCD59 or AdCAGGFP. Corresponding brightfield images are also shown (inset). (B) The average MAC staining intensity per endothelium area of large liver vessels of AdCAGsCD59-injected mice is reduced by 41.4% (***p<0.001) relative to that of AdCAGGFP-injected mice (n = 8).</p

sCD59 is expressed and efficiently secreted from adenovirus <i>in vitro.</i>

<p>(A) Expression cassettes for both sCD59 and GFP or without a transgene were cloned into the deleted E1 region of an E1/E3-deleted adenovirus. (B) ARPE-19 cells infected with AdCAGsCD59 express sCD59 and exhibit robust secretion into the media. CAG, chicken b-actin promoter; pA, polyA; LITR/RITR, left/right inverted terminal repeat; ¥, packaging signal; ΔE1/E3, deleted early regions 1/3; E2/E4, early regions 2/4.</p

Intraperitoneal injection of an adenovirus expressing GFP (AdCAGGFP) shows significant transduction of murine liver at 7 days post-injection.

<p>A representative image of a transverse section of mouse liver showing GFP transduction is presented. DAPI stain of cell nuclei of the same liver section is also shown. GFP+DAPI overlay shows that GFP expression was observed almost exclusively along the peritoneal membrane, with a few cells within the liver expressing GFP.</p

AdCAGsCD59-conditioned media confers significant protection from human serum-mediated cell lysis.

<p>(A) A representative FACS histogram for hepa-1c1c7 cells treated with either NHS or HI-NHS in media conditioned with either AdCAGGFP or AdCAGsCD59. (B) While no significant difference in cell lysis was observed in hepa-1c1c7 cells treated with HI-NHS in media conditioned with either AdCAGGFP or AdCAGsCD59, a significant (34.08±6.40%, **p<0.01) reduction was observed in cell lysis in hepa-1c1c7 cells treated with NHS in media conditioned with AdCAGsCD59 relative to those cells treated with NHS in media conditioned with the control virus, AdCAGGFP. HI-NHS, heat-inactivated normal human serum; NHS, normal human serum. n = 4.</p

Intracardial delivery of mPECAM-1 antibody permits binding to a variety of murine tissues.

<p><u>Liver</u>: In the liver, mPECAM-1 antibody binds endothelial cells along the sinusoids (arrowhead), as well as those of larger blood vessels (arrow). <u>Retina</u>: In the posterior eyecup, mPECAM-1 binds endothelia of the choriocapillaris (arrowhead) and retinal vasculature (arrow). <u>Choroid</u>: A flatmount of the choroid/RPE harvested from Balb/C mice injected intracardially with mPECAM-1 antibody shows more clearly binding of the antibody to the choroidal endothelium. <u>Aorta</u>: In the aorta, mPECAM-1 antibody is observed to bind the endothelial cell layer on the luminal surface (higher magnification of boxed region shown in inset). Intracardial delivery of a generic anti-mouse (GAM) antibody does not result in labeling of endothelial cells in any of the tissues. G, Ganglion Cell Layer; I/O, Inner/Outer Nuclear Layer; C, Choroid.</p

AdCAGpA and AdCAGsCD59 coinjected with AdCAGGFP result in equivalent levels of transduction and at sites distal to the sites of laser burn.

<p>Representative micrographs showing RPE/choroid flatmounts transduced with AdCAGGFP:AdCAGpA (1∶10) and AdCAGGFP:AdCAGsCD59 (1∶10). The region of transduction is demarcated and each of the three laser spots labeled 1–3. No significant difference in area of transduction was observed between AdCAGpA-injected and AdCAGsCD59-injected eyecups. n = 10 eyes (AdCAGsCD59), n = 8 eyes (AdCAGpA). ns, not significant. Scale bar = 200 µm.</p

Delivery of adenovirus to mouse subretinal space does not affect size of laser-induced CNV.

<p>Representative micrographs of FITC-GSL I stained laser-induced CNV spots from each of AdCAGpA-injected and uninjected eyecups. No significant difference was observed in the size of spots induced by laser in AdCAGpA-injected relative to uninjected eyes (p>0.5). n = 12 spots/5 eyes (uninjected), n = 26 spots/9 eyes (AdCAGpA); ns, not significant. Scale bar = 50 µm.</p

AdCAGsCD59 delivered to murine RPE results in a significant reduction in laser-induced CNV.

<p>Representative micrographs showing FITC-GSL I stained laser-induced CNV spots from eyes injected with either AdCAGpA or AdCAGsCD59. AdCAGsCD59 transduction of murine RPE results in a 61.7±19.9% reduction in size of CNV spot relative to eyes injected with AdCAGpA (**p<0.01). n = 33 spots/12 eyes (AdCAGsCD59), n = 26 spots/9 eyes (AdCAGpA). Scale bar = 50 µm.</p

sCD59 Attenuates formation of Membrane Attack Complex on Retina in Diabetic Mice.

<p>(<b>a</b>). Representative retinal sections stained for MAC deposition from un-injected diabetic eyes (n=7) and un-injected non-diabetic eyes (n=6) showing positive MAC staining in the inner limiting membrane in the un-injected diabetic eyes. None of the un-injected non-diabetic eyes (n=0/6) showed staining for MAC, while 43% (n=3/7) of the retinal sections from the diabetic retinas stained positive for MAC. Scale bars = 29μm. Insets: Higher magnification showing increased MAC deposition typically in the inner limiting membrane in un-injected diabetic eyes. (<b>b</b>). Quantification of MAC fluorescence intensity in the inner limiting membrane from un-injected diabetic eyes staining positive for MAC (n=3) showing a 110% increase in MAC deposition, when compared to un-injected non-diabetic eyes (n=6) [p<0.0001]. (<b>c</b>). Representative retinal sections showing reduction in MAC staining in the inner limiting membrane in AAV2/8-sCD59 injected diabetic eyes (n=7) when compared to AAV2/8-pA injected diabetic eyes (n=6). Scale bars = 29μm. Insets: Higher magnification. The reduction in MAC deposition in the AAV2/8-sCD59 injected diabetic eyes was observed as a reduced intensity of MAC staining when compared to AAV2/8-pA injected diabetic eyes. (<b>d</b>). Quantification of MAC fluorescence intensity in the inner limiting membrane of the retina showing a 40% reduction in MAC deposition in AAV2/8-sCD59 injected diabetic eyes (n=7) when compared to AAV2/8-pA injected diabetic eyes (n=6) [p=0.003]. n represents the number of eyes. MAC, membrane attack complex. Note: MAC staining was assessed and quantified from both central as well as peripheral retinal sections in all groups.</p