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Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an <em>Ex Vivo</em> Autoperfused Microflow Chamber Assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

Harvard Medical School, Massachusetts Eye & Ear Infirmary

Purpose. Gene therapy for a number of retinal diseases necessitates efficient transduction of photoreceptor cells. Whereas adenovirus (Ad) serotype 5 (Ad5) does not transduce photoreceptors efficiently, previous studies have demonstrated improved photoreceptor transduction by Ad5 pseudotyped with Ad35 (Ad5/F35) or Ad37 (Ad5/F37) fiber or by the deletion of the RGD domain in the Ad5 penton base (Ad5DeltaRGD). However, each of these constructs contained a different transgene cassette, preventing the evaluation of the relative performance of these vectors, an important consideration before the use of these vectors in the clinic. The aim of this study was to evaluate these vectors in the retina and to attempt photoreceptor-specific transgene expression. Methods. Three Ad5-based vectors containing the same expression cassette were generated and injected into the subretinal space of adult mice. Eyes were analyzed for green fluorescence protein expression in flat-mounts, cross-sections, quantitative RT-PCR, and a modified stereological technique. A 257-bp fragment derived from the mouse opsin promoter was analyzed in the context of photoreceptor-specific transgene expression. Results. Each virus tested efficiently transduced the retinal pigment epithelium. The authors found no evidence that Ad5/F35 or Ad5/F37 transduced photoreceptors. Instead, they found that Ad5/F37 transduced Müller cells. Robust photoreceptor transduction by Ad5DeltaRGD was detected. Photoreceptor-specific transgene expression from the 257-bp mouse opsin promoter in the context of Ad5DeltaRGD vectors was found. Conclusions. Adenovirus vectors may be designed with tropism to distinct cell populations. Robust photoreceptor-specific transgene expression can be achieved in the context of Ad5DeltaRGD vectors.

Adenovirus vectors targeting distinct cell types in the retina.

Sequence variations in complement proteins are associated with age-related macular degeneration (AMD). The terminal pathway of complement results in the formation of the membrane attack complex (MAC) on the cell surface, resulting in their lysis. MAC has been documented on the retinal pigment epithelium (RPE), choroidal blood vessels, and drusen of AMD eyes. Here the investigators test the hypothesis that increasing the expression of decay accelerating factor (CD55) on RPE cells may result in reduced MAC-mediated damage.

Decay accelerating factor (CD55)-mediated attenuation of complement: therapeutic implications for age-related macular degeneration.

A defining feature in proliferative retinopathies is the formation of pathological neovessels. In these diseases, the balance between neovessel formation and regression determines blindness, making the modulation of neovessel growth highly desirable. The role of the immune system in these retinopathies is of increasing interest, but it is not completely understood. We investigated the role of the alternative complement pathway during the formation and resolution of aberrant neovascularization. We used alternative complement pathway-deficient (Fb(-/-)) mice and age- and strain-matched control mice to assess neovessel development and regression in an oxygen-induced retinopathy (OIR) mouse model. In the control mice, we found increased transcription of Fb after OIR treatment. In the Fb(-/-) mice, we prepared retinal flatmounts and identified an increased number of neovessels, peaking at postnatal day 17 (P17; P=0.001). Subjecting human umbilical vein endothelial cells (HUVECs) to low oxygen, mimicking a characteristic of neovessels, decreased the expression of the complement inhibitor Cd55. Finally, using laser capture microdissection (LCM) to isolate the neovessels after OIR, we found decreased expression of Cd55 (P=0.005). Together, our data implicate the alternative complement pathway in facilitating neovessel clearance by down-regulating the complement inhibitor Cd55 specifically on neovessels, allowing for their targeted removal while leaving the established vasculature intact.-Sweigard, J. H., Yanai, R., Gaissert, P., Saint-Geniez, M., Kataoka, K., Thanos, A., Stahl, G. L., Lambris, J. D., Connor, K. M. The alternative complement pathway regulates pathological angiogenesis in the retina.

Article	Year
Adenovirus vectors targeting distinct cell types in the retina. Investigative ophthalmology & visual science\| PubMed ID: 19892875	2010
Decay accelerating factor (CD55)-mediated attenuation of complement: therapeutic implications for age-related macular degeneration. Investigative ophthalmology & visual science\| PubMed ID: 20688727	2010
The alternative complement pathway regulates pathological angiogenesis in the retina. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 24668752	2014

J. Harry Sweigard

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