Autoimmunity in retinal degeneration: autoimmune retinopathy and age-related macular degeneration.

Autoantibody production is associated with a variety of ocular disorders, including autoimmune retinopathy (AIR) and age-related macular degeneration (AMD). A breakdown of immunologic tolerance (ocular immune privilege), including the blood-retinal barrier, anti-immune and anti-inflammatory proteins, and anterior chamber-associated immune deviation may play important roles in these disorders. Although the exact triggers for ocular autoimmunity are unknown, autoimmune targeting of retinal tissue is clearly associated with and may contribute to the pathogenesis of both AIR and AMD. Autoantibody production has long been associated with AIR, a collection of disorders that includes cancer-associated retinopathy, melanoma-associated retinopathy and non-paraneoplastic autoimmune retinopathy. A growing body of evidence indicates that AMD pathogenesis, too, involves ocular inflammation and autoimmunity. Identification and quantification of autoantibodies produced in patients with AIR and AMD may assist with diagnosis, prognosis, and choice of treatments. Animal models that allow investigation of ocular autoimmunity will also be needed to better understand the disease processes and to develop novel therapies. In this review we discuss ocular immune privilege and potential mechanisms of autoimmunity in the eye. We describe how autoimmunity relates to the pathogenesis of AIR and AMD. We explain how the antigen microarray technique is used to detect autoantibodies in patient serum samples, and discuss how current animal models for AMD can be used to investigate autoimmune pathogenesis. Finally, we outline unanswered questions and exciting areas of future study related to autoimmune retinal degeneration.

In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents.

Blood vessels, either in insufficient numbers or in excess, contribute to the pathogenesis of many diseases. Agents that stimulate angiogenesis can improve blood flow in patients with ischemic diseases, whereas anti-angiogenic agents are used to treat disorders ranging from macular degeneration to cancer. In this review I describe in vitro assays that can be used to assess the activity of agents that affect angiogenesis. Means of quantifying endothelial cell matrix degradation, migration, proliferation, apoptosis and morphogenesis are discussed, as are embryoid body, aortic ring and metatarsal assays of vessel outgrowth. Strengths and limitations of these techniques are also addressed.

Wnt1 and Wnt5a affect endothelial proliferation and capillary length; Wnt2 does not.

Blood vessel growth is critical for embryonic development and contributes to pathologies including cancer and diabetic retinopathy. A growing body of evidence suggests that signaling via the Wnt/beta-catenin pathway contributes to angiogenesis, and that paracrine Wnt signaling might alter endothelial cell function. To test the hypothesis that Wnt signaling promotes endothelial cell proliferation and vessel growth, we treated bovine aortic endothelial cells with Wnt1, Wnt2 and Wnt5a derived from coculture with Wnt-expressing fibroblasts. Endothelial cells cultured in the presence of Wnt1 displayed increased Wnt/beta-catenin signaling, proliferation and capillary stability in vitro. Wnt5a, which primarily signals via an alternate Wnt pathway, the Wnt/Ca(++) pathway, decreased both cell number and capillary length. Wnt2, which in other cell types activates the Wnt/beta-catenin pathway, did not activate signaling, affect cell number or increase capillary length. These results suggest that Wnt/beta-catenin and Wnt/Ca(++) signals might have opposing effects on angiogenesis.

Cultured endothelial cells display endogenous activation of the canonical Wnt signaling pathway and express multiple ligands, receptors, and secreted modulators of Wnt signaling.

A growing body of evidence implicates Wnt signaling in the control of angiogenesis. To better understand the role of the Wnt/beta-catenin pathway in endothelial cells (EC), we examined endogenous signaling activity and signaling component expression in vascular cells. We observed stabilization of cytosolic beta-catenin and activation of a T-cell factor (TCF) -luciferase promoter, hallmarks of canonical Wnt signaling activity, in cultured EC. This activity was increased in subconfluent EC, which are known to display characteristics of angiogenic EC, compared with confluent EC, which have a more differentiated phenotype. Endogenous TCF activity was inhibited by transfection with a secreted inhibitor of canonical Wnt signaling. A systematic analysis of Wnt, Fzd, SFRP, and Dkk gene expression in human EC (cultured and freshly isolated), smooth muscle cells (cultured), and aorta demonstrated that numerous Wnt signaling components are expressed by vascular cells. We conclude that Wnt signaling components are expressed and active in cultured EC.

Culture of large vessel endothelial cells on floating collagen gels promotes a phenotype characteristic of endothelium in vivo.

The vascular endothelium in vivo is a remarkably quiescent cell layer that displays a highly differentiated and tissue-specific phenotype. Once established in culture, endothelial cells (EC) are phenotypically different from their in situ counterparts, displaying altered gene expression, increased mitotic index, and decreased cell density. To determine whether manipulating the microenvironment of cells in vitro would lead to a more differentiated phenotype, we cultured bovine aortic EC on floating collagen gels. EC cultured to confluence on floating gels for 24 or 48 hr display mitotic indices nearly identical to those of quiescent endothelium in vivo, nearly two log orders lower than that of EC cultured to confluence on plastic, and cell density on floating gels also resembles that observed for endothelium in vivo. Culture of EC on floating gels leads to decreased expression of platelet-derived growth factor-B, fibronectin, and fibronectin isoform ED-B, and increased levels of connexin40, relative to cells cultured on plastic. We conclude that culture of bovine aortic EC under standard culture conditions results in a phenotype reminiscent of development and/or wound healing, and that culturing them on a floating collagen gel leads to a more differentiated phenotype, reminiscent of that observed for large vessel EC in vivo.