Noninvasive molecular imaging reveals role of PAF in leukocyte-endothelial interaction in LPS-induced ocular vascular injury.

Uveitis is a systemic immune disease and a common cause of blindness. The eye is an ideal organ for light-based imaging of molecular events underlying vascular and immune diseases. The phospholipid platelet-activating factor (PAF) is an important mediator of inflammation, the action of which in endothelial and immune cells in vivo is not well understood. The purpose of this study was to investigate the role of PAF in endothelial injury in uveitis. Here, we use our recently introduced in vivo molecular imaging approach in combination with the PAF inhibitors WEB 2086 (WEB) and ginkgolide B (GB). The differential inhibitory effects of WEB and GB in reducing LPS-induced endothelial injury in the choroid indicate an important role for PAF-like lipids, which might not require the PAF receptor for their signaling. P-selectin glycoprotein ligand-1-mediated rolling of mouse leukocytes on immobilized P-selectin in our autoperfused microflow chamber assay revealed a significant reduction in rolling velocity on the cells' contact with PAF. Rolling cells that came in contact with PAF rapidly assumed morphological signs of cell activation, indicating that activation during rolling does not require integrins. Our results show a key role for PAF in mediating endothelial and leukocyte activation in acute ocular inflammation. Our in vivo molecular imaging provides a detailed view of cellular and molecular events in the complex physiological setting.

Surprising up-regulation of P-selectin glycoprotein ligand-1 (PSGL-1) in endotoxin-induced uveitis.

P-selectin glycoprotein ligand-1 (PSGL-1) is constitutively expressed on leukocytes and was thought to be down-regulated with cell activation. However, this work shows the surprising finding of functional PSGL-1 up-regulation during acute inflammation. PSGL-1 function was studied in our autoperfusion assay, in which blood from a mouse carotid flows through a microchamber coated with a fixed density of P-selectin. Under the inflammatory conditions--uveitis induced by systemic lipopolysaccharide injection--we recorded significantly reduced leukocyte rolling velocity, which suggests PSGL-1 up-regulation; however, flow cytometry showed reduced PSGL-1. When bound leukocytes were released from the vasculature by PSGL-1 blockade, a large peripheral blood leukocyte (PBL) population showed elevated PSGL-1, which could account for the reduced PSGL-1 in the remaining unbound population. In the eye, systemic blockade of PSGL-1 with a monoclonal antibody or recombinant soluble PSGL-1 drastically reduced the severe manifestations of uveitis. Furthermore, PSGL-1 blockade was significantly more effective in reducing retinal leukostasis than was P-selectin blockade. Our results provide surprising evidence for functional PSGL-1 up-regulation in PBLs during acute inflammation. The temporal overlap between PSGL-1 and P-selectin up-regulation reveals an as yet unrecognized collaboration between this receptor-ligand pair, increasing efficiency of the first steps of the leukocyte recruitment cascade.

Vascular adhesion protein-1 blockade suppresses choroidal neovascularization.

Vascular adhesion protein-1 (VAP-1) is an endothelial cell adhesion molecule involved in leukocyte recruitment. Leukocytes and, in particular, macrophages play an important role in the development of choroidal neovascularization (CNV), an integral component of age-related macular degeneration (AMD). Previously, we showed a role for VAP-1 in ocular inflammation. Here, we investigate the expression of VAP-1 in the choroid and its role in CNV development. VAP-1 was expressed in the choroid, exclusively in the vessels, and colocalized in the vessels of the CNV lesions. VAP-1 blockade with a novel and specific inhibitor significantly decreased CNV size, fluorescent angiographic leakage, and the accumulation of macrophages in the CNV lesions. Furthermore, VAP-1 blockade significantly reduced the expression of inflammation-associated molecules such as tumor necrosis factor (TNF) -alpha, monocyte chemoattractant protein (MCP) -1, and intercellular adhesion molecule (ICAM) -1. This work provides evidence for an important role of VAP-1 in the recruitment of macrophages to CNV lesions, establishing a novel link between VAP-1 and angiogenesis. Inhibition of VAP-1 may become a new therapeutic strategy in the treatment of AMD.

In vivo imaging of endothelial injury in choriocapillaris during endotoxin-induced uveitis.

Early detection of ocular inflammation may prevent the occurrence of structural damage or vision loss. Here, we introduce a novel noninvasive technique for molecular imaging and quantitative evaluation of endothelial injury in the choriocapillaris of live animals, which detects disease earlier than currently possible. Using an established model of ocular inflammation, endotoxin-induced uveitis (EIU), we visualized the rolling and adhesive interaction of fluorescent microspheres conjugated to recombinant P-selectin glycoprotein ligand-Ig (rPSGL-Ig) in choriocapillaris using a scanning laser ophthalmoscope (SLO). The number of rolling microspheres in the choriocapillaris peaked 4-10 h after LPS injection. The number of the accumulated microspheres peaked 4 h after LPS injection in the temporal choriocapillaris and 4 and 36 h after LPS injection in the central areas around the optic disk. Furthermore, we semiquantified the levels of P-selectin mRNA expression in the choroidal vessels by reverse transcription-PCR and found its pattern to match the functional microsphere interactions, with a peak at 4 h after LPS injection. These results indicate that PSGL-1-conjugated fluorescent microspheres allow specific detection of endothelial P-selectin expression in vivo and noninvasive assessment of endothelial injury. This technique may help to diagnose subclinical signs of ocular inflammatory diseases.

Inhibition of vascular adhesion protein-1 suppresses endotoxin-induced uveitis.

Inflammatory leukocyte accumulation is a common feature of major ocular diseases, such as uveitis, diabetic retinopathy, and age-related macular degeneration. Vascular adhesion protein-1 (VAP-1), a cell surface and soluble molecule that possesses semicarbazide-sensitive amine oxidase (SSAO) activity, is involved in leukocyte recruitment. However, the expression of VAP-1 in the eye and its contribution to ocular inflammation are unknown. Here, we investigated the role of VAP-1 in an established model of ocular inflammation, the endotoxin-induced uveitis (EIU), using a novel and specific inhibitor. Our inhibitor has a half-maximal inhibitory concentration (IC(50)) of 0.007 microM against human and 0.008 microM against rat SSAO, while its IC(50) against the functionally related monoamine oxidase (MAO) -A and MAO-B is >10 microM. In the retina, VAP-1 was exclusively expressed in the vasculature, and its expression level was elevated during EIU. VAP-1 inhibition in EIU animals significantly suppressed leukocyte recruitment to the anterior chamber, vitreous, and retina, as well as retinal endothelial P-selectin expression. Our data suggest an important role for VAP-1 in the recruitment of leukocytes to the immune-privileged ocular tissues during acute inflammation. VAP-1 inhibition may become a novel strategy in the treatment of ocular inflammatory diseases.