Cholinergic neural activity directs retinal layer-specific angiogenesis and blood retinal barrier formation.

Blood vessels in the central nervous system (CNS) develop unique features, but the contribution of CNS neurons to regulating those features is not fully understood. We report that inhibiting spontaneous cholinergic activity or reducing starburst amacrine cell numbers prevents invasion of endothelial cells into the deep layers of the retina and causes blood-retinal-barrier (BRB) dysfunction in mice. Vascular endothelial growth factor (VEGF), which drives angiogenesis, and Norrin, a Wnt ligand that induces BRB properties, are decreased after activity blockade. Exogenous VEGF restores vessel growth but not BRB function, whereas stabilizing beta-catenin in endothelial cells rescues BRB dysfunction but not vessel formation. We further identify that inhibiting cholinergic activity reduces angiogenesis during oxygen-induced retinopathy. Our findings demonstrate that neural activity lies upstream of VEGF and Norrin, coordinating angiogenesis and BRB formation. Neural activity originating from specific neural circuits may be a general mechanism for driving regional angiogenesis and barrier formation across CNS development.

Systemic low-molecular weight drug delivery to pre-selected neuronal regions.

We describe a procedure for controlled, periodic, reversible modulation of selected regions of the blood-brain barrier (BBB) or the inner-blood-retina barrier (iBRB) based on incorporation into an AAV-2/9 vector of a doxycycline-inducible gene encoding shRNA targeting claudin-5, one of 30 or so proteins constituting the BBB and iBRB. The vector may be introduced stereotaxically into pre-selected regions of the brain or into the retina, rendering these regions permeable to low-molecular weight compounds up to approximately 1 kDa for the period of time during which the inducing agent, doxycycline, is administered in drinking water, but excluding potentially toxic higher molecular weight materials. We report on the use of barrier modulation in tandem with systemic drug therapy to prevent retinal degeneration and to suppress laser-induced choroidal neovascularization (CNV), the latter being the hallmark pathology associated with the exudative, or wet, form of age-related macular degeneration (AMD). These observations constitute the basis of a minimally invasive systemic therapeutic modality for retinal diseases, including retinitis pigmentosa and AMD, where, in early stage disease, the iBRB is intact and impervious to systemically administered drugs.

Hypoxia disrupts the barrier function of neural blood vessels through changes in the expression of claudin-5 in endothelial cells.

The mechanisms underlying the hypoxia-induced disruption of the barrier function of neural vasculature were analyzed with reference to the expression of claudin-5, a component of tight junctions between neural endothelial cells. The movement of claudin-5 from the cytoplasm to the plasma membrane of cultured confluent brain-derived endothelial (bEND.3) cells was closely correlated with the increase in the transendothelial electrical resistance. Inhibition of the expression of claudin-5 by RNAi resulted in a reduction of transendothelial electrical resistance, indicating a critical role of claudin-5 in the barrier property. Hypoxia (1% O(2)) altered the location of claudin-5 in the plasma membrane and the level of claudin-5 protein in bEND.3 cells, and these changes were accompanied by a decrease in the transendothelial electrical resistance. In vivo the claudin-5 molecules were expressed under normoxia in the plasma membrane of retinal microvascular endothelial cells but were significantly reduced under hypoxic conditions. Tracer experiments revealed that the barrier function of hypoxic retinal vasculature with depressed claudin-5 expression was selectively disrupted against small molecules, which is very similar to the phenotype of claudin-5-deficient mice. These in vitro and in vivo data indicate that claudin-5 is a target molecule of hypoxia leading to the disruption of the barrier function of neural vasculature.