Dynamic Increase in Extracellular ATP Accelerates Photoreceptor Cell Apoptosis via Ligation of P2RX7 in Subretinal Hemorrhage

<div><p>Photoreceptor degeneration is the most critical cause of visual impairment in age-related macular degeneration (AMD). In neovascular form of AMD, severe photoreceptor loss develops with subretinal hemorrhage due to choroidal neovascularization (CNV), growth of abnormal blood vessels from choroidal circulation. However, the detailed mechanisms of this process remain elusive. Here we demonstrate that neovascular AMD with subretinal hemorrhage accompanies a significant increase in extracellular ATP, and that extracellular ATP initiates neurodegenerative processes through specific ligation of Purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7; P2X7 receptor). Increased extracellular ATP levels were found in the vitreous samples of AMD patients with subretinal hemorrhage compared to control vitreous samples. Extravascular blood induced a massive release of ATP and photoreceptor cell apoptosis in co-culture with primary retinal cells. Photoreceptor cell apoptosis accompanied mitochondrial apoptotic pathways, namely activation of caspase-9 and translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, as well as TUNEL-detectable DNA fragmentation. These hallmarks of photoreceptor cell apoptosis were prevented by brilliant blue G (BBG), a selective P2RX7 antagonist, which is an approved adjuvant in ocular surgery. Finally, in a mouse model of subretinal hemorrhage, photoreceptor cells degenerated through BBG-inhibitable apoptosis, suggesting that ligation of P2RX7 by extracellular ATP may accelerate photoreceptor cell apoptosis in AMD with subretinal hemorrhage. Our results indicate a novel mechanism that could involve neuronal cell death not only in AMD but also in hemorrhagic disorders in the CNS and encourage the potential application of BBG as a neuroprotective therapy.</p> </div

Clinical characteristics of patients with MH, ERM, and AMD with VH.

<p>The patients underwent pars plana vitrectomy and the collected vitreous samples were subjected to ATP measurement by luciferase assay. There were no significant differences in age or sex ratio among the three groups.</p

Photoreceptor cell apoptosis in primary retinal cell cultures with blood clots.

<p>(A) Schematic image of the double chamber co-culture system of primary retinal cells and blood clots. (B) and (C) The viability of primary retinal cells in the lower chamber was accessed by calcein AM or MitoTracker CMTMRos after 24 h of culture with addition of a clot in the upper chamber (calcein in green, CMTMRos in red, recoverin in blue). The frequency of calcein⁺ or CMTMRos⁺ photoreceptors significantly decreased after incubation with clots. Apyrase treatment significantly rescued photoreceptors. (D<b>)</b> The ATP levels of culture medium in the lower chamber were significantly increased by clot exposure, and reversed by apyrase treatment. (E<b>)</b> The ATP levels in plasma and blood. <i>n = </i>10 per group; **<i>P<</i>0.01. Scale bar: 20 µm.</p

Photoreceptor cell apoptosis by subretinal injection of ATP.

<p>Exogenous ATP was injected into the subretinal space of Wt or P2rx7^−/− mice. TUNEL-positive apoptotic cells developed in the ONL 24 h after the subretinal injection of 1 mM ATP (TUNEL in green and Hoechst 33342 in blue). <i>n = </i>6 per group; **<i>P<</i>0.01. Scale bar: 20 µm.</p

The ATP levels in human vitreous of retinal diseases.

<p>Human vitreous samples were collected during vitreoretinal surgery from patients with MH (<i>n = </i>10), ERM (<i>n</i> = 10), and AMD (<i>n</i> = 15). The ATP levels of vitreous samples were determined by luciferin-luciferase assay (RLU: relative light units). †<i>P</i><0.01.</p

Abundant photoreceptor cell apoptosis in subretinal hemorrhage.

<p>Subretinal injection or vitreous injection of autologous blood was performed in C57BL6 mice. (A) A representative image of hematoxylin/eosin staining in subretinal hemorrhage is shown. Scale bar: 100 µm. (B) Some TUNEL⁺ nuclei were detected in the outer nuclear layer (ONL) in retinal detachment (RD), while abundant TUNEL⁺ nuclei were found in subretinal hemorrhage (SH). TUNEL⁺ cells were less detectable in vitreous hemorrhage (VH) (TUNEL in green, propidium iodide in blue). Scale bar: 20 µm. GCL; ganglion cell layer, IPL; inner plexiform layer, INL; inner nuclear layer, OPL; outer plexiform layer, ONL; outer nuclear layer. (C) The numbers of TUNEL⁺ photoreceptors in RD, VH, and SH. (D) Electron microscopy revealed that numerous photoreceptor cells had undergone apoptosis with chromatin condensation (arrows in the top left panel) or cellular shrinkage (top right panel). Some erythrocytes lost their plasma membranes (black arrowheads in the bottom left panel) and hemolytic debris was observed among erythrocytes (white arrowheads in the bottom left panel) in the subretinal hemorrhage. Minimal hemolytic change was observed in the vitreous hemorrhage (bottom right panel). Scale bars: 2 µm.</p

A P2RX7 antagonist prevents photoreceptor cell apoptosis in a mouse model of subretinal hemorrhage.

<p>(A) After the subretinal injection of autologous blood, photoreceptor cells underwent apoptotic cell death, and AIF-positive staining was observed in TUNEL⁺ photoreceptor nuclei (arrows; AIF in red, TUNEL in green, and Hoechst 33342 in blue). Caspase-9 cleavage was also detected among TUNEL⁺ nuclei (arrowheads; cleaved caspase-9 in red). Scale bar: 10 µm. (B) and (C) TUNEL⁺ apoptotic cells in the absence or presence of 50 µM BBG treatment after experimental subretinal hemorrhage in Wt or P2rx7^−/− mice. <i>n = </i>6 per group; **<i>P<</i>0.01. Scale bar: 20 µm.</p

Correlations of vitreous sCD44 and sVCAM-1 levels in patients with PVR.

<p>There was a strong statistically significant correlation between the vitreous concentration of sCD44 and sVCAM-1 (<i>r</i> = 0.971;<i>P</i><0.0001).</p

Molecular networks associated with the genes expressed in ERMs associated with PVR (PVR-ERM) are shown.

<p>Gene symbols of 10 cell adhesion-related genes (<i>FN1, COL1A2, COL1A1, COL3A1, TIMP3, LGALS1, THBS1, DCN, POSTN, SPARC)</i> from the PVR-ERM cDNA library were queried against the STRING database, and the predicted interactions for genes/proteins were obtained. Filled black circles represent the submitted 10 genes/proteins from the PVR-ERM cDNA library, and the white circles represent potentially expressed 60 genes in PVR-ERMs that are extracted <i>in Silico</i>. Of these, CD44 and VCAM-1 were examine by ELISA and are shown by arrows. The gene names are shown next to the circles. The edges connecting two circles represent the predicted functional associations. An edge is drawn with up to 7 differently colored lines. These lines represent the presence of the seven types of evidence used in predicting the associations. A red line indicates the presence of fusion evidence; a green line-neighborhood evidence; a blue line–co-occurrence evidence; a purple line-experimental evidence; a yellow line-textmining evidence; a light blue line-database evidence; and a black line–co-expression evidence.</p

sCD44 (A) and sVCAM-1 (B) concentrations in the vitreous fluid of patients with secondary ERM and proliferative vitreoretinopathy patients (PVR-ERM).

<p>The levels of both sCD44 and sVCAM-1 were significantly higher in the patients with PVR than in the eyes with secondary ERM (*<i>P</i><0.001).</p