Objective To evaluate the association of subretinal hyper-reflective material (SHRM) with visual acuity (VA), geographic atrophy (GA) and scar in the Comparison of Age related Macular Degeneration Treatments Trials (CATT) Design Prospective cohort study within a randomized clinical trial. Participants The 1185 participants in CATT. Methods Participants were randomly assigned to ranibizumab or bevacizumab treatment monthly or as-needed. Masked readers graded scar and GA on fundus photography and fluorescein angiography images, SHRM on time domain (TD) and spectral domain (SD) optical coherence tomography (OCT) throughout 104 weeks. Measurements of SHRM height and width in the fovea, within the center 1mm2, or outside the center 1mm2 were obtained on SD-OCT images at 56 (n=76) and 104 (n=66) weeks. VA was measured by certified examiners. Main Outcome Measures SHRM presence, location and size, and associations with VA, scar, and GA. Results Among all CATT participants, the percentage with SHRM at enrollment was 77%, decreasing to 68% at 4 weeks after treatment and 54% at 104 weeks. At 104 weeks, scar was present more often in eyes with persistent SHRM than eyes with SHRM that resolved (64% vs. 31%; p<0.0001). Among eyes with detailed evaluation of SHRM at weeks 56 (n=76) and 104 (n=66), mean [SE] VA letter score was 73.5 [2.8], 73.1 [3.4], 65.3 [3.5], and 63.9 [3.7] when SHRM was absent, present outside the central 1mm2, present within the central 1mm2 but not the foveal center, or present at the foveal center (p=0.02). SHRM was present at the foveal center in 43 (30%), within the central 1mm2 in 21 (15%) and outside the central 1mm2 in 19 (13%). When SHRM was present, the median maximum height in microns under the fovea, within the central 1 mm2 including the fovea and anywhere within the scan was 86; 120; and 122, respectively. VA was decreased with greater SHRM height and width (p<0.05). Conclusions SHRM is common in eyes with NVAMD and often persists after anti-VEGF treatment. At 2 years, eyes with scar were more likely to have SHRM than other eyes. Greater SHRM height and width were associated with worse VA. SHRM is an important morphological biomarker in eyes with NVAMD.

Hypoxia is a common environmental stress that regulates gene expression and cell function. A number of hypoxia-regulated transcription factors have been identified and have been shown to play critical roles in mediating cellular responses to hypoxia. One of these is the endothelial PAS-domain protein 1 (EPAS1/HIF2-␣/HLF/ HRF). This protein is 48% homologous to hypoxia-inducible factor 1-␣ (HIF1-␣). To date, virtually nothing is known about the signaling pathways that lead to either EPAS1 or HIF1-␣ activation. Here we show that EPAS1 is phosphorylated when PC12 cells are exposed to hypoxia and that p42/p44 MAPK is a critical mediator of EPAS1 activation. Pretreatment of PC12 cells with the MEK inhibitor, PD98059, completely blocked hypoxiainduced trans-activation of a hypoxia response element (HRE) reporter gene by transfected EPAS1. Likewise, expression of a constitutively active MEK1 mimicked the effects of hypoxia on HRE reporter gene expression. However, pretreatment with PD98059 had no effect on EPAS1 phosphorylation during hypoxia, suggesting that MAPK targets other proteins that are critical for the trans-activation of EPAS1. We further show that hypoxia-induced trans-activation of EPAS1 is independent of Ras. Finally, pretreatment with calmodulin antagonists nearly completely blocked both the hypoxia-induced phosphorylation of MAPK and the EPAS1 trans-activation of HRE-Luc. These results demonstrate that the MAPK pathway is a critical mediator of EPAS1 activation and that activation of MAPK and EPAS1 occurs through a calmodulin-sensitive pathway and not through the GTPase, Ras. These results are the first to identify a specific signaling pathway involved in EPAS1 activation.Regulation of gene expression is a primary response by which cells adapt to changes in the environment. The mechanisms involved in regulation of gene expression in response to hypoxia are beginning to be understood. Transcription factors that are activated by hypoxia include the hypoxia-inducible factor (HIF1-␣), 1 c-fos, and CREB (1-4). HIF1-␣ has been shown to be critical for hypoxia-induced regulation of a number of genes, including glycolytic enzymes, vascular endothelial growth factor, and erythropoieitin (5-7). Recently, endothelial PAS-domain protein 1 (EPAS1, also known as HIF2-␣, HLF, and HRF) was identified as a hypoxia-inducible transcription factor (8 -10). EPAS1 is a basic helix-loop-helix transcription factor, which shares 48% sequence identity with HIF1-␣ (8). EPAS1 protein levels, like HIF1-␣ levels, are relatively low under basal conditions and accumulate upon exposure of cells to hypoxia (11). These factors then translocate to the nucleus and trans-activate target genes containing the sequence 5Ј-GCCCTACGTGCTGTCTCA-3Ј, which is commonly referred to as the hypoxia response element (HRE) (8, 12). EPAS1 is expressed in many tissues and is particularly abundant in the type I oxygen-sensing cells of the carotid body (13). Type I cells act as the primary O 2 sensors in mammals and are responsible for matching changes in art...

Hypoxic/ischemic trauma is a primary factor in the pathology of a multitude of disease states. The effects of hypoxia on the stress-and mitogen-activated protein kinase signaling pathways were studied in PC12 cells. Exposure to moderate hypoxia (5% O 2 ) progressively stimulated phosphorylation and activation of p38␥ in particular, and also p38␣, two stress-activated protein kinases. In contrast, hypoxia had no effect on enzyme activity of p38␤, p38␤ 2 , p38␦, or on c-Jun N-terminal kinase, another stress-activated protein kinase. Prolonged hypoxia also induced phosphorylation and activation of p42/p44 mitogen-activated protein kinase, although this activation was modest compared with nerve growth factor-and ultraviolet light-induced activation. Hypoxia also dramatically down-regulated immunoreactivity of cyclin D1, a gene that is known to be regulated negatively by p38 at the level of gene expression (Lavoie, J. N., L'Allemain, G., Brunet, A., Muller, R., and Pouyssegur, J. (1996) J. Biol. Chem. 271, 20608 -20616). This effect was partially blocked by SB203580, an inhibitor of p38␣ but not p38␥. Overexpression of a kinaseinactive form of p38␥ was also able to reverse in part the effect of hypoxia on cyclin D1 levels, suggesting that p38␣ and p38␥ converge to regulate cyclin D1 during hypoxia. These studies demonstrate that an extremely typical physiological stress (hypoxia) causes selective activation of specific p38 signaling elements; and they also identify a downstream target of these pathways.Mammalian cell function is critically dependent on a continuous supply of oxygen. Even brief periods of oxygen deprivation (hypoxia/ischemia) can result in profound cellular and tissue damage. Thus, it is vital that organisms meet changes in O 2 tension with appropriate cellular adaptations; however, the specific intracellular pathways by which this occurs are not well delineated. The stress-and mitogen-activated protein kinase (SAPK 1 and MAPK) pathways play a critical role in responding to cellular stress and promoting cell growth and survival (1, 2). We therefore investigated the effect of hypoxia on the SAPK and MAPK signaling pathways.SAPKs and MAPKs are the downstream components of three-member protein kinase modules (3). Five homologous subfamilies of these kinases have been identified, and the three major families include p38/SAPK2/RK, JNK/SAPK, and p42/ p44 MAPKs/ERKs (1-6). In general, the stress-activated protein kinases (p38 and JNK) are activated primarily by noxious environmental stimuli such as ultraviolet light, osmotic stress, inflammatory cytokines, and inhibition of protein synthesis (7-10). However, increasing evidence suggests that, at least under certain conditions, these pathways can also be activated by mitogenic and neurotrophic factors (11,12). In contrast, p42/p44 MAP kinases are stimulated primarily by mitogenic and differentiative factors in a Ras-dependent manner (5,13,14), although these enzymes can also be activated by certain environmental stressors (1-3). Thus, we hypothesized that hyp...

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These data suggest that decreases in plasma free-VEGF levels are greater after treatment with aflibercept or bevacizumab compared with ranibizumab at 4 weeks. At 52 and 104 weeks, a greater decrease was observed in bevacizumab versus ranibizumab. Results from 2 subgroups of participants who did not receive injections within at least 1 month and 2 months before collection suggest similar changes in VEGF levels after stopping injections. It is unknown whether VEGF levels return to normal as the drug is cleared from the system or whether the presence of the drug affects the assay's ability to accurately measure free VEGF. No significant associations between VEGF concentration and systemic factors were noted.