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Andrew J. Stempel, Yuzhen Pan, Justine R. Smith, Binoy Appukuttan; Hypoxia-Induced Autophagy in Human Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4288.
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The autophagy-lysosomal pathway allows a cell to degrade non-essential components and potentially survive exposure to physiological or pathological stress. Hypoxic stress is one initiator of autophagy that has been studied extensively in tumor pathobiology. We investigated the ability of human retinal endothelial cells to develop an autophagy response in the setting of oxygen starvation, which is a key feature of human retinal ischemic vasculopathies, including diabetic retinopathy and retinopathy of prematurity.
Confluent immortalized human retinal endothelial cells were cultured under conditions of normoxia (O2 = 21%) or hypoxia (O2 < 1%), achieved in an air-tight Modulator Incubator Chamber (Billups-Rothenberg, Del Mar, CA) flushed with hypoxic gas every 8 hours, for 48 hours. Subsequently, total RNA was isolated with the RNeasy Mini Kit, and cDNA was synthesized from RNA using the RT2 First Strand Kit (both from Qiagen). Relative expression of 84 transcripts with established roles in autophagy, as well as 5 house keeping genes, was determined in quintuplicate using the human RT² Profiler™ PCR Array (Qiagen) and the Chromo4 Thermocycler (Bio-Rad) according to manufacturer’s protocol. Data analysis was performed with the aid of RT2 Profiler PCR Array Data Analysis software (Qiagen). Expression of vascular endothelial growth factor (VEGF)165 and VEGF121 by endothelial cells was determined in parallel to verify hypoxic stress.
An approximate 6-fold increased expression of VEGF165 and VEGF121 was measured for hypoxic human retinal endothelial cells versus control cells (p<0.001), confirming that treated cells had experienced hypoxic stress. After normalization to house keeping genes, 21 autophagy-related transcripts (25%) were significantly (p < 0.05) up-regulated and 11 transcripts were significantly down-regulated in the endothelial cells as a result of exposure to hypoxia, by PCR array. However, increases were greater than 1.5-fold in just 4 genes (i.e., CXCR4, Cathepsin S, DNA-damage regulated autophagy modulator 1 and tumor necrosis factor). Down-regulated transcripts included ATG3, ATG4A, ATG4C and ATG9B, which are involved in autophagic vacuole formation.
Our results suggest that while human retinal endothelial cells may activate autophagy in the presence of hypoxic stress, the response appears to be limited. Overcoming the diminished capacity of these endothelial cells to use autophagy may have therapeutic potential in patients with retinal ischemic vasculopathies.
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