Evaluation of the expression of pigment epitelium derived factor (PEDF) in chronic intermittent hypoxia: validation and choice for best housekeeping genes in the model

Abstract

Pigment-Epitelium Derived Factor (PEDF) is a member of the family of the non- inhibitory serpins, distributed in almost all tissues, including CNS, where it is largely expressed. Comparatively, PEDF has been described as highly active in the Central Nervous System (CNS) acting as antiangiogenic, anti-vasopermeability, neurotrophic, neurogenic and neuroprotector factor. PEDF expression in hypoxia models has already been investigated in neuronal and glial cells, but only in acute sustained hypoxia models. Hypoxia is present in many diseases, including Obstructive Sleep Apnea (OSA). The consequences of hypoxia are largely variable, including complications in angiogenesis, cardiovascular system and the CNS, among many others. The impact of chronic intermittent hypoxia (CIH) in the CNS of adult rats, in PEDF levels is still unknown. The objectives of this work are to evaluate PEDF gene and protein expression in the CNS, as well as evaluate the best housekeeping genes for Real Time PCR for CIH model. 24 Wistar male rats were separated in three groups: (1) Control group ( n = 8); (2) CIH group ( n = 8) was submitted to intermittent hypoxia (IH) cycles of 3 min of 21% to 5% of O2, from 9 to 17 h, during six weeks; (3) CIH + 2 weeks of normoxia group ( n = 8) was submitted to the same treatment as CIH, followed by two weeks of recovery in normoxia. Housekeeping genes (HKG) and PEDF gene expression of Hippocampus, Hypothalamus, Frontal and Temporal Cortices were performed, as well as PEDF Western blot (WB) analysis. HKG stability was analyzed by geNorm Software. PEDF gene and protein levels in the CNS remained unaltered in experimental groups, CIH and CIH + 2 weeks. HKG analysis was performed and the genes classified, by decreasing order of stability: 1) HPRT; 2) β - Actin; 3) GAPDH; 4) β 2-Microglobulin. All the four HKG achieved the criteria for use (M number < 1,5), thus all suitable to use. Although PEDF plays important roles in several processes affected by CIH, its unaltered levels indicate that it is not playing an essential role in CIH. On the other hand, even though CIH is a very widely studied model, our study is the first to explore and validate HKG for use in this model. This work was developed in the Department of Psychobiology of Universidade Federal de São Paulo.