Abstract 51: Effects of Human Immunodeficiency Virus-1 gp120 Viral Proteins on Endothelial Senescence and microRNA Expression

Abstract

Human immunodeficiency virus (HIV)-1 is associated with profound endothelial cell damage and dysfunction. HIV-1 envelope glycoprotein 120 (gp120) is a toxic viral protein released from infected cells and promotes endothelial cell dysfunction. Endothelial cell senescence is a key factor underlying endothelial dysfunction and, in turn, vascular disease risk. It is now clear that microRNAs (miRs) play a pivotal role in regulating cellular senescence. The aim of this study was to determine whether gp120 induces endothelial cell senescence; and if so, whether the cellular expression of senescence-related miRs (mir-34a, -146a and -217) are adversely affected by gp120. Cultured HAECs (3 rd passage) were plated at a density of 5.0x10 5 cells/condition. Cells were treated with media alone or media containing gp120: either lav gp120 (X4; 100ng/mL) or Bal gp120 (R5; 100ng/mL) for 24 h. Thereafter, cells were stained for senescence-associated β-galactosidase (β-gal) for 14 h. Cells from 5-7 random microscope fields were counted and (%) senescence was calculated as the ratio of β-gal positive cells to total cells. To determine miR expression, RNA was extracted from 1.0x10 5 cells and was quantified using RT-PCR. Cellular expression was normalized to RNU6 and calculated as fold change in ΔΔCt from control (N=4, experimental units). Both X4 (32.0±0.7% vs 18.3±1.7%) and R5 (30.2±3.8% vs 18.3±1.7%) significantly increased cellular senescence vs control. Moreover, the magnitude of increase (~65%) in senescent cells was similar between the glycoproteins. Cellular expression of pro-senescence miR-34a (1.60±0.04 fold) was increased (~60%; P<0.05) in response to X4 treatment compared with control. However, expression of miR-146a (1.34±0.36 fold) and miR-217 (1.38±0.36 fold) was not significantly affected by X4. In response to R5, cellular expression of miR-34a (1.23±0.07 fold) was significantly increased (~23%) and miR-146a (1.23±0.07 fold) was significantly decreased (~80%); miR-217 was not influenced by R5 (1.05±0.13 fold). These data indicate that HIV-1 viral protein gp120 induces endothelial cell senescence and adversely affects cellular expression of senescence-associated miRs. miR dysregulation may underlie the pro-senescent effects of gp120.