Reduction of Reactive Oxygen Species by a Growth Hormone‐Releasing Hormone Antagonist in Endothelial Cells

Abstract

INTRODUCTIONGrowth hormone‐releasing hormone (GHRH) is a hypothalamic hormone regulating the secretion of Growth Hormone (GH) from the anterior pituitary gland. The expression of GHRH and its receptors (full length pituitary‐type and splice variants) in tissues such as breast, prostate and lung, suggested that the activities of that hypothalamic hormone is not limited to the GH ‐ insulin‐like growth factor‐I axis. Reactive Oxygen Species (ROS) are involved in inflammation, disrupt inter‐endothelial junctions, promote cytoskeletal remodeling and induce pro‐inflammatory cytokines (e.g. interleukin [IL]‐1β, IL‐6). Those events have been associated to endothelial hyper‐permeability, the hallmark of Acute Respiratory Distress Syndrome (ARDS). The purpose of the present study is to investigate the effects of GHRH and GHRH antagonists (GHRHAnt) in ROS generation of three different endothelial cell lines.METHODSBovine pulmonary artery endothelial cells (BPAEC), human lung microvascular endothelial cells (HMVEC‐L), human cerebral microvascular endothelial cells (hCMEC/D3) and mouse embryonic fibroblasts (NIH/3T3) were maintained at 37°C in a humidified atmosphere of 5% CO2‐ 95% air per suppliers’ instructions. Cells seeded in 96‐well plates (1×104 cells/well, n=4 wells per group) were treated with GHRH (1µM), or GHRHAnt (1µM), or GHRHAnt (1µM) prior to H2O2 (0.1mM) exposure. To measure ROS levels, the cells were incubated with 25µM of DCFDA for 45 min, and fluorescence intensity was measured by a fluorescence microplate reader using excitation and emission wavelength of 485 nm and 535 nm respectively. Protein expression was measured utilizing Western Blot analysis. The densitometry of immunoblots was accomplished by using Image J software (NIH), and Student's t test was used to determine the statistically significant differences among the groups. A value of p < 0.05 was considered significant.RESULTSThe expression of GHRH receptors in BPAEC, hCMEC/D3 and HMVEC‐L was confirmed by Western Blot. NIH/3T3 cells were used as negative controls, since they do not express receptors responding to GHRH. All cells were exposed to vehicle (0.1% DMSO), GHRH (1µM), or GHRHAnt (1µM) for 8 hours. Our results demonstrate that GHRH significantly increases ROS generation in all three endothelial cell lines, and GHRHAnt reduces ROS production. We also measured the effects of GHRHAnt against H2O2‐induced oxidative stress. Cells were treated with vehicle (0.1% DMSO), or GHRHAnt (1µM) for 8 hours, prior to vehicle (PBS) or H2O2 (0.1mM) exposure (8 hours). H2O2 potentiated ROS generation, and that effect was counteracted by GHRHAnt. Those peptides failed to suppress the H2O2‐induced ROS production in NIH/3T3 cells, since they do not express GHRH receptors.CONCLUSIONSOur results reveal that GHRHAnt suppress ROS production in the endothelium, suggesting that those compounds may be of therapeutic value in diseases related to endothelial barrier dysfunction and oxidative stress (e.g. Acute Respiratory Distress Syndrome and sepsis).