No abstract

Glutaminase-1 (GLS1) is a mitochondrial enzyme found in endothelial cells (ECs) that metabolizes glutamine to glutamate and ammonia. Although glutaminolysis modulates the function of human umbilical vein ECs, it is not known whether these findings extend to human ECs beyond the fetal circulation. Furthermore, the molecular mechanism by which GLS1 regulates EC function is not defined. In this study, we show that the absence of glutamine in the culture media or the inhibition of GLS1 activity or expression blocked the proliferation and migration of ECs derived from the human umbilical vein, the human aorta, and the human microvasculature. GLS1 inhibition arrested ECs in the G/G phase of the cell cycle and this was associated with a significant decline in cyclin A expression. Restoration of cyclin A expression via adenoviral-mediated gene transfer improved the proliferative, but not the migratory, response of GLS1-inhibited ECs. Glutamine deprivation or GLS1 inhibition also stimulated the production of reactive oxygen species and this was associated with a marked decline in heme oxygenase-1 (HO-1) expression. GLS1 inhibition also sensitized ECs to the cytotoxic effect of hydrogen peroxide and this was prevented by the overexpression of HO-1. In conclusion, the metabolism of glutamine by GLS1 promotes human EC proliferation, migration, and survival irrespective of the vascular source. While cyclin A contributes to the proliferative action of GLS1, HO-1 mediates its pro-survival effect. These results identify GLS1 as a promising therapeutic target in treating diseases associated with aberrant EC proliferation, migration, and viability.

6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrazolo [1,5-a] pyrimidine (compound C) is a cell-permeable pyrrazolopyrimidine derivative that acts as a potent inhibitor of AMPactivated protein kinase (AMPK). Although compound C is often used to determine the role of AMPK in various physiological processes, it also evokes AMPK-independent actions. In the present study, we investigated whether compound C influences vascular smooth muscle cell (SMC) function through the AMPK pathway. Treatment of rat aortic SMCs with compound C (0.02-10 M) inhibited vascular SMC proliferation and migration in a concentration-dependent fashion. These actions of compound C were not mimicked or affected by silencing AMPK␣ expression or infecting SMCs with an adenovirus expressing a dominant-negative mutant of AMPK. In contrast, the pharmacological activator of AMPK 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside inhibited the proliferation and migration of SMCs in a manner that was strictly dependent on AMPK activity. Flow cytometry experiments revealed that compound C arrested SMCs in the G 0 /G 1 phase of the cell cycle, and this was associated with a decrease in cyclin D1 and cyclin A protein expression and retinoblastoma protein phosphorylation and an increase in p21 protein expression. Finally, local perivascular delivery of compound C immediately after balloon injury of rat carotid arteries markedly attenuated neointima formation. These studies identify compound C as a novel AMPKindependent regulator of vascular SMC function that exerts inhibitory effects on SMC proliferation and migration and neointima formation after arterial injury. Compound C represents a potentially new therapeutic agent in treating and preventing occlusive vascular disease.

AMP‐activated protein kinase (AMPK) is a ubiquitously distributed energy sensing enzyme that is activated by changes in cellular metabolism. Recent studies indicate that AMPK influences endothelial cell (EC) function and vascular homeostasis, but the underlying mechanisms are not fully known. In the present study, we investigated whether AMPK regulates EC proliferation. Treatment of cultured human umbilical vein ECs with serum stimulated a time‐dependent increase in cell number that was inhibited by the AMPK activators, 5‐aminoimidazole‐4‐carboxamide‐1‐â‐D‐ribofuranoside (AICAR), A‐769662, and metformin. The anti‐proliferative action of AICAR was concentration‐dependent, reversed by the adenosine kinase inhibitor 5’‐iodotubercidin, but unaffected by the nitric oxide synthase inhibitor, methyl‐L‐arginine. Infection of ECs with an adenovirus expressing constitutively active AMPKα also blocked EC proliferation. In addition, AMPK activation resulted in a marked decline in DNA synthesis that was associated with the arrest of ECs in the G0/G1 phase of the cell cycle. In conclusion, this study demonstrates that AMPK activation inhibits serum‐stimulated EC proliferation by blocking cell cycle progression. The ability of AMPK to inhibit EC growth may provide another mechanism by which this kinase preserves cellular energy during periods of metabolic stress. Supported by NIH grant HL59976.

Glutaminase (GLS) is a phosphate‐dependent enzyme that converts glutamine to glutamate and ammonia. There are two distinct isoforms of GLS, GLS1 and GLS2, which possess discrete tissue distribution, structural properties, enzyme kinetics, and molecular regulation. Although endothelial cells possess substantial GLS activity, its functional role in these cells remains largely unknown. In the present study, we characterized the expression of GLS in endothelial cells and investigated the effect of GLS on the proliferation of endothelial cells. Human umbilical vein endothelial cells (HUVEC) expressed high levels of GLS1, but GLS2 was not detected in these cells. Treatment of HUVEC with the non‐selective GLS inhibitor 6‐diazo‐5‐oxo‐L‐ornithine or the selective GLS1 inhibitors bis‐2‐(5‐phenylacetamido‐1,3,4‐thiadiazol‐2‐yl)ethyl sulfide or CB‐839 resulted in a concentration‐dependent inhibition of endothelial cell growth. All three GLS1 inhibitors also blocked DNA synthesis in both HUVEC and mouse aortic endothelial cells. Similarly, knockdown of GLS1 expression using a siRNA approach blocked the mitogenic response of endothelial cells. Flow cytometry experiments demonstrated that GLS1 inhibitors or GLS1 silencing arrested endothelial cells in the G0/G1 phase of the cell cycle, as reflected by increases in the percentage of cells in G0/G1 with corresponding decreases in the fraction of cells in S and G2/M phases. Cell cycle arrest by these GLS1 inhibitors was associated with a significant decrease in the expression of cyclin A protein that was paralleled by a reduction in cyclin A mRNA and promoter activity. Finally, adenoviral‐mediated gene transfer of cyclin A largely restored the proliferative response of GLS1‐inhibited endothelial cells. In conclusion, this study demonstrates that endothelial cells selectively express GLS1, and that GLS1 plays a central role in stimulating endothelial cell proliferation and cell cycle progression via the induction of cyclin A. In addition, it identifies GLS1 as an attractive therapeutic target in treating diseases associated with aberrant endothelial cell growth.Support or Funding InformationThis work was supported by the American Heart Association Grant 15GRNT25250015.