HIF1 Induced Glutathione Dysregulation in Platelets Associated with Increased Risk of Vascular Disease in Chuvash Polycythemia.

The von Hippel-Lindau protein-hypoxia-inducible factor pathway is a transcriptional system controlling cellular responses to hypoxia. Hypoxia-inducible factor-1 (HIF-1) is a heterodimer of α and β subunits. Under normoxia, the prolyl residues of the α subunit are hydroxylated allowing the von Hippel-Lindau protein (pVHL) to bind, which targets HIFα for proteasomal degradation. During hypoxia, HIFα does not bind to pVHL and instead forms a transcriptional complex with HIFβ leading to increased expression of a broad range of hypoxia-regulated genes. Patients with Chuvash polycythemia (CP) are homozygous for a 598 C->T mutation in the VHL gene, resulting in a pVHL that causes ineffective degradation of HIFα. Clinically, CP patients suffer from premature mortality related to vascular thrombotic events - an increased risk that is not related to their elevated hematocrit, blood pressure, or known cardio or cerebrovascular risk factors. Lab abnormalities seen in CP include erythrocytosis and an elevated plasma glutathione level (Sergueeva et al, Haematologica. Feb 2008). Glutathione (GSH) plays an essential role in cellular antioxidant protection, and its levels are controlled by two mechanisms. GSH is oxidized to glutathione disulfide (GSSG) but is replenished by reduction of GSSG via glutathione reductase (GSR). GSH is also increased by de novo synthesis, which is regulated at several levels, including GSH feedback inhibition, and glutathione synthetase (GSS) and glutamate cysteine ligase (GCL) activity. To establish the molecular basis of elevated GSH, we examined expression of GSS, GSR, and GCL in the platelets of 11 CP patients and 8 Chuvash controls using qRT-PCR. Analysis revealed a 2-fold increased expression of GCL in CP. GSR and GSS were not statistically different. This data suggests that increased GCL activity might be the mechanism by which GSH is elevated in CP, but whether HIF directly regulates GCL or whether these differences reflect a more global process are presently unknown. Since the promoter of GCL contains an oxidative stressresponse element, transcriptional up regulation of GCL by increased oxidative stress secondary to HIF dysregulation might drive increased GSH synthesis. To examine this question, we measured GSSG in our samples. Although CP patients had elevated GSH (8.00 uM vs. 4.32 uM, p 0.002), the GSH/GSSG ratio (a widely used marker of redox state) showed no differences between CP and controls. Thus, overexpression of GCL and elevated GSH appear not to be compensatory responses to increased oxidative stress in CP. We also found increased GCL expression in VHL mutant mice. To determine if HIF1 might regulate this expression, we next measured GCL expression in HIF1 deficient embryos at embryonic day 9.5 and found decreased expression of GCL. In the homozygote knock-out embryos where HIF1 is absent, GCL expression is decreased, in contrast to CP patients, where HIF1 and GCL expression are both increased. Collectively, our data suggests that HIF1 dysregulates cellular redox homeostasis by upregulating GCL and increasing GSH synthesis in an oxidative-stress-independent manner. The significance of elevated GSH in CP and its possible relationship to increased thromboses remains to be defined. Reactive oxygen species mediate the vascular inflammation seen in the development of atherosclerotic disease, and GSH is an important intracellular scavenger that protects cells against oxidative damage. Yet CP patients have increased GSH as well as increased thrombosis, which is especially provoking since recently published evidence suggests that increased redox potential may be harmful to the cardiovascular system (Rajasekaran, et al Cell 2007). Perhaps the GSH elevation may contribute to the increased vascular disease that constitutes the major cause of mortality in this disorder of hypoxia sensing. The cellular antioxidant defense system is intimately linked to oxidative stress, hypoxia regulation, and vascular homeostasis. Our proposed future studies employing HIF2 knock out mice, model cell systems for HIF1 and HIF2, and the role of GSH in platelet function will be used to further explore the molecular mechanisms that regulate these complex pathways.