Hypoxia-inducible factor pathway activation in restless legs syndrome patients

Background and purpose: These studies tested the hypothesis that hypoxia inducible factor-1 alpha (HIF-1 alpha) pathway activation occurs in substantia nigra neurons and brain microvasculature in patients with restless legs syndrome. Methods: Immunohistochemical analyses of substantia nigra tissue from six RLS and six control subjects were analyzed for HIF-1 alpha, neuronal nitric oxide synthase (nNOS) and nitrotyrosine immunoreactivity. Microvessel lysates were obtained from cortex tissue from four RLS and four control subjects and the lysates were quantified for HIF-2 alpha and vascular endothelial growth factor (VEGF) expression using immunoblot analyses. HIF-1 alpha activation of peripheral blood monocyte cells (PBMCs) (14 RLS and 9 control) was determined through immunoblot analysis of PBMC lysates for EPO. Results: HIF-1 alpha immunoreactivity in substantia nigra neurons was significantly increased in five of six RLS patients as compared with controls. In addition, nNOS and nitrotyrosine expression are up-regulated in the substantia nigra of four of six RLS patients as compared with controls. HIF-2 alpha and VEGF expression are significantly up-regulated in the microvasculature lysates from four RLS cortical brain tissue as compared with controls. Erythropoietin levels are significantly increased in RLS PBMCs. Conclusions: These results demonstrate that the hypoxia pathway is activated in multiple cell types in individuals with RLS. Increased nNOS and nitrotyrosine suggests that nitric oxide is involved in the activation. Activation of the hypoxia pathway can result from or contribute to cellular iron deficiency. These observations suggest a novel direction to explore in RLS that is tied to the iron deficiency model but better explains the findings in postmortem studies.