Hypoxia-inducible factor pathway activation in restless legs syndrome patients.

BACKGROUND AND PURPOSE: These studies tested the hypothesis that hypoxia inducible factor-1α (HIF-1α) 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-1α, 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-2α and vascular endothelial growth factor (VEGF) expression using immunoblot analyses. HIF-1α activation of peripheral blood monocyte cells (PBMCs) (14 RLS and 9 control) was determined through immunoblot analysis of PBMC lysates for EPO. RESULTS: HIF-1α 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-2α 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.

Testicular phenotype in luteinizing hormone receptor knockout animals and the effect of testosterone replacement therapy.

The LH receptor knockout model, developed in our laboratory, was used in determining what FSH alone can do in the absence of LH signaling and whether any of the testicular LH actions are not mediated by androgens. The results revealed that null animals contained smaller seminiferous tubules, which contained the same number of Sertoli cells, spermatogonia, and early spermatocytes as wild-type siblings. The number of late spermatocytes, on the other hand, was moderately decreased, the number of round spermatids was dramatically decreased, and elongated spermatids were completely absent. These changes appear to be due to an increase in apoptosis in spermatocytes. While the number of Leydig cells progressively increased from birth to 60 days of age in wild-type animals, they remained unchanged in null animals. Consequently, 60-day-old null animals contained only a few Leydig cells of fetal type. The age-dependent increase in testicular macrophages lagged behind in null animals compared with wild-type siblings. Orchidopexy indicated that -/- testicular phenotype was not due to abdominal location. Rather, it was mostly due to androgen deficiency, as 21-day testosterone replacement therapy stimulated the growth of seminiferous tubules, decreased apoptosis, and increased the number of late spermatocytes and round spermatids and their subsequent differentiation into mature sperm. The therapy, however, failed to restore adult-type Leydig cells and testicular macrophage numbers to the wild-type levels. In summary, our data support the concept that FSH signaling alone can maintain the proliferation and development of Sertoli cells, spermatogonia, and early spermatocytes. LH actions mediated by testosterone are required for completion of spermatogenesis, and finally, androgen-independent actions of LH are required for the formation of adult-type Leydig cells and recruitment of macrophages into the testes.