Faculty Bibliography

Unesterified cholesterol is a major component of plasma membranes. In the brain of the adult, it is mostly found in myelin sheaths, where it plays a major architectural role. In the newborn mouse, little myelination of neurons has occurred, and much of this sterol comprises a metabolically active pool. In the current study, we have accessed this metabolically active pool and, using LC/MS, have identified cholesterol precursors and metabolites. Although desmosterol and 24S-hydroxycholesterol represent the major precursor and metabolite, respectively, other steroids, including the oxysterols 22-oxocholesterol, 22R-hydroxycholesterol, 20R,22R-dihydroxycholesterol, and the C(21)-neurosteroid progesterone, were identified. 24S,25-epoxycholesterol formed in parallel to cholesterol was also found to be a major sterol in newborn brain. Like 24S- and 22R-hydroxycholesterols, and also desmosterol, 24S,25-epoxycholesterol is a ligand to the liver X receptors, which are expressed in brain. The desmosterol metabolites (24Z),26-, (24E),26-, and 7alpha-hydroxydesmosterol were identified in brain for the first time.

Significant ambiguity exists in the scientific community with regard to the nomenclature of 26-hydroxylated oxysterols. Oxysterols constitute an important class of compounds that have biological roles in the regulation of cholesterol synthesis and as endogenous selective estrogen receptor modulators (SERMs). The ambiguity is attributable to deviations from clearly stated IUPAC rules and is likely to increase as more biologically active oxysterols are identified. This review provides a uniform approach to the naming of 26-hydroxylated sterols for those of current interest and for those on the horizon such as oxysterols of lanosterol that retain the unsaturation at C-24 and C-25 such as (E)-26-hydroxylanosterol. Using this molecule as a starting point, this review hopes to establish a common language to keep all investigators on the same page.

Autoregulation of cholesterol synthesis focuses on the 19 metabolic steps from lanosterol to cholesterol. Although synchronization of their rates of synthesis in all tissues was the paradigm, a known exception occurs in the ovary where a local increase in a sterol intermediate, FF-MAS (follicular fluid meiosis activating sterol), activates meiosis during oocyte maturation. Mutations in the genes that govern synchronization cause an increase in sterol intermediates that follow an alternate, oxysterol, pathway of metabolism. Experimental models in animals imply that oxysterol metabolites are determinants of the dysmorphism that occurs during fetal development in these genetic diseases. These few examples may portend a much broader role for sterol intermediates and their novel oxysterol metabolites in physiologic and pathophysiologic processes

Osteoporosis is an important clinical problem, affecting more than 50% of people over age 50 yr. Estrogen signaling is critical for maintaining proper bone density, and the identification of an endogenous selective estrogen receptor (ER) modulator, 27-hydroxycholesterol (27HC), suggests a mechanism by which nutritional/metabolic status can influence bone biology. With its levels directly correlated with cholesterol, a new possibility emerges wherein 27HC links estrogen and cholesterol signaling to bone homeostasis. In these studies, we found that increasing concentrations of 27HC, both by genetic and pharmacological means, led to decreased bone mineral density that was associated with decreased bone formation and increased bone resorption. Upon manipulation of endogenous estrogen levels, many of the responses to elevated 27HC were altered in such a way as to implicate ER as a likely mediator. In a model of postmenopausal bone loss, some pathologies associated with elevated 27HC were exacerbated by the absence of endogenous estrogens, suggesting that 27HC may act both in concert with and independently from classic ER signaling. These data provide evidence for interactions between estrogen signaling, cholesterol and metabolic disease, and osteoporosis. Patients with high cholesterol likely also have higher than average 27HC, perhaps putting them at a higher risk for bone loss and fracture. More studies are warranted to fully elucidate the mechanism of action of 27HC in bone and to identify ways to modulate this pathway therapeutically

PURPOSE OF REVIEW: To summarize recent findings implicating toxic agents resulting in photooxidation of cholesterol in the etiology of age-related macular degeneration. Understanding the role of these agents and the existing pathways for their neutralization may lead to novel therapeutic approaches. RECENT FINDINGS: The human eye is now known to produce significant quantities of 7-ketocholesterol and related substances as a direct result of photoreceptor function. These substances are highly toxic to retinal cells and the eye has been shown to be unique among human organs in expressing three separate enzymatic pathways that neutralize these agents. Drusen are recently shown to contain significant accumulations of 7-ketocholesterol, likely as a result of failure of these neutralization pathways. In addition to its direct tissue toxicity, which may trigger death of retinal pigment epithelium and photoreceptor cells, ketocholesterol is a potent attractor of macrophages and induces macrophages to express both vascular endothelial growth factor F and metalloproteinases. The role of the former in neovascularization is well understood, whereas the latter is capable of directly inducing breaks in Bruch's membrane. SUMMARY: The toxic role of 7-ketocholesterol and existing pathways for its neutralization may point the way to a unified theory that explains the cause of age-related macular degeneration and points towards novel therapeutic interventions

A major focus for the 21st century are the sterol intermediates in cholesterol synthesis and their metabolites. No longer considered inactive way stations in their transformation to cholesterol, both physiologic and pathophysiologic studies, though early in their development, indicate novel biologic roles for these sterols, and their oxysterol metabolites that bypass cholesterol, the expected end product. A major impetus for further inquiry is the recognition that in genetically determined errors in cholesterol synthesis such as Smith-Lemil-Opitz syndrome, the phenotypic effects on the developing fetus are not solely attributable to the lack of cholesterol but the accumulation of 7-dehydrocholesterol and its 27-hydroxy metabolite. This view is now supported by a new mouse model, the double knockout Insig1 & 2 (insulin-induced genes 1 & 2) in which lack of the protein product results in a greater production of lanosterol compared to cholesterol during fetal life with severe dysmorphic consequences. Further support can be derived from in vitro studies of the Sonic hedgehog signaling pathway, essential for normal morphogenesis in the central nervous system and perhaps other organs, which may require the local presence of oxysterols for full expression. Future studies that can delineate the specific role of a sterol intermediate or its metabolite require a paradigm shift away from the generic use of oxysterols as a class of compounds to a focus on specific sterols that can be expected in tissues and techniques for mimicking the local environment. Another class of oxysterols are those arising by photoxidation, now considered to be an expected event generated by the photons of visible blue light and therefore pari passu with normal vision. The sequence of events from peroxides of cholesterol to hydroxy and keto derivatives is the signature of singlet oxygen as opposed to free radicals and other mechanisms for generating reactive oxygen species. Perhaps surprisingly, the retina expresses CYP 27A1 and CYP 46A1, enzymes with broad substrate specificity for ring-modified sterols, implying that, in addition to a rich blood supply for disposing of potentially toxic oxysterols, they can be detoxified locally. Recognition that the retina has nuclear receptors similar to those found in other tissues raises the possibility that the sterols that are generated may function in their traditional role as ligands for modulating gene expression but other, nonligand, activities can be expected since other proteins such as the oxysterol-binding proteins exist and are considered to have biologic activities. To critically evaluate these potentially new biologic roles for oxysterols a need exists for the synthesis and utilization of the expected naturally occurring metabolites rather than available surrogates that may not be truly representative of their tissue effects and to utilize analytical techniques that can identify their existence at the expected concentrations in tissues

The cardioprotective effects of estrogen are mediated by receptors expressed in vascular cells. Here we show that 27-hydroxycholesterol (27HC), an abundant cholesterol metabolite that is elevated with hypercholesterolemia and found in atherosclerotic lesions, is a competitive antagonist of estrogen receptor action in the vasculature. 27HC inhibited both the transcription-mediated and the non-transcription-mediated estrogen-dependent production of nitric oxide by vascular cells, resulting in reduced estrogen-induced vasorelaxation of rat aorta. Furthermore, increasing 27HC levels in mice by diet-induced hypercholesterolemia, pharmacologic administration or genetic manipulation (by knocking out the gene encoding the catabolic enzyme CYP7B1) decreased estrogen-dependent expression of vascular nitric oxide synthase and repressed carotid artery reendothelialization. As well as antiestrogenic effects, there were proestrogenic actions of 27HC that were cell-type specific, indicating that 27HC functions as an endogenous selective estrogen receptor modulator (SERM). Taken together, these studies point to 27HC as a contributing factor in the loss of estrogen protection from vascular disease

BACKGROUND: Cholesterol elimination occurs through bile acid synthesis that starts within the liver from 7alpha-hydroxylation or in extrahepatic tissues from 27-hydroxylation. This study was aimed at investigating in vivo these two pathways in patients with chronic liver disease. METHODS: Serum concentrations of 7alpha- and 27-hydroxycholesterol were measured in 54 patients (29 with primary biliary cirrhosis and 25 with chronic hepatitis C) and 18 controls. The rate of oxysterol plasma appearance was calculated after intravenous infusions of deuterated 7alpha- and 27-hydroxycholesterol in patients (n=8) and control subjects (n=8) who gave consent. The expression of sterol 27-hydroxylase was evaluated in macrophages isolated from 20 subjects. RESULTS: In patients with liver disease, the rate of plasma appearance of 7alpha-hydroxycholesterol was significantly reduced (1.44+/-0.96 vs. 2.75+/-1.43 mg/hour, p=0.03), the degree of reduction being related with the severity of the disease (p=0.01) whereas that of 27-hydroxycholesterol was unaffected. The rate of plasma appearance of 27-hydroxycholesterol was significantly related to its serum concentrations (r=0.54, p=0.03) and to its release from cultured macrophages ( r=0.85, p=0.03). CONCLUSIONS: In liver disease 7alpha-hydroxylation of cholesterol seems to be impaired while 27-hydroxylation is unaffected. Serum concentrations of 27-hydroxycholesterol are useful to obtain information on the activity of this alternative pathway

PURPOSE OF REVIEW: Recent findings extend the biologic activities of oxysterols as ligands for nuclear receptors to a role in morphogenesis during fetal development and to a role in the metabolism of photooxidation products of cholesterol in the retina. RECENT FINDINGS: A 1000-fold increase of the 27-hydroxy metabolite of 7-dehydrocholesterol in the plasma of children with Smith-Lemli-Opitz syndrome imply that intermediates in cholesterol synthesis follow alternate pathways of metabolism that generate novel oxysterols. A mouse model also finds an increase in sterol intermediates as the proximate cause of dysmorphisms. A role for oxysterols in the effects of Sonic hedgehog protein focuses on their role in normal fetal development. Both CYP27A1 and CYP46A1 are expressed in primate retina indicating that local metabolism of 7-ketocholesterol to nontoxic derivatives is important for preventing retinal degeneration. SUMMARY: Recent data expand the functional roles of oxysterols to fetal development and to the detoxification of oxidation products of cholesterol. This review shifts the focus of attention from studies of their ligand-binding activity to studies of animal models that indicate a number of important biologic effects during fetal development and during the aging process