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It is clearly established that an efficient supply to the brain of α-tocopherol (αTocH), the most biologically active member of the vitamin E family, is of the utmost importance for proper neurological functioning. Although the mechanism of uptake of αTocH into cells constituting the blood–brain barrier (BBB) is obscure, we previously demonstrated that high-density lipoprotein (HDL) plays a major role in the supply of αTocH to porcine brain capillary endothelial cells (pBCECs). Here we studied whether a porcine analogue of human and rodent scavenger receptor class B, type I mediates selective (without concomitant lipoprotein particle internalization) uptake of HDL-associated αTocH in a similar manner to that described for HDL-associated cholesteryl esters (CEs). In agreement with this hypothesis we observed that a major proportion of αTocH uptake by pBCECs occurred by selective uptake, exceeding HDL3 holoparticle uptake by up to 13-fold. The observation that selective uptake of HDL-associated CE exceeded HDL3 holoparticle up to fourfold suggested that a porcine analogue of SR-BI (pSR-BI) may be involved in lipid uptake at the BBB. In line with the observation of selective lipid uptake, RT-PCR and northern and western blot analyses revealed the presence of pSR-BI in cells constituting the BBB. Adenovirus-mediated overexpression of the human analogue of SR-BI (hSR-BI) in pBCECs resulted in a fourfold increase in selective HDL-associated αTocH uptake. In accordance with the proposed function of SR-BI, selective HDL–CE uptake was increased sixfold in Chinese hamster ovary cells stably transfected with murine SR-BI (mSR-BI). Most importantly stable mSR-BI overexpression mediated a twofold increase in HDL-associated [14C]αTocH selective uptake in comparison with control cells. In line with tracer experiments, mass transfer studies with unlabelled lipoproteins revealed that mSR-BI overexpression resulted in a twofold increase in endogenous HDL3-associated αTocH uptake. The results of this study indicate that SR-BI promotes the uptake of HDL-associated αTocH into cells constituting the BBB and plays an important role during the supply of the CNS with this indispensable micronutrient.
Vitamin E is a generic term for a group of compounds known as tocopherols and tocotrienols. α-Tocopherol (αTocH) is the major constituent of the vitamin E family found in mammalian tissues and has the highest biological activity of all the tocopherols and tocotrienols. Plasma αTocH levels are controlled by oral intake, absorption and transfer to newly synthesized lipoproteins, the latter step being under the tight control of the α-tocopherol transfer protein (αTTP) (Arita et al. 1995). In rats, the major site of rat αTTP expression is the liver, however, additional αTTPs have also been identified in lung, heart and brain (Murphy and Mavis 1981), where it is predominantly localized to Bergmann glial cells (Hosomi et al. 1998). Mutations in the gene for αTTP result in a neurologic syndrome of spinocerebellar ataxia (termed ataxia with associated vitamin E deficiency; AVED), characterized by progressive ataxia, areflexia and sensory loss (Ouahchi et al. 1995). In addition to AVED, secondary vitamin E deficiency due to lipid malabsorption (e.g. cholestatic liver disease) or abetalipoproteinemia may be associated with neurological syndromes identical to ataxia (Muller 1995). From the aforementioned findings it is evident that αTocH must be effectively absorbed, transported and delivered to organs and tissues following assembly with newly synthesized lipoproteins and exchange to other circulating lipoprotein classes (Kayden and Traber 1993).
As the hydrophobic αTocH molecule is transported exclusively in association with circulating lipoproteins, the major pathways involved in the delivery of lipoprotein-associated lipids may also be involved in αTocH uptake by organs and tissues. In healthy, fasting human subjects low-density lipoprotein (LDL) carries between 50 and 70% of the circulating αTocH, with the remainder being transported by high-density lipoproteins (HDL) (Sattler et al. 1996). In comparison with humans, the total antioxidant content is approximately fourfold lower in pigs and the αTocH content of porcine LDL is 2–3-fold lower than in humans (Knipping et al. 1990). Uptake of LDL-associated αTocH is facilitated by LDL-receptor mediated pathways (Kayden and Traber 1993), although studies in rabbits lacking functional intact LDL receptors suggest that alternative uptake pathways for lipoprotein-associated αTocH must exist (Cohn et al. 1992). In line with that study it appears that, on a quantitative basis, HDL is a more potent αTocH donor system than LDL (Goti et al. 1998, 2000). Along this line, it is important to note that HDL-associated lipids are metabolized during a process that is fundamentally different from the classical LDL receptor-mediated endocytotic pathway (Goldstein and Brown 1977), namely by selective uptake of HDL lipids (reviewed in Trigatti et al. 2000) without concomitant lipoprotein particle internalization. Selective uptake of HDL-associated lipids, particularly cholesteryl esters (CEs), in rodents is mediated by scavenger receptor class B, type I (SR-BI). Major sites of SR-BI expression are the liver and steroidogenic tissues, however, low level expression in brain has been reported for murine SR-BI (mSR-BI) (Acton et al. 1996). In liver mSR-BI mediates selective uptake of HDL-associated CE and facilitates removal of peripheral, excess cholesterol. Selective uptake of HDL-associated CE by adrenals provides cholesterol that is available for steroidogenesis (Trigatti et al. 2000).
The following observations have prompted us to study whether SR-BI mediates the uptake of HDL-associated αTocH by cells constituting the blood–brain barrier (BBB): (i) HDL is a more effective αTocH donor than LDL (Goti et al. 1998, 2000); (ii) the majority of HDL-associated αTocH is taken up independent of lipoprotein particles by brain endothelial cells (Goti et al. 2000); (iii) the majority of αTocH in mice is transported in the HDL fraction (Sattler et al. 1996); (iv) the adrenal gland, the organ with the highest expression of SR-BI (Acton et al. 1996), has the highest αTocH content on a nmol/g organ basis (Bjornboe et al. 1986); and (v) female mice containing a targeted null mutation for the SR-BI gene are infertile (Trigatti et al. 1999), a major manifestation of vitamin E deficiency in rodents (Evans and Bishop 1922). For these reasons we analysed the expression of SR-BI in a porcine model of the BBB (porcine brain capillary endothelial cells; pBCECs) using RT-PCR, northern and western blot analysis. Furthermore, we assessed the effect of SR-BI overexpression in pBCECs (transient hSR-BI overexpressors generated by adenovirus-mediated transfection) and Chinese hamster ovary (CHO) cells (stable SR-BI overexpressors) on the uptake efficacy of HDL-associated αTocH.
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- Materials and methods
The presence of tight junctions at the BBB provides a gate-keeping function and reduces opportunities for the movement of molecules, and thus require the existence of specific transport/receptor systems for the passage of certain critical compounds into the brain (Rubin and Staddon 1999). In light of the entry of αTocH into the brain it is important to note that the LDL receptor (Dehouck et al. 1997), HB2, a HDL-binding protein (Matsumoto et al. 1997), and scavenger receptors (de Vries et al. 1993) are expressed at the BBB and in the brain. In addition, class A scavenger receptors contributing to the uptake of chemically modified LDL were identified in bovine brain microvessels (Lucarelli et al. 1997) and bovine brain endothelial cells (de Vries et al. 1993). Although lipoprotein receptors are mainly thought of as necessary systems contributing to cholesterol homeostasis, they also deliver vitamins that exert critical functions in the regulation of cellular processes and lipoprotein binding and uptake may be of particular importance for vitamin transport across the BBB.
In a previous report (Goti et al. 2000) we were able to demonstrate that αTocH delivery to the BBB is more effective from HDL particles than from LDL particles. In addition, HDL-associated αTocH is taken up in a holoparticle-independent manner (Goti et al. 1998, 2000), a fact compatible with SR-BI-mediated selective uptake. The physiological significance of SR-BI expression in liver and steroidogenic tissues during HDL metabolism and steroidogenesis was clarified in mice in which the levels of SR-BI expression were manipulated by transgenic approaches (Trigatti et al. 2000). In this study we were able to demonstrate for the first time SR-BI expression in cells constituting the BBB. We identified two immunoreactive pSR-BI bands with apparent molecular masses of 84 and 81 kDa, findings in line with another report (Murao et al. 1997). Whether this is a result of a differential splicing process is not clear. In line with sterol regulatory element binding protein-dependent transcription of SR-BI (Lopez and McLean 1999), we observed cholesterol-dependent regulation of SR-BI expression in our in vitro BBB model.
An important question arising from this study is the physiological relevance of SR-BI expression at the BBB. Using two different SR-BI overexpressing cell systems (adenoviral transfection of pBCECs with hSR-BI and CHO cells stably overexpressing mSR-BI) we obtained clear evidence that the capacity for selective HDL–CE and HDL–αTocH uptake was dependent on SR-BI protein levels. While selective HDL–CE uptake is of utmost importance in liver and steroidogenic tissues (see above), the contribution of this pathway to cholesterol uptake across the BBB might be less significant. The major supply for growth and differentiation of the brain and CNS results from endogenous cholesterol biosynthesis (Turley et al. 1998). This might also explain the major abnormalities during brain development in patients suffering from the Smith-Lemli-Opitz syndrome, which is characterized by Δ7-reductase deficiency (Tint et al. 1994).
Along this line, it is noteworthy that SR-BI mediates bi-directional cholesterol flux, i.e. SR-BI is also involved in cellular cholesterol efflux (Rothblat et al. 1999). In peripheral tissues excess cholesterol stores are mobilized by an ApoA-I and SR-BI dependent pathway and subsequently esterified by the lecithin-cholesterol-acyl transferase reaction (Rothblat et al. 1999). The brain appears to use a different strategy to maintain cholesterol homeostasis, namely conversion of cerebral cholesterol to 24-hydroxycholesterol and subsequent release into the peripheral circulation (Björkhem et al. 1998). As 24-hydroxycholesterol is neurotoxic (Kölsch et al. 1999), it is conceivable that this compound has to follow an efficient elimination route. Whether SR-BI at the BBB contributes to this process in a fashion similar to that described for reverse cholesterol transport is currently under investigation.
The findings of this study, and indirect evidence from other studies (Bjornboe et al. 1986; Sattler et al. 1996; Goti et al. 1998; Trigatti et al. 1999; Orso et al. 2000), strongly support a key function of SR-BI facilitating selective uptake of HDL-associated αTocH at the BBB (and possibly other organs). Selective uptake of αTocH at the BBB is most probably the first step facilitating the supply of the brain with this micronutrient generating a pool of nonlipoprotein-associated αTocH in brain endothelial cells. However, the subsequent steps of paracellular αTocH transport are obscure and we are currently investigating whether specialized lipid domains, such as rafts or caveolae, are involved in the transport of αTocH across the BBB. Another question is the physiological relevance of αTTP expression in brain where it is localized in Purkinje cells (Copp et al. 1999) and surrounding Bergmann glia cells (Hosomi et al. 1998). The authors of the latter study suggested that αTocH uptake by Bergmann glia could occur via the long processes from the basement membrane of the blood vessels. Subsequently, αTTP could facilitate the assembly of αTocH with newly synthesized ApoE-containing lipoprotein-like particles that are secreted by Bergmann glia thus supplying Purkinje cells (and possibly other cerebellar cells) with αTocH. This concept could provide a basis for intercellular αTocH transport in the cerebellum. In contrast, neuronal transport mechanisms of αTocH from the cell body to the axons are largely unknown. However, the expression of several members of the LDL receptor superfamily by neurons (Posse de Caves et al. 2000) may facilitate the uptake (and probably anterograde neuronal transport) of αTocH in association with ApoE-containing lipoproteins. Along this line it is important to note that SR-BI expression was not detectable in cultured rat sympathetic neurons (Posse de Caves et al. 2000). The possibility that cubilin (predominantly expressed in polarized cells; Hammad et al. 1999) and/or megalin could contribute to the neuronal uptake of HDL-associated αTocH warrants further investigation.
Free radicals have been implicated in the pathogenesis of neurodegenerative diseases such as amyothropic lateral sclerosis, Alzheimer's disease and Parkinson's disease (reviewed in Sun and Chen 1998), and it is possible that αTocH which is transported across the BBB to the deeper regions of the CNS beneficially interferes with these oxidative processes. Another prominent feature of neurodegenerative diseases is the presence of activated microglial cells (Aschner et al. 1999). Although microglia may play a role during normal development and repair in the brain, chronic activation of microglia may be injurious. This is most probably a result of the production of pro-inflammatory cytokines and induction of inflammation-related enzymes such as the NADPH–oxidase complex, inducible nitric oxide synthase or cyclooxygenase. Reactive oxygen intermediates that are produced by these enzymatic systems are thought to contribute to neuronal injury although the exact underlying mechanisms are not clear. As microglia are defined as CNS-resident macrophages, some of the microglial enzymatic systems implicated in neuronal injury, such as the NADPH–oxidase complex, are activated by pathways similar to those described for monocytes (Della Bianca et al. 1999), and it seems conceivable that some of the modulatory properties of αTocH during monocyte activation could be extrapolated to microglia activation. Therefore, in analogy to activated monocytes, αTocH could interfere with microglia activation: for example, inhibition of PKC activity by αTocH (Ricciarelli et al. 1998) results in attenuated phosphorylation and translocation of p47phox, one of the cytosolic subunits of the NADPH–oxidase complex in human monocytes (Cachia et al. 1998), thereby decreasing superoxide production. Whether αTocH is able to modulate chemokine production by glial cells as described for monocytes (Deveraj et al. 1996) remains to be elucidated. αTocH-mediated modulation of adhesion molecule expression at the BBB, as observed in activated human monocytes obtained from αTocH-supplemented donors (Deveraj et al. 1996), could affect the entry of leukocytes across the BBB into the brain (Miller 1999). Finally, Heppner et al. (1998) were able to demonstrate a direct effect of αTocH on microglia ramification, a process indicative for immunological deactivation. Whether αTocH is able to induce differential gene expression in the CNS is currently an open question.
In summary, SR-BI-mediated selective uptake of HDL-associated αTocH into the BBB and subsequent redistribution to deeper brain regions by as yet undefined mechanisms could provide an important pathway facilitating a continuous supply of this indispensable micronutrient to the CNS.