Influence of nociceptive stimulation on analgesia nociception index (ANI) during propofol-remifentanil anaesthesia.

BACKGROUND: Measurement of the balance between nociception and anti-nociception during anaesthesia is challenging and not yet clinically established. The Surgical pleth index (SPI), derived from photoplethysmography, was proposed as a surrogate measure of nociception. Recently, the analgesia nociception index (ANI) derived by heart rate (HR) variability was developed. The aim of the present study was to challenge the ability of ANI compared with SPI to detect standardized noxious stimulation during propofol-remifentanil anaesthesia. METHODS: After Ethics approval and informed consent, 25 patients were anaesthetized with propofol [bispectral index (BIS) 30-60]. A laryngeal mask (LMA) was inserted and remifentanil stepwise increased to effect-site concentrations (Ce(remi)) of 0, 2, and 4 ng ml(-1). At each step, tetanic stimulation (STIM) was applied. ANI, SPI, BIS, HR, and mean arterial pressure (MAP) were obtained before and after LMA insertion and each STIM. Analysis was performed using Wilcoxon rank tests and calculation of prediction probabilities (P(K)). RESULTS: ANI and SPI, but not BIS, HR, or MAP, were significantly (P<0.05) changed at all examined steps. ANI response to STIM was (median [IQR]) -24 [-12-35], -30 [-20 - -40] and -13 [-5 - -27] at 0, 2 and 4 ng ml(-1) Ce(remi). However, prediction of movement to STIM was not better than by chance, as P(K) values were 0.41 (0.08) for ANI and 0.62 (0.08) for SPI. CONCLUSIONS: The two variables, ANI and SPI, enabled consistent reflection of stimulation during propofol-remifentanil anaesthesia. Nevertheless, ANI and SPI may improve detection but not prediction of a possible inadequate nociception-anti-nociception balance. Clinicaltrials.gov Identifier. NCT01522508.

Lipoprotein receptors: new roles for ancient proteins.

Lipoprotein receptors used to be viewed simply as the means by which cells were supplied with lipids for energy production and membrane synthesis. This perception has now changed dramatically. Megalin, a member of the low density lipoprotein receptor gene family, turns out to mediate the endocytic uptake of retinoids and steroids, thus helping to regulate their biological function. Other members of this receptor family interact with cytosolic signalling proteins, giving this evolutionarily ancient family of receptors an entirely unexpected new role as transducers of extracellular signals.

Remnant lipoproteins inhibit malaria sporozoite invasion of hepatocytes.

Remnants of lipoproteins, intestinal chylomicrons, and very low density lipoprotein (VLDL), are rapidly cleared from plasma and enter hepatocytes. It has been suggested that remnant lipoproteins are initially captured in the space of Disse by heparan sulfate proteoglycans (HSPGs), and that their subsequent internalization into hepatocytes is mediated by members of the LDL-receptor gene family. Similarly to lipoprotein remnants, malaria sporozoites are removed from the blood circulation by the liver within minutes after injection by Anopheles mosquitoes. The sporozoite's surface is covered by the circumsporozoite protein (CS), and its region II-plus has been implicated in the binding of the parasites to glycosaminoglycan chains of hepatocyte HSPGs. Lactoferrin, a protein with antibacterial properties found in breast milk and neutrophil granules, is also rapidly cleared from the circulation by hepatocytes, and can inhibit the hepatic uptake of lipoprotein remnants. Here we provide evidence that sporozoites, lactoferrin, and remnant lipoproteins are cleared from the blood by similar mechanisms. CS, lactoferrin, and remnant lipoproteins compete in vitro and in vivo for binding sites on liver cells. The relevance of this binding event for sporozoite infectivity is highlighted by our demonstration that apoliprotein E-enriched beta-VLDI and lactoferrin inhibit sporozoite invasion of HepG2 cells. In addition, malaria sporozoites are less infective in LDL-receptor knockout (LDLR -/-) mice maintained on a high fat diet, as compared with littermates maintained on a normal diet. We conclude that the clearance of lipoprotein remnants and sporozoites from the blood is mediated by the same set of highly sulfated HSPGs on the hepatocyte plasma membrane.

The signal sequence receptor has a second subunit and is part of a translocation complex in the endoplasmic reticulum as probed by bifunctional reagents.

Bifunctional cross-linking reagents were used to probe the protein environment in the ER membrane of the signal sequence receptor (SSR), a 24-kD integral membrane glycoprotein (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature [Lond.]. 328:830-833). The proximity of several polypeptides was demonstrated. A 22-kD glycoprotein was identified tightly bound to the 34-kD SSR even after membrane solubilization. The 34-kD polypeptide, now termed alpha SSR, and the 22-kD polypeptide, the beta SSR, represent a heterodimer. We report on the sequence of the beta SSR, its membrane topology, and on the mechanism of its integration into the membrane. Cross-linking also produced dimers of the alpha-subunit of the SSR indicating that oligomers of the SSR exist in the ER membrane. Various bifunctional cross-linking reagents were used to study the relation to ER membrane proteins of nascent chains of preprolactin and beta-lactamase at different stages of their translocation through the membrane. The predominant cross-linked products obtained in high yields contained the alpha SSR, indicating in conjunction with previous results that it is a major membrane protein in the neighborhood of translocating nascent chains of secretory proteins. The results support the existence of a translocon, a translocation complex involving the SSR, which constitutes the specific site of protein translocation across the ER membrane.

Inhibition of hepatic chylomicron remnant uptake by gene transfer of a receptor antagonist.

The low density lipoprotein receptor-related protein (LRP) has been proposed to mediate in concert with the LDL receptor (LDLR) the uptake of dietary lipoproteins into the hepatocytes. This hypothesis was tested by transient inactivation of LRP in vivo. Receptor-associated protein (RAP), a dominant negative regulator of LRP function, was transferred by an adenoviral vector to the livers of mice lacking LDLR (LDLR-/-). The inactivation of LRP by RAP was associated with a marked accumulation of chylomicron remnants in LDLR-/- mice and to a lesser degree in normal mice, suggesting that both LDLR and LRP are involved in remnant clearance.

Direct binding of Reelin to VLDL receptor and ApoE receptor 2 induces tyrosine phosphorylation of disabled-1 and modulates tau phosphorylation.

The large extracellular matrix protein Reelin is produced by Cajal-Retzius neurons in specific regions of the developing brain, where it controls neuronal migration and positioning. Genetic evidence suggests that interpretation of the Reelin signal by migrating neurons involves two neuronal cell surface proteins, the very low density lipoprotein receptor (VLDLR) and the apoE receptor 2 (ApoER2) as well as a cytosolic adaptor protein, Disabled-1 (Dab1). We show that Reelin binds directly and specifically to the ectodomains of VLDLR and ApoER2 in vitro and that blockade of VLDLR and ApoER2 correlates with loss of Reelin-induced tyrosine phosphorylation of Disabled-1 in cultured primary embryonic neurons. Furthermore, mice that lack either Reelin or both VLDLR and ApoER2 exhibit hyperphosphorylation of the microtubule-stabilizing protein tau. Taken together, these findings suggest that Reelin acts via VLDLR and ApoER2 to regulate Disabled-1 tyrosine phosphorylation and microtubule function in neurons.

Cholesterol metabolism and embryogenesis.

Although cholesterol has long been known to be an essential component of cell membranes in vertebrate organisms, recent studies have suggested that cholesterol plays a crucial role in specific processes during embryonic development, including the covalent modification of Hedgehog proteins. Here we review the overlapping developmental phenotypes associated with pharmacologically or genetically induced defects in cholesterol biosynthesis, embryonic cholesterol transport and Hedgehog proteins. Shared aspects of these phenotypes suggest that common mechanisms underlie impaired central nervous system development associated with cholesterol deficiency.

Two receptor systems are involved in the plasma clearance of tissue-type plasminogen activator (t-PA) in vivo.

Tissue-type plasminogen activator (t-PA) is a serine protease, catalyzing the initial step in the fibrinolytic process. Intravenously administered t-PA is rapidly cleared from the circulation by the liver. Two distinct clearance mechanisms, which are mediated by the low density lipoprotein receptor-related protein (LRP) on liver parenchymal cells and by the mannose receptor on liver endothelial cells, have been described. Using competitors and inhibitors of the receptors, we investigated the role of LRP and carbohydrate receptors in t-PA clearance in vivo. To inhibit LRP, the 39-kD protein, which is a potent inhibitor of LRP activity, was overexpressed in the liver of mice using an adenoviral gene transfer technique. Expression of the 39-kD protein resulted in a sustained plasma concentration and an increase in the plasma half-life of 125I-t-PA from less than 1 min to 4-5 min. Blockade of the mannose receptor by intravenous administration of ovalbumin also prolonged the plasma half-life of 125I-t-PA to 3-4 min. The same degree of inhibition of t-PA clearance was also observed after administration of an inhibitor of the fucose receptor, fucosyl-BSA. However, under the conditions established for the complete blockade of the mannose receptor, no additional inhibition of t-PA clearance was observed using fucosyl-BSA, suggesting little or no role for the fucose receptor in the clearance of t-PA. Furthermore, a dramatic increase of the plasma half-life of 125I-t-PA (>> 20 min) was observed in mice overexpressing 39-kD protein and administered ovalbumin +/- fucosyl-BSA. Our results clearly demonstrate that two independent receptor systems, LRP and the mannose receptor, are involved in the hepatic clearance of t-PA.

Inducible inactivation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants.

The multifunctional low density lipoprotein (LDL) receptor-related protein (LRP) has been postulated to participate in a number of diverse physiological and pathological processes ranging from the homeostasis of plasma lipoproteins, atherosclerosis, and fibrinolysis to neuronal regeneration and survival. It has not been possible to demonstrate in vivo the physiological significance of LRP for each of these complex processes by a conventional gene knockout approach because LRP is essential for embryonic development. Here we have used the Cre/loxP recombination system to achieve inducible, tissue-specific and quantitative disruption of the LRP gene in adult mice. Inactivation of LRP in the livers of LDL receptor-deficient mice resulted in the accumulation of cholesterol-rich remnant lipoproteins in the circulation. In normal animals, this caused a compensatory upregulation of the LDL receptor in the liver. Conditional gene targeting has thus allowed us to isolate a specific physiological function of LRP for in vivo analysis and has provided unequivocal evidence for another LDL receptor-independent cholesterol clearance pathway in liver.

Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein receptor-negative mice.

Mice that are homozygous for a targeted disruption of the LDL receptor gene (LDLR-/- mice) were fed a diet that contained 1.25% cholesterol, 7.5% cocoa butter, 7.5% casein, and 0.5% cholic acid. The total plasma cholesterol rose from 246 to > 1,500 mg/dl, associated with a marked increase in VLDL, intermediate density lipoproteins (IDL), and LDL cholesterol, and a decrease in HDL cholesterol. In wild type littermates fed the same diet, the total plasma cholesterol remained < 160 mg/dl. After 7 mo, the LDLR-/- mice developed massive xanthomatous infiltration of the skin and subcutaneous tissue. The aorta and coronary ostia exhibited gross atheromata, and the aortic valve leaflets were thickened by cholesterol-laden macrophages. No such changes were seen in the LDLR-/- mice on a normal chow diet, nor in wild type mice that were fed either a chow diet or the high-fat diet. We conclude that LDL receptors are largely responsible for the resistance of wild type mice to atherosclerosis. The cholesterol-fed LDLR-/- mice offer a new model for the study of environmental and genetic factors that modify the processes of atherosclerosis and xanthomatosis.

Role of the low density lipoprotein receptor in the flux of cholesterol through the plasma and across the tissues of the mouse.

These studies were undertaken to quantify cholesterol balance across the plasma space and the individual organs of the mouse, and to determine the role of the low density lipoprotein receptor (LDLR) in these two processes. In the normal mouse (129 Sv), sterol was synthesized at the rate of 153 mg/d per kg body weight of which 78% occurred in the extrahepatic tissues while only 22% took place in the liver. These animals metabolized 7.1 pools of LDL-cholesterol (LDL-C) per day, and 79% of this degradation took place in the liver. Of this total turnover, the LDLR accounted for 88% while the remaining 12% was receptor independent. 91% of the receptor-dependent transport identified in these animals was located in the liver while only 38% of the receptor-independent uptake wsa found in this organ. When the LDLR was deleted, the LDL-C production rate increased 1.7-fold, LDL-C turnover decreased from 7.1 to 0.88 pools/d, and the plasma LDL-C level increased 14-fold, from 7 to 101 mg/dl. Despite these major changes in the circulating levels of LDL-C, however, there was no change in the rate of cholesterol synthesis in any extrahepatic organ or in the whole animal, and, further, there was no change in the steady-state cholesterol concentration in any organ. Thus, most extrahepatic tissues synthesize their daily sterol requirements while most LDL-C is returned directly to the liver. Changes in LDLR activity, therefore, profoundly alter the plasma LDL-C concentration but have virtually no affect on cholesterol balance across any extrahepatic organ, including the brain.

Elevated levels of SREBP-2 and cholesterol synthesis in livers of mice homozygous for a targeted disruption of the SREBP-1 gene.

The synthesis of cholesterol and its uptake from plasma LDL are regulated by two membrane-bound transcription factors, designated sterol regulatory element binding protein-1 and -2 (SREBP-1 and SREBP-2). Here, we used the technique of homologous recombination to generate mice with disruptions in the gene encoding the two isoforms of SREBP-1, termed SREBP-1a and SREBP-1c. Heterozygous gene-disrupted mice were phenotypically normal, but 50- 85% of the homozygous (-/-) mice died in utero at embryonic day 11. The surviving -/- mice appeared normal at birth and throughout life. Their livers expressed no functional SREBP-1. There was a 1.5-fold upregulation of SREBP-2 at the level of mRNA and a two- to threefold increase in the amount of mature SREBP-2 in liver nuclei. Previous studies showed that SREBP-2 is much more potent than SREBP-1c, the predominant hepatic isoform of SREBP-1, in activating transcription of genes encoding enzymes of cholesterol synthesis. Consistent with this observation, the SREBP-1 -/- animals manifested elevated levels of mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase, farnesyl diphosphate synthase, and squalene synthase. Cholesterol synthesis, as measured by the incorporation of [3H]water, was elevated threefold in livers of the -/- mice, and hepatic cholesterol content was increased by 50%. Fatty acid synthesis was decreased in livers of the -/- mice. The amount of white adipose tissue was not significantly decreased, and the levels of mRNAs for lipogenic enzymes, adipocyte lipid binding protein, lipoprotein lipase, and leptin were normal in the -/- mice. We conclude from these studies that SREBP-2 can replace SREBP-1 in regulating cholesterol synthesis in livers of mice and that the higher potency of SREBP-2 relative to SREBP-1c leads to excessive hepatic cholesterol synthesis in these animals.

Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery.

We employed homologous recombination in embryonic stem cells to produce mice lacking functional LDL receptor genes. Homozygous male and female mice lacking LDL receptors (LDLR-/- mice) were viable and fertile. Total plasma cholesterol levels were twofold higher than those of wild-type litter-mates, owing to a seven- to ninefold increase in intermediate density lipoproteins (IDL) and LDL without a significant change in HDL. Plasma triglyceride levels were normal. The half-lives for intravenously administered 125I-VLDL and 125I-LDL were prolonged by 30-fold and 2.5-fold, respectively, but the clearance of 125I-HDL was normal in the LDLR-/- mice. Unlike wild-type mice, LDLR-/- mice responded to moderate amounts of dietary cholesterol (0.2% cholesterol/10% coconut oil) with a major increase in the cholesterol content of IDL and LDL particles. The elevated IDL/LDL level of LDLR-/- mice was reduced to normal 4 d after the intravenous injection of a recombinant replication-defective adenovirus encoding the human LDL receptor driven by the cytomegalovirus promoter. The virus restored expression of LDL receptor protein in the liver and increased the clearance of 125I-VLDL. We conclude that the LDL receptor is responsible in part for the low levels of VLDL, IDL, and LDL in wild-type mice and that adenovirus-encoded LDL receptors can acutely reverse the hypercholesterolemic effects of LDL receptor deficiency.

Absence of P-selectin delays fatty streak formation in mice.

P-selectin is expressed on activated endothelium and platelets where it can bind monocytes, neutrophils, stimulated T cells, and platelets. Because recruitment of these cells is critical for atherosclerotic lesion development, we examined whether P-selectin might play a role in atherosclerosis. We intercrossed P-selectin-deficient mice with mice lacking the low density lipoprotein receptor (LDLR) because these mice readily develop atherosclerotic lesions on diets rich in saturated fat and cholesterol. The atherogenic diet stimulated leukocyte rolling in the mesenteric venules of LDLR-deficient mice, and the increase in adhesiveness of the vessels was P-selectin-dependent. Most likely due to the reduced leukocyte interaction with the vessel wall, P-selectin-deficient mice on diet for 8-20 wk formed significantly smaller fatty streaks in the cusp region of the aortae than did P-selectin-positive mice. This difference was more prominent in males. At 37 wk on diet, the lesions in the LDLR-deficient animals progressed to the fibrous plaque stage and were distributed throughout the entire aorta; their size or distribution was no longer dependent on P-selectin. Our results show that P-selectin-mediated adhesion is an important factor in the development of early atherosclerotic lesions, and that adhesion molecules such as P-selectin are involved in the complex process of atherosclerosis.

Lipoprotein clearance mechanisms in LDL receptor-deficient "Apo-B48-only" and "Apo-B100-only" mice.

The role of the low density lipoprotein receptor (LDLR) in the clearance of apo-B48-containing lipoproteins and the role of the LDLR-related protein (LRP) in the removal of apo-B100-containing lipoproteins have not been clearly defined. To address these issues, we characterized LDLR-deficient mice homozygous for an "apo-B48-only" allele, an "apo-B100-only" allele, or a wild-type apo-B allele (Ldlr-/- Apob48/48, Ldlr-/-Apob100/100, and Ldlr-/-Apob+/+, respectively). The plasma apo-B48 and LDL cholesterol levels were higher in Ldlr-/-Apob48/48 mice than in Apob48/48 mice, indicating that the LDL receptor plays a significant role in the removal of apo-B48-containing lipoproteins. To examine the role of the LRP in the clearance of apo-B100-containing lipoproteins, we blocked hepatic LRP function in Ldlr-/-Apob100/100 mice by adenoviral-mediated expression of the receptor-associated protein (RAP). RAP expression did not change apo-B100 levels in Ldlr-/-Apob100/100 mice. In contrast, RAP expression caused a striking increase in plasma apo-B48 levels in Apob48/48 and Ldlr-/-Apob48/48 mice. These data imply that LRP is important for the clearance of apo-B48-containing lipoproteins but plays no significant role in the clearance of apo-B100-containing lipoproteins.

Macrophage low-density lipoprotein receptor-related protein deficiency enhances atherosclerosis in ApoE/LDLR double knockout mice.

OBJECTIVE: In vitro studies implicate that the low-density lipoprotein receptor (LDLR)-related protein (LRP) in macrophages has a pro-atherogenic potential. In the present study, we investigated the in vivo role of macrophage specific LRP in atherogenesis independent of its role in the uptake of lipoproteins. METHODS AND RESULTS: We generated macrophage-specific LRP-deficient mice on an apoE/LDLR double-deficient background. Macrophage LRP deletion did not affect plasma cholesterol and triglyceride levels, lipoprotein distribution, and blood monocyte counts. Nevertheless, macrophage LRP deficiency resulted in a 1.8-fold increase in total atherosclerotic lesion area in the aortic root of 18-week-old mice. Moreover, LRP deficiency also resulted in a relatively higher number of advanced lesions. Whereas macrophage and smooth muscle cell content did not differ between LRP-deficient mice and control littermates, a 1.7-fold increase in collagen content and 2.3-fold decrease in relative number of CD3+ T cells were observed in lesions from macrophage specific LRP-deficient mice. CONCLUSIONS: Our data demonstrate that independent of its role in lipoprotein uptake, absence of LRP in macrophages resulted in more advanced atherosclerosis and in lesions that contained more collagen and less CD3+ T cells. In contrast to previous in vitro studies, we conclude that macrophage LRP has an atheroprotective potential and may modulate the extracellular matrix in the atherosclerotic lesions.

Sustained somatic gene inactivation by viral transfer of Cre recombinase.

Transgenic and knockout mice have proven invaluable tools for analyzing physiologically relavant functions of numerous genes. In some cases, however, pleiotropic effects that result from a variable requirement for a particular gene in different tissues, cell types, or stages of embryonic development may complicate the analysis due to a complex phenotype or embryonic lethality. The loxP/Cre-mediated recombination system, which allows tissue-specific gene targeting in the mouse, can be used to overcome these problems. A limitation of current methods is that a mouse carrying a loxP-tagged gene must be crossed with a transgenic mouse expressing the Cre recombinase in an appropriate tissue to obtain the desired gene rearrangement. We have used recombinant adenovirus carrying the Cre recombinase to induce virtually quantitative somatic cell gene disruption in the liver. The targeted gene was the multifunctional low-density lipoprotein receptor-related protein (LRP), a cell surface receptor for alpha 2-macroglobulin and other ligands. Transient expression of Cre following adenoviral infection produced the predicted gene rearrangement, functionally inactivating LRP in the liver. Rearrangement occurred within 6 days after infection and remained stable for at least 28 days. The results demonstrate the suitability of adenoviral Cre gene transfer to induce long-term, quantitative, and temporally controlled gene disruption in the mouse.

Conditional animal models for the study of lipid metabolism and lipid disorders.

The advent of technologies that allow conditional mutagenesis has revolutionized our ability to explore gene functions and to establish animal models of human diseases. Both aspects have proven to be of particular importance in the study of lipid-related disorders. Classical approaches to gene inactivation by conventional gene targeting strategies have been successfully applied to generate animal models like the LDL receptor- and the apolipoprotein E-knockout mice, which are still widely used to study diverse aspects of atherosclerosis, lipid transport, and neurodegenerative disease. In many cases, however, simply inactivating the gene of interest has resulted in early lethal or complex phenotypes which are difficult to interpret. In recent years, additional tools have therefore been developed that allow the spatiotemporally controlled manipulation of the genome, as described in detail in Part I of this volume. Our aim is to provide an exemplary survey of the application of different conditional mutagenesis techniques in lipid research in order to illustrate their potential to unravel physiological functions of a broad range of genes involved in lipid homeostasis.

Lipoprotein receptors: beacons to neurons?

Lipoprotein receptors were originally considered simply as cellular transporters for cholesterol and other lipids. This view is rapidly changing. Signaling functions have recently been recognized in several members of the low-density lipoprotein receptor gene family. These Apolipoprotein E receptors are highly expressed in the developing and in the mature nervous system, in which they regulate crucial developmental processes and might also participate in synaptic neurotransmission.