The ecto-nucleotide pyrophosphatase/phosphodiesterase 2 promotes early osteoblast differentiation and mineralization in stromal stem cells.

The phosphodiesterase enzymes mediate calcium-phosphate deposition in various tissues, although which enzymes are active in bone mineralization is unclear. Using gene array analysis, we found that a member of ecto-nucleotide pyrophosphatase/phosphodiesterase family, ENPP2, was strongly down-regulated with age in stromal stem cells that produce osteoblasts and make bone. This is in keeping with reduced bone formation in older animals. Thus, we hypothesized that ENPP2 is, at least in part, an early mediator of bone formation and thus may reflect reduced bone formation with age. Since ENPP2 has not previously been shown to have a role in osteoblast differentiation, we studied its effect on bone differentiation from stromal stem cells, verified by flow cytometry for stem cell antigens. In these remarkably uniform osteoblast precursors, we did transfection with ENPP2 DsiRNA, scrambled DsiRNA, or no transfection to make cells with normal or greatly reduced ENPP2 and analyzed osteoblast differentiation and mineralization. Osteoblast differentiation down-regulation was shown by alizarin red binding, silver staining, and alkaline phosphatase activity. Differences were confirmed by real-time PCR for alkaline phosphatase (ALPL), osteocalcin (BGLAP), and ENPP2 and by Western Blot for Enpp2. These were decreased, ∼50%, in osteoblasts transfected with ENPP2 DsiRNA compared with cells transfected with a scrambled DsiRNA or not transfected (control) cells. This finding is the first evidence for the role of ENPP2 in osteoblast differentiation and mineralization.NEW & NOTEWORTHY We report the discovery that the ecto-nucleotide pyrophosphatase/phosphodiesterase, ENPP2, is an important regulator of early differentiation of bone-forming osteoblasts.

Age-related decline in bone mineral transport and bone matrix proteins in osteoblasts from stromal stem cells.

We studied osteoblast bone mineral transport and matrix proteins as a function of age. In isolated bone marrow cells from long bones of young (3 or 4 mo) and old (18 or 19 mo) mice, age correlated with reduced mRNA of mineral transport proteins: alkaline phosphatase (ALP), ankylosis (ANK), the Cl-/H+ exchanger ClC3, and matrix proteins collagen 1 (Col1) and osteocalcin (BGLAP). Some proteins, including the neutral phosphate transporter2 (NPT2), were not reduced. These are predominately osteoblast proteins, but in mixed cell populations. Remarkably, in osteoblasts differentiated from preparations of stromal stem cells (SSCs) made from bone marrow cells in young and old mice, differentiated in vitro on perforated polyethylene terephthalate membranes, mRNA confirmed decreased expression with age for most transport-related and bone matrix proteins. Additional mRNAs in osteoblasts in vitro included ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), unchanged, and ENPP2, reduced with age. Decrease with age in ALP activity and protein by Western blot was also significant. Transport protein findings correlated with micro-computed tomography of lumbar vertebra, showing that trabecular bone of old mice is osteopenic relative to young mice, consistent with other studies. Pathway analysis of osteoblasts differentiated in vitro showed that cells from old animals had reduced Erk1/2 phosphorylation and decreased suppressor of mothers against decapentaplegic 2 (Smad2) mRNA, consistent with TGFß pathway, and reduced ß-catenin mRNA, consistent with WNT pathway regulation. Our results show that decline in bone density with age reflects selective changes, resulting effectively in a phenotype modification. Reduction of matrix and mineral transport protein expression with age is regulated by multiple signaling pathways.NEW & NOTEWORTHY This work for the first time showed that specific enzymes in bone mineral transport, and matrix synthesis proteins, in the epithelial-like bone-forming cell layer are downregulated with aging. Results were compared using cells extracted from long bones of young and old mice, or in essentially uniform osteoblasts differentiated from stromal stem cells in vitro. The age effect showed memory in the stromal stem cells, a remarkable finding.

Implications of conformational flexibility, lipid binding, and regulatory domains in cell-traversal protein CelTOS for apicomplexan migration.

Malaria and other apicomplexan-caused diseases affect millions of humans, agricultural animals, and pets. Cell traversal is a common feature used by multiple apicomplexan parasites to migrate through host cells and can be exploited to develop therapeutics against these deadly parasites. Here, we provide insights into the mechanism of the Cell-traversal protein for ookinetes and sporozoites (CelTOS), a conserved cell-traversal protein in apicomplexan parasites and malaria vaccine candidate. CelTOS has previously been shown to form pores in cell membranes to enable traversal of parasites through cells. We establish roles for the distinct protein regions of Plasmodium vivax CelTOS and examine the mechanism of pore formation. We further demonstrate that CelTOS dimer dissociation is required for pore formation, as disulfide bridging between monomers inhibits pore formation, and this inhibition is rescued by disulfide-bridge reduction. We also show that a helix-destabilizing amino acid, Pro127, allows CelTOS to undergo significant conformational changes to assemble into pores. The flexible C terminus of CelTOS is a negative regulator that limits pore formation. Finally, we highlight that lipid binding is a prerequisite for pore assembly as mutation of a phospholipids-binding site in CelTOS resulted in loss of lipid binding and abrogated pore formation. These findings identify critical regions in CelTOS and will aid in understanding the egress mechanism of malaria and other apicomplexan parasites as well as have implications for studying the function of other essential pore-forming proteins.

Epithelial-like transport of mineral distinguishes bone formation from other connective tissues.

We review unique properties of bone formation including current understanding of mechanisms of bone mineral transport. We focus on formation only; mechanism of bone degradation is a separate topic not considered. Bone matrix is compared to other connective tissues composed mainly of the same proteins, but without the specialized mechanism for continuous transport and deposition of mineral. Indeed other connective tissues add mechanisms to prevent mineral formation. We start with the epithelial-like surfaces that mediate transport of phosphate to be incorporated into hydroxyapatite in bone, or in its ancestral tissue, the tooth. These include several phosphate producing or phosphate transport-related proteins with special expression in large quantities in bone, particularly in the bone-surface osteoblasts. In all connective tissues including bone, the proteins that constitute the protein matrix are mainly type I collagen and γ-carboxylate-containing small proteins in similar molar quantities to collagen. Specialized proteins that regulate connective tissue structure and formation are surprisingly similar in mineralized and non-mineralized tissues. While serum calcium and phosphate are adequate to precipitate mineral, specialized mechanisms normally prevent mineral formation except in bone, where continuous transport and deposition of mineral occurs.

Growth and mineralization of osteoblasts from mesenchymal stem cells on microporous membranes: Epithelial-like growth with transmembrane resistance and pH gradient.

Osteoblasts in vivo form an epithelial-like layer with tight junctions between cells. Bone formation involves mineral transport into the matrix and acid transport to balance pH levels. To study the importance of the pH gradient in vitro, we used Transwell inserts composed of polyethylene terephthalate (PET) membranes with 0.4 µm pores at a density of (2 ± 0.4) x 106 pores per cm2. Mesenchymal stem cells (MSCs) prepared from murine bone marrow were used to investigate alternative conditions whereby osteoblast differentiation would better emulate in vivo bone development. MSCs were characterized by flow cytometry with more than 90% CD44 and 75% Sca-1 labeling. Mineralization was validated with paracellular alkaline phosphatase activity, collagen birefringence, and mineral deposition confirming MSCs identity. We demonstrate that MSCs cultured and differentiated on PET inserts form an epithelial-like layer while mineralizing. Measurement of the transepithelial resistance was ∼1400 Ω•cm2 at three weeks of differentiation. The pH value of the media above and under the cells were measured while cells were in proliferation and differentiation. In mineralizing cells, a difference of 0.145 pH unit was observed between the medium above and under the cells indicating a transepithelial gradient. A significant difference in pH units was observed between the medium above and below the cells in proliferation compared to differentiation. Data on pH below membranes were confirmed by pH-dependent SNARF1 fluorescence. Control cells in proliferative medium did not form an epithelial-like layer, displayed low transepithelial resistance, and there was no significant pH gradient. By transmission electron microscopy, membrane attached osteoblasts in vitro had abundant mitochondria consistent with active transport that occurs in vivo by surface osteoblasts. In keeping with osteoblastic differentiation, scanning electron microscopy identified deposition of extracellular collagen surrounded by hydroxyapatite. This in vitro model is a major advancement in modeling bone in vivo for understanding of osteoblast bone matrix production.

Cellular and extracellular matrix of bone, with principles of synthesis and dependency of mineral deposition on cell membrane transport.

Bone differs from other connective tissues; it is isolated by a layer of osteoblasts that are connected by tight and gap junctions. This allows bone to create dense lamellar type I collagen, control pH, mineral deposition, and regulate water content forming a compact and strong structure. New woven bone formed after degradation of mineralized cartilage is rapidly degraded and resynthesized to impart structural order for local bone strength. Ossification is regulated by thickness of bone units and by patterning via bone morphogenetic receptors including activin, other bone morphogenetic protein receptors, transforming growth factor-ß receptors, all part of a receptor superfamily. This superfamily interacts with receptors for additional signals in bone differentiation. Important features of the osteoblast environment were established using recent tools including osteoblast differentiation in vitro. Osteoblasts deposit matrix protein, over 90% type I collagen, in lamellae with orientation alternating parallel or orthogonal to the main stress axis of the bone. Into this organic matrix, mineral is deposited as hydroxyapatite. Mineral matrix matures from amorphous to crystalline hydroxyapatite. This process includes at least two-phase changes of the calcium-phosphate mineral as well as intermediates involving tropocollagen fibrils to form the bone composite. Beginning with initiation of mineral deposition, there is uncertainty regarding cardinal processes, but the driving force is not merely exceeding the calcium-phosphate solubility product. It occurs behind a epithelial-like layer of osteoblasts, which generate phosphate and remove protons liberated during calcium-phosphate salt deposition. The forming bone matrix is discontinuous from the general extracellular fluid. Required adjustment of ionic concentrations and water removal from bone matrix are important details remaining to be addressed.

Support of bone mineral deposition by regulation of pH.

Osteoblasts secrete collagen and isolate bone matrix from extracellular space. In the matrix, alkaline phosphatase generates phosphate that combines with calcium to form mineral, liberating 8 H+ per 10 Ca+2 deposited. However, pH-dependent hydroxyapatite deposition on bone collagen had not been shown. We studied the dependency of hydroxyapatite deposition on type I collagen on pH and phosphate by surface plasmon resonance in 0-5 mM phosphate at pH 6.8-7.4. Mineral deposition saturated at <1 mM Ca2+ but was sensitive to phosphate. Mineral deposition was reversible, consistent with amorphous precipitation; stable deposition requiring EDTA removal appeared with time. At pH 6.8, little hydroxyapatite deposited on collagen; mineral accumulation increased 10-fold at pH 7.4. Previously, we showed high expression Na+/H+ exchanger (NHE) and ClC transporters in osteoblasts. We hypothesized that, in combination, these move protons across osteoblasts to the general extracellular space. We made osteoblast membrane vesicles by nitrogen cavitation and used acridine orange quenching to characterize proton transport. We found H+ transport dependent on gradients of chloride or sodium, consistent with apical osteoblast ClC family Cl-,H+ antiporters and basolateral osteoblast NHE family Na+/H+ exchangers. Little, if any, active H+ transport, supported by ATP, occurred. Major transporters include cariporide-sensitive NHE1 in basolateral membranes and ClC3 and ClC5 in apical osteoblast membranes. The mineralization inhibitor levamisole reduced bone formation and expression of alkaline phosphatase, NHE1, and ClC5. We conclude that mineral deposition in bone collagen is pH-dependent, in keeping with H+ removal by Cl-,H+ antiporters and Na+/H+-exchangers. Periodic orientation hydroxyapatite is organized on type I collagen-coiled coils.

A novel intrinsically fluorescent probe for study of uptake and trafficking of 25-hydroxycholesterol.

Cholesterol homeostasis is regulated not only by cholesterol, but also by oxygenated cholesterol species, referred to as oxysterols. Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), regulate cholesterol homeostasis through feedback inhibition and feed-forward activation of transcriptional pathways that govern cholesterol synthesis, uptake, and elimination, as well as through direct nongenomic actions that modulate cholesterol accessibility in membranes. Elucidating the cellular distribution of 25-HC is required to understand its biological activity at the molecular level. However, studying oxysterol distribution and behavior within cells has proven difficult due to the lack of fluorescent analogs of 25-HC that retain its chemical and physical properties. To address this, we synthesized a novel intrinsically fluorescent 25-HC mimetic, 25-hydroxycholestatrienol (25-HCTL). We show that 25-HCTL modulates sterol homeostatic responses in a similar manner as 25-HC. 25-HCTL associates with lipoproteins in media and is taken up by cells through LDL-mediated endocytosis. In cultured cells, 25-HCTL redistributes among cellular membranes and, at steady state, has a similar distribution as cholesterol, being enriched in both the endocytic recycling compartment as well as the plasma membrane. Our findings indicate that 25-HCTL is a faithful fluorescent 25-HC mimetic that can be used to investigate the mechanisms through which 25-HC regulates sterol homeostatic pathways.

Tracking the subcellular fate of 20(s)-hydroxycholesterol with click chemistry reveals a transport pathway to the Golgi.

Oxysterols, oxidized metabolites of cholesterol, are endogenous small molecules that regulate lipid metabolism, immune function, and developmental signaling. Although the cell biology of cholesterol has been intensively studied, fundamental questions about oxysterols, such as their subcellular distribution and trafficking pathways, remain unanswered. We have therefore developed a useful method to image intracellular 20(S)-hydroxycholesterol with both high sensitivity and spatial resolution using click chemistry and fluorescence microscopy. The metabolic labeling of cells with an alkynyl derivative of 20(S)-hydroxycholesterol has allowed us to directly visualize this oxysterol by attaching an azide fluorophore through cyclo-addition. Unexpectedly, we found that this oxysterol selectively accumulates in the Golgi membrane using a pathway that is sensitive to ATP levels, temperature, and lysosome function. Although previous models have proposed nonvesicular pathways for the rapid equilibration of oxysterols between membranes, direct imaging of oxysterols suggests that a vesicular pathway is responsible for differential accumulation of oxysterols in organelle membranes. More broadly, clickable alkynyl sterols may represent useful tools for sterol cell biology, both to investigate the functions of these important lipids and to decipher the pathways that determine their cellular itineraries.

Delivery of a Protease-Activated Cytolytic Peptide Prodrug by Perfluorocarbon Nanoparticles.

Melittin is a cytolytic peptide derived from bee venom that inserts into lipid membranes and oligomerizes to form membrane pores. Although this peptide is an attractive candidate for treatment of cancers and infectious processes, its nonspecific cytotoxicity and hemolytic activity have limited its therapeutic applications. Several groups have reported the development of cytolytic peptide prodrugs that only exhibit cytotoxicity following activation by site-specific proteases. However, systemic administration of these constructs has proven difficult because of their poor pharmacokinetic properties. Here, we present a platform for the design of protease-activated melittin derivatives that may be used in conjunction with a perfluorocarbon nanoparticle delivery system. Although native melittin was substantially hemolytic (HD50: 1.9 µM) and cytotoxic (IC50: 2.4 µM), the prodrug exhibited 2 orders of magnitude less hemolytic activity (HD50: > 100 µM) and cytotoxicity (IC50: > 100 µM). Incubation with matrix metalloproteinase-9 (MMP-9) led to cleavage of the prodrug at the expected site and restoration of hemolytic activity (HD50: 3.4 µM) and cytotoxicity (IC50: 8.1 µM). Incubation of the prodrug with perfluorocarbon nanoparticles led to stable loading of 10,250 peptides per nanoparticle. Nanoparticle-bound prodrug was also cleaved and activated by MMP-9, albeit at a fourfold slower rate. Intravenous administration of prodrug-loaded nanoparticles in a mouse model of melanoma significantly decreased tumor growth rate (p = 0.01). Because MMPs and other proteases play a key role in cancer invasion and metastasis, this platform holds promise for the development of personalized cancer therapies directed toward a patient's individual protease expression profile.

Side-chain oxysterols modulate cholesterol accessibility through membrane remodeling.

Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), are key regulators of cholesterol homeostasis. New evidence suggests that the alteration of membrane structure by 25-HC contributes to its regulatory effects. We have examined the role of oxysterol membrane effects on cholesterol accessibility within the membrane using perfringolysin O (PFO), a cholesterol-dependent cytolysin that selectively binds accessible cholesterol, as a sensor of membrane cholesterol accessibility. We show that 25-HC increases cholesterol accessibility in a manner dependent on the membrane lipid composition. Structural analysis of molecular dynamics simulations reveals that increased cholesterol accessibility is associated with membrane thinning, and that the effects of 25-HC on cholesterol accessibility are driven by these changes in membrane thickness. Further, we find that the 25-HC antagonist LY295427 (agisterol) abrogates the membrane effects of 25-HC in a nonenantioselective manner, suggesting that agisterol antagonizes the cholesterol-homeostatic effects of 25-HC indirectly through its membrane interactions. These studies demonstrate that oxysterols regulate cholesterol accessibility, and thus the availability of cholesterol to be sensed and transported throughout the cell, by modulating the membrane environment. This work provides new insights into how alterations in membrane structure can be used to relay cholesterol regulatory signals.

Oxysterols are allosteric activators of the oncoprotein Smoothened.

Oxysterols are a class of endogenous signaling molecules that can activate the Hedgehog pathway, which has critical roles in development, regeneration and cancer. However, it has been unclear how oxysterols influence Hedgehog signaling, including whether their effects are mediated through a protein target or indirectly through effects on membrane properties. To answer this question, we synthesized the enantiomer and an epimer of the most potent oxysterol, 20(S)-hydroxycholesterol. Using these molecules, we show that the effects of oxysterols on Hedgehog signaling are exquisitely stereoselective, consistent with the hypothesis that they function through a specific protein target. We present several lines of evidence that this protein target is the seven-pass transmembrane protein Smoothened, a major drug target in oncology. Our work suggests that these enigmatic sterols, which have multiple effects on cell physiology, may act as ligands for signaling receptors and provides a generally applicable framework for probing sterol signaling mechanisms.

Programmable nanoparticle functionalization for in vivo targeting.

The emerging demand for programmable functionalization of existing base nanocarriers necessitates development of an efficient approach for cargo loading that avoids nanoparticle redesign for each individual application. Herein, we demonstrate in vivo a postformulation strategy for lipidic nanocarrier functionalization with the use of a linker peptide, which rapidly and stably integrates cargos into lipidic membranes of nanocarriers after simple mixing through a self-assembling process. We exemplified this strategy by generating a VCAM-1-targeted perfluorocarbon nanoparticle for in vivo targeting in atherosclerosis (ApoE-deficient) and breast cancer (STAT-1-deficient) models. In the atherosclerotic model, a 4.1-fold augmentation in binding to affected aortas was observed for targeted vs. nontargeted nanoparticles (P<0.0298). Likewise, in the breast cancer model, a 4.9-fold increase in the nanoparticle signal from tumor vasculature was observed for targeted vs. nontargeted nanoparticles (P<0.0216). In each case, the nanoparticle was registered with fluorine ((19)F) magnetic resonance spectroscopy of the nanoparticle perfluorocarbon core, yielding a quantitative estimate of the number of tissue-bound nanoparticles. Because other common nanocarriers with lipid coatings (e.g., liposomes, micelles, etc.) can employ this strategy, this peptide linker postformulation approach is applicable to more than half of the available nanosystems currently in clinical trials or clinical uses.

Chloride/proton antiporters ClC3 and ClC5 support bone formation in mice.

Acid transport is required for bone synthesis by osteoblasts. The osteoblast basolateral surface extrudes acid by Na+/H+ exchange, but apical proton uptake is undefined. We found high expression of the Cl-/H+ exchanger ClC3 at the bone apical surface. In mammals ClC3 functions in intracellular vesicular chloride transport, but when we found Cl- dependency of H+ transport in osteoblast membranes, we queried whether ClC3 Cl-/H+ exchange functions in bone formation. We used ClC3 knockout animals, and closely-related ClC5 knockout animals: In vitro studies suggested that both ClC3 and ClC5 might support bone formation. Genotypes were confirmed by total exon sequences. Expression of ClC3, and to a lesser extent of ClC5, at osteoblast apical membranes was demonstrated by fluorescent antibody labeling and electron microscopy with nanometer gold labeling. Animals with ClC3 or ClC5 knockouts were viable. In ClC3 or ClC5 knockouts, bone formation decreased ~40 % by calcein and xylenol orange labeling in vivo. In very sensitive micro-computed tomography, ClC5 knockout reduced bone relative to wild type, consistent with effects of ClC3 knockout, but varied with specific histological parameters. Regrettably, ClC5-ClC3 double knockouts are not viable, suggesting that ClC3 or ClC5 activity are essential to life. We conclude that ClC3 has a direct role in bone formation with overlapping but probably slightly smaller effects of ClC5. The mechanism in mineral formation might include ClC H+ uptake, in contrast to ClC3 and ClC5 function in cell vesicles or other organs.

The structural basis of cholesterol accessibility in membranes.

Although the majority of free cellular cholesterol is present in the plasma membrane, cholesterol homeostasis is principally regulated through sterol-sensing proteins that reside in the cholesterol-poor endoplasmic reticulum (ER). In response to acute cholesterol loading or depletion, there is rapid equilibration between the ER and plasma membrane cholesterol pools, suggesting a biophysical model in which the availability of plasma membrane cholesterol for trafficking to internal membranes modulates ER membrane behavior. Previous studies have predominantly examined cholesterol availability in terms of binding to extramembrane acceptors, but have provided limited insight into the structural changes underlying cholesterol activation. In this study, we use both molecular dynamics simulations and experimental membrane systems to examine the behavior of cholesterol in membrane bilayers. We find that cholesterol depth within the bilayer provides a reasonable structural metric for cholesterol availability and that this is correlated with cholesterol-acceptor binding. Further, the distribution of cholesterol availability in our simulations is continuous rather than divided into distinct available and unavailable pools. This data provide support for a revised cholesterol activation model in which activation is driven not by saturation of membrane-cholesterol interactions but rather by bulk membrane remodeling that reduces membrane-cholesterol affinity.

Psychosine, the cytotoxic sphingolipid that accumulates in globoid cell leukodystrophy, alters membrane architecture.

Globoid cell leukodystrophy (GLD) is a neurological disease caused by deficiency of the lysosomal enzyme galactosylceramidase (GALC). In the absence of GALC, the cytotoxic glycosphingolipid, psychosine (psy), accumulates in the nervous system. Psychosine accumulation preferentially affects oligodendrocytes, leading to progressive demyelination and infiltration of activated monocytes/macrophages into the CNS. GLD is characterized by motor defects, cognitive deficits, seizures, and death by 2-5 years of age. It has been hypothesized that psychosine accumulation, primarily within lipid rafts, results in the pathogenic cascade in GLD. However, the mechanism of psychosine toxicity has yet to be elucidated. Therefore, we synthesized the enantiomer of psychosine (ent-psy) to use as a probe to distinguish between protein-psy (stereo-specific enantioselective) or membrane-psy (stereo-insensitive nonenantioselective) interactions. The enantiomer of psychosine has equal or greater toxicity compared with psy, suggesting that psy exerts its toxicity through a nonenantioselective mechanism. Finally, in this study we demonstrate that psy and ent-psy localize to lipid rafts, perturb natural and artificial membrane integrity, and inhibit protein Kinase C translocation to the plasma membrane. Although other mechanisms may play a role in disease, these data strongly suggest that psy exerts its effects primarily through membrane perturbation rather than through specific protein-psy interactions.

Side-chain oxysterols: from cells to membranes to molecules.

This review discusses the application of cellular biology, molecular biophysics, and computational simulation to understand membrane-mediated mechanisms by which oxysterols regulate cholesterol homeostasis. Side-chain oxysterols, which are produced enzymatically in vivo, are physiological regulators of cholesterol homeostasis and primarily serve as cellular signals for excess cholesterol. These oxysterols regulate cholesterol homeostasis through both transcriptional and non-transcriptional pathways; however, many molecular details of their interactions in these pathways are still not well understood. Cholesterol trafficking provides one mechanism for regulation. The current model of cholesterol trafficking regulation is based on the existence of two distinct cholesterol pools in the membrane: a low and a high availability/activity pool. It is proposed that the low availability/activity pool of cholesterol is integrated into tightly packing phospholipids and relatively inaccessible to water or cellular proteins, while the high availability cholesterol pool is more mobile in the membrane and is present in membranes where the phospholipids are not as compressed. Recent results suggest that oxysterols may promote cholesterol egress from membranes by shifting cholesterol from the low to the high activity pools. Furthermore, molecular simulations suggest a potential mechanism for oxysterol "activation" of cholesterol through its displacement in the membrane. This review discusses these results as well as several other important interactions between oxysterols and cholesterol in cellular and model lipid membranes. This article is part of a Special Issue entitled: Membrane protein structure and function.

Thrombin-targeted liposomes establish a sustained localized anticlotting barrier against acute thrombosis.

The goal of the present work was to design and test an acute-use nanoparticle-based antithrombotic agent that exhibits sustained local inhibition of thrombin without requiring a systemic anticoagulant effect to function against acute arterial thrombosis. To demonstrate proof of concept, we functionalized the surface of liposomes with multiple copies of the direct thrombin inhibitor, d-phenylalanyl-l-prolyl-l-arginyl-chloromethyl ketone (PPACK), which exhibits high affinity for thrombin as a free agent but manifests too rapid clearance in vivo to be effective alone. The PPACK-liposomes were formulated as single unilamellar vesicles, with a diameter of 170.78 ± 10.59 nm and a near neutral charge. In vitro models confirmed the inhibitory activity of PPACK-liposomes, demonstrating a KI' of 172.6 nM. In experimental clots in vitro, treatment of formed clots completely abrogated any further clotting upon exposure to human plasma. The liposomes were evaluated in vivo in a model of photochemical-induced carotid artery injury, resulting in significantly prolonged arterial occlusion time over that of controls (69.06 ± 5.65 min for saline treatment, N = 6, 71.33 ± 9.46 min for free PPACK treated; N = 4, 85.75 ± 18.24 min for precursor liposomes; N = 4, 139.75 ± 20.46 min for PPACK-liposomes; P = 0.0049, N = 6). Systemic anticoagulant profiles revealed a rapid return to control levels within 50 min, while still maintaining antithrombin activity at the injury site. The establishment of a potent and long-acting anticoagulant surface over a newly forming clot with the use of thrombin targeted nanoparticles that do not require systemic anticoagulation to be effective offers an alternative site-targeted approach to the management of acute thrombosis.

Simulation of fusion-mediated nanoemulsion interactions with model lipid bilayers.

Perfluorocarbon-based nanoemulsion particles have become promising platforms for the delivery of therapeutic and diagnostic agents to specific target cells in a non-invasive manner. A "contact-facilitated" delivery mechanism has been proposed wherein the emulsifying phospholipid monolayer on the nanoemulsion surface contacts and forms a lipid complex with the outer monolayer of target cell plasma membrane, allowing cargo to diffuse to the surface of target cell. While this mechanism is supported by experimental evidence, its molecular details are unknown. The present study develops a coarse-grained model of nanoemulsion particles that are compatible with the MARTINI force field. Simulations using this coarse-grained model have demonstrated multiple fusion events between the particles and a model vesicular lipid bilayer. The fusion proceeds in the following sequence: dehydration at the interface, close apposition of the particles, protrusion of hydrophobic molecules to the particle surface, transient lipid complex formation, absorption of nanoemulsion into the liposome. The initial monolayer disruption acts as a rate-limiting step and is strongly influenced by particle size as well as by the presence of phospholipids supporting negative spontaneous curvature. The core-forming perfluorocarbons play critical roles in initiating the fusion process by facilitating protrusion of hydrophobic moieties into the interface between the two particles. This study directly supports the hypothesized nanoemulsion delivery mechanism and provides the underlying molecular details that enable engineering of nanoemulsions for a variety of medical applications.

25-Hydroxycholesterol increases the availability of cholesterol in phospholipid membranes.

Side-chain oxysterols are enzymatically generated oxidation products of cholesterol that serve a central role in mediating cholesterol homeostasis. Recent work has shown that side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), alter membrane structure in very different ways from cholesterol, suggesting a possible mechanism for how these oxysterols regulate cholesterol homeostasis. Here we extend our previous work by using molecular-dynamics simulations of 25-HC and cholesterol mixtures in 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayers to examine the combined effects of 25-HC and cholesterol in the same bilayer. 25-HC causes larger changes in membrane structure when added to cholesterol-containing membranes than when added to cholesterol-free membranes. We also find that the presence of 25-HC changes the position, orientation, and solvent accessibility of cholesterol, shifting it into the water interface and thus increasing its availability to external acceptors. This is consistent with experimental results showing that oxysterols can trigger cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. These effects provide a potential mechanism for 25-HC-mediated regulation of cholesterol trafficking and homeostasis through modulation of cholesterol availability.