David E. Scherrer

Targeted Antiproliferative Drug Delivery to Vascular Smooth Muscle Cells With a Magnetic Resonance Imaging Nanoparticle Contrast Agent Implications for Rational Therapy of Restenosis

Background—Restenosis is a serious complication of coronary angioplasty that involves the proliferation and migration of vascular smooth muscle cells (VSMCs) from the media to the intima, synthesis of extracellular matrix, and remodeling. We have previously demonstrated that tissue factor–targeted nanoparticles can penetrate and bind stretch-activated vascular smooth muscles in the media after balloon injury. In the present study, the concept of VSMC-targeted nanoparticles as a drug-delivery platform for the prevention of restenosis after angioplasty is studied. Methods and Results—Tissue factor–targeted nanoparticles containing doxorubicin or paclitaxel at 0, 0.2, or 2.0 mole% of the outer lipid layer were targeted for 30 minutes to VSMCs and significantly inhibited their proliferation in culture over the next 3 days. Targeting of the nanoparticles to VSMC surface epitopes significantly increased nanoparticle antiproliferative effectiveness, particularly for paclitaxel. In vitro dissolution studies revealed that nanoparticle drug release persisted over one week. Targeted antiproliferative results were dependent on the hydrophobic nature of the drug and noncovalent interactions with other surfactant components. Molecular imaging of nanoparticles adherent to the VSMC was demonstrated with high-resolution T1-weighted MRI at 4.7T. MRI 19F spectroscopy of the nanoparticle core provided a quantifiable approach for noninvasive dosimetry of targeted drug payloads. Conclusions—These data suggest that targeted paramagnetic nanoparticles may provide a novel, MRI-visualizable, and quantifiable drug delivery system for the prevention of restenosis after angioplasty.

Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease

Oxysterols are biomarkers for diagnosis and drug treatment in Niemann-Pick C1 disease. Turning the Tables on Cholesterol A big push in disease research is to identify biochemical markers (biomarkers) in the blood that are early indicators of a disease that is already silently under way. By detecting the disease in its earliest stages, drugs and other therapeutic interventions have the best chance of halting or reversing the course of the disease before major tissue damage has been done. In a new study, Porter and colleagues set out to identify blood biomarkers for Niemann-Pick C1, a childhood neurological disease that is usually fatal. Niemann-Pick C1 disease is caused by mutations in the NPC1 or NPC2 proteins that result in mishandling of cholesterol and lipids in the endolysosomal system of cells. This leads to aberrant deposition of free cholesterol in the central nervous system, the death of neurons, and increasing motor and intellectual impairment, usually resulting in death during adolescence. The early symptoms of the disease are often difficult to distinguish from other childhood diseases, and thus, intervention in the form of a drug such as miglustat often comes too late. This prompted Porter and coworkers to search for possible molecules in the blood that could be used for early diagnosis of the disease and also to monitor the effectiveness of new drugs. On the basis of reports that aberrantly deposited free cholesterol is associated with increased oxidative stress, these investigators reasoned that cholesterol oxidation products (oxysterols) might be the long-sought biomarkers for Niemann-Pick C1 disease. Working in mice lacking the Npc1 gene, the researchers quickly identified two oxysterols that were markedly elevated in the plasma and tissues of the sick mice but not their healthy counterparts. Furthermore, the concentrations of these two oxysterols increased as the disease progressed. Moving into cats carrying an NPC1 mutation, which exhibit similar disease symptoms and progression as human patients, Porter and coworkers were able to decrease elevated concentrations of the two oxysterols and ameliorate disease symptoms by treating the animals with the experimental drug cyclodextrin. But could oxysterols be used as biomarkers in the human disease? The investigators demonstrated that the blood concentrations of two related oxysterol molecules were almost 10 times higher in Niemann-Pick C1 patients than in age-matched healthy controls or those with other diseases such as atherosclerosis or diabetes. Together, these compelling results suggest that the two oxysterol molecules are accurate diagnostic markers of early clinical disease and can be used not only to monitor disease progression but also to demonstrate drug efficacy. Free cholesterol may be at the root of Niemann-Pick C1 disease, but now, there is a way to turn the tables on cholesterol by using its oxidation products to diagnose and treat the disease in its earliest stages. Niemann-Pick type C1 (NPC1) disease is a rare progressive neurodegenerative disorder characterized by accumulation of cholesterol in the endolysosomes. Previous studies implicating oxidative stress in NPC1 disease pathogenesis raised the possibility that nonenzymatic formation of cholesterol oxidation products could serve as disease biomarkers. We measured these metabolites in the plasma and tissues of the Npc1−/− mouse model and found several cholesterol oxidation products that were elevated in Npc1−/− mice, were detectable before the onset of symptoms, and were associated with disease progression. Nonenzymatically formed cholesterol oxidation products were similarly increased in the plasma of all human NPC1 subjects studied and delineated an oxysterol profile specific for NPC1 disease. This oxysterol profile also correlated with the age of disease onset and disease severity. We further show that the plasma oxysterol markers decreased in response to an established therapeutic intervention in the NPC1 feline model. These cholesterol oxidation products are robust blood-based biochemical markers for NPC1 disease that may prove transformative for diagnosis and treatment of this disorder, and as outcome measures to monitor response to therapy.

A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.

Molecular imaging of stretch-induced tissue factor expression in carotid arteries with intravascular ultrasound.

RATIONALE AND OBJECTIVES Molecular imaging with targeted contrast agents enables tissues to be distinguished by detecting specific cell-surface receptors. In the present study, a ligand-targeted acoustic nanoparticle system is used to identify angioplasty-induced expression of tissue factor by smooth muscle cells within carotid arteries. METHODS Pig carotid arteries were overstretched with balloon catheters, treated with tissue factor-targeted or a control nanoparticle system, and imaged with intravascular ultrasound before and after treatment. RESULTS Tissue factor-targeted emulsions bound and increased the echogenicity and gray-scale levels of overstretched smooth muscle cells within the tunica media, versus no change in contralateral control arteries. Expression of stretch-induced tissue factor in carotid artery media was confirmed by immunohistochemistry. CONCLUSIONS The potential for abnormal thrombogenicity of balloon-injured arteries, as reflected by smooth muscle expression of tissue factor, was imaged using a novel, targeted, nanoparticulate ultrasonic contrast agent.

Niemann-Pick C1 protects against atherosclerosis in mice via regulation of macrophage intracellular cholesterol trafficking

Niemann-Pick C1 (NPC1) is a key participant in cellular cholesterol trafficking. Loss of NPC1 function leads to defective suppression of SREBP-dependent gene expression and failure to appropriately activate liver X receptor-mediated (LXR-mediated) pathways, ultimately resulting in intracellular cholesterol accumulation. To determine whether NPC1 contributes to regulation of macrophage sterol homeostasis in vivo, we examined the effect of NPC1 deletion in BM-derived cells on atherosclerotic lesion development in the Ldlr-/- mouse model of atherosclerosis. High-fat diet-fed chimeric Npc1-/- mice reconstituted with Ldlr-/-Npc1-/- macrophages exhibited accelerated atherosclerosis despite lower serum cholesterol compared with mice reconstituted with wild-type macrophages. The discordance between the low serum lipoprotein levels and the presence of aortic atherosclerosis suggested that intrinsic alterations in macrophage sterol metabolism in the chimeric Npc1-/- mice played a greater role in atherosclerotic lesion formation than did serum lipoprotein levels. Macrophages from chimeric Npc1-/- mice showed decreased synthesis of 27-hydroxycholesterol (27-HC), an endogenous LXR ligand; decreased expression of LXR-regulated cholesterol transporters; and impaired cholesterol efflux. Lower 27-HC levels were associated with elevated cholesterol oxidation products in macrophages and plasma of chimeric Npc1-/- mice and with increased oxidative stress. Our results demonstrate that NPC1 serves an atheroprotective role in mice through regulation of LXR-dependent cholesterol efflux and mitigation of cholesterol-induced oxidative stress in macrophages.

Side Chain Oxygenated Cholesterol Regulates Cellular Cholesterol Homeostasis through Direct Sterol-Membrane Interactions

Side chain oxysterols exert cholesterol homeostatic effects by suppression of sterol regulatory element-binding protein maturation and promoting degradation of hydroxymethylglutaryl-CoA reductase. To examine whether oxysterol-membrane interactions contribute to the regulation of cellular cholesterol homeostasis, we synthesized the enantiomer of 25-hydroxycholesterol. Using this unique oxysterol probe, we provide evidence that oxysterol regulation of cholesterol homeostatic responses is not mediated by enantiospecific oxysterol-protein interactions. We show that side chain oxysterols, but not steroid ring-modified oxysterols, exhibit membrane expansion behavior in phospholipid monolayers and bilayers in vitro. This behavior is non-enantiospecific and is abrogated by increasing the saturation of phospholipid acyl chain constituents. Moreover, we extend these findings into cultured cells by showing that exposure to saturated fatty acids at concentrations that lead to endoplasmic reticulum membrane phospholipid remodeling inhibits oxysterol activity. These studies implicate oxysterol-membrane interactions in acute regulation of sterol homeostatic responses and provide new insights into the mechanism through which oxysterols regulate cellular cholesterol balance.

Tissue Factor Pathway Inhibitor Attenuates Procoagulant Activity and Upregulation of Tissue Factor at the Site of Balloon-Induced Arterial Injury in Pigs

Intravenous infusion of recombinant tissue factor pathway inhibitor (rTFPI) for 24 hours decreases neointimal thickening and luminal stenosis 1 month after balloon-induced injury to the carotid arteries in minipigs. This study was designed to determine whether the effect of rTFPI is accounted for by early decreases in procoagulant activity and thrombosis on the injured vessel wall. Vascular injury was induced by balloon hyperinflations in both carotid arteries of anesthetized pigs given no anticoagulant as a control (n=16), an intravenous infusion for 24 hours of rTFPI (0.5 mg/kg bolus and 25 microg. kg(-1). min(-1), n=14), or an intravenous infusion of unfractionated heparin (100 U. kg(-1). h(-1), n=19). Accumulation of radiolabeled autologous platelets was markedly decreased over 24 hours on injured arteries from animals given rTFPI (0.6x10(6)/cm(2)) compared with controls (2.5x10(6)/cm(2), P=0.0004). Deposition of radiolabeled fibrin was also decreased in rTFPI-treated animals (269+/-266 microg/cm(2)) compared with controls (2389+/-1673 microg/cm(2), P=0.04). Similar effects were observed with heparin. However, factor Xa activity, assayed after 24 hours by incubation of the injured arterial segments with the chromogenic substrate S-2222, was decreased more markedly on arteries from rTFPI-treated animals (0.14+/-0.13 OD) than those from heparin-treated animals (0.29+/-0.18 OD) compared with controls (0. 47+/-0.24 OD, P=0.0007). In addition, arteries from rTFPI-treated animals showed a 4-fold lower induction of tissue factor protein compared with controls (P=0.0002). Attenuation of procoagulant activity and tissue factor-mediated thrombin generation in response to injury may account for the promising results with rTFPI in the porcine angioplasty model.

Development of a bile acid–based newborn screen for Niemann-Pick disease type C

A newborn screen for Niemann-Pick disease type C was developed on the basis of discovery of a bile acid marker. Expanding the newborn screen Niemann-Pick disease type C (NPC) is a fatal neurologic disorder caused by the deficiency of an enzyme involved in cholesterol storage. Although this disease was untreatable in the past, new therapeutics are now in clinical trials, but they are most likely to be effective if treatment is started as early as possible, before neurodegeneration has occurred. Jiang et al. identified three bile acids that are greatly increased in the blood of patients with NPC compared to healthy controls. The authors also demonstrated that one of these bile acids can be reliably measured in dried blood spots using mass spectrometry, suggesting that this bile acid test should be evaluated for potential addition to neonatal screening programs. Niemann-Pick disease type C (NPC) is a fatal, neurodegenerative, cholesterol storage disorder. With new therapeutics in clinical trials, it is imperative to improve diagnostics and facilitate early intervention. We used metabolomic profiling to identify potential markers and discovered three unknown bile acids that were increased in plasma from NPC but not control subjects. The bile acids most elevated in the NPC subjects were identified as 3β,5α,6β-trihydroxycholanic acid and its glycine conjugate, which were shown to be metabolites of cholestane-3β,5α,6β-triol, an oxysterol elevated in NPC. A high-throughput mass spectrometry–based method was developed and validated to measure the glycine-conjugated bile acid in dried blood spots. Analysis of dried blood spots from 4992 controls, 134 NPC carriers, and 44 NPC subjects provided 100% sensitivity and specificity in the study samples. Quantification of the bile acid in dried blood spots, therefore, provides the basis for a newborn screen for NPC that is ready for piloting in newborn screening programs.

Rpl13a small nucleolar RNAs regulate systemic glucose metabolism.

Small nucleolar RNAs (snoRNAs) are non-coding RNAs that form ribonucleoproteins to guide covalent modifications of ribosomal and small nuclear RNAs in the nucleus. Recent studies have also uncovered additional non-canonical roles for snoRNAs. However, the physiological contributions of these small RNAs are largely unknown. Here, we selectively deleted four snoRNAs encoded within the introns of the ribosomal protein L13a (Rpl13a) locus in a mouse model. Loss of Rpl13a snoRNAs altered mitochondrial metabolism and lowered reactive oxygen species tone, leading to increased glucose-stimulated insulin secretion from pancreatic islets and enhanced systemic glucose tolerance. Islets from mice lacking Rpl13a snoRNAs demonstrated blunted oxidative stress responses. Furthermore, these mice were protected against diabetogenic stimuli that cause oxidative stress damage to islets. Our study illuminates a previously unrecognized role for snoRNAs in metabolic regulation.

Tracking the Subcellular Fate of 20(S)-Hydroxycholesterol with Click Chemistry Reveals a Transport Pathway to the Golgi

Background: Oxysterols are a class of emerging signaling molecules whose cell biology is poorly understood. Results: A click chemistry-based imaging strategy shows that 20(S)-hydroxycholesterol accumulates in Golgi membranes in a process that depends on ATP and lysosome function. Conclusion: 20(S)-Hydroxycholesterol is transported through a vesicular pathway to the Golgi. Significance: Specific transport pathways may regulate the oxysterol content of cellular membranes. 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.