Veterans Affairs Low-vision Intervention Trial II: One-year Follow-up.

SIGNIFICANCE: One-year follow-up is recommended for patients with macular diseases to assess functional changes associated with disease progression and to modify low-vision (LV) treatment plans, if indicated. PURPOSE: The purpose of this study was to observe 255 patients with macular diseases who received LV rehabilitation (rehabilitation with a therapist) or basic LV services (LV devices dispensed without therapy) during Veterans Affairs Low-vision Intervention Trial II after the trial ended at 4 months until 1-year follow-up. METHODS: The primary outcome measure was visual ability measured with the 48-item Veterans Affairs Low-vision Visual Functioning Questionnaire. Mean visual ability scores for the treatment groups were compared from baseline to 4 months, 4 months to 1 year, and baseline to 1 year. Changes from baseline to 1 year were compared between the two groups. Predictors of changes in visual ability from 4 months to 1 year were assessed using linear regression. RESULTS: Both groups experienced significant improvement in all measures of visual ability from baseline to 1 year but lost visual reading ability during the observation period (LV rehabilitation group, -0.64 [1.2] logit; 95% confidence interval [CI], -0.84 to -0.44 logit; basic LV group, -0.63 [1.4] logit; 95% CI, -0.88 to -0.38 logit), and overall visual ability was lost in the LV rehabilitation group (-0.20 [0.8] logit; 95% CI, -0.34 to -0.06 logit). Loss of visual reading ability in both groups from 4 months to 1 year was predicted by reading ability scores at 4 months, loss of near visual acuity from 4 months to 1 year, and lower EuroQol-5D utility index scores; loss of overall visual ability in the LV rehabilitation group during the same time period was predicted by lower overall ability scores at 4 months. CONCLUSIONS: Visual ability significantly improved in all groups from baseline to 1 year. However, the loss of visual reading ability experienced by both groups from 4 months to 1 year reduced the benefit of the services provided.

Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome.

Monitoring the progress of DNA molecules through a membrane pore has been postulated as a method for sequencing DNA for several decades. Recently, a nanopore-based sequencing instrument, the Oxford Nanopore MinION, has become available, and we used this for sequencing the Saccharomyces cerevisiae genome. To make use of these data, we developed a novel open-source hybrid error correction algorithm Nanocorr specifically for Oxford Nanopore reads, because existing packages were incapable of assembling the long read lengths (5-50 kbp) at such high error rates (between ∼5% and 40% error). With this new method, we were able to perform a hybrid error correction of the nanopore reads using complementary MiSeq data and produce a de novo assembly that is highly contiguous and accurate: The contig N50 length is more than ten times greater than an Illumina-only assembly (678 kb versus 59.9 kbp) and has >99.88% consensus identity when compared to the reference. Furthermore, the assembly with the long nanopore reads presents a much more complete representation of the features of the genome and correctly assembles gene cassettes, rRNAs, transposable elements, and other genomic features that were almost entirely absent in the Illumina-only assembly.

Cdkn2a is an atherosclerosis modifier locus that regulates monocyte/macrophage proliferation.

OBJECTIVE: Common genetic variants in a 58-kb region of chromosome 9p21, near the CDKN2A/CDKN2B tumor suppressor locus, are strongly associated with coronary artery disease. However, the underlying mechanism of action remains unknown. METHODS AND RESULTS: We previously reported a congenic mouse model harboring an atherosclerosis susceptibility locus and the region of homology with the human 9p21 locus. Microarray and transcript-specific expression analyses showed markedly decreased Cdkn2a expression, including both p16(INK4a) and p19(ARF), but not Cdkn2b (p15(INK4b)), in macrophages derived from congenic mice compared with controls. Atherosclerosis studies in subcongenic strains revealed genetic complexity and narrowed 1 locus to a small interval including Cdkn2a/b. Bone marrow (BM) transplantation studies implicated myeloid lineage cells as the culprit cell type, rather than resident vascular cells. To directly test the role of BM-derived Cdkn2a transcripts in atherogenesis and inflammatory cell proliferation, we performed a transplantation study using Cdkn2a(-/-) cells in the Ldlr(-/-) mouse model. Cdkn2a-deficient BM recipients exhibited accelerated atherosclerosis, increased Ly6C proinflammatory monocytes, and increased monocyte/macrophage proliferation compared with controls. CONCLUSION: These data provide a plausible mechanism for accelerated atherogenesis in susceptible congenic mice, involving decreased expression of Cdkn2a and increased proliferation of monocyte/macrophages, with possible relevance to the 9p21 human locus.

Free cholesterol accumulation in macrophage membranes activates Toll-like receptors and p38 mitogen-activated protein kinase and induces cathepsin K.

The molecular events linking lipid accumulation in atherosclerotic plaques to complications such as aneurysm formation and plaque disruption are poorly understood. BALB/c-Apoe(-/-) mice bearing a null mutation in the Npc1 gene display prominent medial erosion and atherothrombosis, whereas their macrophages accumulate free cholesterol in late endosomes and show increased cathepsin K (Ctsk) expression. We now show increased cathepsin K immunostaining and increased cysteinyl proteinase activity using near infrared fluorescence imaging over proximal aortas of Apoe(-/-), Npc1(-/-) mice. In mechanistic studies, cholesterol loading of macrophage plasma membranes (cyclodextrin-cholesterol) or endosomal system (AcLDL+U18666A or Npc1 null mutation) activated Toll-like receptor (TLR) signaling, leading to sustained phosphorylation of p38 mitogen-activated protein kinase and induction of p38 targets, including Ctsk, S100a8, Mmp8, and Mmp14. Studies in macrophages from knockout mice showed major roles for TLR4, following plasma membrane cholesterol loading, and for TLR3, after late endosomal loading. TLR signaling via p38 led to phosphorylation and activation of the transcription factor Microphthalmia transcription factor, acting at E-box elements in the Ctsk promoter. These studies suggest that free cholesterol enrichment of either plasma or endosomal membranes in macrophages leads to activation of signaling via various TLRs, prolonged p38 mitogen-activated protein kinase activation, and induction of Mmps, Ctsk, and S100a8, potentially contributing to plaque complications.

Reduced VLDL clearance in Apoe(-/-)Npc1(-/-) mice is associated with increased Pcsk9 and Idol expression and decreased hepatic LDL-receptor levels.

Niemann-Pick type C1 (NPC1) promotes the transport of LDL receptor (LDL-R)-derived cholesterol from late endosomes/lysosomes to other cellular compartments. NPC1-deficient cells showed impaired regulation of liver_X receptor (LXR) and sterol regulatory element-binding protein (SREBP) target genes. We observed that Apoe(-/-)Npc1(-/-) mice displayed a marked increase in total plasma cholesterol mainly due to increased VLDL, reflecting decreased clearance. Although nuclear SREBP-2 and Ldlr mRNA levels were increased in Apoe(-/-)Npc1(-/-) liver, LDL-R protein levels were decreased in association with marked induction of proprotein convertase subtilisin/kexin type 9 (Pcsk9) and inducible degrader of the LDL-R (Idol), both known to promote proteolytic degradation of LDL-R. While Pcsk9 is known to be an SREBP-2 target, marked upregulation of IDOL in Apoe(-/-)Npc1(-/-) liver was unexpected. However, several other LXR target genes also increased in Apoe(-/-)Npc1(-/-) liver, suggesting increased synthesis of endogenous LXR ligands secondary to activation of sterol biosynthesis. In conclusion, we demonstrate that NPC1 deficiency has a major impact on VLDL metabolism in Apoe(-/-) mice through modulation of hepatic LDL-R protein levels. In contrast to modest induction of hepatic IDOL with synthetic LXR ligands, a striking upregulation of IDOL in Apoe(-/-)Npc1(-/-) mice could indicate a role of endogenous LXR ligands in regulation of hepatic IDOL.

Athsq1 is an atherosclerosis modifier locus with dramatic effects on lesion area and prominent accumulation of versican.

OBJECTIVE: Susceptibility to atherosclerosis is genetically complex, and modifier genes that do not operate via traditional risk factors are largely unknown. A mouse genetics approach can simplify the genetic analysis and provide tools for mechanistic studies. METHODS AND RESULTS: We previously identified atherosclerosis susceptibility QTL (Athsq1) on chromosome 4 acting independently of systemic risk factors. We now report confirmation of this locus in congenic strains carrying the MOLF-derived susceptibility allele in the C57BL/6J-Ldlr(-/-) genetic background. Homozygous congenic mice exhibited up to 4.5-fold greater lesion area compared to noncongenic littermates (P<0.0001). Analysis of extracellular matrix composition revealed prominent accumulation of versican, a presumed proatherogenic matrix component abundant in human lesions but almost absent in the widely-used C57BL/6 murine atherosclerosis model. The results of a bone marrow transplantation experiment suggested that both accelerated lesion development and versican accumulation are mediated, at least in part, by macrophages. Interestingly, comparative mapping revealed that the Athsq1 congenic interval contains the mouse region homologous to a widely-replicated CHD locus on human chromosome 9p21. CONCLUSIONS: These studies confirm the proatherogenic activity of a novel gene(s) in the MOLF-derived Athsq1 locus and provide in vivo evidence for a causative role of versican in lesion development.

Spontaneous atherothrombosis and medial degradation in Apoe-/-, Npc1-/- mice.

BACKGROUND: The formation of an occluding thrombus on a ruptured or eroded atherosclerotic plaque is the hallmark event leading to acute coronary syndromes, myocardial infarction, and sudden death in humans. However, other species are highly resistant to plaque complications, and the specific processes predisposing to plaque destabilization and thrombosis are poorly understood. METHODS AND RESULTS: Mice carrying a null mutation of a gene regulating intracellular cholesterol transport (the Niemann-Pick C1 [Npc1] gene) were crossed with apolipoprotein E (Apoe) knockout mice to examine the effect of Npc1 on atherosclerotic lesion formation. Double-mutant mice showed greater lesion area compared with Apoe-/- littermates. Remarkably, the double mutants also developed large, protruding thrombi associated with the plaques and prominent medial degradation with inflammatory cell infiltration into the adventitia. Genetic studies suggested that the BALB background was permissive for plaque complications compared with C57BL/6J, and a BALB susceptibility locus was mapped by linkage analysis to chromosome 6. Examination of clotting parameters in double-knockout mice revealed that native clotting times were shortened and thrombin-antithrombin complex and soluble CD40 ligand levels were elevated compared with wild-type controls. In addition, cathepsin K was induced in Npc1-/- macrophages, and cathepsin K immunostaining and elastase activity were increased in proximal aortas of double-mutant mice compared with controls. CONCLUSIONS: A defect in intracellular cholesterol trafficking caused by the Npc1 null mutation predisposes to increased lesion formation, atherothrombosis, and medial degradation. Plaque complications may require a procoagulant state and an increased protease activity, leading to plaque destabilization.

Economic Evaluation of Low-Vision Rehabilitation for Veterans With Macular Diseases in the US Department of Veterans Affairs.

Importance: Examining costs and consequences of different low-vision (LV) programs provides important information about resources needed to expand treatment options efficiently. Objective: To examine the costs and consequences of LV rehabilitation or basic LV services. Design, Setting, and Participants: The US Department of Veterans Affairs (VA) Low Vision Intervention Trial (LOVIT) II was conducted from September 27, 2010, to July 31, 2014, at 9 VA facilities and included 323 veterans with macular diseases and a best-corrected distance visual acuity of 20/50 to 20/200. Veterans were randomized to receive basic LV services that provided LV devices without therapy, or LV rehabilitation that added a therapist to LV services who provided instruction and homework on using LV devices, eccentric viewing, and environmental modification. We compared costs and consequences between these groups. Interventions: Low-vision devices without therapy and LV devices with therapy. Main Outcomes and Measures: Costs of providing basic LV services or LV rehabilitation were assessed. We measured consequences as changes in functional visual ability from baseline to follow-up 4 months after randomization using the VA Low Vision Visual Functioning Questionnaire. Visual ability was measured in dimensionless log odds units (logits). Results: Of 323 randomized patients, the mean (SD) age was 80 (10.5) years, 314 (97.2%) were men, and 292 (90.4%) were white. One hundred sixty (49.5%) received basic LV services and 163 (50.1%) received LV rehabilitation. The mean (SD) total direct health care costs per patient were similar between patients who were randomized to receive basic LV services ($1662 [$671]) or LV rehabilitation ($1788 [$864]) (basic LV services, $126 lower; 95% CI, $299 lower to $35 higher; P = .15). However, basic LV services required less time and had lower transportation costs. Patients receiving LV rehabilitation had greater improvements in overall visual ability, reading ability, visual information processing, and visual motor skill scores.

Stimulation of abdominal and upper thoracic muscles with surface electrodes for respiration and cough: Acute studies in adult canines.

OBJECTIVE: To optimize maximal respiratory responses with surface stimulation over abdominal and upper thorax muscles and using a 12-Channel Neuroprosthetic Platform. METHODS: Following instrumentation, six anesthetized adult canines were hyperventilated sufficiently to produce respiratory apnea. Six abdominal tests optimized electrode arrangements and stimulation parameters using bipolar sets of 4.5 cm square electrodes. Tests in the upper thorax optimized electrode locations, and forelimb moment was limited to slight-to-moderate. During combined muscle stimulation tests, the upper thoracic was followed immediately by abdominal stimulation. Finally, a model of glottal closure for cough was conducted with the goal of increased peak expiratory flow. RESULTS: Optimized stimulation of abdominal muscles included three sets of bilateral surface electrodes located 4.5 cm dorsal to the lateral line and from the 8th intercostal space to caudal to the 13th rib, 80 or 100 mA current, and 50 Hz stimulation frequency. The maximal expired volume was 343 ± 23 ml (n=3). Optimized upper thorax stimulation included a single bilateral set of electrodes located over the 2nd interspace, 60 to 80 mA, and 50 Hz. The maximal inspired volume was 304 ± 54 ml (n=4). Sequential stimulation of the two muscles increased the volume to 600 ± 152 ml (n=2), and the glottal closure maneuver increased the flow. CONCLUSIONS: Studies in an adult canine model identified optimal surface stimulation methods for upper thorax and abdominal muscles to induce sufficient volumes for ventilation and cough. Further study with this neuroprosthetic platform is warranted.

Outcomes of the Veterans Affairs Low Vision Intervention Trial II (LOVIT II): A Randomized Clinical Trial.

IMPORTANCE: Randomized clinical trials are needed to compare effectiveness and cost-effectiveness of different low-vision (LV) programs. OBJECTIVE: To determine the value of adding LV rehabilitation with a therapist compared with LV services without intervention. DESIGN, SETTING, AND PARTICIPANTS: A randomized clinical trial was conducted from September 27, 2010, to July 31, 2014, of 323 veterans with macular diseases and best-corrected distance visual acuity (BCDVAbetter-eye) of 20/50 to 20/200. Masked interviewers administered questionnaires by telephone before and after LV treatment. Using an intention-to-treat design, participants were randomized to receive LV devices with no therapy or LV devices with a rehabilitation therapist providing instruction and homework on the use of LV devices, eccentric viewing, and environmental modification. Visual ability was measured in dimensionless log odds units (logits) (0.14-logit change in visual ability corresponds to ability change expected from a 1-line change in visual acuity). INTERVENTIONS: Low-vision devices without therapy and LV devices with therapy. MAIN OUTCOMES AND MEASURES: Comparison of changes (baseline to 4 months) in overall visual ability and in 4 functional domains (reading, visual information, visual motor, and mobility) estimated from responses to the Veterans Affairs Low Vision Visual Functioning Questionnaire (higher scores indicates more ability or less difficulty in performing activities), and comparison of MNREAD changes (baseline to end of treatment) in maximum reading speed, critical print size, and reading acuity (higher number indicates lower visual acuity). RESULTS: Of the 323 participants, 314 were male (97.2%); mean (SD) age, 80 (10.5) years. Basic LV was effective in improving visual ability. However, the LV rehabilitation group improved more in all visual function domains except mobility. Differences were 0.34-logit reading (95% CI, 0.0005 to 0.69; P = .05), 0.27-logit visual information (95% CI, 0.01 to 0.53; P = .04), 0.37-logit visual motor (95% CI, 0.08 to 0.66; P = .01), and 0.27-logit overall (95% CI, 0.06 to 0.49; P = .01). For MNREAD measures, there was more improvement in reading acuity (difference, -0.11 logMAR, 95% CI, -0.15 to -0.07; P < .001) and maximum reading speed (mean increase of 21.0 words/min; 95% CI, 6.4 to 35.5; P = .005), but not critical print size for the LV rehabilitation group (-0.06 logMAR; 95% CI, -0.12 to 0.002; P = .06). In stratified analyses, the LV rehabilitation group with BCDVAbetter-eye worse than 20/63 to 20/200 improved more in visual ability (reading, visual motor, and overall). Differences were 0.56-logit reading ability (95% CI, 0.08-1.04; P = .02), 0.40-logit visual motor (95% CI, 0.03-0.78; P = .04), 0.34-logit overall (95% CI, 0.06-0.62; P = .02). There was no significant difference between treatment groups for those with BCDVAbetter-eye of 20/50 to 20/63. CONCLUSIONS AND RELEVANCE: Both basic LV alone and combined with LV rehabilitation were effective, but the added LV rehabilitation increased the effect only for patients with BCDVAbetter-eye worse than 20/63 to 20/200. Basic LV services may be sufficient for most LV patients with mild visual impairment. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00958360.

Respiratory responses to stimulation of abdominal and upper-thorax intercostal muscles using multiple Permaloc electrodes.

Stimulation of abdominal and upper-thoracic muscles was studied with the long-term goal of improved respiratory care for spinal cord injury (SCI) patients. A 12-channel stimulator and multiple surface and implanted Permaloc electrodes were evaluated in five anesthetized canines. Abdominal stimulation with 100 mA using four bilateral sets of surface electrodes placed on the midaxillary line at the 7th through 13th intercostal spaces and with a closed airway at a large lung volume produced an expiratory tracheal pressure of 109 +/- 29 cm H2O (n = 2, mean +/- standard error of the mean). Similar high pressures were induced with implanted electrodes at the same locations. Upper-thoracic stimulation with 40 mA and four sets of implanted electrodes ventral to the axilla induced inspiratory pressures of -12 +/- 2 cm H2O (n = 5). Combined extradiaphragmatic pacing with an open airway produced a tidal volume of 440 +/- 45 mL (n = 4). The robust respiratory volumes and pressures suggest applications in SCI respiratory care.

Particulate phase cigarette smoke increases MnSOD, NQO1, and CINC-1 in rat lungs.

Loss of antioxidant/oxidant homeostasis perpetuates inflammation in the lungs and may contribute to the development of COPD and lung cancer. Cigarette smoke (CS) is a primary source of airway oxidative stress and recruits inflammatory cells into smokers' lungs. However, whether these consequences are attributable to a specific or the collective fraction of CS is unknown. We investigated whether the particulate or the gas phase of CS would alter expression of the antioxidant enzymes MnSOD and NQO1 or CINC-1. Sprague Dawley rats were exposed to sham (n = 10) or the particulate phase (PP; n = 10) or gas phase (n = 10) of a Kentucky reference cigarette (1R4F) for 2 h/d for 28 d, after which animals were sacrificed and the lower left lobe of the lung was removed. Immunoblots for SOD and NQO1 revealed that lungs exposed to PP had higher MnSOD/actin and NQO1/actin ratios than either sham-or gas phase-treated animals. In contrast, CuZnSOD remained unchanged. In PP-exposed animals, CINC-1 was 3-fold higher than in sham-exposed animals. The increases in MnSOD and NQO1 protein were associated with increases in total SOD, NQO1, and MPO activities. These data provide evidence that the PP of CS alters oxidant/antioxidant homeostasis in the lungs and participates in the pathogenesis of CS-induced lung diseases such as COPD and cancer.

TLR3 deficiency protects against collagen degradation and medial destruction in murine atherosclerotic plaques.

OBJECTIVE: Inflammatory cell activation plays a key role in atherosclerotic plaque growth and acute complications. While secretion of proteases and inflammatory cytokines are likely involved in the development of plaque instability, the precise mechanistic pathways are not well understood. METHODS AND RESULTS: Based on our previous study, we crossed Toll-like receptor 3 (Tlr3)(-/-) mice with a unique BALB-Apoe(-/-)Npc1(-/-) plaque complication-susceptible mouse model, as well as the widely-used B6-Ldlr(-/-) atherosclerosis model, to test the role of TLR3 signaling in the development of plaque instability. TLR3-deficient mice showed no change in aortic root lesion area, but displayed a marked increase in collagen and smooth muscle cell (SMC) content of lesions. Notably, Apoe(-/-)Npc1(-/-)Tlr3(-/-) mice exhibited a 50% reduction in the incidence of medial destruction, a precursor to aortic aneurysm formation. MMP-2 activity was markedly reduced in aortic extracts from Apoe(-/-)Npc1(-/-)Tlr3(-/-) compared to controls, while both MMP-2 and -9 activities were reduced in Ldlr(-/-)Tlr3(-/-) extracts. Consistent with the in vivo data, TLR3 deficiency suppressed MMP-2 activity induced by TNF-α or polyinosine-polycytidylic acid in macrophages from Apoe(-/-)Npc1(-/-) mice. CONCLUSIONS: TLR3 plays a critical role in regulating the degradation of extracellular matrix in lesions, in part by modulation of macrophage MMP-2 and -9 activities.

Hypertrophy of neurons within cardiac ganglia in human, canine, and rat heart failure: the potential role of nerve growth factor.

BACKGROUND: Autonomic imbalances including parasympathetic withdrawal and sympathetic overactivity are cardinal features of heart failure regardless of etiology; however, mechanisms underlying these imbalances remain unknown. Animal model studies of heart and visceral organ hypertrophy predict that nerve growth factor levels should be elevated in heart failure; whether this is so in human heart failure, though, remains unclear. We tested the hypotheses that neurons in cardiac ganglia are hypertrophied in human, canine, and rat heart failure and that nerve growth factor, which we hypothesize is elevated in the failing heart, contributes to this neuronal hypertrophy. METHODS AND RESULTS: Somal morphology of neurons from human (579.54±14.34 versus 327.45±9.17 µm(2); P<0.01) and canine hearts (767.80±18.37 versus 650.23±9.84 µm(2); P<0.01) failing secondary to ischemia and neurons from spontaneously hypertensive rat hearts (327.98±3.15 versus 271.29±2.79 µm(2); P<0.01) failing secondary to hypertension reveal significant hypertrophy of neurons in cardiac ganglia compared with controls. Western blot analysis shows that nerve growth factor levels in the explanted, failing human heart are 250% greater than levels in healthy donor hearts. Neurons from cardiac ganglia cultured with nerve growth factor are significantly larger and have greater dendritic arborization than neurons in control cultures. CONCLUSIONS: Hypertrophied neurons are significantly less excitable than smaller ones; thus, hypertrophy of vagal postganglionic neurons in cardiac ganglia would help to explain the parasympathetic withdrawal that accompanies heart failure. Furthermore, our observations suggest that nerve growth factor, which is elevated in the failing human heart, causes hypertrophy of neurons in cardiac ganglia.