The role of early cerebral edema and hematoma assessment in aneurysmal subarachnoid hemorrhage (a-SAH) in predicting structural brain abnormalities in cognitive impairments-- case controlled study.

BACKGROUND: Early assessment and management of cerebral edema and hematoma following aneurysmal subarachnoid hemorrhage (a-SAH) can significantly impact clinical cognitive outcomes. However, current clinical practices lack predictive models to identify early structural brain abnormalities affecting cognition. To address this gap, we propose the development of a predictive model termed the a-SAH Early Brain Edema/Hematoma Compression Neural (Structural Brain) Networks Score System (SEBE-HCNNSS). METHODS: In this study, 202 consecutive patients with spontaneous a-SAH underwent initial computed tomography (CT) or magnetic resonance imaging (MRI) scans within 24 hours of ictus with follow-up 2 months after discharge. Using logistic regression analysis (univariate and multivariate), we evaluated the association of clinically relevant factors and various traditional scale ratings with cognitive impairment (CI). Risk factors with the highest area under the curve (AUC) values were included in the multivariate analysis and least absolute shrinkage and selection operator (LASSO) analysis or Cox regression analysis. RESULTS: A total of 177 patients were enrolled in the study, and 43 patients were classified with a high SEBE-HCNNSS grade (3 to 5). After a mean follow-up of 2 months, 121 individuals (68.36%) with a-SAH and 3 control subjects developed incident CI. The CT inter-observer reliability of the SEBE-HCNNSS scale was high, with a Kappa value of 1. Furthermore, ROC analysis identified the SEBE-HCNNSS scale (OR 3.322, 95% CI 2.312-7.237, P=0.00025) as an independent predictor of edema, CI, and unfavorable prognosis. These results were also replicated in a validation cohort. CONCLUSION: Overall, the SEBE-HCNNSS scale represents a simple assessment tool with promising predictive value for CI and clinical outcomes post-a-SAH. Our findings indicate its practical utility as a prognostic instrument for risk evaluation after a-SAH, potentially facilitating early intervention and treatment.

Arsenic trioxide elicits anti-tumor activity by inhibiting polarization of M2-like tumor-associated macrophages via Notch signaling pathway in lung adenocarcinoma.

Drug-resistant advanced lung adenocarcinoma (LUAD) is an aggressive malignancy with limited treatment options. A therapeutic strategy for drug-resistant LUAD is to target the tumor associated macrophages (TAMs), because they play an important role in tumor immune escape, progression and metastasis. In this study, we conducted in vivo and in vitro investigation of the inhibitory effect of arsenic trioxide (ATO) on polarization of TAMs educated by LUAD. We found that ATO at a concentration of 4 µM disrupted the Notch-dependent positive feedback loop between LUAD and TAMs. In this loop, ATO inhibited the expression of Jagged1 and Notch1 in LUAD and suppressed M2 polarization via down-regulating Notch-dependent paracrine of CCL2 and IL1ß. As a result, the secretion of M2-derived TGF-ß1 decreased, thus inducing inhibitions of LUAD proliferation, migration, invasion, colony formation and epithelial-mesenchymal transition. In xenograft mouse models, ATO significantly inhibited tumor growth and down-regulated infiltration of M2-like TAMs in tumor tissues. In clinical LUAD biopsy samples, high Jagged1/Notch1 expression positively correlated with tumor-infiltrated M2-like TAMs, leading to poor prognosis. In conclusion, our results identified a novel tumor immunomodulating function for ATO, which can inhibit the polarization of M2-type TAMs to exert anti-tumor effects in the tumor microenvironment. Our results demonstrated the translational potential of repurposing ATO to target TAMs for lung adenocarcinoma treatment.

Human Galectin-7 Gene LGALS7 Promoter Sequence Polymorphisms and Risk of Spontaneous Intracerebral Hemorrhage: A Prospective Study.

Background and purpose: Despite evidence for the role of genetic factors in stroke, only a small proportion of strokes have been clearly attributed to monogenic factors, due to phenotypic heterogeneity. The goal of this study was to determine whether a significant relationship exists between human galectin-7 gene LGALS7 promoter region polymorphisms and the risk of stroke due to non-traumatic intracerebral hemorrhage (ICH). Methods: This two-stage genetic association study included an initial exploratory stage followed by a discovery stage. During the exploratory stage, transgenic galectin-7 mice or transgenic mice with the scrambled sequence of the hairpin structure -silenced down gene LGALS7-were generated and then expressed differentially expressed proteins and galectin-7-interacting proteins were identified through proteomic analysis. During the discovery stage, a single-nucleotide polymorphism (SNP) genotyping approach was used to determine associations between 2 LGALS7 SNPs and ICH stroke risk for a cohort of 24 patients with stroke of the Chinese Han population and 70 controls. Results: During the exploratory phase, LGALS7 expression was found to be decreased in TGLGALS-DOWN mice as compared to its expression in TGLGALS mice. During the discovery phase, analysis of LGALS7 sequences of 24 non-traumatic ICH cases and 70 controls led to the identification of 2 ICH susceptibility loci: a genomic region on 19q13.2 containing two LGALS7 SNPs, rs567785577 and rs138945880, whereby the A allele of rs567785577 and the T allele of rs138945880 were associated with greater risk of contracting ICH [for T and A vs. C and G, unadjusted odds ratio (OR) = 13.5; 95% CI = 2.249-146.5; p = 0.002]. This is the first study to genotype the galectin-7 promoter in patients with hemorrhagic stroke. Genotype and allele association tests and preliminary analysis of patients with stroke revealed that a single locus may be a genetic risk factor for hemorrhagic stroke. Conclusion: A and T alleles of two novel SNP loci of 19q13.2, rs567785577 and rs138945880, respectively, were evaluated for associations with susceptibility to ICH. Further studies with expanded case numbers that include subjects of other ethnic populations are needed to elucidate mechanisms underlying associations between these SNPs and ICH risk.

Novel Subgroups in Subarachnoid Hemorrhage and Their Association With Outcomes-A Systematic Review and Meta-Regression.

Background and Purpose: Subarachnoid hemorrhage (SAH) has long been classified into two main forms, aneurysmal SAH (aSAH) and non-aneurysmal SAH (naSAH), but the related risk factors for aSAH and naSAH are heterogeneous. Our objective was to determine the risk factors for SAH of known or unknown origin with respect to diagnostic evaluation in a large patient cohort. We sought to determine whether our classification system can further predict middle long-term stroke and death. Methods: We performed a systematic review and meta-analysis to identify risk factors for each SAH subtype. The discovery phase analyzed 11 risk factors from case studies in the literature. Kruskal-Wallis, Cox regression, logistic regression, and Kaplan-Meier analyses were used to compare the two groups. Results: A total of 14,904 (34.53%) male and 22,801 (52.84%) female patients were eligible for this study. At a median follow-up of 45.6 months, the 5-years overall survival was 97.768% (95% CI: 0.259-0.292) for aSAH patients and 87.904% (95% CI: 1.459-1.643) for naSAH patients. The 10-years survival rate was 93.870% (95% CI: 2.075-3.086) and 78.115% (95% CI: 2.810-3.156), respectively. Multi-risk factor subgroups showed significant intergroup differences. We identified eight risk factors (drugs, trauma, neoplastic, vessels lesion, inflammatory lesion, blood disease, aneurysm, peri-mesencephalic hemorrhage) using logistic regression, which were optimally differentiated among the aSAH [aSAH-S (AUC: 1), a-d-SAH (AUC: 0.9998), aSAH-T (AUC: 0.9199), aSAH-N (AUC: 0.9433), aSAH-V (AUC: 1), aSAH-I (AUC: 0.9954), a-bd-SAH (AUC: 0.9955)] and naSAH [na-pmSAH (AUC: 0.9979), na-ni-ivl-SAH (AUC: 1), na-t-SAH (AUC: 0.9997), na-ne-SAH (AUC: 0.9475), na-d-SAH (AUC: 0.7676)] subgroups. These models were applied in a parallel cohort, showing eight risk factors plus survival rates to predict the prognosis of SAH. Conclusions: The classification of risk factors related to aSAH and naSAH is helpful in the diagnosis and prediction of the prognosis of aSAH and naSAH patients. Further validation is needed in future clinical applications.

Determinants of neurological disease: Synthesis of systematic reviews.

Systematic reviews were conducted to identify risk factors associated with the onset and progression of 14 neurological conditions, prioritized as a component of the National Population Health Study of Neurological Conditions. These systematic reviews provided a basis for evaluating the weight of evidence of evidence for risk factors for the onset and progression of the 14 individual neurological conditions considered. A number of risk factors associated with an increased risk of onset for more than one condition, including exposure to pesticides (associated with an increased risk of AD, amyotrophic lateral sclerosis, brain tumours, and PD; smoking (AD, MS); and infection (MS, Tourette syndrome). Coffee and tea intake was associated with a decreased risk of onset of both dystonia and PD. Further understanding of the etiology of priority neurological conditions will be helpful in focusing future research initiatives and in the development of interventions to reduce the burden associated with neurological conditions in Canada and internationally.

Systematic reviews of factors associated with the onset and progression of neurological conditions in humans: A methodological overview.

As a component of the National Population Health Study of Neurological conditions, systematic reviews were conducted to identify risk factors associated with the onset and progression of 14 priority neurological conditions. Between 2011 and 2013, electronic databases and grey literature sources were searched to identify systematic reviews and primary studies reporting on the onset and progression of each condition. Inclusion was restricted to studies of humans reported in English or French. Additional condition-specific eligibility criteria were also applied. Titles and abstracts were screened by one reviewer with excluded records verified by a second reviewer. Full-text reports were screened independently by two reviewers. Disagreements were resolved by consensus or third party adjudication. Systematic reviews were quality appraised using the AMSTAR criteria, with only moderate and high quality reviews considered for inclusion. Primary studies were also sought to ensure that evidence from existing systematic reviews was supplemented with recent primary study findings (i.e., those published after the most recent systematic review). Evidence from primary studies was also considered if a systematic review was unavailable or of poor quality. Data were extracted using standardized forms. Where feasible, data were extracted independently by two reviewers. Otherwise, data were extracted by a single reviewer and independent data extraction by a second reviewer was conducted for a randomly selected sample of studies. An updated search was conducted in 2016 to identify systematic reviews published since the initial search in 2011-2013. A summary of the methodology used to conduct the systematic reviews is described. Illustrative results are provided for the risk of amyotrophic lateral sclerosis in relation to occupational exposure to lead and other heavy metals.

Identification of risk factors associated with onset and progression of amyotrophic lateral sclerosis using systematic review and meta-analysis.

Although amyotrophic lateral sclerosis (ALS) was identified as a neurological condition 150 years ago, risk factors related to the onset and progression of ALS remain largely unknown. Monogenic mutations in over 30 genes are associated with about 10% of ALS cases. The age at onset of ALS and disease types has been found to influence ALS progression. The present study was designed to identify additional putative risk factors associated with the onset and progression of ALS using systematic review and meta-analysis of observational studies. Risk factors that may be associated with ALS include: 1) genetic mutations, including the intermediate CAG repeat expansion in ATXN2; 2) previous exposure to heavy metals such as lead and mercury; 3) previous exposure to organic chemicals, such as pesticides and solvents; 4) history of electric shock; 5) history of physical trauma/injury (including head trauma/injury); 6) smoking (a weak risk factor for ALS in women); and 6) other risk factors, such as participating in professional sports, lower body mass index, lower educational attainment, or occupations requiring repetitive/strenuous work, military service, exposure to Beta-N-methylamino-l-alanin and viral infections. Risk factors that may be associated with ALS progression rate include: 1) nutritional status, including vitamin D deficiency; 2) comorbidities; 3) ethnicity and genetic factors; 4) lack of supportive care; and 4) smoking. The extent to which these associations may be causal is discussed, with further research recommended to strengthen the evidence on which determinations of causality may be based.

Clinical application of fast-track surgery with Chinese medicine treatment in the devascularization operation for cirrhotic portal hypertension.

OBJECTIVE: To investigate the clinical effect of fast-track surgery combined with Chinese medicine treatment in devascularization operation for cirrhotic esophageal varices. METHODS: Seventy-two patients with cirrhotic esophageal varices were selected from January 2009 to June 2013, and randomly assigned to a conventional group and a fast-track group (fast-track surgery combined with Chinese medicine treatment) using a randomized digital table, 36 cases in each group. Operation and anesthesia recovery time, postoperative hospitalization and quality of life were recorded and compared between groups during the perioperative period. RESULTS: Compared with the conventional group, the fast-track group had longer operation time (253.6±46.4 min vs. 220.6±51.0 min) and anesthesia recovery time (50.5±15.9 min vs. 23.5±9.6 min; P<0.01); less bleeding (311.3±46.8 mL vs. 356.2±57.5 mL; P<0.01) and less transfusion (1932.3±106.9 mL vs. 2045.6±115.4 mL; P<0.01); as well as faster recovery of gastrointestinal function, shorter postoperative hospitalization and higher quality of life. There were no serious postoperative complications and no further bleeding occurred. CONCLUSION: Fast-track surgery combined with Chinese medicine treatment is a safe and feasible approach to accelerate the recovery of patients with cirrhotic portal hypertension in perioperative period of devascularization operation.

A meta-analysis of observational studies of the association between chronic occupational exposure to lead and amyotrophic lateral sclerosis.

OBJECTIVE: The association between occupational exposure to lead and amyotrophic lateral sclerosis (ALS) was examined through systematic review and meta-analyses of relevant epidemiological studies and reported according to PRISMA guidelines. METHODS: Relevant studies were searched in multiple bibliographic databases through September 2013; additional articles were tracked through PubMed until submission. All records were screened in DistillerSR, and the data extracted from included articles were synthesized with meta-analysis. RESULTS: The risk of developing ALS among individuals with a history of exposure to lead was almost doubled (odds ratio, 1.81; 95% confidence interval, 1.39 to 2.36) on the basis of nine included case-control studies with specific lead exposure information, with no apparent heterogeneity across included studies (I = 14%). The attributable risk of ALS because of exposure to lead was estimated to be 5%. CONCLUSIONS: Previous exposure to lead may be a risk factor for ALS.

Intermediate CAG repeat expansion in the ATXN2 gene is a unique genetic risk factor for ALS--a systematic review and meta-analysis of observational studies.

Amyotrophic lateral sclerosis (ALS) is a rare degenerative condition of the motor neurons. Over 10% of ALS cases are linked to monogenic mutations, with the remainder thought to be due to other risk factors, including environmental factors, genetic polymorphisms, and possibly gene-environmental interactions. We examined the association between ALS and an intermediate CAG repeat expansion in the ATXN2 gene using a meta-analytic approach. Observational studies were searched with relevant disease and gene terms from MEDLINE, EMBASE, and PsycINFO from January 2010 through to January 2014. All identified articles were screened using disease terms, gene terms, population information, and CAG repeat information according to PRISMA guidelines. The final list of 17 articles was further evaluated based on the study location, time period, and authors to exclude multiple usage of the same study populations: 13 relevant articles were retained for this study. The range 30-33 CAG repeats in the ATXN2 gene was most strongly associated with ALS. The meta-analysis revealed that the presence of an intermediate CAG repeat (30-33) in the ATXN2 gene was associated with an increased risk of ALS [odds ratio (OR) = 4.44, 95%CI: 2.91-6.76)] in Caucasian ALS patients. There was no significant difference in the association of this CAG intermediate repeat expansion in the ATXN2 gene between familial ALS cases (OR = 3.59, 1.58-8.17) and sporadic ALS cases (OR = 3.16, 1.88-5.32). These results indicate that the presence of intermediate CAG repeat expansion in the ATXN2 gene is a specific genetic risk factor for ALS, unlike monogenic mutations with an autosomal dominant transmission mode, which cause a more severe phenotype of ALS, with a higher prevalence in familial ALS.

Virtual mutagenesis of isocitrate dehydrogenase 1 involved in glioblastoma multiforme.

BACKGROUND: Site A132Arg mutations potentially impair the affinity of isocitrate dehydrogenase 1 (IDH1) for its substrate isocitrate (ICT), consequently reducing the production of α-ketoglutarate and leading to tumor growth through the induction of the hypoxia-inducible factor-1 (HIF-1) pathway. However, given that the roles of other active sites in IDH1 substrate binding remain unclear, we aimed to investigate IDH1 mutation pattern and its influence on enzyme function. METHODS: Fifteen IDH1 catalytic active site candidates were selected for in silico mutagenesis and protein homology modeling. Binding free energy of the IDH1/ICT complexes with single-site mutations was compared with that of the wild type. The affinity of 10 IDH1 catalytic active sites for the ICT substrate was further calculated. RESULTS: The IDH1 active site included seven residues from chain A (A77Thr, A94Ser, A100Arg, A132Arg, A109Arg, A275Asp, and A279Asp) and three residues from chain B (B214Thr, B212Lys, and B252Asp) that constituted the substrate ICT-binding site. These residues were located within 0.5 nm of ICT, indicating a potential interaction with the substrate. IDH1 changes of binding free energy (ΔE) suggested that the A132Arg residue from chain A contributes three hydrogen bonds to the ICT α-carboxyl and ß-carboxyl groups, while the other nine residues involved in ICT binding form only one or two hydrogen bonds. Amino acid substitutes at A132Arg, A109Arg, and B212Lys sites, had the greatest effect on enzyme affinity for its substrate. CONCLUSIONS: Mutations at sites A132Arg, A109Arg, and B212Lys reduced IDH1 affinity for ICT, indicating these active sites may play a central role in substrate binding. Mutations at sites A77Thr, A94Ser, and A275Asp increased the affinity of IDH1 for ICT, which may enhance IDN1 catalytic activity. Mutant IDH1 proteins with higher catalytic activity than the wild-type IDH1 could potentially be used as a novel gene therapy for glioblastoma multiforme.

Ht31, a protein kinase A anchoring inhibitor, induces robust cholesterol efflux and reverses macrophage foam cell formation through ATP-binding cassette transporter A1.

Macrophage foam cell is the predominant cell type in atherosclerotic lesions. Removal of excess cholesterol from macrophages thus offers effective protection against atherosclerosis. Here we report that a protein kinase A (PKA)-anchoring inhibitor, st-Ht31, induces robust cholesterol/phospholipid efflux, and ATP-binding cassette transporter A1 (ABCA1) greatly facilitates this process. Remarkably, we found that st-Ht31 completely reverses foam cell formation, and this process is ABCA1-dependent. The reversal is also accompanied by the restoration of well modulated inflammatory response to LPS. There is no detectable toxicity associated with st-Ht31, even when cells export up to 20% cellular cholesterol per hour. Using FRET-based PKA biosensors in live cells, we provide evidence that st-Ht31 drives cholesterol efflux by elevating PKA activity specifically in the cytoplasm. Furthermore, ABCA1 facilitates st-Ht31 uptake. This allows st-Ht31 to effectively remove cholesterol from ABCA1-expressing cells. We speculate that de-anchoring of PKA offers a novel therapeutic strategy to remove excess cholesterol from lipid-laden lesion macrophages.

Regulation of cholesterol efflux from macrophages.

PURPOSE OF REVIEW: The lipid efflux pathway is important for both HDL formation and the reverse cholesterol transport pathway. This review is focused on recent findings on the mechanism of lipid efflux and its regulation, particularly in macrophages. RECENT FINDINGS: Significant progress has been made on understanding the sequence of events that accompany the interaction of apolipoproteins A-I with cell surface ATP-binding cassette transporter A1 and its subsequent lipidation. Continued research on the regulation of ATP-binding cassette transporter A1 and ATP-binding cassette transporter G1 expression and traffic has also generated new paradigms for the control of lipid efflux from macrophages and its contribution to reverse cholesterol transport. In addition, the mobilization of cholesteryl esters from lipid droplets represents a new step in the control of cholesterol efflux. SUMMARY: The synergy between lipid transporters is a work in progress, but its importance in reverse cholesterol transport is clear. The regulation of efflux implies both the regulation of relevant transporters and the cellular trafficking of cholesterol.

[Construction and identification of RNA interference lentiviral expression vector of galectin-3 gene].

OBJECTIVE: To construct a recombinant lentiviral U6 plasmids for RNA interference (RNAi) of galectin-3 gene and select the optimal target sequence of galectin-3 gene for RNAi. METHODS: Double-stranded oligo DNAs were designed and synthesized according to the sequence of galectin-3 gene, and ligated into linearized pGCL-GFP/U6 plasmid followed by transformation into competent DH5alpha cells. After PCR and sequence analysis for verification of the positive clones, the plasmid pGCL-GFP/U6 Gal-3shRNA-1 was extracted and transfected into CaCl2-treated 293T cells to obtain the viral vectors containing the RNAi sequence. MCF-7 cells were infected with pGCL-GFP/U6 Gal-3shDNA-1, and at the infection rate over 50%, the cells were harvested to extract the RNA. Real time-PCR was performed to determine the expression level of galectin-3 mRNA in the infected cells. RESULTS: The recombinant vector was successfully constructed as confirmed by sequence analysis. High titer of the virus was obtained, and after infection of MCF-7 cells, RNAi targeting the 1# and 3# sequences in galectin-3 gene resulted in suppression of galectin-3 mRNA expression by 95% and 85%, respectively. CONCLUSION: The recombinant lentiviral U6 plasmid for RNAi of Galectin-3 gene has been successfully constructed, which provides the basis for further study of the role of galectin-3 gene in tumor cells.

Epoxycholesterol impairs cholesteryl ester hydrolysis in macrophage foam cells, resulting in decreased cholesterol efflux.

OBJECTIVE: Strategies to inhibit or reverse cholesterol accumulation in macrophages have been shown to be atheroprotective. Notably, the administration of LXR agonists upregulates key players in the reverse cholesterol transport pathway, including the ABCA1 and ABCG1 transporters. However, the effects of natural LXR activators, oxysterols, on lipid-laden macrophages remains elusive. METHODS AND RESULTS: We assessed the ability of 2 oxysterols, 22(R)-hydroxycholesterol (22-OH) and 24(S),25-epoxycholesterol (epoxycholesterol), to promote cholesterol efflux to apoA-I from LDL- and modified LDL-labeled and loaded macrophages and thus rescue the phenotype associated with the accumulation of cellular cholesterol in these cells. In macrophages labeled with LDL-derived cholesterol, epoxycholesterol treatment enhances ABCA1-mediated cholesterol efflux. In contrast, in AcLDL-loaded macrophages, epoxycholesterol treatment decreases cholesterol efflux to apoA-I, despite a dramatic increase in the expression of ABCA1 in response to epoxycholesterol treatment. We show that the decreased efflux is attributable to impaired cholesterol mobilization from lipid droplets, resulting from decreased cholesteryl ester hydrolase activity. CONCLUSIONS: Epoxycholesterol impairs cholesteryl ester hydrolysis activity in macrophage foam cells, thus reducing the availability of cholesterol for efflux to cholesterol acceptors.

N'-(4-Methoxy-benzyl-idene)-4-nitro-benzo-hydrazide methanol solvate.

The title compound, C(15)H(13)N(3)O(4)·CH(4)O, was synthesized from the reaction of 4-methoxy-benzaldehyde with 4-nitro-benzohydrazide in methanol. The benzene rings of the Schiff base mol-ecule are nearly coplanar, making a dihedral angle of 7.0 (3)°. The methanol solvent mol-ecules are linked to the Schiff base mol-ecules by N-H⋯O, O-H⋯N and O-H⋯O hydrogen bonds, forming chains running parallel to the b axis.

In vivo reverse cholesterol transport from macrophages lacking ABCA1 expression is impaired.

BACKGROUND: ATP-binding cassette transporter A1 (ABCA1) is a key mediator of cholesterol efflux to apoA-I in cholesterol loaded macrophages, a first step of reverse cholesterol transport (RCT) in vivo. Macrophage specific abca1 inactivation or overexpression, respectively, accelerated or suppressed the development of atherosclerosis in mouse models. However, it is yet to be established that the ABCA1 effect is related to specific changes in RCT from the macrophages in vivo. METHODS AND RESULTS: one marrow-derived macrophages from abca1-/- or abca1+/- mice were labeled with 3H-cholesterol-AcLDL or 3H-cholesterol-LDL and injected into abca1+/+ abca1+/- or abca1-/- mice. When injected into abca1+/+ mice, return of 3H-cholesterol from labeled abca1-/- macrophages to serum, liver, bile, and feces was reduced by 50% (P=0.01) compared with control. When labeled wild-type macrophages were injected into abca1-/- mice, as compared with wild-type mice, the return of 3H-cholesterol to serum, liver, bile, and feces was also reduced. CONCLUSIONS: ABCA1 expression in macrophages contributes significantly to in vivo macrophage RCT. The important residual RCT observed from abca1-/- macrophages highlight the functionality of transporters that efflux to HDL.

Differential regulation of ATP binding cassette protein A1 expression and ApoA-I lipidation by Niemann-Pick type C1 in murine hepatocytes and macrophages.

Niemann-Pick type C1 (Npc1) protein inactivation results in lipid accumulation in late endosomes and lysosomes, leading to a defect of ATP binding cassette protein A1 (Abca1)-mediated lipid efflux to apolipoprotein A-I (apoA-I) in macrophages and fibroblasts. However, the role of Npc1 in Abca1-mediated lipid efflux to apoA-I in hepatocytes, the major cells contributing to HDL formation, is still unknown. Here we show that, whereas lipid efflux to apoA-I in Npc1-null macrophages is impaired, the lipidation of endogenously synthesized apoA-I by low density lipoprotein-derived cholesterol or de novo synthesized cholesterol or phospholipids in Npc1-null hepatocytes is significantly increased by about 1-, 3-, and 8-fold, respectively. The increased cholesterol efflux reflects a major increase of Abca1 protein in Npc1-null hepatocytes, which contrasts with the decrease observed in Npc1-null macrophages. The increased Abca1 expression is largely post-transcriptional, because Abca1 mRNA is only slightly increased and Lxr alpha mRNA is not changed, and Lxr alpha target genes are reduced. This differs from the regulation of Abcg1 expression, which is up-regulated at both mRNA and protein levels in Npc1-null cells. Abca1 protein translation rate is higher in Npc1-null hepatocytes, compared with wild type hepatocytes as measured by [(35)S]methionine incorporation, whereas there is no difference for the degradation of newly synthesized Abca1 in these two types of hepatocytes. Cathepsin D, which we recently identified as a positive modulator of Abca1, is markedly increased at both mRNA and protein levels by Npc1 inactivation in hepatocytes but not in macrophages. Consistent with this, inhibition of cathepsin D with pepstatin A reduced the Abca1 protein level in both Npc1-inactivated and WT hepatocytes. Therefore, Abca1 expression is specifically regulated in hepatocytes, where Npc1 activity modulates cathepsin D expression and Abca1 protein translation rate.

Different cellular traffic of LDL-cholesterol and acetylated LDL-cholesterol leads to distinct reverse cholesterol transport pathways.

Endocytosis of LDL and modified LDL represents regulated and unregulated cholesterol delivery to macrophages. To elucidate the mechanisms of cellular cholesterol transport and egress under both conditions, various primary macrophages were labeled and loaded with cholesterol or cholesteryl ester from LDL or acetylated low density lipoprotein (AcLDL), and the cellular cholesterol traffic pathways were examined. Confocal microscopy using fluorescently labeled 3,3'-dioctyldecyloxacarbocyanine perchlorate-labeled LDL and 1,1'-dioctyldecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate-labeled AcLDL demonstrated their discrete traffic pathways and accumulation in distinct endosomes. ABCA1-mediated cholesterol efflux to apolipoprotein A-I (apoA-I) was much greater for AcLDL-loaded macrophages compared with LDL. Treatment with the liver X receptor ligand 22-OH increased efflux to apoA-I in AcLDL-loaded but not LDL-loaded cells. In contrast, at a level equivalent to AcLDL, LDL-derived cholesterol was preferentially effluxed to HDL, in keeping with increased ABCG1. In vivo studies of reverse cholesterol transport (RCT) from cholesterol-labeled macrophages injected intraperitoneally demonstrated that LDL-derived cholesterol was more efficiently transported to the liver and secreted into bile than AcLDL-derived cholesterol. This indicates a greater efficiency of HDL than lipid-poor apoA-I in interstitial fluid in controlling in vivo RCT. These assays, taken together, emphasize the importance of mediators of diffusional cholesterol efflux in RCT.