Defective Lysosomal Lipolysis Causes Prenatal Lipid Accumulation and Exacerbates Immediately after Birth.

Cholesterol and fatty acids are essential lipids that are critical for membrane biosynthesis and fetal organ development. Cholesteryl esters (CE) are degraded by hormone-sensitive lipase (HSL) in the cytosol and by lysosomal acid lipase (LAL) in the lysosome. Impaired LAL or HSL activity causes rare pathologies in humans, with HSL deficiency presenting less severe clinical manifestations. The infantile form of LAL deficiency, a lysosomal lipid storage disorder, leads to premature death. However, the importance of defective lysosomal CE degradation and its consequences during early life are incompletely understood. We therefore investigated how defective CE catabolism affects fetus and infant maturation using Lal and Hsl knockout (-/-) mouse models. This study demonstrates that defective lysosomal but not neutral lipolysis alters placental and fetal cholesterol homeostasis and exhibits an initial disease pathology already in utero as Lal-/- fetuses accumulate hepatic lysosomal lipids. Immediately after birth, LAL deficiency exacerbates with massive hepatic lysosomal lipid accumulation, which continues to worsen into young adulthood. Our data highlight the crucial role of LAL during early development, with the first weeks after birth being critical for aggravating LAL deficiency.

Lysosomal lipoprotein processing in endothelial cells stimulates adipose tissue thermogenic adaptation.

In response to cold exposure, thermogenic adipocytes internalize large amounts of fatty acids after lipoprotein lipase-mediated hydrolysis of triglyceride-rich lipoproteins (TRL) in the capillary lumen of brown adipose tissue (BAT) and white adipose tissue (WAT). Here, we show that in cold-exposed mice, vascular endothelial cells in adipose tissues endocytose substantial amounts of entire TRL particles. These lipoproteins subsequently follow the endosomal-lysosomal pathway, where they undergo lysosomal acid lipase (LAL)-mediated processing. Endothelial cell-specific LAL deficiency results in impaired thermogenic capacity as a consequence of reduced recruitment of brown and brite/beige adipocytes. Mechanistically, TRL processing by LAL induces proliferation of endothelial cells and adipocyte precursors via beta-oxidation-dependent production of reactive oxygen species, which in turn stimulates hypoxia-inducible factor-1α-dependent proliferative responses. In conclusion, this study demonstrates a physiological role for TRL particle uptake into BAT and WAT and establishes endothelial lipoprotein processing as an important determinant of adipose tissue remodeling during thermogenic adaptation.

Lysosomal Acid Lipase Deficiency Controls T- and B-Regulatory Cell Homeostasis in the Lymph Nodes of Mice with Human Cancer Xenotransplants.

Utilization of proper preclinical models accelerates development of immunotherapeutics and the study of the interplay between human malignant cells and immune cells. Lysosomal acid lipase (LAL) is a critical lipid hydrolase that generates free fatty acids and cholesterol. Ablation of LAL suppresses immune rejection and allows growth of human lung cancer cells in lal-/- mice. In the lal-/- lymph nodes, the percentages of both T- and B-regulatory cells (Tregs and Bregs, respectively) are increased, with elevated expression of programmed death-ligand 1 and IL-10, and decreased expression of interferon-γ. Levels of enzymes in the glucose and glutamine metabolic pathways are elevated in Tregs and Bregs of the lal-/- lymph nodes. Pharmacologic inhibitor of pyruvate dehydrogenase, which controls the transition from glycolysis to the citric acid cycle, effectively reduces Treg and Breg elevation in the lal-/- lymph nodes. Blocking the mammalian target of rapamycin or reactivating peroxisome proliferator-activated receptor γ, an LAL downstream effector, reduces lal-/- Treg and Breg elevation and PD-L1 expression in lal-/- Tregs and Bregs, and improves human cancer cell rejection. Treatment with PD-L1 antibody also reduces Treg and Breg elevation in the lal-/- lymph nodes and improves human cancer cell rejection. These observations conclude that LAL-regulated lipid metabolism is essential to maintain antitumor immunity.

Reduced lysosomal acid lipase activity: A new marker of liver disease severity across the clinical continuum of non-alcoholic fatty liver disease?

Lysosomal acid lipase (LAL) plays a key role in intracellular lipid metabolism. Reduced LAL activity promotes increased multi-organ lysosomal cholesterol ester storage, as observed in two recessive autosomal genetic diseases, Wolman disease and Cholesterol ester storage disease. Severe liver steatosis and accelerated liver fibrosis are common features in patients with genetic LAL deficiency. By contrast, few reliable data are available on the modulation of LAL activity in vivo and on the epigenetic and metabolic factors capable of regulating its activity in subjects without homozygous mutations of the Lipase A gene. In the last few years, a less severe and non-genetic reduction of LAL activity was reported in children and adults with non-alcoholic fatty liver disease (NAFLD), suggesting a possible role of LAL reduction in the pathogenesis and progression of the disease. Patients with NAFLD show a significant, progressive reduction of LAL activity from simple steatosis to non-alcoholic steatohepatitis and cryptogenic cirrhosis. Among cirrhosis of different etiologies, those with cryptogenic cirrhosis show the most significant reductions of LAL activity. These findings suggest that the modulation of LAL activity may become a possible new therapeutic target for patients with more advanced forms of NAFLD. Moreover, the measurement of LAL activity may represent a possible new marker of disease severity in this clinical setting.

Deficiency in hormone-sensitive lipase accelerates the development of pancreatic cancer in conditional KrasG12D mice.

BACKGROUND: Hormone sensitive lipase (HSL) is a neutral lipase that preferentially catalyzes the hydrolysis of diacylglycerol contributing to triacylglycerol breakdown in the adipose tissue. HSL has been implicated to play a role in tumor cachexia, a debilitating syndrome characterized by progressive loss of adipose tissue. Consequently, pharmacological inhibitors of HSL have been proposed for the treatment of cancer-associated cachexia. In the present study we used the conditional KrasG12D (KC) mouse model of pancreatic ductal adenocarcinoma (PDAC) with a deficiency in HSL to determine the impact of HSL suppression on the development of PDAC. METHODS: KC;Hsl+/+ and KC;Hsl-/- mice were fed standard rodent chow for 20 weeks. At sacrifice, the incidence of PDAC was determined and inflammation in the mesenteric adipose tissue and pancreas was assessed histologically and by immunofluorescence. To determine statistical significance, ANOVA and two-tailed Student's t-tests were performed. To compare PDAC incidence, a two-sided Fisher's exact test was used. RESULTS: Compared to KC;Hsl+/+ mice, KC;Hsl-/- mice gained similar weight and displayed adipose tissue and pancreatic inflammation. In addition, KC;Hsl-/- mice had reduced levels of plasma insulin and leptin. Importantly, the increased adipose tissue and pancreatic inflammation was associated with a significant increase in PDAC incidence in KC;Hsl-/- mice. CONCLUSIONS: HSL deficiency is associated with adipose tissue and pancreatic inflammation and accelerates PDAC development in the KC mouse model.

Lysosomal Acid Lipase Deficiency, a Rare Pathology: The First Pediatric Patient Reported in Colombia.

BACKGROUND Lysosomal acid lipase deficiency is a rare genetic metabolic lipid storage disease, with a high morbidity, and mortality, in children and adults. It is characterized by a mutation in the LIPA gene that causes an alteration of lipid metabolism, resulting in deposits of cholesterol esters and triglycerides in organs such as the liver, blood vessels, and gastrointestinal tract. Lysosomal acid lipase deficiency is predominantly caused by the mutation c.894G>A, seen in approximately 50-70% of patients. Our objective is to report the first pediatric case of lysosomal acid lipase deficiency in a pediatric patient in Colombia. CASE REPORT The patient is a 14-year-old boy with isolated hepatomegaly since 6 years of age without a family history of dyslipidemia. In the pediatric control, laboratory exams revealed dyslipidemia, and a hepatic biopsy was performed, revealing severe fibrosis with septation and grade 3 microvesicular steatosis (>75%). He was referred to our center and was suspected to have lysosomal acid lipase deficiency. Enzymatic activity was measured, showing absent activity. Confirmatory diagnosis with genetic sequencing showed a pathological homozygous mutation of c.894G>A. CONCLUSIONS Lysosomal acid lipase deficiency can manifest as early- or late-onset, with variable and severe signs and symptoms. The late-onset form has a broad spectrum of manifestations with mild symptoms, leading to under-diagnosis, which increases the actual disease burden. Early diagnosis is essential to initiate enzyme replacement therapy, since the natural disease course can be changed. More studies should be conducted in Latin America to evaluate the prevalence of the disease.

Identification of rare diseases by screening a population selected on the basis of routine pathology results-the PATHFINDER project: lysosomal acid lipase/cholesteryl ester storage disease substudy.

AIMS: Lysosomal acid lipase deficiency (LALD) is an autosomal recessive disorder of cholesterol ester storage associated with hepatic disease, cirrhosis and accelerated atherosclerosis. Its prevalence in the general population, patients with dyslipidaemia and raised transaminases is unclear. This study attempted to identify the prevalence of LALD from patients with abnormal results in laboratory databases. METHODS: Electronic laboratory databases were interrogated to identify from clinical biochemistry records patients with a phenotype of low high-density lipoprotein-cholesterol (≤0.85 mmol/L; 33 mg/dL) and with elevated alanine or aspartate transaminases (≥60 IU/L) on one occasion or more over a 3-year time interval. Patients were recalled, and a dried blood spot sample was collected for lysosomal acid lipase determination by a fluorimetric enzyme assay. Histopathology databases of liver biopsies were interrogated for patients with features of 'microvesicular cirrhosis' or 'cryptogenic cirrhosis' in the report. Histological blocks were sampled, and samples were analysed by next-generation sequencing for the presence of mutations in the LAL gene. RESULTS: Samples were obtained from 1825 patients with dyslipidaemia and elevated transaminases. No cases of LALD were identified. Liver biopsies were obtained from six patients. DNA extraction was successful from four patients. Two patients were homozygous for the LAL c.46A>C;p.Thr16Pro unclassified variant in exon 2. CONCLUSIONS: Pathology databases hold routine information that can be used to identify patients with specific patterns of results or those who had biopsies to allow targeted testing for possible causes of disease. Biochemical screening suggests that the gene frequency of LAL deficiency in adults is less than 1 in 100.

Molecular and clinical characterization of a series of patients with childhood-onset lysosomal acid lipase deficiency. Retrospective investigations, follow-up and detection of two novel LIPA pathogenic variants.

BACKGROUND AND AIMS: Childhood/Adult-onset Lysosomal Acid Lipase Deficiency (LAL-D) is a recessive disorder due to loss of function variants of LAL, the enzyme which hydrolyses cholesteryl esters, derived from internalized apoB containing lipoproteins. The disease is characterized by multi-organ involvement including the liver, spleen, intestine and cardiovascular system. The aim of this study was the clinical and molecular characterization of 14 (13 unrelated) previously unreported patients with childhood-onset LAL-D. METHODS: Data collected included clinical and laboratory investigations, liver imaging, liver biopsy and LIPA gene analysis. The response to lipid-lowering medications, liver transplantation and enzyme replacement therapy (ERT) was reported for some patients. RESULTS: LAL-D was suspected at 4.4 ± 3.3 years of age for the presence of hepatomegaly, elevated serum transaminases and hypercholesterolemia, and was confirmed by liver biopsy/imaging and LAL assay. The follow up period ranged from 3 to 40 years (mean 7.8 ± 4.0 years in 13 cases). Patients treated with statins with or without ezetimibe showed 28% reduction of plasma LDL-cholesterol without a tangible effect on liver enzymes; some patients receiving ERT showed normalized lipoprotein profile and transaminase levels. The common c.894G > A variant was observed in homozygosity or compound heterozygosity in 10 patients. We found seven previously reported variants: p.(Trp140*), p.(Arg218*), p.(Gly266*), p.(Thr288Ile), p.(Leu294Ser), p.(His295Tyr) and p.(Gly342Arg) and two novel variants: p.(Asp345Asn), affecting the LAL catalytic triad, and c.229+3A > C, affecting splicing. Homozygosity for p.(Thr288Ile) or c.229+3A > C was associated with a severe phenotype. CONCLUSIONS: This study provides additional data on the features of childhood-onset LAL-D and describes two novel pathogenic variants of the LIPA gene.

Managing Cardiovascular Risk in Lysosomal Acid Lipase Deficiency.

Lysosomal acid lipase deficiency (LAL-D) is a rare, life-threatening, autosomal recessive, lysosomal storage disease caused by mutations in the LIPA gene, which encodes for lysosomal acid lipase (LAL). This enzyme is necessary for the hydrolysis of cholesteryl ester and triglyceride in lysosomes. Deficient LAL activity causes accumulation of these lipids in lysosomes and a marked decrease in the cytoplasmic free cholesterol concentration, leading to dysfunctional cholesterol homeostasis. The accumulation of neutral lipid occurs predominantly in liver, spleen, and macrophages throughout the body, and the aberrant cholesterol homeostasis causes a marked dyslipidemia. LAL-D is characterized by accelerated atherosclerotic cardiovascular disease (ASCVD) and hepatic microvesicular or mixed steatosis, leading to inflammation, fibrosis, cirrhosis and liver failure. LAL-D presents as a clinical continuum with two phenotypes: the infantile-onset phenotype, formally referred to as Wolman disease, and the later-onset phenotype, formerly referred to as cholesteryl ester storage disease. Infants with LAL-D present within the first few weeks of life with vomiting, diarrhea, hepatosplenomegaly, failure to thrive and rapid progression to liver failure and death by 6-12 months of age. Children and young adults with LAL-D generally present with marked dyslipidemia, hepatic enzyme elevation, hepatomegaly and mixed steatosis by liver biopsy. The average age of the initial signs and symptoms of the later-onset phenotype is about 5 years old. The typical dyslipidemia is a significantly elevated low-density lipoprotein cholesterol (LDL-C) concentration and a low high-density lipoprotein cholesterol (HDL-C) concentration, placing these individuals at heightened risk for premature ASCVD. Diagnosis of the later-onset phenotype of LAL-D requires a heightened awareness of the disease because the dyslipidemia and hepatic transaminase elevation combination are common and overlap with other metabolic disorders. LAL-D should be considered in the differential diagnosis of healthy weight children and young adults with unexplained hepatic transaminase elevation accompanied by an elevated LDL-C level (>160 mg/dL) and low HDL-C level (<35 mg/dL) that is not caused by monogenic and polygenic lipid disorders or secondary factors. Treatment of LAL-D with sebelipase alfa (LAL replacement enzyme) should be considered as the standard of treatment in all individuals diagnosed with LAL-D. Other ASCVD risk factors that may be present (hypertension, tobacco use, diabetes mellitus, etc.) should be managed appropriately, consistent with secondary prevention goals.

Critical role of PPARγ in myeloid-derived suppressor cell-stimulated cancer cell proliferation and metastasis.

Lysosomal acid lipase (LAL) is a key enzyme controlling neutral lipid metabolic signaling in myeloid-derived suppressor cells (MDSCs). MDSCs from LAL-deficient (lal-/-) mice directly stimulate cancer cell proliferation. PPARγ ligand treatment inhibited lal-/- MDSCs stimulation of tumor cell growth and metastasis in vivo, and tumor cell proliferation and migration in vitro. In addition, PPARγ ligand treatment impaired lal-/- MDSCs transendothelial migration, and differentiation from lineage-negative cells. The corrective effects of PPARγ ligand on lal-/- MDSCs functions were mediated by regulating the mammalian target of rapamycin (mTOR) pathway, and subsequently blocking MDSCs ROS overproduction. Furthermore, in the myeloid-specific dominant-negative PPARγ (dnPPARγ) overexpression bitransgenic mouse model, tumor growth and metastasis were enhanced, and MDSCs from these mice stimulated tumor cell proliferation and migration. MDSCs with dnPPARγ overexpression showed increased transendothelial migration, overactivation of the mTOR pathway, and ROS overproduction. These results indicate that PPARγ plays a critical role in neutral lipid metabolic signaling controlled by LAL, which provides a mechanistic basis for clinically targeting MDSCs to reduce the risk of cancer proliferation, growth and metastasis.

Clinical Features of Lysosomal Acid Lipase Deficiency.

OBJECTIVE: The aim of this study was to characterize key clinical manifestations of lysosomal acid lipase deficiency (LAL D) in children and adults. METHODS: Investigators reviewed medical records of LAL D patients ages ≥5 years, extracted historical data, and obtained prospective laboratory and imaging data on living patients to develop a longitudinal dataset. RESULTS: A total of 49 patients were enrolled; 48 had confirmed LAL D. Mean age at first disease-related abnormality was 9.0 years (range 0-42); mean age at diagnosis was 15.2 years (range 1-46). Twenty-nine (60%) were male patients, and 27 (56%) were <20 years of age at the time of consent/assent. Serum transaminases were elevated in most patients with 458 of 499 (92%) of alanine aminotransferase values and 265 of 448 (59%) of aspartate aminotransferase values above the upper limit of normal. Most patients had elevated low-density lipoprotein (64% patients) and total cholesterol (63%) at baseline despite most being on lipid-lowering therapies, and 44% had high-density lipoprotein levels below the lower limit of normal. More than half of the patients with liver biopsies (n = 31, mean age 13 years) had documented evidence of steatosis (87%) and/or fibrosis (52%). Imaging assessments revealed that the median liver volume was ∼1.15 multiples of normal (MN) and median spleen volume was ∼2.2 MN. Six (13%) patients had undergone a liver transplant (ages 9-43.5 years). CONCLUSION: This study provides the largest longitudinal case review of patients with LAL D and confirms that LAL D is predominantly a pediatric disease causing early and progressive hepatic dysfunction associated with dyslipidemia that often leads to liver failure and transplantation.

Hepatocyte-Specific Expression of Human Lysosome Acid Lipase Corrects Liver Inflammation and Tumor Metastasis in lal(-/-) Mice.

The liver is a major organ for lipid synthesis and metabolism. Deficiency of lysosomal acid lipase (LAL; official name Lipa, encoded by Lipa) in mice (lal(-/-)) results in enlarged liver size due to neutral lipid storage in hepatocytes and Kupffer cells. To test the functional role of LAL in hepatocyte, hepatocyte-specific expression of human LAL (hLAL) in lal(-/-) mice was established by cross-breeding of liver-activated promoter (LAP)-driven tTA transgene and (tetO)7-CMV-hLAL transgene with lal(-/-) knockout (KO) (LAP-Tg/KO) triple mice. Hepatocyte-specific expression of hLAL in LAP-Tg/KO triple mice reduced the liver size to the normal level by decreasing lipid storage in both hepatocytes and Kupffer cells. hLAL expression reduced tumor-promoting myeloid-derived suppressive cells in the liver of lal(-/-) mice. As a result, B16 melanoma metastasis to the liver was almost completely blocked. Expression and secretion of multiple tumor-promoting cytokines or chemokines in the liver were also significantly reduced. Because hLAL is a secretory protein, lal(-/-) phenotypes in other compartments (eg, blood, spleen, and lung) also ameliorated, including systemic reduction of myeloid-derived suppressive cells, an increase in CD4(+) and CD8(+) T and B lymphocytes, and reduced B16 melanoma metastasis in the lung. These results support a concept that LAL in hepatocytes is a critical metabolic enzyme in controlling neutral lipid metabolism, liver homeostasis, immune response, and tumor metastasis.

Novel LIPA mutations in Mexican siblings with lysosomal acid lipase deficiency.

Lysosomal acid lipase (LAL) deficiency is an under-recognized lysosomal disease caused by deficient enzymatic activity of LAL. In this report we describe two affected female Mexican siblings with early hepatic complications. At two months of age, the first sibling presented with alternating episodes of diarrhea and constipation, and later with hepatomegaly, elevated transaminases, high levels of total and low-density lipoprotein cholesterol, and low levels of high-density lipoprotein. Portal hypertension and grade 2 esophageal varices were detected at four years of age. The second sibling presented with hepatomegaly, elevated transaminases and mildly elevated low-density lipoprotein and low high-density lipoprotein at six months of age. LAL activity was deficient in both patients. Sequencing of LIPA revealed two previously unreported heterozygous mutations in exon 4: c.253C>A and c.294C>G. These cases highlight the clinical continuum between the so-called Wolman disease and cholesteryl ester storage disease, and underscore that LAL deficiency represents a single disease with a degree of clinical heterogeneity.

Activation of mTOR pathway in myeloid-derived suppressor cells stimulates cancer cell proliferation and metastasis in lal(-/-) mice.

Inflammation critically contributes to cancer metastasis, in which myeloid-derived suppressor cells (MDSCs) are an important participant. Although MDSCs are known to suppress immune surveillance, their roles in directly stimulating cancer cell proliferation and metastasis currently remain unclear. Lysosomal acid lipase (LAL) deficiency causes systemic expansion and infiltration of MDSCs in multiple organs and subsequent inflammation. In the LAL-deficient (lal(-/-)) mouse model, melanoma metastasized massively in allogeneic lal(-/-) mice, which was suppressed in allogeneic lal(+/+) mice owing to immune rejection. Here we report for the first time that MDSCs from lal(-/-) mice directly stimulated B16 melanoma cell in vitro proliferation and in vivo growth and metastasis. Cytokines, that is, interleukin-1ß and tumor necrosis factor-α from MDSCs are required for B16 melanoma cell proliferation in vitro. Myeloid-specific expression of human LAL (hLAL) in lal(-/-) mice rescues these malignant phenotypes in vitro and in vivo. The tumor-promoting function of lal(-/-) MDSCs is mediated, at least in part, through overactivation of the mammalian target of rapamycin (mTOR) pathway. Knockdown of mTOR, Raptor or Rictor in lal(-/-) MDSCs suppressed their stimulation on proliferation of cancer cells, including B16 melanoma, Lewis lung carcinoma and transgenic mouse prostate cancer-C2 cancer cells. Our results indicate that LAL has a critical role in regulating MDSCs' ability to directly stimulate cancer cell proliferation and overcome immune rejection of cancer metastasis in allogeneic mice through modulation of the mTOR pathway, which provides a mechanistic basis for targeting MDSCs to reduce the risk of cancer metastasis. Therefore MDSCs possess dual functions to facilitate cancer metastasis: suppress immune surveillance and stimulate cancer cell proliferation and growth.

Sebelipase alfa over 52 weeks reduces serum transaminases, liver volume and improves serum lipids in patients with lysosomal acid lipase deficiency.

BACKGROUND & AIMS: Lysosomal acid lipase deficiency is an autosomal recessive enzyme deficiency resulting in lysosomal accumulation of cholesteryl esters and triglycerides. LAL-CL04, an ongoing extension study, investigates the long-term effects of sebelipase alfa, a recombinant human lysosomal acid lipase. METHODS: Sebelipase alfa (1mg/kg or 3mg/kg) was infused every-other-week to eligible subjects. Safety and tolerability assessments, including liver function, lipid profiles and liver volume assessment, were carried out at regular intervals. RESULTS: 216 infusions were administered to eight adult subjects through week 52 during LAL-CL04. At week 52, mean alanine aminotransferase and aspartate aminotransferase levels were normal with mean change from baseline of -58% and -40%. Mean changes for low-density lipoprotein, total cholesterol, triglyceride and high-density lipoprotein were -60%, -39%, -36%, and +29%, respectively. Mean liver volume by magnetic resonance imaging and hepatic proton density fat fraction decreased (12% and 55%, respectively). Adverse events were mainly mild and unrelated to sebelipase alfa. Infusion-related reactions were uncommon: three events of moderate severity were reported in two subjects; one patient's event was suggestive of a hypersensitivity-like reaction, but additional testing did not confirm this, and the subject has successfully re-started sebelipase alfa. Of samples tested to date, no anti-drug antibodies have been detected. CONCLUSIONS: Long-term dosing with sebelipase alfa in lysosomal acid lipase-deficient patients is well tolerated and produces sustained reductions in transaminases, improvements in serum lipid profile and reduction in the hepatic fat fraction. A randomized, placebo-controlled phase 3 trial in children and adults is underway (ARISE: NCT01757184).

Myeloid-derived suppressor cells are involved in lysosomal acid lipase deficiency-induced endothelial cell dysfunctions.

The underlying mechanisms that lysosomal acid lipase (LAL) deficiency causes infiltration of myeloid-derived suppressor cells (MDSCs) in multiple organs and subsequent inflammation remain incompletely understood. Endothelial cells (ECs), lining the inner layer of blood vessels, constitute barriers regulating leukocytes transmigration to the site of inflammation. Therefore, we hypothesized that ECs are dysfunctional in LAL-deficient (lal(-/-)) mice. We found that Ly6G(+) cells transmigrated more efficiently across lal(-/-) ECs than wild-type (lal(+/+)) ECs, which were associated with increased levels of PECAM-1 and MCP-1 in lal(-/-) ECs. In addition, lal(-/-) ECs showed enhanced migration and proliferation, decreased apoptosis, but impaired tube formation and angiogenesis. lal(-/-) ECs also suppressed T cell proliferation in vitro. Interestingly, lal(-/-) Ly6G(+) cells promoted in vivo angiogenesis (including a tumor model), EC tube formation, and proliferation. Finally, the mammalian target of rapamycin (mTOR) pathway was activated in lal(-/-) ECs, and inhibition of mTOR reversed EC dysfunctions, including decreasing Ly6G(+) cell transmigration, delaying migration, and relieving suppression of T cell proliferation, which was mediated by decreasing production of reactive oxygen species. Our results indicate that LAL regulates EC functions through interaction with MDSCs and modulation of the mTOR pathway, which may provide a mechanistic basis for targeting MDSCs or mTOR to rejuvenate EC functions in LAL deficiency-related diseases.

Critical role of the mTOR pathway in development and function of myeloid-derived suppressor cells in lal-/- mice.

Lysosomal acid lipase (LAL) is essential for the hydrolysis of cholesteryl esters and triglycerides to generate cholesterol and free fatty acids in cellular lysosomes. Ablation of the lal gene (lal(-/-)) systemically increased expansion of cluster of differentiation molecule 11b (CD11b), lymphocyte antigen 6G (Ly6G) myeloid-derived suppressor cells (MDSCs) that caused myeloproliferative neoplasms in mice. Study of lal(-/-) bone marrow Ly6G(+) MDSCs via transcriptional profiling showed increases in mammalian target of rapamycin (mTOR) signaling pathway transcripts. Injection of mTOR pharmacologic inhibitors into lal(-/-) mice significantly reduced bone marrow myelopoiesis and systemic CD11b(+)Ly6G(+) cell expansion. Rapamycin treatment of lal(-/-) mice stimulated a shift from immature CD11b(+)Ly6G(+) cells to CD11b(+) single-positive cells in marrow and tissues and partially reversed the increased cell proliferation, decreased apoptosis, increased ATP synthesis, and increased cell cycling of bone marrow CD11b(+)Ly6G(+) cells obtained from lal(-/-) mice. Pharmacologic and siRNA suppression of mTOR, regulatory-associated protein of mTOR, rapamycin-insensitive companion of mTOR, and Akt1 function corrected CD11b(+)Ly6G(+) cell in lal(-/-) mice development from Lin(-) progenitor cells and reversed the immune suppression on T-cell proliferation and function in association with decreased reactive oxygen species production, and recovery from impairment of mitochondrial membrane potential compared with control mutant cells. These results indicate a crucial role of LAL-regulated mTOR signaling in the production and function of CD11b(+)Ly6G(+) cells. The mTOR pathway may serve as a novel target to modulate the emergence of MDSCs in those pathophysiologic states in which these cells play an immunosuppressive role.

Hepatic cholesteryl ester accumulation in lysosomal acid lipase deficiency: non-invasive identification and treatment monitoring by magnetic resonance.

BACKGROUND & AIMS: Lysosomal Acid Lipase (LAL) deficiency is a rare metabolic storage disease, caused by a marked reduction in activity of LAL, which leads to accumulation of cholesteryl esters (CE) and triglycerides (TG) in lysosomes in many tissues. We used (1)H magnetic resonance (MR) spectroscopy to characterize the abnormalities in hepatic lipid content and composition in patients with LAL deficiency, and in ex vivo liver tissue from a LAL deficiency rat model. Secondly, we used MR spectroscopy to monitor the effects of an enzyme replacement therapy (ERT), sebelipase alfa (a recombinant human lysosomal acid lipase), on hepatic TG and CE content in the preclinical model. METHODS: Human studies employed cohorts of LAL-deficient patients and NAFLD subjects. Rat experimental groups comprised ex vivo liver samples of wild type, NAFLD, LAL-deficient, and LAL-deficient rats receiving 4weeks of sebelipase alfa treatment. Hepatic (1)H MR spectroscopy was performed using 3T (human) and 7T (preclinical) MRI scanners to quantify hepatic cholesterol and triglyceride content. RESULTS: CE accumulation was identified in LAL deficiency in both human and preclinical studies. A significant decrease in hepatic CE was observed in LAL-deficient rats following treatment with sebelipase alfa. CONCLUSIONS: We demonstrate an entirely non-invasive method to identify and quantify the hepatic lipid signature associated with a rare genetic cause of fatty liver. The approach provides a more favorable alternative to repeated biopsy sampling for diagnosis and disease progression / treatment monitoring of patients with LAL deficiency and other disorders characterised by increased free cholesterol and/or cholesteryl esters.

Lysosomal lipase deficiency: molecular characterization of eleven patients with Wolman or cholesteryl ester storage disease.

Wolman Disease (WD) and cholesteryl ester storage disease (CESD) represent two distinct phenotypes of the same recessive disorder caused by the complete or partial deficiency of lysosomal acidic lipase (LAL), respectively. LAL, encoded by the LIPA gene, hydrolyzes cholesteryl esters derived from cell internalization of plasma lipoproteins. WD is a rapidly progressive and lethal disease characterized by intestinal malabsorption, hepatic and adrenal failure. CESD is characterized by hepatic fibrosis, hyperlipidemia and accelerated atherosclerosis. Aim of the study was the identification of LIPA mutations in three WD and eight CESD patients. The WD patients, all deceased before the first year of age, were homozygous for two novel mutations (c.299+1G>A and c.419G>A) or a mutation (c.796G>T) previously reported as compound heterozygosity in a CESD patient. The two mutations (c.419G>A and c.796G>T) resulting in truncated proteins (p.W140* and p.G266*) and the splicing mutation (c.229+1G>A) were associated with undetectable levels of LIPA mRNA in fibroblasts. All eight CESD patients carried the common mutation c.894G>A known to result not only in a major non-functional transcript with the skipping of exon 8 (p.S275_Q298del), but also in a minor normally spliced transcript producing 5-10% residual LAL activity. The c.894G>A mutation was found in homozygosity in four patients and, as compound heterozygosity, in association with a known (p.H295Y and p.G342R) or a novel (p.W140*) mutation in four other CESD patients. Segregation analysis performed in all patients harboring c.895G>A showed its occurrence on the same haplotype suggesting a common founder ancestor. The other WD and CESD mutations were associated with different haplotypes.

Relative contribution of adipose triglyceride lipase and hormone-sensitive lipase to tumor necrosis factor-α (TNF-α)-induced lipolysis in adipocytes.

TNF-α potently stimulates basal lipolysis in adipocytes, which may contribute to hyperlipidemia and peripheral insulin resistance in obesity. Recent studies show that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) act sequentially in catalyzing the first two steps of adipose lipolysis in response to ß-adrenergic stimulation. Here, we sought to determine their functional roles in TNF-α-induced lipolysis. Silencing of ATGL expression in adipocytes almost completely abolished basal and TNF-α-induced glycerol release. In comparison, the glycerol release under the same conditions was only partially decreased upon reduction in expression of either HSL or the ATGL coactivator CGI-58. Interestingly, overexpression of ATGL restored the lipolytic rates in cells with silenced HSL or CGI-58, indicating a predominant role for ATGL. While expression of ATGL, HSL and CGI-58 remains mostly unaffected, TNF-α treatment caused a rapid abrogation of the ATGL inhibitory protein G0S2. TNF-α drastically decreased the level of G0S2 mRNA, and the level of G0S2 protein could be maintained by inhibiting proteasomal protein degradation using MG-132. Furthermore, coexpression of G0S2 was able to significantly decrease TNF-α-stimulated lipolysis mediated by overexpressed ATGL or CGI-58. We propose that the early reduction in G0S2 content is permissive for TNF-α-induced lipolysis.