Overfeeding Saturated Fat Increases LDL (Low-Density Lipoprotein) Aggregation Susceptibility While Overfeeding Unsaturated Fat Decreases Proteoglycan-Binding of Lipoproteins.

Objective: We recently showed that measurement of the susceptibility of LDL (low-density lipoprotein) to aggregation is an independent predictor of cardiovascular events. We now wished to compare effects of overfeeding different dietary macronutrients on LDL aggregation, proteoglycan-binding of plasma lipoproteins, and on the concentration of oxidized LDL in plasma, 3 in vitro parameters consistent with increased atherogenicity. Approach and Results: The participants (36 subjects; age, 48+/-10 years; body mass index, 30.9+/-6.2 kg/m2) were randomized to consume an extra 1000 kcal/day of either unsaturated fat, saturated fat, or simple sugars (CARB) for 3 weeks. We measured plasma proatherogenic properties (susceptibility of LDL to aggregation, proteoglycan-binding, oxidized LDL) and concentrations and composition of plasma lipoproteins using nuclear magnetic resonance spectroscopy, and in LDL using liquid chromatography mass spectrometry, before and after the overfeeding diets. LDL aggregation increased in the saturated fat but not the other groups. This change was associated with increased sphingolipid and saturated triacylglycerols in LDL and in plasma and reduction of clusterin on LDL particles. Proteoglycan binding of plasma lipoproteins decreased in the unsaturated fat group relative to the baseline diet. Lipoprotein properties remained unchanged in the CARB group. Conclusions: The type of fat during 3 weeks of overfeeding is an important determinant of the characteristics and functional properties of plasma lipoproteins in humans.

Children with familial hypercholesterolemia display changes in LDL and HDL function: A cross-sectional study.

BACKGROUND: The functional status of lipoprotein particles contributes to atherogenesis. The tendency of plasma low-density lipoprotein (LDL) particles to aggregate and the ability of igh-density lipoprotein (HDL) particles to induce and mediate reverse cholesterol transport associate with high and low risk for cardiovascular disease in adult patients, respectively. However, it is unknown whether children with familial hypercholesterolemia (FH) display lipoprotein function alterations. HYPOTHESIS: We hypothesized that FH children had disrupted lipoprotein functions. METHODS: We analyzed LDL aggregation susceptibility and HDL-apoA-I exchange (HAE), and activity of four proteins that regulate lipoprotein metabolism (cholesteryl ester transfer protein, lecithin-cholesterol acyltransferase, phospholipid transfer protein, and paraoxonase-1) in plasma samples derived from children with FH (n = 47) and from normocholesterolemic children (n = 56). Variation in lipoprotein functions was further explored using an nuclear magnetic resonance-based metabolomics profiling approach. RESULTS: LDL aggregation was higher, and HAE was lower in FH children than in normocholesterolemic children. LDL aggregation associated positively with LDL cholesterol (LDL-C) and negatively with triglycerides, and HAE/apoA-I associated negatively with LDL-C. Generally, the metabolomic profile for LDL aggregation was opposite of that of HAE/apoA-I. CONCLUSIONS: FH children displayed increased atherogenicity of LDL and disrupted HDL function. These newly observed functional alterations in LDL and HDL add further understanding of the risk for atherosclerotic cardiovascular disease in FH children.

Plant Stanol Esters Reduce LDL (Low-Density Lipoprotein) Aggregation by Altering LDL Surface Lipids: The BLOOD FLOW Randomized Intervention Study.

OBJECTIVE: Plant stanol ester supplementation (2-3 g plant stanols/d) reduces plasma LDL (low-density lipoprotein) cholesterol concentration by 9% to 12% and is, therefore, recommended as part of prevention and treatment of atherosclerotic cardiovascular disease. In addition to plasma LDL-cholesterol concentration, also qualitative properties of LDL particles can influence atherogenesis. However, the effect of plant stanol ester consumption on the proatherogenic properties of LDL has not been studied. Approach and Results: Study subjects (n=90) were randomized to consume either a plant stanol ester-enriched spread (3.0 g plant stanols/d) or the same spread without added plant stanol esters for 6 months. Blood samples were taken at baseline and after the intervention. The aggregation susceptibility of LDL particles was analyzed by inducing aggregation of isolated LDL and following aggregate formation. LDL lipidome was determined by mass spectrometry. Binding of serum lipoproteins to proteoglycans was measured using a microtiter well-based assay. LDL aggregation susceptibility was decreased in the plant stanol ester group, and the median aggregate size after incubation for 2 hours decreased from 1490 to 620 nm, P=0.001. Plant stanol ester-induced decrease in LDL aggregation was more extensive in participants having body mass index<25 kg/m2. Decreased LDL aggregation susceptibility was associated with decreased proportion of LDL-sphingomyelins and increased proportion of LDL-triacylglycerols. LDL binding to proteoglycans was decreased in the plant stanol ester group, the decrease depending on decreased serum LDL-cholesterol concentration. CONCLUSIONS: Consumption of plant stanol esters decreases the aggregation susceptibility of LDL particles by modifying LDL lipidome. The resulting improvement of LDL quality may be beneficial for cardiovascular health. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01315964.

Susceptibility of low-density lipoprotein particles to aggregate depends on particle lipidome, is modifiable, and associates with future cardiovascular deaths.

Aims: Low-density lipoprotein (LDL) particles cause atherosclerotic cardiovascular disease (ASCVD) through their retention, modification, and accumulation within the arterial intima. High plasma concentrations of LDL drive this disease, but LDL quality may also contribute. Here, we focused on the intrinsic propensity of LDL to aggregate upon modification. We examined whether inter-individual differences in this quality are linked with LDL lipid composition and coronary artery disease (CAD) death, and basic mechanisms for plaque growth and destabilization. Methods and results: We developed a novel, reproducible method to assess the susceptibility of LDL particles to aggregate during lipolysis induced ex vivo by human recombinant secretory sphingomyelinase. Among patients with an established CAD, we found that the presence of aggregation-prone LDL was predictive of future cardiovascular deaths, independently of conventional risk factors. Aggregation-prone LDL contained more sphingolipids and less phosphatidylcholines than did aggregation-resistant LDL. Three interventions in animal models to rationally alter LDL composition lowered its susceptibility to aggregate and slowed atherosclerosis. Similar compositional changes induced in humans by PCSK9 inhibition or healthy diet also lowered LDL aggregation susceptibility. Aggregated LDL in vitro activated macrophages and T cells, two key cell types involved in plaque progression and rupture. Conclusion: Our results identify the susceptibility of LDL to aggregate as a novel measurable and modifiable factor in the progression of human ASCVD.

USF1 deficiency alleviates inflammation, enhances cholesterol efflux and prevents cholesterol accumulation in macrophages.

BACKGROUND: The focus of studies on high-density lipoproteins (HDL) has shifted from HDL-cholesterol (HDL-C) to HDL function. We recently demonstrated that low USF1 expression in mice and humans associates with high plasma HDL-C and low triglyceride levels, as well as protection against obesity, insulin resistance, and atherosclerosis. Here, we studied the impact of USF1 deficiency on HDL functional capacity and macrophage atherogenic functions, including inflammation, cholesterol efflux, and cholesterol accumulation. METHODS: We used a congenic Usf1 deficient mice in C57Bl/6JRccHsd background and blood samples were collected to isolate HDL for structural and functional studies. Lentiviral preparations containing the USF1 silencing shRNA expression vector were used to silence USF1 in human THP-1 and Huh-7 cells. Cholesterol efflux from acetyl-LDL loaded THP-1 macrophages was measured using HDL and plasma as acceptors. Gene expression analysis from USF1 silenced peritoneal macrophages was carried out using Affymetrix protocols. RESULTS: We show that Usf1 deficiency not only increases HDL-C levels in vivo, consistent with elevated ABCA1 protein expression in hepatic cell lines, but also improves the functional capacity of HDL particles. HDL particles derived from Usf1 deficient mice remove cholesterol more efficiently from macrophages, attributed to their higher contents of phospholipids. Furthermore, silencing of USF1 in macrophages enhanced the cholesterol efflux capacity of these cells. These findings are consistent with reduced inflammatory burden of USF1 deficient macrophages, manifested by reduced secretion of pro-inflammatory cytokines MCP-1 and IL-1ß and protection against inflammation-induced macrophage cholesterol accumulation in a cell-autonomous manner. CONCLUSIONS: Our findings identify USF1 as a novel factor regulating HDL functionality, showing that USF1 inactivation boosts cholesterol efflux, reduces macrophage inflammation and attenuates macrophage cholesterol accumulation, linking improved macrophage cholesterol metabolism and inflammatory pathways to the antiatherogenic function of USF1 deficiency.

Tailor-made approach for selective isolation and elution of low-density lipoproteins by immunoaffinity sorbent on silica.

Immunoaffinity procedure was developed for isolation of low density lipoprotein (LDL) from biological samples by using silica-derived immunoaffinity sorbent. Sorbent was prepared by immobilization of monoclonal anti-apoB-100 antibody onto macroporous silica particles, using carefully optimized binding chemistry. Binding capacity of the sorbent towards LDL was determined by batch extraction experiments with solutions of isolated LDL in phosphate-buffered saline, and found to be 8 mg LDL/g. The bound LDL fraction was readily released from the sorbent by elution with ammonia at pH 11.2. The total time needed for isolation procedure was less than 1 h, with LDL recoveries being essentially quantitative for samples containing less than 0.3 mg LDL/mL. With higher concentrations, recoveries were less favorable, most probably due to irreversible adsorption caused by LDL aggreggation. However, reusability studies with isolated LDL at concentration 0.2 mg/mL indicate that the developed immunoaffinity material may be used for multiple binding-release cycles, with minor losses in binding capacity. Finally, the sorbent was successfully applied to isolation of LDL from diluted plasma. Apart from its practical implications for LDL isolation, this study provides crucial insights into issues associated with LDL-sorbent interactions, and may be useful in future efforts directed to development of lipoprotein isolation approaches.

Impact of RYGB surgery on plasma immunoglobulins: association between blood pressure and glucose levels six months after surgery.

We aimed to study levels of natural antibodies in plasma, and their associations to clinical and fecal biomarkers, before and 6 months after Roux-en-Y gastric bypass (RYGB) surgery. Thirty individuals with obesity [16 type 2 diabetic, 14 non-diabetic (ND)] had RYGB surgery. Total plasma IgA, IgG and IgM antibody levels and specific antibodies to oxidized low-density lipoprotein (oxLDL), malondialdehyde-acetaldehyde adducts, Porphyromonas gingivalis gingipain A hemagglutinin domain (Rgp44), and phosphocholine were measured using chemiluminescence immunoassay. Associations between plasma and fecal antibodies as well as clinical markers were analyzed. RYGB surgery reduced blood pressure, and the glycemic state was improved. A higher level of diastolic blood pressure was associated with lower plasma antibodies to oxLDL after surgery. Also, lower level of glucose markers associated with lower level of plasma antibodies to bacterial virulence factors. Antibodies to oxLDL decreased after surgery, and positive association between active serum lipopolysaccharide and specific oxLDL antibodies was detected. Total IgG levels decreased after surgery, but only in ND individuals. Reduced level of total plasma IgG, improved state of hypertension and hyperglycemia and their associations with decreased levels of specific antibodies in plasma, suggest an improved state of systemic inflammation after RYGB surgery.

Lipoprotein(a) induces caspase-1 activation and IL-1 signaling in human macrophages.

Introduction: Lipoprotein(a) (Lp(a)) is an LDL-like particle with an additional apolipoprotein (apo)(a) covalently attached. Elevated levels of circulating Lp(a) are a risk factor for atherosclerosis. A proinflammatory role for Lp(a) has been proposed, but its molecular details are incompletely defined. Methods and results: To explore the effect of Lp(a) on human macrophages we performed RNA sequencing on THP-1 macrophages treated with Lp(a) or recombinant apo(a), which showed that especially Lp(a) induces potent inflammatory responses. Thus, we stimulated THP-1 macrophages with serum containing various Lp(a) levels to investigate their correlations with cytokines highlighted by the RNAseq, showing significant correlations with caspase-1 activity and secretion of IL-1ß and IL-18. We further isolated both Lp(a) and LDL particles from three donors and then compared their atheroinflammatory potentials together with recombinant apo(a) in primary and THP-1 derived macrophages. Compared with LDL, Lp(a) induced a robust and dose-dependent caspase-1 activation and release of IL-1ß and IL-18 in both macrophage types. Recombinant apo(a) strongly induced caspase-1 activation and IL-1ß release in THP-1 macrophages but yielded weak responses in primary macrophages. Structural analysis of these particles revealed that the Lp(a) proteome was enriched in proteins associated with complement activation and coagulation, and its lipidome was relatively deficient in polyunsaturated fatty acids and had a high n-6/n-3 ratio promoting inflammation. Discussion: Our data show that Lp(a) particles induce the expression of inflammatory genes, and Lp(a) and to a lesser extent apo(a) induce caspase-1 activation and IL-1 signaling. Major differences in the molecular profiles between Lp(a) and LDL contribute to Lp(a) being more atheroinflammatory.

The human liver lipidome is significantly related to the lipid composition and aggregation susceptibility of low-density lipoprotein (LDL) particles.

BACKGROUND AND AIMS: The susceptibility of low-density lipoprotein (LDL) to aggregation predicts atherosclerotic cardiovascular disease. However, causes of interindividual variation in LDL lipid composition and aggregation susceptibility remain unclear. We examined whether the lipid composition and aggregation susceptibility of LDL reflect the lipid composition of the human liver. METHODS: Liver biopsies and blood samples for isolation of LDL particles were obtained from 40 obese subjects (BMI 45.9 ± 6.1 kg/m2, age 43 ± 8 years). LDL was isolated using sequential ultracentrifugation and lipidomic analyses of liver and LDL samples were determined using ultra-high performance liquid chromatography-mass spectrometry. LDL aggregation susceptibility ex vivo was analyzed by inducing aggregation by human recombinant secretory sphingomyelinase and following aggregate formation. RESULTS: The composition (acyl carbon number and double bond count) of hepatic triglycerides, phosphatidylcholines, and sphingomyelins (SMs) was closely associated with that of LDL particles. Hepatic dihydroceramides and ceramides were positively correlated with concentrations of the corresponding SM species in LDL as well with LDL aggregation. These relationships remained statistically significant after adjustment for age, sex, and body mass index. CONCLUSIONS: Lipid composition of LDL reflects that of the human liver in obese patients. Changes in hepatic sphingolipid metabolism may contribute to interindividual variation of LDL lipid composition and susceptibility to aggregation.

Lipidomic changes of LDL after consumption of Camelina sativa oil, fatty fish and lean fish in subjects with impaired glucose metabolism-A randomized controlled trial.

BACKGROUND: There is little knowledge on the effects of alpha-linolenic acid (ALA) and n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) on the LDL lipidome and aggregation of LDL particles. OBJECTIVE: We examined if consumption of Camelina sativa oil (CSO) as a source of ALA, fatty fish (FF) as a source of n-3 LCPUFA and lean fish (LF) as a source of fish protein affect the lipidome of LDL as compared to a control diet. METHODS: Participants with impaired glucose tolerance (39 women and 40 men) were randomized to 4 study groups (CSO providing 10 g/d ALA, FF and LF [both 4 fish meals/wk] and control limiting their fish and ALA intake) in a 12-week, parallel trial. Diets were instructed and dietary fats were provided to the participants. The lipidome of LDL particles isolated from samples collected at baseline and after intervention was analyzed with electrospray ionization-tandem mass spectrometry. RESULTS: In the CSO group, the relative concentrations of saturated and monounsaturated cholesteryl ester species in LDL decreased and the species with ALA increased. In the FF group, LDL phosphatidylcholine (PC) species containing n-3 LCPUFA increased. There was a significant positive correlation between the change in total sphingomyelin and change in LDL aggregation, while total PC and triunsaturated PC species were inversely associated with LDL aggregation when all the study participants were included in the analysis. CONCLUSION: Dietary intake of CSO and FF modifies the LDL lipidome to contain more polyunsaturated and less saturated lipid species. The LDL surface lipids are associated with LDL aggregation.

Low-density lipoprotein aggregation predicts adverse cardiovascular events in peripheral artery disease.

BACKGROUND AND AIMS: Peripheral artery disease (PAD) is a systemic manifestation of atherosclerosis that is associated with a high risk of major adverse cardiovascular events (MACE). LDL aggregation contributes to atherosclerotic plaque progression and may contribute to plaque instability. We aimed to determine if LDL aggregation is associated with MACE in patients with PAD undergoing lower extremity revascularization (LER). METHODS: Two hundred thirty-nine patients with PAD undergoing LER had blood collected at baseline and were followed prospectively for MACE (myocardial infarction, stroke, cardiovascular death) for one year. Nineteen age, sex and LDL-C-matched control subjects without cardiovascular disease also had blood drawn. Subject LDL was exposed to sphingomyelinase and LDL aggregate size measured via dynamic light scattering. RESULTS: Mean age was 72.3 ± 10.9 years, 32.6% were female, and LDL-cholesterol was 68 ± 25 mg/dL. LDL aggregation was inversely associated with triglycerides, but not associated with demographics, LDL-cholesterol or other risk factors. Maximal LDL aggregation occurred significantly earlier in subjects with PAD than in control subjects. 15.9% of subjects experienced MACE over one year. The 1st tertile (shortest time to maximal aggregation) exhibited significantly higher MACE (25% vs. 12.5% in tertile 2 and 10.1% in tertile 3, p = 0.012). After multivariable adjustment for demographics and CVD risk factors, the hazard ratio for MACE in the 1st tertile was 4.57 (95% CI 1.60-13.01; p = 0.004) compared to tertile 3. Inclusion of LDL aggregation in the Framingham Heart Study risk calculator for recurrent coronary heart disease events improved the c-index from 0.57 to 0.63 (p = 0.01). CONCLUSIONS: We show that in the setting of very well controlled LDL-cholesterol, patients with PAD with the most rapid LDL aggregation had a significantly elevated MACE risk following LER even after multivariable adjustment. This measure further improved the classification specificity of an established risk prediction tool. Our findings support broader investigation of this assay for risk stratification in patients with atherosclerotic CVD.

The effect of intakes of fish and Camelina sativa oil on atherogenic and anti-atherogenic functions of LDL and HDL particles: A randomized controlled trial.

BACKGROUND AND AIMS: Omega-3 fatty acids are known to have several cardioprotective effects. Our aim was to investigate the effects of intakes of fish and Camelina sativa oil (CSO), rich in alpha-linolenic acid, on the atherogenic and anti-atherogenic functions of LDL and HDL particles. METHODS: Altogether, 88 volunteers with impaired glucose metabolism were randomly assigned to CSO (10 g of alpha-linolenic acid/day), fatty fish (4 fish meals/week), lean fish (4 fish meals/week) or control group for 12 weeks. 79 subjects completed the study. The binding of lipoproteins to aortic proteoglycans, LDL aggregation and activation of endothelial cells by LDL and cholesterol efflux capacity of HDL were determined in vitro. RESULTS: Intake of CSO decreased the binding of lipoproteins to aortic proteoglycans in a non-normalized model (p = 0.006). After normalizing with serum concentrations of non-HDL cholesterol, apolipoprotein B (apoB) or LDL cholesterol, which decreased in the CSO group, the change was no longer statistically significant. In the fish groups, there were no changes in the binding of lipoproteins to proteoglycans. Regarding other lipoprotein functions, there were no changes in any of the groups. CONCLUSIONS: Intake of CSO decreases the binding of lipoproteins to aortic proteoglycans by decreasing serum LDL cholesterol concentration, which suggests that the level of apoB-containing lipoproteins in the circulation is the main driver of lipoprotein retention within the arterial wall. Intake of fish or CSO has no effects on other lipoprotein functions.

LDL aggregation susceptibility is higher in healthy South Asian compared with white Caucasian men.

BACKGROUND: South Asians are more prone to develop atherosclerotic cardiovascular disease (ASCVD) compared with white Caucasians, which is not fully explained by classical risk factors. We recently reported that the presence of aggregation-prone low-density lipoprotein (LDL) in the circulation is associated with increased ASCVD mortality. OBJECTIVE: We hypothesized that LDL of South Asians is more prone to aggregate, which may be explained by differences in their LDL lipid composition. METHODS: In this cross-sectional hypothesis-generating study, LDL was isolated from plasma of healthy South Asians (n = 12) and age- and BMI-matched white Caucasians (n = 12), and its aggregation susceptibility and lipid composition were analyzed. RESULTS: LDL from South Asians was markedly more prone to aggregate compared with white Caucasians. Among all measured lipids, sphingomyelin 24:0 and triacylglycerol 56:8 showed the highest positive correlation with LDL aggregation. In addition, LDL from South Asians was enriched in arachidonic acid containing phosphatidylcholine 38:4 and had less phosphatidylcholines and cholesteryl esters containing monounsaturated fatty acids. Interestingly, body fat percentage, which was higher in South Asians (+26%), positively correlated with LDL aggregation and highly positively correlated with triacylglycerol 56:8, sphingomyelin 24:0, and total sphingomyelin. CONCLUSIONS: LDL aggregation susceptibility is higher in healthy young South Asians compared with white Caucasians. This may be partly explained by the higher body fat percentage of South Asians, leading to sphingomyelin enrichment of LDL. We anticipate that the presence of sphingomyelin-rich, aggregation-prone LDL particles in young South Asians may increase LDL accumulation in the arterial wall and thereby contribute to their increased risk of developing ASCVD later in life.

PCSK9 inhibition alters the lipidome of plasma and lipoprotein fractions.

BACKGROUND AND AIMS: While inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) is known to result in dramatic lowering of LDL-cholesterol (LDL-C), it is poorly understood how it affects other lipid species and their metabolism. The aim of this study was to characterize the alterations in the lipidome of plasma and lipoprotein particles after administration of PCSK9 inhibiting antibody to patients with established coronary heart disease. METHODS: Plasma samples were obtained from patients undergoing a randomized placebo-controlled phase II trial (EQUATOR) for the safe and effective use of RG7652, a fully human monoclonal antibody inhibiting PCSK9 function. Lipoprotein fractions were isolated by sequential density ultracentrifugation, and both plasma and major lipoprotein classes (VLDL-IDL, LDL, HDL) were subjected to mass spectrometric lipidomic profiling. RESULTS: PCSK9 inhibition significantly decreased plasma levels of several lipid classes, including sphingolipids (dihydroceramides, glucosylceramides, sphingomyelins, ceramides), cholesteryl esters and free cholesterol. Previously established ceramide ratios predicting cardiovascular mortality, or inflammation related eicosanoid lipids, were not altered. RG7652 treatment also affected the overall and relative distribution of lipids in lipoprotein classes. An overall decrease of total lipid species was observed in LDL and VLDL + IDL particles, while HDL-associated phospholipids increased. Following the treatment, LDL displayed reduced lipid cargo, whereas relative lipid proportions of the VLDL + IDL particles were mostly unchanged, and there were relatively more lipids carried in the HDL particles. CONCLUSIONS: Administration of PCSK9 antibody significantly alters the lipid composition of plasma and lipoprotein particles. These changes further shed light on the link between anti-PCSK9 therapies and cardiovascular risk.

USF1 deficiency activates brown adipose tissue and improves cardiometabolic health.

USF1 (upstream stimulatory factor 1) is a transcription factor associated with familial combined hyperlipidemia and coronary artery disease in humans. However, whether USF1 is beneficial or detrimental to cardiometabolic health has not been addressed. By inactivating USF1 in mice, we demonstrate protection against diet-induced dyslipidemia, obesity, insulin resistance, hepatic steatosis, and atherosclerosis. The favorable plasma lipid profile, including increased high-density lipoprotein cholesterol and decreased triglycerides, was coupled with increased energy expenditure due to activation of brown adipose tissue (BAT). Usf1 inactivation directs triglycerides from the circulation to BAT for combustion via a lipoprotein lipase-dependent mechanism, thus enhancing plasma triglyceride clearance. Mice lacking Usf1 displayed increased BAT-facilitated, diet-induced thermogenesis with up-regulation of mitochondrial respiratory chain complexes, as well as increased BAT activity even at thermoneutrality and after BAT sympathectomy. A direct effect of USF1 on BAT activation was demonstrated by an amplified adrenergic response in brown adipocytes after Usf1 silencing, and by augmented norepinephrine-induced thermogenesis in mice lacking Usf1. In humans, individuals carrying SNP (single-nucleotide polymorphism) alleles that reduced USF1 mRNA expression also displayed a beneficial cardiometabolic profile, featuring improved insulin sensitivity, a favorable lipid profile, and reduced atherosclerosis. Our findings identify a new molecular link between lipid metabolism and energy expenditure, and point to the potential of USF1 as a therapeutic target for cardiometabolic disease.

Transmembrane prostatic acid phosphatase (TMPAP) interacts with snapin and deficient mice develop prostate adenocarcinoma.

The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP), a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930's. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP) and a transmembrane type-I (TMPAP). The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP(-/-)) with C57BL/6J background. The PAP(-/-) mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells) and in vivo FRET analyses in transient transfected LNCaP cells. The differential gene expression analyses revealed the dysregulation of the same genes known to be related to synaptic vesicular traffic. Both TMPAP and snapin were detected in isolated exosomes. Our results suggest that TMPAP is involved in endo-/exocytosis and disturbed vesicular traffic is a hallmark of prostate adenocarcinoma.