Targeting Fatty Acid Synthase Reduces Aortic Atherosclerosis and Inflammation

Targeting Fatty Acid Synthase Reduces Aortic Atherosclerosis and Inflammation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting Fatty Acid Synthase Reduces Aortic Atherosclerosis and Inflammation Mohamed zayed, Rodrigo Meade, Connor Engel, Larisa Belaygorod, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4139044/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Feb, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Fatty acid synthase (FAS) is predominantly expressed in the liver and adipose tissue. It plays vital roles in de novo synthesis of saturated fatty acid and regulates insulin sensitivity. We previously demonstrated that serum circulating FAS (cFAS) is a clinical biomarker for advanced atherosclerosis, and that it is conjugated to low-density lipoproteins (LDL). However, it remains unknown whether cFAS can directly impact atheroprogression. To investigate this, we evaluated whether cFAS impacts macrophage foam cell formation – a important cellular process leading to atheroprogression. Macrophages exposed to human serum containing high levels of cFAS showed increased foam cell formation as compared to cells exposed to serum containing low levels of cFAS. This difference was not observed using serum containing either high or low LDL. Pharmacological inhibition of cFAS using Platensimycin (PTM) decreased foam cell formation in vitro . In Apoe −/− mice with normal FAS expression, administration of PTM over 16 weeks along with a high fat diet decreased cFAS activity and aortic atherosclerosis without affecting circulating total cholesterol. This effect was also seen in Apoe −/− mice with liver-specific deletion of hepatic FAS. Reductions in aortic root plaque were associated with decreased macrophage infiltration. These findings demonstrate that cFAS can impact arterial atheroprogression. Health sciences/Diseases/Cardiovascular diseases/Vascular diseases/Atherosclerosis Health sciences/Diseases/Cardiovascular diseases/Dyslipidaemias Health sciences/Pathogenesis/Inflammation Health sciences/Medical research/Preclinical research Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Atherosclerosis is one of the major global underlying causes for cardiovascular disease 1 , 2 . Its management predominantly revolves around mitigation of risk factors such as hyperlipidemia with pharmacological therapies that aim to reduce serum circulating lipid levels 3 , 4 . Despite the efficacy of HMG-CoA reductase inhibitors (statins) and PCSK9 inhibitors in reducing serum low-density lipoprotein (LDL) and total cholesterol levels, their impact on cardiovascular events is confined to a modest range of 30–60% 5–9, Moreover, even with effective reduction of total cholesterol using statin monotherapy, individuals contending with cardiovascular co-morbidities continue to be disproportionately susceptible to atheroprogression 10 . This heightened vulnerability translates into a residual major risk of myocardial infarction, stroke, and major peripheral extremity amputations. Recognizing these continued challenges, the American Heart Association (AHA) and European Society of Cardiology (ESC) have underscored the need to further investigate the underlying causes of atheroprogression and have prioritized the exploration of alternative treatment strategies for this recalcitrant and morbid disease process 11 – 14 . Lipids such as cholesteryl esters, triglycerides, and phospholipids, feature saturated fatty acids that wield a significant influence on atheroprogression 1 , 15 . A mounting body of evidence accentuates the pivotal role of saturated fatty acids within atheromatous plaques, amplifying plaque instability and the heightened risk of atheroma rupture 16 . Similarly, while de novo synthesis of fatty acids is integral to lipid homeostasis, the process is essential for orchestrating the transformation of monocytes into foam cells, which play a fundamental role in perpetuating atheroprogression and exacerbating plaque instability and vulnerability 17 . Consequently, the modulation of fatty acid synthesis within evolving atheroma lesions is thought to influence the progression of atherosclerosis. Tissue Fatty Acid Synthase (FAS) is an essential soluble 273kDa intracellular homodimeric enzyme that catalyzes the de novo synthesis of saturated fatty acid through the conversion of acetyl-CoA and malonyl-CoA into palmitate 18 – 20 . It was recently observed that both tissue FAS and serum circulating FAS (cFAS) are elevated in individuals afflicted with severe atherosclerotic cardiovascular disease 21 . Serum cFAS is predominantly produced by the liver and bound to ApoB in LDL particles 22 . Notably, levels of cFAS in serum have a strong correlation with the content of FAS and saturated fatty acids in arterial tissue impacted by atherosclerosis 22 . In macrophages, FAS is also essential for cholesterol influx and cellular stress kinase activation 23 . Here we build upon these prior findings to determine whether targeted inhibition of tissue FAS and/or serum cFAS can impact macrophage foam cell formation and alter the course of in vivo atheroprogression. Method Human serum Native human serum was obtained from an institutional review board (IRB)-approved vascular biobank 24 . As previously described 25 , fresh human serum aliquots were collected from at least 12 hour fasting study participants and concentrated using a 100kDa ultrafiltration centrifuge tube (ThermoFisher Scientific, Waltham, MA), at 15,000g, for 15 minutes. A resultant minimum volume of 250µL of concentrated serum was collected for each patient. Concentrated serum samples were stored in aliquots at -80ºC for subsequent use for in vitro macrophage-foam cell experiments. Serum used for conditioned media studies were obtained from male individuals with similar age demographics and contained either LDL content at low ( 180 mg/dL) 25 , and undetectable levels of cFAS (Table 1 ). Alternatively, media was also conditioned with serum containing either cFAS content at low (0 µg/µl), medium (5–6 µg/µl), or high (> 17 µg/µl), and low levels of LDL (< 90mg/dL; Table 1 ). Serum LDL content was determined by the Washington University in St. Louis Core Laboratory for Clinical Studies (CLCS), utilizing a N-Geneous® LDL cholesterol kit (Sekisui Diagnostics, #7120) using a Roche Cobas c501 analyzer. Serum cFAS content was determined using commercial ELISA according to manufacturer’s instructions (Aviva, OKEH04869) 21 , 22 , 26 . Table 1 Human Serum Lipid Profile. Human serum demographics and lipid concentration from institutional serum biobank that were utilized for foam cell lipid formation experiments. Sex Group Condition Age LDL(mg/dL) cFAS(µg/µl) Male cFAS (n = 3) High 69.3 ± 5.85 63.0 ± 20.5 21.7 ± 4.41 Medium 62.3 ± 18.4 79.6 ± 28.5 5.45 ± 0.12 Low 62.6 ± 6.50 93.0 ± 25.8 0 LDL (n = 3) High 51.3 ± 19.3 183 ± 7.57 0 Medium 66.6 ± 8.32 103 ± 1.15 0 Low 69.3 ± 8.73 35.6 ± 10.1 0 Low density lipoprotein, LDL; circulating fatty acid synthase, cFAS Tissue culture and foam cell formation assessment Human U-937 (ATCC # CRL-1593.2) were cultured for 48 hours in 10% FBS RPMI, and then differentiated to macrophages in 5% FBS RPMI treated with 1µl/ml Phorbol 12-myristate 13-acetate, 95% (Thermo Fisher, # J63916.MB). Monocytes were allowed 24 hours to differentiate into macrophages. Differentiated macrophages were then washed 3x with PBS and incubated for 48 hours in cell culture media RPMI (Thermofisher, # 11875093) conditioned with 10% of human serum containing either low, medium, and high levels of cFAS or LDL on fibronectin treated coverslips ( Supplemental Table 1 & Fig. 1A ). Similarly, macrophages conditioned with serum containing either higher levels cFAS or LDL, were also simultaneously treated with Platensimycin (PTM, 20mM; Cayman Chemical, #15507) for 48 hours. Macrophages were then stained in oil-Red O working solution (3:2 dilution with distilled water of a stock solution of 0.5mg/µl in 100% isopropanol) and hematoxylin and eosin (H&E). Stained coverslips were imaged using a Leica Thunder DM6 B Microsystems inverted fluorescent microscope. Area positively stained with oil-Red O was quantified using ImageJ color threshold toolkit. The intracellular lipid droplet area stained with oil-Red O was expressed as a percentage of the total cell area. Following conditioned media treatments, macrophages were also lysed with standard freeze thaw method in PBS and lysates were standardized to protein concentration using Bradford Protein Assay. Acetyl-CoA concentration was determined by using a commercial ELISA according to the manufacturer’s instructions (MyBioSource, #MBS9309791). Protein was loaded onto Bis-Tris gel (Thermo Fisher Scientific, NW00082BOX) and transferred to polyvinylidene fluoride membranes for content analysis. Anti-FAS 1:500 (Santa Cruz, #c-48357) and anti-β actin 1:10,000 (Abcam, #ab8227) were used for Western blotting. Band densitometry analysis was performed using ImageJ software, and FAS band densities were expressed as ratios relative to the β actin loading control. Animal regulations All animal protocols were approved by a local institutional animal care and use committee (IACUC). Mouse housing, breeding, and experimental procedures were conducted in accordance with national and institutional guidelines and ethics. Mouse models Conditional liver-specific knockdown of Fasn was achieved using previously reported Fasn fl/fl mice that also express an albumin- Cre promoter ( Cre + ) 27 . Liver Fasn fl/fl Cre + mice were crossed with Apoe −/− knockout mice (Jackson lab, strain #002052) to yield Liver Fasn fl/fl Cre + Apoe −/− mice. At 7 weeks of age, Fasn fl/fl Cre + Apoe −/− mice and Fasn +/+ Cre − Apoe −/− littermates were maintained on a continuous 42% high-fat diet for 16 weeks (Inotiv, TD.88137). On a weekly basis, body weights were recorded, and blood serum samples were collected from the tail vein. Similarly, Fasn +/+ Cre − Apoe −/− littermates were maintained on a 42% high-fat diet with and without PTM (100mg/kg/day infused into the diet; generously donated by Dr. Clay F. Semenkovich) for a 16-week treatment period. After 16 weeks, mice were sacrificed and serum, hearts, aorta, liver, and white adipose tissue were collected for immediate analysis, embedded in OCT, or stored at -80°C for later use. FAS enzyme activity and content assay FAS enzyme activity was measured as previously described with some modifications 21 , 22 , 26 . Liver and white adipose tissue were digested in mammalian cell lysis kit (Milipore Sigma, MCL-1KT), and homogenates were centrifuged at 5,000g for 5 min at 4°C. The supernatant and serum were standardized to 30µg of total protein and added to 80µL of assay buffer (1M KPO4 buffer [pH 7], 50mM EDTA [pH 8.0], 50mM DTT, 1.1mM NADPH (Milipore Sigma, N1630), 1mM acetyl CoA (Milipore Sigma, A2056) The rate of NADPH oxidation was monitored at 340nm at 37°C for 30 minutes in the absence, and then in the presence of 10µL of the rate-limiting substrate malonyl-CoA for 10 minutes (1mM;Milipore Sigma, M4263). Data was analyzed by calculating the OD decrease subtracted from the nonspecific background without Malonyl CoA substrate. FAS enzyme was defined as µmoles NADPH consumed·min − 1 ·mg − 1 . An extinction coefficient of 6220 M − 1 cm − 1 was used in the specific activity calculation utilizing Beer’s law as previously described 26 . Tissue and serum FAS protein content were evaluated using commercially ELISA according to manufacturer’s instructions (Aviva, OKEH04869). Aortic atherosclerotic burden assessment Murine hearts were harvested en bloc at time of sacrifice after 16-weeks of diet treatment. The tissue was fixed in OCT compound (Fisher Scientific), and the aortic valve region was sectioned at 10µm thickness. Sections were then fixed in 4% paraformaldehyde (PFA), followed by 60% isopropanol for 5 minutes. Aortic valve sections were then stained in an oil-Red O working solution (3:2 dilution with distilled water from stock solution of 0.5mg/µl in 100% isopropanol). Valve plaque area was quantified in a blinded fashion using ImageJ as percentage of plaque area in the aortic lumen, as previously described 28 . Corresponding sections of aortic value were also stained with 1:50 mouse anti-CD68 antibody (Bio Rad, MCA1957). The primary antibody was detected with a 1:100 secondary antibody, donkey anti-rat IgG labeled with Alexa Fluor 555 (Thermo Fisher Scientific, A78945), followed by DAPI nucleus stain. Stained sections were then imaged on Leica Thunder DM6 B Microsystems inverted fluorescent microscope. The percentage of positively stained CD68 area relative to total aortic lumen was quantified using ImageJ software. Similarly, the entire aorta from the aortic arch to the infrarenal aortic bifurcation were microdissected and resected en bloc at the time of sacrifice. Harvested aortic specimens were fixed in 4% PFA for 24 hours. The tissue was then effaced and stained using oil-Red O, and then imaged with Lecia S9i Microsystem microscope. The area of plaque that positively stained with oil-Red O was taken relative to the aortic valve segment area using ImageJ in a blinded fashion 29 . Tissue histology Murine liver and white adipose tissue were immediately harvested at the time of sacrifice and were embedded in OCT. Tissue was sectioned at 10µm thickness and fixed in 4% PFA and stained by H&E. Sections were also immuno-stained with 2% donkey blocking agent for 1 hour at room temperature, and then with 1:100 primary mouse monoclonal FAS antibody (Santa Cruz, SC-48357), or 1:50 mouse anti-CD68 antibody (Bio Rad, MCA1957). The primary antibody was detected with a 1:100 secondary antibody donkey anti-rat IgG labeled with Alexa Fluor 555 (Thermo Fisher Scientific, A78945), followed by DAPI nucleus stain. Imaging assessments were performed using a Leica Thunder DM6 B Microsystems inverted fluorescent microscope, and staining was quantified using ImageJ software integrated density toolkit 30 . Statical analysis Statistical correlations between continuous variables such as serum cFAS, tissue FAS, content or activity were evaluated using linear regression. Non-parametric two-tailed Man-Whitney tests were used to evaluate the differences between inter- and intra-group analysis. Endpoints obtained over a time course were evaluated using two-way ANOVA with multiple comparisons. All analyses were performed using GraphPad Prism (Prism 9.1 software, GraphPad Software Inc.). p < 0.05 was considered to be statistically significant. All graphical data are presented as mean ± SEM. Results Serum cFAS induces macrophage foam cell formation. Macrophage cytoplasmic lipid droplet accumulation and foam cell formation is a hallmark of atheroprogression 31 . We evaluated whether macrophages conditioned with native human serum either containing high cFAS or LDL can impact foam cell formation in vitro ( Fig. 1A ). Interestingly, we observed a significant correlation between serum cFAS content in the conditioned media and the percentage of macrophages that formed foam cells ( Fig. 1B & 1C ; R 2 = 0.44, p < 0.05). On the other hand, no correlation was observed between serum LDL content and the percentage of macrophage foam cell formation ( Fig. 1D & 1E ; R 2 = 0.05, p = 0.59). Treatment with the FAS inhibitor PTM significantly decreased macrophage-derived foam cell formation when cells were conditioned with serum containing high cFAS (> 17 ug/uL) and low LDL (< 90 mg/dL; Fig. 1F & 1G ; p < 0.01). Macrophages conditioned with serum containing high cFAS, had higher foam cell formation than macrophages conditioned with serum containing high LDL ( Fig. 1G ; p < 0.05). Intracellular FAS activity in macrophages that were conditioned with serum containing high cFAS was higher than macrophages conditioned with serum containing high LDL ( Fig. 1J ; p < 0.05). PTM only reduced intracellular macrophage FAS activity in cells that were conditioned with high cFAS ( Fig. 1H ; p < 0.01). Knockdown and inhibition of FAS alters serum and tissue lipidomics. Fasn +/+ Cre − Apoe −/− and Fasn fl/fl Cre + Apoe −/− were maintained on a high fat diet for 16 weeks. A group of Fasn +/+ Cre + Apoe −/− mice also received PTM throughout this period, and serum, liver, and adipose tissue were collected ( Fig. 2A ). Following 16 weeks of a high-fat diet, Fasn fl/fl Cre + Apoe −/− mice had significantly less weight gain compared to Fasn +/+ Cre − Apoe −/− mice (51% vs 54% increase in weight; p < 0.05; Fig. 2B ). On the other hand, Fasn +/+ Cre − Apoe −/− mice treated with PTM had no significant change in body weight compared to untreated Fasn +/+ Cre − Apoe −/− mice ( Fig. 2B ). All mouse groups demonstrated universal hypercholesteremia with total cholesterol > 1,400 mg/dL at 16 weeks ( Fig. 2C ). Compared to Fasn +/+ Cre − Apoe −/− mice, Fasn fl/fl Cre + Apoe −/− mice treated with and without PTM, demonstrated no significant differences in liver and adipose TGs at 16 weeks ( Fig. 2D & 2E ). On the other hand, FFAs were notably decreased in the liver ( Fig. 2F ; ∆37%), and significantly elevated in adipose tissue of Fasn +/+ Cre − Apoe −/− mice that received PTM treatment ( Fig. 2G ; p < 0.01). Conditional liver FAS knockdown and inhibition impacts serum cFAS and tissue FAS content and activity. We observed a significant decrease in serum cFAS content in Fasn fl/fl Cre + Apoe −/− mice prior to initiation of high-fat diet regimen ( Fig. 3A ; p < 0.05). Similarly, PTM treatment significantly reduced cFAS content in Fasn fl/fl Cre − Apoe −/− mice ( Fig. 3A ; p < 0.05). Interestingly, serum cFAS activity was reduced in Fasn fl/fl Cre + Apoe −/− mice, and PTM-treated Fasn +/+ Cre − Apoe −/− mice ( Fig. 3B ). This reduction was more significant after 9 and 16 weeks with a high-fat diet ( Fig. 3C – 3D ; p < 0.05). After initiation of high-fat diet, Fasn fl/fl Cre + Apoe −/− mice demonstrated a different pattern of FAS content and activity in the hepatic and adipose tissue. As expected, Fasn fl/fl Cre + Apoe −/− mice demonstrated a significant decrease in FAS content and activity in hepatic tissue ( Fig. 3E & 3F ; p < 0.05). Interestingly, Fasn fl/fl Cre + Apoe −/− mice demonstrated a significant increase in FAS content in white adipose ( Fig. 3G ; p < 0.005). PTM treatment did not impact FAS content in the liver, but led to a significant decrease in FAS content and activity in white adipose ( Fig. 3G & 3H ; p < 0.001). Similarly, there was moderate, but not statistically significant, reduction in FAS activity in hepatic tissue of PTM-treated Fasn +/+ Cre − Apoe −/− mice ( Fig. 3F ; p = 0.14). FAS conditional knockdown or pharmacological inhibition reduces atheroprogression. We next evaluated arterial atheroprogression in Fasn fl/fl Cre + Apoe −/− mice and PTM-treated Fasn +/+ Cre − Apoe −/− mice that were maintained on a high fat diet for 16 weeks and aortas harvested for analysis ( Fig. 4A ). Compared to Fasn +/+ Cre − Apoe −/− , Fasn fl/fl Cre + Apoe −/− mice demonstrated a significant reduction in total aortic atherosclerotic plaque formation ( Fig. 4B & C ; p < 0.01). This difference was evident in all aortic segments including the aortic arch, thoracic aorta, and infrarenal aorta ( Fig. 4D - F ; p < 0.05). Similarly, PTM-treated Fasn +/+ Cre − Apoe −/− mice also demonstrated significantly reduced total aortic ( Fig. 4B & C ; p < 0.05), as well as reduced plaque in the aortic arch and infrarenal aortic segments ( Fig. 4D & F ; p < 0.05). Atherosclerotic plaque formation at the aortic valve roots were also evaluated after 16 weeks of a high-fat diet regimen and with and without PTM treatment. Fasn fl/fl Cre + Apoe −/− mice and PTM-treated Fasn +/+ Cre − Apoe −/− mice demonstrated significantly reduced aortic valve root plaque formation ( Fig. 5A & B ; p < 0.01). Similarly, Fasn fl/fl Cre + Apoe −/− mice and PTM-treated Fasn +/+ Cre − Apoe −/− mice demonstrated reduced CD68 + macrophages in the aortic valve wall, while Fasn +/+ Cre − Apoe −/− mice had higher CD68 content in the valve wall atheroma ( Fig. 5C & D ; p < 0.05). Conditional knockdown or inhibition of FAS alters tissue inflammation. As expected, hepatic tissue of Fasn fl/fl Cre + Apoe −/− mice had diminished FAS immunostaining ( Fig. 5A & 5B ; p < 0.05), and reduced CD68 + macrophage content ( Fig. 5A & 5C ; p < 0.05). PTM-treated Fasn +/+ Cre − Apoe −/− mice also demonstrated a modest reduction in liver FAS ( Fig. 5A & 5B ; p = 0.08), and significantly reduced CD68 content ( Fig. 5A & 5C ; p < 0.001). Interestingly, in white adipose tissue, liver Fasn fl/fl Cre + Apoe −/− mice demonstrated increased adipocyte area ( Fig. 5A & 5D ; p < 0.05) and increased FAS content ( Fig. 5A & 5E ; p < 0.0001), but no change in CD68 content ( Fig. 5A & 5F ). In PTM-treated Fasn +/+ Cre − Apoe −/− mice, there was a significant decrease in adipocyte area ( Fig. 5A & 5D ; p < 0.001), modest but significant decrease in FAS content ( Fig. 5A & 5E ; p < 0.05), and a significant decrease in CD68 content ( Fig. 5A & 5G ; p < 0.05). Discussion Our study evaluates the role of tissue FAS and serum cFAS on atheroprogression and tissue inflammation. We observed a significant increase in macrophage foam cell formation when conditioned with serum containing higher cFAS content. On the other hand, treatment with PTM significantly blunted foam cell formation. Similarly, in vivo , conditional knockdown of FAS in the liver, or treatment with PTM, greatly reduced aortic atherosclerotic plaque volume and macrophage content in aortic plaque regions. We also observed that FAS targeting impacted liver-adipose tissue crosstalk. Remarkably, although Fasn fl/fl Cre + Apoe -/- mice exhibited hypercholesteremia while maintained on a 42% high-fat diet, they developed minimal aortic atherosclerotic plaque. Overall, these findings highlight the indispensable roles that tissue FAS and serum cFAS contribute to atheroprogression. Dyslipidemia is a known risk factor for atheroprogression and cardiovascular disease 15,32-34 . Individuals with familial hyperlipidemia are born with dramatically elevated serum LDL cholesterol, develop early atherosclerotic disease onset, and are at higher risk of cardiovascular complications if not intensively treated 35 . Lipid-lowering medications, such as statins (co-enzyme A reductase inhibitors), fibrates, and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, are first-line in the management of hyperlipidemia, and aim to reduce serum circulating LDL or TG content thereby reducing the risk of cardiovascular events such as myocardial infarction (MI), stroke, and major lower extremity amputations resulting from peripheral arterial occlusive disease 36-38 . However, despite reduction of LDL with statins (20-50%) and PCSK9 inhibitors (50-65%), cardiovascular events are still only reduced by 30-60% in patients who are treated with medications within these drug categories 39,40 . This leaves most individuals with significant residual risk of major cardiovascular events and an unclear management strategy to reduce cardiovascular morbidity and mortality 5,41 . These persistent clinical gaps have contributed to a growing suspicion that in addition to traditional lipid mediators like LDL and TGs, there are likely additional key contributors to atheroprogression that are yet to be identified and therapeutically targeted. While individuals with high serum LDL levels (>190 mg/dL) are known to have a higher incidence of MI and stroke, this is indeed not always the case 42 . For example, in The Multi-Ethnic Study of Atherosclerosis (MESA), which evaluated >23,000 over a 16-year period, high serum LDL was observed to not be a risk factor for the incidence of atherosclerotic cardiovascular disease in individuals who had a zero coronary artery calcium (CAC) score on CT angiography 43 . Similarly, in a study of >136,000 patients who were hospitalized for an acute MI, it was observed that nearly 75% of patients had serum LDL levels that would indicate they were not at high risk of cardiovascular events 44 . These studies highlight that beyond LDL cholesterol there are additional serum and/or tissue lipid mediators that can influence whether a patient is either at higher or lower risk for atherosclerotic disease progression. Fatty acids are essential lipids that serve as functional components for TGs, phospholipids, and cholesterol esters. These lipid mediators impact a diverse array of cellular and tissue processes, including cell membrane structure and integrity, as well as serving as biological energy storage units during catabolism 45 . On the other hand, dysregulation of fatty acid synthesis contributes to deleterious conditions such as obesity, non-alcoholic fatty liver disease, and type 2 diabetes 46-48 . In macrophages, fatty acids play key roles in cholesterol uptake, esterification, and lipid efflux 49,50 . However, dysregulation of fatty acid synthesis is know to impact macrophage function, polarization, and phenotypic transformation, 16,20,23 Abrogation of fatty acid synthesis inhibits macrophage cholesterol efflux and foam cell formation 50,51 . This is of particular importance since foam cell accumulation in the arterial intima has been linked to arterial wall atheroma progression and plaque vulnerability 16,17,31,52 . Here we build upon this traditional dogma and demonstrate that on one end serum cFAS plays an important role in macrophage foam cell formation, and on the other end both serum cFAS and endogenous liver FAS play an important role in aortic atherosclerosis. We previously demonstrated that conditional knockdown of Fasn in the liver, but not in skeletal muscle leads to reduced serum cFAS. Additionally, we observed that cFAS co-immunoprecipitated with ApoB in LDL cholesterol serum fractions 22 . These findings previously led us to conclude that cFAS is produced by the liver and is released into the blood stream bound to ApoB in lipoproteins such as LDL. Given the relative concentrations of LDL and cFAS in human serum it is evident that cFAS concentrations are at least an order magnitude less than LDL. Meaning, while cFAS may serve as cargo attached to LDL particles in the serum, not all LDL particles will be saturated with cFAS and vice versa. This is presumably why we observed that human serum had variable content of cFAS and LDL. In our biobanked samples, there were samples that had higher cFAS content (>17ug/uL), and others that essentially undetectable cFAS. Naturally, we also observed serum samples that had very high LDL (>180 mg/dL), while others that had low LDL (<90 mg/dL). Since it was previously reported that there was no correlation between cFAS and LDL content in human serum, we intentionally evaluated the impact of human serum samples with either high and low cFAS or LDL 21 . Like others who demonstrated that LDL alone does not cause macrophage foam cell formation, we also observed that macrophages conditioned with serum containing high LDL, but low cFAS, did not lead to foam cell formation 52-55 . The mechanistic process that facilitates cFAS impact on foam cell formation is currently unknown. However, prior work demonstrates that endogenous FAS in macrophages is essential for the retention of plasma membrane cholesterol, cellular adhesion, and migration, as well as recruitment into adipose tissue that facilitates chronic tissue inflammation induced by nutrient dense diets 50,56-60 . In our study, we similarly observed that pharmacological inhibition of FAS with PTM in mice maintained on 42% high-fat diet dramatically reduced macrophage infiltration in both hepatic and white adipose tissue ( Fig. 5C & 5F ). While conditional knockdown of Fasn in liver tissue also reduced hepatic macrophage infiltration, it did not have as robust of a phenotype in white adipose tissue. Moreover, compared to conditional knockdown of Fasn , treatment with PTM had a more dramatic reduction of macrophages in the liver (127% difference) and white adipose (96% difference) tissue, suggesting that its inhibition of serum cFAS was likely playing a major role in these findings. Pharmacological inhibition of FAS is a topic of multiple prior investigations, particularly since FAS is elevated in malignant tissue, and serum cFAS is also elevated in individuals with certain metastatic tumors 61-64 . Indeed, there are currently FAS inhibitors that are undergoing efficacy testing in phase II human clinical trials and are demonstrating promise 65 . PTM is a commonly used FAS inhibitor that is naturally derived from Streptomyces platensis bacteria. It selectively and competitively binds to both bacterial and mammalian FAS and forms stable complexes with FAS subunits 66 . In Db/Db mice, PTM inhibits de novo fatty acid synthesis and enhances glucose oxidation 67 . Consistent with findings in Db/Db mice, we observed that PTM treatment of Fasn +/+ Cre - Apoe -/- mice supported normal weight gain. Prior studies suggest that this phenotype is observed due to improved hepatic glucose uptake and glycolysis 67 . However, our study demonstrates that PTM clearly also impacts white adipose FAS content and activity, as well as adipocyte lipid storage (expressed as adipocyte area). The remarkable crosstalk between liver and adipose tissue was not only limited to mice treated with PTM, but this was also observed in Fasn fl/fl Cre - Apoe -/- mice, which after 16 weeks of a high-fat diet regimen demonstrated significantly elevated FAS content. Liver and adipose signaling in relation to fatty acid synthesis and macronutrient metabolism has been reported extensively and is a highly orchestrated process. In humans, dietary nutrients, and de novo lipid synthesis in these organ tissue is thought to influence obesity and fatty liver disease 68 . Our findings suggest that cFAS may in part be a vehicle of communication between liver and white adipose tissue. In conclusion, we report that FAS targeting through conditional liver knockdown and targeted pharmacological inhibition, reduces tissue FAS and serum cFAS activity in macrophages, liver, and white adipose tissue. This leads to a significant reduction in atherosclerosis after 16 weeks of a high-fat diet regimen. 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Cell Mol Gastroenterol Hepatol 7(4):749–761 Additional Declarations Yes there is potential Competing Interest. The authors declare the existence of a possible financial/non-financial competing interest. Cite Share Download PDF Status: Published Journal Publication published 19 Feb, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4139044","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":289360839,"identity":"b8168283-dbec-44a0-8941-ca83053c03ae","order_by":0,"name":"Mohamed zayed","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYLACHgYbEMV4gIGBmWgtaQwg1SRpOUyCFvn2M4YP3uacT9xw/vyBAwwV1okNhLQw9uQYG87ddjtxw41koC1n0glrYWbIMZPm3XY7d8MNoMMY2w4T1sLG/8b8N++2c7kbzh8GavlHhBYeiRwzZt5tB3I3HAA6jLGBCC0SEs+KJeduS66feSPZ4EDCsXRjglrk+5M3fni7zc6Y7/zBhw8+1FjLEtTCwMBhgGAnEFYOAuwPiFM3CkbBKBgFIxcAAMHkRD5cpWZnAAAAAElFTkSuQmCC","orcid":"","institution":"Washington University in St. Louis","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"","lastName":"zayed","suffix":""},{"id":289360840,"identity":"188d0a3a-8e64-4d8a-8b11-1e53ee892ace","order_by":1,"name":"Rodrigo Meade","email":"","orcid":"","institution":"Washington University in St. Louis","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rodrigo","middleName":"","lastName":"Meade","suffix":""},{"id":289360841,"identity":"c132294e-33ef-427b-a264-d67234292eef","order_by":2,"name":"Connor Engel","email":"","orcid":"","institution":"Washington University in St. Louis","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Connor","middleName":"","lastName":"Engel","suffix":""},{"id":289360842,"identity":"273b5071-91d7-42cc-8920-e7e000bbba36","order_by":3,"name":"Larisa Belaygorod","email":"","orcid":"","institution":"Washington University in St. Louis","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Larisa","middleName":"","lastName":"Belaygorod","suffix":""},{"id":289360843,"identity":"133fe5d0-e916-462d-8f12-ef85eada4d2c","order_by":4,"name":"Batool Arif","email":"","orcid":"","institution":"Washington University in St. Louis","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Batool","middleName":"","lastName":"Arif","suffix":""},{"id":289360844,"identity":"1d1f295e-5d43-4345-a8a4-36f6c1c69866","order_by":5,"name":"Fong-Fu Hsu","email":"","orcid":"","institution":"Washington University in St. Louis","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fong-Fu","middleName":"","lastName":"Hsu","suffix":""},{"id":289360845,"identity":"064535f8-f566-49b5-bae0-01cf0ad655b7","order_by":6,"name":"Sangeeta Adak","email":"","orcid":"","institution":"Washington University in St. Louis","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sangeeta","middleName":"","lastName":"Adak","suffix":""},{"id":289360846,"identity":"478bc6e3-9b88-466b-bae4-b8cb6d92911c","order_by":7,"name":"Ryan Catlett","email":"","orcid":"","institution":"Washington University in St. Louis","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ryan","middleName":"","lastName":"Catlett","suffix":""},{"id":289360847,"identity":"ab5179af-a089-4300-9a33-a5a3135a439a","order_by":8,"name":"Clay Semenkovich","email":"","orcid":"https://orcid.org/0000-0003-1163-1871","institution":"Washington University in St. Louis","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Clay","middleName":"","lastName":"Semenkovich","suffix":""}],"badges":[],"createdAt":"2024-03-20 18:31:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4139044/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4139044/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-025-07656-1","type":"published","date":"2025-02-19T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56062637,"identity":"e91209a9-4b44-4d70-b2a3-c931dee65d88","added_by":"auto","created_at":"2024-05-08 05:23:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1508902,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecFAS induces macrophage foam cell formation \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(A) Schematic of \u003cem\u003ein vitro\u003c/em\u003e experiments used to evaluate the impact of cFAS or LDL on macrophage foam cell formation. (B) Matured macrophages were conditioned with variable concentrations of cFAS and low LDL, and percentage of foam cells were evaluated. (C) Correlation of Oil Red O positive foam cells and serum cFAS concentration. (D) Matured macrophages were conditioned with variable concentrations of LDL and low cFAS, and percentage of foam cells were evaluated. (E) Correlation of Oil Red O positive foam cells and serum LDL concentration. (F) Matured macrophages were conditioned with either high cFAS or high LDL serum as well as PTM. (G) Foam cell percentage following treatment with conditioned media, and with or without PTM. (H) Cell lysate FAS activity following serum FAS and serum LDL conditioning. * p\u0026lt;0.05, ** p\u0026lt;0.01\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4139044/v1/52455b5bf25b346caa54e948.png"},{"id":56062638,"identity":"151aaae1-96a3-44bb-bd94-6ccb0d2d9546","added_by":"auto","created_at":"2024-05-08 05:23:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":243293,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAS inhibition affects murine lipid homeostasis\u003c/strong\u003e. (A) Schematic of \u003cem\u003ein vivo \u003c/em\u003emurine experiments using \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e- \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+ \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e that were maintained on a high fat diet for 16 weeks. A group of\u003cem\u003e Fasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u0026nbsp; \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003emice also received PTM throughout this period. Serum, liver, and white adipose tissue were collected from all mouse groups. (B) Impact of \u003cem\u003eFasn\u003c/em\u003e knockdown of on murine body weight (n = 5 per group) over 16 week period. (C) Total serum cholesterol for each murine group (n = 3 per group). (D \u0026amp; E) Liver and white adipose tissue triglyceride content (n = 3 per group). (F \u0026amp; G) Liver and white adipose tissue non-esterified free fatty acid content (n = 3). * p\u0026lt;0.05, ** p\u0026lt;0.01\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4139044/v1/ffa507eeccf6281f285caa0e.png"},{"id":56062131,"identity":"8897dcb8-3883-4e2f-adb8-9c482e42f235","added_by":"auto","created_at":"2024-05-08 05:15:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":127382,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConditional liver-specific knockdown of FAS and PTM treatment impacts serum cFAS content and activity. \u003c/strong\u003eSerum specimens from \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e- \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+ \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, and \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e- \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice treated with PTM, were analyzed for cFAS content (A) and cFAS activity (B – D; n = 5 per mouse group). (E \u0026amp; F) The supernatant of digested liver was evaluated for cFAS content and activity (n = 5). (G \u0026amp; H) The supernatant of digested white adipose tissue was evaluated for cFAS content and activity (n = 5). * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4139044/v1/5b4319aa60c274849323e310.png"},{"id":56062136,"identity":"1f2cedcb-1870-4de6-ac17-18a01c7a1f30","added_by":"auto","created_at":"2024-05-08 05:15:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":734581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting FAS reduces aortic atherosclerotic plaque burden.\u003c/strong\u003e (A) Schematic of \u003cem\u003ein vivo \u003c/em\u003emurine experiments using \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e- \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+ \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, and \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e- \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice treated with PTM, that were maintained on a high fat diet for 16 weeks. (B) Representative enfacements of aortic specimens from different mouse groups that were stained with oil red O. Plaque areas are visualized in red. (C) Total aortic plaque assessment in \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (n = 10),\u0026nbsp; \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(n = 10), and \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e that were treated with PTM (n = 8). (D) Plaque burden in the aortic arch segment, (E) thoracic aorta, and (F) infrarenal aorta. * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4139044/v1/a9a1bfb3d1f13c76f0d89cc7.png"},{"id":56062137,"identity":"ee1e8ceb-9c9a-457b-a370-2ddafc2e78f3","added_by":"auto","created_at":"2024-05-08 05:15:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1993254,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting FAS reduced aortic root plaque burden. \u003c/strong\u003e(A) Hearts were isolated from \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e- \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+ \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, and \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e- \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice treated with PTM, that were maintained on a high fat diet for 16 weeks. Aortic valve leaflets were sectioned at 10μm and stained with oil red O. Plaque area is visualized in red. (B) Plaque lesion area percentage was evaluated in each mouse group (n = 3). (C) Aortic valve leaflet sections were also stained with the macrophage marker CD68 (green) and DAPI nuclear stain (blue). * Indicates lumen. (D) integraded density was analyzed to evaluate CD68 content in aortic valve sections of each mouse group (n = 3).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4139044/v1/a95971ac53b88cf494e35d27.png"},{"id":56062132,"identity":"749eb7b0-f5f2-4d6d-aa69-ed26830a0a7b","added_by":"auto","created_at":"2024-05-08 05:15:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5883621,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAS inhibition reduces tissue FAS and inflammation response. \u003c/strong\u003e(A) Liver and white adipose tissue were collected from \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e- \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+ \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, and \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e- \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice treated with PTM, that were maintained on a high fat diet for 16 weeks. Tissue were then sectioned and stained with H\u0026amp;E, and immunostained for FAS and CD68. (B) Quantification of liver FAS staining (n = 5), and (C) liver CD68 staining (n = 5). (D) Average adipocyte vacuole area (n = 5), (E) FAS staining (n = 5), and (F) CD68 staining (n = 5).\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4139044/v1/bf136d8fce0320146854e511.png"},{"id":76740558,"identity":"078c5670-33ad-44f3-b4ab-93346b46e627","added_by":"auto","created_at":"2025-02-20 08:11:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11571725,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4139044/v1/6df4d867-8815-42ec-97b5-7c46e0755ba5.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nThe authors declare the existence of a possible financial/non-financial competing interest.","formattedTitle":"Targeting Fatty Acid Synthase Reduces Aortic Atherosclerosis and Inflammation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAtherosclerosis is one of the major global underlying causes for cardiovascular disease\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Its management predominantly revolves around mitigation of risk factors such as hyperlipidemia with pharmacological therapies that aim to reduce serum circulating lipid levels\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Despite the efficacy of HMG-CoA reductase inhibitors (statins) and PCSK9 inhibitors in reducing serum low-density lipoprotein (LDL) and total cholesterol levels, their impact on cardiovascular events is confined to a modest range of 30\u0026ndash;60%\u003csup\u003e5\u0026ndash;9,\u003c/sup\u003e Moreover, even with effective reduction of total cholesterol using statin monotherapy, individuals contending with cardiovascular co-morbidities continue to be disproportionately susceptible to atheroprogression\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This heightened vulnerability translates into a residual major risk of myocardial infarction, stroke, and major peripheral extremity amputations. Recognizing these continued challenges, the American Heart Association (AHA) and European Society of Cardiology (ESC) have underscored the need to further investigate the underlying causes of atheroprogression and have prioritized the exploration of alternative treatment strategies for this recalcitrant and morbid disease process\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLipids such as cholesteryl esters, triglycerides, and phospholipids, feature saturated fatty acids that wield a significant influence on atheroprogression\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. A mounting body of evidence accentuates the pivotal role of saturated fatty acids within atheromatous plaques, amplifying plaque instability and the heightened risk of atheroma rupture\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Similarly, while \u003cem\u003ede novo\u003c/em\u003e synthesis of fatty acids is integral to lipid homeostasis, the process is essential for orchestrating the transformation of monocytes into foam cells, which play a fundamental role in perpetuating atheroprogression and exacerbating plaque instability and vulnerability\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Consequently, the modulation of fatty acid synthesis within evolving atheroma lesions is thought to influence the progression of atherosclerosis.\u003c/p\u003e \u003cp\u003eTissue Fatty Acid Synthase (FAS) is an essential soluble 273kDa intracellular homodimeric enzyme that catalyzes the \u003cem\u003ede novo\u003c/em\u003e synthesis of saturated fatty acid through the conversion of acetyl-CoA and malonyl-CoA into palmitate\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. It was recently observed that both tissue FAS and serum circulating FAS (cFAS) are elevated in individuals afflicted with severe atherosclerotic cardiovascular disease\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Serum cFAS is predominantly produced by the liver and bound to ApoB in LDL particles\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Notably, levels of cFAS in serum have a strong correlation with the content of FAS and saturated fatty acids in arterial tissue impacted by atherosclerosis\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In macrophages, FAS is also essential for cholesterol influx and cellular stress kinase activation\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Here we build upon these prior findings to determine whether targeted inhibition of tissue FAS and/or serum cFAS can impact macrophage foam cell formation and alter the course of \u003cem\u003ein vivo\u003c/em\u003e atheroprogression.\u003c/p\u003e"},{"header":"Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHuman serum\u003c/h2\u003e \u003cp\u003eNative human serum was obtained from an institutional review board (IRB)-approved vascular biobank\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. As previously described\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, fresh human serum aliquots were collected from at least 12 hour fasting study participants and concentrated using a 100kDa ultrafiltration centrifuge tube (ThermoFisher Scientific, Waltham, MA), at 15,000g, for 15 minutes. A resultant minimum volume of 250\u0026micro;L of concentrated serum was collected for each patient. Concentrated serum samples were stored in aliquots at -80\u0026ordm;C for subsequent use for \u003cem\u003ein vitro\u003c/em\u003e macrophage-foam cell experiments.\u003c/p\u003e \u003cp\u003eSerum used for conditioned media studies were obtained from male individuals with similar age demographics and contained either LDL content at low (\u0026lt;\u0026thinsp;90mg/dL), medium (90\u0026ndash;180 mg/dL), or high (\u0026gt;\u0026thinsp;180 mg/dL)\u003csup\u003e25\u003c/sup\u003e, and undetectable levels of cFAS (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Alternatively, media was also conditioned with serum containing either cFAS content at low (0 \u0026micro;g/\u0026micro;l), medium (5\u0026ndash;6 \u0026micro;g/\u0026micro;l), or high (\u0026gt;\u0026thinsp;17 \u0026micro;g/\u0026micro;l), and low levels of LDL (\u0026lt;\u0026thinsp;90mg/dL; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Serum LDL content was determined by the Washington University in St. Louis Core Laboratory for Clinical Studies (CLCS), utilizing a N-Geneous\u0026reg; LDL cholesterol kit (Sekisui Diagnostics, #7120) using a Roche Cobas c501 analyzer. Serum cFAS content was determined using commercial ELISA according to manufacturer\u0026rsquo;s instructions (Aviva, OKEH04869)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eHuman Serum Lipid Profile.\u003c/b\u003e Human serum demographics and lipid concentration from institutional serum biobank that were utilized for foam cell lipid formation experiments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCondition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLDL(mg/dL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ecFAS(\u0026micro;g/\u0026micro;l)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ecFAS\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e69.3\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;5.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e63.0\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;20.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21.7\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;4.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e62.3\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;18.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e79.6\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;28.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.45\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e62.6\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;6.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e93.0\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;25.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eLDL\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e51.3\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;19.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e183\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;7.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e66.6\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;8.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e103\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e69.3\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;8.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e35.6\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;10.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eLow density lipoprotein, LDL; circulating fatty acid synthase, cFAS\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTissue culture and foam cell formation assessment\u003c/h2\u003e \u003cp\u003eHuman U-937 (ATCC # CRL-1593.2) were cultured for 48 hours in 10% FBS RPMI, and then differentiated to macrophages in 5% FBS RPMI treated with 1\u0026micro;l/ml Phorbol 12-myristate 13-acetate, 95% (Thermo Fisher, # J63916.MB). Monocytes were allowed 24 hours to differentiate into macrophages. Differentiated macrophages were then washed 3x with PBS and incubated for 48 hours in cell culture media RPMI (Thermofisher, # 11875093) conditioned with 10% of human serum containing either low, medium, and high levels of cFAS or LDL on fibronectin treated coverslips (\u003cb\u003eSupplemental Table\u0026nbsp;1\u003c/b\u003e \u0026amp; \u003cb\u003eFig.\u0026nbsp;1A\u003c/b\u003e). Similarly, macrophages conditioned with serum containing either higher levels cFAS or LDL, were also simultaneously treated with Platensimycin (PTM, 20mM; Cayman Chemical, #15507) for 48 hours. Macrophages were then stained in oil-Red O working solution (3:2 dilution with distilled water of a stock solution of 0.5mg/\u0026micro;l in 100% isopropanol) and hematoxylin and eosin (H\u0026amp;E). Stained coverslips were imaged using a Leica Thunder DM6 B Microsystems inverted fluorescent microscope. Area positively stained with oil-Red O was quantified using ImageJ color threshold toolkit. The intracellular lipid droplet area stained with oil-Red O was expressed as a percentage of the total cell area.\u003c/p\u003e \u003cp\u003eFollowing conditioned media treatments, macrophages were also lysed with standard freeze thaw method in PBS and lysates were standardized to protein concentration using Bradford Protein Assay. Acetyl-CoA concentration was determined by using a commercial ELISA according to the manufacturer\u0026rsquo;s instructions (MyBioSource, #MBS9309791). Protein was loaded onto Bis-Tris gel (Thermo Fisher Scientific, NW00082BOX) and transferred to polyvinylidene fluoride membranes for content analysis. Anti-FAS 1:500 (Santa Cruz, #c-48357) and anti-β actin 1:10,000 (Abcam, #ab8227) were used for Western blotting. Band densitometry analysis was performed using ImageJ software, and FAS band densities were expressed as ratios relative to the β actin loading control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnimal regulations\u003c/h2\u003e \u003cp\u003e All animal protocols were approved by a local institutional animal care and use committee (IACUC). Mouse housing, breeding, and experimental procedures were conducted in accordance with national and institutional guidelines and ethics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMouse models\u003c/h2\u003e \u003cp\u003eConditional liver-specific knockdown of \u003cem\u003eFasn\u003c/em\u003e was achieved using previously reported \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice that also express an albumin-\u003cem\u003eCre\u003c/em\u003e promoter (\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e)\u003csup\u003e27\u003c/sup\u003e. Liver \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e mice were crossed with \u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e knockout mice (Jackson lab, strain #002052) to yield Liver \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. At 7 weeks of age, \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e littermates were maintained on a continuous 42% high-fat diet for 16 weeks (Inotiv, TD.88137). On a weekly basis, body weights were recorded, and blood serum samples were collected from the tail vein. Similarly, \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e littermates were maintained on a 42% high-fat diet with and without PTM (100mg/kg/day infused into the diet; generously donated by Dr. Clay F. Semenkovich) for a 16-week treatment period. After 16 weeks, mice were sacrificed and serum, hearts, aorta, liver, and white adipose tissue were collected for immediate analysis, embedded in OCT, or stored at -80\u0026deg;C for later use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFAS enzyme activity and content assay\u003c/h2\u003e \u003cp\u003eFAS enzyme activity was measured as previously described with some modifications\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Liver and white adipose tissue were digested in mammalian cell lysis kit (Milipore Sigma, MCL-1KT), and homogenates were centrifuged at 5,000g for 5 min at 4\u0026deg;C. The supernatant and serum were standardized to 30\u0026micro;g of total protein and added to 80\u0026micro;L of assay buffer (1M KPO4 buffer [pH 7], 50mM EDTA [pH 8.0], 50mM DTT, 1.1mM NADPH (Milipore Sigma, N1630), 1mM acetyl CoA (Milipore Sigma, A2056) The rate of NADPH oxidation was monitored at 340nm at 37\u0026deg;C for 30 minutes in the absence, and then in the presence of 10\u0026micro;L of the rate-limiting substrate malonyl-CoA for 10 minutes (1mM;Milipore Sigma, M4263). Data was analyzed by calculating the OD decrease subtracted from the nonspecific background without Malonyl CoA substrate. FAS enzyme was defined as \u0026micro;moles NADPH consumed\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. An extinction coefficient of 6220 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was used in the specific activity calculation utilizing Beer\u0026rsquo;s law as previously described\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Tissue and serum FAS protein content were evaluated using commercially ELISA according to manufacturer\u0026rsquo;s instructions (Aviva, OKEH04869).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAortic atherosclerotic burden assessment\u003c/h2\u003e \u003cp\u003eMurine hearts were harvested \u003cem\u003een bloc\u003c/em\u003e at time of sacrifice after 16-weeks of diet treatment. The tissue was fixed in OCT compound (Fisher Scientific), and the aortic valve region was sectioned at 10\u0026micro;m thickness. Sections were then fixed in 4% paraformaldehyde (PFA), followed by 60% isopropanol for 5 minutes. Aortic valve sections were then stained in an oil-Red O working solution (3:2 dilution with distilled water from stock solution of 0.5mg/\u0026micro;l in 100% isopropanol). Valve plaque area was quantified in a blinded fashion using ImageJ as percentage of plaque area in the aortic lumen, as previously described\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Corresponding sections of aortic value were also stained with 1:50 mouse anti-CD68 antibody (Bio Rad, MCA1957). The primary antibody was detected with a 1:100 secondary antibody, donkey anti-rat IgG labeled with Alexa Fluor 555 (Thermo Fisher Scientific, A78945), followed by DAPI nucleus stain. Stained sections were then imaged on Leica Thunder DM6 B Microsystems inverted fluorescent microscope. The percentage of positively stained CD68 area relative to total aortic lumen was quantified using ImageJ software.\u003c/p\u003e \u003cp\u003eSimilarly, the entire aorta from the aortic arch to the infrarenal aortic bifurcation were microdissected and resected \u003cem\u003een bloc\u003c/em\u003e at the time of sacrifice. Harvested aortic specimens were fixed in 4% PFA for 24 hours. The tissue was then effaced and stained using oil-Red O, and then imaged with Lecia S9i Microsystem microscope. The area of plaque that positively stained with oil-Red O was taken relative to the aortic valve segment area using ImageJ in a blinded fashion\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eTissue histology\u003c/h2\u003e \u003cp\u003eMurine liver and white adipose tissue were immediately harvested at the time of sacrifice and were embedded in OCT. Tissue was sectioned at 10\u0026micro;m thickness and fixed in 4% PFA and stained by H\u0026amp;E. Sections were also immuno-stained with 2% donkey blocking agent for 1 hour at room temperature, and then with 1:100 primary mouse monoclonal FAS antibody (Santa Cruz, SC-48357), or 1:50 mouse anti-CD68 antibody (Bio Rad, MCA1957). The primary antibody was detected with a 1:100 secondary antibody donkey anti-rat IgG labeled with Alexa Fluor 555 (Thermo Fisher Scientific, A78945), followed by DAPI nucleus stain. Imaging assessments were performed using a Leica Thunder DM6 B Microsystems inverted fluorescent microscope, and staining was quantified using ImageJ software integrated density toolkit\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatical analysis\u003c/h2\u003e \u003cp\u003eStatistical correlations between continuous variables such as serum cFAS, tissue FAS, content or activity were evaluated using linear regression. Non-parametric two-tailed Man-Whitney tests were used to evaluate the differences between inter- and intra-group analysis. Endpoints obtained over a time course were evaluated using two-way ANOVA with multiple comparisons. All analyses were performed using GraphPad Prism (Prism 9.1 software, GraphPad Software Inc.). p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically significant. All graphical data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSerum cFAS induces macrophage foam cell formation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMacrophage cytoplasmic lipid droplet accumulation and foam cell formation is a hallmark of atheroprogression\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. We evaluated whether macrophages conditioned with native human serum either containing high cFAS or LDL can impact foam cell formation \u003cem\u003ein vitro\u003c/em\u003e (\u003cb\u003eFig.\u0026nbsp;1A\u003c/b\u003e). Interestingly, we observed a significant correlation between serum cFAS content in the conditioned media and the percentage of macrophages that formed foam cells (\u003cb\u003eFig.\u0026nbsp;1B \u0026amp; 1C\u003c/b\u003e; R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.44, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On the other hand, no correlation was observed between serum LDL content and the percentage of macrophage foam cell formation (\u003cb\u003eFig.\u0026nbsp;1D \u0026amp; 1E\u003c/b\u003e; R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.05, p\u0026thinsp;=\u0026thinsp;0.59).\u003c/p\u003e \u003cp\u003eTreatment with the FAS inhibitor PTM significantly decreased macrophage-derived foam cell formation when cells were conditioned with serum containing high cFAS (\u0026gt;\u0026thinsp;17 ug/uL) and low LDL (\u0026lt;\u0026thinsp;90 mg/dL; \u003cb\u003eFig.\u0026nbsp;1F \u0026amp; 1G\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Macrophages conditioned with serum containing high cFAS, had higher foam cell formation than macrophages conditioned with serum containing high LDL (\u003cb\u003eFig.\u0026nbsp;1G\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Intracellular FAS activity in macrophages that were conditioned with serum containing high cFAS was higher than macrophages conditioned with serum containing high LDL (\u003cb\u003eFig.\u0026nbsp;1J\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). PTM only reduced intracellular macrophage FAS activity in cells that were conditioned with high cFAS (\u003cb\u003eFig.\u0026nbsp;1H\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown and inhibition of FAS alters serum and tissue lipidomics.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eFasn\u003c/em\u003e \u003csup\u003e \u003cem\u003e+/+\u003c/em\u003e \u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e \u003csup\u003e \u003cem\u003e\u0026minus;\u003c/em\u003e \u003c/sup\u003e \u003cem\u003eApoe\u003c/em\u003e \u003csup\u003e \u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e \u003c/sup\u003e and \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e were maintained on a high fat diet for 16 weeks. A group of \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice also received PTM throughout this period, and serum, liver, and adipose tissue were collected (\u003cb\u003eFig.\u0026nbsp;2A\u003c/b\u003e). Following 16 weeks of a high-fat diet, \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice had significantly less weight gain compared to \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (51% vs 54% increase in weight; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003cb\u003eFig.\u0026nbsp;2B\u003c/b\u003e). On the other hand, \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice treated with PTM had no significant change in body weight compared to untreated \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (\u003cb\u003eFig.\u0026nbsp;2B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAll mouse groups demonstrated universal hypercholesteremia with total cholesterol\u0026thinsp;\u0026gt;\u0026thinsp;1,400 mg/dL at 16 weeks (\u003cb\u003eFig.\u0026nbsp;2C\u003c/b\u003e). Compared to \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice treated with and without PTM, demonstrated no significant differences in liver and adipose TGs at 16 weeks (\u003cb\u003eFig.\u0026nbsp;2D \u0026amp; 2E\u003c/b\u003e). On the other hand, FFAs were notably decreased in the liver (\u003cb\u003eFig.\u0026nbsp;2F\u003c/b\u003e; ∆37%), and significantly elevated in adipose tissue of \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice that received PTM treatment (\u003cb\u003eFig.\u0026nbsp;2G\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003cb\u003eConditional liver FAS knockdown and inhibition impacts serum cFAS and tissue FAS content and activity.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe observed a significant decrease in serum cFAS content in \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice prior to initiation of high-fat diet regimen (\u003cb\u003eFig.\u0026nbsp;3A\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, PTM treatment significantly reduced cFAS content in \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (\u003cb\u003eFig.\u0026nbsp;3A\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Interestingly, serum cFAS activity was reduced in \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, and PTM-treated \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (\u003cb\u003eFig.\u0026nbsp;3B\u003c/b\u003e). This reduction was more significant after 9 and 16 weeks with a high-fat diet (\u003cb\u003eFig.\u0026nbsp;3C \u0026ndash; 3D\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eAfter initiation of high-fat diet, \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice demonstrated a different pattern of FAS content and activity in the hepatic and adipose tissue. As expected, \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice demonstrated a significant decrease in FAS content and activity in hepatic tissue (\u003cb\u003eFig.\u0026nbsp;3E \u0026amp; 3F\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Interestingly, \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice demonstrated a significant increase in FAS content in white adipose (\u003cb\u003eFig.\u0026nbsp;3G\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.005). PTM treatment did not impact FAS content in the liver, but led to a significant decrease in FAS content and activity in white adipose (\u003cb\u003eFig.\u0026nbsp;3G \u0026amp; 3H\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similarly, there was moderate, but not statistically significant, reduction in FAS activity in hepatic tissue of PTM-treated \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (\u003cb\u003eFig.\u0026nbsp;3F\u003c/b\u003e; p\u0026thinsp;=\u0026thinsp;0.14).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFAS conditional knockdown or pharmacological inhibition reduces atheroprogression.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe next evaluated arterial atheroprogression in \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and PTM-treated \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice that were maintained on a high fat diet for 16 weeks and aortas harvested for analysis (\u003cb\u003eFig.\u0026nbsp;4A\u003c/b\u003e). Compared to \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice demonstrated a significant reduction in total aortic atherosclerotic plaque formation (\u003cb\u003eFig.\u0026nbsp;4B \u0026amp; C\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). This difference was evident in all aortic segments including the aortic arch, thoracic aorta, and infrarenal aorta (\u003cb\u003eFig.\u0026nbsp;4D - F\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, PTM-treated \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice also demonstrated significantly reduced total aortic (\u003cb\u003eFig.\u0026nbsp;4B \u0026amp; C\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as well as reduced plaque in the aortic arch and infrarenal aortic segments (\u003cb\u003eFig.\u0026nbsp;4D \u0026amp; F\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eAtherosclerotic plaque formation at the aortic valve roots were also evaluated after 16 weeks of a high-fat diet regimen and with and without PTM treatment. \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and PTM-treated \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice demonstrated significantly reduced aortic valve root plaque formation (\u003cb\u003eFig.\u0026nbsp;5A \u0026amp; B\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and PTM-treated \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice demonstrated reduced CD68\u0026thinsp;+\u0026thinsp;macrophages in the aortic valve wall, while \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice had higher CD68 content in the valve wall atheroma (\u003cb\u003eFig.\u0026nbsp;5C \u0026amp; D\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cb\u003eConditional knockdown or inhibition of FAS alters tissue inflammation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs expected, hepatic tissue of \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice had diminished FAS immunostaining (\u003cb\u003eFig.\u0026nbsp;5A \u0026amp; 5B\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and reduced CD68\u0026thinsp;+\u0026thinsp;macrophage content (\u003cb\u003eFig.\u0026nbsp;5A \u0026amp; 5C\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). PTM-treated \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice also demonstrated a modest reduction in liver FAS (\u003cb\u003eFig.\u0026nbsp;5A \u0026amp; 5B\u003c/b\u003e; p\u0026thinsp;=\u0026thinsp;0.08), and significantly reduced CD68 content (\u003cb\u003eFig.\u0026nbsp;5A \u0026amp; 5C\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eInterestingly, in white adipose tissue, liver \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice demonstrated increased adipocyte area (\u003cb\u003eFig.\u0026nbsp;5A \u0026amp; 5D\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and increased FAS content (\u003cb\u003eFig.\u0026nbsp;5A \u0026amp; 5E\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), but no change in CD68 content (\u003cb\u003eFig.\u0026nbsp;5A \u0026amp; 5F\u003c/b\u003e). In PTM-treated \u003cem\u003eFasn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, there was a significant decrease in adipocyte area (\u003cb\u003eFig.\u0026nbsp;5A \u0026amp; 5D\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), modest but significant decrease in FAS content (\u003cb\u003eFig.\u0026nbsp;5A \u0026amp; 5E\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and a significant decrease in CD68 content (\u003cb\u003eFig.\u0026nbsp;5A \u0026amp; 5G\u003c/b\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u0026nbsp;Our study evaluates the role of tissue FAS and serum cFAS on atheroprogression and tissue inflammation. We observed a significant increase in macrophage foam cell formation when conditioned with serum containing higher cFAS content. On the other hand, treatment with PTM significantly blunted foam cell formation. Similarly, \u003cem\u003ein vivo\u003c/em\u003e, conditional knockdown of FAS in the liver, or treatment with PTM, greatly reduced aortic atherosclerotic plaque volume and macrophage content in aortic plaque regions. We also observed that FAS targeting impacted liver-adipose tissue crosstalk. \u0026nbsp; Remarkably, although \u003cem\u003eFasn\u003csup\u003efl/fl\u0026nbsp;\u003c/sup\u003eCre\u003csup\u003e+\u003c/sup\u003eApoe\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice exhibited hypercholesteremia while maintained on a 42% high-fat diet, they developed minimal aortic atherosclerotic plaque. Overall, these findings highlight the indispensable roles that tissue FAS and serum cFAS contribute to atheroprogression.\u003c/p\u003e\n\u003cp\u003eDyslipidemia is a known risk factor for atheroprogression and cardiovascular disease\u003csup\u003e15,32-34\u003c/sup\u003e. Individuals with familial hyperlipidemia are born with dramatically elevated serum LDL cholesterol, develop early atherosclerotic disease onset, and are at higher risk of cardiovascular complications if not intensively treated\u003csup\u003e35\u003c/sup\u003e. Lipid-lowering medications, such as statins (co-enzyme A reductase inhibitors), fibrates, and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, are first-line in the management of hyperlipidemia, and aim to reduce serum circulating LDL or TG content thereby reducing the risk of cardiovascular events such as myocardial infarction (MI), stroke, and major lower extremity amputations resulting from peripheral arterial occlusive disease\u003csup\u003e36-38\u003c/sup\u003e. However, despite reduction of LDL with statins (20-50%) and PCSK9 inhibitors (50-65%), cardiovascular events are still only reduced by 30-60% in patients who are treated with medications within these drug categories\u003csup\u003e39,40\u003c/sup\u003e. This leaves most individuals with significant residual risk of major cardiovascular events and an unclear management strategy to reduce cardiovascular morbidity and mortality\u003csup\u003e5,41\u003c/sup\u003e. These persistent clinical gaps have contributed to a growing suspicion that in addition to traditional lipid mediators like LDL and TGs, there are likely additional key contributors to atheroprogression that are yet to be identified and therapeutically targeted. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile individuals with high serum LDL levels (\u0026gt;190 mg/dL) are known to have a higher incidence of MI and stroke, this is indeed not always the case\u003csup\u003e42\u003c/sup\u003e. For example, in The Multi-Ethnic Study of Atherosclerosis (MESA), which evaluated \u0026gt;23,000 over a 16-year period, high serum LDL was observed to not be a risk factor for the incidence of atherosclerotic cardiovascular disease in individuals who had a zero coronary artery calcium (CAC) score on CT angiography\u003csup\u003e43\u003c/sup\u003e. Similarly, in a study of \u0026gt;136,000 patients who were hospitalized for an acute MI, it was observed that nearly 75% of patients had serum LDL levels that would indicate they were not at high risk of cardiovascular events\u003csup\u003e44\u003c/sup\u003e. These studies highlight that beyond LDL cholesterol there are additional serum and/or tissue lipid mediators that can influence whether a patient is either at higher or lower risk for atherosclerotic disease progression.\u003c/p\u003e\n\u003cp\u003eFatty acids are essential lipids that serve as functional components for TGs, phospholipids, and cholesterol esters. These lipid mediators impact a diverse array of cellular and tissue processes, including cell membrane structure and integrity, as well as serving as biological energy storage units during catabolism\u003csup\u003e45\u003c/sup\u003e. On the other hand, dysregulation of fatty acid synthesis contributes to deleterious conditions such as obesity, non-alcoholic fatty liver disease, and type 2 diabetes\u003csup\u003e46-48\u003c/sup\u003e. In macrophages, fatty acids play key roles in cholesterol uptake, esterification, and lipid efflux\u003csup\u003e49,50\u003c/sup\u003e. However, dysregulation of fatty acid synthesis is know to impact macrophage function, polarization, and phenotypic transformation,\u003csup\u003e16,20,23\u003c/sup\u003e Abrogation of fatty acid synthesis inhibits macrophage cholesterol efflux and foam cell formation\u003csup\u003e50,51\u003c/sup\u003e. This is of particular importance since foam cell accumulation in the arterial intima has been linked to arterial wall atheroma progression and plaque vulnerability\u003csup\u003e16,17,31,52\u003c/sup\u003e. Here we build upon this traditional dogma and demonstrate that on one end serum cFAS plays an important role in macrophage foam cell formation, and on the other end both serum cFAS and endogenous liver FAS play an important role in aortic atherosclerosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe previously demonstrated that conditional knockdown of \u003cem\u003eFasn\u003c/em\u003e in the liver, but not in skeletal muscle leads to reduced serum cFAS. Additionally, we observed that cFAS co-immunoprecipitated with ApoB in LDL cholesterol serum fractions\u003csup\u003e22\u003c/sup\u003e. These findings previously led us to conclude that cFAS is produced by the liver and is released into the blood stream bound to ApoB in lipoproteins such as LDL. Given the relative concentrations of LDL and cFAS in human serum it is evident that cFAS concentrations are at least an order magnitude less than LDL. Meaning, while cFAS may serve as cargo attached to LDL particles in the serum, not all LDL particles will be saturated with cFAS and vice versa. This is presumably why we observed that human serum had variable content of cFAS and LDL. In our biobanked samples, there were samples that had higher cFAS content (\u0026gt;17ug/uL), and others that essentially undetectable cFAS. Naturally, we also observed serum samples that had very high LDL (\u0026gt;180 mg/dL), while others that had low LDL (\u0026lt;90 mg/dL). Since it was previously reported that there was no correlation between cFAS and LDL content in human serum, we intentionally evaluated the impact of human serum samples with either high and low cFAS or LDL\u003csup\u003e21\u003c/sup\u003e. Like others who demonstrated that LDL alone does not cause macrophage foam cell formation, we also observed that macrophages conditioned with serum containing high LDL, but low cFAS, did not lead to foam cell formation\u003csup\u003e52-55\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe mechanistic process that facilitates cFAS impact on foam cell formation is currently unknown. However, prior work demonstrates that endogenous FAS in macrophages is essential for the retention of plasma membrane cholesterol, cellular adhesion, and migration, as well as recruitment into adipose tissue that facilitates chronic tissue inflammation induced by nutrient dense diets\u003csup\u003e50,56-60\u003c/sup\u003e. In our study, we similarly observed that pharmacological inhibition of FAS with PTM in mice maintained on 42% high-fat diet dramatically reduced macrophage infiltration in both hepatic and white adipose tissue (\u003cstrong\u003eFig. 5C \u0026amp; 5F\u003c/strong\u003e). While conditional knockdown of \u003cem\u003eFasn\u003c/em\u003e in liver tissue also reduced hepatic macrophage infiltration, it did not have as robust of a phenotype in white adipose tissue. Moreover, compared to conditional knockdown of \u003cem\u003eFasn\u003c/em\u003e, treatment with PTM had a more dramatic reduction of macrophages in the liver (127% difference) and white adipose (96% difference) tissue, suggesting that its inhibition of serum cFAS was likely playing a major role in these findings.\u003c/p\u003e\n\u003cp\u003ePharmacological inhibition of FAS is a topic of multiple prior investigations, particularly since FAS is elevated in malignant tissue, and serum cFAS is also elevated in individuals with certain metastatic tumors\u003csup\u003e61-64\u003c/sup\u003e. Indeed, there are currently FAS inhibitors that are undergoing efficacy testing in phase II human clinical trials and are demonstrating promise\u003csup\u003e65\u003c/sup\u003e. PTM is a commonly used FAS inhibitor that is naturally derived from \u003cem\u003eStreptomyces platensis\u0026nbsp;\u003c/em\u003ebacteria. It selectively and competitively binds to both bacterial and mammalian FAS and forms stable complexes with FAS subunits\u003csup\u003e66\u003c/sup\u003e. In \u003cem\u003eDb/Db\u003c/em\u003e mice, PTM inhibits \u003cem\u003ede novo\u003c/em\u003e fatty acid synthesis and enhances glucose oxidation\u003csup\u003e67\u003c/sup\u003e. Consistent with findings in \u003cem\u003eDb/Db\u003c/em\u003e mice, we observed that PTM treatment of \u003cem\u003eFasn\u003csup\u003e+/+\u003c/sup\u003e Cre\u003csup\u003e-\u003c/sup\u003e Apoe\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice supported normal weight gain. Prior studies suggest that this phenotype is observed due to improved hepatic glucose uptake and glycolysis\u003csup\u003e67\u003c/sup\u003e. However, our study demonstrates that PTM clearly also impacts white adipose FAS content and activity, as well as adipocyte lipid storage (expressed as adipocyte area). The remarkable crosstalk between liver and adipose tissue was not only limited to mice treated with PTM, but this was also observed in \u003cem\u003eFasn\u003csup\u003efl/fl\u003c/sup\u003e Cre\u003csup\u003e-\u003c/sup\u003e Apoe\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice, which after 16 weeks of a high-fat diet regimen demonstrated significantly elevated FAS content. Liver and adipose signaling in relation to fatty acid synthesis and macronutrient metabolism has been reported extensively and is a highly orchestrated process. In humans, dietary nutrients, and \u003cem\u003ede novo\u003c/em\u003e lipid synthesis in these organ tissue is thought to influence obesity and fatty liver disease\u003csup\u003e68\u003c/sup\u003e. Our findings suggest that cFAS may in part be a vehicle of communication between liver and white adipose tissue.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, we report that FAS targeting through conditional liver knockdown and targeted pharmacological inhibition, reduces tissue FAS and serum cFAS activity in macrophages, liver, and white adipose tissue. This leads to a significant reduction in atherosclerosis after 16 weeks of a high-fat diet regimen. Additionally, knockdown or inhibition of FAS reduces tissue infiltration in arterial plaque, liver, and adipose tissue. These findings highlight the utility of targeting tissue FAS or serum cFAS for the management of atheroprogression.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare the existence of a possible financial/non-financial competing interest.\u003c/p\u003e \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLibby P, Buring JE, Badimon L et al (2019) Atherosclerosis. Nat Rev Dis Primers 5(1):56\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong P, Fang Z, Wang H et al (2020) Global and regional prevalence, burden, and risk factors for carotid atherosclerosis: a systematic review, meta-analysis, and modelling study. 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Cell Mol Gastroenterol Hepatol 7(4):749\u0026ndash;761\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4139044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4139044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFatty acid synthase (FAS) is predominantly expressed in the liver and adipose tissue. It plays vital roles in \u003cem\u003ede novo\u003c/em\u003e synthesis of saturated fatty acid and regulates insulin sensitivity. We previously demonstrated that serum circulating FAS (cFAS) is a clinical biomarker for advanced atherosclerosis, and that it is conjugated to low-density lipoproteins (LDL). However, it remains unknown whether cFAS can directly impact atheroprogression. To investigate this, we evaluated whether cFAS impacts macrophage foam cell formation \u0026ndash; a important cellular process leading to atheroprogression. Macrophages exposed to human serum containing high levels of cFAS showed increased foam cell formation as compared to cells exposed to serum containing low levels of cFAS. This difference was not observed using serum containing either high or low LDL. Pharmacological inhibition of cFAS using Platensimycin (PTM) decreased foam cell formation \u003cem\u003ein vitro\u003c/em\u003e. In \u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice with normal FAS expression, administration of PTM over 16 weeks along with a high fat diet decreased cFAS activity and aortic atherosclerosis without affecting circulating total cholesterol. This effect was also seen in \u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice with liver-specific deletion of hepatic FAS. Reductions in aortic root plaque were associated with decreased macrophage infiltration. These findings demonstrate that cFAS can impact arterial atheroprogression.\u003c/p\u003e","manuscriptTitle":"Targeting Fatty Acid Synthase Reduces Aortic Atherosclerosis and Inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-08 05:15:50","doi":"10.21203/rs.3.rs-4139044/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c22e0de2-1d6d-484e-8071-20ac912c7a73","owner":[],"postedDate":"May 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30470235,"name":"Health sciences/Diseases/Cardiovascular diseases/Vascular diseases/Atherosclerosis"},{"id":30470236,"name":"Health sciences/Diseases/Cardiovascular diseases/Dyslipidaemias"},{"id":30470237,"name":"Health sciences/Pathogenesis/Inflammation"},{"id":30470238,"name":"Health sciences/Medical research/Preclinical research"}],"tags":[],"updatedAt":"2025-02-20T08:10:56+00:00","versionOfRecord":{"articleIdentity":"rs-4139044","link":"https://doi.org/10.1038/s42003-025-07656-1","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2025-02-19 05:00:00","publishedOnDateReadable":"February 19th, 2025"},"versionCreatedAt":"2024-05-08 05:15:50","video":"","vorDoi":"10.1038/s42003-025-07656-1","vorDoiUrl":"https://doi.org/10.1038/s42003-025-07656-1","workflowStages":[]},"version":"v1","identity":"rs-4139044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4139044","identity":"rs-4139044","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}