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 Fasnfl/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 disease15,32-34. 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 treated35. 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 disease36-38. 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 categories39,40. This leaves most individuals with significant residual risk of major cardiovascular events and an unclear management strategy to reduce cardiovascular morbidity and mortality5,41. 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 case42. 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 angiography43. 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 events44. 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 catabolism45. On the other hand, dysregulation of fatty acid synthesis contributes to deleterious conditions such as obesity, non-alcoholic fatty liver disease, and type 2 diabetes46-48. In macrophages, fatty acids play key roles in cholesterol uptake, esterification, and lipid efflux49,50. 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 formation50,51. This is of particular importance since foam cell accumulation in the arterial intima has been linked to arterial wall atheroma progression and plaque vulnerability16,17,31,52. 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 fractions22. 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 LDL21. 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 formation52-55.
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 diets50,56-60. 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 tumors61-64. Indeed, there are currently FAS inhibitors that are undergoing efficacy testing in phase II human clinical trials and are demonstrating promise65. 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 subunits66. In Db/Db mice, PTM inhibits de novo fatty acid synthesis and enhances glucose oxidation67. 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 glycolysis67. 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 Fasnfl/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 disease68. 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. 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.