ANGPTL3 deficiency impairs lipoprotein production and produces adaptive changes in hepatic lipid metabolism.

Angiopoietin-like protein 3 (ANGPTL3) is a hepatically secreted protein and therapeutic target for reducing plasma triglyceride-rich lipoproteins and low-density lipoprotein (LDL) cholesterol. Although ANGPTL3 modulates the metabolism of circulating lipoproteins, its role in triglyceride-rich lipoprotein assembly and secretion remains unknown. CRISPR-associated protein 9 (CRISPR/Cas9) was used to target ANGPTL3 in HepG2 cells (ANGPTL3-/-) whereupon we observed ∼50% reduction of apolipoprotein B100 (ApoB100) secretion, accompanied by an increase in ApoB100 early presecretory degradation via a predominantly lysosomal mechanism. Despite defective particle secretion in ANGPTL3-/- cells, targeted lipidomic analysis did not reveal neutral lipid accumulation in ANGPTL3-/- cells; rather ANGPTL3-/- cells demonstrated decreased secretion of newly synthesized triglycerides and increased fatty acid oxidation. Furthermore, RNA sequencing demonstrated significantly altered expression of key lipid metabolism genes, including targets of peroxisome proliferator-activated receptor α, consistent with decreased lipid anabolism and increased lipid catabolism. In contrast, CRISPR/Cas9 LDL receptor (LDLR) deletion in ANGPTL3-/- cells did not result in a secretion defect at baseline, but proteasomal inhibition strongly induced compensatory late presecretory degradation of ApoB100 and impaired its secretion. Additionally, these ANGPTL3-/-;LDLR-/- cells rescued the deficient LDL clearance of LDLR-/- cells. In summary, ANGPTL3 deficiency in the presence of functional LDLR leads to the production of fewer lipoprotein particles due to early presecretory defects in particle assembly that are associated with adaptive changes in intrahepatic lipid metabolism. In contrast, when LDLR is absent, ANGPTL3 deficiency is associated with late presecretory regulation of ApoB100 degradation without impaired secretion. Our findings therefore suggest an unanticipated intrahepatic role for ANGPTL3, whose function varies with LDLR status.

Integrin α9ß1 deficiency does not impact the development of atherosclerosis in mice.

Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 (SVEP1) is an extracellular matrix protein that causally promotes cardiovascular disease in humans and mice. However, the receptor mediating the effect of SVEP1 on the development of disease remains unclear. We previously demonstrated that depleting either vascular smooth muscle cell (VSMC)- or myeloid cell-derived integrin α9ß1, the first receptor that was identified to interact with SVEP1, did not phenocopy the disease-abrogating effect of depleting SVEP1. Due to its wide expression in tissues and cell types, here we extend this line of investigation to definitively determine if integrin α9ß1 impacts the development of atherosclerosis. In a mouse model of atherosclerosis, we found that depleting integrin α9ß1 in all cells did not alter plaque size or characteristics of plaque complexity when compared to wild type mice. Further, the significant SVEP1-mediated effects on increase in macrophage content and VSMC proliferation within the atherosclerotic plaque were not altered in animals lacking integrin α9ß1. Together, these findings strongly suggest that integrin α9ß1 is not responsible for mediating the SVEP1-induced promotion of atherosclerosis and support further studies aimed at characterizing other receptors whose interaction with SVEP1 may represent a therapeutically targetable interaction.

A Paradigm For Calling Sequence In Families: The Long Life Family Study.

Over Several years, we have developed a system for assuring the quality of whole genome sequence (WGS) data in the LLFS families. We have focused on providing data to identify germline genetic variants with the aim of releasing as many variants on as many individuals as possible. We aim to assure the quality of the individual calls. The availability of family data has enabled us to use and validate some filters not commonly used in population-based studies. We developed slightly different procedures for the autosomal, X, Y, and Mitochondrial (MT) chromosomes. Some of these filters are specific to family data, but some can be used with any WGS data set. We also describe the procedure we use to construct linkage markers from the SNP sequence data and how we compute IBD values for use in linkage analysis.

Instrumental variable and colocalization analyses identify endotrophin and HTRA1 as potential therapeutic targets for coronary artery disease.

Coronary artery disease (CAD) remains a leading cause of disease burden globally, and there is a persistent need for new therapeutic targets. Instrumental variable (IV) and genetic colocalization analyses can help identify novel therapeutic targets for human disease by nominating causal genes in genome-wide association study (GWAS) loci. We conducted cis-IV analyses for 20,125 genes and 1,746 plasma proteins with CAD using molecular trait quantitative trait loci variant (QTLs) data from three different studies. 19 proteins and 119 genes were significantly associated with CAD risk by IV analyses and demonstrated evidence of genetic colocalization. Notably, our analyses validated well-established targets such as PCSK9 and ANGPTL4 while also identifying HTRA1 and endotrophin (a cleavage product of COL6A3) as proteins whose levels are causally associated with CAD risk. Further experimental studies are needed to confirm the causal role of the genes and proteins identified through our multiomic cis-IV analyses on human disease.

Identification of a leucine-mediated threshold effect governing macrophage mTOR signalling and cardiovascular risk.

High protein intake is common in western societies and is often promoted as part of a healthy lifestyle; however, amino-acid-mediated mammalian target of rapamycin (mTOR) signalling in macrophages has been implicated in the pathogenesis of ischaemic cardiovascular disease. In a series of clinical studies on male and female participants ( NCT03946774 and NCT03994367 ) that involved graded amounts of protein ingestion together with detailed plasma amino acid analysis and human monocyte/macrophage experiments, we identify leucine as the key activator of mTOR signalling in macrophages. We describe a threshold effect of high protein intake and circulating leucine on monocytes/macrophages wherein only protein in excess of ∼25 g per meal induces mTOR activation and functional effects. By designing specific diets modified in protein and leucine content representative of the intake in the general population, we confirm this threshold effect in mouse models and find ingestion of protein in excess of ∼22% of dietary energy requirements drives atherosclerosis in male mice. These data demonstrate a mechanistic basis for the adverse impact of excessive dietary protein on cardiovascular risk.