Faculty Bibliography

BACKGROUND:Cardiometabolic risk factors diabetes, obesity, and hypertension are highly prevalent and contribute to increased cardiovascular disease (CVD). Endothelial dysfunction precedes CVD development. The current study aimed to investigate the EC transcriptome among individuals with varying degree of cardiometabolic risk. METHODS:Adult participants without CVD and various degrees of cardiometabolic risk factor burden (hypertension, diabetes, obesity) were included. Participants underwent brachial vein EC harvesting followed by RNA sequencing. To evaluate the association between cardiometabolic comorbidity burden and outcome transcripts we performed linear regression with multivariable models, adjusting for age, sex, and race/ethnicity. RESULTS:A total of 18 individuals were included in the present analysis (mean age 47 ± 14, 44% female, and 61% White adults). Endothelial cell RNA sequencing revealed 588 differentially expressed transcripts (p-adj <0.05) with excellent discrimination in unsupervised hierarchical clustering analysis. Gene ontology enrichment analysis revealed upregulated pathways associated with T-cell activation (NES = 2.22, p<0.001), leukocyte differentiation (NES= 2.16, p<0.001), leukocyte migration (NES= 2.12, p<0.001), regulation of cell-cell adhesion (NES= 1.91, p=0.006). Downregulated pathways of interest included endothelial cell proliferation (NES= -1.68, p=0.03) and response to interleukin-1 (NES= -1.61, p=0.04). Upregulated genes included VCAM1, CEACAM1, ADAM 17, and CD99L2, all with a log-2-fold change >3 and p-adj <0.05. These genes demonstrated a graded increase in mean normalized counts with increasing number of risk factors. CONCLUSIONS:We demonstrate a proinflammatory and pro-adhesive EC transcriptome associated with increased cardiometabolic risk factor burden offering insight into a potential mechanism linking these risk factors with the development of CVD.

Objective: HbA1c is an insensitive marker for assessing real-time dysglycemia in obesity. This study investigated whether 1-h plasma glucose level (1-h PG) ≥155 mg/dL (8.6 mmol/L) during an oral glucose tolerance test (OGTT) and continuous glucose monitoring (CGM) measurement of glucose variability (GV) better reflected dysglycemia than HbA1c after weight loss from metabolic and bariatric surgery. Methods: This was a prospective cohort study of 10 participants with type 2 diabetes compared with 11 participants with non-diabetes undergoing sleeve gastrectomy (SG). At each research visit; before SG, and 6 weeks and 6 months post-SG, body weight, fasting lipid levels, and PG and insulin concentrations during an OGTT were analyzed. Mean amplitude of glycemic excursions (MAGE), a CGM-derived GV index, was analyzed. Results: The 1-h PG correlated with insulin resistance markers, triglyceride/HDL ratio and triglyceride glucose index in both groups before surgery. At 6 months, SG caused 22% weight loss in both groups. Despite a reduction in HbA1c by 3.0 ± 1.3% in the diabetes group (p < 0.01), 1-h PG, and MAGE remained elevated, and the oral disposition index, which represents pancreatic β-cell function, remained reduced in the diabetes group when compared to the non-diabetes group. Conclusions: Elevation of GV markers and reduced disposition index following SG-induced weight loss in the diabetes group underscores persistent β-cell dysfunction and the potential residual risk of diabetes complications.

OBJECTIVE/UNASSIGNED:HbA1c is an insensitive marker for assessing real-time dysglycemia in obesity. This study investigated whether 1-h plasma glucose level (1-h PG) ≥155 mg/dL (8.6 mmol/L) during an oral glucose tolerance test (OGTT) and continuous glucose monitoring (CGM) measurement of glucose variability (GV) better reflected dysglycemia than HbA1c after weight loss from metabolic and bariatric surgery. METHODS/UNASSIGNED:This was a prospective cohort study of 10 participants with type 2 diabetes compared with 11 participants with non-diabetes undergoing sleeve gastrectomy (SG). At each research visit; before SG, and 6 weeks and 6 months post-SG, body weight, fasting lipid levels, and PG and insulin concentrations during an OGTT were analyzed. Mean amplitude of glycemic excursions (MAGE), a CGM-derived GV index, was analyzed. RESULTS/UNASSIGNED:-cell function, remained reduced in the diabetes group when compared to the non-diabetes group. CONCLUSIONS/UNASSIGNED:-cell dysfunction and the potential residual risk of diabetes complications.

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.

APOC3-Targeting RNAi for HypertriglyceridemiaThis randomized controlled trial examined the safety and side effects of the small interfering RNA ARO-APOC3 in healthy volunteers and patients with hypertriglyceridemia and chylomicronemia. ARO-APOC3 was associated with few adverse events and no dose-limiting toxicities.

Elevated triglycerides and non-high-density lipoprotein cholesterol (HDL-C) are risk factors for atherosclerotic cardiovascular disease (ASCVD). ARO-ANG3 is an RNA interference therapy that targets angiopoietin-like protein 3 (ANGPTL3), a regulator of lipoprotein metabolism. This first-in-human, phase 1, randomized, placebo-controlled, open-label trial investigated single and repeat ARO-ANG3 doses in four cohorts of fifty-two healthy participants and one cohort of nine participants with hepatic steatosis, part of a basket trial. Safety (primary objective) and pharmacokinetics (in healthy participants) and pharmacodynamics (secondary objectives) of ARO-ANG3 were evaluated. ARO-ANG3 was generally well tolerated, with similar frequencies of treatment-emergent adverse events in active and placebo groups. Systemic absorption of ARO-ANG3 in healthy participants was rapid and sustained, with a mean T_max of 6.0-10.5 h and clearance from plasma within 24-48 h after dosing with a mean t_½ of 3.9-6.6 h. In healthy participants, ARO-ANG3 treatment reduced ANGPTL3 (mean -45% to -78%) 85 days after dose. Reductions in triglyceride (median -34% to -54%) and non-HDL-C (mean -18% to -29%) (exploratory endpoints) concentrations occurred with the three highest doses. These early-phase data support ANGPTL3 as a potential therapeutic target for ASCVD treatment. ClinicalTrials.gov identifier: NCT03747224.

A fine balance between uptake, storage and the use of high energy fuels, like lipids, is crucial in the homeostasis of different metabolic tissues. Nowhere is this balance more important and more precarious than in the heart. This highly energy demanding muscle normally oxidizes almost all the available substrates to generate energy, with fatty acids being the preferred source under physiological conditions. In patients with cardiomyopathies and heart failure, changes in the main energetic substrate are observed; these hearts often prefer to utilize glucose rather than oxidizing fatty acids. An imbalance between uptake and oxidation of fatty acid can result in cellular lipid accumulation and cytotoxicity. In this review we will focus on the sources and uptake pathways used to direct fatty acids to cardiomyocytes. We will then discuss the intracellular machinery used to either store or oxidize these lipids and explain how disruptions in homeostasis can lead to mitochondrial dysfunction and heart failure. Moreover, we will also discuss the role of cholesterol accumulation in cardiomyocytes. Our discussion will attempt to weave in vitro experiments and in vivo data from mice and humans and use several human diseases to illustrate metabolism gone haywire as a cause of or accomplice to cardiac dysfunction.

Endothelial cell (EC) CD36 controls tissue fatty acid (FA) uptake. Here we examine how ECs transfer FAs. FA interaction with apical membrane CD36 induces Src phosphorylation of caveolin-1 tyrosine-14 (Cav-1Y14) and ceramide generation in caveolae. Ensuing fission of caveolae yields vesicles containing FAs, CD36 and ceramide that are secreted basolaterally as small (80-100 nm) exosome-like extracellular vesicles (sEVs). We visualize in transwells EC transfer of FAs in sEVs to underlying myotubes. In mice with EC-expression of the exosome marker emeraldGFP-CD63, muscle fibers accumulate circulating FAs in emGFP-labeled puncta. The FA-sEV pathway is mapped through its suppression by CD36 depletion, blocking actin-remodeling, Src inhibition, Cav-1Y14 mutation, and neutral sphingomyelinase 2 inhibition. Suppression of sEV formation in mice reduces muscle FA uptake, raises circulating FAs, which remain in blood vessels, and lowers glucose, mimicking prominent Cd36^-/- mice phenotypes. The findings show that FA uptake influences membrane ceramide, endocytosis, and EC communication with parenchymal cells.

Cholesterol efflux pathways could be exploited in tumor biology to unravel cancer vulnerabilities. A mouse model of lung-tumor-bearing KRAS^G12D mutation with specific disruption of cholesterol efflux pathways in epithelial progenitor cells promoted tumor growth. Defective cholesterol efflux in epithelial progenitor cells governed their transcriptional landscape to support their expansion and create a pro-tolerogenic tumor microenvironment (TME). Overexpression of the apolipoprotein A-I, to raise HDL levels, protected these mice from tumor development and dire pathologic consequences. Mechanistically, HDL blunted a positive feedback loop between growth factor signaling pathways and cholesterol efflux pathways that cancer cells hijack to expand. Cholesterol removal therapy with cyclodextrin reduced tumor burden in progressing tumor by suppressing the proliferation and expansion of epithelial progenitor cells of tumor origin. Local and systemic perturbations of cholesterol efflux pathways were confirmed in human lung adenocarcinoma (LUAD). Our results position cholesterol removal therapy as a putative metabolic target in lung cancer progenitor cells.