Endocrinol Metab.  2024 Jun;39(3):511-520. 10.3803/EnM.2023.1915.

Lipid Variability Induces Endothelial Dysfunction by Increasing Inflammation and Oxidative Stress

• Rhee M
• Lee J
• Lee EY
• Yoon KH
• Lee SH

• KMID: 2556643
• DOI: http://doi.org/10.3803/EnM.2023.1915

Abstract

Background
This study investigates the impact of fluctuating lipid levels on endothelial dysfunction.
Methods
Human aortic and umbilical vein endothelial cells were cultured under varying palmitic acid (PA) concentrations: 0, 50, and 100 μM, and in a variability group alternating between 0 and 100 μM PA every 8 hours for 48 hours. In the lipid variability group, cells were exposed to 100 μM PA during the final 8 hours before analysis. We assessed inflammation using real-time polymerase chain reaction, Western blot, and cytokine enzyme-linked immunosorbent assay (ELISA); reactive oxygen species (ROS) levels with dichlorofluorescin diacetate assay; mitochondrial function through oxygen consumption rates via XF24 flux analyzer; and endothelial cell functionality via wound healing and cell adhesion assays. Cell viability was evaluated using the MTT assay.
Results
Variable PA levels significantly upregulated inflammatory genes and adhesion molecules (Il6, Mcp1, Icam, Vcam, E-selectin, iNos) at both transcriptomic and protein levels in human endothelial cells. Oscillating lipid levels reduced basal respiration, adenosine triphosphate synthesis, and maximal respiration, indicating mitochondrial dysfunction. This lipid variability also elevated ROS levels, contributing to a chronic inflammatory state. Functionally, these changes impaired cell migration and increased monocyte adhesion, and induced endothelial apoptosis, evidenced by reduced cell viability, increased BAX, and decreased BCL2 expression.
Conclusion
Lipid variability induce endothelial dysfunction by elevating inflammation and oxidative stress, providing mechanistic insights into how lipid variability increases cardiovascular risk.

Keyword

Endothelial cells; Dyslipidemias; Palmitic acid; Cardiovascular diseases; Oxidative stress; Inflammation

Figure

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  Fig. 1. Lipid variability increases inflammation in human endothelial cells. Human umbilical vein endothelial cell (HUVEC) and human aortic endothelial cell (HAEC) were exposed to 10% bovine serum albumin as a vehicle control or different concentrations of palmitic acid (PA) for 48 hours: continuous 0, 50, and 100 μM, or alternating 0 and 100 μM every 8 hours (A). The expression of genes (B) and proteins (C, D) associated with inflammation and adhesion molecules were determined in HAEC after PA treatment. Interleukin 6 (IL6) and monocyte chemoattractant protein-1 (MCP1) concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in cell supernatants from HUVEC (E). The nuclear factor κB (NF-κB) subunits were determined in HAEC after PA treatment (F). Nuclear translocation of NF-κB p65 (red fluorescence) and 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei were detected by fluorescence staining in HAEC (G). Scale bar=100 μm. Data are presented as the mean±standard error of the mean of three to five independent experiments. Tnf-α, tumor necrosis factor-α; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; IκBα, IkappaB-alpha. aP<0.05 and bP<0.01 compared to untreated control; cP<0.01 compared to 50 μM; dP<0.01 compared to continuous 100 μM PA.

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  Fig. 2. Lipid variability increases oxidative stress in human endothelial cells. Human aortic endothelial cells (HAECs) were treated with palmitic acid (PA) for 48 hours. Reactive oxygen species production levels were visualized (A) using fluorescence microscopy and quantified (B) after 1 hour incubation with 10 μM 2’,7’-dichlorofluorescin diacetate (DCFH-DA) probe. Scale bar=100 μm. The expression level of mRNA related nitric oxide synthase were measured using real-time polymerase chain reaction in HAEC (C). Data are presented as the mean±standard error of the mean of five independent experiments. aP<0.01 compared to untreated control; bP<0.05 and cP<0.01 compared to 50 μM PA.

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  Fig. 3. Lipid variability leads to mitochondrial dysfunction in human endothelial cells. For real-time measurements of the cellular oxygen consumption rate (OCR), human aortic endothelial cell (HAEC) were plated at 400,000 cells/well in XF24 cell multi-well plate. Cells were incubated with different concentrations of palmitic acid (PA) for 48 hours. Protocol used in data collection and calculations for evaluating OCR values (A). Representative mitochondrial respiration after PA exposure (B). Quantitative analysis of basal respiration, adenosine triphosphate (ATP)-linked respiration, maximal respiration and proton leak (C). Data are presented as the mean±standard error of the mean of five independent experiments. FCCP, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone. aP<0.05 and bP<0.01 compared to untreated control.

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  Fig. 4. Lipid variability induces human endothelial dysfunction. Human aortic endothelial cells (HAECs) were exposed to various concentrations of palmitic acid (PA) for 48 hours. To analyze migration of endothelial cells treated with PA, wound healing assays were performed at indicated doses in HAEC. The dotted lines show the initial area without cells (A, B). Scale bar=200 μm. Data are presented as the mean± standard error of the mean of five independent experiments. HAEC were co-cultured with calcein-acetoxymethyl (AM)-labeled THP1 cells for 2 hours. THP1 monocytes that adhered to HAEC were visualized using a fluorescence microscope and counted (C, D). Scale bar=200 μm. Data are presented as the mean±standard error of the mean of three or five independent experiments. aP<0.01 compared to untreated control; bP<0.05 and cP<0.01 compared to 50 μM PA.

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  Fig. 5. Lipid variability induces apoptosis in human endothelial cells. Cell viability of human aortic endothelial cells (HAECs) after palmitic acid (PA) exposure was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (A). Protein expression levels of BCL-2-associated X protein (BAX) and B cell CLL/lymphoma 2 (BCL2) were estimated by immunoblotting (B). Data are presented as the mean±standard error of the mean of three or five independent experiments. aP<0.05 and bP<0.01 compared to untreated control; cP<0.05 compared to continuous 100 μM PA.

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