Eicosapentaenoic acid reduces pulmonary endothelial cell adhesion protein expression with IL-6

INTRODUCTION: Infectious agents, including SARSCoV- 2, cause pulmonary endothelial cell (EC) dysfunction that leads to acute respiratory distress syndrome (ARDS). EC dysfunction involves increased leukocyte recruitment and cell permeability mediated by various junctional proteins, integrins, and adhesion molecules. The omega-3 fatty acid eicosapentaenoic acid (EPA) and its metabolites modulate inflammation and vascular function. These actions of EPA may contribute to reduced cardiovascular events as reported in outcome trials such as REDUCE-IT. Currently, EPA is being tested in patients at risk for or with COVID-19. This study tested the effects of EPA on protein expression in human pulmonary ECs following challenge by the cytokine IL-6 to simulate conditions encountered in advanced viral infections. METHODS: Human lung microvascular endothelial cells (HMVEC-L) were post-treated with vehicle or EPA (40 μM) in 2% FBS after a 2 hr challenge with IL-6 at 12 ng/ml for 24 h. Proteomic analysis used LC/MS to assess relative expression levels of EC proteins. Only significant (p< 0.05) changes in protein expression between treatment groups >1-fold were analyzed. Specific pathway analysis was carried out using gene set enrichment analysis (GSEA). RESULTS: HMVEC-L treated with EPA following challenge with IL-6 showed significant changes in over 400 proteins compared with IL-6 treatment alone. EPA specifically diminished eleven proteins in the “integrin cell surface interactions” pathway. These pathways proteins included integrins alpha-V, alpha-6, and beta-1, along with PECAM-1, junction adhesion molecule C (JAM3), fibronectin, and ICAM- 2. CONCLUSIONS: EPA reduced expression of pulmonary endothelial adhesion and permeability proteins following IL-6 treatment. The ability of EPA to inhibit EC dysfunction and inflammation may have benefits for patients with or at risk for ARDS due to viruses such as SARS-CoV-2 or sepsis.

Eicosapentaenoic acid increases endothelial function and Heme oxygenase-1 in pulmonary inflammation

INTRODUCTION: Endothelial cell (EC) dysfunction results in reduced nitric oxide (NO) bioavailability leading to inflammation and increased susceptibility to infectious agents. Heme oxygenase-1 (HO-1) produces potent antioxidant and anti-inflammatory products including carbon monoxide. SARS-CoV-2 and influenza affect ECs in multiple vascular beds, including pulmonary tissue. The omega-3 fatty acid eicosapentaenoic acid (EPA) and its metabolites preserve EC function in a manner that may contribute to reduced incident cardiovascular events (REDUCE-IT). Currently, EPA is being tested in patients with or at risk for COVID-19. This study tested the effects of EPA on NO and peroxynitrite (ONOO-) release under conditions of inflammation using lipopolysaccharide (LPS) and the cytokine IL-6. We also measured expression of HO-1 after cell challenge with IL-6. METHODS: Human lung microvascular endothelial cells (HMVEC-L) were pretreated with vehicle or EPA (40 μM) in 2% FBS for 2 h, then challenged with either IL-6 (12 ng/ml) or LPS (200 ng/ml) for 24 h. Cells (including untreated controls) were stimulated with calcium ionophore to measure maximum production of NO and peroxynitrite (ONOO-) using tandem porphyrinic nanosensors. Proteomic analysis was performed using LC/MS to assess relative expression levels. Only significant (p< 0.05) changes in protein expression between treatment groups >1-fold were analyzed. RESULTS: HMVEC-L challenged with LPS and IL-6 showed a pronounced loss of NO release by 22% (p< 0.01) and 18% (p< 0.01), respectively, concomitant with an increase in ONOO- by 28% (p< 0.01) and 26% (p< 0.01), respectively. As a result, the [NO]/[ONOO-] ratio, a marker of eNOS coupling efficiency, decreased by 39% (p< 0.001) and 35% (p< 0.001) with LPS and IL-6, respectively. However, EPA increased this ratio by 39% (p< 0.01) in both LPS and IL-6 treated cells. EPA also caused a 5.7-fold (p = 4.4 × 10-38) increase in expression of HO-1 with IL-6. CONCLUSIONS: These findings indicate that EPA improves NO bioavailability and reduces nitroxidative stress in pulmonary ECs during inflammation with LPS or IL-6. These studies indicate a protective effect of EPA on pulmonary ECs that may reduce inflammatory activation during sepsis, influenza, or advanced COVID-19 that may mediate many aspects of multiorgan system failure.

Eicosapentaenoic acid favorably modulates protein expression in pulmonary endothelial inflammation

INTRODUCTION: SARS-CoV-2 and other viruses can cause endothelial cell (EC) dysfunction in multiple vascular beds, including pulmonary tissue. Infected patients may then develop acute respiratory distress syndrome (ARDS) and cardiovascular (CV) complications. The omega-3 fatty acid eicosapentaenoic acid (EPA) and its bioactive metabolites favorably modulate inflammation and EC function. These benefits of EPA may contribute to reduced CV events as reported in outcome trials (REDUCE-IT). Currently, EPA is being tested in patients with or at risk for COVID-19. This study tested the effects of either EPA pre- or post-treatment on global protein expression in human pulmonary ECs under conditions of inflammation using the cytokine IL-6 to simulate conditions of advanced viral infections. METHODS: Human lung microvascular endothelial cells (HMVEC-L) were pre-treated with either EPA (40 μM) or IL-6 (12 ng/mL) for 2 hr and then treated with IL-6 or EPA, respectively, for 24 hr in media with 2% FBS. Proteomic analysis was performed using LC/MS to assess relative protein expression levels. Only significant (p< 0.05) changes in protein expression between treatment groups >1-fold were analyzed. Expression of soluble intercellular adhesion molecule-1 (sICAM-1) was separately measured with immunochemistry. RESULTS: HMVEC-L pre- and post-treated with EPA during challenge with IL-6 showed significant changes in 100 (49/51 up/down) and 441 (229/212 up/down) proteins, respectively, compared with IL-6 treatment alone. Among the 31 proteins that were significantly modulated by both EPA pre- and post-treatment, thioredoxin reductase 1 increased relative to IL-6 alone, while matrix metalloproteinase 1 and fibronectin both decreased. Other proteins, such as hypoxia up-regulated protein 1, were differentially modulated by EPA relative to IL-6 (increased in pre-treatment, decreased in post-treatment). Finally, EPA significantly reduced sICAM- 1expression by 41% and 12% compared with IL-6 alone in the pre- and post-treatment models, respectively. CONCLUSIONS: These findings indicate that EPA favorably modulates the expression of multiple inflammatory and cytoprotective proteins during inflammation. These studies support a broad anti-inflammatory effect of EPA on pulmonary ECs that may have therapeutic implications for patients at risk for ARDS due to infectious agents including SARS-CoV-2 or other viruses.

Eicosapentaenoic acid reduces pulmonary endothelial caveolae protein and restores nitric oxide

INTRODUCTION: Loss of pulmonary endothelial cell (EC) nitric oxide (NO) bioavailability following infection by SARS-CoV-2 or influenza leads to increased viral uptake, thrombosis and enhanced inflammation. EC production of NO by its synthase (eNOS) is downregulated by proteins in membrane caveolae, including caveolae-associated protein 2. The omega-3 fatty acid eicosapentaenoic acid (EPA) and its bioactive lipid metabolites reduce inflammation and improve EC function in various tissues. These benefits of EPA may contribute to the pronounced cardiovascular event reduction reported in REDUCE-IT. As a result, EPA is now being tested in patients at risk for COVID-19. This study tested the effects of EPA on expression of caveolae-associated protein 2 and NO bioavailability in pulmonary ECs under conditions of inflammation caused by the cytokine interleukin-6 (IL-6). METHODS: Human lung microvascular endothelial cells (HMVEC-L) were pretreated with vehicle or EPA (40 μM) in 2% FBS for 2 h, then challenged with IL-6 at 12 ng/ml for 24 h. Cells (including untreated controls) were stimulated with calcium ionophore to measure maximum production of NO and peroxynitrite (ONOO-) using tandem porphyrinic nanosensors. Proteomic analysis was performed using LC/ MS to capture relative expression levels >1,000 proteins. Only significant (p<0.05) changes in protein expression between treatment groups >2-fold were further analyzed. RESULTS: HMVEC-L challenged with IL-6 showed a pronounced loss of NO bioavailability. EPA treatment increased NO release (17%, p<0.05) and decreased ONOO- release (16%, p<0.05) compared with IL-6 treatment. The [NO]/[ONOO-] ratio, a marker of eNOS coupling efficiency, decreased by 35% (p<0.001) following exposure to IL-6 but EPA treatment increased the ratio by 39% (p<0.001). Improved NO bioavailability correlated with >3-fold reduction in caveolae-associated protein 2 (p<0.05). CONCLUSIONS: EPA restored NO bioavailability and reduced expression of caveolae-associated protein 2 in pulmonary ECs following IL-6 treatment. The ability of EPA to inhibit endothelial inflammatory changes and restore NO bioavailability has therapeutic implications for patients at risk for SARS-CoV-2 infection and other inflammatory states.

Eicosapentaenoic acid reduces pulmonary endothelial inflammation caused by cytokine treatment

INTRODUCTION: Infectious agents like SARS-CoV-2 can cause endothelial cell (EC) dysfunction in multiple vascular beds from different organs in infected patients, including pulmonary tissue that leads to acute respiratory distress syndrome (ARDS). The omega-3 fatty acid eicosapentaenoic acid (EPA) has multifactorial effects that lead to reduced inflammation and improved EC function. These benefits of EPA may contribute to reduced cardiovascular events as reported in REDUCE-IT. EPA is currently being tested in patients at risk for COVID-19 in multiple trials. This study tested the effects of EPA on protein expression in human pulmonary ECs under conditions of inflammation using the cytokine IL-6 to simulate viral infection conditions. METHODS: Human lung microvascular endothelial cells (HMVEC-L) were pretreated with vehicle or EPA (40 μM) in 2% FBS for 2 h, then challenged with IL-6 at 12 ng/ml for 24 h. Proteomic analysis of cell lysates was performed using LC/MS to capture relative expression levels of over 1,000 proteins. Only significant changes in protein expression between treatment groups >2-fold were analyzed. Specific pathway analysis was carried out using gene set enrichment analysis (GSEA). Expression levels of intercellular adhesion molecule-1 (ICAM-1) were measured by Western blot analysis. RESULTS: HMVEC-L pretreated with EPA and then challenged with IL-6 showed reduced release of >60 proteins compared with untreated controls. Among the proteins significantly suppressed were those involved in inflammation, including protein LYRIC, integrin alpha-5, peroxiredoxin-1, macrophage migration inhibitory factor, and lysine-tRNA ligase. GSEA analysis revealed changes in several pathways related to transcription regulation. Exposure to IL-6 also caused a >5-fold increase in ICAM-1 expression compared with vehicle (p<0.001). EPA reduced ICAM-1 expression compared with control by 41% (p<0.05). CONCLUSIONS: These findings indicate that EPA reduces the expression of multiple inflammatory proteins triggered by cytokine treatment. These studies support a broad antiinflammatory effect of EPA on pulmonary ECs that may have therapeutic implications for patients at risk for influenza or SARS-CoV-2 or other pro-inflammatory stimuli.

Eicosapentaenoic acid inhibits ICAM-1 expression in pulmonary endothelial cells with inflammation

INTRODUCTION: Infectious agents like SARS-CoV-2 trigger inflammation in endothelial cells (EC) in multiple vascular beds in infected patients, including pulmonary tissue that leads to acute respiratory distress syndrome (ARDS). Due to its anti-inflammatory effects, the omega-3 fatty acid eicosapentaenoic acid (EPA) is being tested in patients at risk for COVID-19. Additionally, EPA has been shown to significantly reduce cardiovascular events in high risk patients with elevated triglycerides as reported in the REDUCE-IT trial. The objective, then, of this study was to test the ability of EPA to reduce inflammatory changes in pulmonary ECs. METHODS: Human lung microvascular endothelial cells (HMVEC-L) were pretreated with vehicle or EPA (40 μM) in 2% FBS for 2 h, then challenged with lipopolysaccharide (LPS) at 200 ng/mL for 24 h. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in pulmonary ECs was measured with immunochemistry using SDS-PAGE and compared with β- Actin as a control. RESULTS: HMVEC-L challenged with LPS dramatically increased ICAM-1 expression by 1135% (p<0.001) compared with vehicle treatment. Pulmonary ECs pretreated with EPA had reduced expression of ICAM-1 by 47% (p<0.001). LPS modestly increased VCAM-1 expression (45%, p<0.05) in a manner that was reduced by EPA (12%), but it was not significant. CONCLUSIONS: EPA significantly reduced the expression of ICAM-1 in human pulmonary ECs following LPS exposure. These studies indicate a protective effect of EPA on the pulmonary endothelium that may reduce inflammation associated with infectious agents such as SARS-CoV-2.

Eicosapentaenoic acid preserves pulmonary endothelial nitric oxide release following lps challenge

INTRODUCTION: Endothelial cell (EC) dysfunction results in reduced nitric oxide (NO) bioavailability leading to inflammation and thrombus formation. Infectious agents like SARS-CoV-2 and influenza can infect ECs in multiple vascular beds from different organs in infected patients, especially in the lung. The omega-3 fatty acid eicosapentaenoic acid (EPA) and its metabolites can preserve EC function and reduce inflammation. These effects of EPA likely contribute to reduced cardiovascular events as reported in REDUCE-IT. Currently, EPA is being tested in patients at risk for COVID-19. This study tested the effects of EPA on NO bioavailability in pulmonary ECs under conditions of inflammation induced by lipopolysaccharide (LPS). METHODS: Human lung microvascular endothelial cells (HMVEC-L) were pretreated with vehicle or EPA (40 μM) in 2% FBS for 2 h, then challenged with LPS at 200 ng/ml for 24 h. Cells (including untreated controls) were stimulated with calcium ionophore to measure maximum production of NO and peroxynitrite (ONOO-) using tandem porphyrinic nanosensors. RESULTS: HMVEC-L challenged with LPS showed a pronounced loss of NO bioavailability. Cells treated with EPA increased NO release (20%, p<0.05) and decreased ONOO- release (14%, p<0.01) compared with LPS treatment. The [NO]/[ONOO-] ratio, a marker of eNOS coupling efficiency, decreased by 39% (p<0.001) following exposure to LPS. However, EPA increased the ratio by 39% (p<0.01) compared with LPS alone. CONCLUSIONS: These findings indicate that EPA preserves NO bioavailability and reduces nitroxidative stress in pulmonary ECs following LPS treatment. These studies indicate a protective effect of EPA on pulmonary ECs that may reduce the inflammatory changes caused by infectious agents such as influenza or SARS-CoV-2 among other stimuli.