| 1. |
- Zegeye, Mulugeta M, 1986-, et al.
(författare)
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IL-6 trans-signaling regulates vascular endothelial laminin profile and inflammatory responses : Possible mechanism for immune cell recruitment during atherosclerosis?
- 2021
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Ingår i: Atherosclerosis. - : Elsevier. - 0021-9150 .- 1879-1484. ; 331, s. E61-E61
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Background and Aims: We aimed to investigate the role of IL-6 trans-signaling in regulating the release of endothelial inflammatory mediators and endothelial basement mebrane proteins, laminins. We also aimed to investigate the laminin composition in atherosclerotic plaques in relation to the immune cell content.Methods: HUVECs were cultured for in vitro experiments. The BiKE cohort was used to assess expression of laminins in atherosclerotic plaques and healthy vessels. Gene and protein expression was analysed using Microarray/qPCR and proximity extension assay, immunostaining or immunoblotting techniques respectively.Results: The release of 20 inflammatory proteins from ECs was significantly altered (3 downregulated, 17 upregulated) in response to stimulation with IL-6 and sIL-6R (p-value <0.05, FDR 5%). Ingenuity pathway analyses predicted that these proteins enhance lymphocyte binding and transmigration while inhibiting transmigration of granulocytes. ECs treated with combination of IL-6 and sIL-6R upregulated expression of LAMA5 while downregulating LAMA4. Transcriptomic and proteomic analyses showed that both endothelial LAMA4 and LAMA5 were significantly lower in atherosclerotic plaques compared to healthy vessels. In addition, expression of endothelial LAMA5 was significantly lower in plaques from symptomatic patients compared to those from asymptomatic patients. We also showed that endothelial laminins are negatively correlated with immune cell markers.Conclusions: Our findings suggest that IL-6 trans-signaling regulates endothelial inflammatory proteins and laminin production that enhance binding and trans-migration of lymphocytes. In the context of atherosclerosis, expression of laminin alpha chains is altered and also appeared to be associated with plaque stability. Our data also revealed a relationship between laminin chains and immune cell content in atherosclerotic plaques.
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| 2. |
- Kumawat, Ashok Kumar, 1982-, et al.
(författare)
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Inhibition of IL17A Using an Affibody Molecule Attenuates Inflammation in ApoE-Deficient Mice
- 2022
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Ingår i: Frontiers in Cardiovascular Medicine. - : Frontiers Media S.A.. - 2297-055X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- The balance between pro- and anti-inflammatory cytokines released by immune and non-immune cells plays a decisive role in the progression of atherosclerosis. Interleukin (IL)-17A has been shown to accelerate atherosclerosis. In this study, we investigated the effect on pro-inflammatory mediators and atherosclerosis development of an Affibody molecule that targets IL17A. Affibody molecule neutralizing IL17A, or sham were administered in vitro to human aortic smooth muscle cells (HAoSMCs) and murine NIH/3T3 fibroblasts and in vivo to atherosclerosis-prone, hyperlipidaemic ApoE(-/-) mice. Levels of mediators of inflammation and development of atherosclerosis were compared between treatments. Exposure of human smooth muscle cells and murine NIH/3T3 fibroblasts in vitro to alpha IL-17A Affibody molecule markedly reduced IL6 and CXCL1 release in supernatants compared with sham exposure. Treatment of ApoE(-/-) mice with alpha IL-17A Affibody molecule significantly reduced plasma protein levels of CXCL1, CCL2, CCL3, HGF, PDGFB, MAP2K6, QDPR, and splenocyte mRNA levels of Ccxl1, Il6, and Ccl20 compared with sham exposure. There was no significant difference in atherosclerosis burden between the groups. In conclusion, administration of alpha IL17A Affibody molecule reduced levels of pro-inflammatory mediators and attenuated inflammation in ApoE(-/-) mice.
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| 3. |
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| 4. |
- Lindkvist, Madelene, 1984-, et al.
(författare)
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Pleiotropic, Unique and Shared Responses Elicited by IL-6 Family Cytokines in Human Vascular Endothelial Cells
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 23:3
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Tidskriftsartikel (refereegranskat)abstract
- Vascular endothelial cells express glycoprotein 130 (gp130), which is utilized as a signaling receptor by cytokines in the interleukin-6 (IL-6) family. Several IL-6 family cytokines can be found in the circulatory system during physiological or pathological conditions, and may influence endothelial function and response. This study evaluated and compared the cellular and molecular responses induced by IL-6 family cytokines in human endothelial cells. A proteomic analysis showed that IL-6 family cytokines induce the release of a range of proteins from endothelial cells, such as C-C motif chemokine ligand 23, hepatocyte growth factor, and IL-6. Pathway analysis indicated that gp130-signaling in endothelial cells regulates several functions related to angiogenesis and immune cell recruitment. The present investigation also disclosed differences and similarities between different IL-6 family cytokines in their ability to induce protein release and regulate gene expression and intracellular signaling, in regards to which oncostatin M showed the most pronounced effect. Further, this study showed that soluble gp130 preferentially blocks trans-signaling-induced responses, but does not affect responses induced by classic signaling. In conclusion, IL-6 family cytokines induce both specific and overlapping molecular responses in endothelial cells, and regulate genes and proteins involved in angiogenesis and immune cell recruitment.
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| 5. |
- Ljungberg, Liza, 1980-, et al.
(författare)
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Global Transcriptional Profiling Reveals Novel Autocrine Functions of Interleukin 6 in Human Vascular Endothelial Cells
- 2020
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Ingår i: Mediators of Inflammation. - : Hindawi Publishing Corporation. - 0962-9351 .- 1466-1861. ; 2020
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Tidskriftsartikel (refereegranskat)abstract
- Background: Interleukin 6 (IL6) is a multifunctional cytokine produced by various cells, including vascular endothelial cells. IL6 has both pro- and non-/anti-inflammatory functions, and the response to IL6 is dependent on whether it acts via the membrane-bound IL6 receptor alpha (IL6R alpha) (classic signaling) or the soluble form of the receptor (transsignaling). As human endothelial cells produce IL6 and at the same time express IL6R alpha, we hypothesized that IL6 may have autocrine functions.Methods: Knockdown of IL6 in cultured human endothelial cells was performed using siRNA. Knockdown efficiency was evaluated using ELISA. RNA sequencing was employed to characterize the transcriptional consequence of IL6 knockdown, and Ingenuity Pathway Analysis was used to further explore the functional roles of IL6.Results: Knockdown of IL6 in cultured endothelial cells resulted in a 84-92% reduction in the release of IL6. Knockdown of IL6 resulted in dramatic changes in transcriptional pattern; knockdown of IL6 in the absence of soluble IL6R alpha (sIL6R alpha) led to differential regulation of 1915 genes, and knockdown of IL6 in the presence of sIL6R alpha led to differential regulation of 1967 genes (fold change 1.5, false discovery rate<0.05). Pathway analysis revealed that the autocrine functions of IL6 in human endothelial cells are mainly related to basal cellular functions such as regulation of cell cycle, signaling, and cellular movement. Furthermore, we found that knockdown of IL6 activates functions related to adhesion, binding, and interaction of endothelial cells, which seem to be mediated mainly via STAT3.Conclusion: In this study, a large number of novel genes that are under autocrine regulation by IL6 in human endothelial cells were identified. Overall, our data indicate that IL6 acts in an autocrine manner to regulate basal cellular functions, such as cell cycle regulation, signaling, and cellular movement, and suggests that the autocrine functions of IL6 in human endothelial cells are mediated via IL6 classic signaling.
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| 6. |
- Zegeye, Mulugeta M., 1986-, et al.
(författare)
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Activation of the JAK/STAT3 and PI3K/AKT pathways are crucial for IL-6 trans-signaling-mediated pro-inflammatory response in human vascular endothelial cells
- 2018
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Ingår i: Cell Communication and Signaling. - : BioMed Central (BMC). - 1478-811X. ; 16:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: IL-6 classic signaling is linked to anti-inflammatory functions while the trans-signaling is associated with pro-inflammatory responses. Classic signaling is induced via membrane-bound IL-6 receptor (IL-6R) whereas trans-signaling requires prior binding of IL-6 to the soluble IL-6R. In both cases, association with the signal transducing gp130 receptor is compulsory. However, differences in the downstream signaling mechanisms of IL-6 classic- versus trans-signaling remains largely elusive.METHODS: In this study, we used flow cytometry, quantitative PCR, ELISA and immuno-blotting techniques to investigate IL-6 classic and trans-signaling mechanisms in Human Umbilical Vein Endothelial Cells (HUVECs).RESULTS: We show that both IL-6R and gp130 are expressed on the surface of human vascular endothelial cells, and that the expression is affected by pro-inflammatory stimuli. In contrast to IL-6 classic signaling, IL-6 trans-signaling induces the release of the pro-inflammatory chemokine Monocyte Chemoattractant Protein-1 (MCP-1) from human vascular endothelial cells. In addition, we reveal that the classic signaling induces activation of the JAK/STAT3 pathway while trans-signaling also activates the PI3K/AKT and the MEK/ERK pathways. Furthermore, we demonstrate that MCP-1 induction by IL-6 trans-signaling requires simultaneous activation of the JAK/STAT3 and PI3K/AKT pathways.CONCLUSIONS: Collectively, our study reports molecular differences in IL-6 classic- and trans-signaling in human vascular endothelial cells; and elucidates the pathways which mediate MCP-1 induction by IL-6 trans-signaling.
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| 7. |
- Zegeye, Mulugeta M, 1986-, et al.
(författare)
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IL-6 as a Mediator of the Association Between Traditional Risk Factors and Future Myocardial Infarction : A Nested Case-Control Study
- 2021
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Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - : Lippincott Williams & Wilkins. - 1079-5642 .- 1524-4636. ; 41:4, s. 1570-1579
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Studies elucidating the importance of IL (interleukin)-6 trans-signaling associated with risk of future myocardial infarction (MI) are scarce. Additionally, whether elevation in IL-6 explains part of the association between traditional risk factors and future MI has not been explored.Approach and Results: We conducted a nested case-control study including a total of 584 participants (292 cases and 292 controls) from Västerbotten Intervention Programme and MONICA (Multinational Monitoring of Trends and Determinants in Cardiovascular Disease) cohorts. At baseline, plasma cholesterol levels were measured, and clinical characteristics of participants were collected. In this study, we measured the plasma concentration of IL-6, sIL-6R (soluble IL-6 receptor), and sgp130 (soluble-gp130). To estimate extent of IL-6 trans-signaling, we estimated plasma concentration of a novel biomarker, the IL-6 binary complex. IL-6 binary complex concentration was significantly elevated in participants who experienced MI compared with those who did not. Univariate analyses showed that a 2-fold increase in IL-6 binary complex was associated with 2.45× higher risk of future MI (95% CI relative risk, 1.65-3.66, P<0.001). Receiver operating characteristics analyses revealed that the predictive performance of IL-6 binary complex (area under the curve, 0.614) was equivalent to that of IL-6 (area under the curve, 0.603). Furthermore, using Process mediation analyses tool, we found statistically significant indirect effect of smoking and hypertension on future MI that is mediated through increased IL-6 binary complex or plasma IL-6.CONCLUSIONS: IL-6 and IL-6 binary complex concentration in plasma were significantly associated with future MI. Our data additionally imply that both the elevated plasma IL-6, and the IL-6 binary complex concentration could partly explain, and, thus, might hypothetically be functionally associated with the increased risk of MI in smokers and hypertensive participants.
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| 8. |
- Zegeye, Mulugeta M., 1986-, et al.
(författare)
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IL-6 trans-Signaling Impairs Sprouting Angiogenesis by Inhibiting Migration, Proliferation and Tube Formation of Human Endothelial Cells.
- 2020
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Ingår i: Cells. - : MDPI. - 2073-4409. ; 9:6
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Tidskriftsartikel (refereegranskat)abstract
- Sprouting angiogenesis is the formation of new capillaries from existing vessels in response to tissue hypoxia due to growth/development, repair/healing, and also chronic inflammation. In this study, we aimed to elucidate the effect of IL-6, a pleiotropic cytokine with both pro-inflammatory and anti-inflammatory functions, in regulating the sprouting angiogenic response of endothelial cells (ECs). We found that activation of IL-6 trans-signaling inhibited the migration, proliferation, and tube formation ability of ECs. In addition, inhibition of the autocrine IL-6 classic-signaling by depleting endogenous IL-6 from ECs impaired their tube formation ability. At the molecular level, we found that IL-6 trans-signaling in ECs upregulated established endogenous anti-angiogenic factors such as CXCL10 and SERPINF1 while at the same time downregulated known endogenous pro-angiogenic factors such as cKIT and CXCL8. Furthermore, prior activation of ECs by IL-6 trans-signaling alters their response to vascular endothelial growth factor-A (VEGF-A), causing an increased p38, but decreased Erk1/2 phosphorylation. Collectively, our data demonstrated the dual facets of IL-6 in regulating the sprouting angiogenic function of ECs. In addition, we shed light on molecular mechanisms behind the IL-6 trans-signaling mediated impairment of endothelial sprouting angiogenic response.
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| 9. |
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| 10. |
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| 11. |
- Zegeye, Mulugeta M, 1986-, et al.
(författare)
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Interleukin-6 trans-signaling induced laminin switch contributes to reduced trans-endothelial migration of granulocytic cells
- 2023
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Ingår i: Atherosclerosis. - : Elsevier. - 0021-9150 .- 1879-1484. ; 371, s. 41-53
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND AND AIMS: Laminins are essential components of the endothelial basement membrane, which predominantly contains LN421 and LN521 isoforms. Regulation of laminin expression under pathophysiological conditions is largely unknown. In this study, we aimed to investigate the role of IL-6 in regulating endothelial laminin profile and characterize the impact of altered laminin composition on the phenotype, inflammatory response, and function of endothelial cells (ECs).METHODS: HUVECs and HAECs were used for in vitro experiments. Trans-well migration experiments were performed using leukocytes isolated from peripheral blood of healthy donors. The BiKE cohort was used to assess expression of laminins in atherosclerotic plaques and healthy vessels. Gene and protein expression was analyzed using Microarray/qPCR and proximity extension assay, ELISA, immunostaining or immunoblotting techniques, respectively.RESULTS: Stimulation of ECs with IL-6+sIL-6R, but not IL-6 alone, reduces expression of laminin α4 (LAMA4) and increases laminin α5 (LAMA5) expression at the mRNA and protein levels. In addition, IL-6+sIL-6R stimulation of ECs differentially regulates the release of several proteins including CXCL8 and CXCL10, which collectively were predicted to inhibit granulocyte transmigration. Experimentally, we demonstrated that granulocyte migration is inhibited across ECs pre-treated with IL-6+sIL-6R. In addition, granulocyte migration across ECs cultured on LN521 was significantly lower compared to LN421. In human atherosclerotic plaques, expression of endothelial LAMA4 and LAMA5 is significantly lower compared to control vessels. Moreover, LAMA5-to-LAMA4 expression ratio was negatively correlated with granulocytic cell markers (CD177 and myeloperoxidase (MPO)) and positively correlated with T-lymphocyte marker CD3.CONCLUSIONS: We showed that expression of endothelial laminin alpha chains is regulated by IL-6 trans-signaling and contributes to inhibition of trans-endothelial migration of granulocytic cells. Further, expression of laminin alpha chains is altered in human atherosclerotic plaques and is related to intra-plaque abundance of leukocyte subpopulations.
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| 12. |
- Zegeye, Mulugeta M, 1986-, et al.
(författare)
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Soluble LDL-receptor is induced by TNF-α and inhibits hepatocytic clearance of LDL-cholesterol
- 2023
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Ingår i: Journal of Molecular Medicine. - : Springer. - 0946-2716 .- 1432-1440. ; 101:12, s. 1615-1626
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Tidskriftsartikel (refereegranskat)abstract
- Defective LDL-C clearance and hence its elevation in the circulation is an established risk factor for cardiovascular diseases (CVDs) such as myocardial infarction (MI). A soluble LDL-receptor (sLDL-R) has been detected in human plasma which correlates strongly with circulating LDL-C and classical conditions that promote chronic inflammation. However, the mechanistic interplay between sLDL-R, inflammation, and CVDs remains to be investigated. Here, we report that stimulation of HepG2 cells with TNF-α induces the release of sLDL-R into culture supernatants. In addition, TNF-α induces gene expression of peptidases ADAM-17 and MMP-14 in HepG2 cells, and inhibiting these peptidases using TMI 1 significantly reduces the TNF-α induced sLDL-R release. We found that a soluble form of recombinant LDL-R (100 nM) can strongly bind to LDL-C and form a stable complex (KD = E-12). Moreover, incubation of HepG2 cells with this recombinant LDL-R resulted in reduced LDL-C uptake in a dose-dependent manner. In a nested case-control study, we found that baseline sLDL-R in plasma is positively correlated with plasma total cholesterol level. Furthermore, a twofold increase in plasma sLDL-R was associated with a 55% increase in the risk of future MI [AOR = 1.55 (95% CI = 1.10-2.18)]. Nevertheless, mediation analyses revealed that a significant proportion of the association is mediated by elevation in plasma cholesterol level (indirect effect β = 0.21 (95% CI = 0.07-0.38). Collectively, our study shows that sLDL-R is induced by a pro-inflammatory cytokine TNF-α via membrane shedding. Furthermore, an increase in sLDL-R could inhibit hepatic clearance of LDL-C increasing its half-life in the circulation and contributing to the pathogenesis of MI. KEY MESSAGES: TNF-α causes shedding of hepatocytic LDL-R through induction of ADAM-17 and MMP-14. sLDL-R binds strongly to LDL-C and inhibits its uptake by hepatocytic cells. Plasma sLDL-R is positively correlated with TNF-α and cholesterol. Plasma sLDL-R is an independent predictor of myocardial infarction (MI). Plasma cholesterol mediates the association between sLDL-R and MI.
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