Atherosclerosis: The Role of Growth-Differentiation-Factor-15 (GDF-15) in human THP-1 Macrophages Autophagy and Lipid homeostasis

Cardiovascular diseases are the leading cause of death, representing more than 30% of all global deaths. Risk factors for developing atherosclerotic alterations are hypertension, diabetes, obesity, nicotine or alcohol abuse, which are widespread among industrial nations. Atherosclerotic alterations...

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Main Author: Ackermann, Kathrin
Contributors: Kinscherf, Ralf (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2020
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Summary:Cardiovascular diseases are the leading cause of death, representing more than 30% of all global deaths. Risk factors for developing atherosclerotic alterations are hypertension, diabetes, obesity, nicotine or alcohol abuse, which are widespread among industrial nations. Atherosclerotic alterations concern human arteries, where infiltration of modified lipids triggers an immune response, initiating the transmigration of monocytic cells and smooth muscle cells to remove this subendothelial accumulated lipids. In this context, autophagic processes were found highly active in atherosclerotic plaques, especially in lesional macrophages (MΦ). Supporting the MΦ reverse cholesterol transport, autophagy aids to resolve the subendothelial lipid accumulation, promoting plaque reduction and plaque stability. Furthermore, the cytokine Growth-Differentiation-Factor-15 (GDF-15) was found strongly expressed in lesional MΦ, where it is implicated in reverse cholesterol transport as well. These findings suggest a potential link between autophagy and GDF-15 in MΦ lipid homeostasis. This possible implication of GDF-15 in macrophages autophagy and lipid homeostasis was investigated in the present work. Therefore, we diminished or increased GDF-15 protein levels in human THP-1 MΦ, using small interfering RNAs and recombinant GDF-15 protein. Exposing THP-1 MΦ to oxLDL was used to induce foam cell formation. Subsequently, intracellular lipid accumulation and cholesterol levels, autophagy-relevant proteins, as well as autophagic and lysosomal activity were investigated. Aiming to investigate the basal autophagy in macrophages, we altered the GDF-15 protein level without exposing THP-1 MΦ to oxLDL. Diminishing the GDF-15 protein level in THP-1 MΦ led to decreased intracellular lipid accumulations, whereas cholesterol levels remained unaltered. Furthermore, the ATG5 and p62 protein levels decreased, as well as the ATG12 / ATG5-complex levels. Additionally, the p62 accumulation and the autophagy activity were diminished. Since ATG5 and ATG12 are mandatory for the autophagosome formation and p62 accumulation represents autophagic vesicles, these observations indicated to a diminished autophagosome formation, which explained the observed reduction in the autophagy activity. An increased GDF-15 protein level did not influence ATG5 and p62 protein levels or the ATG12 / ATG5-complex. However, it enhanced the p62 and Lamp-1 accumulations and increased the autophagy activity, whereas the lysosome activity remained unaltered. Thus, we concluded that GDF-15 possibly stabilizes the ATG5 protein level or facilitates the ATG12 / ATG5-complex assembly, leading to enhanced build-up in autophagic vesicles and thereby increasing the autophagy activity. To test, whether GDF-15 may operate further upstream in the autophagy signaling pathway, we investigated the Beclin-1 protein level and phosphorylation status of mTOR. Amplified GDF-15 protein levels did not influence Beclin-1 or phosphorylation status of mTOR, which confirmed our conclusion about the impact of GDF-15 on MΦ autophagy being linked to ATG5 protein and/or ATG12 / ATG5-complex. In exposing THP-1 MΦ to oxLDL, we induced foam cell formation. Upon oxLDL-exposure, THP-1 MΦ showed increased lipid accumulation, whereas the cholesterol levels remained unaltered. The p62 and Lamp-1 accumulation were increased, whereas the autophagic and lysosomal activities were diminished. These results were in line with already published data, where oxLDL was reported to induce lipid accumulation and to lead to lysosome expansion and to disturb lysosomal activity. Likewise, our observations of impaired autophagy activity and accumulation of autophagic vesicles have already been described. In proceeding to investigate GDF-15-depending autophagy in foam cells, we exposed THP-1 MΦ with altered GDF-15 protein levels to oxLDL. The combination of an enhanced GDF-15 protein level in THP-1 MΦ and exposure to oxLDL increased the ATG5 and p62 protein levels and the ATG12 / ATG5-complex. Furthermore, increased GDF-15 protein levels in foam cells amplified the p62 and Lamp-1 accumulation, compared to oxLDL-exposure alone. Whereas an enhanced or diminished GDF-15 protein level in combination with oxLDL-exposure had no additional influence on the autophagy and lysosome activity, which remained repressed just like after exposure to oxLDL alone. Conclusively, we found that increased GDF-15 protein levels aggravated the oxLDL-induced accumulation of autophagic vesicles, whereas vice versa decreased GDF-15 protein levels diminished the oxLDl-induced accumulation of autophagic vesicles. This work is the first one to reveal the crucial role of GDF-15 on MΦ autophagy: GDF-15 acts protective during basal autophagy, whereas during excess lipid stimuli, this GDF-15 influence on autophagosome formation aggravates the lipid-induced accumulation of autophagic vesicles. These observations identify GDF-15 as an important regulator in macrophage autophagy and lipid homeostasis, revealing its potential for new therapeutic strategies e.g. in atherosclerosis and cardiovascular diseases or stroke.
Physical Description:124 Pages