AbstractMacrophages within atherosclerotic plaques are heterogeneous, with macrophage-colony stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrated to generate two different macrophage subsets (termed M-Mac and GM-Mac respectively). M-Macs display an elongated shape and are considered anti-inflammatory, due to their proposed roles in immune response suppression and induction of tissue repair. However, GM-Macs exhibit a rounded morphology and are regarded as pro-inflammatory. The actin cytoskeleton modulates cell shape and regulates cellular functions, which in macrophages includes the engulfment of apoptotic cells (efferocytosis), and phagocytosis of modified lipoproteins and subsequent foam cell formation, a hallmark of advanced atherosclerosis.
The key findings within this thesis demonstrate that the actin-perturbing drugs fasudil and pravastatin reduced the F-actin content of human M-Macs and GM-Macs, which was associated with shifting macrophages towards an anti-inflammatory phenotype (increased TGFBI and decreased MMP-12), suggesting actin cytoskeleton remodelling regulates macrophage polarisation. In addition, fasudil or pravastatin decreased oxLDL-induced foam cell formation in human GM-Macs, which was associated with decreased protein expression of the scavenger receptors OLR1 and CD36, alongside increased levels of the cholesterol efflux molecules NCOR1, NCOR2, and PPARα suggesting actin cytoskeleton remodelling regulates foam cell formation. Relatedly, human GM-Macs displayed impaired efferocytosis capacity compared to human M-Macs, which could be restored to M-Mac levels through the addition of fasudil or pravastatin. However, discrepancies were observed between mRNA and protein levels of some key molecules, suggesting the involvement of a post-transcriptional regulatory mechanism, such as SUMOylation. Analysis of advanced human atherosclerotic plaques revealed increased pSTAT5 expression and decreased SUMO protein levels within foam cell macrophage-rich regions, suggesting reduced STAT5 SUMOylation in advanced plaques. Confirmatory in vitro studies showed that blocking STAT5 SUMOylation augmented foam cell formation and efferocytosis capacity in human GM-Mac foam cells while reducing their F-actin content. Preliminary mechanistic studies suggest that sustained STAT5 activation (afforded by blocking its SUMOylation) within human GM-Macs upregulated OLR1 expression and its accumulation at the cell membrane, permitting enhanced efferocytosis effects dependent on actin cytoskeleton remodelling.
Taken together, our results imply an essential role for the actin cytoskeleton in regulating the inflammatory phenotype of macrophages, including the novel regulation by STAT5 SUMOylation, suggesting that targeting actin cytoskeleton remodelling in GM-Macs, may have therapeutic potential for reducing plaque progression and improving stability.
|Date of Award||11 May 2021|
|Supervisor||Jason L Johnson (Supervisor) & Sarah J George (Supervisor)|