Sheila E. Francis

Immune decline in older adults is associated with increased susceptibility to infection and chronic inflammatory diseases. Macrophages are critical innate immune cells that show reduced capacity for phagocytosis and migration with age. Our previous work shows that reduced levels of MYC and USF1 transcription factors are drivers of macrophage age-related functional decline. Here we show that macrophage-specific Myc overexpression improves macrophage migration and, more importantly, is able to improve physical performance at older age in Drosophila, while lifespan remains unaffected. Treatment of human primary macrophages from older individuals with the geroprotective supplement fisetin reverses the decline in MYC expression and improves phagocytosis of pathogens and cell migration functions towards levels seen in younger individuals. Mechanistically, fisetin acts via MYC, by restoring expression levels of MYC targets in human macrophages that are altered with age. Finally, fisetin feeding in older mice improves motor activity and reduces frailty, as well as restoring primary macrophage function and Myc expression in vitro. These findings reveal that restoration of MYC in macrophage ageing is responsible, at least in part, for improvement in physical performance with age and identify this pathway as a rational target to reverse age-related immune decline.

Rupture of unstable atherosclerotic plaques is a major cause of mortality. Endothelial-to-mesenchymal transition associates with advanced atherosclerotic plaques and contributes to plaque progression. We examined the role of Twist1 , a transcription factor that drives endothelial-to-mesenchymal transition, in plaque progression by inducible deletion from endothelial cells in hypercholesterolemic mice ( Twist1 ^ECKO Apo ^-/- ). Single-cell RNA sequencing coupled to endothelial cell-tracking reveals that Twist1 promotes endothelial-to-mesenchymal transition in advanced atherosclerotic plaques. Histological analyses demonstrate that endothelial Twist1 promotes plaque growth and hallmarks of plaque stability (collagen, ACTA2-positive cells) and reduces features of instability (necrosis, macrophage accumulation). Analysis of cultured human aortic endothelial cells shows that TWIST1 contributes to endothelial-to-mesenchymal transition by promoting migration and proliferation through the transcriptional coactivator PELP1. Additionally, TWIST1 promotes endothelial cell proliferation via AEBP1-dependent upregulation of COL4A1. These findings challenge the prevailing view that endothelial-to-mesenchymal transition uniquely destabilizes plaques, by suggesting that TWIST1-driven endothelial-to-mesenchymal transition can promote plaque stability, offering new insights into atherosclerosis pathophysiology and therapeutic potential.

Atherosclerosis progresses from fatty streaks to complex plaques, with rupture leading to life-threatening complications. Endothelial-to-mesenchymal transition (EndMT) is associated with advanced atherosclerotic plaques, but its role in plaque progression remains unclear. To investigate this, we analyzed the role of TWIST1, a key EndMT-driving transcription factor, in plaque development. Using single-cell RNA sequencing of atherosclerotic plaques from hypercholesterolemic mice with inducible deletion of Twist1 from endothelial cells ( Twist1 ^ECKO ApoE ^-/- ) we demonstrate that Twist1 regulates endothelial cell heterogeneity by promoting cell states expressing EndMT markers. Cell tracking confirmed that Twist1 induces EndMT in advanced plaques. Mechanistically, we found that TWIST1 contributes to EndMT by promoting endothelial migration and proliferation through the transcriptional coactivator PELP1. Additionally, TWIST1 induces AEBP1 transcription, which upregulates COL4A1 to drive endothelial proliferation. Analyses of murine brachiocephalic plaques show that endothelial Twist1 promotes plaque growth, collagen deposition, and ACTA2-positive cell accumulation, hallmarks of plaque stability, while reducing necrosis and macrophage infiltration, features of plaque instability. Moreover, TWIST1 expression was associated with asymptomatic human carotid atherosclerosis and predicts favourable clinical outcomes. These findings challenge the prevailing view that EndMT destabilizes plaques, by suggesting that TWIST1-driven EndMT can promote plaque stability, offering new insights into atherosclerosis pathophysiology and therapeutic potential.

SignificanceAlzheimer’s disease does not occur in isolation, and there are many comorbidities associated with the disease, especially diseases of the vasculature. Atherosclerosis is a known risk factor for the subsequent development of Alzheimer’s disease; therefore, understanding how both diseases interact will provide a greater understanding of co-morbid disease progression and aid the development of potential new treatments.AimWe characterize hemodynamic responses and cognitive performance in APP/PS1 Alzheimer’s mice, atherosclerosis mice, and a mixed disease group (APP/PS1 and atherosclerosis) between the ages of 9 and 12 months.ApproachWhisker-evoked hemodynamic responses and recognition memory were assessed in awake mice, immunohistochemistry to assess amyloid pathology, and histology to characterize atherosclerotic plaque load.ResultsWe observed hemodynamic deficits in atherosclerosis mice (versus Alzheimer’s, mixed disease, or wild-type mice), with reduced short-duration stimulus-evoked hemodynamic responses occurring when there was no concurrent locomotion during the stimulation period. Mixed Alzheimer’s and atherosclerosis models did not show differences in amyloid beta coverage in the cortex or hippocampus or atherosclerotic plaque burden in the aortic arch vs relevant Alzheimer’s or atherosclerosis controls. Consistent with the subtle vascular deficits and no pathology differences, we also observed no difference in performance on the object recognition task across groups.ConclusionsThese results emphasize the importance of experimental design for characterizing vascular function across disease groups, as locomotion and stimulus duration impacted the ability to detect differences between groups. Although atherosclerosis did reduce hemodynamic responses, these were recovered in the presence of co-occurring Alzheimer’s disease, which may provide targets for future studies to explore the potentially contrasting vasodilatory mechanisms these diseases impact.

Significance: Behavior regulates dural and cerebral vessels, with spontaneous locomotion inducing dural vessel constriction and increasing stimulus-evoked cerebral hemodynamic responses. It is vital to investigate the function of different vascular network components, surrounding and within the brain, to better understand the role of the neurovascular unit in health and neurodegeneration. Aim: We characterized locomotion-induced hemodynamic responses across vascular compartments of the whisker barrel cortex: artery, vein, parenchyma, draining and meningeal vein. Approach: Using 2D-OIS, hemodynamic responses during locomotion were recorded in 9–12-month-old awake mice: wild-type, Alzheimer’s disease (AD), atherosclerosis or mixed (atherosclerosis/AD) models. Within somatosensory cortex, responses were taken from pial vessels inside the whisker barrel region ([WBR]: “whisker artery” and “whisker vein”), a large vein from the sagittal sinus adjacent to the WBR (draining vein), and meningeal vessels from the dura mater (which do not penetrate cortical tissue). Results: We demonstrate that locomotion evokes an initial decrease in total hemoglobin (HbT) within the draining vein before the increase in HbT within WBR vessels. The locomotion event size influences the magnitude of the HbT increase in the pial vessels of the WBR, but not of the early HbT decrease within the draining veins. Following locomotion onset, an early HbT decrease was also observed in the overlying meningeal vessels, which unlike within the cortex did not go on to exceed baseline HbT levels during the remainder of the locomotion response. We show that locomotion-induced hemodynamic responses are altered in disease in the draining vein and whisker artery, suggesting this could be an important neurodegeneration biomarker. Conclusion: This initial reduction in HbT within the draining and meningeal veins potentially serves as a ‘space saving’ mechanism, allowing for large increases in cortical HbT associated with locomotion. Given this mechanism is impacted by disease it may provide an important target for vascular-based therapeutic interventions.

Significance Alzheimer’s disease does not occur in isolation and there are many comorbidities associated with the disease – especially diseases of the vasculature. Atherosclerosis is a known risk factor for the subsequent development of Alzheimer’s disease, therefore understanding how both diseases interact will provide a greater understanding of co-morbid disease progression and aid the development of potential new treatments. Aim The current study characterizes hemodynamic responses and cognitive performance in APP/PS1 Alzheimer’s mice, atherosclerosis mice, and a mixed disease group (APP/PS1 & atherosclerosis) between the ages of 9 and 12 months. Approach Whisker-evoked hemodynamic responses and recognition memory were assessed in awake mice, immunohistochemistry to assess amyloid pathology, and histology to characterize atherosclerotic plaque load. Results We observed hemodynamic deficits in atherosclerosis mice (vs Alzheimer’s, mixed disease or wild-type mice), with reduced short-duration stimulus-evoked hemodynamic responses occurring when there was no concurrent locomotion during the stimulation period. Mixed Alzheimer’s and atherosclerosis models did not show differences in amyloid beta coverage in the cortex or hippocampus or atherosclerotic plaque burden in the aortic arch vs relevant Alzheimer’s or atherosclerosis controls. Consistent with the subtle vascular deficits and no pathology differences, we also observed no difference in performance on the novel object recognition task across groups. Conclusions These results emphasize the importance of experimental design for characterizing vascular function across disease groups, as locomotion and stimulus duration impacted the ability to detect differences between groups. Whilst atherosclerosis did reduce hemodynamic responses, these were recovered in the presence of co-occurring Alzheimer’s disease which may provide targets for future studies to explore the potentially contrasting vasodilatory mechanisms these diseases impact.

Background: Inflammation is a precursor to atherosclerotic plaque destabilisation, leading to ischaemic events such as stroke. Macrophage phenotypes can be altered by the microenvironment, and certain anti-inflammatory agents may, therefore, stabilise plaques and reduce the risk of recurrent ischaemic events. Methods: Thirteen carotid plaques were obtained from stroke/ Transient Ischaemic Attack (TIA) patients undergoing carotid endarterectomy. An immunofluorescence stain was used to identify common macrophage markers (pan macrophage: CD68, pro-inflammatory: CD86, anti-inflammatory: MRC1), and a novel analysis technique was used to measure the prevalence of macrophage phenotypes in carotid plaques in relation to other histological features of instability. An in vitro model of human blood-derived macrophages was also developed to evaluate the effect of statins and glucocorticoids on macrophage-specific markers using RT-qPCR, Western Blot and immunofluorescence stain. The physiological effect of dexamethasone was further evaluated on macrophages and human carotid plaques cultured ex vivo . Results: The macrophage population (CD68+) in the carotid plaques was dominated by “double-positive” (CD86+MRC1+) macrophages (67.8%), followed by “M1-like” (CD86+MRC1-) (16.5%), “M2-like” (CD86-MRC1+) (8.7%) and “double-negative” (CD86-MRC1-) (7.0%) macrophages. M1-like macrophages were more prevalent in unstable plaque sections than stable ones (p=0.0022). Exposure to dexamethasone increased macrophage MRC1 gene expression in vitro and ex vivo . Dexamethasone also reduced Oxidised Low-Density Lipoprotein Receptor 1 ( OLR1 ) gene and protein expression, leading to a decreased ox-LDL uptake in foam cell assays. This was, in turn, associated with reduced lipid uptake in macrophages, as shown by Oil Red O staining. Conclusions: Human macrophages may be “switched” to a less inflammatory phenotype by exposure to clinically relevant concentrations of glucocorticoid, potentially mediated by a reduction in Oxidised LDL uptake. This effect was not observed following macrophage exposure to statins. Glucocorticoids may have a future role in preventing ischaemic events in patients with advanced atherosclerosis. Graphical Highlights: A high prevalence (68% in this study) of carotid plaque macrophages express both pro-inflammatory (CD86) and anti-inflammatory (MRC1) markers. These may represent a novel macrophage population. Human macrophages may be “reprogrammed” to a less inflammatory phenotype following exposure to glucocorticoids. Dexamethasone increased MRC1 and decreased OLR1 expression in macrophages derived from human blood samples in vitro and in cells derived from cultured human carotid plaque tissue ex vivo. This was associated with reduced oxLDL uptake and reduced lipid accumulation in the macrophages. Dexamethasone has the potential to stabilise carotid atherosclerotic plaques in humans.

Cardiovascular disease remains the leading cause of death worldwide, characterized by atherosclerotic activity within large and medium‐sized arteries. Inflammation has been shown to be a primary driver of atherosclerotic plaque formation, with interleukin‐1 (IL‐1) having a principal role. This review focuses on the current state of knowledge of molecular mechanisms of IL‐1 release from cells in atherosclerotic plaques. A more in‐depth understanding of the process of IL‐1's release into the vascular environment is necessary for the treatment of inflammatory disease processes, as the current selection of medicines being used primarily target IL‐1 after it has been released. IL‐1 is secreted by several heterogenous mechanisms, some of which are cell type–specific and could provide further specialized targets for therapeutic intervention. A major unmet challenge is to understand the mechanism before and leading to IL‐1 release, especially by cells in atherosclerotic plaques, including endothelial cells, vascular smooth muscle cells, and macrophages. Data so far indicate a heterogeneity of IL‐1 release mechanisms that vary according to cell type and are stimulus‐dependent. Unraveling this complexity may reveal new targets to block excess vascular inflammation.

Despite an appetite for change, equality, diversity and inclusivity (EDI)-related issues continue to ripple through the world of research and academia, from inequity at the point of entry into education, through to lack of diversity and equality in senior roles. Many academic institutes and governments are taking action to solve these issues, and we welcome the growing number of inclusive practices in the science communication arena. Building from this, we – at the University of Sheffield, UK – have assessed our own situation, responded to pressures applied by research councils, and listened to our staff and student voice. Our new ‘One University’ initiative puts EDI on a par with research, innovation and education as a core university priority, and our Gender, Disability and Race Action Plans allow us to make measurable and impactful changes. Tackling EDI issues needs a collaborative approach, action at an institutional- or sector-wide level and clear commitment from senior leaders.

Introduction: Despite epidemiological associations between community acquired pneumonia (CAP) and myocardial infarction, mechanisms that modify cardiovascular disease during CAP are not well defined. In particular, largely due to a lack of relevant experimental models, the effect of pneumonia on atherosclerotic plaques is unclear. We describe the development of a murine model of the commonest cause of CAP, Streptococcus pneumoniae pneumonia, on a background of established atherosclerosis. We go on to use our model to investigate the effects of pneumococcal pneumonia on atherosclerosis.Methods: C57BL/6J and ApoE^-/- mice were fed a high fat diet to promote atherosclerotic plaque formation. Mice were then infected with a range of S. pneumoniae serotypes (1, 4 or 14) with the aim of establishing a model to study atherosclerotic plaque evolution after pneumonia and bacteremia. Laser capture microdissection of plaque macrophages enabled transcriptomic analysis.Results: Intratracheal instillation of S. pneumoniae in mice fed a cholate containing diet resulted in low survival rates following infection, suggestive of increased susceptibility to severe infection. Optimization steps resulted in a final model of male ApoE^-/- mice fed a Western diet then infected by intranasal instillation of serotype 4 (TIGR4) S. pneumoniae followed by antibiotic administration. This protocol resulted in high rates of bacteremia (88.9%) and survival (88.5%). Pneumonia resulted in increased aortic sinus plaque macrophage content 2 weeks post pneumonia but not at 8 weeks, and no difference in plaque burden or other plaque vulnerability markers were found at either time point. Microarray and qPCR analysis of plaque macrophages identified downregulation of two E3 ubiquitin ligases, Huwe1 and Itch, following pneumonia. Treatment with atorvastatin failed to alter plaque macrophage content or other plaque features.Discussion: Without antibiotics, ApoE^-/- mice fed a high fat diet were highly susceptible to mortality following S. pneumoniae infection. The major infection associated change in plaque morphology was an early increase in plaque macrophages. Our results also hint at a role for the ubiquitin proteasome system in the response to pneumococcal infection in the plaque microenvironment.