Foam cell-derived 4-hydroxynonenal induces endothelial cell senescence in a TXNIP-dependent manner

Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co-culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP-1 monocyte-derived foam cells, were analysed for the induction of senescence. Senescence associated β-galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4-hydroxnonenal (4-HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4-HNE in the co-culture medium blunted this effect. Furthermore, both foam cells and 4-HNE increased the expression of the pro-oxidant thioredoxin-interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell-induced senescence. Previous studies showed that peroxisome proliferator-activated receptor (PPAR)δ was activated by 4-hydroalkenals, such as 4-HNE. Pharmacological interventions supported the involvement of the 4-HNE-PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell-released 4-HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence

Cathepsin Activity-Based Probes and Inhibitor for Preclinical Atherosclerosis Imaging and Macrophage Depletion

<div><p>Background and Purpose</p><p>Cardiovascular disease is the leading cause of death worldwide, mainly due to an increasing prevalence of atherosclerosis characterized by inflammatory plaques. Plaques with high levels of macrophage infiltration are considered “vulnerable” while those that do not have significant inflammation are considered stable; cathepsin protease activity is highly elevated in macrophages of vulnerable plaques and contributes to plaque instability. Establishing novel tools for non-invasive molecular imaging of macrophages in plaques could aid in preclinical studies and evaluation of therapeutics. Furthermore, compounds that reduce the macrophage content within plaques should ultimately impact care for this disease.</p><p>Methods</p><p>We have applied quenched fluorescent cathepsin activity-based probes (ABPs) to a murine atherosclerosis model and evaluated their use for <i>in vivo</i> imaging using fluorescent molecular tomography (FMT), as well as <i>ex vivo</i> fluorescence imaging and fluorescent microscopy. Additionally, freshly dissected human carotid plaques were treated with our potent cathepsin inhibitor and macrophage apoptosis was evaluated by fluorescent microscopy.</p><p>Results</p><p>We demonstrate that our ABPs accurately detect murine atherosclerotic plaques non-invasively, identifying cathepsin activity within plaque macrophages. In addition, our cathepsin inhibitor selectively induced cell apoptosis of 55%±10% of the macrophage within excised human atherosclerotic plaques.</p><p>Conclusions</p><p>Cathepsin ABPs present a rapid diagnostic tool for macrophage detection in atherosclerotic plaque. Our inhibitor confirms cathepsin-targeting as a promising approach to treat atherosclerotic plaque inflammation.</p></div

Non-invasive imaging of plaques in murine atherosclerosis.

<p>Diabetic, fat-fed mice with a ligated carotid artery were injected with non-quenched probe GB123 or quenched probe GB137 as indicated. Fluorescent molecular tomography (FMT) was used to monitor and follow the pharmacokinetics and signal accumulation in plaques. <b>(a, b)</b> Left images: front overlay of fluorescence and bright field. Middle images: side view of fluorescence alone. These images show strong fluorescence signal (arrows) (GB123 at 4 hours and GB137 at 2 hours post probe injection) around the ligated left carotid artery. Right images show <i>ex vivo</i> fluorescent image of excised heart and carotid arteries (ligated artery is marked).</p

Cathepsin inhibitor induces specific macrophage apoptosis.

<p>Freshly excised human atherosclerotic tissue samples were treated with the cathepsin inhibitor GB111-NH₂ for 24 hours. Serial frozen sections were stained for CD68 and cleaved caspase-3 and visualized by a confocal microscope: DAPI (blue), cleaved caspase-3 (green), CD68 (red), yellow color is overlay of red and green fluorescence. GB111-NH₂ was found to induce specific macrophage cell death (a). Co-localization analysis of CD68 and cleaved Caspase 3 positive cells. Bar graphs present the fraction of apoptotic macrophages out of total CD68 population (b) and the fraction of macrophages out of total apoptotic cells is shown in (c). Data is mean ± SEM (n = 3).</p

Macrophage labeling with fluorescent activity based probe.

<p>Ligated and control carotid arteries from mice treated with GB123 (<b>a</b>) or GB137 (<b>b</b>) (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160522#pone.0160522.g001" target="_blank">Fig 1</a>) were embedded in OCT and serial sectioned. Samples were stained for F4/80, a macrophage marker, and scanned by a confocal microscope: DAPI (blue), Cy5 labeled by probe (red), F4/80 (green), yellow color is overlay of red and green fluorescence. Cathepsin probes were found to co-localize with F4/80 macrophages.</p

Global economic burden of unmet surgical need for appendicitis

Background There is a substantial gap in provision of adequate surgical care in many low- and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially