Colchicine exerts anti-atherosclerotic and -plaque-stabilizing effects targeting foam cell formation.

Colchicine is a broad-acting anti-inflammatory agent that has attracted interest for repurposing in atherosclerotic cardiovascular disease. Here, we studied its ability at a human equivalent dose of 0.5 mg/day to modify plaque formation and composition in murine atherosclerosis and investigated its actions on macrophage responses to atherogenic stimuli in vitro. In atherosclerosis induced by high-cholesterol diet, Apoe-/- mice treated with colchicine had 50% reduction in aortic oil Red O+ plaque area compared to saline control (p = .001) and lower oil Red O+ staining of aortic sinus lesions (p = .03). In vitro, addition of 10 nM colchicine inhibited foam cell formation from murine and human macrophages after treatment with oxidized LDL (ox-LDL). Mechanistically, colchicine downregulated glycosylation and surface expression of the ox-LDL uptake receptor, CD36, and reduced CD36+ staining in aortic sinus plaques. It also decreased macrophage uptake of cholesterol crystals, resulting in lower intracellular lysosomal activity, inhibition of the NLRP3 inflammasome, and reduced secretion of IL-1ß and IL-18. Colchicine's anti-atherosclerotic actions were accentuated in a mouse model of unstable plaque induced by carotid artery tandem stenosis surgery, where it decreased lesion size by 48% (p = .01), reduced lipid (p = .006) and necrotic core area (p = .007), increased collagen content and cap-to-necrotic core ratio (p = .05), and attenuated plaque neutrophil extracellular traps (p < .001). At low dose, colchicine's effects were not accompanied by the evidence of microtubule depolymerization. Together, these results show that colchicine exerts anti-atherosclerotic and plaque-stabilizing effects at low dose by inhibiting foam cell formation and cholesterol crystal-induced inflammation. This provides a new framework to support its repurposing for atherosclerotic cardiovascular disease.

Eukaryotic elongation factor 2 kinase regulates foam cell formation via translation of CD36.

Eukaryotic elongation factor 2 kinase (eEF2K) is an atypical protein kinase that controls protein synthesis in cells under stress. Although well studied in cancer, less is known about its roles in chronic inflammatory diseases. Here, we examined its regulation of macrophage cholesterol handling in the context of atherosclerosis. eEF2K mRNA expression and protein activity were upregulated in murine bone marrow-derived macrophages (BMDMs) exposed to oxidized low-density lipoprotein cholesterol (oxLDL). When incubated with oxLDL, BMDMs from eEF2K knockout (Eef2k-/- ) mice formed fewer Oil Red O+ foam cells than Eef2k+/+ BMDMs (12.5% ± 2.3% vs. 32.3% ± 2.0%, p < .01). Treatment with a selective eEF2K inhibitor, JAN-384, also decreased foam cell formation for C57BL/6J BMDMs and human monocyte-derived macrophages. Disabling eEF2K selectively decreased protein expression of the CD36 cholesterol uptake receptor, mediated by a reduction in the proportion of translationally active Cd36 mRNA. Eef2k-/- mice bred onto the Ldlr-/- background developed aortic sinus atherosclerotic plaques that were 30% smaller than Eef2k+/+ -Ldlr-/- mice after 16 weeks of high cholesterol diet (p < .05). Although accompanied by a reduction in plaque CD36+ staining (p < .05) and lower CD36 expression in circulating monocytes (p < .01), this was not associated with reduced lipid content in plaques as measured by oil red O staining. Finally, EEF2K and CD36 mRNA levels were higher in blood mononuclear cells from patients with coronary artery disease and recent myocardial infarction compared to healthy controls without coronary artery disease. These results reveal a new role for eEF2K in translationally regulating CD36 expression and foam cell formation in macrophages. Further studies are required to explore therapeutic targeting of eEF2K in atherosclerosis.

Non-invasive genetic sexing technique for analysis of short-beaked echidna (Tachyglossus aculeatus) populations.

Identifying male and female echidnas is challenging due to the lack of external genitalia or any other differing morphological features. This limits studies of wild populations and is a major problem for echidna captive management and breeding. Non-invasive genetic approaches to determine sex minimise the need for handling animals and are used extensively in other mammals. However, currently available approaches cannot be applied to monotremes because their sex chromosomes share no homology with sex chromosomes in other mammals. In this study we used recently identified X and Y chromosome-specific sequences to establish a non-invasive polymerase chain reaction-based technique to determine the sex of echidnas. Genomic DNA was extracted from echidna hair follicles followed by amplification of two Y chromosome (male-specific) genes (mediator complex subunit 26 Y-gametolog (CRSPY) and anti-Müllerian hormone Y-gametolog (AMHY)) and the X chromosome gene (anti-Müllerian hormone X-gametolog (AMHX)). Using this technique, we identified the sex of 10 juvenile echidnas born at Perth Zoo, revealing that eight of the 10 echidnas were female. Future use of the genetic sexing technique in echidnas will inform captive management, continue breeding success and can be used to investigate sex ratios and population dynamics in wild populations.

Vasculogenic properties of adventitial Sca-1+CD45+ progenitor cells in mice: a potential source of vasa vasorum in atherosclerosis.

The cellular origins of vasa vasorum are ill-defined and may involve circulating or local progenitor cells. We previously discovered that murine aortic adventitia contains Sca-1+CD45+ progenitors that produce macrophages. Here we investigated whether they are also vasculogenic. In aortas of C57BL/6 mice, Sca-1+CD45+ cells were localised to adventitia and lacked surface expression of endothelial markers (<1% for CD31, CD144, TIE-2). In contrast, they did show expression of CD31, CD144, TIE-2 and VEGFR2 in atherosclerotic ApoE-/- aortas. Although Sca-1+CD45+ cells from C57BL/6 aorta did not express CD31, they formed CD31+ colonies in endothelial differentiation media and produced interconnecting vascular-like cords in Matrigel that contained both endothelial cells and a small population of macrophages, which were located at branch points. Transfer of aortic Sca-1+CD45+ cells generated endothelial cells and neovessels de novo in a hindlimb model of ischaemia and resulted in a 50% increase in perfusion compared to cell-free control. Similarly, their injection into the carotid adventitia of ApoE-/- mice produced donor-derived adventitial and peri-adventitial microvessels after atherogenic diet, suggestive of newly formed vasa vasorum. These findings show that beyond its content of macrophage progenitors, adventitial Sca-1+CD45+ cells are also vasculogenic and may be a source of vasa vasorum during atherogenesis.

Cellular Therapy for Heart Failure.

The pathogenesis of cardiomyopathy and heart failure (HF) is underpinned by complex changes at subcellular, cellular and extracellular levels in the ventricular myocardium. For all of the gains that conventional treatments for HF have brought to mortality and morbidity, they do not adequately address the loss of cardiomyocyte numbers in the remodeling ventricle. Originally conceived to address this problem, cellular transplantation for HF has already gone through several stages of evolution over the past two decades. Various cell types and delivery routes have been implemented to positive effect in preclinical models of ischemic and nonischemic cardiomyopathy, with pleiotropic benefits observed in terms of myocardial remodeling, systolic and diastolic performance, perfusion, fibrosis, inflammation, metabolism and electrophysiology. To a large extent, these salubrious effects are now attributed to the indirect, paracrine capacity of transplanted stem cells to facilitate endogenous cardiac repair processes. Promising results have also followed in early phase human studies, although these have been relatively modest and somewhat inconsistent. This review details the preclinical and clinical evidence currently available regarding the use of pluripotent stem cells and adult-derived progenitor cells for cardiomyopathy and HF. It outlines the important lessons that have been learned to this point in time, and balances the promise of this exciting field against the key challenges and questions that still need to be addressed at all levels of research, to ensure that cell therapy realizes its full potential by adding to the armamentarium of HF management.

Origins and functional evolution of Y chromosomes across mammals.

Y chromosomes underlie sex determination in mammals, but their repeat-rich nature has hampered sequencing and associated evolutionary studies. Here we trace Y evolution across 15 representative mammals on the basis of high-throughput genome and transcriptome sequencing. We uncover three independent sex chromosome originations in mammals and birds (the outgroup). The original placental and marsupial (therian) Y, containing the sex-determining gene SRY, emerged in the therian ancestor approximately 180 million years ago, in parallel with the first of five monotreme Y chromosomes, carrying the probable sex-determining gene AMH. The avian W chromosome arose approximately 140 million years ago in the bird ancestor. The small Y/W gene repertoires, enriched in regulatory functions, were rapidly defined following stratification (recombination arrest) and erosion events and have remained considerably stable. Despite expression decreases in therians, Y/W genes show notable conservation of proto-sex chromosome expression patterns, although various Y genes evolved testis-specificities through differential regulatory decay. Thus, although some genes evolved novel functions through spatial/temporal expression shifts, most Y genes probably endured, at least initially, because of dosage constraints.