OBJECTIVE:Reconstituted high-density lipoproteins (rHDL) improve wound healing in diabetes. We aimed to determine if rHDL elicit anti-inflammatory effects in diabetic wounds, as a mechanism to explain their wound healing benefits. APPROACH:Diabetes was induced using streptozotocin in C57Bl6/J mice. Two full-thickness wounds were placed on the subflanks of diabetic and nondiabetic (ND) mice. Phosphate-buffered saline (PBS) or rHDL (50 µg/wound/day) were applied topically. Wound closure was assessed daily. Inflammatory gene transcripts were measured by qPCR and proteins by Western blotting and enzyme-linked immunosorbent assay in wounds collected at baseline, 24 h, and 3 days postwounding. Wound macrophages were assessed by flow cytometry 7 days postwounding. The fate of fluorescent 3,3-dioctadecyloxacarbocyanine, perchlorate (DiO)-labeled rHDL was tracked by flow cytometry, fluorescent imaging, and microscopy. RESULTS:In diabetic mice, rHDL increased wound closure rates at days 6 (+288%, p < 0.01) and 7 (+639%, p < 0.0001) postwounding, compared with PBS controls. After 3 days, rHDL-treated diabetic wounds had lower Rela (-65%) and C-C motif chemokine ligand 2 (Ccl2) (-59%) mRNA levels and CCL2 protein (29%) than PBS controls, p < 0.05 for all. Wound macrophage content was higher in diabetic than ND wounds, but rHDL did not change macrophage content or polarity. DiO-rHDL were taken up by key wound cells including fibroblasts, macrophages, keratinocytes and endothelial cells, and retained in wounds for at least 48 h. INNOVATION:rHDL exerts anti-inflammatory effects in diabetic wounds early postwounding, which may contribute to its wound healing properties. CONCLUSION:The anti-inflammatory properties of rHDL in diabetic wounds present topical rHDL as a novel treatment option for improving healing in patients with diabetic foot ulcers. [Figure: see text].

Background Lipid-lowering therapy (LLT) is established as a key element in management of patients with coronary artery disease. However, the effect of time of initiation of LLT on outcomes is unclear. Method The study compared outcomes of 5,433 patients from Advara HeartCare's Percutaneous Coronary Intervention (PCI) Registry on the basis of timing of LLT initiation classified as pre- or post-PCI admission. The prevalence of acute coronary syndrome (ACS) as the indication for PCI was compared in groups. In patients who underwent PCI for ACS, the incidence of short- (<30 days) and long-term (>30 days after admission) clinical events (composite of myocardial infarction, cerebrovascular disease, coronary revascularisation, all-cause readmission, and mortality) and first non-fatal cardiovascular events were compared in groups. Results At the time of hospitalisation for PCI, 3,982 (73.7%) were on LLT (PRE-LLT), and 1,418 (26.2%) initiated LLT after admission (POST-LLT). Patients on PRE-LLT were significantly less likely to experience ACS before admission for PCI than were those commencing LLT after discharge (PRE-LLT 32.3% vs POST-LLT 56.9%; p<0.001), even after matching for baseline risk factors. Among these patients with ACS, patients on PRELLT were older than those on POST-LLT (mean 69.5 +/- 9.5 vs 65.0 +/- 10.0 years; p<0.001), and had a higher prevalence of cardiovascular risk factors including diabetes (31.5% vs 9.6%; p<0.001), hypertension (79.7% vs 51.7%; p<0.001), and renal failure (7.6% vs 2.0%; p<0.001). No difference was observed between groups in the risk of short- or long-term (median 2.0 years; interquartile range 1.0-3.0) post-PCI cardiovascular (hazard ratio [HR] 1.08; 0.83-1.40; p=0.55) or overall clinical events (HR 1.11; 0.93-1.32; p=0.26). Conclusions In patients with coronary artery disease, the risk of ACS is reduced by early initiation of LLT before revascularisation is required. Long-term outcomes of patients at high risk prescribed LLT before admission for ACS PCI may not differ from those of patients at lower risk commencing LLT after PCI for ACS.

Converging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these embryonically derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic endothelial-macrophage (EndoMac) progenitor cells in the adventitia of embryonic and postnatal mouse aorta, that are independent of Flt3-mediated bone marrow hematopoiesis and derive from an early embryonic CX3CR1+ and CSF1R+ source. These bipotent progenitors are proliferative and vasculogenic, contributing to adventitial neovascularization and formation of perfused blood vessels after transfer into ischemic tissue. We establish a regulatory role for angiotensin II, which enhances their clonogenic and differentiation properties and rapidly stimulates their proliferative expansion in vivo. Our findings demonstrate that embryonically derived EndoMac progenitors participate in local vasculogenic responses in the aortic wall by contributing to the expansion of endothelial cells and macrophages postnatally. The extraembryonic yolk sac is a major location for developmental hematopoiesis, but it is unclear whether non-bone marrow sources contribute during adulthood. Here they show that embryonically derived endothelial-macrophage progenitor cells located in the aorta are a bipotent source of macrophage and endothelial cells later in life.

Converging evidence indicates developmental overlap whereby some tissue macrophages (MΦs) and endothelial cells (ECs) arise from a common embryonic source, namely yolk sac (YS) erythromyeloid progenitor cells. Although YS-derived MΦs and ECs are widely believed to be maintained after birth by proliferative self-renewal, our group has recently identified YS-derived bipotent endothelial-macrophage (EndoMac) progenitors as an alternative source for their postnatal renewal in tissues. We demonstrated that FACS-isolated and culture-derived progenitors from mouse aorta are Lin-CD45+CD11b-F4/80-Sca-1+c-Kit+ and express fractalkine receptor (CX3CR1) and macrophage colony-stimulating factor (CSF1R). Progenitors from different tissues (aorta, skin, skeletal muscle [SkM]) exhibit important similarities, with subtle tissue-related variations in clonal renewal, angiogenic capacity and gene expression. Finally, with a view to understanding their metabolic regulation, we studied the effects of high glucose and hyperglycaemia on aortic, skin and SkM progenitors using a streptozotocin-induced mouse model of type 1 diabetes and identified glucose-induced reductions in their clonogenicity, differentiation and angiogenic capacity, as well as their DNA integrity, mitochondrial viability and metabolic function. Given that diabetes impairs skin wound healing and ischaemia reperfusion we utilised lineage-mapping techniques, to reveal an attenuation in the rapid burst of proliferative accumulation of YS-derived progenitors in early diabetic skin wounds and ischaemic muscle. This was accompanied by blunting of the expansion of YS-derived MΦs and ECs in later stages of repair. Finally, we showed that progenitors from non-diabetic skin and SkM can engraft, differentiate and promote repair and muscle reperfusion respectively when transplanted into recipient diabetic wounds and ischaemic muscle, whereas these salutary properties are severely diminished for diabetic progenitors. Our findings provide a novel framework to help understand how diabetes impairs wound repair and vascularisation of ischaemic tissue through the lens of YS-derived EndoMac progenitors and the effect of diabetes on their function and reparative potential.

BACKGROUND:Computed tomography coronary angiography (CTCA) is an established modality for the diagnosis and assessment of cardiovascular disease. However, price and space pressure have mostly necessitated outsourcing CTCA to external radiology providers. Advara HeartCare has recently integrated CT services within local clinical networks across Australia. This study examined the benefits of the presence (integrated) or absence (pre-integrated) of this "in-house" CTCA service in real-world clinical practice. METHODS:De-identified patient data from electronic medical records were used to create an Advara HeartCare CTCA database. Data analysis included clinical history, demographics, CTCA procedure, and 30-day outcomes post-CTCA from two age-matched cohorts: integrated (n ​= ​495) and pre-integrated (n ​= ​456). RESULTS:Data capture was more comprehensive and standardised across the integrated cohort. There was a 21% increase in referrals for CTCA from cardiologists observed for the integration cohort vs. pre-integration [n ​= ​332 (72.8%) pre-integration vs. n ​= ​465 (93.9%) post-integration, p ​< ​0.0001] with a parallel increase in diagnostic assessments including blood tests [n ​= ​209 (45.8%) vs. n ​= ​387 (78.1%), respectively, p ​< ​0.0001]. The integrated cohort received lower total dose length product [Median 212 (interquartile range 136-418) mGy∗cm vs. 244 (141.5, 339.3) mGy∗cm, p ​= ​0.004] during the CTCA procedure. 30-days after CTCA scan, there was a significantly higher use of lipid-lowering therapies in the integrated cohort [n ​= ​133 (50.5%) vs. n ​= ​179 (60.6%), p ​= ​0.04], along with a significant decrease in the number of stress echocardiograms performed [n ​= ​14 (10.6%) vs. n ​= ​5 (11.6%), p ​= ​0.01]. CONCLUSION:Integrated CTCA has salient benefits in patient management, including increased pathology tests, statin usage, and decreased post-CTCA stress echocardiography utilisation. Our ongoing work will examine the effect of integration on cardiovascular outcomes.

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-1 beta 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.

Macrophage-derived nitric oxide (NO) plays a critical role in atherosclerosis and presents as a potential biomarker. We assessed the uptake, distribution, and NO detection capacity of an irreversible, ruthenium-based, fluorescent NO sensor (Ru-NO) in macrophages, plasma, and atherosclerotic plaques. In vitro, incubation of Ru-NO with human THP1 monocytes and THP1-PMA macrophages caused robust uptake, detected by Ru-NO fluorescence using mass-cytometry, confocal microscopy, and flow cytometry. THP1-PMA macrophages had higher Ru-NO uptake (+13%, p < 0.05) than THP1 monocytes with increased Ru-NO fluorescence following lipopolysaccharide stimulation (+14%, p < 0.05). In mice, intraperitoneal infusion of Ru-NO found Ru-NO uptake was greater in peritoneal CD11b+F4/80+ macrophages (+61%, p < 0.01) than CD11b+F4/80− monocytes. Infusion of Ru-NO into Apoe−/− mice fed high-cholesterol diet (HCD) revealed Ru-NO fluorescence co-localised with atherosclerotic plaque macrophages. When Ru-NO was added ex vivo to aortic cell suspensions from Apoe−/− mice, macrophage-specific uptake of Ru-NO was demonstrated. Ru-NO was added ex vivo to tail-vein blood samples collected monthly from Apoe−/− mice on HCD or chow. The plasma Ru-NO fluorescence signal was higher in HCD than chow-fed mice after 12 weeks (37.9%, p < 0.05). Finally, Ru-NO was added to plasma from patients (N = 50) following clinically-indicated angiograms. There was lower Ru-NO fluorescence from plasma from patients with myocardial infarction (−30.7%, p < 0.01) than those with stable coronary atherosclerosis. In conclusion, Ru-NO is internalised by macrophages in vitro, ex vivo, and in vivo, can be detected in atherosclerotic plaques, and generates measurable changes in fluorescence in murine and human plasma. Ru-NO displays promising utility as a sensor of atherosclerosis.

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.

Converging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these yolk sac-derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic, self-renewing endothelial-macrophage (EndoMac) progenitor cells in postnatal mouse aorta, heart and lung, that are independent of definitive hematopoiesis and derive from a CX3CR1+ and CSF1R+ yolk sac source. These bipotent progenitors are highly proliferative and vasculogenic, contributing to adventitial neovascularization in the aortic wall and forming perfused blood vessels after adoptive transfer into ischemic tissue. We establish a regulatory role for angiotensin II, which enhances their clonogenic, self-renewal and differentiation properties. Our findings demonstrate that tissue-resident EndoMac progenitors drive local inflammatory and vasculogenic responses by contributing to the renewal and expansion of yolk sac-derived macrophages and endothelial cells postnatally.

Background: Eukaryotic elongation factor 2 kinase (eEF2k) is a unique enzyme that controls protein synthesis and promotes cell survival under stress conditions. Previous studies suggest a role for eEF2k in formation of atherosclerotic plaques, yet cellular mechanisms are still unclear. Here we investigated how eEF2k regulates macrophage handling of atherogenic lipoproteins. Methods: Bone marrow-derived macrophages (BMDMs) were prepared from eEF2k knockout (Eef2k−/−) and wild-type (WT) mice and exposed to different stimuli, including oxidised low-density lipoprotein (ox-LDL). Parallel experiments were performed using BMDMs from C57BL/6 mice and human peripheral blood monocyte-derived macrophages (MDMs) that were treated with a highly selective eEF2k inhibitor, JAN-384. In vivo, Eef2k-LdlrDKO and WT-LdlrKO mice were fed an atherogenic diet for 16 weeks. Results: Despite no differences in viability, proliferation, differentiation or polarisation, Eef2k−/− BMDMs had reduced ability to form oil red-O+ foam cells when incubated with ox-LDL (WT 32.3% ± 2.0% vs. Eef2k−/− 12.5% ± 2.3%, p < 0.01). Similar observations were made for JAN-384 treated C57BL/6 BMDMs and human MDMs (p < 0.05). Mechanistic evaluation revealed 70% lower levels of the CD36 surface marker in Eef2k−/− BMDMs, with eEF2k inhibition having a similar effect. Both eEF2k deficiency and inhibition significantly increased total cholesterol efflux capacity in BMDM-derived foam cells (p < 0.01). In comparison to WT-LdlrKO mice, Eef2k-LdlrDKO mice had lower CD36 expression in circulating monocytes and macrophages after an atherogenic diet (p < 0.01), while foam cell formation was also lower from their BMDMs and peritoneal macrophages (p < 0.01). Conclusion: eEF2k regulates foam cell formation from macrophages, likely through actions on CD36 expression and cholesterol efflux.

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.

Background: Macrophages are integral to vascular biology and disease. Although traditional paradigms assert that vascular macrophages originate from circulating monocytes, recent data suggest that some are seeded in utero from CX 3 CR1 + progenitors, and are maintained postnatally by local self-renewal. We previously identified a novel population of locally-maintained adventitial macrophage progenitor cells (AMPCs) as a source of self-renewing macrophages in adult mouse arteries. Hypothesis: AMPCs express CX 3 CR1 and do not derive from definitive hematopoiesis. Methods: Single-cell disaggregates were prepared from postnatal murine aortas. AMPC content was assessed by macrophage colony-forming unit (CFU-M) assays. Flow cytometry together with fluorescence activated cell sorting (FACS) were used to investigate the immunophenotypic profile of AMPCs and fate-mapping to assess their origins. Results: CFU-M prevalence in C57BL/6J aortic cells was highest in neonatal mice (~100 per 10 5 cells), and diminished progressively with age (~55/10 5 at 3w, ~15/10 5 at 12w, ~5/10 5 at 52w, n>4, P<0.01). Secondary replating of single cells from aortic CFU-M revealed striking self-renewal capacity, with 1 in 10 cells forming new CFU-M (n=8). Undifferentiated CFU-M displayed >95% expression of the stem cell markers Sca-1 and c-Kit, and high levels of CX 3 CR1, but did not acquire the monocyte/macrophage markers CD11b or F4/80 until treated with macrophage-colony stimulating factor (n=6). Mice deficient in CX 3 CR1 (Cx 3 cr1 GFP/GFP ) produced fewer CFU-M than heterozygous (Cx 3 cr1 +/GFP ) littermates (8.7±0.7 vs 15.3±0.7 per 10 5 , P<0.001, n>7). FACS selection confirmed that CFU-M forming AMPCs were exclusively contained within a CX 3 CR1 + subpopulation that did not express either CD11b or F4/80 (n=3). Finally, CFU-M analysis from Flt3 Cre xRosa mT/mG mice demonstrated that AMPCs arise from a FLT3 -ve source, indicating that their origins are independent of definitive hematopoiesis (n=4). Conclusion: Clonogenic, self-renewing murine AMPCs express CX 3 CR1 but not the monocyte/macrophage markers CD11b and F4/80. The high prevalence of AMPCs in neonatal aorta is consistent with prenatal seeding from CX 3 CR1 + progenitors, independent of definitive hematopoiesis.