Targeting macrophages with multifunctional nanoparticles to detect and prevent atherosclerotic cardiovascular disease.

Despite the emergence of novel diagnostic, pharmacological, interventional and prevention strategies, atherosclerotic cardiovascular disease remains a significant cause of morbidity and mortality. Nanoparticle-based platforms encompass diverse imaging, delivery and pharmacological properties that provide novel opportunities for refining diagnostic and therapeutic interventions for atherosclerosis at the cellular and molecular level. Macrophages play a critical role in atherosclerosis and therefore represent an important disease-related diagnostic and therapeutic target, especially given their inherent ability for passive and active nanoparticle uptake. In this review, we discuss an array of inorganic, carbon-based and lipid-based nanoparticles that provide magnetic, radiographic and fluorescent imaging capabilities for a range of highly promising research and clinical applications in atherosclerosis. We discuss the design of nanoparticles that target a range of macrophage-related functions such as lipoprotein oxidation, cholesterol efflux, vascular inflammation and defective efferocytosis. We also provide examples of nanoparticle systems that were developed for other pathologies such as cancer and highlight their potential for repurposing in cardiovascular disease. Finally, we discuss the current state of play and the future of theranostic nanoparticles. Whilst this is not without its challenges, the array of multifunctional capabilities that are possible in nanoparticle design ensures they will be part of the next frontier of exciting new therapies that simultaneously improve the accuracy of plaque diagnosis and more effectively reduce atherosclerosis with limited side effects.

TRIM2 Selectively Regulates Inflammation-Driven Pathological Angiogenesis without Affecting Physiological Hypoxia-Mediated Angiogenesis.

Angiogenesis is a critical physiological response to ischemia but becomes pathological when dysregulated and driven excessively by inflammation. We recently identified a novel angiogenic role for tripartite-motif-containing protein 2 (TRIM2) whereby lentiviral shRNA-mediated TRIM2 knockdown impaired endothelial angiogenic functions in vitro. This study sought to determine whether these effects could be translated in vivo and to determine the molecular mechanisms involved. CRISPR/Cas9-generated Trim2-/- mice that underwent a periarterial collar model of inflammation-induced angiogenesis exhibited significantly less adventitial macrophage infiltration relative to wildtype (WT) littermates, concomitant with decreased mRNA expression of macrophage marker Cd68 and reduced adventitial proliferating neovessels. Mechanistically, TRIM2 knockdown in endothelial cells in vitro attenuated inflammation-driven induction of critical angiogenic mediators, including nuclear HIF-1α, and curbed the phosphorylation of downstream effector eNOS. Conversely, in a hindlimb ischemia model of hypoxia-mediated angiogenesis, there were no differences in blood flow reperfusion to the ischemic hindlimbs of Trim2-/- and WT mice despite a decrease in proliferating neovessels and arterioles. TRIM2 knockdown in vitro attenuated hypoxia-driven induction of nuclear HIF-1α but had no further downstream effects on other angiogenic proteins. Our study has implications for understanding the role of TRIM2 in the regulation of angiogenesis in both pathophysiological contexts.

The FXYD1 protein plays a protective role against pulmonary hypertension and arterial remodeling via redox and inflammatory mechanisms.

Pulmonary hypertension (PH) consists of a heterogenous group of diseases that culminate in increased pulmonary arterial pressure and right ventricular (RV) dysfunction. We sought to investigate the role of FXYD1, a small membrane protein that modulates Na+-K+-ATPase function, in the pathophysiology of PH. We mined online transcriptome databases to assess FXYD1 expression in PH. We characterized the effects of FXYD1 knockout (KO) in mice on right and left ventricular (RV and LV) function using echocardiography and measured invasive hemodynamic measurements under normal conditions and after treatment with bleomycin sulfate or chronic hypoxia to induce PH. Using immunohistochemistry, immunoblotting, and functional assays, we examined the effects of FXYD1 KO on pulmonary microvasculature and RV and LV structure and assessed signaling via endothelial nitric oxide synthase (eNOS) and inflammatory pathways. FXYD1 lung expression tended to be lower in samples from patients with idiopathic pulmonary arterial hypertension (IPAH) compared with controls, supporting a potential pathophysiological role. FXYD1 KO mice displayed characteristics of PH including significant increases in pulmonary arterial pressure, increased muscularization of small pulmonary arterioles, and impaired RV systolic function, in addition to LV systolic dysfunction. However, when PH was stimulated with standard models of lung injury-induced PH, there was no exacerbation of disease in FXYD1 KO mice. Both the lungs and left ventricles exhibited elevated nitrosative stress and inflammatory milieu. The absence of FXYD1 in mice results in LV inflammation and cardiopulmonary redox signaling changes that predispose to pathophysiological features of PH, suggesting FXYD1 may be protective.NEW & NOTEWORTHY This is the first study to show that deficiency of the FXYD1 protein is associated with pulmonary hypertension. FXYD1 expression is lower in the lungs of people with idiopathic pulmonary artery hypertension. FXYD1 deficiency results in both left and right ventricular functional impairment. Finally, FXYD1 may endogenously protect the heart from oxidative and inflammatory injury.

Sex, Endothelial Cell Functions, and Peripheral Artery Disease.

Peripheral artery disease (PAD) is caused by blocked arteries due to atherosclerosis and/or thrombosis which reduce blood flow to the lower limbs. It results in major morbidity, including ischemic limb, claudication, and amputation, with patients also suffering a heightened risk of heart attack, stroke, and death. Recent studies suggest women have a higher prevalence of PAD than men, and with worse outcomes after intervention. In addition to a potential unconscious bias faced by women with PAD in the health system, with underdiagnosis, and lower rates of guideline-based therapy, fundamental biological differences between men and women may be important. In this review, we highlight sexual dimorphisms in endothelial cell functions and how they may impact PAD pathophysiology in women. Understanding sex-specific mechanisms in PAD is essential for the development of new therapies and personalized care for patients with PAD.

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.

Endothelial cell dysfunction: Implications for the pathogenesis of peripheral artery disease.

Peripheral artery disease (PAD) is caused by occluded or narrowed arteries that reduce blood flow to the lower limbs. The treatment focuses on lifestyle changes, management of modifiable risk factors and vascular surgery. In this review we focus on how Endothelial Cell (EC) dysfunction contributes to PAD pathophysiology and describe the largely untapped potential of correcting endothelial dysfunction. Moreover, we describe current treatments and clinical trials which improve EC dysfunction and offer insights into where future research efforts could be made. Endothelial dysfunction could represent a target for PAD therapy.

Biological Sensing of Nitric Oxide in Macrophages and Atherosclerosis Using a Ruthenium-Based Sensor.

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.

Don't Turn Off the Tap! The Importance of Discovery Science to the Australian Cardiovascular Sector and Improving Clinical Outcomes Into the Future.

Despite significant advances in interventional and therapeutic approaches, cardiovascular disease (CVD) remains the leading cause of death and mortality. To lower this health burden, cardiovascular discovery scientists need to play an integral part in the solution. Successful clinical translation is achieved when built upon a strong foundational understanding of the disease mechanisms involved. Changes in the Australian funding landscape, to place greater emphasis on translation, however, have increased job insecurity for discovery science researchers and especially early-mid career researchers. To highlight the importance of discovery science in cardiovascular research, this review compiles six science stories in which fundamental discoveries, often involving Australian researchers, has led to or is advancing to clinical translation. These stories demonstrate the importance of the role of discovery scientists and the need for their work to be prioritised now and in the future. Australia needs to keep discovery scientists supported and fully engaged within the broader cardiovascular research ecosystem so they can help realise the next game-changing therapy or diagnostic approach that diminishes the burden of CVD on society.

The Effects of Exercise on Plaque Volume and Composition in a Mouse Model of Early and Late Life Atherosclerosis.

Background: Exercise is associated with a less atherogenic lipid profile; however, there is limited research on the effect of exercise on atherosclerotic plaque composition and markers of plaque stability. Methods: A total of 110 apolipoprotein (apo)E -/- mice were placed on a chow diet and randomly assigned to control or exercise for a period of 10 weeks, commencing either at 12 weeks of age (the early-stage atherosclerosis, EA group) or at 40 weeks of age (the late-stage atherosclerosis, LA group). At the end of the exercise period, blood was assayed for lipids. Histologic analysis of the aortic sinus was undertaken to assess plaque size and composition that includes macrophage content, monocyte chemoattractant protein (MCP)-1, matrix metalloproteinase-2 (MMP-2), and tissue inhibitors of metalloproteinase 1 and 2 (TIMP-1 and 2). Results: A total of 103 mice (38 EA, 65 LA) completed the protocol. In the EA group, exercise reduced plasma total cholesterol (TC) (-16%), free cholesterol (-13%), triglyceride (TG) (-35%), and phospholipid (-27%) levels, when compared to sedentary control mice (p < 0.01). In the EA group, exercise also significantly reduced plaque stenosis (-25%, p < 0.01), and there were higher levels of elastin (3-fold increase, p < 0.0001) and collagen (11-fold increase, p < 0.0001) in plaques, compared to control mice. There was an increase in plaque MMP-2 content in the exercise group (13% increase, p < 0.05) but no significant difference in macrophage or MCP-1 content. In the LA group, exercise reduced plaque stenosis (-18%, p < 0.05), but there was no significant difference in plaque composition. There was no difference in macrophage, MCP-1, or MMP-2 content in the LA groups. TIMP-1 was lower with exercise in both the EA and LA groups (-59%, p < 0.01 and -51%, p < 0.01 respectively); however, there was no difference in TIMP-2 levels. Conclusion: A 10-week exercise period reduces atherosclerotic plaque stenosis when commenced at both early- and late-stage atherosclerosis. Intervening earlier with exercise had a greater beneficial effect on lipids and plaque composition than when starting exercise at a later disease stage.

The Mechanisms of Restenosis and Relevance to Next Generation Stent Design.

Stents are lifesaving mechanical devices that re-establish essential blood flow to the coronary circulation after significant vessel occlusion due to coronary vessel disease or thrombolytic blockade. Improvements in stent surface engineering over the last 20 years have seen significant reductions in complications arising due to restenosis and thrombosis. However, under certain conditions such as diabetes mellitus (DM), the incidence of stent-mediated complications remains 2-4-fold higher than seen in non-diabetic patients. The stents with the largest market share are designed to target the mechanisms behind neointimal hyperplasia (NIH) through anti-proliferative drugs that prevent the formation of a neointima by halting the cell cycle of vascular smooth muscle cells (VSMCs). Thrombosis is treated through dual anti-platelet therapy (DAPT), which is the continual use of aspirin and a P2Y12 inhibitor for 6-12 months. While the most common stents currently in use are reasonably effective at treating these complications, there is still significant room for improvement. Recently, inflammation and redox stress have been identified as major contributing factors that increase the risk of stent-related complications following percutaneous coronary intervention (PCI). The aim of this review is to examine the mechanisms behind inflammation and redox stress through the lens of PCI and its complications and to establish whether tailored targeting of these key mechanistic pathways offers improved outcomes for patients, particularly those where stent placement remains vulnerable to complications. In summary, our review highlights the most recent and promising research being undertaken in understanding the mechanisms of redox biology and inflammation in the context of stent design. We emphasize the benefits of a targeted mechanistic approach to decrease all-cause mortality, even in patients with diabetes.

Pro-Calcific Environment Impairs Ischaemia-Driven Angiogenesis.

Peripheral arterial disease (PAD) is characterised by accelerated arterial calcification and impairment in angiogenesis. Studies implicate vascular calcification as a contributor to PAD, but the mechanisms remain unclear. We aimed to determine the effect of calcification on ischaemia-driven angiogenesis. Human coronary artery endothelial cells (ECs) were treated with calcification medium (CM: CaCl2 2.7 mM, Na2PO4 2.0 mM) for 24 h and exposed to normoxia (5% CO2) or hypoxia (1.2% O2; 5% CO2 balanced with N2). In normoxia, CM significantly inhibited tubule formation and migration and upregulated calcification markers of ALP, BMP2, and Runx2. CM elevated levels of calcification-protective gene OPG, demonstrating a compensatory mechanism by ECs. CM failed to induce pro-angiogenic regulators VEGFA and HIF-1α in hypoxia and further suppressed the phosphorylation of endothelial nitric oxide synthase (eNOS) that is essential for vascular function. In vivo, osteoprotegerin-deficient mice (OPG-/-), a calcification model, were subjected to hind-limb ischaemia (HLI) surgery. OPG-/- mice displayed elevated serum alkaline phosphatase (ALP) activity compared to wild-type controls. OPG-/- mice experienced striking reductions in blood-flow reperfusion in both 8-week-old and 6-month-old mice post-HLI. This coincided with significant impairment in tissue ischaemia and reduced limb function as assessed by clinical scoring (Tarlov). This study demonstrated for the first time that a pro-calcific environment is detrimental to ischaemia-driven angiogenesis. The degree of calcification in patients with PAD can often be a limiting factor with the use of standard therapies. These highly novel findings require further studies for full elucidation of the mechanisms involved and have implications for the development of therapies to suppress calcification in PAD.

3D-Printed Micro Lens-in-Lens for In Vivo Multimodal Microendoscopy.

Multimodal microendoscopes enable co-located structural and molecular measurements in vivo, thus providing useful insights into the pathological changes associated with disease. However, different optical imaging modalities often have conflicting optical requirements for optimal lens design. For example, a high numerical aperture (NA) lens is needed to realize high-sensitivity fluorescence measurements. In contrast, optical coherence tomography (OCT) demands a low NA to achieve a large depth of focus. These competing requirements present a significant challenge in the design and fabrication of miniaturized imaging probes that are capable of supporting high-quality multiple modalities simultaneously. An optical design is demonstrated which uses two-photon 3D printing to create a miniaturized lens that is simultaneously optimized for these conflicting imaging modalities. The lens-in-lens design contains distinct but connected optical surfaces that separately address the needs of both fluorescence and OCT imaging within a lens of 330 µm diameter. This design shows an improvement in fluorescence sensitivity of >10x in contrast to more conventional fiber-optic design approaches. This lens-in-lens is then integrated into an intravascular catheter probe with a diameter of 520 µm. The first simultaneous intravascular OCT and fluorescence imaging of a mouse artery in vivo is reported.

Associations between adipokines and atrial fibrillation: A systematic review and meta-analysis.

AIMS: Although overweight and obesity are associated with increased risk of atrial fibrillation (AF), the underlying mechanisms are not well characterised. Recent data suggest that this link may be partly due to abnormal adipose tissue-derived cytokines or adipokines. However, this relationship is not well clarified. To evaluate the association between adipokines and AF in a systematic review and meta-analysis. DATA SYNTHESIS: PubMed, Embase, and Web of Science Core Collection were searched from inception through 1st March 2021. Studies were included if they reported any adipokine and AF, with their quality assessed using the Newcastle-Ottawa scale. Data were independently abstracted, with unadjusted and multivariable adjusted estimates pooled in a random-effects meta-analysis. Data are presented for overall prevalent or incident AF and AF subtypes (paroxysmal, persistent, or non-paroxysmal AF). A total of 34 studies, with 31,479 patients, were included. The following adipokines were significantly associated with AF in the pooled univariate data - apelin (risk ratio for prevalent AF: 0.05 [0.00-0.50], p = 0.01; recurrent AF: 0.21 [0.11-0.42], p < 0.01) and resistin (incident AF: 2.05 [1.02-4.1], p = 0.04; prevalent AF: 2.62 [1.78-3.85], p < 0.01). Pooled analysis of multivariable adjusted effect size estimates showed adiponectin as the sole independent predictor of AF incidence (1.14 [1.02-1.27], p = 0.02). Moreover, adiponectin was associated with non-paroxysmal AF (persistent AF: 1.45 [1.08-1.94, p = 0.01; non-paroxysmal versus paroxysmal AF: 3.14 [1.87-5.27, p < 0.01). CONCLUSIONS: Adipokines, principally adiponectin, apelin, and resistin, are associated with the risk of atrial fibrillation. However, the association is not seen after multivariate adjustment, likely reflecting the lack of statistical power. Future research should investigate these relationships in larger prospective cohorts and how they can refine AF monitoring strategies. PROSPERO ID: CRD42020208879.

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.

Multimodality Intravascular Imaging of High-Risk Coronary Plaque.

The majority of coronary atherothrombotic events presenting as myocardial infarction (MI) occur as a result of plaque rupture or erosion. Understanding the evolution from a stable plaque into a life-threatening, high-risk plaque is required for advancing clinical approaches to predict atherothrombotic events, and better treat coronary atherosclerosis. Unfortunately, none of the coronary imaging approaches used in clinical practice can reliably predict which plaques will cause an MI. Currently used imaging techniques mostly identify morphological features of plaques, but are not capable of detecting essential molecular characteristics known to be important drivers of future risk. To address this challenge, engineers, scientists, and clinicians have been working hand-in-hand to advance a variety of multimodality intravascular imaging techniques, whereby 2 or more complementary modalities are integrated into the same imaging catheter. Some of these have already been tested in early clinical studies, with other next-generation techniques also in development. This review examines these emerging hybrid intracoronary imaging techniques and discusses their strengths, limitations, and potential for clinical translation from both an engineering and clinical perspective.

The Role of miR-181c in Mechanisms of Diabetes-Impaired Angiogenesis: An Emerging Therapeutic Target for Diabetic Vascular Complications.

Diabetes mellitus is estimated to affect up to 700 million people by the year 2045, contributing to an immense health and economic burden. People living with diabetes have a higher risk of developing numerous debilitating vascular complications, leading to an increased need for medical care, a reduced quality of life and increased risk of early death. Current treatments are not satisfactory for many patients who suffer from impaired angiogenesis in response to ischaemia, increasing their risk of ischaemic cardiovascular conditions. These vascular pathologies are characterised by endothelial dysfunction and abnormal angiogenesis, amongst a host of impaired signaling pathways. Therapeutic stimulation of angiogenesis holds promise for the treatment of diabetic vascular complications that stem from impaired ischaemic responses. However, despite significant effort and research, there are no established therapies that directly stimulate angiogenesis to improve ischaemic complications such as ischaemic heart disease and peripheral artery disease, highlighting the immense unmet need. However, despite significant effort and research, there are no established therapies that directly stimulate angiogenesis in a clinical setting, highlighting the immense unmet need. MicroRNAs (miRNAs) are emerging as powerful targets for multifaceted diseases including diabetes and cardiovascular disease. This review highlights the potential role of microRNAs as therapeutic targets for rescuing diabetes-impaired angiogenesis, with a specific focus on miR-181c, which we have previously identified as an important angiogenic regulator. Here we summarise the pathways currently known to be regulated by miR-181c, which include the classical angiogenesis pathways that are dysregulated in diabetes, mitochondrial function and axonal guidance, and describe how these relate both directly and indirectly to angiogenesis. The pleiotropic actions of miR-181c across multiple key angiogenic signaling pathways and critical cellular processes highlight its therapeutic potential as a novel target for treating diabetic vascular complications.

Circadian disruption by short light exposure and a high energy diet impairs glucose tolerance and increases cardiac fibrosis in Psammomys obesus.

Type 2 diabetes mellitus (T2DM) increases cardiac inflammation which promotes the development of cardiac fibrosis. We sought to determine the impact of circadian disruption on the induction of hyperglycaemia, inflammation and cardiac fibrosis. METHODS: Psammomys obesus (P. obesus) were exposed to neutral (12 h light:12 h dark) or short (5 h light:19 h dark) photoperiods and fed a low energy (LE) or high energy (HE) diet for 8 or 20 weeks. To determine daily rhythmicity, P. obesus were euthanised at 2, 8, 14, and 20 h after 'lights on'. RESULTS: P. obesus exposed to a short photoperiod for 8 and 20 weeks had impaired glucose tolerance following oral glucose tolerance testing, compared to a neutral photoperiod exposure. This occurred with both LE and HE diets but was more pronounced with the HE diet. Short photoperiod exposure also increased myocardial perivascular fibrosis after 20 weeks on LE (51%, P < 0.05) and HE (44%, P < 0.05) diets, when compared to groups with neutral photoperiod exposure. Short photoperiod exposure caused elevations in mRNA levels of hypertrophy gene Nppa (atrial natriuretic peptide) and hypertrophy transcription factors Gata4 and Mef2c in myocardial tissue after 8 weeks. CONCLUSION: Exposure to a short photoperiod causes impaired glucose tolerance in P. obesus that is exacerbated with HE diet and is accompanied by an induction in myocardial perivascular fibrosis.

Omega-3 fatty acids ameliorate vascular inflammation: A rationale for their atheroprotective effects.

BACKGROUND AND AIMS: Clinical trials have demonstrated reductions in major adverse cardiovascular events with purified high-dose eicosapentaenoic acid (EPA), independent of effects on lipids. We aimed to investigate whether omega-3 fatty acids reduce vascular inflammation, a critical mediator of atherosclerosis, and hypothesised that EPA is superior to docosahexaenoic acid (DHA). METHODS: In a double-blind randomised controlled trial and cell-culture study, 40 healthy volunteers were supplemented with 4 g daily of either EPA, DHA, fish oil (2:1 EPA:DHA), or placebo for 30 days. Serum was incubated with TNF-stimulated human umbilical vein endothelial cells (HUVECs), and markers of acute vascular inflammation (AVI) were measured. The effects of EPA, DHA (600 mg/kg/day), olive oil, or no treatment were also measured in preclinical models of [1] AVI using a periarterial collar (C57Bl/6J; n = 40 mice) and [2] atherosclerosis where ApoE-/- mice (n = 40) were fed a 16-week atherogenic diet. RESULTS: EPA supplementation reduced expression of C-C motif chemokine ligand 2 (CCL2) by 25% compared to placebo (p = 0.03). In the AVI model, EPA reduced vascular expression of VCAM1 by 43% (p = 0.02) and CCL2 by 41% (p = 0.03). Significant inverse correlations were observed between EPA levels and vascular expression of VCAM1 (r = -0.56, p = 0.001) and CCL2 (r = -0.56, p = 0.001). In ApoE-/- mice, EPA reduced aortic expression of Il1b by 44% (p = 0.04) and Tnf by 49% (p = 0.04), with similar inverse correlations between EPA levels and both Il1b (r = -0.63, p = 0.009) and Tnf (r = -0.50, p = 0.04). CONCLUSIONS: Supplementation with EPA, more so than DHA, ameliorates acute and chronic vascular inflammation, providing a rationale for the cardiovascular benefit observed with high dose omega-3 fatty acid administration.

Elevated HDL-bound miR-181c-5p level is associated with diabetic vascular complications in Australian Aboriginal people.

AIMS/HYPOTHESIS: Diabetes is a major burden on Australia's Indigenous population, with high rates of disease and vascular complications. Diabetic vascular complications are associated with impaired ischaemia-driven angiogenesis. MicroRNAs (miRNAs) are key players in the regulation of angiogenesis. HDL-cholesterol (HDL-c) levels are inversely associated with the risk of developing diabetic complications and HDL can carry miRNAs. HDL-miRNA profiles differ in disease states and may present as biomarkers with the capacity to act as bioactive signalling molecules. Recent studies have demonstrated that HDL becomes dysfunctional in a diabetic environment, losing its vasculo-protective effects and becoming more pro-atherogenic. We sought to determine whether HDL-associated miRNA profiles and HDL functionality were predictive of the severity of diabetic vascular complications in Australia's Indigenous population. METHODS: HDL was isolated from plasma samples from Indigenous participants without diabetes ('Healthy'), with type 2 diabetes mellitus ('T2DM') and with diabetes-associated macrovascular complications (specifically peripheral artery disease, 'T2DM+Comp'). To assess HDL angiogenic capacity, human coronary artery endothelial cells were treated with PBS, reconstituted HDL (rHDL, positive control) or isolated HDL and then exposed to high-glucose (25 mmol/l) conditions. The expression levels of two anti-angiogenic miRNAs (miR-181c-5p and miR-223-3p) and one pro-angiogenic miRNA (miR-27b-3p) were measured in the HDL fraction, plasma and treated human coronary artery endothelial cells by quantitative real-time PCR. In vitro endothelial tubule formation was assessed using the Matrigel tubulogenesis assay. RESULTS: Strikingly, we found that the levels of the anti-angiogenic miRNA miR-181c-5p were 14-fold higher (1454 ± 1346%) in the HDL from Aboriginal people with diabetic complications compared with both the Healthy (100 ± 121%, p < 0.05) and T2DM (82 ± 77%, p < 0.05) groups. Interestingly, we observed a positive correlation between HDL-associated miR-181c-5p levels and disease severity (p = 0.0020). Under high-glucose conditions, cells treated with rHDL, Healthy HDL and T2DM HDL had increased numbers of tubules (rHDL: 136 ± 8%, p < 0.01; Healthy HDL: 128 ± 6%, p < 0.01; T2DM HDL: 124 ± 5%, p < 0.05) and branch points (rHDL: 138 ± 8%, p < 0.001; Healthy HDL: 128 ± 6%, p < 0.01; T2DM HDL: 127 ± 5%, p < 0.01) concomitant with elevations in mRNA levels of the key hypoxia angiogenic transcription factor HIF1A (rHDL: 140 ± 10%, p < 0.01; Healthy HDL: 136 ± 8%, p < 0.01; T2DM HDL: 133 ± 9%, p < 0.05). However, this increase in angiogenic capacity was not observed in cells treated with T2DM + Comp HDL (tubule numbers: 113 ± 6%, p = 0.32; branch points: 113 ± 5%, p = 0.28; HIF1A: 117 ± 6%, p = 0.43), which could be attributed to the increase in cellular miR-181c-5p levels (T2DM + Comp HDL: 136 ± 7% vs PBS: 100 ± 9%, p < 0.05). CONCLUSIONS/INTERPRETATION: In conclusion, HDL from Aboriginal people with diabetic complications had reduced angiogenic capacity. This impairment is associated with an increase in the expression of anti-angiogenic miR-181c-5p. These findings provide the rationale for a new way to better inform clinical diagnosis of disease severity with the potential to incorporate targeted, personalised HDL-miRNA intervention therapies to prevent further development of, or to reverse, diabetic vascular complications in Australian Aboriginal people.