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.

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.

Inhibition of Vascular Inflammation by Apolipoprotein A-IV.

Background: Apolipoprotein (apo) A-IV, the third most abundant apolipoprotein in human high density lipoproteins (HDLs), inhibits intestinal and systemic inflammation. This study asks if apoA-IV also inhibits acute vascular inflammation. Methods: Inflammation was induced in New Zealand White rabbits by placing a non-occlusive silastic collar around the common carotid artery. A single 1 mg/kg intravenous infusion of lipid-free apoA-IV or saline (control) was administered to the animals 24 h before collar insertion. The animals were euthanised 24 h post-collar insertion. Human coronary artery cells (HCAECs) were pre-incubated with reconstituted HDLs containing apoA-IV complexed with phosphatidylcholine, (A-IV)rHDLs, then activated by incubation with tumour necrosis factor (TNF)-α. Cell surface vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in the TNF-α-activated HCAECs was quantified by flow cytometry. VCAM-1, ICAM-1 and 3ß-hydroxysteroid-Δ24 reductase (DHCR24) mRNA levels were quantified by real time PCR. Results: Apolipoprotein ApoA-IV treatment significantly decreased collar-induced endothelial expression of VCAM-1, ICAM-1 and neutrophil infiltration into the arterial intima by 67.6 ± 9.9% (p < 0.01), 75.4 ± 6.9% (p < 0.01) and 74.4 ± 8.5% (p < 0.05), respectively. It also increased endothelial expression of DHCR24 by 2.6-fold (p < 0.05). Pre-incubation of HCAECs with (A-IV)rHDLs prior to stimulation with TNF-α inhibited VCAM-1 and ICAM-1 protein levels by 62.2 ± 12.1% and 33.7 ± 5.7%, respectively. VCAM-1 and ICAM-1 mRNA levels were decreased by 55.8 ± 7.2% and 49.6 ± 7.9%, respectively, while DHCR24 mRNA expression increased by threefold. Transfection of HCAECs with DHCR24 siRNA attenuated the anti-inflammatory effects of (A-IV)rHDLs. Pre-incubation of TNF-α-activated HCAECs with (A-IV)rHDLs also inhibited nuclear translocation of the p65 subunit of nuclear factor-κB (NF-κB), and decreased IκBα phosphorylation. Conclusion: These results indicate that apoA-IV inhibits vascular inflammation in vitro and in vivo by inhibiting NF-κB activation in a DHCR24-dependent manner.

A roadmap of strategies to support cardiovascular researchers: from policy to practice.

Cardiovascular disease remains the leading cause of death worldwide. Cardiovascular research has therefore never been more crucial. Cardiovascular researchers must be provided with a research environment that enables them to perform at their highest level, maximizing their opportunities to work effectively with key stakeholders to address this global issue. At present, cardiovascular researchers face a range of challenges and barriers, including a decline in funding, job insecurity and a lack of diversity at senior leadership levels. Indeed, many cardiovascular researchers, particularly women, have considered leaving the sector, highlighting a crucial need to develop strategies to support and retain researchers working in the cardiovascular field. In this Roadmap article, we present solutions to problems relevant to cardiovascular researchers worldwide that are broadly classified across three key areas: capacity building, research funding and fostering diversity and equity. This Roadmap provides opportunities for research institutions, as well as governments and funding bodies, to implement changes from policy to practice, to address the most important factors restricting the career progression of cardiovascular researchers.

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.

Lifestyle Modifies the Diabetes-Related Metabolic Risk, Conditional on Individual Genetic Differences.

Metabolic syndrome is a group of heritable metabolic traits that are highly associated with type 2 diabetes (T2DM). Classical interventions to T2DM include individual self-management of environmental risk factors, such as improving diet quality, increasing physical activity, and reducing smoking and alcohol consumption, which decreases the risk of developing metabolic syndrome. However, it is poorly understood how the phenotypes of diabetes-related metabolic traits change with respect to lifestyle modifications at the individual level. In the analysis, we used 12 diabetes-related metabolic traits and eight lifestyle covariates from the UK Biobank comprising 288,837 white British participants genotyped for 1,133,273 genome-wide single nucleotide polymorphisms. We found 16 GxE interactions. Modulation of genetic effects by physical activity was seen for four traits (glucose, HbA1c, C-reactive protein, systolic blood pressure) and by alcohol and smoking for three (BMI, glucose, waist-hip ratio and BMI and diastolic and systolic blood pressure, respectively). We also found a number of significant phenotypic modulations by the lifestyle covariates, which were not attributed to the genetic effects in the model. Overall, modulation in the metabolic risk in response to the level of lifestyle covariates was clearly observed, and its direction and magnitude were varied depending on individual differences. We also showed that the metabolic risk inferred by our model was notably higher in T2DM prospective cases than controls. Our findings highlight the importance of individual genetic differences in the prevention and management of diabetes and suggest that the one-size-fits-all approach may not benefit all.

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.

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.

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.

The multiple roles of chemokines in the mechanisms of stent biocompatibility.

While the advent of drug-eluting stents has been clinically effective in substantially reducing the rates of major stent-related adverse events compared with bare metal stents, vascular biological problems such as neointimal hyperplasia, delayed re-endothelialization, late stent thrombosis are not eliminated and, increasingly, neoatherosclerosis is the underlying mechanism for very late stent failure. Further understanding regarding the mechanisms underlying the biological responses to stent deployment is therefore required so that new and improved therapies can be developed. This review will discuss the accumulating evidence that the chemokines, small inflammatory proteins, play a role in each key biological process of stent biocompatibility. It will address the chemokine system in its specialized roles in regulating the multiple facets of vascular biocompatibility including neointimal hyperplasia, endothelial progenitor cell (EPC) mobilization and re-endothelialization after vascular injury, platelet activation and thrombosis, as well as neoatherosclerosis. The evidence in this review suggests that chemokine-targeting strategies may be effective in controlling the pathobiological processes that lead to stent failure. Preclinical studies provide evidence that inhibition of specific chemokines and/or broad-spectrum inhibition of the CC-chemokine class prevents neointimal hyperplasia, reduces thrombosis and suppresses the development of neoatherosclerosis. In contrast, however, to these apparent deleterious effects of chemokines on stent biocompatibility, the CXC chemokine, CXCL12, is essential for the mobilization and recruitment of EPCs that make important contributions to re-endothelialization post-stent deployment. This suggests that future chemokine inhibition strategies would need to be correctly targeted so that all key stent biocompatibility areas could be addressed, without compromising important adaptive biological responses.

Lack of Strategic Funding and Long-Term Job Security Threaten to Have Profound Effects on Cardiovascular Researcher Retention in Australia.

BACKGROUND: Cardiovascular disease is the leading cause of death in Australia. Investment in research solutions has been demonstrated to yield health and a 9.8-fold return economic benefit. The sector, however, is severely challenged with success rates of traditional peer-reviewed funding in decline. Here, we aimed to understand the perceived challenges faced by the cardiovascular workforce in Australia prior to the COVID-19 pandemic. METHODS: We used an online survey distributed across Australian cardiovascular societies/councils, universities and research institutes over a period of 6 months during 2019, with 548 completed responses. Inclusion criteria included being an Australian resident or an Australian citizen who lived overseas, and a current or past student or employee in the field of cardiovascular research. RESULTS: The mean age of respondents was 42±13 years, 47% were male, 85% had a full-time position, and 40% were a group leader or laboratory head. Twenty-three per cent (23%) had permanent employment, and 82% of full-time workers regularly worked >40 hours/week. Sixty-eight per cent (68%) said they had previously considered leaving the cardiovascular research sector. If their position could not be funded in the next few years, a staggering 91% of respondents would leave the sector. Compared to PhD- and age-matched men, women were less likely to be a laboratory head and to feel they had a long-term career path as a cardiovascular researcher, while more women were unsure about future employment and had considered leaving the sector (all p<0.05). Greater job security (76%) and government and philanthropic investment in cardiovascular research (72%) were highlighted by responders as the main changes to current practices that would encourage them to stay. CONCLUSION: Strategic solutions, such as diversification of career pathways and funding sources, and moving from a competitive to a collaborative culture, need to be a priority to decrease reliance on government funding and allow cardiovascular researchers to thrive.

The Role of High-Density Lipoproteins in Endothelial Cell Metabolism and Diabetes-Impaired Angiogenesis.

Diabetes mellitus affects millions of people worldwide and is associated with devastating vascular complications. A number of these complications, such as impaired wound healing and poor coronary collateral circulation, are characterised by impaired ischaemia-driven angiogenesis. There is increasing evidence that high-density lipoproteins (HDL) can rescue diabetes-impaired angiogenesis through a number of mechanisms, including the modulation of endothelial cell metabolic reprogramming. Endothelial cell metabolic reprogramming in response to tissue ischaemia is a driver of angiogenesis and is dysregulated by diabetes. Specifically, diabetes impairs pathways that allow endothelial cells to upregulate glycolysis in response to hypoxia adequately and impairs suppression of mitochondrial respiration. HDL rescues the impairment of the central hypoxia signalling pathway, which regulates these metabolic changes, and this may underpin several of its known pro-angiogenic effects. This review discusses the current understanding of endothelial cell metabolism and how diabetes leads to its dysregulation whilst examining the various positive effects of HDL on endothelial cell function.

MicroRNAs as Therapeutic Targets and Clinical Biomarkers in Atherosclerosis.

Atherosclerotic cardiovascular disease remains the leading cause of morbidity and mortality worldwide. Atherosclerosis develops over several decades and is mediated by a complex interplay of cellular mechanisms that drive a chronic inflammatory milieu and cell-to-cell interactions between endothelial cells, smooth muscle cells and macrophages that promote plaque development and progression. While there has been significant therapeutic advancement, there remains a gap where novel therapeutic approaches can complement current therapies to provide a holistic approach for treating atherosclerosis to orchestrate the regulation of complex signalling networks across multiple cell types and different stages of disease progression. MicroRNAs (miRNAs) are emerging as important post-transcriptional regulators of a suite of molecular signalling pathways and pathophysiological cellular effects. Furthermore, circulating miRNAs have emerged as a new class of disease biomarkers to better inform clinical diagnosis and provide new avenues for personalised therapies. This review focusses on recent insights into the potential role of miRNAs both as therapeutic targets in the regulation of the most influential processes that govern atherosclerosis and as clinical biomarkers that may be reflective of disease severity, highlighting the potential theranostic (therapeutic and diagnostic) properties of miRNAs in the management of cardiovascular disease.

High-Energy Diet and Shorter Light Exposure Drives Markers of Adipocyte Dysfunction in Visceral and Subcutaneous Adipose Depots of Psammomys obesus.

Dysfunctional adipose tissue phenotype underpins type 2 diabetes mellitus (T2DM) development. The disruption of circadian rhythms contributes to T2DM development. We investigated the effects of high-energy diet and photoperiod length on visceral and subcutaneous adipose tissue phenotype. Psammomys obesus sand rats exposed to neutral (12 light:12 dark) or short (5 light:19 dark) photoperiod were fed a low- (LE) or high- (HE) energy diet. The HE diet and/or short photoperiod reduced subcutaneous expression of adipocyte differentiation/function markers C/ebpα, Pparδ, Pparγ and Adipoq. Visceral Pparα levels were elevated in the 5:19HE group; however, the HE diet and/or short photoperiod decreased visceral Pparγ and Adipoq expression. 5:19HE animals had elevated Ucp1 yet lower Pgc-1α levels. The HE diet increased visceral Tgf-ß1, Ccl2 and Cd68 levels, suggestive of a pro-inflammatory state. Daily visceral rhythms of these genes were affected by a short photoperiod and/or HE diet. The 12:12HE, 5:19LE or 5:19HE animals had a higher proportion of larger adipocytes, indicating increased adipocyte hypertrophy. Collectively, the HE diet and/or shorter light exposure drives a dysfunctional adipose tissue phenotype. Daily rhythms are affected by a short photoperiod and HE diet in a site-specific manner. These findings provide mechanistic insight on the influence of disrupted circadian rhythms and HE diet on adipose tissue phenotype.

Fenofibrate Rescues Diabetes-Related Impairment of Ischemia-Mediated Angiogenesis by PPARα-Independent Modulation of Thioredoxin-Interacting Protein.

Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, reduces lower limb amputations in patients with type 2 diabetes. The mechanism is, however, unknown. In this study, we demonstrate that fenofibrate markedly attenuates diabetes-related impairment of ischemia-mediated angiogenesis. In a murine model of hindlimb ischemia, daily oral fenofibrate treatment restored diabetes-impaired blood flow recovery, foot movement, hindlimb capillary density, vessel diameter, and vascular endothelial growth factor signaling to nondiabetic levels in both wild-type and PPARα-knockout mice, indicating that these fenofibrate effects are largely PPARα independent. In vitro, fenofibric acid (FFA) rescued high glucose-induced (25 mmol/L) impairment of endothelial cell migration, tubulogenesis, and survival in a PPARα-independent manner. Interestingly, fenofibrate in vivo and FFA in vitro reversed high glucose-induced expression of thioredoxin-interacting protein (TXNIP), an exquisitely glucose-inducible gene previously identified as a critical mediator of diabetes-related impairment in neovascularization. Conversely, adenoviral overexpression of TXNIP abrogated the restorative effects of FFA on high glucose-impaired endothelial cell function in vitro, indicating that the effects of FFA are mediated by TXNIP. We conclude that fenofibrate rescues diabetic impairment in ischemia-mediated angiogenesis, in large part, by PPARα-independent regulation of TXNIP. These findings may therefore explain the reduction in amputations seen in patients with diabetes treated with fenofibrate.

The Role of Chemokines in Wound Healing.

Wound healing is a multistep process with four overlapping but distinct stages: hemostasis, inflammation, proliferation, and remodeling. An alteration at any stage may lead to the development of chronic non-healing wounds or excessive scar formation. Impaired wound healing presents a significant health and economic burden to millions of individuals worldwide, with diabetes mellitus and aging being major risk factors. Ongoing understanding of the mechanisms that underly wound healing is required for the development of new and improved therapies that increase repair. Chemokines are key regulators of the wound healing process. They are involved in the promotion and inhibition of angiogenesis and the recruitment of inflammatory cells, which release growth factors and cytokines to facilitate the wound healing process. Preclinical research studies in mice show that the administration of CCL2, CCL21, CXCL12, and a CXCR4 antagonist as well as broad-spectrum inhibition of the CC-chemokine class improve the wound healing process. The focus of this review is to highlight the contributions of chemokines during each stage of wound healing and to discuss the related molecular pathologies in complex and chronic non-healing wounds. We explore the therapeutic potential of targeting chemokines as a novel approach to overcome the debilitating effects of impaired wound healing.

The regulation of miRNAs by reconstituted high-density lipoproteins in diabetes-impaired angiogenesis.

Diabetic vascular complications are associated with impaired ischaemia-driven angiogenesis. We recently found that reconstituted high-density lipoproteins (rHDL) rescue diabetes-impaired angiogenesis. microRNAs (miRNAs) regulate angiogenesis and are transported within HDL to sites of injury/repair. The role of miRNAs in the rescue of diabetes-impaired angiogenesis by rHDL is unknown. Using a miRNA array, we found that rHDL inhibits hsa-miR-181c-5p expression in vitro and using a hsa-miR-181c-5p mimic and antimiR identify a novel anti-angiogenic role for miR-181c-5p. miRNA expression was tracked over time post-hindlimb ischaemic induction in diabetic mice. Early post-ischaemia when angiogenesis is important, rHDL suppressed hindlimb mmu-miR-181c-5p. mmu-miR-181c-5p was not detected in the plasma or within HDL, suggesting rHDL specifically targets mmu-miR-181c-5p at the ischaemic site. Three known angiogenic miRNAs (mmu-miR-223-3p, mmu-miR-27b-3p, mmu-miR-92a-3p) were elevated in the HDL fraction of diabetic rHDL-infused mice early post-ischaemia. This was accompanied by a decrease in plasma levels. Only mmu-miR-223-3p levels were elevated in the hindlimb 3 days post-ischaemia, indicating that rHDL regulates mmu-miR-223-3p in a time-dependent and site-specific manner. The early regulation of miRNAs, particularly miR-181c-5p, may underpin the rescue of diabetes-impaired angiogenesis by rHDL and has implications for the treatment of diabetes-related vascular complications.

Strikingly Different Atheroprotective Effects of Apolipoprotein A-I in Early- Versus Late-Stage Atherosclerosis.

Preclinical studies have shown benefit of apolipoprotein A-I (apoA-I)/high-density lipoprotein (HDL) raising in atherosclerosis; however, this has not yet translated into a successful clinical therapy. Our studies demonstrate that apoA-I raising is more effective at reducing early-stage atherosclerosis than late-stage disease, indicating that the timing of HDL raising is a critical factor in its atheroprotective effects. To date, HDL-raising clinical trials have only been performed in aged patients with advanced atherosclerotic disease. Our findings therefore provide insight, related to important temporal aspects of HDL raising, as to why the clinical trials have thus far been largely neutral.