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.

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.

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.

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.

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 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.

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.

VEGFR2 is activated by high-density lipoproteins and plays a key role in the proangiogenic action of HDL in ischemia.

High-density lipoproteins augment hypoxia-induced angiogenesis by inducing the key angiogenic vascular endothelial growth factor A (VEGFA) and total protein levels of its receptor 2 (VEGFR2). The activation/phosphorylation of VEGFR2 is critical for mediating downstream, angiogenic signaling events. This study aimed to determine whether reconstituted high-density lipoprotein (rHDL) activates VEGFR2 phosphorylation and the downstream signaling events and the importance of VEGFR2 in the proangiogenic effects of rHDL in hypoxia. In vitro, rHDL increased VEGFR2 activation and enhanced phosphorylation of downstream, angiogenic signaling proteins ERK1/2 and p38 MAPK in hypoxia. Incubation with a VEGFR2-neutralizing antibody attenuated rHDL-induced phosphorylation of VEGFR2, ERK1/2, p38 MAPK, and tubule formation. In a murine model of ischemia-driven neovascularization, rHDL infusions enhanced blood perfusion and augmented capillary and arteriolar density. Infusion of a VEGFR2-neutralizing antibody ablated those proangiogenic effects of rHDL. Circulating Sca1+/CXCR4+ angiogenic progenitor cell levels, important for neovascularization in response to ischemia, were higher in rHDL-infused mice 3 d after ischemic induction, but that did not occur in mice that also received the VEGFR2-neutralizing antibody. In summary, VEGFR2 has a key role in the proangiogenic effects of rHDL in hypoxia/ischemia. These findings have therapeutic implications for angiogenic diseases associated with an impaired response to tissue ischemia.-Cannizzo, C. M., Adonopulos, A. A., Solly, E. L., Ridiandries, A., Vanags, L. Z., Mulangala, J., Yuen, S. C. G., Tsatralis, T., Henriquez, R., Robertson, S., Nicholls, S. J., Di Bartolo, B. A., Ng, M. K. C., Lam, Y. T., Bursill, C. A., Tan, J. T. M. VEGFR2 is activated by high-density lipoproteins and plays a key role in the proangiogenic action of HDL in ischemia.

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.

CC-chemokine class inhibition attenuates pathological angiogenesis while preserving physiological angiogenesis.

Increasing evidence shows that CC-chemokines promote inflammatory-driven angiogenesis, with little to no effect on hypoxia-mediated angiogenesis. Inhibition of the CC-chemokine class may therefore affect angiogenesis differently depending on the pathophysiological context. We compared the effect of CC-chemokine inhibition in inflammatory and physiological conditions. In vitro, the broad-spectrum CC-chemokine inhibitor "35K" inhibited inflammatory-induced endothelial cell proliferation, migration, and tubulogenesis, with more modest effects in hypoxia. In vivo, adenoviruses were used to overexpress 35K (Ad35K) and GFP (AdGFP, control virus). Plasma chemokine activity was suppressed by Ad35K in both models. In the periarterial femoral cuff model of inflammatory-driven angiogenesis, overexpression of 35K inhibited adventitial neovessel formation compared with control AdGFP-infused mice. In contrast, 35K preserved neovascularization in the hindlimb ischemia model and had no effect on physiological neovascularization in the chick chorioallantoic membrane assay. Mechanistically, 2 key angiogenic proteins (VEGF and hypoxia-inducible factor-1α) were conditionally regulated by 35K, such that expression was inhibited in inflammation but was unchanged in hypoxia. In conclusion, CC-chemokine inhibition by 35K suppresses inflammatory-driven angiogenesis while preserving physiological ischemia-mediated angiogenesis via conditional regulation of VEGF and hypoxia-inducible factor-1α. CC-chemokine inhibition may be an alternative therapeutic strategy for suppressing diseases associated with inflammatory angiogenesis without inducing the side effects caused by global inhibition.- Ridiandries, A., Tan, J. T. M., Ravindran, D., Williams, H., Medbury, H. J., Lindsay, L., Hawkins, C., Prosser, H. C. G., Bursill, C. A. CC-chemokine class inhibition attenuates pathological angiogenesis while preserving physiological angiogenesis.

Estrogen Receptor Control of Atherosclerotic Calcification and Smooth Muscle Cell Osteogenic Differentiation.

OBJECTIVE: Vascular calcification is associated with increased risk of myocardial infarction and stroke. The objective of this work was to examine the ability of 17ß-estradiol (E2) to stimulate calcification of vascular smooth muscle cells (VSMC) in vivo, using aged apolipoprotein E-null mice with advanced atherosclerotic lesions, and subsequently to explore underlying mechanisms in vitro. APPROACH AND RESULTS: Silastic E2 capsules were implanted into male and female apolipoprotein E-null mice aged 34 weeks. Plaque and calcified area were measured in the aortic sinus and innominate artery after 8 weeks. Immunohistochemical analysis examined expression of the estrogen receptors (estrogen receptor alpha and estrogen receptor beta [ERß]). VSMC expression of osteogenic markers was examined using digital polymerase chain reaction. Advanced atherosclerotic lesions were present in all mice at the end of 8 weeks. In both male and female mice, E2 increased calcified area in a site-specific manner in the aortic sinus independently of plaque growth or lipid levels and occurred in association with a site-specific decrease in the proportion of ERß-positive intimal cells. Calcified lesions expressed collagen I and bone sialoprotein, with decreased matrix Gla protein. In vitro, E2 suppressed ERß expression and increased VSMC mineralization, demonstrating increased collagen I and II, osteocalcin and bone sialoprotein, and reduced matrix Gla protein and osteopontin. Antagonism or RNA silencing of estrogen receptor alpha, ERß, or both further increased VSMC mineralization. CONCLUSIONS: We have demonstrated that E2 can drive calcification in advanced atherosclerotic lesions by promoting the differentiation of VSMC to osteoblast-like cells, a process which is augmented by inhibition of estrogen receptor alpha or ERß activity.

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 Role of High-Density Lipoproteins in Diabetes and Its Vascular Complications.

Almost 600 million people are predicted to have diabetes mellitus (DM) by 2035. Diabetic patients suffer from increased rates of microvascular and macrovascular complications, associated with dyslipidaemia, impaired angiogenic responses to ischaemia, accelerated atherosclerosis, and inflammation. Despite recent treatment advances, many diabetic patients remain refractory to current approaches, highlighting the need for alternative agents. There is emerging evidence that high-density lipoproteins (HDL) are able to rescue diabetes-related vascular complications through diverse mechanisms. Such protective functions of HDL, however, can be rendered dysfunctional within the pathological milieu of DM, triggering the development of vascular complications. HDL-modifying therapies remain controversial as many have had limited benefits on cardiovascular risk, although more recent trials are showing promise. This review will discuss the latest data from epidemiological, clinical, and pre-clinical studies demonstrating various roles for HDL in diabetes and its vascular complications that have the potential to facilitate its successful translation.

Exploring the Roles of CREBRF and TRIM2 in the Regulation of Angiogenesis by High-Density Lipoproteins.

Angiogenesis, the process of forming new blood vessels, is crucial in the physiological response to ischemia, though it can be detrimental as part of inflammation and tumorigenesis. We have previously shown that high-density lipoproteins (HDL) modulate angiogenesis in a context-specific manner via distinct classical signalling pathways, enhancing hypoxia-induced angiogenesis while suppressing inflammatory-driven angiogenesis. Whether additional novel targets exist to account for these effects are unknown. A microarray approach identified two novel genes, cyclic-adenosine-monophosphate-response-element-binding protein 3 regulatory factor (CREBRF) and tripartite motif-containing protein 2 (TRIM2) that were upregulated by reconstituted HDL (rHDL). We measured CREBRF and TRIM2 expression in human coronary artery endothelial cells following incubation with rHDL and exposure to either hypoxia or an inflammatory stimulus. We found that CREBRF and TRIM2 mRNA were significantly upregulated by rHDL, particularly in response to its phospholipid component 1-palmitoyl-2-linoleoyl-phosphatidylcholine, however, protein expression was not significantly altered. Knockdown of TRIM2 impaired endothelial cell tubulogenesis in vitro in both hypoxia and inflammation, implying a necessary role in angiogenesis. Furthermore, TRIM2 knockdown attenuated rHDL-induced tubule formation in hypoxia, suggesting that it is important in mediating the pro-angiogenic action of rHDL. Our study has implications for understanding the regulation of angiogenesis in both of these pathophysiological contexts by HDL.

Plasma activated coating immobilizes apolipoprotein A-I to stainless steel surfaces in its bioactive form and enhances biocompatibility.

We utilized a plasma activated coating (PAC) to covalently bind the active component of high density lipoproteins (HDL), apolipoprotein (apo) A-I, to stainless steel (SS) surfaces. ApoA-I suppresses restenosis and thrombosis and may therefore improve SS stent biocompatibility. PAC-coated SS significantly increased the covalent attachment of apoA-I, compared to SS alone. In static and dynamic flow thrombosis assays, PAC+apoA-I inhibited thrombosis and reduced platelet activation marker p-selectin. PAC+apoA-I reduced smooth muscle cell attachment and proliferation, and augmented EC attachment to PAC. We then coated PAC onto murine SS stents and found it did not peel or delaminate following crimping/expansion. ApoA-I was immobilized onto PAC-SS stents and was retained as a monolayer when exposed to pulsatile flow in vivo in a murine stent model. In conclusion, ApoA-I immobilized on PAC withstands pulsatile flow in vivo and retains its bioactivity, exhibiting anti-thrombotic and anti-restenotic properties, demonstrating the potential to improve stent biocompatibility.

Reconstituted high-density lipoproteins promote wound repair and blood flow recovery in response to ischemia in aged mice.

BACKGROUND: The average population age is increasing and the incidence of age-related vascular complications is rising in parallel. Impaired wound healing and disordered ischemia-mediated angiogenesis are key contributors to age-impaired vascular complications that can lead to amputation. High-density lipoproteins (HDL) have vasculo-protective properties and augment ischemia-driven angiogenesis in young animals. We aimed to determine the effect of reconstituted HDL (rHDL) on aged mice in a murine wound healing model and the hindlimb ischemia (HLI) model. METHODS: Murine wound healing model-24-month-old aged mice received topical application of rHDL (50 µg/wound/day) or PBS (vehicle control) for 10 days following wounding. Murine HLI model-Femoral artery ligation was performed on 24-month-old mice. Mice received rHDL (40 mg/kg) or PBS, intravenously, on alternate days, 1 week pre-surgery and up to 21 days post ligation. For both models, blood flow perfusion was determined using laser Doppler perfusion imaging. Mice were sacrificed at 10 (wound healing) or 21 (HLI) days post-surgery and tissues were collected for histological and gene analyses. RESULTS: Daily topical application of rHDL increased the rate of wound closure by Day 7 post-wounding (25 %, p < 0.05). Wound blood perfusion, a marker of angiogenesis, was elevated in rHDL treated wounds (Days 4-10 by 22-25 %, p < 0.05). In addition, rHDL increased wound capillary density by 52.6 %. In the HLI model, rHDL infusions augmented blood flow recovery in ischemic limbs (Day 18 by 50 % and Day 21 by 88 %, p < 0.05) and prevented tissue necrosis and toe loss. Assessment of capillary density in ischemic hindlimb sections found a 90 % increase in rHDL infused animals. In vitro studies in fibroblasts isolated from aged mice found that incubation with rHDL was able to significantly increase the key pro-angiogenic mediator vascular endothelial growth factor (VEGF) protein (25 %, p < 0.05). CONCLUSION: rHDL can promote wound healing and wound angiogenesis, and blood flow recovery in response to ischemia in aged mice. Mechanistically, this is likely to be via an increase in VEGF. This highlights a potential role for HDL in the therapeutic modulation of age-impaired vascular complications.

The Role of CC-Chemokines in the Regulation of Angiogenesis.

Angiogenesis, the formation of new blood vessels, is critical for survival and in the regenerative response to tissue injury or ischemia. However, in diseases such as cancer and atherosclerosis, inflammation can cause unregulated angiogenesis leading to excessive neovascularization, which exacerbates disease. Current anti-angiogenic therapies cause complete inhibition of both inflammatory and ischemia driven angiogenesis causing a range of side effects in patients. Specific inhibition of inflammation-driven angiogenesis would therefore be immensely valuable. Increasing evidence suggests that the CC-chemokine class promotes inflammation-driven angiogenesis, whilst there is little evidence for a role in ischemia-mediated angiogenesis. The differential regulation of angiogenesis by CC-chemokines suggests it may provide an alternate strategy to treat angiogenesis associated pathological diseases. The focus of this review is to highlight the significant role of the CC-chemokine class in inflammation, versus ischemia driven angiogenesis, and to discuss the related pathologies including atherosclerosis, cancer, and rheumatoid arthritis. We examine the pros and cons of anti-angiogenic therapies currently in clinical trials. We also reveal novel therapeutic strategies that cause broad-spectrum inhibition of the CC-chemokine class that may have future potential for the specific inhibition of inflammatory angiogenesis.

High-density lipoproteins augment hypoxia-induced angiogenesis via regulation of post-translational modulation of hypoxia-inducible factor 1α.

Increasing evidence suggests that high-density lipoproteins (HDLs) promote hypoxia-induced angiogenesis. The hypoxia-inducible factor 1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway is important in hypoxia and is modulated post-translationally by prolyl hydroxylases (PHD1-PHD3) and E3 ubiquitin ligases (Siah1 and Siah2). We aimed to elucidate the mechanisms by which HDLs augment hypoxia-induced angiogenesis. Preincubation (16 h) of human coronary artery endothelial cells with reconstituted high-density lipoprotein (rHDL) containing apolipoprotein A-I (apoA-I) and phosphatidylcholine (20 µM, final apoA-I concentration), before hypoxia, increased Siah1 (58%) and Siah2 (88%) mRNA levels and suppressed PHD2 (32%) and PHD3 (45%) protein levels compared with hypoxia-induced control levels. After Siah1/2 small interfering RNA knockdown, rHDL was unable to suppress PHD2/3 and failed to induce HIF-1α, VEGF, and tubulogenesis in hypoxia. Inhibition of the upstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway also abrogated the effects of rHDL. Furthermore, knockdown of the scavenger receptor SR-BI attenuated rHDL-induced elevations in Siah1/2 and tubulogenesis in hypoxia, indicating that SR-BI plays a key role. Finally, the importance of VEGF in mediating the ability of rHDL to drive hypoxia-induced angiogenesis was confirmed using a VEGF-neutralizing antibody. In summary, rHDL augments the HIF-1α/VEGF pathway via SR-BI and modulation of the post-translational regulators of HIF-1α (PI3K/Siahs/PHDs). HDL-induced augmentation of angiogenesis in hypoxia may have implications for therapeutic modulation of ischemic injury.

Detection of atherosclerotic plaques with HDL-like porphyrin nanoparticles using an intravascular dual-modality optical coherence tomography and fluorescence system.

Atherosclerosis is the build-up of fatty plaques within blood vessel walls, which can occlude the vessels and cause strokes or heart attacks. It gives rise to both structural and biomolecular changes in the vessel walls. Current single-modality imaging techniques each measure one of these two aspects but fail to provide insight into the combined changes. To address this, our team has developed a dual-modality imaging system which combines optical coherence tomography (OCT) and fluorescence imaging that is optimized for a porphyrin lipid nanoparticle that emits fluorescence and targets atherosclerotic plaques. Atherosclerosis-prone apolipoprotein (Apo)e-/- mice were fed a high cholesterol diet to promote plaque development in descending thoracic aortas. Following infusion of porphyrin lipid nanoparticles in atherosclerotic mice, the fiber-optic probe was inserted into the aorta for imaging, and we were able to robustly detect a porphyrin lipid-specific fluorescence signal that was not present in saline-infused control mice. We observed that the nanoparticle fluorescence colocalized in areas of CD68+ macrophages. These results demonstrate that our system can detect the fluorescence from nanoparticles, providing complementary biological information to the structural information obtained from simultaneously acquired OCT.

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 (NP)-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 levels. 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 NP uptake. In this review, we discuss an array of inorganic, carbon-based, and lipid-based NPs 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 NPs that target a range of macrophage-related functions such as lipoprotein oxidation, cholesterol efflux, vascular inflammation, and defective efferocytosis. We also provide examples of NP 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 NPs. Whilst this is not without its challenges, the array of multifunctional capabilities that are possible in NP 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.