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 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

Chemokine binding protein 'M3' limits atherosclerosis in apolipoprotein E-/- mice

Chemokines are important in macrophage recruitment and the progression of atherosclerosis. The 'M3' chemokine binding protein inactivates key chemokines involved in atherosclerosis (e.g. CCL2, CCL5 and CX3CL1). We aimed to determine the effect of M3 on plaque development and composition. In vitro chemotaxis studies confirmed that M3 protein inhibited the activity of chemokines CCL2, CCL5 and CX3CL1 as primary human monocyte migration as well as CCR2-, CCR5- and CX3CR1-directed migration was attenuated by M3. In vivo, adenoviruses encoding M3 (AdM3) or green fluorescence protein (AdGFP; control) were infused systemically into apolipoprotein (apo)-E-/- mice. Two models of atherosclerosis development were used in which the rate of plaque progression was varied by diet including: (1) a 'rapid promotion' model (6-week high-fat-fed) and (2) a 'slow progression' model (12-week chow-fed). Plasma chemokine activity was suppressed in AdM3-infused mice as indicated by significantly less monocyte migration towards AdM3 mouse plasma ex vivo (29.56%, p = 0.014). In the 'slow progression' model AdM3 mice had reduced lesion area (45.3%, p = 0.035) and increased aortic smooth muscle cell α-actin expression (60.3%, p = 0.014). The reduction in lesion size could not be explained by changes in circulating inflammatory monocytes as they were higher in the AdM3 group. In the 'rapid promotion' model AdM3 mice had no changes in plaque size but reduced plaque macrophage content (46.8%, p = 0.006) and suppressed lipid deposition in thoracic aortas (66.9%, p<0.05). There was also a reduction in phosphorylated p65, the active subunit of NF-κb, in the aortas of AdM3 mice (37.3%, p<0.0001). M3 inhibited liver CCL2 concentrations in both models with no change in CCL5 or systemic chemokine levels. These findings show M3 causes varying effects on atherosclerosis progression and plaque composition depending on the rate of lesion progression. Overall, our studies support a promising role for chemokine inhibition with M3 for the treatment of atherosclerosis.Dhanya Ravindran, Anisyah Ridiandries, Laura Z. Vanags, Rodney Henriquez, Siân Cartland, Joanne T. M. Tan, Christina A. Bursil

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

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.Anisyah Ridiandries, Joanne T. M. Tan, Dhanya Ravindran, Helen Williams, Heather J. Medbury, Laura Lindsay, Clare Hawkins, Hamish C. G. Prosser and Christina A. Bursil

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.Jamie Morton, Shisan Bao, Laura Z. Vanags, Tania Tsatralis, Anisyah Ridiandries, Chung-Wah Siu, Kwong-Man Ng, Joanne T.M. Tan, David S. Celermajer, Martin K.C. Ng, Christina A. Bursil

M3 reduces lipid deposition in descending aortas.

<p>Two models of ‘rapid promotion’ or ‘slow progression’ of atherosclerosis were established in which a HFD or regular chow were fed and AdM3 or AdGFP were infused (<i>n</i> = 10–12/group). See “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173224#sec002" target="_blank">Materials and Methods</a>” for details. Images are representative sections of Oil Red O stained descending thoracic aortas. Oil Red O staining was quantified as a percentage of total thoracic aorta area for the ‘rapid promotion’ model and the ‘slow progression’ model. Data are mean±SEM. *<i>p</i><0.05.</p

M3 reduces plaque macrophage content and p65 activation when the rate of plaque development is more rapid.

<p>Two models of ‘rapid promotion’ or ‘slow progression’ of atherosclerosis were established in which a HFD or regular chow were fed and AdM3 or AdGFP were infused (<i>n</i> = 10–12/group). See “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173224#sec002" target="_blank">Materials and Methods</a>” for details. Upper panels are representative images of Mac-3⁺ macrophages (brown staining) in aortic sinus sections. Quantification of macrophage staining within plaques (μm²) for <b>A.</b> the ‘rapid promotion’ model and <b>B.</b> the ‘slow progression’ model. Phosphorylated p65 levels were measured in aortic arch samples for <b>C.</b> the ‘rapid promotion’ model and <b>D.</b> the ‘slow progression’ model. p65 mRNA levels were determined in aortic arch samples for <b>E.</b> the ‘rapid promotion’ model and <b>F.</b> the ‘slow progression’ model. Data expressed as mean±SEM. *<i>p</i><0.05, **<i>p</i><0.01, ****<i>p</i><0.0001.</p

Summary schematic comparing effects of broad-spectrum inhibition by M3 on atherosclerosis.

<p>The effects of broad-spectrum chemokine inhibition by M3 was demonstrated via two models of atherosclerosis—‘rapid promotion’ and ‘slow progression’. In the rapid promotion model M3 inhibits chemokine activity, causing suppression of inflammatory monocytes, reducing adherence to the endothelium so they accumulate in the circulation rather than enter the plaque. This leads to a reduction in plaque macrophages and a suppression in lipid deposition in the descending thoracic aorta, which develops later, but not in the aortic sinus that would contain plaque of a more advanced stage. In the ‘rapid promotion’ model, aortic phosphorylated p65 was lower which may also have contributed to the reduction in plaque macrophages. In the more gradual slower progressive model, we saw an increase in plaque SMCs, a marker of improved plaque stability, with an overall reduction in atherosclerotic lesion area in the aortic sinus and descending thoracic aorta. Despite the decrease in lesion area, there was no effect on circulating monocytes or plaque macrophage content. This may be explained by the decline in M3 protein and activity at the later time points of this study and the overall lower levels of inflammation in this chow-fed model.</p