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 apolipoprotein A-I mimetic peptide, ETC-642, reduces chronic vascular inflammation in the rabbit

<p>Abstract</p> <p>Background</p> <p>High-density lipoproteins (HDL) and their main apolipoprotein, apoA-I, exhibit anti-inflammatory properties. The development of peptides that mimic HDL apolipoproteins offers a promising strategy to reduce inflammatory disease. This study aimed to compare the anti-inflammatory effects of ETC-642, an apoA-I mimetic peptide, with that of discoidal reconstituted HDL (rHDL), consisting of full-length apoA-I complexed with phosphatidylcholine, in rabbits with chronic vascular inflammation.</p> <p>Results</p> <p>New Zealand White rabbits (n = 10/group) were placed on chow supplemented with 0.2% (w/w) cholesterol for 6-weeks. The animals received two infusions of saline, rHDL (8 mg/kg apoA-I) or ETC-642 (30 mg/kg peptide) on the third and fifth days of the final week. The infusions of rHDL and ETC-642 were able to significantly reduce cholesterol-induced expression of intracellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the thoracic aorta (p < 0.05). When isolated rabbit HDL was pre-incubated with human coronary artery endothelial cells (HCAECs), prior to stimulation with TNF-α, it was found that HDL from ETC-642 treated rabbits were more effective at inhibiting the TNF-α-induced increase in ICAM-1, VCAM-1 and p65 than HDL isolated from saline treated rabbits (p < 0.05). There were, however, no changes in HDL lipid composition between treatment groups.</p> <p>Conclusions</p> <p>Infusion of ETC-642 causes anti-inflammatory effects that are comparable to rHDL in an animal model of chronic vascular inflammation and highlights that apoA-I mimetic peptides present a viable strategy for the treatment of inflammatory disease.</p

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

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

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

M3 inhibits chemokine activity <i>in vitro</i>.

<p>The Boyden chamber migration assay was used to assess <b>A.</b> CCR2-, <b>B.</b> CCR5- and <b>C.</b> CX₃CR1-directed cell migration <i>in vitro</i>. 293T cells were transfected with plasmids encoding CCR2, CCR5 or CX₃CR1. <b>D.</b> Migration of human primary monocytes towards CCL2, CCL5, CX₃CL1 and CXCL12 in Boyden chamber migration assay. Representative images of Calcein AM labelled monocytes migrated to the underside of transwell membranes with and without purified M3 protein (100 ng/ mL) are shown. Data are mean±SEM. *<i>p</i><0.05, **<i>p</i><0.01.</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