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

Abstract 630: Broad-spectrum Inhibition of the CC-Chemokine Class Accelerates Wound Healing.

Introduction: Wound healing is a multistep process involving inflammation, proliferation and remodelling at the wound site. Macrophages are important in the inflammatory stage to remove debris, however excessive macrophage accumulation may prolong the inflammatory response leading to prolonged wound healing, excessive scar formation and loss of function at the injury site. In the wound healing process, the CC-chemokine class is involved in the recruitment of macrophages to the wound site. Inhibition of the CC-chemokine class to reduce macrophage infiltration may present as strategy to increase wound closure and reduce scar formation. Aim: To determine if broad spectrum CC-chemokine inhibitor, 35K, can improve wound healing by reducing the inflammatory response and preventing scar formation. Results: Two full-thickness wounds were created on the dorsum, one each side of the midline, of C57BL6 mice. Wounds were treated topically with PBS or 35K protein daily for 10 days. Wounds treated with 35K closed significantly faster than PBS treated wounds between days 4 - 6 (p&lt;0.05) post-wounding. Laser Doppler measurements revealed 35K significantly increased blood flow at the early (day 3-4) and late stages (day 10) of wound repair. Ten days after wounding, collagen was significantly lower in the 35K treated wounds (25.3% p&lt;0.05). There were also reductions in neovessels (CD31, 39%, p&lt;0.001), arterioles (alpha actin, 48% p&lt;0.01) and macrophages (CD68, 25%), as a percentage of wound area, in 35K treated wounds. In wound tissue collected on day 4 (early stage) post-wounding, 35K treatment had an inhibitory effect on CCL2 and CCL5 protein by 41% (p&lt;0.05) and 36% (p&lt;0.05) respectively, and were reduced by 22% and 66% (p&lt;0.05) respectively at day 10 (late stage) wound healing. Consistent with this, mRNA levels of p65 also decreased in both day 4 (34%, p&lt;0.05) and day 10 (62%, p&lt;0.05) wounds treated with 35K. Conclusion: The CC-chemokine class appears to play a key role in wound repair as topical application of CC-chemokine inhibitor 35K promoted wound healing. This was likely to be via inhibition of inflammation and promotion of blood flow recovery in the early stages of wound repair, thereby resulting in less scar formation. </jats:p

Abstract 423: CC-Chemokine Class Inhibition Attenuates Inflammatory Induced Pathological Angiogenesis Whilst Preserving Ischaemia Driven Physiological Angiogenesis

Introduction: Angiogenesis is critical for survival and in the regenerative response to tissue hypoxia. An imbalance in its regulation causes excessive angiogenesis, exacerbating inflammatory diseases such as cancer and atherosclerosis. Increasing evidence suggests the CC-chemokine class promote inflammatory-driven angiogenesis but, in contrast, have no role in hypoxia-driven angiogenesis. Inhibition of the CC-chemokine class may therefore regulate angiogenesis differently depending on the pathophysiological context. Aim: Using the broad-spectrum CC-chemokine inhibitor ‘35K’, compare the role of the CC-chemokine class in inflammatory versus ischaemia driven angiogenesis. Results: Adenoviruses expressing GFP (AdGFP) or 35K (Ad35K) were injected into mice 3 days prior surgery. Using the murine femoral cuff model of inflammatory angiogenesis, we found a significant decrease in adventitial capillaries (39.7%) and arterioles (75.5%) in the Ad35K group compared to the AdGFP controls, p&lt;0.05. Ad35K mice also had significantly less macrophages (CD68, 32.5%). In contrast, 35K had no effect on markers of neovascularisation including: blood flow recovery and hindlimb capillary density, in the murine hind limb ischaemia model. Consistent with our in vivo findings, in vitro angiogenic assays with endothelial cells, revealed that 35K protein had inhibitory effects on inflammation-induced tubule formation (95.8%), proliferation (88.3%) and migration (15.8%), p&lt;0.05. In hypoxia the inhibitory effects of 35K were more modest for tubule formation (45.2%) and proliferation (43.8%), and migration was completely preserved. Under inflammatory conditions 35K inhibited VEGF (53.4%) and HIF-1α (87.3%) cell protein levels, yet in hypoxia 35K had no effect on VEGF and had a more modest inhibitory effect on HIF-1α (65.4%). Conclusion: Broad-spectrum CC-chemokine inhibition by 35K inhibits inflammation-induced angiogenesis, whilst preserving ischaemia-driven angiogenesis in vitro and in vivo. CC-Chemokine inhibition may be therapeutic strategy to reduce pathological angiogenic diseases without the severe side effects of current therapies caused by complete inhibition in both contexts. </jats:p

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

Confirmation of gene transfer and inhibition of plasma chemokine activity by M3.

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 (n = 10–12/group). See “Materials and Methods” for details. Successful gene transfer and expression following adenoviral delivery was determined by Western immunoblotting. A. Culture media from Ad293 cells infected with AdM3 as well as plasma from mice infused with AdGFP and AdM3 were assessed from 4 to 12 weeks post adenoviral delivery following immunoprecipitation with anti-c-myc agarose beads. B. Chemokine activity was measured using the Boyden chamber migration assay. Calcein-AM labelled monocytes were allowed to migrate towards plasma from mice infused with AdGFP or AdM3 (n = 10–12/group). Data are mean±SEM. *pp<0.0001.</p