Hamish Prosser

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

The role of cholesterol efflux in mechanisms of endothelial protection by HDL.

Purpose of review HDL and their main apolipoprotein (apo) constituent apoA-I are antiatherogenic. This has been predominantly attributed to the ability of apoA-I/HDL to efflux cholesterol from macrophages within atherosclerotic plaques. It is now emerging that a number of the protective properties of HDL may be due to their effects on the endothelium. Recent findings In addition to their well characterized anti-inflammatory and antioxidant effects, apoA-I and HDL regulate several other key biological pathways known to preserve endothelial function and promote vascular repair. The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, and the scavenger receptor B type 1 mediate multiple intracellular signaling pathways as well as the efflux of cholesterol and/or oxysterols in response to apoA-I/HDL. Although cholesterol efflux triggers a host of signaling events in endothelial cells, there is evidence that some of the beneficial actions of HDL may occur independently of efflux. Summary Current data suggest that in endothelial cells ABCA1 and ABCG1 mediate the activation of intracellular signaling pathways primarily through the efflux of cholesterol and oxysterols to apoA-I/HDL. Interaction between HDL and scavenger receptor B type 1 initiates the greatest number of known signaling pathways and there is evidence that some of these are activated independent of efflux.

High-density lipoproteins suppress chemokine expression and proliferation in human vascular smooth muscle cells

The inflammatory chemokines CCL2, CCL5, and CX3CL1 stimulate vascular smooth muscle cell (SMC) proliferation. High‐density lipoproteins (HDLs) exhibit potent cardioprotective and anti‐inflammatory properties. We therefore sought to determine the effect of reconstituted HDLs (rHDLs) on SMC chemokine expression and proliferation and elucidate the mechanisms. Preincubation of primary human SMCs with rHDLs containing apolipoprotein (apo)A‐I and phosphatidylcholine (20 μM, final apoA‐I concentration), before stimulation with TNF‐α, inhibited CCL2 (54%), CCL5 (38%), and CX3CL1 (33%) protein levels. The chemokine receptors CCR2 (29%) and CX3CR1 (22%) were also reduced by rHDLs. Incubation with rHDLs reduced the NF‐κB subunit p65 in the nucleus (39%) and phosphorylated IκBα (28%), both regulators of chemokine expression. Furthermore, rHDLs inhibited the upstream signaling proteins phosphoinositide 3‐kinase (37%) and phosphorylated Akt (pAkt, 49%). Incubation with rHDLs strikingly suppressed SMC proliferation (84%) and ERK phosphorylation (pERK, 29%). Finally, siRNA knockdown of the scavenger receptor SR‐B1 attenuated rHDL‐induced inhibition of SMC chemokine expression, p65, and proliferation, indicating that SR‐B1 plays a key role in mediating these effects. Thus, rHDLs reduce SMC chemokine expression (via NF‐κB/pAkt inhibition) and proliferation (via pERK inhibition). This has important implications for preventing the pathogenesis of neointimal hyperplasia, the main cause of early vein graft/stent failure.—Van der Vorst, E. P. C., Vanags, L. Z., Dunn, L. L., Prosser, H. C., Rye, K.‐A., Bursill, C. A. High‐density lipoproteins suppress chemokine expression and proliferation in human vascular smooth muscle cells. FASEB J. 27, 1413–1425 (2013). www.fasebj.org

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.—Tan, J. T. M., Prosser, H. C. G., Vanags, L. Z., Monger, S. A., Ng, M. K. C., Bursill, C. A. High‐density lipoproteins augment hypoxia‐induced angiogenesis via regulation of post‐translational modulation of hypoxia inducible factor 1α. FASEB J. 28, 206–217 (2014). www.fasebj.org

Multifunctional Regulation of Angiogenesis by High Density Lipoproteins

AIMS High-density lipoproteins (HDL) exert striking anti-inflammatory effects and emerging evidence suggests that they may augment ischaemia-mediated neovascularization. We sought to determine whether HDL conditionally regulates angiogenesis, depending on the pathophysiological context by (i) inhibiting inflammation-induced angiogenesis, but also; (ii) enhancing ischaemia-mediated angiogenesis. METHODS AND RESULTS Intravenously delivered apolipoprotein (apo) A-I attenuated neovascularization in the murine femoral collar model of inflammation-induced angiogenesis, compared with phosphate-buffered saline infused C57BL6/J mice (58%), P < 0.05. Conversely, apoA-I delivery augmented neovessel formation (75%) and enhanced blood perfusion (45%) in the murine hindlimb ischaemia model, P < 0.05. Reconstituted HDL (rHDL) was tested on key angiogenic cell functions in vitro. rHDL inhibited human coronary artery endothelial cell migration (37.9 and 76.9%), proliferation (15.7 and 40.4%), and tubulogenesis on matrigel (52 and 98.7%) when exposed to two inflammatory stimuli: tumour necrosis factor-α (TNF-α) and macrophage-conditioned media (MCM). In contrast, rHDL significantly augmented hypoxia-stimulated migration (36.9%), proliferation (135%), and tubulogenesis (22.9%), P < 0.05. Western blot and RT-PCR analyses revealed that these divergent actions of rHDL were associated with conditional regulation of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and VEGF receptor 2, which were attenuated in response to TNF-α (40.4, 41.0, and 33.2%) and MCM (72.5, 30.7, and 69.5%), but augmented by rHDL in hypoxia (39.8, 152.6, and 15.7%%), all P < 0.05. CONCLUSION HDL differentially regulates angiogenesis dependent upon the pathophysiological setting, characterized by suppression of inflammation-associated angiogenesis, and conversely, by the enhancement of hypoxia-mediated angiogenesis. This has significant implications for therapeutic modulation of neovascularization.

High-Density Lipoproteins Rescue Diabetes-Impaired Angiogenesis via Scavenger Receptor Class B Type I

Disordered neovascularization and impaired wound healing are important contributors to diabetic vascular complications. We recently showed that high-density lipoproteins (HDLs) enhance ischemia-mediated neovascularization, and mounting evidence suggests HDL have antidiabetic properties. We therefore hypothesized that HDL rescue diabetes-impaired neovascularization. Streptozotocin-induced diabetic mice had reduced blood flow recovery and neovessel formation in a hindlimb ischemia model compared with nondiabetic mice. Reconstituted HDL (rHDL) infusions in diabetic mice restored blood flow recovery and capillary density to nondiabetic levels. Topical rHDL application rescued diabetes-impaired wound closure, wound angiogenesis, and capillary density. In vitro, rHDL increased key mediators involved in hypoxia-inducible factor-1α (HIF-1α) stabilization, including the phosphoinositide 3-kinase/Akt pathway, Siah1, and Siah2, and suppressed the prolyl hydroxylases (PHD) 2 and PHD3. rHDL rescued high glucose–induced impairment of tubulogenesis and vascular endothelial growth factor (VEGF) A protein production, a finding associated with enhanced phosphorylation of proangiogenic mediators VEGF receptor 2 and endothelial nitric oxide synthase. Siah1/2 small interfering RNA knockdown confirmed the importance of HIF-1α stability in mediating rHDL action. Lentiviral short hairpin RNA knockdown of scavenger receptor class B type I (SR-BI) in vitro and SR-BI−/− diabetic mice in vivo attenuated rHDL rescue of diabetes-impaired angiogenesis, indicating a key role for SR-BI. These findings provide a greater understanding of the vascular biological effects of HDL, with potential therapeutic implications for diabetic vascular complications.

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‐chemokineclass inhibition attenuates pathological angiogenesis while preserving physiological angiogenesis. FASEB J. 31, 1179–1192 (2017). www.fasebj.org

Erratum. High-Density Lipoproteins Rescue Diabetes-Impaired Angiogenesis via Scavenger Receptor Class B Type I. Diabetes 2016;65:3091–3103

Erratum. High-Density Lipoproteins Rescue Diabetes-Impaired Angiogenesis via Scavenger Receptor Class B Type I. Diabetes 2016;65:3091–3103 DOI: 10.2337/db17-er04b Joanne T.M. Tan, Hamish C.G. Prosser, Louise L. Dunn, Laura Z. Vanags, Anisyah Ridiandries, Tania Tsatralis, Laura Leece, Zoë E. Clayton, Sui Ching G. Yuen, Stacy Robertson, Yuen Ting Lam, David S. Celermajer, Martin K.C. Ng, and Christina A. Bursill In the article listed above, Laura Lecce’s name was erroneously listed as Laura Leece. The authors regret the error.