P731High-density lipoproteins rescue diabetes-impaired angiogenesis by restoring cellular metabolic reprogramming responses to hypoxia

Abstract

Abstract Background We have previously identified that high-density lipoproteins (HDL) rescue hypoxia-induced angiogenesis in diabetes, however the underlying mechanisms remain unknown. A central component of hypoxia-induced angiogenesis is the reprogramming of endothelial cell (EC) metabolism to improve tolerance of hypoxia at the site of injury/repair, including following myocardial infarction. Pyruvate dehydrogenase kinase 4 (PDK4) suppresses mitochondrial respiration in hypoxia to decrease oxygen consumption and preserve cell survival. Diabetes impairs this adaptation to hypoxia, causing cellular dysfunction and may underlie delayed angiogenic responses to ischaemia in hyperglycaemia. Purpose To determine the role of metabolic reprogramming in angiogenesis and diabetes and the effect of reconstituted HDL (rHDL). Methods and results Using an in vitro functional angiogenesis assay, siRNA-mediated knockdown of PDK4 impaired human coronary artery endothelial cell (HCAEC) tubulogenesis in hypoxia by 82% versus siScrambled controls (P<0.0001). HCAECs were treated with rHDL (20μM, apolipoproteinA-I + phosphatidylcholine) or PBS (vehicle) and exposed to glucose (5 or 25 mM, 72 h), then normoxia or hypoxia (1.2% O2, 6 h). PDK4 expression was increased by 65% in hypoxia versus the normoxia control (P<0.05). By contrast, in high glucose PDK4 expression failed to increase in response to hypoxia. Incubation with rHDL rescued this impairment and elevated PDK4 expression by 40% in hypoxia and in high glucose (P<0.01), versus the PBS control. In parallel, rHDL rescued high glucose-impaired tubulogenesis in hypoxia by 64% versus the PBS/normoxia control (P<0.001). We next used a murine model of diabetic wound healing that tracks angiogenesis in the wound bed. We found daily topical application of rHDL (50μg/wound/day) increased wound angiogenesis by 10% (as assessed by Laser Doppler perfusion imaging) and promoted CD31+ neovessel formation by 46% in diabetic wounds, supporting an increased rate of wound closure in diabetic animals of 30% (P<0.05). rHDL treated wounds from diabetic mice had a striking increase in PDK4 gene (180%) and protein expression (350%) in the early-mid stages (24 h and 3-day time points, P<0.05) post-wounding, when the angiogenic response to wound ischaemia is most important. Conclusions We have demonstrated that the key protein controlling the metabolic reprogramming response to hypoxia, PDK4, plays an important role in endothelial cell angiogenesis. We also show that the PDK4 and angiogenesis responses to hypoxia are impaired in high glucose and can be rescued by rHDL. In vivo we find that topical rHDL increases wound angiogenesis and PDK4 levels, explaining the enhanced rate of wound closure in diabetic mice and has implications for improving cardiovascular outcomes for diabetic patients following myocardial infarction.