High density lipoprotein composition and function in healthy pregnancy and preeclampsia

High density lipoprotein (HDL) is well-known for its ability to protect vascular function. It is hypothesised that HDL could be a protective factor for vascular function in healthy pregnancy that fails in preeclampsia resulting in endothelial dysfunction. The aims of this thesis were to determine HDL composition and function throughout healthy pregnancy and in preeclampsia. HDL protein and sphingolipid compositions were determined using proteomic and lipid analysis approaches in healthy pregnancy from pre-pregnancy to postpartum. There were 16 proteins changed in HDL throughout gestation. Higher HDL α-1-antitrypsin content was observed in the first trimester (median LFQ intensity 5.8x108 at 8.4 weeks and 4.2 x10 pre-pregnancy, p=0.044) and may protect the reverse cholesterol transport (RCT) function of HDL. HDL content of sphingosine-1- phosphate (S1P) and ceramides increased in the third trimester (S1P 101.1±9.8, 108.0±19.8 and 128.8±15.6 ng/mL, C18:0 12.0±2.6, 16.9±6.8 and 20.2±6.2 ng/mL, C20:0 30.6±10.6, 40.32±13.0 and 52.6±17.5 ng/mL, C22:0 196.0±101.8, 266.9±119.6 and 362.7±164.2 ng/mL, C24:1 137.2±48.4, 151.8±54.8 and 201.4±60.4 ng/mL for 16, 25 and 35 weeks, respectively), suggesting improved anti-inflammatory, vasodilatory and RCT function of HDL. In preeclampsia, the third trimester HDL protein composition, both late and early onset, was compared to HDL from gestation-matched healthy pregnancy. ApoA-IV was lower in early-onset preeclampsia (median LFQ intensity 3.0x109 and 3.9x109 for early-onset preeclampsia and control, p=0.018), suggesting that HDL may be less efficient at inhibiting excessive inflammation in early-onset preeclampsia. The LFQ intensities of antithrombin-III (9.3x107 and 1.4x108 for late-onset preeclampsia and control, p=0.045) and β-2-glycoprotein 1 (5.9x108and 9.0x108 for late-onset preeclampsia and control, p=0.030) were lower in late-onset preeclampsia which might suggest both activating and inhibiting effects on the coagulation cascade. Next, assays to assess HDL function, including HDL effects on vascular reactivity using wire myography and the anti-clotting potential of HDL, were developed. The wire myography protocol was able to assess HDL effect on vasoconstriction responses of human pregnant visceral adipose tissue arteries, but not vasodilatory responses because vessels contracted to the maximum dose of noradrenaline did not maintain a stable contraction. An antithrombotic functional assay of HDL was developed by adapting an existing thrombin generation assay protocol used for plasma. NaBr carried over from HDL isolation process inhibited thrombin production in the assay, leading to difficulty in standardizing thrombin generation activity measured in HDL samples. Finally, an isolation protocol for HDL was developed where potential extracellular vesicle (EV) contamination in HDL samples was removed using a combination of size-exclusion chromatography and sequential ultracentrifugation. ApoA-I recovery in HDL isolated by this protocol was 39.8%. Comparison of protein composition of EV and HDL (EV-free HDL) isolated by this protocol and HDL isolated by the gold standard method, sequential ultracentrifugation, suggested that the EV proteins that were removed from HDL by this isolation protocol were mostly immunoglobulins and complement proteins. Several essential HDL proteins were partially removed from the HDL particles during the isolation of EV-free HDL, for example, apoC-IV, apoA-IV and paroxonase-1. In conclusion, this thesis provided evidence of pregnancy and preeclampsia-associated changes of HDL protein, S1P and ceramide in a manner that may improve or decrease HDL function. HDL functional assays are needed to validate the relevance of these HDL composition findings

Sphingolipids in HDL – potential markers for adaptation to pregnancy?

Plasma high density lipoprotein (HDL) exhibits many functions that render it an effective endothelial protective agent and may underlie its potential role in protecting the maternal vascular endothelium during pregnancy. In non-pregnant individuals, the HDL lipidome is altered in metabolic disease compared to healthy individuals and is linked to reduced cholesterol efflux, an effect that can be reversed by lifestyle management. Specific sphingolipids such as sphingosine-1-phosphate (S1P) have been shown to mediate the vaso-dilatory effects of plasma HDL via interaction with the endothelial nitric oxide synthase pathway. This review describes the relationship between plasma HDL and vascular function during healthy pregnancy and details how this is lost in pre-eclampsia, a disorder of pregnancy associated with widespread endothelial dysfunction. Evidence of a role for HDL sphingolipids, in particular S1P and ceramide, in cardiovascular disease and in healthy pregnancy and pre-eclampsia is discussed. Available data suggest that HDL-S1P and HDL-ceramide can mediate vascular protection in healthy pregnancy but not in preeclampsia. HDL sphingolipids thus are of potential importance in the healthy maternal adaptation to pregnancy

High-density lipoprotein’s vascular protective functions in metabolic and cardiovascular disease - could extracellular vesicles be at play?

High-density lipoprotein (HDL) is a circulating complex of lipids and proteins known primarily for its role in reverse cholesterol transport and consequent protection from atheroma. In spite of this, therapies aimed at increasing HDL concentration do not reduce the risk of cardiovascular disease (CVD), and as such focus has shifted towards other HDL functions protective of vascular health – including vasodilatory, anti-inflammatory, antioxidant and anti-thrombotic actions. It has been demonstrated that in disease states such as CVD and conditions of insulin resistance such as Type 2 diabetes mellitus (T2DM), HDL function is impaired owing to changes in the abundance and function of HDL-associated lipids and proteins, resulting in reduced vascular protection. However, the gold standard density ultracentrifugation technique used in the isolation of HDL also co-isolates extracellular vesicles (EVs). EVs are ubiquitous cell-derived particles with lipid bilayers that carry a number of lipids, proteins and DNA/RNA/miRNAs involved in cell-to-cell communication. EVs transfer their bioactive load through interaction with cell surface receptors, membrane fusion and endocytic pathways, and have been implicated in both cardiovascular and metabolic diseases – both as protective and pathogenic mediators. Given that studies using density ultracentrifugation to isolate HDL also co-isolate EVs, biological effects attributed to HDL may be confounded by EVs. We hypothesise that some of HDL’s vascular protective functions in cardiovascular and metabolic disease may be mediated by EVs. Elucidating the contribution of EVs to HDL functions will provide better understanding of vascular protection and function in conditions of insulin resistance and potentially provide novel therapeutic targets for such diseases