title: High density lipoprotein composition and function in healthy pregnancy and preeclampsia
Creators: name: Patanapirunhakit, Patamat
Publisher: name: University of Glasgow
Year: 2022

description: 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.