Abstract
High levels of plasma lipoprotein(a) (Lp(a)) are associated with multiple forms of cardiovascular disease (CVD). Lp(a) consists of a low-density lipoprotein (LDL) attached
to the plasminogen homologue, apolipoprotein(a) (apo(a)). Multiple receptors are reported
to mediate Lp(a) uptake, but the effect of each is inconsistent or incomplete. A previous
study from our lab discovered that the plasminogen receptor, plasminogen receptor with a
C-terminal lysine (PlgRKT), promoted Lp(a) uptake in liver cells. An association between
Lp(a) and another plasminogen receptor Annexin A2 was documented in the literature. The
major aim of this study was to determine the metabolism pathway of Lp(a) via plasminogen receptors.
The first aim of this thesis was to determine the endogenous expression of PlgRKT, Annexin
A2 and its conjugating partner S100 calcium-binding protein A10 (S100A10) in human
hepatocellular carcinoma (HepG2) cells and haploid human fibroblast-like (HAP1) cells.
The RT-qPCR and immunocytochemistry results suggested that these receptors are
expressed in both cell lines. This motivated us to further define the mechanism of PlgRKT
and to investigate the role of Annexin A2 and S100A10 in the endocytosis of Lp(a) in the
second aim of the study. HepG2 and HAP1 cells were utilised for overexpression and
knockout of plasminogen receptors. The uptake of Lp(a), LDL, apo(a) and endocytic cargos
were visualised and quantified by confocal microscopy and by western blotting. The uptake
of both Lp(a) and apo(a), but not LDL, was significantly increased in HepG2 and HAP1 cells
overexpressing PlgRKT, Annexin A2 or S100A10. Conversely, both Lp(a) and apo(a), but
not LDL, uptake was significantly reduced in HAP1 cells in which PlgRKT and S100A10
were knocked out. Surface binding studies in HepG2 cells showed that overexpression of
PlgRKT, but not Annexin A2 or S100A10, increased Lp(a) binding. Annexin A2 and
S100A10, on the other hand, appeared to regulate macropinocytosis with both proteins
significantly increasing the uptake of the macropinocytosis marker, dextran, when
overexpressed in HepG2 and HAP1 cells, and a knockout of S100A10 significantly reducing
dextran uptake. Bringing these observations together, the effect of a phosphoinositide -3-
kinase (PI3K) inhibitor, known to inhibit macropinocytosis was tested on Lp(a) uptake.
Results showed a concentration-dependent reduction confirming that Lp(a) uptake was
indeed mediated by macropinocytosis.
Lastly, this thesis examined the effect of drugs known to affect plasminogen receptor
expression and/or macropinocytosis on Lp(a) uptake. These drugs included the macrophage colony-stimulating factor (M-CSF), a known plasminogen receptor regulator. An increase in the expression of the PlgRKT was seen with M-CSF treatment in HepG2 cells. Interestingly, Lp(a) and apo(a) uptake and Lp(a) cell surface binding were significantly increased with M-CSF treatment, while LDL uptake was unaffected, indicating that the apo(a) component of Lp(a) was mediating Lp(a) uptake via PlgRKT and by enhancing the cell surface binding of Lp(a).
A recent study from our lab revealed that the macropinocytosis inhibitor, imipramine, a
tricyclic antidepressant, enhanced Lp(a) uptake. This study also reported that a selective
serotonin reuptake inhibitor (SSRI) antidepressant citalopram, also promoted Lp(a) uptake
similarly. Interestingly, imipramine enhanced the expression of PlgRKT and citalopram
promotes the expression of S100A10. As both antidepressants enhance serotonin levels,
serotonin treatment was also investigated. Serotonin treatment increased Lp(a) uptake by
enhancing the expression of PlgRKT and S100A10, indicating that serotonin promotes Lp(a) uptake by enhancing both cell surface binding and macropinocytosis.
In conclusion, this PhD thesis has uncovered a novel pathway for Lp(a) endocytosis
involving multiple plasminogen receptors that both enhance surface binding and stimulate
macropinocytosis of Lp(a). The involvement of these plasminogen receptors was further
corroborated by testing the effect of different drugs on the expression of plasminogen
receptors or Lp(a) uptake. These novel findings may help to discover new therapeutical
agents to lower circulating Lp(a).