Abstract
The BBB is a biological firewall that carefully regulates the cerebral microenvironment byacting as a physical, metabolic and transport barrier to molecules bound for the brain. This
selectively permeable interface was modelled in this thesis using the recently established
immortalised human cerebral microvascular endothelial cell line (hCMEC/D3) to investigate
interactions with endogenously and exogenously derived molecules of clinical significance.
The endogenous molecules in question are the cationic amino acids (CAA) L-arginine, the
precursor for nitric oxide (NO), and asymmetric dimethylarginine (ADMA), an endogenously
derived analogue of L-arginine that acts as a potent inhibitor of NO production. As well as
being an important vasodilator, NO has regulatory roles in the brain and on the BBB itself.
Transport mechanisms utilised by L-arginine are known, but are not fully understood for ADMA
– particularly so at the BBB. This is of clinical significance giving the emerging role of ADMA in
many brain and cerebrovascular diseases. Understanding these transport mechanisms and
other interactions of ADMA with the BBB is therefore very important for the study of disease
emergence, detection and progression. We discovered in the hCMEC/D3s that high
concentrations of ADMA could induce endothelial dysfunction in a BBB permeability model,
leading to an increase in paracellular permeability to the paracellular marker FITC-dextran
(40kDa). We also illustrated interactions of ADMA with a variety transport proteins, basing the
study design on our observed L-arginine interactions. The CAA transport system y+ was heavily
implicated for both molecules through the use of established inhibitors. Furthermore, the
expression of CAT-1, the best known protein from this group was confirmed in the
hCMEC/D3s. We also found evidence of an efflux transport system for ADMA (but not Larginine),
implicating the neutral and CAA transporter ATB0,+. The protein expression of ATB0,+
was also confirmed in the cells. Intracellular ADMA was even shown to induce transstimulation
of extracellular L-arginine, providing evidence for a role of ADMA in the 'L-arginine
paradox', a phenomenon observed in vivo that administered L-arginine can alleviate the
effects of NO reduction (such as vasoconstriction), despite there being 20-30 times the amount
of L-arginine present to saturate the NO producing enzyme.
In summary, our endogenous molecule findings from this thesis identify the likely transport
mechanisms used by ADMA and implicate ADMA in endothelial dysfunction as well as the ‘Larginine
paradox’. These data are not only important with regards to the brain, but apply to other microvascular endothelia such as those found in peripheral cardiovascular system,
where ADMA remains a major area of investigation.
The exogenous molecules studied during this PhD are drugs currently used to treat the
second-stage of human African Trypanosomiasis (HAT). HAT is a neglected parasitic disease
that continues to persist in sub-Saharan Africa. It is fatal if untreated. Recently, it has been
described that eflornithine and nifurtimox combination therapy (NECT), improves the efficacy
of both drugs compared to their monotherapy, although why this happens remains unclear.
We hypothesised that it may be due to improved CNS delivery, although we failed to show
improvements in accumulation of either eflornithine or nifurtimox with NECT or when the
individual drugs were in combinations with the other anti-HAT drugs. Interestingly, the
combination of eflornithine and pentamidine caused a decrease in eflornithine accumulation,
implicating an unidentified pentamidine-sensitive transport system – possibly an adenosinesensitive
influx transporter. The cellular influx transport mechanisms used by eflornithine has
been suggested to be those used by CAA due to the structural similarity of eflornithine with
the CAA ornithine and so this was studied. We revealed in the hCMEC/D3s that eflornithine
had degrees of sensitivity to a variety of transport mechanisms, in which system y+ appears to
be the principal influx mechanism. Similar anti-HAT drug combination therapy studies with
nifurtimox were performed and also illustrated a significant interaction with pentamidine;
although conversely to eflornithine we demonstrated an increase in nifurtimox accumulation
as a result of nifurtimox-pentamidine combination. Previous in situ observations by our group
suggested nifurtimox was a substrate for efflux transport systems at the BBB that are separate
from P-gp and this too was investigated, identifying the well known drug efflux pump BCRP as
the principal nifurtimox efflux transporter.
With regards to exogenous molecules, we provide evidence of CAA influx mechanisms for
the anti-HAT drug eflornithine and an efflux system for nifurtimox, principally involving BCRP.
We also found that NECT and combination therapy of eflornithine or nifurtimox with the other
anti-HAT drugs did not improve eflornithine or nifurtimox accumulation when compared to
controls – except when pentamidine was combined with nifurtimox. This finding suggests that
nifurtimox-pentamidine combination may improve efficacy of nifurtimox in the field.
Collectively, these data further demonstrate of the suitability of the hCMEC/D3 cell line as a
powerful tool for human in vitro BBB investigation across a range of study areas.
Date of Award | 2012 |
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Original language | English |
Awarding Institution |
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Supervisor | Sarah Thomas (Supervisor) & Giovanni Mann (Supervisor) |