| Summary: | The BBB is a biological firewall that carefully regulates the cerebral microenvironment by acting 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.
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