A new approach to heart valve tissue engineering: mimicking the heart ventricle with a ventricular assist device in a novel bioreactor

The `biomimetic` approach to tissue engineering usually involves the use of a bioreactor mimicking physiological parameters whilst supplying nutrients to the developing tissue. Here we present a new heart valve bioreactor, having as its centrepiece a ventricular assist device (VAD), which exposes the cell-scaffold constructs to a wider array of mechanical forces. The pump of the VAD has two chambers: a blood and a pneumatic chamber, separated by an elastic membrane. Pulsatile air-pressure is generated by a piston-type actuator and delivered to the pneumatic chamber, ejecting the fluid in the blood chamber. Subsequently, applied vacuum to the pneumatic chamber causes the blood chamber to fill. A mechanical heart valve was placed in the VAD`s inflow position. The tissue engineered (TE) valve was placed in the outflow position. The VAD was coupled in series with a Windkessel compliance chamber, variable throttle and reservoir, connected by silicone tubings. The reservoir sat on an elevated platform, allowing adjustment of ventricular preload between 0 and 11 mmHg. To allow for sterile gaseous exchange between the circuit interior and exterior, a 0.2 mu m filter was placed at the reservoir. Pressure and flow were registered downstream of the TE valve. The circuit was filled with culture medium and fitted in a standard 5% CO(2) incubator set at 37 degrees C. Pressure and flow waveforms were similar to those obtained under physiological conditions for the pulmonary circulation. The `cardiomimetic` approach presented here represents a new perspective to conventional biomimetic approaches in TE, with potential advantages. Copyright (C) 2010 John Wiley & Sons, Ltd.

A catecholamine transporter from the human parasite Schistosoma mansoni with low affinity for psychostimulants

The trematode Schistosoma mansoni is the primary cause of schistosomiasis, a devastating neglected tropical disease that affects 200 million individuals. Identifying novel therapeutic targets for the treatment of schistosomiasis is therefore of great public interest. The catecholamines norepinephrine (NE) and dopamine (DA) are essential for the survival of the parasite as they cause muscular relaxation and a lengthening in the parasite and thereby control movement. Here we characterize a novel dopamine/norepinephrine transporter (SmDAT) gene transcript, from S. mansoni. The SmDAT is expressed in the adult form and in the sporocyst form (infected snails) of the parasite, and also in the egg and miracidium stage. It is absent in the cercariae stage but curiously a transcript missing the exon encoding transmembrane domain 8 was identified in this stage. Heterologous expression of the cDNA in mammalian cells resulted in saturable, dopamine transport activity with an apparent affinity for dopamine comparable to that of the human dopamine transporter. Efflux experiments reveal notably higher substrate selectivity compared with its mammalian counterparts as amphetamine is a much less potent efflux elicitor against SmDAT compared to the human DAT. Pharmacological characterization of the SmDAT revealed that most human DAT inhibitors including psychostimulants such as cocaine were significantly less potent in inhibiting SmDAT. Like DATs from other simpler organisms the pharmacology for SmDAT was more similar to the human norepinephrine transporter. We were not able to identify other dopamine transporting carriers within the completed parasite genome and we hypothesize that the SmDAT is the only catecholamine transporter in the parasite and could be responsible for not only clearing DA but also NE. (C) 2011 Elsevier B.V. All rights reserved.

Two allelic isoforms of the serotonin transporter from Schistosoma mansoni display electrogenic transport and high selectivity for serotonin

The human blood fluke Schistosoma mansoni is the primary cause of schistosomiasis, a debilitating disease that affects 200 million individuals in over 70 countries. The biogenic amine serotonin is essential for the survival of the parasite and serotonergic proteins are potential novel drug targets for treating schistosomiasis. Here we characterize two novel serotonin transporter gene transcripts, SmSERT-A and SmSERT-B, from S. mansoni. Southern blot analysis shows that the two mRNAs are the products of different alleles of a single SmSERT gene locus. The two SmSERT forms differ in three amino acid positions near the N-terminus of the protein. Both SmSERTs are expressed in the adult form and in the sporocyst form (infected snails) of the parasite, but are absent from all other stages of the parasite`s complex life cycle. Heterologous expression of the two cDNAs in mammalian cells resulted in saturable, sodium-dependent serotonin transport activity with an apparent affinity for serotonin comparable to that of the human serotonin transporter. Although the two SmSERTs are pharmacologically indistinguishable from each other, efflux experiments reveal notably higher substrate selectivity for serotonin compared with their mammalian counterparts. Several well-established substrates for human SERT including (+/-)MDMA, S-(+)amphetamine, RU 24969, and m-CPP are not transported by SmSERTs, underscoring the higher selectivity of the schistosomal isoforms. Voltage-clamp recordings of SmSERT substrate-elicited currents confirm the substrate selectivity observed in efflux experiments and suggest that it may be possible to exploit the electrogenic nature of SmSERT to screen for compounds that target the parasite in vivo. (C) 2009 Elsevier B.V. All rights reserved.