The SLC1 high-affinity glutamate and neutral amino acid transporter family.

Glutamate transporters play important roles in the termination of excitatory neurotransmission and in providing cells throughout the body with glutamate for metabolic purposes. The high-affinity glutamate transporters EAAC1 (SLC1A1), GLT1 (SLC1A2), GLAST (SLC1A3), EAAT4 (SLC1A6), and EAAT5 (SLC1A7) mediate the cellular uptake of glutamate by the co-transport of three sodium ions (Na(+)) and one proton (H(+)), with the counter-transport of one potassium ion (K(+)). Thereby, they protect the CNS from glutamate-induced neurotoxicity. Loss of function of glutamate transporters has been implicated in the pathogenesis of several diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. In addition, glutamate transporters play a role in glutamate excitotoxicity following an ischemic stroke, due to reversed glutamate transport. Besides glutamate transporters, the SLC1 family encompasses two transporters of neutral amino acids, ASCT1 (SLC1A4) and ASCT2 (SLC1A5). Both transporters facilitate electroneutral exchange of amino acids in neurons and/or cells of the peripheral tissues. Some years ago, a high resolution structure of an archaeal homologue of the SLC1 family was determined, followed by the elucidation of its structure in the presence of the substrate aspartate and the inhibitor d,l-threo-benzyloxy aspartate (d,l-TBOA). Historically, the first few known inhibitors of SLC1 transporters were based on constrained glutamate analogs which were active in the high micromolar range but often also showed off-target activity at glutamate receptors. Further development led to the discovery of l-threo-ß-hydroxyaspartate derivatives, some of which effectively inhibited SLC1 transporters at nanomolar concentrations. More recently, small molecule inhibitors have been identified whose structures are not based on amino acids. Activators of SLC1 family members have also been discovered but there are only a few examples known.

A novel concept for scaffold-free vessel tissue engineering: self-assembly of microtissue building blocks.

Current scientific attempts to generate in vitro tissue-engineered living blood vessels (TEBVs) show substantial limitations, thereby preventing routine clinical use. In the present report, we describe a novel biotechnology concept to create living small diameter TEBV based exclusively on microtissue self-assembly (living cellular re-aggregates). A novel bioreactor was designed to assemble microtissues in a vascular shape and apply pulsatile flow and circumferential mechanical stimulation. Microtissues composed of human artery-derived fibroblasts (HAFs) and endothelial cells (HUVECs) were accumulated and cultured for 7 and 14 days under pulsatile flow/mechanical stimulation or static culture conditions with a diameter of 3mm and a wall thickness of 1mm. The resulting vessels were analyzed by immunohistochemistry for extracellular matrix (ECM) and cell phenotype (von Willebrand factor, alpha-SMA, Ki67, VEGF). Self-assembled microtissues composed of fibroblasts displayed significantly accelerated ECM formation compared to monolayer cell sheets. Accumulation of vessel-like tissue occurred within 14 days under both, static and flow/mechanical stimulation conditions. A layered tissue formation was observed only in the dynamic group, as indicated by luminal aligned alpha-SMA positive fibroblasts. We could demonstrate that self-assembled cell-based microtissues can be used to generate small diameter TEBV. The significant enhancement of ECM expression and maturation, together with the pre-vascularization capacity makes this approach highly attractive in terms of generating functional small diameter TEBV devoid of any foreign material.