Publications

Sphingosine 1-phosphate (S1P) is a biologically active lysophospholipid that serves as a key regulator of cellular differentiation and survival. Immune stimuli increase S1P synthesis and secretion by mast cells and platelets, implicating this molecule in tissue responses to injury and inflammation. Binding of S1P to G(i) protein-coupled receptors activates phosphatidylinositol 3-kinase and Akt in a variety of tissues. To elucidate the mechanisms by which S1P enhances adult cardiac myocyte survival during hypoxia, we used a mouse cell culture system in which S1P(1) receptors were observed to transduce signals from exogenous S1P, an S1P(1) receptor antibody with agonist properties, and the pharmacological agents FTY720 and SEW2871. S1P(1) receptor mRNA and protein were abundantly expressed by adult mouse cardiac myocytes. S1P-S1P(1) receptor axis enhancement of myocyte survival during hypoxia was abolished by phosphatidylinositol 3-kinase inhibition. S1P(1) receptor function was closely associated with activation of Akt, inactivation of GSK-3beta, and reduction of cytochrome c release from heart mitochondria. These observations highlight the importance of S1P(1) receptors on ventricular myocytes as mediators of inducible resistance against cellular injury during severe hypoxic stress.

A very potent and specific inhibitor of mitochondrial NADH:ubiquinone oxidoreductase (complex I), a derivative of NADH (NADH-OH) has recently been discovered (Kotlyar, A. B., Karliner, J. S., and Cecchini, G. (2005) FEBS Lett. 579, 4861-4866). Here we present a quantitative analysis of the interaction of NADH-OH and other nucleotides with oxidized and reduced complex I in tightly coupled submitochondrial particles. Both the rate of the NADH-OH binding and its affinity to complex I are strongly decreased in the presence of succinate. The effect of succinate is completely reversed by rotenone, antimycin A, and uncoupler. The relative affinity of ADP-ribose, a competitive inhibitor of NADH oxidation, is also shown to be significantly affected by enzyme reduction (KD of 30 and 500 microM for oxidized and the succinate-reduced enzyme, respectively). Binding of NADH-OH is shown to abolish the succinate-supported superoxide generation by complex I. Gradual inhibition of the rotenone-sensitive uncoupled NADH oxidase and the reverse electron transfer activities by NADH-OH yield the same final titration point (approximately 0.1 nmol/mg of protein). The titration of NADH oxidase appears as a straight line, whereas the titration of the reverse reaction appears as a convex curve. Possible models to explain the different titration patterns for the forward and reverse reactions are briefly discussed.

The field of tissue engineering has yielded several successes in early clinical trials of regenerative medicine using living cells seeded into biodegradable scaffolds. In contrast to methods that combine biomaterials with living cells, we have developed an approach that uses culture surfaces grafted with the temperature-responsive polymer poly(N-isoproplyacrylamide) that allows for controlled attachment and detachment of living cells via simple temperature changes. Using cultured cell sheets harvested from temperature-responsive surfaces, we have established cell sheet engineering to create functional tissue sheets to treat a wide range of diseases from corneal dysfunction to esophageal cancer, tracheal resection, and cardiac failure. Additionally, by exploiting the unique ability of cell sheets to generate three-dimensional tissues composed of only cultured cells and their deposited extracellular matrix, we have also developed methods to create thick vascularized tissues as well as, organ-like systems for the heart and liver. Cell sheet engineering therefore provides a novel alternative for regenerative medicine approaches that require the re-creation of functional tissue structures.