rIPC (remote ischaemic preconditioning) is a phenomenon whereby short periods of ischaemia and reperfusion of a tissue or organ (e.g. mesentery, kidney) can protect a distant tissue or organ (e.g. heart) against subsequent, potentially lethal, ischaemia. We, and others, have shown that transient limb ischaemia can provide potent myocardial protection experimentally and clinically during cardiac surgery. Nonetheless, our understanding of the signal transduction from remote stimulus to local effect remains incomplete. The aim of the present study was to define the humoral nature of rIPC effector(s) from limb ischaemia and to study their local effects in isolated heart and cardiomyocyte models. Using a Langendorff preparation, we show that infarct size after coronary artery ligation and reperfusion was substantially reduced by rIPC in vivo, this stimulus up-regulating the MAPKs (mitogen-activating protein kinases) p42/p44, and inducing PKCepsilon (protein kinase Cepsilon) subcellular redistribution. Pre-treatment with the plasma and dialysate of plasma (obtained using 15 kDa cut-off dialysis membrane) from donor rabbits subjected to rIPC similarly protected against infarction. The effectiveness of the rIPC dialysate was abrogated by passage through a C18 hydrophobic column, but eluate from this column provided the same level of protection. The dialysate of rIPC plasma from rabbits and humans was also tested in an isolated fresh cardiomyocyte model of simulated ischaemia and reperfusion. Necrosis in cardiomyocytes treated with rIPC dialysate was substantially reduced compared with control, and was similar to cells pre-treated by 'classical' preconditioning. This effect, by rabbit rIPC dialysate, was blocked by pre-treatment with the opiate receptor blocker naloxone. In conclusion, in vivo transient limb ischaemia releases a low-molecular-mass (<15 kDa) hydrophobic circulating factor(s) which induce(s) a potent protection against myocardial ischaemia/reperfusion injury in Langendorff-perfused hearts and isolated cardiomyocytes in the same species. This cardioprotection is transferable across species, independent of local neurogenic activity, and requires opioid receptor activation.

Alzheimer's disease (AD) is caused by the cerebral deposition of ␤-amyloid (A␤), a 38 -43-amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP). Initial studies indicated that final cleavage of APP by the ␥-secretase (a complex containing presenilin and nicastrin) to produce A␤ occurred in the endosomal/lysosomal system. However, other studies showing a predominant endoplasmic reticulum localization of the ␥-secretase proteins and a neutral pH optimum of in vitro ␥-secretase assays have challenged this conclusion. We have recently identified nicastrin as a major lysosomal membrane protein. In the present work, we use Western blotting and immunogold electron microscopy to demonstrate that significant amounts of mature nicastrin, presenilin-1, and APP are co-localized with lysosomal associated membrane protein-1 (cAMP-1) in the outer membranes of lysosomes. Furthermore, we demonstrate that these membranes contain an acidic ␥-secretase activity, which is immunoprecipitable with an antibody to nicastrin. These experiments establish APP, nicastrin, and presenilin-1 as resident lysosomal membrane proteins and indicate that ␥-secretase is a lysosomal protease. These data reassert the importance of the lysosomal/endosomal system in the generation of A␤ and suggest a role for lysosomes in the pathophysiology of AD.

Costello syndrome is characterized by mental retardation, loose skin, coarse face, skeletal deformations, cardiomyopathy, and predisposition to numerous malignancies. The genetic origin of Costello syndrome has not yet been defined. Using immunohistochemistry and metabolic labeling with [3H]-valine, we have established that cultured skin fibroblasts obtained from patients with Costello syndrome did not assemble elastic fibers, despite an adequate synthesis of tropoelastin and normal deposition of the microfibrillar scaffold. We found that impaired production of elastic fibers by these fibroblasts is associated with a functional deficiency of the 67-kD elastin-binding protein (EBP), which is normally required to chaperone tropoelastin through the secretory pathways and to its extracellular assembly. Metabolic pulse labeling of the 67-kD EBP with radioactive serine and further chase of this tracer indicated that both normal fibroblasts and fibroblasts from patients with Costello syndrome initially synthesized comparable amounts of this protein; however, the fibroblasts from Costello syndrome patients quickly lost it into the conditioned media. Because the normal association between EBP and tropoelastin can be disrupted on contact with galactosugar-bearing moieties, and the fibroblasts from patients with Costello syndrome revealed an unusual accumulation of chondroitin sulfate-bearing proteoglycans (CD44 and biglycan), we postulate that a chondroitin sulfate may be responsible for shedding EBP from Costello cells and in turn for their impaired elastogenesis. This was further supported by the fact that exposure to chondroitinase ABC, an enzyme capable of chondroitin sulfate degradation, restored normal production of elastic fibers by fibroblasts from patients with Costello syndrome. We also present evidence that loss of EBP from fibroblasts of Costello syndrome patients is associated with an unusually high rate of cellular proliferation.