Recombinant PAI-1 therapy restores myoendothelial junctions and erectile function in PAI-1-deficient mice.

Myoendothelial junctions are specialised projections of cell : cell contact through the internal elastic lamina between endothelial cells and vascular smooth muscle cells. These junctions allow for endothelial cells and vascular smooth muscle cells to make direct membrane apposition and are involved in cell : cell communication. In this study, we evaluated for the presence of myoendothelial junctions in murine corporal tissue and used plasminogen activator inhibitor (PAI)-1-deficient mice, which lack myoendothelial junctions, to determine whether myoendothelial junctions affect erectile function. Transmission electron microscopy demonstrated the presence of myoendothelial junctions in the corporal tissue of wild-type mice and confirmed the decreased junction numbers in the tissue of PAI-1(-/-) mice. A potential role for myoendothelial junctions in tumescence was established; in that, PAI-1(-/-) mice demonstrated a significantly longer time to achieve maximal intracavernous pressure. Treatment of PAI-1(-/-) mice with recombinant PAI-1 restored the number of myoendothelial junctions in the corporal tissue and also induced a significant decrease in time to maximal corporal pressures. Myoendothelial junctions were similarly identified in the human corporal tissue. These results suggest a critical role for myoendothelial junctions in erectile pathophysiology and therapies aimed at restoring myoendothelial junction numbers in the corporal tissue may provide a novel therapy for erectile dysfunction.

Assessing Spns2-dependent S1P Transport as a Prospective Therapeutic Target.

S1P (sphingosine 1-phosphate) receptor modulator (SRM) drugs interfere with lymphocyte trafficking by downregulating lymphocyte S1P receptors. While the immunosuppressive activity of SRM drugs has proved useful in treating autoimmune diseases such as multiple sclerosis, that drug class is beset by on-target liabilities such as initial dose bradycardia. The S1P that binds to cell surface lymphocyte S1P receptors is provided by S1P transporters. Mice born deficient in one of these, spinster homolog 2 (Spns2), are lymphocytopenic and have low lymph S1P concentrations. Such observations suggest that inhibition of Spns2-mediated S1P transport might provide another therapeutically beneficial method to modulate immune cell positioning. We report here results using a novel S1P transport blocker (STB), SLF80821178, to investigate the consequences of S1P transport inhibition in rodents. We found that SLF80821178 is efficacious in a multiple sclerosis model but - unlike the SRM fingolimod - neither decreases heart rate nor compromises lung endothelial barrier function. Notably, although Spns2 null mice have a sensorineural hearing defect, mice treated chronically with SLF80821178 have normal hearing acuity. STBs such as SLF80821178 evoke a dose-dependent decrease in peripheral blood lymphocyte counts, which affords a reliable pharmacodynamic marker of target engagement. However, the maximal reduction in circulating lymphocyte counts in response to SLF80821178 is substantially less than the response to SRMs such as fingolimod (50% vs. 90%) due to a lesser effect on T lymphocyte sub-populations by SLF80821178. Finally, in contrast to results obtained with Spns2 deficient mice, lymph S1P concentrations were not significantly changed in response to administration of STBs at doses that evoke maximal lymphopenia, which indicates that current understanding of the mechanism of action of S1P transport inhibitors is incomplete.

Interaction between nitric oxide signaling and gap junctions: effects on vascular function.

Nitric oxide signaling, through eNOS (or possibly nNOS), and gap junction communication are essential for normal vascular function. While each component controls specific aspects of vascular function, there is substantial evidence for cross-talk between nitric oxide signaling and the gap junction proteins (connexins), and more recently, protein-protein association between eNOS and connexins. This review will examine the evidence for interaction between these pathways in normal and diseased arteries, highlight the questions that remain about the mechanisms of their interaction, and explore the possible interaction between nitric oxide signaling and the newly discovered pannexin channels. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Endoplasmic reticulum-mediated signalling in cellular microdomains.

The endoplasmic reticulum (ER) is a prime mediator of cellular signalling due to its functions as an internal cellular store for calcium, as well as a site for synthesis of proteins and lipids. Its peripheral network of sheets and tubules facilitates calcium and lipid signalling, especially in areas of the cell that are more distant to the main cytoplasmic network. Specific membrane proteins shape the peripheral ER architecture and influence the network stability to project into restricted spaces. The signalling microdomains are anatomically separate from the cytoplasm as a whole and exhibit localized protein, ion channel and cytoskeletal element expression. Signalling can also occur between the ER and other organelles, such as the Golgi or mitochondria. Lipids made in the ER membrane can be sent to the Golgi via specialized transfer proteins and specific phospholipid synthases are enriched at ER-mitochondria junctions to more efficiently expedite phospholipid transfer. As a hub for protein and lipid synthesis, a store for intracellular calcium [Ca2+ ]i and a mediator of cellular stress, the ER is an important cellular organelle. Its ability to organize into tubules and project into restricted spaces allows for discrete and temporal signalling, which is important for cellular physiology and organism homoeostasis.

Intercellular Ca2+ signaling in alveolar epithelial cells through gap junctions and by extracellular ATP.

Inter- and extracellular-mediated changes in intracellular Ca2+ concentration ([Ca2+]i) can ensure coordinated tissue function in the lung. Cultured rat alveolar epithelial cells (AECs) have been shown to respond to secretagogues with increases in [Ca2+]i and have been shown to be gap junctionally coupled. However, communication of [Ca2+]i changes in AECs is not well defined. Monolayers of AECs were mechanically perturbed and monitored for [Ca2+]i changes. Perturbation of AECs was administered by a glass probe to either mechanically stimulate or mechanically wound individual cells. Both approaches induced a change in [Ca2+]i in the stimulated cell that was propagated to neighboring cells (Ca2+ waves). A connexin mimetic peptide shown to uncouple gap junctions eliminated Ca2+ waves in mechanically stimulated cells but had no effect on mechanically wounded cells. In contrast, apyrase, an enzyme that effectively removes ATP from the extracellular milieu, had no effect on mechanically stimulated cells but severely restricted mechanically wounded Ca2+ wave propagation. We conclude that AECs have the ability to communicate coordinated Ca2+ changes using both gap junctions and extracellular ATP.

Modulation of pulmonary alveolar type II cell phenotype and communication by extracellular matrix and KGF.

The alveolar epithelium consists of two cell types, alveolar type I (AT1) and alveolar type II (AT2) cells. We have recently shown that 7-day-old cultures of AT2 cells grown on a type I collagen/fibronectin matrix develop phenotypic characteristics of AT1 cells, display a distinct connexin profile, and coordinate mechanically induced intercellular Ca(2+) changes via gap junctions (25). In this study, we cultured AT2 cells for 7 days on matrix supplemented with laminin-5 and/or in the presence of keratinocyte growth factor. Under these conditions, cultured AT2 cells display AT2 type morphology, express the AT2-specific marker surfactant protein C, and do not express AT1-specific cell marker aquaporin 5, all consistent with maintenance of AT2 phenotype. These AT2-like cells also coordinate mechanically induced intercellular Ca(2+) signaling, but, unlike AT1-like cells, do so by using extracellular nucleotide triphosphate release. Additionally, cultured cells that retain AT2 cell-specific markers express connexin profiles different from cultured cells with AT1 characteristics. The parallel changes in intercellular Ca(2+) signaling with cell differentiation suggest that cell signaling mechanisms are an intrinsic component of lung alveolar cell phenotype. Because lung epithelial injury is accompanied by extracellular matrix and growth factor changes, followed by extensive cell division, differentiation, and migration of AT2 progenitor cells, we suggest that similar changes may be vital to the lung recovery and repair process in vivo.