These observations suggest that proper retinal blood vessel formation requires an adequate resident microglial population because diminished microglia are associated with decreased vascularity in models of ischemic retinopathy and retinal vascular development. In light of these findings, the traditional definition of microglia as merely immunocompetent cells should be reconsidered to encompass this new function related to blood vessel formation.

Lysophosphatidic acid (LPA) is a bioactive molecule involved in inflammation, immunity, wound healing, and neoplasia. Its pleiotropic actions arise presumably by interaction with their cell surface G protein-coupled receptors. Herein, the presence of the specific nuclear lysophosphatidic acid receptor-1 (LPA 1 R) was revealed in unstimulated porcine cerebral microvascular endothelial cells (pCMVECs), LPA 1 R stably transfected HTC4 rat hepatoma cells, and rat liver tissue using complementary approaches, including radioligand binding experiments, electron-and cryomicroscopy, cell fractionation, and immunoblotting with three distinct antibodies. Coimmunoprecipitation studies in enriched plasmalemmal fractions of unstimulated pCMVEC showed that LPA 1 Rs are dually sequestrated in caveolin-1 and clathrin subcompartments, whereas in nuclear fractions LPA 1 R appeared primarily in caveolae. Immunofluorescent assays using a cell-free isolated nuclear system confirmed LPA 1 R and caveolin-1 co-localization. In pCMVEC, LPA-stimulated increases in cyclooxygenase-2 and inducible nitricoxide synthase RNA and protein expression were insensitive to caveolea-disrupting agents but sensitive to LPAgenerating phospholipase A 2 enzyme and tyrosine kinase inhibitors. Moreover, LPA-induced increases in Ca 2؉ transients and/or iNOS expression in highly purified rat liver nuclei were prevented by pertussis toxin, phosphoinositide 3-kinase/Akt inhibitor wortmannin and Ca 2؉ chelator and channel blockers EGTA and SK&F96365, respectively. This study describes for the first time the nucleus as a potential organelle for LPA intracrine signaling in the regulation of pro-inflammatory gene expression.In the mammalian system, LPA 1 signaling cascades regulate important cellular processes, including gene expression, cell proliferation and growth, cell survival and death, and cell motility and secretion (1-3). These plethora of activities are characteristic features of inflammation that occur in various physiological as well as pathological states (e.g. ontogenic change, wound healing, cancer, etc.) (1-3). In humans, physiological responses induced by LPA arise from specific interactions with at least three genetically identified receptors designated LPA 1 , LPA 2 , and LPA 3 (formerly referred to as EDG 2 , EDG 4 , and EDG 7 receptors, respectively), which belong to the heptahelical transmembrane-spanning G protein-coupled receptor (GPCR) superfamily (4). These receptors show a broad, virtually distinct distribution and may couple in a cell-dependent manner to numerous heterotrimeric G proteins. In this context, LPA 1 and LPA 2 receptors have been shown to interact with G i/o , G q/11/14 , and G 12/13 proteins, whereas the LPA 3 receptor combines with G i/o and G q/11/14 proteins (5). Although many responses induced by extracellular LPA can result from its interaction with plasma membrane GPCRs, there is circumstantial evidence for an intracrine mode of action of LPA. For instance, putative biogenesis (e.g. secretory and cytosolic calcium-depen...

Changes in the levels of transforming growth factor (TGF)-beta cytokines or receptors observed during the progression of several inflammatory and fibrotic disorders have been used to implicate these cytokines in the pathophysiology of these diseases. Although correlative, these studies were inconclusive because they were unable to demonstrate actual continuous TGF-beta-mediated signaling in the involved tissues. We reasoned that the phosphorylation state and subcellular localization of Smad2, the intracellular effector of TGF-beta/activin-mediated signaling, could be used as a marker of active signaling mediated by these cytokines in situ. We therefore used an experimental model of ovalbumin-induced allergic airway inflammation and were able to demonstrate a dramatic increase in the numbers of bronchial epithelial, alveolar, and infiltrating inflammatory cells expressing nuclear phosphorylated Smad2 within the allergen-challenged lungs. This was accompanied by strong upregulation of the activin receptor ALK-4/ActR-IB and redistribution of the TGF-beta responsive ALK-5/TbetaR-I. Although levels of TGF-beta1, TGF-beta2, and TGF-beta3 messenger RNA (mRNA) were marginally altered, the level of activin mRNA was strongly upregulated during the inflammatory response. Our data illustrate the usefulness of antiphosphorylated Smad antibodies in demonstrating active TGF- beta/activin-mediated signaling in vivo and strongly suggest that activin/Smad-mediated signaling could be a critical contributor in the pathophysiology of allergic pulmonary diseases.