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1.  Ocular Inflammation in HLA-B27 Transgenic Mice Reveals a Potential Role for MHC Class I in Corneal Immune Privilege 
Molecular Vision  2015;21:131-137.
HLA-B27 is a major histocompatibility complex class I (MHCI) allele that has been closely associated with the development of ankylosing spondylitis and acute anterior uveitis (AAU), the most common form of uveitis worldwide. We have been characterizing the phenotypes of transgenic mice carrying a human HLA-B27 allele, but that are deficient in endogenous mouse MHCI genes (H-2K−/− and H-2D−/− double knockout, or DKO) to create the HLA-B27/DKO line. In maintaining and expanding this colony, we observed a rare sporadic severe central keratitis that developed in transgenic animals, but that was not present in wild-type (WT) animals.
The corneas of affected HLA-B27/DKO and DKO mice were compared to their WT counterparts by staining with standard histological methods for markers of inflammation and neovascularization. A model of experimental corneal inflammation was subsequently used to test the responses of each genotype to insult.
We identified a previously unreported corneal pathology in naïve HLA-B27/DKO mice, and we describe significantly prolonged CD4+- and CD8+-associated inflammation in these animals following an experimentally induced corneal injury.
These results demonstrate an increased T-cell response in B27/DKO corneas due to the expression of the HLA-B27 allele, suggesting that low MHCI expression in WT corneas is an important contributor to immune privilege.
PMCID: PMC4323692  PMID: 25684978
2.  ROS Detoxification and Proinflammatory Cytokines Are Linked by p38 MAPK Signaling in a Model of Mature Astrocyte Activation 
PLoS ONE  2013;8(12):e83049.
Astrocytes are the most abundant glial cell in the retinal nerve fiber layer (NFL) and optic nerve head (ONH), and perform essential roles in maintaining retinal ganglion cell (RGC) detoxification and homeostasis. Mature astrocytes are relatively quiescent, but rapidly undergo a phenotypic switch in response to insult, characterized by upregulation of intermediate filament proteins, loss of glutamate buffering, secretion of pro-inflammatory cytokines, and increased antioxidant production. These changes result in both positive and negative influences on RGCs. However, the mechanism regulating these responses is still unclear, and pharmacologic strategies to modulate select aspects of this switch have not been thoroughly explored. Here we describe a system for rapid culture of mature astrocytes from the adult rat retina that remain relatively quiescent, but respond robustly when challenged with oxidative damage, a key pathogenic stress associated with inner retinal injury. When primary astrocytes were exposed to reactive oxygen species (ROS) we consistently observed characteristic changes in activation markers, along with increased expression of detoxifying genes, and secretion of proinflammatory cytokines. This in vitro model was then used for a pilot chemical screen to target specific aspects of this switch. Increased activity of p38α and β Mitogen Activated Protein Kinases (MAPKs) were identified as a necessary signal regulating expression of MnSOD, and heme oxygenase 1 (HO-1), with consequent changes in ROS-mediated injury. Additionally, multiplex cytokine profiling detected p38 MAPK-dependent secretion of IL-6, MCP-1, and MIP-2α, which are proinflammatory signals recently implicated in damage to the inner retina. These data provide a mechanism to link increased oxidative stress to proinflammatory signaling by astrocytes, and establish this assay as a useful model to further dissect factors regulating the reactive switch.
PMCID: PMC3871647  PMID: 24376630
3.  Angiogenic Dysfunction in Bone Marrow-Derived Early Outgrowth Cells from Diabetic Animals Is Attenuated by SIRT1 Activation 
Stem Cells Translational Medicine  2012;1(12):921-926.
The study was designed to determine whether diabetes-associated early outgrowth cell (EOC) dysfunction might be attenuated by pharmacological activation of silent information regulator protein 1 (SIRT1), a lysine deacetylase implicated in nutrient-dependent life span extension in mammals. The findings suggest that the impaired angiogenicity of EOCs in diabetes may be a consequence of a reduction in SIRT1 and that its activation may represent a novel therapeutic strategy to augment endothelial repair in this disease.
Impaired endothelial repair is a key contributor to microvascular rarefaction and consequent end-organ dysfunction in diabetes. Recent studies suggest an important role for bone marrow-derived early outgrowth cells (EOCs) in mediating endothelial repair, but the function of these cells is impaired in diabetes, as in advanced age. We sought to determine whether diabetes-associated EOC dysfunction might be attenuated by pharmacological activation of silent information regulator protein 1 (SIRT1), a lysine deacetylase implicated in nutrient-dependent life span extension in mammals. Despite being cultured in normal (5.5 mM) glucose for 7 days, EOCs from diabetic rats expressed less SIRT1 mRNA, induced less endothelial tube formation in vitro and neovascularization in vivo, and secreted less of the proangiogenic ELR+ CXC chemokines CXCL1, CXCL3, and CXCL5. Ex vivo SIRT1 activation restored EOC chemokine secretion and increased the in vitro and in vivo angiogenic activity of EOC conditioned medium derived from diabetic animals to levels similar to that derived from control animals. These findings suggest a pivotal role for SIRT1 in diabetes-induced EOC dysfunction and that its pharmacologic activation may provide a new strategy for the restoration of EOC-mediated repair mechanisms.
PMCID: PMC3659675  PMID: 23283553
Diabetes; Angiogenesis; Early outgrowth cell; SIRT1; Chemokine
4.  Positive influence of AP-2α transcription factor on cadherin gene expression and differentiation of the ocular surface 
The family of transcription factors Activating protein-2 (AP-2) are known to play important roles in numerous developmental events, including those associated with differentiation of stratified epithelia. However, to date, the influence of the AP-2 genes on endogenous gene expression in the stratified epithelia and how this affects differentiation has not been well defined. The following study examines the detailed expression of the AP-2α and AP-2β proteins in the stratified epithelia of the ocular surface, including that in the cornea and developing eyelids. The effect of altered levels of the AP-2α gene on ocular surface differentiation was also examined using a corneal epithelial cell line and AP-2α chimeric mice. Immunolocalization studies revealed that, while AP-2β was broadly expressed throughout all cell layers of the stratified corneal epithelium, AP-2α expression was confined to cell compartments more basally located. AP-2α was also highly expressed in the less differentiated cell layers of the eyelid epidermis. Overexpression of the AP-2α gene in the corneal cell line, SIRC, resulted in a dramatic change in cell phenotype including a clumping growth behavior that was distinct from the smooth monolayer of the parent cell line. Accompanying this change was an up-regulation in levels of the cell adhesion molecule, N-cadherin. Examination of the ocular surface of AP-2α chimeric mice, derived from a mixed population of AP-2α−/− and AP-2α+/+, revealed that a down-regulation in E-cadherin expression is correlated with location of the AP-2α−/− null cells. Together, these findings demonstrate that AP-2α participates in regulating differentiation of the ocular surface through induction in cadherin expression.
PMCID: PMC2517417  PMID: 12694203
ocular surface; cell adhesion; transcription factors; AP-2; cornea; eyelids; differentiation
5.  Transcription Factors Pax6 and AP-2α Interact To Coordinate Corneal Epithelial Repair by Controlling Expression of Matrix Metalloproteinase Gelatinase B 
Molecular and Cellular Biology  2004;24(1):245-257.
Pax6 is a paired box containing transcription factor that resides at the top of a genetic hierarchy controlling eye development. It continues to be expressed in tissues of the adult eye, but its role in this capacity is unclear. Pax6 is present in the adult corneal epithelium, and we showed that the amount of Pax6 is increased at the migrating front as the epithelium resurfaces the cornea after injury (J. M. Sivak, R. Mohan, W. B. Rinehart, P. X. Xu, R. L. Maas, and M. E. Fini, Dev. Biol. 222:41-54, 2000). We also showed that Pax6 controls activity of the transcriptional promoter for the matrix metalloproteinase, gelatinase B (gelB; MMP-9) in cell culture transfection studies. gelB expression is turned on at the migrating epithelial front in the cornea, and it coordinates and effects aspects of epithelial regeneration (R. Mohan, S. K. Chintala, J. C. Jung, W. V. Villar, F. McCabe, L. A. Russo, Y. Lee, B. E. McCarthy, K. R. Wollenberg, J. V. Jester, M. Wang, H. G. Welgus, J. M. Shipley, R. M. Senior, and M. E. Fini, J. Biol. Chem. 277:2065-2072). We define here two positively acting Pax6 response elements in the gelB promoter. Pax6 binds directly to one of these sites through the paired DNA-binding domain. It binds the second site indirectly by interaction with AP-2α, a transcription factor that also exerts control over eye development. Pax6 control of gelB expression was examined in vivo by using a corneal reepithelialization model in mice heterozygous for a Pax6 paired-domain mutation (Sey+/−). A reduced Pax6 dosage in these mice resulted in a loss of gelB expression at the migrating epithelial front. This effect was correlated with an increase in inflammation and the rate of reepithelialization, a finding consistent with the phenotype of gelB knockout mice. Together, these data indicate that Pax6 controls activity of the gelB promoter through cooperative interactions with AP-2α and support an active role for Pax6 in maintenance and repair of the adult corneal epithelium.
PMCID: PMC303332  PMID: 14673159

Results 1-5 (5)