Olga A. Agapova

Differential gene expression in astrocytes from human normal and glaucomatous optic nerve head analyzed by cDNA microarray

Recent advances in cDNA microarray technology have made it possible to analyze expression of several thousand genes at the same time. Using this technique, gene expression in human astrocytes cultured from glaucomatous and normal optic nerve heads (ONH) was compared. One hundred‐fifty genes were differentially expressed more than 5‐fold in glaucomatous cell cultures compared with normal. These genes are involved in a number of biological processes, including signal transduction, cell adhesion and proliferation, ECM synthesis, and degradation. Confirmation of differential gene expression was performed by quantitative RT‐PCR. Western blots and immunohistochemistry demonstrated gene products in cell cultures or in human ONH tissues. Proliferation, adhesion and migration assays tested physiological responses suggested by differential gene expression. Our study suggests that cultured glaucomatous ONH astrocytes retain in culture many phenotypic characteristics that may be relevant to their role in the pathogenesis of glaucoma and, in general to reactive astrocytes in the CNS. Potential applications of these data include the identification and characterization of signaling pathways involved in astrocyte function, studies of the role of steroid‐metabolizing enzymes in the glaucomatous ONH, and further exploration of the role of selected identified genes in experimental animal and in vitro models of glaucoma. GLIA 38:45–64, 2002.

Histone Deacetylase Inhibitors Induce Growth Arrest and Differentiation in Uveal Melanoma

Purpose: Metastasis is responsible for the death of most cancer patients, yet few therapeutic agents are available which specifically target the molecular events that lead to metastasis. We recently showed that inactivating mutations in the tumor suppressor gene BAP1 are closely associated with loss of melanocytic differentiation in uveal melanoma (UM) and metastasis. The purpose of this study was to identify therapeutic agents that reverse the phenotypic effects of BAP1 loss in UM. Experimental Design: In silico screens were done to identify therapeutic compounds predicted to differentiate UM cells using Gene Set Enrichment Analysis and Connectivity Map databases. Valproic acid (VPA), trichostatin A, LBH-589, and suberoylanilide hydroxamic acid were evaluated for their effects on UM cells using morphologic evaluation, MTS viability assays, bromodeoxyuridine incorporation, flow cytometry, clonogenic assays, gene expression profiling, histone acetylation and ubiquitination assays, and a murine xenograft tumorigenicity model. Results: Histone deacetylase (HDAC) inhibitors induced morphologic differentiation, cell-cycle exit, and a shift to a differentiated, melanocytic gene expression profile in cultured UM cells. VPA inhibited the growth of UM tumors in vivo. Conclusions: These findings suggest that HDAC inhibitors may have therapeutic potential for inducing differentiation and prolonged dormancy of micrometastatic disease in UM. Clin Cancer Res; 18(2); 408–16. ©2011 AACR.

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human optic nerve head astrocytes

Glaucomatous optic neuropathy is a common blinding disease characterized by remodeling of the extracellular matrix (ECM) and loss of retinal ganglion cell (RGC) axons at the level of the optic nerve head (ONH). Astrocytes, the major cell type in ONH, may participate in this process by production of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). In normal and glaucomatous ONH, we detected MMP and TIMP expression by immunohistochemistry. Cultured astrocytes were used to characterize expression of MMPs and TIMPs by zymography, Western blot, and RNase protection assay. MMP production was stimulated with phorbol 12‐myristate 13‐acetate (PMA). Astrocytes expressed MMP1, MT1‐MMP, MMP2, TIMP1, and TIMP2 in normal and glaucomatous ONH. MMP2, TIMP1, and TIMP2 localized to RGCs and their axons. Increased MMP1 and MT1‐MMP expression was demonstrated in glaucoma. Cultured astrocytes constitutively expressed MMP2, MT1‐MMP, TIMP1, and TIMP2, whereas MMP3, MMP7, MMP9, and MMP12 were not detectable in tissues or in cultured astrocytes. Our findings demonstrate the presence of specific MMPs and TIMPs in the ONH that may participate in the homeostasis and remodeling of the ECM in glaucoma. Expression of the same MMPs and TIMPs in cultured ONH astrocytes will allow further studies on the mechanisms regulating these enzymes. GLIA 33:205–216, 2001.

Expression of small heat shock proteins and intermediate filaments in the human optic nerve head astrocytes exposed to elevated hydrostatic pressure in vitro

The small heat shock proteins (sHSP), αB‐crystallin and Hsp27 are chaperone molecules that maintain the integrity of intermediate filament (IF) network and prevent unfolding of cellular proteins induced by stress. In the optic nerve head (ONH) of eyes with glaucoma, reactive astrocytes expressed Hsp27, perhaps in response to stress related to elevated intraocular pressure. In this study, we determined the effect of elevated hydrostatic pressure (HP) in the synthesis, distribution and co‐localization of αB‐crystallin and Hsp27 with IF in cultured ONH astrocytes. Astrocyte monolayers were pressurized to 60 mm Hg (92% air 8% CO2) and incubated at 37°C for 6, 24 or 48 hr. Controls were exposed to ambient pressure. Cells were analyzed by immunocytochemistry, Western blot and immunoprecipitation using antibodies to Hsp27, αB‐crystallin, vimentin or GFAP. Control astrocytes seemed flat, polygonal with short processes. αB‐crystallin appeared granular in the perinuclear area and filamentous in the cell periphery. Fine granular Hsp27 was distributed throughout the cytoplasm. GFAP and vimentin co‐localized with Hsp27 in the cytoplasm. Astrocytes exposed to HP were star‐shaped with long processes. Hsp27 was condensed in large granules around the nucleus. GFAP and vimentin co‐localized with Hsp27 and αB‐crystallin in the perinuclear area. Western blot and metabolic labeling detected increased synthesis of Hsp27, GFAP and vimentin but no change in αB‐crystallin. These results indicated that GFAP and vimentin associate with Hsp27 and αB‐crystallin in ONH astrocytes. HP affected the integrity of the cytoskeleton consistent with morphological changes. Small HSP may reinforce and maintain IF integrity in response to HP. J. Neurosci. Res. 66:59–73, 2001.

Differential expression of matrix metalloproteinases in monkey eyes with experimental glaucoma or optic nerve transection.

Extracellular matrix (ECM) remodeling after neuronal injury and reactive gliosis is carried out by activation of matrix metalloproteinases (MMPs) regulated by their tissue inhibitors (TIMPs). In glaucoma, there is a loss of retinal ganglion cells and extensive ECM remodeling (cupping) at the level of the optic nerve head, frequently associated with elevated intraocular pressure. To determine whether ECM remodeling in the glaucomatous optic nerve head occurs in response to loss of axons or to elevated intraocular pressure we compared the patterns of MMP and TIMP expression in the eyes of monkeys with laser-induced glaucoma or with optic nerve transection. MT1-MMP and MMP1 expression was markedly increased in reactive astrocytes in optic nerve heads with experimental glaucoma but not in the optic nerve head of transected eyes. In normal control eyes retinal ganglion cells expressed MMP2, TIMP1 and TIMP2 constitutively, and the proteins were detected in their axons. At the site of transection, MT1-MMP, MMP1, MMP2, TIMP1 and TIMP2 were expressed by reactive astrocytes. Inflammatory cells, fibroblasts and reactive astrocytes at the transected site expressed MMP3 and MMP9, which were undetectable in the retina and optic nerve head in any condition. Constitutive expression of MMP2, TIMP1 and TIMP2 in retinal ganglion cells suggests a role in maintenance of synaptic integrity and plasticity and maintenance of the periaxonal space. Increased MMP1 and MT1-MMP1 expression in the glaucomatous optic nerve head is specific to tissue remodeling due to elevated intraocular pressure and not secondary to loss of axons.

CKD-Induced Wingless/Integration1 Inhibitors and Phosphorus Cause the CKD–Mineral and Bone Disorder

In chronic kidney disease, vascular calcification, renal osteodystrophy, and phosphate contribute substantially to cardiovascular risk and are components of CKD-mineral and bone disorder (CKD-MBD). The cause of this syndrome is unknown. Additionally, no therapy addresses cardiovascular risk in CKD. In its inception, CKD-MBD is characterized by osteodystrophy, vascular calcification, and stimulation of osteocyte secretion. We tested the hypothesis that increased production of circulating factors by diseased kidneys causes the CKD-MBD in diabetic mice subjected to renal injury to induce stage 2 CKD (CKD-2 mice). Compared with non-CKD diabetic controls, CKD-2 mice showed increased renal production of Wnt inhibitor family members and higher levels of circulating Dickkopf-1 (Dkk1), sclerostin, and secreted klotho. Neutralization of Dkk1 in CKD-2 mice by administration of a monoclonal antibody after renal injury stimulated bone formation rates, corrected the osteodystrophy, and prevented CKD-stimulated vascular calcification. Mechanistically, neutralization of Dkk1 suppressed aortic expression of the osteoblastic transcription factor Runx2, increased expression of vascular smooth muscle protein 22-α, and restored aortic expression of klotho. Neutralization of Dkk1 did not affect the elevated plasma levels of osteocytic fibroblast growth factor 23 but decreased the elevated levels of sclerostin. Phosphate binder therapy restored plasma fibroblast growth factor 23 levels but had no effect on vascular calcification or osteodystrophy. The combination of the Dkk1 antibody and phosphate binder therapy completely treated the CKD-MBD. These results show that circulating Wnt inhibitors are involved in the pathogenesis of CKD-MBD and that the combination of Dkk1 neutralization and phosphate binding may have therapeutic potential for this disorder.

Emerging insights into the molecular pathogenesis of uveal melanoma

Uveal melanoma is the most common primary cancer of the eye, and often results not only in vision loss, but also in metastatic death in up to half of patients. For many years, the details of the molecular pathogenesis of uveal melanoma remained elusive. In the past decade, however, many of these details have emerged to reveal a fascinating and complex story of how the primary tumor evolves and progresses. Early events that disrupt cell cycle and apoptotic control lead to malignant transformation and proliferation of uveal melanocytes. Later, the growing tumor encounters a critical bifurcation point, where it progresses along one of two genetic pathways with very distinct genetic signatures (monosomy 3 vs 6p gain) and metastatic propensity. Late genetic events are characterized by increasing aneuploidy, most of which is nonspecific. However, specific chromosomal alterations, such as loss of chromosome 8p, can hasten the onset of metastasis in susceptible tumors. Taken together, this pathogenetic scheme can be used to construct a molecularly based and prognostically relevant classification of uveal melanomas that can be used clinically for personalized patient management.

Selective expression of neural cell adhesion molecule (NCAM)-180 in optic nerve head astrocytes exposed to elevated hydrostatic pressure in vitro.

Glaucomatous optic neuropathy is usually associated with elevated intraocular pressure. Optic nerve head astrocytes may respond to intraocular pressure by stimulation of pressure-sensitive mechanoreceptors on the cell surface. Neural cell adhesion molecule (NCAM) a transmembrane protein, mediates cell adhesion and migration. The NCAM 180 isoform increases in astrocytes of glaucomatous optic nerve head. We characterized the relative expression of NCAM isoforms in human optic nerve head astrocytes grown under elevated hydrostatic pressure. Astrocytes cultured from normal human optic nerve heads were exposed to either atmospheric or continuous hydrostatic pressure of 60 mm Hg, and analyzed at 6-48 h. Changes in cell shape, immunoreactivity, and distribution of GFAP, actin and NCAM were observed in pressure-treated cultures. Newly synthesized (35)S-labeled NCAM protein immunoprecipitated from cell lysates was increased 2-fold within 24 h after exposure to elevated pressure compared to control. The increase in NCAM synthesis was primarily due to the NCAM 180 isoform. A significant increase in NCAM 180 mRNA levels was detected by RT-PCR and Northern blots in cultured optic nerve head astrocytes within 6 h after exposure to elevated pressure. NCAM 180 mRNA and protein synthesis decreased after 24 h and returned to control levels by 48 h. Our data indicate that NCAM 180 transcription and synthesis in astrocytes is stimulated by elevated hydrostatic pressure. Because NCAM 180 interacts with the cytoskeleton through an extended cytoplasmic tail, a selective and transient increase in NCAM 180 in optic nerve head astrocytes exposed to elevated pressure may be relevant to the migration and interactions of reactive astrocytes in glaucoma.

BAP1 deficiency causes loss of melanocytic cell identity in uveal melanoma

BackgroundUveal melanoma is a highly aggressive cancer with a strong propensity for metastasis, yet little is known about the biological mechanisms underlying this metastatic potential. We recently showed that most metastasizing uveal melanomas, which exhibit a class 2 gene expression profile, contain inactivating mutations in the tumor suppressor BAP1. The aim of this study was to investigate the role of BAP1 in uveal melanoma progression.MethodsUveal melanoma cells were studied following RNAi-mediated depletion of BAP1 using proliferation, BrdU incorporation, flow cytometry, migration, invasion, differentiation and clonogenic assays, as well as in vivo tumorigenicity experiments in NOD-SCID-Gamma mice.ResultsDepletion of BAP1 in uveal melanoma cells resulted in a loss of differentiation and gain of stem-like properties, including expression of stem cell markers, increased capacity for self-replication, and enhanced ability to grow in stem cell conditions. BAP1 depletion did not result in increased proliferation, migration, invasion or tumorigenicity.ConclusionsBAP1 appears to function in the uveal melanocyte lineage primarily as a regulator of differentiation, with cells deficient for BAP1 exhibiting stem-like qualities. It will be important to elucidate how this effect of BAP1 loss promotes metastasis and how to reverse this effect therapeutically.

Altered expression of 3α-hydroxysteroid dehydrogenases in human glaucomatous optic nerve head astrocytes

3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) isoforms (AKR1C1-AKR1C4) are aldo-keto reductases that metabolize steroids and other substances in many tissues including the CNS. Here we demonstrated that in glaucomatous human optic nerve heads, increased expression of 3alpha-HSD was localized to reactive astrocytes in the lamina cribrosa. Similar, optic nerve head astrocytes exhibited increased expression of 3alpha-HSD in response to elevated intraocular pressure in a monkey model of experimental glaucoma, but not in monkeys with unilateral optic nerve transection. In vitro, glaucomatous optic nerve head astrocytes expressed higher levels of AKR1C1, AKR1C2, and AKR1C3 mRNA, than normal astrocytes, with significant differential increase of AKR1C2 expression, and exhibited higher enzymatic activity forming 3alpha-androstanediol a well-recognized neurosteroid. Normal astrocytes exposed to elevated hydrostatic pressure selectively increased AKR1C2 expression. Our findings of increased expression of 3alpha-HSDs in glaucomatous optic nerve head astrocytes offer new insights into possible roles for neurosteroids in the pathophysiology of glaucoma.