The activin receptor is stimulated in the skeleton, vasculature, heart, and kidney during chronic kidney disease.

We examined activin receptor type IIA (ActRIIA) activation in chronic kidney disease (CKD) by signal analysis and inhibition in mice with Alport syndrome using the ActRIIA ligand trap RAP-011 initiated in 75-day-old Alport mice. At 200 days of age, there was severe CKD and associated Mineral and Bone Disorder (CKD-MBD), consisting of osteodystrophy, vascular calcification, cardiac hypertrophy, hyperphosphatemia, hyperparathyroidism, elevated FGF23, and reduced klotho. The CKD-induced bone resorption and osteoblast dysfunction was reversed, and bone formation was increased by RAP-011. ActRIIA inhibition prevented the formation of calcium apatite deposits in the aortic adventitia and tunica media and significantly decreased the mean aortic calcium concentration from 0.59 in untreated to 0.36 mg/g in treated Alport mice. Aortic ActRIIA stimulation in untreated mice increased p-Smad2 levels and the transcription of sm22α and αSMA. ActRIIA inhibition reversed aortic expression of the osteoblast transition markers Runx2 and osterix. Heart weight was significantly increased by 26% in untreated mice but remained normal during RAP-011 treatment. In 150-day-old mice, GFR was significantly reduced by 55%, but only by 30% in the RAP-011-treated group. In 200-day-old mice, the mean BUN was 100 mg/dl in untreated mice compared to 60 mg/dl in the treated group. In the kidneys of 200-day-old mice, ActRIIA and p-Smad2 were induced and MCP-1, fibronectin, and interstitial fibrosis were stimulated; all were attenuated by RAP-011 treatment. Hence, the activation of ActRIIA signaling during early CKD contributes to the CKD-MBD components of osteodystrophy and cardiovascular disease and to renal fibrosis. Thus, the inhibition of ActRIIA signaling is efficacious in improving and delaying CKD-MBD in this model of Alport syndrome.

Ligand trap of the activin receptor type IIA inhibits osteoclast stimulation of bone remodeling in diabetic mice with chronic kidney disease.

Dysregulation of skeletal remodeling is a component of renal osteodystrophy. Previously, we showed that activin receptor signaling is differentially affected in various tissues in chronic kidney disease (CKD). We tested whether a ligand trap for the activin receptor type 2A (RAP-011) is an effective treatment of the osteodystrophy of the CKD-mineral bone disorder. With a 70% reduction in the glomerular filtration rate, CKD was induced at 14 weeks of age in the ldlr-/- high fat-fed mouse model of atherosclerotic vascular calcification and diabetes. Twenty mice with CKD, hyperphosphatemia, hyperparathyroidism, and elevated activin A were treated with RAP-011, wherease 19 mice were given vehicle twice weekly from week 22 until the mice were killed at 28 weeks of age. The animals were then evaluated by skeletal histomorphometry, micro-computed tomography, mechanical strength testing, and ex vivo bone cell culture. Results in the CKD groups were compared with those of the 16 sham-operated ldlr-/- high fat-fed mice. Sham-operated mice had low-turnover osteodystrophy and skeletal frailty. CKD stimulated bone remodeling with significant increases in osteoclast and osteoblast numbers and bone resorption. Compared with mice with CKD and sham-operated mice, RAP-011 treatment eliminated the CKD-induced increase in these histomorphometric parameters and increased trabecular bone fraction. RAP-011 significantly increased cortical bone area and thickness. Activin A-enhanced osteoclastogenesis was mediated through p-Smad2 association with c-fos and activation of nuclear factor of activated T cells c1 (NFATc1). Thus, an ActRIIA ligand trap reversed CKD-stimulated bone remodeling, likely through inhibition of activin-A induced osteoclastogenesis.

Ligand trap for the activin type IIA receptor protects against vascular disease and renal fibrosis in mice with chronic kidney disease.

The causes of cardiovascular mortality associated with chronic kidney disease (CKD) are partly attributed to the CKD-mineral bone disorder (CKD-MBD). The causes of the early CKD-MBD are not well known. Our discovery of Wnt (portmanteau of wingless and int) inhibitors, especially Dickkopf 1, produced during renal repair as participating in the pathogenesis of the vascular and skeletal components of the CKD-MBD implied that additional pathogenic factors are critical. In the search for such factors, we studied the effects of activin receptor type IIA (ActRIIA) signaling by using a ligand trap for the receptor, RAP-011 (a soluble extracellular domain of ActRIIA fused to a murine IgG-Fc fragment). In a mouse model of CKD that stimulated atherosclerotic calcification, RAP-011 significantly increased aortic ActRIIA signaling assessed by the levels of phosphorylated Smad2/3. Furthermore, RAP-011 treatment significantly reversed CKD-induced vascular smooth muscle dedifferentiation as assessed by smooth muscle 22α levels, osteoblastic transition, and neointimal plaque calcification. In the diseased kidneys, RAP-011 significantly stimulated αklotho levels and it inhibited ActRIIA signaling and decreased renal fibrosis and proteinuria. RAP-011 treatment significantly decreased both renal and circulating Dickkopf 1 levels, showing that Wnt activation was downstream of ActRIIA. Thus, ActRIIA signaling in CKD contributes to the CKD-MBD and renal fibrosis. ActRIIA signaling may be a potential therapeutic target in CKD.

Retinoblastoma protein prevents enteric nervous system defects and intestinal pseudo-obstruction.

The retinoblastoma 1 (RB1) tumor suppressor is a critical regulator of cell cycle progression and development. To investigate the role of RB1 in neural crest-derived melanocytes, we bred mice with a floxed Rb1 allele with mice expressing Cre from the tyrosinase (Tyr) promoter. TyrCre+;Rb1fl/fl mice exhibited no melanocyte defects but died unexpectedly early with intestinal obstruction, striking defects in the enteric nervous system (ENS), and abnormal intestinal motility. Cre-induced DNA recombination occurred in all enteric glia and most small bowel myenteric neurons, yet phenotypic effects of Rb1 loss were cell-type specific. Enteric glia were twice as abundant in mutant mice compared with those in control animals, while myenteric neuron number was normal. Most myenteric neurons also appeared normal in size, but NO-producing myenteric neurons developed very large nuclei as a result of DNA replication without cell division (i.e., endoreplication). Parallel studies in vitro found that exogenous NO and Rb1 shRNA increased ENS precursor DNA replication and nuclear size. The large, irregularly shaped nuclei in NO-producing neurons were remarkably similar to those in progeria, an early-onset aging disorder that has been linked to RB1 dysfunction. These findings reveal a role for RB1 in the ENS.

BAP1 deficiency causes loss of melanocytic cell identity in uveal melanoma.

BACKGROUND: Uveal 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. METHODS: Uveal 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. RESULTS: Depletion 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. CONCLUSIONS: BAP1 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.

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.

ABCB1 identifies a subpopulation of uveal melanoma cells with high metastatic propensity.

Metastasis of tumor cells to distant organs is the leading cause of death in melanoma. Yet, the mechanisms of metastasis remain poorly understood. One key question is whether all cells in a primary tumor are equally likely to metastasize or whether subpopulations of cells preferentially give rise to metastases. Here, we identified a subpopulation of uveal melanoma cells expressing the multidrug resistance transporter ABCB1 that are highly metastatic compared to ABCB1(-) bulk tumor cells. ABCB1(+) cells also exhibited enhanced clonogenicity, anchorage-independent growth, tumorigenicity and mitochondrial activity compared to ABCB1(-) cells. A375 cutaneous melanoma cells contained a similar subpopulation of highly metastatic ABCB1(+) cells. These findings suggest that some uveal melanoma cells have greater potential for metastasis than others and that a better understanding of such cells may be necessary for more successful therapies for metastatic melanoma.

Id2 deficiency promotes metastasis in a mouse model of ocular cancer.

The inhibitor of DNA binding 2 (Id2) basic helix-loop-helix protein interacts genetically and physically with the pocket proteins (Rb, p107 and p130) and has been implicated as an oncogene. In other studies, however, Id2 has been shown to function as a tumor suppressor. Here, we studied the role of Id2 in a well characterized model of ocular cancer in which the three pocket proteins are inactivated by generating mice lacking one or both Id2 alleles. Id2 deficiency had no impact on tumorigenesis in the eye. Unexpectedly, however, Id2 loss significantly increased the rate of metastasis. Liver metastases in Id2 heterozygotes demonstrated significant decrease of Id2 expression and loss of the remaining Id2 allele, strongly suggesting that Id2 inactivation specifically was required for metastasis in this model. These findings provide new insights into the role of Id2 in metastasis.

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.

Bioinformatic and statistical analysis of the optic nerve head in a primate model of ocular hypertension.

BACKGROUND: The nonhuman primate model of glaucomatous optic neuropathy most faithfully reproduces the human disease. We used high-density oligonucleotide arrays to investigate whole genome transcriptional changes occurring at the optic nerve head during primate experimental glaucoma. RESULTS: Laser scarification of the trabecular meshwork of cynomolgus macaques produced elevated intraocular pressure that was monitored over time and led to varying degrees of damage in different samples. The macaques were examined clinically before enucleation and the myelinated optic nerves were processed post-mortem to determine the degree of neuronal loss. Global gene expression was examined in dissected optic nerve heads with Affymetrix GeneChip microarrays. We validated a subset of differentially expressed genes using qRT-PCR, immunohistochemistry, and immuno-enriched astrocytes from healthy and glaucomatous human donors. These genes have previously defined roles in axonal outgrowth, immune response, cell motility, neuroprotection, and extracellular matrix remodeling. CONCLUSION: Our findings show that glaucoma is associated with increased expression of genes that mediate axonal outgrowth, immune response, cell motility, neuroprotection, and ECM remodeling. These studies also reveal that, as glaucoma progresses, retinal ganglion cell axons may make a regenerative attempt to restore lost nerve cell contact.

Gene expression and functional studies of the optic nerve head astrocyte transcriptome from normal African Americans and Caucasian Americans donors.

PURPOSE: To determine whether optic nerve head (ONH) astrocytes, a key cellular component of glaucomatous neuropathy, exhibit differential gene expression in primary cultures of astrocytes from normal African American (AA) donors compared to astrocytes from normal Caucasian American (CA) donors. METHODS: We used oligonucleotide Affymetrix microarray (HG U133A & HG U133A 2.0 chips) to compare gene expression levels in cultured ONH astrocytes from twelve CA and twelve AA normal age matched donor eyes. Chips were normalized with Robust Microarray Analysis (RMA) in R using Bioconductor. Significant differential gene expression levels were detected using mixed effects modeling and Statistical Analysis of Microarray (SAM). Functional analysis and Gene Ontology were used to classify differentially expressed genes. Differential gene expression was validated by quantitative real time RT-PCR. Protein levels were detected by Western blots and ELISA. Cell adhesion and migration assays tested physiological responses. Glutathione (GSH) assay detected levels of intracellular GSH. RESULTS: Multiple analyses selected 87 genes differentially expressed between normal AA and CA (P<0.01). The most relevant genes expressed in AA were categorized by function, including: signal transduction, response to stress, ECM genes, migration and cell adhesion. CONCLUSIONS: These data show that normal astrocytes from AA and CA normal donors display distinct expression profiles that impact astrocyte functions in the ONH. Our data suggests that differences in gene expression in ONH astrocytes may be specific to the development and/or progression of glaucoma in AA.

Expression of ephrinB1 and its receptor in glaucomatous optic neuropathy.

OBJECTIVE: To determine ephrinB1, ephrinB2 and EphB1 expression in the optic nerve head (ONH) and retina of monkeys with glaucoma and in human ONH astrocytes. METHODS: Using immunohistochemistry, the localisation of ephrinB1, ephrinB2 and EphB1 was determined in the ONH and retina bilaterally in monkeys with monocular laser-induced glaucoma. RT-PCR, western blot and immunocytochemistry were used to study ephrinB1, ephrinB2 and EphB1 expression in cultured human ONH astrocytes from donors with and without glaucoma. RESULTS: There was an increase in ephrinB1 and EphB1 expression in mild to moderate glaucoma. In the ONH, both ephrinB1 and EphB1 were localised to astrocytes and EphB1 was also localised to lamina cribrosa cells and perivascular cells. In the retina, ephrinB1 localised to Muller cells and astrocytes, and EphB1 was found in retinal ganglion cells. In ONH astrocytes in humans with glaucoma, ephrinB1 and EphB1 were up-regulated but barely present in donors without glaucoma. CONCLUSIONS: Ephrins are activated in early and moderate glaucoma in the ONH and retina. We postulate that the up-regulation of Eph/ephrin pathway may play a protective role by limiting axonal damage and inflammatory cell invasion. Loss of ephrin signalling in advanced glaucoma may explain macrophage activation.

A neuroactive steroid 5alpha-androstane-3alpha,17beta-diol regulates androgen receptor level in astrocytes.

Optic nerve head (ONH) astrocytes from patients with glaucomatous optic neuropathy exhibit increased production of 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), a neuroactive metabolite of 5alpha-dihydrotestosterone (5alpha-DHT). To determine whether ONH astrocytes are androgen target cells, and whether 3alpha-diol is capable of regulating astrocyte functions, we studied the response of human ONH astrocytes to 3alpha-diol compared with 17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one (R1881), a synthetic 5alpha-DHT agonist. In ONH astrocytes, both 3alpha-diol and R1881 increased protein levels of androgen receptor (AR) and glial fibrillary acidic protein (GFAP), however, only R1881 also increased the AR mRNA level and astrocyte proliferation. Both R1881 and 3alpha-diol rapidly activate the mitogen-activated protein kinase (MAPK) signaling pathway in ONH astrocytes, as confirmed by phosphorylation of extracellular signal-regulated kinase (ERK). 3Alpha-diol also activates the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. 3Alpha-diol regulates the increase of AR protein level and the phosphorylation through the PI3K/Akt pathway, whereas R1881 regulates them through the MAPK/ERK pathway. Our findings demonstrate that human ONH astrocytes are androgen target cells and respond to androgens by the rapid activation of cell signaling. The activation of the PI3K/Akt pathway by 3alpha-diol may regulate various properties of astrocytes, including cell motility and survival, and may play a role in the formation and maintenance of the reactive phenotype of ONH astrocytes in glaucoma.

Androgen receptor and NFkB expression in human normal and glaucomatous optic nerve head astrocytes in vitro and in experimental glaucoma.

For several decades, clinical and experimental observations suggested a relationship between steroids and glaucoma; however, the possibility that androgens are also involved in the glaucomatous changes in the optic nerve heads (ONH) has not been explored. Our previous findings that glaucomatous ONH astrocytes synthesize androgen-metabolising enzymes and overproduce a neuroactive androgen, 5alpha-androstane-3alpha, 17beta-diol (3alpha-diol) led us to propose that ONH astrocytes are androgen target cells. Androgens modulate different cellular processes through androgen receptor (AR). NFkB is a transcription factor that positively regulates AR transcription. Here, we analysed AR and NFkB expression in normal and glaucomatous ONH astrocytes in vitro, and in vivo in a monkey model of experimental glaucoma (ExpG) by quantitative real time RT-PCR, Western blotting and immunohistochemistry. We demonstrated that in vitro human glaucomatous ONH astrocytes express AR mRNA and protein at higher levels than normal astrocytes and that in vivo ONH astrocytes from eyes with ExpG showed increased nuclear and cytoplasmic AR immunostaining compared to control eyes. In the retina, retinal ganglion cells (RGC) demonstrated cytoplasmic staining both in control and in ExpG eyes. NFkB mRNA expression was higher in glaucomatous ONH astrocytes than in normal and more nuclear NFkB protein was detected in glaucomatous ONH astrocytes. In vivo immunopositive NFkB nuclear staining of ONH astrocytes in ONH and in RGC in retina was detected both in control and in ExpG eyes. We conclude that in addition to our published data, increase of AR and NFkB expression in glaucomatous ONH astrocytes provides strong evidence that androgens play a significant role in the pathophysiology of glaucoma.

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.

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.