H2O2 transit through the mitochondrial intermembrane space promotes tumor cell growth in vitro and in vivo.

Cancer cells experience increased levels of oxidant stress as a consequence of oncogene activation, nucleotide biosynthesis, and growth factor receptor signaling. Mitochondria contribute to this redox stress by generating reactive oxygen species (ROS) along the electron transport chain, which are released to the matrix and the intermembrane space (IMS). Assessing the contribution of mitochondrial ROS in cancer cells is technically difficult, as electron transport chain inhibitors can increase or decrease ROS generation, while they also block oxidative phosphorylation and ATP synthesis. Mitochondria-targeted antioxidant compounds can scavenge ROS in the matrix compartment but do not act on ROS released to the IMS. We assessed the importance of mitochondrial ROS for tumor cell proliferation, survival, and for tumor xenograft growth by stably expressing a hydrogen peroxide (H2O2) scavenger, peroxiredoxin-5, in the mitochondrial IMS (IMS-Prdx5) in 143B osteosarcoma and HCT116 colorectal cancer cell lines. IMS-Prdx5 attenuates hypoxia-induced ROS signaling as assessed independently in cytosol and IMS, HIF-1α stabilization and activity, and cellular proliferation under normoxic and hypoxic culture conditions. It also suppressed tumor growth in vivo. Stable expression of nondegradable HIF-1α only partially rescued proliferation in IMS-Prdx5-expressing cells, indicating that mitochondrial H2O2 signaling contributes to tumor cell proliferation and survival through HIF-dependent and HIF-independent mechanisms.

Role of Hypoxia-Inducible Factors in Regulating Right Ventricular Function and Remodeling during Chronic Hypoxia-induced Pulmonary Hypertension.

Pulmonary hypertension (PH) and right ventricular (RV) hypertrophy frequently develop in patients with hypoxic lung disease. Chronic alveolar hypoxia (CH) promotes sustained pulmonary vasoconstriction and pulmonary artery (PA) remodeling by acting on lung cells, resulting in the development of PH. RV hypertrophy develops in response to PH, but coronary arterial hypoxemia in CH may influence that response by activating HIF-1α (hypoxia-inducible factor 1α) and/or HIF-2α in cardiomyocytes. Indeed, other studies show that the attenuation of PH in CH fails to prevent RV remodeling, suggesting that PH-independent factors regulate RV hypertrophy. Therefore, we examined the role of HIFs in RV remodeling in CH-induced PH. We deleted HIF-1α and/or HIF-2α in hearts of adult mice that were then housed under normoxia or CH (10% O2) for 4 weeks. RNA-sequencing analysis of the RV revealed that HIF-1α and HIF-2α regulate the transcription of largely distinct gene sets during CH. RV systolic pressure increased, and RV hypertrophy developed in CH. The deletion of HIF-1α in smooth muscle attenuated the CH-induced increases in RV systolic pressure but did not decrease hypertrophy. The deletion of HIF-1α in cardiomyocytes amplified RV remodeling; this was abrogated by the simultaneous loss of HIF-2α. CH decreased stroke volume and cardiac output in wild-type but not in HIF-1α-deficient hearts, suggesting that CH may cause cardiac dysfunction via HIF-dependent signaling. Collectively, these data reveal that HIF-1 and HIF-2 act together in RV cardiomyocytes to orchestrate RV remodeling in CH, with HIF-1 playing a protective role rather than driving hypertrophy.

Endothelin2 Induces Expression of Genes Associated with Reactive Gliosis in Retinal Müller Cells.

PURPOSE/AIM OF THE STUDY: Photoreceptor degeneration is normally accompanied by reactive gliosis and gene expression changes in Müller (glial) cells. The signaling pathway involved inducing these changes in Müller cells is not known. It has been proposed that endothelin2 (EDN2) released by degenerating photoreceptors might induce gliotic changes in Müller cells. In the present study, we directly tested the hypothesis by determining whether treatment of Müller cell cultures with EDN2 results in upregulation of genes known to be expressed in activated Müller cells in vivo. MATERIALS AND METHODS: Experiments were carried using an established rat Müller cell line (rMC-1), and gene expression was assessed by qRT-PCR. RESULTS: We observed that EDN2 treatment upregulated transcripts for glial fibrillary acidic protein (Gfap), Serpina3n and endothelin receptor B (EdnrB), three genes associated with reactive gliosis in Müller cells. Ciliary neurotrophic factor (CNTF) treatment similarly led to induction of Gfap, Serpina3n and EdnrB transcripts, whereas glutamate treatment had no significant effect. CONCLUSIONS: The finding supports a role for EDN2 as a signaling agent between photoreceptors and Müller cells.

Regulation of hypoxia-induced pulmonary hypertension by vascular smooth muscle hypoxia-inducible factor-1α.

RATIONALE: Chronic hypoxia induces pulmonary vascular remodeling, pulmonary hypertension, and right ventricular hypertrophy. At present, little is known about mechanisms driving these responses. Hypoxia-inducible factor-1α (HIF-1α) is a master regulator of transcription in hypoxic cells, up-regulating genes involved in energy metabolism, proliferation, and extracellular matrix reorganization. Systemic loss of a single HIF-1α allele has been shown to attenuate hypoxic pulmonary hypertension, but the cells contributing to this response have not been identified. OBJECTIVES: We sought to determine the contribution of HIF-1α in smooth muscle on pulmonary vascular and right heart responses to chronic hypoxia. METHODS: We used mice with homozygous conditional deletion of HIF-1α combined with tamoxifen-inducible smooth muscle-specific Cre recombinase expression. Mice received either tamoxifen or vehicle followed by exposure to either normoxia or chronic hypoxia (10% O2) for 30 days before measurement of cardiopulmonary responses. MEASUREMENTS AND MAIN RESULTS: Tamoxifen-induced smooth muscle-specific deletion of HIF-1α attenuated pulmonary vascular remodeling and pulmonary hypertension in chronic hypoxia. However, right ventricular hypertrophy was unchanged despite attenuated pulmonary pressures. CONCLUSIONS: These results indicate that HIF-1α in smooth muscle contributes to pulmonary vascular remodeling and pulmonary hypertension in chronic hypoxia. However, loss of HIF-1 function in smooth muscle does not affect hypoxic cardiac remodeling, suggesting that the cardiac hypertrophy response is not directly coupled to the increase in pulmonary artery pressure.

Gene expression changes in retinal Müller (glial) cells exposed to elevated pressure.

PURPOSE: Retinal Müller (glial) cells undergo "reactive gliosis", a stress response that is accompanied by changes in their morphology and upregulation of various cellular markers. Reactive gliosis is seen in many retinal diseases and conditions; however, it is not known whether it is a common, stereotypic response or the nature of the response varies with the type of retinal stress. To address this question, we have examined gene expression changes in Müller cells exposed to elevated pressure. MATERIALS AND METHODS: Rat Müller cells (rMC-1) were exposed to elevated pressure, and RNA was extracted and analyzed using Affymetrix GeneChip microarrays to identify pressure-responsive genes. RESULTS: Analysis of microarray data showed that at 6 h, 186 genes had > 1.5-fold change with FDR < 0.01. Of these, 62 genes were up-regulated while 124 genes were down-regulated. At 24 h, 73 genes changed > 1.5-fold. Of these, 37 genes were up-regulated while 36 genes were down-regulated. Ingenuity canonical pathway analysis showed that several signaling and metabolic pathways were significantly changed in Müller cells under high pressure. In addition, among up- and down-regulated genes, we identified eight genes-areg, bmp4, cyp1b1, gpnmb, herc2, msh2, heph, and selenbp1, that have been directly or indirectly associated with elevated intraocular pressure. Two genes, areg and gpnmb, further showed time-dependent changes in mRNA and protein expression. CONCLUSION: The results show that Müller cells in vitro respond to elevated pressure by differential regulation of expressed genes. The transcriptional profile is different from that seen with hypoxia, which indicates that Müller cells respond differentially to different microenvironmental changes in the retina.

Ciliary neurotrophic factor induces genes associated with inflammation and gliosis in the retina: a gene profiling study of flow-sorted, Müller cells.

BACKGROUND: Ciliary neurotrophic factor (CNTF), a member of the interleukin-6 cytokine family, has been implicated in the development, differentiation and survival of retinal neurons. The mechanisms of CNTF action as well as its cellular targets in the retina are poorly understood. It has been postulated that some of the biological effects of CNTF are mediated through its action via retinal glial cells; however, molecular changes in retinal glia induced by CNTF have not been elucidated. We have, therefore, examined gene expression dynamics of purified Müller (glial) cells exposed to CNTF in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Müller cells were flow-sorted from mgfap-egfp transgenic mice one or three days after intravitreal injection of CNTF. Microarray analysis using RNA from purified Müller cells showed differential expression of almost 1,000 transcripts with two- to seventeen-fold change in response to CNTF. A comparison of transcriptional profiles from Müller cells at one or three days after CNTF treatment showed an increase in the number of transcribed genes as well as a change in the expression pattern. Ingenuity Pathway Analysis showed that the differentially regulated genes belong to distinct functional types such as cytokines, growth factors, G-protein coupled receptors, transporters and ion channels. Interestingly, many genes induced by CNTF were also highly expressed in reactive Müller cells from mice with inherited or experimentally induced retinal degeneration. Further analysis of gene profiles revealed 20-30% overlap in the transcription pattern among Müller cells, astrocytes and the RPE. CONCLUSIONS/SIGNIFICANCE: Our studies provide novel molecular insights into biological functions of Müller glial cells in mediating cytokine response. We suggest that CNTF remodels the gene expression profile of Müller cells leading to induction of networks associated with transcription, cell cycle regulation and inflammatory response. CNTF also appears to function as an inducer of gliosis in the retina.

Retinal glucose metabolism in mice lacking the L-glutamate/aspartate transporter.

The conventional view that glucose is the substrate for neuronal energy metabolism has been recently challenged by the "lactate shuttle" hypothesis in which glutamate cycling in glial cells drives all neuronal glucose metabolism. According to this view, glutamate released by activated retinal neurons is transported into Müller (glial) cells where it triggers glycolysis. The lactate released by Müller cells serves as the energy substrate for neuronal metabolism. Because the L-Glutamate/aspartate transporter (GLAST) is the predominant, Na+-dependent, glutamate transporter expressed by Müller cells, we have used GLAST-knockout (GLAST -/-) mice to examine the relationship between lactate release and GLAST activity in the retina. We found that glucose uptake and lactate production by the GLAST -/- mouse retina was similar to that observed in the wild type mouse retina. Furthermore, addition of 1 mM glutamate and NH4Cl to the incubation medium did not further stimulate glucose uptake in either case. When lactate release was measured in the presence of the lactate uptake inhibitor, alpha-cyano-4-hydroxycinnamate, there was no significant change in the amount of lactate released by retinas from GLAST -/- mice compared to the wild type. Finally, lactate release was similar under both dark and light conditions. These results show that lactate production and release is not altered in retinas of GLAST -/- mice, which suggests that metabolic coupling between photoreceptors and Müller cells is not mediated by the glial glutamate transporter, GLAST.

GFAP promoter drives Müller cell-specific expression in transgenic mice.

PURPOSE: In an attempt to identify Müller cell-specific promoters and to better understand the gene regulatory mechanisms in retinal glial cells, the expression of the glial fibrillary acidic protein (GFAP) gene was studied in Müller cell cultures and in GFAP-enhanced green fluorescent protein (EGFP) transgenic mice. METHODS: A transfection assay of GFAP-luciferase constructs carrying a series of nested deletions was performed in an established Müller cell line. For in vivo analysis, transgenic mice were generated by injecting a construct carrying a 2.5-kb, 5' fragment of the mouse GFAP gene linked to the EGFP gene. Isolated retinas from transgenic mice were screened for GFP expression. Subsequently, the identity of the GFP-expressing cells was established by immunostaining cryostat sections of the retina with antibodies against Müller cell antigenic markers. Induction of the transgene and the endogenous GFAP gene was examined by injecting ciliary neurotrophic factor (CNTF) into the eye. RESULTS: The DNA transfection data suggested that proximal 5' sequences of the GFAP gene are sufficient to direct high-level reporter expression in Müller cell cultures. In transgenic mice, GFP fluorescence appeared in radially oriented processes that spanned almost the entire thickness of the retina. Immunostaining with antibodies to cellular retinaldehyde-binding protein (CRALBP) and glutamine synthetase showed that the GFP-expressing cells were Müller cells. GFP-expressing Müller cells were observed in the retinas of both albino and pigmented transgenic mice. In eyes injected with CNTF, both GFP and GFAP levels were highly elevated. These observations suggest that the 2.5-kb, 5' GFAP sequence can direct inducible reporter gene expression in Müller cells. In addition to Müller cells, a few GFP-labeled astrocytes were present in the adult retina. In the developing retina, GFP-expressing astrocytes were first present at the optic nerve head, and as development progressed, the cells gradually moved toward the periphery of the retina and acquired their adult, stellate morphology. CONCLUSIONS: The present study shows that the 2.5-kb, 5' flanking region of the mouse GFAP gene can be used to express GFP, and possibly other genes, specifically in Müller cells in the mouse retina. Furthermore, expression of the transgene can be upregulated by intravitreal injection of CNTF.