Autophagy awakens-the myriad roles of autophagy in head and neck cancer development and therapeutic response.

Autophagy is an evolutionarily conserved cell survival mechanism that degrades damaged proteins and organelles to generate cellular energy during times of stress. Recycling of these cellular components occurs in a series of sequential steps with multiple regulatory points. Mechanistic dysfunction can lead to a variety of human diseases and cancers due to the complexity of autophagy and its ability to regulate vital cellular functions. The role that autophagy plays in both the development and treatment of cancer is highly complex, especially given the fact that most cancer therapies modulate autophagy. This review aims to discuss the balance of autophagy in the development, progression, and treatment of head and neck cancer, as well as highlighting the need for a deeper understanding of what is still unknown about autophagy.

IKAROS and CK2 regulate expression of BCL-XL and chemosensitivity in high-risk B-cell acute lymphoblastic leukemia.

High-risk B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive disease, often characterized by resistance to chemotherapy. A frequent feature of high-risk B-ALL is loss of function of the IKAROS (encoded by the IKZF1 gene) tumor suppressor. Here, we report that IKAROS regulates expression of the BCL2L1 gene (encodes the BCL-XL protein) in human B-ALL. Gain-of-function and loss-of-function experiments demonstrate that IKAROS binds to the BCL2L1 promoter, recruits histone deacetylase HDAC1, and represses BCL2L1 expression via chromatin remodeling. In leukemia, IKAROS' function is impaired by oncogenic casein kinase II (CK2), which is overexpressed in B-ALL. Phosphorylation by CK2 reduces IKAROS binding and recruitment of HDAC1 to the BCL2L1 promoter. This results in a loss of IKAROS-mediated repression of BCL2L1 and increased expression of BCL-XL. Increased expression of BCL-XL and/or CK2, as well as reduced IKAROS expression, are associated with resistance to doxorubicin treatment. Molecular and pharmacological inhibition of CK2 with a specific inhibitor CX-4945, increases binding of IKAROS to the BCL2L1 promoter and enhances IKAROS-mediated repression of BCL2L1 in B-ALL. Treatment with CX-4945 increases sensitivity to doxorubicin in B-ALL, and reverses resistance to doxorubicin in multidrug-resistant B-ALL. Combination treatment with CX-4945 and doxorubicin show synergistic therapeutic effects in vitro and in preclinical models of high-risk B-ALL. Results reveal a novel signaling network that regulates chemoresistance in leukemia. These data lay the groundwork for clinical testing of a rationally designed, targeted therapy that combines the CK2 inhibitor, CX-4945, with doxorubicin for the treatment of hematopoietic malignancies.

O-Linked ß-N-acetylglucosamine (O-GlcNAc) modification: a new pathway to decode pathogenesis of diabetic retinopathy.

The incidence of diabetes continues to rise among all ages and ethnic groups worldwide. Diabetic retinopathy (DR) is a complication of diabetes that affects the retinal neurovasculature causing serious vision problems, including blindness. Its pathogenesis and severity is directly linked to the chronic exposure to high glucose conditions. No treatments are currently available to stop the development and progression of DR. To develop new and effective therapeutic approaches, it is critical to better understand how hyperglycemia contributes to the pathogenesis of DR at the cellular and molecular levels. We propose alterations in O-GlcNAc modification of target proteins during diabetes contribute to the development and progression of DR. The O-GlcNAc modification is regulated through hexosamine biosynthetic pathway. We showed this pathway is differentially activated in various retinal vascular cells under high glucose conditions perhaps due to their selective metabolic activity. O-GlcNAc modification can alter protein stability, activity, interactions, and localization. By targeting the same amino acid residues (serine and threonine) as phosphorylation, O-GlcNAc modification can either compete or cooperate with phosphorylation. Here we will summarize the effects of hyperglycemia-induced O-GlcNAc modification on the retinal neurovasculature in a cell-specific manner, providing new insight into the role of O-GlcNAc modification in early loss of retinal pericytes and the pathogenesis of DR.

Bcl-2 expression is essential for development and normal physiological properties of tooth hard tissue and saliva production.

BACKGROUND: Apoptosis plays a fundamental role in appropriate tissue development and function. Although expression of Bcl-2 has been reported during tooth and submandibular gland (SMG) development, the physiological role Bcl-2 plays during these processes has not been addressed. This study was performed to evaluate the impact of Bcl-2 expression on the formation and properties of tooth hard tissue, and saliva production. METHODS: Twenty-four mice (12 males and 12 females) were divided into three groups of eight (n=8): group A (Bcl-2 +/+), group B (Bcl-2 +/-), and group C (Bcl-2 -/-) and subjected to micro-CT analyses. The mineral content of first molars was analyzed by X-Ray diffraction (XRD) and scanning electron microscopy (SEM) color dot map. The surface microhardness was determined by Vickers test on labial surfaces of incisors. Saliva was collected from different groups of mice after subcutaneous injection of pilocarpine. RESULTS: Samples from Bcl-2 -/- mice showed significantly smaller micro-CT values, lower and poor crystallinity of hydroxyapatite (HA), and lowest surface micro hardness. SMG from Bcl-2 -/- mice showed remarkable reduction in size, consistent with reduced saliva accumulation. CONCLUSIONS: The absence of Bcl-2 expression in SMG did not affect the expression of other Bcl-2 family members. Thus, Bcl-2 expression influence on the formation and properties of tooth hard tissue, and saliva accumulation. SIGNIFICANCE: Bcl-2 expression has a significant impact on the mineralogical content of enamel crystals of tooth structure. Lack of Bcl-2 expression led to impaired production of enamel ACP crystals.

Bone morphogenetic protein 7 regulates reactive gliosis in retinal astrocytes and Müller glia.

PURPOSE: The focus of this study was to determine whether bone morphogenetic proteins (BMPs) trigger reactive gliosis in retinal astrocytes and/or Müller glial cells. METHODS: Retinal astrocytes and the Müller glial cell line MIO-M1 were treated with vehicle, BMP7, or BMP4. Samples from the treated cells were analyzed for changes in gliosis markers using reverse transcriptase - quantitative PCR (RT-qPCR) and western blotting. To determine potential similarities and differences in gliosis states, control and BMP-treated cells were compared to cells treated with sodium peroxynitrite (a strong oxidizing agent that will bring about some aspects of gliosis). Last, mature mice were microinjected intravitreally with BMP7 and analyzed for changes in gliosis markers using RT-qPCR, western blotting, and immunohistochemistry. RESULTS: Treatment of retinal astrocyte cells and Müller glial cells with BMP7 regulated various reactive gliosis markers. When compared to the response of cells treated with sodium peroxynitrite, the profiles of gliosis markers regulated due to exposure to BMP7 were similar. However, as expected, the profiles including the oxidative agent and growth factor were not identical. Treatment of cells with BMP4, however, showed an attenuated response in comparison to peroxynitrite and BMP7 treatment. Injection of BMP7 into the mouse retina also triggered a reactive gliosis response 7 days after injection. CONCLUSIONS: BMP7 induced changes in levels of mRNA and protein markers typically associated with reactive gliosis in retinal astrocytes and Müller glial cells, including glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), a subset of chondroitin sulfate proteoglycans (CSPGs), matrix metalloproteinases (MMPs), and other molecules.

Protein phosphatase 1 (PP1) and Casein Kinase II (CK2) regulate Ikaros-mediated repression of TdT in thymocytes and T-cell leukemia.

BACKGROUND: Ikaros is a DNA-binding protein that acts as master-regulator of hematopoiesis and a tumor suppressor. In thymocytes and T-cell leukemia, Ikaros negatively regulates transcription of terminal deoxynucleotide transferase (TdT), a key protein in lymphocyte differentiation. The signaling pathways that regulate Ikaros-mediated repression of TdT are unknown. Our previous work identified Casein Kinase II (CK2) and Protein Phosphatase 1 (PP1) as regulators of Ikaros DNA binding activity. Here, we investigated the role of PP1 and CK2 in regulating Ikaros-mediated control of TdT expression. PROCEDURES: Ikaros phosphomimetic and phosphoresistant mutants and specific CK2 and PP1 inhibitors were used in combination with quantitative chromatin immunoprecipitation (qChIP) and quantitative reverse transcriptase-PCR (q RT-PCR) assays to evaluate the role of CK2 and PP1 in regulating the ability of Ikaros to bind the TdT promoter and to regulate TdT expression. RESULTS: We demonstrate that phosphorylation of Ikaros by pro-oncogenic CK2 decreases Ikaros binding to the promoter of the TdT gene and reduces the ability of Ikaros to repress TdT expression during thymocyte differentiation. CK2 inhibition and PP1 activity restore Ikaros DNA-binding affinity toward the TdT promoter, as well as Ikaros-mediated transcriptional repression of TdT in primary thymocytes and in leukemia. CONCLUSION: These data establish that PP1 and CK2 signal transduction pathways regulate Ikaros-mediated repression of TdT in thymocytes and leukemia. These findings reveal that PP1 and CK2 have opposing effects on Ikaros-mediated repression of TdT and establish novel roles for PP1 and CK2 signaling in thymocyte differentiation and leukemia.

Thrombospondin-1, BIM and CFH polymorphisms and response to anti-VEGF treatment in neovascular age- related macular degeneration patients.

Age-related macular degeneration (AMD) is a vision threatening disease in older adults. Anti-VEGF treatment is effective for the majority of neovascular AMD (nAMD) patients, although approximately 30% of nAMD patients have an incomplete response for unknown reasons. Here we assessed the contribution of single nucleotide polymorphisms (SNPs) in key angioinflammatory regulatory genes in nAMD patients with an incomplete response compared to those responsive to anti-VEGF treatment. A total of 25 responsive and 30 nAMD patients with an incomplete response to anti-vascular endothelial growth factor (anti-VEGF) treatment were examined for known SNPs that impact the structure and function of thromobospondin-1 (TSP1), Bcl-2-interacting mediator of cell death (BIM) and complement factor H (CFH). Plasma levels of C-C motif chemokine ligand 2 (CCL2/MCP1), TSP1 and VEGF were assessed by ELISA. Patients responsive to anti-VEGF treatment showed a significant increase in the TSP1 rs2228262 AA allele and a trend for the BIM (rs724710) CT allele. Consistent with previous reports, 42% of the patients responsive to anti-VEGF expressed the CC allele for CFH rs1061170. Although the CFH TT allele had similarly low prevalence in both groups, the TC allele tended to be more prevalent in patients with an incomplete response. Patients with an incomplete response also had increased plasma CCL2/MCP1 levels, consistent with the role increased inflammation has in the pathogenesis of nAMD. Our studies point to new tools to assess the potential responsiveness of nAMD patients to anti-VEGF treatment and suggest the potential use of anti-CCL2 for treatment of nAMD patients with an incomplete response to anti-VEGF.

Cyp1B1 expression promotes angiogenesis by suppressing NF-κB activity.

Nuclear factor-κB (NF-κB) is a master regulator of genes that control a large number of cellular processes, including angiogenesis and inflammation. We recently demonstrated that cytochrome P-450 1B1 (Cyp1B1) deficiency in endothelial cells (EC) and pericytes (PC) results in increased oxidative stress, alterations in migration, attenuation of capillary morphogenesis, sustained activation of NF-κB, and increased expression of thrombospondin-2 (TSP2), an endogenous inhibitor of angiogenesis. On the basis of a growing body of evidence that phenethyl isothiocyanate (PEITC) and pyrrolidine dithiocarbamate (PDTC) function as antioxidants and suppressors of NF-κB activation, we investigated their potential ability to restore a normal phenotype in Cyp1B1-deficient (cyp1b1(-/-)) vascular cells. PEITC and PDTC inhibited NF-κB activity and expression in cyp1b1(-/-) EC and PC. We also observed restoration of migration and capillary morphogenesis of cyp1b1(-/-) EC and decreased cellular oxidative stress in cyp1b1(-/-) EC and PC without restoration to normal TSP2 levels. In addition, expression of a dominant-negative inhibitor κBα, a suppressor of NF-κB activation, decreased NF-κB activity without affecting TSP2 expression in these cells. In contrast, knockdown of TSP2 expression resulted in attenuation of NF-κB activity in cyp1b1(-/-) vascular cells. Furthermore, expression of TSP2 in wild-type (cyp1b1(+/+)) cells resulted in increased NF-κB activity. Together, our results demonstrate an important role for TSP2 in modulation of NF-κB activity and attenuation of angiogenesis. Thus Cyp1B1 expression in vascular cells plays an important role in the regulation of vascular homeostasis through modulation of the cellular reductive state, TSP2 expression, and NF-κB activation.

High glucose and diabetes modulate cellular proteasome function: Implications in the pathogenesis of diabetes complications.

The precise link between hyperglycemia and its deleterious effects on retinal and kidney microvasculature, and more specifically loss of retinal perivascular supporting cells including smooth muscle cell/pericytes (SMC/PC), in diabetes are not completely understood. We hypothesized that differential cellular proteasome activity contributes to sensitivity of PC to high glucose-mediated oxidative stress and vascular rarefaction. Here we show that retinal endothelial cells (EC) have significantly higher proteasome peptidase activity compared to PC. High glucose treatment (HGT) increased the level of total ubiquitin-conjugated proteins in cultured retinal PC and EC, but not photoreceptor cells. In addition, in vitro proteasome activity assays showed significant impairment of proteasome chymotrypsin-like peptidase activity in PC, but not EC. The PA28-α/-ß and PA28-ß/-γ protein levels were also higher in the retina and kidney glomeruli of diabetic mice, respectively. Our results demonstrate, for the first time, that high glucose has direct biological effects on cellular proteasome function, and this modulation might be protective against cellular stress or damage induced by high glucose.

Retinal O-linked N-acetylglucosamine protein modifications: implications for postnatal retinal vascularization and the pathogenesis of diabetic retinopathy.

PURPOSE: Hyperglycemia activates several metabolic pathways, including the hexosamine biosynthetic pathway. Uridine diphosphate N-acetylglucosamine (GlcNAc) is the product of the hexosamine biosynthetic pathway and the substrate for O-linked GlcNAc (O-GlcNAc) modification. This modification affects a wide range of proteins by altering their activity, cellular localization, and/or protein interactions. However, the role O-GlcNAcylation may play in normal postnatal retinal vascular development and in the ocular complications of diabetes, including diabetic retinopathy, requires further investigation. METHODS: The total levels of O-GlcNAc-modified proteins were evaluated by western blot analysis of lysates prepared from retinas obtained at different days during postnatal retinal vascularization and oxygen-induced ischemic retinopathy. Similar experiments were performed with retinal lysate prepared from diabetic Ins2(Akita/+) mice with different durations of diabetes and retinal vascular cells cultured under various glucose conditions. The localization of O-GlcNAc-modified proteins in the retinal vasculature was confirmed by immunofluorescence staining. The impact of altered O-GlcNAcylation on the migration of retinal vascular cells was determined using scratch wound and transwell migration assays. RESULTS: We detected an increase in protein O-GlcNAcylation during mouse postnatal retinal vascularization and aging, in part through the regulation of the enzymes that control this modification. The study of the diabetic Ins2(Akita/+) mouse retina showed an increase in the O-GlcNAc modification of retinal proteins. We also observed an increase in retinal O-GlcNAcylated protein levels during the neovascularization phase of oxygen-induced ischemic retinopathy. Our fluorescence microscopy data confirmed that the alterations in retinal O-GlcNAcylation are similarly represented in the retinal vasculature and in retinal pericytes and endothelial cells. Particularly, the migration of retinal pericytes, but not retinal endothelial cells, was attenuated by increased O-GlcNAc modification. CONCLUSIONS: The O-GlcNAc modification pattern changes during postnatal retinal vascular development and neovascularization, and its dysregulation under hyperglycemia and/or ischemia may contribute to the pathogenesis of the diabetic retinopathy and retinal neovascularization.

BIM deficiency differentially impacts the function of kidney endothelial and epithelial cells through modulation of their local microenvironment.

The extracellular matrix (ECM) acts as a scaffold for kidney cellular organization. Local secretion of the ECM allows kidney cells to readily adapt to changes occurring within the kidney. In addition to providing structural support for cells, the ECM also modulates cell survival, migration, proliferation, and differentiation. Although aberrant regulation of ECM proteins can play a causative role in many diseases, it is not known whether ECM production, cell adhesion, and migration are regulated in a similar manner in kidney epithelial and endothelial cells. Here, we demonstrate that lack of BIM expression differentially impacts kidney endothelial and epithelial cell ECM production, migration, and adhesion, further emphasizing the specialized role of these cell types in kidney function. Bim -/- kidney epithelial cells demonstrated decreased migration, increased adhesion, and sustained expression of osteopontin and thrombospondin-1 (TSP1). In contrast, bim -/- kidney endothelial cells demonstrated increased cell migration, and decreased expression of osteopontin and TSP1. We also observed a fivefold increase in VEGF expression in bim -/- kidney endothelial cells consistent with their increased migration and capillary morphogenesis. These cells also had decreased endothelial nitric oxide synthase activity and nitric oxide bioavailability. Thus kidney endothelial and epithelial cells make unique contributions to the regulation of their ECM composition, with specific impact on adhesive and migratory properties that are essential for their proper function.

Caspase-14: a novel caspase in the retina with a potential role in diabetic retinopathy.

PURPOSE: The purpose of this study was to evaluate caspase-14 expression in the retina under normal and diabetic conditions, and to determine whether caspase-14 contributes to retinal microvascular cell death under high glucose conditions. METHODS: Quantitative real-time polymerase chain reaction and western blot analysis were used to evaluate caspase-14 expression in retinal cells, including pericytes (PCs), endothelial cells (ECs), astrocytes (ACs), choroidal ECs, and retinal pigment epithelium (RPE) cells. We also determined caspase-14 expression in the retinas of human subjects with or without diabetic retinopathy (DR) and in experimental diabetic mice. Retinal ECs and PCs were infected with adenoviruses expressing human caspase-14 or green fluorescent protein. Caspase-14 expression was also assessed in retinal vascular cells cultured under high glucose conditions. The number of apoptotic cells was determined with terminal deoxynucleotidyl transferase dUTP nick end labeling staining and confirmed by determining the levels of cleaved poly (ADP-ribose) polymerase-1 and caspase-3. RESULTS: Our experiments demonstrated that retinal ECs, PCs, ACs, choroidal ECs, and RPE cells expressed caspase-14, and DR was associated with upregulation and/or activation of caspase-14 particularly in retinal vasculature. High glucose induced marked elevation of the caspase-14 level in retinal vascular cells. There was a significant increase in the apoptosis rate and the levels of cleaved poly (ADP-ribose) polymerase-1 and caspase-3 in retinal ECs and PCs overexpressing caspase-14. CONCLUSIONS: Our findings indicate that caspase-14 might play a significant role in the pathogenesis of DR by accelerating retinal PC and EC death. Further investigations are required to elaborate the underlying mechanisms.

Congenital pancytopenia and absence of B lymphocytes in a neonate with a mutation in the Ikaros gene.

BACKGROUND: Congenital pancytopenia is a rare and often lethal condition. Current knowledge of lymphoid and hematopoietic development in mice, as well as understanding regulators of human hematopoiesis, have led to the recent discovery of genetic causes of bone marrow failure disorders. However, in the absence of mutations of specific genes or a distinct clinical phenotype, many cases of aplastic anemia are labeled as idiopathic, while congenital immune deficiencies are described as combined immune deficiency. PROCEDURE: We describe the case of a 33-week gestation age male with severe polyhydramnios, hydrops, and ascites who was noted to be pancytopenic at birth. Bone marrow examination revealed a hypocellular marrow with absent myelopoiesis. An immune workup demonstrated profound B lymphopenia, near absent NK cells, and normal T cell number. Due to the similarity of the patient's phenotype with the IKAROS knockout mouse, studies were performed on bone marrow and peripheral blood to assess a potential pathogenic role of Ikaros. RESULTS: DNA studies revealed a point mutation in one allele of the IKAROS gene, resulting in an amino acid substitution in the DNA-binding zinc finger domain. Functional studies demonstrated that the observed mutation decreased Ikaros DNA-binding affinity, and immunofluorescence microscopy revealed aberrant Ikaros pericentromeric localization. CONCLUSIONS: Our report describes a novel case of congenital pancytopenia associated with mutation of the IKAROS gene. Furthermore, these data suggest a critical role of IKAROS in human hematopoiesis and immune development.

Targeted Thrombospondin-1 Expression in Ocular Vascular Development and Neovascularization.

Tight regulation of positive and negative regulators of angiogenesis is essential, particularly in the eye where their dysregulation can lead to vision loss. Thrombospondin-1 (TSP1) is a matricellular protein that negatively regulates angiogenesis and inflammation in the eye. It aids ocular vascular homeostasis such that its loss contributes to increased retinal vascular density and pathologic ocular neovascularization. Our previous studies demonstrated that mice globally lacking TSP1 expression had increased retinal vascular density, decreased hyperoxia-induced retinal vessel loss, and increased choroidal neovascularization. Here we determined the impact to the ocular vasculature of endothelial cell, pericyte, or astrocyte loss of TSP1 expression. Only lack of TSP1 expression in endothelial cells was sufficient to increase choroidal neovascularization with mice lacking expression in pericytes or astrocytes not demonstrating a significant impact. Although the global TSP1 knockout mice demonstrated increased retinal vascular density, individual cell type loss of TSP1 resulted in decreased retinal endothelial cell numbers before and/or after vascular maturation in a cell type specific fashion. Retinas from mice lacking TSP1 expression in endothelial cells, pericytes or astrocytes were not protected from retinal vessel regression in response to hyperoxia as we previously observed in the global knockout. Thus, modulation of TSP1 expression in individual cell types demonstrates a response that is unique to the role TSP1 plays in that cell type of interest, and their coordinated activity is critical for vision.

Caffeine Inhibits Choroidal Neovascularization Through Mitigation of Inflammatory and Angiogenesis Activities.

Adenosine receptors (AR) are widely expressed in a variety of tissues including the retina and brain. They are involved in adenosine-mediated immune responses underlying the onset and progression of neurodegenerative diseases. The expression of AR has been previously demonstrated in some retinal cells including endothelial cells and retinal pigment epithelial cells, but their expression in the choroid and choroidal cells remains unknown. Caffeine is a widely consumed AR antagonist that can influence inflammation and vascular cell function. It has established roles in the treatment of neonatal sleep apnea, acute migraine, and post lumbar puncture headache as well as the neurodegenerative diseases such as Parkinson and Alzheimer. More recently, AR antagonism with caffeine has been shown to protect preterm infants from ischemic retinopathy and retinal neovascularization. However, whether caffeine impacts the development and progression of ocular age-related diseases including neovascular age-related macular degermation remains unknown. Here, we examined the expression of AR in retinal and choroidal tissues and cells. We showed that antagonism of AR with caffeine or istradefylline decreased sprouting of thoracic aorta and choroid/retinal pigment epithelium explants in ex vivo cultures, consistent with caffeine's ability to inhibit endothelial cell migration in culture. In vivo studies also demonstrated the efficacy of caffeine in inhibition of choroidal neovascularization and mononuclear phagocyte recruitment to the laser lesion sites. Istradefylline, a specific AR 2A antagonist, also decreased choroidal neovascularization. Collectively, our studies demonstrate an important role for expression of AR in the choroid whose antagonism mitigate choroidal inflammatory and angiogenesis activities.

Development and characterization of patient-derived xenografts from non-small cell lung cancer brain metastases.

Non-small cell lung cancer (NSCLC) brain metastasis cell lines and in vivo models are not widely accessible. Herein we report on a direct-from patient-derived xenograft (PDX) model system of NSCLC brain metastases with genomic annotation useful for translational and mechanistic studies. Both heterotopic and orthotopic intracranial xenografts were established and RNA and DNA sequencing was performed on patient and matching tumors. Morphologically, strong retention of cytoarchitectural features was observed between original patient tumors and PDXs. Transcriptome and mutation analysis revealed high correlation between matched patient and PDX samples with more than more than 95% of variants detected being retained in the matched PDXs. PDXs demonstrated response to radiation, response to selumetinib in tumors harboring KRAS G12C mutations and response to savolitinib in a tumor with MET exon 14 skipping mutation. Savolitinib also demonstrated in vivo radiation enhancement in our MET exon 14 mutated PDX. Early passage cell strains showed high consistency between patient and PDX tumors. Together, these data describe a robust human xenograft model system for investigating NSCLC brain metastases. These PDXs and cell lines show strong phenotypic and molecular correlation with the original patient tumors and provide a valuable resource for testing preclinical therapeutics.

CYP1B1 and endothelial nitric oxide synthase combine to sustain proangiogenic functions of endothelial cells under hyperoxic stress.

We have recently shown that deletion of constitutively expressed CYP1B1 is associated with attenuation of retinal endothelial cell (EC) capillary morphogenesis (CM) in vitro and angiogenesis in vivo. This was largely caused by increased intracellular oxidative stress and increased production of thrombospondin-2, an endogenous inhibitor of angiogenesis. Here, we demonstrate that endothelium nitric oxide synthase (eNOS) expression is dramatically decreased in the ECs prepared from retina, lung, heart, and aorta of CYP1B1-deficient (CYP1B1(-/-)) mice compared with wild-type (CYP1B1(+/+)) mice. The eNOS expression was also decreased in retinal vasculature of CYP1B1(-/-) mice. Inhibition of eNOS activity in cultured CYP1B1(+/+) retinal ECs blocked CM and was concomitant with increased oxidative stress, like in CYP1B1(-/-) retinal ECs. In addition, expression of eNOS in CYP1B1(-/-) retinal ECs or their incubation with a nitric oxide (NO) donor enhanced NO levels, lowered oxidative stress, and improved cell migration and CM. Inhibition of CYP1B1 activity in the CYP1B1(+/+) retinal ECs resulted in reduced NO levels and attenuation of CM. In contrast, expression of CYP1B1 increased NO levels and enhanced CM of CYP1B1(-/-) retinal ECs. Furthermore, attenuation of CYP1B1 expression with small interfering RNA proportionally lowered eNOS expression and NO levels in wild-type cells. Together, our results link CYP1B1 metabolism in retinal ECs with sustained eNOS activity and NO synthesis and/or bioavailability and low oxidative stress and thrombospondin-2 expression. Thus CYP1B1 and eNOS cooperate in different ways to lower oxidative stress and thereby to promote CM in vitro and angiogenesis in vivo.

Apoptotic rate and metallothionein levels in the tissues of cadmium- and copper-exposed rats.

It is well known that cadmium (Cd) has toxic and carcinogenic effects in rodents and humans, but the effects of Cd on apoptosis are still not clear. Although some studies have shown that Cd has apoptotic potential, other studies have shown that Cd can be antiapoptotic. Parameters such as sensitivity of the exposed organism or cells and the exposure conditions should be important in delineating the effect of Cd on apoptosis. In the present study, we aimed to determine the apoptotic index (AI) of Sprague-Dawley rat tissues that are loaded at a lower Cd concentration than the critical concentration (50 microg/g) for its toxic effects. Metallothionein (MT) levels of tissues were also determined and the experiments repeated with copper (Cu)-exposed rats. We detected decreases in the apoptotic index in liver and lung tissues of Cd-exposed groups accompanied with an increase in MT levels. Also, decreases of AI were detected in the liver tissues of Cu-exposed groups. These findings indicate that Cd can suppress apoptosis in vivo. The possible role of MT expression on the suppression of apoptosis and the importance of free-Cd ion concentration on switching antiapoptotic effects to proapoptotic effects are also discussed.

Bim expression in endothelial cells and pericytes is essential for regression of the fetal ocular vasculature.

Apoptosis plays a central role in developmental and pathological angiogenesis and vessel regression. Bim is a pro-apoptotic Bcl-2 family member that plays a prominent role in both developmental and pathological ocular vessel regression, and neovascularization. Endothelial cells (EC) and pericytes (PC) each play unique roles during vascular development, maintenance and regression. We recently showed that germline deletion of Bim results in persistent hyaloid vasculature, increased retinal vascular density and prevents retinal vessel regression in response to hyperoxia. To determine whether retinal vascular regression is attributable to Bim expression in EC or PC we generated mice carrying a conditional Bim allele (BimFlox/Flox) and VE-cadherin-cre (BimEC mice) or Pdgfrb-cre (BimPC mice). BimEC and BimPC mice demonstrated attenuated hyaloid vessel regression and postnatal retinal vascular remodeling. We also observed decreased retinal vascular apoptosis and proliferation. Unlike global Bim -/- mice, mice conditionally lacking Bim in EC or PC underwent hyperoxia-mediated vessel obliteration and subsequent retinal neovascularization during oxygen-induced ischemic retinopathy similar to control littermates. Thus, understanding the cell autonomous role Bim plays in the retinal vascular homeostasis will give us new insight into how to modulate pathological retinal neovascularization and vessel regression to preserve vision.

Cone-Specific Promoters for Gene Therapy of Achromatopsia and Other Retinal Diseases.

Adeno-associated viral (AAV) vectors containing cone-specific promoters have rescued cone photoreceptor function in mouse and dog models of achromatopsia, but cone-specific promoters have not been optimized for use in primates. Using AAV vectors administered by subretinal injection, we evaluated a series of promoters based on the human L-opsin promoter, or a chimeric human cone transducin promoter, for their ability to drive gene expression of green fluorescent protein (GFP) in mice and nonhuman primates. Each of these promoters directed high-level GFP expression in mouse photoreceptors. In primates, subretinal injection of an AAV-GFP vector containing a 1.7-kb L-opsin promoter (PR1.7) achieved strong and specific GFP expression in all cone photoreceptors and was more efficient than a vector containing the 2.1-kb L-opsin promoter that was used in AAV vectors that rescued cone function in mouse and dog models of achromatopsia. A chimeric cone transducin promoter that directed strong GFP expression in mouse and dog cone photoreceptors was unable to drive GFP expression in primate cones. An AAV vector expressing a human CNGB3 gene driven by the PR1.7 promoter rescued cone function in the mouse model of achromatopsia. These results have informed the design of an AAV vector for treatment of patients with achromatopsia.