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Reactive species derived from cell oxygenation processes play an important role in vascular homeostasis and the pathogenesis of many diseases including retinopathy of prematurity. We show that CYP1B1-deficient (CYP1B1 ؊/؊ ) mice fail to elicit a neovascular response during oxygen-induced ischemic retinopathy. In addition, the retinal endothelial cells (ECs) prepared from CYP1B1 ؊/؊ mice are less adherent, less migratory, and fail to undergo capillary morphogenesis. These aberrant cellular responses were completely reversed when oxygen levels were lowered or an antioxidant added. CYP1B1 ؊/؊ ECs exhibited increased oxidative stress and expressed increased amounts of the antiangiogenic factor thrombospondin-2 (TSP2). Increased lipid peroxidation and TSP2 were both observed in retinas from CYP1B1 ؊/؊ mice and were reversed by administration of an antioxidant. Reexpression of CYP1B1 in CYP1B1 ؊/؊ ECs resulted in down-regulation of TSP2 expression and restoration of capillary morphogenesis. A TSP2 knockdown in CYP1B1 ؊/؊ ECs also restored capillary morphogenesis. Thus, CYP1B1 metabolizes cell products that modulate intracellular oxidative stress, which enhances production of TSP2, an inhibitor of EC migration and capillary morphogenesis. Evidence is presented that similar changes occur in retinal endothelium in vivo to limit neovascularization. (Blood. 2009;113:744-754) IntroductionPathologic angiogenesis is associated with major blinding diseases including retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration (AMD). 1 This neovascularization is driven by the hypoxic stimulus due to loss of existing vessels. The oxygen-induced ischemic retinopathy (OIR) model in mouse recapitulates this condition, whereby exposure to hyperoxia results in loss of existing retinal vessels promoting ischemia-mediated retinal neovascularization. 2 Reactive oxygen species (ROS) play an important role during angiogenesis, and their aberrant production is linked to retinopathy of prematurity and diabetic retinopathy. 3,4 In fact, antioxidants inhibit microvascular degeneration in models of diabetes and OIR. [4][5][6] The cellular mechanisms, which modulate intracellular oxidative stress, however, are not fully characterized.CYP1B1 is a member of the cytochrome P450 family of proteins. It is expressed in extrahepatic epithelial and particularly mesenchymal cells and exhibits a developmentally regulated expression pattern. This family of enzymes catalyzes a wide array of mono-oxygenase reactions targeting both foreign and endogenous lipophilic compounds including fat-soluble vitamins, steroid hormones, and polyunsaturated fatty acid (PUFA) products. 7 Many of these enzymes can function with low specificity to initiate inactivation or excretion pathways, but also function with high specificity and activity to synthesize physiologically active chemicals such as steroid hormones. 8,9 Recent studies indicate that expression of CYP enzymes in the cardiovascular system and their metabolites from arachidonic acid, pla...

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor responsible for mediating the cellular response to the toxic compound 2,3,7,8,-tetrachlorodibenzo-p-dioxin. An essential role for the AhR in cellular biology has been established previously, but no high-affinity endogenous ligand has yet been identified. We have confirmed the presence of a putative endogenous ligand(s) in CV-1 cells through transient transfection with various cytochrome P450 isoforms. Expression of cytochromes P450 1A1, 1A2, or 1B1 reduced AhR-mediated luciferase reporter activity, whereas cytochrome P450 2E1 exhibited no significant effect. Studies with 2,4,3Ј,5Ј-tetramethoxystilbene, a potent and specific inhibitor of cytochrome P450 1B1, was able to partially block cytochrome P450 1B1-mediated reduction in reporter gene activity. These results provide evidence of the existence of a possible feedback mechanism in which AhR-regulated cytochromes P450 from the CYP1A and CYP1B families are able to metabolically alter putative endogenous ligand(s). Several experiments were performed to provide initial characterization of these putative endogenous ligands, including electrophoretic mobility shift assay analyses, which demonstrated that these ligands directly activate the AhR. Soluble extracts from various C57BL/6J and Ahr-null mouse tissues were also analyzed for the presence of AhR activators. Studies revealed that Ahr-null mouse lung tissue had a 4-fold increase in AhR-mediated reporter activity in cells. Quantitative polymerase chain reaction analysis revealed that lung tissue exhibits relatively high constitutive CYP1A1 mRNA levels. These results suggest that there is an autoregulatory feedback loop between the AhR and cytochrome P450 1A1 in mouse lung.

CYP1A1 and CYP1B1 gene and protein expressions vary with time, magnitude, and pattern of shear stress. Increased CYP1A1 gene expression modulates AhR-regulated genes. Based on our in vitro reversing flow data and in vivo immunostained mouse aorta, we suggest that increased expression of both genes reflects an anti-atherogenic endothelial cell phenotype.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that regulates the transcription of certain key enzymes involved in the metabolism of xenobiotic substances including some drugs. The AhR can be activated by a wide range of classes of compounds (e.g. polycyclic aromatic hydrocarbons, benzimidazoles and flavonoids), and interacts with a number of other proteins, including nuclear hormone receptors such as the oestrogen and androgen receptors. Activation of the AhR antagonises the oestrogen receptor and can lead to modulation of its transcriptional activity; thus, activating the AhR may serve as a target for breast cancer therapy. Disruption of normal signalling by drug interactions with the AhR or downstream components of this pathway could result in adverse effects, such as the bioactivation of procarcinogens or the disruption of normal homeostasis. The cytochrome P450s CYP1A1, -1B1, -1A2 and -2S1 are regulated by the AhR, and they are all involved in the metabolism of endogenous substrates as well as xenobiotics. Polymorphisms in the AhR, or polymorphisms in enzymes regulated by the AhR, may cause variations in response to certain drugs in different individuals; this needs to be taken into consideration when administering drugs that interact with this pathway.