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...

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Integration of cell adhesive, survival, and proliferative processes is essential for capillary morphogenesis of endothelial cells (EC) in vitro and vascular development and function in vivo. Unfortunately, the molecular and cellular mechanisms that impact these processes are poorly defined. Here we examined how lack of bim and/or bcl-2 expression impact lung EC function. The absence of bcl-2 or bim had a significant impact on EC adhesion and migration. Lack of bcl-2 expression decreased lung EC migration, whereas lack of bim expression increased migration compared with their wild-type counterparts. Decreased adhesion to fibronectin and vitronectin was observed in both bcl-2-/- and bim-/- lung EC, with bcl-2-/- EC having very little adhesion to either matrix protein. Capillary morphogenesis was greatly diminished in bcl-2-/- EC, which correlated with decreased lung alveolarization in vivo, an angiogenesis-dependent process. We also observed aberrant production of extracellular matrix proteins, eNOS expression, and nitric oxide production in bcl-2-/- lung EC, which could contribute to inability to undergo capillary morphogenesis. The changes in cell adhesion and migration noted in the absence of bim or bcl-2 were independent of their impact on apoptosis. We observed no significant affect on the steady-state rate of apoptosis of lung EC in the absence of bim or bcl-2. Thus, bcl-2 family members, bim and bcl-2, play a central role in modulation of EC proangiogenic properties, which goes beyond their role as simple mediators of mitochondrial homeostasis and apoptosis.

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.

Collectively, these results demonstrate that VPO1 promotes hypoxia-induced proliferation, apoptosis resistance, and migration in PASMCs via the NOX4/VPO1/HOCl/NF-κB signalling pathway.

Kondo S, Tang Y, Scheef EA, Sheibani N, Sorenson CM. Attenuation of retinal endothelial cell migration and capillary morphogenesis in the absence of bcl-2.

Allicin is the major biologically active compounds of freshly crushed garlic. It has been reported to inhibit the proliferation and promote the apoptosis of multiple colorectal cancer cells. However, the anti-colorectal cancer effect of Allicin has not been verified by in vivo studies. In the present study, we investigated the effect of Allicin on azoxymethane/dextran sodium sulfate (AOM/DSS) colorectal cancer mouse model and explore the underlying possible mechanism. Our result showed that Allicin could inhibit colonic tumorigenesis of AOM/DSS mice in vivo. In vitro study showed that Allicin promoted the apoptosis and suppressed the survival and proliferation of HCT116 cells. The molecular mechanism is related to the suppression of STAT3 signaling activation. Thus, our data provide further support for Allicin as a potential favorable supplement for human colorectal cancer.

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