Rebecca C. Garland

Vascular adhesion protein-1 blockade suppresses choroidal neovascularization

Vascular adhesion protein‐1 (VAP‐1) is an endothelial cell adhesion molecule involved in leukocyte recruitment. Leukocytes and, in particular, macrophages play an important role in the development of choroidal neovascularization (CNV), an integral component of age‐related macular degeneration (AMD). Previously, we showed a role for VAP‐1 in ocular inflammation. Here, we investigate the expression of VAP‐1 in the choroid and its role in CNV development. VAP‐1 was expressed in the choroid, exclusively in the vessels, and colocalized in the vessels of the CNV lesions. VAP‐1 blockade with a novel and specific inhibitor significantly decreased CNV size, fluorescent angiographic leakage, and the accumulation of macrophages in the CNV lesions. Furthermore, VAP‐1 blockade significantly reduced the expression of inflammation‐associated molecules such as tumor necrosis factor (TNF) ‐α, monocyte chemoattractant protein (MCP) ‐1, and intercellular adhesion molecule (ICAM) ‐1. This work provides evidence for an important role of VAP‐1 in the recruitment of macrophages to CNV lesions, establishing a novel link between VAP‐1 and angiogenesis. Inhibition of VAP‐1 may become a new therapeutic strategy in the treatment of AMD.—Noda, K., She, H., Nakazawa, T., Hisatomi, T., Nakao, S., Almulki, L., Zandi, S., Miyahara, S., Ito, Y., Thomas, K. L., Garland, R. C., Miller, J. W., Gragoudas, E. S., Mashima, Y., Hafezi‐Moghadam, A. Vascular adhesion protein‐1 blockade suppresses choroidal neovascularization. FASEB J. 22, 2928‐2935 (2008)

Inhibition of vascular adhesion protein-1 suppresses endotoxin-induced uveitis

Inflammatory leukocyte accumulation is a common feature of major ocular diseases, such as uveitis, diabetic retinopathy, and age‐related macular degeneration. Vascular adhesion protein‐1 (VAP‐1), a cell surface and soluble molecule that possesses semi‐carbazide‐sensitive amine oxidase (SSAO) activity, is involved in leukocyte recruitment. However, the expression of VAP‐1 in the eye and its contribution to ocular inflammation are unknown. Here, we investigated the role of VAP‐1 in an established model of ocular inflammation, the endotoxin‐induced uveitis (EIU), using a novel and specific inhibitor. Our inhibitor has a half‐maximal inhibitory concentration (IC50) of 0.007 μM against human and 0.008 μM against rat SSAO, while its IC50 against the functionally related monoamine oxidase (MAO) ‐A and MAO‐B is > 10 μM. In the retina, VAP‐1 was exclusively expressed in the vasculature, and its expression level was elevated during EIU. VAP‐1 inhibition in EIU animals significantly suppressed leukocyte recruitment to the anterior chamber, vitreous, and retina, as well as retinal endothelial P‐selectin expression. Our data suggest an important role for VAP‐1 in the recruitment of leukocytes to the immune‐privileged ocular tissues during acute inflammation. VAP‐1 inhibition may become a novel strategy in the treatment of ocular inflammatory diseases. Noda, K., Miyahara, S., Nakazawa, T., Almulki, L., Nakao, S., Hisatomi, T., She, H., Thomas, K. L., Garland, R. C., Miller, J. W., Gragoudas, E. S., Kawai, Y., Mashima, Y., Hafezi‐Moghadam, A. Inhibition of vascular adhesion protein‐1 suppresses endotoxin‐in‐duced uveitis. FASEB J. 22, 1094–1103 (2008)

Noninvasive molecular imaging reveals role of PAF in leukocyte-endothelial interaction in LPS-induced ocular vascular injury

Uveitis is a systemic immune disease and a common cause of blindness. The eye is an ideal organ for light‐based imaging of molecular events underlying vascular and immune diseases. The phospholipid platelet‐activating factor (PAF) is an important mediator of inflammation, the action of which in endothelial and immune cells in vivo is not well understood. The purpose of this study was to investigate the role of PAF in endothelial injury in uveitis. Here, we use our recently introduced in vivo molecular imaging approach in combination with the PAF inhibitors WEB 2086 (WEB) and ginkgolide B (GB). The differential inhibitory effects of WEB and GB in reducing LPS‐induced endothelial injury in the choroid indicate an important role for PAF‐like lipids, which might not require the PAF receptor for their signaling. P‐selectin glycoprotein ligand‐1‐mediated rolling of mouse leukocytes on immobilized P‐selectin in our autoperfused microflow chamber assay revealed a significant reduction in rolling velocity on the cells’ contact with PAF. Rolling cells that came in contact with PAF rapidly assumed morphological signs of cell activation, indicating that activation during rolling does not require integrins. Our results show a key role for PAF in mediating endothelial and leukocyte activation in acute ocular inflammation. Our in vivo molecular imaging provides a detailed view of cellular and molecular events in the complex physiological setting.—Garland, R. C., Sun, D., Zandi, S., Xie, F., Faez, S., Tayyari, F., Frimmel, S. A. F., Schering, A., Nakao, S., Hafezi‐Moghadam, A. Noninvasive molecular imaging reveals role of PAF in leukocyte‐endothelial interaction in LPS‐induced ocular vascular injury. FASEB J. 25, 1284–1294 (2011). www.fasebj.org

In vivo imaging of endothelial injury in choriocapillaris during endotoxin-induced uveitis

Early detection of ocular inflammation may prevent the occurrence of structural damage or vision loss. Here, we introduce a novel noninvasive technique for molecular imaging and quantitative eval uation of endothelial injury in the choriocapillaris of live animals, which detects disease earlier than cur rently possible. Using an established model of ocular inflammation, endotoxin‐induced uveitis (EIU), we vi sualized the rolling and adhesive interaction of fluores cent microspheres conjugated to recombinant P‐selec‐ tin glycoprotein ligand‐Ig (rPSGL‐Ig) in choriocapillaris using a scanning laser ophthalmoscope (SLO). The number of rolling microspheres in the choriocapillaris peaked 4–10 h after LPS injection. The number of the accumulated microspheres peaked 4 h after LPS injection in the temporal choriocapillaris and 4 and 36 h after LPS injection in the central areas around the optic disk. Furthermore, we semiquantified the levels of P‐selectin mRNA expression in the choroidal vessels by reverse transcription‐PCR and found its pattern to match the functional microsphere interactions, with a peak at 4 h after LPS injection. These results indicate that PSGL‐1‐conjugated fluorescent microspheres allow specific detection of endothelial P‐selectin expression in vivo and noninvasive assessment of endothelial in jury. This technique may help to diagnose subclinical signs of ocular inflammatory diseases.— Miyahara, S., Almulki, L., Noda, K., Nakazawa, T., Hisatomi, T., Nakao, S., Thomas, K. L., Schering, A., Zandi, S., Frimmel, S., Tayyari, F., Garland, R. C., Miller, J. W., Gragoudas, E. S., Masli, S., Hafezi‐Moghadam, A. In vivo imaging of endothelial injury in choriocapillaris during endotoxin‐induced uveitis. FASEB J. 22, 1973–1980 (2008)

Surprising up-regulation of P-selectin glycoprotein ligand-1 (PSGL-1) in endotoxin-induced uveitis

P‐selectin glycoprotein ligand‐1 (PSGL‐1) is constitutively expressed on leukocytes and was thought to be down‐regulated with cell activation. However, this work shows the surprising finding of functional PSGL‐1 up‐regulation during acute inflammation. PSGL‐1 function was studied in our autoperfusion assay, in which blood from a mouse carotid flows through a microcham‐ber coated with a fixed density of P‐selectin. Under the inflammatory conditions—uveitis induced by systemic lipopolysaccharide injection—we recorded significantly reduced leukocyte rolling velocity, which suggests PSGL‐1 up‐regulation; however, flow cytometry showed reduced PSGL‐1. When bound leukocytes were released from the vasculature by PSGL‐1 blockade, a large peripheral blood leukocyte (PBL) population showed elevated PSGL‐1, which could account for the reduced PSGL‐1 in the remaining unbound population. In the eye, systemic blockade of PSGL‐1 with a monoclonal antibody or recombinant soluble PSGL‐1 drastically reduced the severe manifestations of uveitis. Furthermore, PSGL‐1 blockade was significantly more effective in reducing retinal leukostasis than was P‐selectin blockade. Our results provide surprising evidence for functional PSGL‐1 up‐regulation in PBLs during acute inflammation. The temporal overlap between PSGL‐1 and P‐selectin up‐regulation reveals an as yet unrecognized collaboration between this receptor‐ligand pair, increasing efficiency of the first steps of the leukocyte recruitment cascade.— Almulki, L., Noda, K., Amini, R., Schering, A., Garland, R. C., Nakao, S., Nakazawa, T., Hisatomi, T., Thomas, K. L., Masli, S., Hafezi‐Moghadam, A. Surprising up‐regulation of P‐selectin glycoprotein ligand‐1 (PSGL‐1) in endotoxin‐induced uveitis. FASEB J. 23, 929–939 (2009)

Effect of insulin on the induction by dexamethasone of glucose-6-phosphohydrolase and translocase activities in cultured hepatoma cells

The ten-fold increase in glucose-6-phosphatase, previously reported, in 2S FAZA hepatoma cells exposed to dexamethasone, is completely blocked by low concentrations of insulin. At 3 x 10(-10) M insulin, the activity induced by 10(-6) M dexamethasone is reduced by half. The activity of intact microsomes, which reflects translocation of cytoplasmic glucose 6-phosphate into the endoplasmic reticulum, is induced by dexamethasone, but to a lesser extent than the hydrolase. Insulin also prevents this induction.

The effect of p-hydroxymercuribenzoate and congeners on microsomal glucose-6-phosphatase

SummaryIodoacetamide, N-ethylmaleimide, p-hydroxy-mercuribenzoate (p-MB) and HgCl2 were tested as inhibitors of microsomal glucose-6-phosphatase. Iodoacetamide had no effect at 2mm. N-ethylmaleimide inhibited only crude, but not purified microsomal preparations (M2) or crude microsomes exposed to deoxycholate.14C-labelled N-ethylmaleimide was not bound by the M2 protein fraction. p-MB inhibited all types of preparations and the inhibition was not counteracted by detergent. A more detailed study was carried out with the purified M2 fraction (specific activity: 2–4µmoles Pi/min/mg protein). Glucose-6-phosphate hydrolysis was inhibited 50% by 5 × 10−5m p-MB. The inhibition was completely reversible by dithiothreitol except when the enzyme was pre-incubated with p-MB in the absence of substrate. Then p-MB accelerated the temperature-dependent inactivation of glucose-6-phosphatase. Binding studies showed that around 3µmoles14C-p-MB were incorporated into 100 mg M2 protein regardless of the concentration of mercurial in the incubation mixture. That is, over a 25 fold range of p-MB concentration, causing up to 80% inhibition of enzyme activity, no difference was seen in the amount of labelled p-MB which was irreversibly bound to M2 protein. Kinetically p-MB behaved like a reversible inhibitor and this was confirmed by dilution experiments. Several compounds, including some amino acids, antagonized the inhibition by p-MB. The order of effectiveness was EDTA > barbital > tryptophan > histidine > lysine > other amino acids. Glycine, Tris and urea were ineffective competitors of p-MB inhibition. Double reciprocal plots showed that the Km for glucose-6-phosphate was increased and the Vmax reduced in the presence of p-MB. HgCl2 was a more effective inhibitor than p-MB with a Ki of 6 × 10−6m. We conclude that a reaction of p-MB with M2 sulfhydryls does not play a part in the inhibition of enzyme activity. It is suggested that p-MB may interact with one or more amino acid side chains in such a way that enzyme conformation is altered.

Protein synthesis with membrane-bound polysomes and albumin messenger RNA from livers of mutant mice

SummaryInvestigation of deficiencies in serum protein synthesis resulting from deletion-mutations at the albino locus in mice was continued usingin vitro conditions. Previous work showed that although total protein synthesis was only slightly lower in livers from albinos, newly synthesized protein appearing in plasma was 22% of that in controls. It was thought that the disorganized endoplasmic reticulum and Golgi apparatus, characteristic for the liver (and kidney) of these mutants, might be responsible for the observed deficiencies. In the present study mebrane-bound polysomes isolated from the livers of newborn albinos were 55% (c3H/c3H strain) and 62% (c14CoS/c14CoS strain) as efficient as those from normal littermates in incorporating radioactive leucine into protein in a cell-free system. These differences could not be eliminated by the addition of excess liver mRNA, exogenous soluble factors or by the exchange of cell sap between albino and control polysomes. In an earlier study albino liver slices synthesized only 13% (or 17% per mg of total protein synthesized) as much albumin as controls. We have now found that the level of albumin poly(A)+-RNA isolated from albino livers and assayed with a reticulocyte lysate, was almost as high (85%) as in controls. It was concluded that the very low level of albumin synthesis in albino livers did not result from a deficiency of albumin mRNA. Whether the rate-limiting step in synthesis of albumin in mutant livers is at the level of translation or processing for secretion requires further investigation.