Kennard L. Thomas

Acute cardiovascular protective effects of corticosteroids are mediated by non-transcriptional activation of endothelial nitric oxide synthase

Corticosteroids have been shown to exert beneficial effects in the treatment of acute myocardial infarction, but the precise mechanisms underlying their protective effects are unknown. Here we show that high-dose corticosteroids exert cardiovascular protection through a novel mechanism involving the rapid, non-transcriptional activation of endothelial nitric oxide synthase (eNOS). Binding of corticosteroids to the glucocorticoid receptor (GR) stimulated phosphatidylinositol 3-kinase and protein kinase Akt, leading to eNOS activation and nitric oxide–dependent vasorelaxation. Acute administration of pharmacological concentrations of corticosteroids in mice led to decreased vascular inflammation and reduced myocardial infarct size following ischemia and reperfusion injury. These beneficial effects of corticosteroids were abolished by GR antagonists or eNOS inhibitors in wild-type mice and were completely absent in eNOS-deficient (Nos3−/−) mice. The rapid activation of eNOS by the non-nuclear actions of GR, therefore, represents an important cardiovascular protective effect of acute high-dose corticosteroid therapy.

ApoE Deficiency Compromises the Blood Brain Barrier Especially After Injury

BackgroundApolipoprotein E (apoE) mediates lipoprotein uptake by receptors such as the LDL receptor (LDLR). The isoform apoE4 has been linked to Alzheimer’s disease and to poor outcomes after brain injury. Astrocytes that induce blood brain barrier (BBB) properties in endothelium also produce apoE. We decided to investigate the role of apoE in BBB function and in the restoration of BBB after brain injury.Materials and MethodsWild-type (WT) mice and mice deficient in apoE or LDLR were fed normal chow or diets rich in fat and cholesterol. The BBB leakage was determined through injection of Evans blue dye and measurement of the amount of dye extravasated in the brains 3 hours later. Brain injury was induced by applying dry ice directly onto the excised parietal region of the brain. The mice were given 7 days to recover. In some experiments, peroxidase was infused to observe the site of leakage by histology.ResultsWe found 70% more spontaneous leakage of injected Evans blue dye in the brains of apoE−/− mice than in wild type. This increase in permeability appeared selective for the brain. The leaky BBB in apoE−/− mice may provide an explanation for the neurological deficits seen in these animals. In an established model of BBB leakage induced by trauma (cold injury), the apoE−/− mice showed even more compromised BBB function, compared with WT mice, suggesting that apoE is important for BBB recovery. No deficit in BBB was observed in injured LDLR−/− mice, even on Western Diet. In contrast, higher plasma cholesterol levels in apoE−/− mice further increased BBB leakage after injury. We extracted 5x more Evans blue from these brains than from WT. In the injury model, injection of peroxidase resulted in prominent retention of this protein in the cortex of apoE−/− but not in WT.ConclusionsOur results show that the combination of loss of apoE function with high plasma cholesterol and especially brain injury results in dramatic BBB defects in the cortex and may explain in part the importance of apoE in Alzheimer’s disease and in successful recovery from brain injury.

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)

VLA-4 blockade suppresses endotoxin-induced uveitis: in vivo evidence for functional integrin up-regulation

Leukocyte adhesion to the vascular wall is a critical early step in the pathogenesis of inflammatory diseases and is mediated in part by the leukocyte integrin, VLA‐4, which binds to endothelial vascular cell adhesion molecule (VCAM) −1. Here, we investigate VLA‐4s role in endotoxin‐induced uveitis (EIU). At various time points (6–48 h) after EIU induction, the severity of the inflammation was evaluated by quantifying cell and protein content in the aqueous fluid, firm leukocyte adhesion in the retinal vessels, and the number of extravasated leukocytes into the vitreous. Functional activation of VLA‐4 in vivo was investigated in our previously introduced autoperfused micro flow chamber assay. Firm adhesion of EIU leukocytes to immobilized VCAM‐1 under physiological blood flow conditions was significantly increased compared with normal controls (P<0.05), suggesting an important role for VLA‐4 in EIU. VLA‐4 blockade in vivo significantly suppressed all uveitis‐related inflammatory parameters studied, decreasing the clinical score by 45% (P<0.01), protein content in the aqueous fluid by 21% (P<0.01), retinal leukostasis by 68% (P<0.01), and leukocyte accumulation in the vitreous by 75% (P<0.01). Our data provide novel evidence for functional up‐regulation of VLA‐4 during EIU and suggest VLA‐4 blockade as a promising therapeutic strategy for treatment of acute inflammatory eye diseases.—Hafezi‐Moghadam, A., Noda, K., Almulki, L., Iliaki, E. F., Poulaki, V., Thomas, K. L., Nakazawa, T., Hisatomi, T., Miller, J. W., Gragoudas, E. S. VLA‐4 blockade suppresses endotoxin‐induced uveitis: in vivo evidence for functional integrin up‐regulation. FASEB J. 21, 464–474 (2006)

Localization of vascular adhesion protein-1 (VAP-1) in the human eye.

Recently we showed a critical role for Vascular Adhesion Protein-1 (VAP-1) in rodents during acute ocular inflammation, angiogenesis, and diabetic retinal leukostasis. However, the expression of VAP-1 in the human eye is unknown. VAP-1 localization was therefore investigated by immunohistochemistry. Five micrometer thick sections were generated from human ocular tissues embedded in paraffin. Sections were incubated overnight with primary mAbs against VAP-1 (5 microg/ml), smooth muscle actin (1 microg/ml), CD31 or isotype-matched IgG at 4 degrees C. Subsequently, a secondary mAb was used for 30 min at room temperature, followed by Dako Envision + HRP (AEC) System for signal detection. The stained sections were examined using light microscopy and the signal intensity was quantified by two evaluators and graded into 4 discrete categories. In all examined ocular tissues, VAP-1 staining was confined to the vasculature. VAP-1 labeling showed the highest intensity in both arteries and veins of neuronal tissues: retina and optic nerve, and the lowest intensity in the iris vasculature (p < 0.05). Scleral and choroidal vessels showed moderate staining for VAP-1. VAP-1 intensity was significantly higher in the arteries compared to veins (p < 0.05). Furthermore, VAP-1 staining in arteries colocalized with both CD31 and smooth muscle actin (sm-actin) staining, suggesting expression of VAP-1 in endothelial cells, smooth muscle cells or potentially pericytes. In conclusion, immunohistochemistry reveals constitutive expression of VAP-1 in human ocular tissues. VAP-1 expression is nearly exclusive to the vasculature with arteries showing significantly higher expression than veins. Furthermore, VAP-1 expression in the ocular vasculature is heterogeneous, with the vessels of the optic nerve and the retina showing highest expressions. These results characterize VAP-1 expression in human ocular tissues.

Characterization of Azurocidin as a Permeability Factor in the Retina: Involvement in VEGF-Induced and Early Diabetic Blood-Retinal Barrier Breakdown

PURPOSE Azurocidin, released by neutrophils during leukocyte-endothelial interaction, is a main cause of neutrophil-evoked vascular leakage. Its role in the retina, however, is unknown. METHODS Brown Norway rats received intravitreal injections of azurocidin and vehicle control. Blood-retinal barrier (BRB) breakdown was quantified using the Evans blue (EB) dye technique 1, 3, and 24 hours after intravitreal injection. To block azurocidin, aprotinin was injected intravenously before the intravitreal injections. To investigate whether azurocidin plays a role in vascular endothelial growth factor (VEGF)-induced BRB breakdown, rats were treated intravenously with aprotinin, followed by intravitreal injection of VEGF(164). BRB breakdown was quantified 24 hours later. To investigate whether azurocidin may mediate BRB breakdown in early diabetes, aprotinin or vehicle was injected intravenously each day for 2 weeks to streptozotocin-induced diabetic rats, and BRB breakdown was quantified. RESULTS Intravitreal injection of azurocidin (20 microg) induced a 6.8-fold increase in vascular permeability compared with control at 1-3 hours (P < 0.05), a 2.7-fold increase at 3 to 5 hours (P < 0.01), and a 1.7-fold increase at 24 hours (P < 0.05). Aprotinin inhibited azurocidin-induced BRB breakdown by more than 95% (P < 0.05). Furthermore, treatment with aprotinin significantly suppressed VEGF-induced BRB breakdown by 93% (P < 0.05) and BRB breakdown in early experimental diabetes by 40.6% (P < 0.05). CONCLUSIONS Azurocidin increases retinal vascular permeability and is effectively blocked by aprotinin. The inhibition of VEGF-induced and early diabetic BRB breakdown with aprotinin indicates that azurocidin may be an important mediator of leukocyte-dependent BRB breakdown secondary to VEGF. Azurocidin may become a new therapeutic target in the treatment of retinal vascular leakage, such as during diabetic retinopathy.

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)