Ali Hafezi-Moghadam

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.

Tumor Necrosis Factor-α Mediates Oligodendrocyte Death and Delayed Retinal Ganglion Cell Loss in a Mouse Model of Glaucoma

Glaucoma is a widespread ocular disease characterized by a progressive loss of retinal ganglion cells (RGCs). Previous studies suggest that the cytokine tumor necrosis factor-α (TNF-α) may contribute to the disease process, although its role in vivo and its mechanism of action are unclear. To investigate pathophysiological mechanisms in glaucoma, we induced ocular hypertension (OH) in mice by angle closure via laser irradiation. This treatment resulted in a rapid upregulation of TNF-α, followed sequentially by microglial activation, loss of optic nerve oligodendrocytes, and delayed loss of RGCs. Intravitreal TNF-α injections in normal mice mimicked these effects. Conversely, an anti-TNF-α-neutralizing antibody or deleting the genes encoding TNF-α or its receptor, TNFR2, blocked the deleterious effects of OH. Deleting the CD11b/CD18 gene prevented microglial activation and also blocked the pathophysiological effects of OH. Thus TNF-α provides an essential, although indirect, link between OH and RGC loss in vivo. Blocking TNF-α signaling or inflammation, therefore, may be helpful in treating glaucoma.

Delayed Onset of Inflammation in Protease-Activated Receptor-2-Deficient Mice

Endothelial surface expression of P-selectin and subsequent leukocyte rolling in venules can be induced by mast cell-derived histamine and binding of thrombin to protease-activated receptor-1 (PAR1). We hypothesized that activation of endothelial PAR2 by mast cell tryptase or other proteases also contributes to inflammatory responses. Leukocyte rolling flux and rolling velocity were assessed by intravital microscopy of the cremaster muscles of wild-type mice following perivenular micropipette injections of a control (LSIGRL) or PAR2-activating (SLIGRL) oligopeptide. Injection of SLIGRL increased mean rolling leukocyte flux fraction from 34 ± 11 to 71 ± 24% (p < 0.05) and decreased mean rolling velocity from 63 ± 29 to 32 ± 2 μm/s (p < 0.05). No significant changes occurred with control peptide injection. To further evaluate the role of PAR2 in inflammatory responses, PAR2-deficient mice were generated by gene targeting and homologous recombination. Perivenular injections of SLIGRL resulted in only a small increase in rolling leukocyte flux fraction (from 21 ± 8 to 30 ± 2%) and no change in rolling velocity. Leukocyte rolling after surgical trauma was assessed in 9 PAR2-deficient and 12 wild-type mice. Early (0–15 min) after surgical trauma, the mean leukocyte rolling flux fraction was lower (10 ± 3 vs 30 ± 6%, p < 0.05) and mean rolling velocity was higher (67 ± 46 vs 52 ± 36 μm/s, p < 0.01) in PAR2-deficient compared with control mice. The defect in leukocyte rolling in PAR2-deficient mice did not persist past 30 min following surgical trauma. These results indicate that activation of PAR2 produces microvascular inflammation by rapid induction of P-selectin-mediated leukocyte rolling. In the absence of PAR2, the onset of inflammation is delayed.

Interaction of P-selectin and PSGL-1 generates microparticles that correct hemostasis in a mouse model of hemophilia A.

High plasma levels of soluble P-selectin are associated with thrombotic disorders and may predict future cardiovascular events. Mice with high levels of soluble P-selectin have more microparticles in their plasma than do normal mice. Here we show that chimeras of P-selectin and immunoglobulin (P-sel–Ig) induced formation of procoagulant microparticles in human blood through P-selectin glycoprotein ligand-1 (PSGL-1; encoded by the Psgl1 gene, officially known as Selpl). In addition, Psgl1−/− mice produced fewer microparticles after P-sel–Ig infusion and did not spontaneously increase their microparticle count in old age as do wild-type mice. Injected microparticles specifically bound to thrombi and thus could be involved in thrombin generation at sites of injury. Infusion of P-sel–Ig into hemophilia A mice produced a 20-fold increase over control immunoglobulin in microparticles containing tissue factor. This significantly improved the kinetics of fibrin formation in the hemophilia A mice and normalized their tail-bleeding time. P-sel–Ig treatment could become a new approach to sustained control of bleeding in hemophilia.

CD24 mediates rolling of breast carcinoma cells on P-selectin

P‐selectin mediates rolling of neutrophils and other leukocytes on activated endothelial cells and platelets through binding to P‐selectin glycoprotein ligand‐1 (PSGL‐1). Certain PSGL‐1 negative tumor cell lines can bind P‐selectin under static conditions through the GPI‐linked surface mucin, CD24, but the physiological significance of this interaction and whether it can occur under flow conditions is not known. Here, we show that CD24+ PSGL‐1– KS breast carcinoma cells attach to and roll on recombinant P‐selectin under a continuous wall shear stress, although at a lower density and higher velocity than CD24+ PSGL‐1+ cells, such as HL‐60. Adding excess soluble CD24 or removing CD24 from the cell surface with phosphatidylinositol‐phospholipase C (PI‐PLC) significantly reduced KS cell rolling on P‐selectin. The ability of KS cells to roll on P‐selectin was positively correlated with the CD24 expression level. Comparison with three other CD24+ cell lines established that expression of sialyl‐Lewisx antigen was also necessary for CD24‐mediated rolling on P‐selectin. CD24 purified from KS cells supported rolling of P‐selectin transfectants, but not L‐selectin transfectants. Finally, KS cells rolled on vascular endothelium in vivo in a P‐selectin‐dependent manner. Together our data show that CD24 serves as a ligand for P‐selectin under physiological flow conditions. Interaction of tumor cells with P‐selectin via CD24 may be an important adhesion pathway in cancer metastasis.—Aigner, S., Ramos, C. L., Hafezi‐Moghadam, A., Lawrence, M. B., Friederichs, J., Altevogt, P., and Ley, K. CD24 mediates rolling of breast carcinoma cells on P‐selectin. FASEB J. 12, 1241–1251 (1998)

Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis.

Photoreceptor apoptosis is a major cause of visual loss in retinal detachment (RD) and several other visual disorders, but the underlying mechanisms remain elusive. Recently, increased expression of monocyte chemoattractant protein 1 (MCP-1) was reported in vitreous humor samples of patients with RD and diabetic retinopathy as well as in the brain tissues of patients with neurodegenerative diseases, including Alzheimers disease and multiple sclerosis. Here we report that MCP-1 plays a critical role in mediating photoreceptor apoptosis in an experimental model of RD. RD led to increased MCP-1 expression in the Müller glia and increased CD11b+ macrophage/microglia in the detached retina. An MCP-1 blocking antibody greatly reduced macrophage/microglia infiltration and RD-induced photoreceptor apoptosis. Confirming these results, MCP-1 gene-deficient mice showed significantly reduced macrophage/microglia infiltration after RD and very little photoreceptor apoptosis. In primary retinal mixed cultures, MCP-1 was cytotoxic for recoverin+ photoreceptors, and this toxicity was eliminated through immunodepleting macrophage/microglia from the culture. In vivo, deletion of the gene encoding CD11b/CD18 nearly eliminated macrophage/microglia infiltration to the retina after RD and the loss of photoreceptors. Thus, MCP-1 expression and subsequent macrophage/microglia infiltration and activation are critical for RD-induced photoreceptor apoptosis. This pathway may be an important therapeutic target for preventing photoreceptor apoptosis in RD and other CNS diseases that share a common etiology.

Role of Vascular Cell Adhesion Molecule-1 and Fibronectin Connecting Segment-1 in Monocyte Rolling and Adhesion on Early Atherosclerotic Lesions

Atherosclerotic lesion development seems to be inflammatory in nature and involves the recruitment of monocytes to the vessel wall. In this study, we investigated the role of vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (FN) connecting segment-1 containing the amino acid sequence ILDV as functional ligands for alpha(4)beta(1) integrin (VLA-4) in monocyte rolling and adherence to early atherosclerotic lesions. Carotid arteries of apolipoprotein E-deficient mice were isolated and perfused with monocytes or U937 cells. Cell adhesion was reduced 95+/-4% by monoclonal antibodies HP1/2 and HP2/1, which block VLA-4 binding to both VCAM-1 and FN connecting segment-1. mAb HP1/3 preferentially blocked interaction of VLA-4 with FN but not VCAM-1 and decreased adhesion by 30+/-8%. In contrast, blocking VCAM-1 by perfusing the isolated carotid artery with mAb MK-2.7 reduced adhesion by 75+/-12%. Mononuclear cell adhesion to the early atherosclerotic endothelium was inhibited by 68+/-10% in the presence of EILDVPST but not in the presence of control peptide EIDVLPST. When VLA-4 or VCAM-1 was blocked, more mononuclear cells rolled on early lesions at significantly higher (approximately doubled) rolling velocities. These data demonstrate that (1) blockade of VCAM-1 can abrogate the majority (75+/-12%) of VLA-4-dependent monocyte adhesion on early atherosclerotic endothelia and (2) ILDV peptide interferes with VLA-4 binding to both VCAM-1 and FN and may be useful in limiting monocyte adhesion to atherosclerotic lesions.

Rho Kinase Inhibition by Fasudil Ameliorates Diabetes-Induced Microvascular Damage

OBJECTIVE—Leukocyte adhesion in retinal microvasuculature substantially contributes to diabetic retinopathy. Involvement of the Rho/Rho kinase (ROCK) pathway in diabetic microvasculopathy and therapeutic potential of fasudil, a selective ROCK inhibitor, are investigated. RESEARCH DESIGN AND METHODS—Localization of RhoA/ROCK and Rho activity were examined in retinal tissues of rats. Impact of intravitreal fasudil administration on retinal endothelial nitric oxide synthase (eNOS) and myosin phosphatase target protein (MYPT)-1 phosphorylation, intercellular adhesion molecule-1 (ICAM-1) expression, leukocyte adhesion, and endothelial damage in rat eyes were investigated. Adhesion of neutrophils from diabetic retinopathy patients or nondiabetic control subjects to cultured microvascular endothelial cells was quantified. The potential of fasudil for endothelial protection was investigated by measuring the number of adherent neutrophils and terminal transferase-mediated dUTP nick-end labeling–positive endothelial cells. RESULTS—RhoA and ROCK colocalized predominantly in retinal microvessels. Significant Rho activation was observed in retinas of diabetic rats. Intravitreal fasudil significantly increased eNOS phosphorylation, whereas it reduced MYPT-1 phosphorylation, ICAM-1 expression, leukocyte adhesion, and the number of damaged endothelium in retinas of diabetic rats. Neutrophils from diabetic retinopathy patients showed significantly higher adhesion to cultured endothelium and caused endothelial apoptosis, which was significantly reduced by fasudil. Blockade of the Fas-FasL interaction prevented endothelial apoptosis. The protective effect of fasudil on endothelial apoptosis was significantly reversed by Nω-nitro-l-arginine methyl ester, a NOS inhibitor, whereas neutrophil adhesion remained unaffected. CONCLUSIONS—The Rho/ROCK pathway plays a critical role in diabetic retinal microvasculopathy. Fasudil protects the vascular endothelium by inhibiting neutrophil adhesion and reducing neutrophil-induced endothelial injury. ROCK inhibition may become a new strategy in the management of diabetic retinopathy, especially in its early stages.

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.

Role of TGF-β in proliferative vitreoretinal diseases and ROCK as a therapeutic target

Cicatricial contraction of preretinal fibrous membrane is a cause of severe vision loss in proliferative vitreoretinal diseases such as proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR). TGF-β is overexpressed in the vitreous of patients with proliferative vitreoretinal diseases and is also detectable in the contractile membranes. Therefore, TGF-β is presumed to contribute to the cicatricial contraction of the membranes, however, the underlying mechanisms and TGF-βs importance among various other factors remain to be elucidated. Vitreous samples from PDR or PVR patients caused significantly larger contraction of hyalocyte-containing collagen gels, compared with nonproliferative controls. The contractile effect was strongly correlated with the vitreal concentration of activated TGF-β2 (r = 0.82, P < 0.0001). PDR or PVR vitreous promoted expression of α-smooth muscle actin (α-SMA) and phosphorylation of myosin light chain (MLC), a downstream mediator of Rho-kinase (ROCK), both of which were dramatically but incompletely suppressed by TGF-β blockade. In contrast, fasudil, a potent and selective ROCK inhibitor, almost completely blocked the vitreous-induced MLC phosphorylation and collagen gel contraction. Fasudil disrupted α-SMA organization, but it did not affect its vitreal expression. In vivo, fasudil significantly inhibited the progression of experimental PVR in rabbit eyes without affecting the viability of retinal cells by electroretinographic and histological analyses. These results elucidate the critical role of TGF-β in mediating cicatricial contraction in proliferative vitreoretinal diseases. ROCK, a key downstream mediator of TGF-β and other factors might become a unique therapeutic target in the treatment of proliferative vitreoretinal diseases.