In pregnant women infected with Plasmodium falciparum, the parasite-infected red blood cells (IRBCs) sequester in the placenta through chondroitin 4-sulfate (C4S)-mediated adherence. The pattern of IRBC adherence in P. falciparum-infected placenta has been controversial. Moreover, the identity of the chondroitin sulfate proteoglycan (CSPG) receptor, that mediates IRBC adherence, and its location in the placenta have not been established. This study, using immunohistochemical techniques, clearly shows, for the first time, that the low-sulfated CSPGs of the placenta are localized predominantly in the intervillous space. Ex vivo IRBC adherence analyses demonstrate that the IRBCs are adhered to the CSPG receptors in the placenta in a C4S-dependent manner. This IRBC binding pattern was similar to that observed in P. falciparum-infected placentas. These data and the results of dual-fluorescence staining of the endogenous RBCs and syncytiotrophoblasts, and co-localization of CSPG and IRBC adherence unequivocally establish that the low-sulfated CSPGs are the major natural receptors for IRBC adherence in the placenta. Further, it was found that IRBCs adhere mainly in the intervillous space and also at significant levels to the syncytiotrophoblasts. Finally, the ex vivo IRBC adherence method described herein provides a reliable procedure for future studies for the assessment of the efficacy of C4S inhibitors and adhesion inhibitory antibodies.

BackgroundMany retinal diseases are associated with vascular dysfunction accompanied by neuroinflammation. We examined the ability of minocycline (Mino), a tetracycline derivative with anti-inflammatory and neuroprotective properties, to prevent vascular permeability and inflammation following retinal ischemia-reperfusion (IR) injury, a model of retinal neurodegeneration with breakdown of the blood-retinal barrier (BRB).MethodsMale Sprague–Dawley rats were subjected to 45 min of pressure-induced retinal ischemia, with the contralateral eye serving as control. Rats were treated with Mino prior to and following IR. At 48 h after reperfusion, retinal gene expression, cellular inflammation, Evan’s blue dye leakage, tight junction protein organization, caspase-3 activation, and DNA fragmentation were measured. Cellular inflammation was quantified by flow-cytometric evaluation of retinal tissue using the myeloid marker CD11b and leukocyte common antigen CD45 to differentiate and quantify CD11b+/CD45low microglia, CD11b+/CD45hi myeloid leukocytes and CD11bneg/CD45hi lymphocytes. Major histocompatibility complex class II (MHCII) immunoreactivity was used to determine the inflammatory state of these cells.ResultsMino treatment significantly inhibited IR-induced retinal vascular permeability and disruption of tight junction organization. Retinal IR injury significantly altered mRNA expression for 21 of 25 inflammation- and gliosis-related genes examined. Of these, Mino treatment effectively attenuated IR-induced expression of lipocalin 2 (LCN2), serpin peptidase inhibitor clade A member 3 N (SERPINA3N), TNF receptor superfamily member 12A (TNFRSF12A), monocyte chemoattractant-1 (MCP-1, CCL2) and intercellular adhesion molecule-1 (ICAM-1). A marked increase in leukostasis of both myeloid leukocytes and lymphocytes was observed following IR. Mino treatment significantly reduced retinal leukocyte numbers following IR and was particularly effective in decreasing the appearance of MHCII+ inflammatory leukocytes. Surprisingly, Mino did not significantly inhibit retinal cell death in this model.ConclusionsIR induces a retinal neuroinflammation within hours of reperfusion characterized by inflammatory gene expression, leukocyte adhesion and invasion, and vascular permeability. Despite Mino significantly inhibiting these responses, it failed to block neurodegeneration.

Retinal ischemia-reperfusion (IR) induces neurodegenaration as well as blood-retinal barrier (BRB) breakdown causing vascular permeability. Whereas the neuronal death has been extensively studied, the molecular mechanisms related to BRB breakdown in IR injury remain poorly understood. In this study, we investigated the early changes in tight junctional (TJ) proteins in response to IR injury. Ischemia-reperfusion injury was induced in male rat retinas by increasing the intraocular pressure for 45 minutes followed by natural reperfusion. The results demonstrate that IR injury induced occludin Ser490 phosphorylation and ubiquitination within 15 minutes of reperfusion with subsequent vascular permeability. Immunohistochemical analysis revealed a rapid increase in occludin Ser490 phosphorylation and loss of Zonula occludens-1 (ZO-1) protein, particularly in arterioles. Ischemia-reperfusion injury also rapidly induced the activation and phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) at tyrosine 1175. Blocking vascular endothelial growth factor (VEGF) function by intravitreal injection of bevacizumab prevented VEGFR-2 activation, occludin phosphorylation, and vascular permeability. These studies suggest a novel mechanism of occludin Ser490 phosphorylation and ubiquitination downstream of VEGFR2 activation associated with early IR-induced vascular permeability. Flow & Metabolism (2014) 34, 522-531; doi:10.1038/jcbfm.2013; published online 8 January 2014 Journal of Cerebral BloodKeywords: blood-retinal barrier; ischemia-reperfusion injury; occludin phosphorylation; tight junction; vascular endothelial growth factor; vascular permeability INTRODUCTIONRetinal ischemia-reperfusion models several components of various eye disease pathologies such as retinal vein occlusion, diabetic retinopathy, and glaucoma. [1][2][3][4][5][6] The IR model has been widely used for studying retinal neuronal cell damage after ischemic insult 6 and consists of transient ischemia followed by natural reperfusion leading to an inflammatory and neurodegenerative response in the intact retina. 7 Histologic analysis demonstrated that the IR injury causes selective neuronal loss indicated by reduced thickness of retinal layers including the ganglion cell layer, inner nuclear layer, and inner plexiform layer. 8,9 A recent study demonstrated that IR also induced vascular abnormalities such as capillary dropout after 8 to 14 days of reperfusion and concluded these vascular changes occurred after neuronal loss. 6 Recently IR was shown to increase retinal vascular permeability in a vascular endothelial growth factor (VEGF)-dependent manner, suggesting that this model could be used to investigate the mechanisms and drugs targeting VEGFinduced permeability. 3 Vascular endothelia growth factor, a hypoxia-responsive angiogenic and vasopermeability factor, contributes to vascular leakage in multiple retinal pathologies. 10 Although many studies have demonstrated perturbations of retina glial and neuronal elements in IR injury, no stu...

Background:The development of diabetic retinopathy (DR) is incompletely understood. Administered retinylamine is stored in the retinal pigmented epithelium (RPE) where it affects the ocular visual cycle. Results: Retinylamine inhibited vascular and neural lesions of early DR. Conclusion: Both the RPE and visual cycle are novel targets for the inhibition of DR. Significance: Vision-related processes can contribute to DR.

Reactive oxygen species play an important role in the pathogenesis of diabetic retinopathy. We studied the role of adrenergic and serotonin receptors in the generation of superoxide by retina and 661W retinal cells in high glucose and of the α1-adrenergic receptor (AR) on vascular lesions of the retinopathy in experimentally diabetic C57Bl/6J mice (and controls) after 2 and 8 months. Compared with 5 mM glucose, incubating cells or retinal explants in 30 mM glucose induced superoxide generation. This response was reduced or ablated by pharmacologic inhibition of the α1-AR (a Gq-coupled receptor) or Gs-coupled serotonin (5-HT2, 5-HT4, 5-HT6, and 5-HT7) receptors or by activation of the Gi-coupled α2-AR. In elevated glucose, the α1-AR produced superoxide via phospholipase C, inositol triphosphate-induced Ca(2+) release, and NADPH oxidase, and pharmacologic inhibition of these reactions prevented the superoxide increase. Generation of retinal superoxide, expression of proinflammatory proteins, and degeneration of retinal capillaries in diabetes all were significantly inhibited with daily doxazosin or apocynin (inhibitors of α1-AR and NADPH oxidase, respectively), but increased vascular permeability was not significantly affected. Adrenergic receptors, and perhaps other GPCRs, represent novel targets for inhibiting the development of important features of diabetic retinopathy.

PurposeInflammation associated with blood–retinal barrier (BRB) breakdown is a common feature of several retinal diseases. Therefore, the development of novel nonsteroidal anti-inflammatory approaches may provide important therapeutic options. Previous studies demonstrated that inhibition of dipeptidyl peptidase-IV, the enzyme responsible for the degradation of glucagon-like peptide-1 (GLP-1), led to insulin-independent prevention of diabetes-induced increases in BRB permeability, suggesting that incretin-based drugs may have beneficial pleiotropic effects in the retina. In the current study, the barrier protective and anti-inflammatory properties of exendin-4 (Ex-4), an analog of GLP-1, after ischemia-reperfusion (IR) injury were examined.MethodsIschemia-reperfusion injury was induced in rat retinas by increasing the intraocular pressure for 45 minutes followed by 48 hours of reperfusion. Rats were treated with Ex-4 prior to and following IR. Blood–retinal barrier permeability was assessed by Evans blue dye leakage. Retinal inflammatory gene expression and leukocytic infiltration were measured by qRT-PCR and immunofluorescence, respectively. A microglial cell line was used to determine the effects of Ex-4 on lipopolysaccharide (LPS)-induced inflammatory response.ResultsExendin-4 dramatically reduced the BRB permeability induced by IR injury, which was associated with suppression of inflammatory gene expression. Moreover, in vitro studies showed that Ex-4 also reduced the inflammatory response to LPS and inhibited NF-κB activation.ConclusionsThe present work suggests that Ex-4 can prevent IR injury–induced BRB breakdown and inflammation through inhibition of inflammatory cytokine production by activated microglia and may provide a novel option for therapeutic intervention in diseases involving retinal inflammation.

A low-sulfated chondroitin sulfate proteoglycan (CSPG) has been shown to be the receptor for the adherence of Plasmodium falciparum-infected red blood cells (IRBCs) in human placenta. Recently, hyaluronic acid (HA) has been suggested as an additional receptor even though IRBC binding to HA and the presence of HA at locations where IRBCs adhere in the placenta have not been established. In this study, we investigated whether HA is also a receptor for IRBC binding. IRBCs from infected placentas as well as those from different laboratory strains could bind to CSPG but not to HA. In a cell depletion assay, IRBCs from infected placentas could bind quantitatively to CSPG. Although CSPG is present both in the intervillous space and on the syncytiotrophoblast surface, HA is absent in these locations. These data conclusively demonstrate that CSPG, but not HA, is a receptor for IRBC adherence in the placenta. Our data also show, for the first time, that the IRBC-binding CSPG in the placenta is of fetal origin and that, in P. falciparum-infected placentas, the CSPG level is significantly increased, which could exacerbate IRBC adherence and placental pathogenesis. These results have important implications for the development of Of the estimated 2 to 3 million annual fatalities attributable to malaria, Ͼ90% of death is caused by Plasmodium falciparum, the most virulent among the four protozoan parasites that cause malaria in humans.1-3 Although several factors are likely to contribute to the virulence of P. falciparum, it is widely thought that sequestration of parasite-infected red blood cells (IRBCs) in the microvascular capillaries of vital organs plays a central role.3-12 The IRBC sequestration has been reported to be mediated by endothelial cell adhesion molecules, such as thrombospondin, CD36, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin, P-selectin, and platelet endothelial cell adhesion molecule/ CD31. [3][4][5][6][7][8][9][10][11][12] The array of different adhesive mechanisms used by the parasite seems to confer a selective advantage for its efficient survival in the host by switching from one adherent type to another as the host develops adhesion inhibitory antibodies and other phenotype-specific immunity. Thus, in malaria endemic areas, almost all individuals by adulthood develop immunity that effectively controls infection and avoid pathogenesis. However, in the case of women during pregnancy, placenta expresses a new receptor that was previously unavailable for IRBC adherence.

Background Several retinal pathologies exhibit both inflammation and breakdown of the inner blood-retinal barrier (iBRB) resulting in vascular permeability, suggesting that treatments that trigger resolution of inflammation may also promote iBRB restoration. Methods Using the mouse retinal ischemia-reperfusion (IR) injury model, we followed the time course of neurodegeneration, inflammation, and iBRB disruption and repair to examine the relationship between resolution of inflammation and iBRB restoration and to determine if minocycline, a tetracycline derivative shown to reverse microglial activation, can hasten these processes. Results A 90-min ischemic insult followed by reperfusion in the retina induced cell apoptosis and inner retina thinning that progressed for approximately 2 weeks. IR increased vascular permeability within hours, which resolved between 3 and 4 weeks after injury. Increased vascular permeability coincided with alteration and loss of endothelial cell tight junction (TJ) protein content and disorganization of TJ protein complexes. Shunting of blood flow away from leaky vessels and dropout of leaky capillaries were eliminated as possible mechanisms for restoring the iBRB. Repletion of TJ protein contents occurred within 2 days after injury, long before restoration of the iBRB. In contrast, the eventual re-organization of TJ complexes at the cell border coincided with restoration of the barrier. A robust inflammatory response was evident a 1 day after IR and progressed to resolution over the 4-week time course. The inflammatory response included a rapid and transient infiltration of granulocytes and Ly6C+ classical inflammatory monocytes, a slow accumulation of Ly6Cneg monocyte/macrophages, and activation, proliferation, and mobilization of resident microglia. Extravasation of the majority of CD45+ leukocytes occurred from the superficial plexus. The presence of monocyte/macrophages and increased numbers of microglia were sustained until the iBRB was eventually restored. Intervention with minocycline to reverse microglial activation at 1 week after injury promoted early restoration of the iBRB coinciding with decreased expression of mRNAs for the microglial M1 markers TNF-α, IL-1β, and Ptgs2 (Cox-2) and increased expression of secreted serine protease inhibitor Serpina3n mRNA. Conclusions These results suggest that iBRB restoration occurs as TJ complexes are reorganized and that resolution of inflammation and restoration of the iBRB following retinal IR injury are functionally linked.