BackgroundProduction of the chemokine CCL2 by cells of the neurovascular unit (NVU) drives critical aspects of neuroinflammation. Suppression of CCL2 therefore holds promise in treating neuroinflammatory disease. Accordingly, we sought to determine if the compound bindarit, which inhibits CCL2 synthesis, could repress the three NVU sources of CCL2 most commonly reported in neuroinflammation – astrocytes, microglia and brain microvascular endothelial cells (BMEC) – as well as modify the clinical course of neuroinflammatory disease.MethodsThe effect of bindarit on CCL2 expression by cultured murine astrocytes, microglia and BMEC was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Bindarit action on mouse brain and spinal cord in vivo was similarly investigated by qRT-PCR following LPS injection in mice. And to further gauge the potential remedial effects of bindarit on neuroinflammatory disease, its impact on the clinical course of experimental autoimmune encephalomyelitis (EAE) in mice was also explored.ResultsBindarit repressed CCL2 expression by all three cultured cells, and antagonized upregulated expression of CCL2 in both brain and spinal cord in vivo following LPS administration. Bindarit also significantly modified the course and severity of clinical EAE, diminished the incidence and onset of disease, and evidenced signs of disease reversal.ConclusionBindarit was effective in suppressing CCL2 expression by cultured NVU cells as well as brain and spinal cord tissue in vivo. It further modulated the course of clinical EAE in both preventative and therapeutic ways. Collectively, these results suggest that bindarit might prove an effective treatment for neuroinflammatory disease.

BackgroundThere is increasing awareness that, aside from producing cerebrospinal fluid, the choroid plexus (CP) might be a key regulator of immune activity in the central nervous system (CNS) during neuroinflammation. Specifically, the CP has recently been posited to control entry of sentinel T cells into the uninflamed CNS during the early stages of neuroinflammatory diseases, like multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). As the CP is compartmentalized into a stromal core containing fenestrated capillaries devoid of typical blood–brain barrier properties, surrounded by a tight junction-expressing choroidal epithelium, each of these compartments might mount unique responses that instigate the neuroinflammatory process.MethodsTo discern responses of the respective CP stromal capillary and choroidal epithelial tissues during evolving neuroinflammation, we investigated morphology and in situ expression of 93 immune-related genes during early stages of EAE induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Specifically, 3-D immunofluorescent imaging was employed to gauge morphological changes, and laser capture microdissection was coupled to an Immune Panel TaqMan Low Density Array to detail alterations in gene expression patterns at these separate CP sites on days 9 and 15 post-immunization (p.i.). To resolve CP effects due to autoimmunity against MOG peptide, from those due to complete Freund’s adjuvant (CFA) and pertussis toxin (PTX) included in the immunization, analysis was performed on MOG-CFA/PTX-treated, CFA/PTX-treated, and naïve cohorts.ResultsThe CP became swollen and displayed significant molecular changes in response to MOG-CFA/PTX immunization. Both stromal capillary and choroidal epithelial tissues mounted vigorous, yet different, changes in expression of numerous genes over the time course analyzed - including those encoding adhesion molecules, cytokines, chemokines, statins, interleukins, T cell activation markers, costimulatory molecules, cyclooxygenase, pro-inflammatory transcription factors and pro-apoptotic markers. Moreover, CFA/PTX-treatment, alone, resulted in extensive, though less robust, alterations in both CP compartments.ConclusionsMOG-CFA/PTX immunization significantly affects CP morphology and stimulates distinct expression patterns of immune-related genes in CP stromal capillary and epithelial tissues during evolving EAE. CFA/PTX treatment, alone, causes widespread gene alterations that could prime the CP to unlock the CNS to T cell infiltration during neuroinflammatory disease.

The choroid plexus (CP) is considered to be a point of leukocyte entry into the CNS during normal immune surveillance and in neuroinflammatory diseases. The structural and functional alterations within the CP that support this migration are not understood. We used quantitative, high-resolution, 3-dimensional (3-D) fluorescence imaging to analyze CP alterations associated with inflammatory responses in C57/Bl6 mice after the induction of experimental autoimmune encephalomyelitis by immunization with myelin oligodendrocyte glycoprotein (MOG) and complete Freund adjuvant/pertussis toxin (MOG-CFA/PTX) or adjuvants alone (CFA-PTX). The MOG-CFA/PTX and CFA/PTX produced similar effects, although those caused by the former were consistently more marked. Both treatments resulted in the accumulation of serum immunoglobulin G and leukocytes in the CP stroma, consistent with elevated stromal capillary permeability. They also provoked distortions and diminished immunostaining patterns of the tight junction adaptor protein ZO-1 in the choroidal epithelium but no obvious change in the patterns of the tight junction associated protein claudin-2. Only MOG-CFA/PTX triggered visible extravasation of immunoglobulin G and leukocytes across the choroidal epithelium. Our results suggest that CFA/PTX primes the CP for neuroinflammation by inducing structural changes that are exacerbated when there is an immune response to MOG and reinforce the CP as a gateway for leukocytes to enter the CNS by accessing the CSF and leptomeninges.

The angiogenic capacity of local tissue critically regulates the response to ischemic injury. Elevated reactive oxygen species production, commonly associated with ischemic injury, has been shown to promote phosphorylation of the vascular endothelial growth factor receptor 2 (VEGFR2), a critical regulator of angiogenesis. Previous data from our lab demonstrated that diminished levels of the antioxidant glutathione positively augment ischemic angiogenesis. Here, we sought to determine the relationship between glutathione levels and oxidative stress in VEGFR2 signaling. We reveal that decreasing the ratio of GSH to GSSG with diamide leads to enhanced protein S-glutathionylation, increased reactive oxygen species (ROS) production, and enhanced VEGFR2 activation. However, increasing ROS alone was insufficient in activating VEGFR2, while ROS enhanced VEGF-stimulated VEGFR2 activation at supraphysiological levels. We also found that inhibiting glutathione reductase activity is sufficient to increase VEGFR2 activation and sensitizes cells to ROS-dependent VEGFR2 activation. Taken together, these data suggest that regulation of the cellular GSH:GSSG ratio critically regulates VEGFR2 activation. This work represents an important first step in separating thiol mediated signaling events from ROS dependent signaling.

Glutathione is a low molecular weight thiol that is present at high levels in the cell. The high levels of glutathione in the cell make it one of the most abundant antioxidants contributing to cellular redox homeostasis. As a general rule, throughout cardiovascular disease and progression there is an imbalance in redox homeostasis characterized by reactive oxygen species overproduction and glutathione underproduction. As research into these imbalances continues, glutathione concentrations are increasingly being observed to drive various physiological and pathological signaling responses. Interestingly in addition to acting directly as an antioxidant, glutathione is capable of post translational modifications (S-glutathionylation) of proteins through both chemical interactions and enzyme mediated events. This review will discuss both the chemical and enzyme-based S-glutathionylation of proteins involved in cardiovascular pathologies and angiogenesis.

Background: The chemokine CCL2 is a critical mediator of neuroinflammation in diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). CCL2 drives mononuclear cell infiltration into the central nervous system (CNS), alters expression and distribution of microvascular endothelial tight junction proteins, and disrupts the blood-brain and blood-spinal cord barriers. Immunohistochemistry has consistently revealed astrocytes to be a source of this chemokine during neuroinflammation, while providing less uniform evidence that CNS endothelial cells may also express CCL2. Moreover, the relative contributions of these cell types to the CNS pool of CCL2 during MS/EAE are unclear and the aim of this study was to investigate this further. Methods: CCL2 gene expression was determined by qRT-PCR in different populations of CNS cells at different times following EAE induced by immunization with MOG 35-55 peptide and adjuvants, or after injection with adjuvants alone. CNS cells types were isolated by two different protocols: bulk isolation to yield crude microvascular and parenchymal fractions (containing astrocytes, other glia, and neurons), or laser capture microdissection (LCM) to acquire more precisely microvascular endothelial cells, astrocytes or other parenchymal cells.

We sought to investigate the effects of diabetes and hyaluronidase on the thickness of the endothelial glycocalyx layer in the mouse retina. In our study, the retinal circulation of diabetic Ins2(Akita) mice and their nondiabetic littermates were observed via intravital microscopy. The endothelial glycocalyx thickness was determined from the infusion of two fluorescently labeled plasma markers, one of which was a high molecular weight rhodamine dextran (MW=155,000) excluded from the glycocalyx, and the other a more permeable low molecular weight sodium fluorescein (MW=376). In nondiabetic C57BL/6 mice, the glycocalyx thickness also was evaluated prior to and following infusion of hyaluronidase, an enzyme that can degrade hyaluronic acid on the endothelial surface. A leakage index was used to evaluate the influence of hyaluronidase on the transport of the fluorescent tracers from the plasma into the surrounding tissue, and plasma samples were obtained to measure levels of circulating hyaluronic acid. Both diabetes and hyaluronidase infusion significantly reduced the thickness of the glycocalyx in retinal arterioles (but not in venules), and hyaluronidase increased retinal microvascular leakage of both fluorescent tracers into the surrounding tissue. However, only hyaluronidase infusion (not diabetes) increased circulating plasma levels of hyaluronic acid. In summary, our findings demonstrate that diabetes and hyaluronidase reduce the thickness of the retinal endothelial glycocalyx, in which hyaluronic acid may play a significant role in barrier function.

Arterial and venous endothelial cells exhibit differential responses to oxidant stress, and decreases in GSH:GSSG are more exacerbated in venous endothelial cells. Specific pathogenesis in these vascular conduits, with respect to oxidant stress handling, warrants further study, especially considering surgical interventions such as Coronary artery bypass grafting that use both interchangeably.