Exploring the use of high-dose simvastatin as therapy for oxidative stress in disease models of neuroinflammation

Background: There is growing evidence that HMG-CoA reductase inhibitors, otherwise known as the statin family, can exert pleiotropic effects in many areas. Of these, potential neuroprotective effects have gained significant attention. It is well established that microglia, the brain’s resident phagocytes, play a pivotal role in the pathogenesis of neuroinflammation and neurodegeneration. This process is thought to be, in part, due to the presence of chronically activated microglia. In this study, we investigated the potential neuroprotective properties of HMG-CoA reductase using an in vitro model of inflammatory cell activation and in vivo models of posterior uveitis and multiple sclerosis. The focus of this study was to investigate the effect of simvastatin on the microglial cell and its activation products. Emphasis was placed on the production of reactive species released by this cell type and the subsequent damage these cause to biological macromolecules. Methods: The microglial cell line BV2 were treated with simvastatin (1μM; 2 to 120 h), in vitro before being activated for 48 hours with a pro-inflammatory mix of LPS, TNFa and IFNg. Supernatants were taken and nitric oxide levels measured using the Griess assay. The animal model of posterior uveitis, experimental autoimmune uveoretinitis (EAU), was established in wild type C57BL/6 mice through subcutaneous injection of IRBP1-20. Mice were treated orally with simvastatin at 50, 75 or 100 mg/kg. Fundus images were taken before and after treatment administration for evaluation of clinical ocular pathology. Retinal flat mounts were prepared from simvastatin treated mice to assess cellular infiltrates. Experimental autoimmune encephalomyelitis (EAE), an in vivo model of MS, was induced by rMOG subcutaneous immunisation. The effect of simvastatin treatment was assessed clinically and by immunohistochemical analysis of tissue sections to determine cellular infiltrates and levels of oxidative damage to biological macromolecules, consistent with those assessed in EAU. Results: The levels of nitric oxide produced by microglial cells were significantly reduced when exposed to a pre-incubation of simvastatin for 3 48 hours, compared to cells receiving the pro-inflammatory mix alone. In EAU, fundoscopic analysis revealed that high-dose simvastatin halts clinical disease progression in IRBP1-20 induced posterior uveitis. Retinal flat mounts prepared from these cohorts showed a significant decrease in the expression of the innate immune cell surface receptor CD11b. Additionally, histological examination of eye sections displayed a significant reduction in lipid peroxidation as revealed by the marker 4-Hydroxynonenal (4HNE), nitrosylated proteins, as measured by 3-nitrotyrosine and oxidised DNA/RNA as determined by 8-OHdG. In line with this study, results from our EAE model demonstrated an important role for microglial cell number in disease, whilst also providing evidence of simvastatin decreasing oxidative damage to macromolecules in areas of extensive pathology. Discussion: These data provide evidence to support the notion that microglial cell activation may contribute to the pathogenesis of neuroinflammatory disease and that statins may attenuate damage through their ability to inhibit the production of reactive species. Further to this, we provide therapeutic, chemical and physical evidence that simvastatin can provide protection against 1) nitric oxide production in an inflammatory environment 2) clinical disease attenuation in EAU and EAE and 3) reduction in peroxynitrite levels in vivo. Collectively, these data provide evidence that statins may attenuate microglial cell activation by ways of inhibiting the production of reactive species. Thus, the evidence presented in this thesis points to the importance and potential use of simvastatin therapy as a neuroprotective therapeutic agent

CCL4 induces inflammatory signalling and barrier disruption in the neurovascular endothelium

BACKGROUND: During neuroinflammation many chemokines alter the function of the blood-brain barrier (BBB) that regulates the entry of macromolecules and immune cells into the brain. As the milieu of the brain is altered, biochemical and structural changes contribute to the pathogenesis of neuroinflammation and may impact on neurogenesis. The chemokine CCL4, previously known as MIP-1β, is upregulated in a wide variety of central nervous system disorders, including multiple sclerosis, where it is thought to play a key role in the neuroinflammatory process. However, the effect of CCL4 on BBB endothelial cells (ECs) is unknown. MATERIALS AND METHODS: Expression and distribution of CCR5, phosphorylated p38, F-actin, zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin) were analysed in the human BBB EC line hCMEC/D3 by Western blot and/or immunofluorescence in the presence and absence of CCL4. Barrier modulation in response to CCL4 using hCMEC/D3 monolayers was assessed by measuring molecular flux of 70 ​kDa RITC-dextran and transendothelial lymphocyte migration. Permeability changes in response to CCL4 in vivo were measured by an occlusion technique in pial microvessels of Wistar rats and by fluorescein angiography in mouse retinae. RESULTS: CCR5, the receptor for CCL4, was expressed in hCMEC/D3 cells. CCL4 stimulation led to phosphorylation of p38 and the formation of actin stress fibres, both indicative of intracellular chemokine signalling. The distribution of junctional proteins was also altered in response to CCL4: junctional ZO-1 was reduced by circa 60% within 60 ​min. In addition, surface VE-cadherin was redistributed through internalisation. Consistent with these changes, CCL4 induced hyperpermeability in vitro and in vivo and increased transmigration of lymphocytes across monolayers of hCMEC/D3 cells. CONCLUSION: These results show that CCL4 can modify BBB function and may contribute to disease pathogenesis

LRG1 destabilizes tumor vessels and restricts immunotherapeutic potency

Background: A poorly functioning tumor vasculature is pro-oncogenic and may impede the delivery of therapeutics. Normalizing the vasculature, therefore, may be beneficial. We previously reported that the secreted glycoprotein leucine-rich α-2-glycoprotein 1 (LRG1) contributes to pathogenic neovascularization. Here, we investigate whether LRG1 in tumors is vasculopathic and whether its inhibition has therapeutic utility. Methods: Tumor growth and vascular structure were analyzed in subcutaneous and genetically engineered mouse models in wild-type and Lrg1 knockout mice. The effects of LRG1 antibody blockade as monotherapy, or in combination with co-therapies, on vascular function, tumor growth, and infiltrated lymphocytes were investigated. Findings: In mouse models of cancer, Lrg1 expression was induced in tumor endothelial cells, consistent with an increase in protein expression in human cancers. The expression of LRG1 affected tumor progression as Lrg1 gene deletion, or treatment with a LRG1 function-blocking antibody, inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function, resulting in enhanced efficacy of cisplatin chemotherapy, adoptive T cell therapy, and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent to immune active. Conclusions: LRG1 drives vascular abnormalization, and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics. Funding: Wellcome Trust (206413/B/17/Z), UKRI/MRC (G1000466, MR/N006410/1, MC/PC/14118, and MR/L008742/1), BHF (PG/16/50/32182), Health and Care Research Wales (CA05), CRUK (C42412/A24416 and A17196), ERC (ColonCan 311301 and AngioMature 787181), and DFG (CRC1366)

Therapeutic Validation of GEF-H1 Using a De Novo Designed Inhibitor in Models of Retinal Disease

Inflammation and fibrosis are important components of diseases that contribute to the malfunction of epithelia and endothelia. The Rho guanine nucleotide exchange factor (GEF) GEF-H1/ARHGEF-2 is induced in disease and stimulates inflammatory and fibrotic processes, cell migration, and metastasis. Here, we have generated peptide inhibitors to block the function of GEF-H1. Inhibitors were designed using a structural in silico approach or by isolating an inhibitory sequence from the autoregulatory C-terminal domain. Candidate inhibitors were tested for their ability to block RhoA/GEF-H1 binding in vitro, and their potency and specificity in cell-based assays. Successful inhibitors were then evaluated in models of TGFβ-induced fibrosis, LPS-stimulated endothelial cell-cell junction disruption, and cell migration. Finally, the most potent inhibitor was successfully tested in an experimental retinal disease mouse model, in which it inhibited blood vessel leakage and ameliorated retinal inflammation when treatment was initiated after disease diagnosis. Thus, an antagonist that blocks GEF-H1 signaling effectively inhibits disease features in in vitro and in vivo disease models, demonstrating that GEF-H1 is an effective therapeutic target and establishing a new therapeutic approach

CTLA-4 controls follicular helper T-cell differentiation by regulating the strength of CD28 engagement

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is an essential regulator of T-cell responses, and its absence precipitates lethal T-cell hyperactivity. However, whether CTLA-4 acts simply to veto the activation of certain clones or plays a more nuanced role in shaping the quality of T-cell responses is not clear. Here we report that T cells in CTLA-4-deficient mice show spontaneous T-follicular helper (TFH) differentiation in vivo, and this is accompanied by the appearance of large germinal centers (GCs). Remarkably, short-term blockade with anti-CTLA-4 antibody in wild-type mice is sufficient to elicit TFH generation and GC development. The latter occurs in a CD28-dependent manner, consistent with the known role of CTLA-4 in regulating the CD28 pathway. CTLA-4 can act by down-regulating CD80 and CD86 on antigen presenting cells (APCs), thereby altering the level of CD28 engagement. To mimic reduced CD28 ligation, we used mice heterozygous for CD28, revealing that the magnitude of CD28 engagement is tightly linked to the propensity for TFH differentiation. In contrast, other parameters of T-cell activation, including CD62L down-regulation and Ki67 expression, were relatively insensitive to altered CD28 level. Altered TFH generation as a result of graded reduction in CD28 was associated with decreased numbers of GC B cells and a reduction in overall GC size. These data support a model in which CTLA-4 control of immunity goes beyond vetoing T-cell priming and encompasses the regulation of TFH differentiation by graded control of CD28 engagement