Lihua Yao

Secreted phospholipase A2 activity in experimental autoimmune encephalomyelitis and multiple sclerosis

BackgroundThere is increased interest in the contribution of the innate immune system to multiple sclerosis (MS), including the activity of acute inflammatory mediators. The purpose of this study was to test the involvement of systemic secreted phospholipase A2 (sPLA2) enzymes in experimental autoimmune encephalomyelitis (EAE), an MS model, and to determine if enzyme activity is elevated in MS patients.MethodsA non-invasive urinary assay was developed in order to monitor enzymatically active sPLA2 levels in Dark Agouti rats after induction of EAE. Some Rats were treated with nonapeptide CHEC-9, an uncompetitive sPLA2 enzyme inhibitor, during the initial rise in urinary enzyme levels. Body weight and clinical EAE score were measured for 18 days post immunization (PI), after which the rats were sacrificed for H&E and myelin staining, and for ED-1 immunocytochemistry, the latter to quantify macrophages and activated microglia. The urinary sPLA2 assay was also applied to un-timed samples collected from a cross section of 44 MS patients and 14 healthy controls.ResultsMean levels of enzymatically active sPLA2 in the urine increased following immunization and peaked between days 8–10 PI which was just prior to the onset of EAE symptoms. At this time, a transient attenuation of activity was detected in the urine of CHEC-9 treated rats consistent with the activity-dependent properties of the inhibitor. The peptide also reduced or abolished EAE symptoms compared to vehicle-injected controls. Histopathological changes in the spinal cords of the EAE rats correlated generally with clinical score including a significant reduction in ED-1+ cells after peptide treatment. Multiple Sclerosis patients also showed elevations in sPLA2 enzyme activity. Mean levels of sPLA2 were increased 6-fold in the urine of patients with active disease and 4-fold for patients in remission, regardless of immunomodulating therapy.ConclusionThe results suggest that sPLA2 enzymes, traditionally thought to be part the acute phase inflammatory response, are therapeutic targets for MS.

Inhibition of secreted phospholipase A2 by neuron survival and anti-inflammatory peptide CHEC-9

BackgroundThe nonapeptide CHEC-9 (CHEASAAQC), a putative inhibitor of secreted phospholipase A2 (sPLA2), has been shown previously to inhibit neuron death and aspects of the inflammatory response following systemic treatment of rats with cerebral cortex lesions. In this study, the properties of CHEC-9 inhibition of sPLA2 enzymes were investigated, using a venom-derived sPLA2 group I and the plasma of rats and humans as the sources of enzyme activity. The results highlight the advantages of inhibitors with uncompetitive properties for inflammatory disorders including those resulting in degeneration of neurons.MethodsSamples of enzyme and plasma were reacted with 1-Palmitoyl-2-Pyrenedecanoyl Phosphatidylcholine, a sPLA2 substrate that forms phospholipid vesicles in aqueous solutions. Some of the plasma samples were collected from restrained peptide-treated rats in order to confirm the validity of the in vitro assays for extrapolation to in vivo effects of the peptide. The enzyme reactions were analyzed in terms of well-studied relationships between the degree of inhibition and the concentrations of different reactants. We also examined interactions between different components of the reaction mixture on native polyacrylamide gels.ResultsIn all cases, the peptide showed the properties of an uncompetitive (or anti-competitive) enzyme inhibitor with Ki values less than 100 nanomolar. The electrophoresis experiments suggested CHEC-9 modifies the binding properties of the enzyme only in the presence of substrate, consistent with its classification as an uncompetitive inhibitor. Both the in vitro observations and the analysis of plasma samples from restrained rats injected with peptide suggest the efficacy of the peptide increases under conditions of high enzyme activity.ConclusionModeling studies by others have shown that uncompetitive inhibitors may be optimal for enzyme inhibition therapy because, unlike competitive inhibitors, they are not rendered ineffective by the accumulation of unmodified substrate. Such conditions likely apply to several instances of neuroinflammation where there are cascading increases in sPLA2s and their substrates, both systemically and in the CNS. Thus, the present results may explain the efficacy of CHEC-9 in vivo.

Secreted Phospholipase A2 Involvement in Neurodegeneration: Differential Testing of Prosurvival and Anti-Inflammatory Effects of Enzyme Inhibition

There is increased interest in the contribution of secreted phospholipase A2 (sPLA2) enzymes to neurodegenerative diseases. Systemic treatment with the nonapeptide CHEC-9, a broad spectrum uncompetitive inhibitor of sPLA2, has been shown previously to inhibit neuron death and aspects of the inflammatory response in several models of neurodegeneration. A persistent question in studies of sPLA2 inhibitors, as for several other anti-inflammatory and neuroprotective compounds, is whether the cell protection is direct or due to slowing of the toxic aspects of the inflammatory response. To further explore this issue, we developed assays using SY5Y (neuronal cells) and HL-60 (monocytes) cell lines and examined the effects of sPLA2 inhibition on these homogeneous cell types in vitro. We found that the peptide inhibited sPLA2 enzyme activity in both SY5Y and HL-60 cultures. This inhibition provided direct protection to SY5Y neuronal cells and their processes in response to several forms of stress including exposure to conditioned medium from HL-60 cells. In cultures of HL-60 cells, sPLA2 inhibition had no effect on survival of the cells but attenuated their differentiation into macrophages, with regard to process development, phagocytic ability, and the expression of differentiation marker CD36, as well as the secretion of proinflammatory cytokines TNF-α and IL-6. These results suggest that sPLA2 enzyme activity organizes a cascade of changes comprising both cell degeneration and inflammation, processes that could theoretically operate independently during neurodegenerative conditions. The effectiveness of sPLA2 inhibitor CHEC-9 may be due to its ability to affect both processes in isolation. Testing potential anti-inflammatory/neuroprotective compounds with these human cell lines and their conditioned media may provide a useful screening tool prior to in vivo therapeutic applications.

Guiding migration of transplanted glial progenitor cells in the injured spinal cord

Transplantation of glial-restricted progenitors (GRPs) is a promising strategy for generating a supportive environment for axon growth in the injured spinal cord. Here we explored the possibility of producing a migratory stream of GRPs via directional cues to create a supportive pathway for axon regeneration. We found that the axon growth inhibitor chondroitin sulfate proteoglycan (CSPG) strongly inhibited the adhesion and migration of GRPs, an effect that could be modulated by the adhesion molecule laminin. Digesting glycosaminoglycan side chains of CSPG with chondroitinase improved GRP migration on stripes of CSPG printed on cover glass, although GRPs were still responsive to the remaining repulsive signals of CSPG. Of all factors tested, the basic fibroblast growth factor (bFGF) had the most significant effect in promoting the migration of cultured GRPs. When GRPs were transplanted into either normal spinal cord of adult rats or the injury site in a dorsal column hemisection model of spinal cord injury, a population of transplanted cells migrated toward the region that was injected with the lentivirus expressing chondroitinase or bFGF. These findings suggest that removing CSPG-mediated inhibition, in combination with guidance by attractive factors, can be a promising strategy to produce a migratory stream of supportive GRPs.

Anti-Inflammatory Peptide Regulates the Supply of Heat Shock Protein 70 Monomers: Implications for Aging and Age-Related Disease

Reducing the levels of toxic protein aggregates has become a focus of therapy for disorders like Alzheimers and Parkinsons diseases, as well as for the general deterioration of cells and tissues during aging. One approach has been an attempt to influence the production or activity of a class of reparative chaperones called heat shock proteins (HSPs), of which HSP70 is a promising candidate. Manipulation of HSP70 expression results in disposal of misfolded protein aggregates that accumulate in aging and disease models. Recently, HSP70 has been shown to bind specifically to an amino-terminal sequence of a human diffusible survival evasion peptide (DSEP), dermcidin. This sequence includes CHEC-9, an orally available anti-inflammatory and cell survival peptide. In the present study, we found that the CHEC-9 peptide also binds HSP70 in the cytosol of the cerebral cortex after oral delivery in normal rats. Western analysis of non-heat-denatured, unreduced samples suggested that peptide treatment increased the level of active HSP70 monomers from the pool of chaperone oligomers, a process that may be stimulated by potentiation of the chaperones adenosine triphosphatase (ATPase). In these samples, a small but consistent gel shift was observed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a multifunctional protein whose aggregation is influenced by HSP70. CHEC-9 treatment of an in vitro model of α-synuclein aggregation also results in HSP70-dependent dissolution of these aggregates. HSP70 oligomer-monomer equilibrium and its potential to control protein aggregate disease warrant increased experimental attention, especially if a peptide fragment of an endogenous human protein can influence the process.

Uncompetitive Phospholipase A2 Inhibition by CHEC Sequences Including Oral Treatment of Experimental Autoimmune Myeloencephalitis

Catalytic sites of metal dependent phosphatases and phospholipases are characterized by a catalytic histidine (H) and adjacent acidic metal binding residue E (or D). For the secreted phospholipases A2 (sPLA2s), the active sites are stabilized by nearby flanking cystines. CHEC-9 (CHEASAAQC), which displays this organization, is a sPLA2 inhibitor that is an effective treatment for models of traumatic and autoimmune neurodegenerative disorders. We screened modifi- cations of this sequence and found that elimination of the two central alanines followed by cyclization results in another potent sPLA2 inhibitor, CHEC-7 (CHEASQC). CHEC-7, like CHEC-9, had uncompetitive kinetic properties making it ideal for in vivo applications. Subcutaneous injections of CHEC-7 into restrained rats inhibited the transient rise in plasma sPLA2 activity suggesting that this peptide was also effective in vivo. Oral delivery of the peptide attenuated the LPS- induced increases in sPLA2 activity. Thin layer chromatography and mass spectroscopy of representative LPS-treated rats suggested that the peptide also reduced levels of lysophosphatidylcholine, a hydrolytic product of the enzyme reaction. In order to test the efficacy of CHEC-7 for neuroimmune therapies, rats were immunized with spinal cord homogenates to induce experimental autoimmune encephalomyelitis (EAE), followed by daily treatment with oral (1.5 mg/kg) or subcuta- neous (0.1, 0.5, and1.5mg/kg) CHEC-7 and CHEC-9 (n=10 for each different condition). The rats were scored for severity of disease applying a standard EAE scale. Control rats all developed disease over the 23-day course of the experiments. Both CHEC-9 and CHEC-7 produced highly significant reductions in disease severity including via oral delivery. The performance of oral CHEC-7 was notable in that this treatment completely prevented disease in half the rats. The results suggested that continued study of modifications of CHEC sequences would provide stable therapeutic peptides for a variety of inflammatory and autoimmune disorders.

Heptamer Peptide Disassembles Native Amyloid in Human Plasma Through Heat Shock Protein 70

Proteostasis, which includes the repair and disposal of misfolded proteins, depends, in part, on the activity of heat shock proteins (HSPs), a well-known class of chaperone molecules. When this process fails, abnormally folded proteins may accumulate in cells, tissues, and blood. These species are a hallmark of protein aggregation diseases, but also amass during aging, often in the absence of an identified clinical disorder. We report that a neuroprotective cyclic heptapeptide, CHEC-7, which has been applied systemically as a therapeutic in animal neurodegeneration models, disrupts such aggregates and inhibits amyloidogenesis when added in nanomolar concentrations to human plasma. This effect includes aggregates of amyloid beta (Aβ1-40, 1-42), prominent features of Alzheimers disease pathology. The activity of endogenous HSP70, a recently discovered target of the peptide, is required as demonstrated by both antibody blocking and application of pifithrin-μ, an HSP70 inhibitor. CHEC-7 is the first high-affinity compound to stimulate HSP70s disaggregase activity and therefore enable this endogenous mechanism in a human systemic environment, increasing the likelihood of a convenient therapy for protein aggregate disease, including age-related failures of protein repair.