Background and purpose
Cerebral preconditioning provides insights into endogenous mechanisms that protect the brain from ischemic injury. Hypoxia and the anesthetic isoflurane are powerful preconditioning agents. Recent data show that sphingosine 1-phosphate (S1P) receptor stimulation improves outcome in rodent models of stroke. Endogenous S1P levels are controlled by the expression and activity of sphingosine kinases (SPK). We hypothesize that SPK up-regulation mediates preconditioning induced by isoflurane and hypoxia and reduces ischemic injury.
Male wild-type C57BL/J, SPK1−/− and SPK2−/− mice were exposed to isoflurane (IsoPC) or hypoxia preconditioning (HPC) before transient middle cerebral artery occlusion. Infarct volume and neurological outcome were measured 24 hours later. SPK inhibitors (SKI-II and ABC294640) were used to test the involvement of SPK2. Expressions of SPK1, SPK2 and HIF1α were determined. Primary cultures of mouse cortical neurons were exposed to isoflurane before glutamate- or hydrogen peroxide-induced cell death.
IsoPC and HPC significantly reduced infarct volume and improved neurological outcome in wild-type and SPK1−/− mice, but not in SPK2−/− mice. Pretreatment with SKI-II or ABC294640 abolished the IsoPC-induced tolerance. Western blot showed a rapid and sustained increase in SPK2 level, whereas SPK1 level was similar between preconditioned mice and controls. HIF1α was up-regulated in wild-type IsoPC mice, but not in SPK2−/−. IsoPC protected primary neurons against cell death, which was abolished in ABC294640-treated cells.
Applying genetic and pharmacological approaches, we demonstrate that neuronal SPK2 isoform plays an important role in cerebral preconditioning.
Sphingosine kinase 2; preconditioning; isoflurane; hypoxia; cerebral ischemia; neurons; cell death
The efficacy of B cell depletion therapy in rheumatoid arthritis (RA) has driven interest in understanding the mechanism. Because the decrease in autoantibododies in RA does not necessarily correlate with clinical outcome other mechanisms may be operative. We previously reported in proteoglycan-induced arthritis (PGIA), B cell depletion inhibits autoreactive T cell responses. Recent studies in B cell depletion therapy also indicate a role for B cells in suppressing regulatory mechanisms. Here we demonstrate that B cells inhibited both the expansion and the function of T regulatory (Treg) cells in PGIA. Utilizing an anti-CD20 mAb, we depleted B cells from mice with PGIA and assessed the Treg cell population. Compared to control antibody-treated mice, Treg cell percentages were elevated in B cell-depleted mice, with a higher proportion of CD4+ T cells expressing Foxp3 and CD25. On a per cell basis, CD4+CD25+ cells from B cell depleted mice expressed increased amounts of Foxp3 and were significantly more suppressive than those from control Ab-treated mice. The depletion of Treg cells with an anti-CD25 mAb concurrent with B cell depletion therapy restored the severity of PGIA to levels equal to untreated mice. Although titers of autoantibodies did not recover to untreated levels, CD4+ T cell recall responses to the immunizing antigen returned as measured by T cell proliferation and cytokine production. Thus, B cells have the capacity to regulate inflammatory responses by enhancing T effector cells along with suppressing Treg cells.
B cells; T; Autoimmunity; Rheumatoid Arthritis
Dipeptidyl peptidase 4 (DPP4) and angiotensin-converting enzyme (ACE) are important target enzymes in glycemic control and renovascular protection. Here, we studied the effect of NWT-03, an egg protein hydrolysate with DPP4- and ACE-inhibitory activity, on renovascular damage in Zucker diabetic fatty (ZDF) rats. Comparisons were made to rats treated with vildagliptin (VIL), included as a positive control for the effect of DPP4 inhibition.
ZDF rats received NWT-03 (1 g/kg/day) or VIL (3 mg/kg/day) from 10 to 25 weeks of age. Metabolic and renal functions were assessed; the kidney was removed for histological analysis of glomerulosclerosis and expression of pro-inflammatory/fibrotic markers (RT-PCR and Western blotting); and the aorta was removed for studies of endothelium-dependent relaxation (EDR).
Hyperinsulinemic ZDF rats typically developed signs of type-2 diabetes and renovascular damage, as evidenced by albuminuria, glomerulosclerosis, and impaired EDR. Neither NWT-03 nor VIL improved metabolic parameters; for VIL, this was despite a 5-fold increase in glucagon-like peptide (GLP)-1 levels. NWT-03 and VIL both reduced renal interleukin (Il)-1β/Il-13 mRNA expression and glomerulosclerosis. However, only NWT-03 additionally decreased renal tumor necrosis factor (TNF)-α mRNA and P22phox protein expression, reduced albuminuria, and restored aortic EDR. Indomethacin added to the organ bath instantly improved aortic EDR, indicating a role for cyclooxygenase (COX)-derived contractile prostanoids in opposing relaxation in ZDF rats. This indomethacin effect was reduced by NWT-03, but not by VIL, and coincided with decreased renal COX-1/2 protein expression.
Conclusion and Interpretation
Long-term supplementation with the egg protein hydrolysate NWT-03 attenuated renovascular damage in this preclinical rat model of type 2 diabetes. A comparison to the DPP4-inhibitor VIL suggests that the effects of NWT-03 were related to both ACE- and DPP4-inhibitory properties. The development of protein hydrolysates with a multiple-targeting strategy may be of benefit to functional food formulations.
Enterovirus 71 (EV71) is the most important causative agent of hand, foot and mouth disease (HFMD) in children. In most cases, it is a self-limiting illness. However some EV71 infectious cases can develop severe clinical outcomes, such as encephalitis, meningitis, poliomyelitis like paralysis, and even death. To identify the determinants of virulence, the deduced amino acid sequence of polyprotein and nucleotide sequence of 5′-NTR and 3′-NTR in 25 SC-EV71 strains (strains from severe cases) and 31 MC-EV71 strains (strains from mild cases) were analyzed. Results showed four amino acids on two positions (GlyP710/GlnP710/ArgP710 and GluP729) on the DE and EF loop of VP1, one (LysP930) on the surface of protease 2A and four nucleotides on three positions (GP272, UP488 and AP700/UP700) in the 5'-NTR region are associated with EV71 virulent phenotype. Predicted secondary structure of RNA using the consensus sequence of 5'-NTR by RNAStructure showed the mutation of nucleotide at position 488 in strain BJ08-Z004-3 (position 491 in prototype strain BrCr) can result in the discrepancy of an additional pair of nucleotides and thus change the stability of the second structure of IRES. Fragment base content analysis showed that in the region 696 to 714 bp at the 5'-NTR, where the AP700/UP700 was located, the nucleotide constitution ratios differed significantly between SC-EV71 and MC-EV71 strains. In conclusion, comparative genomic analysis showed that virulence of EV71 strains are mainly determined by the amino acids on two positions of VP1, one position of protease 2A and the nucleotides on three positions in 5'-NTR.
Familial hemiplegic migraine type 1, a monogenic migraine variant with aura, is linked to gain-of-function mutations in the CACNA1A gene encoding CaV2.1 channels. The S218L mutation causes severe channel dysfunction, and paroxysmal migraine attacks can be accompanied by seizures, coma, and hemiplegia; patients expressing the R192Q mutation exhibit hemiplegia only. Familial hemiplegic migraine knock-in mice expressing the S218L or R192Q mutation are highly susceptible to cortical spreading depression, the electrophysiological surrogate for migraine aura, and develop severe and prolonged motor deficits after spreading depression. The S218L mutants also develop coma and seizures and sometimes die. To investigate underlying mechanisms for these symptoms, we used multielectrode electrophysiological recordings, diffusion-weighted magnetic resonance imaging, and c-fos immunohistochemistry to trace spreading depression propagation into subcortical structures. We showed that unlike the wild type, cortical spreading depression readily propagated into subcortical structures in both familial hemiplegic migraine type 1 mutants. Whereas the facilitated subcortical spread appeared limited to the striatum in R192Q, hippocampal and thalamic spread was detected in the S218L mutants with an allele-dosage effect. Both strains exhibited increased susceptibility to subcortical spreading depression and reverberating spreading depression waves. Altogether, these data show that spreading depression propagates between cortex, basal ganglia, diencephalon, and hippocampus in genetically susceptible brains, which could explain the prolonged hemiplegia, coma, and seizure phenotype in this variant of migraine with aura.
In the title compound, C22H19NO, the pyridine ring and the adjacent naphthalene ring system are nearly coplanar, making a dihedral angle of 3.3 (1)°, while the pyridine and benzene rings are perpendicular to each other, with a dihedral angle of 89.9 (1)°. The crystal packing is stabilized by intermolecular O—H⋯N hydrogen bonds and C—H⋯π interactions.
Epigenetic factor CTCF (CCCTC binding factor) plays important roles in genetic controls of the cell fate. Previous studies found in corneal epithelial cells that CTCF is regulated by epidermal growth factor (EGF) through activation of NF-κB p65/p50. It also found that CTCF is suppressed in ultraviolet (UV) stress-induced corneal epithelial cells. However, it is still unknown how UV stress down-regulates CTCF affecting the cell fate. In the present study, we report that regulation of CTCF by extracellular stress signals is dependent upon activations of an oxidative stress-regulated protein Bcl-3. We found that activated Bcl-3 was able to bind to the κB sites identified in the CTCF promoter region. Bcl-3 was activated by UV irradiation to interact with NF-κB p50 by forming a Bcl-3/p50 heterodimer complex. The Bcl-3/p50 complex suppressed CTCF promoter activity to down-regulate CTCF transcription. Unlike the effect of EGF, UV stress-induced Bcl-3 activation suppressed CTCF activity without involving the IκBα and p65 pathway. Thus, results of the study reveal a novel mechanism for regulatory control of CTCF in UV stress-induced human corneal epithelial cells, which requires activation and formation of Bcl-3/p50 complex through a noncanonical NF-κB pathway.
The ability to predict endothelial cell migration rates may aid in the design of biomaterials that endothelialize following implantation. However, the complexity of the signaling response to migration-promoting stimuli such as sphingosine 1-phosphate (S1P) makes such predictions quite challenging. A number of signaling pathways impact S1P-mediated cell migration, including the Akt and Src pathways, which both affect activation of the small GTPase Rac. Rac activation promotes the formation of lamellipodia, and thus should be intimately linked to cell migration rates. In immortalized endothelial cells, expression of proteins that inhibit Akt, Src, and Rac (PTEN, CSK, and β2-chimaerin, respectively) was decreased using RNA interference, resulting in increases in the basal level of activation of Akt, Src, and Rac. Cells were scrape-wounded and stimulated with 1 μM S1P. The timecourse of Akt, Src, and Rac activation was followed over 2 h in the perturbed cells, while migration into the scrape wound was measured over 6 h. Rac activation at 120 min post-stimulation was highly correlated with the mean migration rate of cells, but only in cells stimulated with S1P. Using partial least squares regression, the migration rate of cells into the scrape wound was found to be highly correlated with the magnitude of the early Akt peak (e.g., 5–15 min post-stimulation). These results demonstrated that biochemical measurements might be useful in predicting rates of endothelial cell migration.
Endothelial cell; Migration; Modeling; Partial least squares regression; Biochemistry
Pax6 plays an important role in embryonic cell (ES) differentiation during embryonic development. Expression of Pax6 undergoes from a low level to high levels following ES cell differentiation to neural stem cells, and then fades away in most of the differentiated cell types. There is a limited knowledge concerning how Pax6 is regulated in ES cell differentiation. We report that Pax6 expression in mouse ES cells was controlled by CCCTC binding factor (CTCF) through a promoter repression mechanism. Pax6 expression was significantly enhanced while CTCF activity was kept in the constant during ES cell differentiation to radial glial cells. Instead, the interaction of CTCF with Pax6 gene was regulated by decreased CTCF occupancy in its binding motifs upstream from Pax6 P0 promoter following the course of ES cell differentiation. Reduced occupancy of CTCF in the binding motif region upstream from the P0 promoter was due to increased DNA methylations in the CpG sites identified in the region. Furthermore, changes in DNA methylation levels in vitro and in vivo effectively altered methylation status of these identified CpG sites, which affected ability of CTCF to interact with the P0 promoter, resulting in increases in Pax6 expression. We conclude that there is an epigenetic mechanism involving regulations of Pax6 gene during ES cell differentiation to neural stem cells, which is through increases or decreases in methylation levels of Pax6 gene to effectively alter the ability of CTCF in control of Pax6 expression, respectively.
Vasoregression is a hallmark of vascular eye diseases but the mechanisms involved are still largely unknown. We have recently characterized a rat ciliopathy model which develops primary photoreceptor degeneration and secondary vasoregression. To improve the understanding of secondary vasoregression in retinal neurodegeneration, we used microarray techniques to compare gene expression profiles in this new model before and after retinal vasoregression. Differential gene expression was validated by quantitative RT-PCR, Western blot and immunofluorescence. Of the 157 genes regulated more than twofold, the MHC class II invariant chain CD74 yielded the strongest upregulation, and was allocated to activated microglial cells close to the vessels undergoing vasoregression. Pathway clustering identified genes of the immune system including inflammatory signaling, and components of the complement cascade upregulated during vasoregression. Together, our data suggest that microglial cells involved in retinal immune response participate in the initiation of vasoregression in the retina.
The contribution of the pro-inflammatory cytokines IFN-γ and IL-17 to the pathogenesis of experimental arthritis is controversial. In proteoglycan-induced arthritis (PGIA) severe arthritis is dependent on the production of IFN-γ whereas IL-17 is dispensable. In collagen-induced arthritis (CIA) and antigen-induced arthritis (AIA), although high levels of IFN-γ are secreted, disease is exacerbated in IFN-γ or IFN-γ receptor deficient mice due to the ability of IFN-γ to suppress IL-17 expression. In the present study, we investigated the effect of IFN-γ on the IL-17 response and its consequences in PGIA. In PG-immunized IFN-γ−/− mice, despite reduction in arthritis, the PG-specific CD4+ T cell IL-17 response was significantly increased. Elevated IL-17 contributed to development of arthritis as disease in IFN-γ /IL-17−/− was significantly reduced in comparison to either IFN-γ−/− or IL-17−/− mice. A contribution of IFN-γ and IL-17 to the development of arthritis was also identified in T-bet−/− mice. PG-specific CD4+ T cells from T-bet−/− mice produced reduced IFN-γ and elevated concentrations of IL-17. Both IFN-γ and IL-17 contribute to arthritic as T-bet−/− mice lacking IL-17 (T-bet/IL-17−/−) were resistant whereas WT, T-bet−/−, and IL-17−/− mice were susceptible to PGIA. T cell proliferation and autoantibody production did not correlate with development of disease, however, expression of cytokines and chemokines in joint tissues demonstrate that IFN-γ and IL-17 cooperatively contribute to inflammation. These results demonstrate that both IFN-γ and IL-17 have the potential to induce PGIA but it is the strength of the IFN-γ response that regulates the contribution of each of these T helper effector cytokines to disease.
T cells; Cytokines; Chemokines; Rodents; Autoimmunity; Rheumatoid Arthritis
CCR5 and its ligands (CCL3, CCL4 and CCL5) may play a role in inflammatory cell recruitment into the joint. However, recently it has been reported that CCR5 on T cells and neutrophils acts as a decoy receptor for CCL3 and CCL5 to assist in resolution of inflammation. To determine whether CCR5 functions as a pro-inflammatory or anti-inflammatory mediator in arthritis, we examined the role of CCR5 in proteoglycan (PG)-induced arthritis (PGIA).
PGIA was induced by immunization of BALB/c wild type (WT) and CCR5-deficient (CCR5−/−) mice with human PG in adjuvant. The onset and severity were monitored overtime. Met-RANTES was used to block CCR5 in vivo. Arthritis was transferred to SCID mice with spleen cells from arthritis WT and CCR5−/− mice. Cytokines and chemokines were measured by ELISA.
Treatment with the CCR5 inhibitor, Met-RANTES, and CCR5−/− mice developed exacerbated arthritis late in the course of disease. The increase in arthritis severity in CCR5−/− correlated with elevated serum levels of CCL5. However, exacerbated arthritis was not intrinsic to the CCR5−/− lymphoid cells as arthritis transferred into SCID recipients was similar in WT and CCR5−/− mice. CCR5 expression in the SCID was sufficient to clear CCL5 as serum levels of CCL5 were the same in SCID recipients receiving WT or CCR5−/− cells.
These data demonstrate that CCR5 is a key player in controlling the resolution of inflammation in experimental arthritis.
Autoimmunity; Inflammation; Rheumatoid Arthritis; Chemokine and Rodent
Patent foramen ovale and pulmonary arteriovenous shunts are associated with serious complications such as cerebral emboli, stroke, and migraine with aura. The pathophysiological mechanisms that link these conditions are unknown. We aimed to establish a mechanism linking microembolization to migraine aura in an experimental animal model.
We introduced particulate or air microemboli into the carotid circulation in mice to determine whether transient microvascular occlusion, insufficient to cause infarcts, triggered cortical spreading depression (CSD), a propagating slow depolarization that underlies migraine aura.
Air microemboli reliably triggered CSD without causing infarction. Polystyrene microspheres (10μm) or cholesterol crystals (<70μm) triggered CSD in 16 of 28 mice, with 60% of the mice (40% of those with CSD) showing no infarcts or inflammation on detailed histological analysis of serial brain sections. No evidence of injury was detected on magnetic resonance imaging examination (9.4T; T2 weighted) in 14 of 15 selected animals. The occurrence of CSD appeared to be related to the magnitude and duration of flow reduction, with a triggering mechanism that depended on decreased brain perfusion but not sustained tissue damage.
In a mouse model, microemboli triggered CSD, often without causing microinfarction. Paradoxical embolization then may link cardiac and extracardiac right-to-left shunts to migraine aura. If translatable to humans, a subset of migraine auras may belong to a spectrum of hypoperfusion disorders along with transient ischemic attacks and silent infarcts.
Neuronal damage is correlated with vascular dysfunction in the diseased retina, but the underlying mechanisms remain controversial because of the lack of suitable models in which vasoregression related to neuronal damage initiates in the mature retinal vasculature. The aim of this study was to assess the temporal link between neuronal damage and vascular patency in a transgenic rat (TGR) with overexpression of a mutant cilia gene polycystin-2.
Vasoregression, neuroglial changes and expression of neurotrophic factors were assessed in TGR and control rats in a time course. Determination of neuronal changes was performed by quantitative morphometry of paraffin-embedded vertical sections. Vascular cell composition and patency were assessed by quantitative retinal morphometry of digest preparations. Glial activation was assessed by western blot and immunofluorescence. Expression of neurotrophic factors was detected by quantitative PCR.
At one month, number and thickness of the outer nuclear cell layers (ONL) in TGR rats were reduced by 31% (p<0.001) and 17% (p<0.05), respectively, compared to age-matched control rats. Furthermore, the reduction progressed from 1 to 7 months in TGR rats. Apoptosis was selectively detected in the photoreceptor in the ONL, starting after one month. Nevertheless, TGR and control rats showed normal responses in electroretinogram at one month. From the second month onwards, TGR retinas had significantly increased acellular capillaries (p<0.001), and a reduction of endothelial cells (p<0.01) and pericytes (p<0.01). Upregulation of GFAP was first detected in TGR retinas after 1 month in glial cells, in parallel with an increase of FGF2 (fourfold) and CNTF (60 %), followed by upregulation of NGF (40 %) at 3 months.
Our data suggest that TGR is an appropriate animal model for vasoregression related to neuronal damage. Similarities to experimental diabetic retinopathy render this model suitable to understand general mechanisms of maturity-onset vasoregression.
Cell cycle inhibition of neural stem and progenitor cells is critical for maintaining the stability of central nervous system in adults, but it may represent a significant hurdle for neural regeneration after injury. We have previously demonstrated that the cyclin-dependent kinase inhibitor (CKI) p21cip1/waf1 (p21) maintains the quiescence of neural stem-like cells under cerebral ischemia, as similarly shown for the hematopoietic stem cells. Here, we report the distinct role of another CKI member, p27kip1 (p27) in neural progenitor cells (NPCs) from adult brain (subventricular zone and hippocampal subgranular zone) under both homeostatic and ischemic conditions. The basal level of NPC proliferation in the p27−/− mice was higher than that in p27+/+ mice. Upon ischemia, the overall proliferation of NPCs continued to be higher in p27−/− mice than that in p27 +/+ mice. Moreover, the increase of NPC proliferation in p27−/− mice remained until 2 weeks after ischemia whereas it resumed back to the basal level in p27+/+ mice. As a result, newly generated neuronal cells in the granular layer of p27−/− brain were more abundant compared to p27 +/+ controls. These new data demonstrate that p27 functions as a distinct inhibitor for NPC proliferation under homeostatic as well as ischemic conditions.
IL-17 is the hallmark cytokine for the newly identified subset of T helper cells, Th17. Th17 cells are important instigators of inflammation in several models of autoimmune disease; in particular, collagen induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE), which were previously characterized as Th1-mediated diseases. Although high levels of IFN-γ are secreted in CIA and EAE, disease is exacerbated in IFN-γ or IFN-γ receptor deficient mice due to the ability of IFN-γ to suppress IL-17 secretion. However, in proteoglycan-induced arthritis (PGIA), severe arthritis is dependent on the production of IFN-γ. We were therefore interested in determining the role of IL-17 in PGIA. We assessed the progression of arthritis in IL-17-deficient (IL-17−/−) mice and found the onset and severity of arthritis equivalent in wildtype (WT) and IL-17−/− mice. Despite evidence that IL-17 is involved in neutrophil recruitment, synovial fluid from arthritic joints showed a comparable proportion of Gr1+ neutrophils in WT and IL-17−/− mice. IL-17 is also implicated in bone destruction in autoimmune arthritis, however histological analysis of the arthritic joints from WT and IL-17−/− mice revealed a similar extent of joint cellularity, cartilage destruction and bone erosion despite significantly reduced RANKL expression. There were only subtle differences between WT and IL-17−/− in pro-inflammatory cytokine expression, T cell proliferation and autoanibody production. These data demonstrate that IL-17 is not absolutely required for autoimmune arthritis and production of other proinflammatory mediators are sufficient to compensate for the loss of IL-17 in PGIA.
Autoimmunity; Cytokines; Inflammation; Rheumatoid Arthritis and Rodent