Microglial Calcium Release-Activated Calcium Channel Inhibition Improves Outcome from Experimental Traumatic Brain Injury and Microglia-Induced Neuronal Death.

Store-operated Ca2+ entry (SOCE) mediated by calcium release-activated calcium (CRAC) channels contributes to calcium signaling. The resulting intracellular calcium increases activate calcineurin, which in turn activates immune transcription factor nuclear factor of activated T cells (NFAT). Microglia contain CRAC channels, but little is known whether these channels play a role in acute brain insults. We studied a novel CRAC channel inhibitor to explore the therapeutic potential of this compound in microglia-mediated injury. Cultured microglial BV2 cells were activated by Toll-like receptor agonists or IFNγ. Some cultures were treated with a novel CRAC channel inhibitor (CM-EX-137). Western blots revealed the presence of CRAC channel proteins STIM1 and Orai1 in BV2 cells. CM-EX-137 decreased nitric oxide (NO) release and inducible nitric oxide synthase (iNOS) expression in activated microglia and reduced agonist-induced intracellular calcium accumulation in microglia, while suppressing inflammatory transcription factors nuclear factor kappa B (NF-κB) and nuclear factor of activated T cells (NFAT). Male C57/BL6 mice exposed to experimental brain trauma and treated with CM-EX-137 had decreased lesion size, brain hemorrhage, and improved neurological deficits with decreased microglial activation, iNOS and Orai1 and STIM1 levels. We suggest a novel anti-inflammatory approach for managing acute brain injury. Our observations also shed light on new calcium signaling pathways not described previously in brain injury models.

Activated complement protein C5a does not affect brain-derived endothelial cell viability and zonula occludens-1 levels following oxygen-glucose deprivation.

BACKGROUND AND PURPOSE: Ischemic brain injury induces both functional and structural disarray affecting the blood-brain barrier (BBB) which in return aggravates stroke outcomes. Complement system and its bioactive proteins are important molecular responders to ischemia. C5a protein along with its receptor C5a receptor 1 is a key component of this system with potent pro-inflammatory and chemoattractant properties. The purpose of this study is to investigate the role of C5a protein and its receptor which are believed to participate in the inflammatory response that follows ischemic insult. MATERIALS AND METHODS: To mimic an ischemic in vivo event in which C5a may contact brain endothelial cells after injury, we studied oxygen-glucose deprivation (OGD) followed by reperfusion in brain microvascular endothelial cells (b.End. 3) by only added C5a at the time of reperfusion. Cell death and viability were estimated by trypan blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, respectively. Tight junction protein zonula occluden (ZO-1) levels were analyzed by Western blot analysis, and nitric oxide (NO) was assessed using the Griess reagent. RESULTS: Brain-derived endothelial cell was susceptible to OGD-induced injury in a duration-dependent manner as was the presence of ZO-1 protein. However, the addition of C5a protein had no notable effects even when used at high concentrations up to 100 nM. While OGD led to reduction in ZO-1 protein levels, no change was seen following the addition of C5a. Finally, OGD led unexpectedly to small decreases in NO generation, but this was again unaltered by C5a. CONCLUSIONS: Our study suggests that complement system protein C5a may not have a direct role in the disruption of BBB, following brain ischemia. This is in contrary with previous literature that suggests a possible role of this protein in the inflammatory response to ischemia.

Pharmacologic heat shock protein 70 induction confers cytoprotection against inflammation in gliovascular cells.

The inhibition of the 90-kDa heat shock protein (HSP90) leads to upregulation of the 70-kDa-inducible HSP70. HSP70 has been previously shown to be neuroprotective and anti-inflammatory. Geldanamycin (GA) and other HSP90 inhibitors have emerged as promising therapeutic agents in cancer, presumably owing to their ability to upregulate HSP70. However, the effects of HSP90 inhibition in brain inflammation are still unclear. We investigate the effect of a panel of HSP90 inhibitors on endotoxin-activated microglia and eventual protection from brain-derived endothelial cells. Prior studies have shown that GA protects brain cells from oxidative stress. We show here that when astrocytes or microglial BV2 cells were pretreated with GA or other HSP90 inhibitors, endotoxin-induced cell death was reduced in cocultures of BV2 microglia and brain-derived endothelial cells (bEND.3). Endotoxin-stimulated BV2 cells led to increased nitric oxide (NO) and inducible nitric oxide synthase which was prevented by treatment with all HSP90 inhibitors. HSP90 inhibitors also prevented lipopolysaccharide (LPS)-induced BV2 cell death. We also found that HSP90 inhibition blocked nuclear translocation of nuclear factor kappa B and attenuated IκBα degradation, and inhibited LPS-activated JAK-STAT phosphorylation. We show that pharmacologic inhibition of HSP90 with subsequent HSP70 induction protects cells that comprise the cerebral vasculature against cell death owing to proinflammatory stimuli. This approach may have therapeutic potential in neurological conditions with an inflammatory component.

Triggering receptor expressed on myeloid cells 2 (TREM2) deficiency attenuates phagocytic activities of microglia and exacerbates ischemic damage in experimental stroke.

Clearing cellular debris after brain injury represents an important mechanism in regaining tissue homeostasis and promoting functional recovery. Triggering receptor expressed on myeloid cells-2 (TREM2) is a newly identified receptor expressed on microglia and is thought to phagocytose damaged brain cells. The precise role of TREM2 during ischemic stroke has not been fully understood. We explore TREM2 in both in vitro and in vivo stroke models and identify a potential endogenous TREM2 ligand. TREM2 knockdown in microglia reduced microglial activation to an amoeboid phenotype and decreased the phagocytosis of injured neurons. Phagocytosis and infarcted brain tissue resorption was reduced in TREM2 knock-out (KO) mice compared with wild-type (WT) mice. TREM2 KO mice also had worsened neurological recovery and decreased viable brain tissue in the ipsilateral hemisphere. The numbers of activated microglia and phagocytes in TREM2 KO mice were decreased compared with WT mice, and foamy macrophages were nearly absent in the TREM2 KO mice. Postischemia, TREM2 was highly expressed on microglia and TREM2-Fc fusion protein (used as a probe to identify potential TREM2 binding partners) bound to an unknown TREM2 ligand that colocalized to neurons. Oxygen glucose deprivation-exposed neuronal media, or cellular fractions containing nuclei or purified DNA, but not cytosolic fractions, stimulated signaling through TREM2. TREM2-Fc fusion protein pulled down nucleic acids from ischemic brain lysate. These findings establish the relevance of TREM2 in the phagocytosis of the infarcted brain and emphasize its role in influencing neurological outcomes following stroke. Further, nucleic acids may be one potential ligand of TREM2 in brain ischemia.

Microglial P2Y12 deficiency/inhibition protects against brain ischemia.

OBJECTIVE: Microglia are among the first immune cells to respond to ischemic insults. Triggering of this inflammatory response may involve the microglial purinergic GPCR, P2Y12, activation via extracellular release of nucleotides from injured cells. It is also the inhibitory target of the widely used antiplatelet drug, clopidogrel. Thus, inhibiting this GPCR in microglia should inhibit microglial mediated neurotoxicity following ischemic brain injury. METHODS: Experimental cerebral ischemia was induced, in vitro with oxygen-glucose deprivation (OGD), or in vivo via bilateral common carotid artery occlusion (BCCAO). Genetic knock-down in vitro via siRNA, or in vivo P2Y12 transgenic mice (P2Y12-/- or P2Y12+/-), or in vivo treatment with clopidogrel, were used to manipulate the receptor. Neuron death, microglial activation, and microglial migration were assessed. RESULTS: The addition of microglia to neuron-astrocyte cultures increases neurotoxicity following OGD, which is mitigated by microglial P2Y12 deficiency (P<0.05). Wildtype microglia form clusters around these neurons following injury, which is also prevented in P2Y12 deficient microglia (P<0.01). P2Y12 knock-out microglia migrated less than WT controls in response to OGD-conditioned neuronal supernatant. P2Y12 (+/-) or clopidogrel treated mice subjected to global cerebral ischemia suffered less neuronal injury (P<0.01, P<0.001) compared to wild-type littermates or placebo treated controls. There were also fewer microglia surrounding areas of injury, and less activation of the pro-inflammatory transcription factor, nuclear factor Kappa B (NFkB). INTERPRETATION: P2Y12 participates in ischemia related inflammation by mediating microglial migration and potentiation of neurotoxicity. These data also suggest an additional anti-inflammatory, neuroprotective benefit of clopidogrel.

Sphingolipids in cardiovascular and cerebrovascular systems: Pathological implications and potential therapeutic targets.

The sphingolipid metabolites ceramide, sphingosine, and sphingosine-1-phosphate (S1P) and its enzyme sphingosine kinase (SphK) play an important role in the regulation of cell proliferation, survival, inflammation, and cell death. Ceramide and sphingosine usually inhibit proliferation and promote apoptosis, while its metabolite S1P phosphorylated by SphK stimulates growth and suppresses apoptosis. Because these metabolites are interconvertible, it has been proposed that it is not the absolute amounts of these metabolites but rather their relative levels that determine cell fate. The relevance of this "sphingolipid rheostat" and its role in regulating cell fate has been borne out by work in many labs using many different cell types and experimental manipulations. A central finding of these studies is that SphK is a critical regulator of the sphingolipid rheostat, as it not only produces the pro-growth, anti-apoptotic messenger S1P, but also decreases levels of pro-apoptotic ceramide and sphingosine. Activation of bioactive sphingolipid S1P signaling has emerged as a critical protective pathway in response to acute ischemic injury in both cardiac and cerebrovascular disease, and these observations have considerable relevance for future potential therapeutic targets.

Endotoxin-activated microglia injure brain derived endothelial cells via NF-κB, JAK-STAT and JNK stress kinase pathways.

BACKGROUND: We previously showed that microglia damage blood brain barrier (BBB) components following ischemic brain insults, but the underlying mechanism(s) is/are not well known. Recent work has established the contribution of toll-like receptor 4 (TLR4) activation to several brain pathologies including ischemia, neurodegeneration and sepsis. The present study established the requirement of microglia for lipopolysaccharide (LPS) mediated endothelial cell death, and explored pathways involved in this toxicity. LPS is a classic TLR4 agonist, and is used here to model aspects of brain conditions where TLR4 stimulation occurs. METHODS/RESULTS: In monocultures, LPS induced death in microglia, but not brain derived endothelial cells (EC). However, LPS increased EC death when cocultured with microglia. LPS led to nitric oxide (NO) and inducible NO synthase (iNOS) induction in microglia, but not in EC. Inhibiting microglial activation by blocking iNOS and other generators of NO or blocking reactive oxygen species (ROS) also prevented injury in these cocultures. To assess the signaling pathway(s) involved, inhibitors of several downstream TLR-4 activated pathways were studied. Inhibitors of NF-κB, JAK-STAT and JNK/SAPK decreased microglial activation and prevented cell death, although the effect of blocking JNK/SAPK was rather modest. Inhibitors of PI3K, ERK, and p38 MAPK had no effect. CONCLUSIONS: We show that LPS-activated microglia promote BBB disruption through injury to endothelial cells, and the specific blockade of JAK-STAT, NF-κB may prove to be especially useful anti-inflammatory strategies to confer cerebrovascular protection.

Direct protection of cultured neurons from ischemia-like injury by minocycline.

Minocycline, a tetracycline antibiotic, is now known to protect cells via an anti-inflammatory mechanism. We further explored this effect using an in vitro model of ischemia-like injury to neurons. Coculturing neurons with microglia, the brain's resident immune cell, modestly increased cell death due to oxygen and glucose deprivation (OGD), compared to neurons alone. Treatment of cocultures with minocycline decreased cell death to a level significantly lower than that of neurons alone. Treatment of cocultures with minocycline or inhibitors of various immune mediators, also led to decreased cell death. Importantly, treatment of neuron cultures without added microglia with these same inhibitors of tissue plasminogen activator, matrix metalloproteinases, TNF-alpha and inducible nitric oxide synthase as well as minocycline also led to decreased cell death. Thus, anti-inflammatory treatments appear to be directly protective of neurons from in vitro ischemia.

Adult cardiac fibroblasts null for sphingosine kinase-1 exhibit growth dysregulation and an enhanced proinflammatory response.

Cardiac fibroblasts are critical for the maintenance of extracellular matrix deposition and turnover in the normal heart and are key mediators of inflammatory and fibrotic myocardial remodeling in the injured and failing heart. Sphingosine kinase (SphK) activation is a well-recognized determinant of cell fate in cardiac myocytes and other cells, but SphK responses have not previously been studied in cardiac fibroblasts. Initially we found that total SphK activity is over 10-fold higher in cardiac fibroblasts than in adult mouse cardiac myocytes. SphK is composed of two major isoforms, SphK-1 and SphK-2. In fibroblasts isolated from SphK-1 knockout mice, SphK activity was greatly reduced indicating that SphK-1 is the major isoform expressed in these cells. To determine whether SphK regulates cell proliferation and the proinflammatory protein inducible nitric oxide synthase (iNOS), we exposed cultured cardiac fibroblasts to the cytokine interleukin-1beta (IL-1beta) and/or hypoxia. Both hypoxia and IL-1beta alone and in combination enhanced fibroblast SphK activity. In wild-type fibroblasts, hypoxia induced proliferation, but in SphK-1 null fibroblasts this response was blunted even in the presence of serum. In contrast, we found that iNOS expression and NO production were enhanced in SphK-1 null fibroblasts during hypoxia. In wild-type fibroblasts, IL-1beta was only a weak inducer of iNOS and of NO accumulation and hypoxia alone had no significant effect on iNOS activation. However, IL-1beta in combination with hypoxia extensively stimulated iNOS and NO production, and this stimulation was enhanced in SphK-1 null fibroblasts. We conclude that activation of endogenous SphK-1 serves a dual regulatory function: it is required for optimal cardiac fibroblast proliferation but is a negative modulator of proinflammatory responses during hypoxia.

Protein kinase C isozymes in hypertension and hypertrophy: insight from SHHF rat hearts.

Chronic hypertension results in cardiac hypertrophy and may lead to congestive heart failure. The protein kinase C (PKC) family has been identified as a signaling component promoting cardiac hypertrophy. We hypothesized that PKC activation may play a role mediating hypertrophy in the spontaneously hypertensive heart failure (SHHF) rat heart. Six-month-old SHHF and normotensive control Wistar Furth (WF) rats were used. Hypertension and cardiac hypertrophy were confirmed in SHHF rats. PKC expression and activation were analyzed by Western blots using isozyme-specific antibodies. Compared to WF, untreated SHHF rats had increased phospho-active alpha (10-fold), delta (4-fold), and epsilon (3-fold) isozyme expression. Furthermore, we analyzed the effect of an angiotensin II type 1 receptor blocker (ARB) and hydralazine (Hy) on PKC regulation in SHHF rat left ventricle (LV). Both the ARB and Hy normalized LV blood pressure, but only the ARB reduced heart mass. Neither treatment affected PKC expression or activity. Our data show differential activation of PKC in the hypertensive, hypertrophic SHHF rat heart. Regression of hypertrophy elicited by an ARB in this model occurred independently of changes in the expression and activity of the PKC isoforms examined.

Alterations in G protein and MAP kinase signaling pathways during cardiac remodeling in hypertension and heart failure.

The present study was undertaken to elucidate the G-protein and mitogen-activated kinase (MAP kinase) coupled signaling profile in a genetic model of hypertension and congestive heart failure (CHF) that mimics similar disease in humans. At the receptor level, Ang II type 1 receptor (AT1R) increased in left ventricular hypertrophy (LVH) and reverted to normal in CHF, whereas there was a downregulation of the Ang II type 2 receptor (AT2R) in CHF. At the transducer level, Galphaq and Galpha12 protein levels were unchanged during LVH but decreased significantly in CHF. In contrast, Gbeta and Galpha13 protein content were markedly upregulated in CHF. Furthermore, using phospho-specific antibodies in Western blots and in vitro kinase assays, we found at the effector level an upregulation of the small G-protein Rac1 activity during LVH but a decrease during CHF. In parallel, small G-protein Rho activity was significantly increased during LVH but was unchanged in failure. We found at the downstream level that MAP kinase isoforms extracellular signal regulated-kinase (ERK1/2), big mitogen-activated kinase (BMK1/ERK5), C-jun N-terminal-activated kinase (JNKs/SAPKs), and stress-activated kinase (p38) bioactivities were increased during LVH. During CHF, ERK1/2 and JNK1/2 kinase activities were decreased, whereas BMK1/ERK5 kinase activity reverted to normal values. In conclusion, this study demonstrates, for the first time, multistep alterations of G-protein and MAP kinase signaling pathways in LVH and progression to failure in a genetic model of hypertension and failure.