Translating cell survival and cell longevity into treatment strategies with SIRT1.

The sirtuin SIRT1, a class III NAD(+)-dependent protein histone deacetylase, is present throughout the body that involves cells of the central nervous system, immune system, cardiovascular system, and the musculoskeletal system. SIRT1 has broad biological effects that affect cellular metabolism as well as cellular survival and longevity that can impact both acute and chronic disease processes that involve neurodegenerative disease, diabetes mellitus, cardiovascular disease, and cancer. Given the intricate relationship SIRT1 holds with a host of signal transduction pathways ranging from transcription factors, such as forkhead, to cytokines and growth factors, such as erythropoietin, it becomes critical to elucidate the cellular pathways of SIRT1 to safely and effectively develop and translate novel avenues of treatment for multiple disease entities.

The Src homology 2 domain tyrosine phosphatases SHP-1 and SHP-2: diversified control of cell growth, inflammation, and injury.

Interest in the diverse biology of protein tyrosine phosphatases that are encoded by more than 100 genes in the human genome continues to grow at an accelerated pace. In particular, two cytoplasmic protein tyrosine phosphatases composed of two Src homology 2 (SH2) NH2-terminal domains and a C-terminal protein-tyrosine phosphatase domain referred to as SHP-1 and SHP-2 are known to govern a host of cellular functions. SHP-1 and SHP-2 modulate progenitor cell development, cellular growth, tissue inflammation, and cellular chemotaxis, but more recently the role of SHP-1 and SHP-2 to directly control cell survival involving oxidative stress pathways has come to light. SHP-1 and SHP-2 are fundamental for the function of several growth factor and metabolic pathways yielding far reaching implications for disease pathways and disorders such as diabetes, neurodegeneration, and cancer. Although SHP-1 and SHP-2 can employ similar or parallel cellular pathways, these proteins also clearly exert opposing effects upon downstream cellular cascades that affect early and late apoptotic programs. SHP-1 and SHP-2 modulate cellular signals that involve phosphatidylinositol 3-kinase, Akt, Janus kinase 2, signal transducer and activator of transcription proteins, mitogen-activating protein kinases, extracellular signal-related kinases, c-Jun-amino terminal kinases, and nuclear factor-kappaB. Our progressive understanding of the impact of SHP-1 and SHP-2 upon multiple cellular environments and organ systems should continue to facilitate the targeted development of treatments for a variety of disease entities.

Erythropoietin involves the phosphatidylinositol 3-kinase pathway, 14-3-3 protein and FOXO3a nuclear trafficking to preserve endothelial cell integrity.

BACKGROUND AND PURPOSE: Clinical indications for erythropoietin (EPO) in the vascular system reach far beyond the treatment of anemia, but the development of EPO as a non-toxic agent rests heavily upon the cellular pathways controlled by EPO that require elucidation. EXPERIMENTAL APPROACH: We modulated gene activity and examined cellular trafficking of critical pathways during oxidative stress that may work in concert with EPO to protect primary cerebral endothelial cells (ECs) during oxidative stress, namely protein kinase B (Akt1), 14-3-3 protein, the Forkhead transcription factor FOXO3a. KEY RESULTS: Here, we show that preservation of ECs by EPO during oxygen-glucose deprivation (OGD) required the initial activation of the phosphatidylinositol 3-kinase (PI-3K) pathway through Akt1, since specific pharmacological blockade of Akt1 activity or gene silencing of Akt1 prevented EC protection by EPO. EPO subsequently involved a series of anti-apoptotic pathways to activate STAT3, STAT5, and ERK 1/2. Furthermore, EPO maintained the inhibitory phosphorylation and integrity of the 'pro-apoptotic' transcription factor FOXO3a, promoted the binding of FOXO3a to 14-3-3 protein and regulated the intracellular trafficking of FOXO3a. Additionally, gene silencing of FOXO3a during OGD significantly increased EC survival, but did not synergistically improve cytoprotection by EPO, illustrating that EPO relied upon the blockade of the FOXO3a pathway. CONCLUSIONS AND IMPLICATIONS: Our work defines a novel cytoprotective pathway in ECs that involves PI-3 K, STAT3, STAT5, ERK 1/2, 14-3-3 protein and FOXO3a, which can be targeted for the development of EPO as a clinically effective and safe agent in the vascular system.

Winding through the WNT pathway during cellular development and demise.

In slightly over a period of twenty years, our comprehension of the cellular and molecular mechanisms that govern the Wnt signaling pathway continue to unfold. The Wnt proteins were initially implicated in viral carcinogenesis experiments associated with mammary tumors, but since this period investigations focusing on the Wnt pathways and their transmembrane receptors termed Frizzled have been advanced to demonstrate the critical nature of Wnt for the development of a variety of cell populations as well as the potential of the Wnt pathway to avert apoptotic injury. In particular, Wnt signaling plays a significant role in both the cardiovascular and nervous systems during embryonic cell patterning, proliferation, differentiation, and orientation. Furthermore, modulation of Wnt signaling under specific cellular influences can either promote or prevent the early and late stages of apoptotic cellular injury in neurons, endothelial cells, vascular smooth muscle cells, and cardiomyocytes. A number of downstream signal transduction pathways can mediate the biological response of the Wnt proteins that include Dishevelled, beta-catenin, intracellular calcium, protein kinase C, Akt, and glycogen synthase kinase-3beta. Interestingly, these cellular cascades of the Wnt-Frizzled pathways can participate in several neurodegenerative, vascular, and cardiac disorders and may be closely integrated with the function of trophic factors. Identification of the critical elements that modulate the Wnt-Frizzled signaling pathway should continue to unlock the potential of Wnt pathway for the development of new therapeutic options against neurodegenerative and vascular diseases.

Activating Akt and the brain's resources to drive cellular survival and prevent inflammatory injury.

Protein kinase B, also known as Akt, is a serine/threonine kinase and plays a critical role in the modulation of cell development, growth, and survival. Interestingly, Akt is ubiquitously expressed throughout the body, but its expression in the nervous system is substantially up-regulated during cellular stress, suggesting a more expansive role for Akt in the nervous system that may involve cellular protection. In this regard, a body of recent work has identified a robust capacity for Akt and its downstream substrates to foster both neuronal and vascular survival during apoptotic injury. Cell survival by Akt is driven by the modulation of both intrinsic cellular pathways that oversee genomic DNA integrity and extrinsic mechanisms that control inflammatory microglial activation. A series of distinct pathways are regulated by Akt that include the Forkhead family of transcription factors, GSK-3 beta, beta-catenin, c-Jun, CREB, Bad, IKK, and p53. Culminating below these substrates of Akt are the control of caspase mediated pathways that promote genomic integrity as well as prevent inflammatory cell demise. With further levels of progress in defining the cellular role of Akt, the attractiveness of Akt as a vital and broad cytoprotectant for both neuronal and vascular cell populations should continue to escalate.

Targeting WNT, protein kinase B, and mitochondrial membrane integrity to foster cellular survival in the nervous system.

Targeting essential cellular pathways that determine neuronal and vascular survival can foster a successful therapeutic platform for the treatment of a wide variety of degenerative disorders in the central nervous system. In particular, oxidative cellular injury can precipitate several nervous system disorders that may either be acute in nature, such as during cerebral ischemia, or more progressive and chronic, such as during Alzheimer disease. Apoptotic injury in the brain proceeds through two distinct pathways that ultimately result in the early externalization of membrane phosphatidylserine (PS) residues and the late induction of genomic DNA fragmentation. Degradation of DNA may acutely impact cellular survival, while the exposure of membrane PS residues can lead to microglial phagocytosis of viable cells, cellular inflammation, and thrombosis in the vascular system. Through either independent or common pathways, the Wingless/Wnt pathway and the serine-threonine kinase Akt serve central roles in the maintenance of cellular integrity and the prevention of the phagocytic disposal of cells "tagged" by PS exposure. By selectively governing the activity of specific downstream substrates that include GSK-3beta, Bad, and beta-catenin, Wnt and Akt serve to foster neuronal and vascular survival and block the induction of programmed cell death. Novel to Akt is its capacity to protect cells from phagocytosis through the direct modulation of membrane PS exposure. Intimately linked to the activation of Wnt signaling and Akt is the maintenance of mitochondrial membrane potential and the regulation of Bcl-xL, mitochondrial energy metabolism, and cytochrome c release that can lead to specific cysteine protease activation.

Metabotropic glutamate receptors promote neuronal and vascular plasticity through novel intracellular pathways.

During the initial development and maturation of an individual, the metabotropic glutamate receptor (mGluR) system becomes a necessary component for the critical integration of cellular function and plasticity. In addition to the maintenance of cellular physiology, the mGluR system plays a critical role during acute and chronic degenerative disorders of the central nervous system. By coupling to guanosine-nucleotide-binding proteins (G-proteins), the mGluR system employs a broad range of signal transduction systems to regulate cell survival and injury. More commonly, it is the activation of specific mGluR subtypes that can prevent programmed cell death (PCD) consisting of two distinct pathways of genomic DNA degradation and membrane phosphatidylserine (PS) residue exposure. To offer this cellular protection, mGluRs modulate a series of down-stream cellular pathways that include protein kinases, mitochondrial membrane potential, cysteine proteases, intracellular pH, endonucleases, and mitogen activated protein kinases. Prevention of cellular injury by the mGluR system is directly applicable to clinical disability, since immediate and delayed injury paradigms demonstrate the ability of this system to reverse PCD in both neuronal and vascular cell populations. Further understanding of the intricate pathways that determine the protective nature of the mGluR system will provide new therapeutic avenues for the treatment of neurodegenerative disorders.

Effects and mechanisms of triacetylshikimic acid on platelet adhesion to neutrophils induced by thrombin and reperfusion after focal cerebral ischemia in rats.

AIM: To investigate the effect of triacetylshikimic acid (TSA) on the platelet adhesion to neutrophils and P-selectin expression on activated platelet membrane induced by thrombin and reperfusion after focal cerebral ischemia. METHODS: The platelet adhesion to neutrophils was evaluated by rosette assay, and P-selectin expression on platelet membrane was determined by flow cytometry. RESULTS: TSA 10 - 1000 micromol/L markedly inhibited thrombin(0.4 kU/L)-induced platelet adhesion to neutrophils. The platelet adhesion to neutrophils induced by a 21-h reperfusion after middle cerebral artery occlusion for 3 h was also inhibited in a dose-dependent manner by TSA 50 - 200 mg/kg given by ig immediately and at 60 min again after the onset of cerebral ischemia. TSA was also shown to decrease the P-selectin expression on platelet surface induced by thrombin in washed platelet and by adenosine diphosphate (ADP) 5 micromol/L in whole blood. CONCLUSION: Reperfusion after cerebral ischemia and thrombin induced platelet adhesion to neutrophils, which could be reduced by TSA probably due to its inhibition of P-selectin expression on activated platelets.

Cell cycle induction in post-mitotic neurons proceeds in concert with the initial phase of programmed cell death in rat.

Neuronal programmed cell death (PCD) is increasingly becoming recognized as a dynamic process that may be amenable to resolution. Critical to this resolution is the identification of the cellular pathways that modulate the initial stages of apoptotic death. In this regard, we examined whether the activation of a latent cell cycle was associated with the initial phase of PCD. We demonstrate that free radical nitric oxide induced PCD results in the rapid generation of membrane phosphatidylserine residue exposure. This early phase of PCD functions in parallel with an untoward attempt to enter the cell cycle in the same population of post-mitotic neurons. We therefore offer an attractive molecular target to prevent or reverse neuronal PCD by elucidating a novel mechanism through which the majority of neurons meet their demise by attempting to enter a latent cell cycle.

dl-3-n-butylphthalide attenuates reperfusion-induced blood-brain barrier damage after focal cerebral ischemia in rats.

AIM: To study the protective effect of dl-3-n-butylphthalide (NBP) on blood-brain barrier (BBB) damage induced by reperfusion following focal cerebral ischemia. METHODS: Focal cerebral ischemia in rats was performed by inserting a nylon suture into intracranial segment of internal carotid artery to block the origin of middle cerebral artery and reperfusion by withdrawing the nylon suture. Permeability of BBB was determined by extravasation of the protein-bound Evans blue dye to cerebral cortex and further evaluated by immunohistochemical or electronmicroscopic method. RESULTS: Reperfusion for 3 h following focal cerebral ischemia for 3 h produced BBB damage which exhibited the increase in extravasation in cerebral cortex, elevation of the expression of immunoglobulin (IgG), and pore formation in endothelial cell membrane of capillary in cerebral cortex. NBP (5-20 mg.kg-1) decreased the extravasation in a dose-dependent manner. The expression of IgG in cerebral cortex was decreased and the ultrastructure damage of capillaries was alleviated after treatment with NBP. NBP 20 mg.kg-1 also alleviated brain edema caused by 3-h reperfusion following 3-h middle cerebral artery occlusion (MCAO). CONCLUSION: NBP has protective effect on BBB damage induced by reperfusion after MCAO.

dl-3-n-butylphthalide improves regional cerebral blood flow after experimental subarachnoid hemorrhage in rats.

AIM: To investigate the effect of dl-3-n-butylphthalide (dl-NBP) on experimental subarachnoid hemorrhage (SAH) in rats. METHODS: SAH was induced by injection of autologous artery blood 0.35 mL into lateral ventricle. Regional cerebral blood flow (rCBF) in caudate nucleus was determined by hydrogen clearance method. RESULTS: A rapid and marked decrease in rCBF in caudate nucleus was observed 15 min after SAH and the rCBF remained at low level (about 50% pre-SAH value) within 180 min. dl-NBP (50, 100 mg.kg-1, i.g.) increased rCBF 30-180 min after the onset of SAH without significant effect on mean artery blood pressure. dl-NBP 100 mg.kg-1 increased rCBF in caudate nucleus by 26% at 15 min and by 36% at 180 min respectively after SAH. d-NBP but not l-NBP (10 mg.kg-1, i.p.) increased rCBF. CONCLUSION: dl-NBP improves rCBF in caudate nucleus of rats subjected to SAH.

Effects of dl-3-n-butylphthalide on production of TXB2 and 6-keto-PGF1 alpha in rat brain during focal cerebral ischemia and reperfusion.

AIM: To study the effects of dl-3-n-butylphthalide (NBP) on the changes of thromboxane B2 (TXB2) and 6-keto-PGF1 alpha (6-keto-PGF1 alpha) contents in hippocampus, striatum, and cerebral cortex of rats subjected to focal cerebral ischemia followed by reperfusion. METHODS: Focal cerebral ischemia was induced by inserting a nylon suture into intracranial segment of internal carotid artery from external carotid artery and blockade of the origin of middle cerebral artery. For reperfusion, the suture was pulled out to restore the blood flow to the ischemic brain. Determination of TXB2 and 6-keto-PGF1 alpha was performed by RIA method. RESULTS: Reperfusion following focal cerebral ischemia resulted in increases in TXB2 at 5 min and 6-keto-PGF1 alpha at 30 min and a decrease in the ratio of epoprostenol (PGI2)/thromboxane A2 (TXA2) (6-keto-PGF1 alpha/TXB2) at 5 min in hippocampus, striatum, and cerebral cortex. NBP 10 mg.kg-1 reduced the content of TXB2 without decreasing effect on 6-keto-PGF1 alpha. NBP 20 mg.kg-1 reduced both TXB2 and 6-keto-PGF1 alpha in lesser extent than aspirin (Asp, 20 mg.kg-1). NBP 20 or 10 mg.kg-1 elevated the ratio of PGI2/TXA2 after reperfusion, but Asp 20 mg.kg-1 did not increase the ratio except in striatum at 5 min after reperfusion. CONCLUSION: NBP increases the ratio of PGI2/TXA2 which may have beneficial effects on the impaired microcirculation in postischemic brain tissues.