Debjani Tripathy

RANTES upregulation in the Alzheimer's disease brain: A possible neuroprotective role

Numerous studies demonstrate inflammatory proteins in the brain and microcirculation in Alzheimers disease (AD) and implicate inflammation in disease pathogenesis. However, emerging literature suggests that neuroinflammation can also be neuroprotective. The chemokine RANTES has been implicated in neurodegenerative diseases including AD. The objectives of this study are to determine the expression of RANTES in AD microvessels, its regulation in endothelial cells and its effects on neuronal survival. Our data show elevated expression of RANTES in the cerebral microcirculation of AD patients. Treatment of neurons in vitro with RANTES results in an increase in cell survival and a neuroprotective effect against the toxicity of thrombin and sodium nitroprusside. Oxidative stress upregulates RANTES expression in rat brain endothelial cells. Developing strategies to augment neuroprotection and diminish inflammatory activation of multifunctional mediators such as RANTES holds promise for the development of novel neuroprotective therapeutics in AD.

Acetaminophen inhibits neuronal inflammation and protects neurons from oxidative stress

BackgroundRecent studies have demonstrated a link between the inflammatory response, increased cytokine formation, and neurodegeneration in the brain. The beneficial effects of anti-inflammatory drugs in neurodegenerative diseases, such as Alzheimers disease (AD), have been documented. Increasing evidence suggests that acetaminophen has unappreciated anti-oxidant and anti-inflammatory properties. The objectives of this study are to determine the effects of acetaminophen on cultured brain neuronal survival and inflammatory factor expression when exposed to oxidative stress.MethodsCerebral cortical cultured neurons are pretreated with acetaminophen and then exposed to the superoxide-generating compound menadione (5 μM). Cell survival is assessed by MTT assay and inflammatory protein (tumor necrosis factor alpha, interleukin-1, macrophage inflammatory protein alpha, and RANTES) release quantitated by ELISA. Expression of pro- and anti-apoptotic proteins is assessed by western blots.ResultsAcetaminophen has pro-survival effects on neurons in culture. Menadione, a superoxide releasing oxidant stressor, causes a significant (p < 0.001) increase in neuronal cell death as well as in the release of tumor necrosis factor alpha, interleukin-1, macrophage inflammatory protein alpha, and RANTES from cultured neurons. Pretreatment of neuronal cultures with acetaminophen (50 μM) increases neuronal cell survival and inhibits the expression of these cytokines and chemokines. In addition, we document, for the first time, that acetaminophen increases expression of the anti-apoptotic protein Bcl2 in brain neurons and decreases the menadione-induced elevation of the proapoptotic protein, cleaved caspase 3. We show that blocking acetaminophen-induced expression of Bcl2 reduces the pro-survival effect of the drug.ConclusionThese data show that acetaminophen has anti-oxidant and anti-inflammatory effects on neurons and suggest a heretofore unappreciated therapeutic potential for this drug in neurodegenerative diseases such as AD that are characterized by oxidant and inflammatory stress.

Thrombin, a mediator of cerebrovascular inflammation in AD and hypoxia

Considerable evidence implicates hypoxia and vascular inflammation in Alzheimers disease (AD). Thrombin, a multifunctional inflammatory mediator, is demonstrable in the brains of AD patients both in the vessel walls and senile plaques. Hypoxia-inducible factor 1α (HIF-1α), a key regulator of the cellular response to hypoxia, is also upregulated in the vasculature of human AD brains. The objective of this study is to investigate inflammatory protein expression in the cerebrovasculature of transgenic AD mice and to explore the role of thrombin as a mediator of cerebrovascular inflammation and oxidative stress in AD and in hypoxia-induced changes in brain endothelial cells. Immunofluorescent analysis of the cerebrovasculature in AD mice demonstrates significant (p < 0.01–0.001) increases in thrombin, HIF-1α, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases (MMPs), and reactive oxygen species (ROS) compared to controls. Administration of the thrombin inhibitor dabigatran (100 mg/kg) to AD mice for 34 weeks significantly decreases expression of inflammatory proteins and ROS. Exposure of cultured brain endothelial cells to hypoxia for 6 h causes an upregulation of thrombin, HIF-1α, MCP-1, IL-6, and MMP2 and ROS. Treatment of endothelial cells with the dabigatran (1 nM) reduces ROS generation and inflammatory protein expression (p < 0.01–0.001). The data demonstrate that inhibition of thrombin in culture blocks the increase in inflammatory protein expression and ROS generation evoked by hypoxia. Also, administration of dabigatran to transgenic AD mice diminishes ROS levels in brain and reduces cerebrovascular expression of inflammatory proteins. Taken together, these results suggest that inhibiting thrombin generation could have therapeutic value in AD and other disorders where hypoxia, inflammation, and oxidative stress are involved.

Pigment epithelium-derived factor (PEDF) protects cortical neurons in vitro from oxidant injury by activation of extracellular signal-regulated kinase (ERK) 1/2 and induction of Bcl-2

Mitigating oxidative stress-induced damage is critical to preserve neuronal function in diseased or injured brains. This study explores the mechanisms contributing to the neuroprotective effects of pigment epithelium-derived factor (PEDF) in cortical neurons. Cultured primary neurons are exposed to PEDF and H₂O₂ as well as inhibitors of phosphoinositide-3 kinase (PI3K) or extracellular signal-regulated kinase 1/2 (ERK1/2). Neuronal survival, cell death and levels of caspase 3, PEDF, phosphorylated ERK1/2, and Bcl-2 are measured. The data show cortical cultures release PEDF and that H₂O₂ treatment causes cell death, increases activated caspase 3 levels and decreases release of PEDF. Exogenous PEDF induces a dose-dependent increase in Bcl-2 expression and neuronal survival. Blocking Bcl-2 expression by siRNA reduced PEDF-induced increases in neuronal survival. Treating cortical cultures with PEDF 24 h before H₂O₂ exposure mitigates oxidant-induced decreases in neuronal survival, Bcl-2 expression, and phosphorylation of ERK1/2 and also reduces elevated caspase 3 level and activity. PEDF pretreatment effect on survival is blocked by inhibiting ERK or PI3K. However, only inhibition of ERK reduced the ability of PEDF to protect neurons from H₂O₂-induced Bcl-2 decrease and neuronal death. These data demonstrate PEDF-mediated neuroprotection against oxidant injury is largely mediated via ERK1/2 and Bcl-2 and suggest the utility of PEDF in preserving the viability of oxidatively challenged neurons.

Acetaminophen protects brain endothelial cells against oxidative stress

Increasing evidence suggests that acetaminophen has unappreciated anti-oxidant and anti-inflammatory properties. Drugs that affect oxidant and inflammatory stress in the brain are of interest because both processes are thought to contribute to the pathogenesis of neurodegenerative disease. The objective of this study is to determine whether acetaminophen affects the response of brain endothelial cells to oxidative stress. Cultured brain endothelial cells are pre-treated with acetaminophen and then exposed to the superoxide-generating compound menadione (25 microM). Cell survival, inflammatory protein expression, and anti-oxidant enzyme activity are measured. Menadione causes a significant (p<0.001) increase in endothelial cell death as well as an increase in RNA and protein levels of tumor necrosis factor alpha, interleukin-1, macrophage inflammatory protein alpha, and RANTES. Menadione also evokes a significant (p<0.001) increase in the activity of the anti-oxidant enzyme superoxide dismutase (SOD). Pre-treatment of endothelial cell cultures with acetaminophen (25-100 microM) increases endothelial cell survival and inhibits menadione-induced expression of inflammatory proteins and SOD activity. In addition, we document, for the first time, that acetaminophen increases expression of the anti-apoptotic protein Bcl2. Suppressing Bcl2 with siRNA blocks the pro-survival effect of acetaminophen. These data show that acetaminophen has anti-oxidant and anti-inflammatory effects on the cerebrovasculature and suggest a heretofore unappreciated therapeutic potential for this drug in neurodegenerative diseases such as Alzheimers disease that are characterized by oxidant and inflammatory stress.

Hypoxia induces angiogenic factors in brain microvascular endothelial cells.

Hypoxia is increasingly recognized as an important contributing factor to the development of brain diseases such as Alzheimers disease (AD). In the periphery, hypoxia is a powerful regulator of angiogenesis. However, vascular endothelial cells are remarkably heterogeneous and little is known about how brain endothelial cells respond to hypoxic challenge. The objective of this study is to characterize the effect of hypoxic challenge on the angiogenic response of cultured brain-derived microvascular endothelial cells. Brain endothelial cell cultures were initiated from isolated rat brain microvessels and subjected to hypoxia (1% O(2)) for various time periods. The results showed that hypoxia induced rapid (≤ 0.5h) expression of hypoxia-inducible factor 1α (HIF-1α) and that cell viability, assessed by MTT assay, was unaffected within the first 8h. Examination of brain endothelial cell cultures for pro- and anti-angiogenic proteins by western blot, RT-PCR and ELISA revealed that within 0.5 to 2h of hypoxia levels of vascular endothelial growth factor and endothelin-1 mRNA and protein were elevated. The expression of heme oxygenase-1 also increased but only after 8h of hypoxia. In contrast, similar hypoxia exposure evoked a decrease in endothelial nitric oxide synthase and thrombospondin-2 levels. Exposure of brain endothelial cell cultures to hypoxia resulted in a significant (p<0.001) decrease (94%) in tube length, an in vitro index of angiogenesis, compared to control cultures. The data indicate that, despite a shift toward a pro-angiogenic phenotype, hypoxia inhibited vessel formation in brain endothelial cells. These results suggest that in brain endothelial cells expression of angiogenic factors is not sufficient for the development of new vessels. Further work is needed to determine what factors/conditions prevent hypoxia-induced angiogenic changes from culminating in the formation of new brain blood vessels and what role this may play in the pathologic changes observed in AD and other diseases characterized by cerebral hypoxia.

Expression of Macrophage Inflammatory Protein 1-α is Elevated in Alzheimer's Vessels and is Regulated by Oxidative Stress

Inflammatory mediators are highly expressed in the Alzheimers disease (AD) brain. We have shown that in AD the cerebral microcirculation is a rich source of cytokines and chemokines including interleukins (IL) 1beta, IL-6, IL-8, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1. However, the factors that regulate expression of these inflammatory proteins have not been defined. The objective of this study is to compare expression of macrophage inflammatory protein 1-alpha (MIP-1alpha) in brain microvessels isolated from AD patients to vessels from age-matched controls and further to determine whether expression of MIP-1alpha in brain endothelial cells is altered by oxidative stress. The data show that brain AD-derived microvessels express high levels of MIP-1alpha mRNA and release high levels of MIP-1alpha protein compared to brain microvessels isolated from controls. Treatment of brain endothelial cell cultures with menadione, a superoxide releasing compound, hydrogen peroxide, lipopolysacharride, or oxidatively modified low density lipoproteins (LDL) (Ox-LDL, HNE-LDL) results in a dose- dependent increase in MIP-1alpha mRNA levels and MIP-1alpha release into the media. These results suggest that oxidative and lipid insults to the brain microvasculature are likely to contribute to the inflammatory milieu of the AD brain.

Neurovascular Unit and the Effects of Dosage in VEGF Toxicity: Role for Oxidative Stress and Thrombin

Bidirectional communication between neurons and vascular cells is important to the maintenance of the central nervous system (CNS) milieu. Vascular endothelial growth factor (VEGF), through its ability to affect both vascular and neuronal cells, is likely a key protein in this process. Despite considerable literature documenting a neuroprotective function for VEGF, overexpression of this protein has also been shown in a wide variety of CNS diseases, including Alzheimers disease (AD). Increased oxidative stress and elevated thrombin levels have also been documented in AD, specifically in the microvasculature. The aim of the current study is to examine endothelial cells and neurons in vitro to determine the effects of oxidative stress and thrombin on VEGF release as well as the effects of low and high dose VEGF on neuronal viability. The data show that microvessels isolated from AD patients secrete significantly higher levels of VEGF compared to control-derived vessels. Exposure of brain endothelial cells to oxidative stress (sodium nitroprusside, menadione, or hydrogen peroxide) or thrombin significantly increases VEGF expression. Exposure of cultured neurons to oxidative stress increases expression of thrombin. Treating rat cortical neurons with high dose VEGF (≥500 ng/ml) decreases neuronal survival and expression of the anti-apoptotic protein Bcl-2 while increasing proapoptic proteins caspase 3 and phosphorylated p38 MAPK. High dose VEGF also negates the decrease in amyloid-β evoked by low dose VEGF. These results suggest that despite literature supporting neuroprotective effects of this protein, caution is warranted prior to implementation of VEGF as a therapeutic in the brain.

p38 MAPK: A Mediator of Hypoxia-Induced Cerebrovascular Inflammation

Vascular perturbations and hypoxia are increasingly implicated in Alzheimers disease (AD) pathogenesis. Cerebral hypoxia induces a large number of inflammatory proteins in brain endothelial cells via signaling pathways that have not been defined. The p38 mitogen-activated protein kinase (MAPK) signaling system has been implicated in endothelial injury and inflammation. The objective of this study is to examine p38 MAPK levels in the cerebromicrovasulature in AD and AD animal models and determine the role of p38 MAPK signaling in hypoxia-mediated effects on brain endothelial cells. Western blot analysis of isolated human brain microvessels show that the phosphorylated (active) form of p38 MAPK (pp38 MAPK) is increased in vessels derived from AD brains compared to control-derived vessels. Similarly, immunofluorescent analysis reveals an increase in cerebrovascular pp38 MAPK as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in transgenic AD mice. Exposure of brain endothelial cells to hypoxia (2-6 hours) shows a time-dependent increase in pp38 MAPK. Examination of these cultures at 6 hours hypoxia shows that iNOS and COX-2 are significantly elevated and that the selective p38 MAPK inhibitor SB203580 significantly reduces the hypoxia-mediated increase in their expression. Inhibition of p38 MAPK in cultured brain endothelial cells also significantly decreases the hypoxia-induced increase in the inflammatory proteins, matrix metalloproteinase-2 and angiopoietin-2. These data demonstrate that pp38 MAPK is a key regulator of hypoxia in the cerebrovasculature and suggest that control of this signaling pathway could have therapeutic value in AD and other disorders where hypoxia is involved.

Cerebrovascular expression of proteins related to inflammation, oxidative stress and neurotoxicity is altered with aging

BackgroundMost neurodegenerative diseases are age-related disorders; however, how aging predisposes the brain to disease has not been adequately addressed. The objective of this study is to determine whether expression of proteins in the cerebromicrovasculature related to inflammation, oxidative stress and neurotoxicity is altered with aging.MethodsBrain microvessels are isolated from Fischer 344 rats at 6, 12, 18 and 24 months of age. Levels of interleukin (IL)-1β and IL-6 RNA are determined by RT-PCR and release of cytokines into the media by ELISA. Vessel conditioned media are also screened by ELISA for IL-1α, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α, (TNFα), and interferon γ (IFNγ). Immunofluorescent analysis of brain sections for IL-1β and IL-6 is performed.ResultsExpression of IL-1β and IL-6, both at RNA and protein levels, significantly (p < 0.01) decreases with age. Levels of MCP-1, TNFα, IL-1α, and IFNγ are significantly (p < 0.05-0.01) lower in 24 month old rats compared to 6 month old animals. Immunofluorescent analysis of brain vessels also shows a decline in IL-1β and IL-6 in aged rats. An increase in oxidative stress, assessed by increased carbonyl formation, as well as a decrease in the antioxidant protein manganese superoxide dismutase (MnSOD) is evident in vessels of aged animals. Finally, addition of microvessel conditioned media from aged rats to neuronal cultures evokes significant (p < 0.001) neurotoxicity.ConclusionsThese data demonstrate that cerebrovascular expression of proteins related to inflammation, oxidative stress and neurotoxicity is altered with aging and suggest that the microvasculature may contribute to functional changes in the aging brain.