Vijay K. Kalra

Amyloid Peptide-Induced Cytokine and Chemokine Expression in THP-1 Monocytes Is Blocked by Small Inhibitory RNA Duplexes for Early Growth Response-1 Messenger RNA

In Alzheimer’s disease (AD) one finds increased deposition of Aβ and also an increased presence of monocytes/macrophages in the vessel wall and activated microglial cells in the brain. AD patients show increased levels of proinflammatory cytokines by activated microglia. Here we used a human monocytic THP-1 cell line as a model for microglia to delineate the cellular signaling mechanism involved in amyloid peptides (Aβ1–40 and Aβ1–42)-induced expression of inflammatory cytokines and chemokines. We observed that Aβ peptides at physiological concentrations (125 nM) increased mRNA expression of cytokines (TNF-α, and IL-1β) and chemokines (monocyte chemoattractant protein-1 (MCP-1), IL-8, and macrophage inflammatory protein-1β (MIP-1β)). The cellular signaling involved activation of c-Raf, extracellular signal-regulated kinase-1 (ERK-1)/ERK-2, and c-Jun N-terminal kinase, but not p38 mitogen-activated protein kinase. This is further supported by the data showing that Aβ causes phosphorylation of ERK-1/ERK-2, which, in turn, activates Elk-1. Furthermore, Aβ mediated a time-dependent increase in DNA binding activity of early growth response-1 (Egr-1) and AP-1, but not of NF-κB and CREB. Moreover, Aβ-induced Egr-1 DNA binding activity was reduced >60% in THP-1 cells transfected with small interfering RNA duplexes for Egr-1 mRNA. We show that Aβ-induced expression of TNF-α, IL-1β, MCP-1, IL-8, and MIP-1β was abrogated in Egr-1 small inhibitory RNA-transfected cells. Our results indicate that Aβ-induced expression of cytokines (TNF-α and IL-1β) and chemokines (MCP-1, IL-8, and MIP-1β) in THP-1 monocytes involves activation of ERK-1/ERK-2 and downstream activation of Egr-1. The inhibition of Egr-1 by Egr-1 small inhibitory RNA may represent a potential therapeutic target to ameliorate the inflammation and progression of AD.

Ethanol-Induced Expression of ET-1 and ET-BR in Liver Sinusoidal Endothelial Cells and Human Endothelial Cells Involves Hypoxia-Inducible Factor-1α and MicroRNA-199

Chronic alcohol consumption leads to inflammation and cirrhosis of the liver. In this study, we observed that liver sinusoidal endothelial cells (LSEC) derived from ethanol-fed rats showed several fold increases in the mRNA expression of endothelin-1 (ET-1), hypoxia-inducible factor-1α (HIF-1α), and inflammatory cytochemokines compared with control rat LSEC. We also observed the same results in acute ethanol-treated LSEC from control rats and human dermal microvascular endothelial cells. Ethanol-mediated ET-1 expression involved NADPH oxidase and HIF-1α activation. Furthermore, ethanol increased the expression of the ET-1 cognate receptor ET-BR in Kupffer cells and THP-1 monocytic cells, which also involved HIF-1α activation. Promoter analysis and chromatin immunoprecipitation showed that hypoxia response element sites in the proximal promoter of ET-1 and ET-BR were required for the binding of HIF-1α to up-regulate their expression. We showed that microRNAs, miR-199 among several microRNAs, attenuated HIF-1α and ET-1 expression, while anti-miR-199 reversed the effects, suggesting that ethanol-induced miR-199 down-regulation may contribute to augmented HIF-1α and ET-1 expression. Our studies, for the first time to our knowledge, show that ethanol-mediated ET-1 and ET-BR expression involve HIF-1α, independent of hypoxia. Additionally, ethanol-induced ET-1 expression in rat LSEC is regulated by miR-199, while in human endothelial cells, ET-1 expression is regulated by miR-199 and miR-155, indicating that these microRNAs may function as novel negative regulators to control ET-1 transcription and, thus, homeostatic levels of ET-1 to maintain microcirculatory tone.

Curcumin, the active constituent of turmeric, inhibits amyloid peptide‐induced cytochemokine gene expression and CCR5‐mediated chemotaxis of THP‐1 monocytes by modulating early growth response‐1 transcription factor

Epidemiological studies show reduced risk of Alzheimers disease (AD) among patients using non‐steroidal inflammatory drugs (NSAID) indicating the role of inflammation in AD. Studies have shown a chronic CNS inflammatory response associated with increased accumulation of amyloid peptide and activated microglia in AD. Our previous studies showed that interaction of Aβ1−40 or fibrilar Aβ1−42 caused activation of nuclear transcription factor, early growth response‐1 (Egr‐1), which resulted in increased expression of cytokines (TNF‐α and IL‐1β) and chemokines (MIP‐1β, MCP‐1 and IL‐8) in monocytes. We determined whether curcumin, a natural product known to have anti‐inflammatory properties, suppressed Egr‐1 activation and concomitant expression of cytochemokines. We show that curcumin (12.5–25 µm) suppresses the activation of Egr‐1 DNA‐binding activity in THP‐1 monocytic cells. Curcumin abrogated Aβ1−40‐induced expression of cytokines (TNF‐α and IL‐1β) and chemokines (MIP‐1β, MCP‐1 and IL‐8) in both peripheral blood monocytes and THP‐1 cells. We found that curcumin inhibited Aβ1−40‐induced MAP kinase activation and the phosphorylation of ERK‐1/2 and its downstream target Elk‐1. We observed that curcumin inhibited Aβ1−40‐induced expression of CCR5 but not of CCR2b in THP‐1 cells. This involved abrogation of Egr‐1 DNA binding in the promoter of CCR5 by curcumin as determined by: (i) electrophoretic mobility shift assay, (ii) transfection studies with truncated CCR5 gene promoter constructs, and (iii) chromatin immunoprecipitation analysis. Finally, curcumin inhibited chemotaxis of THP‐1 monocytes in response to chemoattractant. The inhibition of Egr‐1 by curcumin may represent a potential therapeutic approach to ameliorate the inflammation and progression of AD.

Alterations in Organization of Phospholipids in Erythrocytes as Factor in Adherence to Endothelial Cells in Diabetes Mellitus

Erythrocytes from patients with diabetes mellitus exhibit increased adherence to cultured human vascular endothelial cells. We investigated the alterations in erythrocyte surface characteristics that may contribute to their abnormal adherence. The organization of phospholipids in the lipid bilayer, as determined by phospholipase A2 treatment and chemical labeling with fluorescamine and trinitrobenzene sulfonic acid (TNBS), is altered in erythrocytes from diabetic patients. Specifically, 12–18% of phosphatidylserine in diabetic erythrocytes (n = 25) is accessible to phospholipase A2 hydrolysis and TNBS labeling, compared to none in normal subjects. These results suggest either a loss in lipid asymmetry or in vivo destabilization of erythrocyte membranes in diabetic patients, causing increased accessibility to phospholipase A2 degradation. The dye merocyanine 540 (MC-540), which is sensitive to the packing of lipids in the bilayer of the membrane, revealed more binding and fluorescence in erythrocytes from diabetic patients than in those from normal subjects. On flow cytometric analysis, 64.5 ± 17.0% red blood cells (RBCs) in diabetic patients, compared to 35.1 ± 25.9% RBCs in normal subjects, showed positive MC-540 binding, indicating significant (P < .001) differences in the packing of lipids in the external leaflet of the bilayer. The results of our study suggest that a loss of lipid asymmetry and/or less ordered packing in the outer leaflet of the diabetic erythrocyte membrane may be responsible for the increased propensity of erythrocytes to adhere to vascular endothelium.

Lipoxygenase metabolites induced expression of adhesion molecules and transendothelial migration of monocyte‐like HL‐60 cells is linked to protein kinase C activation

Studies have shown that, among lipoxygenase metabolites examined, 15(S)‐hydroperoxy‐5,8,11,13‐eicosa‐tetraenoic acid (15[S]‐HPETE), at micromolar concentrations, selectively causes injury to cultured endothelial cells. We investigated whether physiologically relevant concentrations of lipoxygenase metabolites affected the expression of cell adhesion molecules (CAMs) involved in the adhesion of leukocytes and/or the accumulation of leukocytes in the vascular endothelium, these being the initial events in endothelial cell injury. Among lipoxygenase metabolites, 15(S)‐HPETE and 12(S)‐HETE, at nanomolar concentrations, induced surface expression of a subset of cell adhesion molecules (CAM), ICAM‐1, ELAM‐1, and VCAM‐1, in human umbilical vein endothelial cells (HUVEC), which is associated with an increased binding activity of the transcription factor, NF‐κB, to the consensus motif common to the CAM genes in the HUVEC nuclear extracts. Furthermore, 15(S)‐HPETE (1 nM) caused a threefold increase in the rate of transendothelial migration of vitamin D3‐differentiated HL‐60 monocyte‐like cells and showed a thirtyfold increase in the phosphorylation of PECAM‐1, an adhesion molecule involved in endothelial cell‐cell adhesion. Both an antibody to PECAM‐1 and the protein kinase C inhibitor, GF 109203X, reduced 15(S)‐HPETE‐induced transmigration of monocyte‐like HL‐60 cells by approximately 75% and 85%, respectively. Treatment of HUVEC with a phosphatase inhibitor, calyculin A, augmented both the phosphorylation of PECAM‐1 and transmigration of monocyte‐like HL‐60 cells induced by 15(S)‐HPETE. Our results show that 15(S)‐HPETE, at physiological concentrations, induced activation of protein kinase C in HUVEC and leads to the phosphorylation of PECAM‐1, thus facilitating the migration of monocyte‐like HL‐60 cells across the endothelial cell monolayer. It is suggested that phosphorylation/dephosphorylation events in PECAM‐1 are important in regulating the trafficking of monocytes across the endothelial cell monolayer.

Placenta growth factor augments endothelin-1 and endothelin-B receptor expression via hypoxia-inducible factor-1α

Pulmonary hypertension (PHT) develops in sickle cell disease (SCD) and is associated with high mortality. We previously showed that erythroid cells produce placenta growth factor (PlGF), which activates monocytes to induce proinflammatory cytochemokines, contributing to the baseline inflammation and severity in SCD. In this study, we observed that PlGF increased expression of endothelin-1 (ET-1) and endothelin-B receptor (ET-BR) from human pulmonary microvascular endothelial cells (HPMVECs) and monocytes, respectively. PlGF-mediated ET-1 and ET-BR expression occurred via activation of PI-3 kinase, reactive oxygen species and hypoxia inducible factor-1 alpha (HIF-1 alpha). PlGF increased binding of HIF-1 alpha to the ET-1 and ET-BR promoters; this effect was abrogated with mutation of hypoxia response elements in the promoter regions and HIF-1 alpha siRNA and confirmed by chromatin immunoprecipitation analysis. Furthermore, PlGF-mediated ET-1 release from HPMVECs and ET-BR expression in monocytes creates a PlGF-ET-1-ET-BR loop, leading to increased expression of MCP-1 and IL-8. Our studies show that PlGF-induced expression of the potent vasoconstrictor ET-1 and its cognate ET-BR receptor occur via activation of HIF-1 alpha, independent of hypoxia. PlGF levels are intrinsically elevated from the increased red cell turnover in SCD and in other chronic anemia (eg, thalassemia) and may contribute to inflammation and PHT seen in these diseases.

Hypoxia-Mediated Expression of 5-Lipoxygenase–Activating Protein Involves HIF-1α and NF-κB and MicroRNAs 135a and 199a-5p

Hypoxia occurs in a number of pathological states, such as pulmonary, hematological, and cardiovascular disorders. In this study, we examined the molecular mechanism by which hypoxia contributes to increased leukotriene formation. Our studies showed hypoxia augmented the expression of 5-lipoxygenase activating protein (FLAP), a key enzyme in leukotriene formation, in both human pulmonary microvascular endothelial cells and a transformed human brain endothelial cell line. Hypoxia-induced FLAP mRNA expression involved activation of NADPH-oxidase, PI-3 kinase, mitogen-activated protein kinase, NF-κB, and hypoxia-inducible factor (HIF)-1α. Hypoxia-induced FLAP promoter activity was attenuated on mutation of hypoxia-response elements (HREs) and NF-κB binding motif in the FLAP promoter. Hypoxia also augmented binding of HIF-1α to HREs in FLAP promoter as demonstrated by EMSA with nuclear extracts. Furthermore, chromain immunoprecipitation analysis showed HIF-1α bound to HREs in native chromatin obtained from hypoxia-treated cells. Next, we examined the role of HIF-1α regulated microRNAs on FLAP expression. Our studies showed decreased expression of miR-135a and miR-199a-5p in response to hypoxia. However, overexpression of anti–miR-135a and anti–miR-199a-5p oligonucleotides led to a several fold increased FLAP mRNA and protein expression. These studies demonstrate for the first time that hypoxia-mediated FLAP expression is regulated by HREs and NF-κB site in its promoter, and negatively regulated by miR-135a and miR-199a-5p, which target the 3′-UTR of FLAP mRNA. An understanding of these regulatory pathways provides new avenues to ameliorate leukotriene formation and hence reactive airway disease, and inflammation in individuals who have sickle cell disease.

High levels of placenta growth factor in sickle cell disease promote pulmonary hypertension.

Pulmonary hypertension is associated with reduced nitric oxide bioavailability and early mortality in sickle cell disease (SCD). We previously demonstrated that placenta growth factor (PlGF), an angiogenic factor produced by erythroid cells, induces hypoxia-independent expression of the pulmonary vasoconstrictor endothelin-1 in pulmonary endothelial cells. Using a lentivirus vector, we simulated erythroid expression of PlGF in normal mice up to the levels seen in sickle mice. Consequently, endothelin-1 production increased, right ventricle pressures increased, and right ventricle hypertrophy and pulmonary changes occurred in the mice within 8 weeks. These findings were corroborated in 123 patients with SCD, in whom plasma PlGF levels were significantly associated with anemia, endothelin-1, and tricuspid regurgitant velocity; the latter is reflective of peak pulmonary artery pressure. These results illuminate a novel mechanistic pathway linking hemolysis and erythroid hyperplasia to increased PlGF, endothelin-1, and pulmonary hypertension in SCD, and suggest that strategies that block PlGF signaling may be therapeutically beneficial.

Gp120 activates children's brain endothelial cells via CD4

Encephalopathy represents a common and serious manifestation of HIV-1 infection in children, but its pathogenesis is unclear. We demonstrated that gp120 activated human brain microvascular endothelial cells (HBMEC) derived from children in up-regulating ICAM-1 and VCAM-1 expression, IL-6 secretion and increased monocyte transmigration across monolayers. Another novel observation was our demonstration of CD4 in isolated HBMEC and on microvessels of children’s brain cryosections. Gp120-induced monocyte migration was inhibited by anti-gp120 and anti-CD4 antibodies. This is the first demonstration that gp120 activates HBMEC via CD4, which may contribute to the development of HIV-1 encephalopathy in children.

Ethanol Augments RANTES/CCL5 Expression in Rat Liver Sinusoidal Endothelial Cells and Human Endothelial Cells via Activation of NF-κB, HIF-1α, and AP-1

Chronic alcohol consumption leads to liver inflammation and cirrhosis. Alcoholic liver disease patients have increased levels of hepatic RANTES/CCL5. However, less is known about the molecular mechanisms for ethanol-induced RANTES up-regulation. In this study, we observed that liver sinusoidal endothelial cells derived from ethanol-fed rats (E-rLSECs) showed severalfold increases in RANTES and hypoxia-inducible factor 1α (HIF-1α) mRNAs compared with control rLSECs (C-rLSECs). Similar effects were seen in acute ethanol treatment of isolated rLSECs and human dermal microvascular endothelial cells. Ethanol-induced RANTES mRNA expression required ethanol metabolism, p38 MAPK, HIF-1α, and JNK-2, but not JNK-1. EMSA experiments showed increased HIF-1α binding to wild-type hypoxia response elements (HREs; −31 to −9 bp) within the RANTES promoter in response to ethanol. RANTES promoter analysis showed that cis elements proximal to the transcription start site, HRE-1 (nt −22 to −19), HRE-2 (nt −32 to −29), and AP-1 (nt −250 to −244) were required for ethanol-mediated RANTES expression. These results were corroborated by chromatin immunoprecipitation assays showing augmented HIF-1α binding to HRE-1. Additionally, promoter analysis revealed c-Jun, c-Jun/c-Fos, and JunD, but not JunB, bound to the AP-1 site of the RANTES promoter. Ethanol-mediated activation of NF-κB led to HIF-1α activation and concomitant RANTES expression. Plasma of ethanol-fed c-Junflox/flox-Mx-1-Cre mice showed attenuated levels of RANTES compared with ethanol-fed control mice, supporting the role of c-Jun in ethanol-induced RANTES expression. Our studies showed that ethanol-mediated RANTES/CCL5 expression occurs via HIF-1α activation independently of hypoxia. The identification of HIF-1α and AP-1 in ethanol-induced RANTES expression provides new strategies to ameliorate ethanol-induced inflammatory responses.