Jason D. Morrow

Formation of Isoprostane-like Compounds (Neuroprostanes) in Vivo from Docosahexaenoic Acid

F2-isoprostanes are prostaglandin F2-like compounds that are formed nonenzymatically by free radical-induced oxidation of arachidonic acid. We explored whether oxidation of docosahexaenoic acid (C22:6ω3), which is highly enriched in the brain, led to the formation of F2-isoprostane-like compounds, which we term F4-neuroprostanes. Oxidation of docosahexaenoic acidin vitro yielded a series of compounds that were structurally established to be F4-neuroprostanes using a number of mass spectrometric approaches. The amounts formed exceeded levels of F2-isoprostanes generated from arachidonic acid by 3.4-fold. F4-neuroprostanes were detected esterified in normal whole rat brain and newborn pig cortex at a level of 7.0 ± 1.4 ng/g and 13.1 ± 8 ng/g, respectively. Furthermore, F4-neuroprostanes could be detected in normal human cerebrospinal fluid and levels in patients with Alzheimer’s disease (110 ± 12 pg/ml) were significantly higher than age-matched controls (64 ± 8 pg/ml) (p < 0.05). F4-neuroprostanes may provide a unique marker of oxidative injury to the brain and could potentially exert biological activity. Furthermore, the formation of F4-neuroprostane-containing aminophospholipids might adversely effect neuronal function as a result of alterations they induce in the biophysical properties of neuronal membranes.

Regulation of Constitutive Cyclooxygenase-2 Expression in Colon Carcinoma Cells

Cyclooxygenase-2 (COX-2) is not normally expressed in the human large intestine, but its levels are increased in the majority of human colorectal carcinomas. Here we investigate the regulation of constitutive COX-2 expression and prostaglandin production in human colorectal carcinoma cells. Both COX-2 mRNA and protein were expressed in well differentiated HCA-7, Moser, LS-174, and HT-29 cells, albeit at different levels. COX-2 expression was not detected in several poorly differentiated colon cancer cell lines including DLD-1. Transcriptional regulation played a key role for the expression of COX-2 in human colon carcinoma cells, and both the nuclear factor for interleukin-6 regulatory element and the cAMP-response element were responsible for regulation ofCOX-2 transcription. COX-2 mRNA was more stable in HCA-7 cells than in the other cell lines tested. Both transcriptional and post-transcriptional regulation of COX-2 involved the MAP kinase pathway. Modulation of the Akt/protein kinase B or Rho B signaling pathways altered the levels of COX-2 expression. Furthermore, COX-2 protein is degraded through ubiquitin proteolysis, and its half-life was ∼3.5–8 h. HCA-7 cells produced significant quantities of prostaglandin E2 and other prostaglandins. Moser and LS-174 cells also generated prostaglandins, but levels were significantly lower than that observed in HCA-7 cells.

A selective cyclooxygenase 2 inhibitor suppresses the growth of H-ras-transformed rat intestinal epithelial cells

BACKGROUND & AIMS Constitutive expression of cyclooxygenase 2 (COX-2) has been found in 85% of colorectal cancers. Ras mutations are found in 50% of colorectal adenocarcinomas. The aim of this study was to determine the role of COX-2 in ras-induced transformation in rat intestinal epithelial (RIE) cells. METHODS Cell growth was determined by cell counts. The expression of COX-2 was examined by Northern and Western analyses. For tumorigenicity assays, cells were inoculated into dorsal subcutaneous tissue of athymic nude mice. DNA-fragmentation assays were performed to detect apoptosis. RESULTS The expression of COX-2 was increased in RIE-Ras cells at both messenger RNA (9-fold) and protein (12-fold) levels. Prostaglandin I2 levels were elevated 2.15-fold in RIE-Ras cells. Serum deprivation further increased COX-2 expression 3.8-fold in RIE-Ras cells. Treatment with a selective COX-2 antagonist (SC58125) inhibited the growth of RIE-Ras cells through inhibition of cell proliferation and by induction of apoptosis. SC-58125 treatment reduced the colony formation in Matrigel by 83.0%. Intraperitoneal administration of SC-58125 suppressed RIE-Ras tumor growth in nude mice by 60.3% in 4 weeks. SC-58125 treatment also induced apoptosis in RIE-Ras cells as indicated by increased DNA fragmentation. CONCLUSIONS Overexpression of COX-2 may contribute to tumorigenicity of ras-transformed intestinal epithelial cells. Selective inhibition of COX-2 activity inhibits growth of ras-transformed intestinal epithelial cells and induces apoptosis.

Enhanced In Vivo Lipid Peroxidation at Elevated Plasma Total Homocysteine Levels

An elevated plasma total homocysteine level (tHcy) is considered an independent risk factor for atherosclerosis. The mechanisms by which hyperhomocysteinemia induces atherosclerosis are only partially understood, but promotion of LDL oxidation and endothelial injury have been suggested. The purpose of this study was to test the hypothesis that a high plasma tHcy is associated in men with increased in vivo lipid peroxidation, as measured by plasma F2-isoprostane concentrations. We investigated this association in a subset of the participants in the Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) study. Of 256 male participants, a subsample of 100 consecutive men was selected for F2-isoprostane assays. The mean tHcy was 11.0 micromol/L, and the mean F2-isoprostanes was 29.6 ng/L. The simple correlation coefficient for association between tHcy and F2-isoprostane was 0.40 (P<0.001). In a linear regression model, the variables with the strongest associations with F2-isoprostane were tHcy (standardized coefficient 0.33, P<0.001), serum triglycerides (0.21, P=0.042), carbohydrate-deficient transferrin (0.15, P=0.132), and plasma lipid-standardized alpha-tocopherol (-0.11, P=0.252) (R2=0.24, P<0. 001 for model). Plasma F2-isoprostane levels increased linearly across quintiles of tHcy (P<0.001). The unadjusted mean (95% confidence interval) F2-isoprostanes was 47.5% greater in the highest tHcy quintile (37.4, 31.1 to 43.6 ng/L) than in the lowest quintile (25.3, 21.3 to 29.3 ng/L). Adjustment for the strongest other determinants of F2-isoprostane reduced this difference to 28. 2% (P=0.010). Our present data suggest that elevated fasting plasma tHcy is associated with enhanced in vivo lipid peroxidation in men.

Brain Regional Quantification of F-Ring and D-/E-Ring Isoprostanes and Neuroprostanes in Alzheimer’s Disease

Isoprostanes (IsoP) are produced exclusively from free radical damage to arachidonic acid, a fatty acid that is evenly distributed throughout white matter and gray matter, whereas neuroprostanes (NPs) are generated analogously from docosahexaenoic acid (DHA), a fatty acid enriched in gray matter where it is concentrated in neurons. IsoP and NPs derive from endoperoxide intermediates that isomerize to D/E-ring forms or that are reduced to F-ring compounds. We quantified F-ring and D/E-ring IsoP and NPs in temporal and parietal cortex, hippocampus, and cerebellum of nine definite Alzheimers disease (AD) patients and 11 age-matched controls. Total NP levels (F-ring plus D/E-ring), but not total IsoP, were significantly greater in AD than controls (P: < 0.0001); only cerebral regions in AD patients had NPs greater than controls (P: < 0.05). The F-ring to D/E-ring ratio for NPs, but not IsoP, was 40 to 70% lower in all brain regions of AD patients compared to controls (P: < 0.005). These data extend results from in situ techniques, that have localized reactive products of lipid peroxidation primarily to neurons, by quantifying significantly greater free radical damage to the DHA-containing compartments in cerebrum in AD patients than controls, and suggest that one mechanism of increased oxidative stress may be diminished reducing capacity in DHA-containing compartments.

Linkage of hypoalbuminemia, inflammation, and oxidative stress in patients receiving maintenance hemodialysis therapy

BACKGROUND Hypoalbuminemia is a powerful predictor of cardiovascular mortality in maintenance hemodialysis patients. Increased biomarkers of acute-phase inflammation and oxidative stress are highly prevalent and also correlate with cardiovascular morbidity and mortality. The extent to which hypoalbuminemia, biomarkers of inflammation, and biomarkers of oxidative stress are linked in this patient population is unknown. We hypothesized that a high proportion of hypoalbuminemic hemodialysis patients also would manifest increased levels of biomarkers of inflammation and oxidative stress. METHODS We surveyed 600 maintenance hemodialysis patients and identified 18 severely hypoalbuminemic patients (serum albumin level < 3.2 g/dL [32 g/L]) without recent infection or hospitalization. We then identified 18 age-, race-, sex-, and diabetes-matched normoalbuminemic hemodialysis patients, as well as 18 age-, race-, sex-, and diabetes-matched healthy subjects, for cohort comparison. Measurements of plasma interleukin-6 (IL-6) levels, plasma protein reduced thiol content, plasma protein carbonyl content, and plasma free F2-isoprostane levels, as well as serum concentrations of C-reactive protein (CRP) and prealbumin, were performed for study purposes. RESULTS Levels of serum CRP, IL-6, plasma protein thiol oxidation, and protein carbonyl formation were significantly elevated in both hypoalbuminemic and normoalbuminemic hemodialysis patients compared with healthy subjects and also were significantly different in hypoalbuminemic maintenance dialysis patients compared with normoalbuminemic hemodialysis patients. Prealbumin levels were significantly lower in hypoalbuminemic hemodialysis patients than in other groups. CONCLUSION There is a high prevalence of inflammation and oxidative stress in the maintenance hemodialysis population. Levels of inflammatory and oxidative stress biomarkers are increased further in hypoalbuminemic compared with normoalbuminemic dialysis patients. Hypoalbuminemia, acute-phase inflammation, and oxidative stress may act synergistically to increase cardiovascular morbidity and mortality risk in maintenance hemodialysis patients.

Neuroprotective Effects of the Triterpenoid, CDDO Methyl Amide, a Potent Inducer of Nrf2-Mediated Transcription

The NF-E2-related factor-2 (Nrf2)/antioxidant response element (ARE) signaling pathway regulates phase 2 detoxification genes, including a variety of antioxidative enzymes. We tested neuroprotective effects of the synthetic triterpenoid CDDO-MA, a potent activator of the Nrf2/ARE signaling. CDDO-MA treatment of neuroblastoma SH-SY5Y cells resulted in Nrf2 upregulation and translocation from cytosol to nucleus and subsequent activation of ARE pathway genes. CDDO-MA blocked t-butylhydroperoxide-induced production of reactive oxygen species (ROS) by activation of ARE genes only in wild type, but not Nrf2 knockout mouse embryonic fibroblasts. Oral administration of CDDO-MA resulted in significant protection against MPTP-induced nigrostriatal dopaminergic neurodegeneration, pathological alpha-synuclein accumulation and oxidative damage in mice. Additionally, CDDO-MA treatment in rats produced significant rescue against striatal lesions caused by the neurotoxin 3-NP, and associated increases in the oxidative damage markers malondialdehyde, F2-Isoprostanes, 8-hydroxy-2-deoxyguanosine, 3-nitrotyrosine, and impaired glutathione homeostasis. Our results indicate that the CDDO-MA renders its neuroprotective effects through its potent activation of the Nrf2/ARE pathway, and suggest that triterpenoids may be beneficial for the treatment of neurodegenerative diseases like Parkinsons disease and Huntingtons disease.

The Magnitude of Brain Lipid Peroxidation Correlates with the Extent of Degeneration but Not with Density of Neuritic Plaques or Neurofibrillary Tangles or with APOE Genotype in Alzheimer's Disease Patients

Numerous post mortem studies have demonstrated increased accumulation of lipid peroxidation products in diseased regions of Alzheimers disease (AD) brain; however, few have used techniques that quantify the magnitude of lipid peroxidation in vivo. F(2)-isoprostanes (F(2)-IsoPs) are exclusive products of free radical-mediated peroxidation of arachidonic acid, and their quantification has been widely used as an in vivo biomarker of the magnitude of lipid peroxidation. We have determined F(2)-IsoP concentrations in lateral ventricular fluid (VF) from 23 AD and 12 age-matched controls and correlated these with neuropathological and genetic markers of AD. VF F(2)-IsoP levels were significantly elevated in AD patients compared with controls (p < 0.01) and were significantly correlated with three different measures of brain degeneration: reduction in brain weight (p < 0.01), degree of cortical atrophy (p < 0.01), and Braak stage (p = 0.02). When analysis was restricted to AD patients only, VF F(2)-IsoP levels still were significantly correlated to reduction in brain weight and degree of cortical atrophy (p < 0.05). VF F(2)-IsoP concentrations were not related to density of neuritic plaques or neurofibrillary tangles in seven brain regions, or to the number of epsilon4 alleles of the apolipoprotein E gene (APOE). These data suggest that the magnitude of brain lipid peroxidation is closely related to the extent of brain degeneration in AD but is not significantly influenced by the density of neuritic plaques or neurofibrillary tangles, or the number of epsilon4 alleles of APOE.

Inflammation and cerebral amyloidosis are disconnected in an animal model of Alzheimer's disease

The hypothesis that inflammation and beta amyloid deposition are causally linked in Alzheimers disease (AD) was tested in a transgenic mouse model. Untreated beta amyloid plaque-bearing Tg2576 mice did not differ from wild type animals in brain levels of most inflammatory mediators. Indomethacin treatment suppressed brain levels of prostaglandins by 90%, but reduced hippocampal beta amyloid by only 20%, with no effect on cortical beta amyloid. The discordant effects on beta amyloid and cyclooxygenase (COX) suggest that these effects of nonsteroidal anti-inflammatory drugs (NSAIDs) are not causally linked. Further evidence against a causal relationship is seen in an unexpected trend to lower levels of beta amyloid after an inflammatory stimulus [lipopolysaccharide (LPS)].

Lack of Integrin α1β1 Leads to Severe Glomerulosclerosis after Glomerular Injury

Severity of fibrosis after injury is determined by the nature of the injury and host genetic susceptibility. Metabolism of collagen, the major component of fibrotic lesions, is, in part, regulated by integrins. Using a model of glomerular injury by adriamycin, which induces reactive oxygen species (ROS) production, we demonstrated that integrin α1-null mice develop more severe glomerulosclerosis than wild-type mice. Moreover, primary α1-null mesangial cells produce more ROS both at baseline and after adriamycin treatment. Increased ROS synthesis leads to decreased cell proliferation and increased glomerular collagen IV accumulation that is reversed by antioxidants both in vivo and in vitro. Thus, we have identified integrin α1β1 as a modulator of glomerulosclerosis. In addition, we showed a novel pathway where integrin α1β1 modulates ROS production, which in turn controls collagen turnover and ultimately fibrosis. Because integrin α1β1 is expressed in many cell types this may represent a generalized mechanism of controlling matrix accumulation, which has implications for numerous diseases characterized by fibrosis.