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Objective To characterize surgical treatment and outcomes of C1 fractures in a population-based setup. Methods Patients with C1 fracture treated at Kuopio University Hospital Neurosurgery were retrospectively identified from January 1996 to June 2017. C1 fractures were classified according to the AO Spine Upper Cervical and Gehweiler classification systems. Patients were divided into 4 groups based on their treatment: group 1 (underwent C1 surgery as a primary option), group 2 (underwent C1 surgery as a secondary option after initial nonoperative treatment), group 3 (underwent surgery involving the C1 level with main indication being a concomitant cervical spine fracture), and group 4 (C1 fracture treatment was nonoperative). Results We identified 47 patients with C1 fracture (mean age, 60.3 ± 18.2 years; 83.0% men; American Society of Anesthesiologists score, 2.3 ± 0.8). Concomitant cervical spine fractures were present in 89.4% of cases, most commonly in the C2 vertebra (75.4%). In group 2, 3 of 5 fractures changed from AO Spine type A to B in control imaging after nonoperative treatment, indicating fracture instability and requiring secondary surgery. Good C1 fracture alignment was achieved for 10 of 10 followed-up patients in groups 1 and 2, and for 10 of 11 followed-up patients in group 3. Residual neck pain and stiffness were present in all groups. Neurologic symptoms were rare and mild. Conclusions For unstable C1 fractures, surgery is safe treatment with good outcomes. Fractures initially determined as stable may require surgery if alignment is worsened in follow-up imaging. Magnetic resonance imaging is recommended to better detect unstable C1 fractures in diagnostic imaging.

Purpose This study aims to elucidate the incidence of and independent risk factors for spinal cord stimulator implantations for patients who underwent lumbar spine surgery. Methods The PERFormance, Effectiveness, and Cost of Treatment (PERFECT) episodes database, which was established for selected diseases and procedures in Finland, includes all patients who underwent lumbar spine surgery for degenerative spine conditions or spinal cord stimulation (SCS) in Finland from 1986 to 2018. The data on age, sex, hospital diagnoses, surgical procedures, and causes of death were imported from the Finnish national registers into the PERFECT database. Results Between 1986 and 2018, 157,824 patients had their first lumbar spine procedure and for 1769 (1.1%) of them, a subsequent SCS procedure was observed during the follow-up. The cumulative incidence of SCS for persistent or recurrent pain after lumbar disk herniation, spinal stenosis, degenerative disk disease, and spondylolysis and spondylolisthesis surgery at 15 years was 1.2%, 1.0%, 2.7%, and 2.6% respectively. At 15 years, the cumulative incidence of SCS for persistent or recurrent pain after lumbar spine surgery after five or more lumbar spinal operations was 11.9%. Conclusion Repeated surgery was the most prominent significant risk factor for SCS for persistent or recurrent pain after lumbar spine surgery. The risk of SCS for persistent or recurrent pain after lumbar spine surgery increases significantly along with the number of lumbar spine procedures. When considering repeated lumbar spine surgery, careful evaluation of treatment options should take place to ensure good patient outcomes.

BACKGROUND Spinal cord stimulation (SCS) is an effective treatment in failed back surgery syndrome (FBSS). The effect of neuropathic pain medication use on SCS outcome is poorly understood. OBJECTIVE To study the effect of gabapentinoid use on SCS outcome measured by trial success, explantation rate and opioid dose reduction during a 2-yr follow-up. METHODS The study cohort included 203 consecutive FBSS patients who underwent SCS in a single tertiary center during January 1997 to March 2014. Purchase data of gabapentinoids, opioids, tricyclic antidepressants, serotonin and noradrenaline reuptake inhibitors, and benzodiazepines during January 1995 to March 2016 were retrieved from national registries. RESULTS In multivariate Cox regression analysis, patients using gabapentinoids had significantly fewer explantations during the 2-yr follow-up (hazard ratio [HR] 0.2, 95% CI 0.04-0.81, P = .03). In contrast, patients with opioid use of >40 morphine milligram equivalent before implantation had significantly more explantations (HR 6.7, 95% CI 2.5-18, P 50% dose reduction. This indicates that patients with SCS could benefit from concomitant use of gabapentinoids. Prospective randomized trials are warranted to verify this hypothesis.

BACKGROUND Spinal cord stimulation (SCS) is an effective treatment in chronic neuropathic pain, but its efficacy in complex regional pain syndrome (CRPS) needs to be proven. OBJECTIVE To study the outcome of SCS in CRPS as measured by trial success, explantation rate, complications, and changes in opioid and neuropathic pain medication use over a 4-yr follow-up. METHODS We retrospectively reviewed all medical records of 35 consecutive CRPS patients who underwent SCS trials at 2 hospitals during January 1998 to December 2016. The purchase data of opioids and neuropathic pain medication during January 1995 to March 2016 were retrieved from national registries. RESULTS Based on a 1-wk trial, permanent SCS was implanted in 27 (77%) patients. During the median follow-up of 8 yr, 8 (30%) SCS devices were explanted, of which 7 were because of inefficient pain relief. Complications leading to revision occurred in 17 (63%) patients: 8 electrode migrations or stimulation to the wrong area, 1 deep infection, 9 hardware malfunctions, 2 pulse generator discomforts, and 2 SCS replacements. None of the 6 patients using strong opioids discontinued their use during the 2-yr follow-up. The mean opioid dose increased nonsignificantly both in patients with SCS in permanent use (53 ± 150 morphine milligram equivalents morphine milligram equivalent (MME)/day to 120 ± 240 MME/day) and in patients who had SCS explanted (27 ± 72 MME/day to 57 ± 66 MME/day). CONCLUSION Despite the fact that CRPS patients were not able to discontinue or reduce their strong opioid or neuropathic pain medication use, 70% continued to use their SCS device during a median 8-yr follow-up.

Background Idiopathic normal pressure hydrocephalus (iNPH) is a neurodegenerative disease with a characteristic symptom triad of gait disturbance, cognitive decline, and incontinence. Recently, also dysfunctions in upper limbs have been described in iNPH and reported to improve after shunt surgery. We aim to describe the role of upper limb motor function in the clinical assessment of iNPH patients and its influence on activities of daily living (ADL). Methods Seventy-five consecutive patients with probable iNPH were studied pre-operatively and at 3 and 12 months after shunt surgery. The pre-operative evaluation included lumbar drainage of cerebrospinal fluid (tap test). Motor functions were assessed in upper and lower limbs with Grooved Pegboard Test (GPT), Box & Block Test (BBT), Total Score of Gait (TSG), and balance test. ADL was assessed with Barthel’s index and cognition in accordance with the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Results Patients showed improvement in all motor tests and ADL at 3 months after shunt surgery. The improvement remained stable during the 12-month post-operative follow-up. The motor function tests correlated with each other and with ADL. Conclusions A 3-month follow-up period after shunt surgery is adequate to show improvement in motor tasks, and a positive outcome will last for at least 12 months. A shunt-responsive dysfunction of upper limb motor performance plays a major role in ADL of iNPH patients. Therefore, we suggest an evaluation of upper limb motor performance to be included in routine evaluation of iNPH patients.

BackgroundHealth-related quality of life (HRQoL) is severely impaired in persons with idiopathic normal pressure hydrocephalus (iNPH). The HRQoL improves in a number of patients after the placement of a cerebrospinal fluid (CSF) shunt, but long-term follow-up of HRQoL is rare.MethodsExtended follow-up (60 months) of a prospective cohort study involving 189 patients with iNPH who underwent shunt surgery. Preoperative variables were used to predict favorable HRQoL outcome (improvement or non-deterioration) measured by the 15D instrument 5 years after shunting.ResultsOut of the 189 initially enrolled study participants, 88 had completed 5-year HRQoL follow-up (46%), 64 had died (34%), and 37 (20%) failed to complete the HRQoL follow-up but were alive at the end of the study. After initial post-operative HRQoL improvement, HRQoL deteriorated so that 37/88 participants (42%) had a favorable HRQoL outcome 5 years after shunting. Multivariate binary logistic regression analysis indicated that younger age (adjusted OR 0.86, 95% CI 0.77–0.95;p p p ConclusionsThis extended follow-up showed that the self-evaluated HRQoL outcome is associated with iNPH patients’ pre-operative cognitive status, overweight and age. The post-operative deterioration may reflect the natural progression of iNPH, but also derive from aging and comorbidities. It indicates a need for long-term follow-up.

Objective Spinal cord stimulation (SCS) is an effective treatment in failed back surgery syndrome (FBSS). We studied the effect of preimplantation opioid use on SCS outcome and the effect of SCS on opioid use during a two‐year follow‐up period. Materials and methods The study cohort included 211 consecutive FBSS patients who underwent an SCS trial from January 1997 to March 2014. Participants were divided into groups, which were as follows: 1) SCS trial only (n = 47), 2) successful SCS (implanted and in use throughout the two‐year follow‐up period, n = 131), and 3) unsuccessful SCS (implanted but later explanted or revised due to inadequate pain relief, n = 29). Patients who underwent explantation for other reasons (n = 4) were excluded. Opioid purchase data from January 1995 to March 2016 were retrieved from national registries. Results Higher preimplantation opioid doses associated with unsuccessful SCS (ROC: AUC = 0.66, p = 0.009), with 35 morphine milligram equivalents (MME)/day as the optimal cutoff value. All opioids were discontinued in 23% of patients with successful SCS, but in none of the patients with unsuccessful SCS (p = 0.004). Strong opioids were discontinued in 39% of patients with successful SCS, but in none of the patients with unsuccessful SCS (p = 0.04). Mean opioid dose escalated from 18 ± 4 MME/day to 36 ± 6 MME/day with successful SCS and from 22 ± 8 MME/day to 82 ± 21 MME/day with unsuccessful SCS (p

Background: Spinal cord stimulation is an efficient treatment for selected patients with chronic neuropathic pain. Since 2015, neuromodulation nurses at Kuopio University Hospital have been responsible for patient education and stimulator adjustments. Aims: The purpose is to determine what factors professionals perceived to affect patients' wellbeing in the neuromodulation process for chronic neuropathic pain. Methods: Four nurses and four physicians on the neuromodulation team performed a strengths, weaknesses, opportunities and threats analysis of the operational environment. All evaluated the attributes independently, and a multiple-criteria analysis was performed based on the results. Findings: The most important factors were excellent or good treatment results, having neuromodulation nurses, having a well-functioning neuromodulation team, vulnerability because of the limited number of specialists, and the establishment of a pain centre. Conclusion: This study highlights the importance of neuromodulation nurses and teamwork in chronic pain neuromodulation. The establishment of a multidisciplinary pain centre is strongly recommended.

Background Idiopathic intracranial hypertension (IIH) is a rare disease of unknown aetiology related possibly to disturbed cerebrospinal fluid (CSF) dynamics and characterised by elevated intracranial pressure (ICP) causing optic nerve atrophy if not timely treated. We studied CSF dynamics of the IIH patients based on the available literature and our well-defined cohort. Method A literature review was performed from PubMed between 1980 and 2020 in compliance with the PRISMA guideline. Our study includes 59 patients with clinical, demographical, neuro-ophthalmological, radiological, outcome data, and lumbar CSF pressure measurements for suspicion of IIH; 39 patients had verified IIH while 20 patients did not according to Friedman’s criteria, hence referred to as symptomatic controls. Results The literature review yielded 19 suitable studies; 452 IIH patients and 264 controls had undergone intraventricular or lumbar CSF pressure measurements. In our study, the mean CSF pressure, pulse amplitudes, power of respiratory waves (RESP), and the pressure constant (P0) were higher in IIH than symptomatic controls (p < 0.01). The mean CSF pressure was higher in IIH patients with psychiatric comorbidity than without (p < 0.05). In IIH patients without acetazolamide treatment, the RAP index and power of slow waves were also higher (p < 0.05). IIH patients with excess CSF around the optic nerves had lower relative pulse pressure coefficient (RPPC) and RESP than those without (p < 0.05). Conclusions Our literature review revealed increased CSF pressure, resistance to CSF outflow and sagittal sinus pressure (SSP) as key findings in IIH. Our study confirmed significantly higher lumbar CSF pressure and increased CSF pressure waves and RAP index in IIH when excluding patients with acetazolamide treatment. In overall, the findings reflect decreased craniospinal compliance and potentially depleted cerebral autoregulation resulting from the increased CSF pressure in IIH. The increased slow waves in patients without acetazolamide may indicate issues in autoregulation, while increased P0 could reflect the increased SSP.

Objective Idiopathic intracranial hypertension (IIH) is aetiologically unknown disorder that associates with endocrinological disturbances, including dysfunction of hypothalamic-pituitary-adrenal-axis. Neuroendocrinological dysfunctions have also been characterized in psychiatric disorders, and therefore we investigated the presence of psychiatric disorders of patients with IIH in a well-defined cohort. Patients and methods A total of 51 patients with IIH were included. Patient demographics, symptoms, imaging data, ophthalmological and clinical findings were collected. Results At the time of diagnosis the mean age was 32.5years (SD 10.7), the body mass index was 37.1 kg/m2 (SD 7.4), and the opening pressure 29.1 mmHg (SD 6.2). A total of 88.2% of patients were female and 45.1% were diagnosed with a psychiatric co-morbidity prior to IIH diagnosis. The mean follow-up time was 4.4 years (SD 5.4). The overall treatment outcome was significantly poorer on a group of patients with psychiatric diagnosis when compared to individuals without such history (p = 0.001), but there were no differences in the resolution of papilledema (p = 0.405). Patients with IIH and psychiatric disorders had more often empty sella on their imaging at diagnosis when compared to patients without psychiatric co-morbidity (p = 0.044). Conclusion Psychiatric disorders are highly prevalent in patients with IIH and associate with worse subjective outcomes. These findings advocate for monitoring the mental health of patients with IIH and warrant further multidisciplinary research to understand the potentially underlying psychosocial and neuroendocrinological mechanisms.

Accumulating evidence suggests that constitutively active Nrf2 has a pivotal role in cancer as it induces pro-survival genes that promote cancer cell proliferation and chemoresistance. The mechanisms of Nrf2 dysregulation and functions in cancer have not been fully characterized. Here, we jointly analyzed the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE) and the Cancer Genome Atlas (TCGA) multi-omics data in order to identify cancer types where Nrf2 activation is present. We found that Nrf2 is hyperactivated in a subset of glioblastoma (GBM) patients, whose tumors display a mesenchymal subtype, and uncover several different mechanisms contributing to increased Nrf2 activity. Importantly, we identified a positive feedback loop between SQSTM1/p62 and Nrf2 as a mechanism for activation of the Nrf2 pathway. We also show that autophagy and serine/threonine signaling regulates p62 mediated Keap1 degradation. Our results in glioma cell lines indicate that both Nrf2 and p62 promote proliferation, invasion and mesenchymal transition. Finally, Nrf2 activity was associated with decreased progression free survival in TCGA GBM patient samples, suggesting that treatments have limited efficacy if this transcription factor is overactivated. Overall, our findings place Nrf2 and p62 as the key components of the mesenchymal subtype network, with implications to tumorigenesis and treatment resistance. Thus, Nrf2 activation could be used as a surrogate prognostic marker in mesenchymal subtype GBMs. Furthermore, strategies aiming at either inhibiting Nrf2 or exploiting Nrf2 hyperactivity for targeted gene therapy may provide novel treatment options for this subset of GBM.

Background The Kuopio University Hospital (KUH) idiopathic normal pressure hydrocephalus (iNPH) cerebrospinal fluid (CSF) shunting protocol is described together with the initial outcomes of 175 patients with probable iNPH treated according to this protocol from a defined population. Our secondary aim was to display the variety of differential diagnoses referred to the KUH iNPH outpatient clinic from 2010 until 2017. Methods Patients were divided into four groups according to the prognostic tests: tap test (positive or negative) and infusion test (positive or negative). The short-term outcome was compared between groups. The 3-month outcome following shunt surgery was assessed by measuring gait speed improvement, using a 12-point iNPH grading scale (iNPHGS) and the 15D instrument. Results From 341 patients suspected of iNPH, 88 patients were excluded from further research mostly due to deviation from the protocol’s gait assessment guidelines. Hence 253 patients with suspected iNPH were included in the study, 177/253 (70%) of whom were treated with a CSF shunt. A favorable clinical outcome following surgery was observed in 79–93% of patients depending on the prognostic group. A moderate association (Cramer’s V = 0.32) was found between the gait speed improvement rate and the prognostic group (X2, p = 0.003). Patients with a positive tap test had the highest gait speed improvement rate (75%). In addition, an improvement in walking speed was observed in 4/11 patients who had both a negative tap test and a negative infusion test. Other outcome measures did not differ between the prognostic groups. Conditions other than iNPH were found in 25% of the patients referred to iNPH outpatient clinic, with the most prevalent being Alzheimer’s disease. Conclusions Our results emphasize the importance of a systematic diagnostic and prognostic workup especially in cases with an atypical presentation of iNPH. Additional diagnostic testing may be required, but should not delay adequate care. Active surgical treatment is recommended in patients with a high clinical probability of iNPH. Other neurological conditions contributed to most of the non iNPH diagnoses. Electronic supplementary material The online version of this article (10.1186/s12987-019-0142-9) contains supplementary material, which is available to authorized users.

Accumulating evidence suggests that constitutively active Nrf2 has a pivotal role in cancer as it induces pro-survival genes that promote cancer cell proliferation and chemoresistance. The mechanisms of Nrf2 dysregulation and functions in cancer have not been fully characterized. Here, we jointly analyzed the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE) and the Cancer Genome Atlas (TCGA) multi-omics data in order to identify cancer types where Nrf2 activation is present. We found that Nrf2 is hyperactivated in a subset of glioblastoma (GBM) patients, whose tumors display a mesenchymal subtype, and uncover several different mechanisms contributing to increased Nrf2 activity. Importantly, we identified a positive feedback loop between SQSTM1/p62 and Nrf2 as a mechanism for activation of the Nrf2 pathway. We also show that autophagy and serine/threonine signaling regulates p62 mediated Keap1 degradation. Our results in glioma cell lines indicate that both Nrf2 and p62 promote proliferation, invasion and mesenchymal transition. Finally, Nrf2 activity was associated with decreased progression free survival in TCGA GBM patient samples, suggesting that treatments have limited efficacy if this transcription factor is overactivated. Overall, our findings place Nrf2 and p62 as the key components of the mesenchymal subtype network, with implications to tumorigenesis and treatment resistance. Thus, Nrf2 activation could be used as a surrogate prognostic marker in mesenchymal subtype GBMs. Furthermore, strategies aiming at either inhibiting Nrf2 or exploiting Nrf2 hyperactivity for targeted gene therapy may provide novel treatment options for this subset of GBM.

Phospholipids, such as 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), are the major components of cell membranes. Their exposure to reactive oxygen species creates oxidized phospholipids, which predispose to the development of chronic inflammatory diseases and metabolic disorders through endothelial activation and dysfunction. Although the effects of oxidized PAPC (oxPAPC) on endothelial cells have been previously studied, the underlying molecular mechanisms evoking biological responses remain largely unknown. Here, we investigated the molecular mechanisms of oxPAPC function with a special emphasis on NRF2-regulated microRNAs (miRNAs) in human umbilical vein endothelial cells (HUVECs) utilizing miRNA profiling, global run-on sequencing (GRO-seq), genome-wide NRF2 binding model, and RNA sequencing (RNA-seq) with miRNA overexpression and silencing. We report that the central regulators of endothelial activity, KLF2 for quiescence, PFKFB3 for glycolysis, and VEGFA, FOXO1 and MYC for growth and proliferation, are regulated by transcription factor NRF2 and the NRF2-regulated miR-106b∼25 cluster member, miR-93, in HUVECs. Mechanistically, oxPAPC was found to induce glycolysis and proliferation NRF2-dependently, and oxPAPC-dependent induction of the miR-106b∼25 cluster was mediated by NRF2. Additionally, several regulatory loops were established between NRF2, miR-93 and the essential regulators of healthy endothelium, collectively implying that NRF2 controls the switch between the quiescent and the proliferative endothelial states together with miR-93., published version, peerReviewed

A central mechanism in cellular defence against oxidative or electrophilic stress is mediated by transcriptional induction of genes via the ARE (antioxidant-response element), a cis-acting sequence present in the regulatory regions of genes involved in the detoxification and elimination of reactive oxidants and electrophiles. The ARE binds different bZIP (basic-region leucine zipper) transcription factors, most notably Nrf2 (nuclear factor-erythroid 2-related factor 2) that functions as a transcriptional activator via heterodimerization with small Maf proteins. Although ARE activation by Nrf2 is relatively well understood, the mechanisms by which ARE-mediated signalling is down-regulated are poorly known. Transcription factor BACH1 [BTB (broad-complex, tramtrack and bric-a-brac) and CNC (cap'n'collar protein) homology 1] binds to ARE-like sequences, functioning as a transcriptional repressor in a subset of ARE-regulated genes, thus antagonizing the activator function of Nrf2. In the present study, we have demonstrated that BACH1 itself is regulated by Nrf2 as it is induced by Nrf2 overexpression and by Nrf2-activating agents in an Nrf2-dependent manner. Furthermore, a functional ARE site was identified at +1411 from the transcription start site of transcript variant 2 of BACH1. We conclude that BACH1 is a bona fide Nrf2 target gene and that induction of BACH1 by Nrf2 may serve as a feedback-inhibitory mechanism for ARE-mediated gene regulation.

Objective Endothelial lipase (EL) is a new member of triacylglycerol lipase family that has been shown to decrease high-density lipoprotein (HDL) cholesterol levels leading to increased risk of atherosclerosis. Its expression is increased during inflammation and by inflammatory cytokines. Sulforaphane (SFN) is a naturally occurring isothiocyanate present in cruciferous vegetables that has antioxidant and anti-inflammatory effects. Nuclear factor (NF)-κB is one of the molecular targets for SFN-mediated protective effects. Our aim was therefore to assess whether SFN could impact on EL expression via modulation of NF-κB pathway. Methods and results Quantitative PCR and Western blot results demonstrated that SFN inhibited tumor necrosis factor (TNF)-α-mediated induction of EL in human umbilical vein endothelial cells (HUVEC). Lentiviral transduction of HUVEC with mutated form of IκB-α (IκBM) as well as silencing of NF-κB subunit p65 using RNA interference revealed that TNF-α-mediated induction of EL is mediated through NF-κB pathway. In addition, a total of five NF-κB binding sites were found in LIPG gene, which encodes EL. SFN inhibited binding of NF-κB to these sites analyzed by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). SFN also inhibited TNF-α mediated phosphorylation of IκB kinase (IKK) 1/2 and IκB-α. Conclusions Collectively, these results indicate that SFN inhibits EL expression via inhibition of NF-κB which may have a beneficial effect on HDL cholesterol levels.

Electrophilic fatty acid derivatives, including nitrolinoleic acid and nitro-oleic acid (OA-NO2), can mediate anti-inflammatory and pro-survival signaling reactions. The transcription factor Nrf2, activated by electrophilic fatty acids, suppresses redox-sensitive pro-inflammatory gene expression and protects against vascular endothelial oxidative injury. It was therefore postulated that activation of Nrf2 by OA-NO2 accounts in part for its anti-inflammatory actions, motivating the characterization of Nrf2-dependent and -independent effects of OA-NO2 on gene expression using genome-wide transcriptional profiling. Control and Nrf2-small interfering RNA-transfected human endothelial cells were treated with vehicle, oleic acid, or OA-NO2, and differential gene expression profiles were determined. Although OA-NO2 significantly induced the expression of Nrf2-dependent genes, including heme oxygenase-1 and glutamate-cysteine ligase modifier subunit, the majority of OA-NO2-regulated genes were regulated by Nrf2-independent pathways. Moreover, gene set enrichment analysis revealed that the heat shock response is the major pathway activated by OA-NO2, with robust induction of a number of heat shock genes regulated by the heat shock transcription factor. Inasmuch as the heat shock response mediates anti-inflammatory and cytoprotective actions, this mechanism is proposed to contribute to the protective cell signaling functions of nitro-fatty acids and other electrophilic fatty acid derivatives.

Nuclear factor erythroid 2-related factor 2 (Nrf2) coordinates the up-regulation of cytoprotective genes via the antioxidant response element (ARE). In the pathogenesis of Alzheimer's disease (AD) current evidence supports the role of oxidative stress. Considering the protective role of Nrf2 against oxidative injury, we studied Nrf2 and Nrf2–ARE target genes in transgenic AD mice and tested whether Nrf2 could confer neuroprotection against amyloid-beta peptides (Aβ). Nrf2–ARE pathway was attenuated in APP/PS1 transgenic mouse brain at the time of Aβ deposition. Boosting the activity of the Nrf2–ARE pathway by tert-butylhydroquinone treatment or adenoviral Nrf2 gene transfer protected against Aβ toxicity. This neuroprotection was associated with increased expression of Nrf2 target genes and reduced phosphorylation of p66Shc, a marker of increased susceptibility for oxidative stress. The findings suggest that the Nrf2–ARE pathway may be impaired in AD and that induction of the Nrf2–ARE defence mechanism may prevent or delay AD-like pathology.

Background— Reactive oxygen species (ROS) play a major role in vascular inflammation and pathophysiology of many vascular diseases such as atherosclerosis and injury-induced neointima formation after balloon angioplasty. Nuclear factor E2–related factor-2 (Nrf2) is a transcription factor orchestrating antioxidant and cytoprotective responses on oxidative and electrophilic stress, and it has been shown to have antiinflammatory effects in vascular cells in vitro. We therefore postulated that Nrf2 gene transfer would have salutary effects on vascular inflammation after angioplasty. Methods and Results— Transduction of vascular smooth muscle cells (VSMCs) with Nrf2-expressing adenovirus increased the expression of several antioxidant enzymes including heme oxygenase-1 (HO-1) compared with β-galactosidase (AdLacZ)-transduced controls. Moreover, Nrf2 gene transfer also inhibited vascular smooth muscle cell (VSMC) proliferation, and the effect was partially reversed by the HO inhibitor Sn(IV) protoporphyrin. In vivo, adenoviral gene transfer effectively reduced oxidative stress determined by antibody staining against oxidized epitopes of LDL, as well as inhibited vascular inflammation assessed by the macrophage cell count and monocyte chemoattractant protein-1 (MCP-1) staining. However, the antiproliferative effects of Nrf2 in vivo were counterbalanced with diminished apoptosis in neointimal VSMCs, resulting in no change in neointimal hyperplasia. Conclusions— Nrf2 gene transfer or Nrf2-inducing drugs may have therapeutic applications in vascular diseases in which inflammation and oxidative stress play a role. However, the contrasting growth inhibitory and antiapoptotic effects of Nrf2 need to be considered in pathological conditions in which SMC proliferation plays a critical role.

Unsaturated fatty acids are prone to radical reactions that occur in biological situations where extensive formation of reactive oxygen and nitrogen species (ROS and RNS) takes place. These reactions are frequent in inflammatory conditions such as atherosclerosis, and yield a variety of biologically active species, many of which are electrophilic in nature. Electrophilic lipid oxidation and nitration products can influence redox cell signaling via S-alkylation of protein thiols, and moderate exposure to these species evokes protective cell signaling responses through this mechanism. Herein, we review the stress signaling pathways elicited by electrophiles derived from unsaturated fatty acids, focusing on the Keap1-Nrf2 pathway, the heat shock response pathway (HSR), and the unfolded protein response pathway (UPR).

Objective— The mature form of human vascular endothelial growth factor-D (hVEGF-D ΔNΔC ) is an efficient angiogenic factor, but its full mechanism of action has remained unclear. We studied the effects of hVEGF-D ΔNΔC in endothelial cells using gene array, signaling, cell culture, and in vivo gene transfer techniques. Methods and Results— Concomitant with the angiogenic and proliferative responses, hVEGF-D ΔNΔC enhanced the phosphorylation of VEGF receptor-2, Akt, and endothelial nitric oxide synthase. Gene arrays, quantitative reverse transcription–polymerase chain reaction, and Western blot revealed increases in VEGF-A, stanniocalcin-1 (STC1), and neuropilin (NRP) 2 expression by hVEGF-D ΔNΔC stimulation, whereas induction with hVEGF-A 165 altered the expression of STC1 and NRP1, another coreceptor for VEGFs. The effects of hVEGF-D ΔNΔC were seen only under high-serum conditions, whereas for hVEGF-A 165 , the strongest response was observed under low-serum conditions. The hVEGF-D ΔNΔC -induced upregulation of STC1 and NRP2 was also evident in vivo in mouse skeletal muscle treated with hVEGF-D ΔNΔC by adenoviral gene delivery. The importance of NRP2 in hVEGF-D ΔNΔC signaling was further studied with NRP2 small interfering RNA and NRP antagonist, which were able to block hVEGF-D ΔNΔC -induced survival of endothelial cells. Conclusion— In this study, the importance of serum and upregulation of NRP2 and STC1 for VEGF-D ΔNΔC effects were demonstrated. Better knowledge of VEGF-D ΔNΔC signaling and regulation is valuable for the development of efficient and safe VEGF-D ΔNΔC -based therapeutic applications for cardiovascular diseases.

Besides their well-characterized proinflammatory and proatherogenic effects, oxidized phospholipids, such as oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-phosphocholine) have been shown to have beneficial responses in vascular cells via induction of antioxidant enzymes such as heme oxygenase-1. We therefore hypothesized that oxPAPC could evoke a general cytoprotective response via activation of antioxidative transcription factor Nrf2. Here, we show that oxPAPC increases nuclear accumulation of Nrf2. Using the small interfering RNA approach, we demonstrate that Nrf2 is critical in mediating the induction of glutamate-cysteine ligase modifier subunit (GCLM) and NAD(P)H quinone oxidoreductase-1 (NQO1) by oxPAPC in human endothelial cells, whereas the contribution to the induction of heme oxygenase-1 was less significant. The induction of GCLM and NQO1 was attenuated by reduction of electrophilic groups with sodium borohydrate, as well as treatment with thiol antioxidant N -acetylcysteine, suggesting that the thiol reactivity of oxPAPC is largely mediating its effect on Nrf2-responsive genes. Moreover, we show that oxidized phospholipid having a highly electrophilic isoprostane ring in its sn-2 position is a potent inducer of Nrf2 target genes. Finally, we demonstrate that the oxPAPC-inducible expression of heme oxygenase-1, GCLM, and NQO1 is lower in Nrf2-null than wild-type mouse carotid arteries in vivo. We suggest that the activation of Nrf2 by oxidized phospholipids provides a mechanism by which their deleterious effects are limited in the vasculature.

Oxidative stress is important in several pathologies, including cardiovascular diseases such as atherosclerosis and cardiac ischemia-reperfusion injury. An important mechanism for adaptation to oxidative stress is induction of genes through the antioxidant response element (ARE), which regulates the expression of antioxidant and cytoprotective genes via the transcription factor Nrf2 (nuclear factor E2-related factor 2). As Nrf2-regulated genes are induced during oxidant stress occurring, for example, in reperfusion after ischemia, we took a novel approach to exploit ARE for the development of oxidative stress-inducible gene therapy vectors. To this end, one, two or three ARE-containing regions from human NAD(P)H:quinone oxidoreductase-1, glutamate-cysteine ligase modifier subunit and mouse heme oxygenase-1 were cloned into a vector expressing luciferase under a minimal SV40 promoter. The construct, which was the most responsive to ARE-inducing agents, was chosen for further studies in which a lentiviral vector was produced for an efficient transfer to endothelial cells. Heme oxygenase-1 (HO-1), which has well-characterized anti-inflammatory properties, was used as the therapeutic transgene. In human endothelial cells, ARE-driven HO-1 overexpression inhibited nuclear factor-kappaB activation and subsequent vascular cell adhesion molecule-1 expression induced by tumor necrosis factor-alpha. We conclude that the ARE element is a promising alternative for the development of oxidative stress-inducible gene therapy vectors.

Enterolactone is a lignan formed by enterobacteria from precursors in plant foods. Due to its phenolic structure, it can act as an antioxidant, e.g. via direct scavenging of hydroxyl radical. Moreover, many, but not all, phenolic compounds can have indirect antioxidative effects through induction of heme oxygenase-1 (HO-1), which has antiinflammatory functions via production of antioxidants bilirubin and biliverdin as well as carbon monoxide, thereby contributing to cardiovascular health. Our aim was therefore to assess whether enterolactone has indirect antioxidative effects via induction of HO-1 in endothelial cells. The effect of enterolactone on HO-1 mRNA and protein expression in human umbilical vein endothelial cells (HUVEC) was analyzed by quantitative real-time PCR and western blot. The role of nuclear factor-E2-related factor 2 (Nrf2) in HO-1 induction by enterolactone was studied using small interfering RNA (siRNA) and chromatin immunoprecipitation (ChIP) methods. Our results showed that enterolactone induced HO-1 in HUVEC in a time- and concentrationdependent manner. The induction appeared to be mediated via the transcription factor Nrf2, as Nrf2 siRNA abolished the HO-1 induction by enterolactone. We also showed using ChIP that exposure to enterolactone increased the binding of Nrf2 to the promoter region of HO-1. In conclusion, enterolactone increases the expression of HO-1 via Nrf2, which may contribute to its vasculoprotective effects. J. Nutr. 138: 1263‐1268, 2008.