2α Levels Research Articles

Abstract IA007: A novel strategy to trigger ferroptosis in kidney cancer

Abstract Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of the von Hippel Lindau (VHL) gene resulting in constitutive activation of the hypoxia inducible factors, HIF-1α and HIF-2α. VHL loss and HIF activation promote lipid accumulation that increases the sensitivity of ccRCC cells to ferroptosis. Here, we reveal Iron Sulfur Cluster Assembly 2 (ISCA2) as a novel molecular target whose inhibition decreases HIF-1/2α levels and induces ferroptosis specifically in VHL-deficient cells. ISCA2 is a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex and ISCA2 inhibition either pharmacologically or using siRNA triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 inhibition also decreases HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1α translation through unknown mechanisms. Strikingly, pharmacological inhibition of ISCA2 using a novel, orally available ISCA2 inhibitor significantly reduced ccRCC xenograft growth in vivo, decreased HIF-α levels and increased lipid peroxidation, suggesting increased ferroptosis in vivo. Thus, the targeting of ISCA2 may be a promising therapeutic strategy to inhibit HIF-1/2α and to induce ferroptosis in pVHL deficient cells. Citation Format: Mei Yee Koh. A novel strategy to trigger ferroptosis in kidney cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr IA007.

ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma

Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of the von Hippel Lindau (VHL) gene, which results in the constitutive activation of the hypoxia inducible factors, HIF-1α and HIF-2α. Using a high throughput screen, we identify novel compounds that decrease HIF-1/2α levels and induce ferroptosis by targeting Iron Sulfur Cluster Assembly 2 (ISCA2), a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex. ISCA2 inhibition either pharmacologically or using siRNA decreases HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1α translation through unknown mechanisms. Additionally, ISCA2 inhibition triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 levels are decreased in ccRCC compared to normal kidney, and decreased ISCA2 levels are associated with pVHL loss and with sensitivity to ferroptosis induced by ISCA2 inhibition. Strikingly, pharmacological inhibition of ISCA2 using an orally available ISCA2 inhibitor significantly reduced ccRCC xenograft growth in vivo, decreased HIF-α levels and increased lipid peroxidation, suggesting increased ferroptosis in vivo. Thus, the targeting of ISCA2 may be a promising therapeutic strategy to inhibit HIF-1/2α and to induce ferroptosis in pVHL deficient cells.

Cancer-induced Changes In Endoplasmic Stress And Autophagy Responses After Mechanical Overload In ApcMin/+ Mice

Cancer cachexia is characterized by the severe weight and muscle loss. The endoplasmic reticulum (ER) stress and autophagy perpetuate skeletal muscle homeostasis. Tumor cells elicit ER stress and unfolded protein response (UPR) in skeletal muscle. The inhibition of ER stress also activates protein degradation and autophagy, which magnifies muscle mass declination. While UPR moderates mechanical stress, the adaptations in UPR and autophagy in cancer cachexia are unknown. PURPOSE: To determine whether tumor burden alters UPR and autophagy responses to mechanical overload in mice. METHODS: Age-matched male wild-type (WT, n = 5) and ApcMin/+ (Min, n = 5) mice were utilized. Synergist ablation (SA) was performed on the left leg, while the right leg served as an internal control. Seven days after mechanical overload, the plantaris muscles were removed. After tissue homogenization, routine Western blotting was conducted using 80 ~ 120 μg of the total protein. Independent t-test (WT vs. Min) and two-way ANOVA (genotype x treatment) were used for statistical analysis. The coefficient of determination (r^2) was used to examine a significant correlation. RESULTS: Min mice showed a decrease of BW by 9.8% compared to their peak BW. Similarly, sham control plantaris weight in Min mice was smaller than that of WT mice (18.7 ± 0.6 mg vs. 15.1 ± 0.3 mg for WT and Min, respectively). In both mice, mechanical overload increased the plantaris weights, but the response was small in Min mice (43.3 ± 5.8% vs. 21.3 ± 4.6%, p ≤ 0.05). Western blot analysis resulted in Min mice having a reduced phospho-p70S6K after mechanical overload than WT mice (4.2-fold vs. 1.9-fold, p ≤ 0.05). Phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α, 4.2-fold vs. 3.0-fold, p ≤ 0.05) and beclin (4.3-fold vs. 1.8-fold, p ≤ 0.05) levels were reduced in response to the stimulus in Min mice. A significant correlation was observed between p70S6K and eIF2α and between p70S6K and Beclin regardless of mouse genotype. The slopes of the regression curve in Min mice were smaller than WT mice (p ≤ 0.05). CONCLUSIONS: Reduced ER stress and autophagy responses to mechanical overload were associated with decreased mTORC1 signaling in cachectic mice. Supported by Louisiana BORSF (LEQSF(2017-20)-RD-A-22).

ORAI3 contributes to hypoxia-inducible factor 1/2α-sensitive colon cell migration.

Hypoxia is a pivotal initiator of tumor angiogenesis and growth through the stabilization of hypoxia-inducible factors (HIFs). This study set out to examine the involvement of HIF-1α and HIF-2α in colon cancer and ascertained whether ORAI3 was involved in the pathway. Patients and murine models as well as human colorectal adenocarcinoma tumor (CW2) cells were included to examine the levels of ORAI1/3 and HIF-1/2α levels. Calcium imaging was utilized to ascertain the activity of calcium channel. Scratch assay was used to assess the migration capacity of the cells. Tumors from murine colon cancer xenograft models and patients with colon cancer displayed high ORAI1/3 and HIF-1/2α levels. Hypoxia treatment, mimicking the tumor microenvironment in vitro, increased ORAI1/3 and HIF-1/2α expression as well as store-operated Ca2+ entry (SOCE). Of note is that HIF-1/2α silencing decreased SOCE, and HIF-1/2α overexpression facilitated SOCE. Furthermore, ORAI3 rather than ORAI1 expression was inhibited by HIF-1/2α silencing while increased by ML228. Luciferase assay also confirmed that ORAI3 was elevated in the presence of ML228, indicating the linkage between HIF-1/2α and ORAI3. Additionally, colony-forming potential and cell migration capacity were decreased in siHIF-1α and siHIF-2α as well as siORAI3 cells, and the facilitating effect of ML228 on cell migration and colony-forming potential was also decreased in siORAI3 CW-2 cells, which points out the importance of ORAI3 in HIF1/2α pathway. Our findings allow to conclude that both HIF-1α and HIF-2α facilitate ORAI3 expression, hence enhancing colon cancer progression.

Attracting Common Carp to a bait site with food reveals strong positive relationships between fish density, feeding activity, environmental DNA, and sex pheromone release that could be used in invasive fish management.

Measurement of environmental DNA (eDNA) is becoming a common technique to survey for rare and invasive fish due to its sensitivity and specificity. However, its utility is limited by an incomplete understanding of factors governing its sources and fates. Failure to detect eDNA is especially difficult to interpret so surveillance techniques often collect large numbers of samples across broad regions. If, however, fish could be reliably attracted to a single location where their eDNA could be easily measured that would be useful. We conducted a proof‐of‐concept study of this idea using invasive Common Carp. We monitored the distribution of radio‐tagged Carp and their eDNA across a 67 ha lake focusing at the bait site while a pheromone (Prostaglandin F2α; PGF 2α) was also measured to determine their reproductive condition. Prior to baiting, Carp were patchily distributed and while eDNA was occasionally detectable, it was patchy and only loosely associated with moderately dense groups of fish. Further, neither Carp, nor their eDNA were consistently measurable at the bait site and surrounding region, and the pheromone was not measurable at all. However, once baiting commenced, Carp started visiting the bait site and feeding, especially at night, where eDNA levels increased 500‐fold as fish densities doubled and PGF 2α became detectable. Fish presence, eDNA and pheromone concentrations peaked at night after 6 days, strongly suggesting feeding activity was the main driver. While the presence of eDNA precisely coincided with this aggregation, levels had dropped dramatically within 5 m. PGF 2α levels dropped less rapidly and demonstrated the presence of live mature fish. We suggest that food could be used to train fish to come to locations where they otherwise are too scarce to be reliably measured, increasing their eDNA release, making them measurable, and their reproductive condition also discernable by measuring pheromones.

Hypercholesterolemia increases plasma saturated and n-6 fatty acids altering prostaglandin homeostasis and promotes endothelial dysfunction in rabbits.

The present study evaluated the plasma fatty acid levels and the vascular prostaglandin (PG) release in a rabbit model of early hypercholesterolemia with endothelial dysfunction. Rabbits were fed either a control diet (CD) or a diet containing 1 % cholesterol (HD) for 5-6 weeks. The level of fatty acids was measured in plasma. The levels of PG and nitric oxide (NO) released from the aorta were also determined. Vascular morphology of the aorta was characterized by intima and media thickness measurements. The rabbits fed with HD had higher levels of arachidonic acid (ARA) and lower levels of oleic acid. The linoleic acid level was unchanged. PGI(2) and NO were diminished and PGF(2α) levels, the PGI(2)/TXA(2) ratio and the intima/media ratio were increased in rabbits fed with HD. In conclusion, feeding HD for a short period increased ARA plasma levels and unbalanced release of vasodilator/vasoconstrictor PG redirected the pathway to vasoconstrictor metabolite release. These lipid metabolism alterations in addition to the reduced NO levels and the moderate changes in the vascular morphology contributed to the endothelial dysfunction in this animal model. Therefore, the present findings support the importance of early correction or prevention of high cholesterol levels to disrupt the endothelial dysfunction process that leads to cardiovascular disease.

Fluvastatin-induced reduction of oxidative stress ameliorates diabetic cardiomyopathy in association with improving coronary microvasculature

Diabetic cardiomyopathy is associated with increased oxidative stress and vascular endothelial dysfunction, which lead to coronary microangiopathy. We tested whether statin-induced redox imbalance improvements could ameliorate diabetic cardiomyopathy and improve coronary microvasculature in streptozotocin-induced diabetes mellitus (DM). Fluvastatin (10 mg/kg/day) or vehicle was orally administered for 12 weeks to rats with or without DM. Myocardial oxidative stress was assessed by NADPH (nicotinamide adenine dinucleotide phosphate) oxidase subunit p22(phox) and gp91(phox) mRNA expression, and myocardial 8-iso-prostaglandin F(2α) (PGF(2α)) levels. Myocardial vascular densities were assessed using anti-CD31 and anti-α-smooth muscle actin (SMA) antibodies. Fluvastatin did not affect blood pressure or plasma cholesterol, but attenuated increased left ventricular (LV) minimum pressure and ameliorated LV systolic dysfunction in DM rats in comparison with vehicle (LV dP/dt, 8.9 ± 1.8 vs 5.4 ± 1.0 × 10(3) mmHg/s, P < 0.05). Myocardial oxidative stress increased in DM, but fluvastatin significantly reduced p22(phox) and gp91(phox) mRNA expression and myocardial PGF(2α) levels. Fluvastatin enhanced myocardial endothelial nitric oxide synthase (eNOS) protein levels and increased eNOS, vascular endothelial growth factor, and hypoxia-inducible factor-1α mRNA expression. CD31-positive cell densities were lower in DM rats than in non-DM rats (28.4 ± 13.2 vs 48.6 ± 4.3/field, P < 0.05) and fluvastatin restored the number (57.8 ± 18.3/field), although there were no significant differences in SMA-positive cell densities between groups. Fluvastatin did not affect cardiac function, oxidative stress, or vessel densities in non-DM rats. These results suggest that beneficial effects of fluvastatin on diabetic cardiomyopathy might result, at least in part, from improving coronary microvasculature through reduction in myocardial oxidative stress and upregulation of angiogenic factor.

Sex-specific differences in hyperoxic lung injury in mice: Implications for acute and chronic lung disease in humans

Sex-specific differences in pulmonary morbidity in humans are well documented. Hyperoxia contributes to lung injury in experimental animals and humans. The mechanisms responsible for sex differences in the susceptibility towards hyperoxic lung injury remain largely unknown. In this investigation, we tested the hypothesis that mice will display sex-specific differences in hyperoxic lung injury. Eight week-old male and female mice (C57BL/6J) were exposed to 72h of hyperoxia (FiO2>0.95). After exposure to hyperoxia, lung injury, levels of 8-iso-prostaglandin F2 alpha (8-iso-PGF 2α) (LC–MS/MS), apoptosis (TUNEL) and inflammatory markers (suspension bead array) were determined. Cytochrome P450 (CYP)1A expression in the lung was assessed using immunohistochemistry and western blotting. After exposure to hyperoxia, males showed greater lung injury, neutrophil infiltration and apoptosis, compared to air-breathing controls than females. Pulmonary 8-iso-PGF 2α levels were higher in males than females after hyperoxia exposure. Sexually dimorphic increases in levels of IL-6 (F>M) and VEGF (M>F) in the lungs were also observed. CYP1A1 expression in the lung was higher in female mice compared to males under hyperoxic conditions. Overall, our results support the hypothesis that male mice are more susceptible than females to hyperoxic lung injury and that differences in inflammatory and oxidative stress markers contribute to these sex-specific dimorphic effects. In conclusion, this paper describes the establishment of an animal model that shows sex differences in hyperoxic lung injury in a temporal manner and thus has important implications for lung diseases mediated by hyperoxia in humans.

Copper Deficiency Leads to Anemia, Duodenal Hypoxia, Upregulation of HIF-2α and Altered Expression of Iron Absorption Genes in Mice

Iron and copper are essential trace metals, actively absorbed from the proximal gut in a regulated fashion. Depletion of either metal can lead to anemia. In the gut, copper deficiency can affect iron absorption through modulating the activity of hephaestin - a multi-copper oxidase required for optimal iron export from enterocytes. How systemic copper status regulates iron absorption is unknown. Mice were subjected to a nutritional copper deficiency-induced anemia regime from birth and injected with copper sulphate intraperitoneally to correct the anemia. Copper deficiency resulted in anemia, increased duodenal hypoxia and Hypoxia inducible factor 2α (HIF-2α) levels, a regulator of iron absorption. HIF-2α upregulation in copper deficiency appeared to be independent of duodenal iron or copper levels and correlated with the expression of iron transporters (Ferroportin - Fpn, Divalent Metal transporter – Dmt1) and ferric reductase – Dcytb. Alleviation of copper-dependent anemia with intraperitoneal copper injection resulted in down regulation of HIF-2α-regulated iron absorption genes in the gut. Our work identifies HIF-2α as an important regulator of iron transport machinery in copper deficiency.

Role of TNF-α in the mechanisms responsible for preterm delivery induced by Stx2 in rats.

Infections with a strain of Escherichia coli producing Shiga toxins could be one of the causes of fetal morbidity and mortality in pregnant women. We have previously reported that Shiga toxin type 2 (Stx2) induces preterm delivery in pregnant rats. In this study, we evaluate the role of TNF-α, PGs and NO in the Stx2-induced preterm delivery. Pregnant rats were treated with Stx2 (0.7 ng g(-1)) and killed at different times after treatment. Placenta and decidua were used to analyse NOS activity by the conversion of L-[(14)C]arginine into L-[(14)C]citrulline, levels of PGE(2) and PGF(2α) assessed by radioimmunoassay, and cyclooxygenase (COX) proteins by Western blot. TNF-α level was analysed in serum by ELISA and by cytotoxicity in L929 cells. The inhibitor of inducible NOS, aminoguanidine, the COX-2 inhibitor, meloxicam, and the competitive inhibitor of TNF-α, etanercept, were used alone or combined to inhibit NO, PGs and TNF-α production respectively, to prevent Stx2-induced preterm delivery. Stx2 increased placental PGE(2) and decidual PGF(2α) levels as well as COX-2 expression in both tissues. Aminoguanidine and meloxicam delayed the preterm delivery time but did not prevent it. Etanercept blocked the TNF-α increase after Stx2 treatment and reduced the preterm delivery by approximately 30%. The combined action of aminoguanidine and etanercept prevented Stx2-induced preterm delivery by roughly 70%. Our results demonstrate that the increased TNF-α and NO induced by Stx2 were the predominant factors responsible for preterm delivery in rats.

Inflamm-Aging and Arachadonic Acid Metabolite Differences with Stage of Tendon Disease

The contribution of inflammation to the pathogenesis of tendinopathy and high prevalence of re-injury is not well established, although recent evidence suggests involvement of prostaglandins. We investigated the roles of prostaglandins and inflammation-resolving mediators in naturally occurring equine tendon injury with disease stage and age. Levels of prostaglandins E2 (PGE2), F2α (PGF2α), lipoxin A4 (LXA4) and its receptor FPR2/ALX were analysed in extracts of normal, sub-acute and chronic injured tendons. To assess whether potential changes were associated with altered PGE2 metabolism, microsomal prostaglandin E synthase-1 (mPGES-1), prostaglandin dehydrogenase (PGDH), COX-2 and EP4 receptor expression were investigated. The ability of tendons to resolve inflammation was determined by assessing FPR2/ALX expression in natural injury and IL-1β stimulated tendon explants.Alterations in the profile of lipid mediators during sub-acute injury included low PGE2 and elevated LXA4 levels compared to normal and chronic injuries. In contrast, PGF2α levels remained unchanged and were three-fold lower than PGE2. The synthetic capacity of PGE2 as measured by the ratio of mPGES-1:PGDH was elevated in sub-acute injury, suggesting aberrations in tendon prostaglandin metabolism, whilst COX-2 and EP4 receptor were unchanged. Paradoxically low tendon PGE2 levels in early injury may be attributed to increased local clearance via PGDH or the class switching of lipid mediators from the prostaglandin to the lipoxin axis. PGE2 is therefore implicated in the development of tendon inflammation and its ensuing resolution. Whilst there was no relationship between age and tendon LXA4 levels, there was an age-associated decline in FPR2/ALX receptor expression with concurrent increased PGE2 levels in injury. Furthermore, uninjured tendon explants from younger (<10 years) but not older horses (≥10 years) treated with IL-1β responded by increasing FPR2/ALX suggesting aged individuals exhibit a reduced capacity to resolve inflammation via FPR2/ALX, which may present a potential mechanism for development of chronic tendinopathy and re-injury.

Platelet activation in patients with the Raynaud phenomenon

The Raynaud phenomenon (RP) is an exaggerated and reversible vasospasm of small arteries triggered by cold or emotional stress. Primary RP (PRP) term is used when the underlying condition is unknown. An altered regulation in vascular tone and/or release of soluble mediators from activated platelets plays a role in PRP through an increased oxidative stress. We assessed platelet activation and oxidative stress in patients with PRP by measuring platelet PAC-1, an index of glycoprotein (Gp) IIb/IIIa receptor activation, thromboxane A(2) (TXA(2)), an index of platelet activation and 8-epi-prostaglandin F(2α) (8-epi-PGF(2α)), a marker of endogenous in vivo peroxidation. Eighteen asymptomatic patients with PRP (age 41.37 ± 16.94 years; 17 women, 1 man) and 18 healthy subjects (age of 35.11 ± 13.16 years; 16 women, 2 men) were studied. PAC-1 was analysed by flow cytometry while circulating TXB(2) , a stable metabolite of TXA(2) and 8-epi-PGF(2α) levels were assessed by ELISA kit. Our results show a significant platelet activation in PRP patients as indicated by increased PAC-1 expression (65.29 ± 15.24%; P < 0.001), TXB(2) (1477.83 ± 454.04 pg/mL; P= 0.003) and 8-epi-PGF(2α) circulating levels (42.50 ± 14.14 ng/mL; P < 0.001). An inverse correlation between the degree of PAC-1 expression and TXB(2) levels (r=-0.527; P= 0.02) was also found in PRP patients, suggesting that downregulation of GpIIb/IIIa receptor expression may occur during thrombocytopoiesis, as a consequence of the chronic exposure to increased TXB(2) concentration. Our study for the first time shows a marked activation of GpIIb/IIIa receptor in asymptomatic patients with PRP and supports antiplatelet therapy in PRP patients.

A Randomly-Controlled Study on the Cardiac Function at the Early Stage of Return to the Plains after Short-Term Exposure to High Altitude

High altitude acclimatization and adaptation mechanisms have been well clarified, however, high altitude de-adaptation mechanism remains unclear. In this study, we conducted a controlled study on cardiac functions in 96 healthy young male who rapidly entered the high altitude (3700 m) and returned to the plains (1500 m) after 50 days. Ninety eight healthy male who remained at low altitude were recruited as control group. The mean pulmonary arterial pressure (mPAP), left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), cardiac function index (Tei index) were tested. Levels of serum creatine kinase isoform MB (CK-MB), lactate dehydrogenase isoenzyme-1 (LDH-1), endothelin-1 (ET-1), nitrogen oxide (NO), serum hypoxia-inducible factor-1α (HIF-1α), 8-iso-prostaglandin F2α (8-iso PGF2α), superoxide dismutase (SOD) and malonaldehyde (MDA) were measured at an altitude of 3700 m and 1500 m respectively. The results showed that after short-term exposure to high altitude mPAP and Tei index increased significantly, while LVEF and LVFS decreased significantly. These changes were positively correlated with altitude. On the 15th day after the subjects returned to low altitude, mPAP, LVEF and LVFS levels returned to the same level as those of the control subjects, but the Tei index in the returned subjects was still significantly higher than that in the control subjects (P<0.01). We also found that changes in Tei index was positively correlated with mPAP, ET-1, HIF-1α and 8-iso PGF2α levels, and negatively correlated with the level of NO, LVEF, LVFS, CK-MB and LDH-1. These findings suggest that cardiac function de-adapts when returning to the plains after short-term exposure to high altitude and the function recovery takes a relatively long time.

γ-Glutamylcysteine inhibits oxidative stress in human endothelial cells

Aims γ-Glutamylcysteine (GGC) is a dipeptide and substrate for synthesis of the antioxidant glutathione (GSH), whose health promoting properties include reducing risks of oxidative stress-related injuries and diseases. The objective of this study was to investigate the efficacy of GGC on GSH synthesis and oxidative stress in human endothelial cells. Main methods We assessed oxidative stress, GSH, GSH synthetase (GSS) expression, and transcription factor DNA binding levels in human umbilical vein endothelial cells (HUVEC). Key findings We found significantly higher levels of PPARγ DNA binding and lower levels of GSH, GSS protein, NF-κB p65 DNA binding, thiobarbituric acid reactive substances (TBARS), and 8-epi-PGF 2α in a concentration-dependent manner, compared with the control. GSH and GSS protein levels showed a negative correlation with PPARγ DNA binding levels and positive correlation trends with NF-κB p65 DNA binding, TBARS, and 8-epi-PGF 2α levels. A putative binding site for NF-κB was found at 4 227 bases upstream from the transcription start site of GSS gene, but none for PPARs. These findings suggest the involvement of NF-κB in regulation of GSS expression. Subsequent GSH synthesis might be affected by the suppression of GSS expression in tested conditions. Significance Besides its substrate role in GSH synthesis, GGC may play a role in protection against oxidative stress by serving as an antioxidant and modulating the expression of protein(s) related to antioxidant defense. Thus, we speculate that GGC may serve as a novel intra- and intercellular therapeutic dipeptide for oxidative stress-related injuries and diseases.

Interstitial F2-Isoprostane 8-Iso-PGF2αAs a Biomarker of Oxidative Stress after Severe Human Traumatic Brain Injury

Oxidative stress is a major contributor to the secondary injury process after experimental traumatic brain injury (TBI). The importance of oxidative stress in the pathobiology of human TBI is largely unknown. The F(2)-isoprostane 8-iso-prostaglandin F(2α) (8-iso-PGF(2α)), synthesized in vivo through non-enzymatic free radical catalyzed peroxidation of arachidonic acid, is a widely used biomarker of oxidative stress in multiple disease states, including TBI and cerebral ischemia/reperfusion. Our hypothesis is that harvesting of biomarkers directly in the injured brain by cerebral microdialysis (MD) is advantageous because of its high spatial and temporal resolution compared to blood or cerebrospinal fluid sampling. The aim of this study was to test the feasibility of measuring 8-iso-PGF(2α) in MD, ventricular cerebrospinal fluid (vCSF), and plasma samples collected from patients with severe TBI, and to compare the MD signals with MD-glycerol, implicated as a biomarker of oxidative stress, as well as MD-glutamate, a biomarker of excitotoxicity. Six patients (4 men, 2 women) were included in the study, three of whom had a focal/mixed TBI, and three a diffuse axonal injury (DAI). Following the bedside analysis of routine MD biomarkers (glucose, lactate:pyruvate ratio, glycerol, and glutamate), two 12-h MD samples per day were used to analyze 8-iso-PGF(2α) from 24 h up to 8 days post-injury. The interstitial levels of 8-iso-PGF(2α) were markedly higher than the levels obtained from plasma and vCSF (p<0.05), supporting our hypothesis. The MD-8-iso-PGF(2α) levels correlated strongly (p<0.05) with MD-glycerol and MD-glutamate, which are widely used biomarkers of membrane phospholipid degradation/oxidative stress and excitotoxicity, respectively. This study demonstrates the feasibility of analyzing 8-iso-PGF(2α) in MD samples from the human brain. Our results support a close relationship between oxidative stress and excitotoxicity following human TBI. MD-8-iso-PGF(2α) in combination with MD-glycerol may be useful biomarkers of oxidative stress in the neurointensive care setting.

Expression of Concern: Role of LOX/COX pathways in 3‐nitropropionic acid‐induced Huntington's Disease‐like symptoms in rats: protective effect of licofelone

Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by a degeneration of striatal neurons. The possible role of COX and lipoxygenase (LOX) pathways has been well-documented in the pathology of several neurodegenerative disorders including HD. Licofelone is a competitive inhibitor of COX-1- and COX-2 and 5-LOX isoenzymes. Therefore, the present study was designed to investigate possible neuroinflammatory and apoptotic mechanisms in the neuroprotective effect of licofelone against 3-nitropropionic acid (3-NP)-induced HD-like symptoms in rats. Rats were administered 3-NP (10 mg·kg⁻¹ day⁻¹, i.p.) for 14 days. Licofelone (2.5, 5 and 10 mg·kg⁻¹, p.o.) was given once a day, 1 h before 3-NP treatment for 14 days. Body weight and behavioural parameters (locomotor and rotarod activity) were assessed on the 1st, 5th, 10th and 15th day post-3-NP administration. Malondialdehyde, nitrite concentration, endogenous antioxidant enzymes (superoxide dismutase and catalase levels), mitochondrial enzyme complexes, pro-inflammatory compounds (TNF-α, IL-6, NF-κB), PGs (PGE₂ and PGF(2α)) and caspase-3 activity were measured on day 15 in the striatum. Systemic 3-NP treatment significantly reduced body weight, locomotor activity, oxidative defence, mitochondrial enzyme complex activities and increased TNF-α, IL-6, caspase-3 activity, NF-κB and PGE₂ and PGF(2α) levels in the striatum. Licofelone (2.5, 5 and 10 mg·kg⁻¹) significantly attenuated the impairment in behavioural, biochemical and mitochondrial, pro-inflammatory and pro-apoptotic markers as compared with vehicle-treated group. The results demonstrate the involvement of pro-inflammatory compounds and the apoptotic cascade in the neuroprotective effect of licofelone against 3-NP-induced neurotoxicity.

Comparative Study on 15‐Ketodihydro‐PGF2α Plasma Concentrations in Newborn Horses, Donkeys and Calves

The aim of this study was to compare the plasma profiles of 15-ketodihydro-PGF(2α) (PGM) in healthy neonates of three different species from birth until the third week of life. Twenty-four horse foals, 12 donkey foals, and 9 calves were studied. Blood samples were collected at 10, 20 and 30 min after birth, at 3, 24 and 72 h after birth, and at 7, 10, 17 and 21 days of life. All mothers experienced normal gestation lengths and normal, spontaneous deliveries. All newborns were judged mature and viable. Hormone concentrations were higher (p < 0.05) in horse foals 20 and 30 min after birth compared to later samples, and at 10 min in donkey foals compared to later samples (p < 0.05). In calves, higher (p < 0.05) concentrations of PGM were observed 10, 20, 30 min and 3 hours from parturition compared to later samples. These findings may be related to increased fetal placental unit production during parturition, while the relatively high PGF(2α) levels in the days after parturition may be connected with their role in completing organ maturation. Despite the existing differences between these species, the statistical analysis did not discover significant differences in PGM profiles during the first 3 weeks of life in donkey, horse and cattle newborns. The low levels observed 10 days after birth are possibly due to a fast completion of maturational development in these species.

Effects of Resveratrol, a Grape Polyphenol, on Uterine Contraction and Ca2+Mobilization in Ratsin Vivoandin Vitro

Dysmenorrhea is directly related to elevate prostaglandin F (PGF)(₂α) levels. In Western medicine, this condition is treated using nonsteroidal antiinflammatory drugs. Because nonsteroidal antiinflammatory drugs produce many side effects, Chinese medicinal therapy is considered as a feasible alternative for treating dysmenorrhea. Many special physiological components used in Chinese medicine, such as resveratrol, have been isolated and identified. Resveratrol has many physiological functions, such as antioxidation and anticarcinogenic effects. However, the relationship between uterine smooth muscle contraction and resveratrol remains unknown. Here, we studied the in vitro and in vivo effects of resveratrol on uterine smooth muscle contraction. The uterus was separated from a female Sprague Dawley rat, and uterine smooth muscle contraction activity was measured and recorded. The results demonstrated that 1) resveratrol treatment inhibited PGF(₂α)-, oxytocin-, acetylcholine-, and carbachol-induced uterine contractions in rats; 2) resveratrol inhibited uterine contractions stimulated by the Ca²(+) channel activator (Bay K 8644) and depolarization in response to high K(+) (KCl); 3) resveratrol inhibited PGF(₂α)-induced increases in the [Ca²(+)]i in human uterine smooth muscle cells; 4) resveratrol could mimic Ca²(+) channel blockers to block Ca²(+) influx through voltage-operated Ca²(+) channels in the plasma membrane; and 5) resveratrol inhibited PGF(₂α)-induced uterine contractions in rats in vivo. Resveratrol inhibited uterine contractions induced by PGF(₂α) and high K(+) in a concentration-dependent manner in vitro; furthermore, it inhibited Ca²(+)-dependent uterine contractions. Thus, resveratrol consistently suppressed the increases in intracellular Ca²(+) concentrations ([Ca²(+)]i) induced by PGF(₂α) and high K(+) concentrations. It can be assumed that resveratrol probably inhibited uterine contraction by blocking external Ca²(+) influx, leading to decreased [Ca²(+)]i. Therefore, resveratrol can be considered as a feasible alternative therapeutic agent for dysmenorrhea.

Asymmetric dimethylarginine (ADMA) levels are increased in patients with fibromyalgia: Correlation with tumor necrosis factor-α (TNF-α) and 8-iso-prostaglandin F 2α (8-iso-PGF 2α)

Objective The aim of the study was to investigate serum levels of asymmetric dimethylarginine (ADMA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and plasma levels of 8-iso-prostaglandin F 2α (8-iso-PGF 2α) in patients with fibromyalgia. Design and method Twenty-seven patients with fibromyalgia and twenty healthy controls were enrolled in this study. ADMA, TNF-α, IL-6 and 8-iso-PGF 2α levels were measured by enzyme-linked immunosorbent assay (ELISA). Results Serum levels of ADMA and TNF-α and plasma levels 8-iso-PGF 2α were significantly increased in patients with fibromyalgia compared to controls. However, no significant difference was observed in IL-6 levels between the two groups. ADMA concentrations were positively correlated with TNF-α and 8-iso-PGF 2α levels in patients with fibromyalgia. Conclusion This is the first study reporting that ADMA levels are significantly elevated in patients with fibromyalgia in association with increased 8-iso-PGF 2α and TNF-α concentrations. Thereby, ADMA could be suggested as a reliable marker of endothelial dysfunction in patients with fibromyalgia.

Peripartal plasma concentrations of 15-ketodihydro-PGF 2α, cortisol, progesterone and 17-β-estradiol in Martina Franca jennies

The transition from intra- to extrauterine environment represents a very delicate phase, in which the successful coordination of maturation is strictly connected with several hormonal changes during the last weeks of gestation and at parturition. While the peripartal endocrinology in the mare has been deeply investigated, the peripartal hormonal changes in the jenny need further evaluation. The aim of this study is to evaluate the mean 15-ketodihydro-PGF 2α (PGFM), cortisol (C), progesterone (P4), and 17β-estradiol (E2) levels during the peripartal period in this species. Ten Martina Franca jennies, with normal gestational length and parturition, were enrolled. From each jenny, blood was collected twice a day from 10 d before to 7 d after parturition and from the plasma obtained PGFM, C, P4 and E2 were analyzed by RIA. Higher, constant PGFM concentrations were observed in the pre-foaling days compared to the decreasing levels detected the days after delivery, as previously observed in the mare. During the whole period of observation no significant differences in plasma C levels were detected. In contrast to the mare, P4 has always been detectable and the highest level found at −2.5 days was significantly different compared to samples obtained between −10 and −4.5 days and between 1.5 and 7 days after foaling. Finally, E2 showed higher concentrations before foaling, with the highest values between −3 and −1.5 days, decreasing only one day before foaling. A positive correlation was found between PGFM and P4, during the last 4 days of gestation, while a positive correlation between PGFM and E2 was observed during the prepartum. Despite some similarities with the mare exist, differences have been found in P4 and E2 profiles, underlining once more the differences in the physiology of this two species.