Polyethylene Glycol-catalase Research Articles

Hydrogen Peroxide Promotes the Production of Radiation-Derived EVs Containing Mitochondrial Proteins.

In spite of extensive successes, cancer recurrence after radiation treatment (RT) remains one of the significant challenges in the cure of localized prostate cancer (PCa). This study focuses on elucidating a novel adaptive response to RT that could contribute to cancer recurrence. Here, we used PC3 cell line, an adenocarcinoma from a bone metastasis and radio-resistant clone 695 cell line, which survived after total radiation dose of 66 Gy (2 Gy × 33) and subsequently regrew in nude mice after exposure to fractionated radiation at 10 Gy (2 Gy × 5). Clone 695 cells not only showed an increase in surviving fraction post-radiation but also an increase in hydrogen peroxide (H2O2) production when compared to PC3 cells. At the single cell level, confocal microscope images coupled with IMARIS rendering software demonstrate an increase in mitochondrial mass and membrane potential in clone 695 cells. Utilizing the Seahorse XF96 instrument to investigate mitochondrial respiration, clone 695 cells demonstrated a higher basal Oxygen Consumption Rate (OCR), ATP-linked OCR, and proton leak compared to PC3 cells. The elevation of mitochondrial function in clone 695 cells is accompanied by an increase in mitochondrial H2O2 production. These data suggest that H2O2 could reprogram PCa's mitochondrial homeostasis, which allows the cancer to survive and regrow after RT. Upon exposure to RT, in addition to ROS production, we found that RT induces the release of extracellular vesicles (EVs) from PC3 cells (p < 0.05). Importantly, adding H2O2 to PC3 cells promotes EVs production in a dose-dependent manner and pre-treatment with polyethylene glycol-Catalase mitigates H2O2-mediated EV production. Both RT-derived EVs and H2O2-derived EVs carried higher levels of mitochondrial antioxidant proteins including, Peroxiredoxin 3, Glutathione Peroxidase 4 as well as mitochondrial-associated oxidative phosphorylation proteins. Significantly, adding isolated functional mitochondria 24 h prior to RT shows a significant increase in surviving fractions of PC3 cells (p < 0.05). Together, our findings reveal that H2O2 promotes the production of EVs carrying mitochondrial proteins and that functional mitochondria enhance cancer survival after RT.

S1P (Sphingosine-1-Phosphate)-Induced Vasodilation in Human Resistance Arterioles During Health and Disease.

Preclinical studies suggest that S1P (sphingosine-1-phosphate) influences blood pressure regulation primarily through NO-induced vasodilation. Because microvascular tone significantly contributes to mean arterial pressure, the mechanism of S1P on human resistance arterioles was investigated. We hypothesized that S1P induces NO-mediated vasodilation in human arterioles from adults without coronary artery disease (non-coronary artery disease) through activation of 2 receptors, S1PR1 (S1P receptor 1) and S1PR3 (S1P receptor 3). Furthermore, we tested whether this mechanism is altered in vessels from patients diagnosed with coronary artery disease. Human arterioles (50-200 µm in luminal diameter) were dissected from otherwise discarded surgical adipose tissue, cannulated, and pressurized. Following equilibration, resistance vessels were preconstricted with ET-1 (endothelin-1) and changes in internal diameter to increasing concentrations of S1P (10-12 to 10-7 M) in the presence or absence of various inhibitors were measured. S1P resulted in significant dilation that was abolished in vessels treated with S1PR1 and S1PR3 inhibitors and in vessels with reduced expression of each receptor. Dilation to S1P was significantly reduced in the presence of the NOS (NO synthase) inhibitor Nω-nitro-L-arginine methyl ester and the NO scavenger 2-4-(carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Interestingly, dilation was also significantly impaired in the presence of PEG-catalase (polyethylene glycol-catalase), apocynin, and specific inhibitors of NOX (NADPH oxidases) 2 and 4. Dilation in vessels from patients diagnosed with coronary artery disease was dependent on H2O2 alone which was only dependent on S1PR3 activation. These translational studies highlight the inter-species variation observed in vascular signaling and provide insight into the mechanism by which S1P regulates microvascular resistance and ultimately blood pressure in humans.

Crotalus atrox Induced Cellular Injury Triggers an Oxidative Stress Gene Response in HEK‐293T Cells

Wound and regeneration involves a series of complex processes that can induce oxidative stress by elevating intracellular reactive oxygen species (ROS) levels. In this study, we used RNA‐seq and qPCR to examine the molecular signature of Human Embryonic Kidney cells (HEK 293T) stimulated with Crotalus atrox venom. NGS data was assembled and annotated using BLASTx and UNIPROT. Results showed 243 genes were differentially expressed (106 upregulated, 137 downregulated) between Crotalus atrox stimulated and non‐stimulated cells (p<0.05). HEK 293T stimulated with Crotalus atrox venom for 4 hours showed an increase in the expression of oxidative stress response genes (e.g., TOMM6, EGR (1,2,3), HSP70 (1A, 1B), UPK3BL (1,2)), and protein synthesis (ETV5, EMP1) (fc>2.0). Significantly downregulated (log2(fc)<‐1.20) genes were associated with protein coding (e.g., ABCG1, ABCA1, PPARA, HMGA2, TLCD5). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified 8 significant KEGG pathways, which included an amphetamine addiction pathway (p<0.03). Genes identified in this pathway such as FosB proto‐oncogene (FosB) and activity regulated cytoskeleton protein (ARC) are connected to oxidative stress responses. Quantitative PCR (qPCR) was used to evaluate several genes identified from RNA‐seq data. Cells stimulated with Crotalus atrox venom for 4 hours induced an up‐regulation of HSPA1A (2.49±0.49), HSPA1B (2.04±0.12), and cytochrome P450 1A1 (CYP1A1) (3.24±0.33), matching NGS data trends. Pre‐incubation with the zinc chelator tris(2‐pyridylmethyl) amine (TPA) prior to venom stimulation induced a significant up‐regulation in HSPA1B (13.2±0.04). Extending Crotalus atrox venom stimulation to 8 hours increased the gene expression of metallothionein 1X (MT1X, 4.68±0.26) and peroxiredoxin‐4 (PRDX4, 3.34±0.11) compared to non‐stimulated cells. Interestingly, pre‐incubating cells with polyethylene glycol (PEG) catalase then stimulating cells with Crotalus atrox venom for 8 hours produced PRDX4 and MT1X gene expression values that were not significantly different from the non‐stimulated control. In contrast, glutathione peroxidase‐1 (GPx‐1) was significantly downregulated (0.27±0.16) after an 8 hour stimulation with Crotalus atrox venom, compared to non‐stimulated cells. Our data suggests Crotalus atrox induced cellular injury involves several gene families connected to oxidative stress responses.

Oxidative Stress-induced Autophagy Compromises Stem Cell Viability.

Stem cell therapies have emerged as a promising treatment strategy for various diseases characterized by ischemic injury such as ischemic stroke. Cell survival after transplantation remains a critical issue. We investigated the impact of oxidative stress, being typically present in ischemically challenged tissue, on human dental pulp stem cells (hDPSC) and human mesenchymal stem cells (hMSC). We used oxygen-glucose deprivation (OGD) to induce oxidative stress in hDPSC and hMSC. OGD-induced generation of O2•- or H2O2 enhanced autophagy by inducing the expression of activating molecule in BECN1-regulated autophagy protein 1 (Ambra1) and Beclin1 in both cell types. However, hDPSC and hMSC pre-conditioning using reactive oxygen species (ROS) scavengers significantly repressed the expression of Ambra1 and Beclin1 and inactivated autophagy. O2•- or H2O2 acted upstream of autophagy, and the mechanism was unidirectional. Furthermore, our findings revealed ROS-p38-Erk1/2 involvement. Pre-treatment with selective inhibitors of p38 and Erk1/2 pathways (SB202190 and PD98059) reversed OGD effects on the expression of Ambra1 and Beclin1, suggesting that these pathways induced oxidative stress-mediated autophagy. SIRT3 depletion was found to be associated with increased oxidative stress and activation of p38 and Erk1/2 MAPKs pathways. Global ROS inhibition by NAC or a combination of polyethylene glycol-superoxide dismutase (PEG-SOD) and polyethylene glycol-catalase (PEG-catalase) further confirmed that O2•- or H2O2 or a combination of both impacts stems cell viability by inducing autophagy. Furthermore, autophagy inhibition by 3-methyladenine (3-MA) significantly improved hDPSC viability. These findings contribute to a better understanding of post-transplantation hDPSC and hMSC death and may deduce strategies to minimize therapeutic cell loss under oxidative stress.

Mechanism of the switch from NO to H2O2 in endothelium-dependent vasodilation in diabetes.

Coronary microvascular dysfunction is prevalent among people with diabetes and is correlated with cardiac mortality. Compromised endothelial-dependent dilation (EDD) is an early event in the progression of diabetes, but its mechanisms remain incompletely understood. Nitric oxide (NO) is the major endothelium-dependent vasodilatory metabolite in the healthy coronary circulation, but this switches to hydrogen peroxide (H2O2) in coronary artery disease (CAD) patients. Because diabetes is a significant risk factor for CAD, we hypothesized that a similar NO-to-H2O2 switch would occur in diabetes. Vasodilation was measured ex vivo in isolated coronary arteries from wild type (WT) and microRNA-21 (miR-21) null mice on a chow or high-fat/high-sugar diet, and B6.BKS(D)-Leprdb/J (db/db) mice using myography. Myocardial blood flow (MBF), blood pressure, and heart rate were measured in vivo using contrast echocardiography and a solid-state pressure sensor catheter. RNA from coronary arteries, endothelial cells, and cardiac tissues was analyzed via quantitative real-time PCR for gene expression, and cardiac protein expression was assessed via western blot analyses. Superoxide was detected via electron paramagnetic resonance. (1) Ex vivo coronary EDD and in vivo MBF were impaired in diabetic mice. (2) Nω-Nitro-L-arginine methyl ester, an NO synthase inhibitor (L-NAME), inhibited ex vivo coronary EDD and in vivo MBF in WT. In contrast, polyethylene glycol-catalase, an H2O2 scavenger (Peg-Cat), inhibited diabetic mouse EDD ex vivo and MBF in vivo. (3) miR-21 was upregulated in diabetic mouse endothelial cells, and the deficiency of miR-21 prevented the NO-to-H2O2 switch and ameliorated diabetic mouse vasodilation impairments. (4) Diabetic mice displayed increased serum NO and H2O2, upregulated mRNA expression of Sod1, Sod2, iNos, and Cav1, and downregulated Pgc-1α in coronary arteries, but the deficiency of miR-21 reversed these changes. (5) miR-21-deficient mice exhibited increased cardiac PGC-1α, PPARα and eNOS protein and reduced endothelial superoxide. (6) Inhibition of PGC-1α changed the mRNA expression of genes regulated by miR-21, and overexpression of PGC-1α decreased the expression of miR-21 in high (25.5mM) glucose treated coronary endothelial cells. Diabetic mice exhibit a NO-to-H2O2 switch in the mediator of coronary EDD, which contributes to microvascular dysfunction and is mediated by miR-21. This study represents the first mouse model recapitulating the NO-to-H2O2 switch seen in CAD patients in diabetes.

Anti-inflammatory and anti-fibrotic effects of modafinil in nonalcoholic liver disease

Small- and intermediate-conductance Ca2+-activated K+ channels, KCa2.3 and KCa3.1, are involved in cellular signaling processes associated with inflammation and fibrosis. KCa2.3 and KCa3.1 are upregulated by proinflammatory cytokines and profibrotic growth factors. Cyclic AMP, which downregulates KCa2.3 and KCa3.1, is elevated by modafinil in cells; accordingly, we investigated whether modafinil exerts anti-inflammatory and anti-fibrotic responses via KCa2.3- and KCa3.1-mediated pathways in high-fat diet (HFD)- or thioacetamide-induced liver disease models in mice. Modafinil was administered orally in the form of a racemate, (R)-isomer, or (S)-isomer. We also determined whether the treatment targeted the profibrotic activity of hepatic stellate cells using immortalized human hepatic stellate cells (LX-2 cells). Modafinil improved HFD- or thioacetamide-induced changes compared to the control, leading to a reduced inflammatory response, collagen deposition, and α-smooth muscle actin expression both in vivo and in vitro. However, modafinil did not relieve HFD-induced steatosis. There were no significant differences in the effects of the (R)- and (S)-isomers of modafinil. KCa2.3 and KCa3.1 were upregulated and catalase was downregulated in liver tissues from thioacetamide- or HFD-induced liver disease models or in TGF-β-treated LX-2 cells. TGF-β-induced upregulation of KCa2.3, KCa3.1, collagen, and α-smooth muscle actin and downregulation of catalase were reversed by modafinil, polyethylene glycol catalase, N-acetylcysteine, siRNA against KCa2.3 or KCa3.1, and Epac inhibitors. Our investigation revealed that modafinil attenuated inflammatory and fibrotic progression via KCa2.3- and KCa3.1-mediated pathways in nonalcoholic hepatitis, suggesting that inhibiting KCa2.3- and KCa3.1-mediated signaling may serve as a novel therapeutic approach for inflammatory and fibrotic liver diseases.

Pro-Oxidant Effect of Resveratrol on Human Breast Cancer MCF-7 Cells is Associated with CK2 Inhibition

Casein kinase 2 (CK2) plays a critical role in the proliferation and apoptosis of cancer cells. Resveratrol is a bioactive compound with anticancer and anti-inflammatory effects. This study investigated the pro-oxidant cytotoxic effects of resveratrol in association with the inhibition of CK2 activity on human breast carcinoma cells MCF-7. We showed that resveratrol and TBB, an inhibitor of CK2, decreased cell viability in a concentration dependent manner with an IC50 value of 238 µM and 106 µM after 24 h, of treatment, respectively. Resveratrol and TBB decreased CK2 activity by 1.6 and 1.4-fold, respectively, and both significantly decreased mitochondrial membrane potential. However, only resveratrol increased reactive oxygen species (ROS) levels by 1.7-fold as opposed to TBB, which did not affect ROS levels. Indeed, incubating MCF-7 cells with the antioxidant polyethylene glycol-catalase (PEG-CAT) preserved cell viability from the cytotoxic effects of resveratrol, but not from TBB toxicity. This effect seemed to be related to PEG-CAT ability to prevent CK2 inhibition induced by resveratrol incubation. In conclusion, this study demonstrated that the cytotoxic effect of resveratrol on MCF-7 cells might be associated with its pro-oxidant action, which inhibited CK2 activity, affecting cell viability and mitochondrial function.

Differential responses of resistance arterioles to elevated intraluminal pressure in blacks and whites.

Black Americans have an earlier onset, higher average blood pressure, and higher rates of hypertension-related mortality and morbidity, compared to whites. The racial difference may be related to microvasculature, the major regulatory site of blood pressure. The goal of this study was to compare the response of resistance vessels to high intraluminal pressure between black and white participants. A total of 38 vessels were obtained from human fat samples [21 black, 17 white; mean age 32 ± 12 yr and body mass index (BMI) 26.9 ± 4.9; between-group P ≥ 0.05] and included in this study. Internal diameter was measured in response to the flow induced by various pressure gradients (Δ10, Δ20, Δ40, Δ60, and Δ100 cmH2O), and flow-induced dilation (FID) was calculated before and after high intraluminal pressure (150 cmH2O). Before high intraluminal pressure, FID was not different between blacks and whites (P = 0.112). After exposure to high intraluminal pressure, FID was reduced at every pressure gradient in vessels from blacks (P < 0.001), whereas FID did not change in white participants except at Δ100 cmH2O. When incubated with the hydrogen peroxide (H2O2) scavenger polyethylene glycol-catalase (PEG-catalase), the FID response in vessels from black, but not white, individuals was significantly reduced and the magnitude was higher at normal pressure relative to high pressure. Our findings suggest that the vessels from self-identified black individuals are more susceptible to microvascular dysfunction following transient periods of high intraluminal pressure compared to whites and show greater dependence on H2O2 as a main contributor to FID at normal pressures.NEW & NOTEWORTHY Microvascular function regulates blood pressure and may contribute to racial differences in the incidence and prevalence of hypertension and other cardiovascular diseases. Here, we show that using an ex vivo model of resistance arterioles isolated from human gluteal fat tissue, flow-induced dilation is not different between black and white participants. However, when exposed to transient increases in intraluminal pressure, the flow-induced dilation in resistance arterioles from black participants demonstrated greater reductions relative to their white counterparts, indicating a higher sensitivity to pressure change in the microvasculature.

Investigation of Sphingosine‐1‐Phosphate‐induced Vasodilation in the Human Microvasculature

The sphingolipid sphingosine-1-phosphate (S1P) has emerged as a regulator of microvascular tone in animals, however its role in the human microcirculation remains unknown. Pre-clinical studies suggest that S1P-induced activation of two endothelial-specific receptors, S1PR1 and S1PR3,elicits nitric-oxide (NO)-mediated vasodilation. As opposed to S1PR1, the S1PR3 pathway also activates NADPH oxidase (NOX) to produce reactive oxygen species (ROS). We therefore hypothesized that S1P induces vasodilation in the human microvasculature through activation of S1PR1 and S1PR3 in both a ROS-independent and ROS-dependent manner, respectively. Human microvessels (100-200µm in diameter) were prepared for videomicroscopy. Following equilibration, arterioles were pre-constricted with endothelin-1 to 30-70% of their passive diameters. Luminal diameter was measured and recorded in regular intervals (1 min) in response to increasing concentrations of S1P (10-12 to 10-6 M) in the presence or absence of the S1PR1 receptor antagonist W146 (10-5M), S1PR3 antagonist CAY10444 (10-5 M), nitric oxide synthase inhibitor Nω-nitro-ʟ-arginine (L-NAME, 10-4 M), NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO, 10-4 M), polyethylene glycol-catalase (peg-Cat, 500U/ml), NADPH oxidase inhibitor apocynin (3x10-4 M), NOX-2 inhibitor GSK2795039 (10-6 M) or the NOX-4 inhibitor GKT137831 (10−6 M). S1P induced vasodilation in a dose-responsive manner with a maximum dilation of 56.5%±4.9, n=12 (mean±SEM). Dilation was abolished during inhibition of S1PR1 (1.5%±5.2, n=4) and was reduced during inhibition of S1PR3 (19.7%±8.7, n=6). Both L-NAME and c-PTIO inhibited S1P-induced dilation (13.0%±7.5, n=4 and 11.2%±3.3, n=4, respectively). Interestingly, dilation was nearly completely inhibited by peg-Cat (11%±6.6, n=4), apocynin (12.5%±4.4, n=3) and the NOX-4 inhibitor (7.2%±3.1, n=4). Dilation was also partly reduced during inhibition of NOX-2 (25.6%±7.8, n=4). These data suggest that S1P-induced dilation occurs through activation of S1PR1 and S1PR3 through formation of NO and NOX-4-generated H2O2. These translational studies highlight the inter-species variation observed in vascular signaling and provide insight into the mechanism by which S1P regulates microvascular resistance in humans.

Restoration of afferent arteriolar autoregulatory behavior in ischemia-reperfusion injury in rat kidneys.

Renal autoregulation is critical in maintaining stable renal blood flow (RBF) and glomerular filtration rate (GFR). Renal ischemia-reperfusion (IR)-induced kidney injury is characterized by reduced RBF and GFR. The mechanisms contributing to renal microvascular dysfunction in IR have not been fully determined. We hypothesized that increased reactive oxygen species (ROS) contributed to impaired renal autoregulatory capability in IR rats. Afferent arteriolar autoregulatory behavior was assessed using the blood-perfused juxtamedullary nephron preparation. IR was induced by 60 min of bilateral renal artery occlusion followed by 24 h of reperfusion. Afferent arterioles from sham rats exhibited normal autoregulatory behavior. Stepwise increases in perfusion pressure caused pressure-dependent vasoconstriction to 65 ± 3% of baseline diameter (13.2 ± 0.4 μm) at 170 mmHg. In contrast, pressure-mediated vasoconstriction was markedly attenuated in IR rats. Baseline diameter averaged 11.7 ± 0.5 µm and remained between 90% and 101% of baseline over 65-170 mmHg, indicating impaired autoregulatory function. Acute antioxidant administration (tempol or apocynin) to IR kidneys for 20 min increased baseline diameter and improved autoregulatory capability, such that the pressure-diameter profiles were indistinguishable from those of sham kidneys. Furthermore, the addition of polyethylene glycol superoxide dismutase or polyethylene glycol-catalase to the perfusate blood also restored afferent arteriolar autoregulatory responsiveness in IR rats, indicating the involvement of superoxide and/or hydrogen peroxide. IR elevated mRNA expression of NADPH oxidase subunits and monocyte chemoattractant protein-1 in renal tissue homogenates, and this was prevented by tempol pretreatment. These results suggest that ROS accumulation, likely involving superoxide and/or hydrogen peroxide, impairs renal autoregulation in IR rats in a reversible fashion.NEW & NOTEWORTHY Renal ischemia-reperfusion (IR) leads to renal microvascular dysfunction manifested by impaired afferent arteriolar autoregulatory efficiency. Acute administration of scavengers of reactive oxygen species, polyethylene glycol-superoxide dismutase, or polyethylene glycol-catalase following renal IR restored afferent arteriolar autoregulatory capability in IR rats, indicating that renal IR led to reversible impairment of afferent arteriolar autoregulatory capability. Intervention with antioxidant treatment following IR may improve outcomes in patients by preserving renovascular autoregulatory function and potentially preventing the progression to chronic kidney disease after acute kidney injury.

Critical Interaction Between Telomerase and Autophagy in Mediating Flow-Induced Human Arteriolar Vasodilation.

Coronary artery disease (CAD) is associated with a compensatory switch in mechanism of flow-mediated dilation (FMD) from nitric oxide (NO) to H2O2. The underlying mechanism responsible for the pathological shift is not well understood, and recent reports directly implicate telomerase and indirectly support a role for autophagy. We hypothesize that autophagy is critical for shear stress-induced release of NO and is a crucial component of for the pathway by which telomerase regulates FMD. Approach and Results: Human left ventricular, atrial, and adipose resistance arterioles were collected for videomicroscopy and immunoblotting. FMD and autophagic flux were measured in arterioles treated with autophagy modulators alone, and in tandem with telomerase-activity modulators. LC3B II/I was higher in left ventricular tissue from patients with CAD compared with non-CAD (2.8±0.2 versus 1.0±0.2-fold change; P<0.05), although p62 was similar between groups. Shear stress increased Lysotracker fluorescence in non-CAD arterioles, with no effect in CAD arterioles. Inhibition of autophagy in non-CAD arterioles induced a switch from NO to H2O2, while activation of autophagy restored NO-mediated vasodilation in CAD arterioles. In the presence of an autophagy activator, telomerase inhibitor prevented the expected switch (Control: 82±4%; NG-Nitro-l-arginine methyl ester: 36±5%; polyethylene glycol catalase: 80±3). Telomerase activation was unable to restore NO-mediated FMD in the presence of autophagy inhibition in CAD arterioles (control: 72±7%; NG-Nitro-l-arginine methyl ester: 79±7%; polyethylene glycol catalase: 38±9%). We provide novel evidence that autophagy is responsible for the pathological switch in dilator mechanism in CAD arterioles, demonstrating that autophagy acts downstream of telomerase as a common denominator in determining the mechanism of FMD.

Hypoxia-inducible factor-1 mediates pancreatic β-cell dysfunction by intermittent hypoxia.

The role of hypoxia-inducible factor (HIF)-1 in pancreatic β-cell response to intermittent hypoxia (IH) was examined. Studies were performed on adult wild-type (WT), HIF-1α heterozygous (HET), β-cell-specific HIF-1-/- mice and mouse insulinoma (MIN6) cells exposed to IH patterned after blood O2 profiles during obstructive sleep apnea. WT mice treated with IH showed insulin resistance, and pancreatic β-cell dysfunction manifested as augmented basal insulin secretion, and impaired glucose-stimulated insulin secretion and these effects were absent in HIF-1α HET mice. IH increased HIF-1α expression and elevated reactive oxygen species (ROS) levels in β-cells of WT mice. The elevated ROS levels were due to transcriptional upregulation of NADPH oxidase (NOX)-4 mRNA, protein and enzymatic activity, and these responses were absent in HIF-1α HET mice as well as in β-HIF-1-/- mice. IH-evoked β-cell responses were absent in adult WT mice treated with digoxin, an inhibitor of HIF-1α. MIN6 cells treated with in vitro IH showed enhanced basal insulin release and elevated HIF-1α protein expression, and these effects were abolished with genetic silencing of HIF-1α. IH increased NOX4 mRNA, protein, and enzyme activity in MIN6 cells and disruption of NOX4 function by siRNA or scavenging H2O2 with polyethylene glycol catalase blocked IH-evoked enhanced basal insulin secretion. These results demonstrate that HIF-1-mediated transcriptional activation of NOX4 and the ensuing increase in H2O2 contribute to IH-induced pancreatic β-cell dysfunction.

Pegylated-Catalase Is Protective in Lung Ischemic Injury and Oxidative Stress

Lung transplant ischemia-reperfusion injury is typified by toxic metabolites and oxygen free radicals leading to worse graft function. Catalase is an enzyme involved in oxidative-stress detoxification. We hypothesize that direct delivery of highly concentrated polyethylene glycol-catalase (PEG-CAT) during normothermic exvivo lung perfusion (EVLP) significantly reduces ischemia-reperfusion injury. To demonstrate protection, primary culture porcine endothelial cells were treated with PEG-CAT (0 to 1250 U/mL) in a model of oxidative stress (400 μM H2o2). Invivo, rat lungs were subjected to 0 hours or 1 hour of warm ischemic injury and 2 hours of EVLP with or without PEG-CAT. Perfusate was collected throughout the perfusion duration and tissue was collected at the end. Tissue and perfusate underwent analysis for markers of apoptosis and a biometric signature of lung health. Uptake of PEG-CAT into primary endothelial cells was demonstrated with Alexa Fluor 488-labeled PEG-CAT. Oxidatively stressed cells pretreated with PEG-CAT had significantly decreased cytotoxicity and caspase 3/7 activity and increased cell viability and cell membrane integrity. In a rat model of warm ischemia with EVLP, PEG-CAT improved allograft viability as measured by indications of cell membrane integrity (lactate dehydrogenase and hyaluronic acid), presence of vasoconstrictive peptides (endothelin-1 and big endothelin-1) released from endothelial cells, and reduced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling). Invitro and exvivo, PEG-CAT protects against oxidative stress-induced cytotoxicity, maintains cellular metabolism, and mitigates lung ischemia-reperfusion in an experimental model. Together, these data suggest that PEG-CAT is a potential therapeutic target for donor organs at risk for ischemia-reperfusion injury.

Sphingosine‐1‐Phosphate Receptor 1 Mediated Vasodilation in Human Arterioles

Precise regulation of vascular resistance is critical to maintain proper tissue perfusion. Recent data in animal models have shown that sphingolipids, a ubiquitous class of bioactive lipid messengers, can influence vasomotor tone via dilation or constriction. It has been suggested that sphingosine‐1‐phosphate (S1P) elicits vasodilation through generation of endothelial cell nitric oxide (NO) via activation of sphingosine‐1‐phosphate receptor 1 (S1PR1). We therefore examined the hypothesis that S1P is a regulator of human microvascular tone and elicits dilation via S1PR1‐induced increase in NO. Arterioles from healthy human adipose tissue were dissected and prepared for videomicroscopy, equilibrated and pre‐constricted with endothelin 1. Changes in arteriolar luminal diameter were recorded in response to increasing concentrations of S1P (10−12 to 10−6M) in the presence or absence of the S1PR1 receptor antagonist W146 (10−5M), nitric oxide synthase (NOS) inhibitor Nω‐nitro‐ l‐arginine (L‐NAME, 10−4M) or hydrogen peroxide (H2O2) scavenger polyethylene glycol‐catalase (peg‐Cat, 500U/ml). Administration of exogenous S1P induced vasodilation in healthy vessels to a maximal dilation of 63.7%±5.0, n=8 (mean±SEM). As with previous animal studies, vascular constriction was observed with higher, but still physiological levels of S1P (10−6 M). Vasodilation due to S1P was reduced following inhibition of S1PR1 compared to S1P alone (1.8%±4, n=5). In the presence of L‐NAME, S1P‐induced dilation decreased (9%±7.9, n=6). Interestingly, addition of peg‐Cat, an H2O2 scavenger, also decreased S1P‐induced dilation (11.1%±6.6, n=3). Together these data suggest that S1PR1 is a critical mechanistic component of S1P‐induced vasodilation in the human vasculature. Further, the S1P vasodilatory pathway may require formation of H2O2 in addition to NO.Support or Funding InformationThis research was supported by National Institute of Health (NHLBI) K08 HL141562‐02 (JKF)

Inhibitor of myogenic differentiation family isoform a, a new positive regulator of fibronectin production by glomerular mesangial cells.

Overproduction of extracellular matrix proteins, including fibronectin by mesangial cells (MCs), contributes to diabetic nephropathy. Inhibitor of myogenic differentiation family isoform a (I-mfa) is a multifunctional cytosolic protein functioning as a transcriptional modulator or plasma channel protein regulator. However, its renal effects are unknown. The present study was conducted to determine whether I-mfa regulated fibronectin production by glomerular MCs. In human MCs, overexpression of I-mfa significantly increased fibronectin abundance. Silencing I-mfa significantly reduced the level of fibronectin mRNA and blunted transforming growth factor-β1-stimulated production of fibronectin. We further found that high glucose increased I-mfa protein content in a time course (≥48 h) and concentration (≥25 mM)-dependent manner. Although high glucose exposure increased I-mfa at the protein level, it did not significantly alter transcripts of I-mfa in MCs. Furthermore, the abundance of I-mfa protein was significantly increased in the renal cortex of rats with diabetic nephropathy. The I-mfa protein level was also elevated in the glomerulus of mice with diabetic kidney disease. However, there was no significant difference in glomerular I-mfa mRNA levels between mice with and without diabetic nephropathy. Moreover, H2O2 significantly increased I-mfa protein abundance in a dose-dependent manner in cultured human MCs. The antioxidants polyethylene glycol-catalase, ammonium pyrrolidithiocarbamate, and N-acetylcysteine significantly blocked the high glucose-induced increase of I-mfa protein. Taken together, our results suggest that I-mfa, increased by high glucose/diabetes through the production of reactive oxygen species, stimulates fibronectin production by MCs.

48 - Matrix metalloproteinase-2-induced EGFR transactivation and increased ROS and vascular contraction

Imbalanced matrix metalloproteinase (MMP)-2 activity may result of enhanced reactive oxygen species (ROS) formation and promotes cardiovascular dysfunction. We show for the first time that MMP-2 is upstream of increased ROS formation and activates signaling mechanisms impairing redox balance. Incubation of vascular smooth muscle cells (VSMC) with recombinant MMP-2 increased ROS formation assessed with dihydroethidium (DHE) by flow cytometry. This effect was blocked by the NADPH oxidase inhibitor apocynin or by polyethylene glycol-catalase (PEG-catalase), and by MMP inhibitors (doxycycline or GM6001). Next, we showed in HEK293 cells that MMP-2 transactivates heparin-binding epidermal growth factor (HB-EGF) leading to EGF receptor (EGFR) activation and increased ROS concentrations. This effect was prevented by the EGFR kinase inhibitor Ag1478, and by phospholipase C (PLC) or protein kinase C (PKC) inhibitors (A778 or chelerythrine, respectively), confirming the involvement of EGFR pathway in MMP-2-induce responses. Next, we showed that intraluminal exposure of aortas to MMP-2 increased vascular MMP-2 levels detected by immunofluorescence and gelatinolytic activity (by in situ zimography) in association with increased ROS formation. This effect was inhibited by MMP inhibitors (phenanthroline or doxycycline) and by apocynin or PEG-catalase. MMP-2 also increased aortic contractility to phenylephrine and this effect was prevented by MMP inhibitor GM6001 and by apocynin or PEG-catalase, showing again that increased ROS formation mediates functional effects of MMP-2. These results show that MMP-2 activates the EGFR and triggers downstream signaling pathways increasing ROS formation and promoting vasoconstriction. These findings may have various implications for cardiovascular diseases.

Matrix metalloproteinase-2-induced epidermal growth factor receptor transactivation impairs redox balance in vascular smooth muscle cells and facilitates vascular contraction

Increased reactive oxygen species (ROS) formation may enhance matrix metalloproteinase (MMP)-2 activity and promote cardiovascular dysfunction. We show for the first time that MMP-2 is upstream of increased ROS formation and activates signaling mechanisms impairing redox balance. Incubation of vascular smooth muscle cells (VSMC) with recombinant MMP-2 increased ROS formation assessed with dihydroethidium (DHE) by flow cytometry. This effect was blocked by the antioxidant apocynin or by polyethylene glycol-catalase (PEG-catalase), and by MMP inhibitors (doxycycline or GM6001). Next, we showed in HEK293 cells that MMP-2 transactivates heparin-binding epidermal growth factor (HB-EGF) leading to EGF receptor (EGFR) activation and increased ROS concentrations. This effect was prevented by the EGFR kinase inhibitor Ag1478, and by phospholipase C (PLC) or protein kinase C (PKC) inhibitors (A778 or chelerythrine, respectively), confirming the involvement of EGFR pathway in MMP-2-induce responses. Next, we showed that intraluminal exposure of aortas to MMP-2 increased vascular MMP-2 levels detected by immunofluorescence and gelatinolytic activity (by in situ zimography) in association with increased ROS formation. This effect was inhibited by MMP inhibitors (phenanthroline or doxycycline) and by apocynin or PEG-catalase. MMP-2 also increased aortic contractility to phenylephrine and this effect was prevented by MMP inhibitor GM6001 and by apocynin or PEG-catalase, showing again that increased ROS formation mediates functional effects of MMP-2. These results show that MMP-2 activates the EGFR and triggers downstream signaling pathways increasing ROS formation and promoting vasoconstriction. These findings may have various implications for cardiovascular diseases.

Short-term high-glucose treatment decreased abundance of Orai1 protein through posttranslational mechanisms in rat mesangial cells.

The short-term effect of high-glucose (HG) treatment on store-operated Ca2+ entry in mesangial cells (MCs) is not well-known. The aim of the present study was to determine whether and how HG treatment for a short period altered protein abundance of Orai1, the channel mediating store-operated Ca2+ entry in MCs. Rat and human MCs were exposed to HG (25 mM) for 2, 4, 8, and 24 h, and the abundance of Orai1 protein was significantly decreased at the time points of 8 and 16 h. Consistently, HG treatment for 8 h significantly reduced store-operated Ca2+ entry in rat MCs. However, HG treatment for the same time periods did not alter the levels of Orai1 transcript. Cycloheximide, a protein synthesis inhibitor, did not affect the HG-induced decrease of Orai1 protein, suggesting a posttranslational mechanism was involved. However, the HG effect on Orai1 protein was significantly attenuated by MG132 (a ubiquitin-proteasome inhibitor) and NH4Cl (a lysosomal pathway inhibitor). Furthermore, HG treatment for 8 h stimulated ubiquitination of Orai1 protein. We further found that polyethylene glycol-catalase, an antioxidant, significantly blunted the HG-induced reduction of Orai1 protein. In support of involvement of reactive oxygen species in the HG effects, hydrogen peroxide (H2O2) itself significantly decreased abundance of Orai1 protein and increased the level of ubiquitinated Orai1. Taken together, these results suggest that a short-term HG treatment decreased abundance of Orai1 protein in MCs by promoting the protein degradation through the ubiquitination-proteasome and -lysosome mechanisms. This HG-stimulated posttranslational mechanism was mediated by H2O2.

MicroRNA-21 Contributes to Reduced Microvascular Function in Binge Drinking Young Adults.

Binge drinking is associated with increased risk for cardiovascular (CV) disease. MicroRNA-21 (miR21) is up-regulated in the setting of excessive alcohol consumption and CV disease. Therefore, the goal of this study was to examine the vasodilatory responses to flow and acetylcholine (ACh) in the absence and presence of an anti-miR21 inhibitor in the microcirculation of young adult repeated binge drinkers (BDs). Gluteal subcutaneous adipose tissue biopsies were obtained from young adults (18 to 30years, n=35 vessels from BDs and n=28 vessels from abstainers). Resistance arteries (RAs) were isolated, incubated with anti-miR21 or a negative control (NC) to miR21 (12hours; 50nM), and lumen diameters measured with video microscopy. miR21 of adipose tissues was determined by quantitative polymerase chain reaction. Flow-induced dilation and ACh-induced dilation (AChID) were reduced in BDs as compared to abstainers. The miR21 inhibitor but not the NC abrogated these effects in BDs, but did not affect vasodilation in abstainers. Nitric oxide synthase inhibition with L-NAME reduced vasodilation in abstainers but not in BDs. In BDs, vasodilation was reduced by L-NAME in the presence of anti-miR21 but not the NC. Scavenging the reactive oxygen species, hydrogen peroxide with polyethylene glycol catalase reduced dilation in BDs but did not affect the restored dilation by the miR21 inhibitor. Maximum dilation to papaverine (endothelium independent) was similar between groups and unaffected by pharmacological inhibition. Finally, vascular endogenous miR21 was increased in BDs compared to abstainers. Endogenous miR21 is increased in RAs of young BDs, leading to reduced flow and AChID in the microcirculation.

Telomerase reverse transcriptase protects against angiotensin II-induced microvascular endothelial dysfunction.

A rise in reactive oxygen species (ROS) may contribute to cardiovascular disease by reducing nitric oxide (NO) levels, leading to loss of NO's vasodilator and anti-inflammatory effects. Although primarily studied in larger conduit arteries, excess ROS release and a corresponding loss of NO also occur in smaller resistance arteries of the microcirculation, but the underlying mechanisms and therapeutic targets have not been fully characterized. We examined whether either of the two subunits of telomerase, telomerase reverse transcriptase (TERT) or telomerase RNA component (TERC), affect microvascular ROS production and peak vasodilation at baseline and in response to in vivo administration to angiotensin II (ANG II). We report that genetic loss of TERT [maximal dilation: 52.0 ± 6.1% with vehicle, 60.4 ± 12.9% with Nω-nitro-l-arginine methyl ester (l-NAME), and 32.2 ± 12.2% with polyethylene glycol-catalase (PEG-Cat) ( P < 0.05), means ± SD, n = 9-19] but not TERC [maximal dilation: 79 ± 5% with vehicle, 10.7 ± 9.8% with l-NAME ( P < 0.05), and 86.4 ± 8.4% with PEG-Cat, n = 4-7] promotes flow-induced ROS formation. Moreover, TERT knockout exacerbates the microvascular dysfunction resulting from in vivo ANG II treatment, whereas TERT overexpression is protective [maximal dilation: 88.22 ± 4.6% with vehicle vs. 74.0 ± 7.3% with ANG II (1,000 ng·kg-1·min-1) ( P = not significant), n = 4]. Therefore, loss of TERT but not TERC may be a key contributor to the elevated microvascular ROS levels and reduced peak dilation observed in several cardiovascular disease pathologies. NEW & NOTEWORTHY This study identifies telomerase reverse transcriptase (TERT) but not telomerase RNA component as a key factor regulating endothelium-dependent dilation in the microcirculation. Loss of TERT activity leads to microvascular dysfunction but not conduit vessel dysfunction in first-generation mice. In contrast, TERT is protective in the microcirculation in the presence of prolonged vascular stress. Understanding the mechanism of how TERT protects against vascular stress represents a novel target for the treatment of vascular disorders.