Stress Invivo Research Articles

Luteolin ameliorates hyperuricemic nephropathy by activating urate excretion and Nrf2/HO-1/NQO1 antioxidant pathways in mice.

Luteolin is a natural flavonoid, which exists in many plants, including onions, broccoli, carrots, peppers, celery, olive oil, and mint. Luteolin is a dietary flavonoid with potent uric acid-lowering and antioxidant bioactivities. To date, the mechanism by which luteolin alleviates hyperuricemia nephropathy (HN) still needs to be better defined. This study aims to evaluate the therapeutic efficacy of luteolin in a preclinical mouse model and invitro. Luteolin was administered in the HN mice induced by the combination of potassium oxonate and hypoxanthine to evaluate the potential renoprotective effects invivo. The NRK-52E cells were stimulated with adenosine for invitro evaluation. Hematoxylin and eosin staining, biochemical analysis, immunoblotting, immunofluorescence, and immunohistochemistry were performed for the histopathologic and mechanistic investigations. The results suggest that luteolin attenuated tubular dilation and epithelial atrophy in the renal tissue of HN mice. Further, luteolin improved biochemical indicators concerning renal functions and oxidative stress invivo. Mechanistically, luteolin reduced the renal expressions of KIM-1 and caspase-3. Luteolin activated renal SIRT1/6 cascade and its downstream Nrf2-mediated antioxidant pathway. Furthermore, luteolin elevated the renal expressions of ATP-binding cassette subfamily G isoform 2 protein (ABCG2) and organic anion/cation transporters. In addition, livers of luteolin-treated HN mice exhibited robust inhibition of xanthine oxidase. Together, our study shows that luteolin alleviates renal injury in the HN mice by activating urate excretion and Nrf2/HO-1/NQO1 antioxidant pathways and inhibiting liver xanthine oxidase activity. Thus, luteolin may be a potential agent for the treatment of HN.

Tauroursodeoxycholic acid ameliorates renal injury induced by COL4A3 mutation

COL4A3/A4/A5 mutations have been identified as critical causes of Alport syndrome and other genetic chronic kidney diseases. However, the underlying pathogenesis remains unclear, and specific treatments are lacking. Here, we constructed a transgenic Alport syndrome mouse model by generating a mutation (Col4a3 p.G799R) identified previously from one large Alport syndrome family into mice. We observed that the mutation caused a pathological decrease in intracellular and secreted collagen IV α3α4α5 heterotrimers. The mutant collagen IV α3 chains abnormally accumulated in the endoplasmic reticulum and exhibited defective secretion, leading to persistent endoplasmic reticulum stress invivo and invitro. RNA-seq analysis revealed that the MyD88/p38 MAPK pathway plays key roles in mediating subsequent inflammation and apoptosis signaling activation. Treatment with tauroursodeoxycholic acid, a chemical chaperone drug that functions as an endoplasmic reticulum stress inhibitor, effectively suppressed endoplasmic reticulum stress, promoted secretion of the α3 chains, and inhibited the activation of the MyD88/p38 MAPK pathway. Tauroursodeoxycholic acid treatment significantly improved kidney function invivo. These results partly clarified the pathogenesis of kidney injuries associated with Alport syndrome, especially in glomeruli, and suggested that tauroursodeoxycholic acid might be useful for the early clinical treatment of Alport syndrome.

FoxM1 coordinates cell division, protein synthesis, and mitochondrial activity in a subset of β cells during acute metabolic stress

Pancreatic β cells display functional and transcriptional heterogeneity in health and disease. The sequence of events leading to β cell heterogeneity during metabolic stress is poorly understood. Here, we characterize β cell responses to early metabolic stress invivo by employing RNA sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing(ATAC-seq), single-cell RNA-seq (scRNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and real-time imaging to decipher temporal events of chromatin remodeling and gene expression regulating the unfolded protein response (UPR), protein synthesis, mitochondrial function, and cell-cycle progression. We demonstrate that a subpopulation of β cells with active UPR, decreased protein synthesis, and insulin secretary capacities is more susceptible to proliferation after insulin depletion. Alleviation of endoplasmic reticulum (ER) stress precedes the progression of the cell cycle and mitosis and ensures appropriate insulin synthesis. Furthermore, metabolic stress rapidly activates key transcription factors including FoxM1, which impacts on proliferative and quiescent β cells by regulating protein synthesis, ER stress, and mitochondrial activity via direct repression of mitochondrial-encoded genes.

Advanced oxidation protein products induce Paneth cells defects by endoplasmic reticulum stress in Crohn's disease

Paneth cells (PC) play a key role in the innate immune response of intestine epithelium, and PC defects contribute to the pathogenesis of Crohn's disease (CD). In this study, we utilized active CD tissues and advanced oxidation protein products (AOPP)-challenged C57BL/6 mouse model to investigate the effect of AOPP on PC defects in CD. We found that AOPP accumulated in active CD tissues and was negatively associated with lysozyme expression, while positively correlated with the presence of ER stress markers. Furthermore, AOPP treatment induced PC defects mainly through excessive ER stress invivo, and AOPP also caused mitochondria-associated ER membranes formation and mitochondrial dysfunction. In addition, the effects of AOPP could be attenuated by the administration of ER stress inhibitor, TUDCA. These findings suggest a pathogenic role of AOPP contributing to PC defects and may provide the basis for developing new strategies to managing CD.

Potential of Tamarind Shell Extract against Oxidative Stress In Vivo and In Vitro

Tamarind shell is rich in flavonoids and exhibits good biological activities. In this study, we aimed to analyze the chemical composition of tamarind shell extract (TSE), and to investigate antioxidant capacity of TSE in vitro and in vivo. The tamarind shells were extracted with 95% ethanol refluxing extraction, and chemical constituents were determined by ultra-performance chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS). The free radical scavenging activity of TSE in vitro was evaluated using the oxygen radical absorbance capacity (ORAC) method. The antioxidative effects of TSE were further assessed in 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH)-stimulated ADTC5 cells and tert-butyl hydroperoxide (t-BHP)-exposed zebrafish. A total of eight flavonoids were detected in TSE, including (+)-catechin, taxifolin, myricetin, eriodictyol, luteolin, morin, apigenin, and naringenin, with the contents of 5.287, 8.419, 4.042, 6.583, 3.421, 4.651, 0.2027, and 0.6234 mg/g, respectively. The ORAC assay revealed TSE and these flavonoids had strong free radical scavenging activity in vitro. In addition, TSE significantly decreased the ROS and MDA levels but restored the SOD activity in AAPH-treated ATDC5 cells and t-BHP-exposed zebrafish. The flavonoids also showed excellent antioxidative activities against oxidative damage in ATDC5 cells and zebrafish. Overall, the study suggests the free radical scavenging capacity and antioxidant potential of TSE and its primary flavonoids in vitro and in vivo and will provide a theoretical basis for the development and utilization of tamarind shell.

Residual strains in ascending thoracic aortic aneurysms: The effect of valve type, layer, and circumferential quadrant

The stress distribution in ascending thoracic aortic aneurysms is determined by the mechanical properties, geometry, loading conditions, and zero-stress state of the aneurysmal aorta. Our objective was to fully characterize the zero-stress state of the aneurysmal aorta in twelve tricuspid aortic valve patients and eight (age/aortic diameter-matched) bicuspid aortic valve patients, for which little data are available. Opening angles and residual stretches were measured for the intact wall and individual layers according to quadrant and were similar in the two patient groups. The intact-wall and medial opening angles were comparable; their circumferential but not their axial ones peaked in the left lateral quadrant, with non-significant regional differences in the other layers. The intima’s circumferential opening angles were the highest of all layers (∼300 deg) and the adventitia’s the lowest (∼165 deg), with lesser layer differences in the axial opening angles. Upon radially cutting aortic rings, the released circumferential residual stretches were tensile (of large magnitude) externally and compressive (of small magnitude) internally, unlike the axial residual stretches released when cutting intact-wall strips, whose magnitude was small externally and large internally. Nevertheless, large circumferential compressive residual stretches were released in the adventitia upon layer dissection, counteracting the large circumferential tensile stretches of the intact wall externally. Moreover, the large axial tensile residual stretches of the intima counteracted the large axial compressive stretches of the intact wall internally. These layer-specific residual stretches may moderate the in-vivo stress gradients across wall thickness, serving as a protective mechanism against aortic dissection or rupture.

Asiatic Acid Fabricated Nanoconstructs to Mitigate Amyloid Beta1-42 Induced Injury in SH-SY5Y Cells In-Vitro and Ameliorates Cognitive Impairment by Dual Cholinesterase Inhibition and Attenuation of Oxidative Stress In-Vivo.

Asiatic acid (AA) is reported for its neuroprotective potential in Alzheimer's disease (AD). This present work aimed to develop AA loaded nanostructured lipid carriers (AAN) for targeting the delivery of AA into the brain and ameliorating the cognitive deficits in AD rats. AAN was optimized using the Box-Behnken design, considering 3 factors (soya lecithin, tween 80, and high pressure homogenizer (HPH) pressure) as independent variables while particle size (PS), zeta potential (ZP) and entrapment efficiency (EE) were dependent variables. Cytotoxicity assay and internalization studies of AAN were evaluated in SH-SY5Y cells and further neuroprotective efficiency on intracellular amyloid beta (Aβ) aggregation was evaluated in Aβ 1-42 treated cells with thioflavin T (ThT). The behavioral acquisition effects were evaluated in Aβ 1-42 (5µg/ 5 µL, intracerebroventricular (ICV), unilateral) induced AD model followed by the histology and quantification of neurotransmitters levels. The optimized AAN revealed desired PS (44.1 ± 12.4nm), ZP (- 47.1 ± 0.017 mv) and EE (73.41 ± 2.53%) for brain targeting delivery of AA. In-vitro, AAN exhibited better neuroprotective potential than AA suspension (AAS). AA content was 1.28 folds and 2.99 folds heightened in plasma and brain respectively after the i.p. administration of AAN as compared to AAS. The results of pharmacodynamic studies manifested the AAN treatment significantly (p < 0.05) ameliorated the cognitive deficits. Hence, developed AAN has neuroprotective potential and should be further considered as an unconventional platform in preclinical model for the management of AD.

EFFECT OF GLYCOSAMINOGLYCANS ON OPENING ANGLE OF INTACT &amp; CROSSLINKED AORTAS

EFFECT OF GLYCOSAMINOGLYCANS ON OPENING ANGLE OF INTACT &amp; CROSSLINKED AORTAS

SLC22 Transporters in the Fly Renal System Regulate Response to Oxidative Stress In Vivo.

Several SLC22 transporters in the human kidney and other tissues are thought to regulate endogenous small antioxidant molecules such as uric acid, ergothioneine, carnitine, and carnitine derivatives. These transporters include those from the organic anion transporter (OAT), OCTN/OCTN-related, and organic cation transporter (OCT) subgroups. In mammals, it has been difficult to show a clear in vivo role for these transporters during oxidative stress. Ubiquitous knockdowns of related Drosophila SLC22s—including transporters homologous to those previously identified by us in mammals such as the “Fly-Like Putative Transporters” FLIPT1 (SLC22A15) and FLIPT2 (SLC22A16)—have shown modest protection against oxidative stress. However, these fly transporters tend to be broadly expressed, and it is unclear if there is an organ in which their expression is critical. Using two tissue-selective knockdown strategies, we were able to demonstrate much greater and longer protection from oxidative stress compared to previous whole fly knockdowns as well as both parent and WT strains (CG6126: p < 0.001, CG4630: p < 0.01, CG16727: p < 0.0001 and CG6006: p < 0.01). Expression in the Malpighian tubule and likely other tissues as well (e.g., gut, fat body, nervous system) appear critical for managing oxidative stress. These four Drosophila SLC22 genes are similar to human SLC22 transporters (CG6126: SLC22A16, CG16727: SLC22A7, CG4630: SLC22A3, and CG6006: SLC22A1, SLC22A2, SLC22A3, SLC22A6, SLC22A7, SLC22A8, SLC22A11, SLC22A12 (URAT1), SLC22A13, SLC22A14)—many of which are highly expressed in the kidney. Consistent with the Remote Sensing and Signaling Theory, this indicates an important in vivo role in the oxidative stress response for multiple SLC22 transporters within the fly renal system, perhaps through interaction with SLC22 counterparts in non-renal tissues. We also note that many of the human relatives are well-known drug transporters. Our work not only indicates the importance of SLC22 transporters in the fly renal system but also sets the stage for in vivo studies by examining their role in mammalian oxidative stress and organ crosstalk.

Zanthoxylum alatum Roxb. seed extract ameliorates stress aggravated DSS-induced ulcerative colitis in mice: Plausible role on NF-κB signaling axis

Ethnopharmacological relevanceZanthoxylum alatum (ZA) Roxb (family: Rutaceae) plant has been traditionally used for multiple indications by local healers among different communities of South Asian countries mainly in India and Bangladesh. The extracts of ZA have reported strong anti-inflammatory and anti-oxidant activities, but no scientific report is available on its efficacy in intestinal inflammatory disorders like ulcerative colitis. Aim of the studyThe overall objective of our study was to evaluate the anti-inflammatory potency of hydro-ethanolic extract of Zanthoxylum alatum seed (ZAHA) using both in-vitro NF-κB-luciferase translocation assay and in-vivo stress aggravated dextran sodium sulfate (DSS)-induced ulcerative colitis model. Materials and methodsThe in-vitro anti-inflammatory effect of ZAHA extract was evaluated by luciferase assay in HEK293 cells. Parameters such as body weights, behavioural, colonoscopy, colon lengths and spleen weights were measured and recorded in stress aggravated DSS-induced colitis model in C57BL/6 mice. Biochemical, histological and immunoblot analysis in the colon tissues were determined to prove its anti-inflammatory and anti-oxidant activities. Characterization of the extract was done by LC-MS/MS study. ResultsInitial in vitro NF-κB-luciferase translocation assay showed that the hydroalcoholic extract of ZA (ZAHA) showed potent inhibitory activity for NF-κB translocation by TNF-α stimulation and hence this particular extract was further evaluated in stress aggravated DSS-induced ulcerative colitis model in C57BL/6 mice. Treatment of ZAHA for two weeks at a dose of 200 mg/kg significantly ameliorated the stress aggravated DSS-induced colitis in mice. Histological alterations, infiltration of inflammatory cells, and the levels of IL-1β, IL-6, TNF-α in colon tissue and serum samples were significantly decreased in ZAHA treatment groups compared to the stress aggravated DSS induced colitis animals. Moreover, the protein expressions of p-NF-κB, p-IκBα, p-STAT3, COX-2, and TNF-α were significantly reduced in colon tissues of ZAHA treated groups and also increased anti-oxidant markers like SOD-1, Nrf2 significantly when compared with disease control group. Characterization of the extract further by LC-MS/MS revealed the presence of several active compounds which could be responsible for its anti-inflammatory activity. ConclusionsThus from the above findings it can be concluded that ZAHA ameliorates stress aggravated DSS-induced ulcerative colitis due to its anti-inflammatory and anti-oxidant activity.

Potential Effects of Poloxamer 188 on Rat Isolated Brain Mitochondria after Oxidative Stress In Vivo and In Vitro.

Outcome after cerebral ischemia is often dismal. Reperfusion adds significantly to the ischemic injury itself. Therefore, new strategies targeting ischemia/reperfusion (I/R) injury are critically needed. Poloxamer (P)188, an amphiphilic triblock copolymer, is a highly promising pharmacological therapeutic as its capability to insert into injured cell membranes has been reported to protect against I/R injury in various models. Although mitochondrial function particularly profits from P188 treatment after I/R, it remains unclear if this beneficial effect occurs directly or indirectly. Here, rat isolated brain mitochondria underwent oxidative stress in vivo by asphyxial cardiac arrest or in vitro by the addition of hydrogen peroxide (H2O2) after isolation. Mitochondrial function was assessed by adenosine triphosphate synthesis, oxygen consumption, and calcium retention capacity. Both asphyxia and H2O2 exposure significantly impaired mitochondrial function. P188 did not preserve mitochondrial function after either injury mechanism. Further research is indicated.

Protective Effects of Fisetin on Hepatic Ischemia-reperfusion Injury Through Alleviation of Apoptosis and Oxidative Stress

Hepatic ischemia-reperfusion injury (IRI) is the main leading cause of morbidity and mortality of patients after liver surgery and transplantation. Fisetin, a kind of flavonoid, has been reported to protect against myocardial and cerebral IRI. However, the effects of fisetin on liver IRI were poorly investigated. C57BL/6 mice were used to establish the liver IRI model invivo. Intraperitoneal injection of fisetin was performed one hour before IR treatment (1h ischemia and 6h reperfusion). Invitro experimental study was conducted using AML-12 hepatocytes with 1h hypoxia and 12h reoxygenation (HR) treatment. Tissue damage was evaluated through serum AST and ALT levels and hematoxylin-eosin (HE) staining. Cell apoptosis was assessed by TUNEL staining and protein levels of Bax, Bcl-2, cleaved-caspase-3, and cleaved-PARP. Oxidative stress was evaluated by ROS and MDA levels and the activity of SOD and GSH-Px. Immunohistochemistry and immunofluorescence assay were performed to observe the translocation of Nrf2 from the cytoplasm into the nucleus. The histopathological assessment showed that fisetin attenuated IR-induced liver damage obviously. Besides, fisetin served a protective role in IR liver to alleviate cell apoptosis and oxidative stress invivo and invitro. Introduction of high concentration of fisetin promoted the translocation of Nrf2 from the cytoplasm into the nucleus, increasing protein expression of its downstream elements, at least HO-1 in IR liver tissues and hepatocytes after HR. Inhibition of Nrf2 could reverse the effects of fisetin on cell viability, cell apoptosis, and also oxidative stress of HR hepatocytes, suggesting that Nrf2 signaling was necessary in fisetin-mediated regulations of liver IRI. Fisetin alleviates liver damage, cell apoptosis, and oxidative stress induced by liver IRI, at least through Nrf2/HO-1 signaling pathway, suggesting that fisetin could be considered as a targeted drug for liver IRI treatment.

Translational Repression of G3BP in Cancer and Germ Cells Suppresses Stress Granules and Enhances Stress Tolerance

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress invivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.

Interpersonal Stress Severity Longitudinally Predicts Adolescent Girls' Depressive Symptoms: the Moderating Role of Subjective and HPA Axis Stress Responses.

In recent decades, stress response models of adolescent depression have gained attention, but it remains unclear why only certain adolescents are vulnerable to the depressogenic effects of stress while others are not. Building on evidence that affective and physiological responses to stress moderate the impact of stress exposure on depression, the current study examined whether the interaction between severity of interpersonal stress, subjective affective reactivity, and hypothalamic-pituitary-adrenal (HPA) axis reactivity to an acute, in-vivo psychosocial stressor prospectively predicted depressive symptoms nine months later. Hypotheses were examined with a clinically-oversampled group of 182 adolescent girls (aged 12-16) to ensure an examination of the widest possible range of risk. Self-report measures of affect and salivary cortisol samples were collected before and after an in-vivo stress task to assess affective reactivity and HPA axis reactivity, respectively. Severity of interpersonal stress between baseline and nine months was assessed using a semi-structured interview and was objectively coded for severity and content theme (i.e., interpersonal vs. non-interpersonal). Results indicate that experiences of severe interpersonally-themed stress predict elevated levels of depressive symptoms longitudinally only for adolescent girls with elevated affective reactivity to stress, and suggest that these deleterious effects of stress are most exacerbated for girls with elevated physiological responses to stress. Findings suggest that it may be critical to examine both affective and physiological stress responses when assessing risk for depression in adolescents.

An apportionment method for the oxidative potential of atmospheric particulate matter sources: application to a one-year study in Chamonix, France

Abstract. Inhaled aerosolized particulate matter (PM) induces cellular oxidative stress in vivo, leading to adverse health outcomes. The oxidative potential (OP) of PM appears to be a more relevant proxy of the health impact of the aerosol rather than the total mass concentration. However, the relative contributions of the aerosol sources to the OP are still poorly known. In order to better quantify the impact of different PM sources, we sampled aerosols in a French city for one year (2014, 115 samples). A coupled analysis with detailed chemical speciation (more than 100 species, including organic and carbonaceous compounds, ions, metals and aethalometer measurements) and two OP assays (ascorbic acid, AA, and dithiothreitiol, DTT) in a simulated lung fluid (SLF) were performed in these samples. We present in this study a statistical framework using a coupled approach with positive matrix factorization (PMF) and multiple linear regression to attribute a redox-activity to PM sources. Our results highlight the importance of the biomass burning and vehicular sources to explain the observed OP for both assays. In general, we see a different contribution of the sources when considering the OP AA, OP DTT or the mass of the PM10. Moreover, significant differences are observed between the DTT and AA tests which emphasized chemical specificities of the two tests and the need of a standardized approach for the future studies on epidemiology or toxicology of the PM.

Dark Rearing Does Not Prevent Rod Oxidative Stress In Vivo in Pde6brd10 Mice

PurposeIn cyclic light-reared Pde6brd10 mice, rod cell oxidative stress contributes to the degenerative phenotype. Dark rearing Pde6brd10 mice slows but does not prevent atrophy. This suggests that outer retinal oxidative stress occurs in Pde6brd10 mice independent of light exposure, a hypothesis tested in this study.MethodsMouse strains Pde6brd10 and C57Bl/6 (wild type) were dark reared until postnatal day (P) 23 (P23) or P30. In subgroups of dark-reared mice, (1) layer-specific excessive free radical production (i.e., an oxidative stress biomarker) in vivo via QUEnch-assiSTed magnetic resonance imaging (QUEST MRI) was indicated by a significant reduction in the greater-than-normal spin-lattice relaxation rate R1 (1/T1) with methylene blue, (2) superoxide production was measured ex vivo in whole retina (lucigenin), and (3) retinal layer spacing and thickness were assessed in vivo (optical coherence tomography, MRI).ResultsIn P23 male Pde6brd10 mice, only the outer superior retina showed oxidative stress in vivo, as measured by QUEST MRI; a lucigenin assay confirmed supernormal superoxide production. In contrast, at P30, no evidence for retinal oxidative stress was observed. In P23 female Pde6brd10 mice, no retinal oxidative stress was apparent; however, at P30, oxidative stress was observed in superior inner and outer nuclear layers. Male and female Pde6brd10 mice at P23 had normal retinal thicknesses, whereas at P30, modest thinning was noted in inferior and superior retina.ConclusionsWe confirmed that outer retinal oxidative stress occurs in male and female dark-reared Pde6brd10 mice. Male and female Pde6brd10 mice demonstrated similar degrees of retinal thinning, but with unexpectedly distinct spatial and temporal retinal oxidative stress patterns.

Functional morphology in the pages of the AJPA

Functional morphology in the pages of the <i>AJPA</i>

Evaluating the interaction of a tracheobronchial stent in an ovine in-vivo model.

Tracheobronchial stents are used to restore patency to stenosed airways. However, these devices are associated with many complications such as stent migration, granulation tissue formation, mucous plugging and stent strut fracture. Of these, granulation tissue formation is the complication that most frequently requires costly secondary interventions. In this study a biomechanical lung modelling framework recently developed by the authors to capture the lung in-vivo stress state under physiological loading is employed in conjunction with ovine pre-clinical stenting results and device experimental data to evaluate the effect of stent interaction on granulation tissue formation. Stenting is simulated using a validated model of a prototype covered laser-cut tracheobronchial stent in a semi-specific biomechanical lung model, and physiological loading is performed. Two computational methods are then used to predict possible granulation tissue formation: the standard method which utilises the increase in maximum principal stress change, and a newly proposed method which compares the change in contact pressure over a respiratory cycle. These computational predictions of granulation tissue formation are then compared to pre-clinical stenting observations after a 6-week implantation period. Experimental results of the pre-clinical stent implantation showed signs of granulation tissue formation both proximally and distally, with a greater proximal reaction. The standard method failed to show a correlation with the experimental results. However, the contact change method showed an apparent correlation with granulation tissue formation. These results suggest that this new method could be used as a tool to improve future device designs.

Modeling Acute ER Stress in Vivo and in Vitro.

The endoplasmic reticulum (ER) is a critical organelle that synthesizes secretory proteins and serves as the main calcium storage site of the cell. The accumulation of unfolded proteins at the ER results in ER stress. Although the association between ER stress and the pathogenesis of many metabolic conditions have been well characterized using both in vivo and in vitro models, no standardized model concerning ER stress exists. Here, we report a standardized model of ER stress using two well-characterized ER stress-inducing agents, thapsigargin and tunicamycin. Our aim in this current study was 2-fold: to characterize and establish which agent is optimal for in vitro use to model acute ER stress and to evaluate which agent is optimal for in vivo use. To study the first aim we used two well-established metabolic cell lines; human hepatocellular carcinoma (HepG2s) and differentiated mouse adipocytes (3T3-L1). In the second aim we utilized C57BL/6J mice that were randomized into three treatment groups of sham, thapsigargin, and tunicamycin. Our in vitro results showed that tunicamycin worked as a rapid and efficacious inducer of ER stress in adipocytes consistently, whereas thapsigargin and tunicamycin were equally effective in inducing ER stress in hepatocytes. In regards to our in vivo results, we saw that tunicamycin was superior in not only inducing ER stress but also recapturing the metabolic alterations associated with ER stress. Thus, our findings will help guide and inform researchers as to which ER stress agent is appropriate with regards to their model.

Auranofin Induces a Reversible In-Vivo Stress Response That Correlates With a Transient Clinical Effect In Patients With Chronic Lymphocytic Leukemia

Auranofin (AF) is an oral disease-modifying anti-rheumatic agent. Using a high throughput screening assay of 2,816 FDA-approved drugs against primary tumor cells in vitro, we have previously identified AF as one of five drugs with selective anti-Chronic Lymphocytic Leukemia (CLL) activity. We have shown that AF induces oxidative stress and apoptosis in CLL cells in vitro independent of classic prognostic markers (ASH Meeting Abstract 865, 2012). Here, we evaluated the in vivo effects of AF on CLL using blood samples collected from six patients treated with single agent AF at the NIH as part of a multi-center clinical trial led by the University of Kansas Cancer Center (NCT01419691).Five CLL patients and one patient with Small Lymphocytic Lymphoma (SLL) were enrolled in this open-label phase II study of AF in relapsed/refractory patients. Patients were started on AF 6 mg daily administered orally on 28-day cycles, with a dose escalation to 9 mg after the first cycle if no grade ≥2 toxicity occurred. AF was generally well tolerated. The best response was stable disease. Sequential blood samples were obtained prior to, and during the first cycle on drug. CLL cells were isolated by density gradient centrifugation. To study the in vivo effect of AF on redox balance we used dihydroethidium (DHE) and concomitantly measured cell viability using 3,3′-dihexyloxacarbocyanine iodide (DiOC6) in CD19 gated fresh cells by flow cytometry. Within 24 hours of the first dose AF induced an average 1.8 fold increase in DHE+ CLL cells indicating increased levels of ROS. Concomitantly there was a similar increase in apoptosis as shown by Annexin V staining. The increase in ROS production and apoptosis was transient; by day 7 all these changes had reverted to baseline or were even below baseline in three patients. A concomitant and equally transient decrease in the absolute lymphocyte count (ALC) and lactate dehydrogenase (LDH) level was also observed.To investigate the in vivo effect of AF on tumor biology, total RNA (2.5 μg) from CLL cells of three patients treated with AF was profiled on Human Genome U133 Plus 2.0 arrays (Affymetrix). Three time points were analyzed: baseline (D0), after one dose of AF (D1), and a week later (D7). Compared to baseline, there were 182 genes (29-up, 153-down) whose expression changed >1.5-fold at P<.01 on D1, but only 14 genes at D7, suggesting a transient in vivo effect of auranofin on the CLL cells. Next we used a previously characterized gene signature that is regulated by the transcription factor NRF2, which is a key regulator of the cellular response to oxidative stress. We have previously shown that this NRF2 gene signature is upregulated in CLL cells treated with AF in vitro. Surprisingly, we observed that after 24 hours of in vivo therapy, AF induced a statistically significant decrease in the expression of the NRF2 signature in CLL cell and that these changes resolved by D7. To search for biologic processes leading to the gene expression changes, we used Gene Set Enrichment Analysis (GSEA). After 24 hours of in vivo treatment with AF, CLL cells displayed upregulation of genes in pathways involved in drug resistance (such as excretion and transport channels). In contrast, there was a decrease in the expression of genes involved in oxygen metabolism, DNA repair, and protein folding. With the exception of changes in genes regulating redox homeostasis, all these changes reverted back to baseline by D7.Taken together, in vivo AF appears to transiently decrease anti-oxidant defenses regulated by NRF2 concomitant with an increase in cellular ROS levels and induction of some degree of cellular apoptosis. However, in vivo an adaptive response emerged that mobilized potentially compensatory mechanisms, including induction of pathways that may prevent intracellular accumulation of AF and a decrease in cellular processes that generate ROS, such as protein folding. The transient nature of the cellular response in vivo is consistent with the limited clinical activity seen in our patients. Plans are underway to determine the AF maximum tolerated dose in CLL patients. We will continue to evaluate the in vivo effects of AF on CLL cells at higher doses.Supported by the Intramural Research Program of NHLBI and NCATS, NIH; a grant from The Leukemia and Lymphoma Society Therapy Acceleration Program to The Learning Collaborative™, as well as philanthropic support.We thank our patients for participating in these research studies. Disclosures:Off Label Use: Auranofin is not FDA approved for use in CLL.