bluuebunny/crossref_metadata_2025 huggingface dataset:
ATP loss and purinergic receptor signaling contribute to early transcriptional responses activated by JNK in liver regeneration after partial hepatectomy in the rat
Authors: Juskeviciute Egle , Crumm Sara , Correnti Jason , Hoek Jan B.
Published: June 2021

A complex program of gene expression changes drives the onset of liver regeneration after 70% partial hepatectomy (PHx) in the rat. However, the signals that initiate this response have not been well characterized. We reported recently (Crumm et al., 2008) that a rapid loss of adenine nucleotides (AdN) after PHx generates early stress signals that contribute to the onset of liver regeneration. Conditions that prevent the loss of AdN or that inhibit purinergic receptor activation suppress early signaling responses. This study focuses on the potential role of connexin (Cx) hemi‐channels in AdN loss and downstream signaling events. To specifically block different Cx isoforms, we used gap peptides. Pretreating animals with gap peptide specific for Cx26/32, but not for Cx43 or pannexin‐1 inhibited AdN loss. Pretreatment with the nitric oxide synthase inhibitor L‐NAME or the alpha‐adrenergic antagonist phentolamine also completely prevents loss of and upon PHx. However, pretreatment with the purinergic receptor antagonist suramin does not prevent AdN loss. All these treatments suppress Jun N‐terminal kinase (JNK) phosphorylation after PHx. In the analysis of downstream targets of JNK contributing to the early gene expression changes after PHx these inhibitors also decreased phospho‐cJun transcriptional activity at AP1 and CREB binding sites.

Supported by NIH AA008714/AA018873 to JBH.
Interferon‐β induces cellular senescence in vascular smooth muscle cells
Authors: Byeon Hye‐eun , Rhee Dong‐Kwon , Pyo Suhkneung
Published: June 2021

Interferon‐¥â(IFN‐¥â) is one of the type‐¥° interferon with potent antiviral and anti‐proliferative activities. Previous studies have documented that IFN‐¥â induces p53‐dependent senescence. However, the mechanism of IFN‐¥â‐induced cellular senescence remains unclear. In the present study, we examined the mechanism by which IFN‐¥â induced cellular senescence in vascular smooth muscle cells (VSMCs). Cells treated with IFN‐¥â (500~2000U/ml) under serum free condition was arrested in G1 phase of cell cycle. In addition, treatment with IFN‐¥â resulted in increased in senescence‐associated ¥â‐galactosidase (SA‐¥â‐gal) activity and PAI‐1 expression. Western blot analysis revealed that IFN‐β treatment up‐regulated p53 expression and its phosphorylation, whereas mdm2 expression was decreased. We also examined the effect of IFN‐β treatment on other cell cycle related proteins such as p21 and CDK2. The expression of p21 was increased in cells treated with IFN‐β accompanied by down‐regulation of CDK2, which supported the G1 phase arrest. Moreover, the decreased mRNA level of TERT (Telomerase Reverse Transcriptase) by IFN‐β was confirmed by RT‐PCR. Taken together, the present data suggest that IFN‐β regulates the levels of cell cycle‐related proteins and G1 phase cell cycle arrest, leading to induction of the senescence‐like arrest of VSMCs.
Crosstalk between ALK1 and Notch signaling in the vascular defects associated with HHT
Authors: Kim Jai‐Hyun , Hughes Christopher C.W.
Published: June 2021

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder involving the vascular system, and is caused by mutations in endoglin (HHT1) and ALK‐1 (HHT2), receptors for the TGFb/BMP family of ligands. The ALK‐5 and ALK‐1 activates the downstream Smad2/3 and Smad1/5, respectively and regulates transcription of specific genes involved in angiogenesis. Notch works in conjunction with other angiogenic pathways to pattern and stabilize the vasculature. The arterial‐venous cell fate is determined before blood circulation starts, and pathways upstream of the arterial specific marker EphrinB2 and its receptor EphB4 are involved in this process. Specifically, the Notch and TGFb‐ALK1 pathways are crucial for AV differentiation. In order to investigate the role of Notch and ALK1 on the expression of the EphrinB2 gene, we cloned the promoter region and found that NICD and ALK1 up‐regulate the EphrinB2 promoter synergistically. A single RBP site is required for notch activity and the promoter also contains multiple TGFb/BMP response elements. We have also investigated the role of ALK1 in EC sprout formation using an in vitro angiogenesis model and find that ALK1 knock down disrupts normal sprout formation. These data support the concept of functional interaction between Notch signaling and BMP/ALK1 signaling. This work was supported by the HHT Foundation International.
In vitro preliminary findings on the effect of biodegradable composite material in fibroblasts‐stimulated angiogenesis
Authors: Pirraco Ana , Rocha Ana Sofia , Vieira André , Vieira Joana , Guedes Rui , Soares Raquel
Published: June 2021

The current ligaments treatment injuries has several drawbacks that prompted attempts to use synthetic materials in ligament replacements. Although fibroblasts do not comprise a large volume of ligament tissue, they have an important role in ligament tissue regeneration as they are able to model ECM and angiogenesis, therefore affecting the healing process.

The aim is to appraise the biocompatibility of different biodegrable ligament composites (PGA‐PCL, PLA‐PCL, PDO, PGA) with the fibroblast‐mediated angiogenic process in vitro.

HFF1 fibroblast cells were cultured on plates coated with each of the composite and cell viability was examined. Media from HFF1 cells incubated with composite material was removed and used to appraise the effect on angiogenesis (aortic ring assay and capillary‐like structures formation assay).

No significant difference in cell viability was found for any composite tested. Aortic ring results suggested no disturbance of the angiogenic process. These findings were corroborated by the formation of cord‐like structures assay, as no significant difference in the number of structures was found in comparison to controls.

Overall, our preliminary findings indicate that the composites do not promote nor prevent angiogenesis, being thus a good tool for the development of ligament composites. Supported by FCT (PTDC/EME‐PME/70155/2006)
Nicotine stimulates retinal angiogenesis via α7‐nicotinic receptor and matrix metalloproteinases (MMP)‐mediated signaling pathway
Authors: Buckley Adam W. , Brown Kathleen C. , Dom Aaron M. , Weller Donald W. , Shah Yashoni H. , Egleton Richard D. , Dasgupta Piyali
Published: June 2021

Proliferative retinopathies are among the leading causes of blindness in the elderly. These diseases are characterized by aberrant retinal angiogenesis, which leads to compromised function of the retina and ultimately loss of vision. Smoking is one of the major risk factors for the development of proliferative retinopathies, such as age related macular degeneration (ARMD) and diabetic retinopathy. Our study examined whether nicotine stimulates retinal angiogenesis if administered at concentrations commonly found in the plasma of smokers. Results indicated that treatment with nicotine enhanced angiogenic‐tubule formation in Matrigel duplex assays using primary human retinal microvascular endothelial cells. Nicotine also induced sprouting angiogenesis in retinal explants isolated from Zucker rats. The proangiogenic effects of nicotine were mediated by α7 nicotinic acetylcholine receptors (nAChRs) and downstream activation of MMP‐2 and MMP‐9. Most importantly, antagonists of α7‐nAChRs potently inhibit nicotine‐induced retinal angiogenesis in both duplex assays and rat retinal explants. Our results suggest that α7‐nAChR may be a valuable molecular target for therapy of ARMD. Funding for our study was supported by the American Retina Foundation, the INBRE Summer Program, the Sigma Xi GIAR, an MU‐ADVANCE Fellowship, and the Flight Attendant Medical Research Institute YCSA Grant.
C‐Reactive Protein Impairs eNOS protein‐protein interactions
Authors: Valleggi Simona , Devaraj Sridevi , Jialal Ishwarlal
Published: June 2021

C Reactive Protein (CRP) is an acute‐phase protein and a cardiovascular risk marker. CRP levels are inversely correlated with endothelial vasoreactivity. CRP decreases eNOS expression and activity in vitro and in vivo. We have shown that CRP mediates eNOS uncoupling. In this study, we tested the effect of CRP on eNOS interaction and association with docking proteins, a fundamental step in regulating eNOS activity. Human Aortic Endothelial cells were incubated in absence and presence of CRP (25ug/ml for 18hrs) followed by stimulation with VEGF (100ng/mL for 0–20 minutes). Association between eNOS and Caveolin‐1 (Cav1), Dynamin II (DynII), Porin and Heat Shock Protein 90 (Hsp90) was assessed by immunoprecipitating cell lysates with eNOS followed by blotting for Hsp90, DynII, Porin and Cav1, using eNOS as loading control (n=3–5 separate experiments). CRP treatment results in a 87% decrease in eNOS‐Hsp90 association, 26% decrease in eNOS‐Porin association and 33% increase in eNOS‐Cav1 association. No significant changes have been observed in eNOS‐DynII association. Thus, we provide further evidence for CRP inducing endothelial dysfunction in HAECs via changes in eNOS association with docking proteins such as Hsp90, Cav1 and Porin.
Effect of Radiation on Human Intestinal Microvascular Endothelial Cells (HIMEC): Role of Akt/mTOR and NFκB
Authors: Rafiee Parvaneh , Binion David G , Wellner Michael , Otterson Mary F
Published: June 2021

Radiation therapy is an essential modality in the treatment of colorectal cancers. Radiation exerts an anti‐angiogenic effect on tumors, inhibiting endothelial proliferation and survival in the tumor microvasculature. However, damage from low levels of irradiation can induce a paradoxical effect, stimulating survival in endothelial cells. We used HIMEC to define effects of low level radiation on these gut specific endothelial cells. 2 Gy of irradiation activates NFκB and the PI3K/Akt pathway, which is involved in cell cycle re‐entry and cell survival in HIMEC. A downstream target of PI3K/Akt is mammalian target of rapamycin (mTOR), which contributes to cell proliferation and angiogenesis. The aim of this study was to investigate the signaling molecules involved in the radio‐sensitizing effects of curcumin on HIMEC subjected to low level of irradiation. We have demonstrated that exposure of HIMEC to low level of irradiation induced mTOR and Akt phosphorylation, which was attenuated by curcumin, rapamycin and LY294002. Activation of NFκB by low level of irradiation was inhibited by curcumin and SN‐50. Curcumin also induced apoptosis by induction of caspase‐3 cleavage in irradiated HIMEC. In conclusion, curcumin significantly inhibited NFκB and attenuated the effect of irradiation‐induced pro‐survival signaling through the PI3k/Akt/mTOR and NFκB pathways in these gut specific endothelial cells. Curcumin may be a potential radio‐sensitizing agent for enhanced anti‐angiogenic effect in colorectal cancer radiation therapy.
Lipid raft‐dependent NADPH oxidase regulates polychlorinated biphenyls‐induced expression of cell adhesion molecules in human brain endothelial cells
Authors: Eum Sung Yong , Choi Yean Jung , Andras Ibolya E , Hennig Bernhard , Toborek Michal
Published: June 2021

Exposure to persistent organic pollutants, such as polychlorinated biphenyls (PCBs), can lead to expression of cell adhesion molecules (CAMs) of the brain endothelium leading to chronic neuroinflammation. In the present study, we have explored whether NADPH oxidase (NOX) modulates ortho‐substituted PCBs‐induced upregulation of CAMs in brain endothelial cells. Superoxide production, as measured by dihydroethidium (DHE), was increased in the cells exposed to PCB153. Exposure to PCB153 induced phosphorylation of cytosolic components including p47 and Rac and translocation into the lipid rafts domain for NOX activation. Upregulation of ICAM‐1 and VCAM‐1 was impeded by inhibitors to NAPDH oxidase, Rac1, and nitric oxide synthase. Moreover, isolation of lipid raft fraction using non‐detergent sucrose gradient method confirmed predominant distribution of ICAM‐1 in lipid raft domain. Results of the present study indicate that accumulation of ROS through lipid raft‐dependent NADPH oxidase regulates PCB153‐mediated induction of CAMs in brain endothelial cells. Due to its role in leukocyte infiltration, induction of CAMs may contribute to PCBs‐induced cerebrovascular disorders and neurotoxic effects in the CNS. Supported in part by NIH grants (P42 ES 07380, MH63022, MH072567, and NS39254) and the American Heart Association (09SDG2300037).
Pharmacological and toxicological evaluation of methanolic extracts from leaves, steam and roots of a Cnidoscolus plant
Authors: Arjona Carlos A. , Rodríguez‐Fragoso Lourdes , Rosado‐Vallado Miguel , Fitz‐Aranda Claudia , Mendoza‐Rivera Brissa , Reyes‐Esparza Jorge
Published: June 2021

The aim of this work was to evaluate antifungal activities, and the effect on viability, cell proliferation and embryotoxicity of a Cnidoscolus plant antifungal activities were evaluated through microdilution with broth method in different yeast strain. Effect on cell viability and the effect on cell proliferation were evaluated on two cell lines (renal and hepatic cells) at 24 h. Embryotoxicity was evaluated on chicken embryos (Gallus gallus). Leaves and roots extracts had antifungal activity against Candida albicans (752 y 10231 ATCC) with concentrations of 0.1 to 10000μg/mL. Both extracts were cytotoxic and decrease cellular proliferation at fourth day with concentrations higher than 50μg/mL. These methanolic extracts did not affect embryo viability with concentrations lowers to 100μg/mL However, they affected embryo development when the extracts were 100μg/mL or higher. It was observed a minor development, affectation of CNS, reduction of somites, absence or hypoplasia of optical and optical placodes. These results shown that the leaves and roots methanol extracts might have different components which are responsible for the antifungal activity and/or toxic activity.
Efficacy of Ozone Therapy Against the Formation of Peritoneal Adhesion in Rat
Authors: Uysal Bulent , Demirbag Suzi , Cayci Tuncer , Poyrazoglu Yavuz , Topal Turgut , Guven Ahmet , Korkmaz Ahmet
Published: June 2021

INTRODUCTION

Peritoneal adhesion (PA) is a consequence of wound healing that sometimes causes severe postoperative complications. Despite all the preventive measures and agents, PA has still not been eliminated completely. The aim of this study was to evaluate the effect of the medical ozone therapy (MOT) on the development of postoperative peritoneal and uterine tube adhesions.

MATERIAL AND METHOD

Twenty one female Wistar‐Albino type rats were used in this study. The rats were randomly divided into three groups of sham‐operated, PA and PA+MOT. After performing a lower midline abdominal incision, a standard uterine lesion was created by electrocauterization in the PA and PA+MOT groups. The PA+MOT group received 0.7 mg/kg per day ozone/oxygen mixture intraperitoneally for a total of 3 days. At the 7th day, peritoneal washing fluids of rats were obtained for TNF‐alpha evaluation. Then all of the rats were killed, and uterine adhesions were staged according to the Leach scoring system. Uterine tissue samples were harvested for biochemical and histopathological evaluation.

RESULTS

In the PA+MOT group, the total adhesion score, oxidative stress markers including malondialdehyde, superoxide dismutase and glutathione peroxides were significantly lower than the PA group (P < 0.05). In addition, TNF‐alpha levels of peritoneal washing fluid were found decreased in the PA+MOT group than the PA group.

CONCLUSION

Medical Ozone Therapy significantly reduces the prevalence of peritoneal and uterine tube adhesion formation in rats by possible decreasing oxidative stress.
Inflammatory Complications of Leptin
Authors: Vaughan Tamisha , Li Liwu
Published: June 2021

Leptin, a key adipokine involved in regulating food intake and body weight, has been recently implicated in the exacerbation of inflammation. Elevated leptin levels are correlated with increased inflammation in obese individuals with cardiovascular complications. However, the underlying molecular mechanism is poorly understood. In this report, we demonstrated that leptin alone failed to induce the expression of inflammatory cytokines such as IL‐6 in murine macrophages and human monocytic cells. Instead, leptin significantly synergized with LPS in inducing the expression of IL‐6. The key inflammatory signaling molecule, Interleukin‐Receptor Associate Kinase 1 (IRAK‐1), is partially involved in mediating the effects of both LPS and leptin. IRAK‐1 deficient macrophages exhibit significantly lower expression of IL‐6 following LPS or LPS plus leptin stimulation. Mechanistically, we observed that leptin led to stability of IRAK‐1 both transcriptional and protein expression. Taken together, our data reveal that leptin primarily serves as a helper, instead of an initiator of inflammation during the pathogenesis of obesity related inflammatory diseases. The mechanism by which leptin mediates changes in the immune function is not known. In this present study, we aim to test the hypothesis that leptin is involved in activation proinflammatory mediators through stabilizing IRAK‐1.
Short term incubation of bovine neutrophils with Escherichia coli lipopolysaccharide increases their production of reactive oxygen species and formation of extracellular traps in a dose‐dependent manner but has no effect on their chemotactic and antimicrobial capacities
Authors: Revelo Xavier , Waldron Matthew
Published: June 2021

The objective of this study was to investigate the effects of Escherichia coli lipopolysaccharide (LPS) on the function of blood neutrophils (PMNL) harvested from Holstein cows (n = 7). PMNL were incubated with 0, 1, 25 and 50 μg/mL of LPS for 120 min and the generation of reactive oxygen species (ROS), formation of extracellular traps (NETs), chemotactic and antimicrobial capacities were determined. LPS increased the total and intracellular production of ROS in a dose‐dependent manner. Areas under the curves indicated that 50 μg/mL of LPS enhanced intracellular ROS in non‐stimulated PMNL by 184% whereas 25 and 50 μg/mL of LPS increased intracellular ROS in stimulated cells by 79 and 145%, respectively. These effects were partially inhibited by diphenyleneiodonium chloride, a NADPH oxidase blocker. Treatment of neutrophils with 25 and 50 μg/mL of LPS resulted in a 105% increase of NETs release by non‐stimulated PMNL. LPS had no effect on PMNL chemotaxis and did not alter their capacity to kill Staphylococcus aureus. These results suggest that LPS “primes” the neutrophils toward enhanced immunity by increasing the generation of ROS and the expression of NETs. Internal funds.
Biological effects of HIV‐1 glycoprotein gp120's interaction with CD4+ T cells
Authors: Foster Monique , Tuen Michael , Hioe Catarina
Published: June 2021

HIV (human immunodeficiency virus) access to CD4+ T cells depends on the interaction of gp120 (viral envelope glycoprotein) with CD4+ receptor molecules on the targeted cells. Gp120 projects from the surface of HIV like small spikes and is the only extracellular protein on the virus envelope. The contact made between CD4+ T cells and gp120 is essential for virus interaction, but it also can mediate other effects, such as apoptosis or activation of T cells. In our study, we evaluated whether HIV‐1 glycoprotein interaction with CD4+T cells leads to apoptosis or activation of T cells. To do this, we allowed CD4+ T cells to interact with gp120/ICAM‐1 on filter of transwells and analyzed the cells migrating through the wells after interaction with gp120 + ICAM‐1 or ICAM‐1 alone. The data shows: gp120 temporarily stops the CD4+ T cells from migrating, and then resumes migration after one hour, the cells interaction with gp120 + ICAM‐1 did not display any change in expression of apoptosis markers and with stimulation of gp120, CD4+ T cells could still proliferate in response to IL‐2 and allogenic stimulation. These data indicate that the CD4‐‐gp120 interaction, on its own, is not likely to make CD4+ T cells more susceptible to apoptosis. Rather, this interaction may activate the cells to support HIV‐1 infection more efficiently.
The Effects of Cell Lysates of Bifidobacterium longum (BL730) and Lactobacillus casei (E5) on in vitro Stimulation of Mouse Immune Cells
Authors: Lee Woo‐Sik , Hwang Seong‐Gu , Abanto Oliver Diaz , Park Seung Yong
Published: June 2021

The present study was conducted to investigate in vitro mechanisms involved in the immunostimulatory effects of Bifidobacterium longum and Lactobacillus casei. RAW 264.7 macrophage cell line, primary cultures of BALB/c peritoneal macrophages and neutrophils were treated with different concentrations of B. longum lysates (BL) or L. casei lysates (LC). Nitric oxide (NO) production and IL‐6 cytokine expression were measured using nitrite assay and sandwich ELISA, respectively. Neutriphils and macrophages were treated with or without BL or LC, and the phagocytotic activities were measured using FACS. The antioxidant activity of BL and LC were analysed using DPPH assay. Both lysates induced NO production in both cell line and primary cultured cells. The NO production in primary cultures of macrophages and neutrophils were significantly higher (p<0.05) when treated with 500 μg/mL of LC than those treated with the same dose of BL. The BL stimulated IL‐6 cytokine expression significantly higher than LC in both cell line and primary cultured cells. The LC enhanced phagocytotic activity of neutrophils more strongly than BL. The free radical scavenging activity of LC was significantly higher (p<0.05) than that of BL. The results of the present study suggest that both B. longum and L. casei can modulate immune response in mouse, but with different mode of actions.
Regulation of VEGFs expression in human retinal cells by cytokines: implications for the role of inflammation in age‐related macular degeneration
Authors: Nagineni Chandra N , Kommineni Vijay K , William Abitha , Hooks John J , Detrick Barbara
Published: June 2021

Chronic inflammation has been proposed to be associated with the pathogenesis of age‐related macular degeneration (AMD). Choroidal neovascularization (CNV) observed in exudative form of AMD results in loss of vision. The objective of this study is to investigate the effects of inflammatory mediators on VEGFs expression in retinal cells. We used human retinal pigment epithelial (HRPE) and choroidal fibroblast cells (HCHF) prepared from adult donor eyes. The effects of inflammatory cytokine (IC) mix (IFN‐γ + TNF‐α + IL‐1β) on global gene expression (Affymetrix GeneChip) profiles in HRPE cells, revealed 10 and 9 fold increase in the expression of VEGF‐A and VEGF‐C respectively. The microarray results were validated by HRPE secretion of VEGFs proteins. IL‐1β is the most potent inducer of VEGFs secretion followed by IFN‐γ and TNF‐α. Combination of any of these cytokines was more effective in enhancing VEGFs secretion in a dose dependent manner. NFκB and JAK‐STAT pathway, but not HIF‐1α, Sp‐1, Sp‐3 and STAT‐3, transcription factors were involved in IC action. VEGFs secretion by HCHF cells was also significantly enhanced by IC. Our results show that three pro‐inflammatory cytokines induce the secretion of VEGFs by HRPE and HCHF cells. These findings indicate that in AMD the presence of pro‐inflammatory cytokines in chronic inflammation promote choroidal and retinal neovascularization.
Expression profiling of toll‐like receptor mRNAs in various tissues of Toda and Murrah buffaloes
Authors: Raj G Dhinakar , Venkatachalam Ramaswamy , Angamuthu Raja , Gopalan Tirumurugaan Krishnaswamy , Rathnassamy Vignesh Ambothi , Sakhthivel Dhanasekaran , Kathaperumal Kumanan
Published: June 2021

Toda buffalo is a native genetically isolated semi‐wild breed, maintained by the aboriginal Toda tribes, present in the Nilgiri Hills of Tamil Nadu, India. The available population is only 1800. It is a brown medium sized animal with long horns set wide apart in a circular fashion. This breed resembles the swamp buffaloes, but based on karyological studies, it is classified under the river buffalo. The expression profile of toll‐like receptor (TLR) 1–10 mRNAs in spleen, mesenteric lymph node, lung, skin, jejunum and rectum were assessed using TaqMan real time PCR with GAPDH as endogenous control from Toda and Murrah buffaloes (n=4). The Ct values for different TLR mRNA in the tissues were compared among each breed with spleen expression levels as calibrator as well as between the two breeds and analyzed using the relative expression software tool (REST 2008‐V2.0.7). The Toda buffaloes showed higher expression levels of TLR 1 and 10 mRNA in the lymph node, TLR 1 in jejunum and TLR 1, 5, 6, 7 and 10 mRNAs in the skin. However, in Murrah buffaloes, lowered expression levels of TLR 10 mRNA in lung, TLR 4 in lymph node and TLR 10 in rectum and skin were observed. The Toda buffaloes showed significantly higher levels of TLR9 mRNA in the lymph node and TLR 1,5,6,9 and 10 mRNAs in the skin than Murrah. Thus, higher expression levels of a wide repertoire of TLR mRNA were detected in the skin of Toda buffaloes. The resistance of the Toda buffalo to protozoan parasites such as Theileria or Babesia, as is known anecdotally, could be related to the stronger innate immune responses mediated by TLRs in the skin.

This work has been funded under the National Agricultural Innovative Project, Indian Council of Agricultural Research, New Delhi
The Effect of Quercetin in Cultured Human RPE Cells under Oxidative Stress and in Ccl2/Cx3cr1 Double Deficient Mice
Authors: Cao Xiaoguang , Tuo Jingsheng , Shen Defen , Liu Melissa , Chan Chi‐Chao
Published: June 2021

PURPOSE

To investigate mechanisms for the protective effects of quercetin in cultured human retinal pigment epithelium (RPE) cells and in Ccl2−/−/Cx3cr1−/− (DKO) mice, which spontaneously develop retinal lesions mimicking the characteristics of age‐related macular degeneration.

METHODS

Cultured ARPE‐19 cells were exposed to 1mM H2O2 with or without 50μM quercetin for 2 hours. DKO and C57/B6 (WT) mice were treated with 25mg/kg/day quercetin via intraperitoneal injection for two months.

RESULTS

Quercetin protected RPE cells from H2O2‐induced injury, increased the Bcl‐2/Bax transcript ratio, decreased Bax, FADD, Caspase‐3, −9, TNF‐α, iNOS and COX‐2 mRNA levels, and lowered COX and NO levels. DKO mice had a lower antioxidative capacity, higher serum NO, COX and PGE2 levels, and higher ocular bax and caspase3 mRNA levels. Quercetin decreased serum oxidative and inflammatory molecules but not ocular pro‐apoptotic or pro‐inflammatory transcript levels; the retinal lesions in the DKO mice remained unaffected.

CONCLUSIONS

Quercetin protects human RPE cells from oxidative stress in vitro by directly inhibiting the apoptosis pathway and exerting anti‐inflammatory effects. Quercetin has no effect on retinal lesions in DKO mice due to insufficient suppression of ocular apoptosis or inflammation.

The study was supported by the Intramural Research Program of the National Eye Institute, NIH.
Innate resolution of traumatic injury‐induced skeletal muscle inflammation is initiated within 24 hours and involves MKP‐1 induction, Hsf‐1 disinhibition, and Hsp27 expression
Authors: Szelenyi Eric , Warren Gordon , Urso Maria
Published: June 2021

Heat shock protein (Hsp) 27 contributes to the innate resolution of inflammation. Expression of this cytokine is tightly controlled at the level of heat shock factor (Hsf)‐1 activation which is negatively regulated by ERK1 phosphorylation. A central regulator of inflammatory fate, MAPK phosphatase (MKP)‐1 is postulated to positively regulate Hsf‐1 activation via ERK deactivation. To explore this notion in a skeletal muscle injury model, mouse tibialis anterior muscles were subjected to traumatic freeze injury and protein expression and phosphorylation levels from both unilateral injured and contralateral control legs were analyzed at 3, 10, 24, 48, and 72 h post‐injury (PI). At 10 and 24 h PI, a 40% induction (p<.05) of MKP‐1 protein expression was observed with concomitant ERK1/2 deactivation (325% decrease, p<.01) and Hsf‐1 disinhibition (50% decrease; p<.05). An abolishment of constitutive p38 phosphorylation was also seen and sustained through 72 h PI (p<.01). Significant negative correlations were observed amongst Hsp27 protein expression with ERK1/2 (R=−0.65; p<.01) and p38 (R=−0.73; p<.01) phosphorylation. These results support a role of MKP‐1 in suppressing pro‐ and initiating anti‐inflammatory processes through ERK1/2 deactivation and p38 repression. Additionally, MKP‐1 is implicated in the positive regulation of Hsp27 expression through indirect Hsf‐1 disinhibition.
Reduced bioavailability of tetrahydrobiopterin impairs neovascularization after hind limb ischemia
Authors: Santhanam Anantha Vijay R , d'Uscio Livius V , Smith Leslie A , Katusic Zvonimir S
Published: June 2021

Impaired endothelial function in cardiovascular disorders is mediated, in part, by uncoupling of eNOS caused by reduced availability of tetrahydrobiopterin (BH4). Endothelial progenitor cells (EPCs), originating from bone marrow, contribute to repair of vascular system by either neovascularization following tissue ischemia or re‐endothelialization after endothelial injury. The contribution of BH4 to the angiogenic function has not been studied. In the present study, we investigated the role of BH4 in angiogenesis and mobilization of cells enriched in EPCs in hph‐1 mutant mice, a genetic model of GTP‐cyclohydrolase I deficiency. HPLC analysis revealed that enzymatic activity of GTP‐cyclohydrolase I is reduced by 65% in bone marrow of hph‐1 mice (22±4 pmol neopterin/mg/h; P<0.05 vs. wild‐type mice: 63±4 pmol/mg/h). Basal counts of cells co‐expressing CD34 and VEGFR2 (Flk‐1) were reduced in bone marrow and peripheral blood of hph‐1 mice. Fourteen days after hind limb ischemia, recovery of blood flow was significantly reduced in hph‐1 mice as compared to blood flow in ischemic limb of wild type mice. Our findings suggest that reduced bioavailability of BH4 impairs mobilization of cells enriched in EPCs. This effect may help to explain impaired angiogenesis in hph‐1 mice.
Betaine‐homocysteine methyltransferase pharmacogenetics: human liver genotype‐phenotype association study
Authors: Feng Qiping , Kalari Krishna , Fridley Brooke L , Jenkins Gregory , Zhang Jianping , Johnson Monica Nye , Weinshilboum Richard M
Published: June 2021

Objectives

Betaine‐homocysteine methyltransferase (BHMT) catalyzes the remethylation of homocysteine. BHMT in the human liver is responsible for up to 25% of homocysteine metabolism. Knowledge of the relationship between genetic polymorphisms and BHMT phenotypes could enhance our understanding of the role of this reaction in drug metabolism.

Methods

Levels of BHMT enzyme activity and immunoreactive protein were measured in 268 human liver biopsies. Twelve tag BHMT SNPs from our own gene resequencing and HapMap data were used to genotype DNA from these samples. Genotype‐phenotype association analysis was then performed. BHMT enzyme activity averaged 0.077 ± 0.028 units/μg, with a 12 fold variation.

Results

BHMT protein levels averaged 6.9 ± 3.3 units/μg, with a 50‐fold variation. Three SNPs (rs41272270, rs16876512, and rs6875201), located 28kb upstream, in the 5′‐UTR and in intron 1 of BHMT, respectively, were significantly correlated with both phenotypes (p<10−8 for enzyme activity and p<10−5 for protein level). rs41272270 was associated with altered protein binding during electrophoresis mobility shift assay.

Conclusions

Three tag SNPs were significantly associated with human hepatic BHMT enzyme activity and immunoreactive protein. These observations may help us understand the regulation of BHMT in the human liver and its role in drug metabolism. Supported by NIH U01 GM61388, R01 GM28157, R01 CA132780.
Sudden death in mice with increased Go signaling
Authors: Kehrl Jason , Charbeneau Raelene , Dalton Hans , Haung Xinyan , Neubig Richard
Published: June 2021

The inhibitory G protein Gαo is the most abundant G protein in the brain, yet its signaling mechanism is incompletely understood. To better understand the mechanism and physiological role of Gαo signaling, we generated a knock–in mouse with a mutation (G184S) that enhances Gαo‐mediated signaling. Homozygous GαoG184S/G184S mice die shortly after birth. Born at Mendelian ratios, none survive 24 hours. The heterozygous GαoG184S/+ mice are viable and appear normal up to ~12 weeks of age. After this time, they show a strain‐dependent lethality ‐ mice on a 129/SvJ background live more than a year but after back‐crossing onto a C57BL/6J background about half die between 12 and 30 weeks of age. Weight, grooming, and behavior appear normal up to 24 hours prior to death. To identify novel genes that may modify Gαo signaling or physiological functions involved in sudden death, we undertook a genome‐wide SNP analysis. Potential modifiers of this phenotype were identified on Chromosome 17. Future studies will use this model to attempt to understand both Gαo‐mediated signaling and the potential causes and significance of this novel sudden model. Supported by RO1‐GM39561 (RRN) and 5T32GM008322 (JMK).
Differential activation of rat and human pregnane X receptor by terpene trilactones
Authors: Lau Aik‐Jiang , Chang Thomas K. H
Published: June 2021

Pregnane X receptor regulates the expression of genes (e.g. CYP3A) involved in various biological functions. Among the terpene trilactones, ginkgolide A induces CYP3A in rat and human hepatocytes, but conflicting data exist for bilobalide. We compared the effect of five individual terpene trilactones on rat PXR (rPXR) and human PXR (hPXR) activity. As assessed by a luciferase reporter gene assay in rPXR‐ or hPXR‐transfected human hepatoma cells (HepG2), control analysis showed that pregnenolone 16α‐carbonitrile activated rPXR with an EC50 of 1.0 ± 0.2 μM (mean ± S.E.M., n = 4) and Emax of 17 ± 2 fold, whereas rifampicin activated hPXR with an EC50 of 0.4 ± 0.1 μM and Emax of 12 ± 2 fold. Ginkgolide A (30–100 μM) activated rPXR (7–8 fold; Emax = 9 ± 1 fold) to a lesser extent than hPXR (9–11 fold; Emax = 12 ± 1 fold), and the EC50 values were 12 ± 1 and 16 ± 1 μM for rPXR and hPXR activation, respectively. The increase in rPXR activity (2–8 fold) by ginkgolide B (3–100 μM) was less than that of hPXR (3–15 fold). Ginkgolide C (60–100 μM) activated rPXR to a somewhat lesser extent than hPXR, but ginkgolide J and bilobalide had no effect. In summary, several of the ginkgolides activated rPXR and hPXR differentially. Ginkgolide A and ginkgolide B were stronger activators than ginkgolide C, ginkgolide J, and bilobalide, suggesting a relationship between the structure of terpene trilactones and activation of rPXR and hPXR. [Supported by CIHR and MSFHR]
Losartan is a Substrate of Organic Anion Transporting Polypeptide 2B1
Authors: Flynn Colleen Ann , Hagenbuch Bruno A. , Reed Gregory A.
Published: June 2021

Losartan, an angiotensin II receptor antagonist, is used as a probe of CYP2C9 activity both alone and in probe drug cocktails. Although losartan conversion to E3174 provides an index of CYP2C9 activity, transporters may also play a role in losartan disposition. Therefore, we tested whether losartan would affect organic anion transporting polypeptide (OATP) 2B1‐mediated transport. Uptake ofthe model substrate estrone‐3‐sulfate (E3S) was measured using a CHOcell line stably expressing OATP2B1 in theabsence and presence of losartan at pH 7.4 and pH 5.5. OATP2B1‐mediated uptake of E3S was strongly inhibited by losartan at both pHs, with IC50 values of 8.2 μM at pH 7.4 and 2.8 μM at pH 5.5, indicating that losartan could be a substrate of OATP2B1. To test this, losartan transport was measured at pH 5.5 and 7.4. Uptake was higher at pH 5.5 as compared to pH 7.4 and initial kinetics at pH 7.4 showed saturable uptake (Km = 16.6 μM). These resultsdemonstrate that losartan is a novel substrate of OATP2B1 at both pH 7.4 and 5.5, with increased transport capacity at the lower pH. We are currently characterizing the OATP2B1‐mediated losartan transport and will also examine this transporter as a site for drug‐drug interactions.
Oral absorption and pharmacokinetics of Methadone HCl and intestinal multidrug resistance gene expression in horses
Authors: Linardi Renata Lehn , Barker Steven A , Short Charles , Kousoulas Konstantin G , Huang Li‐Ju , Stokes Ashley M
Published: June 2021

This study aimed to determine the pharmacokinetics (PK) and oral bioavailability of methadone in horses to evaluate a possible influence of transport proteins on drug absorption through a relative quantitation of multidrug resistance gene/P‐glycoprotein (MDR1/P‐gp) gene expression in horse tissues.

Six healthy horses received methadone by intravenous, oral, or nasogastric tube (NG) routes. Methadone serum concentration was measure and PK analysis performed. Total RNA was extracted from tissues from 10 horses and TaqMan RT‐PCR was used to quantify the gene of interest using Relative Standard Curve Method and fold‐differences in nucleic acid across tissues. Significance was determined with a P‐value < 0.05.

Methadone was rapidly absorbed and eliminated with no side effects observed. Concentration was higher and occurred sooner after oral compared to the NG administration. Bioavailability was approximately 90% and 28% for oral and NG routes, respectively. Compared to the oral mucosa tissue, MDR1/P‐gp mRNA expression levels were significantly higher in the esophagus, stomach, small intestine, and liver.

Methadone absorption from the small intestine appears limited possibly due to high expression levels of MDR1/P‐gp gene. High bioavailability after oral administration is supported by the low gene expression levels in tissues like oral mucosa and sublingual.

Funding was provided by VCS and EHSP Grants, LSU.
Identification of critical cysteine residues of Keap1 in arsenic‐sensing and suppression of Nrf2
Authors: He Xiaoqing , Ma Qiang
Published: June 2021

Arsenic activates Nrf2 to induce phase II and antioxidative genes. Here we analyzed arsenic‐Keap1 cysteine thiol interactions for Nrf2 activation. Arsenic‐based Nrf2 activators FlAsH and phenylarsine oxide (PAO) were used to probe binding of arsenic to Keap1. FlAsH emitted strong fluorescence upon binding to purified Keap1, and arsenic, tert‐butyhydroquinone (tBHQ), or the thiol reactive 2, 3–dimercaptopropanol inhibited FlAsH binding. Purified or endogenous Keap1 was effectively pulled down by PAO affinity beads in vitro and from hepa1c1c7 cells, and arsenic, tBHQ, free PAO, or cadmium blocked Keap1 pull down. Furthermore arsenic and free PAO significantly reduced the free thiol contents of purified or endogenous Keap1 in cells. Therefore, arsenic, FlAsH, PAO, tBHQ, and cadmium bind to Keap1 cysteine thiols competitively. All domains of Keap1 bound PAO, but the linker region exhibited highest binding activity. The function of arsenic‐Keap1 interaction was evaluated in a reconstituted system that mimics endogenous Nrf2 regulation. Mutation of C273 or C288 in linker region resulted in higher level expression of Nrf2 protein in the absence of inducers. Mutation of C151 abolished Nrf2 activation by arsenic. Overexpression of C273A, C288A, or C151A altered the basal and arsenic‐induced expression of Nqo1 consistent with regulated protein levels of Nrf2. The study demonstrates important roles of C273 and C288 in the suppression of Nrf2 by Keap1 under basal conditions and critical function of C151 in arsenic sensing and responsiveness. Our findings support the notion that arsenic binds to different sets of Keap1 cysteine residues to regulate divergent functions in Nrf2 signaling.
Lactoferrin Protects Against Acetaminophen‐Induced Liver Injury in Mice
Authors: Yin Hao , Cheng Linling , Holt Michael , Hail Numsen , MacLaren Robert , Ju Cynthia
Published: June 2021

Acetaminophen‐induced liver injury (AILI) is a significant health problem and represents the most frequent cause of drug‐induced liver failure in the United States. The development and implementation of successful therapeutic intervention strategies have been demanding, due to significant limitations associated with the current treatment for AILI. Lactoferrin (Lac), a glycoprotein present in milk, has been demonstrated to possess a multitude of biological functions. Our study demonstrated a profound protective effect of Lac in a murine model of AILI, which was not dependent on its iron binding ability, inhibition of acetaminophen (APAP) metabolism, or a direct cytoprotective effect on hepatocytes. Instead, Lac treatment significantly attenuated APAP‐induced liver sinusoidal endothelial cell dysfunction and ameliorated hepatic microcirculation disorder. This protective effect of Lac appeared to be dependent on hepatic resident macrophages (Kupffer cells, KC). Collectively, our data indicated that Lac, through activation of KC, inhibited APAP‐induced liver sinusoidal endothelial cell damage and improved hepatic congestion, thereby protecting against AILI. These findings reveal the significant therapeutic potential of Lac during AILI and other types of liver diseases.
Network Analysis of the Cytoprotective Effect of CDDO‐IM against Oxidant Stress in Human Umbilical Vein Endothelial Cells (HUVEC)
Authors: Wang Xinyu , Bynum James , Stavchansky Salomon , Bowman Phillip
Published: June 2021

1[2‐cyano‐3,12‐dioxooleana‐1,9(11)‐dien‐28‐oyl]imidazole (CDDO‐IM), a derivative of oleanolic acid, has been demonstrated to possess anti‐inflammatory activity and to prevent tumor progression in mice if present when tumor cells were administered. CDDO‐IM (kindly supplied by Dr. Michael Sporn, Dartmouth University) was compared to the phenolic cytoprotectant caffeic acid phenethyl ester (CAPE), and CDDO‐IM at 200 nM was more effective than CAPE at 20 μM in protecting HUVEC from oxidant stress produced by menadione. Since CDDO‐IM possesses no antioxidant activity we tested it for transcriptional activation with whole genome microarray and found that about 700 genes were up or down‐regulated by CDDO‐IM. In addition to up‐regulating heme oxygenase‐1, a well‐known cytoprotective gene, it also induced members of the heat shock protein family. Submission of genes altered in their expression by greater than two fold up‐regulation to Ingenuity Pathway Analysis (IPA) produced networks related to cellular development, growth and proliferation, cell signaling, and canonical pathways including NRF2‐mediated oxidative stress response and PPAR signaling indicating that cytoprotection involves multiple pathways in addition to the well described phase II enzyme induction.
Cytoprotection of human endothelial cells from oxidative stress by polyphenols: the role of gene expression versus direct antioxidant effect
Authors: Wang Xinyu , Bynum James , Stavchansky Salomon , Dubick Michael , Hackman Robert , Keen Carl , Bowman Phillip
Published: June 2021

Polyphenols have been implicated in protecting cells against oxidative stress. Several polyphenols including caffeic acid phenethyl ester (CAPE), curcumin, resveratrol, caffeic acid, catechin, and Oligonol™, a commercial source of a mixture of polyphenolics were investigated for their cytoprotective effects in an in vitro model of menadione‐induced oxidative stress in human umbilical vein endothelial cells and for the transcriptional activity. CAPE, curcumin, and resveratrol showed good dose‐dependent cytoprotection against menadione‐induced cytotoxicity, whereas oligonol, (+)‐catechin, and caffeic acid did not. The results of ‘direct’ antioxidant capacities of those compounds by 2, 7‐dichlorofluorescin assay indicated that most compounds tested showed good free radical scavenging abilities except resveratrol. However, ‘direct’ antioxidant activity did not correlate well with their cytoprotective effects. Gene expression analysis with whole genome microarrays and submission of statistically significant results to Ingenuity Pathway Analysis showed that a number of genes were up‐ or down‐regulated by these compounds in unique and common patterns, which may account for their beneficial effects, in particular the heat shock protein family and IL‐8 signaling pathway.
Specificity of Brain Structures Sensitive to Initiating Nerve Agent‐Induced Seizures
Authors: Shih Tsung‐Ming , Skovira Jacob W , McDonough John H
Published: June 2021

Seizures not only complicate the treatment of nerve agent poisoning, but can also lead to extensive brain damage if uncontrolled. An understanding of the brain structures that initiate seizure activity would be beneficial in identifying targets for therapeutic intervention following nerve agent exposure. This study identified brain structures within the forebrain and parahippocampal regions sensitive to initiating nerve agent‐induced seizure activity and determined whether this sensitivity was ubiquitous throughout each structure. Rats were surgically prepared to record brain electroencephalographic (EEG) activity and implanted bilaterally with guide cannulae toward a designated area of the brain. On the day of the experiment, animals were injected bilaterally with 1 microliter (μl) VX (0.88 μg) through an injection cannula inserted into the guide cannulae and continuously monitored for EEG seizure activity. The amygdala, and the endopiriform, piriform, and rhinal cortices, were all sensitive to initiating seizure activity to varying degrees. Specificity was also observed between different injection sites within these structures in regard to seizure initiation. The results show a hierarchy of brain structures sensitive to initiating nerve agent‐induced seizures and within each structure a regional specificity that should be further examined as a target for anticonvulsant therapies.
Lysophospholipid signaling in human neural progenitors
Authors: Callihan Phillip , Gelineau‐van Waes Janee , Stice Steven L , Riley Ronald T , Hooks Shelley B
Published: June 2021

Recent studies have shown that the fungal metabolite Fumonisin B1 (FB1) and the immunomodulatory drug FTY720 cause neural tube defects in mice. Both drugs inhibit ceramide synthase, resulting in an accumulation of sphingosine 1‐phosphate (S1P) and sphinganine 1‐phosphate (Sa1P) which are agonists for G‐protein coupled S1P receptors (S1PR). S1PRs are key regulators of neural development, suggesting that FB1 or FTY720 may cause developmental toxicity by altering signaling through S1PR. We have recently described S1P signaling pathways in human embryonic stem cell‐derived neuroepithelial cells (hES‐NEP). The goal of this project is to define the effects of FTY720 and FB1 on hES‐NEP cells, and establish this system as a platform in which to define the mechanism of lysophospholipid signaling in neural development. In the current study, we show that treatment of hES‐NEP with FB1 or FTY720 recapitulates the alterations in lipid profiles observed in‐vivo, resulting in marked increases in Sphinganine and Sa1P. Further, FTY720 is phosphorylated to the S1P receptor agonist FTY720‐P in hES‐NEP cells. Like S1P, both Sa1P and FTY720‐P stimulate activation of phospholipase C and inhibition of adenylyl cyclase, but with distinct rank order potencies and sensitivity to receptor and G‐protein inhibitors, suggesting these metabolites have unique signaling properties in human neural progenitors.

Funding for this work is provided by the NIH and the University of Georgia.