bluuebunny/crossref_metadata_2025 huggingface dataset:
β1 adrenergic receptor‐initiated dilation in rat cerebral arteries is mediated by Shaker‐type KV1 channels and is attenuated in angiotensin II‐induced hypertension
Authors: Joseph Biny K , Rusch Nancy J , Rhee Sung W
Published: June 2021

The β1 adrenergic receptor (β1AR), a protein ubiquitously expressed in the cardiovascular system, initiates vasodilation in the cerebral circulation. For example, rat cerebral arteries dilate robustly to isoproterenol (ISO) and norepinephrine (NE), and these responses are blocked by atenolol, a β1AR blocker. However, the identity of the ion channel(s) that mediate the β1AR‐initiated dilation is unclear. In this study, we report that the dilator responses to ISO and NE of isolated, cannulated rat cerebral arteries perfused at 60 mm Hg are abolished by 1 μM correolide, a selective blocker of Shaker‐type voltage‐gated (KV1) channels. To examine the effect of hypertension on this dilation, we used Sprague‐Dawley rats subcutaneously infused with angiotensin II (Ang II, 500 ng/kg/min) for two weeks. Cerebral arteries from Ang II hypertensive rats dilated significantly less to ISO and NE compared to the saline‐infused control rats, suggesting a loss of β1AR‐initiated dilation. Real‐time PCR revealed a reduction in gene expression of pore‐forming α1.2 and α1.5 subunits of KV1 channels in cerebral arteries of Ang II hypertensive rats. Collectively, our study provides evidence for: i) a functional coupling of KV1 channels and β1AR in rat cerebral arteries, and ii) a coordinated loss of this vasodilator pathway in the Ang II rat model of hypertension that may result in cerebral blood flow abnormalities. AHA 0715570Z (BKJ)
The Surprising Pharmacology of Vascular α2‐Adrenoceptors
Authors: Crassous Pierre‐Antoine , Flavahan Sheila , Flavahan Nicholas
Published: June 2021

Mouse tail arteries express smooth muscle constrictor α1 and α2‐adrenoceptors (α‐ARs). Although pretreatment with the α2‐AR antagonist rauwolscine (3x10‐8M) does not inhibit constriction to α‐AR agonists cirazoline, phenylephrine or norepinephrine, it causes powerful transient dilation when given during constriction to these agonists. Experiments were performed to analyze the mechanism of this dilation in isolated tail arteries. The transient relaxation to rauwolscine was mimicked by another α2‐AR antagonist (RX821002, 3x10‐8M), was dependent on agonist activity at α2‐ARs (norepinephrine = phenylephrine > cirazoline), and did not occur during AR‐independent constriction to U46619. However, during combined constriction to the α2‐AR agonist UK14304 plus U46619, rauwolscine abolished the α2‐AR‐component and caused a powerful transient dilation of the U46619‐component of constriction. A similar dilation was observed after rapid removal of UK14304 from the perfusate. Endothelial denudation abolished these dilator responses. In conclusion, α2‐AR activation is associated with arterial constriction that reverses rapidly, and endothelium‐dependent dilation that reverses slowly. α2‐AR antagonists therefore cause transient dilation that is greater than the contribution of α2‐ARs to the constriction. This interesting phenomenon provides a rapid and sensitive test of agonist selectivity.
Analysis of leptin receptor and vascular reactivity changes in aorta and isolated heart rat with low weight at birth and with high hypercaloric diet/ Diabetes type 2 model.
Authors: Castillo‐Hernandez Maria del Carmen , Manuel‐Apolinar Leticia , Guevara‐Balcazar Gustavo , Velazquez‐Dominguez J. Antonio , Zarate‐Treviño Arturo , Castillo‐Henkel Carlos
Published: June 2021

This work study the expression of leptin ObRb receptor and the functional changes in the vascular reactivity in thoracic and abdominal aorta and isolated heart, obese and control Sprague Dawley rats were study, in one group was induced intrauterine malnutrition (E) due to the 50% food restriction in the pregnant rats and was compare with another group with any food restriction, control (C). The newborns with low weight at birth (BPN) group E had been feed with hypercaloric diet (60% lipids) until 6 months older. Recognize the vascular effect of angiotensin II and phenylephrine in thoracic and abdominal rings and isolated heart rat, and in the other hand we analyze the RNA total expression and RT‐PCR for ObRb receptor.

Results

Intrauterine malnutrition showed BPN in the group E (p<0.01) compare with group C, and at 6 months older the difference between 2 groups was ±100g for group E with the hypercaloric diet. Group E showed a hyperglycemic (p<0.01), like an increment in the leptin receptor ObRb expression (p<0.01) compared with the group C. On the other hand both groups showed differences in the vascular reactivity and heart responses for angiotnesin II, it's possible that those could be by the changes in the leptin receptor expression in the obese rat.
Opposing effects of chronic stress and leptin on adult hippocampal neurogenesis
Authors: Garza Jacob , Guo Ming , Zhang Wei , Lu Xin‐Yun
Published: June 2021

Adult hippocampal neurogenesis has been implicated in the therapeutic mechanism of antidepressants. Here we show that the adipocyte‐derived peptide leptin promotes neurogenesis in the adult hippocampus under basal conditions and reverses chronic stress‐induced impairment of neurogenesis. Chronic administration of leptin increased cell proliferation, but had no significant effect on differentiation and survival of newly proliferated cells in the dentate gyrus of the hippocampus. Analysis of signal transduction pathways in cultured hippocampal neural progenitor cells revealed that leptin stimulated phosphorylation of Akt and STAT3, but not Erk1/2. Pretreating the cells with specific inhibitors of Akt or STAT3 attenuated leptin‐stimulated cell proliferation in a dose‐dependent manner, suggesting that these pathways may mediate the effects of leptin on neurogenesis. In a chronic stress model of depression, we demonstrated that neurogenesis was suppressed by chronic stress, which was accompanied by depression‐like behavioral deficits. Chronic leptin treatment was able to reverse the chronic stress‐induced suppression of neurogenesis and behavioral deficits. Taken together, these results support an important role for leptin in adult hippocampal neurogenesis under both physiological and pathological conditions.
In vitro evaluation of the neuroprotective effects of Lepidium meyenii (maca) in crayfish neuronal and rat neuroblastoma cell lines
Authors: Nguyen Diane , Pino‐Figueroa Alejandro , Maher Timothy J.
Published: June 2021

Oxidative stress, considered one of the major factors leading to cell damage or death, is characteristic of many neurodegenerative diseases. Extracts of the Peruvian plant Lepidium meyenii (maca) containing compounds such as macamides and others, have been shown in preliminary in vivo studies to exert neuroprotective effects. In this study, the neuroprotective effects of the pentane extract and various isolated macamides were evaluated on neurotoxicity induced by hydrogen peroxide in two cultured animal cell lines: Orconectes limosus (crayfish), OLGA‐PH‐J/92, and rat neuroblastoma, NG108‐15, cell lines. Determination of cell viability used the MTS assay and spectrophotometric measurement. Experiments in crayfish neurons showed that various concentrations of the pentane extract and the macamides attenuated hydrogen‐peroxide induced cell oxidative stress in vitro, by 25‐52% compared to control. Studies on rat neuroblastoma cells using various incubation parameters showed a reduction of oxidative damage by a range of 11‐66% (depending on experimental method used). These in vitro studies support the in vivo neuroprotective effects observed in ischemia‐damaged rats. Additional studies are needed to further characterize the pharmacology of these compounds for their potential as a clinically useful approach to the treatment of neurodegenerative diseases. Supported by: SURF MCPHS.
Integration of dilator and constrictor pathways for arteriolar reactivity in the metabolic syndrome
Authors: Frisbee Jefferson C. , Brock Robert W. , Hollander John M.
Published: June 2021

Previous study suggests that, with evolution of the metabolic syndrome, arteriolar reactivity is altered such that endothelium‐dependent dilation is attenuated and specific constrictor responses (adrenergic and myogenic) are enhanced, and may act to constrain hyperemia at low‐moderate metabolic demand. This study investigated interactions between dilator and constrictor reactivity in isolated resistance arterioles of obese Zucker rats (OZR). Dilation in OZR to acetylcholine, arachidonic acid (AA) and reduced PO2 was blunted vs. controls, although adenosine dilation was constrained only by structural alterations to the arteriolar wall. Increased adrenergic tone (phenylephrine) or intralumenal pressure (ILP) blunted dilation in both strains (OZR>LZR). Treatment of arterioles with TEMPOL (anti‐oxidant) or SQ‐29548 (PGH2/TxA2 receptor antagonist) improved dilation to AA, hypoxia and acetylcholine with increased ILP, but had minimal effect with increased adrenergic tone. Adenosine dilation was maintained in both strains with increased ILP or adrenergic tone. These results suggest that constrictor effects of adrenergic tone and dilator influences of adenosine are robust contributors to integrated vascular tone, and that endothelium‐dependent stimuli and myogenic tone may act as modifiers via the impact of oxidant stress on NO availability and AA metabolism. (NIH R01 DK64668, AHA EIA 0740129N)
Endothelial cell morphology creates preferred regions for leukocyte transmigration at venular convergences
Authors: Sumagin Ronen , Sarelius Ingrid H
Published: June 2021

Leukocyte transmigration (TEM) occurs mainly via endothelial cell (EC) junctions, but whether there is a preference for two‐ or tri‐cellular junctions is not clear. Using intravital confocal microscopy of anesthetized mouse cremaster muscle in combination with immunofluorescence labeling we showed that leukocyte TEM in venular convergences was 8‐fold higher than in straight venular regions. Interestingly, due to the difference in EC size, shape and orientation, the number of tri‐cellular junctions in converging venules is significantly higher than in straight regions (20.7±1.2 vs 12.43±1.1/6000μm2 respectively). The majority (55±1.8%) of adhered leukocytes in venular convergences were found at the tri‐cellular junctions, compared to only 21±2.8% in straight regions. Importantly, 45.1±4% of leukocytes in these straight regions and 59.3±2% in venular convergences used the tri‐cellular junctions for TEM, suggesting tri‐cellular junctions as the preferred route. Leukocyte crawling distances were shorter (17.8±2.4μm vs 26.2±2.6μm) and the TEM efficiency was higher in venular convergences (32.8±6.1 %/total adhered) compared to straight venular regions (14.6±6.1, %/total adhered). We also found that 42.2±4% of the tri‐cellular junctions in both regions were surrounded by ICAM‐1 enriched areas, supporting their role as portals for leukocyte TEM. Supported by NIH HL 18208, HL 75186 and AHA 0615677T
Retrograde shear stress modulates rat mesentery microvessel permeability and endothelial adhesion structures
Authors: Adamson R H , Sarai R K , Weinbaum S , Curry F E
Published: June 2021

Vascular endothelial cells (EC) orient their long axis parallel to the fluid flow direction both in vivo and in culture. The EC surface glycocalyx transduces the fluid flow shear stress that mediates nitric oxide production (Florian et al. Circ Res 2003) and EC cytoskeletal rearrangement (Thi et al. PNAS 2004). To test that in vivo shear stress regulates EC adhesion structures and permeability we compared final hydraulic permeability (Lp) measured in rat mesentery venular microvessels perfused orthograde with Lp measured in vessels perfused retrograde, both for about 3 hours. Shear stress ranged from 0.2 to 17 dyne/cm2 in the retrograde group and from 3.2 to 36 in the orthograde group. For retrograde vessels Lp (range 0.4 to 30 x 10−7 (cm/s*cmH2O)) was significantly correlated (p < 0.05) to mean shear stress, with a linear regression slope 1.5 x 10−7 (cm/s*cmH2O)/(dyne/cm2) and Lp intercept ‐0.6 x 10−7 (cm/s*cmH2O). For orthograde vessels the Lp (2.0 ± 0.6 x 10−7 (cm/s*cmH2O)) was not correlated with mean shear stress. We also examined the continuity of the adhesion protein VE cadherin (VE) and that of the tight junction protein occludin (Oc). Significantly more discontinuities were observed for both VE and Oc in the retrograde group than in the orthograde group. In vivo reversal of flow direction stimulates increased Lp correlated with shear stress and induces rearrangement of cytoskeletal structures. HL44485
Mechanisms Underlying Mechanical Dysfunction After Rewarming From Hypothermia
Authors: Tveita Torkjel , Han Young‐Soo , Prakash Y.S. , Sieck Gary C
Published: June 2021

Rewarming patients following profound hypothermia may result in acute heart failure and high mortality (50‐80%); however underlying pathophysiological mechanisms are largely unknown. We characterized myocardial dysfunction induced by hypothermia/rewarming (H/R) by measuring intracellular Ca2+ concentration ([Ca2+]i) and twitch force in intact electrically stimulated left ventricular (LV) rat papillary muscles loaded with Fura‐2AM. In skinned papillary muscle fibers, we assessed maximal Ca2+‐activated force (Fmax) and Ca2+ sensitivity of force. Following H/R, peak twitch force decreased by 30‐40%, but [Ca2+]i was not significantly altered. In skinned preparations, maximal Fmax was decreased by ~30%, while the pCa required for 50% of Fmax (pCa50) was reduced by ~0.14. Interestingly, in rewarmed papillary muscle, both total cTnI phosphorylation and protein kinase A (PKA)‐mediated cTnI phosphorylation at Ser23/24 were significantly increased compared to controls. We conclude that after H/R myocardial contractility is significantly reduced as evidenced by reduced twitch force and Fmax. Reduced myocardial contractility is attributed to decreased Ca2+ sensitivity of force that likely results from increased cTnI phosphorylation.
Effects on ventilation of Lung Volume Reduction Surgery in Emphysema
Authors: Bartels Matthew N , Gerardo Renee E , Layton Aimee M , Downey John A , Demeersman Ronald E
Published: June 2021

AIM

To evaluate efficiency of ventilation during exercise in emphysema after lung volume reduction surgery (LVRS) compared to medical treatment (MED).

METHODS

41 patients with emphysema were randomized into LVRS (33) or MED (8). Data were compared from a cardiopulmonary exercise test (CPX) using a 5 or 10 watt protocol. CPX was performed at baseline and 12 months post randomization. T‐test with a Bonferroni correction was used determine statistical differences at p<0.01.

RESULTS

Baseline demographics and exercise evaluations were the same between groups. LVRS demonstrated improved work capacity [37 (17) vs 50 (18) Watts, p<0.01], improved PetCO2 [42.9 (6.0) to 38.4 (3.8) mmHg, p<0.001)], and increased ventilation [(VE) (24.9 (7.2) vs 31.6 (10.6) liters/min, p<0.005)] while no change was found in the MED group. At 12 months LVRS improved versus MED for VE [(31.6 (10.6) vs 22.1 (6.9) liters/min, p<0.01)], and Wattage [50 (18) vs 33 (15) Watts, p<0.005], with a trend in PetCO2 [38.4 (3.8) vs 47.1 (8.7) mmHg, p<0.001].

DISCUSSION

LVRS improved ventilation allowing improved exercise capacity. These improvements may be due to removal of alveolar dead space in the LVRS group with improved pulmonary mechanics and improved ventilatory efficiency. Further elucidation of the mechanisms of improvement needs to be done with more detailed analysis of ventilatory mechanics with exercise. Supported by the VIDDA foundation
Physical Training does not Influence Vasodilatation Responses in Individuals with M235T AGT and I/D ECA Gene Polymorphisms
Authors: Dias Rodrigo Goncalves , Alvim Rafael O. , Rene Cleber R. , Alves Guilherme B. , Pereira Alexandre C. , Krieger Jose E. , Negrao Carlos E.
Published: June 2021

We hypothesized that: 1)Reflex muscle vasodilatation to exercise is attenuated in individuals with 235T or D alleles and 2)Physical training would improve muscle vasodilatation responses in these individuals.

Methods

108 healthy individuals were genotyped for the M235T AGT (MM=20; MT=49; TT=39) and I/D ACE (DD=38; DI=48; II=22) genes variants. Mean blood pressure (MBP) and forearm blood flow (FBF, plethysmography) were evaluated at rest and at 30% handgrip exercise. Physical training was performed for 17 weeks (3 running sessions/week).

Results

Resting forearm vascular conductance (FVC) and MBP were similar among AGT or ACE genotypes. During exercise, FVC increased significantly and similarly among MM, MT and TT (3.6±0.4; 3.3±0.2; 3.6±0.2 units, respectively, P=.91) and DD, DI and II (3.4±0.2; 3.6±0.3; 3.3±0.3 units, respectively, P=.92). Physical training significantly increased peak VO2(P=.001). Resting FVC was unchanged by physical training. Physical training improved slightly and similarly FVC responses to exercise in MM, MT and TT (4.1±0.5; 3.8±0.2; 3.8±0.3 units, respectively, P=.81) and DD, DI and II (3.6±0.2; 4.0±0.3; 3.7±0.4 units, respectively, P=.38).

Conclusions

M235T AGT and the I/D ACE gene polymorphisms do not influence reflex muscle vasodilatation to exercise and the vascular response to physical training in healthy individuals.
Endothelial Progenitor Cell Adhesion Mediated by α4β1 and α5β1 Integrins is Critical to the Restoration of Lung Vascular Endothelial Barrier Function in a Model of Endotoxin‐induced Acute Lung Injury
Authors: Wary Kishore K , Vogel Steven M , Zhao Yidan D , Malik Asrar B
Published: June 2021

Here we tested the hypothesis that integrin‐mediated adhesion event is a critical requirement for the endothelial barrier protection induced by endothelial progenitor cells (EPCs). We used bone‐marrow (BM)‐derived mononuclear (MNCs) as a source for EPCs, and characterized for EPC marker expression including CD34, VE‐cadherin, Flk1, and integrins. To model Acute Lung Injury (ALI), the recipient mice were γ‐irradiated then challenged intraperitonially with bacterial endotoxin Lipopolysaccharide (LPS). Mice were administered with either control endothelial cells (ECs), EPCs, EPCs incubated with adhesion blocking anti‐integrin monoclonal antibodies, or EPCs transduced with specific shRNA integrin construct. The end points assessed were: (a) survival, (b) lung water content, (c) capillary filtration coefficient (Kfc), (d) H&E staining of lung sections, and (e) confocal image analysis of EPC retention. We found that treatment with EPCs afforded 50% survival (P<0.001) and restored lung fluid balance (Kfc, P<0.001) in α4 and α5 integrins‐dependent manner. Confocal image analyses showed increased retention of Red Fluorescent Protein (RFP)‐tagged EPCs until 8 weeks and overall improved lung morphology. Thus, the treatment with EPCs reduced the extent of LPS‐induced lung microvessel injury, restored lung fluid balance, and increased survival in α4β1 and α5β1 integrins dependent manner.
Effects of endothelin‐1 on endothelial progenitor cell function
Authors: Diehl Kyle J , Westby Christian M , Weil Brian R , MacEneaney Owen J , Morgan Richard G , Mestek Michael L , Kushner Erich J , Van Engelenburg Alexander M , DeSouza Christopher A
Published: June 2021

Circulating endothelial progenitor cells (EPCs) play an important role in maintaining and repairing the vascular endothelium and restoring functional activity. Endothelin (ET)‐1, a potent vasoconstrictor peptide, is released by the endothelium and contributes to endothelial damage and atherogenesis. The effects of ET‐1 on EPC function are unknown. We tested the hypothesis that ET‐1 impairs EPC function. Peripheral blood samples were collected from 10 healthy adult humans. Cells with phenotypic EPC characteristics were isolated and EPC clonogenic (colony forming unit assay) and migratory (Boyden chamber) capacity as well as apoptotic susceptibility (TUNEL assay) were determined in the absence and presence and ET‐1 (100 pM). The number of EPC colony‐forming units (42±12 vs. 39±11) and migratory capacity (910±146 vs. 936±148) was not significantly different between the ET‐1 treated and untreated cells. In response to staurosporine (1 mM), EPCs treated with ET‐1 demonstrated a 20% increase (P<0.05) in cellular apoptosis. The proapoptotic effect of ET‐1 was abolished with ET receptor blockade as well as with apocynin, an NADPH inhibitor. These results indicate that ET‐1 does not affect EPC colony formation or migratory capacity but does increase EPC susceptibility to apoptosis through a NADPH‐dependent mechanism. Increased EPC apoptosis may contribute to the proatherogenic effects of ET‐1.
V. Amygdalina Extracts Inhibit the Growth of Estrogen Receptor Positive/Negative Human Breast Carcinoma Cells In Vitro
Authors: Robinson Brittany , Gesham Lecia , Izevbigie Ernest , Cameron Joseph A
Published: June 2021

Tamoxifen (TAM) is one of the most effective chemotherapies against estrogen receptor‐ positive (ER+) breast cancer (BC). While Paclitaxel or Taxol (TAX) is effective against estrogen receptor‐negative (ER‐) BC. Health disparities exist in breast cancer mortality; although the incidence of BC is highest in White Women (WW), African American women (AAW) have higher mortality rate than other racial or ethnic groups in the U.S. Evidence suggests that aqueous extracts of Vernonia amygdalina (VA) retard the growth of ER+ human BC cells. The effects of VA have not been well characterized in ER‐ cells. The objective of this study was to investigate the effects of VA in ER‐ cells. Although, MDA MB‐231 cell line is commonly used for ER‐ cells studies, the human ductal carcinoma cells (BT‐549) have been reported to express little or no ER‐α and thus represent a suitable model to study ER‐ cell growth. Hence MCF‐7 (ER+) and BT‐549 cells were selected for this study. Mitosis was determined by DNA synthesis assays and confirmed cell counts using a hemacytometer. The results indicate that increasing concentrations of VA abrogated cell growth in a concentration‐dependent (10, 100, and 1000 µg/ml) fashion. Estrogen treatment (20 ng/ml) stimulated DNA synthesis in MCF‐7 but not in BT‐549 cells. The estrogen‐stimulated DNA synthesis observed in MCF‐7 cells was reversed by either VA or TAM. These data suggest that both ER+ and ER‐ breast cancer patients may benefit from VA consumption. Supported in part by RCMI‐NIH Grant G122RR13459‐07S1, NCMHD‐NIH Grant P20MD000534‐01 and NIGMS‐NIH Grant R25 GM50117.
Development of a cell culture system to assess the effect of Cryptosporidium parvum infection on bystander cells
Authors: Foster Derek Michael , Stauffer Stephen Howard , Gookin Jody L.
Published: June 2021

Research has shown that the uninfected bystander cells in a C. parvum infected monolayer are at times preferentially apoptotic when compared to infected cells, and our goal was to further elucidate this effect in a nontumorgenic porcine intestinal cell line, IPEC‐J2. The effect of infective dose and duration of infection on the monolayer were assessed by determining the number of organisms, each life stage of C. parvum, total IPEC cells, infected cells, organisms per infected cell, total apoptotic cells, and infected, apoptotic cells. Coculture of infected monolayers with uninfected monolayers was used to assess the bystander effect on transepithelial electrical resistance and cell fate.

The percentage of IPEC cells that became infected was similar with each dose, ranging from 37‐41% and peaked at 48 hours with the higher doses leading to multiple organisms per infected cell. All doses similarly decreased the total number of cells per high power field with no difference in the effect on bystander cells. During the first 24 hours, bystander cells appeared to be preferentially lost and accounted for the decrease in total cell numbers with significantly more apoptotic, bystander cells than apoptotic, infected cells per field. Early in C. parvum infections, bystander cells appear to play a significant role in the pathophysiology of disease.
Oxidative stress Attenuates Epithelial Differentiation and Accelerates Cell Migration in Caco‐2 cells
Authors: Dunagan Mitzi , Rao R.K.
Published: June 2021

Oxidative stress plays a crucial role in carcinogenesis and tumor metastasis. Numerous studies demonstrated that H2O2 destabilizes intercellular junctions, a process that is involved in cell migration. In the present study, we evaluated the effect of H2O2 on epithelial differentiation and cell migration in Caco‐2 cells. Epithelial differentiation was induced by 3D growth of Caco‐2 cells on matrigel and cell migration analyzed by scratch wound method. H2O2 (100‐250 μM) was administered to the medium. Src kinase inhibitor, PP2 (3 μM) and PI3‐kinase inhibitor, LY294002 (25 μM) were added 30 min prior to H2O2. The role of c‐Src was determined by stable expression of constitutively active c‐Src(pY418). Cell morphology recorded by phase contrast microscopy and the intercellular junctions assessed by confocal microscopy for occludin and ZO‐1. Results show that H2O2 dose‐dependently accelerated cell migration, which was attenuated by PP2 or LY294002, suggesting that H2O2 promotes cell migration by a c‐Src and PI3‐kinase‐dependent mechanism. H2O2‐induced cell migration was associated with redistribution of occludin and ZO‐1 from the intercellular junctions, which was also attenuated by PP2 and LY294002. Expression of c‐Src(pY418) significantly accelerated cell migration. Caco‐2 cells on Matrigel grew to form glandular structure enclosing a cavity. However, in the presence of H2O2 Caco‐2 cells grew into an irregular clump of cells with no cavity; this was associated with irregular distribution of occludin and ZO‐1. These results suggest that oxidative stress may prevent epithelial differentiation and promote cell migration.
EGF Prevents Hydrogen Peroxide‐Induced Tyrosine Phosphorylation and Redistribution of Tight Junction and Adherens Junction Proteins in Human Colonic Mucosa
Authors: Seth Ankur , Sheth Parimal , Rao R.K.
Published: June 2021

Oxidative stress destabilizes intercellular junctions and promotes tumor metastasis. Previous studies demonstrated that H2O2 disrupts tight junctions (TJs) and adherens junctions (AJs) by a Tyr‐kinase‐dependent mechanism in Caco‐2 cells. In the present study, we evaluated the effect of H2O2 on TJs and AJs in human colonic mucosa. These studies were conducted according to the approved IRB protocol. Human colonic biopsies were incubated with 30 nM epidermal growth factor (EGF) 10 min prior to H2O2 (20 μM) for 1 hour. TJ and AJ integrity was evaluated by analyzing the distribution of occludin, ZO‐1, E‐cadherin and β‐catenin by immuofluorescence confocal microscopy. Triton‐insoluble and soluble fractions were immunoblotted for TJ and AJ proteins. Tyr‐phosphorylation of was determined by immunoprecipitation and immunoblot analysis. Results show that H2O2 induces a redistribution of occludin, ZO‐1, E‐cadherin and β‐catenin from the intercellular junctions into the intracellular compartments. H2O2 induced a significant reduction of these TJ and AJ proteins in the Triton‐insoluble fraction, while increasing the levels of these proteins in Triton‐soluble fraction. Tyr‐phosphorylation of TJ and AJ proteins was increased by H2O2. Pretreatment of tissues with EGF attenuated H2O2‐induced Tyr‐phosphorylation and redistribution of TJ and AJ proteins from the intercellular junctions and Triton‐insoluble factions. These results indicate that oxidative stress induces Tyr‐phosphorylation and redistribution of TJ and AJ proteins in human colonic mucosa and EGF prevents these effects of H2O2.
Interaction of RNA‐binding protein CUG‐BP1 with c‐Myc mRNA 3′‐untranslated region represses its translation following polyamine depletion
Authors: Liu Lan , Rao Jaladanki N , Zou Tongtong , Xiao Lan , Yu Tingxi , Wang Pengyuan , Zhang Xian , Turner Douglas J , Wang Jian‐ying
Published: June 2021

Polyamines are required for normal gut mucosal growth by regulating c‐Myc expression, but the mechanism underlying polyamines remains unclear. The RNA‐binding protein CUG‐BP1 binds to GC‐rich sequence of the given mRNAs and regulates translation of its target transcripts. This study determined if polyamines regulate c‐Myc translation by altering CUG‐BP1 interaction with c‐Myc 3′‐untranslated region (3'UTR).

Methods

Studies were conducted in intestinal crypt cells (IEC‐6 line). RNA‐binding was examined by biotin pull‐down and RNP‐IP assays. CUG‐BP1 function was investigated by its silencing and overexpression; c‐Myc translation was examined by the chimeric Luc‐Myc‐3'UTR reporter.

Results

CUG‐BP1 overexpression increased its association with c‐Myc 3'UTR, repressed c‐Myc translation, and decreased its protein level (by ~60%). CUG‐BP silencing reduced its binding to c‐Myc 3'UTR, promoted c‐Myc translation, and induced its protein (by ~5‐fold). Polyamine depletion by inhibiting ODC increased [CUG‐BP1/c‐Myc mRNA] complex, which was associated with repressed c‐Myc translation. CUG‐BP1 silencing rendered the c‐Myc mRNA translatable and prevented decrease in c‐Myc protein in polyamine‐deficient cells.

Conclusion

These results indicate that polyamine depletion represses c‐Myc translation by inducing CUG‐BP1 interaction with c‐Myc 3′‐UTR. (This work was supported by NIH and VA grants)
Acidification of damaged cells is a consequence of damage to the gastric epithelium, and may contribute to surface pH increases after damage
Authors: Demitrack Elise Susan , Montrose Marshall H
Published: June 2021

Focal damage of the gastric surface epithelium causes a transient rise in extracellular pH (pHo) over the damage site, prior to epithelial repair. To identify sources of the pHo rise we measured intracellular pH (pHi) of cells in and around the damaged area. The exposed gastric mucosa of anesthetized mice was loaded with 5 μM SNARF‐5F‐AM, and tissue imaged on a two‐photon microscope stage, during tissue superfusion with buffered pH 3 saline. A 2‐3 cell region of the gastric epithelium was photodamaged (710 nm, 350 mW for 5‐10 sec). Damage was monitored as loss of NAD(P)H autofluorescence and disrupted cell structure (confocal reflectance). pHi was measured near the damage (5‐10 μm), far from the damage (100‐150 μm), or in the actual damaged area. Before damage, initial pHi values were similar in all cell groups (~pH 7.1). After damage, pHi values were similar in near and far cells (7.17 ± 0.06 and 7.15 ± 0.04), but decreased to 6.45 ± 0.08 in damaged cells (n=4). Adding 20 μM iv EIPA or 100 μM luminal SITS did not significantly change initial pHi values in any cell type. After damage, EIPA selectively acidified damaged cells vs control (6.00 ± 0.09 vs. 6.45 ± 0.08, n=4), but SITS did not affect damaged cell acidification (6.59 ± 0.3, n=4). We conclude that damaged gastric epithelial cells may activate basolateral Na/H exchange as part of transepithelial alkali secretion that causes the luminal pHo increase seen after epithelial damage.
Silibinin inhibits CYP2E1‐dependent ethanol metabolism, oxidative stress and cell proliferation in a rat model of hepatocellular carcinoma (HCC)
Authors: Brandon‐Warner Elizabeth , Sugg J. Andrew , Schrum Laura W , McKillop Iain H
Published: June 2021

Many deleterious effects of ethanol (EtOH) are attributed to oxidative stress following metabolism, generating renewed interest in anti‐oxidants as potential therapeutic agents. Previous studies have identified EtOH metabolism as important in regulating EtOH‐dependent increases in cell proliferation using a rat model of hepatocellular carcinoma (HCC). The aim of the current study was to determine the effect of silibinin (a flavinoligand derived from Silymarin marianum) on ethanol metabolism, oxidative stress and cell proliferation using an H4IIE cell culture model of HCC. Exposure of H4IIE cells to EtOH lead to dose‐dependent increases in CYP2E1 protein expression in the absence of significant changes in acetaldehyde or aldehyde dehydrogenase (ADH/ALDH) expression. These data revealed significantly increased EtOH metabolism, oxidative stress/lipid peroxidation in H4IIE cells exposed to EtOH in the absence of silibinin. Treatment of H4IIE cells with silibinin prior to EtOH abrogated the effects of ethanol on CYP2E1 expression with no evident change in ADH/ALDH. In doing so silibinin significantly decreased the rate of EtOH metabolism, oxidative stress and, EtOH‐dependent HCC proliferation. In conclusion, these data demonstrate silibinin inhibits EtOH‐dependent CYP2E1 induction in HCC cells thereby inhibiting the effect of EtOH on HCC mitogenesis. This research was funded by NIH‐grants AA‐16858 (IHM) & AA14891 (LWS).
Differential Effects of EGCG, a Green Tea Antioxidant, on Vascular Endothelial Growth Factor (VEGF) Expression and Proliferation in Cultured ER‐positive and ER‐negative Human Breast Cancer Cells
Authors: Young Emily , Wells Jeremy , Tucker Kevan B , Chawla Simran , Gu Jian‐Wei
Published: June 2021

Estrogen receptor (ER)‐positive breast cancers have a better prognosis than ER‐negative breast cancers, which are more aggressive. The present study determines whether EGCG, a green tea antioxidant, exerts different effects on VEGF expression and proliferation in cultured ER‐positive (MCF‐7) and ER‐negative (MDA‐MB‐231) human breast cancer cells. MCF‐7 and MDA‐MB‐231 cells were cultured using RPMI 1640 media with 10% FBS. ELISA showed that VEGF and VEGF receptor‐2 were more highly expressed in ER‐negative cells than ER‐positive cells (>8‐fold, P<0.01; n=8). 3H‐thymidine incorporation showed that EGCG at 10, 25, and 50 µg/ml caused a dose‐related inhibition in the proliferation of MCF‐7 cells, by 29, 54, and 85%, compared to the control, respectively (P<0.01). However, the same concentrations of EGCG did not inhibit the proliferation of MDA‐MB‐231 cells. EGCG at 50 µg/ml significantly decreased VEGF expression (1752±49 vs. 2254±91 pg/mg; P<0.01) in cultured MCF‐7 cells, compared to the control. However, EGCG at 10 to 50 µg/ml did not inhibit VEGF expression in cultured MDA‐MB‐231 cells. These results suggest that highly expressed VEGF and its receptor may be involved in the aggressive malignant signaling systems of ER‐negative breast cancers, and that EGCG, a green tea antioxidant, can be used to treat ER‐positive human breast cancers, which are sensitive to EGCG. (HL51971 & AA013821)
Effect of previous inflammation on the susceptibility of the colon to subsequent physical restraint stress‐induced changes
Authors: Santiago Olga I. , Cruz Mirella L. , Cuevas Marielly , Isidro Raimond A. , Appleyard Caroline B.
Published: June 2021

Stress can influence the systemic and local immune systems of the intestine, and we have previously shown the susceptibility of the colon to psychological water avoidance stress. Aim: Determine the effect of previous intestinal inflammation on restraint stress‐induced changes in the colon.

Methods

Four groups of female rats were used: normal; control‐RS (restraint stress ‐ stress alone); colitis, (trinitrobenzene sulfonic acid 30 mg in 50% ETOH ic) which was allowed to heal for 2 weeks); and colitis‐RS (colitis, then RS 2 weeks later). RS was induced by placing the animals in a wire restrainer for 3hrs, during 3 consecutive days. The colons were examined for damage and levels of IL‐10, IL‐4 and IFN gamma, colonic mast cells and bacterial levels in MacConkey, blood agar and lactobacillus media were measured.

Results

Prior inflammation caused increases in colonic propulsive activity, mast cells, macroscopic and microscopic damage in the animals exposed to RS. There were lower numbers of lactobacillus. No significant changes in total bacterial load or cytokine levels were found.

Conclusion

Prior inflammation increases the susceptibility of the colon to respond to physical stress.
Alterations in hepatic macronutrient metabolism in response to short term high fat feeding in rats
Authors: Sunny Nishanth E , Satapati Santhosh , Fu Xiaorong , He TianTeng , Liu Shu‐Hao , Burgess Shawn C
Published: June 2021

Insulin resistance (IR) is coupled to obesity or high fat feeding by ectopic lipid metabolism and accumulation of intermediates that disrupt insulin signaling. We recently reported that hepatic mitochondrial fat metabolism is dysfunctional in Zucker Diabetic Fatty (ZDF) rats. The objective of this study was to test whether similar effects on hepatic mitochondrial metabolism occur in lean (ZDF+/−) rats following a short term (7d) high fat (60% fat calories) challenge. Conscious, unrestrained rats were infused with stable isotope tracers followed by NMR or LC‐MS isotopomer analysis of plasma metabolites to determine hepatic fluxes. Fasting hepatic ketogenesis and fat oxidation is impaired in obese diabetic rats. In contrast, a short term high fat diet increased fasting ketone concentrations and did not affect TCA cycle flux or GNG. Ureagenesis, an energy intensive pathway that spans both cytosolic and mitochondrial compartments, was also lower in fat fed lean ZDF+/− rats compared to obese ZDF−/− rats. We conclude that short‐term high fat feeding elicits a physiological response in several hepatic mitochondrial and cytosolic pathways that facilitates whole body metabolism of lipid rich macronutrients, but that this adaptation is fundamentally different from the patho‐physiological features of hepatic fat metabolism in obese diabetic rats.
Effects of chromium picolinate on reactivity of aortas from spontaneously hypertensive rats (SHR)
Authors: Mozaffari Mahmood , Liu Jun , Wimborne Hereward , Abebe Worku
Published: June 2021

Chromium picolinate [Cr(Pic)3] is a nutritional supplement promoted as adjuvant therapy for type 2 diabetes. Further, hypertension is a coexisting disorder in type 2 diabetic patients. Although Cr(Pic)3 improves [Ca2+]i recovery in insulin‐treated rat vascular smooth muscle cells, its effects on vascular reactivity remain to be established. Thus possible effects of Cr(Pic)3 on vascular reactivity and blood pressure were assessed using SHR, which also display insulin resistance. Male SHR were fed with diets lacking or containing 10 mg/kg Cr(Pic)3 from 6 to12 weeks of age (n=6/group); blood pressure (BP) was measured prior to isometric tension measurements. The treatment caused a slight (p=0.07) increase in BP. Endothelium‐denuded aortic rings from both groups of rats contracted similarly to norepinephrine and KCl. Relaxation of endothelium‐intact aortas from Cr(Pic)3‐treated rats to acetylcholine was enhanced compared to controls. This effect of acetylcholine was inhibited by L‐NAME. Aortas from both control and Cr(Pic)3‐fed SHR relaxed similarly to sodium nitroprusside. In conclusion, Cr(Pic)3 does not alter either the contractile or relaxant properties of the vascular smooth muscle in SHR. However, the treatment improves vascular reactivity in SHR via endothelium‐mediated effects, which may be associated with increased production and/or release of NO. Supported by NIH.
L‐Arginine infusion attenuates postprandial lipemia in healthy elderly
Authors: Puga Guilherme Morais , Meyer Christian , Alexander Jeffrey , Mandarino Lawrence , Everman Sarah , Katsanos Christos
Published: June 2021

Increased plasma concentration of L‐arginine enhances the production of nitric oxide (NO), which has been proposed to increase fat oxidation in skeletal muscle. The purpose of this study was to determine whether L‐arginine infusion lowered the increase in plasma triglycerides (TG) and increased the rate of oxidation of ingested fat in healthy elderly subjects following a fatty meal. On two separate days in the morning after an overnight fast, nine healthy elderly (8 males and 1 female, age, 68 ± 4 yr; body mass, 85.7 ± 10.8 kg; % body fat, 27 ± 3%; triglycerides, 78.1 ± 32.5 mgµdL−1; glucose, 91.9 ± 7.4 mgµdL−1) ingested whipping cream (0.4 g of fat per kg body weight) enriched with [13C]‐triolein. Plasma TG concentrations were measured in plasma from blood samples collected every hour during an 8‐h period following meal ingestion. Expired gases were measured every 2 hours to determine whole‐body fat oxidation. Subjects rested in bed for the duration of the trials. On one of these days, in a random order, 300 ml of 10% L‐arginine (arginine day) was infused during the first hour after the meal, whereas on the other day (control day) the same volume of saline was infused. Arginine infusion decreased the incremental TG area under the curve: control day, 99.7 ± 28.7 mgµdL−1µ8h−1; arginine day, 22.8 ± 24.2 mgµdL−1µ8h−1. The rate of oxidation of ingested fat was not different between the arginine and control days: 4.7 ± 0.4 μmolµkgFFM−1µ8h−1 versus 4.2 ± 0.5 µmol·kgFFM−1·8h−1 respectively. In conclusion, L‐arginine infusion attenuates postprandial lipemia in healthy elderly subjects, but this not mediated by an increase in the rate of oxidation of ingested fat.
Mitochondrial reactive oxygen species are required for hypoxia‐induced degradation of keratin intermediate filaments
Authors: Ridge Karen M , Litvan Juan , Ni Na
Published: June 2021

Hypoxia can cause stress and structural changes to the epithelial cytoskeleton. The intermediate filament network is known to reorganization in response to stress. We examined whether rats exposed to hypoxia (8% O2; 24 h) had altered keratin IF expression in their alveolar epithelial cells (AEC). There was a significant decrease in keratin protein abundance in hypoxic AEC as compared to AEC isolated from normoxic, control rats. To define the potential mechanisms regulating this process we studied changes to the keratin IF network in human A549 cells exposed to 1.5% oxygen; 24 h. We observed a time dependent disassembly/degradation of keratin 8 and 18 proteins which was associated with an increase in reactive oxygen species (ROS). Hypoxia‐treated A549 cells deficient in mitochondrial DNA or A549 cells treated with a small interfering RNA against the Rieske iron‐sulfur protein of mitochondrial complex III did not have increased levels of ROS, nor was the keratin IF network disassembled/degraded. The catalase/superoxide dismutase (SOD) mimetic (EUK‐134) prevented the hypoxia‐mediated keratin IF degradation as did the overexpression of SOD1, but not SOD2. Accordingly, we provide evidence that hypoxia promotes the disassembly/degradation of the keratin IF network via mitochondrial complex III‐generated reactive oxygen species.
High K (HK) intake enhances the inhibitory effect of Arachidonic Acid (AA) to ENaC by stimulating CYP Epoxygenase and inhibiting soluble Epoxide Hydrolase (sEH)
Authors: Sun Peng , Wang Wenhui
Published: June 2021

We used the patch‐clamp technique to study the effect of AA on ENaC in the CCD in rat on HK diet or on Na‐deficient diet (Na‐D). AA inhibits ENaC activity and is mediated by 11,12‐EET. However, the sensitivity of ENaC to AA is higher in rats on HK diet (ED‐50, 3 µM) than that on Na‐D (ED‐50, 5 µM). Moreover, HK intake significantly increased the expression of CYP2C23 and 11,12 EET content in the CCD. The inhibitory effect of 11,12‐EET to ENaC is enhanced in rats on HK diet which decreases ED‐50 of 11,12‐EET from 70 nM in rats on Na‐D to 30 nM. However, inhibiting sEH, which converts EET to DHET (a weak inhibitor of ENaC), increased significantly the sensitivity of ENaC to 11,12‐EET in the rats on Na‐D to the extent similar to that on HK. Finally, the observation that HK intake suppresses the metabolic rate of converting EET to DHET in the kidney suggests that HK intake may inhibit sEH and, consequently, enhance the sensitivity of ENaC to EET. We conclude that HK intake enhances the inhibitory effect of AA and 11,12‐EET on ENaC, increasing CYP epoxygenase activity and decreasing sEH activity are partially responsible for augmenting the inhibition of AA and 11,12‐EET, respectively.
Characterization of label retaining intestinal cells: demonstration of quiescence
Authors: Garrison Aaron P. , von Allmen Doug C. , Speck Karen E. , Dekaney Christopher M. , Helmrath Michael A.
Published: June 2021

Background

Retention of DNA analogues is a long‐accepted marker of intestinal stem cells (ISC). To date, characterization of the proliferative status and the potential contribution of secretory lineages to this population of cells in non‐damaged intestine have not been published. In this study we utilized our reproducible murine model of ISC expansion following ileo‐cecal resection (ICR) to test the hypothesis that label retaining cells (LRC) are undifferentiated, proliferating cells that co‐label with known ISC markers.

Methods

The thymidine analogue IdU was administered orally to control and ICR mice (n = 2‐4) for 7d preceding and following surgery. 8wks later, a second analogue (CldU) was given 3d prior to harvest. Tissue was analyzed using dual fluorescent antibodies to IdU, CldU, Lysozyme, Chromogranin A (ChrgA), PCNA, and potential markers of ISC DCAMKL‐1 and p‐ser552 β‐catenin.

Results

Following ICR, IdU+ cells occurred in 91/237 (0.38) crypts, compared to 63/248 (0.26) crypts in controls. 94% of the IdU+ cells were Lysozyme or ChrgA+, whereas 100% in the control group were of secretory lineage. All LRC were negative for CldU, PCNA, DCAMKL‐1, and p‐ser552 β‐catenin.

Conclusions

Contrary to our hypothesis, most LRC are either long‐lived Paneth or enteroendocrine cells. The rare non‐secretory LRC are quiescent and did not co‐label with ISC markers.

Research support: NIH T32 GM08450‐14 (APG), NIH R03 DK078796‐01 (MAH)
The Dysferlin homolog fer‐1 is required for efficient cholinergic synaptic transmission in C. elegans
Authors: Krajacic Predrag , Mosqueira Matias , Hermanowski Jane , Lozynska Olga , Shen Xiaoning , Arratia Paulo , Khurana Tejvir S. , Lamitina Todd
Published: June 2021

Limb Girdle Muscular Dystrophy 2B (LGMD2B) is a progressive and incurable muscle disorder. Mutations in the Dysferlin gene cause LGMD2B, but the molecular mechanisms underlying disease pathogenesis are unclear. To better understand the pathogenesis of this disease, we studied the C. elegans Dysferlin homolog fer‐1. Previous studies have characterized fer‐1 only with regards to its roles in spermatogenesis. We found that fer‐1 is also expressed in muscle, where it regulates the localization of post‐synaptic acetylcholine receptors at the neuromuscular junction, suggesting a new role for Dysferlin as a regulator of cholinergic synaptic transmission. Consistent with these data, fer‐1 mutants were resistant to drugs that increase cholinergic signaling, and electrophysiological approaches confirm that fer‐1 mutants have a defect in acetylcholine‐dependent synaptic signaling. Acute administration of a cholinesterase inhibitor improved the excitability and functional properties of fer‐1 muscle. Given the strong evolutionary conservation in synaptic physiology between C. elegans and humans, these data suggest that disruption of cholinergic signaling may contribute to LGMD2B pathology and that drugs enhancing cholinergic signaling might be an effective therapeutic strategy for treatment.

Supported by NIH Training Grant in Muscle Biology (T32 AR‐053461‐02) and ITMAT (UL1RR024134) grant.
Genetic Analysis of Signaling Genes for Affects on Egg Production in the Domestic Chicken (Gallus gallus)
Authors: Flack Brenda R. , Hyde Carrie , Rhoads Douglas
Published: June 2021

We are screening for genetic variants of hormone and receptor genes that contribute to diversity in egg production in the female broiler chicken (Gallus gallus). Years of intensive selection for growth characteristic have resulted in defects in reproductive performance including reduction in total number of eggs laid. We obtained blood samples and total egg data for three elite commercial broiler lines. We analyzed approximately 80 to 200 hens from each line using microsatellite loci from the chromosomal locus of several candidate genes. Genes selected are known to be involved in signaling for ovary function: Follicle Stimulating Hormone Beta subunit (FSHâ) and receptor (FSHR), Luteinizing Hormone Receptor (LHR), Insulin‐like Growth Factor 1 (IGF1) and receptor (IGF1R), Anti‐Mûllerian Hormone (AMH), and Prolactin (PL). Genotypes were determined and analyzed for allele and genotype frequencies. Our results show that particular haplotypes (alleles) of the regions of FSHR and PL are statistically disproportionate with respect to egg production representing potential Quantitative Trait Loci (QTLs) for this trait. QTL effects are highly dependent on the line analyzed. Our genetic data suggests that egg production could be increased in selected lines by including these loci.