bluuebunny/crossref_metadata_2025 huggingface dataset:
Oral L‐arginine treatment significantly reduces blood pressure without altering the renal T cell profile in the DOCA salt model of hypertension
Authors: Gillis Ellen E. , Crislip G. Ryan , Sullivan Jennifer C.
Published: June 2021

The immune system plays a critical role in the development of hypertension. Our lab has previously reported that there is a sex difference in the renal T cell profile in DOCA salt hypertension, with males having more pro‐inflammatory Th17 cells, whereas females have more anti‐inflammatory regulatory T cells (Tregs). DOCA treated males also have a greater blood pressure (BP) response to DOCA than females, and we propose this is due to the relative abundance of Tregs in the female. We have also previously reported that an intact nitric oxide (NO) system is required for Treg maintenance in hypertension, and females have greater NO levels than males. In the current study, we hypothesized that treatment with L‐arginine, a substrate for NO production, would increase renal Tregs and attenuate the sex difference in hypertension in male and female DOCA treated rats. Briefly, male and female Sprague Dawley rats (n=4–6, 10 wks of age) were uni‐nephrectomized and subcutaneously implanted with a DOCA pellet (200 mg/rat, 60‐d time release) and given 0.9% NaCl to drink ad libitum. A subset of rats were treated with L‐arginine (350 mg/kg/d, via drinking water). BP was measured by tail cuff plethysmography at baseline and after 3 wks of DOCA treatment. After 3 wks of treatment, kidneys were processed for flow cytometric analysis of T cells (CD3+CD4+FOXp3+ Tregs, CD3+CD4+RORγ+ Th17 cells, and IL‐10+CD3+ T cells). BP was comparable between treatment groups within each sex at baseline, although males had a higher BP than females (Table, effect of sex, p<0.001, 2‐Way ANOVA). DOCA treatment increased BP in all groups compared to baseline values (Table, effect of DOCA, p=0.002). L‐arginine treatment attenuated DOCA‐induced increases in BP in both sexes, although this effect was greater in females (Table, effect of L‐arginine, p=0.02, interaction of sex and L‐arginine, p=0.03). Females had a more anti‐inflammatory renal T cell profile, with greater levels of Tregs and IL‐10+ cells, whereas males had more pro‐inflammatory Th17 cells (Table, effect of sex, p<0.05 for Tregs, Th17, and IL‐10). Interestingly, despite the significant BP effect observed, L‐arginine treatment did not impact the renal T cell profile in either sex (Table, effect of L‐arg, p>0.05 for Tregs, Th17, and IL‐10). Future studies will examine additional mechanisms by which L‐arginine supplementation lowers BP in both sexes. The DOCA salt model is characterized by high levels of oxidative stress, which is known to impair the NO‐NOS signaling pathway by decreasing the amount of L‐arginine available to produce NO via eNOS.

Support or Funding Information

This work was supported by NIH 5R01HL12709102

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dietary Saturated and Unsaturated Fat and Fructose Induce Distinct Patterns of Hepatic Steatosis in Male Mice
Authors: Song Ming , Shi Hongxue , Jin Jian , Cave Matt , McClain Craig
Published: June 2021

Objective

Nonalcoholic fatty liver disease (NAFLD) is a major cause of liver disease, with a global prevalence of 25.24%. The etiology of NAFLD is complex, and dietary nutrients and their interactions play a critical role. The aim of this study was to determine the effect of different types of dietary fat‐fructose interactions on the pathogenesis of NAFLD and to explore the potential underlying mechanism(s).

Methods

Male adult C57BL/6J mice were assigned to six groups and fed with diets (AIN‐93G based) enriched with different types of fat (with or without fructose added in the water), such as saturated fat (SF‐‐beef tallow), n−6 polyunsaturated fat (n6USF‐‐corn oil), or n−3 polyunsaturated fat (n3USF‐‐menhaden fish oil), respectively, for 20 weeks. 42% of calories was derived from the fat. Tap water or tap water containing 10% fructose (w/v) was given ad lib during the entire experiment. Body weight and energy efficiency ratio were monitored. Liver histology, fat accumulation, and liver enzymes, as well as glucose tolerance test were evaluated.

Results

Both SF and n6USF fed mice developed obesity and hepatic steatosis, but with distinct phenotypes. n3USF fed mice with or without fructose exhibited a lean shape with normal liver histology and liver enzymes. There was no difference between SF and n6USF fed mice in terms of body weight (BW). Mice fed with SF displayed more severe hepatic steatosis and liver injury as well as hepatic fibrosis compared to n6USF fed mice. Fructose feeding led to significantly increased liver weight and liver weight:body weight ratio in SF fed mice, but not in the n6USF or n3USF fed mice. While the energy efficiency ratio was the highest in n6USF fed mice, the hepatic triglyceride content in n6USF fed mice was significantly lower than that of SF fed mice. Interestingly, n6USF fed mice displayed marked glucose intolerance which was worsened by fructose feeding, and this effect was not obvious in SF fed mice.

Conclusion

Our data demonstrated that dietary SF‐fructose and n6USF‐fructose interactions differentially contribute to the development of obesity and hepatic steatosis. n3USF plays a beneficial role in the prevention of obesity and fatty liver, even with the consumption of excess fructose. The underlying mechanism(s) are currently under investigation.

Support or Funding Information

Supported by grants from NIH and Veterans Administration

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ROLE OF ESTRADIOL RECEPTOR BETA (ERβ) ON BRAIN MITOCHONDRIAL FUNCTIONS AND CARDIO‐RESPIRATORY CONTROL IN AGED FEMALE MICE
Authors: LAOUAFA Sofien , ROUSSEL Damien , MARCOUILLER François , SOLIZ Jorge , BAIRAM Aida , JOSEPH Vincent
Published: June 2021

We tested the hypothesis that ERβ is involved in brain mitochondrial function and cardio‐respiratory control in aged female mice. We used aged (17–18 months‐old) ERβKO and wild‐type controls (WT) female mice to assess arterial blood pressure (via a tail‐cuff sensor) and indices of respiratory pattern (sighs and apneas – recorded by whole body plethysmography at rest). We also measured respiratory parameters at rest and in response to brief (<10 minutes) exposure to hypoxia (12% O2) or hypercapnia (5% CO2). Because ERβ is localized in mitochondria, and because estradiol and ERβ agonist increase mitochondrial O2 consumption, we used fresh brain cortex tissue (~2 mg) permeabilized with saponin to assess mitochondrial respiration with high‐resolution respirometry (O2k‐ Oroboros) and in‐vitro activity of the complex I of the electron transport chain. In ERβKO female mice, mean arterial blood pressure was lower than in old WT mice (88±4 vs 110±3 mmHg, P<0.05), the frequency of sighs was similar in ERβKO than in WT micez (26±3 vs 19±2 sighs/h P=ns) and the frequency of apneas much lower (4±2 vs 26±9 apnea/h, P=0.01). In ERβKO mice, minute ventilation is reduced in response to hypoxia (−50%, P<0.01) and hypercapnia (−39%, P<0.01) compared to WT mice. Concerning mitochondrial function, with substrates of complex I (Pyruvate/Glutamate/Malate) and in presence of ADP, ERβKO mice have a reduced O2 consumption compared to WT mice (60±7 vs 98±4 pmol O2/s/mg tissue, p<0.05). With the substrate of complex II (succinate) and ADP, there was no difference between ERβKO and WT mice. In vitro activity of complex I is 70% lower in ERβKO mice compared to WT (P<0.01). We conclude that the deletion of ERβ reduces chemoreflex functions and reduces mitochondrial function in the central nervous system. This role ERβ may be relevant in pathological or physiological conditions in which O2 homeostasis is compromised (sleep apnea, altitude, lung diseases).

Support or Funding Information

CIHR, Région Rhône Alpes and consulat de France in Québec

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sirt3 Restores Lipid Homeostasis in Bovine Hepatocytes Exposed to NEFA
Authors: Liu Lei , Xing Dongmei , Su Jianming , Peng Tao , Wang Jianguo , Wen Lixin , Cheng Tianyin
Published: June 2021

Introduction

Sirt3, a major deacetylase in mitochondria, plays a pivotal role in the regulation of lipid metabolism in mice and humans. Dynamic changes of Sirt3 expression are observed in ruminants during the transition period, a time when fat mobilization initiates and non‐esterified fatty acids (NEFA) sore in plasma, and also a time when most of lipid metabolism disorders in ruminants take place, like fatty liver and ketosis etc. However, the rationale between Sirt3 and fat metabolism disturbance in dairy cows has not been characterized yet. The current study aims to investigate interrelations of Sirt3 and NEFA on expressions of key factors involved in fat metabolism in bovine hepatocytes.

Methods

Hepatocytes from a calf were isolated, and primarily cultured by a modified 2 steps of collagenase perfusion method. NEFA ranging from 0 mM to 1.2 mM were added, and their effects on the expression of Sirt3 was studied. Sirt3 overexpression was induced by adenovirus (AdMAX system), and expressions of key fat metabolism factors, namely acetyl‐CoA carboxylase‐1 (ACC‐1), fatty acid synthase (FAS), carnitine palmitoyltransterase‐1 (CPT‐1), CPT‐2, acyl‐CoA oxidase (ACO), apolipoprotein B (ApoB) and ApoE were investigated. The level of supernatant very low‐density lipoprotein (VLDL) content and cellular triacylglycerol (TG) contents were also measured. Then 1.2 mM NEFA and Sirt3 overexpression vectors were co‐incubated with hepatocytes. All above mentioned parameters were also tested.

Results

The mRNA level of Sirt3 was significantly up‐regulated by 0.3 mM NEFA, but was significantly down‐regulated by 1.2 mM NEFA (p<0.01). Sirt3 overexpression significantly induced expressions of ACC‐1, CPT‐1, CPT‐2, ACO, ApoB and ApoE (p<0.01), and elevated cellular TG contents (p<0.01). However, Ad‐Sirt3 treatment did not change mRNA level of FAS and supernatant VLDL level compared with controls. Ad‐Sirt3 + 1.2 mM NEFA treatment increased the mRNA level of Sirt3 by 4‐fold compared to Ad‐GFP controls (p<0.01), and regained expressions of CPT‐1, CPT‐2 and ACO previous inhibited by 1.2 mM NEFA. Moreover, cellular TG contents were lowered in Ad‐Sirt3 + 1.2 mM NEFA group compared 1.2 mM NEFA treatments (p<0.01).

Conclusion

Those data indicated that Sirt3 was involved in the regulation of lipid metabolism in bovine hepatocytes exposed to high level NEFA by restoring lipid homeostasis. Further investigations are needed to explore the potential role of Sirt3 as a therapeutic target for maintenance of perinatal health in dairy cows.

Support or Funding Information

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mapping Hair Cortisol Concentrations Across Body Sites to Study Health Disparities
Authors: Marquez Hilda F. , Samayoa Cathy , Marquez‐Magaña Leticia
Published: June 2021

Perceived discrimination, poverty, and environmental stress stimulates the release of cortisol, the primary stress hormone, through the hypothalamic–pituitary–adrenal (HPA) axis. Chronic stress, a long‐term form of stress, increases the risk of various diseases, including; diabetes, hypertension, and inflammatory and autoimmune disorders among all populations. Unfortunately, ethnic minorities experience higher levels of chronic stress, and bear the burden of health disparities. Hair cortisol is a retrospective biological marker for cortisol exposure and can be used as an integrated measure of long‐term HPA activity and chronic stress. However, studies that use hair from the scalp may face collection challenges due to the symbolic and spiritual meaning that is carried by locks of hair among populations particularly affected by health disparities. Therefore, identifying alternatives for the measurement of chronic cortisol exposure would enable inclusion of diverse population in health disparities research. The purpose of this study is to determine if hair across body sites can be used for cortisol analysis to investigate chronic stress among populations affected by health disparities. I hypothesized that hair from arms, legs, and pelvic area will be correlated to scalp hair cortisol levels, and could be used to assess chronic cortisol levels. In this study, participants were asked to provide hair samples from the following four regions of the body; the scalp, arms, legs, and pelvic region. Hair samples were processed, and cortisol was extracted using the established methanol method. Cortisol was quantified using an Enzyme‐Linked Immunosorbent Assay. A total of eight participants were recruited, five females and three males. Participants represented 4 different ethnic groups and were of recruited from a University. 25% of participants provided samples from all four hair sites, while 62.5% provided all but pubic hair. Average cortisol levels ranged from 236 pg/ml in the scalp to 1020 pg/ml in the legs. Linear correlation analysis was conducted to determine the relationship between hair cortisol concentration (HCC) from the scalp and other areas of the body. Our preliminary analysis shows trends suggesting that arm and leg hair may be culturally appropriate samples for hair cortisol analysis. In conclusion, this study revealed cortisol levels across body hair sites, and highlights the need to incorporate culturally acceptable biomarkers to address and eliminate health disparities.

Support or Funding Information

CCSF/SMCCD/SFSU BRIDGES TO THE BACCALAUREATE PROGRAM: R25 GM050078

SFSU SF BUILD: ENABLING STUDENTS TO REPRESENT IN SCIENCE‐ TL4 GM118986

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Inflammatory Reflex Controls Inflammation in Response to Viral Challenges
Authors: Martelli Davide , Farmer David G. S. , Komegae Evilin N. , McKinley Michael Joseph , McAllen Robin Michael
Published: June 2021

The nervous and immune systems talk to and influence each other. In particular, the nervous system helps maintain inflammation within appropriate limits. It is known that a neural reflex, termed the inflammatory reflex, is engaged and inhibits inflammation in response to lipopolysaccharide, a component of the wall of gram negative bacteria. This reflex has its efferent motor arm in the splanchnic nerves. Viral infections are different from bacterial infections and the innate immune system detects and responds differently to them.

The aim of this study was to test the hypothesis that the inflammatory reflex is involved in the control of inflammation triggered by viruses as well as bacteria.

For this purpose, we investigated whether the injection of polyinosinic:polycytidylic acid (Poly I:C; 1mg/kg i.v.), which mimics viral challenge, is able to activate splanchnic sympathetic nerve activity (SSNA) in urethane anesthetized rats. We also studied the effect of bilateral section of the greater splanchnic nerves on the systemic inflammatory response 90 minutes after the injection of the same dose of Poly I:C.

Our results showed that poly I:C induces an activation of the SSNA that lasted for at least 7 hours. Furthermore, splanchnic nerve section resulted respectively in 3‐fold higher and 4‐fold lower levels of the pro‐inflammatory cytokine plasma tumor necrosis factor α (TNF) and the anti‐inflammatory cytokine interleukin 10.

These results show that a viral challenge activates the inflammatory reflex in the same way as a bacterial challenge. Viral infections can lead to secondary bacterial infections. Because viral stimulation of the inflammatory reflex would suppress innate immune function, this may be why the host's ability to fight off a secondary attack by bacteria is compromised.

Support or Funding Information

This work was supported by project grant number 1098887 from the National Health and Medical Research Council (NHMRC) of Australia and from the Victorian Government Operational Infrastructure Support Program. E. N. Komegae was the recipient of a postdoctoral fellowship from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP 2016/1555‐6).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An increase in prefrontal oxygenation at the start of voluntary cycling exercise was observed independently of exercise effort and muscle mass
Authors: Asahara Ryota , Endo Kana , Liang Nan , Matsukawa Kanji
Published: June 2021

We have reported using near‐infrared spectroscopy that an increase in prefrontal oxygenated‐hemoglobin concentration (Oxy‐Hb) at the start of cycling exercise has relation to central command, defined as a feedforward signal descending from higher brain centers. The final output of central command evokes the cardiovascular responses depending on exercise effort. If the prefrontal cortex may output the final signal of central command toward the autonomic nervous system, the prefrontal oxygenation should increase depending on exercise effort. To test the hypothesis, we investigated the effects of exercise intensity and muscle mass on prefrontal oxygenation by longitudinal comparisons in 13 subjects. The subjects performed one‐ or two‐legged cycling at various relative intensities (20%, 35%, 45%, and 60% of maximal voluntary exercise intensity) for 1 min. The prefrontal Oxy‐Hb and cardiovascular variables were simultaneously measured during exercise. The increase in cardiac output and the decrease in total peripheral resistance at the start of one‐ and two‐legged cycling were augmented (P<0.05) in proportion to exercise intensity and/or muscle mass recruitment. However, the increase in prefrontal Oxy‐Hb seemed independent of exercise intensity. When the rating of perceived exertion was equal, the increase in prefrontal Oxy‐Hb was not different (P>0.05) between one‐and two‐legged cycling. Furthermore, no correlations (P>0.05) were found between the increase in prefrontal Oxy‐Hb and exercise intensity or rating of perceived exertion. It is likely that increased prefrontal oxygenation at the start of cycling exercise does not represent the final output of central command descending to the autonomic nervous system.

Support or Funding Information

This study was funded by Grant‐in‐Aid (15H03061) for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aquaporin 1 regulates pulmonary arterial smooth muscle cell apoptosis
Authors: Yun Xin , Jiang Haiyang , Huetsch John , Damarla Mahendra , Shimoda Larissa
Published: June 2021

Pulmonary arterial hypertension (PAH) is a devastating disease, characterized by functional and structural vascular changes that lead to right heart failure and death. PAH pathogenesis involves remodeling of distal small diameter pulmonary arteries, including the development of vaso‐occlusive lesions featuring proliferation, migration and reduced apoptosis of pulmonary arterial smooth muscle cells (PASMCs). Patients often present with late disease, where targeting apoptosis to reverse remodeling would be of benefit; unfortunately, no such therapy currently exists. Apoptosis is a complex process, often involving disruption of the mitochondrial membrane and release of cytochrome c to activate the downstream effector, caspase 3, the final executioner of apoptosis. Formerly, we showed that the water channel protein, aquaporin 1 (AQP1), is a key molecule mediating PASMC proliferation and migration by increasing β‐catenin levels. Others have shown in different cell types that activating β‐catenin prevent apoptosis and improved cell survival. Therefore, in this study, we explored whether AQP1 also regulates PASMC survival and/or susceptibility to apoptosis. Primary cultured rat distal PASMCs were isolated from the SU5416‐hypoxia (SuHx) rat model of PAH or control rats. PASMCs from SuHx rats exhibited lack of apoptosis, measured by: 1) visualizing nuclear morphology via Hoechst 33342 staining; 2) cleaved caspase 3 expressions; and 3) caspase 3 activity, both at baseline and in response to the apoptotic stimuli staurosporine (ST) or H2O2. AQP1 mRNA and protein expression were significantly elevated in PASMCs from SuHx rats. Not surprisingly, β‐catenin levels were also elevated in these cells. Compared to non‐targeting siRNA, depleting AQP1 induced apoptosis at baseline in both control and SuHx rat PASMCs. Furthermore, reduction of AQP1 sensitized SuHx PASMCs to apoptotic stimuli. Our results indicate that AQP1 is a critical regulator of apoptosis in PASMCs, controlling caspase 3 activation, potentially through a pathway involving β‐catenin. Moreover, these findings suggest the possibility that targeting AQP1 might provide an attractive therapeutic option to both stop the progression of, and even reverse, vascular remodeling in PAH.

Support or Funding Information

HL126514

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exposure to Leukotriene E4 Induces Hypersensitivity of Vagal Pulmonary Sensory Neurons in Rats
Authors: Gu Qihai (David) , Emamifar Nick , Gilbert Carolyn , McLaurin Hugh
Published: June 2021

Leukotrienes E4 (LTE4), the most stable and abundant cysteinyl leukotrienes (CysLTs), has been generally studied less than other two CysLTs (LTC4 and LTD4), likely because it is only a weak agonist for the classical type 1 (CysLT1) and type 2 (CysLT2) receptors for CysLTs. However, recent identification of GRP99 protein as a high affinity receptor for LTE4 suggests that LTE4 may play a more pivotal role in the pathogenesis of airway inflammatory diseases such as asthma. The present study is carried out to investigate the effect of LTE4 on the chemo‐sensitivity of pulmonary afferent neurons in both whole‐animal ventilatory and single neuron electrophysiological recordings. In anesthetized spontaneously breathing rats, intratracheal instillation of LTE4 (2 nmol in 100 μl saline) significantly potentiates chemoreflexes evoked by right‐atrial injection of capsaicin, a specific agonist for transient receptor potential vanilloid receptor 1 (TRPV1). In patch‐clamp recordings with isolated rat vagal pulmonary sensory neurons, pretreatment with LTE4 concentration‐dependently enhances capsaicin‐induced TRPV1‐mediated whole‐cell inward currents. Our patch‐clamp experiments further demonstrate that the zinc‐evoked TRPA1‐mediated current is also markedly enhanced in these neurons. In summary, our data show that exposure to LTE4 leads to hypersensitivity of vagal pulmonary sensory neurons and augments pulmonary chemoreflexes in rats.

Support or Funding Information

Supported by MUSM and Navicent Health Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Constitutive Model of Ovine Left and Right Ventricles Biaxial Mechanical Properties
Authors: Liu Wenqiang , Labus Kevin M. , Nguyen‐Truong Michael , McGilvray Kirk , Puttlitz Christian M. , Wang Zhijie
Published: June 2021

Ventricular dysfunction is the most common cause of heart failure, which contributes significantly to mortality and morbidity in the modern society. To better understand the mechanism of the left‐ and right‐ventricle (LV and RV) failure, especially cardiac diastolic dysfunction, it is important to investigate the myocardial biaxial mechanical properties. Prior research has been focused on LV alone and has been limited to small animal models. Hence, there is a lack of study on the RV including the fundamental mechanical differences between LV and RV. Furthermore, there is a lack of data on the mechanical properties in large animal hearts (e.g., sheep) which better mimics the human heart. Our goal here was to characterize the biaxial mechanical properties of healthy sheep LV and RV tissue through constitutive modeling. In this study, a constitutive model (modified Ogden model) that is commonly used for the anisotropic hyperelastic tissue's analysis was applied to capture the biaxial mechanical behavior of the heart obtained by ex vivo experiments. The biaxial mechanical behavior was distinct between the RV and LV in both circumferential and longitudinal directions. We found that the model fit the experimental curve better for RV than LV (Figure). Furthermore, the LV and RV had different model parameters including infinitesimal shear modulus μ, nonlinearity α and anisotropy k (Table). Our results indicated that overall, the RV is stiffer in the longitudinal direction than the circumferential direction (positive k), whereas the LV is stiffer in the circumferential direction (negative k). The RV exhibited a greater degree of anisotropy than the LV, based on the magnitude of the k parameter. The RV has slightly more linear behavior (a smaller α) than the LV (a larger α). To date, this is the first study to investigate and compare biaxial mechanical behavior between LV and RV in sheep using a constitutive modeling approach. The study of the cardiac mechanics will deepen the understanding of physiological and pathological mechanisms of the myocardium. Future work will involve developing a better model for LV biaxial behavior and include more structure parameters such as collagen fiber orientation and distribution.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing Exogenous Cell Engraftment for Cystic Fibrosis Cell Therapy
Authors: Lee Rhianna Elizabeth , Miller Sean Michael , Mascenik Teresa Mary , Tarran Robert , Randell Scott Howard
Published: June 2021

Cell therapy is a potential method for treating cystic fibrosis (CF), although cell engraftment into the highly resistive, pseudostratified human bronchial epithelium (HBE) is a major challenge. Transient epithelial injury is an approach to improve cell uptake, but the extent of injury required and the ability of cells grown by alternative methods to engraft are poorly understood. Our overarching hypothesis is that compounds such as polidocanol, an FDA approved drug used to sclerose varicose veins, can increase cell engraftment by targeting luminal barrier cells. To test this hypothesis, we engrafted exogenous, adenovirus‐EGFP transduced primary human airway epithelial cells into well‐differentiated air‐liquid interface (ALI) HBE cell cultures following treatment with varying doses of polidocanol. Quantitation of cell engraftment demonstrated that 0.125% polidocanol increased engraftment without causing overall ALI cell culture lysis. Further confocal microscopy characterization of the polidocanol injury indicates that engraftment does not require epithelial denudation to the level of the basal lamina. Primary HBE ALI cultures were then injured with polidocanol and the recovery of transepithelial electrical resistance was tracked over time after addition of no cells, conventionally‐grown cells, or cells grown with the conditionally reprogrammed cell (CRC) method. Cell engraftment expedites epithelial restoration, and CRC‐grown cells encourage recovery sooner than conventionally‐grown cells. CF ALI HBE cell cultures engrafted with non‐CF cells were tested for functional CFTR recovery in Ussing chambers. CRC HBE cells engraft, persist and recapitulate CFTR function more effectively than conventionally‐grown cells. Our results suggest that transient polidocanol injury and the CRC culture method may be a viable approach to enhance cell engraftment for CF cell therapy.

Support or Funding Information

Supported by CFF grants RANDELXX015 and BOUCHE15R0CF2015, and NIH grants U01HL111018 and P30DK065988.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increased global lysine acetylation in diabetic vasculature is associated with downregulation of SIRT‐1 in vascular smooth muscle cells.
Authors: Johnson Christina Mary , Burke Cameron , Carrillo‐Sepulveda Maria Alicia
Published: June 2021

Vascular complications are the main problems faced by patients with Type 2 Diabetes Mellitus (T2DM). In addition to endothelial dysfunction, vascular smooth muscle cell (VSMC) dysfunction is postulated to play a crucial role in the development of diabetic vascular complications. Recent studies from our laboratory have demonstrated that acetylation of lysine residues, a posttranslational modification, is increased in vascular tissue exposed to metabolic disorders, such as diabetes. We hypothesized that diabetic conditions would dysregulate lysine deacetylases and acetyltransferases in VSMC, consequently leading to increased global lysine acetylation in diabetic blood vessels. Using male adult Goto‐Kakizaki (GK) rats (16‐weeks‐old) as a model of non‐obese type 2 diabetes and their respective Wistar controls, we found that these diabetic rats exhibited high levels of fasting blood glucose (191 ± 12 vs. 98±7 mg/dL vs. controls, p<0.05), as well as hemoglobin A1C (6.82 ± 0.93 vs. 5.2 ±0.2 % controls, p<0.01). Through direct measurement of blood pressure via right carotid catheterization, we also found greater systolic blood pressure in the GK group (166.57 ±24.27 vs. 124.58 ±17.07 controls, p<0.01). This effect was accompanied by high levels of global lysine acetylation in GK blood vessels (3.3 fold of increase vs. control, p<0.001). To test our hypothesis that VSMC is a major cell type in blood vessels contributing to increased vascular lysine acetylation in diabetic conditions, we utilized isolated aortic VSMC from GK rats in primary cultures. After euthanasia with isoflurane (via nasal 5% in 100% O2), thoracic aortas from GK rats were dissected and VSMC were isolated by an enzymatic digestion method. Phenotypically, VSMC from GK rats exhibited an enhanced proliferative phenotype in comparison to VSMC from Wistar control rats (Figure 1). Strikingly, we found that VSMC from GK rats exhibited a marked decrease in SIRT‐1 deacetylase expression (3.5‐fold decrease vs. control, p<0.05). Moreover, expression of the acetyltransferase PCAF was increased in VSMC from GK rats (1.5‐fold increase vs. control, p<0.05). Taken together, our in vitro results suggest that downregulation of SIRT‐1 and upregulation of PCAF may mediate the increased levels of global lysine acetylation in diabetic blood vessels, confirming our hypothesis. To further this investigation, we will identify the proteins that are specifically acetylated in diabetic VSMC through mass spectrometry analysis, as well as the effects on global lysine acetylation when overexpressing SIRT‐1 in VSMC.

Support or Funding Information

In‐House Grant, NYIT

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Resuscitation with Anaerobically Stored Blood
Authors: Williams Alexander Thomas , Yoshida Tatsuro , Dunham Andrew , Cabrales Pedro
Published: June 2021

Background

Hemorrhagic shock (HS) severely decreases oxygen (O2) delivery and induces cardiovascular collapse. In parallel to controlling the hemorrhage, doctors respond by infusing large volumes of red blood cells (RBCs) to restore blood volume, O2 carrying capacity, and hemodynamic stability. The quality of the transfused RBCs determines the recovery from HS, and extent of sequelae prompted by the HS. This study compares the ability to recover from HS with conventionally stored RBCs, anaerobically (O2 saturation <10%) stored RBCs, and anaerobic/hypercapnic (O2 saturation <10% & pCO2 (@37°C) ~70 mmHg) stored RBCs.

Methods

Packed red blood cells (pRBCs) stored in AS‐3 after leukorfiltration were created from donor rats. pRBC were randomly stored under 1) conventional; 2) anaerobic; 3) anaerobic/hypercapnic conditions. Sprague‐Dawley rats (150–200g) were hemorrhaged (50% of blood volume), held in hypovolemia for 30 minutes, and resuscitated to recover blood pressure to 90% pre‐hemorrhage with pRBC stored for 3 weeks. Systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation. Vital organ inflammation, oxygenation, and function were evaluated post resuscitation. Data were analyzed using two‐way ANOVA, followed by the appropriate post hoc analyses.

Results

Conventionally stored RBCs were susceptible to both biochemical and mechanical changes during storage compared to anaerobic and anaerobic/hypercapnic storage. 24‐hour post transfusion recovery of anaerobically and anaerobic/hypercapnic stored RBCs was significantly higher compared to conventionally stored RBCs (P<0.05). Shock impaired cardiac function and oxygen delivery and resuscitation with pRBCs restored cardiac output. Resuscitation with conventionally stored cells resulted in severe cardiac ischemia. Resuscitation with either anaerobic or anaerobic/hypercapnic required significantly lower volume (1.9 and 2 fewer units than conventional storage) of pRBC to preserve hemodynamics during resuscitation (P < 0.05). Animals resuscitated with conventionally stored RBCs showed impaired ability to deliver O2 to tissues, increased lung, liver, and spleen inflammatory markers, and decreased liver and kidney function, compared to animals resuscitated with either anaerobic or anaerobic/hypercapnic pRBC.

Conclusion

Studies indicate that anaerobic or anaerobic/hypercapnic storage of RBC can improve clinical outcomes from hemorrhagic shock in a rat model, as evidenced by hemodynamic stability, O2 delivery, and vital organ inflammation and function.

Support or Funding Information

NIH Grants from the National Heart Lung and Blood Institute, T32‐HL105373, P01‐HL110900, R01‐HL126945. Financial support was received for Hemanext for the completion of the study. Hemanext did not participate in the implementation of the study.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Vascular Localization of Zika Virus Following in vivo Infection
Authors: Brown Isola A.M. , DeLalio Leon J. , Liu Shufeng , Isakson Brant E. , Wang Tony T.
Published: June 2021

Zika virus (ZIKV) is an emerging blood‐borne pathogen that produces flu‐like symptoms in infected individuals and is responsible for microcephaly and other neonatal birth defects. Despite this growing public health concern, the molecular mechanisms involved in ZIKV infection are still poorly understand. We have recently shown that African and South American derived ZIKV strains infect human endothelial cells in culture (Liu et al., Circ Res, 2016). Still, the role of the vasculature and endothelium during in vivo infection are not known. We investigated a role for the vasculature in ZIKV infection and hypothesize that ZIKV expression in infected animals will localize to vascular beds.

We established ZIKV infection using an in vivo animal model of infection in 4‐week old C57Bl/6 mice. Mice received 6.4×106 plaque forming units (PFUs) of either the PRVABC59 or DAK strains of ZIKV, isolated in Puerto Rico and Senegal respectively. Mice were sacrificed at 5 days post‐infection and we used immunohistochemistry to detect ZIKV expression in various vascular beds including brain, liver, aorta and mesenteric. ZIKV was labeled using a novel murine monoclonal antibody D1‐4G2‐4‐15 against the viral coat protein E‐protein.

At 5 days post‐infection (dpi), mice treated with the DAK strain have decreased body weight compared to Day 0, while mice treated with the PRV are resistant to this weight loss. At 5 dpi, both groups of mice have increased brain ZIKV viral loads. However, the viral load in DAK mice was four‐fold that of PRV‐treated mice. Mice treated with DAK show increased morbidity with 0% survival at 7 dpi while the PRV strain is non‐lethal up to 15 dpi. At 5 dpi, the ZIKV viral coat protein E protein was detectable in arteries and veins of the cerebral circulation from brains of mice treated with either ZIKV strain and co‐localized with the endothelial marker claudin‐5. Interestingly, cerebral capillaries were found to have the DAK ZIKA, whereas the PRV did not.

The co‐localization of ZIKV with endothelial markers suggests a role in the pathology of in vivo ZIKV infection. These findings improve our understanding of the pathology of ZIKV infection and hint at potential strain variation that may be mediated by endothelium.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bardoxolone activates cardiac Nrf2, increases antioxidant expression and lowers arterial pressure in rats with heart failure
Authors: Zhang Andi , Tian Changhai , Gao Lie , Zucker Irving H.
Published: June 2021

Chronic heart failure (CHF) is associated with increased levels of oxidant stress in many tissues. High levels of reactive oxygen species (ROS) impairs cardiac, neural and renal function. Increased ROS in CHF is, in part, due to low levels of a variety of antioxidant enzymes. Many of these enzymes are regulated by the activity of the transcription factor Nuclear factor E2‐related factor 2 (Nrf2), which binds to antioxidant response elements (ARE) on multiple genes. Activation of Nrf2 has been shown to modulate a variety of disease states as well as several negative effects of the aging process. We hypothesized that pharmacological activation of systemic Nrf2 would provide beneficial hemodynamic effects in a rodent model of CHF. Male Sprague Dawley rats were subjected to left coronary artery ligation or sham surgery, and were divided into four groups (Sham+Vehicle, Sham+Nrf2 activator, CHF+Vehicle and CHF+Nrf2 activator) based on echocardiographic analysis 4 weeks post myocardial infarction. The Nrf2 activator, bardoxolone was administered daily for another 2 weeks by intraperitoneal injection (i.p) injection. Control animals were treated with the saline vehicle. Hemodynamics were evaluated by ventricular catheterization (Millar) and echocardiography (Vevo 2100). Animals were sacrificed, tissues taken and kept at −80 °C until analyzed. Our results demonstrated that Bardoxolone did not cause any changes in LV ejection fraction in either group (72.1±1.3 sham‐vehicle vs 73.0±1.2 sham‐bardoxolone; 32.9± 0.8 CHF‐vehicle vs 33.5±0.8 CHF‐bardoxolone). However, Bardoxolone significantly reduced LV systolic pressure and mean arterial pressure in CHF rats (LVSP; Vehicle 138.0±8.0 vs bardoxolone 117.0±3.9 mm Hg, p=0.04; MAP; Vehicle 97.8±2.9 vs bardoxolone 87.5±4.5 mm Hg, p=0.07). In addition, Bardoxolone had no effect on LVEDP or LV dp/dt max or min in rats with CHF. Molecular studies further revealed that In CHF rats Nrf2 mRNA was increased in the non‐infarcted areas of the heart by 2.5‐fold. Importantly, both mRNA and protein levels of NAD(P)H dehydrogenase [quinone] 1 (NQO1), a known target for Nrf2, was significantly increased by bardoxolone. Co‐IP experiments in skeletal muscle also showed that Bardoxolone reduced NF‐kB and increased Nrf2 binding to the CREB‐Binding Protein, respectively. These data suggest that activation of Nrf2 may provide beneficial antioxidant effects on cardiac muscle in CHF and reduces afterload. The mechanisms for the peripheral effects have not been determined.

Support or Funding Information

Supported by PO1 HL 62222 and an APS summer undergraduate fellowship

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stretching Single Titin Molecules from Failing Human Hearts at Cardiac Cycle Reveals Titin's Role in Cardiac Kinetic Reserve
Authors: Chen Mei‐pian , Kiduko Salome A. , Saad Nancy S. , Canan Benjamin D. , Kilic Ahmet , Mohler Peter J. , Janssen Paul M.L.
Published: June 2021

Diastolic dysfunction occurs not only in diastolic heart failure, but also ubiquitously in systolic heart failure. A main determinant of diastolic passive tension, the elastic sarcomeric protein titin, has been shown to be associated with heart failure, with unresolved mechanisms. Heart failure patients commonly experience symptoms only during physical activities or stress. To determine whether titin is playing a role in the mechanisms of heart failure in terms of its known impaired property of frequency‐dependent acceleration of relaxation (FDAR), we studied the FDAR responses in live left ventricular cardiomyocytes and single‐molecule force spectroscopy (SMFS) of titin elastic domains (PEVK‐distal Ig, distal Ig) from failing (n=11) and non‐failing (n=7) human hearts. To access SMFS on native titin, we developed a novel approach using atomic force microscopy to detect titin passive tension (TPT) based on frequency‐modulated cardiac cycle. Mean TPT was shown to be reduced upon an increased heart rate, which was significantly blunted in failing human hearts. The results from titin distal Ig domain were significantly correlated with the frequency‐dependent relaxation kinetics of human cardiomyocytes from the corresponding hearts, which was shown to be impaired in failing hearts. Correlation studies suggested that the higher the TPT, the faster the cardiomyocytes relaxed, but lower the potential of myocytes to speed up relaxation at a higher heart rate; such poorer FDAR response was also associated with a less reduction or a bigger increase in TPT upon a raised heart rate. Our study established a novel approach in detecting tension physiologically on native titin domains. The data from human hearts suggested that the regulation of kinetic reserve in cardiac relaxation and its pathological changes are associated with the intensity and dynamic changes of passive tension on titin.

Support or Funding Information

This project is supported by RO1HL113084 to Janssen PML, and T32NS779844 to Chen MP

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic high fat diet disrupts renal molecular clock
Authors: Zhang Dingguo , Speed Joshua S. , Colson Jackson C. , Bailey Shannon M. , Young Martin E. , Gamble Karen L. , Pollock Jennifer S. , Pollock David M.
Published: June 2021

Kidney function follows a strong circadian rhythm that is tightly regulated by clock genes. We previously reported that high salt diet induced dyssynchrony in renal clock gene expression in the cortex and medulla. However, whether changes in other dietary factors pose threats to renal circadian rhythms remain largely unknown. The current study was designed to test the hypothesis that high fat diet disrupts renal clock gene expression with 2 mouse models. First, C57Bl/6J male mice (8 weeks old, n=3) were fed normal fat (NF) or high fat (HF) diet for 20 weeks. During the last 2 weeks of the protocol, mice underwent either time restricted feeding, where food access was allowed only during lights‐off, active period (7 pm–7 am), or sham feeding procedure. Restricted feeding did not cause significant changes in body weight or food consumption. Renal cortex was collected in 4‐hour increments throughout a 24‐hour period and clock gene expression measured by qPCR. We found that Per2 expression has a robust circadian rhythm with a peak expression at zeitgeber time (ZT) 13. We did not observe any significant differences among groups. Second, Period2Luciferase (Per2Luc) mouse model was utilized to monitor real‐time molecular clock rhythm. Male and female Per2Luc mice (n=4) were fed NF or HF diet with sham or time restricted feeding. Kidneys were dissected into three parts (cortex, outer and inner medulla) at the end of study and cultured for 3 days to measure bioluminescent rhythms. We found that HF diet lengthened the period of luciferase rhythm by 2 hours (NF: 23.98 hours vs. HF: 26.17 hours, p=0.02) in the cortex. No significant differences were observed in the groups in renal outer medulla or inner medulla. Restricted feeding did not cause any significant changes in any groups. These data suggest that clock gene expression in the kidney follows a circadian rhythm that can be disrupted by chronic high fat diet.

Support or Funding Information

P01 HL136267 Integrating novel mechanisms controlling sodium excretion and blood pressure UAB School of Medicine AMC21 Circadian Disruption and Susceptibility to Target Organ Damage

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hypertension Precedes Metabolic Syndrome in the ALMS1 (Alstrom Syndrome 1) Knockout Rat
Authors: King‐Medina Keyona N. , Ortiz Pablo A.
Published: June 2021

We previously found that Alstrom syndrome 1 protein (ALMS1) is expressed in the kidney where it regulates thick ascending limb (TAL) NaCl reabsorption by controlling NKCC2 endocytosis. In humans, inactivating mutations in the ALMS1 gene causes obesity, insulin resistance and hypertension (metabolic syndrome). We found that deletion of ALMS1 in (Dahl salt‐sensitive) rats induced hypertension (measured by radio‐telemetry and tail‐cuff plethysmography). Moreover, the ALMS1 Knockout (ALMS1 KO) rats are hyperphagic and develop progressive obesity over time. Obesity is a risk factor for hypertension and insulin resistance. However, it is unclear whether the increase in blood pressure (BP) occurs prior to or after the development of metabolic alterations (obesity and insulin resistance) in the ALMS1 KO rats. We hypothesize that hypertension in the ALMS1 KO rats is primarily caused by a renal defect and not secondary to the metabolic syndrome. In order to study this we obtained young (shortly after weaning) ALMS1 KO and wild‐type (WT) rats (age range: 5–7 weeks old) and measured body weight, baseline blood glucose, glucose tolerance (IP injection of 2 kg/g of BW glucose) and systolic blood pressure (SBP) by tail‐cuff over time. ALMS1 KO (n=6) and WT (n=6) rats were given 30 g per day of regular chow (0.4% Na) with free access to water. At 5–7 weeks of age ALMS1 KO rats had similar body weights (ALMS1 KO: 209±13 g vs. WT: 182±15 g, N.S.). Baseline glucose was similar (ALMS1 KO: 88±3 vs. WT: 75±7 mg/dL, N.S.) and glucose tolerance test produced similar peaks and area under the curve. Importantly, the SBP was higher in ALMS1 KO rats (ALMS1 KO: 156±5 vs. WT: 143+3 mmHg, p<0.05). Four weeks later (9–11 weeks old), body weight began to diverge between strains (ALMS1 KO: 326±10 g vs. WT: 282±17 g, p<0.05). Baseline glucose was higher in ALMS1 KO (ALMS1 KO: 84±2 vs. WT: 71±3 mg/dL, p<0.05) and glucose tolerance test produced a higher peak in ALMS1 KO (ALMS1 KO: 376±27 vs. WT: 305±17 mg/dL, p<0.05), as well as a larger area under the curve (ALMS1 KO: 11915±1349 vs. WT: 9176±916 mg*min/dL). Lastly, the SBP continued to be elevated in the ALMS1 KO rats (ALMS1 KO: 160±3 vs. WT: 144±3 mmHg, p<0.05). Therefore, our data show that the genetic deletion of ALMS1 causes hypertension in rats and that the increase in blood pressure precedes the development of obesity and insulin resistance in this model. This is a new rat model of metabolic syndrome with established hypertension that does not require a high salt diet. Ongoing experiments will test whether caloric restriction prevents or decrease the hypertension and metabolic syndrome in this rat strain.

Support or Funding Information

NIH F31 Diversity Grant: 5F31HL123285 (King‐Medina)and NIH‐NIDDK Grant: 5R01DK107263 (Ortiz)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.