bluuebunny/crossref_metadata_2025 huggingface dataset:
The Association between Suppressed Transformation of Fibroblasts to Myofibroblasts and Fibrolysis Induced by Copper Supplementation in Monkeys of Myocardial Ischemic Infarction
Authors: Xiao Ying , Liu Yinjie , Han Pengfei , Luo Qin , Wang Heng , Kang Y. James
Published: June 2021

Myocardial ischemia is associated with cardiac tissue copper (Cu) loss and fibrosis. Cu supplementation diminishes cardiac fibrosis and improves cardiac function. The transformation of fibroblasts to myofibroblasts is highly responsible for the pathogenesis of cardiac fibrosis. This study was undertaken to test the hypothesis that suppression of the transformation from fibroblasts to myofibroblasts is responsible for Cu‐induced reduction of cardiac fibrosis. Rhesus monkeys of 4–5 years old were subjected to coronary artery ligation to induce myocardial infarction (MI). At the fourth week after the surgery, an ultrasound‐directed Cu‐albumin microbubble organ‐specific Cu delivery technique was used to treat myocardial infarct monkeys twice a week for four weeks. This treatment significantly increased Cu concentrations in the infarct area, reduced the infarct size, and loosened collagen cross‐linking network. Total fibroblasts labeled with vimentin were increased in the infarct area, and this increase was not changed by Cu supplementation. In contrast, myofibroblasts, labeled with both vimentin and α‐smooth muscle actin (a‐SMA), were also significantly increased in the infarct area, but Cu supplementation significantly reduced this increase, along with decreased mRNA levels of type I and III collagens. This decrease in collagen production was accompanied by a significant increase in metalloproteinase‐1 (MMP‐1) and the MMP‐1 producing fibroblasts (vimentin+ and MMP‐1+ cells). In addition, primary cultures of neonatal rat cardiac fibroblasts were treated with 0.1 μM angiotensin II (Ang II) to increase cellular α‐SMA concentrations, mimicking the transformation from fibroblasts to myofibroblasts. This increase in α‐SMA production was significantly suppressed by addition of 5 μM CuSO4 in the cultures. These results suggest that Cu inhibits the transformation of fibroblasts to myofibroblasts, leading to the reduction of collagen synthesis and the increase in collagen degradation, and their combination results in fibrolysis in the infarcted myocardium.

Support or Funding Information

This study was supported by National Science Foundation of China (81230004 and 81300109).

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 NLRP3 mediates the recruitment of lymphocytes in the cavernosal tissue and erectile function impairment in DOCA/salt mice.
Authors: Fais Rafael Sobrano , Rodrigues Fernanda Luciano , da Silva Josiane Fernandes , Tostes Rita C. , Carneiro Fernando Silva
Published: June 2021

Introduction

Erectile dysfunction (ED) is associated with risk factors for the development of cardiovascular diseases such as hypertension. Increased activity of the immune system is closely linked to the development of hypertension and ED. Arterial hypertension is strongly influenced by pro‐inflammatory effects of NLRP3. NLRP3 activity promotes vascular changes, cardiac and renal hypertrophy and increase in blood pressure. In addition, NLRP3 is involved in the recruitment of cells of the immune system. Increasing evidence shows that increased lymphocyte infiltrate is involved in the genesis and/or aggravation of various cardiovascular diseases.

Hypothesis

NLRP3 increases lymphocyte infiltrate, promotes impairment of corpora cavernosa (CC) reactivity and ED in DOCA/salt hypertensive mice.

Objective

Evaluate the role of NLRP3 in lymphocyte recruitment in CC, impairment of erectile function and changes in CC reactivity in DOCA/salt mice.

Methods

(CEUA 213/2016). Male C57BL/6 (WT) and NLRP3 deficient mice (NLRP3−/−) were either treated with vehicle or induced to DOCA/salt hypertension. Fluorescence to FAM‐FLICA and histochemistry were performed to analyze respectively the caspase‐1 activity and the number of lymphocytes in CC. The intracavernous pressure (ICP) was measured. Reactivity of CC to acetylcholine (ACh) and sodium nitroprusside (SNP) was performed.

Results

DOCA/salt hypertension increased the caspase‐1 activity in the CC. In addition, we observed increased lymphocyte infiltrate in the CC (WTsham: 18.5 ± 1.0; WTdoca: 33.5 ± 1.4, n=6), impaired erectile function (decreased ICP in WTdoca in the frequencies of 4, 8, 12 and 16 Hz) and reduced relaxation for ACh (pD2 WTsham: 7.2 ± 0.08, n=7; WTdoca: 6.5 ± 0.12, n=7) and SNP (pD2 WTsham: 7.2 ± 0.07, n=6; WTdoca: 6.7 ± 0.06, n=7) in the CC of WT mice. The absence of NLRP3 prevented the increase in the caspase‐1 activity and in the lymphocyte infiltrate (NLRP3−/−sham: 12.0 ± 1.2; NLRP3−/−doca: 17.6 ± 1.9, n=5) in the CC. Furthermore, NLRP3 absence normalized the erectile function, the reactivity to ACh (pD2 NLRP3−/−sham: 7.0 ± 0,14, n=6; NLRP3−/−doca: 6.9 ± 0.16, n=7) and to SNP (pD2 NLRP3−/−sham: 8.2 ± 0.08, n=6; NLRP3−/−doca: 8.5 ± 0.07, n=7) in DOCA/salt hypertension.

Conclusion

NLRP3 mediates the lymphocyte recruitment, impairment in erectile function and CC reactivity, promoted by DOCA/salt hypertension.

Support or Funding Information

Financial support: FAPESP, CNPq, CAPES, CRID.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aldosterone‐Induced Hypertension is T Lymphocyte‐Dependent and Attenuated by Activation of the G Protein‐Coupled Estrogen Receptor 1
Authors: Dinh Quynh Nhu , Vinh Antony , Kim Hyun Ah , Diep Henry , Broughton Bradley , Chrissobolis Sophocles , Drummond Grant , Sobey Christopher
Published: June 2021

The G protein‐coupled estrogen receptor 1 (GPER), a membrane‐localised estrogen receptor, may modulate some of the effects of aldosterone. G‐1, a GPER agonist, can promote T cell‐mediated anti‐inflammatory actions and acutely lower blood pressure. This study aimed to test the effects of G‐1 and G‐15 (GPER antagonist) on aldosterone‐induced hypertension in male and female mice, and to examine the cellular mechanisms involved. C57Bl6 and RAG1‐deficient mice were treated with vehicle, aldosterone/salt (0.72 mg/kg/d SC plus 0.9% NaCl for drinking) ± G‐1 (0.03 mg/kg/d SC) and/or G‐15 (0.3 mg/kg/d SC) for 14 d. Some females were ovariectomised 7 d prior to treatment. T cells were adoptively transferred via IV injections 21 d prior to treatment. In males, aldosterone/salt caused a sustained increase in blood pressure of ~25 mmHg within 7 d and this increase was attenuated by ~50% in the presence of G‐1 (n=11–13, P<0.05). G‐15 alone did not alter hypertension caused by aldosterone/salt but it prevented the anti‐hypertensive effect of G‐1. By contrast, the pressor response to aldosterone/salt was delayed in females where an increase in blood pressure was only observed at 14 d; however co‐administration of aldosterone/salt with G‐15 resulted in a marked increase of ~20 mmHg from 7 d (n=6–8, P<0.05). Administration of aldosterone/salt produced a robust increase in blood pressure from d 7 in ovariectomised females (n=5–9, P<0.05), similar to that observed in males. T and B cells in kidneys of male and female C57Bl6 mice were found to express GPER. In male RAG1‐deficient mice, aldosterone/salt had virtually no effect on blood pressure (n=8, P<0.05). However, adoptive transfer of 7×106 T cells from male C57Bl6 into male RAG1‐deficient mice fully restored the pressor response to aldosterone/salt, which could be attenuated by co‐treatment with G‐1 (n=8–14, P<0.05). This study indicates that activation of GPER by G‐1 and/or endogenous estrogen profoundly attenuates aldosterone/salt‐induced hypertension in mice. T cells are key contributors to aldosterone/salt‐induced hypertension, and therapeutic activation of GPER on T cells may reduce hypertension caused by elevated levels of aldosterone.

Support or Funding Information

NHMRC grant

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 reactive oxygen species in gestational diabetes and the impact on the developing fetus
Authors: Arriaga Diana
Published: June 2021

Gestational diabetes mellites (GDM) is characterized by elevated levels of glucose in a pregnant woman's bloodstream that occurs from an overproduction of hormones. Under GDM, elevated glucocorticoids (GC) and insulin like growth factor (IGF)‐1 hormones in the placenta may possibly lead to an increase in the amount of reactive oxygen species (ROS) in the embryo due to an increase in glucose utilization and mitochondrial activity. Overproduction of ROS can modify the genome, thus predisposing offspring's to diabetes and obesity later. The aim of the study is to investigate the mechanism by which GDM leads to an increased production of ROS and evaluate the epigenetic changes that occur. I hypothesize that under diabetic conditions, the embryo will have an increase in ROS production due to increased hormone levels, resulting in epigenetic changes in the offspring. An in vivo model for GDM will be established by feeding female mice a high‐fat diet for four weeks. Controls will be fed a standard diet. Fasting blood glucose levels and glucose tolerance tests will monitor the development of diabetes. Mating between a diabetic or control female will occur with a normal male. At 6.5 and 17.5 days of gestation and 7 days post birth, maternal, fetal and placental blood and tissue will be collected. Biochemical assays will evaluate tissue ROS levels and epigenetic changes. Blood analysis will measure hormone and glucose levels. Results from this study will hopefully lead to the development of treatments to lower the incidence of diabetes in future generations.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of extracellular vesicles released by hypoxic adipocytes in Insulin action
Authors: Mora Silvia , Mleczklo Justyna , Ortega Francisco , FernandezReal Jose Manuel , Falcon‐Perez Juan Manuel
Published: June 2021

Extracellular vesicles (EVs) are released by many cells and provide a mechanism for intercellular communication in an autocrine and paracrine fashion. EVs are also released during pathological states. Adipocytes release EVs, however, the impact of these vesicles in glucose metabolism and insulin action has not been thoroughly investigated. We sought to investigate whether EVs released by metabolically challenged adipocytes exposed to hypoxia or to macrophage conditioned media affect insulin responsiveness and glucose transport. EVs were isolated from the culture supernatant of adipocytes cultured under normoxia, hypoxia (1% oxygen) or exposed to macrophage conditioned media (15%v/v). EVs were isolated from plasma of lean and obese individuals. EVs were used to treat cells and glucose transport and activation of insulin signalling cascades was measured. EVs released from hypoxic adipocytes but not those released by adipocytes exposed to macrophage conditioned media impaired insulin‐stimulated 2‐deoxyglucose uptake and reduced insulin‐stimulated Ser473 phosphorylation of AKT. ERK signalling was intact. Insulin stimulated glucose transport in muscle cells was also inhibited. EVs from obese individuals decreased insulin stimulated 2‐deoxyglucose uptake in adipocytes (p=0.0159). EVs from obese subjects contained distinct miRNA signatures. These findings indicate that EVs released by stressed adipocytes impair insulin action.

Support or Funding Information

University of Livepool, MINECO and FEDER

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inflammation and Kidney Injury in Diabetic African American Men
Authors: Cao Lei , Boston Ava M. , Jegede Olugbemiga Ebenezer , Harrison Scott Henry , Newman Robert Howard , Ongeri Elimelda Moige
Published: June 2021

Chronic kidney disease is the most common complication of diabetes. Without interventions, diabetic kidney disease (DKD) progresses to end stage renal disease (ESRD). In the United States, minority ethnic groups (e.g. African Americans) are disproportionately burdened by DKD. While genetic factors contribute to the disparities in DKD, little is known about the underlying cellular and molecular mechanisms. Several studies have shown that inflammation plays a role in the pathophysiology of DKD and the fibrosis observed in diabetic nephropathy. The goal of the current study was to determine the association between specific inflammation markers and diabetic kidney injury in diabetic African American men, an underrepresented sub‐population with high risk for diabetes and DKD. To this end, diabetic patients with (n=21) and without (n=88) diagnosed kidney disease were recruited at the Cone Health Community Health and Wellness Center in Greensboro, NC. Age‐matched, non‐diabetic subjects (n=81) served as controls. Assays for albumin and creatinine were used for biochemical assessment of kidney function. The diabetic patients were then stratified into three groups based on their albumin to creatinine ratios (ACR); normoalbuminuria (ACR<30 mg/g), microalbuminuria (30 mg/g<ACR<300 mg/g), and macroalbuminuria (ACR>300 mg/g). Enzyme‐linked immunosorbent assays were used to measure the plasma and urinary levels of five inflammatory markers; two markers of system‐wide markers (adiponectin and C‐reactive protein (CRP)); and three markers involved in renal inflammation (tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), intercellular cell adhesion molecule‐1 (ICAM‐1)). Plasma levels of the pro‐inflammatory cytokines TNF‐α and IL‐6 were significantly higher in diabetic patients with micro‐ and macroalbuminuria when compared to non‐diabetic controls and diabetic patients with normoalbuminuria. Urinary levels for ICAM‐1 and the anti‐inflammatory cytokine, adiponectin, were higher in diabetic patients with macroalbuminuria than the other three groups. Plasma CRP was elevated in diabetic subjects compared to non‐diabetic controls, but did not correlate with the extent of diabetic kidney injury. In contrast, urinary ICAM‐1, plasma TNF‐α, and plasma adiponectin had moderate positive correlations with ACR. The plasma levels for ICAM‐1, IL‐6, and CRP were not significantly correlated with the ACR. The data suggest that certain inflammation pathways play a role in the pathogenesis of DKD in African American men.

Support or Funding Information

This study was supported by the Minority Men's Health Initiative (MMHI) through NIH/NIMHD Center Award # U54MD008621 (CFDA 93.307), Sub‐Awards # HU140400 (to RHN and EMO) and HU150006 (to EMO and SHH); and the NIH/NIGMS Award Number SC3GM102049 to EMO.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
nNOS in Embryonic Kidney Contributes to Glomerular Maturation
Authors: Der Balint , Gyarmati Georgina , Riquier‐Brison Anne , Shroff Urvi , Peti‐Peterdi Janos
Published: June 2021

The regulatory role of neuronal nitric oxide synthase (nNOS) in renal and glomerular hemodynamics is well established. In addition, nNOS is involved in cellular migration in other organs, such as the brain. The highest level of nNOS expression occurs on day 6 after birth (P6), when the mouse kidney is still developing. The purpose of this study was to explore a novel, non‐traditional role of nNOS in renal development and glomerular maturation. To determine the timepoints and localization of nNOS expression in embryonic kidney, fluorescent reporter nNos+/−‐Cre+/+/mTmG+/+ mice were induced with tamoxifen at different timepoints during pregnancy. The specific inhibitor of nNOS, 7‐Nitroindazole (7‐NI) was administered daily by ip. injections (20 mg/kg) from E16.5 to P7 in pregnant Pod+/+‐Cre+/+/Confetti+/+ mice. Fetal kidneys were harvested after birth at P7 and further processed for histological analysis. During embryonic kidney development, nNOS expression is detectable in the medullary region as early as E16.5. At E19.5, nNOS is expressed in macula densa cells at the juxtaglomerular apparatus and in a few cells of the medullary collecting duct. At P3, nNOS was localized in the same cell types as in earlier stages. Pharmacological inhibition of nNOS for 10 days by daily 7‐NI treatment did not alter the kidney/body weight ratio compared to vehicle treatment, albeit the 7‐NI treated animal group had growth retardation (n=4 each). The diameter of glomeruli was 20% smaller, and the cumulative podocyte number in glomeruli was decreased by 40% in 7‐NI treated animals (n=4). Albeit 7‐NI treatment resulted in the reduction of body weight likely due to nonrenal effects, the embryonic presence of nNOS in macula densa cells and the negative impact of nNOS inhibition on glomerular diameter and podocyte number suggest a novel, non‐traditional role for nNOS in embryonic kidney development and glomerular maturation.

Support or Funding Information

Research support NIH NIDDK R01 DK64324

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutant Actn4 alters the distribution of intracellular stress within podocytes and contributes to their mechanical failure in response to stretch
Authors: Feng Di , Notbohm Jacob , He Shijie , Wang Minxian , Krishnan Ramaswamy , Pollak Martin
Published: June 2021

Alpha‐actinin‐4 (ACTN4 in humans, Actn4 in mice) crosslinks actin filaments in podocytes, providing them mechanical resilience as they help maintain the glomerular filtration barrier. Although human disease‐causing mutant ACTN4 leads to brittle reconstituted actin networks in vitro, how this affects the ability of podocytes to withstand the external mechanical challenges within the glomerulus remains unknown. We studied primary podocytes isolated from wild‐type (WT) and disease‐causing mutant Actn4 knock‐in mice. We previously reported that, after transient stretch, WT cells recovered their baseline contraction and actin cytoskeleton breakages, whereas mutant podocytes developed irrecoverable reductions in their contraction and irreparable disruptions in their cytoskeletons. We now report that after stretch, when challenged in a centrifugal detachment assay, mutant podocytes (11/23) had a higher probability of detachment than WT (2/19) (odds ratio =7.42, 95% CI 1.27, 80.75; p=0.017). Through an analysis of baseline biophysical properties, we found that the actin filaments of mutant Actn4 podocytes displayed a more uniform distribution of orientation (aligned more parallel to each other) compared with the filaments of WT podocytes (which displayed more random orientation). We then computed baseline intracellular stress in terms of two constituent components: the magnitude of mean principal stress as tension (σ), and the orientation of first principal stress as principal orientation (θ). The spatial distribution of intracellular stress within the podocytes was measured using the autocorrelation function C(r). The distance over which the C(r) decays to a value of 0.5 was extrapolated as a singular value from the autocorrelation curves and is termed correlation length, or r1/2. The greater the r1/2 value, the greater the distance over which intracellular stress maintains uniformity. We found that mutant podocytes displayed a more uniform distribution of intracellular stress across the cell, both in terms of magnitude (r1/2 σ mean ± SE: mutant 18.0 ± 0.8 μm vs. WT 13.4 ± 0.8 μm) and orientation (r1/2 θ mean ± SE: mutant 34.7 ± 2.2 μm vs. WT 25.9 ± 2.3 μm). Moreover, the more uniform the intracellular stress was distributed at baseline, the less likely mutant podocytes recovered their baseline contraction after periodic stretch. Taken together, our findings provide novel insight into the mechanism by which ACTN4 mutation alters the alignment of the cytoskeleton and impairs the resiliency of the podocyte, leaving it vulnerable to mechanical stretch and more prone to detachment in its glomerular environment.

Support or Funding Information

R37DK059588 and T32DK007199

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cerebrovascular Reactivity in Habitually Exercising Healthy Adults
Authors: Miller Kathleen B. , Harvey Ronée E. , Howery Anna J. , Greisch Mackenzie L. , Carl Alexandra E. , Joyner Michael J. , Barnes Jill N.
Published: June 2021

Cerebrovascular reactivity to hypercapnia declines with age and low reactivity may be a biomarker of cognitive decline. Central elastic arteries that perfuse the brain stiffen with age, increasing their transmission of pulsatile mechanical forces into the cerebral microvasculature and impairing cerebral microvessel function. Habitual physical activity, however, has been shown to reduce central arterial stiffness and is associated with better cognitive function. Therefore, the purpose of this study was to evaluate cerebrovascular reactivity in young and older healthy adults who habitually exercise. We hypothesized that older adults who habitually exercised would have similar cerebrovascular reactivity when compared with young adults. However, because the underlying mechanisms of cerebral blood flow regulation may differ with age, we also evaluated blood pressure and central hemodynamic measures. We recruited 25 young (YA: age = 27 ± 1 y) and 20 older (OA: age = 59 ± 1 y) habitual exercisers. Middle cerebral artery velocity (MCAv) was recorded using transcranial Doppler ultrasound. In order to assess cerebrovascular reactivity, mean arterial pressure (MAP), end‐tidal carbon dioxide (ETCO2), and MCAv were continuously recorded at rest and during stepwise elevations of inhaled CO2. Cerebrovascular conductance index (CVCi) was calculated as MCAv/MAP. Reactivity was calculated as the linear relationship between the change in ETCO2 and the change in each variable of interest (MCAv, CVCi and MAP). Central arterial stiffness (carotid‐femoral pulse wave velocity, PWV) and central hemodynamics (augmentation index, AIx) were assessed by applanation tonometry. Older adults had higher MAP (YA: 85 ± 2 mmHg; OA: 90 ± 3 mmHg; p<0.05) and AIx (YA: 0 ± 3 %; OA: 20 ± 2 %; p<0.05) at rest compared with young adults. PWV was not statistically different between groups (YA: 4.9 ± 0.3 cm/s; OA: 5.7 ± 0.5 cm/s; p=0.18). MCAv and CVCi reactivity to hypercapnia were similar between young and older adults (MCAv reactivity, YA: 2.0 ± 0.2 cm/s/mmHg; OA: 2.2 ± 0.1 cm/s/mmHg; p=0.46, CVCi reactivity, YA: 0.018 ± 0.002 cm/s/mmHg2; OA: 0.016 ± 0.001 cm/s/mmHg2; p=0.42); however, older adults demonstrated higher MAP reactivity to hypercapnia (YA: 0.3 ± 0.1 a.u.; OA: 0.8 ± 0.1 a.u.; p<0.05). PWV and AIx were not associated with MCAv, CVCi, or MAP reactivity to hypercapnia (p>0.05). Our results suggest that habitual exercise preserves cerebrovascular reactivity; however, MAP reactivity is augmented with aging allowing older adults to sufficiently increase cerebral blood flow during hypercapnia.

Support or Funding Information

Supported by National Institute of Health grant HL118154

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kinin B1 Receptor Knockdown Prevents DOCA‐Salt Hypertension by Modulating Mitochondrial Oxidative Stress in the Brain
Authors: Sriramula Srinivas
Published: June 2021

The imbalance between reactive oxygen species (ROS) production and anti‐oxidant capacity in the central nervous system results in oxidative stress and is implicated in the pathogenesis of hypertension. Deoxycorticosterone acetate (DOCA)‐salt hypertension is associated with increased expression of the pro‐inflammatory kinin B1 receptor (B1R) in the brain. We previously reported that DOCA‐salt hypertension is attenuated in global B1R knockout (B1RKO) mice. However, the functional role of B1R in inducing oxidative stress within the brain during neurogenic hypertension has not been fully studied. In this study, we tested the hypothesis that the beneficial effects of B1R knockdown are mediated by reduction in mitochondrial oxidative stress in the brain. DOCA‐salt treatment (1mg/g body wt. DOCA, 1% saline in drinking water for 3 weeks) resulted in significantly lower blood pressure (BP; telemetry) level in B1RKO mice (121 ±2 mmHg, n=12) compared to wild‐type controls (WT) (138 ±3 mmHg, n=12). DOCA‐salt treatment resulted in decreased catalase (79.1 ±5.6 vs. 150.3 ±10.4 mmol/min/ml, p<0.01), total superoxide dismutase (SOD, 4.19 ±0.3 vs. 5.97 ±0.3 U/ml, p<0.01), and the mitochondrial antioxidant manganese SOD (0.67 ±0.09 vs. 1.58 ±0.05 U/ml, p<0.01) activities in hypothalamic homogenates of WT mice, which were prevented in B1RKO mice (134.8 ±4.6 mmol/min/ml, 5.89 ±0.1 U/ml, and 1.68 ±0.22 U/ml, respectively, p<0.01). WT mice treated with DOCA‐salt showed increased oxidative stress as indicated by increased total ROS levels in the hypothalamus as measured using electron paramagnetic resonance spectrometer. This increase in ROS production was not observed in B1RKO mice treated with DOCA‐salt. Furthermore, DOCA‐salt hypertension resulted in decreased phosphorylation of eNOS‐ser1177 in the PVN (35 ±5 % decrease, n=9, p<0.05 vs WT), which was prevented in B1RKO mice. In addition, the gene expression of PPAR Gamma coactivator (PGC) 1‐Alpha and Uncoupling protein‐2 (UCP‐2), two important genes involved in mitochondrial function, were decreased in the PVN of DOCA‐salt treated WT mice but not in B1RKO mice. Together, these data provide evidence that kinin B1R knockdown reduces mitochondrial oxidative stress, improves antioxidant and nitric oxide signaling, and there by attenuates the development of DOCA‐salt hypertension.

Support or Funding Information

The American Heart Association (15SDG25720021)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CVS role of TRPV: from single channels to HRV assessment with Artificial Intelligence.
Authors: O'Brien Fiona , Williams Bryan M , Pratt Harry M. , Barrett‐Jolley Richard
Published: June 2021

The paraventricular nucleus (PVN) of the hypothalamus is an important autonomic control centre in the brain with roles in stress, metabolism, cardiovascular function and homeostasis. Identified PVN neurones project directly to the spinal cord and can modulate cardiovascular function via sympathetic control, however, the underlying mechanisms are not fully understood. Work from our group has highlighted roles for TRPV4 in osmosensing and thermoregulation in the PVN. To investigate whether TRPV4 channels may play a role in modulating cardiovascular function, we have used a combination of in vivo, in vitro and in silico techniques.

Heart rate, blood pressure (BP) and tail blood volume were recorded from CD1 mice using noninvasive tail plethysmography at varying cage temperatures. To record cardiovascular parameters and internal body temperature in unrestrained animals, we employed radio‐telemetry in Wistar rats. To analyse ECG signals, we began by comparing an artificially intelligent (AI) 1D Convolutional Neural Network beat detection model (in Python‐Keras) with traditional peak detection methods. We used heart rate variability analyses (HRVA) to investigate autonomic nervous system influences with and without a TRPV4 agonist. To characterise TRPV4 channel gating, we used patch‐clamp electrophysiology on PVN neurones from mouse hypothalamic brain slices. Results are given as mean ± SEM; significances were assessed by one‐way ANOVA or Student's t‐tests where appropriate.

Intraperitoneal administration of the selective TRPV4 antagonist GSK 2193874 (300 μg/kg) resulted in an increased temperature evoked vasodilation, which was not observed in controls (n=3, 5 p<0.01). In radio telemetry experiments, the blind AI detected beat frequency significantly correlated (Rho 0.88, p<0.001, n=10) to the human supervised peak detection methods in the physiological range (<800bpm, >400bpm) and was therefore used for the remaining analysis. Intraperitoneal administration of the the TRPV4 agonist GSK 1016790A (50 μg/kg) caused no change in mean HR, but a significant increase in Low Frequency (LF) power, consistent with an effect on the autonomic system. In brain slice experiments on PVN neurones, ion channels that fit the TRPV4 ‘gating profile, for example the slope conductance (58±9 pS), reversal potential (‐8±3 mV), sensitivity to 4αDD and gating kinetics were sensitive to temperature changes (n=4‐6, p<0.05).

Our electrophysiology results illustrate that temperature changes lead to activation of TRPV4‐ like channels on PVN neurones. We show that inhibition of TRPV4 channels has profound effects on thermosensing responses such as vasodilation, further iimplicating its role in thermoregulation and modulation of autonomic outflow from the PVN.

Support or Funding Information

This work was funded by the BBSRC.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Second hand smoke exposure depresses neuronal excitability and enhances 4‐AP sensitive channel function of cardiac vagal neurons in the nucleus ambiguus
Authors: Sun Jun‐Qing , Pan Shi‐Yue , Karey Emma , Chen Chao‐Yin
Published: June 2021

BACKGROUND

Second hand smoke (SHS) is a significant risk factor for cardiac dysfunction including arrhythmias and sudden cardiac death. Previously we showed that environmentally relevant concentrations of SHS reduce heart rate variability (HRV), an index of autonomic function. We also showed that SHS exposure significantly reduces cardiac vagal neuronal excitability in the nucleus ambiguus (NA), the final vagal cardiac output regulating HRV. We further showed that SHS‐induced attenuation of SK channel activity that may serve as a partial compensatory mechanism to the reduction in overall neuronal excitability. Here, we sought to test the hypothesis that SHS exposure, at an environmentally relevant concentration that reduces HRV, depresses neuronal excitability of cardiac vagal neurons in the NA by enhancing 4‐AP sensitive potassium channel function.

METHODS

Adult male C57BL6/J mice underwent open chest surgeries to retrograde label cardiac vagal neurons by applying a fluorescent dye (DiI) to the sinoatrial node. After two weeks of recovery from surgery, mice were randomly assigned to either filtered air (FA) or SHS (3 mg/m3, 6 hr/d, 5 d/wk) for 4‐, 8‐ or 12‐weeks. Whole‐cell current clamp recordings in brainstem slices were performed on anatomically identified cardiac vagal neurons in the NA with fluorescent labeling.

RESULTS

SHS exposure significantly (p < 0.05) reduced spiking response to depolarizing current injections after 4‐ and 12‐weeks – but not 8‐weeks – of exposure. Perfusion with 1 mM of 4‐AP, a voltage‐activated potassium channel blocker, similarly increased spiking response by 5–10 spikes/s across all time points. The 4‐AP induced changes in spiking responses were not different between SHS and FA (p > 0.05). SHS exposure also significantly (p < 0.05) increased voltage and current threshold for action potential generation for all time points tested (4‐, 8‐, and 12‐weeks). 4‐AP significantly lowered the voltage threshold; these effects were significantly (p < 0.05) bigger in the SHS group (−1.2 ± 0.5 mV vs. −3.7 ± 0.4 mV, FA vs. SHS respectively). In contrast, 4‐AP similarly lowered current threshold for action potential generation in both FA and SHS groups (−80 ± 13 pA vs. −103 ± 12 pA, FA vs. SHS, respectively).

CONCLUSIONS

We conclude that environmentally relevant SHS exposure induces time‐dependent changes in neuronal excitability of cardiac vagal neurons. Some of these changes may be the result of increased voltage‐activated potassium channel function.

Support or Funding Information

R01 ES025229; T32 HL086350

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stress Dampening and Anxiolytic Effects of Overexpressing Angiotensin Converting Enzyme 2 in Female Mice
Authors: Cahill Karlena M. , Alleyne Amy R. , Smeltzer Michael D. , Raizada Mohan K. , de Kloet Annette D. , Krause Eric G.
Published: June 2021

Accumulating evidence has revealed intricate roles for a number of components of the renin‐angiotensin system (RAS) in the progression or alleviation of stress‐related disorders. Along these lines, the ‘pro‐stress' actions of Angiotensin‐II are largely thought to be mediated by the angiotensin type‐1 receptor (AT1R). On the other hand, a counter regulatory limb of the RAS that depends on the conversion of Angiotensin‐II to Angiotensin‐(1‐7) by angiotensin‐converting enzyme 2 (ACE2) has been postulated to exert stress‐dampening actions. We have previously found that stimulating this enzyme has potent anxiolytic and hypothalamic‐pituitary‐adrenal (HPA) axis dampening effects in male mice; however, the impact of stimulating this counter regulatory limb on stress‐related responses in females is yet to be explored. That being said, females are often considered to be more susceptible to stress‐related mood disorders, making it essential that novel therapeutic options for females are also uncovered. Therefore, in this set of studies, we tested the hypothesis that ACE2 has anxiolytic and endocrine stress‐dampening effects in female mice. We used the Cre/Lox system to generate female mice that ubiquitously overexpress ACE2. Female ACE2 knock‐in (ACEKI) mice and their wild‐type littermate controls were tested in the elevated‐plus maze (EPM) and open field maze (OFM) to evaluate the impact of the genetic manipulation on anxiety‐like behavior. Mice were then subjected to an acute 30 min restraint stress challenge and blood samples were collected for the evaluation of HPA axis reactivity. As expected, overexpression of ACE2 leads to elevated ACE2 expression and activity in all tissues examined (i.e., hypothalamus, pituitary, blood, and brain). In regards to anxiety‐like behavior, a statistically significant increase (p < 0.05) in time spent in the open arms was observed in the ACE2KI mice relative to controls, demonstrating an anxiolytic effect of ACE2 overexpression. Total distance traveled in the EPM and OFM were not different between the groups, indicating that the locomotor activity of the female mice is unchanged by ACE2 overexpression. Finally, ACE2KI female mice also exhibited significantly reduced HPA axis activity relative to their wild‐type littermate counterparts (p < 0.05). Collectively, the results indicate that augmenting ACE2 activity reduces stress reactivity and anxiety‐like behavior in female mice.

Support or Funding Information

NIH:HL125805 and AHA GRNT33660969 (AdK), NIH: HL122494 (EGK), NIH HL033610 and HL102033 (MKR).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thrombin action on NST astrocytes disrupts glycemic and respiratory control
Authors: Hermann Gerlinda E. , Rogers Richard C.
Published: June 2021

Burns and severe trauma can produce a post‐injury “metabolic self‐destruction” characterized by catabolic metabolism and hyperglycemia. The severity of the hyperglycemia is highly correlated with post‐trauma morbidity and mortality. No mechanism has been posited to connect severe trauma with a loss of autonomic control over metabolism. However, traumatic injury causes other failures of autonomic function; one of those, gastric stasis and ulceration (“Cushing's Ulcer”) has recently been connected with the generation of thrombin. As a protease, thrombin (produced as a consequence of tissue injury) acts on G‐protein‐coupled protease‐activated receptors (PAR). Our previous work established that PAR1 receptors located on astrocytes in the autonomically critical nucleus of the solitary tract (NST) are responsible for modulating gastric control circuit neurons. The NST is at the center of a homeostatic control nexus that receives vast quantities of visceral afferent data from cranial nerve afferents including the vagus. This region of the hindbrain is also outside the “blood‐brain barrier” and is accessible to large molecules from the circulation. The NST uses this neural and chemical information to regulate gastrointestinal, metabolic, cardiorespiratory, endocrine, and behavioral functions. Astrocytes in the hindbrain have been also been implicated as important detectors of low glucose or glucose utilization and when activated, these astrocytes trigger CRR. There may be a convergence between the effects of thrombin to derange hindbrain gastrointestinal control and the hindbrain circuitry that initiates counter‐regulatory responses (CRR) to increase glycemia in reaction to critical hypoglycemia. We tested the hypothesis that administration of thrombin to the floor of the fourth ventricle (4V) or directly into the NST could trigger increases in glycemia through the mediation of astrocytes. Pre‐exposure of the 4V to fluorocitrate (FC; a selective blocker of astrocyte metabotropic signaling) was used to evaluate astrocyte involvement in thrombin‐triggered changes in glycemia. Our results suggest that thrombin acts within the NST to increase glycemia through an astrocyte dependent mechanism. Blockade of purinergic, but not glutamatergic, gliotransmission pathways interrupted the effect of thrombin to increase glycemia. Our studies also revealed that thrombin, acting in the NST, produces a rapid, dramatic and potentially lethal suppression of respiratory rhythm that was also a function of purinergic gliotransmission. These results suggest that the critical connection between traumatic injury and a general collapse of autonomic regulation involves thrombin action on astrocytes.

Support or Funding Information

NIH NIDDK 108765, NIH NINDS 96113

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of the cognitive functions of rat pulps and mothers exposed to environmental enrichment, exercise training or social isolation before and during pregnancy
Authors: Okudan Nilsel , Belviranli Muaz
Published: June 2021

The aim of this study was to examine effects of social isolation, enriched environment and exercise on learning in rats. The study included 36 female Wistar rats. The rats were randomly divided into four different groups; control (n = 6), exercise (n = 10), social isolation (n = 10) and the enriched environment groups (n = 10). The social isolation group and the enriched environment group were housed under their specific conditions and the exercise group and the control group were housed in standard conditions during 6 weeks. The rats in the exercise group swam for 60 min per day, 5 days a week for 6 weeks. After 6 weeks, the rats were evaluated in the Morris water maze. At the end of 6 weeks, brain and blood samples were taken from the rats under anesthesia and the hippocampus tissue was dissected. It was frozen rapidly with liquid nitrogen and stored until use at −80 ° C for biochemical analysis. BDNF and NGF levels were measured in these samples. In conlusion, while enriched environment was a positive effect on spatial learning, social isolation was a negative effect on spatial learning and increase thigmotactic behaviors. According to the analysis results NGF and BDNF levels in the hippocampus and plasma did not change with environmental conditions and exercise. Time of exposure to social isolation, procedures of the enriched environment, time of exposure to the environment, type and duration of exercise and gender may affect the results.

Support or Funding Information

This work was supported by the Selcuk University Scientific Research and Project Coordinatorship (project number: 14401089).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neuroprotective signaling mechanisms of telomerase in neuronal cells against oxidative stress
Authors: Park Sookyoung , Hong Yonggeun
Published: June 2021

Canonically, telomerase was viewed as an enzyme with only one action: maintenance and extension of telomeres to prevent cellular senescence/aging. Novel roles for telomerase, particularly its catalytic subunit telomerase reverse transcriptase (TERT), in normal brain function and protection of CNS cells have recently emerged. However, little is known about whether telomerase also exerts neuroprotective effects in neuronal cells against oxidative stress and how its activity may be modulated by known neuroprotective agents such as estrogen (17β‐estradiol, E2). Human origin SH‐SY5Y neuroblastoma cells were differentiated into cholinergic phenotype by 10 μM retinoic acid over 6 days following, then E2 and hydrogen peroxide were treated respectively. E2 treatment reduced apoptotic changes including Bax:Bcl‐2 ratio and apoptosis‐related proteases caspase‐3, as well as neuronal cell death induced by oxidative stress. In addition, E2 treatment enhanced TERT expression and activity in neuronal cells. To characterize changes in NF‐κB and I‐κB expression/translocation, their protein levels were compared in cytosolic/nuclear fractions. The change in NF‐κB:I‐κB ratio was shown in neuronal cells following oxidative stress, then E2 attenuated NF‐κB transcriptional activity in neuronal cells. To determine whether TERT is required for the neuroprotective effects of estrogen in neuronal cells against oxidative stress, hTERT siRNA was employed. Specific knockdown of hTERT in E2‐treated cells showed significant apoptotic changes following oxidative stress, indicating cytoprotective effects of estrogen was lost in cells treated with hTERT knockdown. Taken together, these findings suggest that TERT is an essential mediator of estrogen neuroprotection.

Support or Funding Information

NRF‐2014R1A1A3051724 to S.P., NRF‐2017R1A2A2A01067169 to Y.H., KGM4611714 to Y.H., Korea.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
5‐HT2B Receptor Expression in the Phrenic Motor Nucleus of Rats with Intrapleural CTB‐Saporin‐Induced Phrenic Motor Neuron Death
Authors: Borkowski Lauren F. , Nichols Nicole L.
Published: June 2021

Intrapleural injection of cholera toxin B fragment conjugated to saporin (CTB‐SAP) selectively kills respiratory motor neurons, and mimics aspects of neuromuscular disorders and neurodegenerative diseases including phrenic motor neuron death and decreased phrenic motor output. In this model, respiratory plasticity (acute intermittent hypoxia induced phrenic long‐term facilitation; pLTF) is enhanced following 7 days (d) of CTB‐SAP compared to control treatment. In contrast, pLTF in 28d CTB‐SAP treated rats is comparable to pLTF in controls, suggesting that the underlying mechanisms of plasticity at both time points may differ. pLTF in naïve rats predominately requires 5‐HT2 receptors; however, there is an alternative mechanism for pLTF that requires A2A receptors. Our preliminary data suggest that A2A receptors are increased in the phrenic motor nucleus of 7d, not 28d, CTB‐SAP treated rats (Craig et al., 2017), suggesting that 7d CTB‐SAP rats utilize the alternative mechanism for enhanced pLTF. However, it remains unknown how CTB‐SAP induced phrenic motor neuron death affects 5‐HT2A/B receptor expression, and if 5‐HT2A/B receptors are utilized for pLTF in CTB‐SAP treated rats. Here, we tested the hypothesis that 5‐HT2B receptor expression in the phrenic motor nucleus is decreased in 7d CTB‐SAP treated rats, but is not different in 28d CTB‐SAP treated rats when compared to controls using immunohistochemistry. 5‐HT2B receptor expression was studied in Sprague Dawley rats 7 and 28 days following bilateral intrapleural injections of: 1) CTB‐SAP (25μg), or 2) un‐conjugated CTB and SAP (control). Our preliminary data indicate that following CTB‐SAP injection, total 5‐HT2B receptor positive pixels and C4 fractional area are decreased in the phrenic motor nucleus and non‐phrenic ventral horn at 7d, not 28d, vs. controls (n=3 for each group; p<0.05). This suggests 5‐HT2B receptors recover to control levels and are potentially utilized for pLTF in 28d CTB‐SAP treated rats. Future studies will be focused on whether 5‐HT2A receptor expression is similarly affected, whether 5‐HT2A/B receptor expression is impacted on other cell types (i.e., glial cells), and which 5‐HT receptor isoform is required for pLTF following CTB‐SAP‐induced phrenic motor neuron death. This project increases our understanding of the expression of receptors associated with respiratory plasticity and its implications for breathing following motor neuron death.

Support or Funding Information

Supported by NIH K99/R00 HL119606

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Extracellular Vesicles: An Additional Compartment for the Second Messenger, cyclic Adenosine Monophosphate (cAMP)
Authors: Sayner Sarah L. , Maulucci Marcy , Scruggs April , Zhou Chun , Bauer Natalie
Published: June 2021

Rationale

Cyclic AMP is generated by adenylyl cyclase to initiate intracellular signaling leading to diverse cellular and tissue responses. Compartmentalization of the cAMP signal within the cellular environment is critical to maintain signaling specificity; however, cAMP is also released into the surrounding extracellular space. Many cell types release extracellular vesicles that encapsulate a variety of functional proteins and RNAs/miRNAs; however, little is known about whether they encapsulate signaling molecules, such as cyclic adenosine monophosphate (cAMP), which could contribute to the extracellular cAMP pool.

Methods

To test the hypothesis that endothelial cells release extracellular vesicles containing cAMP, media was collected from unstimulated aortic and pulmonary endothelial cells and subject to low‐speed and subsequently high‐speed centrifugation. To determine whether cAMP in these extracellular vesicles increased upon cell stimulation, pulmonary microvascular endothelial cells (PMVEC) were stimulated over time to increase cellular cAMP and extracellular vesicles analyzed for cAMP levels. Finally, isoproterenol and rolipram was perfused through the isolated rat lung to determine whether extracellular vesicles isolated from the intact organ also contain cAMP.

Results

While cAMP was detected in extracellular vesicles derived from various endothelial cell types, cAMP was higher in PMVECs compared to systemic endothelial cells (aorta) or endothelial cells from pulmonary conduit vessels, pulmonary artery endothelial cells. Further, stimulation of PMVECs with agents that increase near membrane cAMP (isoproterenol or forskolin) in the presence of the phosphodiesterase inhibitor, rolipram, led to an increase in cAMP in extracellular vesicles over time (upto 60 minutes). In PMVECs, cell lysates showed a maximum increase in cAMP 15 minutes after treatment, while the maximum increase in extracellular vesicle‐cAMP was not observed until 60 minutes after treatment. Extracellular vesicles from the perfusate of isoproterenol and rolipram stimulated isolated lungs contained elevated cAMP compared to unstimulated controls.

Conclusion

Extracellular vesicles released from both the systemic as well as pulmonary vasculature contain cAMP, and cAMP within extracellular vesicles can be increased upon cell stimulation. While cAMP is rapidly packaged into extracellular vesicles within 5‐minute of cell stimulation, the maximal increase is delayed. Isolated lungs can also be stimulated to release extracellular vesicles with increased cAMP. Thus, extracellular cAMP while already accepted to be free in the cytosol is also encapsulated within extracellular vesicles.

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