bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypothalamic Corticotrophin Releasing Hormone Signal Contributes to Elevated Sympathetic Outflow in primary hypertension
Authors: Zhang Hua , Zhou Jing‐Jing , Shao Jian‐Ying , Sheng Zhaofu , Zheng Peiru , Li De‐Pei
Published: May 2022

Corticotropin‐releasing hormone (CRH) is a neuropeptide involved in regulating neuroendocrine and autonomic function. CHR mRNA and protein expression in the hypothalamic paraventricular nucleus (PVN) are significantly increased in patients with primary hypertension. However, the role of CRH signals in elevated sympathetic outflow in primary remains unclear. Here, we showed that CRH receptor 1 (CRHR1) protein level rats was significantly increased in the PVN tissue in spontaneously hypertensive (SHR) compared with Wistar‐Kyoto (WKY). Upregulated CRHR1 level in the PVN was not significantly affected by lowing arterial blood pressure (ABP) by celiac ganglionectomy in SHRs. Immunocytochemistry staining revealed that CRHR1 immunoreactivities were in PVN neurons projecting to the rostral ventrolateral medulla (RVLM). Bath application of CRH induced a significantly greater increase in the firing rate of PVN‐RVLM projection neurons in SHRs than in WKY rats. Blocking CRHRs with astressin or CRHR1 with antalarmin significantly decreased firing rate of PVN‐RVLM projection neurons in SHR but not in WKY, while blocking CRHR2 with antisauvagine‐30 did not alter the firing rate of these neurons in SHR. Both antalarmin and antisauvagine‐30 did not change the frequency and amplitude of miniature inhibitory post‐synaptic currents or miniature excitatory post‐synaptic currents recorded in brain slice preparation of SHR and WKY rats. In addition, bilateral microinjection of CRH into the PVN produced a greater dose‐dependent increases in arterial blood pressure (ABP), renal sympathetic activity (RSNA), and heart rate (HR) in anesthetized SHRs than in WKY rats. Microinjection of CRHR1 antagonist NBI35965 into the PVN significantly reduced basal ABP, RSNA, and HR in SHRs but did not change ABP, RSNA, and HR in WKY rats. These data suggested that the upregulation of CRHR1 in the PVN is critically involved in elevated sympathetic outflow and hyperactivity of sympathetic‐related neurons in hypothalamus in primary hypertension.
Kidney‐Specific BMAL1 Knockout is Protective in High Salt Diet‐Induced Renal Inflammation
Authors: Crislip Gene R. , Costello Hannah M. , Juffre Alexandria , Cheng Kit‐Yan , Wingo Charles S. , Scindia Yogesh M. , Gumz Michelle L.
Published: May 2022

The circadian clock factor BMAL1, particularly within the kidney, has been shown to influence systemic blood pressure (BP) control. A diet high in salt is linked to immune cell infiltration in the kidney and development of hypertension. Previously, we generated renal distal segment BMAL1 knockout mice (KS‐BMAL1 KO) using Ksp‐cadherin Cre. Male KS‐BMAL1 KO exhibit lower BP than control floxed BMAL1 Cre negative mice (CNTL). In this study, we tested the effect of a low potassium, high salt (0KHS; 4% NaCl) diet in order to determine if KS‐BMAL1 KO are protected from salt‐sensitive hypertension. The goal of this study was to test the hypothesis that male KS‐BMAL1 KO display reduced BP and a lower renal inflammatory phenotype than CNTL in response to a 0KHS diet. BP was measured via telemeter implants (N=7‐8). Panels of inflammatory and injury markers were measured using Millipore multiplex immunoassay kits in renal cortical and medullary tissue from KS‐BMAL1 KO and CNTL on a normal diet and following 10 days of 0KHS diet (N=6‐7). Additionally, whole kidney tissue from mice treated with 0KHS for 10 days was digested and flow cytometric analysis was performed (N=5‐6). KS‐BMAL KO did not exhibit the increase in BP as seen in control mice following 0KHS (CNTL: 125±2 to 132±1; KO: 123±2 to 122±2 mmHg; Interaction P<0.05). CNTL had a greater increase in cortical Kidney Injury Molecule‐1 levels following 0KHS than KS‐BMAL1 KO (CNTL: 31±5 to 975±103; KO: 20±6 to 651±61 pg/mg; Interaction P=0.02). Additionally, CNTL displayed a greater increase in cortical interleukin (IL) 6 following 0KHS than CNTL (CNTL: 0.5±0.04 to 9±1; KO: 0.4±0.07 to 5±0.9 pg/mg; Interaction P=0.04). Medullary IL6 levels were more than 15 times greater following 0KHS but no genotype effect was seen. Although cortical interferon gamma (IFNγ) levels increased more than 8‐fold following 0KHS, there were no differences between genotypes. However, CNTL had a greater increase in cortical IL17 following 0KHS than CNTL (CNTL: 0.8±0.03 to 2.5±0.2; KO: 0.8±0.04 to 1.9±0.1 pg/mg; Interaction P=0.01). CNTL also had greater medullary IL17. Furthermore, CNTL had nearly 2 times greater CD45+ total leukocytes and neutrophils than KS‐BMAL1 KO following 0KHS. BMAL1 in renal distal segments contributes to BP regulation. Additionally, KS‐BMAL1 KO exhibit a reduced proinflammatory phenotype following 0KHS compared to CNTL. These data suggest that BMAL1 may play a role in the renal inflammatory response to 0KHS. Whether BMAL1 protects against inflammation because of or in addition to the lower BP phenotype remains to be determined.
Intermittent Cocaine Self‐Administration but not Passive Cocaine Infusions Reduces H‐Current in Putative Dopaminergic Neurons of the Ventral Tegmental Area
Authors: Vélez‐Acevedo Omaris , Calo‐Guadalupe Cristhian , Bosque‐Cordero Karl , Consuegra‐García Daisy , Vázquez‐Torres Rafael , Jiménez‐Rivera Carlos
Published: May 2022

Intermittent Access (IntA) cocaine self‐administration is a protocol suggested to better simulate human drug use patterns due to its temporal dynamics of drug administration. IntA is also known to produce incentive salience and psychomotor sensitization. Dopaminergic (DA) neurons display a prominent mixed cation current conductance known as the hyperpolarization‐activated cyclic nucleotide current, or Ih, which contributes to neural processes such as resting membrane potential, firing frequency modulation, and synaptic integration. Previous results from our laboratory demonstrated that Ih amplitude is reduced significantly after cocaine sensitization. This Ih reduction resulted in an increased temporal summation, mean depolarization, and excitatory postsynaptic potential (EPSP) amplitude, all factors related to an enhanced excitability state. Since the cocaine sensitization model involves noncontingent drug injections administered by the experimenter, it is crucial to determine if electrophysiological changes in ventral tegmental area (VTA) DA cells are present when drugs are self‐administered (contingent). In the present study, we explored if DA neurons present an alteration in Ih after exposure to IntA. We hypothesize that VTA DA cells’ Ih modulation is dependent on the associative learning processes acquired during operant conditioning. Using the whole‐cell patch‐clamp technique in brain slices, we investigated the effects of cocaine IntA, and passive cocaine infusions (yoked controls) on Ihamplitude and rebound excitability. Our results demonstrate that an IntA protocol, but not passive cocaine infusions, produces a significant Ih amplitude reduction (P<0.05). No differences in rebound action potentials (APs) were observed. A depolarizing current protocol showed a significant increase in the number of APs (P<0.05). These results suggest that Ih modulation and intrinsic activity regulation are dependent on associative learning to drug cues.
Sex‐specific colonic mitochondrial dysfunction in the indomethacin‐induced inflammatory bowel disease model in rats
Authors: Hoang Ngoc , Brooks Karen , Edwards Kristin S.
Published: May 2022

Inflammatory bowel disease (IBD) characterized by inflammation of the gastrointestinal tract. It includes Crohn’s disease and ulcerative colitis. In 2015, 3 million U.S. adults have been diagnosed with IBD and many people go undiagnosed due to mild symptoms. Women appear to have more severe and recurring symptoms of IBD compared to men, most likely due to hormonal fluctuations. Several studies show mitochondrial alterations in IBD patients. Our hypothesis is mitochondrial dysfunction in IBD could have a sex‐specific effect. Male and female rats received two doses of indomethacin (7.5 mg/kg) 24 hour apart to induce IBD. Colon tissues were collected for mitochondrial isolation two days after injections. Mitochondrial respiration and reactive oxygen species (mtROS) production were measured simultaneously by an Oroboros Fluorespirometer using glutamate/malate, succinate, oleate (long‐chain fatty acid; LCFA), or octanoate (medium chain fatty acid; MCFA). Complex IV activity, a marker of oxidative phosphorylation capacity, was measured. Data were normalized to mitochondrial content using citrate synthase (CS) activity, which confirms that the observed changes are not due to loss of mitochondrial content. CS activity showed no change in females but a significant decrease in male IBD rats compared to controls (2425 ± 200.2 vs. 3586 ± 214.4 nmol citrate/min/g tissue, p<0.05). When respiration was normalized to CS activity, males showed no significant difference compared to controls with complex I and complex II‐driven respiration. However, IBD females showed a significant decrease in Complex I‐driven respiration (0.4258 ± 0.02903 vs 0.5437 ± 0.03939 nmol e‐/nmol citrate, p<0.5) and Complex II ‐driven respiration (0.7489 ± 0.035118 vs. 0.9029 ± 0.04664 nmol e‐/nmol citrate, p<0.05). For LCFA oxidation, males (0.6912 ± 0.07261 vs. 0.2554 ± 0.01185 nmol e‐/nmol citrate, p<0.05) and females (0.3984 ± 0.03192 vs. 0.2389 ± 0.02243 nmol e‐/nmol citrate, p<0.05) showed a significant increase in activity compared to controls. Both males and females showed no significant decrease in LCFA or MCFA oxidation. Males and females both showed a significant increase in mtROS with all substrates. Complex IV activity was not different than controls in males but female activity was significantly decreased (1.099 ± 0.09693 vs. 1.379 ± 0.05922 nmol e‐/nmol citrate, p<0.05). Alterations to mitochondrial function and mitochondrial content observed in the indomethacin‐induced IBD rat model suggests a link between mitochondrial dysfunction and IBD. There are differences in male and female rats suggesting a potential mechanism for the observed differences in male and female patients. Our study provides a better understanding of the role mitochondria in the development of IBD. It also opens an avenue for the development of strategies to re‐establish normal mitochondrial function that could provide more options for preventive and therapeutic interventions for IBD.
Circulating human sPRR increased blood pressure in female mice fed a low‐fat diet
Authors: Nichols Kellea , Poupeau Audrey , Arthur Gertrude , Yiannikouris Frederique
Published: May 2022

Essential hypertension and obesity‐hypertension have been associated with elevated plasma soluble prorenin receptor (sPRR) in men. Additionally, our laboratory previously found that the infusion of mouse sPRR elevates systolic blood pressure (SBP) in high‐fat (HF) fed male mice through activation of the sympathetic nervous system but did not elevate SBP in HF‐fed female mice. Interestingly, the infusion of mouse sPRR increased renal and hepatic angiotensinogen (AGT) and plasma renin concentration in female mice fed a standard diet. However, the mechanistic role of sPRR on the renin angiotensin system (RAS) to regulate blood pressure in lean female mice remains to be investigated. Moreover, there is critical gap concerning the functional role of human sPRR on blood pressure in women. Therefore, in the present study, we aim to determine whether human sPRR participates to blood pressure control in lean female mice.

A transgenic mouse model expressing the human form of the soluble prorenin receptor (HsPRR‐Myc‐tag) was developed. Human sPRR‐transgenic female mice were bred with male mice expressing Alb/Cre recombinase to generate mice selectively expressing human sPRR (Alb‐HsPRR) in the liver and control littermates (CTL). Control and Alb‐HsPRR female mice were fed a LF‐diet for 8 months (n=11/groups). Body weight was assessed weekly and body composition was examined monthly. Blood pressure was assessed by radiotelemetry. Human sPRR‐Myc‐tag was detected in the liver of Alb‐HsPRR female mice and plasma sPRR levels increased by 50‐fold (CTL: 3.6±0.5 ng/ml, Alb‐HsPRR: 190.5±24.4 ng/ml; P<0.05), which validated the humanized mouse model.

Elevated circulating human sPRR did not change body weight (CTL: 22.2±0.4g, Alb‐HsPRR: 23.0±0.3g) or fat mass (CTL: 2.5±0.2, Alb‐HsPRR:3.1±0.2g). Liver‐derived human sPRR significantly elevated SBP in Alb‐HsPRR compared to control female mice (Night SBP: CTL, 130.5±1.2 mmHg; Alb‐HsPRR, 135.9±2 mmHg; P<0.05) and acute injection of Angiotensin II exacerbated the SBP elevation. Interestingly, the decrease in blood pressure mediated by Losartan was not different between Alb‐HsPRR and control female mice (Night □SBP: CTL, ‐13.1±2.2 mmHg; Alb‐HsPRR, ‐14.8±2.7 mmHg; P>0.05). Plasma AGT and renin activity were similar between Alb‐HsPRR and control littermate female mice suggesting that the elevation of SBP was not mediated by the circulating renin angiotensin system. Therefore, whether the sympathetic nervous system is involved in human sPRR‐mediated increase of SBP remains to be examined. Altogether, these results support an important role of circulating human sPRR in blood pressure control in women.
Identifying Therapeutic Inhibitors of TDP43 Phase‐Separation
Authors: Rubien Jack , Lavorando Ellen , Padilla George , Shorter James
Published: May 2022

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease for which there has been little improvement in treatment over the past 25 years. ALS is linked to mutations in dozens of genes, but despite its genetic complexity, 97% of patients share a distinct molecular pathology: cytoplasmic inclusions of TAR DNA‐Binding protein 43 (TDP‐43). Further, TDP‐43 aggregates are toxic to healthy neurons, suggesting that TDP‐43 aggregates contribute to neurodegeneration in ALS. Due to the near‐ubiquity of TDP‐43 inclusions in ALS patients and their contribution to ALS pathology, prevention or dissolution of these TDP‐43 inclusions presents a promising therapeutic strategy. In cells, TDP43 can form both solid aggregates or liquid droplets, through liquid‐liquid phase separation (LLPS). Here, we screened a library of small molecules and RNAs to identify inhibitors of TDP‐43 LLPS using an in vitro assay which quantifies TDP‐43 liquid droplet formation. Hits from this screen were further characterized based on their ability to reverse and inhibit both TDP‐43 droplets and aggregates to obtain functional profiles for each promising inhibitor. Pairing functional data of inhibitors with structural information regarding their binding to TDP‐43 enables a close‐dissection of the interactions that drive TDP‐43 self‐assembly. Thus, small molecule screens which target protein self‐assembly present a powerful approach for both developing novel therapeutics and identifying the interactions that govern self‐assembly; these inform each other and may lead to the discovery of novel therapeutic agents which target protein self‐assembly to treat severe diseases, like ALS.
Uncovering the role of neuropeptide receptor GPR83 in nociception
Authors: Hanlon Jack P. , Young Ryan E. , Devi Lakshmi A. , Fakira Amanda K.
Published: May 2022

Nociception, the perception of noxious stimuli, is a critical function for the sensation of pain that protects us from damaging ourselves and helps protect the body following an injury. While pain is important, many people suffer from chronic pain due to a dysregulation of the mechanisms that detect noxious stimuli. G‐protein coupled receptors (GPCRs) are critical regulators of cell function making them frequent targets of therapeutic agents. The recent identification of the neuropeptide PEN as an endogenous ligand for the GPCR, GPR83, opens the possibility of exploring the neurobiological function of this receptor. Recent studies have provided evidence that GPR83 is expressed in a specific ascending spinal tract and that these neurons are highly sensitive to mechanical stimuli. New tools to study GPR83 include small molecule agonists and antagonists. Using these compounds, we have identified that GPR83 agonists blunt morphine antinociception while antagonists enhance morphine antinociception. Since morphine is the most effective therapeutic for pain this suggests that GPR83 is involved in the pain pathway however, it is unknown whether there is a direct impact of GPR83 on nociception. Moreover, the potential of targeting GPR83 to alleviate chronic pain has not been investigated. We have preliminary data suggesting that GPR83 antagonists increase sensitivity to mechanical stimuli while having no impact on thermal stimuli. Our study aims to further characterize GPR83’s role in nociception and to identify whether this receptor has any direct impact on pain itself, using the Complete Freud’s Adjuvant (CFA) model of inflammatory pain.
Trimethylamine N‐oxide might impair cholinergic system by affecting the structural integrity of Acetylcholinesterase
Authors: Kumari Kritika , Singh Laishram R.
Published: May 2022

Trimethylamine N‐oxide (TMAO), in humans, is obtained from gut microbiota and is known to accumulate in human serum as well as cerebrospinal fluid at high levels. There are several reports for the involvement of serum TMAO in various human diseases including, atherosclerosis, neurodegeneration, cancer etc. One important aspect in neurodegeneration is it's strong association with memory loss. However, the exact molecular mechanism is not yet explored. Keeping in mind that impairment in memory or onset of Alzheimer's is due to decline in cholinergic system of the brain, we thought TMAO could have potentially targeted the cholinergic system. Acetylcholinesterase (AChE) is an important enzyme responsible for the breakdown of neurotransmitter, acetylcholine and therefore plays a crucial role in neuronal plasticity. Recently, our laboratory discovered that TMAO inhibits folding of proteins specifically that are rich in proline content. Since AChE is also a protein characterized by the presence of large number of proline residues, we thought it is worthwhile to investigate if TMAO inhibits it's activity. In the present article we investigated the effect of TMAO on the structure and functional activity of AChE. We explored that TMAO is a specific inhibitor of AChE. The results indicate that TMAO might induce decline in the cholinergic system by impairing the function of AChE.
Fluoropyrimidine Bioactivation and Metabolism by the Gut Microbiome
Authors: Guthrie Benjamin G. H. , Turnbaugh Peter J.
Published: May 2022

Despite decades of research into why populations of cancer patients differentially respond to the same treatment, variation in response to therapy regimens remains a significant challenge in the oncology field. However, our understanding of the causal mechanisms of interpatient variability remains lacking; up to 35% of patients require dose adjustments and 10% of patients are removed from the fluoropyrimidine capecitabine altogether. This research seeks to further our understanding of the microbiome’s role in fluoropyrimidine treatment by identifying gut bacterial strains responsible for capecitabine activation as well as biochemically characterizing drug inactivating enzymes from the human gut microbiome. Growth curve data show that knockouts of bacterial enzymes homologous to mammalian enzymes known to be involved in capecitabine metabolism increase bacterial resistance to capecitabine. Additionally, an unbiased transposon mutagenesis screen identifies enzymes known to increase resistance as well as enzymes both within and outside of nucleotide metabolism. These two pieces of data suggest that bacteria have the capacity to metabolize capecitabine, upending the paradigm that activation of this prodrug happens exclusively in the liver and tumor. Confirmation of these metabolites with high resolution triple quadrupole linear ion trap mass spectrometry is ongoing. Separately, previous studies indicate that gut bacteria are capable of degrading 5‐fluorouracil, however it is not clear how efficient these pathways are or to what extent they occur in the host. Enzyme kinetics of homologues of this enzyme within the microbiome show that degradative reaction for uracil is essentially irreversible. Additionally, this enzymatic characterization has identified the non‐enzymatic enantiomer of the degraded fluoropyrimidine as a potential inhibitor for this enzyme, which might allow for co‐dosing and inhibition of bacterial metabolism. Metagenomic sequencing of the degradative enzymes within patient samples is ongoing and will potentially reveal the main drivers of this activity in patients. These avenues of research will uncover new bacterial metabolism pathways of xenobiotics, compare these metabolic pathways between microbiota and host, and have the potential to lead to new advances in disrupting microbial metabolism of chemotherapeutics.
Supraphysiological Levels of Testosterone Induce Cardiac Dysfunction via NLRP3 Inflammasome Activation in Macrophages
Authors: Alves Juliano V. , Costa Rafael M. , Omoto Ana C. M. , Oliveira‐Neto Jose T. , Silva Josiane F. , Machado Mirele R. , Rosa Ualter G. , Bonato Vania L. D. , Tostes Rita C. A.
Published: May 2022

Introduction

Testosterone (Testo) modulates vascular tone and cardiac performance. Athletes who use Testo at supraphysiological doses exhibit increased blood pressure, higher inflammatory marker levels, vascular dysfunction, and cardiac hypertrophy. NLRP3 inflammasome activation, as part of the innate immune system response, contributes to proinflammatory cytokines production, leading e.g to cardiac dysfunction.

Hypothesis

We hypothesized that supraphysiological levels of Testo promotes cardiac dysfunction via generation of mitochondrial reactive oxygen species (mROS) and activation of the NLRP3 inflammasome.

Methods

Male, 12 week‐old wild type (WT) and NLRP3 knockout (NLRP3‐/‐) mice were used. Mice were treated with testosterone propionate [Testo‐P (10 mg/kg)] or vehicle for 30 days. Cardiac function was evaluated using echocardiography. After in vivo experiments, western blot and ELISA were performed to evaluate NLRP3 inflammasome components. ROS generation was evaluated by lucigenin. Bone marrow‐derived macrophages (BMDMs) were isolated, primed with lipopolysaccharide (LPS [500 ng/mL 4 h]) and stimulated with Testo [10‐7 M], for 4, 6, 12 and 24 h. ROS generation was evaluated by DHE fluorescence and MitoSOX.

Results

Echocardiography showed cardiac dysfunction in WT mice treated with Testo‐P, characterized by reduced ejection fraction, shortening fraction, cardiac output and systolic volume. Testo‐P also increased interventricular septum, left ventricle posterior wall and decreased left ventricle internal diameter. All these effects were observed in NLRP3‐/‐mice. Furthermore, WT mice treated with Testo‐P showed increased cardiac expression of NLRP3 receptor, active Caspase‐1 and IL‐1β levels. These effects were prevented in NLRP3‐/‐. In addition, increased ROS generation was observed in the left ventricle of WT mice, but not NLRP3‐/‐ mice, treated with TP. Testo‐P‐treated WT mice showed increased macrophage infiltrate in the left ventricle, and this effect was not seen in the WT vehicle mice. In in vitro experiments, BMDMs primed with LPS and stimulated with Testo showed increased expression of the NLRP3 receptor and IL‐1β levels after 12 and 24 h. In addition, Testo‐stimulated BMDMs exhibited increased mROS generation.

Conclusion

Supraphysiological levels of testosterone induce cardiac dysfunction via mROS generation and NLRP3 inflammasome activation. This study was approved by the Ethics Committee on Animal Experimentation of the Ribeirao Preto Medical School (020/2021).
Dose dependent effects of green tea extracts in the skeletal development of a Down syndrome mouse model
Authors: Martínez Abadías Neus , Llambrich Sergi , González Rubèn , Albaigès Julia M. , Wouters Jens , Roldán Jorge , Salassa Sara , Van Bulck Vicky , Wouters Kaat , Callaerts‐Vegh Zsuzsanna , Sharpe James , Dierssen Mara , Vande Velde Greetje
Published: May 2022

Down syndrome (DS) is a congenital disorder caused by trisomy of chromosome 21. The genetic imbalance results in intellectual disability, but also alters the development of different body systems, including the cranial and postcranial skeleton. To normalize altered development and to rescue skeletal phenotypes, green tea extracts enriched in EGCG (GTE‐EGCG) have been proposed for therapy. In this study, we longitudinally followed the skeletal postnatal development in a mouse model for DS, Ts65Dn, and assessed the effects of both a high dose and a low dose of pre‐ and postnatal GTE‐EGCG treatment. We administered a green tea extract containing 45% EGCG at doses of 100 or 30 mg/kg/day to pregnant Ts65Dn females, starting at embryonic day 9 and continued until adulthood. We longitudinally scanned wild‐type and Ts65Dn mice at postnatal days 3, 14 and 29 using in vivo micro‐computed tomography (µCT).

Quantitative morphometric analyses showed that TS mice have a significantly different skull and mandibular shape than WT mice, as well as reduced bone mineral density (BMD) of the premaxilla and humerus bones that were detected already after birth. Moreover, Ts65Dn mice presented shorter faces, humerus and scapula, with significant differences appearing later in development, at the juvenile stage. Treatment with GTE‐EGCG generally deviated both WT and TS mice from normal growth trajectories, exacerbating some morphological anomalies, such as skull brachycephaly and reduced bone dimensions and mineral density. However, the effects of pre‐ and postnatal GTE‐EGCG administration were dose‐dependent, and low dose GTE‐EGCG treatment presented rescuing effects in facial shape. Our results underscore the complex nature of the DS phenotype and the need of further research to characterize the diverse effects of GTE‐EGCG treatment on different systems and structures and to fine‐tune dose and timing to achieve optimal safety and therapeutic potential.
Neutrophil Extracellular Traps Contribute to Perivascular Adipose Tissue Dysfunction in High‐Fat Diet Obese Mice
Authors: Costa Rafael M. , Neves Karla , Lopes Rhéure , Alves Juliano , Fedoce Aline , Silva Josiane , Rodrigues Daniel , Prado Douglas , Schnaider Ayda , Cunha Fernando , Alves José Carlos , Tostes Rita
Published: May 2022

Background

Obesity triggers functional changes in the perivascular adipose tissue (PVAT), favoring the release of vasoconstrictor factors and the activation of contractile mechanisms in vascular smooth muscle cells. In the PVAT of obese individuals, there is infiltration of immune cells, including neutrophils. A neutrophil defense mechanism is the formation of neutrophil extracellular traps (NETs), a cytotoxic framework that allows maintenance and expansion of inflammatory processes. We hypothesize that PVAT dysfunction in obese mice is due to increased neutrophil infiltration, NETs formation and inflammation.

Methods

Six weeks‐old male Balb/C mice received control (CD) or high fat diet (HFD) for 18 weeks. Vascular function was assessed in endothelium‐intact mesenteric arteries in the presence or absence of PVAT. Mice were treated with DNAse type 1 (10 mg/kg for 14 days, every other day). Molecular mechanisms were investigated. Experiments were approved by the Ethics Committee on Animal Research of the Ribeirao Preto Medical School, University of Sao Paulo (protocol nº 209/2016)

Results

Values are expressed relatively to the contraction triggered by 120 mM KCl [Emax (% KCl)]. In Balb/C mice, the PVAT decreased the contractile response in control arteries [Control PVAT (‐): 123.2±8.1 n=6; Control PVAT (+): 64.4±4.0 n=6]. However, HFD promoted partial loss of PVAT anti‐contractile effect [HFD PVAT (‐): 127.9±6.9 n=6; HFD PVAT (+): 98.3±7.4 n=6]. In PVAT of obese mice there was a greater amount of neutrophils, compared to PVAT from control mice [Control: 8.0% n=5; HFD: 18.9% n=5]. In PVAT of obese mice there was greater formation of NETs, and production of interleukin 6 (IL‐6), compared to PVAT from control mice [NETs in pg/ml – Control: 30.5±2.7 n=6; HFD: 71.4±3.2 n=6; IL‐6 in pg/100 mg of PVAT – Control: 45.6±1.9 n=6; HFD: 98.4±3.1 n=6]. DNAse treatment reduced the formation of NETs and IL‐6 in the PVAT of obese mice [NETs in pg/ml – HFD+DNAse: 18.3±1.2 n=6; IL‐6 in pg/100 mg of PVAT – HFD: 48.1±3.4 n=6]. DNAse treatment restored PVAT anticontractile function in obese mice [HFD+DNAse PVAT (+): 71.3±7.4 n=6]. Adipose tissue of obese humans exhibited greater formation of NETs and production of IL‐6 compared to adipose tissue of eutrophic humans [NETs in pg/ml – Eutrophic: 20.5±3.7 n=7; Obese: 79.2±1.3 n=7; IL‐6 in pg/100 mg of adipose tissue – Eutrophic: 9.7±2.9 n=7; Obese: 27.7±2.6 n=7].

Conclusion

These results indicate that, in obesity, there is a greater neutrophils infiltration in the PVAT and, consequently, greater formation of NETs and inflammation, events that contribute to PVAT dysfunction.
FTIR Spectroscopy Reveals Molecular Changes in BPC 157 Treated Blood Vessels
Authors: Gamulin Ozren , Krajacic Maria , Oroz Katarina , Coric Luka , Dretar Vilim , Strbe Sanja , Seiwerth Sven , Sikiric Predrag
Published: May 2022

From the rats treated with stable gastric pentadecapeptide BPC 157 (10ng/kg ip) or saline (5ml/kg ip) at 5 min before sacrifice, the spectra of thoracic aorta tissue samples were compared. From each rat, 20 samples were cut and FTIR spectra were recorded.

Vibrational spectra of the samples were recorded with a Perkin‐Elmer Spectrum GX spectrometer equipped with liquid N2 refrigerated Mercury Cadmium Telluride (MCT) detector. Data were acquired in 450 ‐ 4000 cm‐1 spectral range, in transmission mode with a resolution of 4 cm‐1.

First, all recorded spectra were baseline corrected and normalized. Then we utilized the principal component analysis (PCA) to examine the possibility of separating BPC treated and control samples on a PCA score‐score graph. Using 100 spectra of BPC treated and 100 spectra of control samples, a PCA model was made. As seen in Figure 1, there is a clear distinction, mostly due to the PC1 score, between spectra of BPC treated (red) from the control group (green).

To identify the parts of FTIR spectra that contributed the most to the separation between BPC treated and the control group of spectra differential spectrum was calculated and compared with PC1 loadings.

Differential spectrum and PC loadings show that main bands responsible for separation of the BPC treated and control sample spectra are in the range from 930‐1200 cm‐1, then vibrational bands at 1660 cm‐1,1730 cm‐1, 1120 cm‐1 and in the range from 2840 – 3090 cm‐1. In the first range, there are three vibrational bands at 968 cm‐1, 1087 cm‐1 and 1240 cm‐1 that belong to P‐O symmetric and asymmetric vibrations in nucleic acids respectably, and vibrational band 1170 cm‐1 that belongs to C‐O and C‐O‐C stretching vibrations of carbohydrate. The vibrational band at 1660cm‐1 belongs to amid I stretching vibrations of proteins, and the last range of spectral lines belongs to C‐H symmetric and asymmetric C‐H, C‐H2 and C‐H3 vibrations in lipids. The line at 1730 cm‐1 that is very pronounced in control group spectra is C‐O symmetric vibration in fatty acids. Most prominent difference between BPC treated and control group was observed region of C‐H vibrations of lipids and vibrational line at 1730 cm‐1 of fatty acids.

Thus, different FTIR spectra evidenced that BPC treatment causes changes of the tissue at molecular level.
Anatomical Study of Abdominal Wall Muscles Innervation
Authors: Alblowi Abdulrahman , Malikov Serguei , Perez Manuel , Labrousse Marc , Settembre Nicla
Published: May 2022

Background

A variety of surgical specialties use different abdominal wall incisions to access the abdominal cavity. A thorough understanding of the anatomy, distribution, communication and course of these nerves are essential for surgeons. Anatomical knowledge remains limited. The anatomic relationships to other landmarks such as bone structures, ligaments, and muscles are important in prevention of peri‐operative complications such as abdominal muscle weakness, flank bulge, and incisional hernias. Imaging modalities do not provide many details about abdominal wall nerves.

Purpose

The goal of this study is to provide a better understanding of the directions, course, communications, and branches of abdominal muscle wall nerves in order to minimize the risk of nerve injury.

Method

Four frozen‐preserved cadavers with no abdominal wall scars visible were dissected layer by layer. The ninth, tenth, eleventh, and twelfth intercostal nerves were dissected from the midline to the posterior axillary line.

Result

The ninth, tenth, eleventh, and twelfth thoracolumbar nerves run in the same plane, between the internal oblique muscle and the transverse muscle, with varying directions and course. The T9 intercostal nerve has a horizontal course between the muscles, whereas the T10 and T11 and T12 have an oblique course toward the midline. Eleventh intercostal nerve, and twelfth subcostal nerves are bigger compared to the other nerves, with several communications and branches.

Conclusion

The anatomy of nerves in abdominal muscles appears to be more complex and variable.

Eleventh intercostal nerve and twelfth subcostal nerves appear to have larger contribution to the abdominal wall muscle innervation. They have a complex and variable network of communication. Preserving these nerves during surgical incisions, operation, and closure could help in the prevention of postoperative abdominal wall complications.
Alpha‐ketoglutarate promotes goblet cell differentiation and alters metabolome in a mouse model of colitis
Authors: Bravo Iniguez Alejandro , Tian Qiyu , Du Min , Zhu Meijun
Published: May 2022

Background

Alpha‐ketoglutarate (aKG) is a key intermediate of the tricarboxylic acid (TCA) cycle. Previously, we demonstrated that aKG supplementation mitigates inflammation, enhances epithelial barrier integrity, and promotes oxidative phosphorylation in dextran sulfate sodium (DSS)‐induced colitis of mice. Intestinal epithelial homeostasis is regulated by cell energetics and metabolites from the microbiome. Given the importance of the mucosal layer in maintaining gut epithelial barrier function and microbiome composition, we hypothesized that dietary aKG facilitates the differentiation of secretory lineage epithelial cells and improves the gut metabolome.

Methods

C57BL/6 male mice received drinking water with or without 1% aKG for three weeks, when mice were subjected to DSS induction for 7 days followed by 7 days recovery. The GC‐MS analysis was used to profile the cecal metabolomic changes due to aKG supplementation. In addition, colonic tissues were sampled for both biological and histochemical analyses.

Results

GC‐MS metabolomics revealed separation of the cecal metabolites between control (CON) and aKG supplemented (aKG) mice. In total, the contents of 40 metabolites were found to be different between CON and aKG groups. These metabolites include amino acids, fatty acids, and the B vitamin, niacin, succinic acid (butanedioic acid), the conjugate acid of succinate. Putrescine, a polyamine produced by intestinal microbiota and methysuccinic acid were among the metabolites with the largest fold change increased by dietary aKG. Amino acids including essential amino acids and those involved in the TCA cycle such as threonine and aspartic acid were elevated in the aKG group, of which, glycine displayed a 10‐fold increase. On the other hand, ornithine, an intermediate of the urea cycle, and amide products such as oleamide and urea were significantly decreased in aKG group. Pathway analysis revealed that the urea cycle, along with and arginine and proline metabolism and glutathione metabolism were altered due to aKG supplementation. Furthermore, aKG supplementation increased goblet cells based on Alcian blue staining, associated with increased mRNA expression of Mucin 2 and goblet cell differentiation markers Trefoil factor 3 and Krüppel‐like factor 4.

Conclusion

Together, dietary aKG enhances intestinal epithelial differentiation to favor goblet cell differentiation, which correlates with improvement in metabolome.
Endogenous NRG4 promotes ST3GAL4 expression, leukocyte infiltration, and inflammation in IL‐10R neutralization colitis
Authors: Bernard Jessica K. , Schumacher Michael , Liu Cambrian , Katada Kay , Frey Mark
Published: May 2022

Background

The ErbB family of receptor tyrosine kinases are essential for the regulation of colonic epithelial cell proliferation, differentiation, survival, and wound healing. We previously showed that ErbB4 is induced as a compensatory response by intestinal inflammation; treatment with exogenous NRG4 (an ErbB4 ligand) protects colonocytes against cytokine‐induced apoptosis and ameliorates murine experimental colitis. Exogenous NRG4 can further alleviate intestinal inflammation by suppressing the activity of pro‐inflammatory macrophages. However, the role of endogenous NRG4 in the intestine has not been described.

Hypothesis

Based on our previous studies, we hypothesized that NRG4 deletion would exacerbate intestinal inflammation.

Methods

Colitis was induced in 5‐week‐old wild‐type (WT) and NRG4 knockout (KO) mice by i.p. injection with 1 mg/kg IL‐10R neutralizing antibody, weekly for four weeks. Inflammation was assessed by fecal lipocalin‐2 ELISA, plasma myeloperoxidase ELISA, and total spleen weight. Disease was assessed by histological scoring of paraffin embedded colon rolls. Cytokine (TNF, IL‐1b, IFNg, and IL‐6) expression was determined by qPCR on RNA from full thickness distal colon homogenates. ST3GAL4 expression was assessed by qPCR and immunohistochemistry. Intestinal immune cell populations were identified and analyzed by mass cytometry (CyTof) using FlowJo software.

Results

Fecal lipocalin expression was elevated in all mice after IL‐10R antibody treatment. Surprisingly, WT mice displayed significantly more fecal lipocalin than NRG4 KO cage mates. Similarly, after 4 weeks, WT mice had significantly higher plasma myeloperoxidase (2‐fold, p<0.001), increased spleen weight (57% more, p<0.01), and more histological injury and inflammation (72% higher score, p<0.05) versus their KO cage mates. Furthermore, mRNA levels for IFNg (3.7‐fold difference, p<0.01) and IL‐6 (1.6‐fold difference, p<0.05) were significantly higher in WT versus KO cage mates. CyTof analysis identified a reduction in CD8+ T cell populations, as well as alterations in overall T cell populations. Expression of ST3GAL4, a sialyltransferase that contributes to inflammatory leukocyte recruitment, was significantly reduced in NRG4‐null colons and epithelia (>90% reduction, p<0.01 and p<0.001 respectively), suggesting a mechanism for altered T cell populations.

Conclusion

In contrast to the protective effects of exogenous NRG4, endogenous NRG4 appears to participate in recruitment of CD8+ T cells and intestinal inflammation, possibly through regulation of ST3GAL4.
Dual COX‐2/sEH Inhibition and Immune Checkpoint Blockade Regress Bladder Cancer Tumors in Mice
Authors: Rothenberger Eva , Wang Weicang , Kipper Franciele C. , Kelly Abigail , Hwang Sung Hee , Duncan Madeline , Bielenberg Diane R. , Henderson Paul T. , Cimino George , Zimmermann Maike , Hammock Bruce D. , Panigrahy Dipak
Published: May 2022

Unresolved inflammation plays a critical role in bladder cancer initiation and progression. Acute inflammation leads to eicosanoid‐driven cytokine production. Controlling the local and systemic inflammatory response in bladder cancer is critical to preventing bladder cancer progression. Chemotherapy (e.g. gemcitabine and cisplatin), the standard of care for advanced bladder cancer, disrupts the resolution of inflammation and is only partially effective in preventing tumor recurrence after treatment. Although immunotherapy induces a durable response in 15 to 30% of patients in advanced bladder cancer, a majority of patients do not respond due to drug resistance mechanisms. Therefore, there is a critical unmet medical need to improve chemotherapy and immunotherapy in bladder cancer. To address this, we have developed dual COX‐2/sEH inhibitors (e.g. PTUPB). This lead compound is representative of a newly patented class of “dual acting” molecules that target two important enzymes in the arachidonic acid cascade with nanomolar binding affinity: cyclooxygenase‐2 (COX‐2) and soluble epoxide hydrolase (sEH). Oral dosing of PTUPB is well‐tolerated in animal models. Since chemotherapy and immunotherapy both induce tumor‐promoting inflammation via an eicosanoid and cytokine storm, we hypothesized that dual COX‐2/sEH inhibition using PTUPB would enhance immunotherapy in experimental bladder cancer via anti‐inflammatory mechanisms. When syngeneic (MB49) bladder tumors reached ~200 mm3 in immunocompetent mice, treatment was initiated with PTUPB (oral, 30‐60 mg/kg), anti‐CTLA‐4 (200 µg initial dose, 100 µg Q3D), anti‐PD1 (200 µg Q3D), gemcitabine (75 mg/kg Q3D), cisplatin (5 mg/kg Q5D), or various combinations thereof. After 9 days of treatment, mice were sacrificed and bladder tumor tissues were analyzed for sEH and COX‐2 expression, as well as endoplasmic reticulum (ER) stress. Here we demonstrate that chemotherapy stimulated sEH expression in tumor tissue which was counter‐regulated by PTUPB. Importantly, PTUPB enhanced immunotherapy in the MB49 bladder cancer model. While monotherapy treatment with PTUPB, gemcitabine/cisplatin, anti‐PD1 or anti‐CTLA4 suppressed tumor growth compared to control, the tumors eventually escaped by the end of the study. Dual COX‐2/sEH inhibition in combination with chemotherapy (gemcitabine and cisplatin) and/or immune checkpoint blockade (anti‐CTLA‐4 or anti‐PD1) induced tumor regression via synergistic anti‐tumor activities. Chemotherapy induced the expression of ER stress response genes (e.g. BiP and CHOP), COX‐2, and pro‐inflammatory cytokines in bladder cancer tissue which were repressed by PTUPB. Taken together, our results demonstrate dual COX‐2/sEH inhibition as a novel therapeutic approach to enhance immunotherapy in bladder cancer without overt toxicity.
Impact of Smoking On Immunomodulation Of Dental Pulp Stem Cells
Authors: Tahrani Leyla , Vang David , Han Xiaoyuan , Xiao Nan
Published: May 2022

COVID‐19 has caused a global pandemic since 2019. After infecting the host cells, SARS‐CoV‐2 shows ability to hijack the host immune system and launch a cytokines‐storm. Smoking is reported to predispose patients to the respiratory system damage and lead to increased vulnerability to SARS‐CoV‐2 and more severe outcomes. Stem cells (SCs) are proposed as an alternative therapy of COVID‐19, especially the severe cases.

Objective

to investigate the impact of smoking and vaping on the inflammatory response of dental pulp stem cells (DPSCs).

Hypothesis

cigarette smoking and vaping inhibit the immunomodulatory, antimicrobial, and regenerative properties of the DPSCs and will reduce the success of stem cell therapy of COVID‐19.

Methods

DPSCs were treated with various doses of cigarette smoke condensate (CSC) or nicotine for 24‐96 hours. Cell lysate and concentrated cell culture medium were collected at the end of the treatment. Expression of inflammatory cytokines were determined using human cytokine array, ELISA and quantitative PCR.

Results

the level of TGF‐b2□ TGF‐b3, pulmonary and activation‐regulated chemokine (PARC or CCL‐18), tissue inhibitor of metalloproteinases (TIMP)‐1 and LIGHT (TNF superfamily member 14) significantly decreased after 48 hours exposed to 100mg/ml CSC. Interestingly, nicotine induced a different pattern of change of cytokine release. ELISA and quantitative PCR also showed that CSC treatment led to elevation of pro‐inflammatory cytokine IL‐6 level and reduction of anti‐inflammatory cytokine TGF‐b1 level in DPSC.

Conclusion

our current results indicated that smoking changes the cytokine expression profile in DPSCS, which has been used in clinical trials in treating COVID‐19. In addition to sharing many similarities with bone marrow mesenchymal SCs, DPSCs are also easy to access and show low immunogenicity reaction, making them valuable source to battle COVID‐19. We will further investigate the mechanism of how the SCs attenuate the cytokine storm induced by COVID‐19.
Cocaine affects astrocyte activation and iNOS expression
Authors: Fandaros Marina , Liu Yanzuo , Hua Yueming , Pan Yingtian , Rubenstein David A. , Du Congwu , Yin Wei
Published: May 2022

It is estimated that there are more than two million people addicted to cocaine in the United States. How cocaine affects neuronal functions has been extensively studied. Astrocytes, an important member of the neurovascular unit, also play significant roles in brain pathology. However, their role in cocaine use disorder is unclear. Astrocytes are glial cells that support the neurons and maintain the blood‐brain barrier. Astrocytes can express inducible nitric oxide synthase (iNOS) that is responsible for the transient production of nitric oxide (NO). Both iNOS and NO have been reported to be actively involved in cocaine addiction. We hypothesize that cocaine can cause astrocyte activation and inflammation, resulting in iNOS upregulation, which can in turn, affect both neuronal and brain microvascular endothelial functions. To test this hypothesis, an in vitro astrocyte culture model and an in vivo chronic cocaine mouse model were used. In the in vitro model, human astrocytes from the cerebrocortex were treated with cocaine (10μM) (or lipopolysaccharide at 2μg/mL as the positive control) for 24 hours. iNOS expression was measured using solid‐phase ELISA. Astrocyte activation was assessed by immunofluorescence microscopy for Glial Fibrillary Acidic Protein (GFAP) expression. Changes in astrocyte morphology were quantified using a Sholl analysis. In the in vivo model, wild type C57BL/6 mice were treated with cocaine (30 mg/kg per day through intraperitoneal injection) for two weeks. Mouse brain tissue, especially in the prefrontal cortex, was collected and fixed with paraformaldehyde before slicing. Astrocyte activation and number were quantified using immunofluorescence microscopy (of GFAP). Astrocyte iNOS expression was measured using an iNOS ELISA kit (using collected mouse brain homogenate). The results demonstrated that lipopolysaccharide (LPS, the endotoxin) significantly increased iNOS expression after 24hr treatment. Cocaine, in comparison, had a more significant impact in iNOS upregulation in early passages of cultured astrocytes. Sholl analysis demonstrated that cocaine and LPS both induced significant morphological changes and reduced the number of astrocyte processes. Results obtained from the in vivo studies indicated that chronic cocaine treatment significantly decreased the number of astrocytes in the mouse prefrontal cortex, while increasing iNOS expression. In summary, astrocytes can respond to cocaine stimulation in vitro and in vivo, likely through iNOS‐medicated inflammatory responses. Therefore, investigation in the role of astrocytes in cocaine addiction may bring insight to new therapeutic target to treat cocaine use disorder.