bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Glycoproteins on the Surface of Cow's Milk Exosomes that Mediate the Uptake of Exosomes into Human Colon Carcinoma Caco‐2 Cells
Authors: Sukreet Sonal , Zhang Hanyuan , Adamec Jiri , Cui Juan , Zempleni Janos
Published: November 2023

Background

MicroRNAs (miRNAs) play essential roles in gene regulation. A substantial fraction of miRNAs in tissues and body fluids is encapsulated in exosomes (exos), thereby conferring protection against degradation. Traditionally, miRNAs have been considered endogenous regulators of gene expression. Recently, we have made the paradigm‐shifting discovery that humans absorb dietary miRNAs from cow's milk (encapsulated in exos) by endocytosis, and dietary miRNAs have biological activity in humans. Inhibitor and competitor studies implicated glycoproteins on the surface of exos and cells in intestinal transport.

Objective

Identify glycoproteins on cow's milk exos as putative substrate recognition feature in intestinal transport.

Methods

Exos were isolated from cow's milk using ultra centrifugation; identity, purity, and integrity were confirmed using nanoparticle tracker, western blot, and transmission electron microscopy. Where applicable, exos were treated with the proteases Glu‐C (targeting glu), trypsin (targeting arg and lys), or proteinase K (non‐specific); controls were not treated with proteases. For transport studies, exos were labeled using fluorophore (FM‐4), and uptake rates were analyzed in human intestinal Caco‐2 cells. Surface peptides released by treatment with trypsin were identified using LC/MS‐MS and Mascot, and glycoproteins were identified using SwissProt. One way Anova and Bonferroni's Multiple comparison was used to test for statistical significance.

Results

We identified 417 exosomal surface proteins, including N‐glycans (4), O‐glycans (2), and C‐glycans (2). When exos were treated with Glu‐C or trypsin, rates (Vmax) decreased compared with controls (arbitrary units): 88±1 for control, 40±0.6 for trypsin, 19±0.3 for Glu‐C (P<0.05 vs. control). For comparison, we identified 504 proteins in breast milk exos (6‐N‐glycans, 4 O‐glycans, and 3 C‐glycans). The identities of surface glycoproteins were distinct in bovine and human milk exos.

Conclusion

Eight glycoproteins were identified on surface of cow's milk exos, which might be essential for intestinal transport. We propose that glycan features, as opposed to protein features, are important for exo recognition by intestinal cells.

Future directions

We will use site‐directed mutagenesis and gene knockout protocols in cell cultures and mice to provide unambiguous evidence for the importance of distinct glycoprotein features on the mucosal surface in exo recognition.

Support or Funding Information

Supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award number 2015‐67017‐23181, NIH 1P20GM104320 and P30GM103335, the Gerber Foundation, the Egg Nutrition Center, the University of Nebraska Agricultural Research Division (Hatch Act), and USDA multistate group W3002.
AMPK Enhances Enterocyte Migration and in vivo Intestinal Barrier Function
Authors: Sun Xiaofei , Xue Yansong , Du Min , Zhu Meijun
Published: November 2023

Impairment in gut epithelial integrity and barrier function is associated with many diseases, such as inflammatory bowel disease, obesity and diabetes. Inadequate mucosal wound healing is associated with defective enterocyte migration along the crypt‐villus axis. AMP‐activated protein kinase (AMPK), an energy sensor, has been suggested to mediate epithelial barrier function and enhances wound healing processes in cultured cells. However, the causal relationship between AMPK and intestinal barrier function remains underdetermined. The objective of this study was to provide direct evidence of the regulatory role of AMPK on intestinal barrier function and enterocyte migration in vivo. To test, mice with AMPKα1‐floxed gene were cross‐bred with VilCre mice to specifically knock out AMPKα1 gene in intestinal epithelial cells where villin is expressed. The migration of enterocyte and intestinal barrier function were compared between AMPK VilCre knockout (KO) and wild type (WT) male mice at the age of 2.5 months. AMPK VilCre KO mice had increased intestinal epithelial paracellular permeability to FITC‐dextran compared with WT mice, indicating a leaking gut. In addition, mucosal thickness and the migration of BrdU‐labeled enterocytes were decreased in AMPK VilCre KO mice. CDX2 is the key transcription factor committing cells to the intestinal epithelial lineage. Immunofluorescence staining further showed an abnormal expression pattern of CDX2 in AMPK VilCre mice, where CDX2 expression was extended from crypt to the middle of villi, while CDX2 expression resided along the epithelial cells in WT mice. Taken together, these data clearly show that AMPK is required for proper intestinal barrier function and enterocyte migration in vivo possibly through regulating cdx2 expression.

Support or Funding Information

NIH R15HD073864
AMPK Improves Intestinal Epithelial Differentiation via Regulating CDX2
Authors: Sun Xiaofei , Yang Qiyuan , Du Min , Zhu Meijun
Published: November 2023

Background

Gut epithelium forms the largest and critical barrier against the external environment, which is constantly renewed. The homeostasis of intestinal barrier is based on a delicate regulation of epithelial proliferation and differentiation. AMP‐activated protein kinase (AMPK) regulates gene expression and cellular differentiation, and its inactivation is as a major etiological factor for many diseases. However, the role of AMPK in intestinal epithelial barrier function has been only sparsely studied.

Objective

The objective of this study was to evaluate the regulatory role of AMPK on intestinal epithelial differentiation and uncover its underlying mechanism.

Results

AICAR, an AMPK activator, improved the intestinal epithelial barrier function of Caco‐2 cells as indicated by increased transepithelial electrical resistance (TEER) and reduced paracellular FITC‐dextran permeability. Consistently, AICAR treatment enhanced the expression of epithelial differentiation markers, E‐cadherin and Villin, Alkaline Phosphatase activity, and the formation of tight junction based on Calcium switching assay and ZO‐1 immunostaining. Transfection of Caco‐2 cells with AMPK wild type (WT) plasmid, which enhances AMPK activity, improved epithelial barrier function and epithelial differentiation, while K45R, a dominant negative mutant, impaired. In addition, AMPK enhanced the expression of CDX2, the key transcription factor committing stem cells to intestinal epithelial lineage, which was associated with enrichment of H3K4me3, a permissive histone modification in the cdx2 promoter, while reducing inhibitory histone modification, H3K27me3. Immunofluorescence staining revealed a lower level of CDX2 expression in Caco‐2 cells with AMPK dominant negative mutant compared to that with AMPK WT.

Conclusion

AMPK enhances intestinal barrier function and epithelial differentiation through inducing the expression of CDX2.

Support or Funding Information

NIH R15HD073864
Activation Of The Constitutive Androstane Receptor Enhances Wound Healing – A New Link Between Xenobiotic Sensing And Intestinal Mucosal Homeostasis?
Authors: Hudson Grace , Zamponi Alexandra , Alston Laurie , Chang Thomas , Hirota Simon Andrew
Published: November 2023

Background

Compounds released from the intestinal microbiota may play a role in maintaining mucosal homeostasis, but little is known about the receptors that sense and respond to these compounds. Recently a cytosolic xenobiotic sensor, called the pregnane X receptor (PXR), was identified as a receptor for microbial metabolites, and shown to play a key role in regulating intestinal epithelial barrier and wound healing following injury. In the current study, we sought to determine whether the constitutive androstane receptor (CAR), which shares structural, functional and pharmacological features with the PXR, plays a role in regulating wound healing and mucosal repair.

Methods

Caco‐2 intestinal epithelial cells were used to model wound healing. Wounds formed using Ibidi culture inserts were treated with CITCO, a selective human CAR agonist, and imaged for 24 hours. Wound closure was quantified and cell migration tracks assessed. To interrogate the intracellular events involved in the CITCO‐induced responses, we assessed the role of p38 MAPK and ERK1/2 signaling in our assays, along with proliferation and migration (xCELLigence impedance‐based migration assay). To determine the role of the CAR in vivo, we assessed recovery from dextran sulphate sodium (DSS)‐induced colitis (2.5% W/V in drinking water; 5 days exposure, followed by 9 days of recovery) in CAR‐deficient mice and WT mice treated with TCPOBOP (3 mg/kg; IP; daily during recovery phase), a selective rodent CAR agonist.

Results

Activation of the CAR enhanced wound closure in Caco‐2 monolayers, which was associated with increased cell migration, but not proliferation. The enhancement of cell migration was dependent on p38 MAPK signaling. Treating WT mice with a CAR agonist accelerated their recovery from DSS‐induced colitis. In contrast, CAR‐deficient mice exhibited delayed recovery.

Conclusions

Our data suggest that the CAR regulates the cellular events that contribute to wound healing and mucosal restitution following injury. While the microbe‐derived compounds that activate the CAR have yet to be identified, its close relative, the PXR, has been shown to respond to such ligands. Taken together, we posit that xenobiotic receptors play a key role in regulating the response to injury and may provide new therapeutic targets to enhance mucosal repair.

Support or Funding Information

Crohn's & Colitis Canada; Canadian Foundation for Innovation; The Dr. Lloyd Sutherland Investigatorship in IBD/GI Research
Chronic Alcohol Consumption Alters Rhythms in Key Enzymes and Transcription Factors Involved in Lipid Metabolism: Potential Role of the Circadian Clock
Authors: Valcin Jennifer Allison , Udoh Uduak Sunday , Swain Telisha , Bailey Shannon Marie
Published: November 2023

Chronic alcohol consumption disrupts hepatic lipid metabolism causing fatty liver disease or steatosis. The molecular and cellular mechanisms responsible for alcohol‐induced fat accumulation in hepatocytes remain unclear. New studies indicate that many metabolic processes, including lipid metabolism, are influenced by the molecular circadian clock. At the core of this molecular oscillator are the two transcription factors BMAL1 and CLOCK that drive rhythmic expression of other clock and clock‐regulated metabolic genes. We previously reported that chronic alcohol consumption disrupts the function of the molecular circadian clock in the liver. However, the impact this has on downstream clock‐regulated pathways, like lipid metabolism, is unknown. The purpose of this study was to determine whether chronic alcohol disrupts clock‐controlled rhythmic expression of triglyceride metabolism genes and transcription factors that regulate lipid metabolism. For these studies, male hepatocyte‐specific BMAL1 knockout (HBK) mice and their wild‐type (WT) control littermates were kept under a 12:12 hour light‐dark cycle and pair‐fed control or alcohol‐containing diets for 5 weeks. Livers were collected at 4‐hour intervals for a 24 hour period and gene expression was measured by qPCR. In WT alcohol‐fed mice, we observed a dampening in the diurnal rhythms in several triglyceride metabolism genes. Alcohol significantly decreased the diurnal gene expression rhythm of patatin‐like phospholipase domain‐containing 3 (Pnpla3), an enzyme that mediates triglyceride breakdown. This result suggests that the alcohol‐mediated decrease in Pnpla3 may contribute, in part, to triglyceride accumulation in liver. Rhythmic expression of Pnpla3 was also completely abolished in liver of both control and alcohol‐fed HBK mice, suggesting a role of the clock in Pnpla3 regulation. The diurnal rhythm in acetyl CoA carboxylase 2 (Acc2) was also decreased in livers of WT alcohol‐fed mice with further dampening observed in livers of HBK alcohol‐fed mice. Interestingly, the diurnal rhythm of Acc2 was phase shifted in the HBK control‐fed compared to WT controls. In addition, diurnal rhythms in sterol regulatory element binding protein 1c (Srebp1c) and Acc1, both of which are involved in fatty acid synthesis, were also dampened in livers of WT and HBK alcohol‐fed mice. Overall, our findings indicate that chronic alcohol consumption disrupts normal diurnal rhythms of lipid metabolism processes, suggesting that alterations in the balance of these tightly regulated oscillations likely contribute to the alcoholic steatosis.

Support or Funding Information

This work is supported by NIH grant R01 AA020199
TRPV4 Modulates CYP2E1 Function in Nonalcoholic Steatohepatitis Progression
Authors: Seth Ratanesh Kumar , Das Suvarthi , Dattaroy Diptadip , Alhasson Firas A , Michelotti Gregory A , Nagarkatti Mitzi , Nagarkatti Prakash , Bell Phillip D , Liedtke Wolfgang B , Diehl Anna Mae , Chatterjee Saurabh
Published: November 2023

Emerging evidence shows that oxidative stress via the activation of cytochrome p450 2E1 (CYP2E1) is key to the progression of inflammation in Nonalcoholic steatohepatitis (NASH). However, the molecular mediators and its pathways that regulate CYP2E1 mediated oxidative stress in NASH remain obscure. In the present study, the CYP2E1 substrate pyrazole or bromodichloromethane (BDCM) were used to induce CYP2E1 mediated oxidative stress, inflammation and NASH pathology in high‐fat diet fed mice. Results showed that the transient receptor potential vanilloid channel 4 (TRPV4) mRNA expression and protein levels were significantly elevated in parallel to increases in CYP2E1 and correlated well with increased lipid peroxidation, IL1β, MCP1, TNFα and HMGB1 levels in NASH livers and in Kupffer cells. TRPV4 knockout (KO) mice showed exacerbated inflammation, increased CYP2E1 protein (2 fold higher than wild type), lipid peroxidation (4‐Hydroxynonenal, 4‐HNE >4 fold), inflammatory cytokines (>25 fold), infiltration of leukocytes, sinusoidal endothelial dysfunction (SED) marker genes, HMGB1 levels, decreased phosphorylated endothelial nitric oxide synthase (pNOS3) and nitric oxide (NO) release. Mechanistically, diallyl sulfide (CYP2E1 inhibitor) or NO donor DETANONOate administration to TRPV4 KO mice completely abrogated enhanced inflammation and NASH pathophysiology. Use of NO donor significantly decreased CYP2E1‐mediated lipid peroxidation, proinflammatory genes and SED markers in TRPV4 KO mice suggesting that TRPV4 might regulate CYP2E1‐function via NOS3 phosphorylation and increased NO release. In vitro, CYP2E1‐mediated lipid peroxidation and levels of HMGB1, a damage‐associated molecular patterns (DAMPs) was increased in HepaRG cells in the presence of TRPV4 antagonist but had no significant effect on NO release. Since the CYP2E1‐mediated release of 4‐HNE and DAMPs from necrosed hepatocytes is known, the resident macrophages were primed with 4‐HNE. NOS‐3 phosphorylation, NO release and Phospho‐AKT levels from Kupffer cells were decreased with TRPV4 antagonist treatment while there was a concomitant increase in MCP‐1 and TNF‐α levels. Interestingly, Kupffer cells treated with TRPV4 agonist reversed decreased pNOS3 and pAKT levels. The results obtained shows that TRPV4 modulated CYP2E1‐mediated oxidative stress in hepatocytes, inflammation and endothelial injury by increasing NOS3‐mediated release of nitric oxide in the microenvironment via the AKT pathway. Based on the above, targeting TRPV4 or its downstream signaling cascade might be a promising therapeutic strategy in NASH.

Support or Funding Information

This study was supported by NIH pathway to independence award 7R00ES019875‐03 to Saurabh Chatterjee
Reduced Jejunal Basal Chloride Secretion In Diabetes Is Reversed By Genistein Diet
Authors: Al‐Nakkash Layla , Masood Faisal , Catmull Shawn
Published: November 2023

Obesity and diabetes are often associated with disturbances in intestinal function, i.e. slowed gastrointestinal transit. The objective of this study was to characterize jejunal secretory function in a clinically relevant model of diabetes and obesity: the leptin‐deficient ob/ob mouse. We measured transepithelial short circuit current (Isc), across freshly isolated segments of jejunum from 11‐week old ob/ob and lean C57Bl/6J male and female mice. Separate segments of jejunum were frozen for western blot determination of key proteins involved in secretory transport. Basal Isc was significantly decreased in ob/ob female mice (82±7 μA/cm2, n=9, P<0.05) versus leans (107±14 μA/cm2, n=9), and genistein‐diet reversed this (112±12 μA/cm2, n=17, P< 0.05). Basal Isc was significantly decreased in ob/ob male mice (54±7 μA/cm2, n=11), versus leans (85±12, n=10, μA/cm2, P<0.05) and genistein reversed this (109±10 μA/cm2, n=7, P<0.05). Inhibition with clotrimazole and ouabain (both 100 μM, basolateral) was significantly reduced in all ob/ob mice compared to leans (P<0.05). Expression of total CFTR (normalized to GAPDH) was significantly decreased 2‐fold (P<0.05) in all ob/ob mice compared to leans. Our data suggests that the reduced basal jejunal Isc in all ob/ob mice is a consequence of reduced CFTR expression, decreased activity of the basolateral KCa channel and Na+/K+‐ATPase (males also exhibit reduced NKCC1 expression), each of which likely contributes towards this phenotype in jejunum of the ob/ob mouse. Genistein diet has beneficial effects on basal Isc: in males mediated by increased Na+/K+‐ATPase‐sensitive Isc and increased NKCC1expression, and, in females mediated by increased KCa‐sensitive Isc and increased adenosine expression. Improved understanding intestinal dysfunctions in the ob/ob jejunum, may allow for the development of novel drug targets to treat obesity and diabetes, and may also be of benefit in CF‐related diabetes.

Support or Funding Information

Supported by: Soy Health Research Program, Diabetes Action Research and Education Foundation, and MWU intramural funds.
Disruption of Serotonin Signaling Results in Loss of Coordinated Tone Development and Subsequent Inhibition of Peristalsis in Guinea Pig Distal Colon
Authors: McGill Mahalia , Klinger‐Lawrence MaryBeth , Herrera Gerald
Published: November 2023

This project was designed to examine the effects of the selective serotonin reuptake inhibitor, Fluoxetine, on colonic motility using the isolated guinea pig distal colon. The Gastrointestinal Motility Monitor system (“GIMM”, Catamount Research and Development Inc., St. Albans, VT) was used to analyze peristaltic motility during fecal pellet propulsion, as well as to conduct spatiotemporal analysis of motility patterns. Peristaltic motility in colonic segments was significantly slowed by Fluoxetine at concentrations of 20, 30 and 40 μM (p<0.05 vs. vehicle). Indeed, in colonic segments treated with 40 μM Fluoxetine, peristaltic motility was completely abolished (at least 1 of 3 trials with no peristaltic motility) in 4 out of 5 experimental preparations. This striking disruption in peristaltic motility by Fluoxetine was associated with a pronounced change in spatiotemporal motility patterns. In the presence of 40 μM Fluoxetine, average colonic diameter during the motility trial was substantially increased relative to pre‐treatment diameter (36.6 ± 21.5 % increase from baseline diameter, n=5). In vehicle‐treated colonic segments, average diameter remained relatively unchanged from pre‐treatment levels (0.5 ± 3.5 % change from baseline diameter, n=11). These findings suggest that serotonin signaling in the wall of the gut is required for normal peristaltic propulsive motility, and that disruption of serotonin signaling using Fluoxetine results in a loss of coordinated tone in the wall of the gut. Understanding the exact role of serotonin in normal gut motility is essential to gaining insights into novel therapeutic strategies for treating bowel dysfunction.

Support or Funding Information

This study was supported by Catamount Research and Development
Notch and mTOR Regulate Proliferation of Human Gastric Cancer Cells
Authors: Wong Gabriela , Demitrack Elise S , Samuelson Linda C
Published: November 2023

Background

The Notch signaling pathway regulates proliferation and differentiation of stem cells in the stomach. We have recently shown in the mouse that constitutive Notch activation stimulates proliferation of LGR5‐expressing antral stem cells and that inhibition of mTORC1 attenuates the Notch‐mediated increase in proliferation. The goal of this study was to test whether Notch and mTOR regulate proliferation of human gastric cancer (GC) cells.

Methods

AGS, MKN45 and NCI‐N87 human GC cell lines were treated with vehicle (DMSO), DAPT (Notch inhibition), rapamycin (mTORC1 inhibition), or both for 5 consecutive days. Proliferation was measured each day using the CellTiter 96 Proliferation Assay. Gene expression analysis of Notch pathway components, and stem and differentiated cell markers was performed using qRT‐PCR.

Results

Notch inhibition did not significantly affect growth of AGS cells, but significantly inhibited growth of MKN45 and NCI‐N87 cells after 2 days of DAPT treatment. Rapamycin treatment significantly reduced proliferation of all 3 cell lines. Proliferation was further reduced in all 3 cell lines with combined Notch and mTOR inhibition, suggesting the necessity of both pathways to sustain GC cell growth. Gene expression analysis revealed that AGS and MKN45 GC cells express Notch1 and Notch2 receptors. In AGS cells, Notch inhibition reduced mRNA abundance of the stem cell marker and Notch target gene olfactomedin 4 (OLFM4). Furthermore, Notch inhibition in AGS cells significantly increased expression of the deep mucous cell markers TFF2 and SPDEF. These findings are in accordance with the role of Notch to stimulate proliferation and repress cellular differentiation in the mouse stomach.

Conclusion

Notch and mTOR are required to sustain growth of human GC cells in vitro. Further research to understand these mechanisms might benefit GC therapies by identifying specific pathways that directly impact cancer cell growth.

Support or Funding Information

APS Undergraduate Summer Research Fellowship; NIH F32‐DK093349; NIH P01‐DK062041 and NCI P50‐CA130810
The Impact of Dietary Soy on Gut Microbiome
Authors: Gill India R , Uno Jennifer K
Published: November 2023

The metabolism of daidzein, a major compound of soy, produces equol in the presence of intestinal bacteria. Equol is more biologically active and its role in human health remains controversial. It has been shown to have links to tumor regression, tumor progression, cardiovascular health and neurologic function. Soymeal is a common product used in the mouse chow of research labs. Given its controversial role in host health, mouse facilities at the University of North Carolina at Chapel Hill switched to a non soy‐based diet. The purpose of this study is to examine the impact that the removal of soy has on bacterial communities. We expect to see a decrease in phyla such as bifidobacteria that play a role in the metabolism of isoflavones like daidzein. Mice were fed a soy‐based diet (Prolab Isopro RMH 3000) and a non soy‐based diet (Harlan Laboratories). Bacterial DNA was extracted from stool and phyla were examined and quantified by qPCR. In soy fed mice, we saw a slight decrease in universal gut bacteria as well as increases in bifidobacteria and lactobacillus. Bifidobacteria was the only phyla that changed significantly, with a 13.63 fold increase (n=7, p=0.05). In order to determine if soy is directly responsible for the microbial changes, we employed the use of zebrafish. Zebrafish were first treated with amoxicillin to clear bacterial flora and bacteria were allowed to recolonize in the presence of or absence of soy. We saw 12.5% and 13.5% decrease in lactobacillus and bifidobacteria, respectively (n=5, p=0.046 and p= 0.05). There was no significant change in total bacteria. This indicates that dietary soy can cause a significant change in the composition of the gut microbiome. This study warrants further research into the role soy plays in regulating microbial communities and emphasizes the need for diet regulation in research facilities.

Support or Funding Information

Elon University
Obesity Prevention by Calorie Restraint Using the Bathroom Scale Model Prevents Hypertension for Life in Nonhuman Primates
Authors: Hansen Barbara Caleen , Linden Ellen H , Newcomb Jennifer D.
Published: November 2023

Obesity prevention via application of the “bathroom scale” model of long term calorie restriction/calorie titration in nonhuman primates (NHPs) prevents the slow motion development of features of the metabolic syndrome, specifically dyslipidemia, and now hypertension. This has enabled time‐linked analysis of the natural history of emergent features in the search for mechanisms underlying the metabolic syndrome. The present study is a follow on of the first defining of normal NHP blood pressure (BP) (121.8 ± 1.6/63.5 ±1.1 mmHg systolic/diastolic respectively), with a Mean Arterial Pressure (MAP) of 85.6 ± 1.1 mmHg, as measured by the GE pro 100V2 instrument. We sought to determine the consequences of obesity, type 2 diabetes, and obesity prevention on the progression of hypertension. BP levels were found to be highly similar to those hypertension levels defined by the American Heart Association JNC7 report in humans, however, mean diastolic BP was significantly lower in NHPs relative to reported normal human levels. (63.5 vs ~ 80), albeit that the 80 is not the mean of normal but rather a threshold above which is abnormal, with mean of normal in humans undefined. We have found progressive increases in hypertension in NHPs with increasing body weight over the adult monkey range 7 to 25 kg (r=0.01, p=0.006). We also examined 1) the relationship of developing hypertension to T2DM onset/dx in 64 monkeys, 2) to the consequences of long‐term diabetes with and without obesity, and 3) then determined the relationship of diabetes prevention by calorie restriction (CR) to hypertension (N=7). Long term CR of NHPs for ~ 23+ years from early adulthood through old age entirely prevented all levels of hypertension compared to ad libitum fed controls (N=192), and in fact CR significantly lowered blood pressure levels (CR: SBP 116.5, DBP 58.2 & MAP 82.9 mm Hg; ad lib controls: SBP 131.3, DBP 66.6 & MAP 92.3 mm Hg). CR monkeys showed 11, 12 and 10% lower systolic, diastolic and mean arterial BPs respectively (p's<0.01). Elderly CR monkeys had a non‐significant difference in BP compared to the above referenced normal adults of similar weight with no CR. In addition systolic BP defined a greater prevalence of hypertension in adult NHPs (67%) compared to diastolic BP (21%) in adult rhesus monkeys. The life time prevalence of T2DM in nonhuman primates appears, based on current long term data, to be approximately 65%. Despite their similar prevalence, the overlap of diabetes and hypertension is not fully concordant, although both relate to obesity in NHPs. Non‐similar trajectories suggest separate underlying regulatory mechanisms. In conclusion, this large animal model naturally develops in middle age all features of human hypertension, including the high prevalence and wide range of severity, together with associations with obesity, age and diabetes.

Support or Funding Information

National Institute on Aging HHSN263200800022C
IL‐1β Participates in Sensitization of Bronchopulmonary C‐fibers Induced by Prenatal Nicotinic Exposure (PNE) in Rat Pups
Authors: Zhao Lei , Zhuang Jianguo , Xu Fadi
Published: November 2023

Maternal smoking during pregnancy has the highest risk for inducing sudden infant death syndrome (SIDS) characterized by cardiorespiratory failure including aggravated apnea. We recently found that PNE promoted hypoxia‐induced central apnea by sensitizing the bronchopulmonary C‐fibers (PFCs), i.e., prolonged PCF firing and apneic response to right atrial bolus injection of capsaicin, a selective stimulant to PCFs (Zhuang et al., AJP 2015), but the relevant mechanisms have not been fully understood. IL‐1β is a potent stimulant to PCFs and its concentration in serum is substantially increased in the majority of SIDS victims (Vennemann et al., Int J Legal Med. 2012). Thus we asked whether PNE increased IL‐1β release into the lungs and IL‐1 type I receptor (IL1R1) expression in nodose/jugular (N/J) ganglion, especially the vagal pulmonary C‐neurons, and if so, whether the prolonged apneic response to capsaicin by PNE would be shortened by pretreatment of IL‐1Ra (an IL1R1 antagonist). To this end, we collected BALF to detect gene and protein levels of IL‐1β released into the airways and lungs, harvested N/J ganglion for determining IL1R1 gene and protein expression, and extracted vagal pulmonary C‐neurons retrograde‐traced by DiI for analysis of IL1R1 mRNA. Subsequently, in anesthetized and spontaneously breathing pups, we compared the respiratory responses to atrial bolus injection of capsaicin before and after IL‐1R1 blockade. Our results showed that PNE obviously elevated IL‐1β in BALF, and upregulated IL1R1 gene and protein expression of N/J ganglion, particularly IL1R1 mRNA in pulmonary C neurons. Moreover, the PNE‐induced prolongation of the apneic response to capsaicin was significantly shortened by ~55% after IL‐1R1 blockade. Our results suggest that PNE promotes IL‐1β release into the lungs and IL1R1 expression in vagal pulmonary C‐neurons to sensitize PCFs, contributing to the prolongation of the PCF‐mediated apnea.

Support or Funding Information

Supported by HL119683 and HL107462
Short‐Term Modulation of the Ventilatory Response to Exercise is Preserved in Obstructive Sleep Apnea
Authors: Bernhardt Vipa , Bassett J Todd , Moran Raksa B , Marines‐Price Rubria , Mitchell Gordon S , Lee Won Y , Babb Tony G
Published: November 2023

Background

The ventilatory response to exercise is temporarily augmented in response to environmentally (e.g., breathing apparatus) or physiologically altered conditions (e.g., respiratory disease), maintaining constant relative arterial PCO2 regulation from rest to exercise; this augmentation is called short‐term modulation (STM) of the ventilatory response to exercise. Obesity and/or obstructive sleep apnea could affect the exercise ventilatory response and STM capacity due to the additional mechanical or ventilatory load on the respiratory system and/or the repetitive intermittent hypoxia during sleep. We hypothesized that: 1) the exercise ventilatory response is augmented in obese OSA patients compared with otherwise healthy obese adults, and 2) the capacity for STM with added dead space is diminished in obese OSA patients.

Methods

Nine obese adults with OSA (age: 39 ± 6 yr, BMI: 40 ± 5 kg/m2, AHI: 25 ± 24 events/hr [range 6–73], mean ± SD) and 8 obese adults without OSA (age: 38 ± 10 yr, BMI: 37 ± 6 kg/m2, AHI: 1 ± 2) completed three, 20‐min bouts of constant‐load submaximal cycling exercise (8 min rest, 6 min at 10 and 30 W) with or without added external dead space (200 or 400 ml; 20 min rest between bouts). Steady state measurements were made of O2, CO2, minute ventilation ( E) and end‐tidal PCO2 (PETCO2). The exercise ventilatory response was defined as the slope of the E‐ CO2 relationship (Δ E/Δ CO2).

Results

In control (i.e. no added dead space), the exercise ventilatory response was not significantly different between non‐OSA and OSA groups (slope: 30.5 ± 4.2 and 30.5 ± 3.8, p > 0.05); PETCO2 did not differ from rest to exercise in either group (p > 0.05). In the trials with added external dead space, no significant differences between groups were observed. Δ E/Δ CO2 increased with increasing dead space (p < 0.05); PETCO2 increased with increasing dead space and exercise intensity (interaction p < 0.05); however, the change in PETCO2 from rest to exercise remained small (<2 mmHg) in both groups, demonstrating STM.

Conclusions

Contrary to our hypotheses: 1) the exercise ventilatory response is not different between obese OSA patients and otherwise healthy obese adults, and 2) the capacity for STM with added dead space is not different between groups. Thus, the capacity for STM is preserved in OSA.

Support or Funding Information

King Charitable Foundation Trust, Texas Health Presbyterian Hospital, NIH Grant R01 HL096782
Synchrony Between Ventral Respiratory Column Expiratory Neurons is Altered during Fictive Swallow in Cat
Authors: Zhou Guannan , Tsai Hsiu‐Wen , Samuel Immanuel , Morris Kendall , Nuding Sarah , Segers Lauren , Gestreau Christian , Lindsey Bruce , Bolser Donald , Davenport Paul
Published: November 2023

Swallow and breathing are both fundamental functions for mammals. Anatomically, breathing and swallow share a common passageway, the pharynx. Precise coordination between swallow and breathing is essential for passing the bolus down into the esophagus, while preventing it entering the airways. The swallow central pattern generator and the respiratory CPG share common neural components. We hypothesized that the ventral respiratory column (VRC) respiratory functional network architecture alters during fictive swallow to produce the swallow breathing pattern. We stimulated the superior laryngeal nerve of paralyzed, decerebrated, ventilated cats to elicit swallow. Extracellular neuronal activity from multiple neurons was recorded simultaneously with microelectrode arrays during fictive swallow and breathing. A spike‐synchronization with bootstrap analysis was performed on the data, allowing us to identify temporal correlations (spike synchrony) between different neuron spike trains. SLN elicited fictive swallows changed synchrony of expiratory neurons (E neurons) in the VRC. During fictive swallows, the incidence of synchronized pairs of neurons decreased, although some pairs were more synchronized. The synchronized spike activity indicates a common swallow‐related synaptic input to these VRC E neurons in the respiratory neural network. The alterations of synchrony during fictive swallow suggest that the VRC E neurons were transiently “retasked” to generate a coordinated swallow breathing pattern.
Influence of electrical and natural stimulation of pharyngeal/laryngeal sensory afferents on the swallow motor pattern
Authors: King Suzanne , Bolser Donald C. , Rose Melanie J. , Poliacek Ivan , Shen Tabitha Y. , Musselwhite M. Nicholas , Pitts Teresa
Published: November 2023

Electrical stimulation of the superior laryngeal nerve (ES‐SLN) is a method that is a mainstay to study swallow. Although its efficiency in evoking a swallow motor response has been well proven, it is a deterministic stimulus that is unlike natural stimulation (mechanical/chemical) of the diverse afferent beds lying in aerodigestive tract. We hypothesized that swallowing muscle activity induced by injection of water into the oropharynx would contrast those produced by ES‐SLN. Swallow was evoked in anesthetized spontaneously breathing cats via injection of a 3cc bolus of water, unilateral ES‐SLN (5 and 20Hz), or a combination of 5Hz ES‐SLN with water. Electromyography (EMG) activity (duration, amplitude) was measured concurrently in thyrohyoid, thyropharyngeus (ThPh), mylohyoid, geniohyoid, cricopharyngeus, parasternal, and posterior cricoarytenoid muscles. Significant stimulus‐related effects were observed in ThPh when a stimulus was presented individually, as indicated by increases in mean amplitude with 5Hz ES‐SLN compared to water alone. No differences were found between ES‐SLN (either frequency) compared to a combination of ES‐SLN with water. Additionally, an inverse relationship was apparent between the frequency of the ES‐SLN and the amplitude of ThPh activity. Current results suggest: a) stimulus modality influences the motor pattern of the pharyngeal phase of swallow in an anesthetized animal model and b) frequency dependence of afferent feedback on the spatiotemporal features of some, but not all, swallow‐related motor outputs. Supported by HL 111215, HL 103415, P30‐GM103507.

Support or Funding Information

Supported by HL 111215, HL 103415, P30‐GM103507.
The exploration of novel systemic biomarkers of chronic obstructive pulmonary disease with different levels of the severity
Authors: Ishihara Yoko , Nakao Motoyuki
Published: November 2023

Introduction

The burden of chronic obstructive pulmonary disease (COPD) is glowing worldwide. COPD is characterized by progressive airflow limitation associated with lung and systemic inflammation, and is caused mainly by cigarette smoking and second‐hand smoke. The present study assessed serum cytokine/chemokine profiles in outpatient with COPD, and in the subject free of respiratory diseases. The purpose of this study is to find out useful biomarker of COPD.

Method

Subjects were recruited at the hospital, clinic and healthcare center in Yamaguchi, Nagasaki and Kumamoto prefecture, Japan. 48 patients with COPD and age‐ and sex‐matched control subjects were involved in this study. Serum samples were analyzed using MAGPIX system magnetic‐bead based multi‐analyte (29 analyte) panels.

Result

In the subjects, 35 patients with bronchial asthma occurred with COPD was included. The prevalence of comorbidities such as hypertension and diabetes were not significantly different except for allergic rhinitis, which was only found in the patients group. When compared with the control group, IL‐6, IL‐15, TNFβ, and IP‐10 (CXCL10/interferon‐γ‐inducible protein 10) levels were significantly higher in the patient group. IL‐6 and IL‐15 was not detected in the control group, and TNFβ was only found one subject in the control group. Interestingly, EGF levels were significant higher in the control group than in the patients group although EGF level has been reported to be increased in COPD patients. When the subjects were stratified by the severity of COPD, only IP‐10 showed positive association with the severity of COPD. Current or ex‐smoking also increased IP‐10 levels in the patient group although the difference was not significant because of small sample number of never smokers in the patient group. When the subjects were divided by smoking status, IP‐10 levels in current or ex‐smokers were higher than those in never‐smokers but the difference was not significant.

Conclusions

Results obtained in this study showed that differential expression of biomarkers is present in the patients with COPD, and in the control subjects. Large part of these biomarkers showed higher levels by the presence of COPD or experience of smoking. IP‐10 levels were also influenced by the smoking experience, however, the significance was only found in the patient group. These results suggest that serum IP‐10 is one of the promising candidates for novel biomarker of COPD and its severity.

Support or Funding Information

This study was supported by Grants‐in‐Aid for Scientific Research (B) 20406001, 23406001 and (C) 26340053, and a “MEXT‐Supported Program for the Strategic Research Foundation at Private Universities” from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Blocking of EDA‐Fibronectin Attenuates Airway Hyperresponsivenss in a Murine Model of Acute and Chronic Allergen‐Induced Airway Remodeling
Authors: Assayag Miri , Berkman Neville
Published: November 2023

Rationale

Airway remodeling refers to changes in the structural components of the airway wall such as subepithelial fibrosis, increased myofibroblasts and increased airway smooth muscle. Airway hyperresponsiveness (AHR) is a defining feature of asthma and may be attributable to the presence of airway remodeling. Fibronectin is a 440‐kd dimeric major ECM glycoprotein that plays a regulatory role during embryogenesis, wound healing and maintenance of tissue integrity. EDA‐FN is a splice variant of fibronectin. Increased levels of EDA‐FN were reported in the lungs of asthmatic patients and of allergen exposed mice. We have previously shown that EDA −/− mice exposed to chronic allergen challenge have attenuated airway fibrosis and AHR.

Aim

To determine whether blocking the EDA‐FN in‐vivo in wild type mice (genetically “intact”) attenuates the development of airway remodeling and airway hyperresponsiveness in a murine model of acute and chronic allergen‐induced “asthma”.

Methods

BALB/c female mice (6 groups, 5–10/group) were exposed to saline (Sal) or ovalbumin (OVA) (intraperitoneal sensitization followed by inhalations on alternate days for 1 or 4 weeks). Three of the groups received intraperitoneal EDA‐FN blocking antibody (every 5 days) beginning prior to sensitization. AHR in response to methacholine (Flexivent), bronchoalveolar lavage (BAL) cell counts and histological evaluation (H&E, Masson Trichrome) was performed 24h after the last inhalation followed by collagen content and cytokine expression in the lung tissue.

Results

OVA treated mice showed a significant increase in AHR (airway resistance) in both the acute (relative to baseline: 4.12±0.48) and chronic (3.93±0.36) groups in comparison with saline treated mice (2±0.1) and this increase was abolished by EDA‐FN blocking antibody in acute OVA treated mice (2.76±0.24, p<0.03), but not in the chronic model (OVA 3.93±0.36 vs OVA+Ab EDA‐FN 4.06±0.35). OVA exposed mice had marked BAL eosinophilia (acute: 77.5±2.8% and chronic 57.5±2.8%) that was significantly lowered by anti EDA‐FN treatment (58.8±3% acute and 39.3±3.2% chronic, p<0.001 for both acute and chronic) while antibody treatment alone was similar to saline. Histologic evaluation also showed less peribronchial inflammation in the acute model in response to blocking antibody but no reduction in the chronic model. The increase in lung collagen content seen in response to chronic OVA‐exposure was completely abolished in the presence of anti EDA‐FN antibody (93.4±4.6 mg collagen/50 mg tissue vs. 56.3±2.9 mg collagen/50 mg tissue, p<0.001 compared to SAL 66±7.6 mg collagen/50 mg tissue).

Conclusions

In a murine model of allergen induced “asthma”, inhibition of EDA‐FN attenuates airway hyperresponsiveness, airway fibrosis and airway inflammation, however these effects vary, depending on chronicity of allergen and antibody exposure.

Support or Funding Information

Israel Science Foundation
Estrogen Interferes with Cigarette Smoke Exposure‐Induced Proliferation in Human Airway Smooth Muscle Cells
Authors: Wang Shengyu , Goorhouse Katelyn J , Freeman Michelle R , Pabelick Christina M , Prakash Y.S. , Sathish Venkatachalem
Published: November 2023

Introduction

Asthma is acutely aggravated and chronically worsened by cigarette smoke (CS), as well as secondhand smoke exposure. In this regard, studies show significant differences between women vs. men in their response to the detrimental effects of CS, suggesting that female sex steroids (particularly estrogen) may be involved. Airway smooth muscle (ASM) cell proliferation and airway remodeling is a key feature of asthma and COPD. Surprisingly, there is little information regarding sex steroid effects on cigarette smoke‐induced changes of airway structure and function. In the current study, we hypothesize that estrogen interferes with CSE exposure‐induced proliferation, thereby contributing to airway smooth muscle remodeling.

Methods and Results

Human ASM tissues (epithelium‐denuded) and isolated ASM cells were from lung samples of patients with known smoking histories undergoing thoracic surgery for non‐infectious, focal pathology. Exposure of human ASM to cigarette smoke extract (CSE, Kentucky research cigarette 1RF4) 1%CSE or 2%CSE for 24h significantly increased ASM proliferation (proliferation assay: Cell Titer Aueous One, and Cyquant). Exposure of physiological concentrations of 17‐β‐estradiol (E2, 1nM) did not alter ASM proliferation at baseline. Interestingly, E2 pre‐treatment (1 h prior to CSE exposure) significantly reduced CSE‐induced human ASM proliferation. This was further confirmed by Western blot analysis for proliferative markers PCNA and Cyclin E. In addition, to explore the mechanistic bases of differential effects of E2 on ASM proliferation under basal and CSE‐exposed conditions, the estrogen receptor profile in ASM tissue was screened in parallel for patients with and without smoking histories. Human ASM tissue from smokers samples showed substantial increased ERα and β expression compared to non‐smokers. Inhibition of ERα and β prevented the effects of estrogen on CSE‐induced increases in proliferation.

Conclusions

Collectively, estrogen exposure decreased human ASM proliferation induced by CSE. ER expression and signaling is enhanced in CSE induced inflamed ASM, with a potential beneficial role in reducing remodeling with CS exposure. These results will lay the foundation for examining the role of estrogen signaling in pathogenesis of cigarette smoke‐induced airway remodeling and possible sex based therapeutic targets in smoking‐related diseases.

Support or Funding Information

Supported by Flight Attendants Medical Research Institute (VS) and NIH R01 grant HL123494 (VS)
EP1 Receptor Agonists Augment Hyperoxia‐Induced Airway Hyperresponsiveness
Authors: Sopi Ramadan B , Neziri Burim , Mladenov Mitko , Vela Driton , Lajçi Njomza , Thaqi Qendrim , Bislimi Kemajl , Jakupaj Muharrem , Lajçi Azem
Published: November 2023

Neonatal hyperoxia is associated with airway hyperresponsiveness. It was shown that hyperoxia upregulates prostaglandin E2 synthesis, which has contrasting physiological effects depending on subtypes of receptors and other conditions. We tested the hypothesis that EP1 receptors are involved in airway hyperresponsiveness induced by hyperoxia. Tracheal cylinders were obtained from 12 days Wistar rat pups exposed to hyperoxia (≥ 95% O2) or room air for seven days. These cylinders were used to study contractile responses of airway smooth muscle evoked by methacholine (MCh, 10−8 – 10−4 M) or electrical field stimulation (EFS, 0.5 – 62 Hz) in absence or presence of EP1 agonists (17‐phenyl‐trinor‐PGE2 (analog of PGE2), 1 μM; or PGE2, 1 μM) or an EP1 antagonist (SC19920, 10 μM). MCh and EFS induced dose‐dependent and frequency‐dependent contractile responses of airway smooth muscle, respectively. The contractile responses were significantly higher in preparations obtained from animals exposed to hyperoxia as compared to those obtained from controls exposed to room air, and these enhanced contractile responses in hyperoxic tissues were augmented in presence of EP1 agonists (17‐PGE2 or PGE2), but significantly attenuated by the EP1 antagonist (SC19920). The results of this study revealed that PGE2 – EP1 receptor signaling pathway contributes to hyperoxia‐induced airway hyperresponsiveness and we speculate that the use of EP1 antagonists might be an effective therapeutic approach to reverse the hyperoxia‐induced enhanced contractile responses of airway smooth muscle.

Support or Funding Information

Supported by MEST