bluuebunny/crossref_metadata_2025_split huggingface dataset:
Claudin‐23 Strengthens the Colonic Epithelial Barrier by Regulating Claudin‐3 and ‐4 proteins in the Tight Junction Plasma Membrane
Authors: Raya‐Sandino Arturo , Lozada‐Soto Kristen M. , Garcia‐Hernandez Vicky , Luissint Anny‐Claude , Koval Michael , Parkos Charles A. , Nusrat Asma 
Published: May 2022

Colonic epithelial barrier function is controlled by differential expression of Claudin (CLDN) proteins in the crypt‐luminal axis. We recently identified the expression of a CLDN family member, CLDN‐23, in intestinal epithelial cells (IECs). However, the role of CLDN‐23 in regulating epithelial barrier function has not been identified. Analysis of the human and murine intestinal crypt‐luminal axis revealed increased CLDN‐23 expression in differentiated IECs facing the lumen compared to the proliferative crypt‐base IECs. Similarly, CLDN‐23 expression was higher in differentiated human and murine IECs cultured as a monolayer on permeable supports compared to proliferative IECs cultured in a 3D matrigel matrix. To investigate the contribution of CLDN‐23 in regulating IEC barrier function in vivo, we generated inducible intestinal‐epithelia specific (Villin‐CreERT2) Cldn23 knockout mice (Cldn23ERΔIEC). Functional analysis displayed increased intestinal mucosal permeability to 4kDa FITC dextran, indicating compromised epithelial barrier function. To confirm this observation, we created human IECs in which CLDN‐23 expression was either silenced or enhanced in vitro. CLDN‐23 loss increased paracellular permeability to 4kDa FITC dextran and reduced transepithelial electrical resistance (TEER) consistent with a leaky barrier. Conversely, CLDN‐23 overexpression resulted in improved barrier function. Altogether, these data suggest that CLDN‐23 controls the IEC barrier function.Evaluation of CLDN protein interactions employing co‐culture of HeLa cells expressing CLDN‐23 with those expressing either CLDN‐2, CLDN‐3, or CLDN‐4 showed that CLDN‐23 interacts in trans with barrier‐forming CLDN‐3 and CLDN‐4, but not with pore‐forming CLDN‐2. Using in situ proximity ligation assay, we observed close association in cis between CLDN‐23 and barrier‐forming CLDN‐3 and CLDN‐4 in the TJ plasma membrane, but not with CLDN‐2. These findings suggest that CLDN‐23 interacts in both cis and trans with barrier‐forming CLDN‐3 and CLDN‐4 in differentiated IECs. Interestingly, the overexpression of CLDN‐23 in human IECs revealed up‐regulation of barrier‐forming CLDN‐3 protein expression in the TJ while decreasing the pore‐forming CLDN‐2 protein at this site. In conclusion, we identified that CLDN‐23 strengthens the colonic barrier properties by orchestrating the composition of CLDN protein interactions in epithelial TJs.
The Role of Scleraxis in Inducing Vascular Stiffness
Authors: Al‐Hattab Danah S. , Moffat Teri , Ledingham Allison , Czubryt Michael P. 
Published: May 2022

Vascular fibrosis (VF) is one of the most significant pathologies related to blood vessel dysfunction. VF results in an impairment of vascular tone due to an increase in extracellular matrix proteins (ECM) and vascular smooth muscle synthesis within the vessel wall, thus increasing vascular stiffness and reducing lumen diameter. VF is both caused by and exacerbates numerous vascular diseases including diabetes, atherosclerosis and in particular, hypertension, which accounts for 13% of deaths globally (WHO World Health Report, 2002). Multiple studies have investigated various therapeutic approaches to interfere with VF, but to date this condition remains untreated, thus novel approaches to target fibrosis development and progression are urgently required.Our lab identified the transcription factor scleraxis as a novel master regulator of cellular phenotype conversion. We demonstrated that scleraxis is sufficient to induce fibroblast to myofibroblast phenotype conversion, a critical step in the development of fibrosis in many tissue types. Scleraxis also induces Epithelial to Mesenchymal Transition (EMT) and cell migration as part of development and cell differentiation. Some studies have shown that mechanical stretch in tendons increases expression of scleraxis, while additional studies reported that stretch in rabbit aorta induces the vascular smooth muscle proliferative phenotype. Our preliminary data has revealed that scleraxis is expressed in VSMCs in the arterial wall, and we have found elevated scleraxis expression in high pressure versus low pressure regions of vessels. We thus hypothesize that scleraxis is sufficient and necessary to induce vascular fibrosis.Our data shows that scleraxis overexpression increases thickness of the aortic vascular wall in scleraxis overexpression mice, via histological sections, thus contributing to the progression of vascular fibrosis. In vitro studies show that scleraxis overexpression in Human Aortic Smooth Muscle Cells (HAOSMC) induces their proliferation and migration using different assays (CCk8/Edu+ proliferation assay, Transwell migration assay, gene expression assays). Scleraxis overexpression also induces hypertrophy of HAOSMC shown via immunostaining. This suggests that scleraxis overexpression induces VSMC plasticity from contractile to synthetic‐proliferative phenotype, thus inducing VSMC hyperplasia.New & Noteworthy: Our findings indicate that scleraxis plays a role in VSMC plasticity via scleraxis‐ mediated transcriptional regulation of VSMC proliferation and migration. This work has potential relevance to many related diseases that feature VSMC activation, including atherosclerosis and restenosis.
Does Cell Proliferation Affect Embryonic Flexure and Torsion?
Authors: Wang Dereck , Ramasubramanian Ashok 
Published: May 2022

Two vital and simultaneous morphogenetic events that occur during early embryo development are the flexure and torsion (F&T) of the embryonic body axis. Both contribute to development of the fetal position, which can be observed in avian and mammalian embryos. Correct progression of F&T is crucial for the proper development of the heart and other organs. Embryonic flexure can be divided into three segments: cranial, cervical, and thoracic. In this study, we focus on cervical flexure and torsion of the embryonic body axis. Differential cell proliferation, the difference in the rate of division of cells between regions in the developing embryo, is one mechanism that could influence F&T. Goodrum & Jacobson (1981) observed thickening of the dorsal midbrain relative to the ventral midbrain during cranial flexure. Miller & White (1998) have shown using mouse embryos that differential growth could cause torsion of an axis confined to an attached sphere.We performed studies inhibiting cell proliferation. Fertile white leghorn chick eggs were incubated to approximately Hamburger and Hamilton (HH) Stage 12 or 13. At these stages, F&T have just begun. We then added aphidicolin, a mitotic inhibitor, to whole embryo culture. Embryos were observed at 0, 5, and 10 hours to see if F&T were repressed. We hypothesized that the addition of aphidicolin would inhibit progression of F&T. We developed quantitative schemes to score the extent of F&T progression from HH 12 to HH 15. Embryos were scored from 0 to 4 based on the degree of cervical flexure and were also scored from 0 to 4 based on the degree of torsion. Afterward, the difference in F&T scores of same embryos at time t=0 h and t=10 h was calculated to determine the extent of both. These scores were then averaged for multiple embryos.Compared with controls (n=46), embryos incubated in 400 µM aphidicolin (n=18) flexed and rotated at a significantly lower rate. For flexion, the mean difference in scores for control and treated embryos were 1.85 and 1.22, respectively. For torsion, the mean difference in scores for control and treated embryos were 1.62 and 1.25, respectively. These differences were statistically significant. It is noted that global application of aphidicolin did impact the overall health of embryos. However, our preliminary results indicate that differential cell proliferation could play a role in rotation and flexure progression in the developing chicken embryo.References:Goodrum, G. R., & Jacobson, A. G. (1981). Cephalic flexure formation in the chick embryo. Journal of Experimental Zoology, 216(3), 399‐408. doi:10.1002/jez.1402160308Miller, S. A., & White, R. D. (1998). Right‐Left Asymmetry of Cell Proliferation Predominates in Mouse Embryos Undergoing Clockwise Axial Rotation. The Anatomical Record, 250(1), 103‐108. doi:10.1002/(sici)1097‐0185(199801)250:13.0.co;2‐s
Distinct Wnt Signaling Thresholds Required for Polyposis in the Proximal and Distal Stomach
Authors: Delgado Elizabeth , Keeley Theresa M. , Hibdon Elise S. , McGowan Kevin P. , Gillilland Merritt G. , Colacino Justin A. , Stoffel Elena , Samuelson Linda C. 
Published: May 2022

ObjectiveThe Wnt pathway is critical for gastrointestinal stem cell proliferation. Patients with Familial Adenomatous Polyposis (FAP) have germline mutations in the adenomatous polyposis coli (APC)gene, resulting in dysregulated Wnt signaling and a polyposis syndrome with stark regional differences in disease severity. In the stomach, the proximal corpus contains numerous polyps, while few polyps are located in the distal antrum. Here, we tested the hypothesis that there are regional differences in sensitivity to Wnt signaling along the gastric proximal‐distal axis, and that a ‘just‐right’ level of Wnt signaling is required for gastric polyposis.MethodsTo model gastric polyp formation in FAP patients, we used mice with an inducible Apcloss‐of‐function mutation. Gastric tissue responses were studied one month after maximal Wnt activation in homozygous Sox2‐CreERT2; Apc580flox/flox(APC‐/‐) and submaximal Wnt activation in heterozygous Sox2‐CreERT2; Apc580flox/+ (APC+/‐) mice. To gain insight into regional differences in Wnt response, RNA sequencing was performed on corpus and antral organoids treated with the Wnt inhibitor XAV939.ResultsIn the distal stomach, we observed hyperproliferation and tissue expansion in homozygous APC‐/‐ mice with no change in heterozygous APC+/‐mice. In contrast, the proximal stomach did not show an effect in homozygous APC‐/‐ mice but showed a modest increase in proliferation in heterozygous mice. PCR analysis of APC‐/‐ tissue confirmed APC gene deletion in the antrum, but not corpus, suggesting regional differences in maintenance of homozygous mutant stem cells. Following acute Wnt inhibition, corpus and antral organoids shared many Wnt‐regulated genes. However, Gene Ontology Enrichment analysis showed genes associated with cell differentiation, cell population proliferation, and cell cycle regulation were upregulated in antral organoids but downregulated in corpus organoids. KEGG analysis indicated downregulation of gastric cancer genes in both tissue organoids; however, only antral organoids showed downregulation of signaling pathways regulating pluripotency.ConclusionThe corpus and antrum exhibit differences in the Wnt signaling threshold required to initiate polyposis. Whereas submaximal levels of Wnt signaling are sufficient to initiate growth in the corpus, maximal levels are needed for antral hyperproliferation. Notably, ‘high‐Wnt’ stem cells are retained in the antrum and lost from the corpus, suggesting regional differences in Wnt regulation of gastric stem cells. Our findings in mouse highlight how heterozygous APC mutations may be sufficient for proximal stomach polyposis—a clinically relevant observation, as the vast majority of FAP patient gastric polyps are benign sessile growths located in the proximal stomach, with less frequent antral adenomatous polyps more closely associated with gastric cancer.
Lithium‐associated nephrotoxicity: role of GDF15‐mediated collecting duct cells proliferation
Authors: Tabibzadeh Nahid , Cheval Lydie , Morla Luciana , Chevillard Lucie , Megarbane Bruno , Bienaime Frank , Vidal‐Petiot Emmanuelle , Crambert Gilles 
Published: May 2022

Lithium (Li+) salts are the most effective treatment of bipolar disorder (BD), affecting 1 to 4% of the population worldwide. However, this efficacy is counterbalanced by adverse long‐term events, mostly renal effects. Lithium treatment discontinuation is usually not an option as it substantially increases suicidal risk. Understanding the sequence of events in the kidneys might thus improve therapeutic strategies while maintaining this treatment.Li+‐induced nephrogenic diabetes insipidus (NDI) is the earliest and the most frequent side effect of chronic lithium use, leading to polyuria and polydipsia. Li+ treatment decreases AQP2 expression both in vivo and in vitro. Li+ treatment also induces a microcystic tubulointerstitial nephropathy eventually leading to end‐stage kidney disease. The mechanisms of tubular toxicity and microcyst formation have been related to a proliferative effect of Li+. The time course, the site and the mechanisms of this proliferation are still a matter of debate.Previous studies from our lab have shown that acidosis‐induced collecting duct cell proliferation depended on the overexpression of GDF15 by principal cells, especially in the early phase of linear proliferation along the tubular axis.Our objective during this project is to determine the pathways leading to planar and cystic proliferation of collecting duct cells, in particular GDF15 signaling, and their role in lithium nephrotoxicity, and to identify potential biomarkers that could help to predict the course of Li‐induced nephropathy.We have established a rat and a mouse experimental model treated with therapeutic concentrations of Li+, displaying as expected polyuria and polydipsia, with microcystic tubular dilations and a global proliferation of collecting duct cells (both principal and intercalated) on kidney slices and on microdissected collecting ducts, with an increase in the intercalated/principal cells ratio (figure). We examined the effect of Li+ in GDF15‐/‐ and wild type (WT) mice and found significantly lower polyuria and polydipsia in GDF15‐/‐ mice, along with a higher urine osmolality, and decreased intercalated/principal cell ratio compared to WT mice (figure).As collecting duct cell proliferation is mediated by GDF15, we studied urinary GDF15 levels (GDF ELISA kit) in Li+‐treated patients with proven diabetes insipidus and found them significantly increased compared to control patients (p<0.01) (figure).In conclusion, these results suggest a direct relationship between lithium‐induced proliferation signals, collecting duct cell dysfunction and GDF15 expression. Further investigation is needed to determine if GDF15 signaling might represent a therapeutic target in Li+‐induced nephrotoxicity.
Reinforcing Effects of Fentanyl and a Fentanyl Analog Found in Illicit Drug Markets
Authors: McGriff Shelby A. , Thorndike Eric , Chojnacki Michael , Baumann Michael , Schindler Charles 
Published: May 2022

HYPOTHESISIllicitly manufactured fentanyl and fentanyl analogs are the main drivers of the worsening opioid epidemic. Although the pharmacology of fentanyl is well characterized, there is little information about the reinforcing effects of newly emerging fentanyl analogs that are contributing to opioid overdose. Here, we compare the effects of fentanyl and the clandestine fentanyl analog, cyclopropylfentanyl, on drug self‐administration behavior in male and female rats.METHODSSprague‐Dawley rats fitted with intravenous (i.v.) jugular catheters were placed in experimental chambers containing two nose poke holes. Active nose poke responses resulted in delivery of a constant volume of drug (0.2 mL) over a period of 2 s on a fixed‐ratio 1 schedule, followed by a 20 s timeout. Acquisition doses for the compounds were 0.01 mg/kg/inj for fentanyl and cyclopropylfentanyl, while 0.1 mg/kg/inj was used for heroin. Following 10 days of acquisition training, dose‐effect testing began with three additional doses, each of which were tested for 3 days. Subjects then returned to the original training dose until responses stabilized and underwent saline extinction.RESULTSAcquisition of fentanyl self‐administration was achieved by both male and female rats, with significant differences between active versus inactive nose pokes for both sexes over the last 4 days of training (p<0.05). A similar profile of acquisition was seen for heroin and cyclopropylfentanyl. Both fentanyl and cyclopropylfentanyl showed a typical inverted‐U dose‐effect function during dose‐effect testing. Maximal response rates were similar among all drugs, with fentanyl showing maximum responding at 0.001 mg/kg/inj, cyclopropylfentanyl at 0.003 mg/kg/inj, and heroin at 0.003 mg/kg/inj.CONCLUSIONBoth male and female rats showed comparable rates of fentanyl acquisition. Further comparison between heroin, fentanyl, and cyclopropylfentanyl show that these compounds support self‐administration, indicating that emerging fentanyl analogs display significant abuse liability and may contribute to compulsive use in humans.
Investigating the Molecular Mechanisms Driving 7α,25‐dihydroxycholesterol‐GPR183‐Induced Hypersensitivity
Authors: Braden Kathryn , Campolo Michela , Chen Zhoumou , Giancotti Luigi , Doyle Timothy M. , Esposito Emanuela , Zhang Jinsong , Cuzzocrea Salvatore , Arnatt Christopher K. , Salvemini Daniela 
Published: May 2022

Neuropathic pain is a debilitating health concern and there is an urgent need for non‐opioid analgesic targets. Our group has identified GPR183 as a novel potential therapeutic target for neuropathic pain. GPR183 is a G‐protein coupled receptor that promotes the migration of immune cells in response to its ligand, 7α,25‐dihydroxycholesterol (7α,25‐OHC). We have shown that GPR183 is upregulated in the dorsal horn spinal cord during neuropathic pain states in rodents and intrathecal injections of 7α,25‐OHC is able to induce allodynia in mice in a GPR183‐dependent manner. However, the mechanism by which GPR183 activation leads to pain is unknown. These studies aim to elucidate the molecular signaling pathways that contribute to 7α,25‐OHC‐induced hypersensitivity. Based on previous literature, we hypothesized that GPR183 activation in the spinal cord would activate mitogen‐activated protein kinases (MAPKs), extracellular signal‐regulated kinase (ERK) and p38, leading to the production of neuroexcitatory cytokines contributing to hypersensitivity.MethodsTo investigate the signaling pathways downstream of GPR183 activation in the spinal cord, we performed unbiased RNA‐Sequencing following intrathecal injections of 7α,25‐OHC with or without the GPR183 antagonist, SAE‐14, in male and female ICR mice. We then performed follow‐up studies giving pharmacological inhibitors of the identified pathways prior to intrathecal 7α,25‐OHC to confirm functional relevance. We also took spinal cord tissue for western blot and enzyme‐linked immunoassay (ELISA) analysis following intrathecal injections.ResultsThrough our unbiased RNA‐sequencing we found that many of the genes that were differentially regulated by GPR183 activation contain binding sites for NF‐κB or transcription factors that are regulated by the MAPK’s, ERK or p38. Additionally, we found that small molecule inhibitors of these pathways delayed 7α,25‐OHC‐induced mechanical allodynia. Importantly, the GPR183 antagonist, SAE‐14, blocked the 7α,25‐OHC‐induced upregulation of these pathways, including the upregulation of neuroexcitatory cytokines interleukin‐1β and tumor necrosis factor α.ConclusionThese findings provide novel insight into how spinal GPR183 signaling produces hypersensitivity and can contribute to neuropathic pain states.
Activation of satellite glia in stellate ganglia from chronic heart failure rats
Authors: Tu Huiyin , Zhang Dongze , Wadman Michael C. , Li Yu‐Long 
Published: May 2022

Cardiac sympathetic overactivation is linked to ventricular arrhythmogenesis and is associated with mortality in patients with chronic heart failure (CHF). However, the sites and mechanisms responsible for increased cardiac sympathetic activity are still poorly understood. Our previous studies showed that CHF‐elevated proinflammatory cytokine levels and macrophage expansion in stellate ganglia are critical factors for cardiac sympathoexcitation. Here we tested if activation of satellite glial cells (SGCs) contributes to macrophage expansion in stellate ganglia from CHF rats.Rat CHF was induced by surgical ligation of the left anterior descending coronary artery. SGCs and postsynaptic sympathetic neurons were respectively identified by anti‐S100B and anti‐tyrosine hydroxylase (TH) antibodies with immunofluorescence staining. As a marker of SGC activation, glial fibrillary acidic protein (GFAP) was measured by immunofluorescence double staining and Western blot analysis.Sympathetic neurons in stellate ganglia from both sham and CHF rats were totally surrounded by S100B‐positive SGCs. However, However, a few GFAP‐postive SGCs covered sympathetic neurons in sham stellate ganglia, whereas more GFAP‐positive SGCs surrounded sympathetic neurons in CHF stellate ganglia. Western blot data showed that GFAP was elevated in stellate ganglia from CHF rats, compared to sham rats.From these results, we conclude that activation of SGCs in stellate ganglia is higher in CHF rats than that in sham rats, which might contribute to the neuroinflammation and sympathoexcitation in stellate ganglia.
L‐Citrulline Attenuates Inflammation Induced Phenotypic Changes in Neonatal Rat Alveolar Type 1 Cells
Authors: Mohammadi Atefeh , Gauda Estelle B. 
Published: May 2022

IntroductionBronchopulmonary dysplasia (BPD) is a chronic lung disease initiated by inflammation and oxidative stress during the early stages of lung development, disrupting alveolar growth. BPD damages alveolar Type 1 (AT1) epithelial cells, causing decreased alveolarization and impaired gas exchange. Recently, it was shown that AT1 cells undergo cell reprogramming into surfactant producing alveolar Type 2 cells (AT2) in hyperoxic and hypoxic lung injury models, causing significant structural damage in the neonatal lung. While current treatments have improved neonatal care, they cause significant side effects. L‐citrulline, a naturally occurring amino acid, protects the newborn rat lung from injury, preserving alveolar growth in various neonatal BPD models. Due to the significance of inflammation in BPD, we sought to investigate the effect of inflammatory stimuli, lipopolysaccharide (LPS) and tumour necrosis factor α (TNF‐α), and L‐citrulline treatment on AT1 cell programming in vitro. We hypothesize that L‐citrulline treatment will prevent inflammation‐induced AT1 cell reprogramming in vitro.MethodsAT1 cells were isolated from newborn rats on postnatal day 4 using fluorescence activated cell sorting. AT1 cells were pre‐treated with L‐citrulline (4mM/mL or 10mM/mL) for 1hr prior to LPS (10µg/mL), TNF‐α (5ng/mL), or a combination of LPS and TNF‐α for 24hrs in vitro. Whole cell lysates were prepared for immunoblotting. AT1 cells were also assayed for the level of injury‐induced cell viability using the MTT assay.ResultsAT1 cell lysates from the LPS+TNF‐α group had significantly decreased RTI‐40 protein expression compared to the control group (p<0.04 LPS+TNF‐α vs. control, n=6). In the presence of L‐citrulline, RTI‐40 protein expression of cells exposed to LPS+ TNF‐α was maintained (p<0.02 L‐citrulline+LPS+TNF‐α vs. LPS+TNF‐α, n=4) (Figure 1). AT1 cell viability in LPS, TNF‐α, and the combined exposure group was comparable to the control group (n=8) (Figure 2).ConclusionOur preliminary data strongly suggests that L‐citrulline treatment mitigates inflammation‐induced cell reprogramming in neonatal AT1 cells. Our findings show that the injury‐induced decrease in RTI‐40 expression is not due to reduced AT1 cell viability, rather due to phenotypic changes. Given the importance of AT1 cells in alveolarization, gas exchange, and BPD pathogenesis, characterizing the cell specific effects of L‐citrulline on AT1 cell reprograming is considerably critical and highly relevant to identifying a safe and effective treatment for premature infants that can mitigate the effects of inflammation on the developing lung.