bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell‐Free Hemoglobin Increases Lung Alveolar‐Capillary Barrier Permeability Via TLR4
Authors: Shaver Ciara M , Putz Nathan D , Bastarache Julie A , Ware Lorraine B 
Published: November 2023

ObjectiveElevated levels of cell‐free hemoglobin (CFH) are present in the airspace of patients with ARDS and are associated with disruption of the alveolar‐capillary barrier. Intra‐tracheal delivery of CFH is sufficient to disrupt alveolar permeability in the lung. However, the mechanisms by which this occurs are unknown. TLR4 expressed on macrophages is a key mediator of the inflammatory effects of CFH, but the role TLR4 in CFH‐induced lung epithelial injury has not been studied. We hypothesized that TLR4 would be a critical mediator of CFH‐induced alveolar‐capillary barrier permeability.MethodsTLR4KO or wild‐type (WT) control mice were treated with intra‐tracheal ultrapure endotoxin‐free CFH (100μg). After 24 hours, bronchoalveolar lavage (BAL) was performed. Inflammation was assessed by manual cell counts with differentials and by cytokine expression (ELISA). Alveolar‐capillary barrier function was assessed by BAL protein (BCA assay). Lung epithelial injury was assessed by BAL RAGE (ELISA). For in vitro studies, MLE‐12 mouse lung epithelial cells were treated with CFH (1mg/mL) for 24 hours and paracellular permeability measured by electrical cell‐substrate impedance sensing (ECIS) in the presence or absence of TLR4 inhibitors.ResultsAs compared to WT mice, TLR4KO mice had attenuated alveolar‐capillary barrier disruption in response to CFH as measured by BAL protein (476±71μg/mL vs. 291±58, p<0.001) (Figure 1). TLR4KO mice also had a 76% reduction in BAL RAGE levels (p<0.001), suggesting that TLR4 mediates lung epithelial cell injury in response to CFH. In addition, TLR4KO had markedly reduced airspace inflammation (p<0.001). To further study whether CFH had direct effects on lung epithelial cells, MLE‐12 cells were exposed to CFH in the presence or absence of TLR4 inhibitors (antibody or small molecules). Whereas CFH caused decreased resistance across the epithelial monolayer, inhibition of TLR4 binding or signaling had no effect on CFH‐mediated permeability in vitro.ConclusionsCell‐free hemoglobin disrupts the alveolar‐capillary barrier in the mouse lung and disrupts paracellular permeability in cultured lung epithelial cell monolayers. TLR4 is a critical mediator of lung epithelial cell injury in vivo and contributes to alveolar‐capillary barrier integrity. Inhibition of TLR4 on epithelial cells did not impact paracellular permeability in vitro. Together, these results demonstrate that CFH has both TLR4‐dependent and TLR4‐independent effects on lung epithelial cells.Support or Funding InformationNIH HL103836, HL126671 and HL087738; Vanderbilt Faculty Search Scholars; American Thoracic Society
NADPH‐Oxidase Signaling is Dysregulated in Thromboxane Analog‐Induced Intrauterine Growth Restricted Mice Versus Appropriately Grown Mice
Authors: Robbins Mary E , Cardona Herminio J , Taylor Joann M , Perez Marta , Fung Camille , Farrow Kathryn Noel 
Published: November 2023

BackgroundInfants with intrauterine growth restriction (IUGR) are at increased risk for bronchopulmonary dysplasia (BPD) and BPD‐associated pulmonary hypertension (PH), but this remains poorly understood. NADPH‐oxidase (Nox) have been implicated as a source of oxidative stress in both BPD and PH by others. More recently, oxidative stress has been proposed to be increased in IUGR pregnancies.ObjectiveTo determine if Nox signaling is disrupted in IUGR mice relative to appropriately grown mice.MethodsPregnant C57Bl6 mice had a micro‐osmotic pump containing U‐46619 (2000 ng/h) or vehicle (controls) implanted at E12.5 through term. U‐46619 dams had gestational hypertension. U‐46619 pups weighing ≤1.266 g at birth (<10th percentile) were considered growth restricted (GR). GR and control mice were placed in 21% O2 (room air, RA) or 75% O2 (chronic hyperoxia, CH) within 24h of birth and sacrificed at 14d. Lungs were inflation fixed and stained with hemotoxylin and eosin or harvested for tissue. At 14d, lung morphometry was assessed. Lung Nox 1, Nox2, and Nox4 expression was measured by Western blot, normalized to β‐actin.ResultsCH‐GR mice had significantly increased alveolar area and decreased alveolar counts relative to CH‐control mice (1426±219 vs. 1023±71 μm2; 26±3.4 vs. 34±2 alveoli per hpf), and RA‐GR had a trend towards increased alveolar area and significantly decreased alveolar counts relative to RA‐control mice (738±30 vs. 611±32 μm2; 48±1.5 vs. 55±2 alveoli per hpf). The RA‐GR mice had significantly less lung Nox1 and Nox4 protein expression relative to RA‐control mice, but there was no difference in lung Nox2 expression between GR and control mice in RA. Hyperoxia increased lung Nox2 and Nox4 expression in control mice, but not GR mice.ConclusionsGR mice have disrupted lung Nox protein expression relative to control mice. GR mice have less Nox1 and Nox4 in RA and less Nox2 and Nox4 in CH. Unexpectedly, these findings suggest less oxidative stress in the GR mice even though they have more alveolar simplification both in RA and CH. The mechanism of Nox dysregulation remains to be elucidated in these mice. Others have implicated epigenetic regulatory mechanisms for the Noxs. Given aberrant promotor DNA methylation, histone modifications along the body of the gene, and nucleosome positioning have been demonstrated to prevent upregulation of other genes in our IUGR model, we speculate that these epigenetic mechanisms may also be involved in the inability to upregulate the Noxs after IUGR.Support or Funding InformationNIH HL109478
Increased Activation of M2 Macrophages in 1 Year‐Old Rat Lungs Following Neonatal Hyperoxia Exposure
Authors: Yoder Hannah E. , Braun Rudolf K. , Sobotik Adelaide , Tetri Laura H. , Goss Kara N. , Eldridge Marlowe W. 
Published: November 2023

RationaleMacrophages are activated in response to insult. M1 macrophage polarization is associated with inflammation and tissue deterioration, whereas the M2 macrophage corresponds to wound repair and angiogenesis and has an anti‐inflammatory phenotype. Lipopolysaccharide (LPS) or interferon gamma (IFNγ) promote M1 differentiation and up‐regulate pro‐inflammatory cytokines like interleukin‐ (IL‐) 1β, IL‐6, and IL‐12 whereas M2 differentiation is regulated by IL‐4. Since hyperoxia exposure induces inflammation leading to the pathology seen in bronchopulmonary dysplasia (BPD) through induction of M1 macrophages, we tested the macrophage population in lung tissue of 1 year‐old rats. We postulated an increased activation of M2 macrophages in lungs of hyperoxia‐exposed rats, suggesting a continued wound healing response. Collagen levels were also assessed. We expected to see an increase in collagen levels of hyperoxic rats, suggesting that there is persistent phenotype after one year.MethodsNewborn rats were exposed to 85% hyperoxia (Hx) or normoxia (Nx) (21%) for 14 days and then kept at Nx conditions. At one year, the lung was lavaged with PBS, the left lung was inflated and fixed (4% formaldehyde). Cells were isolated using a 100 μm cell strainer, red blood cells were lysed (ammonium chloride), and the cells frozen in freezing medium. For flow cytometry, the cells were stained for CD45, CD24, CD163, CD80, CD86, CD204, and CD206. Analysis was done with an LSR‐2. Collagen levels were assessed using Masson's trichome‐stained lung sections. A blinded third party divded each image into grids and ranked each square on a scale of 0–3 (0=none, 3=severe) for fibrosis.ResultsAnalysis of CD80 and CD86 showed an increased proportion of the M1 macrophages in the Hx lung compared to the Nx lung (11.7% higher in Hx). The Hx M1 showed more of activation marker CD163 compared to the Nx M1 in the CD24− cell population (12% higher in Hx). Similarly, the proportion of CD204+ M2 was increased within the CD45+ cell population of Hx (6.3% higher in Hx). Comparing M1 to M2 macrophages, Hx exposure shifted the ratio of macrophages toward an M2 response. These findings were supported by the histologic assessment of collagen showing increased lung collagen in Hx compared to Nx rats (p=0.008).ConclusionOne year after neonatal Hx exposure, adult rat lungs demonstrate a macrophage population shift towards M2, suggesting a continued anti‐inflammatory response. Further, the increased level of collagen in Hx rats suggests that there is persistent damage after one year. The data indicates that humans born preterm with BPD may have long term alterations in inflammatory and repair pathways.Support or Funding InformationNHLBI, R01 HL115061‐03, Suppl (Eldridge) & NHLBI, R01 HL115061 (Eldridge)
Comparison of treatment efficacy in the hypoglossal motor system of AAV‐DES‐coGAA and novel vector AAV‐DES‐IGFIIcoGAA in Gaa−/− mice
Authors: Doyle Brendan Michael , Turner Sara M.F. , Poirier Amy , Jorgensen Marda L , Falk Darin J. , Doerfler Phillip A. , Byrne Barry J. , Fuller David D. 
Published: November 2023

Pompe disease is a neuromuscular disorder characterized by a systemic lack of the glycogen metabolizing enzyme acid‐alpha glucosidase. The result is extensive intracellular lysosomal glycogen accumulation and disruption of cellular architecture and function. The hypoglossal (XII) motor system is particularly susceptible to pathology in Pompe disease and both tongue and XII motoneurons show substantial pathology early in the disease progression. Patients often present with macroglossia and tongue weakness, and animal models show pathology in tongue muscles and XII motoneurons. Initial clinical testing indicates that intramuscular gene therapy with adeno‐associated virus (AAV) is safe in Pompe patients and recent work in animal models demonstrates that AAV serotype 9 (AAV9) can produce persistent transgene expression in both tongue muscle and XII motoneurons following a single tongue injection. In ongoing studies, we are using Pompe mice (Gaa−/−) to evaluate the impact of a modified GAA protein with expression being driven by the Desmin promoter. The GAA protein has been modified to allow for IGFII receptor mediated uptake (IGFIIcoGAA) to improve cellular and lysosomal uptake. Initial tests in Gaa−/− mice indicate increased GAA activity without a concomitant increase in antibody response following AAV9‐IGFIIcoGAA delivery to the tibialis anterior muscle. To determine if the new vector offers improved efficacy in the tongue motor system, adult Gaa−/− mice were injected into the base of the tongue with 7μL (1e11 vg/μL) of AAV9‐DES‐coGAA or AAV9‐DES‐IGFIIcoGAA, diluted to 20 μL in lactated ringers (LR), or an equivalent sham injection (20 μL LR). Tongue, medulla and XII nerve tissues were harvested 3‐mo post‐injection. Tissues were paraffin embedded, cut at 7μm and stained with Periodic acid‐Schiff (PAS) to visualize glycogen. Initial qualitative evaluation of tongue histology confirms that both vectors achieved significant clearance of glycogen from tongue myofibers. This was illustrated by the absence of PAS staining at the site of injection and the absence of the prototypical Gaa−/− muscle histopathology characterized by vacuolization and disrupted cellular architecture. Ongoing work is focused on evaluating XII nerve and motoneurons for quantitative comparison of tissues from the three treatment groups.Support or Funding Information2R01HD052682‐06A1 (DDF and BJB)
State‐Dependent Expiratory Abdominal Activity During Hypercapnia in Adult Unanaesthetized Rats
Authors: Leirao Isabela P , Silva Carlos AS , Gargaglioni Luciane H , da Silva Glauber SF 
Published: November 2023

Expiration at rest is a passive process. Expiratory muscles (ex: abdominals) are recruited in situations where respiratory demand is increased, such as during hypercapnia, leading to emergence of active expiration (A.E.). The present study tested: (i) the hypothesis that A.E. occurs in a state‐dependent manner (sleep‐wake cycle) during hypercapnia exposure in adult rats and (ii) the functional effects of A.E. in the pulmonary ventilation (VE). Wistar rats (280–330g) were implanted with electrodes for electroencephalography (EEG) and electromyography (EMG) of the neck, diaphragm (DIA) and abdominal (ABD) muscles. A.E. events were considered as rhythmic contraction of the abdominal muscle interposed to diaphragm (i.e., the expiration period). VE was measured by the barometric method. At least eight days after surgery, animals were submitted to the experimental protocol. Animals were allowed to acclimate in the chamber (~30 min). After this period, EEG and EMG wires were connected and animals were exposed (~60 min) to room air and hypercapnia (7% CO2). EEG, EMG and VE were recorded. The results demonstrate that regardless of state, there was no A.E. during room air. During hypercapnia, A.E. occurred in both states, wakefulness and sleep. The DIA‐to‐ABD ratio indicated that the occurrence of A.E. was predominant during non‐REM sleep. When A.E. was present, the VE had a positive correlation (p<0.05) with peak amplitude of ABDEMG activity. The higher the amplitude of ABDEMG, the higher was VE values. Furthermore, in both wakefulness and sleep, VE was also higher (p<0.05) in periods with A.E. compared to periods in which there were no A.E. events These data indicate the predominant expression of A.E. during sleep and its functional role to support VE in conditions of increased respiratory demand.Support or Funding InformationSao Paulo Research Foundation (FAPESP #2013/17606‐9)
Aging‐associated Abnormalities of Diaphragm Contractile Function in Wistar‐Kyoto Rats
Authors: Novielli Nicole M , Foster Andrew J , Huber Jason S , Murrant Coral L , Simpson Jeremy A 
Published: November 2023

Aging presents a decline of respiratory function and is a significant contributor to aging‐associated morbidity and mortality. Specifically, reduced diaphragm muscle function can impair inspiratory force, promote insufficient ventilation during increased physical demand, and contribute to poor airway clearance. Rodent models of aging provide a means to directly assess and characterize abnormalities in diaphragm function; however, rodent models of aging can also possess concomitant pathological states, which should not be disregarded. The Wistar‐Kyoto rat (WKY) can exhibit modifications in cardiovascular function with advanced age. Both cardiovascular and respiratory functions are linked, and can deteriorate over a lifespan. Thus, we used a comprehensive evaluation of cardiac and respiratory function to investigate age‐related abnormalities of hemodynamic and respiratory muscle function. Diaphragm strips from young (~0.4yrs; n= 7, 14 strips) and aged (~2.3yrs; n=8, 14 strips) male WKY were used to investigate in vitro diaphragm force‐frequency (1–120Hz) relationship, fatigue and recovery. Aged diaphragm demonstrated ~30% decrease in force production at lower ranges of stimulation frequencies (i.e., 1–40Hz), compared to young diaphragm. Peak rate of force production (dT/dtmax, index of contractility) was also reduced by up to ~37% (p<0.05) at lower stimulation frequencies, compared to young diaphragm. In response to fatiguing stimulation, young and aged diaphragm force was equivalently reduced. While both aged and young diaphragm recovered to the same level of force, aged diaphragm recovered to it's maximal force ~30% faster than young diaphragm, where increases in force (normalized to maximal force) were up to 10% greater in aged diaphragm over time (p<0.05). In effort to investigate whether changes in force‐frequency relationship between adult and aged rats was specific to respiratory muscle, we also investigated the contractile properties of soleus (predominantly type I; slow‐twitch) and extensor digitorum longus (EDL; predominantly type IIb; fast‐twitch) muscles. Aged soleus demonstrated ~50% reduction in force production over 20–90Hz stimulation frequencies, compared to young soleus (p<0.05). In contrast, force‐frequency curves were similar between young and aged EDL. Herein, these findings demonstrate a right‐ward shift in the force‐frequency curve generated by aged diaphragm, accompanied by impaired rate of force production at lower stimulus frequencies. Maximal force production was conserved between young and aged diaphragm, thus mechanistic impairments of aged diaphragm are likely attributable to augmented calcium release, prior to the onset of deficits in myofilament function with aging.Support or Funding InformationCIHR and NSERC
Altered Neuromuscular Junction Protein Expression in the Diaphragm of Mechanically Ventilated Patients
Authors: Smuder Ashley J , Levine Sanford , Powers Scott K 
Published: November 2023

Mechanical ventilation (MV) is a life‐saving intervention used to sustain alveolar ventilation by inflating the lungs via positive pressure, thus eliminating muscular activity of the diaphragm and leading to the rapid development of diaphragm weakness. This condition is referred to as ventilator‐induced diaphragm dysfunction (VIDD), and results from MV‐induced diaphragm atrophy and contractile dysfunction. The diaphragm is the primary inspiratory muscle in humans and diaphragm weakness is predicted to be a major factor that contributes to the inability to wean patients from the ventilator. Therefore, understanding the mechanisms responsible for VIDD is critical in order to develop a clinical intervention to protect against VIDD. In this regard, contractile activity of the diaphragm is regulated by the transmission of action potentials from the phrenic motoneuron. Transmission occurs at the neuromuscular junction (NMJ), and impairment of the NMJ can result in muscle weakness. Therefore, we tested the hypothesis that prolonged MV results in inactivity‐induced alterations to NMJ protein expression. Diaphragm muscle biopsies were obtained from 8 patients undergoing surgery (control subjects) and 8 brain‐dead organ donors (case subjects). Control subjects were mechanically ventilated for 2–3 hours whereas case subjects were ventilated for 18–69 hours. To determine if prolonged MV alters protein expression of proteins involved in the NMJ, we measured the expression of proteins critical to NMJ function in the diaphragm. Our results reveal that MV results in a statistically significant increase in both mRNA (+5.02 ± 1.45 fold) and protein (+183.8 ± 26.5 percent) abundance of the receptor tyrosine kinase, MuSK. In contrast, prolonged MV promoted a significant decrease in Agrin mRNA (−0.42 ± 0.09 fold) and protein (−77.0 ± 6.52 percent) expression. In addition, analysis of mRNA expression revealed significant increases in the acetylcholine receptor (AchR) subunits AchRα (+5.34 ± 1.25 fold) and AchRβ (+2.21 ± 0.56 fold) in the diaphragms of case subjects. This data suggests that MV results in NMJ instability, and that proper signaling at the NMJ may be required to prevent diaphragmatic contractile dysfunction following prolonged MV.Support or Funding InformationSupported by NIH R01 AR064189 awarded to SKP
Mechanical And Chemical Factors Required For Maintaining Cardiac Rhythm In Drosophila Melanogaster Larva
Authors: de Castro Clara , Titlow Josh , Majeed Zana R , Malloy Cole , King Kayla , Cooper Robin 
Published: November 2023

Cardiac‐intrinsic mechanisms for regulating heart rate (HR) in response to stretch have been characterized in many different organisms. However, the molecular mechanisms for mechanosensitive regulation are not yet fully understood. Drosophila melanogaster larva, with its simple and genetically tractable circulatory system, is well‐suited to investigate the functional role of stretch‐activated channels (SACs) and chemical modulators in regulating heart rate. Here we establish an exposed heart preparation that delivers continuous mechanical stimulation in the presence of pharmacological manipulations. Continuous mechanical stimulation substantially stabilized the HR over 2hr incubation compared to stagnant incubation with the same saline. The addition of a chemical modulator cocktail further enhanced HR stability in perfused preparations. To determine the role of mechanosensitive regulation in prolonged HR stability, we assayed various pharmacological SAC blockers, e.g., amiloride, gadolinium, ruthenium red, streptomycin, and tarantula toxin. The relative efficacy of these blockers on mechanically stabilized HR further establishes Drosophila larva as an appropriate system for investigating cardiac mechanosensation and provides novel insight into the underlying SACs.Support or Funding InformationPersonal Funds (RLC)
Low Phosphate Diet Prevents Klotho Deficiency‐induced Aortic Aneurysm
Authors: Wang Qiongxin , Lin Yi , Ali Quaisar , Sun Zhongjie 
Published: November 2023

BackgroundMutation of Klotho gene causes aberrant mineral metabolism characterized by severe hyperphosphatemia and hypercalceamia and early death. However, the causative link between klotho deficiency and aortic aneurysm (AA) is unknown.Methods and ResultsKlotho‐floxed alleles (Klotho flox/flox) and transgenic tamoxifen‐inducible kidney specific CreERT2 mice were crossed to generate double‐transgenic mice (klotho flox/flox; KspCad CreER T2+/−). Tamoxifen (60 mg/kg/day, IP, for a total of 5 consecutive days) was given to induce kidney‐specific deletion of klotho gene. The aortas were intact in the control mice, whereas the thoracic and abdominal AA were observed in all the klotho knockout mice (100% incidence) on normal diet (ND) which suffered hyperphosphatemia and hypertension after 15 days of tamoxifen‐induced gene deletion. The histological staining exhibited severe aortic destruction in klotho knockout mice on ND, including aortic calcification and expansion, elastin degradation, collagen deposition and the absence of vascular smooth muscle cells (VSMCs) (residues <5%) substituted by macrophages and osteoclasts. Moreover, significantly increased expression and activity of matrix metallopeptidase (MMP) 2 and 9 were detected in the aortas of klotho knockout mice on ND compared to control mice. However, low phosphate diet (LPD) aattenuated hyperphosphatemia, hypertension, structure damage of aortas, and resultant AA formation by klotho deficiency. In cultured mouse vascular aortic smooth muscle cells (MOVAS), inorganic phosphate and calcium intensively induced osteoblast transition, MMP2 secretion and cell death by upregulating RUNX2, CREB phosphorylation and cleavage of caspase 3.ConclusionsWe conclude that klotho deficiency instigates AA formation through hyperphosphatemia‐driven aortic calcification and elastin degradation.