bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Rationale

Macrophages 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.

Methods

Newborn 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.

Results

Analysis 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).

Conclusion

One 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 Information

NHLBI, 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 Information

2R01HD052682‐06A1 (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 Information

Sao 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 Information

CIHR 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 Information

Supported 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 Information

Personal Funds (RLC)
Low Phosphate Diet Prevents Klotho Deficiency‐induced Aortic Aneurysm
Authors: Wang Qiongxin , Lin Yi , Ali Quaisar , Sun Zhongjie
Published: November 2023

Background

Mutation 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 Results

Klotho‐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.

Conclusions

We conclude that klotho deficiency instigates AA formation through hyperphosphatemia‐driven aortic calcification and elastin degradation.
Suppression of DAPK1 benefits to ischemia brain injury via mechanism(s) of ER stress mediated apoptotic pathway
Authors: Kim Joo‐Heon , Won Jinyoung , Hong Yonggeun
Published: November 2023

Stroke occurs as results of arterial occlusion in the brain, and leads to irreversible neuronal injury. Activation of death‐associated protein kinase 1 (DAPK1) induces endoplasmic reticulum (ER) stress and triggers apoptotic signal pathway. Here, we examined whether DAPK1 is involved in ER stress mediated apoptotic cell death in focal cerebral ischemia. To demonstrate the effect of DAPK1 on ER stress dependent cell death, the experimental animals were subjected to middle cerebral artery occlusion and injected with oxazolone‐based DAPK1 inhibitor. The experimental animals exhibited increase of total infarct volume and neurologic damage after focal cerebral ischemia. Molecular mechanism related with ER stress response and apoptosis was differently expressed in DAPK1 inhibitor‐treated group compared vehicle group. While the expression of BCL‐2 related to anti‐apoptosis was up regulated, the expression of BAX and caspase‐3 related to pro‐apoptosis was down regulated in DAPK1 inhibitor group compared to vehicle group. It was shown that neuronal apoptosis combined with altered expression of ER stress sensor marker including XBP1, ATF6 and GRP78/BiP in lesion. These results indicated that suppression of DAPK1 induces apoptotic cell death and protects against ischemic neuronal damage. Thus, DAPK1 would be a novel therapeutic approach for ischemic injury because suppression of DAPK1 induces neuronal cell rescue and promotes neural regeneration.

Support or Funding Information

NRF□2013R1A2A2A01067169, KGM4611613 to Y.H., J‐H.K. was supported by development fund foundation (2015) from GSNU. Republic of Korea
Involvement of NF2 Signaling Pathway in β‐Adrenergic Receptor‐Stimulated Cardiac Myocyte Apoptosis
Authors: Dalal Suman , Connelly Barbara A. , Singh Mahipal , Singh Krishna
Published: November 2023

β‐adrenergic receptor (β‐AR) stimulation induces cardiac myocyte apoptosis in vitro and in vivo. Tumor suppressor protein NF2 (neurofibromin 2, also known as merlin) is a member of the ezrin/radixin/moesin (ERM) family of proteins. Post‐translational modifications such as phosphorylation and sumoylation affect NF2 activity, subcellular localization and function. Role of NF2 in inhibition of cell growth and apoptosis is well established in a variety of cell types. However, the role of NF2 in β‐AR‐stimulated cardiac myocyte apoptosis remains to be investigated. Here we provide evidence that treatment of adult rat ventricular myocytes (ARVMs) with β‐AR agonist (isoproterenol, ISO, 10 μM) increases phosphorylation (serine 518) and sumoylation of NF2. Activation of adenylyl cyclase using forskolin mimicked the effects of ISO. ISO‐mediated increase in phosphorylation of NF2 was inhibited by protein kinase A inhibitor (H89) and β1‐AR receptor antagonist (CGP 20712A). Knockdown of NF2 using siRNA decreased ISO‐mediated increase in the number of apoptotic ARVMs (CTL, 8.3±0.6; ISO, 23.2±1.0*; negSiRNA+ISO, 21.3±1.2*; NF2‐SiRNA+ISO, 12.0±1.5$; *p<0.05 vs CTL; $p<0.05 vs ISO and negSiRNA+ISO, n=3–4). Adenoviral‐mediated expression of WT‐NF2 led to increased phosphorylation and sumoylation of NF2, and apoptosis. It also activated JNKs, a pro‐apoptotic kinase. Treatment with ISO and forskolin, or adenoviral‐mediated expression of NF2 increased phosphorylation (inactivation) of YAP, a downstream target of NF2. In H9C2 cardiomyocytes, knockdown of NF2 using siRNA decreased YAP phosphorylation. These data suggest that β‐AR stimulation affects post‐translational modification of NF2 via the involvement of PKA pathway, and NF2 plays a pro‐apoptotic role in β‐AR‐stimulated myocyte apoptosis.

Support or Funding Information

Supported by Department of Veterans Affairs (BX002332), NIH R15HL129140 (NHLBI) and Institutional Research and Improvement account.
The Role of CIP4 in SH‐SY5Y Cell Proliferation and Differentiation
Authors: Suthivanich Phichaya , Saengsawang Witchuda
Published: November 2023

Cdc42‐interacting protein 4 (CIP4) is a protein in the F‐BAR family, which plays important roles in various cellular events including endocytosis and membrane protrusion in several cell types. CIP4 has been shown to coordinate membrane dynamics and cytoskeletal reorganization in several cell types. CIP4 is expressed in several tissues including the developing brain. However, the role of CIP4 in the brain is still unclear. Here we showed that CIP4 was highly expressed in mouse brain during an early stage of development (embryonic day 10.5), which is a period that neural progenitor cells actively proliferate and differentiate. We then determined the roles of CIP4 during cell proliferation and differentiation using neuroblastoma (SH‐SY5Y) cells as a model. We showed that CIP4 level was significantly increased in differentiated cells, which was induced by retinoic acid compared with un‐differentiated cells (proliferating cells). Overexpression of CIP4 resulted in a decrease in cell proliferation. In addition, CIP4 knockdown completely abolished the ability of retinoic acid to induce differentiation of SH‐SY5Y cells. Together these results suggest that CIP4 may play an important role during an early stage of brain development, possibly by inhibiting cell proliferation and promoting cell differentiation.

Support or Funding Information

This work was supported by Faculty of Science, Mahidol University and a grant from Mahidol University to WS.
Circulating Progenitor Cell Quantity and Colony‐Forming Capacity in Lean and Obese Adults
Authors: Niemiro Grace M , Allen Jacob M , Mailing Lucy J , Holscher Hannah D , Woods Jeffrey A , De Lisio Michael
Published: November 2023

Obesity is associated with chronic low‐grade inflammation and decreased tissue repair capacity. Circulating progenitor cells (CPCs) are a heterogeneous cell population involved in tissue repair, and include hematopoietic stem/progenitor cells (HSPCs) that are precursors to all mature hematopoietic cells. Rodent models have shown that acute inflammatory stimuli induce myeloid differentiation of HSPCs via Toll‐like receptor 4 (TLR4) signaling. However, the effects of a chronic inflammatory condition, like obesity, on CPC content and differentiation has not been previously examined in humans. The purpose of the present cross‐sectional study was to compare the quantity of various CPC populations, HSPCs expressing TLR4, and HSPC colony forming unit (CFU) capacity between obese (OB) and healthy weight (HW) adults. Participants were divided into HW (n=19) or OB (n=15) based on body mass index. Blood was collected for quantification of the following CPC populations: CPCs (CD34+), hematopoietic stem/progenitor cells (HSPCs: CD34+/CD45dim), HSPCs expressing TLR4 (TLR4+HSPCs: CD34+/CD45dim/TLR4+), bone marrow‐derived mesenchymal stromal cells (BM‐MSCs: CD45−/CD34+/CD31−/CD105+, adipose‐derived MSCs (AT‐MSCs;CD45−/CD34+,CD31−/CD105+), endothelial progenitor cells (EPCs: CD45−/CD34+/CD31+), endothelial cells (ECs; CD34+/CD31−), common myeloid progenitors (CMPs: Lineage−/CD34+/CD38+/CD123low/CD45RA−), and common lymphoid progenitors (CLP: Lineage−/CD34+/CD38+/CD10+) by flow cytometry. Granulocyte (CFU‐G), macrophage (CFU‐M), and mixed (CFU‐GM) colony forming capacity was also evaluated. HSPCs expressing TLR4 were lower (P<0.05) in the OB group compared to HW. Granulocyte, macrophage, and granulocyte‐macrophage colonies were greater (P<0.05) in the OB group compared to HW. Adipose tissue derived MSCs in the OB group trended to be lower (P=0.12) compared to HW. There were no differences between groups in the other cell populations analyzed. Here we show that HSPCs from OB have increased myeloid lineage CFU capacity. Enhanced myeloid CFU could have been due to increased differentiation of TLR4+HSPCs leading to an overall decrease in TLR4+HSPC quantity. Together, these data indicate that obesity is associated with increased myeloid differentiation of HSPCs, perhaps via differentiation of TLR4+HSPCs, which likely exacerbates the chronic inflammation observed in obesity.
Xenotransplantation of human mesenchymal stem cells alters host gene expression following prolonged high fat feeding
Authors: Newell Christopher , Hittel Dustin S. , Nyamandi Vongai , Khan Aneal , Shearer Jane
Published: November 2023

Background

Initially identified as plastic‐adherent cells, human mesenchymal stem cells (hMSCs) are non‐hematopoietic stem cells which are capable of differentiating into a multitude of cell lineages. Over the past decade the number of clinical trials involving hMSCs has steadily increased, resulting in hMSCs being the most commonly used cells in tissue engineering and regenerative medicine. Known to preferentially home to sites of injury and inflammation, administered hMSCs have exhibited promotion of host tissue repair through donor cell engraftment, release of cell signaling factors, and transfer of healthy organelles. As such, enhanced hMSC engraftment to host tissues and improved therapeutic potential may be induced by a state of low‐grade inflammation. Here we induce low‐grade inflammation through excessive nutrient consumption, achieved through prolonged high fat feeding.

Purpose

To examine the impact of hMSC treatment on the liver tissue of mice following a prolonged high fat feeding regiment.

Methods

At 4 weeks of age, mice were placed on a high fat diet (60% kcal fat) for 20 weeks in order to elicit an obese phenotype. Following dietary manipulation, mice were randomly divided into two groups: hMSC treatment (HFM; n = 8) or saline control (HFS; n = 8). Mitochondrial metabolism, microarray gene expression profiling, and metabolomic profiling of host liver tissue were specifically highlighted for their important role in the obese phenotype.

Results

As a measure of mitochondrial metabolic activity, maximal complex I oxygen consumption (supported by the addition of ADP) was higher in HFM animals (p<0.05). The marker of mitochondrial abundance, citrate synthase, was also elevated following hMSC administration (p<0.05). Microarray gene expression profiling identified 226 genes with significant differential expression between groups ‐ fold change (±1.2) and p‐value (<0.01). Specific gene networks and pathway analysis commonly identified cellular signalling and actin cytoskeleton regulation as upregulated processes in HFM mice (p<0.01).

Conclusions

Our data indicate that xenogeneic hMSC treatment improves metabolic capacity of host liver and results in widespread host gene shifting. The upregulation of cell signaling and actin cytoskeleton regulation may provide mechanistic insight into how hMSCs impart their ability to migrate to areas of inflammation and transfer organelles to host tissues.

Support or Funding Information

This study was supported by NSERC (JS). This research was supported by PhD funding to CN from MitoCanada and Alberta Innovates – Health Solutions.
Sex Differences in Intestinal Epithelial Crypt Cell Proliferation
Authors: Zhou Weinan , Suriyavirun Natcha , Dailey Megan J.
Published: November 2023

The intestinal epithelium plays an essential role in ingestive behaviors and immune homeostasis. The epithelium is continually renewed every few days by the proliferation and differentiation of stem cells located in the intestinal crypts. Studies have shown that the luminal nutrient content drives changes in the stem cell proliferative process, but it is unknown whether there are male‐female differences in these nutrient‐driven changes. To determine whether sex differences affect crypt cell proliferation, we utilized primary small intestinal epithelial crypts isolated from male and female C57BL/6J mice to investigate the differences in the gene expression of steroid hormone receptors, crypt cell proliferation, and crypt cell proliferation in response to steroid hormones. We found that estrogen receptor α was expressed in crypts from both male and female mice, while estrogen receptor β, androgen receptor, and progesterone receptor were not expressed in crypts from either sex. Crypt cell proliferation was higher in crypts isolated from female mice compared with crypts isolated form male mice, however, 17β‐Estradiol had no effect on crypt cell proliferation in either sex. These results suggest that sex differences may influence crypt cell proliferation, but these differences may not be driven by steroid hormones.

Support or Funding Information

USDA Hatch ILLU‐538‐926
Body on‐chip and 3D culture: An overview
Authors: Klingelhutz Aloysius John , Gourronc Francoise , Chaly Anna , Ankrum James
Published: November 2023

The development of better methods to grow human tissue in conditions that more closely recapitulate normal physiology and anatomy (i.e. biomimetic technology) will greatly enhance drug and toxin testing as well as advance our understanding of biology. Two dimensional (2D) cell culture has been extremely valuable for research, but cells grown in 2D culture are not subject to the same mechanical and physiologic conditions as cells in tissue. While animal studies are an alternative, rodents and humans often exhibit large differences with regard to their responses to drugs and toxins. Furthermore, animal studies can be expensive. Growing cells in 3D conditions is challenging but advances are being made that allow researchers to do so. One relatively straightforward method relies on hanging drop technology to develop 3D spheroids. These spheroids often exhibit differential growth and differentiation as compared to 2D culture. Methods to generate and grow spheroids as well as our experience with the development of human adipose spheroids will be discussed. Tissue on‐chip technology in a microfluidics platform raises the possibility of incorporating 3D tissue into a system that allows one to carefully regulate what the tissues are exposed to and to measure what they secrete. Advances are being made in development of tissue on‐chip culture systems for lung, cardiac, and liver, as well as other tissues. These approaches could greatly advance the ability to test how different factors affect physiology. In addition, it may also be possible to make connections between different tissue types (i.e. body on‐chip), thus allowing an assessment of how one cell or tissue type affects another. Innovative methods for 3D culture and tissue/body on‐chip technology will be reviewed.

Support or Funding Information

Development of a human white fat biomimetic, University of Iowa Vice President for Research Internal Funding Initiation; Role of aryl hydrocarbon receptor in adipogenesis and diabetes, University of Iowa Fraternal Order of Eagles Diabetes Research Center Pilot Award
Macrophage migration inhibitory factor (MIF) but not its homologue D‐dopachrome tautomerase (D‐DT) promotes fibroblast motility in a CD44/CD74‐independent manner
Authors: Szczesniak Pawel , Meinhardt Andreas , Klug Jörg
Published: November 2023

Macrophage migration inhibitory factor (MIF) and D‐dopachrome tautomerase (D‐DT) are paralogous pro‐inflammatory cytokines with chemokine‐like functions. Whereas cytosolic MIF plays a role in cell cycle, secreted MIF and D‐DT activate second messenger signalling through a CD44/CD74 receptor complex. To differentiate between receptor‐ and non‐receptor‐mediated events, COS‐7/M6 fibroblasts deficient in CD44/CD74 (WT) were employed to generate stable cell lines expressing CD44 and CD74. Chemokinesis (random single‐cell motility) was assessed in cells stimulated with native recombinant MIF/D‐DT, enzyme‐null recombinant MIF, inhibitors of MIF's tautomerase activity, and inhibitors of clathrin‐ and non‐clathrin‐dependent endocytosis. In the presence of CD44/CD74, both MIF and D‐DT stimulated chemokinesis but only MIF enhanced chemokinesis in the absence of receptors. The stimulatory effect of MIF on migration depended on its tautomerase activity and lipid raft/caveolae‐mediated, but not clathrin‐mediated endocytosis. To observe a direct effect of MIF and D‐DT on actin dynamics, actin polymerization was quantified in an in vitro actin assembly assay. MIF but not D‐DT decreased the rate of F‐actin assembly. By decreasing the rate of cell extract‐driven actin assembly, MIF phenocopies the function of F‐actin capping proteins. Moreover, stimulation with MIF increased the number of prominent F‐actin stress fibres in COS‐7/M6 WT. Taken together, MIF stimulates fibroblast chemokinesis independent of its receptors, likely by directly modulating the actin cytoskeleton. MIF‐mediated chemokinesis appears to depend on lipid raft/caveolae‐mediated endocytosis. D‐DT triggers chemokinesis only in the presence of MIF/D‐DT receptors – CD44/CD74 – implying D‐DT requires the classical receptor‐driven second‐messenger transduction pathway.

Support or Funding Information

Intramural funding.
Lipid signaling regulates fibroblast migration and the actin cytoskeleton in response to stiffness and microtopography
Authors: Mkrtschjan Michael , Gaikwad Snehal , Dommaraju Sagar , Li Jieli , Russell Brenda
Published: November 2023

Cell migration depends on mechanotransduction—the conversion of mechanical cues from the microenvironment to internal biochemical cellular signals, such as by protein phosphorylation. Lipid signals are more difficult to study than proteins, but also play a key role in mechanotransduction pathways. In this study, we apply bioengineering approaches to manipulate physical cues of topography and stiffness in order to reprogram cells, assessing the effects of lipid signaling (PIP2) on the actin cytoskeleton. Primary cardiac fibroblasts were grown on polyacrylamide or polydimethylsiloxane (PDMS) with stiffness of 10, 100, or 400 kPa and glass (>1 GPa). Micropost topography was fabricated from PDMS (400 kPa) with a 75 μm‐spaced tetragonal array. An increase in actin assembly rate was determined by the kfrap kinetic constant from fluorescence recovery after photobleaching, and was significantly higher for cells grown on the microtopography than on the flat surface, P<0.05, n=7. To test the role of lipid signaling, PIP2 availability was reduced by neomycin (500 μM) scavenging, or the PIP2 level was increased by PI3K inhibition with wortmannin (1 μM). The localization of actin and lamellipodin (a PIP2 / actin binding protein) depended on stiffness and topography and was further changed by drugs. The actin cytoskeleton had more prominent stress fibers extending to lamella edge with neomycin treatment; the lamellipodin distribution is being assessed. Fibroblast migration is known to decelerate as the surface stiffens, and was measured here through a gap created by removal of a barrier or by the traditional scratch assay. Migration velocity at all stiffnesses was significantly faster with reduction of PIP2 availability with neomycin treatment compared to untreated fibroblasts (P<.05, n= 6). With wortmannin treatment, the velocity is similar to the untreated cells. We conclude that primary cardiac fibroblasts respond to stiffness and microtopography via lipid signaling and PIP2 to regulate cell migration and the architecture of the actin cytoskeleton. Therefore, the use of drugs or manipulation of the microenvironment might be beneficial for control of fibrosis in disease.

Support or Funding Information

HL62426.
Phyllanthus taxodiifolius Beille Extract Disrupted Microtubule Dynamics in Glioma Cells
Authors: Kwanthongdee Jaturon , Saengsawang Witchuda , Patoomratana Tuchinda
Published: November 2023

Glioma is the most common, life threatening, malignant primary brain tumor. This tumor has the ability to spread and invade through surrounding areas in the brain. Cell migration and adhesion are the most important processes involved in cancer cell spreading and invasion. Phyllanthus taxodiifolius Beille extract has been studied for its anti‐cancer action in breast and lung cancer cells. However, its mechanisms of action are still unclear. In addition, the effect of Phyllanthus taxodiifolius Beille extract on brain cancer cell viability and invasiveness, especially in glioma cells, has never been determined. Here we investigated the effect of Phyllanthus taxodiifolius Beille extract on glioma cell migration and adhesion using wound‐healing cell migration and cell spreading assays, respectively. We also determined its effects on microtubule structure and dynamics using both in vitro microtubule polymerization assay, and live cell imaging techniques with Total Internal Fluorescence Microscope (TIRFM). The results showed that Phyllanthus taxodiifolius Beille extract reduced glioma cell viability, disrupted cell adhesion and delayed cell migration. In addition, the extract rapidly disrupted microtubule structure and dynamics in glioma cells. Real time imaging of microtubule end‐binding protein (EB3) in glioma cells demonstrated that the extract disrupted the binding and dynamics of EB3 at the tips of microtubules. This study provided supporting evidences for the anti‐cancer properties of Phyllanthus taxodiifolius Beille extract. It is also the first study to demonstrate that the Phyllanthus taxodiifolius Beille extract interfered microtubule structure and dynamics, and suggested the possibility to further study and develop Phyllanthus taxodiifolius Beille derived compounds into a novel anti‐cancer drug targeting microtubules.

Support or Funding Information

This work was supported by a grant from the Center of Excellence on Environmental Health and Toxicology (EHT) to WS. We also gratefully acknowledge support from the Faculty of Science and Faculty of Graduate Studies, Mahidol University (to WS and JK) and support from Sritrangtong scholarship program (to JK).
ATP and extracellular calcium regulates differentially the phagocytosis of S. aureus and C. glabrata by macrophages.
Authors: Pérez Gabriela , Hernandez‐Silva Cesar , Arreola Jorge , Pérez‐Cornejo Patricia
Published: November 2023

Phagocytosis is a fundamental function of innate immune cells; many receptors have been shown to mediate phagocytosis of bacterial and fungal pathogen. However, if the mechanism of phagocytosis is common among bacterial and fungal pathogens is poorly defined. We studied the effects of extracellular ATP and extracellular Ca2+ on uptake of Staphylococcus aureus and Candida glabrata in phagocytosis by J774 macrophages. Our findings show that phagocytosis of S. aureus was blocked by ATP and the P2X7 receptor agonist BzATP. A438079, an antagonist of P2X7 receptors, partially reverted the effects of ATP on bacterial phagocytosis. This data suggests that other P2X or P2Y receptors might be important for phagocytosis de S. aureus. In contrast, phagocytosis of C. glabrata remained unaffected by ATP stimulation. To determine if P2X‐mediated Ca2+ entry into macrophages was blocking the engulfment of bacteria, we measured phagocytic activity in the absence or presence of 2 mM extracellular Ca2+ with or without ATP. Ca2+, in the absence of ATP, was required for engulfment of C. glabrata but not S. aureus. Adding ATP inhibited phagocytosis of S. aureus regardless of Ca2+, suggesting that Ca2+ entry was not important for inhibiting phagocytosis. On the other hand, phagocytosis of normal or hyper‐adherent C. glabrata mutants had an absolute requirement for extracellular Ca2+ due to yeast adhesion to macrophages mediated by Ca2+‐dependent adhesion proteins. Addition of extracellular Ba2+ did not replace Ca2+, this indicate the high selectivity for Ca2+ in the phagocytic process of C. glabrata. We conclude that Ca2+ entry no role in phagocytosis of S. aureus; while the effect of Ca2+ on C. glabrata phagocytosis was mediated for calcium‐dependent adhesins.

Support or Funding Information

This work was supported by CONACYT (219949 joint grant to PPC and JA) and FAI (C16‐FAI‐09‐37.37 to GP)
Postpartum Cervical Remodeling Entails Acute Alteration in Expression of Mechanical‐ and Immune‐Related Factors
Authors: Stanley Robert Lee , Burton Catherine , Shah Sonam , Yang Eileen , Sampathkumar Prashanth , Rao Sanjana , Del Castillo Val , Douglas Veronica , Frank Martin , Mowa Chishimba Nathan
Published: November 2023

Because the cervix undergoes marked mechanical trauma during birth and remains briefly open postpartum, it requires a speedy and complete tissue repair to avoid immediate and long term obstetrical complications, such as infection and possibly preterm labor, respectively. Here, we attempt to unravel the key biological changes of postpartum cervical repair in mice cervix during the first 48h using a proteome‐wide technology, proteomics, as well as real time PCR and confocal immunofluorescence. The proteomics data revealed variable expression of several intermediate filaments, cytoskeletal modulators, and proteins with immune‐ and/or wound healing properties. Real time PCR verified the proteomics and confocal immunofluorescence data of 13 molecules. We conclude that postpartum cervical repair involves expression of multiple factors, notably immune‐ and mechanical‐related factors that could potentially be used as markers for determining the phase of postpartum cervical repair in obstetrical complications, such as cervical lacerations.

Support or Funding Information

Appalachian State University Office of Student Research
AGE‐RAGE Signaling Is Intersected by Rap1a Crosstalk in Fibroblasts in Diabetic Hearts
Authors: Stewart James A. , Langford Jaslyn , Zhao Jia , Worsham Rebecca
Published: November 2023

Rap1a is a member of the Ras GTPase superfamily that acts as a molecular switch coupling extracellular events to intracellular signaling. The purpose of this study was to identify a role for Rap1a in the AGE/RAGE signaling cascade. We hypothesized that Rap1a GTPase intersects the downstream signaling modulators of the AGE/RAGE signaling cascade to promote extracellular remodeling and fibroblast function in the diabetic heart. Initial observations demonstrated significantly elevated Rap1a protein expression levels in cardiac fibroblasts isolated from diabetic hearts. Therefore, a series of studies were performed to alter Rap1a activity and protein levels to determine the influence of Rap1a in AGE/RAGE signaling cascade. Cardiac fibroblasts were isolated from diabetic (db/db) fibroblasts and non‐diabetic (het) fibroblasts. A Rap1a‐EPAC agonist was used to increase Rap1a activity and protein levels resulting in elevated AGE/RAGE signaling markers, such as PKC‐ζ and ERK1/2 phosphorylation, α‐SMA, and RAGE protein levels. Furthermore, silencing Rap1a protein expression decreased PKC‐ζ and ERK1/2 phosphorylation, α‐SMA, and RAGE as a result of Rap1a siRNA treatment. These studies demonstrated that Rap1a crosstalk occurred in the AGE/RAGE cascade. Rap1a involvement in the AGE/RAGE cascade identifies a new molecular mechanism, which could further potentiate fibroblast differentiation and ECM remodeling in diabetes. Deactivation of this mechanism represents a potential therapeutic approach to regulate fibroblast phenotype changes, collagen accumulation, and RAGE expression.

Support or Funding Information

This research was supported by the American NIH Academic Research Enhancement Award (R15)(PA‐13‐313, JAS), and Department of Defense Peer Reviewed Medical Research Program ‐ Investigator‐Initiated Research Award PR150906 ‐ GRANT12018632, JAS.
Effects of Essential Oil on Fear Memory and the Immune Response: A Potential Alternative Therapy for Post Traumatic Stress Disorder (PSTD)
Authors: Moshfegh Cassandra , Swiercz Adam P , Hopkins Lauren , Hurr Chansol , Young Colin N , Marvar Paul J
Published: November 2023

Chronic stress plays an integral role in activation and exacerbation of inflammation in the peripheral immune system. Peripherally circulating immune cells are also capable of crossing the blood‐brain barrier and can promote inflammation in the central nervous system (CNS), which has been suggested to contribute to mental health disorders, such as post traumatic stress disorder (PTSD). However, the association between fear memory and the immune system is not well characterized. Moreover, there are only two FDA‐‐approved medications currently for PTSD. Therefore we sought to examine orange essential plant oil (OEPO), previously been found to have CNS depressant‐‐like effects, on fear memory and immune cell activation in a mouse model of PTSD (Pavlovian Fear Conditioning). The treatment group (n=8) was administered 25% OEPO via olfactory exposure prior to and after fear conditioning. Mice exposed to 25% OEPO showed no difference in percent freezing during fear acquisition compared to controls. However, when tested for extinction retention 48 hours later the treatment group experienced a significant decrease in freezing behavior (17.1 ± 1.6 % (p < 0.01) versus control (3.67 ± 2.13%), suggesting that OEPO affects extinction of fear memory in mice. Next we examined the peripheral and central immune response following extinction of fear memory using flow cytometry analysis. A significant decrease in the number of effector memory T cells CD62loCD44hi 7.955(± 0.48)% was observed in the treatment group relative to control 12.05(± 0.708)% (p < 0.05). To evaluate the effect of OEPO on the central immune response, we performed an immunological challenge with intraperitoneal injected lipopolysaccharide (LPS) and assessed immune cell populations in the meningeal compartment. Our preliminary evidence suggest that LPS injected mice treated with OEPO for 2hrs have decreased CD45+CD11b+ cells 54.2% compared to LPS injected mice treated with water 68.7%. These data suggest that OEPO may modulate the neuroimmune response during fear memory recall. Further studies are required to determine the neuroimmune mechanism(s) responsible for the extinction of fear memory in OEPO treated mice.

Support or Funding Information

NIH R00 HL107675‐‐03