bluuebunny/crossref_metadata_2025 huggingface dataset:
Erythropoietin impacts the stress‐related respiratory dysfunction in mice: neonatal maternal separation and sex dimorphism
Authors: Elliot‐Portal Elizabeth , Kinkead Richard , Soliz Jorge
Published: November 2023

Neonatal maternal separation (NMS) is an established model of early life adversity that mimics stressful conditions experienced by newborn and infants. NMS disrupts brain development and as adults, individuals show sex‐dependent abnormal neuroendocrine response, predisposing them to physiological/psychological disorders. Concerning the neural control of respiration, studies in rats revealed that NMS causes respiratory instability and increased levels of corticosterone that remained during adulthood. Moreover, increased ventilatory response to hypoxia (HVR) but decreased to hypercapnia (HcVR) was observed in male animals, while decreased HVR but increased HcVR was observed in female rats.

Although it is primarily recognized for its capacity to stimulate erythrocyte production, erythropoietin (Epo) is a cytokine that is also produced in the brain (by neurons and astrocytes), and plays role in development, maintenance, protection and repair of the nervous system. Studies from our lab demonstrated that cerebral Epo is a potent and sex‐specific respiratory stimulant that enhances the ability to tolerate hypoxic conditions at neonatal and adult ages. As cerebral Epo also attenuates the activation of the hypothalamic‐pituitary‐adrenal (HPA) axis, here we hypothesized that Epo protects against the stress‐induced cardio‐respiratory dysfunction produced by NMS. To test this hypothesis, circulating corticosterone level of adult (3 months) male and female Tg21 mice, was increased by subcutaneous implantation of beeswax pellets. 14 days later basal ventilation, HVR (10%O2 in N2) and HcVR (5% CO2, 21%O2 in N2) was evaluated by whole‐body plethysmography. Our preliminary results showed that corticosterone does not alter basal ventilation nor HVR in female and male Tg21 mice. However, corticosterone decreased HcVR in female Tg21 mice, meanwhile males showed an increased HcVR. These results suggest that Epo modulates the cortiscosterone‐induced cardiorespiratory dysfunctions.

Support or Funding Information

Canadian Institutes of HealthResearch (MOP 130258), the Star Foundation for Children's HealthResearch. The salary of Jorge Soliz is supported by the “Fonds de recherche du Quebec‐Santé” (FRQ‐S).
Ventilatory and Neurochemical Effects of Microdialysis of a μ‐opioid Receptor Agonist (DAMGO) into the hypoglossal nucleus of awake and sleeping goats
Authors: Langer Thomas M , Neumueller Suzanne , Burgraff Nicholas James , Pan Lawrence , Hodges Matthew R , Forster Hubert V
Published: November 2023

Unilateral dialysis of [D‐Ala2, N‐MePhe4, Gly‐ol]‐enkephalin (DAMGO) into the ventral respiratory column (VRC) of awake and sleeping goats causes a decrease in breathing. This observed decrease is transient, perhaps because of a concomitant local increase in GABA. However, data of others suggest opioids acting on the hypoglossal nucleus (HN) may contribute to opioid induced respiratory depression (ORD) (J Physiol. 2009 Jun 1; 587(Pt 11): 2677–2692). These data suggest neuromodulator compensation may not occur in motor nuclei such as the HN. Accordingly, herein we chronically implanted unilateral microtubules into the HN of adult goats. We then dialyzed DAMGO during both awake and NREM sleep states. We found that dialysis of DAMGO decreased tidal volume and pulmonary ventilation in a majority of goats during both awake and NREM sleep states. In contrast to findings in the VRC, DAMGO dialysis into the HN does not change GABA or any other neuromodulator measured herein in the effluent dialysate. Accordingly, findings to date do not support the concept of neuromodulatory compensation occurring in the HN.

Support or Funding Information

National Heart, Lung, and Blood Institute grants HL‐25739, HL‐112996, HL‐007852, and by the Department of Veterans Affairs
Exposure to chronic intermittent hypoxia does not elicit aberrant respiratory plasticity in adult guinea pig
Authors: O'Connor Karen M. , Lucking Eric F. , Burns David P. , O'Halloran Ken D.
Published: November 2023

Chronic intermittent hypoxia (CIH), a major pathological feature of obstructive sleep apnoea, enhances carotid body (CB) chemosensory responses to hypoxia producing enhanced basal breathing and ventilatory responsiveness to hypoxia, increased respiratory instability, autonomic dysfunction, cardiac arrhythmias, and hypertension. CIH may also affect metabolic rate. We sought to explore if CIH induces ventilatory and/or metabolic changes in guinea‐pigs, a species that presents with hypoxia insensitive carotid bodies.

Duncan Hartley adult male guinea pigs were exposed to repeated 5 minute cycles consisting of 90s hypoxia (FiO2=0.05 nadir) and 210s normoxia (FiO2 = 0.21) for 8 hours per day for 2 weeks (CIH, n=8). Sham animals (n=8) were subjected to the same environmental cues but were constantly exposed to room air. Guinea pigs had free access to food and water throughout the exposure. Using whole‐body plethysmography, ventilation and metabolism were measured during air breathing (FiO2 = 0.21), hypoxia (FiO2 = 0.10) and hypercapnia (FiCO2 = 0.05) in unanesthetised, unrestrained animals at quiet rest. Data are presented as mean±SD and were analysed using unpaired Student t tests or Mann‐Whitney U tests.

Baseline ventilation and metabolism in normoxia were unaffected by exposure to CIH. As such, the ventilatory equivalent (VE/VCO2) was similar in CIH exposed and sham animals: 41.2±10.7 versus 35.6±5.2 (p=0.201; sham versus CIH). Exposure to acute hypoxia (FiO2 = 0.10 for 10 mins) did not alter VE, tidal volume (VT) or respiratory frequency (f), both in sham (47±4ml/min/100g; 0.5±0.04ml/100g and 103±9bpm, respectively) and CIH exposed animals (45±5ml/min/100g; 0.5±0.006ml/100g and 98±5bpm, respectively) compared with respective baseline values. Metabolism (VCO2) during acute hypoxia was not significantly different between the two groups (1.01±0.09 versus 1.16±0.2ml/min/100g). Nonetheless, VE/VCO2 was significantly decreased in CIH exposed animals compared with sham (46.6±2.9 versus 39.5±2.5; p=0.0001). In response to hypercapnia, CIH exposed animals had a significantly blunted tidal volume response compared with sham animals (1.1±0.01ml/100g versus 0.84±0.01ml/100g; sham versus CIH). However, VE/VCO2 during acute hypercapnia was not different between the two groups.

Our data reveals that CIH exposure does not affect basal breathing, nor does it enhance hypoxic ventilatory responsiveness, which has been described in rats and mice with hypoxia sensitive carotid bodies. Our study employing a species with hypoxia insensitive carotid bodies suggests a critical role for CIH‐induced plasticity in peripheral oxygen sensing mechanisms within the carotid body in the manifestation of aberrant respiratory control.

Support or Funding Information

Department of Physiology, School of Medicine, University College Cork
Aerobic Fitness Influences Autonomic Support of Blood Pressure in Premenopausal, but not Postmenopausal Women
Authors: Baker Sarah E , Limberg Jacqueline K , Nicholson Wayne T , Curry Timothy B , Barnes Jill N , Joyner Michael J
Published: November 2023

Background

The risk of hypertension increases as women reach menopause. Our group has recently reported that autonomic support of blood pressure (BP) is increased in post‐menopausal women. Given regular exercise is known to decrease the risk of developing hypertension, we investigated the role of aerobic fitness on autonomic support of BP in women. We hypothesized that trained premenopausal, but not postmenopausal, women would have less autonomic support of BP than age‐matched untrained women.

Methods

Twelve healthy, young premenopausal (27±1yrs) and 10 older postmenopausal (56±1yrs) women completed VO2max tests on cycle ergometers. The participants were then stratified by fitness level based on VO2max percentiles from the American College of Sports Medicine's Guidelines (≤50th percentile = untrained; >50th percentile = trained; premenopausal: 7 untrained, 5 trained; postmenopausal: 5 untrained, 5 trained). On a separate day muscle sympathetic nerve activity (MSNA, microneurography, peroneal nerve), heart rate (electrocardiography), and blood pressure (brachial arterial catheter) were recorded before and during ganglionic blockade using trimethaphan camsylate (dose:1.5–7mg/min). Stroke volume (SV), cardiac output (CO), and total peripheral resistance (TPR) were calculated from the blood pressure waveform (WinCPRS).

Results

Muscle sympathetic nerve activity was higher in trained young women than in untrained young women (burst incidence: 35±11 vs. 12±2 bursts/100 heart beats, p=0.03). Young trained women had a smaller reduction in mean arterial pressure (MAP) with trimethaphan than young untrained women (ΔMAP:−5±3 vs. −15±4mmHg, p=0.01), despite a larger reduction in total peripheral resistance (ΔTPR: −6±1 vs. −2±1mmHg*min/L, p=0.02). The trained young women were able to maintain their MAP due to a greater increase in cardiac output (ΔCO: 1.4±0.4 vs. −0.3±0.3L/min, p= 0.01). In older postmenopausal women, MSNA and the fall in BP, CO, and TPR with trimethaphan were not different between trained and untrained individuals (p= 0.81, 0.16, 0.52 and 0.09, respectively).

Conclusion

Baseline MSNA was higher in trained premenopausal women when compared to untrained premenopausal women; however, aerobic fitness did not influence baseline MSNA in postmenopausal women. In young trained women, the fall in MAP following ganglionic blockade was only one‐third of the response seen in young untrained women. Baseline MSNA and the change in MAP, CO, and TPR with ganglionic blockade were not different between trained and untrained postmenopausal women. These data suggest aerobic fitness reduces autonomic support of blood pressure in young trained women, but not in trained postmenopausal women.

Support or Funding Information

Support: HL83947 (MJJ); HL131151 (SEB), DK 7352‐37 (SEB), HL118154 (JNB)
Central Hemodynamic Response to Acute Sympathetic Activation via Cold Pressor Test in Healthy Aging
Authors: Pearson James , Borner Anastasiya , Murray Kyle
Published: November 2023

The incidence of cardiovascular related events, such as myocardial infarction and stroke, increase in aged individuals. This cardiovascular risk is elevated further during the winter months when environmental temperatures decrease. Increased sympathetic nerve activity accompanies decreases skin surface temperature. This increase in sympathetic nerve activity may have implications for the control of central hemodynamics and arterial stiffness. We aimed to investigate the influence of acute sympathetic activation via cold pressor test upon central hemodynamics and arterial stiffness in young and healthy aged individuals.

Eleven young (Young: 7 males, 27±7yrs, 175±9cm, 77.4±13.8 kg) and eleven healthy aged individuals (Aged: 6 males, 57±12 yrs, 172±8 cm, 75.8±23 kg) completed a cold pressor test (CPT) where the left hand was immersed up to the wrist in 2–4°C water for 3 minutes while supine. All participants were free of cardiovascular acting medications and disease. Central hemodynamics and arterial stiffness, including subendocardial viability ratio (SEVR %), were estimated via radial tonometry linked to software with a generalized transfer function (SphygmoCor®, AtCor Medical). Aortic pulse wave reflection was referenced to a heart rate of 75 bpm (augmentation index; Aix75; %). Aortic pulse wave velocity (aPWV; m/s) was estimated using an automated sphygmomanometer (Mobil‐O‐Graph®; I.E.M). Mean arterial pressure (MAP; mmHg) and heart rate (HR; bpm) were estimated using photoplethysmography (NexFin®, BMEYE). Measurements were obtained at baseline prior to, during and at the end of the cold pressor test.

Baseline MAP was not different between Young and Aged (90±10 vs. 91±10 mmHg, P > 0.05). Baseline AIx75 and aPWV were both different between Young and Aged (AIx75: −2±16 vs. 15±9% and aPWV: 5.4±0.6 vs.7.9±1.4 m/s, both P < 0.01). At the end of CPT, MAP increased relative to baseline in both Young and Aged (106±8 and 106±11 mmHg; both P < 0.01) but was not different between groups (P > 0.05). At the end of CPT AIx75 increased relative to baseline in both Young (+18±13% to 15±14%, P = 0.0003) and Aged (+12±6% to 26±7%; P = 0.0001). AIx75 was higher in the aged individuals at the end of the cold pressor test (P = 0.027). Relative to baseline SEVR was unchanged at the end of CPT in Young (150±35 to 155±42%; P > 0.05) but decreased in Aged (157±26 to 135±36%; P = 0.0055).

Acute sympathetic activation via cold pressor test resulted in a greater aortic pulse wave reflection and reduced subendocardial viability ratio in healthy aged relative to young individuals. This suggests that acute sympathetic activation increases myocardial strain and may help explain the increased incidence of myocardial infarction in aged individuals during winter months.
Dissecting the Role of EZH2 and H3K27me3 in Endometriosis
Authors: Colon‐Caraballo Mariano , Soto‐Vargas John , Urrutia Raul , Flores Idhaliz
Published: November 2023

Background

Endometriosis, defined as the growth and development of endometrial like tissue, (i.e., glands and stroma) outside the uterine cavity, is a gynecological condition that affects 10% of women in their reproductive age. Cumulative evidence has demonstrated that candidate genes which are related with cellular behaviors characteristic of endometriosis are epigenetically regulated by DNA methylation and histone acetylation. However, the specific role of histone methylation and the enzymes responsible for setting these histone marks (e.g., histone methyltransferases‐HMT's) in the regulation of these candidate genes is still unknown. EZH2 is an HMT that sets the H3K27me3 histone mark, a repressor of the transcriptional machinery. We have previously reported that endometriotic lesions are characterized by high levels of H3K27me3; therefore, we aimed to assess basal and hormone‐regulated protein expression levels of EZH2 isoforms (α and β) and also elucidate if H3K27me3 is enriched within or near promoter regions of candidate genes.

Methods

Western Blot (WB) was performed to determine basal and ovarian steroid EZH2 protein levels in cell lines. Immunohistochemistry (IHC) of an endometriosis‐TMA was used to assess EZH2 α and β protein levels in tissue samples. Chromatin immunoprecipitation (ChIP‐qPCR) with an H3K27me3 antibody was conducted using endometrial and endometriotic samples. Scratch Assay was performed to determine the effect of EZH2 inhibition on proliferative cellular behavior

Results

WB results suggest that endometriotic cells expressed higher levels of EZH2α, compared to non‐endometriotic cells. Also, hormonal treatments did not modulate EZH2 isoform expression in any of the cell lines tested. IHC indicated that in endometriotic lesions the glands and stroma had higher EZH2α and β protein levels compared to eutopic endometrium from patients. ChIP‐qPCR results showed higher levels of H3K27me3 levels within promoter regions of candidate genes in at least some of the endometriotic lesions. Pharmacological inhibition of EZH2 reduced the proliferative rate of the endometriotic cells.

Conclusions

The present study is the first in reporting that EZH2 isoform protein expression is elevated in endometriosis but not regulated by steroid hormones. We also showed that H3K27me3 is elevated in the promoter region of candidate genes in some lesions. Elucidating the expression profile and function of HMTs may support the use of novel histone methyltransferases inhibitors (HMTi) as a possible treatment for this enigmatic disease.

Support or Funding Information

R01HD050509, NCI1U56CA126379‐01, R25GM082406.
A potential role for the novel human helicase, G4‐Resolvase 1 in HIV‐1 RNA packaging
Authors: Cintron Kevin
Published: November 2023

HIV‐1 is a retrovirus that infects human immune cells and is responsible for causing aquired immune deficiency syndrome (AIDS). An estimated 34 million people have died from AIDS‐related causes so far, including 1.2 million in 2014(Worldwide). After the viral HIV particle enters the host cell cytoplasm, its RNA is then reverse transcribed into double‐stranded DNA and integrated into the host genome. The provirus is transcribed to produce viral RNA, which is then, exported from nucleus to cytoplasm. The full length and un‐spliced RNA is used for packaging or translated for viral proteins. Previous work has demonstrated that an array of human proteins bind to HIV‐1 RNA. One such protein is G4‐Resolvase1 (G4R1) (aliases: DHX36, RHAU), a multifunctional helicase required for eliciting an interferon immune response to viral nucleic acid. Additionally, G4R1 was discovered as the primary protein in human cells responsible for unwinding G‐quadruplex DNA and RNA structures. G‐quadruplexes are stable nucleic acid secondary structures that form within G‐rich strands of DNA or RNA. These structures are enriched in gene promoters of the HIV and have been known to be involved in transcriptional regulation by antagonizing transcription and also potentially affecting translational expression. We hypothesize that G4R1 affects HIV transcription and subsequent packaging of RNA into viral particles. To test our hypothesis, we utilized HeLa cells containing a tetracycline‐inducible siRNA targeted to knockdown G4R1 protein expression. Thus far, I have confirmed G4R1 knockdown in HeLa cells using western blot analysis and ongoing work includes testing the effect of G4R1 knockdown on HIV RNA packaging and interferon immune response to HIV expression.
Characterization of Tetratricopeptide 39c (Ttc39c) in Skeletal Muscle
Authors: Hayes Caleb , Waddell David
Published: November 2023

Ttc39c has been identified as a novel gene in skeletal muscle that is differentially regulated under conditions of neurogenic atrophy in wild‐type mice compared to Muscle RING Finger 1 (MuRF1) knockout mice. MuRF1 is a ubiquitin E3 ligase that is known to be an important mediator of muscle wasting; however, evidence presented here suggests that MuRF1 may also function as a regulator of atrophy‐induced gene expression. Western blot analysis comparing undifferentiated and differentiated C2C12 cell lysates demonstrates that Ttc39c protein is expressed in proliferating myoblasts, but is reduced in differentiated myotubes. The transcriptional regulation of two distinct transcripts (termed 001 and 004) of Ttc39c have been examined by cloning the alternative promoter regions of each transcript into to a reporter plasmid. These reporter constructs were then transfected into muscle cells along with expression plasmids for MyoD and myogenin, two myogenic regulatory factors (MRFs) that regulate the expression of muscle‐specific genes by binding canonical E‐box elements (5′‐CANNTG‐3′). The reporter gene constructs for both Ttc39c transcripts show significant transcriptional activity in cultured muscle cells and are transcriptionally responsive to MRF ectopic expression. Furthermore, a conserved E‐box element in the Ttc39c‐004 proximal promoter was also identified, mutated, and analyzed for its role in the regulation of Ttc39c‐004 expression. The mutation of the conserved E‐box rendered the Ttc39c‐004 reporter gene inactive, demonstrating that the element is necessary for Ttc39c‐004 expression. Finally, the 004 transcript reporter construct was also transfected into muscle cells along with an expression plasmid for MuRF1. MuRF1 overexpression repressed Ttc39c‐004 reporter activity, while overexpression of a MuRF1 RING domain mutant failed to repress the Ttc39c‐004 reporter. The identification and characterization of novel genes that are activated during neurogenic atrophy helps improve our understanding of the molecular genetic events that lead to muscle wasting and could eventually lead to new therapeutic targets in the future.

Support or Funding Information

The work was support by University of North Florida Transformational Learning Opportunity grants to D.W.
Striatal patch compartment lesions reduce habitual behaviors
Authors: Jenrette Terrell , Logue Jordan , Horner Kristen Ashley
Published: November 2023

The striatum is associated with habit formation and reward association. The striatum is composed of the patch and matrix compartments, and previous studies have shown that enhanced activation of the patch compartment relative to the matrix compartment is related to inflexible behaviors, such as stereotypy. Habitual behaviors are also inflexible in nature, but whether enhanced activation of the patch compartment contributes to habitual behavior is not known. Our experiment analyzed the role of the patch compartment in habit formation, by using a targeted neurotoxin to ablate the neurons of the patch compartment prior to sucrose self‐administration. Rats were bilaterally infused in the striatum with the neurotoxin dermorphin‐saporin (DERM‐SAP) to ablate the neurons of the patch compartment or unconjugated saporin (SAP, as a control) and allowed to recover for eight days. The rats were then trained to self‐administer sucrose using a random interval paradigm proven to generate habitual sucrose consumption. We then associated sucrose consumption with a negative stimulus (LiCl). Subjects were reintroduced to the self‐administration chamber and rats that continued to lever press, despite learning to associate sucrose with a negative stimulus, were considered to exhibit habitual behavior. Our data shows that DERM‐SAP pre‐treatment reduced sucrose self‐administration upon reintroduction to the self‐administration chamber in animals that received LiCl treatment, indicating that habit formation was attenuated in these animals. This data demonstrates that the patch compartment is necessary for the development of habitual behavior.

Support or Funding Information

NIH DA0235303 (KAH)

Mercer University Seed Grant (KAH)
Environmental Pollutant PBDE‐209 Regulates NO/cGMP Signaling through Activating NMDA Receptors In Neuronal Cells
Authors: Chen Jingsi , Li Xiuying , Li Xiaomei , Chen Min , Chen Dunjin
Published: November 2023

Decabromodiphenyl ether (PBDE‐209) is a persistent and important environmental pollutant that was once incorporated into many domestic products as a fire retardant. Once PBDE‐209 was thought to be safe. This is because it accumulated only at 500nM or lower concentrations in the human body and was rapidly metabolized. However, recent epidemiological studies showed that perinatal exposure to the compound causes neurodevelopmental abnormalities, such as delayed cognitive behavior development. Moreover, PBDE‐209 has been found to be toxic to cultured neurons, significantly impairing neuronal differentiation. However, the molecular mechanisms through which PBDE‐209 causes these untoward effects in neurons are unclear.

Nitric oxide (NO) and cGMP signaling play an important role in regulating neuronal maturation and abnormalities in this pathway have been linked to neuronal dysfunction and cell death. Accordingly, we tested whether relevant levels of PBDE‐209 disrupts nitric oxide (NO) and cGMP signaling in HT‐22 cells, a model neuronal cell line. We measured NO production and cGMP levels in these cells due to exposure to 100nM, 200nM, and 400nM PBDE‐209 in culture media. Whereas 100nM PBDE‐209 did not alter the levels of these mediators, we found that 200nM and 400nM PBDE‐209 increased NO production and that 400nM PBDE209 increased cGMP levels. After pretreatment with L‐nitro‐arginine methylester (L‐NAME), a nitric oxide synthase (NOS) inhibitor, NO production and cGMP levels were decreased in the PBDE‐209 exposed cells. This result suggested that NOS mediates the effects of PBDE‐209 on NO/cGMP signaling. Next, we tested the role of N‐methyl‐D‐aspartate (NMDA) receptors on PBDE‐209's regulation of NO/cGMP using MK‐801, a NMDA receptor inhibitor. We determined that PBDE‐209 increases NO production, cGMP levels, and neuronal nitric oxide synthase (nNOS) isoform expression through the activity of the NMDA receptor. Moreover, this NMDA receptor‐mediated effect of PBDE‐209 was observed to regulate cGMP levels through inhibiting the expression of phosphodiesterase (PDE) 5, which hydrolyzes cGMP, and PDE2, which is highly abundant in the hippocampus and metabolizes cAMP in addition to cGMP. Under certain conditions, cGMP mediates apoptosis in neuronal cells. Therefor, we measured the effect of PBDE‐209 on the Bcl‐2/Bax ratio, which was indicative of apoptosis induction, using immunoblotting. We found that PBDE‐209‐stimulated NMDA receptor activity increases the Bcl‐2/Bax ratio in the neuronal cell line.

Our studies implicate the NMDA‐NO/cGMP pathway in pathogenic mechanisms through which PBDE‐209 induces neurotoxicity.

Support or Funding Information

A Chinese National Nature Science Grant (Youth Project: 81402652) supported these research studies.
Heterodimerization of Organic Anion Transporting Polypeptide 1B3 can affect function and surface expression
Authors: Zhang Yuchen , Hagenbuch Bruno
Published: November 2023

OATP1B3 is a drug transporter expressed at the basolateral membrane of human hepatocytes, along with other transporters, including OATP1B1, OCT1 and NTCP. It has a broad spectrum of substrates including endogenous compounds and numerous drugs. Thus, modulation of OATP1B3 function and expression has the potential to affect drug efficacy and disposition. Previous studies in our lab demonstrated that OATP1B3 can form homo‐ and heterodimers, both in cell models and in frozen human liver tissue. However, functional consequences of these interactions have not been investigated so far. In this study, we used transient transfection of HEK293 cells with FLAG‐tagged OATP1B3 in combination with a His‐tagged non‐functional OATP1B3 mutant, or with His‐tagged OATP1B1 or NTCP, surface biotinylation, and cell‐based uptake assays to characterize the functional consequences of the dimerization. Co‐transfection of mutant and wild‐type OATP1B3 resulted in a similar uptake of both estrone‐3‐sulfate and estradiol‐17β‐glucuronide after normalization for surface expression as co‐transfection of empty vector with wild‐type OATP1B3, suggesting that the homodimer of OATP1B3 works as two independent functional subunits. Uptake of the OATP1B3‐specific substrate CCK8 decreased significantly when OATP1B3 was co‐expressed with NTCP, while co‐expression with OATP1B1 had no effect. In contrast, uptake of low concentration estrone‐3‐sulfate by OATP1B1 and taurocholic acid by NTCP was not affected by co‐expression with OATP1B3. Expression of OATP1B3 on the surface of HEK293 cells was reduced when OATP1B3 was co‐expressed with NTCP suggesting that the functional decrease might be a result of disturbed protein production and/or translocation to the plasma membrane. These results indicate that co‐expression of NTCP with OATP1B3 can affect OATP1B3‐mediate drug transport and as a consequence can have an effect on drug therapy.

Support or Funding Information

NIH grants GM103549 and GM077336
Dystroglycan regulates the Notch pathway to influence muscle stem cell maintenance
Authors: Mizrachi Mark , Wang Felix , Sharma Himanshu , Prives Joav , Colognato Holly
Published: November 2023

The extracellular matrix receptor dystroglycan provides a critical structural linkage across the plasma membrane, bridging the extracellular matrix and cytoskeleton in skeletal muscle. Due to this key role, dystroglycan loss‐of‐function leads to membrane instability and muscle degeneration in a subset of muscular dystrophies termed dystroglycanopathies. However it is increasingly recognized that dysfunction in muscle stem cells, and the accompanying failed muscle regeneration, is also a critical factor in muscular dystrophy pathogenesis. Muscle stem cells rely upon Notch, a signaling receptor that regulates stem cell fate determination, to maintain the stem cell pool and to appropriately regulate muscle progenitor production and differentiation by regulating the expression of the transcription factors Pax7 and MyoD. We recently discovered that dystroglycan can suppress Notch signaling in neural stem cells to regulate neural progenitor cell production. We therefore hypothesized that dystroglycan may also regulate Notch signaling in muscle stem cells, termed satellite cells, during muscle regeneration. To test this hypothesis we used the C2C12 myoblast cell line as a model system for muscle satellite cells in which we could perturb dystroglycan expression or dystroglycan function, followed by an examination of Notch signaling and muscle stem and progenitor cell phenotypes. Our preliminary experiments indicate that dystroglycan does indeed regulate Notch signaling in muscle satellite cells although, surprisingly, dystroglycan appears to promote Notch signaling. Dystroglycan loss‐of‐function leads to decreased expression of canonical Notch target genes, decreased expression of Pax7, a transcription factor required for muscle stem cell maintenance, and precocious expression of MyoD, a transcription factor required for muscle cell differentiation. Together these data suggest a key role for dystroglycan in utilizing the Notch signaling pathway to regulate the balance between muscle stem cell and progenitor cell populations. These data further indicate that disturbances in Notch signaling and stem cell dysfunction may play a significant role in dystroglycanopathies and related muscular dystrophies.

Support or Funding Information

ASPET Summer Undergraduate Research Fellowship
Cancer Cells with De Novo or Acquired Resistance to Carfilzomib Remain Dependent on the Proteasome for Their Survival and Growth
Authors: Lee Min Jae , Driscoll James , Kim Kyung Bo
Published: November 2023

Although the use of proteasome inhibitors (PIs)—bortezomib (Velcade®, Btz) and carfilzomib (Kyprolis®, Cfz)—in treating patients with multiple myeloma (MM) has successfully improved clinical outcomes, a subset of PI‐naïve patients fails to respond to these inhibitors, and almost all patients who do respond eventually acquire resistance to FDA‐approved PIs. Therefore, new therapeutic agents are urgently needed for these patients. In this study, we aim to develop novel, effective PIs for treating MM patients who do not respond or who no longer respond to Cfz therapies. We found that both H727 cancer cells with de novo Cfz resistance and RPMI 8226 multiple myeloma cells with acquired Cfz resistance remain highly sensitive to PYR‐41 (ubiquitin activating enzyme E1 inhibitor), and WP‐1130, P5091, and b‐AP15 (deubiquitinase inhibitors), suggesting that the ubiquitin‐proteasome system is essential to the survival of these Cfz‐resistant cancer cells. Additionally, silencing of proteasome α7 subunit, a component required for the assembly of active 20S proteasome, induced almost complete cell death, indicating that active proteasomes remain indispensable for the survival of Cfz‐resistant H727 cells. Based on these results, we tested the activities of cell‐permeable PIs with a variety of pharmacophores against Cfz‐resistant cancer cells. Among PIs we tested, P1' analogues of Cfz—UK101 and UK102—showed promising anticancer efficacies. Remarkably, UK‐101 and UK‐102 also displayed encouraging efficacies against primary MM cells that are resistant to both Btz and Cfz. In summary, we report that the proteasome remains essential to cancer cells that are resistant to Cfz, and that proteasome inhibition may still be a valid therapeutic strategy for patients who do not respond to Cfz therapy.

Support or Funding Information

This project is supported by a grant from NIH (R01 CA188354).
Adipose Tissue Constituents and the Adipokine Resistin Impair Vascular Endothelial Function in Obesity via Elevated Arginase Activity
Authors: Atawia Reem T. , Chen Jijun , Toque Haroldo A. , Yiew Kan Hui , Caldwell Ruth B. , Caldwell Robert W.
Published: November 2023

Objective

Define the molecular mechanisms underlying obesity‐induced endothelial dysfunction with an emphasis on the role of arginase (ARG) as a target for the better management of disease progression.

Introduction

Arginase (ARG) is expressed in endothelial cells (EC) in 2 isoforms (ARG1 and ARG2) and can contribute to vascular and endothelial dysfunction by limiting arginine availability for nitric oxide (NO) synthesis. Obesity is an important factor in several cardiovascular comorbidities and is associated with vascular endothelial dysfunction. Obesity is characterized by adipose tissue dysregulation resulting in increased secretion of pro‐inflammatory cytokines and adipokines such as resistin, which induces insulin resistance and endothelial dysfunction. Mechanisms underlying adipose tissue dysregulation‐induced endothelial dysfunction is not well understood.

Methods

To determine the effect of obesity on adipocyte dysregulation, mature adipocytes were extracted from visceral adipose tissues of diet‐induced obese (DIO) and lean mice to determine gene expression of pro‐inflammatory cytokines and adipokines. Additionally, Mouse aortic endothelial cells (MAEC) and aortic rings from lean mice were exposed to conditioned media (CM) prepared from visceral adipose tissues of DIO and lean mice or resistin (100 ηg/ml) for 24 hr to assess endothelial and vascular functions.

Results

Adipocytes from DIO mice showed increased mRNA expression of the pro‐inflammatory cytokines (TNF‐α and MCP‐1) and the adipokine resistin, compared to lean mice cells. MAEC exposed to CM from DIO mice showed increased mRNA expression of ARG1 and ARG2 (34% and 77%, respectively, p<0.05) accompanied by decreased NO production by 32%, p<0.01. Aortic rings incubated with CM from DIO mice exhibited a marked reduction in endothelium‐dependent vasorelaxation (EDV) to acetylcholine and increased contractile responses to phenylephrine (PE). Moreover, CM from DIO mice caused a 143% increase in resistin levels compared to CM from lean mice. Exposure of MAEC to resistin resulted in increased expression of ARG1 mRNA and decreased NO production (30% and 39%, respectively, p<0.05). Exposure of aortic rings to resistin impaired EDV. These dysfunctions were largely prevented by co‐treatment with the specific ARG inhibitor ABH (100 μM). The resistin‐induced increase in ARG1 expression in MAEC involves activation of the p38 mitogen‐activated protein kinase pathway as co‐treatment with p38 inhibitor (SB202190) prevented both effects in response to resistin.

Conclusion

Adipose tissue constituents from DIO mice and the pro‐inflammatory adipokine resistin impair EC‐dependent vasorelaxation via elevated arginase expression/activity

Support or Funding Information

NIH Grant EY11766 & NIH HL70215
C‐terminus of caveolin‐1 plays a critical role in endothelial cell migration and cell metabolism
Authors: Siamwala Jamila H , Bunn David A , Nguyen Uyen , Miyanohara Atsushi , Roth David M , Patel Hemal H
Published: November 2023

Caveolins are evolutionary conserved plasma membrane proteins necessary for the formation of caveolae in diverse cell types. Caveolae are involved in intracellular transport, extracellular secretion, cell signaling and cellular homeostasis. Caveolin‐1 (Cav‐1) mutations in the C‐terminus are associated with pulmonary hypertension, progeria like syndrome, and lipodystrophy. However, the mechanisms by which C‐terminal Cav‐1 mutations cause disease etiology is unclear. Pulmonary hypertension is characterized by endothelial cell dysfunction in pulmonary arteries. We hypothesized that Cav‐1 mutant protein (c.478_479delTT) alters endothelial cell metabolism and migration. Primary endothelial cells were treated with HIV1‐CMV‐GFP or HIV1‐CMV‐hCav1‐159 lentiviral vector representing the C‐terminus‐mutated Cav‐1 protein for 48hr followed by wound healing assay and mitochondrial structure and function assessed by electron microscopy and Seahorse extracellular flux assay, respectively. We also assessed for biochemical markers such as focal adhesion kinase protein expression (FAK) involved in cell migration. Cells treated with Cav‐1 mutant virus showed 60% inhibition of endothelial cell migration compared to GFP controls. Electron microscopy revealed small and rounded mitochondria with disorganized cristae in cells treated with Cav‐1 mutant. The maximal respiration was reduced in cells treated with Cav‐1 mutant. FAK expression was downregulated in cells treated with Cav‐1 mutant suggesting a FAK mediated inhibition of endothelial cell migration.

Conclusions

The C‐terminus of Cav‐1 may be an as of yet poorly understood but critical regulator of endothelial function and metabolism.

Support or Funding Information

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Glutaminase 1 Promotes the Proliferation of Endothelial Cells via the Induction of Cyclin A
Authors: Durante William , Liu Xiao‐ming , Yates Benjamin , Yu Yajie , Peyton Kelly J.
Published: November 2023

Glutaminase (GLS) is a phosphate‐dependent enzyme that converts glutamine to glutamate and ammonia. There are two distinct isoforms of GLS, GLS1 and GLS2, which possess discrete tissue distribution, structural properties, enzyme kinetics, and molecular regulation. Although endothelial cells possess substantial GLS activity, its functional role in these cells remains largely unknown. In the present study, we characterized the expression of GLS in endothelial cells and investigated the effect of GLS on the proliferation of endothelial cells. Human umbilical vein endothelial cells (HUVEC) expressed high levels of GLS1, but GLS2 was not detected in these cells. Treatment of HUVEC with the non‐selective GLS inhibitor 6‐diazo‐5‐oxo‐L‐ornithine or the selective GLS1 inhibitors bis‐2‐(5‐phenylacetamido‐1,3,4‐thiadiazol‐2‐yl)ethyl sulfide or CB‐839 resulted in a concentration‐dependent inhibition of endothelial cell growth. All three GLS1 inhibitors also blocked DNA synthesis in both HUVEC and mouse aortic endothelial cells. Similarly, knockdown of GLS1 expression using a siRNA approach blocked the mitogenic response of endothelial cells. Flow cytometry experiments demonstrated that GLS1 inhibitors or GLS1 silencing arrested endothelial cells in the G0/G1 phase of the cell cycle, as reflected by increases in the percentage of cells in G0/G1 with corresponding decreases in the fraction of cells in S and G2/M phases. Cell cycle arrest by these GLS1 inhibitors was associated with a significant decrease in the expression of cyclin A protein that was paralleled by a reduction in cyclin A mRNA and promoter activity. Finally, adenoviral‐mediated gene transfer of cyclin A largely restored the proliferative response of GLS1‐inhibited endothelial cells. In conclusion, this study demonstrates that endothelial cells selectively express GLS1, and that GLS1 plays a central role in stimulating endothelial cell proliferation and cell cycle progression via the induction of cyclin A. In addition, it identifies GLS1 as an attractive therapeutic target in treating diseases associated with aberrant endothelial cell growth.

Support or Funding Information

This work was supported by the American Heart Association Grant 15GRNT25250015.
Helium Mediated Regulation of Endothelial Permeability: Involvement of Connexin 43, VE‐Cadherin and Caveolin‐1
Authors: Konkel Moritz , Smit Kirsten Fernande , Hollmann Markus Werner , Patel Hemal H , Albrecht Martin , Weber Nina Claudia
Published: November 2023

Background

Caveolae are lipid enriched invaginations of the plasma membrane while Caveolins are structural proteins essential for the formation of these invaginations. Caveolins modulate signal transduction pathways in various cell types. The noble gas helium, a potent cardioprotectant, leads to decreased protein levels of Caveolin‐1 (Cav‐1) in the human endothelium and an increased level of Cav‐1 in the supernatant of human umbilical vein endothelial cells (HUVEC). Moreover, helium regulates actin filaments and decreases permeability of HUVEC monolayers.

We hypothesized that (1) an upregulation of the adhesion molecules Connexin 43 and VE‐Cadherin might be involved in the decreased permeability of the endothelium after helium treatment and (2) that Cav‐1 plays a crucial role in the helium induced decrease of endothelial permeability.

Methods

HUVEC were isolated from fresh umbilical cords, pooled and grown upon confluent monolayers. Cells were used at passages 3–5. Cells were either exposed to 20 min of helium (5% CO2, 25% O2, 70% He) or control gas (5% CO2, 25% O2, 70% N2) in a specialized gas chamber. Expression of adhesion molecules Connexin 43 and VE‐Cadherin was measured at 6, 12 and 24 hours using IR‐Western blot analyses.

In order to investigate how the protective effect of helium is related to Caveolin we transfected HUVEC with small interfering RNA (siRNA) for Cav‐1 72 h before the respective experiment.

Permeability was measured using Fluorescein isothiocyanate labelled bovine serum albumin (FITC‐BSA) flow‐throw after 6, 12 and 24 hours.

Results

In the helium group, protein analyses in cell lysate showed a significantly higher expression of Connexin 43 and VE‐Cadherin at 6 and 12 hours in comparison to the control group (for Connexin 43 in helium treated cells at 6h: 1.31±0.39 and 12h: 1.27±0.37. For Connexin 43 in control cells at 6h: 0.90±0.32 and 12h: 0.82±0.26. For VE‐Cadherin in helium treated cells at 6h: 1.19±0.28 and 12h: 1.14±0.37. For VE‐Cadherin in control cells at 6h: 0.87±0.11 and 12h: 0.85±0.14: all treatments and time points vs. control 6h and control 12h, respectively: P<0.05).

Interestingly, cell permeability measurements of Cav‐1 siRNA transfected HUVEC showed no significant differences between the helium and control group suggesting a key role of Cav‐1 in endothelial permeability.

Conclusion

These findings suggest that an upregulation of the adhesion molecules Connexin 43 and VE‐Cadherin might be involved in the decreased permeability of the endothelium after helium treatment and that Cav‐1 plays a crucial role in the helium induced decrease of endothelial permeability.

Support or Funding Information

This project was in part funded by a grant fromthe IARS (International Anesthesia Research Society) and the SCA (Society of Cardiovascular Anesthesiologists) to NC Weber
Tetramethylpyrazine Prevents sEH Dysregulation to Protect Coronary Endothelial Function Through an anti‐ER Stress Dependent Mechanism
Authors: Mak Shiu‐Kwong , Sun Wen‐Tao , Fu Jie , Yang Qin
Published: November 2023

Activation of soluble epoxide hydrolase (sEH) is associated with endothelial dysfunction in cardiovascular disease such as hypertension, though mechanisms underlying sEH dysregulation are inadequately understood and whether endoplasmic reticulum (ER) stress is involved is yet to be studied. Tetramethylpyrazine (TMP) is a major bioactive ingredient of Chinese herb Chuanxiong that has been long known for cardiovascular benefits. However, whether TMP may protect vascular endothelial cells from ER stress and whether it may regulate sEH expression remain unknown. This study therefore aimed to investigate whether ER stress participates in angiotensin II (Ang‐II)‐induced sEH dysregulation in coronary endothelium and whether anti‐ER stress mechanism is involved in the vasoprotective effect of TMP through the regulation of sEH. Endothelial cells (PCECs) were isolated by enzymatic digestion from porcine coronary arteries (PCAs) and primary cell cultures were used for western blot, ELISA, and reverse‐transcription PCR analysis. Isometric force study of PCAs was performed in a myograph for assessment of endothelial dilator function. Ang‐II enhanced sEH expression and lowered 11,12‐EET level in PCECs, along with increased expression of ER stress markers, i.e., GRP78, ATF6, p‐PERK, and p‐IRE1. The increases of sEH mRNA and protein were also observed in PCECs exposed to tunicamycin, a chemical ER stress inducer. Treatment of PCECs with ER stress inhibitors suppressed Ang‐II‐induced upregulation of sEH and loss of 11,12‐EET. PCAs subjected to Ang‐II or tunicamycin exposure exhibited impaired endothelium‐dependent relaxation, which was improved by inhibitors of ER stress or sEH. TMP showed comparable inhibitory effect to that of ER stress inhibitors on Ang‐II‐induced ER stress and dysregulation of sEH and EET. sEH expression was also normalized in tunicamycin‐exposed cells that underwent TMP treatment. TMP was similarly efficient as ER stress inhibitor in the restoration of endothelial vasodilator function in Ang‐II‐ and tunicamycin‐exposed PCAs. The present study suggests that ER stress mediates Ang‐II‐induced sEH upregulation in coronary endothelium. TMP has potent anti‐ER stress capacity through which TMP normalizes sEH expression and confers protective effect against Ang‐II on endothelial function of coronary arteries. These findings added new mechanistic insight into the cardiovascular benefits of TMP and may suggest a potential of broadening the use of this traditional Chinese medicine in the treatment of diseases whose pathogenesis involves ER stress.

Support or Funding Information

Supported by RGC/GRF CUHK14118414; Lui Che Woo Institute of Innovative Medicine ‐ CARE theme 8303303, and CUHK Direct Grant 4054273.
Beneficial Effects of (−)‐Epicatechin on Biomarkers of Endothelial Cell Aging
Authors: Mansour Christina , Ceballos Guillermo , Ramirez‐Sanchez Israel , Villarreal Francisco
Published: November 2023

Aging is known to contribute to the development of endothelial dysfunction and as such facilitates the development of cardiovascular diseases (CVD). The loss of the ability of endothelial cells to produce physiological amounts of nitric oxide (NO) partly underlies the detrimental effects of aging. Thus, the identification of agents that can mitigate aging induces loss of endothelial cell function (i.e. NO production) are of great interest. Several reports indicate that the administration of the cacao flavanol (−)‐epicatechin (Epi), can partly reverse the deleterious effects of aging in organs such as the heart and kidneys. However, less is known on its effect on aged endothelium. To address this question, we implemented a primary cell model of aging that is based on the use of high passage number (31–35) bovine coronary endothelial cells (BCAEC) in culture. We specifically evaluated NO production through the endothelial NO synthase (NOS) pathway, markers of cell senescence (β‐galactosidase) and mitochondrial biogenesis (sirtuin‐1, mitofilin, oxidative phosphorylation complexes II, IV and V, Tfam and citrate synthase activity). BCAEC at low passage number (8–13 passage number) were used as “young” controls. Cells were treated with either vehicle (water) of Epi (100 nM) for 48 h and media and cells collected for testing using kits (NO, citrate synthase) or Western blots. All experiments were performed independently a minimum of 3 times in triplicate. All results were statistically analyzed. Results indicate that senile cells loose their capacity to produce NO by ~33% (p<0.05) and that Epi treatment can essentially fully restore their production levels to those noted for vehicle treated young cells. These results paralleled to those noted for the phosphorylation of eNOS. While β‐galactosidase levels doubled (100% increase) in aged cells, those treated with Epi only evidenced about a 40% increase (p<0.05). The levels of phosphorylated sirtuin‐1, mitofilin, oxidative phosphorylation complexes and Tfam were all reduced by ~40% with aging (p<0.05) and were largely restored by Epi treatment. Altogether, results strongly suggest that Epi can partly reverse markers of endothelial cell aging by restoring critical components of vascular function. These effects may partly explain the epidemiological evidence for the beneficial effects of cocoa consumption on the incidence of CVD.

Support or Funding Information

NIH DK98717