bluuebunny/crossref_metadata_2025 huggingface dataset:
Disruption of membrane caveolae limits pulmonary vasoconstriction induced by agonists in pulmonary hypertension rats (1089.8)
Authors: Jiao Hai‐Xia , Mu Yun‐Ping , Wang Rui‐Xing , Lin Da‐Cen , Sham James , Lin Mo‐Jun
Published: June 2021

A rise in cytosolic Ca2+ in pulmonary artery smooth muscle cells (PASMCs) is the major trigger for pulmonary vasoconstriction. Previous studies of pulmonary hypertension (PH) show that canonical transient receptor potential (TRPC) is an important constituent of store‐operated Ca2+ channel (SOCC); TRPC genes are upregulated and store‐operated Ca2+ entry (SOCE) is augmented in PASMCs of monocrotaline (MCT)‐treated and chronic hypoxic (CH) rats. Since TRPC and caveolin may functionally colocalize in caveolae in PASMCs and coordinate to regulate pulmonary vascular tone, we sought to examine the effects of caveolae disruption on agonist‐induced contraction of isolated pulmonary arteries (PAs) of CH‐ and MCT‐induced PH rats. Rats developed severe PH and right ventricular hypertrophy within three weeks after CH‐(10% O2) exposure or single intraperitoneal injection of MCT at a dose of 60 mg/kg. The tension of PA rings induced by various agonists were compared with and without the caveolae‐disrupting agent methyl‐beta‐cyclodextrin (MCD) or cholesterol repletion with a cholesterol:MCD mixture (1:5). The MCD‐pretreatment attenuated PA contraction induced by cyclopiazonic acid (CPA), endothelin‐1(ET‐1) and phenylephrine (PE), but had no effect on KCl‐induced vasoconstriction in control groups. The cholesterol:MCD mixture rescued PA‐contraction induced by CPA, ET‐1 and PE. Compared with the control group, MCD‐pretreatment resulted in further inhibition of the CPA‐induced contraction of PAs of both CH‐ and MCT‐treated rats, as well as the PE‐induced contraction of PAs of MCT‐treated rats, but had no effect on KCl‐induced contraction in the CH‐ and MCT‐ groups. These results suggest that the activity of voltage‐dependent Ca2+ channels does not depend on the integrity of associated with caveolae, but the SOCC may be closely associated with the cholesterol‐enriched membrane lipid domains of PAs. Moreover, the enhanced SOCE of PAs caveolae in PH rats.

Grant Funding Source: NSFC31171104 and FJMU09ZD010
Role of mitochondrial KATP channels in PKCβ‐dependent constriction of small pulmonary arteries (1090.10)
Authors: Snow Jessica , Norton Charles , Sands Michelle , Jernigan Nikki , Gonzalez Bosc Laura , Walker Benjimen , Resta Thomas
Published: June 2021

PKCβ mediates vasoconstriction through a unique signaling pathway in the pulmonary circulation that involves mitochondrial reactive oxygen species (mitoROS). We hypothesized that PKCβ elicits these responses through activation of vascular smooth muscle mitochondrial KATP channels (mitoKATP). To test this hypothesis, we examined constriction in endothelium‐disrupted, pressurized rat pulmonary arteries (~150 μm diameter) and mitoROS production (by MitoSOX fluorescence) in cultured rat and human pulmonary arterial smooth muscle cells (PASMC) in response to the PKC agonist phorbol 12‐myristate 13‐acetate (PMA). PMA caused vasoconstriction and mitoROS production that were significantly attenuated (p<0.05) by both the PKCβ inhibitor LY‐333,531 (10 nM) and PKCβ siRNA. Consistent with our hypothesis, the specific mitoKATP channel antagonist 5‐hydroxydecanoate (5‐HD; 500 μM) inhibited vasoconstriction and mitoROS production to both PMA and the mitoKATP activator diazoxide (10‐5‐ 10‐3 M). Consistent with a role for mitoKATP in PKCβ‐dependent mitoROS generation, PMA caused mitochondrial membrane potential depolarization (detected by tetramethylrhodamine methyl ester fluorescence), which was prevented by 5‐HD (p<0.05 vs. vehicle) in both pulmonary arteries and human PASMC. We conclude that PKCβ enhances mitoROS production via mitoKATP activation in PASMC to mediate vasoconstriction.

Grant Funding Source: Supported by NIH R01 HL088192 and American Heart Association grants 1POST5260026 and 13GRNT14
Interleukin‐33 in pulmonary arterial hypertension: a role in disease pathogenesis? (1090.4)
Authors: Aroonsakool Nakon , Titone Dominic , Li Jinghong , Dumouchel Justin , Lombardi Sandra , Kim Nick , Poch David , Bigby Timothy , Murray Fiona
Published: June 2021

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance that leads to right ventricular failure. PAH can be a primary disease (idiopathic, IPAH) or secondary to conditions such as connective tissue disease. Interleukin‐33 (IL‐33), which is the ligand for the ST2 receptor, is a cytokine that acts as an alarmin. Since PAH is associated with the infiltration of inflammatory cells and increased cytokines, we aimed to investigate the role of IL‐33 in PAH. Using an IL‐33 ELISA we found that IL‐33 is increased in the serum of PAH patients, in particular IPAH [1798 +/‐846 pg/ml (n=6) vs. 24 +/‐ 13 pg/ml (n=6), p<0.01], compared to control subjects. We found that the mRNA expression of IL‐33 is increased ~8‐fold in peripheral blood mononuclear cells isolated from PAH‐patients and PAH‐animal models compared to control; Il‐33 is up‐regulated in the lung of animal models of PAH. Injury of pulmonary artery endothelial cells (PAEC) released IL‐33, thus implying that the increased circulating IL‐33 with PAH may occur if the membrane integrity of PAEC is compromised. Administration of IL‐33 (500 ng/day intracheally for four days) induced pulmonary arterial remodeling in WT and IL‐33 KO mice, but not ST2 KO mice. Our data demonstrate that IL‐33 is increased in PAH, which via ST2‐dependent signaling, contributes to remodeling of the PA and hence the pathophysiology of the disease.

Grant Funding Source: Supported by NIH
Circulating microparticles from pulmonary arterial hypertension stimulate ICAM‐1 and E‐selectin expression in pulmonary artery endothelial cells (1090.6)
Authors: Hargett Leslie , O'Neill Kealan , Yarbrough Thomas , Bauer Natalie
Published: June 2021

Circulating Microparticles from Pulmonary Arterial Hypertension stimulate ICAM‐1 and E‐selectin expression in Pulmonary Artery Endothelial CellsHargett L1, O’Neill K, Yarbrough T1, and Bauer N1,2. the Center for Lung Biology1 and Department of Pharmacology 2, University of South Alabama, College of Medicine, Mobile AL 36688.

Microparticles (MPs) are submicron vesicles released by various cells that serve as mediators of inflammation. MPs harbor adhesion molecules and can stimulate the expression n of adhesion molecules in target cells. MPs are increased in pulmonary arterial hypertension (PAH). The hallmark PAH arterial vasculopathy involves inflammatory cell infiltration. Rho kinase (ROCK) is implicated in the expression of adhesion molecules in the pulmonary endothelium. We propose that MPs, generated as a result of PAH, activate ROCK and increasing the expression of adhesion molecules in pulmonary endothelium. MPs were isolated from the blood of normal and PAH rats (Su/Nx/Hx model). Pulmonary artery endothelial cells (PAECs) were treated with media, MPs from normal rats, or MPs from PAH rats. ICAM‐1 and E‐selectin, as assessed by western and flow cytometry, were increased in the PAECs. ROCK activity, as indicated by phosphorylated MYPT1696 western, was increased in PAH‐MP treated PAECs. Our results indicate that MPs from 8 week PAH rats increase the expression of ICAM‐1 and E‐selectin selectively in PAECs 6 hours after treatment. Also, preliminary data show increased phosphorylated MYPT1 in response to MPs from 8 week PAH rats. In conclusion, MPs from PAH rats increase adhesion molecule expression and may enhance ROCK activity in PAECs in vitro. As our studies progress, an inflammatory role for MPs in PAH could be revealed.

This research is funded by AHA 11 (SDG7390037) Grant and NHLBI T32 (HL076125) Training Grant.
Repetitive acute intermittent hypoxia downregulates inflammatory gene expression in the rat ventral cervical spinal cord (1091.7)
Authors: Watters Jyoti , Roopra Avtar , Bowen Kellie , Vinit Stephane , Macfarlane Peter , Mitchell Gordon
Published: June 2021

Intermittent hypoxia exerts pathologic and beneficial effects in the CNS, depending on the dose and duration. Whereas hundreds of hypoxic episodes per day mimic sleep apnea and promote neuroinflammation, “low dose” protocols, such as repetitive acute intermittent hypoxia (rAIH; 10 episodes of 10.5% O2 per day, 3 days/week for 4 weeks), upregulate growth/trophic factors and promote spinal motor plasticity. Because rAIH increases protein levels of key molecules associated with spinal respiratory motor plasticity, we hypothesized that the mRNAs for these and other molecules contributing to neuronal plasticity would be similarly increased. Nimblegen neuronal gene microarrays were used to profile transcript levels in the spinal ventral horn (C4‐C6) of male Lewis rats exposed to normoxia or rAIH (n=3 arrays/condition). Surprisingly, there was little evidence of increased mRNA levels of pro‐plasticity genes, and these results were confirmed with qRT‐PCR for specific genes of interest. Instead, there were significant decreases in inflammatory genes including cyclooxygenase‐2, tumor necrosis factor family members, chemokines and interleukins 1β and 6. To explore the cause of these changes, we used whole genome Vista analyses to identify the transcription factor binding sites over‐represented in the upstream 5kb promoter regions of genes affected by rAIH. Genes with binding sites for nuclear factor kappa B (NF‐kB) were the most over‐represented (p<0.005), suggesting that rAIH may decrease NF‐kB transcriptional activity. Thus, in contrast to chronic intermittent hypoxia, rAIH may be anti‐inflammatory, depleting spinal NF‐kB target genes.

Grant Funding Source: Supported by NIH HL111598, HL080209, HL69064 and NS065067
Ventilatory sensitivity to CO2 is altered by external breathing resistance (1092.10)
Authors: Shykoff Barbara , Warkander Dan
Published: June 2021

CO2 retention by divers underwater or by subjects exercising while breathing on a resistive circuit is uncorrelated with resting ventilatory sensitivity to CO2. We hypothesized that work of breathing might modulate sensitivity. Read’s rebreathing method was used to measure resting ventilatory sensitivity to CO2 with no added resistance, inspiratory, expiratory, or both resistances. External resistors were two plugs with holes 0.56”, measured to cause work of breathing per tidal volume of 1 kPa with minute ventilation 100 L/min. Ventilation was measured with a turbine flow meter. CO2 in the mouthpiece was monitored using a sector mass spectrometer. Preliminary data are available from 7 healthy Navy divers. Sensitivities are given in (L/min)/Torr for the initial increase in CO2; some subjects showed increased sensitivity once CO2 climbed. Median values were 1.9 without added resistance, 3.9 with inspiratory resistance, 2.3 with expiratory resistance, and 3.2 with both resistances. However, minima were 0.5 without resistance, 0.12 with inspiratory resistance, and negative with expiratory or both resistances. For all conditions, some subjects showed normal sensitivities only after CO2 had climbed above 52 to 60 Torr, and some showed no ventilatory response. In this small sample, resting ventilatory sensitivity to CO2 changed unpredictably with external resistance.

Grant Funding Source: Supported by Naval Sea Systems Command Deep Submergence Biomedical Program
The thyrotropin‐releasing hormone analog Taltirelin augments the hypercapnic ventilatory response in CO2‐insensitive Brown Norway but not Sprague Dawley rats (1092.6)
Authors: Hodges Matthew , Novotny Tyler , Echert Ashley , Puissant Madeleine , Mouradian Gary
Published: June 2021

Thyrotropin‐releasing hormone (TRH) is an excitatory neuropeptide that may modulate the hypercapnic ventilatory response (HCVR). We have found reduced expression of TRH in the medullary raphe in Brown Norway (BN) compared to Sprague Dawley (SD) rats. Herein we tested the hypothesis that the reduced TRH in the BN rat contributes to their blunted HCVR by measuring ventilation (VE), arterial blood gases, and rectal temperature (TR) in BN and SD rats during exposures to room air (RA) or hypercapnia (7% CO2) before and 1 hr after IP injections of saline, or 0.3 mg/kg or 1.0 mg/kg of the TRH analog Taltirelin (TAL). VE in RA did not differ among strains during all control studies, but VE increased after low and high dose TAL in both strains (p蠄0.05), with a greater increase in BN rats (p<0.05). The slope of the relationship between VE and PaCO2 breathing RA and 7% CO2 (ΔVE/ΔPaCO2) was lower in BN (0.4 ± 0.3) vs. SD (10.2 ± 1.5) rats after saline (p<0.05), and increased in BN (6.3 ± 1.0; p<0.05) but not SD (8.2 ± 1.2; p=0.15) rats after high dose TAL, where ΔVE/ΔPaCO2 no longer differed among the strains (p=0.207). TR also significantly increased in BN rats after high dose TAL (+1.1 ± 0.1°C; p<0.001), but was unaffected in SD rats (+0.1 ± 0.2°C; p=0.523). These data suggest an increased sensitivity to TRH receptor stimulation in BN rats, consistent with the concept that reduced endogenous TRH expression contributes to their blunted HCVR.

Grant Funding Source: Supported by NIH HL097033
Generation of multiciliated cells in functional airway epithelium from human iPSC (1094.5)
Authors: Firth Amy , Menon Tushar , Dargitz Carl , Qualls Susan , Wright Rebecca , Khanna Ajai , Verma Inder
Published: June 2021

Despite a significant improvement in the availability of therapeutic options to treat lung diseases, pulmonary disease still remains a major cause of morbidity and mortality around the world. Currently there are limited opportunities to study human lung disease either in vivo and in vitro. Using induced pluripotent stem cells (iPSC) we have generated a reproducible differentiation protocol to make mature post‐mitotic multiciliated cells in a functional airway epithelium.

iPSC were generated from human skin biopsies and differentiated via FOXA2+SOX17+ definitive endoderm (>90% efficiency) to FOXA2+NKx2.1+ anterior foregut endoderm, FOXA2+NKx2.1+SOX2+ (~50% efficiency) pulmonary endoderm and then matured in an air liquid interface. Robust multiciliogenesis occurred when Notch signaling was inhibited and was confirmed by; i) the assembly of multiple pericentrin stained centrioles at the apical surface, ii) expression of transcription factor FOXJ1 and iii) presence of multiple acetylated tubulin labeled cilia projections in individual cells. The presence of NKx2.1+CC10+ Clara cells, MUC5A/C+ goblet cells and FOXA2+p63+ basal cells was also confirmed showing we are generating a complete polarized epithelial cell layer comprised of all relevant cell types. Functional cAMP activated and CFTRinh‐172 sensitive CFTR currents were recorded in isolated epithelial cells by whole cell patch clamp technique. Furthermore, we have corrected the deltaF508 mutation in the CFTR gene (>80% of all cases of CF) using a combination of CRISPR‐Cas9 endonuclease‐mediated genome editing and piggyBac transposase technologies, in the CF patient‐derived iPSC.

The generation of mature multiciliated cells in a human iPSC differentiated respiratory epithelium and the ability to correct disease causing mutations provides a significant advancement toward modeling a number of human respiratory diseases in vitro.

Grant Funding Source: Supported in part by CIRM and the Berger Foundation
The role of Na/K‐ATPase in cardiac stem cells (1094.6)
Authors: Shi Huilin , Drummond Christopher , Tian Jiang
Published: June 2021

It has been shown that Na/K‐ATPase is involved in cardiac remodeling in 5/6th partial nephrectomized (PNx) mice and rats. Our recent work observed that genetic reduction of the Na/K‐ATPase α1 subunit results in higher cardiac c‐kit positive cell numbers. To delineate the mechanisms, we isolated sphere forming cardiac stem cells from Na/K‐ATPase α1 heterozygous mice and their wild type littermates to compare their proliferation capacity. It was found that heart tissue from α1 heterozygous mice produces more phase‐bright cells than that from WT mice during the same time period, indicating a faster growth rate with reduction of Na/K‐ATPase. The formation of first generation cardiospheres (CSps) on poly‐D‐lysine coated surfaces is also faster with cells isolated from α1 heterozygous mice. To test the proliferation rate in in vitro culture, the purified primary CSps derived cells (PCDCs) were obtained by growing first generation CSps on fibronectin coated surfaces. These cells show positive staining of progenitor cell markers c‐kit, Oct4 and Nkx2.5, but negative staining for the cardiac marker α‐actinin. The results showed that PCDCs from both wild type and heterozygous mice can form cardiospheres at a similar rate which are of a similar size. These data suggest that reduction of Na/K‐ATPase may regulate cell growth in vivo or in the early stage of stem cell purification but has no significant effect on its capacity of sphere forming once selected and cultured in vitro.

Grant Funding Source: Suported by NIH grant R01 HL‐105649
Inhibitory effects of J10 on lung cancer cell motility: an in vitro and in vivo study (1095.14)
Authors: Wu Chieh‐Hsi , Chen Shuo‐Chueh , Chien Yi‐Chung , Pan Chun‐Hsu , Chen Chih‐Yi , Sheu Jyh‐Horng
Published: June 2021

There are many major causes of cancer death, including metastasis of cancer. Dihydroaustrasulfone alcohol, which isisolated from marine coral, has shown antioxidant activity, but has not been reported to have an anti‐cancer effect. We first discovered that dihydroaustrasulfone alcohol provided a concentration‐dependent inhibitory effect on the migration and motility of human non‐small cell lung carcinoma (NSCLC) A549 cells by trans‐well and wound healing assays. The results of a zymography assay and Western blot showed that dihydroaustrasulfone alcohol suppressed the activities and protein expression of matrix metalloproteinase (MMP)‐2 and MMP‐9. Further investigation revealed that dihydroaustrasulfone alcohol suppressed the phosphorylation of ERK1/2, p38, and JNK1/2. Dihydroaustrasulfone alcohol also suppressed the expression of PI3K and the phosphorylation of Akt. Furthermore, dihydroaustrasulfone alcohol markedly inhibited tumor growth in Lewis lung cancer (LLC)‐bearing mice. We concluded that Dihydroaustrasulfone alcohol is a new pure compound with anti‐migration and anti‐tumor growth activity in lung cancer and might be applied to clinical treatment in the future.

Grant Funding Source: Supported by National Science Council, Taiwan with the Grant No: NSC 101‐2320‐B‐039 ‐058 a
Increases in protein expression of the alpha7beta1 integrin pathway following 8 weeks of acetaminophen administration in the rat soleus (1095.17)
Authors: Gallagher Philip , Graham Zachary , Broderick Tom , Carroll Chad
Published: June 2021

Acetaminophen (APAP) has been shown to increase muscle mass following chronic exercise. Proteins of the alpha7beta1 integrin complex can detect stretch along the sarcolemma and activate signaling pathways that are responsible for cytoskeletal rearrangement and muscle hypertrophy. The purpose of this study was to determine if 8 weeks of aerobic exercise with or without APAP would effect expression of alpha7beta1 integrin, the FAK/cSrc/ERK1/2 cascade and certain hypertrophic proteins of the PI3K pathway in rat soleus muscle. Breifly, 10 week‐old male Wistar rats were randomly assigned to either a sedentary+placebo group (SED+PLAC; n=6), a sedentary+APAP group (SED+APAP; n=6), an exercise+placebo group (EX+PLAC; n=6) or an exercise+APAP (EX+APAP; n=6). Rats ran in progressive volumes (up to 60min/day) at 20m/min with an 8° grade for 8 weeks. Rats in the APAP groups received 200mg/kg/day of APAP via oral gavage. Protein expression was measured using Western immunoblotting. We found that rats that were administered APAP had higher levels of the 70 kDa alpha7 integrin subunit, total FAK, cSrc and p70s6k independent of exercise. Rats in the SED+APAP group had higher expression of total 4eBP1 compared to all other groups and rats in the EX+APAP group has higher expression of phosphorylated cSrc(416) compared to all other groups. Levels of phosphorylated 4eBP1(37/46) in the SED+APAP were higher compared to SED+PLAC. Finally, there were no differences between any group in the levels of expression for the full length alpha7 subunit, the beta1 integrin subunit, alpha‐actinin, total and phosphorylated ERK1/2(202/204), phosphorylated FAK(397) and phosphorylated p70s6k(389). In conclusion, consumption of APAP is associated with large changes in expression of multiple proteins of the focal adhesion independent of aerobic exercise in the rat soleus. Furthermore, APAP consumption is related to increases in expression of proteins of the hypertrophic PI3K pathway.
The significance of IL‐10 stimulation in cardiac innate responses (1095.18)
Authors: Bagchi Ashim , Akolkar Gauri , Mandal Soma , Gao Xiaoling , Yang Xi , Singal Pawan
Published: June 2021

It is known that the cardioprotective role of IL‐10 may involve innate signaling via the activation of patterns recognition receptors, such as Toll‐like receptor 4 (TLR4). We have previously demonstrated that myeloid differentiation gene factor 88 (MyD88) plays a key role in this innate response to IL‐10. In the present study, we further characterized downstream details in IL‐10 stimulation of innate signaling using IL‐10 knockout (KO) mice. A significant increase in the expression of TLR2 was noticed in the KO mice hearts whereas TLR4 expression was not different from the wildtype (WT). There was a significant downregulation in MyD88 expression. The expression of interleukin‐1 receptor associated kinases ‐1 (IRAK‐1) was slightly but significantly less in IL‐10 KO hearts with almost no difference in IRAK‐4 expression. On the contrary, IRAK‐M (an inhibitor of MyD88‐ dependent TLR4 signaling pathway) and IRAK‐2 levels were higher in the KO mice compared to WT. Levels of TNF‐α in IL‐10 KO mice were higher both in the heart and blood. IL‐1β was elevated in the blood of IL‐10 KO mice; however, there was no change in cardiac expression. TLR2‐ mediated TNF‐α activation in IL‐10 KO mice led to an increase in apoptosis as evident from increased expression of pro‐apoptotic protein, Bax and Bax/Bcl‐xL ratio. In the KO hearts, the increased proteolytic enzyme activity of caspase3/7 as well as fibrosis in the heart suggested that TLR2‐mediated IRAK‐M/IRAK‐2 activation might enhance TGF‐β and TNF‐α. Thus an increase apoptosis as well as fibrosis in the IL‐10 KO mice suggest that this cytokine is an important player in restoring cardiac cells from damage.

Grant Funding Source: Supported by the Canadian Institutes of Health Research
Long‐term atorvastatin treatment inhibited pro‐survival signaling, reduced mitochondrial function and altered ultrastructural integrity in cardiac myocytes (1095.6)
Authors: Zemljic‐Harpf Alice , Godoy Joseph , Schilling Jan , Kassan Adam , Schwarz Anna , Asfaw Elizabeth , Alvarez Erika , Dalton Nancy , Niesman Ingrid , Patel Hemal
Published: June 2021

Introduction: Statins are widely used to lower low‐density lipoprotein cholesterol (LDL‐C) for prevention of cardiovascular disease. The FDA warns that statins may induce skeletal muscle side effects. Skeletal muscle biopsies from patients with statin myopathy showed defects in mitochondrial ultrastructure. Impaired mitochondrial function was postulated as a key cause of statin‐induced myopathy. Long‐term statin effects on cardiac muscle are currently unclear.

Methods and Results: Neonatal cardiac myocytes were treated with either atorvastatin (lipophilic) or pravastatin (hydrophilic) for up to 48 hours (both at 1‐10 μM). Atorvastatin, but not pravastatin, inhibited RhoA activation, reduced mTOR/AKT signaling, and induced apoptosis. Further, atorvastatin treatment reduced protein expression of caveolin, dystrophin and epidermal growth factor receptor in cardiac myocytes. Both statins induced ER stress responses, but only atorvastatin increased CHOP initiated apoptotic cell death. Assessment of mitochondrial function after 48 hours of statin therapy revealed reduced oxygen consumption rates in atorvastatin treated cardiac myocytes, when compared to pravastatin or vehicle treated cells. The extracellular acidification rate profile was unchanged between groups at all time points. When mice underwent long‐term atorvastatin or pravastatin (5mg/kg/day for 7 months) treatment, both statins reduced LDL‐C, but only atorvastatin increased creatine kinase serum levels. Atorvastatin therapy in mice altered mitochondrial ultrastructure and induced protein aggregate formation in the heart. Atorvastatin, but not pravastatin, increased mortality in cardiomyopathic mice, when compared to vehicle treated controls.

Conclusion: Atorvastatin reduced mitochondrial function, altered cardiac ultrastructure and induced apoptosis in cardiac myocytes. Our data suggest that long‐term atorvastatin treatment impaired mitochondrial integrity in cardiac muscle.
Effect of altered endogenous melatonin concentration by conditional light control on cellular organization in injured spinal cord (1096.2)
Authors: Jin Yunho , Hong Yunkyung , Park Kanghui , Hong Yonggeun
Published: June 2021

Spinal cord injury(SCI) causes primary and secondary damage, leading to neurologic deficits. We aimed to determine the effect of altered melatonin level in cerebrospinal fluid(CSF) by conditional light control on cellular organization and motor recovery of SCI rats. Rats were divided into 3 groups and pre‐adapted under each condition for at least 2 weeks prior to operation; L/D(12/12‐hr light/dark), LL(24‐hr constant light), DD(24‐hr constant dark). After microsurgery, we collected injured cord segments and CSF. Rats under DD condition exerted higher BBB scores than that of L/D from day 7 through 28, while LL condition induced reduction of BBB scores than L/D on day 21 and 28. Melatonin concentration in CSF was the highest in DD on day 3, which was concomitant with NeuN upregulation in injured cord lesions. Elevated melatonin concentration in DD condition was sustained day 7 only. Although NeuN expression was significantly increased in all of groups at day 28 compared to day 3, its expression was the lowest in LL condition. Olig2 was also raised in DD condition on day 28. These results suggest that higher endogenous melatonin concentration in CSF may prevents neuronal cell loss but also promotes oligodendrocyte differentiation in injured cord region, leading to motor recovery in SCI rats.

Key words: Spinal cord injury, Cerebrospinal fluid, Endogenous melatonin, Light control, NeuN
Inhibition of IClswell impairs TNFα‐dependent activation of NF‐κB (1096.3)
Authors: Ettinger Nicholas , Nguyen Hong‐Ngan , Manlove Courtney , Lamb Fred
Published: June 2021

Cytokines such as TNFα drive inflammation via transcription factors including NF‐κB. Biologic agents targeting TNFα modulate disease, but require I.V. administration and cause immune suppression. Thus there is a need to develop oral, titratable therapies. In smooth muscle cells, NF‐κB activation by TNFα depends upon intracellular, endosomal ROS production by Nox1 and deficiency of ClC‐3 disrupts ROS production at this site. TNFα also activates IClswell via endosomal ROS. We hypothesized that IClswell activity is required for NF‐κB activation by TNFα. We used a luciferase‐NF‐κB reporter to quantify responses to TNFα (10ng/ml). Activation of HEK293 cells was reduced by both Tamoxifen (10μM, 13.9±3.5 to 6.4±2.2 fold) and DCPIB (30μM, 41.5±4.9 to 8.2±0.8 fold, N=6). In addition, inhibition of this channel in HUVECs with tamoxifen resulted in a 40% decrease in NFκB activation (N=6). These data suggest that IClswell activation by TNFα may play a role in NF‐κB signaling. IClswell has also been hypothesized to allow exit of ROS from the endosome to facilitate downstream cell signaling. Thus, we tested whether inhibition of ROS production would also lead to a decrease in NFκB activation. Inhibition of ROS production by TNFα with DPI resulted in a five‐fold increase in relative NFκB activation (N=4). Identification of the specific role of this anion conductance and localization of its activity to the plasma membrane or endosomes is the subject of ongoing investigation.

Grant Funding Source: NIH 5T32HD068256‐03
Role of cardiac fibroblasts in myocardial dysfunction in mice with sepsis: effect of NLRP3 inflammasome activation (1096.7)
Authors: Zhang Wenbo , Xu Xuemei , Kao Raymond , Mele Tina , Martin Claudio , Rui Tao
Published: June 2021

Background: Inflammasomes are intracellular platforms which promote maturation and releasing of IL‐1β and IL‐18. Cardiac fibroblast is the most abundant non‐cardiomyocytes within the heart. The aim of the present study was to evaluate whether sepsis activates NLRP3 inflammasome in cardiac fibroblasts and subsequently affects function of cardiomyocytes through IL‐1β thereby results in myocardial dysfunction. Materials and Methods: NLPR3, procaspase‐1, caspase‐1 p10, pro‐IL‐1β and IL‐1β in cardiac fibroblasts were assessed with Western blot or ELISA. Cardiomyocyte intracellular cAMP was assessed with direct immunoassay kit. Mouse model of sepsis was induced by intraperitoneal injection of LPS. Mouse myocardial function was evaluated with a mouse pressure‐volume loop analysis system. Results: Treatment of cardiac fibroblasts with LPS resulted in activation of capase‐1 (increased p10) and induced maturation and releasing of IL‐1β. Genetically (siRNA) and pharmacologically (Glyburide) inhibition of NLRP3 inflammasome in cardiac fibroblasts blocked the LPS‐induced activation of the caspase‐1, as well as the maturation and releasing of the IL‐1β. Inhibition of NLRP3 inflammasome in cardiac fibroblasts prevented the decrease in intracellular cAMP in cardiomyocyte conditioned with supernatants of cardiac fibroblasts with LPS. Inhibition of the NLRP3 inflammasome attenuated the myocardial dysfunction in mice with sepsis. Conclusions: Activation of NLRP3 inflammasome in cardiac fibroblasts results in increased IL‐1β production which mediates the cardiac fibroblast‐cardiomyocyte interaction and promotes the induction of myocardial dysfunction in mice with sepsis. (HSFO GIA 2012‐000212 to TR).

Grant Funding Source: HSFO
Modulation of the CX3CL1/CX3CR1 signaling pathway by acetylsalicylic acid (aspirin) in human trophoblast (1096.9)
Authors: Szukiewicz Dariusz , Alkhalayla Habib , Stangret Aleksandra
Published: June 2021

Aspirin inhibits cyclooxygenase (COX), acting stronger on the COX‐1 than the COX‐2. COX‐independent antiinflammatory effects of aspirin were also reported. Signaling pathway related to the chemokine CX3CL1 and its receptor CX3CR1 was proposed as a new target for aspirin. CX3CL1 modulates inflammatory response within the feto‐placental unit, influencing both adhesion and chemotaxis. HT produces CX3CL1 and expresses the CX3CR1. We examined the effects of aspirin on CX3CL1 production and CX3CR1 expression in HT obtained from normal term pregnanies (N = 24). The cytotrophoblasts were isolated using Kliman’s method based on trypsin, DNase and a 5‐70% Percoll gradient centrifugation, and cultured in normoxia. Secretion of CX3CL1 was augmented by lipopolysaccharide (1μg/ml). Next, the cultures were continuously exposed to aspirin, given in 6 different concentrations (1mM to 6mM) in respective groups. CX3CL1 levels in the culture media were measured using ELISA. After 5 days the cultures were fixed and immunostained for CX3CR1. CX3CR1 expression was examined by quantitative morphometry. Aspirin administered in higher doses (4.0mM and above) produced significant (p < 0.05) inhibition of CX3CL1 production in HT. This effect was inversely proportional to significantly increased (p < 0.02) expression of CX3CR1. Thus, influencing CX3CL1/CX3CR1, aspirin interferes with placental chemokine signaling network.

Grant Funding Source: Supported by WUM grant: 2M2‐W2‐13.
Protease‐activated receptor ‐3 signals independent of protease‐activated receptor‐1 to regulate endothelial nitric oxide synthase in human endothelial cells (1097.1)
Authors: Tillery Lakeisha , Motley‐Johnson Evangeline
Published: June 2021

Protease‐activated receptor‐3 (PAR‐3) is widely recognized as a non‐signaling elusive member of a G protein‐coupled receptor family known to regulate the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway via phosphorylation of its regulatory sites Thr495 and Ser1177. The eNOS/NO pathway is responsible for vascular smooth muscle cell relaxation and disruption of this pathway leads to endothelial dysfunction, laminar shear stress and vascular inflammation, a precursor to cellular induced hypertension. Using human coronary artery endothelial cells (HCAECs) as a model, we demonstrated that PAR‐3 is highly co‐expressed with PAR‐1 and ‐2 and can be activated using a synthetic peptide ligand, TFRGAP. We hypothesize that PAR‐3 can signal independently of PAR‐1 and ‐2 to regulate eNOS via site‐ specific phosphorylation and induce pro‐inflammation via the NFkB pathway. Analysis using eNOS specific antibodies showed that 500uM of TFRGAP lead to the phosphorylation of the positive site of eNOS‐Ser1177 and the negative site ‐Thr495, 30 seconds and 15 minutes respectively. Pretreatment of HCAECs with 10uM SCH79797, a selective PAR‐1 inhibitor, did not block the increase in phosphorylation at both eNOS regulatory sites stimulated by TFRGAP. Similar results where found in a stable PAR‐1 deficient cell line of HCAECs which confirmed the signaling potential of PAR‐3 using thrombin and the PAR‐3 activating peptide. Our data suggests that PAR‐3 is able to regulate eNOS/NO signaling via site‐specific phosphorylation that is independent of PAR‐1 activation. In additional studies cytokine production was measured using an ELISA after PAR‐3 activation. Data from these studies will lead to a better understanding of endothelial dysfunction and vascular inflammation while identifying potential drug targets for cardiovascular diseases.

Grant Funding Source: The project [poster] described was supported by CTSA award No. UL1TR000445
Dynamic measurements of [K+]i in cardiac differentiated P19 cells with a quantum dots (QD) potassium nanobiosensor, K‐QD (1097.5)
Authors: Wong Lid , Mao Hua
Published: June 2021

We reported the first dynamic measurements of [K+]i in intact differentiated P19 cells using a K‐QD nanobiosensor. K‐QD was synthesized by conjugating a 15‐crown‐5 K+ receptor to water soluble QD emitted at 630 nm. In Vitro spectral characterization of K‐QD confirms the sensitivity and specificity of K‐QD that is operated within the physiological K+ ranged from 4 to 130 mM. Differentiation of P19 cells was induced with 1% DMSO. A photon counting fluorescence microscope system with 405 nm excitation and 630nm emission was used to perform experiments with K‐QD loaded in the differentiated P19 cells using cation liposome. In four studies, 10‐8M to 10‐5M cumulative dose responses of [K+]i of each of the following 10 agents were determined. They were (i) quinidine, TEA and glibenclamide for the inhibition of the efflux of K+; (ii) bumetanide, ouabain, and acetylstrophanthidin for the inhibition of the influx of K+; (iii) levocromokalim and NS1619 for the activation of the K+ efflux; and (iv) nifedipine and verapamil for the inhibition of the calcium channels. Five experiments were performed per agent. The predictable and consistent responses induced by all these agents suggest that the application of K‐QD to determine the [K+]i responses in the cardiac differentiated P19 cells could be a robust platform for K+ channel compound screening, drug discovery and toxicology.

Grant Funding Source: NIDDK R44‐DK084600
NHE8’s role in male fertility involves LHR protein trafficking (1097.8)
Authors: Xu Hua , Chen Huacong , Li Jing , Ghishan Fayez
Published: June 2021

We reported last year that NHE8, the newest member of the sodium/hydrogen exchanger family, plays an important role in male fertility. NHE8 has abundant expression in the male reproductive organs, particularly in Leydig cells and epididymal duct cells. Male mice lacking NHE8 are infertile. Loss of NHE8 expression also resulted in reduced luteinizing hormone receptor (LHR) expression in male mice. Furthermore, luteinizing hormone (LH)‐stimulated cAMP production was reduced in isolated NHE8KO Leydig cells. After NHE8 expression was silenced by NHE8 siRNA in Leydig cells, reduced LHR expression and dramatically reduced cAMP production after LH stimulation were noted. Confocal microscopic imaging showed that LHR protein was targeted to early endosome in Leydig cells in the absence of NHE8 function instead of the plasma membrane. Therefore, male infertility in NHE8KO mice is due to the disruption of LHR protein trafficking, which results in an interruption in the precise hormone regulation that governs normal sperm development. This study is supported by NIH grant R01DK073638.

Grant Funding Source: Supported by NIH grant R01DK073638
Apical NH4Cl acts to reduce intracellular Na concentration in mTAL via cellular depolarization (1098.6)
Authors: Guha Avirup , Sun Jingping , O'Connor Paul
Published: June 2021

We have recently demonstrated that both cellular acidification and low intracellular [Na] promote NADPH oxidase activity in mTAL via activation of the voltage‐gated proton channel Hv1. NH4+ is normally present in the luminal fluid and its concentration increases during acidosis. It is known that apical uptake of NH4+ promotes intracellular acidification in mTAL, however the effect of apical NH4Cl on intracellular [Na] has not been studied. The goal of the current study was to determine the effect of increasing apical [NH4Cl] on intracellular [Na] in mTAL. We hypothesize that increasing apical NH4Cl concentration reduces intracellular [Na]. Measurements were performed using a FluoStar Omega fluorescent plate reader in cultured mTAL cells loaded with the Na sensitive dye CoroNa Green (n=6). Addition of NH4Cl to the media (HBSS pH 7.40) dose dependently reduced intracellular [Na] in cultured mTAL. Both the NKCC2 inhibitors furosemide and bumetanide lowered intracellular [Na] but did not prevent the action of NH4Cl to further reduce intracellular [Na]. Depolarization of mTAL using KCl reduced intracellular [Na] and greatly blunted the response to 8mM NH4Cl (‐62%, P<0.0002). We conclude that physiologically relevant increases in apical [NH4Cl] (4‐8mM) lower intracellular [Na] in mTAL and that these changes are mediated predominately by cellular depolarization. We speculate that during acidosis, increased NH4Cl delivery to the mTAL promotes NADPH oxidase activity in part by lowering intracellular [Na].

Grant Funding Source: AHA SDG 10SDG4150061
Ion regulation and stress tolerance during oscillating salinity in two color morphs of the green crab, Carcinus maenas (1099.6)
Authors: Himes Anthony , Balschi Wingcue , Frederich Markus
Published: June 2021

Carcinus maenas occurs in two color morphs: green after molting and red after prolonged intermolt. The ability to ion regulate in C. maenas is well documented in green morphs at low salinity, but no studies have focused on fluctuating salinity levels, like that of a tidal environment. We investigated 1) how the ability to ion regulate differ between the two morphs, 2) how oscillating salinity changes affect both morphs, and 3) if these changes vary with sex.

Red and green morphs of both sexes were exposed to constantly low salinity (12 ppt) or oscillating salinity, changing from 12 to 32 ppt every 6 h. We assessed hemolymph osmolarity, running endurance on a treadmill, reaction time, and mRNA and protein expression of key ion transporters in the gills.

Green morphs are better ion regulators than red morphs, with higher mRNA expression of cytosolic carbonic anhydrase (cCA), Na+K+ATPase (NKX), and Na+H+ antiporter (NHX). Greater mRNA expression of cCA, NHX, and NKX in crabs exposed to constantly low salinity shows that low salinity is more stressful than oscillating salinity. Little difference was noted between sexes.

Our data show that the well‐documented ion regulatory capabilities need to be viewed in a color morph specific context. Oscillating salinity being less strenuous than constantly low salinity is in agreement with the conditions in the crabs’ natural habitat as they are rarely exposed to constantly low salinity.

Grant Funding Source: NSF‐IOB0640478 to M.F., APS undergraduate summer research fellowship to A.H.
Feeding on the wing: energy allocation and prioritization in the hawk moth Manduca sexta (1100.3)
Authors: Arcas Sam , Davidowitz Goggy , Contreras Heidy
Published: June 2021

Independently, feeding and exercise have been documented as energetically costly activities. However, how simultaneous exercise and digestion affect energy allocation and exercise performance remains unclear. Using a simple model, we make predictions of how energy may be prioritized during exercise after consumption of food varying in quality. We used Manduca sexta (Sphingidae) to understand how organisms, adapted to “feeding on the wing”, prioritize and resolve a seeming conflict from these two simultaneous competing demands (digestion and activity). Previously, we showed that there was an additive effect of digestion to the metabolic cost of forward flight in M. sexta. In this study we focus on hovering flight to eliminate the effect of flight speed on metabolic rate. Adult, male M. sexta were fed one of 5 treatments (water, 5, 15, 25, 35% sucrose) and then required to sustain hovering flight until exhaustion. We recorded time to exhaustion as a measure of flight performance. Metabolic cost of flight was measured using flow through respirometry.
Calcium availability regulates calcium transport by the chorioallantoic membrane of an oviparous squamate (1100.8)
Authors: Jackson Ariella , Stewart James , Pyles Rebecca , Ecay Tom
Published: June 2021

Embryos of Pantherophis guttatus (the corn snake) develop within calcareous eggshells. Together, the yolk and eggshell provide all the calcium needed to support development through hatching. Hatchlings receive 25% of their calcium from the eggshell and removing shell calcium by physically peeling off the outer calcareous layers retards embryonic growth (length and mass) and reduces expression of calcium transport proteins by the chorioallantois, the epithelial tissue that catalyzes shell calcium transport. We hypothesize that chorioallantois calcium transport is regulated by calcium availability and predict that supplementing peeled eggs with external calcium will increased calcium transport protein expression and reverse the effects of shell peeling on hatchling growth. Paired peeled and intact eggs were incubated in either distilled water or water plus calcium chloride (2mM, 5mM, or 20mM) and incubated to the last developmental stage prior to hatching. At that time, the chorioallantois was assayed for expression of carbonic anhydrase II (CAII) and calbindin‐D28K by immunoblotting. Preliminary data show that expression of calbindin‐D28K is positively correlated with calcium supplementation in peeled eggs. CAII expression also increased, but not with the same linear concentration dependence. We conclude that chorioallantois expression of CAII and calbindin‐D28K is regulated by shell calcium but with different sensitivities. Further, 20mM calcium supplementation in peeled eggs yields calbindin‐D28K expression levels comparable to intact eggs. However, calcium supplementation did not reverse the growth effects of eggshell peeling suggesting that additional eggshell properties impact embryonic growth. (AJJ supported by an NSF‐REU fellowship)
The (pro)renin receptor blocker peptide effects on regulation of body weight and glucose homeostasis in mice with diet‐induced obesity (1101.10)
Authors: Shamansurova Zulaykho , Tan Paul , Bisotto Sandra , Michel Catherine , Nguyen Thi , Schiller Peter , Lavoie Julie
Published: June 2021

Introduction. The Prorenin receptor [(P)RR] is a recently discovered member of the renin‐angiotensin system (RAS). Adipose tissue (AT) is a 2nd major source of circulating angiotensinogen during obesity and (P)RR effects in adipose tissue have not been clearly identified yet. In this study, we investigated the relationship between (P)RR and body weight (BW) and expression of gene and proteins in the insulin signaling pathway in AT.

Material and methods. 12 week‐old C57BL/6 male mice were placed on a high‐fat/high‐carbohydrate (HF/HC) or normal diet (ND) and treated with a (P)RR blocker [(P)RR‐B] or saline for 10 weeks. BW and food intake was assessed weekly. At the end of treatment, mice were euthanized, plasma was collected for the measurement of glucose (G) and insulin (I); AT were separated, weighed and flash frozen. The GLUTs gene expressions were measured by q‐PCR; PKB proteins detected by Western‐Blot.

Results.(P)RR‐B administration lowered BW in mice on HF/HC as a result of lower body fat mass. Circulating levels of G,I and the G/I ratio were lowered by the (P)RR‐B on both diet. The GLUTs gene expression in AT was affected by HF/HC and improved with (P)RR‐B administration. Moreover, the PKB phosphorylation in AT was affected with HF/HC, where (P)RR‐B administration improve PKB phosphorylation in both diet.

Conclusion. The pharmacologic blockade of the (P)RR lowered BW in mice on a HF/HC, improved glucose homeostasis potentially by improving GLUTs gene expression profile and PKB phosphorylation in AT.

Grant Funding Source: Supported by CDA, MDRC
Post‐mortal CO2 release by insects at high temperatures (1101.14)
Authors: Heinrich Erica , Gray Emilie , Vorhees Ashley , Bradley Timothy
Published: June 2021

Researchers utilizing thermolimit respirometry to study insect thermal tolerance have previously reported an unexplained surge of carbon dioxide release by insects following death at high temperatures. This phenomenon has been referred to as the “post‐mortal peak” (PMP). In some insects, the CO2 release rate during the PMP may be up to 50% higher than the maximal rate achieved in the live insect. We have observed the PMP in fruit flies, mosquitoes, crickets, cockroaches, and beetles. While it has been verified that the PMP occurs after death, the cause of death does play a role in the appearance of the PMP. We have observed the PMP only when an insect dies due to high temperature stress. Furthermore, the PMP does not occur in the absence of atmospheric oxygen, or if the dead insect is subjected to cyanide prior to the initiation of the peak. On the basis of those results, we hypothesize that the CO2 released in the PMP derives from the insect’s mitochondria. We are currently simultaneously measuring CO2 release and oxygen uptake during thermal ramping in order to clarify the source and function of the PMP. Understanding this event may provide insights into the physiological implications of heat stress in insects and the timeline of biochemical events before and after death at high temperatures.

Grant Funding Source: Supported by The National Science Foundation
Skeletal muscle fibers in dystrophin‐deficient mice express intercellular adhesion molecule‐1 (1102.12)
Authors: Torres‐Palsa María José , Koziol Matthew , Goh Qingnian , Peterson Jennifer , Pizza Francis
Published: June 2021

Duchenne muscular dystrophy (DMD) is an X‐linked inherited disease resulting from the absence of the cytoskeletal protein dystrophin in skeletal and cardiac muscle cells. Without dystrophin, skeletal muscles incur repeated cycles of damage and regeneration. In a murine model of DM (i.e. mdx mice), myeloid cells (neutrophils and macrophages) contribute to the onset and progression of DMD. The objective of this investigation was to determine if intercellular adhesion molecule‐1 (ICAM‐1), a protein of the inflammatory response, is expressed by myofibers in mdx mice. We hypothesize that the expression of ICAM‐1 by myofibers in dystrophin‐deficient muscle serves as a mechanism by which myeloid cells exacerbate the muscle pathology. Western blot revealed increased expression of ICAM‐1 in mdx compared to control wild‐type mice. We found ICAM‐1 expressed on the membrane of necrotic, injured, and regenerating myofibers of mdx mice via immunolabeling. Myofibers of control wild‐type mice did not express ICAM‐1. These observations may indicate that skeletal muscle fiber expression of ICAM‐1 serves as a mechanism by which neutrophils and macrophages attach to skeletal muscle and exacerbate the myopathy in the mdx mice.