bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pressurized Pulmonary Arteries from the TWIK‐2 Knockout Mouse Exhibit Increased Vascular Reactivity
Authors: Kitagawa Melanie G , Durgan David J , Reynolds Julia , Bryan Robert M , Pandit Lavannya M 
Published: November 2023

TWIK‐2 is a member of the two‐pore domain potassium channel family. C57/BL6‐SV129 mice lacking functional TWIK‐2 channels develop pulmonary hypertension at 20 but not 10 weeks of age. We hypothesize that pulmonary arteries from TWIK‐2 KO mice show perturbations in vascular reactivity when compared to wild‐type (WT) mice.Right ventricular systolic pressure, a surrogate for pulmonary artery (PA) pressure, was measured in mice using isoflurane anesthesia. Right main PAs were harvested from 20 to 22‐week‐old WT and TWIK‐2 knockout (KO) mice and mounted between two glass micropipets. Each PA was bathed in Krebs buffer gassed with 20% O2 and 5%CO2 to achieve a pH of 7.4. Development of spontaneous tone was assessed by comparing PA diameters at pressures of 5, 15, and 30mmHg in 3 mM Ca+2 Krebs buffer to that in Ca+2‐free buffer. Constrictor responses were compared in PAs from WT and KO mice when phenylephrine (PE) was added to the bath at log increments from 10−10 M to 10−5 M. Following the constriction with PE, each PA was exposed to 10−5M acetylcholine (Ach) abluminally to measure endothelial‐mediated vasodilation. The data was analyzed using a repeated measure two‐way ANOVA or Mann‐Whitney Rank Sum test as appropriate.PA pressures were 22 ± 3 and 34 ± 1 mmHg in WT and KO mice respectively (p=0.01). In the presence of Ca+2, PA diameters were similar between genotype (898 ± 40 and 936 ± 40 μm for WT and KO respectively at 5 mmHg) and increased linearly 21 ± 3 μm / mmHg (R2 = 0.90) at 15 and 30 mmHg. In Ca+2‐free buffer, the diameters were not increased compared to that in the presence of Ca+2 at the three pressures studied. Thus, the right main branch of the PA does not develop spontaneous tone and dilates passively with increases in pressure. Concentration response curves to PE showed a similar maximal constrictor response; however, there was a significant interaction between genotype and PE concentration (p=0.013) and the EC50, the concentration for half of the maximal response, was 42‐fold less in KO (1.68 X 10 −10 M) compared to WT (7.03 X 10−9 M, p=0.029). Endothelial‐mediated dilations to acetylcholine (~11% of maximal diameter) were similar between genotype. The increased sensitivity to PE indicates the potential for an enhanced constrictor response in PAs from KO mice. This enhanced constrictor response in PAs from TWIK‐2 KO mice may have a role in the development of pulmonary hypertension.Support or Funding InformationVeterans Affairs Career Development Award Program (L.M. Pandit)
Reduced Membrane Cholesterol Following Chronic Hypoxia Limits Depolarization‐induced Ca2+ Entry In Pulmonary Arterial Endothelial Cells
Authors: Zhang Bojun , Naik Jay , Jernigan Nikki , Resta Thomas , Walker Benjimen 
Published: November 2023

Chronic hypoxia (CH)‐induced pulmonary hypertension is associated with diminished production of endothelium‐derived Ca2+‐dependent vasodilators such as nitric oxide. Interestingly, ATP‐induced endothelial Ca2+ entry as well as membrane cholesterol (Chol) are decreased in pulmonary arteries from rats exposed to CH (4 wk, PB = 380 torr) compared to normoxic control rats. Furthermore, Chol supplementation restores ATP‐induced Ca2+ entry to the level of controls, suggesting that reduced membrane Chol following CH limits the activity of ion channels required for agonist‐mediated Ca2+ entry. Depolarization‐induced Ca2+ entry is a major component of the response to ATP and is also decreased after CH. However, the effect of Chol on this component of Ca2+ entry is unknown. We hypothesized that depolarization‐induced entry also requires membrane Chol, and that CH attenuates this response by decreasing membrane Chol. To test this hypothesis, we administered Chol (2 mM) or epicholesterol (EpiCh; epimeric form of cholesterol, 2 mM) to acutely isolated pulmonary arterial endothelial cells (PAEC) from control and CH rats to either supplement or replace native Chol. The efficacy of membrane Chol manipulation was confirmed by filipin staining in isolated PAECs. Ca2+ influx in response to a depolarizing stimulus of KCl (60 mM) was assessed by Mn2+‐quenching of fura‐2 fluorescence. Whereas KCl‐induced Ca2+ entry was observed in cells from control rats, exposure to CH nearly abolished this response. EpiCh treatment greatly reduced KCl‐induced Ca2+ influx in PAECs from control rats (p<0.05 vs. vehicle) without further effect in those from CH animals. Chol treatment, however, restored depolarization‐induced Ca2+ entry in PAECs from CH rats (p<0.05 vs. vehicle). Ca2+ influx in response to KCl was inhibited (p<0.05 vs. vehicle) by the T‐type Ca2+ channel antagonist, mibefradil (10 μM), but not by the L‐type Ca2+ channel inhibitor, diltiazem (50 μM). We conclude that membrane Chol is required for depolarization‐induced Ca2+ entry through T‐type channels in PAECs and that reduced Ca2+ entry after CH may be due to loss of this key regulator.Support or Funding InformationAHA Southwest Affiliate Award #15GRNT21080001
G Protein‐Coupled Receptor (GPCR) Expression in Pulmonary Artery Smooth Muscle Cells of Human and Experimental Models of Pulmonary Arterial Hypertension
Authors: Aroonsakool Nakon , Sriram Krishna , Michkov Alexander V. , Insel Paul A. 
Published: November 2023

Pulmonary Arterial Hypertension (PAH) occurs as a primary (idiopathic) disease (IPAH) and secondary to various conditions. Both types are characterized by increased pulmonary vascular resistance and remodeling of the pulmonary artery, which derives in part from the proliferation of pulmonary artery smooth muscle cells (PASMCs), and can lead to right ventricular (RV) heart failure. Current treatments for PAH are inadequate, especially in terms of impact on mortality; thus, new therapies are needed. Animal models that mimic features of human PAH have been used to assess pathophysiology and evaluate treatments for PAH and include the exposure of rodents to chronic hypoxia (in the absence or presence of the VEGFR inhibitor SU5416) or treatment of rats with monocrotaline. Our studies are testing the hypothesis that previously unrecognized G protein‐coupled receptors (GPCRs) may contribute to PAH pathophysiology and may be new therapeutic targets. To begin to test this hypothesis, we used TaqMan® GPCR arrays as an unbiased approach to identify and quantify GPCRs in PASMCs of humans, rats and mice, including from patients with PAH and experimental PAH animal models. Development of PAH and RV hypertrophy in the animal models was confirmed by immunostaining/histology and quantified using the Fulton index (the [right ventricle]/[left ventricle + septum] weight). We found that PASMCs express >100 GPCRs and that the GPCR expression profiles of mouse and rat PASMCs are similar to that of human PASMCs. Moreover, we find numerous changes (of at least 2‐fold) in GPCR expression of PASMCs from PAH patients compared to control PASMCs. GPCRs with altered expression include receptors that link to G proteins whose signaling effectors (e.g., cAMP, Ca++, etc.) are predicted to regulate PASMC proliferation and tone. These results show that PASMCs express many more GPCRs than previously known and that the GPCR expression profile differs in PAH‐PASMCs. GPCRs with altered expression may contribute to altered function in PAH and thus may be novel therapeutic targets for this disease.Support or Funding InformationSupported by DOD AWARD: No. W81XWH‐14‐1‐0372
Calcineurin ‐NFAT Signaling Molecules Regulate Proliferation, Migration and Apoptosis in PASMCs of Pulmonary Hypertensive Rats induced by Monocrotaline
Authors: Lin Mo‐Jun , He Rui‐Lan , Liu Xiao‐Ru , Gui Long‐Xin , Wang Rui‐Xing , Mu Yun‐Ping , Sham James S.K. 
Published: November 2023

Pulmonary arterial hypertension (PAH) is characterized by increased resting intracellular free Ca2+ concentration of pulmonary arterial smooth muscle cells (PASMCs), angiogenesis and vascular remodeling. Previous studies of PAH show that canonical transient receptor potential (TRPC) genes are upregulated and store‐operated Ca2+ entry is augmented in PASMCs of monocrotaline (MCT)‐treated rats. While increased intracellular Ca2+ can further activate calcineurin (CaN) and make nuclear factor of activated T cells (NFAT) dephosphorylation and nuclear translocation. Since CaN and NFAT may functionally regulate the transcription of a variety of cytokines, we sought to examine the effects of CaN‐NFAT signaling molecules on proliferation, apoptosis and migration in PASMCs of control and MCT‐induced rats. Rats developed severe PAH and right ventricular hypertrophy within three weeks after single intraperitoneal injection of MCT at a dose of 60 mg/kg. MCT treatment can upregulated mRNA and protein expression of STIM1/2‐ORAI1/2‐CaNBβ‐NFATc2/3/4 on PAs, as well as developed proliferation and migration and decreased apoptosis on PASMCs. Pretreatment with Cyclosporine A (CsA) inhibited proliferation and migration on PASMCs of control (CON) and MCT rats, in a dose dependence manner (15, 30 60μM), but inhibited apoptosis only on high concentration (60μM). Proliferation, migration and apoptosis resistance of PASMCs in CON and MCT rats were obviously inhibit by siRNA of NFATc2 and NFATc4. Pretreatment with siRNA of NFATc3 were no effect to proliferation, migration and apoptosis resistance of PASMCs in CON rats, but can obviously suppressed proliferation and migration, promoted apoptosis of PASMCs in MCT rats. Our results hence show that MCT treatment caused significant alterations in the expression of STIM1/2‐ORAI1/2‐CaNβ‐NFATc2/3/4 signaling molecules. NFATc2/4 are essential factor for PASMCs growth, however, NFATc2/34 may contribute to pulmonary vascular remodeling in PAH induced by MCT.Support or Funding InformationThis work is supported by NSFC31171104, NSFC 31371165, NSFC 31571179 and FJMU09ZD010
HDAC6‐Hsp90 interplay in Pulmonary Arterial Hypertension
Authors: Chabot Sophie , Boucherat Olivier , Ruffenach Grégoire , Breuils‐Bonnet Sandra , Tremblay Eve , Provencher Steeve , Bonnet Sébastien 
Published: November 2023

BackgroundPulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by the elevation of mean pulmonary arterial pressure. Pulmonary arterial smooth muscle cells (PASMCs) from PAH patients exhibit a cancer‐like metabolic‐dependent pro‐proliferative, pro‐migratory and anti‐apoptotic phenotype leading to development of progressive pulmonary artery remodeling. The histone deacetylase 6 (HDAC6) is mainly cytoplasmic and involved primarily in “non‐histone” functions. HDAC6 interacts and deacetylates various proteins such as α‐tubulin and Hsp90 promoting proto‐oncogene activation such as Survivin (implicated in PAH) to carry out cancerous functions. We hypothesize that HDAC6/Hsp90 axis is up‐regulated in PAH and contributes to the development of the cancer‐like phenotype through in part Survivin stabilization.Method/ResultsUsing a translational molecule‐cell‐organ‐animal and multidisciplinary approach, we demonstrated that both HDAC6 and HSP90 are up‐regulated (immunofluorescence and immunoblot; p<0.01) in the lungs and PASMCs isolated from PAH patients (n=5) compared to age‐matched controls (n=5). HDAC6 inhibition via a pharmacological (Tubastatin A) or molecular (siHDAC6) approach dose dependently increased α‐tubulin acetylation and decreased PAH‐PASMC migration (wound healing assay; p<0.05), resistance to apoptosis (Annexin‐V; p<0.05) and proliferation (Ki67; p<0.01). Increased apoptosis following Tubastatin A treatment was associated with mitochondrial membrane depolarization (TMRM) of PAH‐PASMCs and inhibition of the oxygen consumption rate in a concentration‐dependent manner (Seahorse XFe 24 system). All these effects were associated with a down‐regulation of Survivin. Similarly, inhibition of Hsp90 (AT13387 or siHsp90) reverses the cancer‐like phenotype and this effect was associated with a down‐regulation of HDAC6, suggesting that HDAC6 up‐regulation in PAH is Hsp90‐mediated. In vivo, HDAC6 inhibition in monocrotaline‐induced PAH decreases both mean pulmonary artery pressure (right heart catheterization, p<0.01) and right ventricular hypertrophy (Fulton index).ConclusionWe provide evidence that Hsp90 and HDAC6 are specifically up‐regulated in human PAH and contributes to the proliferative, migratory and anti‐apoptotic phenotype seen in PAH‐PASMCs. As HDAC6 inhibitors have been tested in cancer, this offers a short‐term new therapeutic avenue for PAH patients.Support or Funding InformationCanada Research Chairs and CIHR grants to S. Bonnet and S. Provencher supported this work
JNK2 Regulates Vascular Remodeling in Pulmonary Hypertension
Authors: Das Mita , Zawada W. Michael , West James , Stenmark Kurt 
Published: November 2023

Structural alterations of pulmonary arterial (PA) wall are the key pathological features of pulmonary hypertension (PH). Although such vascular abnormalities correlate with heightened phosphorylation of c‐Jun N‐terminal kinases 1/2 (JNK1/2) in rat models of PH, contribution of each individual JNK isoform to the pathophysiology of PH is unknown. Hence, we hypothesized that activation of either one, or both JNK isoforms regulates PA wall thickening in PH. Comparing JNK1/2 activation in lungs of healthy individuals vs lungs of PH patients showed robust JNK1/2 phosphorylation (an index of JNK activation) accompanied by remarkable vascular thickening only in the disease‐affected lungs. In PH patients’ lungs, increased JNK1/2 phosphorylation paralleled marked reduction in MAP kinase phosphatase 1 (a key JNK dephosphorylator) expression. Association of JNK1/2 activation with vascular wall thickening was then confirmed in a well‐established neonatal calf model of hypoxia‐induced PH. To ascertain the role of each JNK isoform in PH pathophysiology, wild‐type (WT), JNK1 null (JNK1−/−), and JNK2 null (JNK2−/−) mice were exposed for six weeks to either normoxia (ambient air) for control or chronic hypoxia (10% O2) to develop PH. Hypoxia stimulated an increase in JNK1/2 phosphorylation that associated with the development of PH like pathology i.e. excessive accumulation of extracellular matrix, small vessel (20–100 μm diameter) muscularization with medial wall thickening, and up‐regulation of small vessel density in WT mouse lungs. Similar to the hypoxia‐exposed WT lungs, increase in deposition of extracellular matrix and structural remodeling of the vascular wall were also detected in hypoxic JNK1−/− lungs. In contrast, JNK2−/− mice lacked such hypoxia‐induced up‐regulation of matrix accumulation as well as vascular abnormalities in the lung. However, right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH) were increased to a similar magnitude by hypoxia among all three genotypes. Our findings suggest that JNK2 regulates hypoxia‐induced PA structural thickening and that selectively modulating JNK2 actions might quell vascular abnormalities in PH.Support or Funding InformationNational Institutes of Health HL64917
Ubiquitin Proteasome System in Pulmonary Hypertension
Authors: Wade Brandy E , Hart C Michael , Sutliff Roy L 
Published: November 2023

Pulmonary Hypertension (PH) is characterized by a sustained increase in pulmonary arterial pressure. Increased pulmonary vascular pressure and resistance result in right ventricle (RV) hypertrophy and can ultimately lead to RV failure and death. The mechanisms leading to vascular cell proliferation and vascular remodeling in PH are incompletely defined. Current therapeutic approaches that stimulate vasodilation inadequately address the cellular proliferation and vascular remodeling that underlies the pathogenesis of PH. To more directly address mechanisms of cell proliferation in PH, this project focuses on the ubiquitin proteasome system (UPS) which plays an important role incellular homeostasis by regulating protein stability. Current evidence demonstrates changes in UPS activity in PH; however, the pathways and proteins influenced by these changes are poorly defined. Mice exposed to normoxic or hypoxic conditions for 3 weeks were used to identify proteins with altered ubiquitination in response to hypoxia exposure. Lungs were collected, and the PTMScan® Ubiquitin Remnant Motif (K‐ɛ‐GG) Kit was used to precipitate ubiquitinated proteins for analysis by mass spectrometry (MS). 245 proteins were identified with a ±2 fold change. In silico analysis identified several proteins that promote cell proliferation. Tax1bp1, Hspa8, Hspb1, Fhl1, Gnb1 interact with important cellular proliferation pathways and exhibit decreased ubiquitination in lungs from mice exposed to hypoxia, suggesting an increase in their stability in hypoxia. These results using MS and in silico analysis suggest that hypoxia‐induced changes in the UPS may impact proteins involved in pathways that regulate cellular proliferation by increasing or decreasing their stability.Support or Funding InformationThis work was supported by NIH RO1 HL 102167 and the T32 Training Program in Academic Pulmonary Medicine (T32HL076118).
Enabling a Viable Pipeline of NHLBI Extramural Investigators in Fiscally Challenging Times
Authors: Maric‐Bilkan Christine , Charette Marc , Oh Young , Schum Paula , Gao Yunling , McDonald Cheryl , Reid Diane , Robinson Valerie , Stanley Dennis , Fuentes Kelin , Olive Michelle , Eblen Matthew , Pearson Katrina , Tolunay Eser , Galis Zorina 
Published: November 2023

A major goal of the National Heart, Lung, and Blood Institute (NHLBI) is to maintain a viable and robust pipeline of extramural investigators. We have examined data on NIH funding trends among research project grant (RPG) awardees using economic and demographic data between fiscal year 1998 and fiscal year 2014. We have divided the investigators who received RPG awards during this period into three age groups: “early‐stage” (24–40 years), “mid‐career” (41–55 years) and ”established” (56 years and greater). We observed that there has been a low level, but progressively steady shift, in the distribution of NHLBI RPG awardees. The cause of this shift appears to be due, in part, to a parallel shift in the demographics of NHLBI RPG applicants. The combined result of these shifts has implications for the overall distribution of NHLBI RPG direct cost dollars. The magnitude of these effects is significantly increased during periods of flat or slightly declining levels of RPG funding. The three main drivers of these trends, 1) the shifting demographics of NHLBI RPG applicants, 2) the changing distribution of RPG direct cost dollars, and 3) the adverse effects of prolonged periods of level or slightly declining research funding, will be presented in detail along with suggested proposals that are intended to ensure the NHLBI will maintain its steady pipeline of qualified investigators now and for the foreseeable future.Support or Funding InformationNone
Tactical post hoc Exam Key Review
Authors: Price Joseph A. 
Published: November 2023

The immediate task in reviewing course exam (typically one best answer multiple choice) results is to make any key revisions in order to release the grades on that exam in an often controversial, emotionally debated, inconsistent, and too often misguided process. Here I show a tactical approach to post hoc exam review that will deal with poorly performing test items, and/or a too low class average, and do so in a manner fair to the students as a class. The specific method shown in the flow chart is grounded on and extension of established educational and psychometrics literature and principles, and in a novel way takes a seemingly complicated subjective process and organizes decisions into an easy to follow but consistent, effective solution, based on specific predetermined criteria while preserving instructor's options. This psychometrics driven method is based on the answers to two questions: “What portion of the class should get an item correct?”, and “How many students is it acceptable to fail?”, and on an implementation of a philosophy of education. It is particularly useful for newer medical school faculty and course directors.Support or Funding InformationNo extramural support.
An Active Learning Activity Utilizing a Headline News Story to Engage Students, Enhance Interest, and Improve In‐class Learning of Cell Cycle Regulation
Authors: Dirks‐Naylor Amie J 
Published: November 2023

An active learning activity was utilized to engage students and enhance in‐class learning of cell cycle regulation in a Pharm.D. integrated biological sciences course. The aim of the study was to determine the effectiveness and perception of the in‐class activity. Following completion of lecture on the topic of cell cycle regulation, students completed a 10‐question multiple choice quiz before and after engaging in the activity. The activity involved reading of a headline news article published by ScienceDaily.com entitled, “One Gene Lost Equals One limb Regained”. The name of the gene was deleted from the article and, thus, the end goal of the activity was to determine the gene of interest by the description in the story. The activity included compiling a list of all potential gene candidates before sufficient information was given to identify the gene of interest (p53). A survey was completed to determine student perceptions of the activity. Quiz scores improved by an average of 20% after the activity (40.1 ± 1.95 vs 59.9 ± 2.14; p< 0.0001; N= 96). Students enjoyed the activity, found the news article interesting, and believed that the activity improved their understanding of cell cycle regulation. The majority of students agreed that the in‐class activity peaked their interest for learning the subject matter and also agreed that if they understand a concept during class, they are more likely to want to study that concept outside of class. In conclusion, the activity improved in‐class understanding and enhanced interest in cell cycle regulation.Support or Funding InformationNone