bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hydrogen sulfide activates Ca2+ sparks to induce cerebral arteriole dilation
Authors: Liang Guo Hua , Xi Qi , Leffler Charles W. , Jaggar Jonathan H. 
Published: June 2021

Hydrogen sulfide (H2S) is a gaseous vasodilator produced by endothelial cells. Mechanisms by which H2S induce vasodilation are unclear. We tested the hypothesis that H2S dilates piglet cerebral arterioles by modulating local and global intracellular Ca2+ signals in smooth muscle cells. High‐speed confocal imaging revealed that H2S increased Ca2+ spark frequency ~1.43 fold and decreased global intracellular Ca2+ concentration ([Ca2+]i) by ~37 nM in smooth muscle cells of intact cerebral arteries. In contrast, H2S did not alter Ca2+ wave frequency. In voltage‐clamped (–40 mV) cells, H2S increased the frequency of Ca2+ spark‐induced transient Ca2+‐activated K+ (KCa) currents ~1.8 fold, but did not alter the amplitude of these events. H2S did not alter the activity of single KCa channels recorded in the absence of Ca2+ sparks in arteriole smooth muscle cells. H2S increased SR Ca2+ load ([Ca2+]SR), measured as caffeine (10 mM)‐induced [Ca2+]i transients, ~1.5 fold. H2S dilated pressurized cerebral arterioles. Iberiotoxin, a KCa channel blocker, and ryanodine, a ryanodine receptor (RyR) channel inhibitor, reduced H2S‐induced vasodilation by ~38 and ~37%, respectively. In summary, our data indicate that H2S elevates [Ca2+]SR, leading to Ca2+ spark activation in cerebral arteriole smooth muscle cells. The subsequent elevation in transient KCa current frequency leads to a reduction in global [Ca2+]i and vasodilation.NIH/NHLBI
Phenylephrine stimulates the production of reactive oxygen species (ROS) in endothelial cells from hypertensive but not in normotensive rat aorta
Authors: Silva Bruno R , Pernomian Laena , Bendhack Lusiane Maria 
Published: June 2021

The endothelial cells (EC) play important role in the vascular tone control. The production of ROS is increased in hypertension that can modify the contractile response to agonists. This study aimed to evaluate the role of EC on the phenylephrine (PE)‐induced contraction in normotensive (2K) and renal hypertensive (2K‐1C) rat aorta and the effect of PE on the production of ROS in EC from both rat groups. Concentration‐effect curves were constructed for PE in 2K and 2K‐1C intact endothelium (E+) or denuded aortas (E−). The efficacy (Emax) and potency (pD2) were compared. Isolated EC were incubated with the ROS‐sensitive dye DHE and the fluorescence intensity (FI) was measured by flow citometry. EC were stimulated with PE (0.1μM) for 10 min with the antioxidants superoxide scavenger Tiron or Catalase. The contraction was similar in 2K and 2K‐1C E− aortas. In E+ aortas the Emax was lower in 2K‐1C rings (1.2±0.2g, n=7 p<0.001) than in 2K (2.2±0.1g, n=5), but the potency was not different between them. Basal FI of DHE on EC was similar in 2K (43388.5±869.8, n=4) and 2K‐1C (37373.0±536.2, n=4), which was increased by PE only in 2K‐1C (68911.5±4146.3, n=4 p<0.001). The enhancement of ROS was reversed to the basal levels by the antioxidants. Our results suggest that the lower contraction induced by PE in intact endothelium 2K‐1C aorta can be due to the EC increased production of ROS stimulated by PE.Supported by FAPESP, CAPES and CNPq.
Mechanisms that decrease human Neprilysin gene expression in cigarette smoke‐induced pulmonary vascular disease
Authors: Wick Marilee J. , Loomis Zoe L. , Hersh Louis B. , Dempsey Edward C. 
Published: June 2021

We previously demonstrated that lung neprilysin (NEP) may be protective against cigarette smoke (CS)‐induced pulmonary vascular remodeling, a major contributor to pulmonary hypertension (PHTN) in lung disorders/diseases, such as chronic obstructive pulmonary disease (COPD). We found decreased NEP levels in COPD lung, at least some of which are due to changes in NEP transcription that may involve epigenetic mechanisms, but not ROS‐induced NEP gene damage or NEP gene promoter methylation. The goal of the present study is to identify CS‐induced mechanisms that decrease NEP gene transcription. We now present evidence that histone proteins may be modified (methylated, deacetylated) and decrease NEP transcription in response to CS. This is shown with human PA SMCs in ChIP experiments with antibodies to modified histone proteins (H3ac, H3K9me2), and with the compounds 5‐Aza‐2′deoxycytidine, Trichostatin A, or Valproic Acid. CS‐specific changes in regulatory or transcription factor (HIF‐1 and 2, NFkB) binding to the NEP gene that could decrease NEP transcription are also investigated in ChIP experiments with PA SMCs. Our results may suggest new ways to increase endogenous pulmonary NEP levels to treat or prevent smoke or hypoxia‐induced pulmonary vascular remodeling.Support: NHLBI RO3‐HL‐095430, NHLBI PPG P05 HL014985 and VA Merit.
Anti‐endothelial cell antibodies (AECA) in pulmonary arterial hypertension (PAH)
Authors: Morrow Rebekah , Fagan Karen A. , Cioffi Donna L. 
Published: June 2021

PAH is a fatal disease that results in right heart failure. PAH pathology is unclear, but endothelial activation and immune responses contribute to vascular remodeling. AECA are found in many autoimmune disorders and both idiopathic and connective tissue disease‐associated PAH. In autoimmune vasculitides, AECA activate endothelial cells through increased reactive oxygen species (ROS) production. While the effects of AECA on endothelial cells have been studied in some diseases, the effects of AECA from PAH on pulmonary endothelial cells are unknown. We sought to determine whether AECA bind and increase ROS in pulmonary endothelial cells.MethodsWestern blots were performed using membrane fractions of human pulmonary and systemic endothelial cells. Blots were treated with serum from PAH patients or healthy controls. Fluorescence microscopy was used to assess the effects of AECA on ROS production in cells treated with IgG from PAH patients or healthy controls.ResultsWhile some bands were found in both PAH and healthy subjects, a band of 37 kD was seen in pulmonary microvascular endothelial cells when blots were treated with PAH serum. Cells treated with IgG from healthy controls showed no increase in ROS but PAH IgG elicited an increase. These data suggest that some AECA from PAH bind to pulmonary endothelial cells and may lead to ROS generation. Supported by 5R00HL089361 and 5T32HL076125‐8.
DIDS causes Src‐dependent inhibition of Na,K‐ATPase in the ciliary epithelium and slows aqueous humor secretion by the intact porcine eye
Authors: Delamere Nicholas A , Mandal Amritlal , Shahidullah Mohammad 
Published: June 2021

Aqueous humor secretion is the result of net ion transport that involves the coordinated operation of Na,K‐ATPase in the nonpigmented ciliary epithelium (NPE) with several other basolateral transporters and channels in the ciliary epithelium bilayer. Here, we report on the ability of DIDS to reduce aqueous humor secretion and present studies to examine the mechanism.In an intact pig eye preparation the rate of aqueous secretion was significantly reduced by DIDS added to the blood‐side perfusate. In primary cultured NPE, DIDS inhibited ouabain‐sensitive 86Rb uptake and activated Src family kinase (SFK) and ERK1/2 as judged by Western blot analysis. PP2, a SFK inhibitor, prevented the DIDS effect on 86Rb uptake, SFK and ERK1/2. DIDS was found to reduce cytoplasmic pH in BCECF‐loaded NPE. Parallel studies showed PP2 eliminates activation of SFK and ERK1/2 that occurs upon pH reduction.The ability of DIDS to suppress aqueous humor secretion is consistent with inhibition of Na,K‐ATPase in the NPE following a drop in cytoplasmic pH and SFK and ERK1/2 activation.Funding: NIH Grant EY06915
Neonatal Bladder Inflammation Reduces Stress‐Induced Opioidergic Inhibition of Visceromotor Responses to Urinary Bladder Distension in Adult Rats
Authors: Ness Timothy J , DeWitte Cary R , Robbins Meredith T 
Published: June 2021

ObjectiveThe present study sought to determine the effect of the opioid antagonist naloxone on stress‐induced analgesia and stress‐induced hyperalgesia (SIH) mechanisms in adult rats which experienced bladder inflammation as neonates.MethodsFemale rat pups (P14–16) were anesthetized and treated with intravesical 1% zymosan (ZY− rats). Control pups were only anesthetized (AN− rats). As adults, rats were separated into acute footshock (‐FS‐; 15 min, 1 mA, 1 S duration) or non‐footshock (‐NFS‐) groups & further subdivided into naloxone‐treated (1 mg/kg; ‐NAL) and saline‐treated (−SAL) subgroups. These rats were anesthetized immediately following FS or NFS treatments, drugs given i.p.and visceromotor responses (VMR) to phasic, graded urinary bladder distension (UBD) determined.ResultsThere was no significant effect of naloxone treatment on UBD‐related SIH observed in ZY‐rats (VMRs of ZY‐FS‐NAL rats = ZY‐FS‐SAL rats > ZY‐NFS‐NAL rats = ZY‐NFS‐SAL rats). In contrast, naloxone treatment revealed a reactive opioidergic suppression of SIH in AN‐rats (VMRs of AN‐FS‐NAL rats > other AN− groups).ConclusionsThese data support the assertion that neonatal bladder inflammation results in the failure of an opioidergic inhibitory system which is activated by acute stress, the results of which is magnification of the concurrent pronociceptive processes related to visceral SIH. Supported by DK51413
Some Morphologic and Growth Features of Juxta‐alveolar Smooth Muscle Cell Lines from High Altitude Hypoxia Sheep
Authors: John Theresa Adebola 
Published: June 2021

OBJECTIVESWe derived smooth muscle cells (SMCs) from lung terminal parenchyma and studied the effect of high altitude hypoxia (HAH) on their monolayer morphology and growth profiles.METHODSLungs were obtained from adult sheep kept at 12, 470 ft altitude, PaO2 ~60 Torr for ~4 months and age matched control sheep kept at sea level. SMCs migrated from terminal lung parenchyma were cultured as monolayers, characterized by alpha‐smooth muscle actin immunofluorescence, and studied for monolayer morphologies and tritiated (3H) thymidine incorporation growth profiles.RESULTSHAH‐SMC morphologic varieties were: apoptotic, sub‐confluent, spindle‐form, hill and valley, swirl‐form, and rhomboid monolayers and control SMC varieties were swirl and hill‐and‐valley formations at confluence, jointly from vascular and bronchiolar termini. The normal growth pattern, typified by intrapulmonary 7th generation vein SMCs, showed mean peak 3H‐thymidine incorporation of 2829.46±294.24 counts per minute (cpm) at 72h. Comparatively, HAH‐SMCs profiles were varied: normal, subprolific, prolific, retarded, and senescent. At extremes, mean peak cpm by prolific cells were 7775.42±475.77 and by senescent cells were 26.71.CONCLUSIONChronic HAH caused morphologic and functional changes, possibly combined pathologic formations and adaptive responses, in juxta‐alveolar SMCs.NHLBI MIRS HL‐059435 to T. A. John
Angiotensin II (AngII) regulates activity of kinases and phosphatases in neurons
Authors: Basu Urmi , Alikunju Saleena , Zimmerman Matthew C. 
Published: June 2021

AngII signaling in the brain plays critical role in regulating cardiovascular function. AngII increases neuronal firing rate and modulates ion channel activity, which may be controlled by cellular kinases and phosphatases. Here, we tested the hypothesis that AngII stimulation of central neurons influences protein levels and activity of calcium/calmodulin kinaseII (CaMKII), protein kinase C (PKC), and protein phosphatase 2A (PP2A). Mouse CATH. a neurons were stimulated with AngII (100nm; 10 min‐24 hr) and PP2A and PKCδ protein levels were measured by Western blot analysis. CaMKII activity was assessed by detecting its phosphorylated levels and PP2A activity by a ser/thr Malachite Green phosphatase assay. AngII significantly increased phosphorylated CaMKII levels after 2 and 24 hr of stimulation (3.17 and 3.19‐fold increase vs. vehicle, P<0.05) and PKC δ protein levels after 24 hr (2.63‐fold increase vs. vehicle, P<0.05). AngII modestly decreased PP2A protein levels after 0.5–24 hrs of stimulation. PP2A activity decreased within 10 min and up to 1 hr of AngII stimulation (818 μg phosphate released at 10 mins and 794 μg at 1 hr vs. 1666 μg in vehicle). Together, these data indicate that in neurons AngII inhibits PP2A activity while increasing PKCδ, and activating CaMKII. Future studies will determine the role of these kinases and PP2A in modulating ion channel activity in AngII‐stimulated neurons.AHA0930204N
Versican expression and turnover in hepatic stellate cell activation
Authors: Maurice Sean B , Crick Cody L , Kim Wan-Cheol , Law Christine , Norrie Minetta , Kim Sang-Eun , Winwood Paul 
Published: June 2021

The pathophysiologic processes of extracellular matrix deposition and turnover in liver injury are central to our understanding of liver fibrosis and the development of antifibrotic therapies. Here we report expression of versican and the proteoglycan degrading enzymes ADAMTS1 and 4 by hepatic stellate cells (HSCs) activated to a myofibroblastic phenotype in tissue culture.HSCs were isolated from C57B/6 mice by pronase/collagenase digestion and density gradient centrifugation and activated by culture on plastic. Versican expression was detected in HSCs at all stages of activation in culture. Expression increased gradually from days 1 through to 7, then more dramatically, up to 5 fold, by days 10 and 14. Immunofluorescence microscopy confirmed versican production by activated HSCs. Western blotting of activated HSC media demonstrated secretion of both intact versican and versican proteolytic products. Expression of the known versican processing enzymes, ADAMTS1 and 4, was detected at all time points but was suppressed during early stages of HSC activation (days 2 through 7) relative to day 1 levels.Versican synthesis and turnover appear to be important components of HSC activation. Its regulated turnover may play a role in modulating normal and fibrotic remodeling in the liver. The reduction in ADAMTS expression could contribute to the deposition of a versican rich matrix in which tissue remodeling occurs.Grant Funding Source: Northern Medical Program
Investigating the developmental processes of nasal contour shape
Authors: Wheat Amber D. 
Published: June 2021

Nasal contour shape (NCS) is considered to be a useful indicator of ancestry in humans, which would imply that it is minimally affected by environmental factors over short periods of time. However, it is unclear whether differences in NCS are largely determined by genetics, exhibit responses to environmental factors (e.g., plasticity resulting from climatic factors), or are a combination of the two. One method for examining this looks at the integration of cranial shape. High correlations between NCS and other regions of the cranium, such as the vault and cranial base, may indicate ontogenetic constraint. This study tests three hypotheses: basicranial dimensions are correlated with nasal contour shape; cranial vault shape correlates with nasal contour shape; human groups significantly vary in the shape of the nasal contours. Seventy‐six crania of adult humans from three populations were utilized: Arikara, American “blacks,” and American “whites.” Preliminary results unexpectedly indicate no significant differences in NCS exist between population groups. Phenotypic correlations were calculated and results indicate that nasal contour shape is not significantly correlated with cranial vault shape or basicranial dimensions.Grant Funding Source : AAA
FGF‐INDUCED ACTIVATION OF THE LIMB SPECIFIC SHH REGULATORY REGION (LSSRR)
Authors: Bailey Traci W , Pira Charmaine U , Oberg Kerby C 
Published: June 2021

Regeneration of amputated embryonic chick wings can be induced by Fgf, and upregulation of Shh is an essential step in this process. During development, a 1.8 kb conserved non‐coding region up‐stream of Shh functions as a limb‐specific Shh regulatory region (LSSRR). We hypothesize that LSSRR is involved in Fgf‐induced limb regeneration. To test our hypothesis, we electroporated (EP) an LSSRR‐green fluorescent protein (GFP)‐reporter construct into stage 23 chick limb bud mesoderm and then implanted an Fgf‐soaked bead to induce Shh expression. Green fluorescence was detected around the Fgf bead after 24 hr, supporting a role for LSSRR in the up‐regulation of Shh by Fgf. To determine the sequences within the LSSRR that were required for Shh up‐regulation, we isolated three separate candidate sequences within the LSSRR that contained Hox or RAR/NFκB binding site. The candidate sequences were ligated into a ptk‐GFP reporter plasmid and then EP into the presumptive wing mesoderm of stage 14 chick embryos. All three candidate sequences showed fluorescence at 48 hr, indicating enhancer activity with these minimal fragments. We conclude that during chick wing regeneration, Fgf utilizes the LSSRR in upregulating Shh and that multiple enhancer elements within the LSSRR may contribute to Shh regulation.Grant Funding Source : Departmental Funding
Negative Regulation of Enhancer‐Associated RNA in Macrophages
Authors: Lam Michael T , Cho Han , Heinz Sven , Benner Christopher , Kaikkonen Minna U , Tanaka-Oishi Yumiko , Kosaka Mika , Lee Cindy , Evans Ronald M , Glass Christopher K 
Published: June 2021

Recent discoveries of genome‐wide transcription at non‐coding regions suggest an unrecognized role of non‐coding RNAs. One class of these non‐coding RNAs is transcribed from cell‐type specific enhancer elements (eRNA). Here we show how transcription of eRNAs is negatively regulated by Rev‐Erb α and β, a subclass of the nuclear receptor family characterized as dedicated transcriptional repressors. Genome‐wide localization of Rev‐Erb α and β in macrophages by chromatin‐immunoprecipitation couple with sequencing showed that 70% of their binding sites are located in enhancer elements distal to protein coding genes. Approximately 55% of these distal regions are enriched for RNA Polymerase II. To test whether these enhancer elements express RNAs, we performed global run‐on sequencing with confirmation using RT‐PCR and detected nascent RNA transcripts at Rev‐Erb bound enhancers. Transcription of eRNA is de‐repressed in Rev‐ Erb deficient macrophages, whereas constitutive expression of Rev‐Erb represses eRNA transcription. Moreover, changes in eRNA expression are positively correlated to changes in expression of nearby protein coding genes. These findings suggest a mechanism whereby Rev‐Erb regulates gene expression by repressing eRNA from distal enhancer elements.Grant Funding Source : NIH
Developing Henna‐Haematoxylin stain as alternative to Nissl stain in delineating the cytoarchitecture of the neocortex and archicortex of the cerebrum
Authors: Alawa Judith Nkiruka , Gideon Olugbenga , Adetiba Bamidele , Alawa Clement B. 
Published: June 2021

Demonstration of neuronal distribution patterns such as cerebral cortical layers or zones of neuronal cell loss usually requires specific stains such as Nissl stains which are not easily obtainable in developing countries. This study aims at testing henna as a counterstain to haematoxylin in delineating the cortical layers in the cerebrum. Paraffin‐embedded serial sections (5μm) of cerebral cortex of Wistar rat fixed in Bouin's fluid and stained with haematoxylin and henna (0.5% w/v aqueous crude extract of Lawsonia inermis leaves dissolved in 5% aqueous alcohol and mordanted with potassium alum) was observed to be effective in mapping out the six cortical layers of the neocortex and the three layers of the archicortex distinctly. It also stained the cytoplasm of the perikarya and glial cells brown and in addition demonstrated the thalamic nucleus in the central core of the cerebrum as a dense aggregation of neuronal cell bodies criss‐crossed by bundles of fibres. Thus, henna used as a counterstain to haematoxylin demonstrated reliability as a neurological stain to delineate the cellular layers in the archicortex (hippocampus) and the neocortex and therefore could serve as a readily available and cheaper alternative to Nissl stains for developing or third world countries.Grant Funding Source: Ahmadu Bello University Research Grant
Distribution of urea transporter B (UT‐B) in the mice brain
Authors: ran jianhua , Sun Shanquan , He Guiqiong 
Published: June 2021

The study was performed to describe morphologic features of UT‐B and speculated its functional roles in brain. Immunohistochemistry and immunofluorescence double staining were performed to detect the cellular localization and cell type of this transporter. UT‐B is widespread expressed in astrocytes in cerebral cortex, hippcampus, cerebellum, olfactory bulb as well as in some special glia, such as Bergmann glia, Müller cells, ependymal cells and epithelial cells of the choroid plexus. Additionally, UT‐B expressed in the neuron of cerebral cortex, the hippocampus, the mitral cells in the olfactory bulb, indicated it new role in the brain. Furthermore, UT‐B positive signaling was detected in NG2 cells. Wide distribution of UT‐B in the brain indicated that UT‐B may make an important role in controlling urea transport between neurons and astrocytes sequence throughout the astrocytic network and guiding urea to blood vessels and the cerebral spinal fluid (CSF) to keep homeostasis in the brain.Supported by the National Natural Science Foundation of China (30500171), and the Natural Science Foundation of Chongqing (CSTC2010BB5098).
The death of neurons derived from familial Alzheimer's disease pluripotent stem cells is no different from the death of neurons derived from normal controls
Authors: Shaner Mason A , Yuan Shauna H , Goldstein Lawrence S 
Published: June 2021

Neuronal death, a pathological hallmark of Alzheimer's Disease (AD) is seen in patients whose presenilin‐1 (PS1) mutation causes their autosomal dominant AD. We assayed markers of the apoptotic death of neurons induced from pluripotent stem cells obtained from human PS1 and non‐demented controls. Our investigation included immunofluorescent imaging, analyzing TUNEL, MAP2, Caspase 3, and GABA staining for multiple samples from different PS1 individuals at different length of culture conditions. We found no significant differences between the PS1 and control groups. These baseline observations of an in‐vitro model suggest that expression of the AD phenotype requires factors beyond standard culturing conditions. This model can be used to explore stresses on the control and PS1 cell populations that will differentiate them, providing a disease‐in‐a‐dish model for those biochemical alterations likely to express the premature apoptotic phenotype of autosomal dominant AD. Research support was provided by CIRM (California Institute of Regenerative Medicine) and HHMI (Howard Hughes Medical Institute).Grant Funding Source : CIRM (California Institute of Regenerative Medicine), HHMI (Howard Hughes Medical Institute)
AChR Clustering is Increased by Neuregulin
Authors: Boothe Sarah M. , Grow Wade A. 
Published: June 2021

In skeletal muscle development, agrin is released by the approaching motor neuron prior to innervation and the formation of the neuromuscular synapse. Agrin induces the clustering of existing acetylcholine receptors (AChRs) at the site of the future neuromuscular synapse through an agrin signaling pathway. Neuregulin is also released by the approaching motor neuron and induces the production of additional AChRs, especially at the nuclei near the future neuromuscular synapse. Our lab investigates myogenesis and neuromuscular synapse formation using the C2C12 myotube cell culture model. C2C12 myoblasts proliferate in growth medium, and then fuse into myotubes when switched to low‐serum differentiation medium (DM) for 72 hours. Myotubes are stimulated to cluster AChRs by the addition of agrin for the last 16 hours in DM. Our hypothesis was that neuregulin would increase AChR clustering in C2C12 myotubes as previously demonstrated in another cell line. Concentrations of agrin or neuregulin as low as 10 ng/ml was sufficient to increase AChR clustering. We conclude that AChR clustering in C2C12 myotubes is increased either by utilizing existing AChRs or the production of additional AChRs.Grant Funding Source: None