bluuebunny/crossref_metadata_2025 huggingface dataset:
Asymptomatic AD is associated with hypertrophy of CA1‐hippocampus neurons in the NUN study
Authors: Iacono Diego , Pletnikova Olga , Rudow Gay , Patel Ela , Tudor Dianna , Markesbery William , Snowdon David , Troncoso Juan C
Published: June 2021

Objective: a) to measure differences in the volume of CA1 hippocampal neurons, and their nuclei and nucleoli in four different groups: subjects with Alzheimer's Disease (AD) pathology and no cognitive deficits (ASYMAD), mild cognitive impairment (MCI), definite AD (AD) and age‐matched controls (C).

Introduction: the pathology of AD evolves decades before the disease becomes clinically manifest. Consequently, it is common to find substantial amounts of Aβ ‐plaques (NP) and neurofibrillary tangles (NFT) in autopsy brains of older subjects with normal cognition briefly before death (ASYMAD).

Methods: we used designed‐based stereology to measure the volume of neuronal cell bodies, nuclei, and nucleoli in CA1. We examined postmortem brains of 13 C, 10 ASYMAD, 5 MCI, 10 AD subjects from the NUN study.

Results: We found a significant hypertrophy of the neuronal cell bodies, nuclei, and nucleoli in CA1 of ASYMAD subjects compared to controls (p<0.01) and MCI (p<0.01). The hypertrophy of CA1 neurons in ASYMAD may represent a very early pathologic effect of A‐beta and tau on neurons or a reflection of cellular mechanisms that prevent the progression of the disease into dementia. Studies of the correlation of ApoE genotypes and ASYMAD are in progress.
Reversible posterior leukoencephalopathy syndrome: Report of 3 autopsy cases
Authors: Takei Hidehiro , Bhattacharjee Meenakshi B , Fuller Gregory , Powell Suzanne Z
Published: June 2021

Background: Reversible posterior leukoencephalopathy syndrome (RPLS) is an increasingly recognized clinicoradiological entity associated with multiple pathologies, the most common being hypertensive crisis. Because of the “reversible” nature of this syndrome, few autopsy/histological findings have been reported to date.

(Case 1) A 48 y/o woman with a history of allogeneic renal transplant and hypertension presented with headache, blurred vision, confusion and died of pneumonia. (Case 2) A 48 y/o man who had a history of malignant lymphoma treated developed seizures and died of fungal pneumonia. (Case 3) A 74 y/o woman with a past medical history significant for hypertension, idiopathic thrombocytopenic purpura treated with splenectomy followed by Rituximab and steroid died of multiorgan failure. Radiologically, these 3 patients showed MR image findings characteristic of RPLS. Postmortem examination of these brains revealed leukoencephalopathic changes including diffuse white matter rarefaction with mild gliosis (Case 1) and relatively well‐circumscribed perivascular rarefied foci without gliosis (Cases 2, 3).

Discussion: Our cases support the two proposed pathophysiologic mechanisms for PRLS; a breakdown in cerebrovascular autoregulation with ensuing interstitial accumulation of fluid (Cases 1), and cerebral vasospasm with resulting ischemia within the involved territories (Cases 2, 3).
Subarachnoid hemorrhage induces reactive gliosis and brain injury
Authors: Murakami Kentaro , Dumont Travis , Tranmer Bruce I. , Wellman George C.
Published: June 2021

A wide range of CNS pathologies are known to induce reactive gliosis, including head trauma and injury, ischemic stroke, brain tumors and neurodegenerative diseases. Little is known, however, regarding the development of reactive gliosis following subarachnoid hemorrhage (SAH). The goals of this study are to characterize reactive gliosis following SAH and to determine the cellular basis of this phenomenon. Reactive gliosis was examined by the following criteria: (1) astroglial hypertrophy (detected by increased immunostaining of glial fibrillary acidic protein), (2) dedifferentiation of astrocytes (detected by re‐expression of nestin) and (3) increased metabolism of astrocytes (monitored by enhanced S‐100B production). Our results demonstrate that brain stem and cortex obtained from a rabbit SAH model exhibit these characteristics of reactive gliosis. As astrocytes have been shown to play a critical role in cerebral blood microcirculation, reactive gliosis following SAH could have a significant impact on the ability of astrocytes to communicate vasodilatory signals to parenchymal arterioles in response to neuronal activity. This work was supported by the Totman Medical Research Trust Fund, the Peter Martin Brain Aneurysm Endowment, AHA and the NIH (R20 RR16435 & R01 HL078983).
TDP‐43 immunoreactivity in anoxic, ischemic and proliferating lesion of the central nervous system
Authors: Lee Edward B. , Lee Virginia M.‐Y. , Trojanowski John Q. , Neumann Manuela
Published: June 2021

TDP‐43 proteinopathies are a newly categorized group of neurodegenerative diseases characterized by progressive cognitive and motor impairments associated with the abnormal accumulation and mislocalization of the nuclear TAR‐DNA‐binding protein‐43 (TDP‐43) protein in neurons and glia. Little is known about the expression and distribution of TDP‐43 in normal and pathologic states. A panel of clinical neuropathologic specimens was examined for TDP‐43 expression by immunohistochemistry. TDP‐43 inclusions like those seen in neurodegenerative frontotemporal dementia and motor neuron disease were not seen in a variety of anoxic, ischemic and proliferating lesions. However, TDP‐43 was found in Rosenthal fibers and eosinophilic granular bodies associated with low grade tumors and reactive brain tissue. Furthermore, cytoplasmic TDP‐43 was seen in M‐phase tumor cells, but not in mitotic spindles. These findings expand our knowledge of the distribution and localization of TDP‐43 in pathologic specimens, and indicate that the TDP‐43 inclusions seen in frontotemporal dementias and motor neuron diseases are specific to a neurodegenerative process.

Supported by an intradepartmental grant from the Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania (to EBL) and a grant by the Federal Ministry of Education and Research (BMBF: “Degenerative dementias: Target identification, validation and translation into treatment strategies” to MN).
Intracranial mycotic aneurysm: a case report
Authors: Rishi Kirtee , Li Jianyi , Cherqui Alice , Takei Hidehiro , Powell Suzanne Z
Published: June 2021

Intracranial mycotic aneurysms are extremely rare and commonly associated with infective endocarditis. The unusual location of these aneurysms should alert the clinician to the possibility of a mycotic aneurysm. We report an unusual case of a 46 year old woman with previous history of insulin dependent diabetes and an ophthalmic artery aneurysm clipped 10 years ago. She underwent a successful resection of a right frontoparietal meningioma, however, two weeks post‐operatively she presented with subarachnoid hemorrhage (SAH). An angiogram was performed and it was negative. She remained hospitalized and developed recurrent SAH two weeks later. A repeat angiogram revealed a distal basilar artery 1cm fusiform aneursym with irregular excrescences. She expired within 3 days of the last angiogram and a brain only autopsy was performed. The skull base showed extensive hemorrhage in the posterior fossa, SAH, right uncal and upward cerebellar herniation and a 2.5cm basilar tip aneurysm. Coronal sections of brain showed intraventricular hemorrhage with softening and necrosis of the brainstem. Histopathologic examination showed extensive acute and chronic inflammation with septae fungal hyphae present in the thrombi and the wall of the basilar tip aneurysm. A high index of clinical suspicion for mycotic aneurysm following surgical intervention may result in early treatment and improved patient outcome.
Cardiac pathology in a case of limb‐girdle muscular dystrophy type 2I (LGMD‐2I)
Authors: Margeta Marta , Winder Thomas L , Moore Steven A
Published: June 2021

LGMD‐2I is caused by mutations in fukutin‐related protein (FKRP) that lead to abnormal glycosylation of α‐dystroglycan in skeletal muscle. Here, we report cardiac pathology in a 17‐year old boy who initially presented with progressive proximal leg weakness exacerbated by illness or exertion. The skeletal muscle biopsy showed a mild muscular dystrophy with normal staining for dystrophin, sarcoglycans, utrophin, merosin, and β‐dystroglycan. Staining with α‐dystroglycan glycoepitope antibodies IIH6 and VIA4‐1 varied from absent to nearly normal, a mosaic pattern characteristic for dystroglycanopathies. Direct sequencing of FKRP gene detected the common point mutation c.826C>A (p.L276I) in both alleles, thus establishing the diagnosis of LGMD‐2I. At the age of 17, the patient developed severe congestive heart failure and underwent cardiac transplantation. The heart showed biventricular hypertrophy and dilatation with interstitial fibrosis and extensive fatty replacement. Immunoperoxidase staining demonstrated a complete absence of IIH6 staining – a pattern similar to, but more severe than, the one observed in the patient's skeletal muscle. While clinical cardiac involvement is common in LGMD‐2I, this is the first direct demonstration of cardiac pathology and severe impairment of heart α‐dystroglycan glycosylation in this disease. Supported by NS053672.
Bradykinin Induces Matrix Metalloproteinase‐9 Expression and Cell Migration through a PKC‐δ‐dependent ERK/Elk‐1 Pathway in Astrocytes
Authors: Tsai Hsin‐Wen , Hsieh Hsi‐Lung , Yang Chuen‐Mao
Published: June 2021

Many reports have shown that matrix metalloproteinase (MMP)‐9 plays an important role in brain inflammation and diseases. In our previous study, bradykinin (BK) has been shown to induce proMMP‐9 expression via MAPKs and NF‐κB in rat brain astrocytes (RBA‐1). However, the molecular mechanisms and physiological roles underlying BK‐induced MMP‐9 expression in RBA‐1 remain unclear. Here we reported that BK induced proMMP‐9 expression and promoted RBA‐1 cell migration, via a B2 BK receptor‐activated protein kinase C‐δ (PKC‐δ)‐dependent signaling pathway. Activation of PKC‐δ led to phosphorylation and translocation of extracellular signal‐regulated kinase 1/2 (ERK1/2) and then activated a transcription factor Elk‐1. Phospho‐Elk‐1 bound to MMP‐9 promoter and thereby induced transcription of MMP‐9. The rat MMP‐9 promoter containing an Elk‐1 cis‐binding site (Ets domain), that located at nucleotides −511 to −506 was identified as a crucial domain linking to BK action. Moreover, BK induced recruitment of p300 (as a transcriptional co‐activator) to the MMP‐9 promoter, leading to the acetylation of histone H4 in chromatin and facilitating MMP‐9 gene transcription. Taken together, these results suggested that in RBA‐1 cells, activation of ERK1/2 by a PKC‐δ‐dependent event mediated through Elk‐1 pathway is essential for MMP‐9 gene up‐regulation and cell migration induced by BK.
Role of delta receptor efficacy as a determinant of delta/mu opioid interactions in rhesus monkeys
Authors: Negus S. Stevens , Rice Kenner C.
Published: June 2021

Delta opioid agonists have been reported to selectively enhance the antinociceptive effects of mu opioid agonists without enhancing other, potentially untoward mu agonist effects such as sedation, respiratory depression or abuse potential. The purpose of the present study was to examine the role of delta receptor efficacy as a determinant of delta/mu interactions in rhesus monkeys in two different behavioral procedures: (a) an assay of schedule‐controlled responding maintained by food reinforcement, and (b) an assay of thermal nociception. The effects of the selective mu agonist fentanyl were examined in combination with the high‐efficacy delta agonist SNC243A, the intermediate‐efficacy agonist MSF61, or the delta antagonist naltrindole. Fentanyl was tested in combination with three different proportions of each delta opioid. Fentanyl/SNC243A interactions were superadditive in the assay of antinociception and additve in the assay of schedule‐controlled responding. Fentanyl/MSF61 interactions were additive in both procedures, and fentanyl/naltrindole interactions were additive or subadditive in both procedures. These results suggest that high efficacy at delta receptors is required for synergistic delta/mu interactions in assays of antinociception in rhesus monkeys. Supported by R01 DA11460 from NIDA/NIH.
G‐protein coupled receptor kinase 2(GRK2) is involved in μ‐receptor signaling in the mouse ileum but not colon
Authors: Ross Gracious R , Gabra Bichoy R , Dewey William L , Akbarali Hamid I
Published: June 2021

Repeated administration of morphine results in tolerance to antinociception but not constipation. Tolerance develops to the effects of morphine in the ileum but not the colon. The objective of this study was to identify the intracellular signaling mechanisms responsible for these differences in these two organs. Morphine (100 nM) inhibited field‐stimulation evoked (EFS) cholinergic contractions in longitudinal muscle preparations and produced atropine‐insensitive contractions in the circular muscle from ileum and colon. In the presence of the GRK2 inhibitor, methyl 5‐[2‐(5‐nitro‐2‐furyl)vinyl]‐2‐furoate (100 μM), morphine‐mediated inhibition of EFS contractions in the longitudinal muscle and direct contractions of circular muscle were abolished in the ileum but not the colon. The concentration‐response to acetylcholine in both tissues was not affected by the GRK2 inhibitor. Cell lysates from morphine treated ileum and colon were immunoprecipitated with anti‐GRK2 antibody. Immunoblot with anti‐μ‐receptor (MOR) antibody showed a band of ~70 kDa in the ileum but not the colon demonstrating interaction of MOR with GRK2 in the ileum but not colon and may form the basis for the differential tolerance development between the ileum and colon. Supported by DK46367, DA01647, DA020836
Morphine in Combination with Metabotropic Glutamate Receptor Antagonists on Schedule‐Controlled Responding and Thermal Nociception
Authors: Fischer Bradford D , Zimmerman Eric I , Picker Mitchell J , Dykstra Linda A
Published: June 2021

The present study examined the interactive effects of morphine in combination with metabotropic glutamate (mGlu) receptor antagonists on schedule‐controlled responding and thermal nociception. Drug interaction data were examined with isobolographic and dose‐addition analysis. Morphine, the mGlu1 receptor antagonist JNJ16259685, the mGlu5 receptor antagonist MPEP, and the mGlu2/3 receptor antagonist LY341495 all decreased rates of schedule‐controlled responding. JNJ16259685/morphine, MPEP/morphine, and LY341495/morphine mixtures produced additive effects on this endpoint. Morphine also produced dose‐dependent antinociception in the assay of thermal nociception, whereas JNJ16259685, MPEP, and LY341495 failed to produce an effect. In this assay, JNJ16259685/morphine and LY341495/morphine mixtures produced supra‐additive effects, whereas MPEP/morphine mixtures produced additive effects. These results suggest that an mGlu1 and an mGlu2/3 receptor antagonist, but not an mGlu5 receptor antagonist, selectively enhances the antinociceptive effects of morphine. In addition, these data confirm that the behavioral effects of drug mixtures depend on the endpoint under study. Supported by grants R01‐DA02749 and T32‐DA07244.
Effects of agonists with mixed efficacy profiles at the mu and kappa opioid receptor on amphetamine‐mediated dopamine release
Authors: Bosse Kelly E , Bradbury Faye A , Jutkiewicz Emily M , Husbands Steve M , Gnegy Margaret E , Traynor John R
Published: June 2021

Although studies have demonstrated that non‐selective, mixed kappa/mu opioid agonists attenuate stimulant self‐administration with fewer side effects than selective kappa agonists, less is known about the contribution of relative mu or kappa efficacy in the effectiveness of these compounds. Consequently, a series of agonists with similar binding affinities but varying levels of efficacy in the [35S]GTPγS assay at the mu and kappa receptor were compared for ability to alter amphetamine (AMPH)‐mediated dopamine release from rat striatal slices. Addition of the selective kappa agonist U50488H to striatal slices reduced AMPH‐induced dopamine release, while addition of the selective mu agonist DAMGO enhanced the amount of dopamine released in response to AMPH. For mixed agonists the modulation of AMPH‐mediated dopamine release coincided with the predominant efficacy of the agonist at the mu or kappa receptor. However, butorphanol increased AMPH‐mediated dopamine release, despite having a relative lower efficacy at the mu vs. kappa receptor. The kappa inhibitory effect of butorphanol was only observed upon insurmountable antagonism of the mu receptor with clocinnamox. These results demonstrate that the effect of mixed compounds to modulate AMPH‐stimulated dopamine release is dependent on the balance of kappa opioid inhibitory effects and mu opioid stimulatory effects. Supported by DA 04087, 07315, 19728.
Investigating DAT uptake inhibitors as potential replacement therapies for cocaine
Authors: Batman Angela , Dutta Aloke , Reith Maarten , Beardsley Patrick M.
Published: June 2021

There are no approved therapies for cocaine dependence. Our objective is to develop potential replacement medications that possess the subjective effects of cocaine but with longer durations of action and the ability to reduce cocaine self administration.

Cocaine blocks the dopamine (DAT), serotonin (SERT), and norepinephrine (NET) transporters. We have reported that our lead compound, D84, a 3‐hydroxy substituted analogue of 1‐[2‐(diphenylmethoxy)ethyl]‐4‐(3‐phenylpropyl) piperazine (GBR 12935), is one of the most potent and selective inhibitors of the DAT so far disclosed [Ki's (nM): 4.05 (DAT):1274 (SERT):164 (NET)]. D84 possesses several of our target properties and here we continue its evaluation, as well as that of new analogues. D84 dose‐dependently (9.6–30.4 mg/kg) reduces cocaine self‐administration in rats. Previously undisclosed compounds, D225, D232, and D233, elevated locomotor activity for several hours, and occasioned cocaine's discriminative stimulus effects. Compound D232 was almost 10 fold more potent than D233 in this latter regard, perhaps attributable to its greater SERT potency. The results show these new GBR 12935 analogues possess features desirable for a cocaine replacement therapy.

This research was supported by NIDA Grant DA012449, A. Dutta (PI).
The effect of paraquat on the expression of D2 dopamine receptor in CAD cells
Authors: Kim Ok‐Jin J , Megaris Michael Paul , Azadani Zahra Crystal
Published: June 2021

We identified a protein kinase Czeta interacting protein (ZIP1) as an interacting protein of the D2 dopamine receptor (DAR) through a yeast two hybrid assay. We confirmed that the functional interaction between D2 DAR and ZIP1 in human embryonic kidney 293 (HEK 293) cells by using immunoprecipitation/western blot analysis, cAMP assays and radioligand binding assays. Significantly, we also demonstrated that D2 DAR and ZIP1 were coimmunoprecipitated from mouse brain and colocalized in mouse brain. The role of ZIP1 has not been clearly elucidated although overexpression of ZIP1 resulted in the reduction of membrane associated D2 DAR in HEK 293T cells. Our studies also demonstrated that ZIP1 was induced by an herbicide paraquat in HEK 293T cells and Cath.a.Differentiated (CAD) dopaminergic neuronal cells. The expression of membrane associated D2 DAR was reduced in CAD cells stably transfected with D2 DAR (CAD‐D2 DAR) in the presence of paraquat by radioligand binding assays. Subsequent radioligand binding assay and Western blot analysis studies also appeared to indicate that paraquat reduces the expression of membrane associated D2 DAR in the presence of ZIP1 in CAD‐D2 DAR cells. The further characterization of the effect of paraquat on the expression and trafficking of D2 DAR are still in the progress.

This work was supported by the University of Kansas General Research Fund to OJK and Undergraduate Research Assistantship fund to MM.
Selective regulation of chemokine CXCL6 by estrogen receptorβ (ERβ)
Authors: Scotland Ramona Sumintra , Roy Bornali , Duchene Johan , Ahluwalia Amrita
Published: June 2021

Endothelium‐derived chemokines are integral mediators of inflammatory cell recruitment and have been implicated in the pathogenesis of cardiovascular disease (CVD). Estrogens are thought to underlie the protective effects of female sex against CVD, although the exact mechanisms of this effect are uncertain. We investigated the hypothesis that estrogens inhibit endothelial chemokine production in response to an inflammatory stimulus.

qPCR of human umbilical vein endothelial cells (HUVECs) confirmed presence of ERα (46kD variant), ERβ and GPR30 mRNA. IL‐1β (1ng/ml, 8h) treatment elevated CXCL1 (Groα, 370‐fold), CXCL5 (ENA78, 310‐fold), CXCL6 (GCP2, 1030‐fold), CXCL8 (IL8, 150‐fold), and CCL2 (MCP1, 30‐fold) expression in HUVECs. Whilst pre‐treatment of cells with the ERα agonist 4,4′,4″(4‐Propyl‐[1H]‐pyrazole‐1,3,5‐triyl)trisphenol (PPT, 1nM, 16h) had no effect on IL‐1β‐induced chemokine expression, treatment with the ERβ agonist 2,3‐bis(4‐hydroxyphenyl)‐propionitrile (DPN, 1nM, 16h) suppressed CXCL6 expression only (P<0.01, n=7). This effect was abolished by 1h pre‐treatment with ER antagonist ICI 182,780 (100nM, n=5).

Thus our results show that activation of ERß but not ERα selectively inhibits endothelial CXCL6 expression; a response that likely contributes to the anti‐inflammatory actions of estrogens in vivo.

Supported by Wellcome Trust.
Synthesis and theoretical calculations of 5‐aminosalicylic acid derivatives as potential analgesic agents
Authors: Correa Jose , Ramirez Eduardo , Badillo Abigail , Gomez Carlos , Fabila Humberto L , Trujillo Jose G
Published: June 2021

5‐Aminosalicylic acid is one of the drugs most commonly used for inflammatory bowel disease treatment, although its use is limited due to side effects. The aim of this work was to synthesize four 5‐ASA derivatives (1–4) and analyze their pharmacological effects. The compound structures were elucidated by spectral (IR and 1H and 13C‐NMR) analysis, and their analgesic effects and lethal doses 50 (LD50) were evaluated in the mouse model. In addition, their Log Ps and affinities for both cyclooxygenase enzymes (COX I and COX II) were evaluated through theoretical calculations.

All compounds showed analgesic activities from 0.1 mg/Kg to 16 mg/Kg in the mouse model. The imides showed more affinity by COX enzymes and their Log Ps were the highest. The docking calculations showed that all compounds have good affinities for COX I and COX II ((1 ?M), making ????, van der Waals interactions and hydrogen bonds. The toxicities of all compounds were low, judging by the LD50.

Finally, the docking analysis show that the compounds act on COX enzymes and their analgesic effects could be mediated in part by the inhibition of these enzymes.
Transmembrane Domain IV of the Gallus gallus VT2 Vasotocin Receptor is Essential for Forming a Heterodimer with the Corticotrophin Releasing Hormone Receptor
Authors: Mikhailova Marina Victorovna , Blansett Jonathan , Jacobi Sandie , Mayeux Philip R. , Cornett Lawrence E.
Published: June 2021

Corticotropin releasing hormone receptor (CRHR) and the VT2 arginine vasotocin receptor (VT2R) are vital links in the hypothalamic‐pituitary‐adrenal axis that enable a biological response to stressful stimuli in avian species. CRHR and VT2R are both G‐protein coupled receptors, and have been shown by us to form a heterodimer via fluorescent resonance energy transfer (FRET) analysis in the presence of their respective ligands, corticotrophin releasing hormone (CRH) and arginine vasotocin (AVT). The dimerization interface of the heterodimer is unknown, but computational analyses predict that transmembrane domains (TMs) as likely sites of the interaction. In this study, we constructed chimerical VT2Rs, tagged with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), by replacing the 4th transmembrane region (TM4) of VT2R with TM4 of the β2‐adrenergic receptor (β2AR). The VT2R/β 2AR‐chimera was expressed in HeLa cells and membrane trafficking confirmed using confocal microscopy. VT2R/β2AR‐chimera functionality was demonstrated by the mobilization of intracellular Ca2+ with the addition of AVT. FRET analysis was then performed on VT2R/β2AR‐chimera/CRHR pairs, and the calculated distance was observed to be >10 nm apart, indicating that heterodimerization was disrupted by mutating TM4. Therefore, TM4 is an essential part of the dimerization interface between the VT2R and CRHR.
β2‐adrenergic receptor mediated generation of reactive oxygen species is a component required for signal transduction, desensitization, and homodimerization
Authors: Moniri Nader H. , Daaka Yehia
Published: June 2021

The β2‐adrenergic receptor (β2‐AR) has previously been shown to stimulate the formation of reactive oxygen species (ROS). The aim of the current study was to assess the importance of ROS on β2‐AR signaling cascades. Using HEK‐293 cells, which endogenously express β2‐AR, along with pharmacological inhibitors of various stages of ROS‐generating machinery, we examined the role of ROS generation on β2‐AR signaling, desensitization and homodimerization. Our results demonstrate that treatment of cells with the antioxidant NAC, the flavin‐containing oxidase inhibitor DPI, or the Rac1 inhibitor NSC23766, decreases β2‐AR‐mediated ROS generation, and importantly, that ROS inhibition abrogates β2‐AR‐mediated cAMP formation and PKA activation, as well as receptor phosphorylation and internalization. Additionally, our data demonstrate that β2‐AR‐β‐arrestin interactions, as well as β2‐AR‐β2‐AR homodimerization are decreased upon treatment of cells with ROS inhibitors. Taken together, these results suggest that intracellular ROS, which are generated upon agonist‐stimulation of β2‐AR, serve to stabilize receptor conformations that facilitate partner‐protein interactions which regulate downstream signaling, desensitization and homodimerization. Experiments to elucidate mechanisms of β2‐AR‐mediated ROS generation are currently underway in our laboratory.
Chronic EPI Treatment Fails to Down‐Regulate Del (301–303) 2B‐AR in Normal or GRK3 Over‐Expressing Neuronal Cells
Authors: Eikenburg Douglas C. , Salim Samina , Gokhale Aarti , Taneja Manish
Published: June 2021

A polymorphism of the human α2B‐AR (Del301‐303) (MUT‐α2B‐AR) results in reduced G‐protein coupled receptor kinase (GRK)‐mediated receptor phosphorylation, impaired short‐term desensitization and increased risk for MI. Recently, we have created NG108 cells stably transfected to express haemeagglutinin (HA)‐epitope‐tagged WT and MUT‐α2B‐ARs. In cell lines expressing the WT and MUT‐α2B‐AR at comparable levels (100 fmol/mg), the human polymorph, MUT‐α2B‐AR, displays reduced agonist‐stimulated phosphorylation and does not down‐regulate in response to epinephrine (EPI, 20μ‐1mM) treatment while the WT‐α2B‐AR is phosphorylated as well as down‐regulated upon EPI treatment. In addition, employing immunofluorescence labeling, we have observed that WT‐α2B‐AR internalizes upon EPI treatment whereas MUT‐α2B‐AR does not. Finally, we have studied the effect of EPI treatment on MUT‐α2B‐AR in cells stably over‐expressing GRK3 3.2 fold. Even 1 mM EPI treatment in GRK3 over‐expressing cells failed to down‐regulate the MUT‐α2B‐AR. These results suggest that a small mutation in the α2B‐AR eliminates agonist‐induced down‐regulation, an effect that cannot be reversed by increasing agonist concentration or increasing GRK3 expression. These results support a connection between early desensitization events such as phosphorylation and later α2‐AR down‐regulation. (supported by AHA, Tex. Affiliate and NARSAD)
The mechanisms related to the cardioprotective effects of protein kinase C epsilon (PKC ε‐) peptide inhibitor in ischemia/reperfusion (I/R) injury when given at reperfusion
Authors: Chen Qian , Kay Helen Young , Teng Jane , Burkhart Samantha , Zambrano Christopher , Krass Samuel , Adams Jovan , Grilli Christopher , Guglielmello Giuseppe , Brue Richard , Young Lindon Howard
Published: June 2021

PKC is a key enzyme involved in I/R injury. Previously, we showed that PKC ε− significantly restored postreperfused cardiac function up to 99% in polymorphonuclear leukocytes (PMNs, 200×106) induced myocardial I/R injury rat model. In this study, we further investigated the effect of PKC ε− on cardiac I/R injury and the underlying mechanisms. Compared to I/R+PMN hearts, we found that PKC ε− treated I/R+PMN hearts exhibited less cell injury and significantly lower PMN vascular adherence/infiltration in 45 min posterperfused heart tissue by using triphenyltetrazolium chloride staining, hemoxylin & eosin stain and myeloperoxidase assay. Furthermore, expression of endothelial adhesion molecules (i.e., intercellular adhesion molecule‐1; ICAM‐1) which contributes to PMN adherence/infiltration was also significantly decreased by 47±5% (P<0.05). Moreover, the NOS inhibitor, NG‐nitro‐L‐arginine methyl ester, did not block the cardioprotection of PKC ε−. However, PKC ε− (0.4 mg/Kg) significantly decreased hydrogen peroxide release during reperfusion in an in vivo femoral I/R model. Therefore, cardioprotection of PKC ε− is possibly related to attenuating ICAM‐1 expression, PMN adherence/infiltration and oxidative stress during reperfusion.

This study was supported by NHLBI Grant 1R15HL‐76235‐01 and Center For Chronic Disorders Of Aging at PCOM.
Suppression of the mitochondrial permeability transition (MPT) with NIM811 mitigates storage/reperfusion injury and improves graft survival after rat liver transplantation (LT)
Authors: Theruvath Tom P , Ramshesh Venkat K , Zhong Zhi , Lemasters John J
Published: June 2021

We examined whether N‐methyl‐4‐isoleucine cyclosporin (NIM811) prevents mitochondrial dysfunction via the MPT and improves graft function after rat LT. METHODS. LT was performed after 18 h cold storage and vehicle or NIM811 treatment of explants and recipients. Serum ALT, necrosis, and apoptosis at 6 h after LT and 30‐day survival were assessed. At 4 h after LT, recipients were infused with membrane potential‐indicating rhodamine 123 (Rh123) and cell death‐indicating propidium iodide (PI), and fluorescence was then assessed by intravital multiphoton microscopy. RESULTS. After LT with vehicle and NIM811, ALT increased to 5242 U/L and 2159 U/L, respectively (p<0.01). Necrosis and TUNEL also decreased from 21% and 21 cells/field, respectively, after vehicle to 9% and 5 cells/field after NIM811, (p<0.05). Long‐term graft survival increased from 27% after vehicle treatment to 70% after NIM811 (p<0.05). After vehicle, 4.3 hepatocytes/HPF contained depolarized mitochondria not retaining Rh123 without PI labeling, whereas nonviable hepatocytes with depolarized mitochondria were 3.3 cells/HPF. After NIM811, viable hepatocytes with depolarized mitochondria decreased to 0.7 cells/HPF (p<0.05), and nonviable hepatocytes decreased to 0.4 cells/HPF (p<0.05). In CONCLUSION, NIM811 attenuated graft injury and prolonged survival by blocking the MPT and preventing mitochondrial depolarization.
Ethanol preconditioning prevents ischemia‐reperfusion‐induced mitochondrial dysfunction in intestinal mucosa by an adenosine‐dependent mechanism in rats
Authors: Kalogeris Theodore J. , Wang Qun , Gaskin F. Spencer , Wang Meifang , Korthuis Ronald J.
Published: June 2021

Ethanol preconditioning (EPC) protects tissues from ischemia‐reperfusion (IR)‐induced injury by an unknown mechanism. Whereas ischemic preconditioning prevents IR‐induced mitochondrial dysfunction, it is unknown whether EPC has a similar protective effect. We tested this question in rats subjected to intestinal IR (20 min ischemia, 60 min reperfusion), 24 h after either control or ethanol (0.63 ml/kg) gavage, assaying mucosal mitochondrial respiration (MR), enterocyte mitochondrial membrane potential (MMP), and levels of mitochondrial cytochrome c. Compared with sham‐treated rats, IR decreased enterocyte mitochondrial respiratory function, membrane potential and cytochrome c content by 77%, 67%, and 61%, respectively. These effects were completely reversed in EPC rats. The protective effects of ethanol on MR and MMP were prevented in rats administered DMPX (adenosine A2 receptor antagonist), 10 min prior to EPC treatment; ethanol's protective effect on MMP was reproduced by administration of CPCA, an adenosine A2 receptor agonist. These results support the hypothesis that EPC prevents intestinal mucosal mitochondrial dysfunction through an adenosine‐dependent mechanism. Supported by AA14945 and DK43785.
Effects of vascular endothelial growth factor (VEGF) and angiotensin II (Ang II) on endothelial nano‐channels
Authors: Bodor Csaba , Nagy Janos , Jenei Attila , Balogh Zsófia , Masszi András , MirzaHosseini Shahrokh , Rosivall László
Published: June 2021

The juxtaglomerular portion of afferent arteriole (AA) is fenestrated and these nano‐channels may serve as a pathway for the renin into the circulation. VEGF and Ang II were examined as possible regulators of the formation of endothelial fenestration in human umbilical vein endothelial cell culture. Atomic force microscopy (AFM) was used to study the morphology of the fenestrae and dynamism of its appearance. Permeability of the endothelial monolayer, as a functional indication of fenestration was measured using 40 kDa FITC‐labeled dextran. With AFM 12.1±2.5 fenestrae/8x8 μm field of the endothelial cell surface were observed. About 90 percent of fenestrae had diameter less than 200 nm. The opening of individual fenestrae showed gyrus‐like upfolding. Measurements revealed funnel shaped nano‐channels. 48 hours treatments with both Ang II (10−7 M) and VEGF (100 ng/ml) induced significant increases in the number of channels. Ang II significantly increased endothelial permeability too. The permeability increasing effects of VEGF were more pronounced and appeared within 15 minutes. In summary we have visualized and characterized the individual endothelial nano‐channel utilizing the AFM. In addition we have demonstrated that Ang II and VEGF increase the number of fenestrae and the permeability of the HUVEC monolayer, therefore both agents could be important regulators of endothelial fenestration in the AA.
The Link Between Chronic Renal Blood Flow and Arterial Pressure Control by Angiotensin II in Mice.
Authors: Labazi Hicham , Banes‐Berceli Amy K.L. , Brands Michael W.
Published: June 2021

Angiotensin II (AngII) can cause significant decreases in renal blood flow, but the sustained effect on renal blood flow and the relationship to the hypertensive response to AngII are not known. This study measured that relationship by infusing AngII (sc.) via osmotic minipump in C57BL/6 mice instrumented either with chronic renal artery flow probes or with DSI arterial pressure telemetry devices. All mice were fed a high salt diet (4% NaCl) throughout the study, and they were allowed at least one week of recovery from surgery before control measurements. Mice in the 90 ng/min mean arterial pressure (MAP) group (n=4) had a baseline MAP of 115±2 mmHg, and AngII infusion increased MAP to 158±3 mmHg. The 90 ng/min renal blood flow (RBF) mice (n=4) had a rapid decrease in RBF (measured 4 hrs/day) that was sustained for the 7‐day period, averaging 45.2±11.9 % of control. RBF returned rapidly to control levels during the recovery period. In the 20 ng/min mice, AngII increased MAP from 118±3 to 152±3 mmHg (n=3), and the RBF response (n=2) was the same as in the 90 ng/min mice. A “slow‐pressor” dose of AngII (6 ng/min, n=3) did not change RBF over a 14‐day infusion period. These results show that maximal hypertensive doses of AngII cause marked and sustained decreases in RBF, but that slower‐developing hypertension may not be linked to a renal vasoconstrictor mechanism. (AHA SDG ABB, HL 56259, HL 74167 MWB).
MicroRNA‐21 Increases Aldosterone Secretion and Proliferation in H295R Human Adrenocortical Cells
Authors: Romero Damian G. , Plonczynski Maria W. , Carvajal Cristian A. , Gomez‐Sanchez Elise P. , Gomez‐Sanchez Celso E.
Published: June 2021

MicroRNAs (miRNAs) are endogenous small non‐coding RNAs which decrease the expression levels of specific genes by translational repression, sequestration and degradation of their mRNAs. Angiotensin II (AII) is an important modulator of adrenal zona glomerulosa steroidogenesis and proliferation. Since each miRNA may regulate the expression level of multiple genes, thereby resembling the mechanism of action and regulatory networks of transcription factors, we hypothesize that specific miRNAs may be involved in AII‐mediated adrenocortical cell responses. We screened more than 200 miRNAs for miRNAs regulated by AII in the human adrenocortical cell line H295R and found that mir‐21 expression levels were specifically and time dependently upregulated by AII reaching a 4.4‐fold (n=3, p<0.05) induction after 24 h. AII upregulated mir‐21 was biologically active as determined by its ability to decrease 40 % (n=6, p<0.05) the expression of a fusion mRNA reporter system with mir‐21 target sequences. Plasmid‐driven overexpression of mir‐21 increased aldosterone secretion (27.7(4.1 vs. 16.4(0.7 pg/(g prot.day, n=3, p<0.05), but not cortisol (226(26 vs. 218(13 pg/(g prot.day, n=3, p<0.05) and cell proliferation (132.6(1.5 vs. 100.0(6.0 % control, n=6, p<0.05). Alterations in mir‐21 expression levels or function may be involved in dysregulation of AII signaling and abnormal adrenal aldosterone secretion in humans
INSULAR CORTEX NORADRENERGIC NEUROTRANSMISSION MODULATES THE BAROREFLEX IN RATS.
Authors: Alves Fernando Henrique Ferrari , Crestani Carlos Cesar , de Aguiar Correa Fernando Morgan
Published: June 2021

Previous study from our laboratory indicated that bilateral microinjections of CoCl2 into the IC evoked reversible decrease in the bradycardiac reflex, suggesting that synapses in the IC modulates the parasympathetic component of the baroreflex. Thus the aim of the present study was to verify the participation of the IC‐noradrenergic neurotransmission in the modulation of the parasympathetic limb of the baroreflex.

Microinjection of selective noradrenergic á1‐adrenoceptor antagonist WB4101 into the IC caused a shift of the reflex threshold pressure bradycardiac responses to MAP increases toward higher MAP values (F=5, p<0.05), without affecting baroreflex gain (F=0.08, p>0.05). The effect of WB 4101 was reversible and the HR response returned to pre‐injection levels after 60 min (p>0.05). However, bilateral microinjection of the selective noradrenergic á2‐receptors antagonist RX821002 (F= 0,006, p> 0.05) did not affected baroreflex responses.

Our data indicate that noradrenergic á1‐receptors in the IC are involved in the modulation of the parasympathetic baroreflex component. Financial support: FAEPA, CNPq
Cerebral blood flow reactivity to CO2 during exercise
Authors: Hayashi Naoyuki , Ogoh Shigehiko , Ainslie Philip N , Miyamoto Tadayoshi
Published: June 2021

The arterial CO2 partial pressure (PaCO2) is associated with cerebral blood flow (CBF). However, the relationship between PaCO2 and CBF during exercise remains unclear. To investigate the mechanisms relevant to a control of the CBF during exercise, we measured the blood velocity in the middle cerebral artery (MCAV) in six males under various PaCO2 conditions by controlling inhaled CO2 fraction (FICO2 = 0, 3.5, and 5.0 %) and controlling ventilation (VE = 9 ‐ 38 L/min at rest and 12 ‐ 68 L/min during exercise) for 12 min at rest and during 40 W ergometer cycling. The reactivity of MCAV to PaCO2 change at rest was 1.08 ± 0.48 and 0.96 ± 0.17 cm/s/mmHg obtained from FICO2 and VE control, respectively, while that during exercise was 1.41 ± 0.43 and 1.00 ± 0.30 cm/s/mmHg. The reactivity during exercise was significantly greater when obtained from FICO2 control trial than that obtained from VE control. The range of PaCO2 was lower in VE control than in FICO2 control. These results suggest that the MCAV reactivity to PaCO2 is greater in high PaCO2 range during exercise, implying a possibility that increased MCAV reactivity has a role in washing‐out CO2 against great amount of CO2 product during exercise.
Perinatal exposure to PBDEs elevate systolic blood pressure in response to hyperosmotic stimulation in aged adult rats
Authors: Shah Ashini , Gaertner Mark , Coburn Cary , Kodavanti Prasada Rao , Watson‐Siroboe Abeena , Shahidizadeh Anoush , Whitley Rebecca , Gillard Elizabeth R , Curras‐Collazo Margarita
Published: June 2021

Polybrominated diphenyl ethers (PBDEs) are flame retardants used widely throughout the US. These chemicals are persistent throughout the environment and accumulate in biota. In previous work we showed that PBDEs and the structurally similar compounds PCBs disrupt the somatodendritic and/or peripheral release of vasopressin from the supraoptic nucleus of the hypothalamus (Coburn et al, 2005; 2007). Vasopressin hormone output into the circulation produces vasoconstriction and antidiuresis. Because of the interaction between PBDEs and VP we hypothesized that there should be measurable effects of PBDEs on blood pressure (BP) regulation. PBDE‐treated rats were exposed perinatally to the PBDE industrial mixture, DE‐71 (mothers given 1.7 or 30.6 mg/kg/d, GD 6‐PND 21 by oral gavage) and then tested as aged adults (14–18 months). BP was measured using tail cuff sphygmometry. First, the rats were tamed until their baseline BP was stable (compared restraint vs anesthesia). Rats were divided into two groups and subjected to an ip injection (0.6cc/100g) of 0.15 M (normosmotic) or 3.5 M NaCl (hyperosmotic). PBDE treatment alone did not change basal BP at 30 min or 3 h. Hyperosmotic PBDE‐treated rats showed a significant increase in systolic BP at 3 h post injection. In comparison, hyperosmotic controls (PBDE‐naïve) showed no significant rise in BP at any timepoint. These results suggest that exposure to environmental PBDEs may permanently disrupt cardiovascular function. Supported by UC Mexus (MC) and American Heart Association (RW), UC Dean's Research Fellowship (AS), MARC program (MG), EPA (PK)
Sympathetic innervation of the skin in type 2 diabetes mellitus
Authors: Strom Nicholas Alan , Roberts Shelly K , Kingsley‐Berg Shirley A , Charkoudian Nisha
Published: June 2021

In humans, sympathetic nerves in the skin provide tonic vasoconstrictor innervation. Although neural changes in type 2 diabetes mellitus (T2DM) are well recognized, it is unclear to what extent the activity of these vasoconstrictor nerves is altered in individuals with relatively uncomplicated T2DM. We tested the hypothesis that skin sympathetic nerve activity (SSNA) is decreased at rest in individuals with T2DM compared to healthy controls (C), and that the increase in SSNA with whole body cooling is smaller in T2DM than in C. We measured SSNA using microneurography, skin blood flow using laser‐Doppler flowmetry (LDF), and arterial pressure using Finometer in T2DM and C subjects at baseline and during 3–5 minutes of whole body cooling. LDF was measured on the foot at the site of innervation of the measured SSNA signal. Whole body cooling was followed by 35 min of local warming of the skin to 42°C to determine maximum cutaneous vascular conductance. In preliminary studies (N=3 per group), SSNA at baseline (T2DM: 22 ± 8 vs. C: 30 ± 1 bursts/min) and during whole body cooling (T2DM: 28 ± 11 vs. C: 43 ± 2 bursts/min; deltas: T2DM: + 6 ± 3 vs. C: +13 ± 1 bursts/min) tended to be lower in T2DM compared to C. Our data suggest that changes in control of SSNA in T2DM contribute to altered thermoregulatory responses in these individuals.

Supported by NIH R01 HL73884 and CTSA UL1‐RR24150.
Valsalva ratios and insulin sensitivity
Authors: Barlow Matthew A. , Sharma Arti , Gonzalez Leticia , Garcia Tiffany , Smith Michael L. , Yoshishige Darice , Caffrey James L.
Published: June 2021

The chronic metabolic disorders, obesity, diabetes, hypertension and heart disease are approaching epidemic proportions in the U.S. These disorders are more frequent, occur earlier and are often more severe in ethnic minorities. This ongoing study is designed to evaluate differences in the hormonal and cardiovascular status of African, Caucasian and Hispanic American adults in various states of health and disease. The study hopes to develop a database of less‐invasive methodologies to identify people most at‐risk for diabetes and heart disease early, before the diseases develop. The study also plans to evaluate whether cellular and humoral markers differ among ethnic groups. Estimated insulin sensitivity among the study population was as predicted for the general population. Relationships between blood glucose and insulin delineated healthy normals, diabetics and a transitional group of mildly hyperglycemic and euglycemic subjects with estimated insulin sensitivities very similar to that of diabetics. In approximately half of the subjects, glucagon declined sharply as expected during glycemic challenge. However, glucagon remained unchanged or was elevated in the remaining subjects. Autonomic reflex responses based on standardized valsalva ratios indicated approximately half of the subjects were below historical norms. The valsalva ratios correlated tightly with insulin sensitivity.
Electron microscopy of serotonin in arterial smooth muscle tissue
Authors: Priestley Jessica R. , Thompson Janice , Ni Wei , Watts Stephanie
Published: June 2021

5‐Hydroxytryptamine (5‐HT) is a powerful vasoconstrictor. Data from immunocytology and contractile experiments suggest that 5‐HT is an actively handled pool within an artery. We hypothesized that 5‐HT would be located intracellularly in discrete vesicles in arterial smooth muscle cells and is also potentially associated with proteins. Following ultra‐thin sectioning of samples, immunogold staining was performed to visualize serotonin in pargyline‐treated (100 mg/kg) rat superior mesenteric arteries (SMA) with rabbit anti‐5HT (Sigma) 1:50 and followed by 10nm gold‐conjugated goat anti‐rabbit (Sigma) 1:50. Sections were observed under an electron microscope (JEOL100 CXII, Japan Electron Optics Laboratories). We found 5‐HT located in the SMA smooth muscle cell, but not packaged in vesicles. 5‐HT appears to associate with protein such as actin in smooth muscle tissue. These findings were validated by western analyses of arterial homogenates which indicate the presence of several serotonylated proteins using a 5‐HT antibody. While the physiological relevance of this remains unknown, the finding of intracellular 5‐HT suggests 5‐HT may have a function once inside a smooth muscle cell.
Vascular guidance tunnels direct endothelial cell‐pericyte interactions and basement membrane matrix deposition
Authors: Stratman Amber Nicole , Koh Wonshill , Davis Michael J , Davis George E.
Published: June 2021

Endothelial cell (EC) tube morphogenesis in 3D extracellular matrices is controlled by EC intracellular vacuolation and vesicular fusion leading to lumen formation and interconnection of neighboring cells to form multicellular tube networks. These latter events require proteolysis in collagen matrices by MT1‐MMP. The EC generated proteolyzed spaces, termed vascular guidance tunnels, represent a physical imprint in the matrix allowing for cellular movement and tube remodeling within these spaces in an MMP‐independent manner. Over a period of days, time‐lapse and immunofluorescence microscopy shows marked recruitment of pericytes into these vascular guidance tunnels. The creation of a 2D plane within a 3D matrix environment allows for enhanced EC‐pericyte interactions, proteolytic independent movement and pericyte proliferation. Furthermore, it is only when pericytes are present within the vascular guidance tunnels that deposition of the basement membrane components collagen type IV, laminin, nidogens 1/2, and perlecan occurs. In the absence of pericytes, ECs produce each of these components, although they are not locally deposited. Pericyte proliferation within these tunnel spaces increases the ratio of pericytes to ECs leading to basement membrane matrix deposition and EC tube stabilization.