bluuebunny/crossref_metadata_2025 huggingface dataset:
Ramalin inhibits LPS‐induced autophagic response in macrophages through the downregulation of iNOS expression
Authors: Park Hye‐Jin , Yim Joung Han , Lee Hong Kum , Pyo Suhkneung
Published: June 2021

Activation of macrophages is essential for the host defense system and accumulating evidence suggests that autophagy is an important component in macrophage activity. Prolonged activation of macrophages, however, becomes detrimental and contributes to the progression of chronic inflammatory diseases with abnormal cell death. Therefore, controlling the overactivation of macrophages may be a new therapeutic strategy for various inflammatory diseases. Here we showed that ramalin, an antioxidant compound from the Antarctic lichen Ramalina terebrata, inhibited both LPS‐induced autophagy and iNOS in macrophages. To verify the role of NO in autophagy, we compared autophagy‐related gene expression in LPS‐stimulated and SNP‐stimulated macrophages by western blotting. Interestingly, ramalin did not suppress NO and autophagy in macrophages exposed to NO donor, SNP. In addition, inhibition of NO production by SMT significantly reduced LPS‐induced autophagy. These finding imply that ramalin might suppress LPS‐induced autophagy by inhibiting NO secretion. Ramalin also showed inhibitory effect on LPS‐induced NF‐κB and MAPK activation. Overall, these finding indicate that ramalin suppresses autophagy in LPS‐stimulated macrophages by preventing NO production and downregulating both NF‐κB and MAPK activation.
CDP‐choline protects against sepsis‐induced acute tissue injury in rats.
Authors: Yilmaz Mustafa Sertac , Sevim Cigdem , Altinbas Burcin , Ozyigit Musa Ozgur , Savci Vahide , Yalcin Murat
Published: June 2021

In the present study, we aimed to determine whether CDP‐choline can improve the outcome of cecal ligation and incision (CLI) in rats. Male Sprague‐Dawley rats were randomly divided into the following four experimental groups: sham, CLI and treated with saline or CDP‐choline. Saline (1 ml/kg) or CDP‐choline (50 and 100 mg/kg) was intravenously injected just after the CLI. Mean arterial pressure (MAP) and heart rate (HR) of all animals were evaluated through the experiment. At 6h after CLI; lung, liver, kidney and spleen tissues were collected. Optical microscopy was performed to examine the pathologic changes. All tissues were stained with hematoxylin‐eosin before examination under light microscopy.

In CLI‐animals, MAP decreased significantly throughout observation time compared with sham animals. First significant changes between both groups were observed 3 h after the respective surgical procedure. There was no any significant change observed in HR of the animals. Treatment with CDP‐choline at doses of 50 and 100 mg/kg, i.v. significantly attenuated the CLI‐induced tissue inflammation compared to control animals.

In conclusion, intravenously injected CDP‐choline prevents tissue injury induced by CLI. The protective effect of CDP‐choline is not related to its pressor effect.

This study was supported by The Commission of Scientific Research Projects of Uludag University, Project number: UAP(T)‐2011/13.
Pentadecapeptide BPC 157 and rat osteoarthritis
Authors: Sikiric Predrag , Krstonijevic Zoran , Sever Marko , Lojo Nermin , Drmic Domagoj , Zenko Anita , Brcic Luka , Zoricic Ivan , Rasic Zarko , Rucman Rudolf , Seiwerth Sven
Published: June 2021

Rat osteoarthritis (transected anterior cruciate and medial collateral ligament, medial meniscectomy) exhibited large cartilage destruction, still preserved bone (4 weeks), proximal tibia (cartilage and bone) totally destructed (8 and 12 weeks), and severely impaired knee function. This was rescued with stable gastric pentadecapeptide BPC 157, 10 μg, 10 ng (/kg) either ip once daily or in drinking water (0.16 μg; 0.16 ng/ml/12ml/day) till the sacrifice (at week 4, 8, 12). Knee articular (tibial and femoral) surface similar to nonoperative animals at 4 weeks, only few cartilage lesions at 8 and 12 weeks, thus, cartilage and bone preservation confirmed by X ray exam. Functionally, all parameters analysed (walking, leg pressure force, knee mobility (o)) were improved and rat's leg length maintained. Unlike general failure in controls, BPC 157‐rats walk as non‐operated rats, exhibiting better leg pressure force: i.e., BPC 157‐rats (10μg/kg in drinking water) after 4 weeks 18+/−0.4N (operated) and 20+/−0.7N (nonoperated leg) vs. controls 12+/−1.3 N (operated) and 20+/− 0.5 N (non operated leg); after 12 weeks BPC 157‐rats exhibited 19+/− 0,3N (operated) and 20+/−0.6N (nonoperated), controls only 7+/− 1.1N (operated) and 20+/−1.4N (nonoperated). BPC 157‐rats constantly exhibited improved knee mobility (73 +/− 3.7o (BPC 157) vs. 10 +/−1.3o (controls, 8 weeks)). Supported by Grant 108–1083570‐3635, Croatia
Alternatively‐spliced DNase I acts as dominant‐negative inhibiting cisplatin toxicity to kidney cells
Authors: Zhdanov Dmitry , Wang Xiaoying , Fahmi Tariq , Shah Sudhir V. , Basnakian Alexei G.
Published: June 2021

DNase I is apoptotic endonuclease that promotes toxic acute kidney injury (AKI) by cisplatin. It is the most studied, the most abundant, and the most active endonuclease in mammalian tissues. However, the regulation of this enzyme is not understood. We found that AKI by cisplatin in mice is associated with the generation of truncated, alternatively‐spliced DNase I isoform that lacks exon 4. The isoform was named Δ4DNase I. Expression of Δ4DNase I was associated with low DNase I protein expression, low DNase and nuclear endonuclease activity in mouse organs. Since it is missing some DNA‐ and actin‐binding sites with the skipped exon, Δ4DNase I has decreased DNA‐degrading and actin‐binding activity. Overexpression of Δ4DNase I suppresses DNase I and some other endonucleases as measured using real‐time RT‐PCR, Western blotting and activity assays. Δ4DNase I overexpression also protects NRK‐52E cells from cisplatin injury in vitro, and, thus, seems to be important mechanism of DNase I regulation during cisplatin AKI. NIH R01DK078908 and VA Merit Review grants to AGB and SVS.
High Fat Diet Failed to Induce NALP3 Inflammasome Activation and Glomerular Injury in Apoptosis‐Associated Speck‐like Protein (ASC) Knockout Mice
Authors: Boini Krishna M. , Xia Min , Abais Justine M. , Zhang Yang , Li Pin‐Lan
Published: June 2021

Inflammasome, an intracellular inflammatory machinery, plays a key role in the pathogenesis of obesity and associated disorders. The present study was performed to determine the role of key inflammasome adaptor molecule ASC in high fat diet (HFD)‐ induced inflammasome activation and glomerular injury. ASC gene knockout (Asc−/−) and wild type (Asc+/+) mice were fed a HFD or normal chow (ND) for 8 weeks to produce obesity and associated glomerular injury. HFD significantly increased the body weight, blood glucose levels and NADPH‐dependent superoxide (O2•−) production in Asc+/+mice compared to ND fed mice. However, such HFD‐induced body weight gain, blood glucose levels and O2•− production were attenuated in Asc−/− mice. Confocal microscopic analysis showed that the HFD enhanced the colocalization of NALP3 with ASC or Caspase‐1, the essential components of NALP3 inflammasomes, in glomeruli of Asc+/+ than in Asc−/− mice. Consistent with decreased inflammasome formation, the caspase‐1 activity and IL‐1β production were also significantly attenuated in Asc−/− mice fed a HFD. Morphological examinations showed that HFD induced more profound injury in glomeruli of Asc+/+ mice than Asc−/− mice(the glomerular damage index (GDI) in Asc+/+: 2.2 ± 0.1 vs. Asc−/−: 1.2 ± 0.1). This decreased GDI in Asc−/− mice on the HFD was accompanied by attenuated proteinuria and albuminuria. Fluorescent immunohistochemical examinations using podocin and desmin as a podocyte markers showed that the inflammasome formation induced by the HFD were mostly located in podocytes as demonstrated by co‐localization of podocin with NALP3 or desmin with NALP3. In conclusion, normal expression and function of ASC gene is critical to the formation and activation of NALP3 inflammasomes and associated glomerular injury in obesity (supported by NIH grants DK54927 and HL‐75316).
Epithelial‐to‐Mesenchymal Transition Induced by Accumulation of Autophagosomes in Podocytes
Authors: Xia Min , Li Cai‐Xia , Li Cao , Abais Justine M. , Zhang Yang , Boini Krishna M. , Li Pin‐Lan
Published: June 2021

Mature podocytes are highly differentiated cells in the kidney and changes in this differentiated status of podocytes such as epithelial‐to‐mesenchymal transition (EMT) or dedifferentiation contribute to the development of glomerular injury or sclerosis. The present study was designed to test the hypothesis that EMT occurs in podocytes if its autophagic process is impaired. In conditionally immortalized mouse podocytes, we demonstrated that blockade of autophagic flux by lysosomal V‐ATPase inhibitor, bafilomycin A1 or by silencing of its isoform using V‐ATPase V0 siRNA significantly induced EMT, as shown by decreased expression of epithelial markers, p‐cadherin and ZO‐1, and increased expression of mesenchymal markers, FSP‐1 and α‐SMA. This enhanced EMT was accompanied by reduction of podocin production, a podocyte function. All these changes were confirmed by confocal microscopy, real time RT‐PCR and Western blot analysis. Correspondingly, both bafilomycin A1 and V‐ATPase V0 siRNA blocked autophagic flux that resulted in a large increase in autophagosomes in podocytes, as shown by increased expression of microtubular associated protein 1 light chain 3‐II (LC3B‐II). In particular, a dramatic increase in the level of cytoplasmic scaffold protein P62 was observed when autophagic flux was blocked. In contrast, a specific inhibitor of autophagosome formation, spautin‐1 had no effect on podocyte EMT under control condition, but it significantly reversed bafilomycin A1‐induced effects on both EMT and LC3B‐II or P62 expression. These results suggest that the maturation and normal function of podocytes are critically controlled by autophagic process and that lysosome dysfunction and consequent impairment of autophagic flux may induce EMT in these podocytes (supported by NIH grants HL057244, HL‐75316 and DK54927).
One teacher's journey from the bench to the classroom
Authors: Cruzan Jeff
Published: June 2021

The story of my transition from the research bench to teaching high school math and science is (I have been told) inspirational and compelling, and I'd like to tell it to you. It's a tale of survival, personal growth, amazing highs and low lows. My seven‐year journey has led me to my current position at a private prep school and to a term on the board of public education of my Massachusetts community. Teaching is both the most difficult and most rewarding job I've ever had; I wouldn't trade my decision to leave the research bench for anything. Especially in high schools, the teaching profession needs people like you, with deep backgrounds in their field and a love of science that can bring the subject to life for kids.
Deficiency of glia maturation factor suppresses 1‐methyl‐4‐ phenylpyridinium induced toxicity in primary cultures of mouse astrocytes
Authors: Khan Mohammad M , Javed Hayate , Zaheer Asgar
Published: June 2021

Glia maturation factor (GMF), a brain specific protein, is isolated, sequenced and cloned in our laboratory, predominantly localized in astrocytes. In the present studies, we demonstrate that deficiency of GMF in astrocytes limits the production of reactive oxygen species (ROS) and suppresses the NF‐kB mediated inflammatory responses in 1‐methyl‐4‐phenylpyridinium (MPP+)‐induced toxicity. Primary astrocytes obtained from wild type (Wt) and GMF‐KO mice were treated with 5, 10 and 20 ìM MPP+ for 24, 48 and 72 hr. Our results show decrease production of ROS and nitric oxide in GMF‐KO astrocytes following MPP+ treatment. In contrast, astrocytes derived from Wt mice exhibited increase level of ROS along with enhance level of nitric oxide. Additionally, we found decrease activity of nuclear factor‐êB, and reduce levels of proinflammatory cytokines/chemokines in GMF‐KO astrocytes compared to Wt astrocytes. The overall data suggests that GMF‐KO astrocytes were more resistant to MPP+ toxicity compare to Wt astrocytes. These findings provide the first evidence that GMF deficiency protects astrocytes from MPP+‐induced toxicity by reducing the ROS production along with diminished levels of NF‐kB and proinflammatory cytokine/chemokine expression. (Supported by VA Merit Review award and NINDS grant NS073670).
Perturbations in Intracellular Ca2+ Concentrations and DNA Damage are Coupled to the Activation of PARP‐1 During ROS‐induced Necrotic Cell Death
Authors: Ramirez Frances M , Lau Serrine S , Monks Terrence J
Published: June 2021

2,3,5‐Tris(glutathion‐S‐yl)hydroquinone (TGHQ) generated reactive oxygen species (ROS), cause an increase in intracellular calcium concentrations (iCa2+), DNA strand breaks, and hyperactivation of poly(ADP‐ribose)polymerase (PARP)‐1, resulting in necrotic cell death. The relationship between Ca2+ channels and PARP‐1 was therefore examined in renal proximal tubule epithelial cells (HK‐2) treated with TGHQ. 2‐APB, a store‐operated Ca2+ channel (SOCs) inhibitor, decreased PAR ribosylation, indicating that activation of Ca2+ channels has a direct effect on PARP‐1 activity in ROS‐induced cell death. siRNA knockdown of poly(ADP‐ribose)glycohydrolase indicated that free ADPR does not activate Ca2+ channels in HK‐2 cells. Pharmacological inhibition of the sarcoplasmic‐endoplasmic reticulum calcium pump (SERCA), and knockdown of STIM1/Orai (CRAC channel) revealed the increase in iCa2+ responsible for crosstalk with PARP‐1 does not originate from the ER. Moreover, Ca2+‐free media experiments excluded extracellular Ca2+ as the culprit. Thus PARP‐1 is not hyperactivated via PARG metabolism, and the Ca2+ sources activating PARP‐1 may include mitochondrial stores. We are using calcium imaging to further investigate the elusive relationship between specific Ca2+ channels and PARP‐1, as well as the mechanism(s) by which PARP‐1 and Ca2+ interact to contribute to necrotic cell death.
X‐ray Crystal Structure of the Cytochrome P450 2B4 Active Site Mutant F297A in Complex with Clopidogrel: Insights into Compensatory Rearrangements of the Binding Pocket
Authors: Shah Manish B , Jang Hyun‐hee , Stout C. David , Halpert James R
Published: June 2021

Prior X‐ray crystal structures of rabbit cytochrome P450 2B4 and human P450 2B6 have revealed the pivotal role of side‐chain rearrangement of active site residues F206 and F297 in accommodating various inhibitors or substrates. To explore further the structural basis of ligand binding, the crystal structure of 2B4 F297A was solved in complex with the drug clopidogrel. The structure demonstrated the reorientation of ligand in the active site such that the acetoxy group is oriented toward the heme, whereas the thiophene moiety now extends to the void region of A297 on the I‐helix. Interestingly, movement of the I‐helix and several amino acid side chains within the active site was observed in apparent response to the altered binding orientation. Results of flexible docking using the 2B4 wild type or the F297A‐virtual mutant positioned either the thiophene or chlorophenyl group closer to heme. However, docking of clopidogrel using the F297A structure or a virtual mutant where the I‐helix was also repositioned, oriented clopidogrel preferentially with either the acetoxy or the chlorophenyl group closest to heme. The study provides insight into how the altered active site adapts to accommodate the substrate in a distinct orientation and has important implications for understanding the role of active site residues in functional differences among related P450 enzymes. Supported by NIH grant ES03619.
Organization of cytochrome P450 system components in the endoplasmic reticulum
Authors: Park Ji Won , Reed James R. , Brignac‐Huber Lauren M. , Cawley George F. , Eyer Marilyn , Backes Wayne L.
Published: June 2021

P450s are membrane‐bound proteins found in the endoplasmic reticulum (ER). The ER is known to be heterogeneous, containing both ordered and disordered regions. Ordered microdomains are more tightly packed and not readily solubilized with non‐ionic detergents and are called detergent resistant membranes (DRM). The goal of this study is to determine if these microdomains affect P450 localization. The localization of P450 system proteins was examined in rabbit liver microsomes after incubation with 1% Brij 98, followed by sucrose gradient centrifugation. Unlike CYP1A2 (which localizes in the DRM), CYP2E1 proteins localized in non‐DRM and CYP2B4 was found to be equally distributed between both domains. Importantly, for P450 to function, it is necessary for this heme protein to form a complex with its electron donor CPR; however, CPR is found largely in the DRM. Consequently, for CYP2E1 to function, a mechanism for complex formation is required, such as translocation of either CYP2E1 or CPR between the lipid domains, or the interaction of CYP2E1 with the small amount of residual CPR localized in the disordered region, which would limit its activity. These results demonstrate that the components of the P450 system are organized in specific membrane regions, and not randomly distributed throughout in the ER membrane. Supported by ES004344
Cellular retinoic acid binding proteins (CRABPs) channel retinoic acid to CYP26A1
Authors: Nelson Cara Hartz , Lutz Justin D , Isoherranen Nina
Published: June 2021

The aim of this study was to characterize the interaction between cellular retinoic acid binding proteins I and II (CRABPI and CRABPII) and CYP26A1 using recombinant CYP26A1 microsomes and purified CRABPs. To characterize the interactions of CRABPs with CYP26A1, the kinetics of 4‐OH‐RA formation from free retinoic acid (RA) or holoCRABP was characterized in the presence and absence of albumin. The effect of apoCRABP on CYP26A1 activity was measured with varying ratios of CRABPs and RA. In the absence of albumin, both holoCRABPI and II had significantly lower Vmax values than free RA but the Km for holoCRABPII was also significantly less than the Km values for holoCRABPI or free RA. In contrast, albumin decreased 4‐OH‐RA formation by >;90% and no RA metabolism was observed by CYP3A4 and CYP2C8 using holoCRABPs as substrate. Both apoCRABPI and II inhibited the metabolism of RA by CYP26A1. The kinetic results were globally fitted to a model that accounts for direct CRABP‐CYP26 interaction and channeling and holoCRABPII and apoCRABPII were found to have Kd values of 86 nM and 47 nM to CYP26A1, respectively. The CRABP binding to CYP26A1 was found to decrease the kcat by a α of 0.55. These data demonstrate that CRABPs channel RA to CYP26A1 and are not acting as a protein sink, like albumin. In addition, the inhibition of CYP26A1 by apoCRABPs suggests that CRABPs interact directly with CYP26A1 and regulate RA concentrations.
Listeria monocytogenes inlA/inlB as possible drug delivery systems
Authors: Broeker Nathan Phillip
Published: June 2021

The internalin proteins (inlA and inlB) of Listeria monocytogenes induce phagocytosis by normally non‐phagocytic human cells. This unique attribute makes inlA/inlB attractive as a possible therapeutic drug delivery system. inlB binds to the membrane protein “Met” present on non‐phagocytic cells and results in actin rearrangement leading to uptake of the bacterial pathogen. However, inlA binds to E‐cadherin on host epithelial cells, which mediate entry into the host cell through the recruitment of α‐catenin and β‐catenin. Either or both of these proteins may provide a means of drug delivery by conjugation of drug to the proteins. Previous work in our lab has resulted in the successful cloning and over‐expression of inlB. Current studies focus on the cloning and over‐expression of inlA and purification of native inlB. Both inlA and inlB have been partially purified with current work focused on labeling and eukaryotic uptake studies. This publication was made possible by grants from the National Center for Research Resources (5P20RR016469) and the National Institute for General Medical Science (NIGMS) (8P20GM103427), a component of the National Institutes of Health (NIH) and its contents are the sole responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.”
Class III β‐tubulin expression and function by human vascular pericytes in vitro
Authors: Stapor Peter C , Lee Murfee Walter
Published: June 2021

Class III β‐tubulin was recently identified as a marker of angiogenic pericytes in adult rat mesenteric microvascular networks. Upregulation of class III β‐tubulin by pericytes temporally correlates with capillary sprouting. While its function in pericytes remains unknown, class III β‐tubulin expression by tumor cells has been associated with resistance to tubulin‐binding agents. The objectives of this study were to determine whether human pericytes express class III β‐tubulin in vitro, and whether the presence of class III β‐tubulin correlates with resistance to paclitaxel, a tubulin‐binding agent. Human placental pericytes (hPCs) were plated in 6‐well culture dishes or on glass slides and grown to confluence. Confluent monolayers of hPCs in the 6‐well plates were scratched and immediately treated with control, 1nM paclitaxel‐or 10 nM paclitaxel‐supplemented media. Cultured pericytes positively labeled for class III β‐tubulin. Starting at 6 hours post scratch, the area closure for both paclitaxel groups was significantly less than the control. For unscratched cells cultured on glass slides, 10 nM paclitaxel treatments caused a decrease in cell proliferation indicated by the percentage of Ki‐67positive nuclei. These results suggest that cultured vascular pericytes display an angiogenic phenotype and that class III β‐tubulin expression by pericytes might not be associated with resistance to tubulin‐binding agents.
Blunted temporal activity of microvascular perfusion heterogeneity in metabolic syndrome: a new attractor for peripheral vascular disease?
Authors: Butcher Joshua T , Goodwill Adam G , Stanley Shyla C , Frisbee Jefferson C
Published: June 2021

A key outcome for peripheral vascular disease (PVD) is a progressive decay in skeletal muscle fatigue resistance with elevated metabolic demand. We have demonstrated that PVD in obese Zucker rats (OZR) is partially due to increased perfusion distribution heterogeneity (γ) at successive microvascular bifurcations, with its cumulative impact being an increasingly heterogeneous distribution of perfusion across terminal arterioles, leading to greater tissue ischemia. To minimize this outcome, a likely compensatory mechanism against increased γ should be an increased temporal switching at bifurcations to blunt downstream deficits. Using in situ cremaster muscle, we determined that temporal activity was lower in OZR than in LZR, and this difference was present in both proximal (1A‐2A) and distal (3A‐4A) arteriolar bifurcations. While adrenoreceptor blockade (phentolamine) improved temporal activity in 1A‐2A bifurcations in OZR, this was without impact in the distal microcirculation, where only interventions against oxidant stress (TEMPOL) and thromboxane A2 activity (SQ‐29548) were effective. Analysis of the attractor for γ indicated that it was elevated in OZR, and also exhibited a reduced range, suggesting that the ability of the microcirculation to respond to any challenge is highly restricted, and may represent the major contributor to the poor muscle performance in OZR.
Flow‐Induced Structural Adaptation of Tumor Vasculature by Selective Micro‐Laser Ablation
Authors: Gruionu Gabriel , Gruionu Lucian , Munn Lance L.
Published: June 2021

The effects of anti‐angiogenesis therapy on tumor vasculature are transient, and the exact mechanisms of vessel remodeling are not known. Here we developed experimental and theoretical models of tumor vascular remodeling for customizing the dose and timing of vessel‐targeted drugs. We used a multiphoton laser to interrupt vessel segments downstream from a bifurcation and observed remodeling in the adjacent branches in normal skin vasculature and AK4.4 pancreatic tumors implanted in the mouse dorsal skin fold chamber. We assessed blood flow and cellular dynamics using fluorescence stereomicroscopy and optical frequency‐domain imaging. The theoretical simulation of the vascular network dynamics is based on a previous network model developed by Gruionu et al (Am J Physiol, 2005). A finite element model was used to simulate oxygen and nutrient delivery in the surrounding tissue. The laser microsurgery caused significant remodeling in both arterial (mean: 151%, max: 229%) and venous (mean: 153%, max: 581%) sides of the circulation. The predictions of the model showed good correlation with the observed changes in the dorsal skinfold chamber. The models will help us define the contribution of local stimuli to remodeling of tumor vascular beds and generate predictions for altering tumor blood flow and development of future therapies. Supported by NIH R01CA149285 and CNCS (Romania) PN‐II‐ID‐PCE‐2011–3‐0664.
Aging attenuates spontaneous endothelial Ca2+ events with altered perivascular nerve function in mouse mesenteric arteries in vivo
Authors: Westcott Erika Boerman , Segal Steven S
Published: June 2021

Perivascular nerve activity has been linked to local endothelial cell (EC) Ca2+ events through α1‐adrenoreceptor (AR) activation, but the effects of aging on perivascular nerves and EC Ca2+ are unclear. We tested the hypothesis that aging alters local EC Ca2+ signals in vivo. Superfused (pH, 7.4; 36°C) mesenteric arteries (MAs) of anesthetized Young (3–6 month; n=9) and Old (24–26 month; n=5) Cx40BAC‐GCaMP2 mice were imaged (30 frames/s) using spinning disk confocal intravital microscopy. In Young, ECs exhibited spontaneous Ca2+ events [0.31 ± 0.03 Hz; amplitude (F/Fo): 1.31 ± 0.012; duration: 0.11 ± 0.004 s]. In Old, Ca2+ events were nearly absent. Electrical field stimulation (EFS) produced frequency‐dependent MA constriction with Old >; Young by ~20%. Blocking sensory nerves (capsaicin, 10 μM) enhanced MA constriction during EFS by ~20% in Young (P < 0.05) but not Old indicating loss of sensory inhibition of sympathetic vasoconstriction in Old. Cumulative activation (1 nM to 10 μM) of α1ARs (phenylephrine) but not α2ARs (UK14304) exhibited reduced sensitivity (P<0.05) in Old vs. Young. Immunolabeling confirmed sympathetic and sensory nerves were maintained in Old thus functional changes were not due to loss of perivascular innervation. The loss of local EC Ca2+ signals in Old may reflect α1AR desensitization and thereby reduce EC feedback in regulating smooth muscle tone. (NIH R01HL086483, R37HL041026).
Interference with PPARγ in endothelium accelerates angiotensin II‐mediated vascular dysfunction
Authors: Hu Chunyan , Faraci Frank M. , Sigmund Curt D.
Published: June 2021

Subjects carrying dominant‐negative mutations in peroxisome proliferator‐activated receptor γ (PPARγ) exhibit severe hypertension. We previously showed that transgenic mice (E) expressing the PPARγ V290M mutation in endothelium exhibited an augmented pressor response to angiotensin II (Ang II). Herein, we hypothesize that vascular dysfunction induced by Ang II is exacerbated in E mice. Therefore, non‐transgenic (NT) and E mice were infused with a subpressor dose of Ang II (120 ng•kg−1•min−1, osmotic pumps) or saline for 2 weeks and carotid artery function was assessed using a wire myograph. There was no difference in acetylcholine (ACh)‐induced relaxation between the saline‐treated groups. Ang II caused significantly impaired relaxation to ACh in E mice (at 10−4M: 55.7±3.3% in E vs 70.0 ± 3.8% in NT, p<0.05). Vasorelaxation to sodium nitroprusside (SNP) was significantly impaired in Ang II infused E mice suggesting possible smooth muscle dysfunction. Tempol, a superoxide scavenger, significantly improved the ACh response in E. Serotonin‐induced contraction was increased after Ang II in NT and E, but the response was more pronounced in E (at 10−6 M: 0.22±0.02g in E vs 0.14±0.02g in NT, p<0.05). We conclude that interference with endothelial PPARγ function accelerates Ang II‐mediated vascular dysfunction through an oxidative stress‐dependent mechanism. (Support: NHLBI)
Post‐nodal lymphatic vessels adapt to sustained high flow conditions by becoming weaker pumps
Authors: Dongaonkar Ranjeet M. , Nguyen Tam L. , Quick Christopher M. , Laine Glen A. , Wilson Emily , Heaps Cristine , Hardy Joanne , Stewart Randolph H.
Published: June 2021

Edemagenic challenges can make passive lymph flow so high that active lymphatic vessel pumping may not be necessary to propel lymph. Indeed, an acute increase in lymph flow is reported to decrease lymphatic contraction frequency and strength in vitro. However, the lymphatic response to a chronic increase in flow in vivo is unknown. We hypothesized that lymphatic vessels become weaker pumps when exposed to a sustained increase in lymph flow. We surgically occluded bovine mesenteric veins to create mesenteric venous hypertension and high lymph flow conditions. Three days after surgery, post‐nodal mesenteric lymphatic vessels (MVH) were isolated and compared to vessels from sham surgery animals (SHAM) to quantify changes in function, biomechanics, calcium homeostasis and gene expression. Compared to SHAM, MVH vessels exhibited a lower pump index, active tension, K+ induced cytosolic [Ca2+], as well as a lower expression of SERCA type 3, voltage‐dependent Ca2+ channel, and voltage‐gated K+ channel regulatory genes. In short, post‐nodal lymphatic vessels adapt to high flow conditions by becoming weaker pumps. The present work is the first to integrate experimental approaches at the tissue, cellular, and molecular levels to relate the mechanisms of adaptation to biomechanical and functional responses to clinically‐relevant edemagenic stimuli. (Supported by NIH and AHA)
In vivo assessment of blood pooling and muscle pump dynamics under orthostatic stress
Authors: Leguy Carole , Blaber Andrew , Kuemmel Jakob , Beck Luis , Zange Jochen , Rittweger Joern
Published: June 2021

A better understanding of blood pooling to the lower limbs and muscle pumping (increasing venous return) is essential to prevent orthostatic intolerance. The goal of this study is to assess the dynamics of these physiological responses under orthostatic stress. Repeated 70 degrees Head Up Tilt (HUT) of 3 minutes were performed on a group of 15 healthy volunteers. After 1st and 2nd minute of HUT, the subjects performed a 3 s calf Muscle Contraction (MC) at 30% of maximal electromyographic activity. Calf muscles EMG, ECG and finger blood pressure were recorded continuously. Blood volume flow (BVF) of the femoral artery was assessed with ultrasound from 1 min before to 1 min after HUT. A peak BVF of 620 mL/min was measured after HUT, whereas peak BVF after MC reached 950 mL/min. Plateau BVF in the femoral artery during HUT increased slightly after the MC possibly due to a metabolic response of the leg muscle. Finally, mean arterial pressure immediately after tilting and after MC decreased. We therefore conclude that the muscle pump through its effect on venous pressure of the lower limbs is a fundamental aspect in blood pressure regulation during orthostatic stress.
High‐intensity aerobic training restores the ability to modulate sympathetic vasoconstriction in contracting skeletal muscle of hypertensive and healthy middle‐aged individuals
Authors: Mortensen Stefan P , Nyberg Michael , Gliemann Lasse , Thaning Pia , Hellsten Ylva
Published: June 2021

In young individuals, contracting skeletal muscle can override sympathetic vasoconstrictor activity (functional sympatholysis). To investigate the role of exercise training on functional sympatholysis in hypertension, we measured leg hemodynamics before and after an 8 week period of high intensity exercise training (3–4 times/week) in 8 untrained hypertensive (46±2 years) and 8 healthy controls (47±5 years) during leg exercise and arterial ATP infusion with and without infusion of tyramine. There was no difference in the vasodilatory response to infused ATP between groups. Tyramine induced a similar vasoconstrictor response in both groups and did not alter the vasodilatory response to ATP in either group. Before the training period, exercise hyperemia was lower in the hypertensive subjects (P<0.05) and tyramine lowered exercise hyperemia in both groups (P<0.05). After the training period, exercise hyperemia was similar in both groups and tyramine did not change exercise hyperemia in any of the groups. These results demonstrate that functional sympatholysis is impaired in the leg of both untrained healthy and hypertensive middle‐aged individuals and that exercise training normalizes functional sympatholysis. The vasodilatory and sympatholytic effect of plasma ATP does not appear to be affected by hypertension or the physical activity level.

Supported by the Danish Research Council
Chronic recordings of cardiac contractility in conscious rats via telemetry
Authors: Guild Sarah‐Jane , McCormick Daniel , Lim Matthew , Pauly Bob , Kirton Robert , Budgett David , Kondo Masahiro , Stehlin Ellyce , Barrett Carolyn , Rigel Dean F , Malpas Simon
Published: June 2021

Measurements of cardiac contractility in conscious freely moving animals are uncommon yet could offer considerable opportunity for understanding cardiovascular disease progression and treatment. We have utilized a new high‐fidelity solid‐state pressure sensor telemetry device for chronically measuring left ventricular pressure (LVP) and dP/dt in rats. The pressure sensor catheter tip (2‐Fr) was inserted into the left ventricular chamber through the apex of the heart and the telemeter body implanted in the abdomen. Data was measured up to 85 days after implant. The average daytime dP/dt max was 9706 ±264 mmHg/s, ranging from 8186 to 11382 mmHg/s (n=24). A circadian variation in dP/dt max and heart rate (HR) was observed with an average increase during the night phase in dP/dt max of 2198 ±280 mmHg/s and in HR of 60 ±5 bpm. The alpha‐adrenergic blocker propranolol, alpha‐adrenergic‐agonist isoproterenol, alpha1‐adrenergic agonist dobutamine, Ca2+ channel blocker verapamil and the calcium sensitizer levosimendan were administered repeatedly throughout the implant period, inducing changes in dP/dt max of −6000 to +13,000 mmHg/s from control and dose‐dependent time course changes in contractility. We propose that the chronic measurement of LVP in conscious rats can provide a valuable tool in cardiovascular research.
Ang (1–7) Has a Greater Contribution to the Blood Pressure Lowering Effects of AT1 Receptor Blockade in Female Spontaneously Hypertensive Rats (SHR) Compared to Males
Authors: Zimmerman Margaret , Sullivan Jennifer
Published: June 2021

Ang (1–7) contributes to the blood pressure (BP) lowering effects of renin angiotensin system inhibitors in male SHR. We have previously published that female SHR have greater Ang (1–7) levels than males, however, the functional implications of this with regards to BP control remains largely unknown. Therefore, the goal of this study was to test the hypothesis that female SHR have a larger Ang (1–7) contribution to the BP lowering effects of the angiotensin receptor blocker (ARB) candesartan than male SHR. BP was measured via telemetry in 12 week old male and female SHR (n=4–8). Animals were pre‐treated with either the Ang (1–7) antagonist A‐779 (4 days; 48 μg/kg/hr via osmotic minipump) or vehicle. Candesartan was then co‐administered (0.5 mg/kg/day via drinking water) for a week. A‐779 alone did not alter basal BP in either sex. In male SHR, candesartan‐mediated decreases in BP were comparable in vehicle (15% decrease: 143±2 to 122±3 mmHg; p<0.05) and A‐779 treated (12% decrease: 147±3 to 129±4 mmHg; p<0.05). In contrast, A‐779 attenuated candesartan‐mediated decreases in BP (5% decrease: 127±1 to 121±2 mmHg; NS) in female SHR compared to vehicle treatment (10% decrease: 128±1 to 115±1 mmHg; p<0.05). In conclusion, female SHR have a greater Ang (1–7) contribution to ARB‐mediated decreases in BP than males. This study was funded by National Institutes of Health grant 1R01 HL093271–01A1 to J.C.S.
Sex difference in low dose of angiotensin (ANG) II sensitizing effect on pressor effect of subsequent high dose of ANG II
Authors: Xue Baojian , Zhang Zhongming , Guo Fang , Hay Meredith , Johnson Alan Kim
Published: June 2021

Previous studies using an Induction‐Delay‐Expression (I‐D‐E) experimental design have demonstrated that one‐week ANG II pre‐treatment can sensitize the brain to produce an enhanced hypertensive response to subsequent ANG II. It has been shown that estrogen can act through the CNS to attenuate ANG II hypertension. The present study was to test whether there is a sex difference in the sensitizing effects of ANG II and the involvement of central estrogen in this sensitization process. Male, intact and ovariectomized (OVX) female rats were implanted for telemetered blood pressure (BP) recording. During I low doses of ANG II alone or concurrent administration with icv estrogen was given for 1 week. After1 week (D) rest, slow pressor doses of ANG II was given for 2 weeks (E).

In males and OVX females, ANG II had no sustained effect on BP during I and D. However, during E ANG II‐induced hypertension was greater in the groups receiving ANG II during I in comparison to those groups receiving vehicle. Central administration of estrogen during I blocked low dose of ANG II‐induced sensitization. In intact females, low dose of ANG II actually induced a decrease in BP during I and D. Subsequent high doses of ANG II during E did not result in an enhanced increase in BP in either group. The results indicate that estrogen can play a protective role during the sensitization process and attenuate the development of ANG II‐induced hypertension.
L‐arginine Transporters: A New Treatment Target in Obesity Induced Hypertension?
Authors: Rajapakse Niwanthi W , Karim Florian , Head Geoffrey A , Kaye David M
Published: June 2021

Cationic amino acid transporter‐1 (CAT‐1) is the predominant L‐arginine transporter expressed in endothelial cells. We hypothesised endothelial CAT‐1 over‐expression can ameliorate obesity induced hypertension.

With the use of telemetry, baseline mean arterial pressure (MAP) and responses of MAP to the ganglionic blocker, pentolinium (5 mg/kg) were measured in conscious wild type mice (WT; n = 10) and mice over expressing endothelial CAT1 (CAT+; n=15) fed a normal or a high fat diet for 20 weeks.

Both genotypes of mice developed obesity when placed on a high fat diet (Pdiet ≤ 0.007). MAP was greater by 12 ± 2 mmHg (Pdiet < 0.001) in WT mice fed a high fat diet, compared to those fed a normal fat diet. In contrast, MAP was not significantly different in CAT+ mice fed a normal or a high fat diet (Pdiet*genotype= 0.002). Reduction in MAP to pentolinium was greater in WT mice fed a high fat diet (−27 ± 2 mmHg), compared to WT mice fed a normal fat diet (−16 ± 2 mmHg; Pdiet = 0.004) suggesting that the obesity induced hypertension is due to increased activity of the sympathetic nervous system. In CAT+ mice, MAP responses to pentolinium were similar in mice fed a normal or a high fat diet (Pdiet = 0.7; Pdiet*genotype=0.01). These data suggest that endothelial CAT1 over expression can separate obesity from hypertension by buffering the activity of the sympathetic nervous system.
RyR2/Ca2+ release channel is crucial for pulmonary arterial hypertension
Authors: Zheng Yun‐Min , Wang Yong‐Xiao
Published: June 2021

Pulmonary arterial hypertension (PAH) is a devastating lung disease. However, the underlying molecular mechanisms remain to be fully elucidated. Ryanodine receptor‐2 (RyR2) is important for cardiovascular functions. In this study, thus, we sought to investigate the potentially crucial role of this Ca2+ release channel in PAH. Our investigations demonstrate that RyR2 gene knockout (KO) abolishes the increased right ventricular pressure and weight (i.e., PAH) in mice after chronic hypoxia (hypoxic exposure for 3 weeks). The chronic hypoxic increase in pulmonary arterial constriction and muscle layer are both eliminated in RyR2 KO mice. RyR activity, Ca2+ release and basal [Ca2+]i in pulmonary artery smooth muscle cells (PASMCs) are all augmented in control, but not in RyR2 KO mice. These exciting animal studies may have a general significance in humans, as RyR activity is significantly enhanced in PASMCs of chronic obstructive lung disease (COPD) patients with PAH. Fascinatingly, treatment with tetracaine (a classic RyR antagonist that is clinically used in anesthesia) blocks chronic hypoxia‐induced PAH and cellular responses. In conclusion, our findings for the first time provide breakthrough evidence that chronic hypoxia causes RyR2 activation, Ca2+ release, pulmonary arterial constriction/remodeling, and ultimately PAH; and RyR antagonists may become new and effective drugs in the treatment of PAH.
PVN Gαi2 protein‐gated signal transduction ‐ a renal nerve dependent mechanism required for sodium homeostasis and blood pressure regulation in Sprague‐Dawley rats
Authors: Wainford Richard David , Kuwabara Jill Toshiko
Published: June 2021

Aim

We examined the role of PVN Gαi2 proteins, which are upregulated by high salt intake, in maintaining salt‐resistance.

Methods

Naïve, bilateral renal denervated (RDNX) or sham RDNX Sprague‐Dawley rats (N=6/gp) received a bilateral PVN infusion of a scrambled (S) or Gαi2 oligodeoxynucleotide (ODN‐300ng/side/day) and a 7‐day normal 0.4% (NS) or high 8% NaCl (HS) diet. On day‐7 MAP, 24h Na+ balance, FENa, plasma norepinephrine (NE), plasma renin activity (PRA), urinary angiotensinsogen (UAGT), and natriuresis to i.v. hydrochlorothiazide (HCTZ‐1.5mg/kg) were assessed.

Results

HS‐intake increased FENa, suppressed plasma NE, PRA, UAGT & natriuresis to HCTZ (P<0.05) without altering MAP in PVN S infused rats. HS intake in intact & sham RDNX rats receiving a PVN Gαi2 infusion evoked elevated plasma NE (NE [nmol/L]; S intact HS 43±4 vs Gαi2 intact HS 108±8, P<0.05), increased natriuresis to HCTZ (peak ΔUNaV to HCTZ [μeq/min]; S intact HS 7.4±0.6 vs Gαi2 intact HS 14.7±0.9, P<0.05), hypertension (MAP [mmHg]; S intact HS 128±2 vs Gαi2 intact HS 147±3, P<0.05), reduced FENa (FENa [%]; S intact HS 0.9±0.1 vs Gαi2 intact HS 0.2±0.1, P<0.05) and suppressed PRA and UAGT. RDNX in HS maintained PVN Gαi2 infused rats prevented hypertension (MAP [mmHg]; Sham RDNX HS Gαi2 149±3 vs RDNX HS Gαi2 132±4, P<0.05), elevated plasma NE, sodium retention and enhanced natriuresis to HCTZ (peak ΔUNaV to HCTZ [μeq/min]; Sham RDNX HS Gαi2 14.8±1 vs RDNX HS Gαi2 8.5±0.7, P<0.05).

Conclusion

Renal nerve dependent dysregulation of sympathetically driven, but not RAAS mediated, sodium retaining mechanisms, potentially involving altered NCC regulation, occurred following PVN Gαi2‐protein down‐regulation. Therefore PVN Gαi2 protein‐gated pathways represent a central mechanism required to regulate NE mediated sodium homeostasis and maintain salt‐resistance ‐ HL107330.
Mitochondrial superoxide in pro‐hypertensive T‐cell activation
Authors: Nazarewicz Rafal R , Dikalova Anna E , Bikineyeva Alfiya T , Harrison David G , Dikalov Sergey I
Published: June 2021

Activation of T‐cells and increased superoxide production are crucial in the development of hypertension (J Exp Med. 2007; 204:2449). Interestingly, depletion of mitochondrial superoxide dismutase (SOD2) increases hypertension while SOD2 overexpression blunts hypertension (Circ Res. 2010; 107:106). We hypothesized that mitochondrial superoxide plays an important role in T‐cell activation and pro‐hypertensive immune response. In order to test this hypothesis we investigated the effect of mitochondria targeted SOD2 mimetic mitoTEMPO and SOD2 expression (SOD2+/− or SOD2 overexpression) on T cell activation and pro‐hypertensive immune effect by adaptive transfer of T‐cells in RAG1 KO mice. Preliminary analysis of spleenocytes from hypertensive mice infused with angiotensin II showed decrease in mitochondrial membrane potential, increase in mitochondrial superoxide and cellular ATP that is consistent with attenuated mitochondrial function and activation of redox dependent glycolysis. Indeed, treatment of spleenocytes with mitochondria targeted antioxidant mitoTEMPO significantly blunted these changes. Since T‐cells are essential in pro‐hypertensive immune response, we isolated T‐cell fraction from spleenocytes and studied the role of mitochondrial superoxide in T‐cells proliferation and cytokine production. T‐cells isolated from hypertensive mice showed three fold increased cell proliferation and TNF‐α secretion which was blocked by treatment of T‐cells with mitoTEMPO. Therefore, this work suggets that mitochondrial superoxide is essential in T‐cells metabolic changes, activation and pro‐hypertensive immune response.
Soluble Fms‐like tyrosine kinase‐1 release from human umbilical vascular endothelial cells is decreased by intracellular 5‐aminoimidazole‐4‐carboxamide‐3‐ ribonucleoside accumulation
Authors: Banek Christopher Todd , Gillham Haley , Needham Karen , Gilbert Jeffrey S
Published: June 2021

Recent studies from our laboratory have suggested the potent AMP kinase (AMPK) activator 5‐aminimidazole‐4‐carboxamide‐3‐ ribnucleotide (AICAR) lowers blood pressure and restores angiogenic balance (e.g. increased ratio of VEGF (vascular endothelial growth factor) to sFlt‐1 (soluble VEGF receptor 1) in rats with placental ischemia‐induced hypertension, but the mechanisms remain unknown. The purpose of this study was to model placental ischemia with human placental trophoblast (BeWo) and umbilical vascular endothelial (HUVEC) cells, and determine if AICAR modifies angiogenic balance via adenosine receptor or adenosine transporter mediated mechanisms. Cells were cultured at 37°C for 12 hours in atmospheric (20%), physiologic normoxic (8%) or hypoxic (1.5%) conditions, and treated with AICAR (2mM), purinergic (P1) receptor (8‐(p‐sulfophenyl) theophylline, 8‐SPT) and adenosine transporter (dipyridamole, DPM) blockers (100μM). HUVECs treated with AICAR showed a decreased (P<0.05) secreted sFlt‐1 in all O2 conditions, and DPM blocked this effect and not by 8‐SPT. AMPKα phosphorylation was increased (P<0.05) in hypoxic HUVECs with AICAR, and DPM blocked this effect. BeWos were unaffected by AICAR. Through modeling placental ischemia in vitro, we have demonstrated AICAR decreases sFlt‐1 secretion by HUVECs and this is mediated by increased intracellular AICAR and not via P1 receptor signaling.
Early life stress induces altered expression of epigenetic chromatin modification enzymes in aorta and renal vessels
Authors: Ho Dao H. , Loria Analia S. , Pollock Jennifer S.
Published: June 2021

Early life stress (ELS) is linked to increased risk of adult cardiovascular disease (CVD). Epigenetic pathways are important in cardiovascular maintenance. The current aim is to elucidate whether ELS alters chromatin modification enzyme expression in aorta and renal vessels in adult male Wistar Kyoto rats. Maternal separation (MatSep), a model of ELS, consisted of separating pups from the mother for 3h/day from postnatal day 2 to 14. Unseparated pups served as controls. Gene expression of 84 chromatin remodeling enzymes was assessed in thoracic aorta and isolated renal vessels of 16 week old rats by large‐scale qRT‐PCR array. mRNA expression of histone deacetylase 2 (HDAC2) and protein arginine N‐methyltransferase 2 were significantly decreased in aortas of MatSep rats (0.78±0.05 and 0.76±0.11 fold change relative to control rats (RFC), respectively; p<0.02; n=3/group). MatSep increased renal vessel mRNA expression of genes involved in DNA methylation (DNA methyltransferase 1; RFC=1.2±0.001), and histone phosphorylation (cyclin‐dependent kinase 2; RFC=1.35±0.09), methylation (histone‐lysine‐N‐methyltransferase SUV39H1; RFC=2.49±0.82), and acetylation (HDAC3 and HDAC10; RFC=1.29±0.11 and 1.41±0.06, respectively; p<0.05; n=3/group). Future studies will test whether ELS‐mediated CVD in adulthood is due to derangement of epigenetic pathways in vascular tissue.

NIH F32 HL116145; NIH P01 HL69999