bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel findings in membrane depolarization‐induced force development in vascular smooth muscle (865.11)
Authors: Ets Hillevi , Moreland Robert
Published: June 2021

A unique phenomenon of vascular smooth muscle (VSM) is its ability to maintain force for a prolonged period of time. The exact mechanism responsible is not known but studies suggest a critical role for L‐type Ca2+ channels (LTCC). The purpose of this study was to investigate the consequences of prolonged LTCC opening with an agonist, Bay K8644, to elucidate the intracellular mechanisms responsible for maintaining VSM tone. Smooth muscle tissue isolated from swine carotid arteries was mounted for isometric force recording. Tissues were stimulated with Bay K8644 alone or following pretreatment with one of the following inhibitors: nifedipine (LTCC), ML‐7 (MLC kinase), bisindolylmaleimide (PKC), U0126 (MAP kinase), or H‐1152 (Rho kinase). The following results were obtained: • Bay K8644 increased MLC phosphorylation, PKC activity, MAP kinase activity, and force development but partially inhibited ROCK activity; CPI‐17 phosphorylation was not changed.

• Bay K8644‐dependent activation of PKC inhibited MAP kinase activity; the Bay K8644 partial inhibition of ROCK activity was reversed by inhibition of LTCC with nifedipine .

• Inhibition of MAP kinase activity significantly increased MLC kinase activity and inhibition of MLC kinase activity greatly increased MAP kinase activity.

These results indicate that the maintenance of force in VSM involves an intricate signaling network with multiple modulatory steps.
The large conductance Ca2+‐activated K+ channel: a key intermediate in cholinergic regulation of human urinary bladder smooth muscle excitability (865.5)
Authors: Parajuli Shankar , Hristov Kiril , Cheng Qiuping , Malysz John , Rovner Eric , Petkov Georgi
Published: June 2021

We investigated whether muscarinic receptor (MR) activation increases human urinary bladder smooth muscle (UBSM) excitability by directly or indirectly inhibiting the large conductance Ca2+‐activated K+ (BK) channels. We used the MR agonist, carbachol, to determine the changes in BK channel activity in freshly‐isolated human UBSM cells using the perforated whole cell and single BK channel patch‐clamp recordings. Carbachol inhibited the amplitude and frequency of BK channel‐mediated spontaneous transient outward currents and spontaneous transient hyperpolarizations, both triggered by ryanodine receptor‐dependent Ca2+ release. Carbachol also caused UBSM cell depolarization, which was not observed in the presence of iberiotoxin, a BK channel inhibitor. The potential direct effects of carbachol on BK channels were examined under conditions of removing all major cellular Ca2+ sources for BK channel activation with pharmacological inhibitors (thapsigargin, ryanodine, and nifedipine). In the presence of these inhibitors, carbachol did not affect the single BK channel open probability and mean BK channel conductance (cell‐attached configuration) or the whole cell BK currents. The data support the concept that MR activation triggers indirect BK channel inhibition mediated by intracellular Ca2+, thus increasing human UBSM cell excitability.

Grant Funding Source: Supported by NIH R01 DK084284 to Georgi V. Petkov.
Novel insight into human urinary bladder function: the role of transient receptor potential melastatin 4 channels (865.6)
Authors: Hristov Kiril , Parajuli Shankar , Smith Amy , Malysz John , Malysz John , Petkov Georgi
Published: June 2021

Transient receptor potential melastatin 4 (TRPM4) channels have been recently identified in urinary bladder smooth muscle (UBSM) in rodents. Here, we applied a combined experimental approach to study the role of TRPM4 channels in human UBSM function. We used RT‐PCR, Western blot, immunohistochemistry, immunocytochemistry, patch‐clamp electrophysiology, and isometric tension recordings of human UBSM isolated strips from donor patients. mRNA message for TRPM4 channels in whole tissue and single UBSM cells was detected. Western blot and immunohistochemistry revealed TRPM4 protein expression in whole UBSM tissue. Immunocytochemistry further confirmed TRPM4 protein expression in DSM single cells. Voltage‐clamp experiments showed that the TRPM4 channel inhibitor 9‐phenanthrol decreased the transient inward cation currents (TICCs) recorded at ‐70 mV. Inhibition of IP3‐receptors with xestospongin‐C also abolished the TICCs activity in human UBSM. 9‐phenanthrol hyperpolarized the UBSM membrane potential and attenuated the spontaneous phasic, carbachol‐induced and nerve‐evoked contractions in UBSM isolated strips. The results reveal that TRPM4 channels are present in human UBSM and are critical regulators of human UBSM excitability and contractility. UBSM TRPM4 channels should be considered further as new targets overactive bladder treatment.

Grant Funding Source: Supported by R01 DK084284 to G. V. Petkov
Angiotensin II activates the proteasome and stimulates vascular smooth muscle cell proliferation (866.10)
Authors: Martin Doug , Wang Xuejun , Li Yifan , Ford Hannah , Kjellsen Barton , Li Shuai
Published: June 2021

Growing evidence implicates an important role of the proteasome in cardiovascular disease. However, the role of the proteasome in hypertension is understudied. We showed previously that proteasome inhibition attenuated angiotensin hypertension‐induced aortic hypertrophy in vivo. This study tested the hypothesis that proteasome inhibition attenuates angiotensin‐induced vascular smooth muscle proliferation.

Experiments were performed on a rat embryonic aortic cell line, A7r5 (ATCC) and human umbilical artery vascular smooth muscle primary cells (Cell Applications) (HUAVSM). The cells were treated for 48 hours with vehicle (0.01% DMSO), angiotensin II (AngII) (100 nM), a proteasome inhibitor, bortezomib (Bort) (5 nM) or both (AngII+Bort). Cell number and viability were assessed by flow cytometry. Cells were also stained for Ki67, a marker of proliferating cells, and DAPI to stain DNA. Adenoviral infection was used to co‐express a green fluorescent protein fused with a ubiquitination signal sequence (GFPu) and a red fluorescent protein (RFP) in the cell. The GFPu to RFP ratio was taken as an inverse index of ubiquitin‐proteasome system (UPS) activity.

Compared to vehicle, Ang II treatment increased UPS activity. Cell number was increased by AngII in both A7r5 and HUAVSM cells. AngII treatment increased Ki67 staining compared to vehicle in both A7r5 and HUAVSM cells. Treatment with Bort alone did not affect cell viability or cell number in either A7r5 or HUAVSM cells. On the other hand, Bort treatment abolished the AngII‐induced increase in UPS activity, cell number and Ki67 staining.

Collectively, these data suggest that AngII‐induced vascular smooth muscle cell proliferation is mediated, at least in part, via activation of the UPS. This effect may contribute to AngII hypertension induced vascular remodeling.

Grant Funding Source: Supported by HL072166, HL085629 (X.J.W.), HL109964 (Y.L.), NIGMS: P20GM103443 SD BRIN (D.M., H.F.)
RNA‐Seq for comprehensive analysis of differences in gene programs between conduit and resistance arteries (866.5)
Authors: Dippold Rachael , Fisher Steven
Published: June 2021

Smooth muscle can be phenotypically categorized as tonic (eg. conduit arteries) versus phasic, or mixed properties (eg. microcirculatory arteries). Characterization of the gene expression programs that underlie the phenotypic differences between conduit and microcirculatory arteries is important to understanding the differing pathologic responses of these vessels. We performed RNA‐Seq on rat aortas vs mesenteric arteries (MAs) to identify differences in their gene programs >900 gene transcripts are differentially expressed between the two groups. Gene ontology analysis reveals an enrichment of genes involved in processes that are indicative of the oxidative “slow” phenotype in the aorta and of the “fast” phenotype in the MAs. The expression of PP1 regulatory subunits fit these phenotypes: PP1 glycogen‐targeting subunit (GL) is more highly expressed in the AO (FC=7.6), while the MP/PP1 myosin‐targeting subunit (Mypt1) is more highly expressed in the MAs (FC=3.4). We have also investigated the splicing programs of the two vessel types, with a focus on genes involved with contractility as well as factors that regulate transcription and splicing. Together our results suggest broad differences in the gene programming of conduit and resistance arteries supportive of the tonic and phasic, or mixed, smooth muscle phenotypes.
Vascular oxidative stress promotes aortic stiffening (867.2)
Authors: Wu Jing , Itani Hanaa , Chen Wei , Roberts L. Jackson , Harrison David
Published: June 2021

Oxidative stress correlates with arterial stiffness in clinical studies. Reactive oxygen species (ROS) contribute to blood pressure elevation, vascular smooth muscle hypertrophy and vascular dysfunction in hypertension. We hypothesized that vascular ROS induce aortic stiffening, which in turn promotes systolic hypertension with aging. We created mice overexpressing p22phox in smooth muscle (tgsm/p22phox mice), which have a constitutive increase in vascular ROS production. Six month old tgsm/p22phox mice were normotensive at baseline (118±6 systolic, mmHg), but had increased aortic stiffness as indicated by the leftward shift on stress‐strain curves. Aortic collagen level, as detected by hydroxyproline content, was increased in tgsm/p22phox mice compared to age‐matched C57Bl/6 mice (76.3±11.9 vs. 43.2±7.2 ng/μg total protein). At 9 months of age, aortic stiffening did not further increase but these mice developed mild hypertension (137±11 systolic), demonstrating that excessive ROS first promotes aortic stiffening and subsequently hypertension. Tempol, a superoxide dismutase mimetic, normalized blood pressure (117±10 systolic), reduced vascular collagen deposition (37.5±9.5 ng/μg) and prevented aortic stiffening in these mice, suggesting a role of vascular ROS in aging‐induced aortic stiffening. ROS oxidize arachidonic acid to produce isoketals, known to cross‐link lysines and promote protein cross‐linking, which could lead to vascular stiffening. Immunohistochemistry indicated a marked increase of isoketals in the aorta and peri‐aortic fat of tgsm/p22phox mice, which was prevented by 2‐hydroxybenzylamine (2‐HOBA), a scavenger of isoketals. 2‐HOBA also reduced blood pressure, prevented collagen deposition and aortic stiffening in tgsm/p22phox mice treated from 3 months to 9 months of age. We conclude that ROS promote aortic stiffening and that this might in part be due to isoketal formation and protein crosslinking.

Grant Funding Source: Supported by NIH R01 grant HL105294
Altered wall mechanics of conduit arteries of obese Zucker rats with progression of metabolic syndrome (867.8)
Authors: Chantler Paul , Fournier Sara , DeVallance Evan , Butcher Joshua , Frisbee Jefferson
Published: June 2021

The metabolic syndrome (MetSyn) in obese Zucker rats (OZR) comes with a wide array of distinct vasculopathies including impaired endothelium‐dependent dilation and more modest alterations to VSM‐dependent responses (constrictor or dilator). While OZR do not develop atherosclerotic plaques, the presence of these alterations to wall mechanics in conduit arteries would have significant implications for pulse wave propagation, thrombo‐embolytic events, and responses to cardiovascular events such as stroke. To address this, we studied the mechanics of ex vivo carotid arteries (CA) from 17 week old OZR, where vascular tone was abolished. OZR exhibited obesity, severe insulin resistance, moderate hypertension and dyslipidemia. In response to elevated intraluminal pressure, CA from OZR exhibited a smaller expansion than CA from LZR. However, wall thickness and cross‐sectional area was reduced in CA from OZR vs. LZR. At low intraluminal pressures, incremental distensibility of CA from OZR was reduced vs. LZR. The circumferential stress vs. strain relation for CA from OZR was left‐shifted vs. LZR, with an increased β (slope) coefficient (p<0.01). These results suggest that CA stiffness in OZR is increased over that in LZR and that this loss of arterial distensibility could contribute to poor cardiovascular outcomes in MetSyn.

Grant Funding Source: Supported by National Institutes of Health and American Heart Association
Aliskiren associate with L‐arginine improves aorta vascular reactivity of renovascular hypertension rats (867.9)
Authors: Santuzzi Cintia Helena , Tiradentes Renata , Mengal Vinicius , Gouvea Sonia , Abreu Glaucia
Published: June 2021

We evaluate the effects of aliskiren + L‐arginine on the vascular reactivity and systolic blood pressure (SBP) in aortic rings in renovascular hypertensive rats. Animals were randomly assigned to one of five groups: sham, 2 kidney one clip hypertension (2K1C), 2K1C treated with aliskiren (ALSK), 2K1C treated with L‐arginine (L‐arg), and 2K1C treated with aliskiren plus L‐arginine (ALSK+ L‐arg). All Treatments were started 7 days after 2K1C hypertension and the treatments were conducted for 21 days: 2K1C had the following effects: increase in SBP; increased of contractile response to phenylephrine (1 nM‐100 mM) of aortic rings; impairment of the relaxation induced by acetylcholine (0.1 nM‐300 mM) and unaffected relaxation induced by sodium nitroprusside (0.01 nM‐0.3 mM). The treatment with ALSK+L‐arg was able to decreased the contractile response to phenylephrine and improve the acetylcholine relaxation. Endothelium removal and NG‐nitro‐L‐arginine methyl ester (100 mM) increased the response to phenylephrine in 2K1C rats more than in ALSK+L‐arg treated group rats. Losartan (10 mM) reduced the response to phenylephrine in all groups and enalapril (10 mM) reduced the response only in 2K1C and ALSK groups. The endothelial nitric oxide synthase expression increased in 2K1C and L‐arg group, inducible nitric oxide synthase increased only in 2K1C group, neuronal nitric oxide synthase increased in L‐arg and ALSK+L‐arg group and AT1 expression increased in 2K1C and L‐arg group and AT2 expression increased only in ALSK+L‐arg, but no difference found in ACE expression. Results suggest that ALSK+L‐arg treatment promoted the preservation of aortic endothelial function and this can be one of the factors that contributed to the normalization of SBP in this group, furthermore, these data suggest an important modulation of the renin‐angiotensin system associated with direct effects of L‐arginine to enhance vasodilation and modulate the protein expression of nitric oxide synthase in aorta of 2K1C rats.
The role of miR‐301a and the influence of extracellular environment surface tensions on the dichotomous phenotype shown in primary human bone marrow‐derived mesenchymal stem cells (868.4)
Authors: Müller Alison , Li Yun , Klassen Jessica , Freed Darren
Published: June 2021

It has been established that the microenvironment of mesenchymal stem cells (MSCs) is influential in determining differentiation. Expression of non‐coding RNAs, such as microRNAs, has been found to be altered in response to various stimuli and may thus contribute to stem cell differentiation. Previous research in our lab has shown that miR‐301a mediates a dichotomous phenotype in primary human MSCs. As MSCs have been found to directly contribute to cardiac remodeling post‐MI, we sought to understand how migration from bone marrow to cardiac tissue could influence MSC differentiation. The objective of this study is to analyze how varying matrix stiffness would influence miR‐301a expression and MSC differentiation. We analyzed mRNA levels of c‐kit, Dicer, MYH9 and 10 and found that these have increased expression on fibronectin‐coated 2kPa plates simulating native bone marrow stiffness, however this is blunted on 15kPa plates, simulating left ventricle myocardium stiffness. When over‐expressing miR‐301a, we found that there is a significant decrease in c‐kit, Dicer, MYH9 and 10. Interestingly, after quantifying miR‐301a expression on these coated plates we found that there is a substantial increase on 15kPa fibronectin‐coated plates. These observations indicate that matrix stiffness may be influencing MSC differentiation via miR‐301a.
Respiratory virus co‐infection enhances Pseudomonas aeruginosa biofilm growth on airway epithelial cells (869.1)
Authors: Bomberger Jennifer , Lashua Lauren , Fischer Douglas , Hendricks Matthew
Published: June 2021

Clinical observations link respiratory virus infection and Pseudomonas aeruginosa colonization in chronic lung disease, including cystic fibrosis and chronic obstructive pulmonary disease. The development of P. aeruginosa into highly antibiotic resistant biofilm communities promotes airway colonization and accounts for disease progression in chronic lung disease patients. We hypothesized that respiratory viral co‐infection promotes P. aeruginosa biofilm growth and colonization. In the presence of respiratory syncytial virus (RSV) co‐infection, we observed a significant increase in P. aeruginosa biofilm formation on airway epithelial cells (AECs). RSV infection induces a potent antiviral innate immune response in AECs through interferon (IFN)‐λ signaling. IFN‐λ signaling is capable of recapitulating the RSV‐induced bacterial biofilm formation. We have isolated the biofilm stimulatory activity to purified exosomes in conditioned media obtained from AECs infected with RSV. Iron is essential to P. aeruginosa growth and biofilm maturation, and chelation of iron inhibits biofilm growth on human AECs. We observed increased levels of iron in conditioned media from RSV‐infected AECs and chelation of iron in the conditioned media blocked biofilm stimulation. RSV infection did not alter iron transporter abundance in the plasma membrane, but dramatically enhanced the epithelial transcytosis and apical secretion of exosome‐associated, iron‐bound transferrin. This represents the first study that links viral co‐infection, and the resulting iron transport defect, to the development of biofilms and chronic bacterial infections.

Grant Funding Source: Supported by R00HL098342 and P30DK072506 (JMB)
Azithromycin induces sestrin2 expression through Nrf2 signaling pathway in lung epithelial cells stimulated with cigarette smoke extract (869.11)
Authors: Yang Yu , Cuevas Santiago , Armando Ines , Jose Pedro
Published: June 2021

Azithromycin has been reported to reduce exacerbation of chronic obstructive pulmonary disease (COPD) based on its antioxidant and anti‐inflammatory effects. The aim of this study was to identify the underlying molecular mechanisms. We found that decreased intracellular Nrf2 protein level (‐55±6%) and downstream target NQO1 (‐45±4%), increased reactive oxygen species (ROS) production (1.6‐fold) and interleukin 8 (IL‐8) secretion (2.1‐fold) in A549 cells exposed to cigarette smoke extract (CSE) (200ug/ml, 24h). Pretreatment with azithromycin (5µg/ml) suppressed ROS production (‐29±4%) and IL‐8 (‐45±6%) induced by CSE. Furthermore, azithromycin promoted Nrf2 nuclear translocation. Sestrin2, an antioxidant enzyme, was increased after azithromycin treatment in a time and concentration dependent manner. Additionally, azithromycin reduced hyper‐oxidized peroxiredoxins, the downstream target of sestrin2. Knockdown of Nrf2 resulted in suppression of azithromycin‐induced sestrin2 expression (‐35±3%). Silencing sestrin2 abolished azithromycin‐induced decrease in peroxiredoxins hyper‐oxidation and partially attenuated the inhibitory effect of azithromycin on ROS production and IL‐8 expression (by 26% and 41%, respectively). Our results suggest that one of the antioxidant and anti‐inflammatory effects of azithromycin in COPD may be, in part, mediated by induction of sestrin2 via Nrf2.

Grant Funding Source: HL068686
Pseudomonas aeruginosa quorum‐sensing homoserine lactone inhibits O2 consumption and stimulates H+ secretion by airway epithelia (869.2)
Authors: Machen Terry , Schwarzer Christian , Fischer Horst
Published: June 2021

P. aeruginosa biofilms accumulate in the airways of cystic fibrosis patients and produce the quorum‐sensing molecule N‐(3‐oxododecanoyl)‐homoserine lactone (C12) that depolarizes mitochondria. We tested whether C12 and P. aeruginosa biofilms change O2 consumption and H+ secretion into the extracellular fluid of JME (CF nasal epithelia, ΔF508CFTR) and CFTR‐corrected JME cells studied in Seahorse X24‐well dishes. C12 caused dose‐dependent (min = 1 µM, max = 50 µM) inhibition of O2 consumption (to 0.6x control) and increases in H+ secretion (to 1.3x control), similar to effects of mitochondrial poisons antimycin + rotenone. As measured by pH‐stat, C12 increased H+ secretion into the apical solution of human tracheal epithelial (HTE) cells in primary culture by 0.3 µeq.cm‐2.hr‐1, similar to rates measured using Seahorse. H+ secretion by HTE cells was blocked by Zn2+. PAO1wt biofilms inhibited O2 consumption and increased H+ secretion by JME cells, while PAO1lasI biofilm and planktonic PAO1wt did not. JME and CFTR‐corrected JME cells behaved similarly during all treatments. These data indicated: (i) P. aeruginosa biofilms, most likely operating through C12, inhibit O2 consumption and stimulate H+ secretion by airway epithelia. (ii) H+ secretion occurs through a Zn2+‐sensitive H+ channel in the apical membrane. (iii) Although there is no direct role for CFTR in these processes, a downward spiral in CF is predicted: increased acidity in CF ASL (no HCO3 secretion) → reduced antimicrobial factors → P. aeruginosa accumulation in biofilms → inhibit mitochondrial metabolism and increase anaerobic glycolysis in epithelia → increased epithelial secretion of H+ → increased acidity in ASL → reduced antimicrobial factors → increased P. aeruginosa accumulation.

Grant Funding Source: NIH, CFRI
Respiratory syncytial virus‐induced chronic airway inflammation and AHR (CAI‐CAHR) is associated with upregulation of Th2‐like cytokines via TLRs‐TRIF but not TSLP signaling pathway (870.2)
Authors: Zang Na , Zhou Na , Deng Yu , Xu Fadi , Liu Enmei
Published: June 2021

We have shown that TLRs‐TRIF pathway is responsible for the RSV infection‐induced CAI‐CAHR. This pathway is reportedly involved in Th2 responses that are predominate in the patients with severe RSV infection. Thus, we hypothesized that the RSV‐induced CAI‐CAHR was associated with upregulation of Th2‐like cytokines dependent on TLRs‐TRIF pathway. Mice at day 7, 14, 21, 30 and 60 postinfection were placed in a whole body plethysmograph to define AHR or euthanized to determine Th2‐like cytokines (in BALF) and airway inflammation (inflammatory cells in BALF and histopathology). RSV infection induced CAI‐CAHR concomitant with significant (All P < 0.05 or 0.01) elevation of IL‐4, IL‐5, IL‐10, IL‐13, TNF‐α and TSLP from day 14 to 60 postinfection with the peak at day 21. The same protocols were performed in other mice after giving resveratrol (inhibitor of TRIF, ip) daily from day 0 to day 21 postinfection. Resveratrol treatment strikingly reduced the RSV infection‐induced Th2‐like cytokines and CAI‐CAHR. As Th2‐like cytokines are also controlled by TSLP in asthmatic patients and animals, anti‐TSLP neutralized antibody (ip, on day 14 after postinfection) was utilized in the subsequent study. This treatment failed to alter the peak Th2‐like cytokines and CAI‐CAHR. Our results suggest that RSV infection‐induced CAI‐CAHR is associated with upregulation of Th2‐like cytokines via TLRs‐TRIF but not TSLP signaling pathway.
Beta adrenergic pathway independent relaxation of human airway smooth muscle (870.9)
Authors: Komalavilas Padmini , Brophy Colleen , Cheung‐Flynn Joyce
Published: June 2021

Severe acute asthma, defined as asthma that is refractory to current anti‐inflammatory agents and bronchodilators, accounts for an estimated two‐million emergency department visits annually. β2‐adrenergic receptor (β2AR) agonists (β‐agonists) are widely used for the treatment of bronchospasm associated with acute asthma attacks, however, some patients develop refractory responses to long term usage of β –agonists. It also causes adverse effects in patients with a genetic polymorphism (Arg/Arg genotype, about a sixth of the US population) in the adrenergic receptor creating a need for additional therapeutics for managing asthma. We have shown earlier that β‐agonist induced airway smooth muscle (ASM) relaxation is associated with increases in the phosphorylation of HSP20. In this study we evaluate the effect of a transducible phosphopeptide mimetic of HSP20 protein (P20 peptide) on the relaxation of human ASM. Freshly isolated human ASM was pretreated with either a β‐agonist, isoproterenol (ISO), or the P‐20 peptide followed by a bronchoconstrictor, carbachol and force was measured in a tissue bath system. ISO blocked carbachol induced contraction in human ASM by activating the β2AR pathway. P‐20 peptide also blocked carbachol induced contraction of ASM, but in a β2AR independent manner. P20 peptide also relaxed human ASM precontracted with carbachol. These results suggest that the P20 peptide targets molecular events downstream of the β‐agonists‐cAMP pathway and that P20 peptide may be developed as a therapeutic to alleviate bronchospasm for patients who are refractory to β‐agonist, thus reducing the morbidity, mortality, and costs associated with managing asthma.

Grant Funding Source: Supported by NIH RO1 70715
The orexin receptor 1 in the locus coeruleus modulates the hypercapnic ventilatory response in unanesthetized rats (872.12)
Authors: Vicente Mariane , Dias Mirela , Bicego Kenia , Gargaglioni Luciane
Published: June 2021

Orexin modulates CO2‐drive to breath and there is an extensive population of orexin receptor 1 (OX1R) in the Locus coeruleus (LC). We tested the hypothesis that OX1R located in the LC is involved in chemoreception. To this end, we injected SB‐334867 (OX1R antagonist, 5 mM) into the LC of male Wistar rats. Pulmonary ventilation (VE), body temperature (Tb, dataloggers), mean arterial pressure (MAP) and heart rate (HR) were measured in air followed by hypercapnia (7% CO2) in unanesthetized rats. Hypercapnia caused an increase in VE, which resulted from increases in respiratory frequency (fR) and tidal volume (VT), but did not affect MAP, fH or Tb in all groups. SB‐334867 caused a significant attenuation (70%), of the hyperpnea induced by hypercapnia (n=9) compared with its vehicle (n=6; P< 0.05). This effect was due to both decreased VT and f. Basal ventilation, MAP, HR or Tb, were not affected by OX1R antagonism. These results suggest that projections of orexin‐containing neurons to the LC contribute via orexin R1 to the hypercapnic chemoreflex control in unanesthetized rats.

Grant Funding Source: FAPESP and CNPq
Conditional knockdown of Kir4.1 in astrocytes blunts the hypercapnic respiratory response in awake mice (872.7)
Authors: Hawkins Virginia , Kuo Fushan , Bellemare Laura , Perez Diana , Dubreuil Todd , Mulkey Daniel
Published: June 2021

Central chemoreception is the mechanisms by which the brain regulates breathing in response to changes in tissue CO2/H+. The retrotrapezoid nucleus (RTN) has been identified as a key locus for central respiratory control. Neurons in this region sense CO2/H+ in part by inhibition of TASK‐2 channels. RTN astrocytes are also chemosensitive and CO2‐evoked release of the gliotransmitter ATP most likely occurs via hemichannels. However, RTN astrocytes also respond to bath acidification with ~8 mV depolarization, which is mediated by inhibition of a Kir4.1‐like conductance. The contribution of this pH sensitive current in RTN astrocytes to central respiratory drive is yet to be determined. Here we generate a tamoxifen inducible, astrocyte specific Kir4.1 knockout (ciKir4.1KO) by crossing GFAP‐CreERT2 and a Kir4.1 floxed mouse lines. Whole‐body plethysmography experiments performed on 6 week old mice show that ciKir4.1KO animals breathe normally control conditions but exhibit a reduced tidal volume response to CO2 compared to tamoxifen treated GFAP‐CreERT2 Kir4.1 wild type (i.e., control) mice. These results suggest that Kir4.1 channels in astrocytes contribute to the CO2 ventilatory response in awake mice.

Grant Funding Source: NIH HL104101 (DKM)
GAL‐160 is a potent orally bioavailable ventilatory stimulant in rats (873.3)
Authors: Hewitt Matthew , Gruber Ryan , Ideo Courtney , Puskovic Veljko , Mardirosian Saro , Peng Sean , Golder Francis , MacIntyre D
Published: June 2021

GAL‐160 is a novel chemoreceptor modulator entering early clinical development as a therapeutic for sleep‐disordered breathing. Here, we describe GAL‐160’s properties as a ventilatory stimulant in rats. The effects of bolus GAL‐160 (0.01 – 10 mg/kg IV) on breathing, mean arterial pressure (MAP) and heart rate (HR, as inferred from pulse rate) were measured using whole‐body plethysmography and blood pressure telemetry in conscious rats. GAL‐160 dose‐dependently increased minute volume (Emax ~400% above baseline; ED50 0.05 mg/kg) by increasing tidal volume and respiratory rate. At the highest dose tested, and in comparison to vehicle‐treated controls GAL‐160 produced no significant changes in mean arterial MAP (7.1 ± 2.3%) or HR (‐8.3 ± 5.7%). GAL‐160 (20 mg/kg PO) increased minute volume (Emax ~ 100% above vehicle) which remained elevated for up to 75 minutes post‐gavage with no effect on MAP (0.1 ± 0.5%) or HR (7.6 ± 0.7%) compared to vehicle. The pharmacokinetic‐pharmacodynamic relationship for GAL‐160 was evaluated following IV infusion and using changes in PaCO2 as a marker for ventilatory stimulation. The GAL‐160 plasma concentration associated with a statistically significant reduction in PaCO2 (10% decrease relative to baseline) was 380 ng/ml. Collectively, these data demonstrate that GAL‐160 is a potent, orally bioavailable ventilatory stimulant with negligible effects on cardiovascular function.
Acute leptin evokes physiological responses in rat carotid body type I cells (873.4)
Authors: Pye Richard , Ricker Ellen , Jurcsisn Jennifer , Barr Barbara , Wyatt Christopher
Published: June 2021

Leptin is a central stimulant of respiration and also contributes to obesity related sympathoactivation. Recently leptin receptors have been identified on type I cells from the carotid body. Our most recent data indicate that activation of these receptors with leptin initiates calcium signaling events and increases outward macroscopic currents in isolated type I cells.

The acute (10 min) application of leptin (200 ng.ml‐1) evoked calcium signaling responses in isolated type I cells. Cells significantly increased their Fura 2 F340/F380 ratio by 0.09 ± 0.015 ratio units (n =24, P < 0.001). Leptin also increased the peak membrane currents in a subset (65%) of type I cells. At +10mV leptin (200ng.ml‐1) increased peak macroscopic currents from 123 ± 19 pA.pF‐1 to 198 ± 28 pA.pF‐1 (n = 6, P < 0.02). The remaining 35% of type I cells were unresponsive to this concentration of leptin. Interestingly, leptin does not alter the rate of oxidative phosphorylation in isolated type I cells as measured by reduction of C12‐resazurin to C12‐resorufin.

These data suggest that changes in leptin signaling may potentially influence carotid body function at the level of the type I cell. Given the involvement of the carotid bodies in the control of breathing and sympathetic tone, this finding may be especially pertinent to obesity related pathologies such as obesity hypoventilation syndrome and essential hypertension.

Grant Funding Source: Supported by NIH‐1RO1HL091836 & NIH‐ 1S10RR025075‐01
Sepsis progression and multiple organ dysfunction in rats: carotid body involvement (873.5)
Authors: Fernández Ricardo , Cortes Paula , Nardocci Gino , Navarro Cristina , Rodriguez Jorge , Reyes Edison
Published: June 2021

Sepsis syndromes involve pathological processes often leading to multiple organ dysfunction (MOD) and death. Sepsis progresses to MOD due uncontrolled release of inflammatory mediators. Lately, increasing evidence supports the idea that the carotid body both regulates the immune status and plays a protective role during sepsis syndromes. In anaesthetized (pentobarbitone, 60 mg/kg IP) male Sprague‐Dawley rats (BW 100±20 g) we measured plasma levels of cytokines, chemokines and MOD‐markers, 90‐min after IP administration of either saline or 15 mg/kg LPS in control (SHAM surgery; intact carotid/sinus nerves) and in bilateral carotid chemo/baro‐denervated rats (BCN). Respiratory frequency (fR) and heart rate (fH) were augmented in SHAM‐LPS group, but systolic blood pressure (PS) dropped. BCN decreases the survival time of rats and blunts the fR response to LPS but enhancing both, the fH increase and the PS fall. SHAM‐LPS and BCN‐LPS rats shown increased plasma levels of IL‐1β, IL‐10, and TNF‐α; however, the increase in both ILs was lower in BCN‐LPS, which also had augmented IL‐2 levels. Unchanged cortisol plus increased levels of epinephrine can be related to increased plasma levels of TNF‐α. Finally, BCN‐LPS induced an early onset of MOD identified by increased plasma levels of creatine kinase, amylase, creatinine, neutrophil gelatinase‐associated lipocaline, total bilirrubin and alanine aminotransferase. We propose that carotid body would modify the inflammatory response during sepsis syndromes through a communication network involving neural, humoral, and cytokine components, and also would play a protective role in the sepsis progression to MOD.

Grant Funding Source: Supported by: FONDECYT 1120976 and UNAB DI‐354‐13/R (to RF) and UDD CI 23400098 (to PR)
Role of the carotid chemoreceptors in insulin‐mediated sympathoexcitation in humans (873.7)
Authors: Limberg Jacqueline , Mozer Michael , Joyner Michael , Curry Timothy
Published: June 2021

Objective: Given recent evidence from rodents supporting a novel role for the carotid bodies in insulin‐mediated sympathoexcitation, we sought to determine whether carotid chemoreceptor desensitization would reduce muscle sympathetic nerve activity (MSNA) during hyperinsulinemia in humans. Methods: Young, healthy subjects (n=4, 30±2 yrs) breathed hyperoxic gas (End‐tidal O2 = 100%, PaO2 505±13 mmHg) for 15 min at baseline and during steady‐state hyperinsulinemia (5±1 vs 53±1 µU/mL). Hyperinsulinemia was achieved using a 90‐min hyperinsulinemic (1 mU/kg FFM/min), euglycemic (108±4 mg/dL) clamp. CO2 was maintained at resting levels (PaCO2 41±1 mmHg). Changes in heart rate, blood pressure, minute ventilation, and MSNA (microneurography of the peroneal nerve) were assessed. Results: Hyperinsulinemia increased MSNA [burst frequency (p<0.01), burst incidence (p=0.02), total activity (p=0.02)]. Hyperoxia decreased heart rate (p=0.01) and minute ventilation (p=0.11), and increased mean blood pressure (p=0.04). Consistent with our hypothesis, the effect of hyperoxia on MSNA (Δ) was different between baseline and hyperinsulinemia (burst frequency, p=0.03; burst incidence, p=0.10). Conclusion: These data are the first to suggest the carotid chemoreceptors may contribute to insulin‐mediated sympathoexcitation in humans.

Grant Funding Source: Supported by: NIH R01 DK090541 (MJJ), NIH T32 DK07352 (JKL)
Overexpression of GRK5 prevents angiotensin II blockade of peak potassium currents and normalizes Kv4.3 expression in neurons (874.12)
Authors: Becker Bryan , Haack Karla , Zucker Irving
Published: June 2021

The Angiotensin II (Ang II)‐ Ang II type 1 Receptor (AT1R) signaling axis has been shown to decrease peak potassium (K+) currents (Ito) and Kv4.3 (a voltage gated K+ channel subunit) expression. G protein coupled receptor kinase 5 (GRK5) phosphorylates AT1R and desensitizes it. We hypothesized that GRK5 overexpression can blunt the Ang II‐mediated decrease in Ito and normalize Kv4.3 expression in CATH.a neurons. Neurons were transfected with either mock or pcDNA3‐GRK5 plasmid and stimulated with either vehicle or 100 nM Ang II for 4 hours. Neurons treated with Ang II exhibited decreased Ito relative to control (95.8 ± 16.6 vs 149.4 ± 18.9 pA/pF; p<0.05). This effect was attenuated by GRK5 overexpression (179.0 ± 34.5 pA/pF). Time to peak current was significantly increased in AngII neurons relative to control (45.5 ± 14.5 vs 21.3 ± 5.1 ms; p<0.05). GRK5 overexpression normalized this response (21.4 ± 5.6 ms). Western blot analyses indicated that GRK5 overexpression significantly increased basal Kv4.3 levels compared to sham (1.5 ± 0.3 vs 1.0 ± 0.1) but did not prevent the Ang II‐mediated decrease in Kv4.3 (0.5 ± 0.2 vs 0.7 ± 0.2). siRNA knockdown of GRK5 decreased basal Kv4.3 levels compared to sham (0.3 ± 0.1 vs 1.0 ± 0.1) and Ang II did not decrease Kv4.3 expression further. These data suggest that GRK5 overexpression blunts the Ang II‐mediated decrease in Ito. GRK5 may contribute to the regulation of Kv4.3.

Grant Funding Source: Supported by NIH grant P01‐HL62222
Early surge in oxidative stress in the brain subfornical organ during angiotensin‐II‐induced hypertension (874.7)
Authors: Dong Frederick , Young Colin , Davisson Robin
Published: June 2021

Angiotensin‐II (Ang‐II)‐dependent hypertension is characterized by oxidative stress, endoplasmic reticulum (ER) stress, and transcription factor activation in the subfornical organ (SFO). However, the relative temporal pattern of these responses remains unclear. As a first step, we tested the hypothesis that reactive oxygen species (ROS) levels increase early in the SFO during Ang‐II‐induced hypertension. Male C57B1/6 mice were implanted with osmotic minipumps for low dose infusion of Ang‐II (600 ng/kg/min) and were sacrificed at day 0, 3, 5, 7, and 14 (n=3‐4/timepoint). Brains were collected, and SFO cryosections were obtained and stained with dihydroethidium to quantify ROS production. No changes in SFO ROS levels were found at day 3 of Ang‐II, relative to day 0 (0.7±0.1 fold day 0, p>0.05). However, a surge in ROS production within the SFO occurred at day 5 (1.7±0.2 fold day 0, p<0.05), prior to an increase in arterial pressure, and this response was maintained for up to 14 days (1.5±0.1 fold day 0, p<0.05). Interestingly, we have also found a surge in nuclear factor‐ĸ‐B activation at day 5 (3.7±1.4 fold day 0; p<0.05), as well as upregulation of ER stress biomarkers at day 7. These findings demonstrate for the first time an early pre‐hypertensive increase in oxidative stress within the SFO that coincides with transcriptional activation and the occurrence of ER stress.

Grant Funding Source: Supported by HL63887, HL96571, HL84207, AHA13POST14410020, K99HL166776
Silencing of Nox2 and Nox4 in the subfornical organ prevents renal but not aortic T cell infiltration during angiotensin II‐induced hypertension (874.9)
Authors: Lob Heinrich , Song Jiunn , Butler Scott , Peterson Jeffrey , Mark Allyn , Davisson Robin
Published: June 2021

Reactive oxygen species (ROS) in the SFO are crucial for Ang II HTN and have been linked to aortic T cell activation. Signals from the SFO regulate renal sympathetic nerve activity (RSNA). Increased RSNA contributes to renal T cell activation. Here we compared the effects of silencing the two NADPH oxidase (Nox) isoforms (Nox2, Nox4) found in the SFO on aortic and renal T cell activation during Ang II HTN. We targeted the SFO of C57Bl/6 mice with adenoviruses encoding siRNAs directed selectively against Nox2 and Nox4 (AdsiNox2 + AdsiNox4). One week later osmotic minipumps were implanted to infuse Ang II (600 ng/kg/min) for 14 days. Silencing of both Nox2 and Nox4 significantly reduced SFO ROS and blunted Ang II‐HTN. Flow Cytometry revealed that despite normal arterial pressure, silencing of Nox2 and Nox4 in the SFO did not prevent aortic T cell infiltration in response to Ang II (AdLacZ Veh: 0.2 ± 0.06 x 104, Ang II: 5.6 ± 2.0 x 104; AdsiNox2+AdsiNox4 Veh: 0.09 ± 0.02 x 104, Ang II: 9.1 ± 3.8 x 104 cells/aorta; n=5‐8, p<0.05 vs AdLacZ). In contrast, renal T cell infiltration was blunted in these animals (AdLacZ Veh: 0.2 ± 0.05 x 104, Ang II: 2.1 ± 0.4 x 104; AdsiNox2+AdsiNox4 Veh: 0.4 ± 0.1 x 104, Ang II: 0.4 ± 0.9 x 104; n=5, p<0.05). These findings demonstrate that Nox‐derived ROS in the SFO are important for renal T cell infiltration and kidney inflammation, and suggest that this may be important for the development of Ang II HTN.

Grant Funding Source: 12SDG9160019, HL63887, HL96571, HL84207
Neural reflex control of immunity: the splanchnic anti‐inflammatory pathway (875.1)
Authors: Martelli Davide , Yao Song , McKinley Michael , McAllen Robin
Published: June 2021

Exaggerated or insufficient inflammatory responses can both be life threatening, and the nervous system helps maintain inflammation within appropriate limits. It is believed that the parasympathetic vagus nerve acts via the splenic sympathetic nerve to down‐regulate the cytokine response to immune challenge. Here we report that the systemic inflammatory response in rats, measured by plasma levels of tumour necrosis factor‐α 90 minutes after intravenous lipopolysaccharide (LPS, 60μg/kg), was unaffected by vagotomy but was enhanced five‐fold if the splanchnic sympathetic nerves were cut. Plasma corticosterone levels were unaffected, so did not contribute. Electrophysiological recordings demonstrated that efferent neural activity in the splanchnic nerve and its splenic branch was increased at least four‐fold after LPS. Furthermore, we show that, under these conditions, the increase in the efferent neural activity in the splenic sympathetic nerves is entirely driven by the splanchnic nerves, not the vagi. Thus, immune challenge triggers a powerful inhibitory reflex that suppresses the inflammatory response. Its efferent pathway is in the splanchnic sympathetic nerves, not the vagi as previously thought.

Grant Funding Source: Supported by NHMRC project grant 1051102
Decreased T‐regulatory cells are associated with blood pressure elevation and sympathoexcitaton in hypertensive rats (875.10)
Authors: Katsuki Masato , Hirooka Yoshitaka , Kishi Takuya , Sunagawa Kenji
Published: June 2021

Background: Previous reports have suggested that T‐regulatory cells (Tregs) prevent angiotensin II (AngII)‐induced hypertension and organ damage. Abnormal sympathoexcitation is a major factor in the pathogenesis of hypertension. The aim of the present study was to determine whether Tregs are involved in the development of hypertension, and whether blockade of brain AngII type 1 receptors (AT1R)‐induced sympathoinhibition affects Tregs or not.

Methods and Results: We examined Tregs in stroke‐prone spontaneously hypertensive rats (SHRSP) at 3 week (early pre‐hypertensive stage), 5 week (late pre‐hypertensive stage), or 15 week (established hypertensive stage) old, and in age‐matched normotensive Wistar‐Kyoto rats (WKY). CD4+CD25+Foxp3+ cells in the spleen and blood did not differ between WKY and early pre‐hypertensive SHRSP, whereas significantly decreased both in late pre‐hypertensive and established hypertensive SHRSP (p<0.05). In established hypertensive SHRSP, intracerebroventricular infusion of losartan (AT1R blocker) for 2 weeks was done to cause depressor response with central sympathoinhibition, and Foxp3 mRNA expression in the spleen was significantly higher than in vehicle‐treated SHRSP.

Conclusion: Decreases in T‐Regulatory cells precede blood pressure elevation in SHRSP, thereby Tregs play a key role in the development of hypertension associated with sympathoexcitation.
Brain ACE2 overexpression reduces DOCA‐salt hypertension independently of endoplasmic reticulum stress (875.3)
Authors: Xia Huijing , Queiroz Thyago , Lazartigues Eric
Published: June 2021

Endoplasmic reticulum (ER) stress was recently reported to contribute to hypertension. We previously observed that ACE2 in the paraventricular nucleus (PVN) is pivotal for blunting DOCA‐salt hypertension. To assess the contribution of ER stress, the PVN was collected from non‐transgenic (NT, n=4) and transgenic mice overexpressing brain ACE2 (SA, n=4), submitted to sham or 3 wk DOCA‐salt. ER stress (Bip, ATF4) and autophagy (LC3, LAMP2) markers were measured by Western blot. Baseline expression of Bip (1±0.07 vs. 0.96±0.11), ATF4 (1±0.06 vs. 0.99±0.02), LC3 II/I (1±0.02 vs. 1.16±0.11) and LAMP2 (1±0.08 vs. 0.97±0.05) were similar in NT and SA mice. Surprisingly, DOCA‐salt treatment did not alter these markers in NT (1.13±0.13; 1.14±0.05; 1.14±0.09; 1.13±0.09, respectively) or SA (1.04±0.04; 1.19±0.09; 1.1±0.07; 1.18±0.06, respectively) mice, despite a significant increase in blood pressure (BP, telemetry) in NT and a smaller rise in SA mice. To further assess the role of ER stress in DOCA‐salt hypertension, NT mice (n=8) were infused icv with TUDCA (5 µg/day, 21 days), an ER stress inhibitor, during DOCA‐salt treatment. DOCA‐salt alone significantly increased BP (127±5 vs. 101±2 mmHg, P<0.05), however, this increase was not affected by TUDCA (123±4 mmHg). Our data suggest that ER stress does not contribute to DOCA‐salt hypertension and that ACE2 blunts this neurogenic hypertension independently of ER stress.

Grant Funding Source: Supported by AHA (12EIA830004)
Angiotensin II contributes to sympathetically‐mediated hypertension in autonomic failure (875.4)
Authors: Arnold Amy , Okamoto Luis , Gamboa Alfredo , Shibao Cyndya , Raj Satish , Robertson David , Biaggioni Italo
Published: June 2021

Angiotensin (Ang) II contributes to hypertension by direct vasoconstriction, and indirectly by promoting sympathetic activation. The importance of the latter to blood pressure regulation remains controversial, particularly in clinical populations. As a novel approach to test the hypothesis that Ang II promotes sympathetically‐mediated hypertension, we compared depressor responses to the Ang II AT1 receptor antagonist losartan in central autonomic failure (AF) patients in whom supine hypertension is driven by residual sympathetic tone to peripheral AF patients with noradrenergic denervation and sympathetic‐independent hypertension. Single dose losartan (50 mg, PO) was administered at bedtime to 10 peripheral and 7 central AF patients, with supine blood pressure measured q2 hours for 12 hours in a randomized, double‐blind, placebo‐controlled study. There were no differences in plasma Ang II levels between groups (42±3 central vs. 43±5 pg/ml peripheral AF; p=0.734); however, peak depressor responses to losartan were significantly greater in central (‐43±10 mmHg) compared to peripheral AF patients (‐19±8 mmHg; p<0.05). These findings suggest that Ang II contributes to residual sympathetic tone in central autonomic failure, and through this mechanism to hypertension. Overall, these patients provide a unique model to assess the importance of Ang II‐sympathetic interactions in human hypertension.

Grant Funding Source: Supported by AHA grant 11POST7330010 and NIH grants HL056693, 5U54NS065736 and UL1TR000445.
Astrocyte‐specific angiotensin II type 1 receptor knockout by using cre‐loxp system attenuates left ventricular remodeling after myocardial infarction (875.9)
Authors: Isegawa Kengo , Hirooka Yoshitaka , Kishi Takuya , Sunagawa Kenji
Published: June 2021

Background: Brain angiotensin II type 1 receptor (AT1R)‐induced pathways (i.e., apoptosis, oxidative stress, and sympathoexcitation) worsen left ventricular (LV) remodeling after myocardial infarction (MI). Although astrocytes were recently suggested to have multiple and important functions in neurovascular unit, normal astrocytes have little AT1R. We hypothesized that MI‐induced abnormal expression of astrocyte AT1R, not neuron, would be a primary cause of worsening LV remodeling.

Methods and Results: We generated astrocyte‐specific AT1R knockout mice (astrocyte/AT1R (‐/‐)). In astrocyte/AT1R (+/+) mice at 1 month after MI, we examined brainstem tissues by double immunohistostaining for glial fibrillary acidic protein (GFAP) and AT1R, and determined GFAP positive cells had morphologic changes with increased AT1R expression compared to sham mice without MI. In astrocyte/AT1R (‐/‐) at 1 month after MI, GFAP positive cells with AT1R expression were not observed. LV size at 1 month after MI was smaller in astrocyte/AT1R (‐/‐) than in astrocyte/AT1R (+/+) mice. Heart weight of astrocyte/AT1R (‐/‐) at 1 month after MI was lighter than that of astrocyte/AT1R (+/+) mice.

Conclusion: Targeted deletion of AT1R in astrocytes attenuated LV dilatation after MI. These findings suggest that MI‐induced abnormal expression of AT1R in astrocytes is potential primary cause of worsening LV remodeling after MI.