bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Novel Inhibitors of P‐glycoprotein Mediated Multidrug Resistance
Authors: Follit Courtney A. , McCormick James W. , Nanayakkara Amila K. , Wise John G. , Vogel Pia D.
Published: November 2023

Multidrug resistance (MDR) in cancers due to the overexpression of multidrug transporter proteins such as P‐glycoprotein (P‐gp) is a leading cause of chemotherapy failure. P‐gp uses ATP binding and hydrolysis to export a broad range of structurally dissimilar compounds, including many of the most commonly used chemotherapeutics. While this mechanism helps to protect tissues from toxins, the overexpression of P‐gp presents a major obstacle to cancer pharmacotherapy by preventing chemotherapeutics from reaching effective concentrations in the cancer cells. Reversing MDR by inhibiting P‐gp mediated efflux has been attempted for many years. The search for MDR reversing agents has resulted in a number of hit and lead compounds, many of which competitively inhibited chemotherapeutic transport by P‐gp, but to date none have succeeded in becoming FDA‐approved drugs. By searching for compounds that interact preferentially with the ATP binding domains of P‐gp, instead of the drug binding domain, we aim to identify inhibitors of P‐gp with novel mechanisms of action. We predict that these properties will be more suitable for future development of co‐therapeutics to reverse MDR.

In in silico studies using targeted molecular dynamics and massively parallel drug docking of millions of drug‐like molecules, we have identified hundreds of potential inhibitors predicted to have high affinity for the ATP binding domain of P‐gp but low affinity for its drug binding domain. We previously reported that four of these compounds inhibited P‐gp catalyzed ATP hydrolysis (Brewer FK, Follit CA, Vogel PD, Wise JG (2014) Molecular Pharmacology, 86(6), 716–726) and three of those reversed the P‐gp mediated MDR phenotype of prostate cancer cells in culture (Follit CA, Brewer FK, Wise JG, Vogel PD (2015) Pharmacology Research & Perspectives 3(5): e00170). Here, we describe our continued search for potent P‐gp inhibitors using a cell‐based assay for initial screening of predicted P‐gp inhibitors that reports on the metabolic activity of the cells. “Hits” that were found to reverse MDR in a prostate cancer cell line after assaying ~100 computationally identified compounds, were further evaluated by live cell imaging. The majority of inhibitors identified in the metabolic activity assays also demonstrated inhibition of P‐gp‐catalyzed efflux of P‐gp substrates in the cell imaging experiments, supporting the usefulness of the metabolic activity assay as a medium‐throughput primary screen.

Support or Funding Information

This work is supported by NIH NIGMS [R15GM09477102] to JGW/PDV, SMU Dissertation Fellowship, SMU University Research Council, SMU Center for Drug Discovery, Design and Delivery, The Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.
Cyclic AMP response element binding (CREB) protein modulates glycolytic genes and maintains endothelial monolayer integrity.
Authors: Sheikh Rayees Rafiq , Rajput Charu Rafiq , Mehta Dolly Rafiq
Published: November 2023

The vascular endothelium lining the intima of blood vessels plays a pivotal role in regulating vascular smooth muscle tone, host defense and tissue fluid hemostasis. Cyclic AMP response element binding (CREB) protein, transcribes genes which regulate inflammation, cell proliferation, survival and carbohydrate metabolism. Herein, using endothelial‐cell specific CREB null mice, we tested the hypothesis that CREB reprograms metabolism in endothelial cells (ECs) and thereby maintains endothelial monolayer integrity. We observed that EC‐CREB deletion increased vascular leak under basal conditions and augmented it further after endotoxin challenge, a well‐known inducer of vascular injury. Interestingly, impairment of CREB expression in lungs and pulmonary ECs increased the expression of hexokinase‐II (HK2), a key inducer of glycolysis. In contrast, the expression of mitochondrial uncoupling protein 2 (UCP2), which inhibits oxidative cycle, was decreased. Endotoxin further augmented the alteration in HK2 and UCP2 in EC‐CREB null lungs and CREB depleted ECs. These data demonstrate a novel role of CREB in preventing glycolytic pathway entry into oxidative cycle in ECs and in maintaining vascular homeostasis.
SENP1 mediated deSUMOylation of KLF15 in Human Pulmonary Microvascular Endothelial Cells: A critical pathway for the development of hypoxic pulmonary hypertension
Authors: Pandey Deepesh , Nomura Yohei , Hori Daijiro , Bhatta Anil , Leucker Thorsten , Shimoda Larissa Akimi , Berkowitz Dan , Romer Lew
Published: November 2023

Rationale

Apart from hypoxia inducible factor (HIF), there are very limited studies describing master transcriptional regulators of critical genes important in endothelial dysfunction during hypoxia.

Objective

Kruppel Like Factor 15 has recently been shown to be critical for activation of proinflammatory processes in vascular smooth muscle contributing to atherogenesis. Although KLF15 is abundantly expressed in vascular endothelium the role of KLF15 in the regulation of vascular endothelial function remains poorly understood. Here we tested the hypothesis that KLF15 is a critical regulator of Arg2 transcription in hypoxia exposed human pulmonary microvascular endothelial cells.

Approach and Results

Adenovirally mediated overexpression of KLF15 in human pulmonary microvascular endothelial cells (HPMEC) and in rat pulmonary arteries decreased Arg2 mRNA, protein abundance, and activity. HPMECs exposed to 48 hours of hypoxia exhibited a robust decrease in KLF15 protein levels (that was sustained after 24 hours of reoxygenation) and a reciprocal increase in Arg2 protein and mRNA expression. Chromatin immunoprecipitation (CHIP) indicated direct binding of KLF15 to the Arg2 promoter, which was relieved when HPMEC were exposed to hypoxia, Further, overexpression of KLF15 reversed hypoxia‐induced augmentation of arginase activity and decrements in Nitric Oxide production in HPMEC, and also reversed hypoxia‐induced endothelial dysfunction in isolated rat pulmonary artery rings. Finally, we have elucidated the mechanism by which hypoxia regulates KLF15 abundance/stability and altered cytoplasmic vs. nuclear compartmentalization in HPMEC, a process that involves de‐Sumoylation of KLF15 at lysine 116 residue by SENP1.

Conclusions

KLF15 is a critical regulator and repressor of endothelial Arg2 expression, and thereby, NO and pulmonary endothelial function/dysfunction. Overexpression or activation of KLF15 may represent novel therapeutic strategies for pulmonary hypertension

Support or Funding Information

This work was supported by American Heart Association Scientist Development Grant to Deepesh Pandey (16SDG27340010), 2017 StAAR award from Department of Anesthesiology and Critical Care Medicine, Johns Hopkins School of Medicine. NIH‐R01HL124213 to Dan Berkowitz.
Endothelial HIF‐1α Is Required for Endothelial Regeneration and Vascular Repair Following Sepsis Challenge
Authors: Huang Xiaojia , Zhang Xianmin , Zhao Youyang
Published: November 2023

Background

Endothelial repair is critical for the maintenance of vascular homeostasis after inflammatory injury. However, little is known about the intrinsic pathways that trigger endothelial regeneration and inflammation resolution.

Methods and Results

Employing the mice with Tie2Cre‐mediated disruption of HIF‐1α (encoding hypoxia inducible factor 1, HIF‐1α) (Hif1αTie2) in endothelial cells, we demonstrated that endothelial regeneration requires activation of HIF‐1α signaling in endothelial cells, which subsequently regulates the expression of the reparative transcription factor Forkhead box M1 (FoxM1). Hif1αTie2 mice exhibited impaired vascular repair, persistent lung inflammation, and increased mortality rate after sepsis challenge. Selective restoration of either HIF‐1α or FoxM1 in pulmonary vascular endothelial cells in HIF1αTie2 mice restored endothelial proliferation and reversed the defective vascular repair. Activation of HIF‐1α via a pharmacological approach potentiated endothelial regeneration and vascular repair.

Conclusions

These studies defined a critical role of endothelial HIF‐1α in activating the intrinsic FoxM1‐dependent endothelial regeneration program following inflammatory vascular injury. Thus, activation of HIF‐1α‐mediated endothelial regeneration is a promising strategy for treatment of inflammatory vascular diseases such as acute respiratory distress syndrome.

Support or Funding Information

This work was supported by NIH grants R01HL123957, R01HL125350.
Intestinal epithelial barrier function is disrupted by acrolein, an environmental/dietary pollutant and lipid metabolite
Authors: Chen Wei‐Yang , Zhang Jingwen , Barve Shirish , McClain Craig , Joshi‐Barve Swati
Published: November 2023

The gastrointestinal epithelial barrier function is critical for maintaining selective gut permeability that limits the entry of bacteria and pathological bacterial components (e.g., endotoxin, lipopolysaccharide‐LPS) into systemic circulation, which can result in inflammation and injury. Clinical and experimental studies suggest that intestinal barrier dysfunction (“leaky gut”) can predispose to or enhance chronic inflammatory intestinal and non‐intestinal disorders, such as diabetes, IBD, alcoholic liver disease and autoimmune diseases, etc. Many environmental and dietary factors are thought to alter intestinal epithelium leading to gut permeability and bacterial translocation. Acrolein is an environmental and dietary pollutant, and a lipid‐derived metabolite. This study evaluates the effects of acrolein on intestinal epithelial barrier function and permeability, both in vitro and in vivo. Our data demonstrate that oral acrolein exposure in mice caused intestinal barrier permeability leading to increased translocation of bacterial endotoxin/lipopolysaccharide into the blood. Also, acrolein adversely affected the barrier integrity of a polarized Caco‐2 monolayer, as demonstrated by a decrease in transepithelial electrical resistance (TEER), with a corresponding increase in barrier permeability to a fluorescent dye (FD‐4). Acrolein exposure resulted in the downregulation and delocalization/redistribution of key tight junction proteins (TJP, e.g., zonula occludens‐1, claudin‐1 and occludin) that critically regulate epithelial paracellular permeability. In addition to TJP alterations, acrolein also caused epithelial cell death by apoptosis, which is known to be a significant mechanism that contributes to intestinal barrier dysfunction and permeability in inflammatory bowel diseases. Overall, we demonstrate that exposure to acrolein has a major impact on the intestinal epithelium that involves (i) decrease/redistribution and discontinuous localization of TJPs, and (ii) epithelial cell apoptosis, thereby resulting in loss of barrier integrity/function and permeability. Our findings highlight the adverse consequences of environmental and dietary pollutants/factors on intestinal barrier dysfunction that may contribute to gut permeability and bacterial translocation.

Support or Funding Information

Supported by NIH and Veterans Administration.
Importance of Axial Length in the Detection of Carotid Artery Stiffness Induced by a High Fat Diet
Authors: Abshire Caleb Michael , Reverte‐Ribo Virginia , Rosales‐Martinez Carla , Zimmerman Margaret A , Miller Kristen S , Prieto Minolfa , Lindsey Sarah H
Published: November 2023

Cardiovascular disease (CVD) is the leading cause of death worldwide and in the United States. Arterial stiffness, characterized by reduced compliance in large conducting arteries, is a major risk factor for CVD. Individuals with cardiometabolic risk factors such as obesity and insulin resistance have stiffer arteries and an increased risk of developing CVD. Recent evidence suggests that ex vivo arterial stiffening can be more fully characterized by performing pressure myography at multiple fixed lengths. Therefore, the aims of this study were to characterize the effect of a high fat diet on murine carotid stiffness using biaxial mechanical phenotyping.

At 4 weeks of age, some C57BL/6 male mice were placed the DIO Rodent Purified Diet with 45% Energy from fat (HFD, TestDiet 58V8) while control animals remained on a normal diet. At 39 weeks of age, common carotid arteries were harvested and cannulated for assessment of biaxial mechanical properties using pressure myography. After equilibration and pre‐conditioning, in vivo carotid artery length was estimated by finding the length at which the artery caused minimal change to longitudinal force when subjected to pressures ranging from 70–150 mmHg. Pressure‐diameter and force‐length curves were obtained at the estimated in vivo length as well as 5% above and below the estimated in vivo length. Statistical analysis was performed using 2‐way ANOVA with Graphpad Prism.

Baseline diameter of carotid arteries pressurized at 10 mmHg tended to be larger in HFD (388 ± 7 μm, n=5) versus control arteries (360 ± 13 μm, n=4, P=0.08). Pressure‐diameter data obtained at the estimated in vivo length showed a significant interaction between diet and pressure (P<0.001) with the largest differences evident at pressures at and above 70 mmHg (P<0.05). HFD significantly decreased arterial compliance, a measure of the change in diameter at each 10 mmHg pressure step (P<0.05), especially at lower pressures. Finally, arterial distensibility was calculated as the percent change in outer diameter from the original diameter obtained at 10 mmHg. HFD significantly decreased distensibility (P<0.05), with the greatest differences found at pressures above 40 mmHg (P<0.05). Interestingly, the impact of diet on arterial compliance was significantly attenuated when experiments were performed at an axial stretch above or below the estimated in vivo length.

These data show a significant impact of HFD on carotid stiffness in male C57BL/6 mice. Of note, this difference was only detected when experimental conditions closely approximated the in vivo state. Additional analysis of this data will determine the impact of HFD and axial length on the force‐length and stress‐strain relationships. In conclusion, our study shows the importance of axial length in the analysis of diet‐induced carotid artery stiffness.
Antihyperuricemic and xanthine oxidase inhibitory activities of the ethanolic extract of Tribulus arabicus and its fractions
Authors: Abu‐Gharbieh Eman , Shehab Naglaa G.
Published: November 2023

Published data concerning the composition and bioactivity of Tribulus arabicus are very limited. This study aimed to investigate the bioactive contents of the Tribulus arabicus grown in the United Arab Emirates, assess xanthine oxidase inhibitory activities of its ethanolic extract, and fractions; hexane, chloroform and n‐butanol and to evaluate their antihyperuricemic in vivo. LD50 of the ethanolic extract confirmed its safety up to 5g/kg. The ethanolic extract and its fractions were evaluated in vitro for their XO inhibitory activity and the in vivo for anti‐hyperuricemic activity using potassium oxonate‐induced hyperuricemia model in mice. Two different doses (250 and 500 mg/kg) of the ethanolic extract were used and one dose (100 mg/kg) was used for each fraction. Allopurinol was used as a positive control.

Tribulus arabicus ethanolic extract together with its hexane and n‐butanol fractions exhibited a significant inhibitory effect on xanthine oxidase activity (IC50 20.4, 12.6 and 15.3 μg/mL respectively), while only a modest reduction in enzymatic activity was noticed with chloroform fraction with IC50 45.8 μg/mL. Furthermore, oral administration of the ethanolic extract at doses of 250 and 500 mg/kg reduced significantly the serum urate levels in oxonate‐induced hyperuricemic mice by 30.1 and 64.6% respectively. Hexane and n‐butanol fractions showed a significant reduction in uric acid levels by 51.7 and 54.1% respectively.

Results suggest that the ethanolic extract of Tribulus arabicus and its hexane and n‐butanol fractions might have great potential as a hypouricemic agent. This activity is attributed to the high content of phenolic and triterpenoidal compounds.
Anti‐inflammatory effects of Tibouchina granulosa in experimental model of endometriosis
Authors: Guilhon Carolina Carvalho , Minho Alan , Mattos Romulo , Nasciutti Luiz Eurico , Fernandes Patricia Dias
Published: November 2023

Tibouchina granulosa is an ornamental tree commonly found in Brazil and some species of the genus are used in popular medicine. Researches from our group have shown that the tea from the leaves present a significant wound healing effect and acute anti‐inflammatory activity. Endometriosis is a debilitating gynecologic disease characterized by the implantation of endometrial tissue in ectopic locations. So far there is no treatment to the pathology itself, most of the women have to go through a hormone therapy or surgery. The aim of this study was to analyze if the ethanol extract (EE) from the leaves of T. granulosa, and ethyl acetate fraction (EAF) could be used as a therapy to treat endometriosis.

After implantation and establishment of autologous endometrium onto the peritoneum abdominal wall of Swiss webster female mice (25–35g, n = 8–30), the animals were divided in the following groups: vehicle group that received water (p.o.), EE (30 or 100 mg/kg), EAF (10, 30 or 100 mg/kg) for 15 consecutive days. At last day mice were euthanized, the size of the lesion and the quantification of cytokines in serum and in the tissue were performed and compared among the groups. Statistic was done by ANOVA followed by post‐test of Newman‐Keuls's. Protocols for animal use received the number #ICBDFBC‐015 (COBEA/UFRJ/Brazil).

Vehicle‐treated group doubled the ectopic lesion size and the explants from the animals treated with all doses of the EE and EAF have maintained its size (1.75 ± 0.49 to vehicle‐treated group versus 1.00 ± 0.39 and 1.21 ± 0.37 to 30 or 100 mg/kg‐EE treated mice and 1.08 ± 0.33, 0.90 ± 0.35, 1.07 ± 0.41 to 10, 30 or 100 mg/kg‐EAF treated groups, respectively). Levels of tumor necrosis factor‐α (TNF‐α) in serum and in ectopic focus were significantly reduced in the EAF‐treated group (100 mg/kg dose) (229.98 ± 61.5 pg/ml in vehicle‐treated group versus 151.8 ± 54.2 pg/ml in EAF‐treated group and 6,686.9 ± 1,061.3 pg/ml protein in vehicle‐treated group versus 2,469.7 ± 2,223.1 pg/ml protein in EAF‐treated group, respectively). The chemokine monocyte chemoattractant protein‐1 (MCP‐1) levels in explant was significantly inhibited with 30 and 100 mg/kg EAF doses (2,505.6 ± 829.6 pg/ml protein in control group versus 1,294.3 ± 312.7 pg/ml of protein and 593.4 ± 141.9 pg/ml of protein to 30 and 100 mg/kg, respectively).

Taken together our data suggest that Tibouchina granulosa extract from leaves and it fraction in ethyl acetate have anti‐inflammatory effects in this experimental model of endometriosis.

Support or Funding Information

Acknowledgements: Instituto Vital Brazil (donation of animals), CAPES, CNPq and FAPERJ (financial support).
Phospholamban is degraded through ubiquitin‐proteasome pathway by pVHL in failing hearts
Authors: ASAHI MICHIO , Yokoe Shunichi
Published: November 2023

The ubiquitin‐proteasome system (UPS) plays an essential and pivotal role for intracellular protein degradation and protein homeostasis. The von Hippel‐Lindau (VHL) is a tumor suppressor that forms E3 ubiquitin ligase complex, which negatively regulates the hypoxia‐inducible factor‐1a (HIF‐1a) by promoting its polyubiquitination. Although it is reported that protein VHL (pVHL) is involved in the stabilization of heart function via HIF1‐a regulation, the role of pVHL in UPS‐mediated protein degradation in the heart is not fully understood. Here, we show that phospholamban (PLN), which acts as a negative inhibitor of sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA2a), was co‐immunoprecipitated with pVHL in both PLN‐overexpressing HEK293 cells and mouse hearts with dilated cardiomyopathy (transgenic mice expressing an Arg9 to Cys point mutation in PLN, TgPLNR9C mice). Furthermore, the overexpression of VHL into HEK293 cells decreased PLN expression levels under the H2O2 stress condition, whereas knockdown of VHL by transient transfection with VHL‐siRNA increased PLN expression level both under the normal and the H2O2 stress conditions. Taken together with our previous study in which we showed that PLN is ubiquitinated at Lys3 residue, and polyubiquitination of PLN was significantly increased with its lower expression in TgPLNR9C mice compared to control, we propose that increased oxidative stress may increase the binding of pVHL with PLN to induce its degradation in failing hearts.
Poloxamer 188 Decreases Hypoxia/Reoxygenation‐Induced LDH Release from Isolated Cardiomyocytes
Authors: Salzman Michele M. , Hackel Benjamin J. , Bates Frank S. , Bartos Jason A. , Yannopoulos Demetris , Riess Matthias L.
Published: November 2023

Introduction

Even when the best cardiopulmonary resuscitation (CPR) is delivered, many patients who experience a cardiac arrest die or suffer severe cardiac damage. Reintroduction of blood flow at the start of CPR after systemic ischemia may cause additional injury to the heart beyond that caused by the ischemia itself. Determining the mechanism of action of a compound administered during CPR on the cellular (e.g., cardiomyocyte) dysfunction caused by ischemia/reperfusion (I/R) could improve CPR practices in the future.

Hypothesis

Poloxamer 188 (P188), with its unique hydrophilic/lipophilic chemical properties, protects isolated cardiomyocytes against hypoxia/reoxygenation (H/R; simulated I/R) injury.

Methods

Isolated cardiomyocytes (human, HCMs) underwent 3 hours hypoxia (1% O2; serum‐& glucose‐free medium; 37°C) then 2 hours reoxygenation (21% O2; complete medium; 37°C) ± P188 [0.1μM–2mM]. Endpoints were markers of cell proliferation, cell viability (mitochondrial dehydrogenase activity), and cellular injury (lactate dehydrogenase [LDH] release). Statistics: ANOVA and SNK post hoc comparisons, p<0.05 *vs normoxia, † vs vehicle.

Results

There was significantly decreased cell proliferation (15,300±488 vs *13,396±102 cells/well, n = 6) and viability (0.570±0.039 vs *0.482±0.034 abs/well, n = 6), as well as significantly increased LDH release (0.337±0.054 vs *0.975±0.242 abs/well, n = 6), following H/R compared to cells in control/normoxic conditions. P188 did not attenuate the decreased cell proliferation nor viability caused by H/R, but did significantly decrease LDH release in the H/R conditions (†0.520±0.101 vs 0.975±0.242 abs/well, n = 6), albeit not to control/normoxic levels, at a concentration of 1mM. The other P188 concentrations used (0.1μM, 1μM, and 2mM) did not have a significant effect.

Conclusions

P188 (1mM) significantly decreased H/R‐induced LDH release from isolated HCMs, but did not attenuate the decreased cell proliferation and viability caused by H/R. P188 protection of cardiomyocytes may require additional components, such as the preservation of nitric oxide release from P188‐protected cardiac endothelial cells.

Support or Funding Information

This work was supported by NIH grant (RO1 HL095122) and institutional funds.
Cardiac Na+/Ca2+ exchanger (NCX1) inhibitors and enhancers in the heart
Authors: Watanabe Yasuhide , Kimura Junko
Published: November 2023

We have been examined acute effects of several agents on cardiac Na+/Ca2+ exchange current (INCX) in guinea pig cardiac ventricular cells by using the whole‐cell voltage clamp method. So far we found agents which inhibit or stimulate the function of cardiac Na+/Ca2+ exchanger (NCX1). Among antiarrhythmic drugs, amiodarone, bepridil, dronedarone, cibenzoline, azimilide, carvedilol and aprindine inhibited INCX in a concentration‐dependent manner. 2,3‐buanedione monoxim (BDM), KB‐R7943, SEA0400 and SN‐6 also inhibited INCX in a concentration‐dependent manner. The properties of the NCX1 blockers are as follows; In the presence of trypsin in the pipette solution, the inhibitory effects of amiodarone, bepridil and BDM on INCX were attenuated, suggesting that these drugs inhibit NCX1 from the intracellular side. In contrast, aprindine, azimilide, dronedarone, cibenzoline, carvedilol, KB‐R7943, SEA0400, SN‐6 and YM‐244769 were trypsin‐insensitive NCX1 inhibitors. It is reported that the binding sites of KB‐R7943, SEA0400 SN‐6 and YM‐244769 (four benzyloxyphenyl derivatives) are intracellular XIP (exchanger inhibitory peptide) region. KB‐R7943, SEA0400 SN‐6 and YM‐244769 suppressed the uni‐directional outward INCX more potently than uni‐directional inward INCX. The mechanism of this mode‐dependency is unknown but may be related to intracellular Na+ concentration. On the other hand nicorandil and flecainide stimulated INCX in a concentration‐dependent manner. The enhancing effect of NCX1 by nicorandil may be contributed to bind intracellular cGMP increasing to regulatory sites of intracellular large loop between 5th and 6th transmembrane segments of NCX1. However, it is unknown about the enhancing mechanisms of dofetilide and flecainide on NCX1 function.
2‐Methoxyestradiol protect against cardiac hypertrophy induced by abdominal aortic constriction
Authors: Almaayah Zaid H. , El‐Kadi Ayman O.S.
Published: November 2023

Numerous experimental studies have supported the evidence that 2‐methoxyestradiol (2ME) is a biologically active metabolite and mediates multiple effects on the cardiovascular systems that are largely independent of estrogen receptor. 2ME is a major cytochrome P450 1B1 (CYP1B1) metabolite and has been reported to have a vasoprotective and anti‐inflammatory actions. However, whether 2ME would prevent cardiac hypertrophy induced by abdominal aortic constriction (AAC) has not been investigated yet. Therefore, the overall objectives of the present study are to elucidate the potential anti‐hypertrophic effect of 2ME and to explore the mechanism(s) involved. For this purpose, Sprague–Dawley rats were subjected to either sham or abdominal aortic constriction surgery to induce cardiac hypertrophy and treated with 2ME or vehicle. Thereafter, cardiac hypertrophy parameters were determined using echocardiography whereas, protein expression level and the formation of mid‐chain hydroxyeicosatetraenoic acid (mid‐chain HETEs) were measured using western blot analysis and liquid chromatography‐electron spray ionization‐mass spectrometry, respectively. Our results showed that 2ME significantly inhibited cardiac hypertrophy induced by AAC as evidenced by a decrease in the heart weight to tibial length ratio and left ventricular morphology such as intraventricular septum, left ventricular internal diameter and left ventricular posterior wall. Mechanistically, the protective effect of 2ME against cardiac hypertrophy induced by AAC was mediated through a significant inhibition of CYP1B1 and its associated cardiotoxic metabolites mid‐chain HETEs. Our study provides the first evidence that 2ME prevents cardiac hypertrophy induced by AAC through CYP1B1 and mid‐chain HETEs‐dependent mechanisms.

Support or Funding Information

This work was supported by a grant from the CIHR to A.O.S.E. Z.H.M. is the recipient Izaak Walton Killam and Alberta Innovates‐Health solution Scholarships.
Impact of Bradykinin B1 Receptor Blockade on Angiotensin II‐Induced Cardiac and Aortic Remodeling
Authors: McCormick Brittney , Huot‐Marchand Julie‐Emilie , deBlois Denis , Hale Taben
Published: November 2023

Angiotensin II (AngII) is a growth factor known to induce vascular and cardiac hypertrophy and fibrosis. AngII has been shown to regulate bradykinin B1 receptor (B1R) expression in the heart and aorta and we have recently shown that concomitant B1R antagonism could prevent AngII‐induced cardiac fibrosis. The present study investigated the impact of AngII and bradykinin B1R antagonism on blood pressure and left ventricular (LV) and aortic hypertrophy. Male Sprague‐Dawley rats were treated for 4 weeks with vehicle or AngII (200 ng/kg per min) in the presence or absence of the B1R antagonist R‐954 (400 μg/kg per day). In addition, bromodeoxyuridine (BrdU) was infused to assess cellular proliferation. At sacrifice, mean arterial pressure (MAP) was assessed via carotid artery cannulation in anesthetized rats. The LV and aorta were weighed, and the aortic and cardiomyocyte cross sectional area (CSA) were measured. Additionally, LV DNA content was determined and aortic cellular proliferation was assessed via immunohistochemistry to measure BrdU incorporation. Relative to vehicle, AngII increased MAP (59%), aortic cell proliferation (144%), and aortic CSA (34%), ‐ effects that were not offset by B1R antagonism. However, B1R antagonism did attenuate the AngII‐induced increase in aortic mass. Similarly, AngII increased LV hypertrophy (35%) and total DNA content (44%) relative to control, both of which were prevented by concomitant B1R antagonism. Cardiomyocyte CSA was not different across treatment groups. This study reveals tissue‐specific effects of bradykinin B1R antagonism. Although AngII induced significant remodeling in both the heart and aorta, concomitant B1R blockade only prevented cell accumulation and remodeling in the LV. Future studies will investigate the mechanism underlying this effect to further elucidate the role of the bradykinin B1R and its interaction with AngII in remodeling heart and aorta.

Support or Funding Information

Canadian Male Sexual Health Council
Acute myocardial dysfunction after ionizing radiation exposure. Fumbles on the way to Mars
Authors: Ciccarelli Michele , Franco Antonietta , Sorriento Daniela , Fiordelisi Antonella , Gambardella Jessica , Trimarco Bruno , Iaccarino Guido
Published: November 2023

In time of programming interstellar journeys, the exposition to ionizing radiation (IR) attempts to the health of travelers. IR causes cardiac dysfunction due to the increased reactive oxygen species production (ROS). Mitochondria, being the primary source of radical oxygen species (ROS) and of energetic substrates for the myocardium, are the possible mechanisms of IR related cardiac dysfunction. To evaluate the possible involvement of mitochondria to mediate the effects of IR exposure on cardiac function, in C57BL/6 mice subjected to IR exposure by X‐ray (4 Gy), we performed echocardiography at basal, and 3 and 24 hours post IR exposure. Mice were sacrificed and heart samples collected at each time point for analysis of mitochondria morphology by transmission electron microscopy (TEM) and biochemistry. At 3 h post‐IR, we observed increased left ventricle diastolic diameter (LVDd: 39.5±0.3 vs 35±0.15 mm; p<0.05 vs Basal) with reduced ejection fraction (EF: 45%±2 vs 66%±4; p<0.05 vs Basal). At 24h post‐MI, we found a recovered LVDd (36.2±0.4 vs 35±0.15 mm; ns) and ameliorated cardiac contractile function (EF: 60%±3 vs 66%±4; ns). Morphological analysis by TEM (Fig 1) reveled that, at 3 hours post IR exposure, mitochondria were reduced in number, showing cristae disarrangement with intra‐vacuolization and fragmentation. At 24 h post‐IR mitochondria number and morphology are recovered with normal cristae and absence of vacuolization. Morphological changes were accompanied by molecular modifications involved in mitochondria induced apoptosis (Cyt C), mitophagy (p62) and mitochondrial mass (SOD). In particular, Cyt C release was dramatically increased into cytosolic fraction at 3 h post IR and reduced at 24 h. Similarly, p62 was increased in response to stress at 3 h with a partial reduction at 24 h. Inversely, SOD level was reduced at 3h but normalized at 24h as compared to basal, indicating recovering of mitochondrial mass. Mitochondria damage was also evaluated by expression of the different complexes (I to IV) composing the mitochondria respiratory chain through RT‐PCR. At 3 h post‐IR complex I,II,III and IV are all reduced as well ATPase expression. At 24 hours post‐IR, complexes expression and ATPase were recovered. A recently described modulatori of mitochondrial survival, GRK2, was investigated in related in vitro model. In H9C2, over‐expression of GRK2, induced a rightward shift in time for mitochondrial alterations and ROS production, which occurred after 8 hours after IR. Opposite, GRK2 silencing lead to anticipated mitochondrial impairment and no recovery occurred at 8h. Mechanistically, IR exposure produced interaction of GRK2 with MFN1 and 2, key molecules of mitochondrial recovery process, at 3h post IR. IR induces an acute cardiomyopathy that is associated to an altered mitochondrial morphology and function.
Reliability of Heart Rate Variability as an Assessment of Cardiac Sympathetic Activity in Humans
Authors: Wakeham Travis R. , Fonkoue Ida T. , Durocher John J. , Cooke William H. , Carter Jason R.
Published: November 2023

Spectral analysis of heart rate variability (HRV) has been widely used as an indirect, non‐invasive method for assessing cardiac autonomic function in numerous psychophysiological and clinical research studies. However, there is debate whether or not the low‐frequency (LF) component of the analysis provides accurate insight into cardiac sympathetic activity in humans. The purpose of this study was to evaluate the reliability of the LF component of HRV compared to that of peripheral muscle sympathetic nerve activity (MSNA), which remains the only direct method to assess sympathetic neural outflow in humans and has been consistently shown to have high intra‐individual reproducibility. Sixteen adults (9 men and 7 women, 21 ± 1 years, body mass index of 23 ± 1 kg/m2) were examined, and completed three experimental sessions across two laboratory visits. Experimental sessions one and two were conducted during the first laboratory visit and separated by 45 minutes of supine rest, while the third session was completed approximately one month after the first laboratory visit. Each session included a five‐minute supine baseline that included recordings of MSNA via microneurography and R‐R intervals using a three‐lead electrocardiogram. Intraclass correlation analyses (Cronbach's α coefficient) were performed to determine the consistency of HRV and MSNA across the three sessions. The spectral analysis revealed that the high‐frequency (HF) component, which is primarily associated with cardiac parasympathetic activity, was highly reliable (Cronbach's α = 0.90, P < 0.001). In contrast, the LF component, which is often utilized as a noninvasive estimate of cardiac sympathetic activity, was not significantly consistent across the three sessions (Cronbach's α = 0.45, P = 0.078). As previously reported (Fonkoue & Carter, Am J Physiol Regul Integr Comp Physiol, 2015), MSNA was highly consistent across the three sessions (Cronbach's α = 0.84, P < 0.001). In summary, our findings demonstrate that unlike MSNA, which has been consistently shown to be a stable and reliable measurement of peripheral sympathetic activity, the LF component of HRV is not a stable and reliable measurement of cardiac sympathetic activity in humans.
CMT‐3, an anti‐inflammatory, non‐antibiotic tetracycline, attenuates AngII‐induced hypertension
Authors: Sharma Ravindra K , Santisteban Monica , Lobaton Gilberto O , Richards Elaine M , Raizada Mohan K
Published: November 2023

Background

Our previous studies have established that activation of microglial cells and neuro‐inflammation in autonomic brain regions, particularly in the paraventricular nucleus (PVN) of the hypothalamus, plays a critical role in hypertension (HTN). This conclusion is primarily based on the evidence that the tetracycline antibiotic minocycline (Mino) inhibits microglial activation and attenuates HTN. Since Mino has both anti‐inflammatory and antibiotic properties, we sought to determine the property responsible for its antihypertensive effects using the chemically modified tetracycline‐3 (CMT‐3), that has no antibiotic activity.

Objective

We hypothesized that central administration of CMT‐3, would inhibit microglial activation, neuro‐inflammation and attenuate HTN.

Methods

Six‐week‐old male SD rats (n=8–12/group) were implanted with telemetry transducers. Ten days after surgery, 2 osmotic minipumps were implanted to subcutaneously infuse AngII (200ng/kg/min,) or vehicle and intracerebroventricularly infuse CMT‐3 (3.5μg/hr) or vehicle. Mean arterial pressure (MAP) was recorded weekly; after 4 weeks of AngII treatment, spectral analysis of was performed for autonomic nerve activity and anti‐Iba1 immunostaining was carried out to quantitate activated microglia in the brain.

Results

CMT‐3 significantly attenuated the AngII‐induced increase in MAP both in light: (Con:102±3, CMT‐3:101±4, AngII:158±23, AngII+CMT‐3:125±20 mmHg) and dark cycles (Con:102±2, CMT‐3: 102±3, AngII: 174±20, AngII+CMT‐3:145±28 mmHg). The heart to body weight ratio was similarly decreased (Con:2.9±0.3, CMT‐3:2.9±0.1, AngII:3.9±0.4, AngII+CMT‐3:3.3±0.2 mg/g; p<0.05) with CMT‐3. CMT‐3 normalized AngII perturbed autonomic activity, as measured by LF/HF of SBP (Con:1.8±0.7, CMT‐3:2.0±1.6, AngII:5.4±4, AngII+CMT‐3:2.9±1.9; p<0.001)). Furthermore, AngII increased activated microglial cells ~2.5‐fold, predominantly in the PVN, and CMT‐3 significantly attenuated this increase (p<0.01).

Conclusion

These data indicate that inhibition of neuro‐inflammation is key in correction of autonomic dysregulation and attenuation of HTN. They suggest that the anti‐inflammatory, rather than the antibiotic property, of Mino mediates these effects. Thus, CMT‐3 may be a better therapeutic candidate for neurogenic resistant hypertension in humans as it circumvents the problems associated with continuous antibiotic administration.

Support or Funding Information

This work is supported by NIH grant R01HL033610
The Effect of a Patent Foramen Ovale and Sex on the Hypercapnic Ventilatory Response
Authors: Hardin Alyssa M. , Boulet Lindsey M. , Davis James T. , Chang Alex J. , Foster Glen E. , Lovering Andrew T.
Published: November 2023

A patent foramen ovale (PFO) is a right‐to‐left intracardiac shunt pathway present in approximately 35% of the general healthy population. Individuals with a PFO (PFO+) exhibit higher alveolar to arterial oxygen difference (AaDO2) values at rest than subjects without a PFO (PFO−), indicating that PFO+ subjects have a source of right‐to‐left shunt significant enough to decrease gas exchange efficiency at rest. Additionally, PFO+ subjects have blunted ventilatory acclimatization to hypoxia compared to PFO− subjects. While the presence of a PFO is known to influence hypoxic chemosensitivity, it has yet to be determined if the presence of a PFO has an effect on the hypercapnic ventilatory response (HCVR).

Thirty subjects (15 female) matched for height, weight, sex and age participated in this study. Subjects completed two hypercapnic breathing trials in a randomized and balanced order, separated by ≥ 40 minutes of rest between trials. At the start of each trial, subjects began breathing room air until steady‐state values for end‐tidal oxygen and carbon dioxide (PETO2 and PETCO2) were established. PETO2 and PETCO2 were then controlled utilizing a dynamic end‐tidal forcing system (AirForce). During the hyperoxic hypercapnia (HH) trial, PETO2 was clamped at 250 mmHg and PETCO2 was increased in a stepwise fashion to target values of +3 mmHg, +6 mmHg and +9 mmHg of each subject's baseline PETCO2. Each stage consisted of a 90 second steady‐state data collection period after each increase. The procedure for the normoxic hypercapnia (NH) trial was identical to the HH trial except that PETO2 was clamped at the resting baseline value for each subject and PETCO2 increased as above. Hypercapnic ventilatory response (HCVR) was calculated as the change in minute ventilation (VE) divided by the change in PETCO2 (L/min/Torr CO2).

PFO+ subjects demonstrated a blunted, significantly lower HCVR than PFO− subjects during both the HH (PFO−: 1.80 L/min/Torr CO2 ± 0.17, PFO+: 1.19 L/min/Torr CO2 ± 0.14, p < 0.05) and NH (PFO−: 1.79 L/min/Torr CO2 ± 0.23, PFO+: 1.24 L/min/Torr CO2 ± 0.26, p < 0.05) trials. These data suggest that PFO+ subjects have a blunted ventilatory response to acute exposure to hypercapnia. Within the subset of female subjects, PFO+ females had a significantly lower HCVR than PFO− females in the HH trial (PFO−: 1.85 L/min/Torr CO2 ± 0.27, PFO+: 1.06 L/min/Torr CO2 ± 0.13, p < 0.05) and NH trial (PFO−: 1.81 L/min/Torr CO2 ± 0.28, PFO+: 1.15 L/min/Torr CO2 ± 0.11, p < 0.05). However, no differences were observed between the male PFO+ and PFO− subjects in the HH (PFO−: 1.77 L/min/Torr CO2 ± 0.37, PFO+: 1.35 L/min/Torr CO2 ± 0.24, p > 0.05) or NH (PFO−: 1.76 L/min/Torr CO2 ± 0.24, PFO+: 1.33 L/min/Torr CO2 ± 0.28, p > 0.05) trials. These results suggest that female PFO+ subjects have a blunted HCVR compared to female PFO− subjects.
Mitochondrial development is impaired in hyperoxic rats and this may underpin the blunting of the acute hypoxic ventilatory response
Authors: Fayyad Tariq H , Rakoczy Ryan J , Hartpence Annah J , Wyatt Christopher N
Published: November 2023

Carotid body (CB) responses to hypoxia are low at birth and increase over time to mature responses. Using an in vitro rat CB‐carotid sinus nerve (CSN) preparation, Kholwadwala and Donnelly (1992) demonstrated that the CSN activity in response to hypoxia increased from low levels to robust adult responses after two weeks. This time course of maturation was paralleled by an increase in TASK channel sensitivity to hypoxia in the O2‐sensing Type I cells (Kim et al, 2011). Previous studies have indicated that a fall in Type I cell mitochondrial volume and an increase in the rate of oxidative phosphorylation may underpin the development of the hypoxic ventilatory response (Paulet et al, 2012).

Our most recent studies addressed the phenomenon that hyperoxia delays the maturation of the acute hypoxic response (Bavis, RW et al, 2010). Our initial findings suggest that hyperoxia caused the juvenile non‐oxygen‐sensing Type I cells to remain locked in a mitochondrial rich state and that as the rat developed this mitochondrial rich state impaired development of the hypoxic response. Thus there appears to be a correlation between mitochondrial volume and the ability to respond to hypoxia in rat Type I cells.

Support or Funding Information

NIH RO1 HL091836
Phox2b‐negative Parafacial Neurons Simultaneously Regulate Active Expiration and Airway Patency Under Hypercapnia in in situ Preparations of Rats
Authors: Moraes Davi J.A. , de Britto Alan A.
Published: November 2023

Hypercapnia produces active expiration in rats and the recruitment of late‐expiratory (late‐E) neurons located in the parafacial Respiratory Group (pFRG) of the ventral medullary brainstem. We tested the hypothesis that hypercapnia produces active expiration and concomitant cranial respiratory motor responses controlling the oropharyngeal and upper airway patency by pFRG late‐E neurons disinhibition, but not via synaptic excitation. Phrenic nerve, abdominal nerve (AbN), cranial respiratory motor nerves, subglottal pressure, and medullary and spinal neurons/motoneurons were recorded in in situ preparations of juvenile rats. Hypercapnia evoked AbN active expiration (n=12), exaggerated late‐E discharges in cranial respiratory motor outflows (n=12), and glottal dilatation (n=6) via late‐E synaptic excitation of abdominal (n=8), hypoglossal (n=6) and laryngeal (n=12) motoneurons. Simultaneous rhythmic late‐E activity in previously silent Phox2b‐negative pFRG neurons was also evoked by hypercapnia (n=12). In addition, hypercapnia‐evoked AbN active expiration, neural and neuronal late‐E activities were eliminated by pFRG inhibition (n=18), but not after blockade of synaptic excitation (n=9). On the other hand, pFRG inhibition did not affect either hypercapnia‐induced inspiratory increases in respiratory motor outflows (n=12) or CO2 sensitivity of the more medial Phox2b‐positive neurons in the retrotrapezoid nucleus (RTN; n=8), despite elimination of AbN active expiration. Our data suggest that neither RTN Phox2b‐positive nor other CO2‐sensitive brainstem neurons activate Phox2b‐negative pFRG late‐E neurons under hypercapnia to produce AbN active expiration and concomitant cranial motor respiratory responses controlling the oropharyngeal and upper airway patency. Hypercapnia seems to produce pFRG late‐E neuronal disinhibition in in situ preparations of juvenile rats to activate abdominal, hypoglossal and laryngeal motoneurons.

Support or Funding Information

FAPESP: SÃO PAULO RESEARCH FOUNDATION
Mitochondrial Hsp90 exerts pro‐survival functions in Pulmonary Arterial Hypertension
Authors: BOUCHERAT Olivier , BREUILS‐BONNET Sandra , CHABOT Sophie , MELOCHE Jolyane , VITRY Géraldine , LAMBERT Caroline , NADEAU Valérie , TREMBLAY Eve , SUTENDRA Gopinath , MICHELAKIS Evangelos , CHAE Young , ALTIERI Dario , PAULIN Roxanne , PROVENCHER Steeve , BONNET Sébastien
Published: November 2023

RATIONALE

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease with a poor prognosis and no therapeutic option. Although the causal pathomechanisms contributing to remodeling of the pulmonary vascular bed in PAH are still unclear, several features, including hyperproliferation and resistance to apoptosis of pulmonary smooth muscle cells (PASMCs) sustained by oncogenic pathways activation and metabolic alterations, have led to the emergence of the cancer‐like concept. The molecular chaperone heat shock protein 90 (Hsp90), by interacting with its client proteins, is directly associated with malignant growth and proliferation. In addition to be highly expressed in the cytosol, Hsp90 exists in a subcellular pool compartimentalized in the mitochondria (mtHsp90) of tumor cells, but not in normal cells, where it promotes cell survival.

OBJECTIVE

We hypothesized that Hsp90 up‐regulation in PAH triggers PASMC proliferation and resistance to apoptosis.

METHODS AND RESULTS

We showed that Hsp90 is up‐regulated in lungs and PASMCs isolated from distal pulmonary arteries from PAH patients compared to control donors. Using pharmacological inhibitors (AT13387 and 17‐AAG), we demonstrated that cytosolic Hsp90 stabilizes the expression of numerous clients proteins overexpressed in PAH that promote cell growth and survival. More importantly, we demonstrated that Hsp90 is specifically expressed in PAH‐PASMCs mitochondria (immunoblot, dual immunofluorescence and immunogold electronic microscopy), and not in healthy cells. Whereas cytosolic Hsp90 inhibition displays a lack of absolute specificity for PAH‐PASMCs, selective inhibition of mtHsp90 activity using Gamitrinib decreased PAH‐PASMC proliferation (Ki67 labeling) and resistance to apoptosis (Annexin V assay) without affecting control cells. In PAH‐PASMCs, mtHSP90 accumulation prevents the accumulation of mitochondrial DNA damage and maintains bioenergetics functions (Seahorse). In the fawn‐hooded rat and monocrotaline‐induced models of PAH, mtHsp90 inhibition reduces PA remodeling thus improving pulmonary hemodynamic parameters.

CONCLUSION

We demonstrated for the first time that accumulation of mtHsp90 is a cardinal feature of PAH‐PASMCs, contributing to the development of vascular lesions.

Support or Funding Information

ENTELLIGENCE young investigator program