bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatial expression of Receptor for Advanced Glycation End‐Products (RAGE) in diverse tissue and organ systems differs following exposure to secondhand cigarette smoke
Authors: Gassman Jason R. , Lewis Joshua B , Milner Dallin , Monson Troy , Broberg Dallin , Bodine Jared , Lewis Adam , Dunaway Todd , Egbert Kaleb , Chavarria Bryan , Thomas Daniel , Arroyo Juan A , Reynolds Paul R
Published: November 2023

The receptor for advanced glycation end‐products (RAGE) is a pattern recognition receptor initially identified in the lung and known to be expressed by many cell types including smooth muscle cells, fibroblasts, macrophages/monocytes and epithelial cells. RAGE is capable of binding a variety of endogenous ligands including advanced glycation end‐products (AGEs), S100/calgranulins, amyloid‐β peptide, and HMGB1. Furthermore, exogenous entities such as tobacco smoke particulates also have been demonstrated to activate RAGE and as such, RAGE signaling has been implicated as a major contributor to a variety of inflammatory states. In the present study we examined the expression patterns of RAGE in pulmonary and systemic tissues from mice exposed to room air (RA) and then compared expression with mice following exposure to daily secondhand smoke for up to 6 months via a nose‐only exposure system (Scireq, Montreal, Canada). Following sacrifice, real time PCR revealed interesting expression patterns for RAGE mRNA in tissues and organs such as eyes, lungs, heart, kidney, spleen, gastrocnemius, liver, brain, small intestine, adipose tissue, and dermis. RT‐PCR analysis revealed that augmented RAGE expression is a cellular response to secondhand smoke exposure. Confirmatory studies aimed at characterizing protein levels were also conducted using immunoblot and immunohistochemistry. In conclusion, our data reveal that as already detected in several studies involving the pulmonary apparatus, elevated RAGE expression is a distinct response to smoke exposure and its expression likely elevates the pro‐inflammatory RAGE signaling axis in cells and tissues responding to exposure.

Support or Funding Information

This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).
Targeting Of Endocytic Pathways By The Legionella pneumophila Effector Protein AnkX
Authors: Romero Barbara Pamela , Modla Shannon , Neunuebel Ramona
Published: November 2023

Legionella pneumophila is an opportunistic bacterium that infects human alveolar macrophages causing a severe type of pneumonia known as Legionnaires’ disease. L. pneumophila remains enclosed in a membrane‐derived vacuole throughout infection. Its ability to survive and replicate in this compartment is due to the presence of a type IV secretion system that facilitates the translocation of over 300 effector proteins into the host cytosol. Many of these proteins subvert vesicular trafficking to hijack vesicles from the endoplasmic reticulum‐to‐Golgi trafficking pathway to camouflage the Legionella‐containing vacuole (LCV). The major players involved in remodeling the LCV are still largely unknown. Previously, it was shown that the L. pneumophila effector AnkX is translocated into the host cell during infection and facilitates avoidance of pathogen degradation through an unknown mechanism. We hypothesize that during infection AnkX interferes with host endocytic pathways to prevent fusion of the LCV with endocytic compartments, which would lead to degradation via phagosome maturation. Here we show evidence to support this hypothesis. We performed intracellular localization studies of mammalian cells ectopically expressing fluorescently‐tagged AnkX. Using total internal reflection fluorescence (TIRF) microscopy we showed that EmGFP‐AnkX localizes mainly at the plasma membrane in a punctate pattern. TIRF live cell microscopy revealed that these punctae are moving in a linear trajectory with a dynamic resembling that of the retrograde flow of actin. Structured illumination microscopy of HeLa cells cotransfected with EmGFP‐AnkX and mCherry‐LifeAct suggested that AnkX may interact with actin cables. Electron microscopy of COS1 cells transfected with EmGFP‐AnkX coupled with immunogold labeling confirmed AnkX's localization at the plasma membrane. Our preliminary data suggest that AnkX localizes to recycling endosomes, but other endocytic compartments may be targeted by AnkX as well. Furthermore, our data show that AnkX is also present on nascent and mature endosomes. Future studies will focus on probing AnkX's colocalization with endocytic vesicles during infection.
Tyrosine Phosphorylation of the Transcription Factor Yin Yang 1
Authors: Daraiseh Susan , Kassardjian Ari , Rizkallah Raed , Alexander Karen , Hurt Myra
Published: November 2023

Yin Yang 1 (YY1) is a multifunctional zinc finger transcription factor that can activate or suppress gene expression depending on both the promoter and co‐factors it recruits. In addition to the transcriptional functions of YY1, it has been found to regulate a broad spectrum of biological processes such as development, apoptosis, DNA repair, autophagy, oncogenesis and X‐chromosome inactivation. Several of the functions carried out by YY1 have been found to be regulated by post translation modifications. Our previous work has shown the modulation of the function of YY1 through phosphorylation at amino acid residues serine and threonine. However, little is known about the molecular mechanisms by which tyrosine phosphorylation regulates YY1's function. YY1 has six tyrosine residues within its 414 amino acid sequence. PhosphoSitePlus® (PSP) has identified two tyrosine phosphopeptides through a global proteome study that have matched peptide sequences to YY1, specifically showing that tyrosine residue 251 and 254 are phosphorylated. In order to further understand and characterize the signaling pathways that YY1 is involved in, we have developed two rabbit poly‐clonal phosphospecific antibodies against each tyrosine residue; (α‐pY251) and (α‐pY254), respectively. Both antibodies are able to detect phosphorylated YY1 in vivo and in vitro. Additionally, we have utilized Group Based Prediction System (GPS 3.0) to predict kinases that phosphorylate YY1 through matching experimental data to theoretical spectra. Based on the high‐throughput proteomic and computational data we have used our phosphospecific antibodies in a combination of biochemical assays to identify several protein tyrosine kinases that phosphorylate YY1. Our findings will have great importance for understanding cellular signaling pathways that regulate this multifunctional protein.
Molecular mechanisms underlying angiotensin II‐induced angiotensin converting enzyme 2 internalization and degradation
Authors: Ogunlade Blessing , Sriramula Srinivas , Lazartigues Eric , Guidry Jessie J , Filipeanu Catalin M
Published: November 2023

Angiotensin converting enzyme II (ACE2), the newest identified member of renin‐angiotensin system (RAS), catalyses the conversion of angiotensin (ang)‐II to ang‐(1‐7) which plays a compensatory role through Mas1 receptor in the cardiovascular system. We previously reported that decreased ACE2 expression and activity leads to the development of ang‐II‐mediated hypertension in mice, that ang‐II stimulated ACE2 internalization and degradation in lysosomes and that inhibition of this process prevents the hypertensive actions of ang‐II. On the other hand, severe acute respiratory syndrome coronavirus is internalized together with ACE2, clearly indicating a regulated endocytotic mechanisms for this enzyme. However, the molecular mechanisms controlling this event remain largely unknown and the aim of the present study was to clarify these aspects.

We first performed a proteomic study in HEK293T cells co‐transfected with ACE2 and AT1R and found that ang‐II treatment (100 nM, 4 h) significantly changed the interactions of ACE2 only with fascin, an actin‐bundling protein. Confocal microscopy experiments confirmed that ang‐II strongly enhanced the co‐localization of the two proteins in the perinuclear space. In addition, we demonstrated that ang‐II treatment augmented ubiquitination of ACE2 and amino acid analysis indicated that in the C‐terminus the enzyme has only five putative lysine ubiquitination sites. Six ACE2 mutants were constructed, five in which each individual ubiquitination site was replaced by arginine, and the sixth mutant (ACE2‐L5R) included all five mutations. In basal conditions in HEK293T cells co‐transfected with AT1R, ACE2 wild‐type activity was 3536.88 ±331.63FU/min/μg protein. Ang‐II treatment (100 nM, 4h) significantly attenuated enzymatic activity (3298.60±255.89). Remarkably, every mutant displayed enhanced enzymatic activity in basal conditions of at least 195±45%, with the highest increase observed in the case of ACE2‐L5R mutant being 285±61%. Furthermore, none of these ACE2 mutants displayed decreased enzymatic activity after ang‐II treatment. The present results demonstrate that ACE2 is constitutively ubiquinated and that this posttranslational modification is augmented by increased levels of ang‐II, contributing to lysosomal degradation of the enzyme in fascin‐dependent manner and overall diminishing its compensatory role against overactive RAS. These data indicate that interfering with the mechanisms contributing to ang‐II‐mediated ACE2 degradation may constitute an alternative therapy in cardiovascular diseases.

Support or Funding Information

Supported by NIH‐P20‐GM103514 (EL, JJG and CMF), R01‐HL093178 (EL), and startup funds from Howard University (CMF)
Hydrogen sulfide inhibits Orai3‐mediated store‐operated Ca2+ entry by targeting extracellular cysteines
Authors: Fresquez Adriana Maria , White Carl
Published: November 2023

The store operated calcium entry (SOCE) pathway is activated by interactions between the endoplasmic reticulum localized STIM1 and STIM2 proteins and the Orai 1, 2 and 3 plasmalemmal Ca2+ channels. We recently discovered that the gaseous signaling molecule hydrogen sulfide (H2S) selectively inhibits SOCE mediated by STIM1 and Orai3. We showed that STIM1/Orai3 regulation has physiological importance in the innate immune system where endogenously produced H2S limits maximal activation of SOCE and proinflammatory cytokine production. The purpose of the current study was to define the mechanism by which H2S modifies STIM1/Orai3 function. It is known that H2S impinges on protein function by targeting disulfide bridges. Sequence analysis of human Orai1, 2 and 3 revealed two cysteines (C226 and C232) unique to the human and mouse Orai3 protein. We hypothesized that C226 and C232 are the site of action for H2S on Orai3 and C226 and C232 forms a disulfide bridge. We tested this by assessing the effect of mutating the C226 and C232 residues to serine on H2S‐regulation of SOCE. This was done by transiently transfecting wild‐type or mutant human Orai3 into HEK293 cells stably expressing human STIM1 and measuring SOCE by Ca2+ imaging. Treatment with the H2S donor GYY4137 (500 μM) inhibited the SOCE mediated by STIM1/Orai3. Mutating either C226 or C232 alone had no effect on SOCE under control conditions, however, the ability of GYY4137 to inhibit SOCE was lost in the mutants. A double mutation of both C226 and C232 also had no effect on SOCE under control conditions and GYY4137 again failed to inhibit SOCE. In addition, pre‐incubation with the reductant dithiothreitol (5 mM) completely blocked the inhibitory effect of GYY4137. These data are consistent with a model in which H2S inhibits SOCE mediated by STIM1/Orai3 by targeting a disulfide bridge between C226 and C232.

Support or Funding Information

Rosalind Franklin University of Medicine and Science
The role of SNARE proteins in targeting KCa3.1 to the basolateral membrane of epithelial cells
Authors: Farquhar Rachel , Hamilton Kirk Lee
Published: November 2023

Targeting epithelial membrane proteins to a specific membrane is crucial for proper epithelial cell function. KCa3.1, a calcium‐activated, intermediate‐conductance potassium channel, is targeted to the basolateral membrane (BLM) in epithelial cells. A consequence of mutations of KCa3.1 is impaired ion and fluid transport of erythrocytes resulting in hereditary xerocytosis. Surprisingly, the mechanism of membrane targeting of KCa3.1 is not well understood. Recently, we reported that KCa3.1 is targeted to the BLM of epithelial cells by a Myosin‐Vc‐dependent pathway (Farquhar et al. FASEB J. 30:1224.41, 2016) and by a Rab1‐ and Rab8‐dependent process (Bertuccio et al. PlosONE 9:e92013, 2014). Here, we extend our work to examine the role of the SNARE proteins VAMP3, syntaxin 4 (STX‐4) and SNAP‐23 in the targeting of KCa3.1 to the BLM of Fischer rat thyroid (FRT) epithelial cells. We used our FRT epithelial cell line stably expressing KCa3.1‐BLAP/Bir‐A‐KDEL (cells grown on filters), immunoblot (IB), siRNA and Ussing chamber experiments in this study. Our IB approach was to streptavidin label channels that arrive at the BLM to determine the effects of siRNA knockdown of specific SNARE proteins on the targeting to and function (KCa3.1 current) of KCa3.1 at the BLM of the epithelial cells. Using siRNA, we showed that knockdown of VAMP3 reduced the BLM expression of KCa3.1 by 49 ± 8% (P ≤ 0.05, n = 3) compared with control cells. With Ussing chamber experiments, we used 1‐EBIO (100 μM, an agonist of KCa3.1) to stimulate current via KCa3.1. Knockdown of VAMP3 reduced the KCa3.1 current by 86 ± 9% (P ≤ 0.05, n = 3) compared with control cells. Similarly, knockdown of STX‐4 lowered the BLM expression of KCa3.1 by 84 ± 5% (P ≤ 0.05, n = 3) and reduced the KCa3.1 current by 82 ± 8% (P ≤ 0.05, n = 2). Finally, siRNA knockdown of SNAP‐23 reduced the expression of KCa3.1 at the BLM by 52 ± 11% (P ≤ 0.05, n = 6) and reduced the KCa3.1 current by 1.2 ± 0.3 μA (P ≤ 0.05, n = 6) compared with control cells. These data suggest that VAMP3, STX‐4 and SNAP‐23 are critical for the targeting KCa3.1 to BLM of polarized epithelial cells.

Support or Funding Information

This work was supported by UORG and Dean's Fund grants from the University of Otago and an Aims grant from the Department of Physiology (KLH).
The characterization of SPX‐101, a new peptide promoter of ENaC internalization, revealed basic mechanistic understanding of ENaC internalization and downstream signaling events.
Authors: Walker Matthew Paul , Sesma Juliana , Wu Bryant , Stuhlmiller Timothy , Scott David
Published: November 2023

Cystic Fibrosis (CF) is a life‐altering disease that affects over 70,000 people worldwide. CF is defined by mutations in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) causing an improperly functioning protein or loss of protein expression. Loss of CFTR activity causes an imbalance in ion levels leading to defects in mucus clearance and acidification of the lung. A major effect of lung acidification is the loss SPUNC1 activity. SPLUNC1 is secreted to the airway surface liquid and regulates ENaC through direct binding to the extracellular region of the channels. This interaction is lost at low pH, resulting in ENaC accumulation on the membrane and dehydration of the lung. Restoring SPLUNC1 regulation of ENaC is an attractive target for CF therapy, as it will increase lung hydration allowing for increased mucus clearance and normalization of normal lung function. SPX‐101 is a SPLUNC1‐derived peptide that internalizes ENaC regardless of pH. SPX‐101 can be nebulized directly into the lung allowing for efficient drug delivery into the target organ while achieving low systemic circulation. SPX‐101 has shown significant efficacy in CF animal models and safety in pre‐clinical toxicology and phase 1 clinical trials.

Mechanistically, SPX‐101 internalized ENaC in a dose dependent manner in CF‐derived human bronchial epithelial cell (HBEC), decreased amiloride sensitive current, and increased airway surface liquid. SPX‐101 induced ENaC ubiquitination causing a robust and prolonged decrease in the membrane levels of ENaC alpha, beta, and gamma. ENaC internalization was achieved through activation of a pro‐internalization kinase axis followed by a compensatory increase in signaling mediators responsible for regulating ENaC stability. This internalization and downstream signaling activation is markedly different than ENaC channel blockers such as amiloride or derivatives of the parent molecule. Amiloride and its derivatives do not induce ENaC internalization or regulate downstream kinases, suggesting that these downstream signaling events are caused by internalization and/or SPX‐101 induced changes in ENaC structure rather than changes in sodium secretion. Together this work provides the molecular basis for the activity of the first in class peptide promoter of ENaC internalization, SPX101, and provided valuable insight into the mechanisms by which ENaC proteins are regulated on the cell surface.
Antibody‐antibiotic conjugates: a new platform for treatment of serious bacterial infections
Authors: Brown Eric J
Published: November 2023

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long‐term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. We have shown that intracellular S. aureus in mice can establish infection even in the presence of vancomycin, and we have developed a novel therapeutic that effectively kills intracellular S. aureus. This antibody‐antibiotic conjugate consists of an anti‐S. aureus antibody that recognizes a highly expressed epitope on wall teichoic acid, conjugated to a potent antibiotic that is activated only after its release from the antibody in the proteolytic environment of the phagolysosome. The antibody‐antibiotic conjugate is superior to vancomycin for treatment of bacteremia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections. This paradigm may have implications for treatment of other bacterial infections.

Support or Funding Information

This work was funded by Genentech, Inc.
Engineered bacteriophage therapeutics against multidrug‐resistant pathogens
Authors: Barbu Magda , DelRosario Yvette , Hubby Bolyn
Published: November 2023

Since the introduction of penicillin over 70 years ago, the number of patient deaths caused by infectious diseases has dramatically decreased and antibiotics have become among the most commonly prescribed drugs. However, the selective pressure imposed by antibiotic overuse has led to the emergence of bacterial strains resistant to virtually all antimicrobials and has renewed interest in the development of lytic bacteriophage (or phage) therapeutic and prophylactic agents. The advantages of using phage to combat infectious diseases include minimal disruption of resident flora, lack of cross‐resistance with antibiotics, low toxicity and self‐limiting dosing. Despite these attractive features, translational development of natural phage has been hindered mainly by the difficulty of accessing bacterial hosts within biofilms, the rapid emergence of resistant bacteria to a single phage, and above all, by the narrow host specificity of phage compared to antibiotics. The need for customized and complex combinations of natural phage to achieve adequate host range activity has made their development as licensed therapeutics very difficult. Genetic engineering of phage genomes can overcome these hurdles; however, broadly applicable methods for efficient construction of defined mutations in virulent phage genomes are still in their infancy. Thus, we developed a completely cell‐free phage engineering method that allows rapid and iterative editing of viral genomic DNA. In parallel, we sequenced and annotated the genomes of ~300 MDR P. aeruginosa clinical isolates, and subsequently determined the susceptibility of each isolate to specific phages. Through bioinformatics analysis in combination with our engineering platform, we have successfully collapsed the host range of a family into a representative phage. We further engineered wide host range phage to express secondary payloads, such as biofilm degrading enzymes and antimicrobial moieties, and demonstrated that these phages have improved activity compared to natural phage in P. aeruginosa models of infection.

Support or Funding Information

Synthetic Genomics Vaccines, Inc.

NIAID R21AI121531
L‐Sulforaphane Decreased Contractile Response in Mesenteric Arteries in a Rat Model of Gestational Hypertension
Authors: Cushen Spencer C , Osikoya Oluwatobiloba , Goulopoulou Styliani
Published: November 2023

Background

Maternal hypertension is a state of inflammation characterized by oxidative stress. Exposure of pregnant rats to the Toll‐like Receptor 9 activator, ODN2395, induces hypertension and upregulates vascular oxidative stress. The transcription factor, Nuclear Factor, Erythroid 2 Like 2 (Nrf2), is a regulator of antioxidant response, which is overexpressed in placentas from patients with preeclampsia. However, the role of Nrf2 in maternal vascular dysfunction is unknown.

Hypothesis

L‐sulforaphane, an Nrf2 activator, will have anti‐contractile effects on arteries from pregnant rats treated with CpG oligonucleotides (a model of gestational hypertension).

Methods

Pregnant Sprague‐Dawley rats were treated with synthetic unmethylated CpG oligonucleotides (ODN2395, 100μg/intraperitoneal injection) or saline (Control) on gestational day 14, 16, and 18 (term=21–22 days). Blood pressure was measured before pregnancy and on gestational day 19 using the tail cuff method. The contractile responses of mesenteric resistance arteries to a thromboxane A2 (TxA2) mimetic, U46619, in the presence or absence of Nrf2 activator, L‐sulforaphane (L‐S, 40 μM), were assessed by wire myography on gestational day 21.

Results

Dams treated with ODN2395 had greater systolic blood pressure on gestational day 19 compared to control rats (Control, n=9: 100±4 mmHg vs. ODN2395, n=7: 119±4 mmHg, p=0.007). One‐hour incubation with L‐sulforaphane did not affect contractile responses to U46619 in arteries from control or ODN2395‐treated rats. Incubation with L‐sulforaphane for 3 hours reduced the contractile response to U46619 in mesenteric arteries from both ODN2395 and control rats (Peak contraction as %Max KCl (120mM), Control Veh: 120.5%±4.85; Control L‐S: 39.1%±7.16; ODN2395 Veh: 111.1%±4.19; ODN2395 L‐S: 42.6%±2.68).

Conclusion

Pregnancy is a state of oxidative stress and this may explain the anti‐contractile effects of L‐sulforaphane in arteries from normal, healthy rats. In preeclampsia, levels of oxidative stress are greater compared to normotensive pregnancies and thus, systemic treatment with L‐sulforaphane or other Nrf2 activators may improve poor cardiovascular outcomes in pregnancies with preeclampsia.

Support or Funding Information

University of North Texas Health Science Center; American Heart Association
Arterial Pulse Wave Velocity is Increased in Prematurely‐Born Adults and Rats Given Neonatal Supplemental Oxygen
Authors: Peters Matthew , Hoover Michael , Sturgeon Madison , Vellookunnel Shilpa , Chandrasekar Shreya , Schaeffer Gilbert V. , Sindler Amy L. , Pierce Gary L. , Stauss Harald M. , Farrell Emily T. , Eldridge Marlowe W. , Bates Melissa
Published: November 2023

Thirteen percent of infants are born prematurely and this population is at increased risk of cardiovascular disease (CVD) as they age. We hypothesized that compared with term‐born controls (CON), individuals born prematurely would have premature aortic stiffening, indicating greater CVD risk.

To test this hypothesis in humans, aortic pulse wave velocity (aPWV) was used to determine aortic stiffness in young adults born preterm between 1989 and 1991 (PT, 21–23 years) and age‐matched controls (CON). Intra‐arterial brachial artery pressure waveforms from 9 PT and 11 CON subjects were converted to central aortic pressure waveforms using a published transfer function. aPWV was then derived from the time delay between the forward and reflected waves and the effective reflecting distance, estimated based on individual anthropometric data. To test the hypothesis in a rodent model that perinatal supplemental oxygen (O2) promotes aortic stiffening, newborn rat pups were exposed to 80% O2 for 14 days, or 21% O2 as a control.

aPWV was higher in PT (10.1±2.6 m/s) compared with CON (7.2±0.8 m/s, p=0.025). aPWV did not correlate with systolic blood pressure in either group (PT R2=0.02, p=0.73; CON R2=0.17, p=0.20). At age 12 months, aortic stiffness, determined by aPWV, was higher in 80% O2‐exposed rats (487.1±27.8 cm/s) compared with controls (371.1±13.25 cm/s, p=0.02).

These data suggest that individuals born prematurely experience accelerated vascular aging of the aorta and that exposure to supplemental O2 as part of clinical care in premature infants may be a potential mechanism mediating this novel observation.

Support or Funding Information

University of Iowa Start‐Up Fund
Smooth Muscle Cells Are the Source of Heart Failure‐Causing Methylglyoxal in Diabetes Mellitus
Authors: Bidasee Keshore R , Alomar Fadhel , Fang Rui , Cox Jesse L , Rozanski George J , Singh Jaipaul
Published: November 2023

Heart failure/diabetic cardiomyopathy (DC) is an established case of morbidity and mortality in individuals with diabetes mellitus (DM). Yet, its molecular triggers remain undefined. Supra‐physiologic levels of the dicarbonyl species methylglyoxal (MG) has emerged as a leading candidate since it triggers several of the pathobiologies reported in DC. Why/how MG increases in the heart in DM remains undefined. Here left ventricular tissues from DM patients (Type 1 and Type 2 DM) and Type 1 DM rats were used to assess protein levels of the MG synthesizing and degradation enzymes, vascular adhesion protein‐1 (VAP‐1) and glyoxalase‐I (Glo‐I), respectively. Type 1 DM rats were also used to determine if targeted increase of Glo‐I in the heart would be cardio‐protective. In left ventricular tissues from patients and rats with DM, VAP‐1 was upregulated in smooth muscle cells (SMCs) and Glo‐I was reduced in SMCs and ventricular myocytes. MG and its metabolite argpyrimidine were also upregulated in left ventricular tissues. Increasing Glo‐I protein in SMCs and myocytes of Type 1 DM rats using a custom‐designed adeno‐associated virus blunted myocyte dysregulation and DC progression. Glo‐I restoration also attenuated VAP‐1 upregulation, lowered MG production, prevented microvascular leakage, and reduced mitochondria ROS generation, NF‐κB activation and TNF‐α production. It also blunted argpyrimidine modification and dysregulation of the sarcoplasmic reticulum Ca2+ cycling proteins, RyR2 and SERCA2a. From these new data we conclude that the myocyte‐dysregulating and heart failure‐causing increase in MG during DM arises from increased expression of VAP‐1 in ventricular SMCs and from decreased expression of Glo‐I in SMCs and ventricular myocytes. They also suggest that lowering SMCs‐generated MG could also be a new therapeutic strategy to blunt heart failure/DC development in DM.

Support or Funding Information

GTRP 1053, UNMC, UNeMED
Cavin‐3 deficiency reduces caveolae abundance in vascular and urinary bladder smooth muscle cells
Authors: Zhu Baoyi , Swärd Karl , Ekman Mari , Uvelius Bengt , Rippe Catarina
Published: November 2023

We investigated the effect of cavin‐3 (protein kinase C delta binding protein, Prkcdbp) deficiency on vascular and urinary bladder function. Small mesenteric arteries and the urinary bladder were isolated from mice lacking cavin‐3 (Prkcdbp−/−, knockout: KO) and from control littermates (Prkcdbp+/+, wild type: WT). One group of mice was used for blood pressure measurements, whereas another set of mice underwent bladder outlet obstruction followed by analysis of protein expression using western blotting. Our studies revealed that loss of cavin‐3 resulted in a 40% reduction of the caveola protein cavin‐1 (polymerase I and transcript release factor, Ptrf) in vascular and bladder smooth muscle. Electron microscopy demonstrated that loss of cavin‐3 was accompanied by a reduction of caveolae by 40% in smooth muscle, while the abundance of caveolae in endothelial cells was unchanged. Vascular contraction in response to cirazoline (α1‐adrenergic agonist) was normal, but nitric‐oxide dependent relaxation was enhanced. The latter effect was associated with elevated expression of soluble guanylyl cyclase, and yet blood pressure was similar in WT and KO mice. Contraction of the urinary bladder was not affected by loss of cavin‐3, and the proteomic response to outlet obstruction, including STAT3 (signal transducer and activator of transcription 3) phosphorylation, induction of synthetic markers, and repression of contractile markers were identical, the only exception being a curtailed induction of the Golgi protein GM130 (Golgi matrix protein 130 kD). In conclusion, loss of cavin‐3 thus reduces the number of caveolae in smooth muscle and partly destabilizes cavin‐1, but the functional consequences are modest and include an elevated vascular sensitivity to nitric oxide and a somewhat disturbed Golgi homeostasis in a situation of severe cellular stress.

Support or Funding Information

This study was funded by The Swedish Research Council (Vetenskapsrådet, http://www.vr.se/, K2015‐65X‐22662‐01‐3/KS), the Crafoord Foundation (http://en.crafoord.se/, CR), and the Royal Physiographic Society (Kungliga Fysiografiska Sällskapet, http://www.fysiografen.se/, CR).
Transient Receptor Potential Vanilloid 4 Channel Mediates Endothelium‐Dependent Vasodilation in Parenchymal Arterioles
Authors: Diaz‐Otero Janice M , Jackson William F , Dorrance Anne M
Published: November 2023

Chronic hypertension increases the risk of vascular dementia development and damage caused by cerebral ischemia. This may be partially the result of impaired endothelium‐dependent dilation of parenchymal arterioles (PAs) that regulate perfusion of the white matter. Transient receptor potential (TRP) channels such as the TRP vanilloid 4 (TRPV4) regulate endothelium‐dependent vasodilation and calcium homeostasis in peripheral arteries and large cerebral arteries. However, the role of TRPV4 in PA vasodilation has not been studied. We hypothesized that TRPV4 activation is a key regulator of endothelium‐dependent dilation in the PAs from normotensive and hypertensive rats. PAs vasodilation from 14–15 week old male normotensive Sprague Dawley (SD) rats and stroke‐prone spontaneously hypertensive rats (SHRSP) were studied by pressure myography. Data are presented as mean ± SEM. The presence of TRPV4 in the PAs was confirmed by immunostaining. The PAs from SHRSP had increased myogenic tone (30±5 vs 51±6 %tone, n=8; SD vs SHRSP; p = 0.02) and impaired carbachol (CbCh)‐induced dilation (35±7 vs 25±7 %dilation, n=4; SD vs SHRSP; p = 0.001) compared to the SD rats. After the development of myogenic tone (60 mmHg) PAs were incubated with the TRPV4 antagonist, GSK2193874 (GSK2, 10−7M) for 10 minutes, then CbCh‐mediated dilation (10−9–10−5M) was assessed. In normotensive rats, TRPV4 antagonism prevented the CbCh‐induced vasodilation (54±7 vs −7±14 %dilation at 10−5M, n = 4; CbCh vs CbCh+GSK2; p =0.008). In the SHRSP, inhibition of TRPV4 caused a loss of myogenic tone in the PAs (46±9 vs 23±12 %tone, n = 4; before vs. after GSK2; p = 0.1) that was not observed in SD rats, but this was not significantly different. TRPV4 antagonism also prevented endothelium‐dependent vasodilation in the PAs from SHRSP (46±1 vs −30±20 %dilation at 10−5M, n = 4; CbCh vs CbCh+GSK2; p = 0.02). These data show that TRPV4 plays an important role in mediating endothelium‐dependent dilation in cerebral arterioles from normotensive and hypertensive rats. TRPV4 may also contribute to augmented myogenic tone in SHRSP PAs, a role not observed in PAs from SD rats; this could be due to changes in the expression or activity of TRPV4 in the smooth muscle cells. Our data suggest that TRPV4 may have a critical role in mediating the hypertension‐associated changes in the vascular function of cerebral arterioles. The impaired vasodilation and changes in myogenic tone may compromise the regulation of cerebral blood flow during hypertension. TRPV4 could be a potential therapeutic target to improve vascular function of cerebral arterioles during hypertension and to decrease the risk of stroke and vascular dementia development that accompany hypertension.

Support or Funding Information

Supported by PO1‐HL070687
Effects of High Glucose and Major Metabolic Substrates on Alpha‐adrenergic Contractions in Rat Mesenteric and Deep Femoral Arteries
Authors: Yoo Hae Young , Vorn Rany
Published: November 2023

Hyperglycemia causes vascular damage and increased risk of cardiovascular diseases. It has been demonstrated that chronic exposure to high glucose impaired endothelial functions. However, specific effects of short‐term exposure to high glucose on vascular reactivity are controversial. Moreover, the combined effects of other metabolic substrates such as free fatty acids (FFA) on vascular reactivity remain poorly understood. Here we investigate the effects of short‐term exposure to high glucose with or without major metabolic substrates including FFAs termed “nutrition with fatty acid” (NF) solution, on mesenteric (MA) and deep femoral arteries (DFA) of rats. Arterial ring segments were mounted in a double‐wire myograph. Contraction in response to phenylephrine (PhE) was determined in control (5 mM) and high glucose (23 mM, HG) environments over a 30 min period. Our results showed that PhE‐induced vasocontraction was enhanced by pre‐incubation of HG solution in both arteries. A combined incubation with HG and palmitic acid (100 mM) induced similar sensitization of PhE‐contractions in both arteries. In contrast, high K+‐induced contractions were not affected by HG. Interestingly, pre‐incubation with NF solution decreased PhE‐induced contraction in DFA but increased the contraction in MA. In NF solution, the HG‐induced facilitation of PhE‐contraction was not observed in MA. Furthermore, the PhE‐induced contraction of DFA was attenuated by HG in NF solution. Our results demonstrate that the sensitization of PhE‐induced arterial contraction by HG is differentially affected by other metabolic substrates. The conversation of skeletal arterial contractility by HG in NF solution requires careful interpretation of the previous in vitro studies where only glucose is included in physiological salt solutions. Further studies are required to elucidate the mechanism underlying the inconsistent effect of NF solution on MA and DFA.

Support or Funding Information

This study was supported by a National Research Foundation of Korea grant funded by the Ministry of Science, ICT, & Future Planning (2015R1C1A1A01054038).
Impaired autophagy contributes to the vascular dysfunction in type 2 diabetes
Authors: Choi Soo‐Kyoung , Byeon Seonhee , Kwon Youngin , Lee Young‐Ho
Published: November 2023

Vascular dysfunction is a major complication in type 2 diabetes. It has been suggested that dysregulation of autophagy is associated with various cardiovascular diseases. However, the relationship between autophagy and type 2 diabetic vascular dysfunction remains unclear. Thus, in this study, we examined whether impaired autophagy is involved in vascular dysfunction of type diabetic mice. Ten to 12‐week old male type 2 diabetic (db−/db−) mice and their control (db−/db+) mice were used. The expressions of LC3□ and beclin‐1 (markers of autophagy) were decreased in mesenteric artery, aorta and heart from type 2 diabetic mice compared to control mice.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science, ICT & Future Planning) (No. 2016R1C1B1006504).
Comparisons of Proximal Aortic Pulse Wave Velocity Measurements: Carotid‐Femoral vs. Heart‐Brachial PWV
Authors: Sugawara Jun , Tomoto Tsubasa , Tanaka Hirofumi
Published: November 2023

The proximal aorta (e.g., the ascending aorta and aortic arch) is the most elastic segment of the arterial tree directly connected with the left ventricle and plays the dominant role in buffering the cyclic mechanical forces of cardiac pulsations. Carotid‐femoral pulse wave velocity (cfPWV) is considered as the reference standard measurement for large elastic artery stiffness but the most proximal segments of the aorta are omitted in its measurement. The primary aim of the present study was to compare two indices of arterial stiffness that differ in the contribution of proximal aortic segments. To do so, using the cross‐sectional study design, we compared cfPWV (mainly reflecting stiffness of the abdominal aorta) and heart‐brachial PWV (hbPWV; including the proximal aortic stiffness) in relation to aging‐related change and to central hemodynamic indices. In a total of 190 adults (92 men and 98 women) aged 53±15 yrs were studied. Pulse transition times were obtained between carotid and femoral pressure waveforms (via applanation tonometry) for cfPWV and between the second heart sound and the dicrotic notch of brachial pressure waveforms (via air‐plethysmography) for hbPWV, respectively. Arterial path lengths were measured directly by MRI. Central aortic hemodynamic indices were estimated from carotid arterial pressure waveform by general transfer function‐based pulse wave analysis. cfPWV and hbPWV were significantly correlated with age (r=0.68 and r=0.79, both P<0.0001). Aging‐related increases (calculated from the slope of regression lines) were 9.9%/decade in cfPWV and 14.7%/decade in hbPWV. Aortic augmentation index (an index of left ventricular after load) was significantly correlated with cfPWV (r=0.45) and more strongly with hbPWV (r=0.50). Likewise, aortic systolic tension‐time integral and diastolic pressure‐time integral (indices of myocardial oxygen demand and supply) were correlated with hcPWV (r=0.49 and r=0.53, P<0.0001 for both) and more strongly with hbPWV (r=0.50 and r=0.57, P<0.0001 for both). Our findings suggest that a measure of PWV including the ascending aorta exhibits a greater age‐related increase and stronger associations with central hemodynamic indices than cfPWV. Prospective data linking hbPWV to clinical endpoints are needed.

Support or Funding Information

This study was supported by JSPS KAKENHI Grant Number JP25702045 and JP26670116.
Role of Receptor Activator of Nuclear Factor kB Ligand (RANKL) in Coronary Smooth Muscle Contraction and Medial Calcification
Authors: Barnard April , Noblet Jillian , Goodwill Adam , Kiel Alex , Sturek Joshua , Tune Johnathan
Published: November 2023

Recent evidence indicates that coronary perivascular adipose tissue (PVAT) secretes osteogenic factors including receptor activator of nuclear factor kB ligand (RANKL) which may contribute to the initiation and progression of coronary calcification. This study was designed to examine the vascular effects of RANKL on vasomotor function and arterial calcification in ex vivo cultured epicardial coronary arteries from healthy swine. Isolated arterial rings (~3mm length) were cultured with or without RANKL (300 pM) or the RANKL endogenous antagonist osteoprotegrin (OPG; 30 pM) for acute, 2 or 8 days in osteogenic media (DMEM supplemented with 30% fetal bovine serum, 3.8 mM phosphate, 7.5 U/ml alkaline phosphatase). Isometric tension studies of acutely treated arteries demonstrated no effect of RANKL or OPG on coronary arteries contractions to KCl (0 – 60mM) or relaxations to bradykinin (1 nM – 1μM). In contrast, KCl contractions were attenuated ~25% by RANKL incubation for 2 days (P = 0.02) and 8 days of culture (P = 0.002). OPG exposure had no effect on contractile function of cultured rings. Vascular smooth muscle viability following 8 day of culture was verified by concentration‐dependent relaxation to adenosine (10nM – 10μM), which was similar in all treatment groups. Von Kossa staining revealed time dependent increases in medial coronary calcification following 2–8 days of culture with RANKL (P = 0.037). Coronary calcification in arteries exposed to OPG were similar to untreated time controls (P = 0.853). Taken together, these data suggest the potential of RANKL to promote trans‐differentiation and calcification of coronary vascular smooth muscle.
Acute nitrate supplementation lowers retrograde shear stress in post‐menopausal women
Authors: Somani Yasina , Kim Jin‐Kwang , Burke Olivia , Moore David , Grine Kristen , Jiang Dongsheng , Flanagan Michael , Proctor David
Published: November 2023

Purpose

Shear patterns in large conduit arteries are an important determinant of vascular health. More specifically, high levels of retrograde and oscillatory shear stress are associated with reduced endothelium‐mediated dilation. In the present study, we employed a double‐blind randomized cross‐over design to examine the effects of an acute dose of dietary nitrate supplementation on resting brachial artery shear patterns in post‐menopausal women.

Methods

Ten post‐menopausal women (age: 60–64 years; resting blood pressure: 125 ± 12/78 ± 10 mmHg) consumed beetroot juice (BrJnitrate; 140 mL) or a placebo (BrJplacebo; 140 mL of nitrate‐depleted beetroot juice, James White Beet‐It Sport; IND#119978) on two visits separated by one week. 120 minutes following juice consumption, brachial artery diameter and velocities were measured using Doppler ultrasound. Brachial artery diameter was assessed using edge detection software and oscillatory shear was calculated based on retrograde and antegrade shear rate.

Results

A lower retrograde shear rate was observed following BrJnitrate vs. BrJplacebo consumption (−4.2 ± 1.6 vs. −7.9 ± 1.9 s−1, p < 0.05). Although statistical significance was not reached, lower oscillatory shear was exhibited following BrJnitrate vs. BrJplacebo consumption (0.10 ± 0.03 vs. 0.21 ± 0.06 a.u., p = 0.055).

Conclusion

Our findings suggest that acute nitrate supplementation improves conduit shear patterns in older women, a population at heightened risk of cardiovascular disease. The lower retrograde shear stress, as well as the downward trend observed in oscillatory shear, suggests that nitrate supplementation may be vasoprotective in post‐menopausal women.

Support or Funding Information

Penn State Ambulatory Research Fund (Jaffe Endowment)
Impact of air temperature on core temperature regulation during exercise using a simulated burn injury model
Authors: Cramer Matthew N. , Moralez Gilbert , Kouda Ken , Gagnon Daniel , Crandall Craig G.
Published: November 2023

Following a burn injury, excision of injured skin and subsequent grafting lead to attenuated sweating rates and thereby a diminished capacity for evaporative heat loss, resulting in exacerbated elevations in core temperature and greater risk of heat illness during physical activities. Since the capacity for evaporation is dependent on the absolute area of non‐injured body surface area (BSA), and the heat load imposed on the body reflects both metabolic and environmental sources of heat gain, the detrimental effect of a burn injury on core temperature regulation during exercise at a given intensity is likely dependent on the interaction between the size of a burn injury and the prevailing air temperature. To test this possibility, four healthy males (23 ± 3 years, 80.1 ± 11.1 kg, 1.98 ± 0.15 m2) visited the laboratory on eight occasions to complete 1 h of cycling at a fixed rate of metabolic heat production (~6 W/kg; indirect calorimetry) in a 39°C or 24°C environment (20% relative humidity) with a simulated burn injury of 0%, 20%, 40%, or 60% BSA. Burn injuries were simulated by covering skin on the torso, arms, and legs with highly absorbent, vapor‐impermeable material that impedes sweat evaporation. Core temperature was measured in the gastrointestinal tract (Tgi). Elevations in Tgi at 24°C were not different across all simulated burn injury levels (0%: 0.81 ± 0.13°C, 20%: 0.70 ± 0.20°C, 40%: 0.78 ± 0.14°C, 60%: 0.65 ± 0.16°C; P ≥ 0.32). At 39°C, the increase in Tgi was not different between 0% (0.83 ± 0.24°C), 20% (0.96 ± 0.35°C), and 40% (0.99 ± 0.15°C) simulated burn injuries (P ≥ 0.60), but the rise in Tgi with a 60% simulated burn injury (1.84 ± 0.16°C) was greater than the other “injury” levels (P<0.05). Consistent with this observation, elevations in Tgi were exacerbated at 39°C versus 24°C only with a 60% simulated burn injury (P<0.01). In summary, core temperature reached similarly elevated levels following 1 h of moderate‐intensity exercise with simulated burn injuries of 20%, 40%, and 60% of BSA under temperate environmental conditions, and with simulated burn injuries of 20% and 40% in hot conditions. However, exercise in the heat at the same intensity with a 60% BSA simulated burn injury resulted in a greatly exacerbated elevation in Tgi. Therefore, burn survivors with injuries spanning 60% of total BSA may be exposed to a greater risk of hyperthermia and heat illness when working at a fixed intensity in a hot environment.

Support or Funding Information

Funding: Department of Defense – US Army, W81XWH‐15‐1‐0647
Weight Loss‐Induced Changes in Muscle Protein Turnover in Young Men: Effects of Dietary Protein Intake and Resistance Exercise
Authors: Hector Amy , McGlory Chris , Damas Felipe , Phillips Stuart
Published: November 2023

A negative consequence of diet‐induced weight loss is the loss of lean body mass (LBM), particularly muscle mass, which is induced by an imbalance between rates of muscle protein synthesis (MPS) and muscle protein breakdown (MPB). Increased protein intake and resistance exercise during diet‐induced weight loss promote retention of LBM [1] by stimulating MPS. Currently, the relative contribution of MPS to hypoenergetic diet‐induced LBM loss, and the effect of protein intake and resistance exercise on these variables is not well characterized. In a single blind investigation, adult men (n=24, BMI=28.6±0.6kg/m2, age=22±1y) underwent 10 days of a controlled diet with a 40% reduction in energy intake to create an energy deficit, while performing unilateral resistance exercise and consuming either lower protein (LP) (1.2g/kg/d protein, n=12) or higher protein (HP) (2.4g/kg/d protein, n=12). Pre‐ and post‐intervention testing included DXA, primed constant infusion of ring‐[13C6]‐phenylalanine to measure acute MPS in the fasted state and skeletal muscle biopsies to measure gene and protein expression. Total weight loss (−1.7±0.1kg) and fat mass loss (−0.7±0.1kg) did not differ between groups; however, appendicular lean mass of the exercised leg was preserved compared to the rested leg in both groups (exercise= −0.1±0.1kg, rest= −0.4±0.1kg, p<0.05). Mixed MPS was significantly reduced in both groups following weight loss (HP= −14±4% and LP= −27±2%, p<0.05). In the exercised leg, mixed MPS was preserved in the lower protein group and elevated above baseline in the higher protein group (14±6% p<0.05) 48 hrs following the last exercise bout. Gene expression of TRAF6 was increased in the rested leg of the lower protein group (p<0.05), but there was no change in TRAF6 protein expression at any time point in either group. Gene expression of other targets in the ubiquitin‐proteasome and autophagic‐lysosomal pathways (Atrogin‐1, MuRF‐1, Foxo3a, LC3, Gabarapl1, Ulk2, Beclin1) did not change with weight loss in either group. Protein expression of p‐Foxo3a (Ser253) and p‐TSC2 (Thr1462) also did not change with weight loss in either group. These results suggest that reductions in MPS are the likely reason for LBM loss during the early phase of diet‐induced weight loss. Resistance exercise can help to preserve (LP) or even elevate (HP) rates of fasted MPS, which, in concordance with our earlier work [1], promotes LBM retention.

Support or Funding Information

Supported by NSERC and OGS