bluuebunny/crossref_metadata_2025 huggingface dataset:
Attenuation of the Deleterious Course and Gastric Lesions after Bilateral Nephrectomy in Rats, NO‐system relation. BPC 157, L‐arginine, L‐NAME
Authors: Drmic Domagoj , Sucic Mario , Sever Anita Zenko , Kolenc Danijela , Andrijasevic Vedran , Suran Jelena , Seiwerth Sven , Sikiric Predrag
Published: November 2023

Aim

Chronic renal insufficiency might be associated with increased peptic ulcer presentation. Recently, stable gastric pentadecapeptide BPC 157 (an antiulcer peptide tested in clinical trials, for inflammatory bowel disease and multiple sclerosis, effective against a variety of different ulcerogens), counteracted gastric lesions and other disturbances, after bilateral nephrectomy in rats. Now, we demonstrate that this effect might be NO‐system related.

Materials and methods

Rats were subjected to bilateral nephrectomy, sacrificed either at 24h or 48h. Medication (/kg i.p.) BPC 157 (10μg, 10ng), L‐arginine (100mg), L‐NAME (5mg) alone and/or combined, was given immediately after nephrectomy. Assessment included gastric lesions, gross clinical presentation, urea, creatinine, AST, ALT, bilirubin, K, Na, Mg, Ca, inorganic phosphate, values in serum as described (Sikiric et al, Curr Pharm Des. 2014;20(7):1126–35) as well as brain lesion presentation, as described (Sikiric et al., Curr Neuropharm. 2015, in press).

Results

Comparison of gastric lesions (means±SD, sum of the longest lesions diameters, mm) in the nephrectomized rats (10.5±1.5 (24h), 15.0±3.0 (48h)), revealed that the gastric lesions were either consistently attenuated (vs. *P<0.05, at least) in BPC 157‐rats (1.8±0.5 (24h), 1.0±0.5 (48h)), L‐arginine‐rats (5.8±0.8 (24h), 5.0±0.7 (48h)), BPC 157+L‐arginine‐rats (0.8±0.5 (24h), 1.3±0.4 (48h)), L‐arginine+L‐NAME‐rats (4.9±0.4 (24h), 5.3±0.5 (48h)), BPC 157+L‐NAME‐rats (1.3±0.4 (24h), 1.2±0.5 (48h)), BPC 157+L‐arginine+L‐NAME‐rats (1.6±0.4 (24h), 1.4±0.3 (48h)) or aggravated (vs. *P<0.05, at least) in L‐NAME‐rats (20.5±1.7(24h), 25.0±2.0 (48h)). The influence (decreasing/increasing) on the otherwise increased values of the urea, creatinine, AST, inorganic phosphates as well as on rat edema formation (controls rats were edematous (scored 0–3) (Min/Med/Max, scored 3/3/3)) (attenuation/aggravation) was in accordance with the influence on gastric lesions(attenutation/aggravation) while K, Na, Mg, Ca values in serum were not affected.

Conclusions

Applied after nephrectomy, the otherwise deleterious course was attenuated after BPC 157, and/or L‐arginine administration, while they were aggravated by L‐NAME admisitration, an effect also counteracted by BPC 157 and L‐arginine, portraying it as NO‐related. Gastric lesions seem to be a particular indicator of the severity of the course of events which ensue after nephrectomy.

Support or Funding Information

This research was supported by Grant 108‐1083570‐3635, Ministry of Science, Education and Sports, Republic of Croatiaand by Grant BM099, University of Zagreb, Republic of Croatia.
Amygdaloid Corticotropin Releasing Factor as the Key Mediator of Chronic Visceral Pain in Females with a History of Early Life Stress
Authors: Prusator Dawn K , Meerveld Beverley Greenwood‐Van
Published: November 2023

In females. early life stress (ELS) has been identified as a risk factor for the development of functional pain disorders such as irritable bowel syndrome (IBS) in adults. Previously, our laboratory has shown that unpredictable ELS results in female specific heightened visceral pain behaviors in adulthood. The central amygdala (CeA) has been identified as a key brain nucleus in the processing of stress and pain, however, the role of the CeA in modulating pain following ELS has not been investigated. Therefore, we hypothesized that ELS increases visceral sensitivity in adulthood via altered CeA function, specifically through altered corticotropin releasing factor (CRF) expression.

Neonatal rats underwent classical conditioning using unpredictable odor‐shock pairings or odor only as a control. In adulthood, CRF expression in the CeA was quantified via qRT‐PCR. CRF knockdown occurred via cannulae implanted into the CeA for infusion of antisense or random sense oligodeoxynucleotides (ASO/RSO n=6–7/group). Visceral sensitivity was assessed via visceromotor response (VMR) quantified as the number of abdominal contractions in response to graded pressures (0–60mmHg) of isobaric colorectal distension (CRD). The role of CRF receptor 1 was assessed via administration of a selective CRF receptor 1 antagonist CP 376395 5μg/0.5μl administered bilaterally (n=6/group).

Expression of CRF was upregulated in the CeA following unpredictable ELS (p<0.01) compared to odor only controls. Administration of the RSO non‐targeting control sequence did not alter visceral hypersensitivity previously observed in the unpredictable group (60mmHg: 35.3±0.6) compared to odor only controls (26.2± 0.5). Knockdown of CRF in the CeA via ASO infusion significantly decreased the number of pain responses in unpredictable females as quantified by VMR to CRD (27.6±0.9, p< 0.01), while ASO treatment in the control group did not significantly alter pain responses (23.5±1.4). Administration of CRF1R antagonist mirrored the effects of CRF ASO infusion by significantly decreasing the VMR to CRD in adult females experiencing unpredictable ELS (26.8±1.1, p<0.01) to that of odor only control (27.0±1.1)

In summary, this study suggests a novel consequence of ELS on visceral pain responses in adulthood that are centrally driven via CRF and directly related to the context, predictable or unpredictable, of the ELS experienced. Furthermore, this study identifies for the first time a mechanism by which females develop chronic visceral pain following ELS.

Support or Funding Information

This work was supported by a department of Veterans Affairs Merit Grant to BG‐VM #01BX001195
Three‐dimensional Monolayer Stresses of Neutrophil Diapedesis
Authors: Yeh Yi‐Ting , Serrano Ricardo , Li Yi‐Shuan , del Alamo Juan Carlos , Chien Shu , Lasheras Juan Carlos
Published: November 2023

Neutrophil extravasation is a key step for immune defense and inflammation. Upon activation, neutrophils adhere firmly to the vascular endothelial cells (ECs), breach cell‐cell junctions, transmigrate across endothelium, and cross the basement membrane (BM) in the process of diapedesis. The biochemical mechanisms for diapedesis at EC junctional sites are well defined, but the biomechanical mechanisms remain largely undefined. The movement of neutrophils across the endothelium junction involves 3‐dimensional (3‐D) interactions, and hence it is important to understand the 3‐D cellular forces generated by neutrophil‐EC interaction in order to comprehend the biomechanical regulation of the diapedesis process. We hypothesize that the neutrophil‐EC interaction induces 3‐D cellular stresses to breach the cell junction, and serves as a crucial determinant for diapedesis. We have developed a 3‐D monolayer stress microscopy method (3‐D MSM) to measure and analyze the spatiotemporal evolution of 3‐D monolayer stresses during diapedesis. Our results show significant 3‐D deformations of the EC monolayer during neutrophil diapedesis that induce a mechanical disruption of the cell‐cell junctions, quantified with 3‐D MSM. The increase in barrier function induced by Angiopoietin‐1 and FGF treatments was associated with an augmentation of 3‐D displacements during diapedesis, whereas the negative modulation of junction integrity by thrombin treatment resulted in the reduction of 3‐D displacements. These results indicate that alterations of EC junction integrity lead to changes of diapedesis efficiency. By using the knockdown technique, we identified the adherens junction protein VE‐Cadherin as a key regulator for modulating EC junction integrity and the 3‐D displacements during diapedesis. In summary, we have developed a new approach to study mechanical regulation during diapedesis and provided new insights into the biomechanical mechanisms that control mechanical stress dynamics between neutrophils and the endothelium junction.
A new carbon monoxide (CO)‐releasing molecule (CORM401) suppresses PMN migratory potential through an F‐actin‐dependent mechanism
Authors: Inoue Ken , Patterson Eric , Chung Kyukwang , Potter Richard F , Capretta Alfredo , Fraser Douglas D , Cepinskas Gediminas
Published: November 2023

Background

Our previous work suggested that a newly synthesized Mn2+‐based water‐soluble carbon monoxide (CO)‐releasing molecules (CORM401) interfered with PMN recruitment by inhibiting transendothelial migration across the endothelium. In this study, we further investigated the effects of CORM401 on PMN chemotaxis by an F‐actin‐dependent mechanism.

Methods

Freshly isolated human PMN were placed into a μ‐Slide chemotaxis chamber and allowed to migrate in response to an fMLP (10μM) gradient for 50 min in the absence or presence of 100 μM CORM401 or inactive CORM401(iCORM401). PMN migration was recorded and analyzed for migration directionality and velocity. To assess the effect of CORM401 on fMLP‐stimulated PMN function, F‐actin polymerization was measured by flow cytometry. In addition, the phosphorylation of MAPK and p21‐activated kinases (PAK) in PMN were examined by immunoblotting.

Results

Pre‐treating PMN with CORM401 significantly reduced their directionality and mean velocity (p<0.01) in response to fMLP compared to controls. CORM401 significantly reduced F‐actin polymerization in fMLP‐stimulated PMN (p<0.01). Pre‐treating PMN with CORM401 did not result in decreased MAPK phosphorylation, but resulted in decreased PAK phosphorylation (p<0.05).

Conclusion

CORM401 suppresses neutrophil migratory potential through an F‐actin‐dependent mechanisms. This study showed for the first time that the use of CORM401 has an effect on F‐actin‐dependent mechanisms of PMN.

Support or Funding Information

HSFO 393, IRF‐25‐12
Administration of Metformin During Early Reperfusion Decreases Cardiac Injury Through Inhibition of MPTP Opening
Authors: Lesnefsky Edward , Thompson Jeremy , Hu Ying , Chen Qun
Published: November 2023

BACKGROUND

Ischemia damages the electron transport chain (ETC), including at complex I. The transient, reversible blockade of complex I during early reperfusion decreases cardiac injury. Our studies in isolated mitochondria showed that high dose Metformin (MET) partially inhibits complex I in mitochondria that were damaged by ischemia. In contrast to previous chronic pretreatment studies, we asked if partial blockade of complex I only at the onset of reperfusion using high dose MET (2 mM), without pretreatment, would decrease cardiac injury. Opening of the mitochondrial permeability transition pore (MPTP) is a key effector of ETC‐mediated injury during reperfusion.

METHODS

Buffer‐perfused mouse hearts (C57BL/6) were subjected to 25 min ischemia and 30 min (mitochondrial isolation) or 60 min (infarct size) reperfusion with or without MET (2 mM) treatment for the first 5 min. of reperfusion only. Infarct size was used to evaluate myocardial injury. Opening of MPTP was evaluated by calcium retention capacity (CRC, nmol Ca2+/mg) in isolated mitochondria. Oxidative phosphorylation (OXPHOS) was measured.

RESULTS

High dose MET treatment at reperfusion decreased infarct size and improved contractile function compared to untreated hearts. MET decreased susceptibility to MPTP opening (improved CRC). OXPHOS rate was not improved, since much of the damage occurs during ischemia, before MET treatment during reperfusion. Thus, MET treatment decreases MPTP opening during reperfusion.

CONCLUSION

Administration of MET only during reperfusion protects hearts during ischemia‐reperfusion by partial blockade of complex I with decreased MPTP opening. Regional treatment of myocardium with high dose MET in the cardiac cath lab at the time of stent deployment for acute myocardial infarction is a clinically translatable approach to cardiac protection via modulation of the ischemia‐damaged ETC during early reperfusion.

Support or Funding Information

This work was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs, a CCTR grant from Virginia Commonwealth University, a Scientist Development Grant from the American Heart Association and the Pauley Heart Center of Virginia Commonwealth University.

Isolated hearts
Infarct size
LVDP
LVEDP

IR
37±4
7±1
47±4

MET + IR
15±3*
18±8*
38±2*

Isolated mito
OXPHOS (nAO/min/mg)
CRC

substrates
Glu + Mal
Succ + Rot

TC (n=5)
403±39
530±34
723±37

IR (n=6)
254±30†
396±44†
350±12†

MET+IR (n=G)
216±25†
367±18†
467±22*†

Mean ± SEM; LVDP‐developed pressure mmHg; LVEDP‐end of diastolic pressure; Complex I substrates are glutamate (Glu) + malate (Mal); complex II substrates are succinate (Succ) + rotenone (ROT);

p<0.05 vs. ischemia‐reperfusion (IR);

p<0.05 vs. TC (time control).
Lymphatic Endothelial Cell Response to X‐Ray Irradiation
Authors: Narayanan Anand , Ford John , Zawieja David
Published: November 2023

INTRODUCTION

The lymphatics play a key role in the redistribution of interstitial fluids, cytokines, and immune cells via lymph formation. Severe impairment in lymphatic function causes swelling and inflammation, diagnosed as lymphedema. Secondary lymphedema is a known side effect cancer patients experience after radiation therapy; however, the pathogenesis is unknown. Furthermore astronauts develop edema while in space, being exposed to chronic, low‐dose, high LET background radiation. The lymphatics response radiation has not been extensively investigated. This is one of the first investigations characterizing lymphatic endothelium responses to radiation via viability and cell morphological measures.

METHODS

Primary, rat mesenteric lymphatic endothelial cells were exposed to a broad range of X‐ray (250 kVp, ~0.75 Gy/min) doses: 0, 0.5, 1, 1.5, 2, 4, 16 Gy, and travel sham. Cell viability was analyzed by XTT viability kit between 6–268 hours post‐irradiation at multiple time points. 24 and 72‐hours post‐irradiation, immunofluorescence was performed for β‐catenin, F‐actin, VE‐cadherin, DRAQ7, and measured by confocal microscopy (20× + 63× average z‐stack projections, 0.5 μm slices). No measurable differences were seen between travel sham and 0 Gy; therefore, statistical comparisons of treatment groups were made with 0 Gy group by 1‐way ANOVA and nonparametric Wilcoxon test (p<0.05).

RESULTS

Cell viability dropped in > 1 Gy groups at day 3, but became comparable to 0 Gy values after day 7. Cell morphometry showed radiation sensitivity at all doses. Notably there was a loss of cell‐to‐cell adhesion with irradiated cells increasing in surface area coverage, marked reduction in nuclei per equivalent field of view, and increased cellular roundness. This was coupled with decreased β‐catenin & VE‐cadherin intensity and altered F‐actin anisotropy leading to a loss of intercellular contact.

CONCLUSIONS

Lymphatics show radiation sensitivity in the context of these cell culture experiments. Our results may have functional implications of lymphatics in tissue, with potential endothelial barrier dysfunction leading to leakage and edema. These preliminary experiments will build the framework for future investigations towards clinical and space‐flight relevant lymphatic radiation exposure response.

Support or Funding Information

AN is supported by the NASA‐National Space Biomedical Research Institute Predoctoral Fellowship through NCC 9–58.
The Ca2+‐activated Cl− channel TMEM16A(ANO1) modulates, but is not required for, pacemaking in mouse lymphatic vessels
Authors: Gui Peichun , Zawieja Scott , Li Min , Bulley Simon , Jaggar Jonathan , Rock Jason , Davis Michael
Published: November 2023

An unidentified electrical pacemaker within the lymphatic vessel wall initiates the spontaneous contractions of lymphatic muscle (LM) required for normal lymph transport. The current hypothesis for pacemaking is that intracellular Ca2+ puffs within LM activate a Ca2+‐activated‐Cl− channel (CACC), initiating small depolarizations (STDs) that summate to trigger action potential (APs). The best candidate for the CACC is TMEM16A (ANO1), a channel implicated in pressure‐mediated constriction of vascular smooth muscle. We explored its role in the pacemaking of isolated, pressurized lymphatic vessels from mice, rats and humans. Contractions were always preceded by APs, which were similar in shape among all three species, pointing to a common ionic mechanism. AP firing was preceded not by STDs but by slow, constant diastolic depolarization. Ca2+ puffs/waves occurred spontaneously in many LM cells, but were not synchronized between cells or correlated with contractions. Ca2+ entry following the AP spike was mediated by L‐ and T‐type Ca2+ channels (VGCCs), generating a ~synchronized, global Ca2+ flash over the entire vessel. SERCA inhibitors abolished puffs, but not flashes or pacemaking, whereas nifedipine abolished flashes and pacemaking. TMEM16AGFP mice expressed GFP in every LM cell, as confirmed by PCR and Western blotting. Whole cell recordings from mouse LM cells revealed an outwardly‐rectifying Ca2+‐activated current, with a reversal potential around zero in symmetric [Cl−] solutions that shifted with changes in the Cl− equilibrium potential. Both broad‐spectrum (niflumic acid, DIDS) and “selective” (T16A‐01) CACC inhibitors were of limited value as they inhibited VGCCs at concentrations lower than those required to block TMEM16A. We therefore used an inducible tissue‐specific mouse model to delete TMEM16A from LM. SMMHC‐CreERt2;Rosa26mTmG mice were bred to determine the efficiency of Cre expression in LM, which was >95% in inguinal‐axillary lymphatics and ~99% in popliteal lymphatics. LM cells from SMMHC‐CreERt2;TMEM16Afl/fl mice showed significantly reduced levels of whole‐cell Cl− current compared to TMEM16Afl/fl controls. Spontaneous contractions persisted in pressurized lymphatics from SMMHC‐CreERt2; TMEM16Afl/fl mice, albeit at a significantly reduced frequency, and frequency did not increase normally with pressure elevation. These findings suggest that pacemaking is controlled by the interaction of several LM plasma membrane ionic currents rather than an intracellular Ca2+ event, but that TMEM16A contributes to a baseline excitatory current.

Support or Funding Information

NIH R01 HL‐122578
microRNA‐204, SIRTUIN1, and Caveolin‐1 regulate vascular endoplasmic reticulum stress and endothelium‐dependent vasorelaxation
Authors: Kassan Modar , Vikram Ajit , Li Qiuxia , Kim Young‐Rae , Kumar Santosh , Jacobs Julia , Irani kaikobad
Published: November 2023

Background

Endoplasmic reticulum (ER) stress leads to vascular endothelial dysfunction in disease states such as obesity, diabetes and hypertension. The role of microRNAs in vascular ER stress is not fully understood. Here we examined the role of vascular microRNA‐204 (miR‐204) in inducing endothelial ER stress and dysfunction by governing the expression of the SIRTUIN1 (SIRT1) lysine deacetylase and the Caveolin‐1 (Cav1).

Methods & results

miR‐204 mimic decreased expression of SIRT1 and Cav1, and promoted ER stress in endothelial cells. Tunicamycin induced endothelial ER stress was also mediated by miR‐204. Moreover, knockdown of SIRT1 induced ER stress and reduced Cav1 in endothelial cells, both of which were rescued by a miR‐204 antagomir. Tunicamycin‐induced ER stress upregulated miR‐204, and downregulated SIRT1 and Cav1, in endothelial cells. Similarly and in endothelium of aortas and mesenteric arteries of mice. Antagonism of miR‐204, or SIRT1 overexpression, rescued endothelial cells from tunicamycin‐induced ER stress. Similarly, antagonism of miR‐204 in vivo with anti‐miR‐204 mitigated tunicamycin‐induced vascular ER stress, decline in vascular SIRT1 and Cav1, and impairment of endothelium‐dependent vasorelaxation. Moreover, tunicamycin‐induced vascular ER stress and endothelial dysfunction were exacerbated in mice with conditional deletion of endothelial SIRT1. Additionally, overexpressing Cav1 ex‐vivo, with adenovirus, in tunicamycin‐induced vascular ER stress or in mice with conditional deletion of endothelial SIRT1, reduced ER stress markers and miR‐204 in endothelial cells and was associated with an improvement in vascular endothelial dependent relaxation. Finally, Vascular miR‐204 was upregulated with high‐fat diet feeding, anti‐miR‐204 prevented vascular ER stress stimulated by high‐fat diet feeding.

Conclusions

These findings show an important role for miR‐204 as a mediator of vascular ER stress, and ER stress‐induced endothelial dysfunction, via downregulation of endothelial SIRT1 and cav1.

Support or Funding Information

T32‐ Institutional Training Grant (HL007121)
Macrophages Drive Microvascular Dysfunction in Obesity by Depleting Vessel Hydrogen Sulfide
Authors: Candela Joseph John , White Carl
Published: November 2023

Reduction in endogenous hydrogen sulfide (H2S) has emerged as a contributor to cardiovascular diseases. Its role in obesity‐induced microvasculature dysfunction is not known. Obesity is associated with chronic inflammation and recruitment of macrophages to the Perivascular Adipose Tissue (PVAT). This study was designed to test the hypothesis that vasorelaxation of the microvasculature is impaired in a diet‐induced mouse model of obesity via macrophages impinging on H2S in the microvasculature. Mesenteric arterioles were isolated from lean and obese mice and mounted in a pressure myography chamber. Vascular function was assessed using phenylephrine and acetylcholine. Vessels were loaded with the H2S indicator SF7‐AM and smooth muscle and endothelial layers imaged. The phenotype of macrophages was assessed using flow cytometry. Macrophages from obese mice took on an M1 phenotype compared to lean controls. Arterioles from obese mice showed lower concentrations of H2S compared to lean mice. Vasorelaxation in response to acetylcholine was impaired in arterioles from obese mice compared to lean mice which were reversed with exogenous H2S. Co‐culturing macrophages from obese mice with arterioles from lean or obese mice resulted in decreased endogenous hydrogen sulfide levels in the arterioles compared to when they were cultured with macrophages from lean controls. Arterioles from lean or obese mice showed impaired vasorelaxation when cultured with macrophages from obese mice compared to when they were cultured with macrophages from lean mice. These data supports a model in which H2S biosynthesis is compromised in the obese microvasculature via crosstalk with macrophages which results in impaired vessel function.

Support or Funding Information

Supported by the American Heart Association and a Chicago Medical School/Advocate Lutheran General Hospital Translational Research Pilot Grant.
Changes in miRNA expression patterns and vascular aging phenotypes in mice with developmental liver‐specific knockdown of IGF‐1
Authors: Tarantini Stefano , Giles Cory B , Wren Jonathan D , Ashpole Nicole M , Valcarcel‐Ares Marta Noa , Tucsek Zsuzsanna , Sonntag William E , Csiszar Anna , Ungvari Zoltan
Published: November 2023

The endocrine changes that occur during development are highly conserved across mammalian species and include dramatic increases in the anabolic hormone IGF‐1 during adolescence. There is increasing experimental and clinical evidence that alterations in IGF‐1 levels during development regulate multiple aspects of the aging process and affect the incidence of multiple age‐related diseases, including cardiovascular and cerebrovascular diseases. The present study was designed to characterize the effect of developmental IGF‐1 deficiency on the vascular aging phenotype. To achieve that goal early‐onset, isolated endocrine IGF‐1 deficiency was induced mice by developmental knockdown of IGF‐1 specifically in the liver using Cre‐lox technology (Igf1f/f mice crossed with mice expressing albumin‐driven Cre recombinase). The animals were studied at an age representing ~75% of maximal lifespan potential, which corresponds to the biological age of a ~67 year old human. To assess vascular health endothelium‐dependent vasorelaxation and vascular ROS production were tested. Due to the emergence of miRNAs as important regulators of vascular aging phenotype, miRNA expression profile in the aorta of mice with developmental IGF‐1 deficiency was also tested. We found that aging was associated with endothelial dysfunction, which was unaffected by developmental IGF‐1 deficiency. We identified 19 miRNAs that were differentially expressed in the aortas of aged mice and 11 miRNAs that were differentially expressed miRNAs in aortas of mice with developmental IGF‐1 deficiency. GO terms enriched among predicted targets for miRNAs differentially expressed with age in the aorta include cytokine production, chromatin silencing, intracellular protein transport and regulation of apoptotic cell death. GO terms enriched among predicted targets for miRNAs differentially expressed with developmental IGF‐1 deficiency in the aorta include regulation of apoptotic cell death, blood vessel remodeling and intracellular protein transport. Collectively, developmental IGF‐1 deficiency is associated with a vascular miRNA expression profile later in life that likely plays a role in development of age‐related vascular pathologies.

Support or Funding Information

This work was supported by grants from the American Heart Association and the National Institute on Aging
Neuromodulation of Cranial Nerves for Migraine and Trigeminal Neuropathy Pain: Cardiac Effects
Authors: White Carter R. , Snodgrass Danielle , Yazdizadeh Maryam , Yan‐Go Frisca , Jen Joanna , Harper Rebecca K. , Sauerland Eberhardt K. , Harper Ronald M.
Published: November 2023

Neuromodulation of afferent cranial nerve activity is useful for alleviating migraine and trigeminal pain, which is often poorly managed by pharmacologic interventions. Synchronous stimulation of trigeminal, vagal, glossopharyngeal and cervical nerve afferents shows promise for such intervention, but potential effects on the cardiovascular system must be considered, since several of the cranial nerves exert significant effects on heart rate and blood pressure. The objective was to determine changes in heart rate (HR), HR variability, and blood pressure at two stimulation amplitude levels to cranial nerves. Fourteen subjects with confirmed migraine or trigeminal neuropathy pain were fitted with vibrating devices placed in the ear canals bilaterally. Heart rate (beats/min), inter‐beat intervals (RR, msec), beat‐by‐beat systolic and diastolic blood pressures (mm Hg) were measured continuously during baseline, and during low and high amplitude, and post stimulation periods. Mean HR declined during low and high stimulation; baseline, low, high, and post‐session were 76, 73, 72, 74 bpm respectively, p < .001 (Single‐Factor Repeated Measures ANOVA), with lengthening of mean RR intervals (802, 829, 839, 821 msec, respectively). HR and RR intervals recovered during the post stimulation period to pre‐session values. Mean systolic and diastolic blood pressure declined with stimulation (mean systolic: 130, 124, 125, and 130 mm Hg, respectively; mean diastolic: 79, 77, 77, and 79 mm Hg, respectively), and recovered post‐stimulation. HR variability patterns changed from pronounced baseline variation from respiratory and slower variation influences to reduced slower, but increased respiratory‐related variation, representing a shift to parasympathetic influences during stimulation; these values recovered post‐stimulation. No instances of potentially dangerous bradycardia or tachycardia, or extreme changes in blood pressure appeared. We conclude that HR, HR variability, and blood pressure change during stimulation, and move toward values considered “protective.”

Support or Funding Information

Private Funding was used for this project.
Hemodynamic effects of Enalapril and B‐type natriuretic peptide on the progression of pacing induced cardiac myopathy in conscious dogs
Authors: Qanud Khaled , Shnayderman Nadine , Ochoa Manuel , Xu Xiaobin , Hintze Thomas
Published: November 2023

Purpose

Enalapril and B‐type natriuretic peptide (BNP) are used in cardiovascular clinical practice as a common anti‐hypertensive drugs and a diagnostic marker of heart failure (HF). BNP is secreted from ventricular cardiomyocytes in response to high cardiac filling pressures and has anti‐hypertensive actions by stimulating natriuresis and diuresis. Typically, BNP is elevated in HF but studies suggest that a resistance to endogenous BNP causes this. Thus BNP could be potentially therapeutic as an exogenous source. Few studies have elucidated the changes in hemodynamics and echocardiographic parameters and the outcome on the pulmonary component during HF. Here we tested whether the above mentioned parameters could be favorably affected by Enalapril, BNP and the possible combined treatment resulting in slowing the progression or improving the tolerance to HF.

Methods

Twelve chronically instrumented dogs underwent left ventricular pacing at 210 beats/min for 3 weeks and at 240 beat/min thereafter. Starting at day 10 of pacing, the dogs were randomized into three groups to receive either Enalapril (1 mg/kg) orally, BNP (5–25 mg/kg) by sc injection, or placebo. The transitions from compensated dysfunction to HF occurred between the 3rd and 4th week of pacing, with end‐stage HF occurring at 28±1 days.

Results

We found that dP/dtmax, LVEDP and arterial BP remained unchanged between the control and treated groups. However, Arterial PO2 was better preserved in the Enalapril group compared to the placebo group. Arterial PO2 typically falls during the decompensated stages of HF and is indicative of pulmonary edema. By week 4, arterial PO2 was significantly higher in the Enalapril group (88±2.8 mmHg) compared to the placebo group (78±3.0 mmHg, p<0.05). Echocardiography showed that EF was significantly maintained in the Enalapril group compared to the BNP (weeks 1, 3) and placebo (weeks 2, 3) groups indicating that, although small, the EF may be better maintained. At week 3, EF was 37±2.5% and 39±1.6% for the BNP and placebo groups, respectively, whereas the Enalapril group had an EF of 45±1.6% (p<0.05). In addition, end systolic diameters were significantly decreased with Enalapril compared to the placebo (weeks 2–4). No significant hemodynamic or echocardiographic changes were detected in comparison of compensated dysfunction and end stage heart failure of the groups.

Conclusion

Our data suggests that Enalapril and BNP treated dogs maintained a preserved cardiac systolic function and hemodynamic tolerance during end stage HF, as reflected by clinical parameters and blood values (PO2). Although the data did not reflect significant changes between BNP and placebo, it did not worsen the progression of HF and could be considered safe.
Medical Device Materials and Blood Pressure Alterations in Mice
Authors: Swiercz Adam , Sherman Meredith , Muselimyan Narine , Swift Luther , Marvar Paul , Posnack Nikki Gillum
Published: November 2023

Di‐2‐ethylhexyl phthalate (DEHP) is a plasticizer that is used to impart flexibility to polyvinyl chloride products. Patients have an increased exposure to phthalates through contact with DEHP‐containing medical devices, including: storage bags containing blood, plasma, intravenous fluids, total parenteral nutrition, tubing associated with their administration, nasogastric tubes, enteral feeding tubes, catheters, extracorporeal membrane oxygenation (ECMO) circuits, hemodialysis tubing, respiratory masks and endotracheal tubes. Human health concerns pertaining to DEHP exposure are linked to its endocrine‐disrupting properties. Accordingly, increased exposure has been associated with cancer, metabolic disturbances, reproductive and neurological disorders, and cardiovascular disease. As an example, epidemiological studies have shown a link between DEHP exposure and elevated systolic blood pressure in adolescents. Despite bans and restrictions on the use of DEHP‐containing medical devices in other countries, there is currently no mandate from the Food & Drug Administration for the use of DEHP‐free devices and storage containers. The objective of this study was to quantify the impact of in vivo DEHP exposure on cardiovascular function; thereby, providing additional information for regulatory decisions by the scientific, medical and regulatory community. Healthy C57BL/6 male mice were implanted with radiotelemetry transmitters; briefly, the transmitter catheter was placed in the carotid artery and biopotential leads were routed subcutaneously to collect electrocardiogram (ECG) signals. After surgical recovery, pre‐exposure data was collected, and thereafter, animals were exposed to 0.2 mg/g DEHP or control diet for 4‐weeks. We observed a significant increase in systolic pressure in DEHP‐treated (145 ± 3 mmHg) vs control animals (136 ± 1 mmHg). We also detected an increase in diastolic and mean arterial pressure in DEHP‐treated (119 ± 5 and 132 ± 3 mmHg, respectively) vs control animals (107 ± 2 and 121 ± 2 mmHg). Our previous reports have shown that DEHP diminishes cardiac contractility, which suggests that these effects on blood pressure are likely attributed to alterations in parasympathetic tone and/or an increase in vascular resistance.

Support or Funding Information

This work was supported by the National Institutes of Health (K99ES023477) and the Clinical and Translational Institute at Children's National (UL1TR000075 and KL2TR000076, from the NIH National Center for Advancing Translational Sciences).
Daunorubicin‐induced heart failure elevates hippocampal TNF‐a and caveolin‐1 in rabbits
Authors: Zhang Zheng , Alas Basheer , Zemljic‐Harpf Alice , Ichikawa Yasuhiro , MicKirnan M. Dan , Hammond H. Kirk , Patel Hemal H. , Roth David M. , Head Brian P.
Published: November 2023

Introduction

Anthracyclines are chemotherapy agents used to treat various cancers. Daunorubicin (Dau), a member of the anthracycline family, is cardiotoxic leading to heart failure. Heart failure, resulting in reduced cardiac output, affects multiple organs including the brain. Cognitive and mood impairments including short‐term memory loss and depression as well as changes in hippocampal structure have been observed in heart failure patients. It has been reported that tumor necrosis factor‐α (TNF‐α) is elevated in the brains of heart failure rats which indicates the critical role TNF‐α plays in neuroinflammation in heart failure. We have shown a protective role for caveolin‐1 (cav‐1) in neuronal injury and cav‐1 has been reported to be elevated by TNF‐α in endothelial cells. Here in the Dau induced heart failure rabbits, we probed the expression of hippocampal TNF‐α and its downstream singling molecules including caspase‐3, NF‐kB, phosphorylated NF‐kB and cav‐1.

Methods

New Zealand rabbits were administered weekly, intravenous Dau injections for 9 weeks at a dose of 3 mg/kg. Echocardiography was performed pre‐injection and weekly six weeks after the initial injection. Left Ventricle Fractional Shortening (LVFS) was measured by echocardiography. Rabbits were sacrificed when LVFS fell below 20%, hippocampi were collected, and homogenized. Western blots were performed to measure TNF‐α, caspase‐3, NF‐kB, phosphorylated NF‐kB and caveolin‐1 expression. Liver and lung wet/dry ratio were measured as indications of systemic and pulmonary congestion, as is expected in heart failure.

Results

LVFS was significantly reduced in Dau treated animals (control; 35%, n=4 vs Dau; 16%, n=4, p<0.001). The wet/dry ratio of lung (control; 2.3, n=4 vs Dau; 4.0, n=4, p<0.05) and liver (control; 2.5, n=4 vs Dau; 3.4, n=4, p=0.001) was higher in the Dau treated group. Western blots showed that the Dau treated group (n=4) had higher TNF‐α (p<0.05) and cav‐1 (p=0.004) in the hippocampus compared to the control group (n=4). No significant changes of caspase‐3, NF‐kB, or phosphorylated NF‐kB were observed.

Conclusions

Our data show for the first time that Daunorubicin induced‐heart failure elevates hippocampal TNF‐α and cav‐1 protein expression.
Doxorubicin may damage cardiomyocytes more than cardiac progenitor cells
Authors: Edwards John , Mitry Maria , Laurent Dimtri
Published: November 2023

Long term survival of childhood cancers is now more than 70%. Unfortunately, adult survivors of childhood cancer are at risk for treatment‐related adverse health outcomes. Survivors with a median time from diagnosis of 25 years found 56% had cardiac dysfunction. Analysis of heart transplantation patients found doxorubicin as the underlying cause in 2–3% of all cases. Doxorubicin is a topoisomerase poison that accumulates in the nucleus and the mitochondria. Doxorubicin impacts heavily on the mitochondria as significant mtDNA depletion and mtDNA rearrangements have been reported. Unfortunately nearly all preclinical studies have only determined short effects. Very few studies have examined the long term effects of doxorubicin or used doses that were comparable to clinical treatment protocols. We have developed a murine model for late onset doxorubicin induced heart failure in which the onset was delayed and mimicked that seen clinically (~18 human years). Doxorubicin was injected (1–45 mg/kg, i.p.) at 3 weeks of age. We observed no change in cardiovascular function 2–4 months post‐injection, but a significant decrease in ejection shortening was observed after 6–8 months post‐injection. Low doses of doxorubicin (1–15 mg/kg) did not significantly alter cardiovascular function even out to 16 months post injection. Using flow cytometry we examined cell populations from the left ventricle, circulating non‐RBC cells, and bone marrow. Despite degradation of cardiovascular function we observed significant increases (p<.05) in c‐kit+, SSEA1+, and Nkx2.5+ cells within the heart. In contrast, significant decreases of the c‐kit+, SSEA1+ cells were found in the circulating cells, while no changes were observed in the cell populations from the bone marrow compartment. We assessed mtDNA damage and observed significant increases in mtDNA damage in the ventricular cells and but not ventricular c‐kit+ cells. In cultured neonatal cardiomyocytes, doxorubicin increased mtDNA damage and promoted cell death below the threshold that significantly increase intramitochondrial superoxide levels. Measuring mitochondrial topoisomerase function we observed that H2O2 significantly increased DNA relaxation of supercoiled DNA suggesting that low levels of ROS alter mitochondrial topoisomerase function to promote DNA strand breakage. These findings suggest the heart attempted to regenerate by increased replacement of healthy cardiac progenitor cells but that degradation cardiomyocytes may have been the underlying cause of cardiovascular dysfunction. This suggests that the ability of the heart to direct self repair may be limited and that although the stem cell compartment attempted to compensate for cardiomyocyte loss, the degradation of cardiac function indicated that it was not successful.

Support or Funding Information

Supported in part by NIH RO3HD065551
The Adhesive Epidermal Growth Factor (EGF) Repeats of Platelet SCUBE1 Are Critical for Thrombus Formation
Authors: Wu Meng‐Ying , Liao Wei‐Ju , Yang Ruey‐Bing
Published: November 2023

SCUBE1 (signal peptide‐CUB‐EGF domain‐containing protein 1) is the founding member of a secreted and member‐tethered SCUBE protein family. SCUBEs are composed of ~ 1,000 amino acids with 5 domains: an NH2‐terminal signal peptide sequence, 9 tandem repeats of EGF‐like motifs, a large N‐glycosylated spacer region followed by 3 cysteine‐rich repeats and one CUB domain at the COOH terminus. We previously showed that SCUBE1, a plasma and platelet‐associated protein, might be involved in platelet‐platelet and platelet‐subendothelial matrix interactions through its adhesive EGF‐like motifs in vitro (Cardiovasc Res., 71:486–95, 2006), and genetic ablation of plasma SCUBE1 protected mice against thrombosis (Arterioscler Thromb Vasc Biol., 34:1390–8, 2014). However, whether the adhesive module of the EGF‐like repeats present in platelet SCUBE is essential for platelet adhesion and thrombosis remains largely unknown in vivo. Here, we generated a new mutant (Δ2) mice lacking the EGF‐like repeats to evaluate its functional importance in platelet biology in vivo. While the Δ2‐deficient mice had normal hematological parameters and expression of major platelet receptors, the Δ2 animals showed impaired platelet aggregation induced by ADP, collagen, and thrombin and protected against lethal thromboembolism. In flow chamber assay, washed Δ2 platelets showed marked decrease in its adhesion, aggregation and thrombus formation on collagen‐coated surface under arterial shear conditions. Most importantly, hematopoietic reconstitution of +/+ bone marrow into Δ2/Δ2 mice restore the platelet adhesive function and thrombus formation in vivo. Together, our data demonstrate for the first time that the platelet‐derived SCUBE1 plays a critical role in arterial thrombosis via its adhesive EGF‐like module in vivo, and suggest targeting these adhesive domains of SCUBE1 as potential anti‐thrombotic strategy.
Role of Metabolic and Inflammatory Biomarkers on Femoral Artery Responsiveness in Trained Rats Fed with Hight‐Fat Diet
Authors: Delbin Maria A , Sponton Amanda CS , Araujo Hygor N , da Silva Carmem P Valgas , de Moraes Camila , Zanesco Angelina
Published: November 2023

Atherosclerosis is considered the main cause of peripheral artery disease (PAD). In addition, exercise training has been prescribed to patients with PAD to diminish the pain and to improve blood flow. However, the insight mechanisms of these beneficial effects are not clear. The aim of this study was to examine the femoral artery responsiveness in trained rats fed with high‐fat diet (CEUA 3203‐1). Male Wistar rats were divided into: sedentary (SD), high‐fat diet sedentary (HF‐SD) and high‐fat diet trained (HF‐TR) groups. For sixteen weeks, animals from SD group were fed with standard chow and animals from HF‐SD and HF‐TR were fed with high‐fat diet (56% carbohydrate, 18% protein and 26% fat). After eight weeks, HF‐TR initiated the aerobic exercise training (AET) on treadmill, 5 days/week, 60 min/session and at 50–60% of maximum speed. Concentration‐response curves (CRC) to acetylcholine (ACh), sodium nitroprusside (SNP), phenylephrine (PHE) and the thromboxane A2 analogue (U46619) were obtained in femoral artery. Vascular protein expressions of eNOS, Cu/Zn‐SOD, Mn‐SOD, Ec‐SOD, adiponectin and leptin receptors were determined. Vascular nitric oxide (NO) production and reactive oxygen species (ROS) generation were evaluated. Plama inflammatory biomarkers were also evaluated. AET was effective in reducing body weight and epididymal fat. The maximal response (EMAX) in the CRC to ACh was reduced in femoral artery from HF‐SD group (17%) that was accompanied by reduced eNOS (40%) and Cu/Zn‐SOD (30%) protein expressions as well as NO production, associated with an increase in ROS generation. A positive effect was observed in HF‐TR group on relaxing response to ACh as well as the eNOS and Cu/Zn‐SOD protein expressions were restored. Increased NO production and reduction in ROS generation were also found. No changes in CRC to SNP, PHE and U46619 were observed in all groups. Regarding metabolic parameters, insulin concentration was restored at control values in trained animals (SD: 3±1; HF‐SD: 6±1; HF‐TR: 3±0.5 ng/ml). Inflammatory mediators were also normalized in HF‐TR group (leptin: 9±2 ng/ml; TNF‐α: 0.25±0.02 pg/ml; MCP‐1: 48±12 pg/ml and PAI‐1: 23±9 pg/ml) compared with HF‐SD (leptin: 41±9; TNF‐α: 0.41±0.04, MCP‐1: 138±23 and PAI‐1: 120±23). In contrast, blood glucose, total cholesterol and adiponectin were not different in all groups. Interestingly, the protein expression of leptin receptor was significantly reduced in HF‐SD (30%) group and normalized in HF‐TR group. Our findings show that AET for eight weeks promoted protective effects on metabolic and inflammatory mediators that it was reflected in improvement of endothelium‐dependent relaxation in femoral artery from rats fed high fat‐diet.

Support or Funding Information

CNPq and FAPESP
Putative Sarcomeric Targets of Doxorubicin Induced Matrix Metalloproteinase‐2 Activity in Cardiomyocyctes
Authors: Roczkowsky Andrej , Chan Brandon Y.H. , Hughes Bryan G. , Schulz Richard
Published: November 2023

Anthracyclines, such as doxorubicin, are used to treat a variety of cancers. However, doxorubicin's therapeutic utility is limited because it causes dose‐dependent myocardial injury, which may lead to heart failure in up to 11% of patients. Doxorubicin triggers this injury, in part, by stimulating cellular oxidative stress. Oxidative stress is known to activate matrix metalloproteinase‐2 (MMP‐2), an extra‐ and intra‐cellular protease, which has been implicated in many heart pathologies. Activated intracellular MMP‐2 has been shown to proteolyze sarcomeric proteins α‐actinin, troponin I, and the regulatory protein glycogen synthase kinase‐3β (GSK‐3β) in cardiac tissue or cells under oxidative stress, impairing contractile function. We hypothesized that intracellular MMP‐2 plays a role in doxorubicin cardiotoxicity by proteolyzing these substrates. Neonatal rat ventricular myocytes (NRVM) and human fibrosarcoma (HT1080) cells were treated with doxorubicin (0.5 μM) ± selective MMP‐2 inhibitors ARP‐100 (1 μM) or ONO‐4817 (1 μM) for 2–48 hr. Cell death was evaluated by lactate dehydrogenase release, MMP‐2 activity was measured by gelatin zymography, and MMP‐2 protein and its protein target levels were analyzed by immunoblot. Doxorubicin was used at a concentration which caused 15% cell death in neoplastic HT1080 cells but none in NRVM after 24 hr. In NRVMs, 24 hr doxorubicin increased intracellular MMP‐2 activity up to 311% relative to vehicle control, and this was attenuated by 60% with ARP‐100 or ONO‐4817. 24 hr doxorubicin‐induced MMP‐2 activation is caused, in part, by a 213% increase in MMP‐2 protein levels. Doxorubicin reduced troponin I levels by 40%, however, this was not normalized by ARP‐100 or ONO‐4817. There were no significant changes in α‐actinin or GSK‐3β levels. Doxorubicin at an antitumour concentration activates myocardial MMP‐2 in part by increasing MMP‐2 protein levels. The role of doxorubicin‐induced MMP‐2 activation on the reduction of cellular troponin I levels needs further investigation. Other potential MMP‐2 targets in the sarcomere (eg. titin, myosin light chain‐1) and in other cellular compartments need to be analyzed to better understand the pathophysiological actions of doxorubicin in cardiac myocytes.

Support or Funding Information

Canadian Institutes of Health Research Foundation Scheme Grant (to RS); Women and Children's Health Research Institute Summer Studentship (to AR).
Nitric Oxide Synthase and Soluble Guanylyl Cyclase Mediate Endothelial Responses in Aged Female Endothelial Nitric Oxide Synthase (eNOS)‐Deficient Mice
Authors: Didion Sean P.
Published: November 2023

We have shown previously that endothelial responses are normal in adult heterozygous eNOS deficient male and female mice and with aging endothelial function is markedly impaired in male eNOS+/−, but not female eNOS+/−, mice. The goal of the present study was to determine whether the preserved responses observed in aged female heterozygous eNOS+/− mice are due to NOS‐ and soluble guanylyl cyclase (sGC)‐dependent mechanisms. Endothelium‐dependent and ‐independent responses of carotid arteries from adult (6 months of age) and old (22–24 months of age) female eNOS+/+ and eNOS +/− mice were examined in the presence of vehicle (saline), L‐NNA (a NOS inhibitor), or ODQ (an inhibitor of sGC). Carotid artery responses to acetylcholine were similar (P>0.05) in adult and old female eNOS +/+ and eNOS+/− mice and were significantly inhibited (P<0.05) by L‐NNA and ODQ. Responses to nitroprusside were also similar in both groups of mice and were inhibited (P<0.05) by ODQ but not L‐NNA. The degree of inhibition produced by L‐NNA and/or ODQ was similar (P>0.05) in adult and old mice irrespective of genotype. These findings demonstrate that endothelial responses in old female eNOS+/− mice are not affected by aging and despite a 60% reduction in eNOS protein expression maintain their dependency on NOS and sGC.

Support or Funding Information

Supported by NIH HL‐089884 and HL‐107632.