bluuebunny/crossref_metadata_2025 huggingface dataset:
Administration of SGLT2 inhibitor empagliflozin against TNF‐α induced endothelial dysfunction in human venous and arterial endothelial cells.
Authors: Uthman Laween , Homayr Anna , Hollmann Markus W. , Zuurbier Coert J. , Weber Nina C.
Published: June 2021

Introduction

Type 2 diabetes is commonly associated with vascular inflammation and endothelial dysfunction. Clinical trials have shown unexpected cardiovascular benefits of SGLT2 inhibitors (SGLT2i), a new class of glucose‐lowering agents, in diabetic individuals. We recently showed a direct cardiac class effect of SGLT2i; SGLT2i inhibit sodium hydrogen exchanger 1 (NHE‐1), reduce [Na+]c and induce vasodilation in the healthy heart. Here, we studied if the SGLT2i empagliflozin (EMPA) improves endothelial function during TNF‐α induced endothelial inflammation.

Methods

Human umbilical vein endothelial cells (HUVECs) and human coronary arterial endothelial cells (HCAECs) were pre‐incubated for 2h with either 1 μM, 3 μM EMPA or vehicle and subsequently exposed to 10 ng/mL TNF‐α in the presence of 1 μM (EMPA1), 3 μM EMPA (EMPA3) or vehicle. ROS levels (intracellular H2O2) were measured with a commercial kit and permeability was studied in a trans‐well assay using FITC‐albumin. VCAM‐1 and ICAM‐1 levels were determined using FACS analysis. Expression of (p)eNOS and caveolin1 was detected with western blot.

Results

EMPA treated HUVECs showed a tendency of reduced ROS after 48 h of TNF‐α stimulation (in nM; control 13.3±7.8, TNF 21.5±10.3 (p=0.067 vs. control) and TNF+EMPA1 16.5±6.2 (p=0.604 vs. control)). After 24 h, TNF‐α stimulation significantly increased permeability and EMPA administration did not restore this in HUVECs (albumin leakage in μg/mL: control 36.8±1.8, TNF 61.3±3.8, EMPA1 63.3±2.0). VCAM‐1 and ICAM‐1 levels were increased in both TNF and TNF+EMPA treated HUVECs after 4 h (TNF 6.4‐fold and 7.9‐fold, EMPA1 6.3‐fold and 7.6‐fold, EMPA3 5.7‐fold and 6.8‐fold respectively vs. control). Caveolin‐1 expression was 3.3‐fold increased after 6h of TNF‐α stimulation. Again, the presence of EMPA did not prevent this induction of adhesion molecule expression. Expression of peNOS/GAPDH and eNOS/GAPDH was downregulated after 24 h in both TNF (50% and 44% respectively) and EMPA1+TNF (60% and 49% respectively) conditions compared to control. Interestingly, HCAECs treated with TNF+EMPA showed recovered peNOS/GAPDH expression (TNF 69% and EMPA1 111% vs. control).

Conclusion

EMPA administration exhibited a trend of reduced ROS levels, but it did not affect permeability in HUVECs exposed to TNF‐α. (P)eNOS and caveolin expression remained unaffected in HUVECs, however (p)eNOS expression was recovered in HCAECs treated with EMPA. These findings may suggest endothelial cell type specific effects of EMPA during inflammation.

Support or Funding Information

None.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New Approach in Characterize siRNA Delivery Platform for Clinical Translation
Authors: miti tatiana , Pan Hua , Wickline Samuel A.
Published: June 2021

Study Objectives

A cationic peptide‐based nanotechnology system for siRNA delivery recently developed in our lab not only overcomes limitations of serum instability, endosomal entrapment and vascular constraint at the disease sites of interest, but also exhibits an exceptional safety profile (Hou et al, ACS Nano and Biomaterials; 2014). This delivery system has been applied in mechanistic and preclinical studies in the fields of cardiovascular diseases (Vendrov AE, J Am Heart Assoc. 2017; Lozhkin A, J Mol Cell Cardiol 2017) and arthritis (Yan, PNAS 2016; Zhou JCI 2014) with ongoing anti‐cancer applications on more than 6 different malignancies. Here we sought to leverage biophysical approaches to delineate certain critical physical properties responsible in part for the success of this siRNA delivery system.

Methods and Results

Total reflectance Fourier‐transform infrared spectroscopy (ATR‐FTIR) was used for the first time to demonstrate efficient packing of siRNA into peptide‐based nanoparticles. siRNA and p5RHH have different characteristic absorbance bands within the near IR spectrum, 1700 – 1500 cm−1 for p5RHH and 1250 −1000 cm−1 for siRNA. A net increase in these regions' band intensities corresponds to changes in local concentration of p5RHH/RNA on the FTIR‐ATR crystal. At 6°C and 400:4 μM p5RHH:siRNa ratios, nanoparticles of ~ 355 ± 141 nm radius form and then sediment to the bottom of test tubes (DLS measurements, data not shown). The local increased concentration on the FTIR crystal is due to the slow settling of nanoparticles where the measured spectrum represents a true read out of the nanoparticles composition with minimal contribution from the bulk material. Figure 1 demonstrates dominant bands in the spectrum in the 1700 −1500 cm−1 region (Amide I and II), while siRNA shows low (due to low concentration used) intensity (but distinguishable) peaks in the 1250 −1000 cm−1 with an additional small peak around 1710 cm−1, corresponding to the C=O vibration of guanine. Absorbance bands within these regions exhibit 3 (p5RHH) and 8 (siRNa) fold augmented peaks and the ~1710 cm−1 peak emerges upon nanoparticle formation and settling down on the ATR crystal, strongly indicating a high RNA content in the nanoparticles.

Conclusion

This approach confirms siRNA‐peptide condensation and provides evidence‐based guidance for optimizing these and other nanoparticle formulations for more quantitative real time monitoring of siRNA‐peptide complex formation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DNA‐PK Is Activated in Human Atherosclerotic Plaques And Its Partial Inhibition by Gene Heterozygosity Reduces Atherogenesis in Mouse Model of The Disease
Authors: Ghonim Mohamed A. , Pyakurel Kusma , Luu Hanh , Dean Matthew , Bazan Hernan , Boulares Hamid
Published: June 2021

Atherosclerosis is a chronic inflammatory condition with serious implications including coronary heart disease, cerebrovascular and thromboembolic disorders. Rupture of plaques, the subsequent formation of thrombi, and the consequent occlusion of arteries are the most important events in the onset of cardiovascular complications associated with atherosclerosis. The ultimate clinical goal to prevent plaque rupture and thrombosis is the stabilization and potential regression of vulnerable plaques. DNA‐PK is composed of a 450‐kDa catalytic subunit (DNA‐PKcs) and two DNA‐binding subunits (Ku70/Ku80). Although DNA‐PK is critical for repairing DSBs through non‐homologous end joining, only less than 1% is needed to achieve such function. We have recently reported that partial inhibition of DNA‐PK pharmacologically by NU7441 or genetically by gene heterozygosity reduces asthma‐like traits in 2 animal models of the disease in part by blocking lung VCAM‐1 expression, production of Th2 cytokines through down regulating the expression of several inflammatory genes. Since persistent inflammation is critical for plaque instability and rupture, we sought to examine the role of DNA‐PK in human atherogenesis and determine whether its activation correlates with factors of inflammation and/or markers of DNA damage/repair and to investigate whether inhibition of the enzyme promotes plaque regression in a mouse model of atherosclerosis. We report that DNA‐PKcs is activated in inflammatory as well as structural cells within human atherosclerotic plaques of carotid arteries. Reduction of DNA‐PKcs by gene heterozygosity, which reduces expression and activity of the enzyme by ~50%, promoted a significant reduction of the atherosclerotic burden in high fat diet‐fed (HFD) ApoE−/− mice. Such protective action was accompanied by a significant reduction in LDL levels and an increase in HDL. These effects were achieved without interference or activation of the DNA damage/repair pathways or the development of severe combined immunodeficiency (SCID) syndrome. Overall, our results support the notion that DNA‐PK is a key player in atherogenesis and that it may constitute a novel therapeutic target for the modulation of the disease.

Support or Funding Information

NIH/COBRE 1P30GM106392‐01: COBRE Phase III Transitional Center Grant‐ Mentoring in Cardiovascular Biology

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Insulin Resistance after Long‐Term Glucocorticoid Action as the Mechanism of Transformation of Gastroprotective Effect Glucocorticoids into Proulcerogenic Consequences
Authors: Filaretova Ludmila , Morozova Olga , Podvigina Tatiana
Published: June 2021

Our previous findings suggest that an initial action of endogenous glucocorticoids, including stress‐produced ones, as well as exogenous glucocorticoids, even used at pharmacological doses, is physiological gastroprotective. Prolongation of the hormonal action, even after single glucocorticoid injection, may lead to transformation of gastroprotective hormonal effect to proulcerogenic one. Single injection of dexamethasone or cortisol, but not corticosterone, resulted in transformation of initially gastroprotective hormonal action to proulcerogenic one. Glucocorticoid‐induced long‐term maintenance of blood glucose level accompanied with catabolic effects as well as glucocorticoid‐induced blood corticosterone deficiency in rats may be the reasons of the transformation of gastroprotective action of glucocorticoids to their proulcerogenic effect. The present study was design for further understanding of the mechanisms of transformation of initially gastroprotective action of glucocorticoids to proulcerogenic effect. In experiments in rats we verified the hypothesis that a development of insulin resistance after prolongation of glucocorticoid action may be one of the mechanisms of such transformation. For this, the effects of dexamethasone, cortisol and corticosterone (at pharmacological doses) on insulin sensitivity were investigated at different time points after their single administration. Insulin sensitivity was expressed as a percentage of blood glucose reduction comparatively to its baseline levels. Insulin was injected at a dose 2 IU/kg (i.p.). Blood glucose levels were determined before and 1.5 hours after insulin injection. Prolongation of dexamethasone or cortisol action resulted in a decrease in insulin sensitivity (development of insulin resistance) at the time points when their proulcerogenic effect appeared. However, the sensitivity to insulin was not changed comparing with appropriate controls at those time points when the gastroprotective effect of glucocorticoids appeared. Moreover, prolongation of corticosterone action did not lead either to the transformation of its gastroprotective effect to proulcerogenic one, or to the appearance of insulin resistance. The data obtained suggest that the development of insulin resistance after prolongation glucocorticoid action is one of the mechanisms of transformation of their gastroprotective action into proulcerogenic consequences.

Support or Funding Information

The study was supported by grant of Russian Science Foundation (RSF) №14‐15‐00790.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impaired Platelet Mitochondrial Bioenergetics Is Associated with Hemodynamic Dysfunction in Patient with Peripheral Arterial Occlusive Disease
Authors: Lin Ming‐Lu , Wang Jong‐Shyan
Published: June 2021

Peripheral arterial occlusive disease (PAOD) is a clinical manifestation of systemic atherosclerosis. Circulating platelets can detect and respond to systemic metabolic and inflammatory stressors. The present study aimed to explore whether platelet mitochondrial bioenergetics are associated with the inflammatory indices and hemodynamic functions in patients with PAOD. Sixty‐four individuals diagnosed with PAOD were enrolled randomly in this study. Platelet mitochondrial O2 consumption rate (OCR) were measured using high‐resolution respirometry. The results demonstrated that PAOD patients with neutrophil‐to‐lymphocyte ratio (NLR)>3.0 and platelet‐to‐lymphocyte ratio (PLR)>150 revealed (i) lesser VO2peak and the distance to maximal claudication pain, (ii) lower ankle‐brachial pressure index and pulse wave velocity, and (iii) inferior the maximal and reserve OCR capacities in platelet mitochondria than those with NLR<3.0 and PLR<150. Therefore, hemodynamic dysfunction and exercise intolerance are associated with impaired platelet mitochondrial bioenergetics in inflammatory PAOD patients.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Recovery of Functional Vasodilation During Skeletal Muscle Regeneration
Authors: Fernando Charmain A. , Cornelison D DW. , Segal Steven S.
Published: June 2021

Functional vasodilation underlies the increase in skeletal muscle blood flow to supply the metabolic demands of contracting myofibers. Following injury, myofibers degenerate and then regenerate through satellite cell activation, thereby recapitulating myogenesis. While the microvasculature is also disrupted, the recovery of functional vasodilation during myofiber regeneration is unexplored. The goal of this study was to evaluate the time course and magnitude of the recovery of functional vasodilation in microvascular resistance networks at defined time points during skeletal muscle regeneration (confirmed with histology of muscle cross‐sections). In male C57BL/6J mice (4 months old; n = 5 per group), the gluteus maximus muscle (GM) was injured by local injection of barium chloride solution (1.2% solution, 75 μl). At 5, 10, 21 and 35 days (d) post injury, the resistance network of the GM was evaluated (versus Control) in feed arteries (FA), first‐ (1A), second‐ (2A) and third‐order (3A) arterioles using intravital microscopy in anesthetized mice (pentobarbital sodium; 60 mg/kg). For each branch order, maximal internal diameter (ID; topical sodium nitroprusside, 10−4 M) was not different through 35d (mean values: FA, ~62 μm; 1A, ~55 μm; 2A, ~43 μm; 3A, ~30 μm). Under resting conditions, the ID of all branch orders was greater than Control at 5d and remained elevated in FA at 10d (P<0.05). Respective resting IDs recovered to Control at 21d (FA, ~45 μm; 1A, ~32 μm; 2A, 22 μm; 3A, ~11 μm). Thus, while spontaneous vasomotor tone was initially attenuated throughout the network, it recovered by 21d in all branch orders (FA, ~27%; 1A, ~40%; 2A, ~50%; 3A, 63%). Steady state vasodilation to rhythmic twitch contractions (4 Hz, 30s) was impaired at 5d (with degenerated myofibers), attenuated at 10d (primarily new myofibers), improved through 21d (maturing myofibers) and by 35d was not different from Control. Rapid onset vasodilation to tetanic contraction (100 Hz, 500 ms) was also impaired at 5d, attenuated at 10d (P<0.05), improved through 21d and by 35d was not different from Control across respective branch orders. We suggest that elevated resting diameters with diminished vasomotor tone during the initial stages of regeneration promote tissue perfusion as myofiber recovery begins. During maturation of nascent myofibers, restoration of vasomotor tone underscores the recovery of functional vasodilation to both rhythmic and tetanic contractions. Nevertheless, vasomotor tone recovers sooner than the ability of regenerating myofibers to elicit functional vasodilation.

Support or Funding Information

AHA 17PRE33410260, NIH R37 HL041026

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Inflammation and Pyroptosis mitigate Diabetic Retinopathy via Suppressing Receptor Activated NF‐kB Ligand (RANK‐l)
Authors: Homme Rubens Petit Petit
Published: June 2021

Diabetic Retinopathy (DR) characterized by leaky blood vessels causing vision distortion. DR generally manifests as an asymptomatic condition on its onset, however; in most cases, it is usually diagnosed in its proliferative phase. Currently, there is no cure and the available therapeutic efforts are generally ineffective. Current therapeutic methods are palliative in nature and only a subset of patients benefited. Many studies have shown an association of inflammation with DR pathology. The vitreous and the retina of DR individuals exhibit the presence of inflammatory markers such as TNF‐a, iNOS, and IL‐1b. An increase in inflammation has been shown to impair the tight cellular junctions via degradations of connexins (43 & 26) and Occludin and ZO‐1. This eventually leads to BRB breakdown, thus the occurrence of leaky blood vessels that are responsible for vision loss in DR patients. Receptor‐activated of NF‐kB ligand (RANK‐l) is known to regulates survival of T‐cell, which can break down the BBB for non‐neural antigen activation. We hypothesize that inflammation induces an increase in T cells expression which remains activated via RANK‐l, gradually pyroptosis resulted in the breakdown of BRB. Akita mice were treated with 5 mg/kg body weight of JSH‐23 for two weeks, they were injected intraperitoneal every other day. The level of inflammatory mediators was measured (e.g. iNOS), T cells, RANK‐l, and pyroptosis of retinal cells. Fluorescence angiography was performed as well to analyze the leakiness of retinal vessels. Eye pressure was also measured in Akita mice. The preliminary result indicated a significantly higher pressure in the eye of Akita mice compared to control. Further analyses should indicate a decrease in inflammatory mediators, microvascular leakage, pyroptosis, and eye pressure in treated Akita mice relative to non‐treated, thus vision improvement.

Support or Funding Information

Mahavir Singh, DVM, Ph.DA.;

Akash George, Ph.D.;

Naira Metreveli;

Suresh C Tyagi, Ph.D.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Blood‐based biomarkers as early predictor of mortality in experimental hemorrhagic shock
Authors: Shi Jianru , Dai Wangde , Carreno Juan , Hale Sharon L. , Kloner Robert A.
Published: June 2021

Background

Hemorrhagic shock remains a major cause of mortality and morbidity on the battle field. Appropriate use of blood‐based biomarkers is clinically important to manage patients and predict death versus survival in the early stage of hemorrhagic shock. In continuation of our previous study, blood‐based early biomarkers were tested in 94 rats undergoing hemorrhagic shock.

Methods

Sprague Dawley rats (both genders) were anesthetized with either intraperitoneal ketamine and xylazine or with Isoflurane. Rats were intubated and ventilated with room air. After heparinization, hemorrhagic shock was induced by withdrawing blood to a fixed mean blood pressure of 30 or 40 mmHg for 30 minutes, 35 minutes, 45 minutes, or 60 minutes and then the shed blood was reinfused. Arterial blood samples were collected 1–3 hours after resuscitation with shed blood. The rats were allowed to survive for 6 weeks.

Results

The pH, bicarbonate (HCO3), oxygen saturation (SO2%), base excess and calcium levels were significantly lower in rats that died (n=63) versus survivors (n=31). Rats that died had significantly elevated anion gap, potassium, sodium and chloride levels compared to rats that survived (Table). PO2, PCO2, hematocrit, hemoglobin, glucose and lactate levels are comparable between rats that died versus survived.

Conclusions

The low pH, HCO3, SO2%, base excess and calcium coupled with high anion gap and high potassium, sodium and chloride levels may help predict long term prognosis in hemorrhagic shock.

Support or Funding Information

This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs, through the Peer Reviewed Medical Research Program under Award NO. W81XWH‐16‐1‐0606. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by Department of Defense.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cerebral microhemorrhages impair gait coordination in mice
Authors: Tarantini Stefano , Yabluchanskiy Andriy , Fulop Gabor Aaron , O'Connor Daniel , Sonntag William Edmund , Csiszar Anna , Ungvari Zoltan
Published: June 2021

Impairment in gait coordination and the consequent risk of falling are important problems in the rapidly aging population of the Western world: clinically diagnosable gait abnormalities affect approximately 35% of adults over age 70. While musculoskeletal mechanisms and processes have been extensively studied, factors affecting central neural control of gait coordination in older adults without overt neurological diseases are much less understood. Recent studies raise the possibility that in addition to causing cognitive decline, age‐associated cerebromicrovascular pathologies also contribute to mobility disability. However, the specific microvascular mechanisms underlying the loss of gait coordination in aging are not completely understood. Cerebral microhemorrhages (CMHs) are associated with rupture of small intracerebral vessels and cause focal disruption of neuronal networks. While there is growing evidence that CMHs predict cognitive impairment, the association between CMHs and gait abnormalities have not been investigated. The present study was designed to provide proof‐of‐concept that CMHs are causally linked to progressive gait dysfunction. To achieve this goal, we adapted a model of hypertension‐induced CMHs in mice (treatment with angiotensin II plus the NO synthase inhibitor L‐NAME). Computerized gait analysis was performed daily (Noldus CatWalk). CMHs were verified histologically. Mice with CMHs showed significantly altered gait function as measured by stride length, stride time and stride length variability, changes in phase‐dispersion and gait speed. CMH burden also correlated changes in the regularity index (a measure of inter‐paw coordination). Collectively, hypertension‐induced CMHs result in detectable impairment of gait function in mice. Further studies to analyze association of CMHs with gait abnormalities in human patients are warranted.

Support or Funding Information

This work was supported by grants from the American Heart Association, the National Center for Complementary and Alternative Medicine, and the National Institute on Aging

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increasing endothelial adenosine via adenosine kinase inhibition augments conducted vasodilation in HFpEF
Authors: Davila Alec C. , Christiansen Stephanie , Patel Vijay , Kamath Vinayak , Li Jie , Su Huabo , Huo Yuqing , Weintraub Neal , Bagi Zsolt
Published: June 2021

Dysfunction of coronary microvessels impairs myocardial perfusion, a pathology that has been recently implicated in the development of heart failure with preserved ejection fraction (HFpEF). We sought to determine if HFpEF exerted effects on conducted vasodilation, which critically coordinates vascular resistance longitudinally across the arteriole wall to ensure adequate tissue perfusion. Obese and hypertensive ZSF1 rats, a model of HFpEF, develop left ventricular (LV) hypertrophy and diastolic dysfunction by 20‐wk of age. Conducted vasodilation was examined in isolated and pressurized skeletal muscle arterioles of lean and obese ZSF‐1 rats before (14–16 wk) and after (20–22 wk) the development of LV diastolic dysfunction. In addition, we obtained human coronary arterioles from patients with or without HFpEF. Conducted vasodilation was induced by focal application of acetylcholine, while arteriolar diameter was measured at local and remote sites (500 and 1000 μm away from the focal stimulation) using videomicroscopy. We found a significantly reduced conducted vasodilation in obese ZSF1 rats, at 14–16 wk and 20–22 wk of age, and similar results in HFpEF patients. Impaired conducted vasodilation was associated with an increased expression of adenosine kinase (ADK), the major adenosine metabolizing enzyme. Increasing intracellular adenosine levels via a pharmacological ADK inhibitor, ABT‐702, restored conducted vasodilation in isolated arteries of ZSF1 rats and HFpEF patients. Moreover, mice with endothelium‐specific deletion of ADK exhibited enhanced conducted vasodilation and were protected against the development of transverse aortic constriction‐induced LV hypertrophy and diastolic dysfunction. We concluded that increased vascular ADK expression lead to an impaired conducted vasodilation in HFpEF, which can be restored by increasing endothelial adenosine levels via ADK inhibition.

Support or Funding Information

National Institute of Aging (R01AG054651) and the American Heart Association (GRNT33680171)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regression of pressure overload‐induced cardiac hypertrophy by TETA‐mediated myocardial copper supplementation in rats
Authors: Liu Jiaming , Liu Yinjie , Sun Xiaorong , Fan Suzhen , Ding Xueqin , Qiu Liying , Han Pengfei , Kang Y. James
Published: June 2021

Sustained pressure overload leads to cardiac hypertrophy and copper efflux from the heart. Dietary copper supplementation replenishes copper in the heart and reverses cardiac hypertrophy. On the contrary, a copper selective chelator, trientine (TETA), reverses left ventricular hypertrophy associated with diabetes. The present study was undertaken to test the hypothesis that TETA functions as a copper chaperone delivering copper to the heart leading to regression of cardiac hypertrophy. Adult male Sprague‐Dawley rats were subjected to transverse aortic constriction (TAC) to induce cardiac hypertrophy. Eight weeks after the surgery cardiac hypertrophy was developed and myocardial copper was reduced. TETA was then administrated by gavage at two different doses (21.9 mg/kg·day and 87.6 mg/kg·day) for six weeks. The results showed that at lower dose, TETA replenished copper contents in the heart, accompanied by a decrease in blood plasma and an increase in urine of copper concentrations. At higher dose, TETA did not replenish copper contents in the heart, but markedly increased copper concentrations in the urine, along with a decrease in serum copper concentrations. Corresponding to myocardial copper repletion, lower dose TETA reversed cardiac hypertrophy, as judged by a reduction in the left ventricle wall thickness (LVAWd and LVPWd) and a decrease in the heart size (heart weight/tibia length), and diminished cardiac fibrosis, as reflected by a decrease in collagen I content. TETA at higher dose not only did not regressed cardiac hypertrophy, but caused cardiac hypertrophy in sham‐operated rats. This study demonstrates that TETA, at lower dose, functioned as a copper chaperone delivering copper to the heart of copper deprivation and, at higher dose, simply remained its chelator function removing copper from the body by urinary excretion.

Support or Funding Information

Supported by National Science Foundation of China (81230004 and 81300109).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Ryanodine Type 2 Receptors in Elementary Ca2+ Signaling in Arteries and Vascular Adaptive Responses
Authors: Kassmann Mario , Szijártó István András , García‐Prieto Concha F. , Fan Gang , Schleifenbaum Johanna , Anistan Yoland‐Marie , Tabeling Christoph , Shi Yu , le Noble Ferdinand , Witzenrath Martin , Huang Yu , Markó Lajos , Nelson Mark T. , Gollasch Maik
Published: June 2021

Objective

Cardiovascular disease is the most common cause of death worldwide, and hypertension is the major risk factor. Resistance arteries can adapt their vessel diameter independently by pressure and by flow. Ryanodine receptors (RyRs) are major Ca2+ release channels in the sarcoplasmic reticulum (SR) membrane of myocytes contributing to contractility. Vascular smooth muscle cells (VSMCs) exhibit three different RyR isoforms (RyR1, 2, 3), but the impact of individual RyR isoforms on vascular adaptive responses is largely unknown.

Methods

We generated tamoxifen‐inducible smooth muscle cell (SM) specific RyR2 deficient mice and tested the hypothesis that VSMC RyR2s play a specific role in elementary Ca2+ signaling and adaptive vascular responses to vascular pressure and/or flow.

Results

Targeted deletion of the Ryr2 gene resulted in a complete loss of SR released Ca2+ sparks and the associated Ca2+ activated potassium (BKCa) channel currents, leading to increased myogenic arterial tone in peripheral arteries and systemic blood pressure. RyR2 deficiency enhanced the pulmonary artery pressure response to sustained hypoxia, but had no effect on flow‐dependent effects, including blood flow recovery in ischemic hind limbs.

Conclusion

Our results identify a specific contribution of RyR2 in mediating VSMC Ca2+ sparks relevant for regulating myogenic tone (systemic circulation) and arterial adaptation in response to changes in pressure (hypoxic lung model), but not flow. VSMC expressed RyR2 deserves scrutiny as a therapeutic target for the treatment of vascular responses in hypertension and chronic vascular diseases.

Support or Funding Information

This study was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to M.G and M.W. (SFB‐TR84 C3 and C6), and the Deutsche Akademische Austauschdienst (DAAD) to M.G. and I.S, and the NIH (R37 DK053832, R01 HL121706, PO1 HL095488, R01 HL131181), Fondation Leducq, and the Totman Medical Research Trust to M.T.N., and by the DZHK (German Centre for Cardiovascular Research) and by the BMBF (German Ministry of Education and Research) to M.G. and M.K. We thank Dr. Kirill Essin for help in Ca2+ spark recordings using line scan imaging. CFG‐P is recipient of Ministerio de Educación Cultura y Deporte, Fundación San Pablo‐CEU and Banco Santander fellowships.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelial cell PKD2 (TPP1) channels are essential for flow‐mediated vasodilation
Authors: Mackay Charles E. , Fernandez‐Pena Carlos , Bulley Simon A. , Jaggar Jonathan H.
Published: June 2021

The endothelium detects shear stress generated by blood flow and transduces this mechanosensory signal to regulate arterial function. Intravascular flow stimulates an increase in arterial diameter, a process termed flow‐mediated vasodilation. The underlying mechanisms by which endothelial function is regulated by flow are poorly understood. Endothelial cells (ECs) express several non‐selective cation channels, including PKD2 (also termed TRPP1), but their involvement in flow‐mediated vasodilation is unclear. Here, we generated novel inducible, endothelial cell‐specific PKD2 knockout (PKD2endo−/−) mice to examine the functional significance of this channel in flow‐mediated vasodilation in resistance‐size mesenteric arteries. Immunofluorescence in intact arteries confirmed that PKD2 was present in endothelial cells of PKD2fl/fl mice, but absent in endothelial cells of PKD2endo−/− mice. Pressure (80 mmHg)‐ and depolarization (60 mM K+)‐induced vasoconstriction and passive diameter were similar in PKD2fl/fl and PKD2endo−/− arteries. In contrast, vasodilation to intravascular flow (15 dyn/cm2) in PKD2endo−/− arteries was ~20 % of that in PKD2fl/fl arteries. Endothelium denudation abolished flow‐mediated vasodilation. L‐NNA, a nitric oxide synthase (NOS) inhibitor, reduced flow‐mediated vasodilation by ~30% in PKD2fl/fl arteries. In contrast, TRAM 34 and apamin, IK and SK channel blockers respectively, did not alter flow‐mediated vasodilation. Microelectrodes were used to measure the membrane potential of pressurized, myogenic arteries. In the absence of luminal flow, the membrane potential of PKD2fl/fl and PKD2endo−/− arteries was similar (~−43 mV). Flow hyperpolarized PKD2fl/fl arteries by ~ 6.8 mV, but PKD2endo−/− arteries by only ~1.2 mV, an attenuation of 82 %. Taken together, our data suggest that flow stimulates PKD2 channels in endothelial cells, leading to membrane hyperpolarization, NOS activation and vasodilation.

Support or Funding Information

NIH/NHLBI

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Knocking Out Endothelial TRPV4 Channels Enhances Vasoconstriction and Exacerbates Endothelial Dysfunction in Low‐Flow Vascular Injury
Authors: McFarland Stuart Joseph , Taylor Mark S. , Weber David S.
Published: June 2021

Atherosclerosis remains a leading killer of men and women. It is associated with reduced blood flow, progressive endothelial dysfunction and vascular remodeling. Preventing pathologic endothelial changes is crucial for stopping the disease. Transient receptor potential cation channels (TRPV4) play a key role in endothelial responses to shear stress and local mediators by controlling dynamic endothelial Ca2+ signals. In the current study, we assessed the specific impact of endothelial TRPV4 channels on carotid artery function in both wild type (WT) control and endothelial‐specific TRPV4 knockout transgenic mice (KO). Following tissue harvest, carotid arteries were cut into rings for Isometric force myography. Depolarization‐induced contractions (60 mM KCl) were larger in KO than WT arteries (1.14 ± 0.14 mN vs. 0.52 ± 0.08 mN, P<0.0005, N=6) and a similar trend was noted for contractions induced by the α1 adrenergic receptor agonist phenylephrine (0.68 ± 0.12 mN vs. 0.43 ± 0.08 mN, P<0.1, N=6). The calculated EC50 indicated no significant difference (1.7×10−7 vs 1.5×10−7 M, n=6) in phenylephrine sensitivity. Net relaxation to acetylcholine was increased in KO vs. WT but EC50 values were not significantly different (5.5×10−7 vs 5.9×10−7, n=6). Using confocal imaging, endothelial Ca2+ was evaluated in carotid artery segments that were cut open longitudinally. The dynamic Ca2+ events that drive endothelium dependent vasorelaxation were altered in KO vs. WT arteries, exhibiting a lower frequency of these events but also events with larger mean amplitude and duration. A carotid artery partial ligation model was used to mimic the low flow predicted with atherosclerosis. Following 2 weeks of reduced flow, KO arteries demonstrated enhanced contractility and impaired ACh‐induced relaxation compared to similarly ligated WT arteries. Overall, our findings suggest that loss of endothelial TRPV4 channels inherently augments vasoconstriction and exacerbates endothelial dysfunction under conditions of chronic low flow.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NaHS Induced ANP Secretion via PI3K/Akt/NO/cGMP and KATP Channel Pathway
Authors: Yu Lamei , Park Byung Mun , Kim Suhn Hee
Published: June 2021

The third gasotransmitter, hydrogen sulfide (H2S), is related to the pathogenesis of cardiovascular diseases. However, it's still not clear about the relation between H2S and cardiac hormone, atrial natriuretic peptide (ANP). The purpose of this study is to investigate the effects of H2S donor on ANP secretion and seek its mechanism using normal and isoproterenol (ISP)‐treated hypertrophied rat atria. Different doses of H2S donors were perfused into isolated beating rat atria, and atrial pressure and ANP secretion were measured. NaHS (1, 10, 50, 100 μM) augmented high stretch‐induced ANP secretion and decreased systolic atrial pressure (SAP) dose‐dependently. Pretreatment with an inhibitor or antagonist for phosphatidylinositol 3‐kinase (PI3K; wortmannin), protein kinase B (Akt; API‐2), nitric oxide synthase (NOS; L‐NAME), soluble guanylyl cyclase (sGC; ODQ), and KATP channel (glibenclamide) blocked the augmentation of ANP secretion induced by NaHS. The high stretch‐induced ANP secretion was stimulated by Na2S but was not changed by GYY4137 and sodium thiosulfate. H2S synthesis enzyme inhibitor (DL‐propargylglycine, PAG) did not show any significant changes in atrial parameters. However, the response of ANP secretion to NaHS markedly attenuated and PAG suppressed ANP secretion in ISP‐treated rat atria. The expression of eNOS protein was decreased but the expression of cardiomyocyte‐specific H2S producing enzyme, cystathione γ‐lyase, was not changed in ISP‐treated rat ventricles. These findings clarify that NaHS stimulates ANP secretion through PI3K/Akt/NO/cGMP and KATP channel pathway. The modification of ANP secretion by NaHS and H2S synthesis enzyme inhibitor suggests the possible role of endogenous H2S in the pathogenesis of cardiac hypertrophy.

Support or Funding Information

Supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NO 2017‐R1A2B‐4002214).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Exercise and Peripheral Opioid Receptor Stimulation on Anxiety‐Like Behaviors in a Rat Model of Chronic Pain
Authors: Stocks Silas , Daniels Abigale B. , Ford Caleb M. , Horton Molly , Williams Emily R. , Leal Anna K.
Published: June 2021

Patients with fibromyalgia are more likely to suffer from anxiety. In the acidic saline‐induced chronic pain model, rats exhibit long‐lasting, wide‐spread pain along with anxious behaviors. Exercise training has been shown to decrease pain in both patients with fibromyalgia and in this rat model of chronic pain; however the effect of exercise on anxiety and the contribution of peripheral opioid receptors to this effect are unknown. All experiments were performed in female Sprague‐Dawley rats that demonstrated chronic widespread pain due to acidic saline injections and all rats underwent moderate‐intensity treadmill training (4 weeks; 5 days/week; 30 min/day at 15 m/sec). Anxiety‐like behaviors were determined using the open field test. Control rats (n=4) were observed in the open field (90 × 90 cm) for two minutes before (PRE) and after (POST) each exercise bout. Time spent moving was recorded: decreased motility demonstrated anxiety. A separate group of rats (NAL; n=5) received injections of the nonspecific opioid receptor antagonist naloxone (120 μg in 100 μL saline) in both gastrocnemius muscles ten minutes prior to treadmill running in addition to open field testing. Results showed that treadmill training alone had no effect on control rats' average time spent moving in the open field: 32.5 ± 4.2 s (PRE) and 31.2 ± 4.4 s (POST) for week 1; 35.1 ± 4.2 s (PRE) and 31.7 ± 3.8 s (POST) for week 2; 32.7 ± 4.3 s (PRE) and 38.3 ± 4.8 s (POST) for week 3; and 35.6 ± 4.6 s (PRE) and 34.5 ± 3.1 s (POST) for week 4. However, NAL rats demonstrated increased anxiety‐like behaviors indicated by decreased average time spent moving for all weeks tested: 25 ± 3.9 s (PRE) and 14.2 ± 3.2 s (POST) for week 1; 34.6 ± 3.4 s (PRE) and 18.9 ± 2.8 s (POST) for week 2; 35 ± 4.1 s (PRE) and 16.5 ± 3.2 s (POST) for week 3; and 38 ± 4.5 s (PRE) and 17.9 ± 2.9 s (POST) for week 4. In NAL rats, PRE and POST times were significantly different for all four weeks (p<0.05). PRE times were not different between control and NAL groups, however, POST times were significantly different between control and NAL groups for all weeks. Our data suggests that endorphins released during exercise act on peripheral opioid receptors to reduce anxiety‐like behaviors associated with the exercise bout in a rat model of chronic pain. These findings support the use of exercise to reduce anxiety and chronic pain in people with fibromyalgia.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cyrotherapy Reduces Macrophage Infiltration and Inflammatory Mediators Following Muscle Injury
Authors: Rafferty Patrick , McMahon Katherine , Arrigale Marlena , Carpenter April
Published: June 2021

Cryotherapy following muscle injury is a commonly used treatment in sports medicine. However, there are conflicting reports on the efficacy of cryotherapy to reduce inflammation. The infiltration of macrophages and macrophage produced growth factors are now considered important mediators of the healing process. The purpose of our study was to evaluate the effects of cryotherapy on inflammation associated with muscle regeneration in mice. Following BaCl2 injury, we observed a significant reduction in macrophage infiltration in mice that received cryotherapy (20 min of ice therapy every 2hr during consecutive 12 hr wake cycles) over a 48 hr period. qPCR analysis of mRNA expression for wnt proteins and inflammatory mediators revealed a statistically significant reduction in wnt 2b, 3, 5a, 11, 16 and NFk‐β, TNF‐α, TGF‐β, and IGF‐1. Given that wnt proteins are important during development of muscle and in many tissue regeneration models, we believe this supports the notion that the reduction in inflammation afforded by cryotherapy may correspond with a delay in muscle healing. Studies are ongoing to determine the long‐term effects of cryotherapy on macrophage‐mediated muscle regeneration and extracellular matrix remodeling.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of reflex and mechanical decreases in skin blood flow on exogenous cholinergic‐induced eccrine sweating in humans
Authors: Wilson Thad E. , Fang Milie M. , Dazé Robert P. , Narra Seetharam C. , Metzler‐Wilson Kristen
Published: June 2021

Human skin blood flow (SkBF) and sweating increase concurrently during increases in skin sympathetic nerve activity and subsequent neurotransmitter release to these cutaneous appendages. Previous studies have blocked sweating without affecting SkBF with the use of the muscarinic agonist atropine, but few have altered SkBF without affecting sweating because standard methods such as introducing an α‐adrenergic agonist decrease SkBF but also can engage eccrine sweat gland receptors. Thus, this study aimed to utilize non‐adrenergic mechanical perturbations to decrease SkBF during intradermal perfusion of a cholinergic agonist. Three intradermal microdialysis fibers were placed in dorsal forearm skin of 4 women and 3 men (age 24±1 yr, height 173±4 cm, weight 80±8 kg) to perfuse 2 doses of pilocarpine nitrate (0.01 and 1.66 mg/ml, corresponding to the ED50 and EMAX from previous dose‐response modeling) or the vehicle (lactated Ringer's). Forearm SkBF (laser‐Doppler flowmetry) was decreased for 3 min by engaging the venoarteriolar response (arm lowered ~30 cm from heart level; CVAR), venoarteriolar response plus decreased perfusion pressure (venous occlusion by proximal cuff inflation; CVAR with ΔPP), and ischemia (arterial occlusion by proximal cuff inflation); sweat rate was measured by perfusing anhydrous medical air through a ventilated capsule (capacitance hygrometry). Drug infusions significantly increased SkBF (+87±18 and +57±16 units) and sweating (+0.37±0.20 and +0.64±0.15 mg/ml/min, for pilocarpine ED50 and EMAX, respectively) compared to vehicle control in which SkBF minimally increased (+8±4 units) and there was no alteration in sweating (−0.02±0.09). Decreased SkBF was observed with CVAR (−26±14, −33±9, and −38±9%), CVAR with ΔPP (−62±7, −42±20 and −61±8%), and ischemia (−99±1, −92±5 and −93±2% for pilocarpine ED50, pilocarpine EMAX, and vehicle control, respectively). Despite these significant decreases in SkBF, changes in eccrine sweat rate were minimal (CVAR = −1±12, +3±3, and −2±5%; CVAR with ΔPP = +3±7, −2±4, and −3±4%; and ischemia = +5±10, +5±5, and +6±5% for pilocarpine ED50, pilocarpine EMAX, and vehicle control, respectively). These data suggest that very acute decreases in SkBF do not proportionally decrease cholinergic‐induced sweating in human forearm skin.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Overexpression of Nrf2 Targeting Glutamatergic Neurons in the RVLM Ameliorates Sympathetic Regulation in Mice With Chronic Heart Failure
Authors: Gao Lie , Lobl Marissa B. , Zucker Irving H.
Published: June 2021

Nrf2 is a key transcription factor that regulates redox status by up‐regulating a broad array of antioxidant genes. Previous data from our laboratory demonstrated that enhanced oxidative stress in the RVLM contributed to the sympatho‐excitation in rabbits with chronic heart failure (HF). Accordingly, we hypothesized that overexpression of Nrf2 in the RVLM will suppress sympathetic tone via enhancing antioxidant defense. Thirty C57BL/6 mice were randomized into four groups: Sham‐lentiviral GFP (7); Sham‐lentiviral GFP Nrf2 (7); HF‐lenti GFP (8); and HF‐lenti GFP Nrf2 (8). HF was induced by coronary artery ligation while sham surgery was used as the control. Nrf2 was upregulated selectively in glutamatergic neurons of RVLM by microinjection of Ca2+/calmodulin‐dependent protein kinase‐II (CaMKII) promoter‐driven Nrf2 lentivirus and a corresponding GFP vector was used as the control. We found that (1) Nrf2 protein was significantly downregulated in the RVLM of HF mice, which was restored by Nrf2 gene transfer. (2) The increased urinary NE excretion in HF was attenuated by Nrf2 overexpression (Sham‐GFP: 575.2 ± 265.0, HF‐GFP, 1119.6 ± 270.5, HF‐Nrf2: 812.1 ± 133.0 ng/24hr. *p < 0.05, n = 7–8/group). (3) Radio telemetry experiments (in the conscious state) showed that Nrf2 overexpression enhanced spontaneous baroreflex gain and increased PE induced bradycardia in HF (Decline of HR: Sham‐GFP: −53.7 ± 4.8; HF−GFP: −4.7 ± 5.1; HF‐Nrf2: −25.1 ± 2.3 % of Max. *P < 0.05 HF‐GFP vs Sham‐GFP and HF‐Nrf2 vs HF‐GFP, n = 7–8/group), and attenuated stress‐induced pressor responses in both Sham (surgical‐stress: 101.5 ± 5.9 vs 129.7 ± 7.2 mmHg, restraint‐stress: 118.8 ± 6.8 vs 151.5 ± 8.2 mmHg. *P < 0.05, n = 7/group) and HF mice (surgery‐stress: 111.06 ± 5.87 vs 135.83 ± 7.24 mmHg, restrain‐stress: 102.8 ± 8.2 vs 119.2 ± 6.3 mmHg. *P < 0.05, n = 8/group). No difference in baseline blood pressure and heart rate were observed among these groups. (4) Acute experiments (in the anesthetized state) revealed a decrease in basal renal sympathetic nerve activity (Sham‐GFP: 28.6 ± 5.2, HF‐GFP: 64.7 ± 8.3, HF‐Nrf2: 44.0 ± 6.5 % of Max. *P < 0.05 HF‐GFP vs Sham‐GFP and HF‐Nrf2 vs HF‐GFP, n = 7–8/group) and improvement of baroreflex sensitivity (Maximal Gain: Sham‐GFP: 2.9 ± 0.1, HF‐GFP: 1.1 ± 0.2, HF‐Nrf2: 1.8 ± 0.3 % of mmHg. **p < 0.01 HF‐GFP vs Sham‐GFP; *P < 0.05 HF‐Nrf2 vs HF‐GFP, n = 7–8/group) in HF mice by Nrf2 overexpression. Based on the above data, we conclude that upregulating Nrf2 expression in glutamatergic neurons of the RVLM ameliorates sympathetic regulation in mice with HF and attenuates stress‐induced pressor responses in both sham and HF mice.

Support or Funding Information

Supported by NIH grant P01‐HL62222

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.