bluuebunny/crossref_metadata_2025_split huggingface dataset:
Helium Mediated Regulation of Endothelial Permeability: Involvement of Connexin 43, VE‐Cadherin and Caveolin‐1
Authors: Konkel Moritz , Smit Kirsten Fernande , Hollmann Markus Werner , Patel Hemal H , Albrecht Martin , Weber Nina Claudia 
Published: November 2023

BackgroundCaveolae are lipid enriched invaginations of the plasma membrane while Caveolins are structural proteins essential for the formation of these invaginations. Caveolins modulate signal transduction pathways in various cell types. The noble gas helium, a potent cardioprotectant, leads to decreased protein levels of Caveolin‐1 (Cav‐1) in the human endothelium and an increased level of Cav‐1 in the supernatant of human umbilical vein endothelial cells (HUVEC). Moreover, helium regulates actin filaments and decreases permeability of HUVEC monolayers.We hypothesized that (1) an upregulation of the adhesion molecules Connexin 43 and VE‐Cadherin might be involved in the decreased permeability of the endothelium after helium treatment and (2) that Cav‐1 plays a crucial role in the helium induced decrease of endothelial permeability.MethodsHUVEC were isolated from fresh umbilical cords, pooled and grown upon confluent monolayers. Cells were used at passages 3–5. Cells were either exposed to 20 min of helium (5% CO2, 25% O2, 70% He) or control gas (5% CO2, 25% O2, 70% N2) in a specialized gas chamber. Expression of adhesion molecules Connexin 43 and VE‐Cadherin was measured at 6, 12 and 24 hours using IR‐Western blot analyses.In order to investigate how the protective effect of helium is related to Caveolin we transfected HUVEC with small interfering RNA (siRNA) for Cav‐1 72 h before the respective experiment.Permeability was measured using Fluorescein isothiocyanate labelled bovine serum albumin (FITC‐BSA) flow‐throw after 6, 12 and 24 hours.ResultsIn the helium group, protein analyses in cell lysate showed a significantly higher expression of Connexin 43 and VE‐Cadherin at 6 and 12 hours in comparison to the control group (for Connexin 43 in helium treated cells at 6h: 1.31±0.39 and 12h: 1.27±0.37. For Connexin 43 in control cells at 6h: 0.90±0.32 and 12h: 0.82±0.26. For VE‐Cadherin in helium treated cells at 6h: 1.19±0.28 and 12h: 1.14±0.37. For VE‐Cadherin in control cells at 6h: 0.87±0.11 and 12h: 0.85±0.14: all treatments and time points vs. control 6h and control 12h, respectively: P<0.05).Interestingly, cell permeability measurements of Cav‐1 siRNA transfected HUVEC showed no significant differences between the helium and control group suggesting a key role of Cav‐1 in endothelial permeability.ConclusionThese findings suggest that an upregulation of the adhesion molecules Connexin 43 and VE‐Cadherin might be involved in the decreased permeability of the endothelium after helium treatment and that Cav‐1 plays a crucial role in the helium induced decrease of endothelial permeability.Support or Funding InformationThis project was in part funded by a grant fromthe IARS (International Anesthesia Research Society) and the SCA (Society of Cardiovascular Anesthesiologists) to NC Weber
Tetramethylpyrazine Prevents sEH Dysregulation to Protect Coronary Endothelial Function Through an anti‐ER Stress Dependent Mechanism
Authors: Mak Shiu‐Kwong , Sun Wen‐Tao , Fu Jie , Yang Qin 
Published: November 2023

Activation of soluble epoxide hydrolase (sEH) is associated with endothelial dysfunction in cardiovascular disease such as hypertension, though mechanisms underlying sEH dysregulation are inadequately understood and whether endoplasmic reticulum (ER) stress is involved is yet to be studied. Tetramethylpyrazine (TMP) is a major bioactive ingredient of Chinese herb Chuanxiong that has been long known for cardiovascular benefits. However, whether TMP may protect vascular endothelial cells from ER stress and whether it may regulate sEH expression remain unknown. This study therefore aimed to investigate whether ER stress participates in angiotensin II (Ang‐II)‐induced sEH dysregulation in coronary endothelium and whether anti‐ER stress mechanism is involved in the vasoprotective effect of TMP through the regulation of sEH. Endothelial cells (PCECs) were isolated by enzymatic digestion from porcine coronary arteries (PCAs) and primary cell cultures were used for western blot, ELISA, and reverse‐transcription PCR analysis. Isometric force study of PCAs was performed in a myograph for assessment of endothelial dilator function. Ang‐II enhanced sEH expression and lowered 11,12‐EET level in PCECs, along with increased expression of ER stress markers, i.e., GRP78, ATF6, p‐PERK, and p‐IRE1. The increases of sEH mRNA and protein were also observed in PCECs exposed to tunicamycin, a chemical ER stress inducer. Treatment of PCECs with ER stress inhibitors suppressed Ang‐II‐induced upregulation of sEH and loss of 11,12‐EET. PCAs subjected to Ang‐II or tunicamycin exposure exhibited impaired endothelium‐dependent relaxation, which was improved by inhibitors of ER stress or sEH. TMP showed comparable inhibitory effect to that of ER stress inhibitors on Ang‐II‐induced ER stress and dysregulation of sEH and EET. sEH expression was also normalized in tunicamycin‐exposed cells that underwent TMP treatment. TMP was similarly efficient as ER stress inhibitor in the restoration of endothelial vasodilator function in Ang‐II‐ and tunicamycin‐exposed PCAs. The present study suggests that ER stress mediates Ang‐II‐induced sEH upregulation in coronary endothelium. TMP has potent anti‐ER stress capacity through which TMP normalizes sEH expression and confers protective effect against Ang‐II on endothelial function of coronary arteries. These findings added new mechanistic insight into the cardiovascular benefits of TMP and may suggest a potential of broadening the use of this traditional Chinese medicine in the treatment of diseases whose pathogenesis involves ER stress.Support or Funding InformationSupported by RGC/GRF CUHK14118414; Lui Che Woo Institute of Innovative Medicine ‐ CARE theme 8303303, and CUHK Direct Grant 4054273.
Beneficial Effects of (−)‐Epicatechin on Biomarkers of Endothelial Cell Aging
Authors: Mansour Christina , Ceballos Guillermo , Ramirez‐Sanchez Israel , Villarreal Francisco 
Published: November 2023

Aging is known to contribute to the development of endothelial dysfunction and as such facilitates the development of cardiovascular diseases (CVD). The loss of the ability of endothelial cells to produce physiological amounts of nitric oxide (NO) partly underlies the detrimental effects of aging. Thus, the identification of agents that can mitigate aging induces loss of endothelial cell function (i.e. NO production) are of great interest. Several reports indicate that the administration of the cacao flavanol (−)‐epicatechin (Epi), can partly reverse the deleterious effects of aging in organs such as the heart and kidneys. However, less is known on its effect on aged endothelium. To address this question, we implemented a primary cell model of aging that is based on the use of high passage number (31–35) bovine coronary endothelial cells (BCAEC) in culture. We specifically evaluated NO production through the endothelial NO synthase (NOS) pathway, markers of cell senescence (β‐galactosidase) and mitochondrial biogenesis (sirtuin‐1, mitofilin, oxidative phosphorylation complexes II, IV and V, Tfam and citrate synthase activity). BCAEC at low passage number (8–13 passage number) were used as “young” controls. Cells were treated with either vehicle (water) of Epi (100 nM) for 48 h and media and cells collected for testing using kits (NO, citrate synthase) or Western blots. All experiments were performed independently a minimum of 3 times in triplicate. All results were statistically analyzed. Results indicate that senile cells loose their capacity to produce NO by ~33% (p<0.05) and that Epi treatment can essentially fully restore their production levels to those noted for vehicle treated young cells. These results paralleled to those noted for the phosphorylation of eNOS. While β‐galactosidase levels doubled (100% increase) in aged cells, those treated with Epi only evidenced about a 40% increase (p<0.05). The levels of phosphorylated sirtuin‐1, mitofilin, oxidative phosphorylation complexes and Tfam were all reduced by ~40% with aging (p<0.05) and were largely restored by Epi treatment. Altogether, results strongly suggest that Epi can partly reverse markers of endothelial cell aging by restoring critical components of vascular function. These effects may partly explain the epidemiological evidence for the beneficial effects of cocoa consumption on the incidence of CVD.Support or Funding InformationNIH DK98717
Vitamin D and Its Therapeutic Targets in Gastric Cancer
Authors: Cho Chi Hin , Li Ming Xing , Hu Wei 
Published: November 2023

Backgrounds and objectivesVitamin D3 has been widely used for bone health in humans. Epidemiological studies indicated that vitamin D3 deficiency was correlated with cancer risk. Previous studies also demonstrated that vitamin D3 could potentiate the antitumor effect in a variety of cancer types. Our research focuses on studying the therapeutic effects of vitamin D3 in gastric cancer and its relationship with cathelicidin, an inducible host defense and antimicrobial and anticancer peptide by the vitamin both in vitro and in vivo.MethodsMTT assay was used to detect the gastric cancer cell viability. Flow cytometry was used to detect the cell cycle distribution of gastric cancer cell. An animal model of orthotopic gastric cancer was established in nude mice to evaluate the anticancer actions of vitamin D3 for in vivo study.Results1α,25(OH)2 vitamin D3 (1α,25(OH)2D3), the active form of vitamin D3, could significantly inhibit the proliferation and induce cell cycle arrest through stimulating the expression of p21 in gastric cancer cells (TMK1) but not in the normal gastric epithelial cells (HFE145). CDK2 and cyclin E expressions were decreased by 1α,25(OH)2D3 in TMK1 cells. This vitamin analogue also successfully induced the expression of cathelicidin (LL37) and provoked autophagy in these cancer cells. Knockdown of VDR by siRNA abolished the anti‐proliferative effect of 1α,25(OH)2D3. Decrease the expression of LL37 by siRNA abolished the autophagic effect of 1α,25(OH)2D3 but not the anti‐proliferative effect on cells. These findings indicate that the antitumor effect of vitamin D3 in TMK1 cells is independent of LL37 and autophagy. We also found that daily oral administration with vitamin D3 significantly inhibited tumor growth in mouse stomachs. Such treatment also increased VDR and p21 expressions and induced LCB3 II level in the gastric tumor tissues.ConclusionOur results suggest that vitamin D3 inhibits gastric cancer in a VDR‐dependent manner followed by the increased expression of p21 in gastric cancer cells. As this vitamin is currently used as a dietary supplement mainly for bone diseases in humans, it could also function as a novel therapeutic agent or prophylactic treatment for gastric cancer in the future.Support or Funding InformationThis study was partly supported by the General Research Fund from the Hong Kong Research Grant Council (CUHK 463613) and the Health and Medical Research Fund from the Food and Health Bureau (13120062) in Hong Kong and the National Natural Science Foundation of China (No. 81473269).
Rbfox1 Regulates Calcium Signaling to Prevent Pressure Overload Induced Cardiac Dysfunction
Authors: Umar Sadiq , Zhu Wuqiang , Bender Ingrid , Van Berlo Jop 
Published: November 2023

RNA splicing and processing provide important means of genetic control to diversify gene function, and fine‐tune protein production. RNA regulation is a critical step in the expression of most genes, and its misregulation often results in human disease. Rbfox1 is a RNA binding protein that regulates alternative splicing and RNA stability, and plays an important role in regulating alternative splicing (AS) in neurons, skeletal muscle, and the heart. Recently, Rbfox1 was identified as a critical regulator of Mef2 splicing to protect the heart from failure. However, its role in protecting the heart from failing is likely not limited to Mef2 splicing. To begin to study the role of Rbfox1 in heart failure, we generated cardiomyocyte specific Rbfox1 gene‐deletion mice (cRbfox1−/−). Cardiomyocyte specific deletion of Rbfox1 was confirmed by Western blotting and immunohistochemistry. cRbfox1−/− mice showed mild hypertrophy and depressed cardiac function under homeostatic conditions, which did not deteriorate with age. Pressure overload by trans‐aortic constriction (TAC) caused exaggerated cardiac hypertrophy and accelerated heart failure in cRbfox1−/− compared to wildtype mice. Since Rbfox1 is a known RNA binding protein, we performed RNA sequencing and found significant changes in calcium regulated genes. We measured calcium dynamics and noted a significantly delayed Calcium reuptake into the Sarcoplasmic Reticulum, which is likely caused by decreased SERCA2a expression on Western blot. In conclusion, Rbfox1 is important to maintain cardiac function by regulating calcium signaling. We are currently unraveling the mechanisms via which Rbfox1 regulates Serca2a function.Support or Funding InformationAcknowledgement: This work was supported by National Institutes of Health grants and Lillehei Scholar Award (to JVB).
β‐Arrestin‐Biased β2‐Adrenergic Receptor Signaling Provides Cardioprotection in Ischemia/Reperfusion Injury
Authors: Grisanti Laurel A , de Lucia Claudio , Gao Erhe A , Koch Walter J , Benovic Jeffrey L , Tilley Douglas G 
Published: November 2023

β‐Adrenergic receptors (βAR) are important regulators of cardiac function in the normal and failing heart. Activation of the β1AR subtype increases contractility and cardiomyocyte death whereas the β2AR can promote survival. In response to cardiac injury, increased catecholamines activate and downregulate β1AR thus, β‐adrenergic receptor antagonists or β‐blockers are commonly prescribed in to restore β1AR expression and preserve contractility. However, promoting the pro‐survival effects of β2AR, which are known to occur in part through β‐arrestin (ARR)‐dependent signaling, may be a beneficial therapeutic strategy. Pepducins have been developed based on the intracellular loop (ICL) domains of β2AR to activate either Gαs‐ or βARR‐dependent β2AR signaling pathways. We hypothesized that pepducin‐mediated engagement of βARR‐dependent β2AR signaling in the heart would be therapeutically advantageous following ischemia/reperfusion (I/R) injury. To test this, wild‐type (WT) C57BL/6 mice received three intracardiac injections of either a βARR‐biased pepducin (ICL1‐9) or a scrambled control pepducin at the time of ischemia (30 min) followed by reperfusion. Assessment of cardiomyocyte death 24h post‐I/R using TUNEL staining showed a decrease in cell death in animals treated with ICL1‐9 when compared to scrambled pepducin, correlating with decreased infarct size and improved cardiac function as measured by echocardiography. Assessment of cardiac function at later time points showed that the cardioprotective effects of ICL1‐9 were preserved over time and resulted in decreased cardiac remodeling. Although βARR1KO mice displayed similar cell death, infarct size, and contractility changes following I/R as their WT counterparts, they did not manifest a cardioprotective response from ICL1‐9 treatment, indicating that βARR signaling is essential in relaying ICL1‐9‐dependent cardioprotection. These results demonstrate that pharmacologically‐mediated activation of βARR‐biased β2AR signaling provides a therapeutic benefit in the context of I/R‐induced injury and may provide an improved strategy for the treatment of acute cardiac injury.Support or Funding InformationNIH‐HL‐105414‐DGT
adrenergic Receptor‐Stimulated Cardiac Myocyte Apoptosis: Role of Cytochrome P450 ω‐Hydroxylase Cytochrome P450 ω‐Hydroxylase
Authors: Guo Lirong , Zhong Tiecheng , O'Rourke Stephen T. , Sun Chengwen 
Published: November 2023

Prolonged or excessive β‐adrenergic activation leads to cardiomyocyte loss and heart dysfunction. However, the underlying cellular mechanisms are still unclear. Therefore, we first confirmed the effect of isoproterenol (ISO), a β‐adrenergic receptor agonist, on cardiac toxicity using TUNEL and Caspase activity assays in cultured rat cardiomyocytes. ISO treatment significantly increased cardiomyocyte apoptosis. Persistent ISO stimulation of cardiomyocytes also increased the expression of CYP4A3, a major CYP450 ω‐hydroxylase that produces 20‐Hydroxyeicosatetraenoic acid (20‐HETE), in a time‐dependent manner. Next, we examined the effect of ISO and 20‐HETE on cardiomyocyte apoptosis using annexin V and propidium iodide staining. Treatment with either 20‐HETE or ISO significantly increased cardiomyocyte apoptosis, and inhibition of 20‐HETE production using 17‐ODYA, a CYP450 ω‐hydroxylase inhibitor, dramatically attenuated ISO‐induced cardiomyocyte apoptosis. To determine the apoptotic pathway involved, the mitochondrial membrane potential (ΔΨm) was measured by detecting the ratio of JC‐1 green/red emission intensity. The results demonstrated that 17‐ODYA significantly abolished ISO‐induced disruption in ΔΨm and that 20‐HETE alone induced a marked disruptive effect on ΔΨm in cardiomyocytes. 20‐HETE‐induced ΔΨm disruption and apoptosis were attenuated by KN93, a CaMKII blocker, suggesting that this action of 20‐HETE is mediated by a CaMKII‐dependent mechanism. Taken together, these results demonstrate that 20‐HETE treatment induces significant apoptosis via mitochondrial‐dependent pathways, and that inhibition of 20‐HETE production using 17‐ODYA attenuates ISO‐induced cardiomyocyte apoptosis. Thus, CYP450 ω‐hydroxylase could be a novel target to protect against cardiac damage induced by excessive β‐adrenergic activation or hypersympathoexcitability.Support or Funding InformationThis work was supported by Grants from the National Institute of Neurological Disorders and Stroke, National Institutes of Health (NIH, NS55008), American Heart Association (10GRNT3170012), Norman Bethune Program of Jilin University (2015221), and Education Department of Jilin Province (2015498).
Activation of GPER (G protein‐coupled estrogen receptor) increases Gs activity via forming a plasma membrane complex with AKAP/SAP97
Authors: Yu Xuan , Stallone John N. , Heaps Cristine L. , Han Guichun 
Published: November 2023

Evidence suggest that G protein‐coupled estrogen receptor (GPER) is emerging as a therapeutic target in cardiovascular diseases. Activation of GPER elicits many cardiovascular protective effects, including lowering blood pressure, reducing ischemia/reperfusion‐induced cardiac infarct size and decreasing the process of atherosclerosis in animal models. However, the understanding of the mechanisms underlying GPER‐mediated actions is still limited. The objective of this study is to test the hypothesis that activation of GPER by selective agonist G‐1 increases Gs activation by forming a plasma membrane complex with AKAP/SAP97. Isometric tension studies, Gs activity assays, immunoprecipitation studies and Western blots were performed by using porcine coronary arteries, primary cultured porcine coronary artery smooth muscle cells (PCASMCs) and HA‐tagged GPER gene stably transfected HEK293 cells (GPER‐HEK293). First, we measured the Gs activity in both GPER‐HEK293 cells and PCASMCs with selective GPER agonist G‐1 (1 μM); and observed that treatment with G‐1 significantly elevated the Gs‐GTP level. Next, we explored the association of GPER with Gs in immunoprecipitation studies of both GPER‐HEK293 cells and PCASMCs. Similar to Gs activity assays, activation of GPER with G‐1 promoted the association of activated GPER with Gs, and it was in a concentration dependent manner in PCASMCs. We further observed effects of GPER activation on cAMP production in PCASMCs and demonstrated that G‐1 significantly stimulated cAMP production. Last, we investigated the interaction of both Protein Kinase A‐anchoring Protein (AKAP5) and membrane‐associated guanylate kinase SAP97 to GPER by applying FMP‐API‐1 (100 μM) in isometric tension studies to disrupt the interaction between AKAPs and PKA, and detecting AKAP and SAP97 in immunoprecipitation studies in PCASMCs. Pretreatment with FMP‐API‐1 attenuated G‐1‐induced relaxation (10 – 3,000 nM) of endothelium‐denuded coronary arteries preconstricted with PGF2α (1 μM). In immunoprecipitation studies, G‐1 clearly enhanced the interaction of both AKAP5 and SAP97 to GPER. Therefore, we conclude that activation of GPER with G‐1 stimulates the production of cAMP, which is due to GPER coupling to Gs via forming a plasma membrane signaling complex with AKAP/SAP97.Support or Funding InformationAHA, Diversity of G Protein
Altered regulation of cardiac sympathetic nerve activity during hypertension
Authors: Ramchandra Rohit , McBryde Fiona , George Bindu , Paton Julian F , Mahesh Darvina 
Published: November 2023

Hypertension remains one of the strongest predictors of premature death and an increase in sympathetic nerve activity has been implicated in the pathogenesis of hypertension. In this context, increased sympathetic drive to the heart promotes arrhythmias and sudden death in patients, but the level of cardiac sympathetic nerve activity (CSNA) in hypertension has been assessed only by indirect methods. We tested the hypothesis that directly recorded CSNA is increased in renovascular hypertension.Renovascular hypertension was induced by surgical clipping of one renal artery. Following three weeks post‐clip, electrodes were surgically implanted into cardiac sympathetic nerve fascicles of adult sheep. Following recovery from surgery, resting levels of mean arterial pressure, heart rate and cardiac sympathetic nerve activity were recorded. In conscious sheep, the renal clipping procedure induced hypertension relative to sham operated controls (106 ± 4 vs. 86 ± 6 mmHg, p<0.05, n=6 per group). However, no significant change in heart rate was observed between the two groups. Direct cardiac sympathetic nerve activity recording in conscious sheep showed that burst frequency of cardiac sympathetic nerves was higher in renovascular hypertensive sheep compared to sham control ewes (39 ± 2.6 vs. 28 ± 1.8 bursts per minute, p < 0.05). In addition, our data suggest that baroreflex control of CSNA at lower pressures was impaired (upper plateau of 226 ± 23 vs 386 ± 64 % of control).Our data includes the first ever recordings of cardiac sympathetic nerve activity in hypertension and suggest a potential role for CSNA in mediating the chronic elevation in arterial pressure in hypertensive animals.Support or Funding InformationThe University of Auckland Faculty Research Development Fund