Abstract 3669: Transcoronary Gene Transfer of Serca2a Enhances Coronary Blood Flow through an Increase of eNOS Activity in Endothelial Cells

Congestive heart failure (CHF) is associated with impaired endothelium-dependent nitric oxide (NO)-mediated vasodilatation. Clinical trials using AAV1-SERCA2a myocardial gene delivery in CHF patients are currently underway. We hypothesized that transcoronary SERCA2a gene transfer can increase NO production by endothelial cells (EC). To assess the effects of SERCA2a gene transfer on coronary flow and endothelial function in vivo, we used a preclinical porcine volume-overload heart failure model (severe mitral valve regurgitation (MR)). Two months after MR creation, animals underwent intracoronary injection of AAV1-SERCA2a or saline. Four months post MR creation, the average coronary flow peak velocity (APV) was measured in the mid portion of the left anterior descending artery (LAD), and adjusted to the diameter of coronary artery and the weight of the left ventricle. Compared to saline treated animals, SERCA2a has significantly increased the adjusted coronary flow (ml/min/g) (0.467 ± 0.09, n=6 vs. 0.886 ± 0.18, n=7; P<0.01) but no difference was observed compared to sham operated animals (1.003 ± 0.2137, n=4; P=NS). Confocal immunofluorescence of swine coronary artery sections demonstrated that SERCA2a is expressed in endothelial and vascular smooth muscle cells; however, eNOS was expressed only in EC in all animal groups. Western blot analyses showed down-regulation of SERCA2a and eNOS expression in LAD, LCA and RCA from failing saline-injected animals. In coronary arteries from SERCA2a-transduced animals, expression levels of SERCA2a and eNOS were similar to those in sham-operated animals. To further investigate the effect of SERCA2a gene transfer on endothelial function we used cultured human coronary artery EC. RT-PCR analysis, western blot and e-NOS promoter/ luciferase reporter assay have shown that SERCA2a gene transfer increased expression and Ser-1177 phosphorylation of e-NOS. Furthermore, cGMP production was increased in SERCA2a-infected EC, confirming its possible functional effect on eNOS activity. In conclusion, our results demonstrate that transcoronary SERCA2a gene transfer improves endothelial function in failing hearts through regulation of eNOS expression and activity.

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Regulation of capillary tubules and lipid formation in vascular endothelial cells and macrophages via extracellular vesicle-mediated microRNA-4306 transfer

Journal of International Medical Research ◽

10.1177/0300060518809255 ◽

2018 ◽

Vol 47 (1) ◽

pp. 453-469 ◽

Cited By ~ 1

Author(s):

Ying Yang ◽

Hui Luo ◽

Can Zhou ◽

Rongyi Zhang ◽

Si Liu ◽

...

Keyword(s):

Endothelial Cells ◽

Coronary Artery ◽

Vascular Endothelial Cells ◽

Density Lipoprotein ◽

Extracellular Vesicle ◽

Luciferase Reporter ◽

Vascular Endothelial ◽

Human Coronary Artery ◽

Lipid Formation

Objective This study aimed to examine regulation of capillary tubules and lipid formation in vascular endothelial cells and macrophages via extracellular vesicle-mediated microRNA (miRNA)-4306 transfer Methods Whole blood samples (12 mL) were collected from 53 patients, and miR-4306 levels in extracellular vesicles (EVs) were analyzed by reverse transcription-polymerase chain reaction. Human coronary artery vascular endothelial cells (HCAECs) and human monocyte-derived macrophages (HMDMs) were transfected with a scrambled oligonucleotide, an miR-4306 mimic, or an anti-miR-4306 inhibitor. The direct effect of miR-4306 on the target gene was analyzed by a dual-luciferase reporter assay. Results EV-contained miR-4306 released from HMDMs was significantly upregulated in coronary artery disease. Oxidized low-density lipoprotein (ox-LDL)-stimulated HMDM-derived EVs inhibited proliferation, migration, and angiogenesis abilities of HCAECs in vitro. However, ox-LDL-stimulated HCAEC-derived EVs enhanced lipid formation of HMDMs. The possible mechanism of these findings was partly due to EV-mediated miR-4306 upregulation of the Akt/nuclear factor kappa B signaling pathway. Conclusions Paracrine cellular crosstalk between HCAECs and HMDMs probably supports the pro-atherosclerotic effects of EVs under ox-LDL stress.

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Abstract 557: Type 2 Diabetes is Associated With Impaired High-Density Lipoprotein Endothelial Protective Function

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.557 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Tomas Vaisar ◽

Erica Couzens ◽

Arnold Hwang ◽

Andrew N Hoofnagle ◽

Carolyn E Barlow ◽

...

Keyword(s):

Type 2 Diabetes ◽

Endothelial Cells ◽

Endothelial Function ◽

Lipid Profile ◽

High Density ◽

High Density Lipoproteins ◽

Protective Function ◽

Enos Activity ◽

Sphingosine 1 Phosphate

Aim: One of the hallmarks of diabetes is impaired endothelial function. High density lipoproteins (HDL) can exert protective effects on endothelium stimulating NO production and protecting from inflammation. Previous study suggested that HDL in obese people with diabetes and metabolic syndrome and markedly low HDL-C lost endothelial protective function. We aimed to test whether type 2 diabetes impairs HDL endothelium protective functions in people with otherwise normal lipid profile. Methods: In a case-control study (n=40 per group) nested in the Cooper Center Longitudinal Study, we isolated HDL and measured its ability to stimulate activity of endothelial nitric oxide synthase (eNOS; phosphorylation of Ser1177) in endothelial cells and the ability of HDL to suppress inflammatory response of endothelial cells (NFkB activation). Additionally, we also measured by LCMS levels of sphingosine-1-phosphate (S1P) and plasma P-selectin by ELISA. Results: The HDL in people with type 2 diabetes lost almost 40% of its ability to stimulate eNOS activity (P<0.001) and 20% of its ability to suppress inflammation in endothelial cells ( P <0.001) compared to non-diabetic controls despite similar BMI and lipid profile (HDL-C, LDL-C, TC, TG).The ability of HDL to stimulate eNOS activity was negatively associated with plasma levels of P-selectin, an established marker of endothelial dysfunction (r=–0.32, P <0.001). Furthermore, sphingosine-1-phosphate (S1P) levels were decreased in plasma of people with diabetes ( P =0.017) and correlated strongly with HDL-mediated eNOS activation. Conclusions: Collectively, our data suggest that HDL in individuals with type 2 diabetes loses its ability to maintain proper endothelial function independent of HDL-C, perhaps due to loss of S1P, and may contribute to development of diabetic complications.

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Abstract 17140: Nitric Oxide Production is Decreased in the Left Ventricle of Diabetic Subjects Undergoing Coronary Artery Bypass Grafting

Circulation ◽

10.1161/circ.138.suppl_1.17140 ◽

2018 ◽

Vol 138 (Suppl_1) ◽

Author(s):

Angela Kosta ◽

Sergey V Ryzhov ◽

Robert S Kramer ◽

Reed D Quinn ◽

Douglas Sawyer ◽

...

Keyword(s):

Nitric Oxide ◽

Flow Cytometry ◽

Endothelial Cells ◽

Coronary Artery ◽

Coronary Artery Bypass Grafting ◽

Artery Bypass ◽

Left Ventricular ◽

Nitric Oxide Production ◽

Bypass Grafting ◽

Enos Expression

Introduction: Dysregulation of endothelial nitric oxide synthase (eNOS) and generation of nitric oxide (NO) are critical early indicators for diabetes-induced endothelial dysfunction and cardiovascular complications. We hypothesize that levels of NO production and eNOS expression by endothelial cells are decreased in DM subjects when compared to non-DM subjects. Methods: The study cohort consisted of 9 non-DM subjects and 6 DM subjects undergoing myocardial biopsy at the time of coronary artery bypass grafting surgery. The non-myocyte cell suspensions from the left ventricle (LV), right atrial appendages (RAA), and skeletal muscle (SKM) tissue were analyzed by flow cytometry to measure production of nitric oxide in subpopulations of endothelial and non-endothelial cells. Cells in suspension were incubated with DAF-2DA in the presence or absence of NO synthase inhibitor, L-NAME. Flow cytometry was used to determine production of NO in subpopulations of endothelial and non-endothelial cells from biopsies. Measurements of eNOS and phospho-eNOS (ser1177) were performed using western blot. Results: Basal Nitric oxide production was measurable in non-diabetic subjects \ in 55%, 80% and 65% of unstimulated endothelial cells obtained from RAA, LV and SKM biopsies, compared to 40%, 40%, and 66%, respectively in diabetic subjects ( P < 0.02, DM vs Non-DM). No differences were found in the number of NO-producing non-endothelial cells between DM and non-DM subjects. The level of eNOS showed a trend towards decreased protein expression in DM subjects compared to non-DM. Conclusions: Generation of NO by endothelial cells and level of eNOS expression are decreased in left ventricular endothelial cells of DM patients compared to non-DM. Left ventricular biopsies can be used safely for assessment of NO dysregulation and endothelial dysfunction, and whether these can be improved with interventions targeting diabetic cardiovascular disease.

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Gene transfer of extracellular superoxide dismutase improves endothelial function in rats with heart failure

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00108.2005 ◽

2005 ◽

Vol 289 (2) ◽

pp. H525-H532 ◽

Cited By ~ 34

Author(s):

Shinichiro Iida ◽

Yi Chu ◽

Joseph Francis ◽

Robert M. Weiss ◽

Carol A. Gunnett ◽

...

Keyword(s):

Heart Failure ◽

Superoxide Dismutase ◽

Risk Factor ◽

Endothelial Dysfunction ◽

Gene Transfer ◽

Endothelial Function ◽

Mesenteric Artery ◽

Extracellular Superoxide Dismutase ◽

Protective Effects ◽

Sod Activity

Oxidative stress is associated with endothelial dysfunction in heart failure. The goals of this study were to determine whether 1) gene transfer of extracellular superoxide dismutase (ecSOD) reduces levels of superoxide and improves endothelial function in the aorta and mesenteric artery in rats with heart failure, and 2) the heparin-binding domain (HBD) of ecSOD, by which ecSOD binds to cells, is required for protective effects of ecSOD. Seven weeks after coronary ligation, in rats with heart failure and sham-operated rats, we injected adenoviral vectors intravenously that express ecSOD, ecSOD with deletion of the HBD (ecSODΔHBD), or a control vector. Four days after injection of viruses, responses to acetylcholine, ADP, and sodium nitroprusside were examined in rings of the aorta and mesenteric artery. ecSOD bound to endothelium and increased SOD activity in the aorta after gene transfer of ecSOD, not ecSODΔHBD. Gene transfer of ecSOD, but not ecSODΔHBD, reduced levels of superoxide and improved relaxation to acetylcholine and ADP in the aorta and mesenteric artery from rats with heart failure. Improvement of relaxation to acetylcholine in the mesenteric artery from rats with heart failure after gene transfer of ecSOD was mediated in part by hydrogen peroxide. The major finding of this study is that the HBD of ecSOD is necessary for protection against endothelial dysfunction in rats with heart failure. We speculate that a common gene variant in the HBD of ecSOD, which is a risk factor for ischemic heart disease, may be a risk factor for vascular maladaptation and endothelial dysfunction in heart failure.

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SIRT1-mediated deacetylation of NF-κB inhibits the MLCK/MLC2 pathway and the expression of ET-1 thus alleviating the development of coronary artery spasm

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00366.2020 ◽

2020 ◽

Author(s):

Bo-Wen Wu ◽

Mi-Shan Wu ◽

Yu Liu ◽

Meng Lu ◽

Jin-Dong Guo ◽

...

Keyword(s):

Coronary Artery ◽

Western Blot ◽

Light Chain ◽

Blot Analysis ◽

Western Blot Analysis ◽

Myosin Light Chain ◽

Coronary Artery Spasm ◽

Luciferase Reporter ◽

Sirt1 Expression

Coronary artery spasm (CAS) is an intense vasoconstriction of coronary arteries that cause total or subtotal vessel occlusion. The cardioprotective effect of sirtuin-1 (SIRT1) has been extensively highlighted in coronary artery diseases. The aims within this study include the investigation of the molecular mechanism by which SIRT1 alleviates CAS. SIRT1 expression was first determined by RT-qPCR and Western blot analysis in an endothelin-1 (ET-1)-induced rat CAS model. Interaction among SIRT1, nuclear factor-kappaB (NF-κB), myosin light chain kinase/myosin light chain-2 (MLCK/MLC2), and ET-1 was analyzed using luciferase reporter assay, RT-qPCR and Western blot analysis. After ectopic expression and depletion experiments in vascular smooth muscle cells (VSMCs), contraction and proliferation VSMCs, and expression of contraction-related proteins (α-SMA, calponin, and SM22α) were measured by collagen gel contraction, EdU assay, RT-qPCR and Western blot analysis. The obtained results showed that SIRT1 expression was reduced in rat CAS models. However, overexpression of SIRT1 inhibited the contraction and proliferation of VSMCs in vitro. Mechanistic investigation indicated that SIRT1 inhibited NF-κB expression through deacetylation. Moreover, NF-κB could activate the MLCK/MLC2 pathway and up-regulate ET-1 expression by binding to their promoter regions, thus inducing VSMC contraction and proliferation in vitro. In vivo experimental results also revealed that SIRT1 alleviated CAS through regulation of the NF-κB/MLCK/MLC2/ET-1 signaling axis. Collectively, our data suggested that SIRT1 could mediate the deacetylation of NF-κB, disrupt the MLCK/MLC2 pathway and inhibit the expression of ET-1 to relieve CAS, providing a theoretical basis for the prospect of CAS treatment and prevention.

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Coronary flow velocity reserve predicts adverse prognosis in women with angina and no obstructive coronary artery disease: results from the iPOWER study

European Heart Journal ◽

10.1093/eurheartj/ehaa944 ◽

2020 ◽

Author(s):

Jakob Schroder ◽

Marie M Michelsen ◽

Naja D Mygind ◽

Hannah E Suhrs ◽

Kira B Bove ◽

...

Keyword(s):

Heart Failure ◽

Coronary Artery Disease ◽

Coronary Artery ◽

Coronary Flow ◽

Adverse Outcome ◽

Obstructive Coronary Artery Disease ◽

Composite Outcome ◽

Coronary Flow Velocity Reserve ◽

Coronary Flow Velocity ◽

Artery Disease

Abstract Aims Many patients with angina, especially women, do not have obstructive coronary artery disease (CAD) yet have impaired prognosis. We investigated whether routine assessment of coronary microvascular dysfunction (CMD) is feasible and predicts adverse outcome in women with angina and no obstructive CAD. Methods and results After screening 7253, we included 1853 women with angina and no obstructive CAD on angiogram who were free of previous CAD, heart failure, or valvular heart disease in the prospective iPOWER (Improving Diagnosis and Treatment of Women with Angina Pectoris and Microvascular Disease) study. CMD was assessed by Doppler echocardiography in the left anterior descending artery as coronary flow velocity reserve (CFVR). Patients were followed for a composite outcome of cardiovascular death, myocardial infarction (MI), heart failure, stroke, and coronary revascularization. CFVR was obtained in 1681 patients (91%) and the median CFVR was 2.33 (quartiles 1–3: 2.00–2.74). During a median follow-up of 4.5 years, 96 events occurred. In univariate Cox regression, CFVR was associated with the composite outcome {hazard ratio (HR) 1.07 [95% confidence interval (CI) 1.03–1.11] per 0.1 unit decrease in CFVR; P < 0.001}, primarily driven by an increased risk of MI and heart failure. Results remained significant in multivariate analysis [HR 1.05 (95% CI 1.01–1.09) per 0.1 unit decrease in CFVR; P = 0.01]. In exploratory analyses, CFVR was also associated with the risk of repeated hospital admission for angina and all-cause mortality. Conclusion Assessment of CFVR by echocardiography is feasible and predictive of adverse outcome in women with angina and no obstructive CAD. Results support a more aggressive preventive management of these patients and underline the need for trials targeting CMD.

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Visfatin activates eNOS via Akt and MAP kinases and improves endothelial cell function and angiogenesis in vitro and in vivo: translational implications for atherosclerosis

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.90780.2008 ◽

2009 ◽

Vol 296 (6) ◽

pp. E1440-E1449 ◽

Cited By ~ 60

Author(s):

Fina Lovren ◽

Yi Pan ◽

Praphulla C. Shukla ◽

Adrian Quan ◽

Hwee Teoh ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Endothelial Function ◽

Vein Graft ◽

Map Kinases ◽

Enos Expression ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal ◽

Enos Phosphorylation

Improving endothelial nitric oxide synthase (eNOS) bioactivity and endothelial function is important to limit native, vein graft, and transplant atherosclerosis. Visfatin, a NAD biosynthetic enzyme, regulates the activity of the cellular survival factor, Sirt1. We hypothesized that visfatin may improve eNOS expression, endothelial function, and postnatal angiogenesis. In human umbilical vein (HUVEC) and coronary artery endothelial cells, we evaluated the effects of recombinant human visfatin on eNOS protein and transcript expression and mRNA stability, in the presence and absence of visfatin RNA silencing. We also assessed visfatin-induced protein kinase B (Akt) activation and its association with src-tyrosine kinases, phosphorylation of Ser1177within eNOS in the presence and absence of phosphatidylinositol 3-kinase (PI 3-kinase) inhibition with LY-294002, and evaluated the contributory role of extracellular signal-regulated kinase 1/2. Finally, we determined the impact of visfatin on HUVEC migration, proliferation, inflammation-induced permeability, and in vivo angiogenesis. Visfatin (100 ng/ml) upregulated and stabilized eNOS mRNA and increased the production of nitric oxide and cGMP. Visfatin-treated HUVEC demonstrated greater proliferation, migration, and capillary-like tube formation but less tumor necrosis factor-α-induced permeability; these effects were decreased in visfatin gene-silenced cells. Visfatin increased total Akt and Ser473-phospho-Akt expression with concomitant rises in eNOS phosphorylation at Ser1177; these effects were blocked by LY-2940002. Studies with PP2 showed that the nonreceptor tyrosine kinase, src, is an upstream stimulator of the PI 3-kinase-Akt pathway. Visfatin also activated mitogen-activated protein (MAP) kinase through PI 3-kinase, and mitogen/extracellular signal-regulated kinase inhibition attenuated visfatin-elicited Akt and eNOS phosphorylation. Visfatin-filled Matrigel implants showed an elevated number of infiltrating vessels, and visfatin treatment produced significant recovery of limb perfusion following hindlimb ischemia. These results indicate a novel effect of visfatin to stimulate eNOS expression and function in endothelial cells, via a common upstream, src-mediated signaling cascade, which leads to activation of Akt and MAP kinases. Visfatin represents a translational target to limit endothelial dysfunction, native, vein graft and transplant atherosclerosis, and improve postnatal angiogenesis.

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circSnx12 Is Involved in Ferroptosis During Heart Failure by Targeting miR-224-5p

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.656093 ◽

2021 ◽

Vol 8 ◽

Author(s):

Haoyuan Zheng ◽

Lin Shi ◽

Changci Tong ◽

Yunen Liu ◽

Mingxiao Hou

Keyword(s):

Heart Failure ◽

Western Blot ◽

Iron Metabolism ◽

Iron Homeostasis ◽

Pressure Overload ◽

The Body ◽

Luciferase Reporter ◽

Qrt Pcr ◽

Cerna Network ◽

Echocardiographic Measurement

Circular RNA (circRNA) is a subclass of non-coding RNAs that enables the circular transcripts resistant to the exonuclease digestion. Iron homeostasis is essential for the body to maintain normal physiological functions. At present, the relationship among circRNA, iron metabolism and heart failure remains largely unknown. This study aimed to explore the regulatory mechanism of circRNA and iron metabolism in heart failure. We obtained circRNA, miRNA and mRNA data from public databases and built a ceRNA network. The prediction results were verified in the myocardial tissues of pressure overload-induced heart failure mice through the use of histopathological staining methods, iron and malondialdehyde (MDA) measurement tests, quantitative real-time PCR (qRT-PCR), Western blot analysis and luciferase reporter assay. A total of 4 genes related to iron metabolism and oxidative stress were identified, and a ceRNA network involving 7 circRNAs, 7 miRNAs, and 4 mRNAs was constructed using bioinformatics tools. The results of qRT-PCR and Western blot analyses indicated that the expression level of FTH1 was similar with that predicted by bioinformatics analysis. Echocardiographic measurement showed that heart failure mice have lower fractional shortening and ejection fraction. Moreover, the myocardium of heart failure mice displayed obvious fibrosis as well as increased levels of iron and MDA compared to control mice. Besides, circSnx12 could act as an endogenous sponge to bind with miR-224-5p, and the 3'UTR region of FTH1 also had miRNA binding sites. A circRNA-miRNA-mRNA regulatory network was successfully constructed by identifying differentially expressed genes related to iron metabolism. This new approach reveals potential circRNA targets for the treatment of heart failure.

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Glossogyne tenuifolia Extract Increases Nitric Oxide Production in Human Umbilical Vein Endothelial Cells

Pharmaceuticals ◽

10.3390/ph14060577 ◽

2021 ◽

Vol 14 (6) ◽

pp. 577

Author(s):

Chin-Feng Hsuan ◽

Thung-Lip Lee ◽

Wei-Kung Tseng ◽

Chau-Chung Wu ◽

Chi-Chang Chang ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Western Blot ◽

Umbilical Vein ◽

No Production ◽

Enos Expression ◽

Human Umbilical Vein ◽

Whole Plant ◽

Umbilical Vein Endothelial Cells ◽

Glossogyne Tenuifolia

The vascular nitric oxide (NO) system has a protective effect in atherosclerosis. NO is generated from the conversion of L-arginine to L-citrulline by the enzymatic action of endothelial NO synthase (eNOS). Compounds with the effect of enhancing eNOS expression are considered to be candidates for the prevention of atherosclerosis. In this study, extracts from the aerial, root, and whole plant of Glossogyne tenuifolia (GT) were obtained with ethanol, n-hexane, ethyl acetate (EA), and methanol extraction, respectively. The effects of these GT extracts on the synthesis of NO and the expression of eNOS in human umbilical vein endothelial cells (HUVECs) were investigated. NO production was determined as nitrite by colorimetry, following the Griess reaction. The treatment of HUVECs with EA extract from the root of GT and n-hexane, methanol, and ethanol extract from the aerial, root, and whole plant of GT increased NO production in a dose-dependent manner. When at a dose of 160 μg/mL, NO production increased from 0.9 to 18.4-fold. Among these extracts, the methanol extract from the root of GT (R/M GTE) exhibited the most potent effect on NO production (increased by 18.4-fold). Furthermore, using Western blot and RT–PCR analysis, treatment of HUVECs with the R/M GTE increased both eNOS protein and mRNA expression. In addition, Western blot analysis revealed that the R/M GTE increased eNOS phosphorylation at serine1177 as early as 15 min after treatment. The chemical composition for the main ingredients was also performed by HPLC analysis. In conclusion, the present study demonstrated that GT extracts increased NO production in HUVECs and that the R/M GTE increased NO production via increasing eNOS expression and activation by phosphorylation of eNOS at serine1177.

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