Morphological and Immunohistochemical Features of the Heart under the Effect of Hydrogen Sulfide Balneotherapy.

The effects of hydrogen sulfide baths and sulfide-silt mud applications on the abdominal wall were studied in outbred white rats. Heart preparations stained with hematoxylin and eosin were analyzed and the mean numbers of cells expressing Ki-67, Nkx-2.5, and mesenchymal stem cell markers were determined. Hydrogen sulfide balneotherapy promoted the development of blood capillaries in rat hearts and an increased the expression of CD34. A decrease in the regenerative potential of the myocardium was found due to a decrease in the content of proliferating cells and cells expressing CD73, CD90, and CD105, and also due to the absence of cells stained for cardiomyogenic differentiation marker Nkx-2.5.

Cardiomyocyte-derived small extracellular vesicles can signal eNOS activation in cardiac microvascular endothelial cells to protect against Ischemia/Reperfusion injury.

Rationale: The crosstalk between cardiac microvascular endothelial cells (CMECs) and cardiomyocytes (CMs) has emerged as a key component in the development of, and protection against, cardiac diseases. For example, activation of endothelial nitric oxide synthase (eNOS) in CMECs, by therapeutic strategies such as ischemic preconditioning, plays a critical role in the protection against myocardial ischemia/reperfusion (I/R) injury. However, much less is known about the signals produced by CMs that are able to regulate CMEC biology. Here we uncovered one such mechanism using Tongxinluo (TXL), a traditional Chinese medicine, that alleviates myocardial ischemia/reperfusion (I/R) injury by activating CMEC eNOS. The aim of our study is to identify the signals produced by CMs that can regulate CMEC biology during I/R. Methods:Ex vivo, in vivo, and in vitro settings of ischemia-reperfusion were used in our study, with the protective signaling pathways activated in CMECs identified using genetic inhibition (p70s6k1 siRNA, miR-145-5p mimics, etc.), chemical inhibitors (the eNOS inhibitor, L-NNA, and the small extracellular vesicles (sEVs) inhibitor, GW4869) and Western blot analyses. TritonX-100 at a dose of 0.125% was utilized to inactivate the eNOS activity in endothelium to investigate the role of CMEC-derived eNOS in TXL-induced cardioprotection. Results: We found that while CMEC-derived eNOS activity was required for the cardioprotection of TXL, activation of eNOS in CMECs by TXL did not occur directly. Instead, eNOS activation in CMECs required a crosstalk between CMs and CMECs through the uptake of CM-derived sEVs. We further demonstrate that TXL induced CM-sEVs contain increased levels of Long Intergenic Non-Protein Coding RNA, Regulator Of Reprogramming (Linc-ROR). Upon uptake into CMECs, linc-ROR downregulates its target miR-145-5p leading to activation of the eNOS pathway by facilitating the expression of p70s6k1 in these cells. The activation of CMEC-derived eNOS works to increase survival in both the CMECs and the CMs themselves. Conclusions: These data uncover a mechanism by which the crosstalk between CMs and CMECs leads to the increased survival of the heart after I/R injury and point to a new therapeutic target for the blunting of myocardial I/R injury.

Tongxinluo attenuates oxygen-glucose-serum deprivation/restoration-induced endothelial barrier breakdown via peroxisome proliferator activated receptor-α/angiopoietin-like 4 pathway in high glucose-incubated human cardiac microvascular endothelial cells.

BACKGROUND: Traditional Chinese medicine Tongxinluo (TXL) has been widely used to treat coronary artery disease in China, since it could reduce myocardial infarct size and ischemia/reperfusion injury in both non-diabetic and diabetic conditions. It has been shown that TXL could regulate peroxisome proliferator activated receptor-α (PPAR-α), a positive modulator of angiopoietin-like 4 (Angptl4), in diabetic rats. Endothelial junction substructure components, such as VE-cadherin, are involved in the protection of reperfusion injury. Thus, we hypothesized cell-intrinsic and endothelial-specific Angptl4 mediated the protection of TXL on endothelial barrier under high glucose condition against ischemia/reperfusion-injury via PPAR-α pathway. METHODS: Incubated with high glucose medium, the human cardiac microvascular endothelial cells (HCMECs) were then exposed to oxygen-glucose-serum deprivation (2 hours) and restoration (2 hours) stimulation, with or without TXL, insulin, or rhAngptl4 pretreatment. RESULTS: TXL, insulin, and rhAngptl4 had similar protective effects on the endothelial barrier. TXL treatment reversed the endothelial barrier breakdown in HCMECs significantly as identified by decreasing endothelial permeability, upregulating the expression of JAM-A, VE-cadherin, and integrin-α5 and increasing the membrane location of VE-cadherin and integrin-α5, and these effects of TXL were as effective as insulin and rhAngptl4. However, Angptl4 knock-down with small interfering RNA (siRNA) interference and PPAR-α inhibitor MK886 partially abrogated these beneficial effects of TXL. Western blotting also revealed that similar with insulin, TXL upregulated the expression of Angptl4 in HCMECs, which could be inhibited by Angptl4 siRNA or MK886 exposure. TXL treatment increased PPAR-α activity, which could be diminished by MK886 but not by Angptl4 siRNA. CONCLUSION: These data suggest cell-intrinsic and endothelial-specific Angptl4 mediates the protection of TXL against endothelial barrier breakdown during oxygen-glucose-serum deprivation and restoration under high glucose condition partly via the PPAR-α/Angptl4 pathway.

Ficus deltoidea suppresses endothelial activation, inflammation, monocytes adhesion and oxidative stress via NF-κB and eNOS pathways in stimulated human coronary artery endothelial cells.

BACKGROUND: Ficus deltoidea (FD) has been shown to have antidiabetic, anti-inflammatory, antinociceptive and antioxidant properties. However, its effects on key events in the pathogenesis of atherosclerosis are unknown. AIM: To investigate the endothelial activation, inflammation, monocyte-endothelial cell binding and oxidative stress effects of four FD varieties. METHODS: Human coronary artery endothelial cells (HCAEC) were incubated with different concentrations of aqueous ethanolic extracts of FD var. trengganuensis (FDT), var. kunstleri (FDK), var. deltoidea (FDD) and var. intermedia (FDI), together with LPS. Protein and gene expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), endothelial-leukocyte adhesion molecule-1 (E-selectin), interleukin-6 (IL-6), Nuclear factor-κB (NF-κB) p50 and p65 and endothelial nitric oxide synthase (eNOS) were measured using ELISA and QuantiGene plex, respectively. Adhesion of monocyte to HCAEC and formation of reactive oxygen species (ROS) were detected by Rose Bengal staining and 2'-7'-dichlorofluorescein diacetate (DCFH-DA) assay. RESULTS: FDK exhibited the highest inhibition of biomarkers in relation to endothelial activation and inflammation, second in reducing monocyte binding (17.3%) compared to other varieties. FDK (25.6%) was also the most potent at decreasing ROS production. CONCLUSION: FD has anti-atherogenic effects, possibly mediated by NF-κB and eNOS pathways; with FDK being the most potent variety. It is potentially beneficial in mitigating atherogenesis.

Cardioprotective Effects of Puerarin-V on Isoproterenol-Induced Myocardial Infarction Mice Is Associated with Regulation of PPAR-Υ/NF-κB Pathway.

Puerarin is a well-known traditional Chinese medicine which has been used for the treatment of cardiovascular diseases. Recently, a new advantageous crystal form of puerarin, puerarin-V, has been developed. However, the cardioprotective effects of puerarin-V on myocardial infarction (MI) heart failure are still unclear. In this research, we aim to evaluate the cardioprotective effects of puerarin-V on the isoproterenol (ISO)-induced MI mice and elucidate the underlying mechanisms. To induce MI in C57BL/6 mice, ISO was administered at 40 mg/kg subcutaneously every 12 h for three times in total. The mice were randomly divided into nine groups: (1) control; (2) ISO; (3) ISO + puerarin injection; (4⁻9) ISO + puerarin-V at different doses and timings. After treatment, cardiac function was evaluated by electrocardiogram (ECG), biochemical and histochemical analysis. In vitro inflammatory responses and apoptosis were evaluated in human coronary artery endothelial cells (HCAECs) challenged by lipopolysaccharide (LPS). LPS-induced PPAR-Υ/NF-κB and subsequently activation of cytokines were assessed by the western blot and real-time polymerase chain reaction (PCR). Administration of puerarin-V significantly inhibits the typical ST segment depression compared with that in MI mice. Further, puerarin-V treatment significantly improves ventricular wall infarction, decreases the incidence of mortality, and inhibits the levels of myocardial injury markers. Moreover, puerarin-V treatment reduces the inflammatory milieu in the heart of MI mice, thereby blocking the upregulation of proinflammatory cytokines (TNF-α, IL-1ß and IL-6). The beneficial effects of puerarin-V might be associated with the normalization in gene expression of PPAR-Υ and PPAR-Υ/NF-κB /ΙκB-α/ΙΚΚα/ß phosphorylation. In the in vitro experiment, treatment with puerarin-V (0.3, 1 and 3 µM) significantly reduces cell death and suppresses the inflammation cytokines expression. Likewise, puerarin-V exhibits similar mechanisms. The cardioprotective effects of puerarin-V treatment on MI mice in the pre + post-ISO group seem to be more prominent compared to those in the post-ISO group. Puerarin-V exerts cardioprotective effects against ISO-induced MI in mice, which may be related to the activation of PPAR-γ and the inhibition of NF-κB signaling in vivo and in vitro. Taken together, our research provides a new therapeutic option for the treatment of MI in clinic.

In vitro systems toxicology-based assessment of the potential modified risk tobacco product CHTP 1.2 for vascular inflammation- and cytotoxicity-associated mechanisms promoting adhesion of monocytic cells to human coronary arterial endothelial cells.

Cigarette smoking causes cardiovascular diseases. Heating tobacco instead of burning it reduces the amount of toxic compounds in the aerosol and may exert a reduced impact on health compared with cigarette smoke. Aqueous extract from the aerosol of a potential modified risk tobacco product, the Carbon Heated Tobacco Product (CHTP) 1.2, was compared in vitro with aqueous extract from the smoke of a 3R4F reference cigarette for its impact on the adhesion of monocytic cells to artery endothelial cells. Human coronary artery endothelial cells (HCAEC) were treated for 4 h with conditioned media from human monocytic Mono Mac 6 (MM6) cells exposed to CHTP1.2 or 3R4F extracts for 2 h or directly with those extracts freshly generated. In vitro monocyte-endothelial cell adhesion was measured concomitantly with inflammatory, oxidative stress, cytotoxicity, and death markers. Furthermore, transcriptomics analyses enabled to quantify the level of perturbation in HCAECs, and provide biological interpretation for the underlying molecular changes following exposure to 3R4F or CHTP1.2 extract. Our systems toxicology study demonstrated that approximately 10-15-fold higher concentrations of the CHTP 1.2 aerosol extract were needed to elicit similar effects as the 3R4F smoke extract on cardiovascular disease-relevant inflammation and cytotoxicity-related mechanisms and markers investigated in vitro.

Different transcriptional profiling between senescent and non-senescent human coronary artery endothelial cells (HCAECs) by Omeprazole and Lansoprazole treatment.

Recent evidence suggests that high dose and/or long term use of proton pump inhibitors (PPIs) may increase the risk of adverse cardiovascular events in older patients, but mechanisms underlying these detrimental effects are not known. Taking into account that the senescent endothelial cells have been implicated in the genesis or promotion of age-related cardiovascular disease, we hypothesized an active role of PPIs in senescent cells. The aim of this study is to investigate the changes in gene expression occurring in senescent and non-senescent human coronary artery endothelial cells (HCAECs) following Omeprazole (OPZ) or Lansoprazole (LPZ) treatment. Here, we show that atherogenic response is among the most regulated processes in PPI-treated HCAECs. PPIs induced down-regulation of anti-atherogenic chemokines (CXCL11, CXCL12 and CX3CL1) in senescent but not in non-senescent cells, while the same chemokines were up-regulated in untreated senescent cells. These findings support the hypothesis that up-regulated anti-atherogenic chemokines may represent a defensive mechanism against atherosclerosis during cellular senescence, and suggest that PPIs could activate pro-atherogenic pathways by changing the secretory phenotype of senescent HCAECs. Moreover, the genes coding for fatty acid binding protein 4 (FABP4) and piezo-type mechanosensitive ion channel component 2 (PIEZO2) were modulated by PPIs treatment with respect to untreated cells. In conclusions, our results show that long-term and high dose use of PPI could change the secretory phenotype of senescent cells, suggesting one of the potential mechanisms by which use of PPI can increase adverse outcomes in older subjects.

Cacao Polyphenols Potentiate Anti-Platelet Effect of Endothelial Cells and Ameliorate Hypercoagulatory States Associated with Hypercholesterolemia.

Platelets are related to the formation of blood clots that play a crucial role in thrombosis and other cardiovascular diseases. Cocoa, derived from Theobroma cocoa, has been widely used as functional food for improving cardiovascular health. In the present study, the direct and indirect effects of cacao polyphenols (CPs) were investigated on human platelet aggregation associated with endothelial cells (ECs) senescence. In addition, the effect of CPs on high-fat diet- (HFD-) induced hypercoagulatory states in rats was evaluated. CPs directly inhibited the human platelet aggregation induced by thromboxane analogue, U46619, and treatment of CPs on senescent endothelial cells markedly restored inhibitory effect of ECs on platelet aggregation. Nitric oxide (NO) from ECs is known as modulator of platelet aggregation and CPs increased eNOS activity in ECs and coronary artery. In animal model, increased TG level induced by high fat diet (HFD) was significantly decreased by CPs administration. In addition, the HFD animal had shorter bleeding time, and CPs administration attenuated the HFD-induced changes. Taken together, the present study indicates that CPs have potent anti-platelet effects most likely by direct and indirect effect via ECs and have the potential for lowering the risk of cardiovascular disease-related hypercoagulation due to hypercholesterolemia.

The Redox-sensitive Induction of the Local Angiotensin System Promotes Both Premature and Replicative Endothelial Senescence: Preventive Effect of a Standardized Crataegus Extract.

Endothelial senescence, characterized by an irreversible cell cycle arrest, oxidative stress, and downregulation of endothelial nitric oxide synthase (eNOS), has been shown to promote endothelial dysfunction leading to the development of age-related vascular disorders. This study has assessed the possibility that the local angiotensin system promotes endothelial senescence in coronary artery endothelial cells and also the protective effect of the Crataegus extract WS1442, a quantified hawthorn extract. Serial passaging from P1 to P4 (replicative senescence) and treatment of P1 endothelial cells with the eNOS inhibitor L-NAME (premature senescence) promoted acquisition of markers of senescence, enhanced ROS formation, decreased eNOS expression, and upregulation of angiotensin-converting enzyme (ACE) and AT1 receptors. Increased SA-ß-gal activity and the upregulation of ACE and AT1R in senescent cells were prevented by antioxidants, an ACE inhibitor, and by an AT1 receptor blocker. WS1442 prevented SA-ß-gal activity, the downregulation of eNOS, and oxidative stress in P3 cells. These findings indicate that the impairment of eNOS-derived nitric oxide formation favors a pro-oxidant response triggering the local angiotensin system, which, in turn, promotes endothelial senescence. Such a sequence of events can be effectively inhibited by a standardized polyphenol-rich extract mainly by targeting the oxidative stress.

Salvianolic Acid B Down-regulates Matrix Metalloproteinase-9 Activity and Expression in Tumor Necrosis Factor-α-induced Human Coronary Artery Endothelial Cells.

BACKGROUND: Salvianolic acid B (Sal B) is a bioactive water-soluble compound of Salviae miltiorrhizae, a traditional herbal medicine that has been used clinically for the treatment of cardiovascular diseases. This study sought to evaluate the effect of Sal B on matrix metalloproteinase-9 (MMP-9) and on the underlying mechanisms in tumor necrosis factor-α± (TNF-α±)-activated human coronary artery endothelial cells (HCAECs), a cell model of Kawasaki disease. METHODS: HCAECs were pretreated with 1-10 αµmol/L of Sal B, and then stimulated by TNF-α± at different time points. The protein expression and activity of MMP-9 were determined by Western blot assay and gelatin zymogram assay, respectively. Nuclear factor-κB (NF-κB) activation was detected with immunofluorescence, electrophoretic mobility shift assay, and Western blot assay. Protein expression levels of mitogen-activated protein kinase (c-Jun N-terminal kinase [JNK], extra-cellular signal-regulated kinase [ERK], and p38) were determined by Western blot assay. RESULTS: After HCAECs were exposed to TNF-α±, 1-10 αµmol/L Sal B significantly inhibited TNF-α±-induced MMP-9 expression and activity. Furthermore, Sal B significantly decreased IκBα± phosphorylation and p65 nuclear translocation in HCAECs stimulated with TNF-α± for 30 min. In addition, Sal B decreased the phosphorylation of JNK and ERK1/2 proteins in cells treated with TNF-α± for 10 min. CONCLUSIONS: The data suggested that Sal B suppressed TNF-α±-induced MMP-9 expression and activity by blocking the activation of NF-κB, JNK, and ERK1/2 signaling pathways.

Ginsenoside Re enhances small-conductance Ca(2+)-activated K(+) current in human coronary artery endothelial cells.

AIMS: Ginsenosides, active components in ginseng, have been shown to increase nitric oxide (NO) production in aortic endothelial cells. This effect was reversed by tetraethylammonium (TEA) inhibition of endothelial Ca(2+)-activated K(+) (KCa) channels. The objectives of this study, therefore, were to test 1) whether vasorelaxing ginsenoside Re could affect KCa current, an important regulator of NO production, in human coronary artery endothelial cells (HCAECs); and 2) whether small-conductance KCa (SKCa) channel was the channel subtype involved. MAIN METHODS: Ionic currents of cultured HCAECs were studied using whole-cell patch clamp technique. KEY FINDINGS: Ginsenoside Re dose-dependently increased endothelial outward currents, with an EC50 of 408.90±1.59nM, and a maximum increase of 36.20±5.62% (mean±SEM; p<0.05). Apamin, an SKCa channel inhibitor, could block this effect, while La(3+), a nonselective cation channel (NSC) blocker, could not. When NSC channel, inward-rectifier K(+) channel, intermediate-, and large-conductance KCa channels were simultaneously blocked, ginsenoside Re could still increase outward currents significantly (35.49±4.22%; p<0.05); this effect was again abolished by apamin. Repeating the experiments when Cl(-) channel was additionally blocked gave similar results. Finally, we demonstrated that ginsenoside Re could hyperpolarize HCAECs; this effect was reversed by apamin. These data clearly indicate that ginsenoside Re increased HCAEC outward current via SKCa channel activation, and NSC channel was not involved. SIGNIFICANCE: This is the first report to demonstrate that ginsenoside Re could increase SKCa channel activity in HCAECs. This can be a mechanism mediating ginseng's beneficial actions on coronary vessels.

Enhancement of voltage-gated K+ channels and depression of voltage-gated Ca2+ channels are involved in quercetin-induced vasorelaxation in rat coronary artery.

Quercetin is one of the most common flavonoids in the human daily diet. Its affects the coronary artery, especially L-type voltage-gated Ca2+ channels and voltage-gated K+ channels in the arterial smooth muscle cells, which are poorly understood. The present experiments were designed to study the myogenic effect of quercetin and its possible underlying mechanisms in the rat coronary artery. A wire myograph was used to observe the myogenic effects. Arterial smooth muscle cells were freshly isolated from the rat coronary artery and the intracellular free Ca2+ concentration was measured with molecular probe fluo-4-AM. The effects of quercetin on L-type voltage-gated Ca2+ channels and voltage-gated K+ channels were studied using a whole-cell patch clamp. Quercetin (3-30 µM) produced a depression and relaxation on the contraction induced by KCl or the thromboxane A2 analog 9,11-Dideoxy-9α,11α-methanoepoxy prostaglandin F 2α . The vasorelaxation was attenuated by 4-aminopyridine, a specific voltage-gated K+ channel inhibitor, but was not affected by the NG-nitro-L-arginine methylester ester (a nitric oxide synthesis inhibitor), glibenclamide (a ATP-activated K+ channel inhibitor), iberiotoxin (a Ca2+-activated K+ channel inhibitor), BaCl2 (an inward rectifier K+ channel inhibitor), or by endothelium denudation. At the same concentrations, quercetin reduced the KCl-induced elevation of the intracellular free Ca2+ concentration, inhibited the inward Ca2+ currents through L-type voltage-gated Ca2+ channels, and increased the outward K+ currents through voltage-gated K+ channels in the rat coronary artery smooth muscle cells. Collectively, our results demonstrate that quercetin possesses vasospasmolytic effects in RCA and suggest that depression of the Ca2+ influx through L-type voltage-gated Ca2+ channels and augmentation of voltage-gated K+ channel activity in the myocytes may underlie coronary relaxation.

Postprandial triglyceride-rich lipoproteins promote invasion of human coronary artery smooth muscle cells in a fatty-acid manner through PI3k-Rac1-JNK signaling.

SCOPE: The aim was to investigate the effect of postprandial triglyceride-rich lipoproteins (TRLs) with different fatty acid compositions on human coronary artery smooth muscle cell (hCASMC) invasion and to identify the molecular pathways involved. METHODS AND RESULTS: TRLs were isolated from the plasma of healthy volunteers after the ingestion of single meals enriched in MUFAs, saturated fatty acids (SFAs), or PUFAs. hCASMC invasion was analyzed using transwell chambers with Matrigel. TRLs-SFAs provoked the highest invasion, followed by TRLs-MUFAs and TRLs-PUFAs. Inhibition studies with Orlistat showed that invasion was dependent on the fatty acid composition of the TRLs. Fatty acids incorporated into the cell membranes strongly associated with cell invasion. Pull-down assays showed that TRLs-SFAs were able to increase Rac1 activity via inhibition of RhoA-dependent signaling. Chemical inhibition and siRNA studies showed that Rac1, PI3k, JNK, and MMP2 regulates TRL-SFA-induced hCASMC invasion. CONCLUSION: We demonstrate for the first time that TRLs induce hCASMCs invasion in a fatty acid dependent manner. This effect in TRLs-SFAs is mediated by the PI3k-Rac1-JNK, RhoA, and Rac1-MMP2 pathways. The ingestion of MUFA, compared to other dietary fatty acids such as SFA, could be considered as a nutritional strategy to reduce the atherosclerotic plaque formation.

Arterial territory-specific phosphorylated retinoblastoma protein species and CDK2 promote differences in the vascular smooth muscle cell response to mitogens.

Despite recent advances in medical procedures, cardiovascular disease remains a clinical challenge and the leading cause of mortality in the western world. The condition causes progressive smooth muscle cell (SMC) dedifferentiation, proliferation, and migration that contribute to vascular restenosis. The incidence of disease of the internal mammary artery (IMA), however, is much lower than in nearly all other arteries. The etiology of this IMA disease resistance is not well understood. Here, using paired primary IMA and coronary artery SMCs, serum stimulation, siRNA knockdowns, and verifications in porcine vessels in vivo, we investigate the molecular mechanisms that could account for this increased disease resistance of internal mammary SMCs. We show that the residue-specific phosphorylation profile of the retinoblastoma tumor suppressor protein (Rb) appears to differ significantly between IMA and coronary artery SMCs in cultured human cells. We also report that the differential profile of Rb phosphorylation may follow as a consequence of differences in the content of cyclin-dependent kinase 2 (CDK2) and the CDK4 phosphorylation inhibitor p15. Finally, we present evidence that siRNA-mediated CDK2 knockdown alters the profile of Rb phosphorylation in coronary artery SMCs, as well as the proliferative response of these cells to mitogenic stimulation. The intrinsic functional and protein composition specificity of the SMCs population in the coronary artery may contribute to the increased prevalence of restenosis and atherosclerosis in the coronary arteries as compared with the internal mammary arteries.

Genistein inhibits cell proliferation and stimulates apoptosis in human coronary artery endothelial cells.

BACKGROUND/AIMS: Isoflavone genistein is a plant-derived compound structurally similar to estradiol, which behaves weakly estrogenic or anti-estrogenic in a cell- and concentration-dependent manner. Genistein has been hypothesized to have beneficial effects on vascular diseases, although the mechanism has been unclear. Here, we investigated whether genistein may play a role in atherogenesis by regulating human coronary artery endothelial cell (HCAEC) survival. METHODS: HCAECs obtained from 48- to 53-year-old women (n = 3) were used and immunocytochemistry, cell proliferation assay and apoptosis assay were carried on HCAECs treated by genistein. RESULTS: Immunocytochemistry confirmed that HCAECs in culture express predominantly ESR2. Cell proliferation assay revealed that following 72 h of genistein treatment, HCAEC proliferation decreased in a concentration-dependent (10(-10) to 10(-6)M) manner compared to control (p < 0.01). The anti-proliferative effect of genistein is inhibited by estradiol. Genistein (10(-8)M) also induced a time-dependent increase in the number of apoptotic HCAECs after 24-, 48- and 72-hour treatments as detected by TUNEL and morphological analyses. CONCLUSION: These findings suggest that genistein acts as an anti-proliferative agent on HCAECs. The anti-proliferative and proapoptotic effects of genistein on vascular cells underlie the proposed anti-atherogenic and cardioprotective role of genistein.

Increased endothelin-1 responsiveness in human coronary artery smooth muscle cells exposed to 1,25-dihydroxyvitamin D(3).

Low blood concentrations of 25-hydroxyvitamin D(3) are associated with increased mortality, while some studies suggest improved cardiovascular outcomes with vitamin D(3) supplementation in chronic kidney disease. However, the physiological effects of vitamin D(3) on the cardiovascular system remain poorly understood making it difficult to determine whether vitamin D(3) supplementation might provide cardiovascular benefit or even cause harm. Thus here we investigated the effects of chronic 1,25-dihydroxyvitamin D(3) treatment on intracellular signaling in human coronary artery smooth muscle cells (HCASMCs) and found that 1,25-dihydroxyvitamin D(3) significantly potentiated endothelin (ET-1) signaling. Specifically, 1,25-dihydroxyvitamin D(3) (24-h pretreatment) caused a more than threefold enhancement in both ET-1-induced intracellular calcium mobilization and extracellular signal-regulated kinase (ERK) activation. This 1,25-dihydroxyvitamin D(3)-elicited signaling enhancement was not observed for either vasopressin or carbachol. With the use of endothelin receptor (ETR) isoform-selective antagonists, ETRA was found to be primarily responsible for the 1,25-dihydroxyvitamin D(3)-induced ET-1 responsiveness and yet ETRA mRNA expression and protein abundance were unaltered following 1,25-dihydroxyvitamin D(3) treatment. While there was an increase in ETRB mRNA expression in response to 1,25-dihydroxyvitamin D(3), the protein abundance of ETRB was again unchanged. Finally, ETRA/ETRB heterodimerization was not detected in HCASMCs in either the absence or presence of 1,25-dihydroxyvitamin D(3). Together, these data show for the first time that 1,25-dihydroxyvitamin D(3) enhances endothelin responsiveness in HCASMCs and that the effect is mediated through ETRA.

Ginkgo biloba extract and aspirin synergistically attenuate activated platelet-induced ROS production and LOX-1 expression in human coronary artery endothelial cells.

AIM: In the present study, we investigated whether the therapeutic dosages of Ginkgo biloba extract (EGb) and Aspirin (ASP) might synergistically suppress oxidative stress through regulating the expressions of LOX-1 and phosphorylated p38MAPK (p-p38MAPK) in human coronary artery endothelial cells (HCAECs) ex vivo. METHODS: HCAECs were stressed with activated platelets (2×10(8)/ml) and followed by ASP (1, 2 or 5 mmol/l), EGb (4, 40 or 400 µg/ml) and combinational (1 mmol/l ASP and 40µg/ml EGb) treatments in three groups for 12 h. Superoxide anion in HCAECs was measured with H2DCF-DA probe. The expressions of LOX-1 and p-p38MAPK were examined by Western blot. RESULTS: After stimulation of activated platelets, intracellular superoxide anion was increased about 3-folds in HCAECs. Both ASP and EGb reduced superoxide anion in HCAECs in a dosage dependent manner. Combinational administration of ASP and EGb showed synergistic effect. By Western blot analysis, we were able to show that activated platelets markedly enhanced the expressions of LOX-1 and p-p38MAPK. Both ASP and EGb only inhibited LOX-1 expression in a concentration-dependent manner, but not p-p38MAPK. As expected, the combination of ASP and EGb markedly reduced not only the expression of LOX-1 but also the phosphorylation of p38MAPK. CONCLUSIONS: Both EGb and ASP attenuate the oxidative stress of HCAECs stimulated by activated platelets ex vivo. It appears that the synergistic effect of EGb and ASP may correlate with the inhibition of ROS production, LOX-1 expression and p38MAPK phosphorylation.

Phytochemical activation of Nrf2 protects human coronary artery endothelial cells against an oxidative challenge.

Activation of NF-E2-related factor 2 (Nrf2) is a potential therapeutic intervention against endothelial cell oxidative stress and associated vascular disease. We hypothesized that treatment with the phytochemicals in the patented dietary supplement Protandim would induce Nrf2 nuclear localization and phase II antioxidant enzyme protein in human coronary artery endothelial cells (HCAECs), protecting against an oxidant challenge in an Nrf2- dependent manner. Protandim treatment induced Nrf2 nuclear localization, and HO-1 (778% of control ± 82.25 P < 0.01), SOD1 (125.9% of control ± 6.05 P < 0.01), NQO1 (126% of control ± 6.5 P < 0.01), and GR (119.5% of control ± 7.00 P < 0.05) protein expression in HCAEC. Treatment of HCAEC with H(2)O(2) induced apoptosis in 34% of cells while pretreatment with Protandim resulted in only 6% apoptotic cells (P < 0.01). Nrf2 silencing significantly decreased the Protandim-induced increase in HO-1 protein (P < 0.01). Nrf2 silencing also significantly decreased the protection afforded by Protandim against H(2)O(2)- induced apoptosis (P < 0.01 compared to no RNA, and P < 0.05 compared to control RNA). These results show that Protandim induces Nrf2 nuclear localization and antioxidant enzyme expression, and protection of HCAEC from an oxidative challenge is Nrf2 dependent.

Natural product extract of Dicksonia sellowiana induces endothelium-dependent relaxations by a redox-sensitive Src- and Akt-dependent activation of eNOS in porcine coronary arteries.

BACKGROUND/AIMS: The consumption of polyphenol-rich food is associated with a decreased mortality from coronary diseases. This study examined whether a standardized hydroalcoholic extract of Dicksonia sellowiana (HEDS) triggered endothelium-dependent relaxations in porcine coronary artery rings and characterized the underlying mechanism. METHODS: The phosphorylation level of Src, Akt and eNOS was assessed by Western blot analysis, the formation of reactive oxygen species by dihydroethidine staining and the level of eNOS Ser1177 phosphorylation by immunohistochemical staining in sections of coronary arteries. RESULTS: HEDS-induced endothelium-dependent relaxations were strongly reduced by Nω-nitro-L-arginine, an eNOS inhibitor, and by its combination with charybdotoxin plus apamin, inhibitors of endothelium-derived hyperpolarizing factor-mediated responses. These relaxations were markedly reduced by MnTMPyP (a membrane-permeant mimetic of superoxide dismutase), polyethylene glycol catalase (PEG-catalase; a membrane-permeant analog of catalase), and by wortmannin (an inhibitor of PI3-kinase). HEDS-induced sustained phosphorylation of Akt and eNOS in endothelial cells was abolished by MnTMPyP, PEG-catalase and wortmannin. Oral administration of HEDS induced a significant decrease of mean arterial pressure in spontaneously hypertensive rats. CONCLUSION: These findings indicate that HEDS caused endothelium-dependent relaxations of coronary artery rings through the redox-sensitive activation of the endothelial PI3-kinase/Akt pathway leading to the subsequent activation of eNOS by phosphorylation. HEDS also has antihypertensive properties.

Cysteine, histidine and glycine exhibit anti-inflammatory effects in human coronary arterial endothelial cells.

The activation of nuclear factor-kappa B (NF-κB) in vascular endothelial cells may be involved in vascular pathogeneses such as vasculitis or atherosclerosis. Recently, it has been reported that some amino acids exhibit anti-inflammatory effects. We investigated the inhibitory effects of a panel of amino acids on cytokine production or expression of adhesion molecules that are involved in inflammatory diseases in various cell types. The activation of NF-κB was determined in human coronary arterial endothelial cells (HCAECs) because NF-κB modulates the production of many cytokines and the expression of adhesion molecules. We examined the inhibitory effects of the amino acids cysteine, histidine and glycine on the induction of NF-κB activation, expression of CD62E (E-selectin) and the production of interleukin (IL)-6 in HCAECs stimulated with tumour necrosis factor (TNF)-α. Cysteine, histidine and glycine significantly reduced NF-κB activation and inhibitor κBα (IκBα) degradation in HCAECs stimulated with TNF-α. Additionally, all the amino acids inhibited the expression of E-selectin and the production of IL-6 in HCAECs, and the effects of cysteine were the most significant. Our results show that glycine, histidine and cysteine can inhibit NF-κB activation, IκBα degradation, CD62E expression and IL-6 production in HCAECs, suggesting that these amino acids may exhibit anti-inflammatory effects during endothelial inflammation.