Bradykinin Inhibits Oxidative Stress-Induced Cardiomyocytes Senescence via Regulating Redox State

Background

Cell senescence is central to a large body of age related pathology, and accordingly, cardiomyocytes senescence is involved in many age related cardiovascular diseases. In consideration of that, delaying cardiomyocytes senescence is of great importance to control clinical cardiovascular diseases. Previous study indicated that bradykinin (BK) protected endothelial cells from senescence induced by oxidative stress. However, the effects of bradykinin on cardiomyocytes senescence remain to be elucidated. In this study, we investigated the effect of bradykinin on H₂O₂-induced H9C2 cells senescence.

Methods and Results

Bradykinin pretreatment decreased the senescence induced by H₂O₂ in cultured H9C2 cells in a dose dependent manner. Interestingly, 1 nmol/L of BK almost completely inhibited the increase in senescent cell number and p21 expression induced by H₂O₂. Since H₂O₂ induces senescence through superoxide-induced DNA damage, we also observed the DNA damage by comet assay, and BK markedly reduced DNA damage induced by H₂O₂, and moreover, BK treatment significantly prevented reactive oxygen species (ROS) production in H9C2 cells treated with H₂O₂. Importantly, when co-incubated with bradykinin B2 receptor antagonist HOE-140 or eNOS inhibitor N-methyl-L-arginine acetate salt (L-NAME), the protective effects of bradykinin on H9C2 senescence were totally blocked. Furthermore, BK administration significantly prevented the increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity characterized by increased ROS generation and gp91 expression and increased translocation of p47 and p67 to the membrane and the decrease in superoxide dismutase (SOD) activity and expression induced by H₂O₂ in H9C2 cells, which was dependent on BK B2 receptor mediated nitric oxide (NO) release.

Conclusions

Bradykinin, acting through BK B2 receptor induced NO release, upregulated antioxidant Cu/Zn-SOD and Mn-SOD activity and expression while downregulating NADPH oxidase activity and subsequently inhibited ROS production, and finally protected against cardiomyocytes senescence induced by oxidative stress.     

Hypoxia-induced expression of bradykinin type-2 receptors in endothelial cells triggers NO production,cell migration,and angiogenesis

Bradykinin receptors are differentially expressed in the coronary vascular endothelium of rat and human hearts during the pathogenesis of heart failure, but the mechanisms responsible for this regulation have remained vague. Here we show by quantitative real-time PCR, Western blot analysis, and immunohistochemistry, that hypoxia triggers the expression of bradykinin type-2 receptors (BK-2Rs) in cultured human coronary artery endothelial cells (HCAECs), in isolated rat cardiac microvascular endothelial cells (RCMECs), and in rat hearts subjected to ligation of the left anterior descending coronary artery. Mild hypoxia (5% O₂) induced a fourfold temporal increase in BK-2R mRNA expression in HCAECs, which was also observed at the protein level, whereas severe hypoxia (1% O₂) slightly inhibited the mRNA expression of BK-2Rs. In addition, HOE-140, a BK-2R antagonist, inhibited mRNA and protein expression of BK-2Rs. The BK-2Rs induced by mild hypoxia were biologically active, that is, capable of inducing intracellular production of nitric oxide (NO) upon activation of HCAECs with bradykinin (BK), a response attenuated by HOE-140. In rat hearts recovering from myocardial infarction, BK-2Rs were upregulated in the endothelium of vessels forming at the border zone between fibrotic scar tissue and healthy myocardium. Furthermore, in an in vitro wound-healing assay, RCMEC migration was increased under mild hypoxic culture conditions in the presence of BK and was attenuated with HOE-140. Our present results show that mild hypoxia triggers a temporal expression of functional BK-2Rs in human and rat endothelial cells and support a role for BK-2Rs in hypoxia-induced angiogenesis. J. Cell. Physiol. 221: 359–366, 2009. © 2009 Wiley-Liss, Inc.     

Salvianolic acid B inhibits hydrogen peroxide-induced endothelial cell apoptosis through regulating PI3K/Akt signaling

Background

Salvianolic acid B (Sal B) is one of the most bioactive components of Salvia miltiorrhiza, a traditional Chinese herbal medicine that has been commonly used for prevention and treatment of cerebrovascular disorders. However, the mechanism responsible for such protective effects remains largely unknown. It has been considered that cerebral endothelium apoptosis caused by reactive oxygen species including hydrogen peroxide (H₂O₂) is implicated in the pathogenesis of cerebrovascular disorders.

Methodology and Principal Findings

By examining the effect of Sal B on H₂O₂-induced apoptosis in rat cerebral microvascular endothelial cells (rCMECs), we found that Sal B pretreatment significantly attenuated H₂O₂-induced apoptosis in rCMECs. We next examined the signaling cascade(s) involved in Sal B-mediated anti-apoptotic effects. We showed that H₂O₂ induces rCMECs apoptosis mainly through the PI3K/ERK pathway, since a PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) blocked ERK activation caused by H₂O₂ and a specific inhibitor of MEK (U0126) protected cells from apoptosis. On the other hand, blockage of the PI3K/Akt pathway abrogated the protective effect conferred by Sal B and potentated H₂O₂-induced apoptosis, suggesting that Sal B prevents H₂O₂-induced apoptosis predominantly through the PI3K/Akt (upstream of ERK) pathway.

Significance

Our findings provide the first evidence that H₂O₂ induces rCMECs apoptosis via the PI3K/MEK/ERK pathway and that Sal B protects rCMECs against H₂O₂-induced apoptosis through the PI3K/Akt/Raf/MEK/ERK pathway.     

Endothelial Japanese encephalitis virus infection enhances migration and adhesion of leukocytes to brain microvascular endothelia via MEK‐dependent expression of ICAM1 and the CINC and RANTES chemokines

Currently, the underlying mechanisms and the specific cell types associated with Japanese encephalitis‐associated leukocyte trafficking are not understood. Brain microvascular endothelial cells represent a functional barrier and could play key roles in leukocyte central nervous system trafficking. We found that cultured brain microvascular endothelial cells were susceptible to Japanese encephalitis virus (JEV) infection with limited amplification. This type of JEV infection had negligible effects on cell viability and barrier integrity. Instead, JEV‐infected endothelial cells attracted more leukocytes adhesion onto surfaces and the supernatants promoted chemotaxis of leukocytes. Infection with JEV was found to elicit the elevated production of intercellular adhesion molecule‐1, cytokine‐induced neutrophil chemoattractant‐1, and regulated‐upon‐activation normal T‐cell expressed and secreted, contributing to the aforementioned leukocyte adhesion and chemotaxis. We further demonstrated that extracellular signal‐regulated kinase was a key upstream regulator which stimulated extensive endothelial gene induction by up‐regulating cytosolic phospholipase A₂, NF‐κB, and cAMP response element‐binding protein via signals involving phosphorylation. These data suggest that JEV infection could activate brain microvascular endothelial cells and modify their characteristics without compromising the barrier integrity, making them favorable for the recruitment and adhesion of circulating leukocytes, thereby together with other unidentified barrier‐disrupting mechanisms contributing to Japanese encephalitis and associated neuroinflammation.     

Bradykinin receptor‐1 activation induces inflammation and increases the permeability of human brain microvascular endothelial cells

Neuroinflammatory disorders such as Alzheimer's and Parkinson's diseases are characterised by chronic inflammation and loss of vascular integrity. Bradykinin 1 receptor (B1R) activation has been implicated in many neuroinflammatory diseases, but the contribution of B1R to inflammation and vascular breakdown is yet to be determined. As a result, the present study evaluated the effect of B1R stimulation using Des‐Arg‐9‐BK on the cytokine profile and junctional properties of human cerebral microvascular endothelial cells (hCMVECs). Results showed that stimulation of B1R receptors increased secretion of pro‐inflammatory cytokines, interleukin‐6 (IL‐6), IL‐8, intracellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1) and monocyte chemoattractant protein‐1 (MCP‐1), but decreased the expression of vascular endothelial growth factor (VEGF), a cytokine and growth factor required for maintenance of the vasculature. B1R stimulation also resulted in the loss of occludin expression at tight junctions with no change in VE‐cadherin expression. There was also a significant increase in permeability to Evans blue albumin, suggesting an increase of vascular permeability. Taken together, these results suggest that B1R activation that occurs in neuroinflammatory diseases may contribute to both the inflammation and loss of blood‐brain barrier integrity that is characteristic of these diseases.     

Lipopolysaccharides can protect mesenchymal stem cells (MSCs) from oxidative stress-induced apoptosis and enhance proliferation of MSCs via Toll-like receptor(TLR)-4 and PI3K/Akt

Apoptosis of implanted mesenchymal stem cells (MSCs) limits the efficiency of MSC therapy. Recent studies showed the ligands of Toll-like receptors (TLRs) could control the function of these cells. We have investigated the effect of lipopolysaccharides (LPS), a ligand of TLR4, on the survival of MSCs and explored the roles of TLR4 and PI3K/Akt. H₂O₂/serum deprivation(H₂O₂/SD) induced apoptosis of MSCs but LPS-preconditioning (1.0 μg/ml) protected MSCs from H₂O₂/SD-induced apoptosis and promoted their proliferation. Western blotting showed that 1.0 μg/ml LPS enhanced phosphorylation of both Akt at Ser 473 and nuclear factor-kappa B (NF-κB) p65 at Ser 536. However, the protective effects of LPS on survival were not observed in TLR4^lps-del MSCs. The results suggest appropriate treatments with LPS can protect MSCs from oxidative stress-induced apoptosis and improve the survival of MSCs via the TLR4 and PI3K/Akt pathway.     

Collagen peptide modified carboxymethyl cellulose as both antioxidant drug and carrier for drug delivery against retinal ischaemia/reperfusion injury

Oxidative stress can cause injury in retinal endothelial cells. Carboxymethyl cellulose modified with collagen peptide (CMCC) is of a distinct antioxidant capacity and potentially a good drug carrier. In this study, the protective effects of CMCC against H₂O₂‐induced injury of primary retinal endothelial cells were investigated. In vitro, we demonstrated that CMCC significantly promoted viability of H₂O₂‐treated cells, efficiently restrained cellular reactive oxygen species (ROS) production and cell apoptosis. Then, the CMCC was employed as both drug and anti‐inflammatory drug carrier for treatment of retinal ischaemia/reperfusion (I/R) in rats. Animals were treated with CMCC or interleukin‐10‐loaded CMCC (IL‐10@CMCC), respectively. In comparisons, the IL‐10@CMCC treatment exhibited superior therapeutic effects, including better restoration of retinal structural thickness and less retinal apoptosis. Also, chemiluminescence demonstrated that transplantation of IL‐10@CMCC markedly reduced the retinal oxidative stress level compared with CMCC alone and potently recovered the activities of typical antioxidant enzymes, SOD and CAT. Therefore, it could be concluded that CMCC provides a promising platform to enhance the drug‐based therapy for I/R‐related retinal injury.     

Mechanism of dihydrofolate reductase downregulation in melanoma by 3‐O‐(3,4,5‐trimethoxybenzoyl)‐(−)‐epicatechin

In our search to improve the stability and cellular absorption of tea polyphenols, we synthesized 3‐O‐(3,4,5‐trimethoxybenzoyl)‐(?)‐epicatechin (TMECG), which showed high antiproliferative activity against melanoma. TMECG downregulates dihydrofolate reductase (DHFR) expression in melanoma cells and we detail the sequential mechanisms that result from this even. TMECG is specifically activated in melanoma cells to form a stable quinone methide (TMECG‐QM). TMECG‐QM has a dual action on these cells. First, it acts as a potent antifolate compound, disrupting folate metabolism and increasing intracellular oxidized folate coenzymes, such as dihydrofolate, which is a non‐competitive inhibitor of dihydropterine reductase, an enzyme essential for tetrahydrobiopterin (H₄B) recycling. Such inhibition results in H₄B deficiency, endothelial nitric oxide synthase (eNOS) uncoupling and superoxide production. Second, TMECG‐QM acts as an efficient superoxide scavenger and promotes intra‐cellular H₂O₂ accumulation. Here, we present evidence that TMECG markedly reduces melanoma H₄B and NO bioavailability and that TMECG action is abolished by the eNOS inhibitor N_ω‐nitro‐L ‐arginine methyl ester or the H₂O₂ scavenger catalase, which strongly suggests H₂O₂‐dependent DHFR downregulation. In addition, the data presented here indicate that the simultaneous targeting of important pathways for melanoma survival, such as the folate cycle, H₄B recycling, and the eNOS reaction, could represent an attractive strategy for fighting this malignant skin pathology. J. Cell. Biochem. 110: 1399–1409, 2010. © 2010 Wiley‐Liss, Inc.     

MEK/ERK pathway mediates cytoprotection of salvianolic acid B against oxidative stress‐induced apoptosis in rat bone marrow stem cells

To improve the survival and/or differentiation of grafted BMSCs (bone marrow stem cells) represents one of the challenges for the promising cell‐based therapy. Considerable reports have implicated Sal B (salvianolic acid B), a potent aqueous extract of Salvia miltiorrhiza, in enhancing the survival of cells under various conditions. In this study, we investigated the effect of Sal B on H₂O₂‐induced apoptosis in rat BMSCs, focusing on the survival signalling pathways. Results indicated that the MEK [MAPK (mitogen‐activated protein kinase)/ERK (extracellular‐signal‐regulated kinase) kinase] inhibitor (PD98059) and 10 μM Sal B remarkably prevented BMSCs from H₂O₂‐induced apoptosis through attenuating caspase‐3 activation, which is accompanied by the significant up‐regulation of Bcl‐2. In addition, the ROS (reactive oxygen species) accumulation was also reduced after Sal B treatment. Furthermore, Sal B inhibited the ERK1/2 phosphorylations stimulated by H₂O₂. Taken together, our results showed that H₂O₂‐induced apoptosis in BMSCs via the ROS/MEK/ERK1/2 pathway and Sal B may exert its cytoprotection through mediating the pathway.     

Repression of Kisspeptin1 weakens hydrogen peroxide‐caused injury in HTR8 cells via adjusting PI3K/AKT/mTOR pathway

Kisspeptin1 (KISS1) is a tumor metastatic suppressor, and its increased expression is validated in human placenta trophoblast cells. Nonetheless, the actions of KISS1 in hydrogen peroxide (H₂O₂)‐impaired human trophoblast HTR8 cells still remain imprecise. This research aims to uncover whether KISS1 can mitigate H₂O₂‐triggered cell injury. HTR8 cells were pretreated with 250 μM H₂O₂ for 4 hours; the autophagic markers (Beclin‐1 and LC3B), cell viability, invasion and apoptosis were appraised. Real‐time quantitative polymerase chain reaction and Western blot trials were enforced for the valuation of KISS1 mRNA and protein levels. After si‐KISS1 transfection and 3‐MA manipulation, the aforesaid biological processes were reassessed for ascertaining the influences of repressed KISS1 in H₂O₂‐impaired HTR8 cells. Phosphoinositide 3‐kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway was eventually estimated. H₂O₂ enhanced Beclin‐1 and LC3B expression, restricted cell viability, and invasion, and meanwhile caused apoptosis. The elevation of KISS1 evoked by H₂O₂ was observed in HTR8 cells. In addition, silencing KISS1 was distinctly annulled the function of H₂O₂ in HTR8 cells. Eventually, we observed that the repression of KISS1 triggered the activation of PI3K/AKT/mTOR in HTR8 cells under H₂O₂ management. The diverting research unveiled that KISS1 repression eased H₂O₂‐caused HTR8 cells injury via mediating PI3K/AKT/mTOR pathway.     

Distinct Kinin-Induced Functions Are Altered in Circulating Cells of Young Type 1 Diabetic Patients

Aims/Hypothesis

We aimed to understand early alterations in kinin-mediated migration of circulating angio-supportive cells and dysfunction of kinin-sensitive cells in type-1 diabetic (T1D) patients before the onset of cardiovascular disease.

Methods

Total mononuclear cells (MNC) were isolated from peripheral blood of 28 T1D patients free from cardiovascular complications except mild background retinopathy (age: 34.8±1.6 years, HbA_1C: 7.9±0.2%) and 28 age- and sex-matched non-diabetic controls (H). We tested expression of kinin receptors by flow cytometry and migratory capacity of circulating monocytes and progenitor cells towards bradykinin (BK) in transwell migration assays. MNC migrating towards BK (BK^mig) were assessed for capacity to support endothelial cell function in a matrigel assay, as well as generation of nitric oxide (NO) and superoxide (O₂ ⁻*) by using the fluorescent probes diaminofluorescein and dihydroethidium.

Results

CD14^hiCD16^neg, CD14^hiCD16^pos and CD14^loCD16^pos monocytes and circulating CD34^pos progenitor cells did not differ between T1D and H subjects in their kinin receptor expression and migration towards BK. T1D BK^mig failed to generate NO upon BK stimulation and supported endothelial cell network formation less efficiently than H BK^mig. In contrast, O₂ ⁻* production was similar between groups. High glucose disturbed BK-induced NO generation by MNC-derived cultured angiogenic cells.

Conclusions/Interpretation

Our data point out alterations in kinin-mediated functions of circulating MNC from T1D patients, occurring before manifest macrovascular damage or progressed microvascular disease. Functional defects of MNC recruited to the vessel wall might compromise endothelial maintenance, initially without actively promoting endothelial damage, but rather by lacking supportive contribution to endothelial regeneration and healing.     

Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway

Kallistatin, a plasma protein, protects against vascular and organ injury. This study is aimed to investigate the role and mechanism of kallistatin in endothelial senescence. Kallistatin inhibited H₂O₂‐induced senescence in human endothelial cells, as indicated by reduced senescence‐associated‐β‐galactosidase activity, p16^INK4a and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked H₂O₂‐induced superoxide formation, NADPH oxidase levels and VCAM‐1, ICAM‐1, IL‐6 and miR‐34a synthesis. Kallistatin reversed H₂O₂‐mediated inhibition of endothelial nitric oxide synthase (eNOS), SIRT1, catalase and superoxide dismutase (SOD)‐2 expression, and kallistatin alone stimulated the synthesis of these antioxidant enzymes. Moreover, kallistatin's anti‐senescence and anti‐oxidant effects were attributed to SIRT1‐mediated eNOS pathway. Kallistatin, via interaction with tyrosine kinase, up‐regulated Let‐7g, whereas Let‐7g inhibitor abolished kallistatin's effects on miR‐34a and SIRT1/eNOS synthesis, leading to inhibition of senescence, oxidative stress and inflammation. Furthermore, lung endothelial cells isolated from endothelium‐specific kallistatin knockout mice displayed marked reduction in mouse kallistatin levels. Kallistatin deficiency in mouse endothelial cells exacerbated senescence, oxidative stress and inflammation compared to wild‐type mouse endothelial cells, and H₂O₂ treatment further magnified these effects. Kallistatin deficiency caused marked reduction in Let‐7g, SIRT1, eNOS, catalase and SOD‐1 mRNA levels, and elevated miR‐34a synthesis in mouse endothelial cells. These findings indicate that endogenous kallistatin through novel mechanisms protects against endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway.     

Grape seed extract enhances eNOS expression and NO production through regulating calcium‐mediated AKT phosphorylation in H2O2‐treated endothelium

GSE (grape seed extract) has been shown to exhibit protective effects against cardiovascular events and atherosclerosis, although the underlying molecular mechanisms of action are unknown. Herein, we assessed the ability of GSE to enhance eNOS (endothelial nitric oxide synthase) expression and NO (nitric oxide) production in H₂O₂ (hydrogen peroxide)‐treated HUVECs (human umbilical vein endothelial cells). GSE enhanced eNOS expression and NO release in H₂O₂‐treated cells in a dose‐dependent manner. GSE inhibited intracellular ROS (reactive oxygen species) and reduced intracellular calcium in a dose‐dependent manner in H₂O₂‐treated cells, as shown by confocal microscopy. ROS was inhibited in cells pretreated with 5.0 μM GSE, 2.0 μM TG (thapsigargin) and 20.0 μM 2‐APB (2‐aminoethoxydiphenyl borate) instead of 0.25 μM extracellular calcium. In addition, GSE enhanced eNOS expression and reduced ROS production via increasing p‐AKT (AKT phosphorylation) with high extracellular calcium (13 mM). In conclusion, GSE protected against endothelial injury by up‐regulation of eNOS and NO expression via inhibiting InsP₃Rs (inositol 1,4,5‐trisphosphate receptors)‐mediated intracellular excessive calcium release and by activating p‐AKT in endothelial cells.     

Cytochemical demonstration of an enzymatic production of hydrogen peroxide on the surface of isolated thyroid capillaries

Summary By means of a cytochemical technique, hydrogen peroxide formation was located on the endothelial cell surface (predominantly the luminal aspect) of capillaries obtained by collagenase digestion of rat thyroid. The cyanide-insensitive H₂O₂ formation required aerobic conditions and NAD(P)H as substrate. FAD could also stimulate the reaction, but not xanthine. The cytochemical reaction was blocked by a non-penetrating protein inhibitor. The observations are interpreted as evidence of a plasmalemma-bound H₂O₂-generating enzyme. The findings indicate that microvascular endothelial cells are involved in the release of activated oxygen species, which might have important pathophysiologic implications.     

Atherogenic levels of low-density lipoprotein increase hydrogen peroxide generation in cultured human endothelial cells: Possible mechanism of heightened endocytosis

Cultured human umbilical vein endothelial cells (EC) exposed to atherogenic low-density lipoprotein (LDL) levels have augmented reactive oxygen species generation. Confluent EC were incubated with 30–330 mg/dl LDL cholesterol and cellular hydrogen peroxide (H₂O₂) generation measured. EC incubated with 30 and 90 mg/dl LDL cholesterol showed similar low level H₂O₂ production. In contrast, EC exposed to 180 and 330 mg/dl LDL cholesterol have a marked, dose-related elevation in H₂O₂ generation. Subsequent studies have explored if direct EC exposure to H₂O₂ promotes cellular functional changes similar to those induced by high LDL levels (> 160 mg/dl cholesterol). Confluent EC were incubated with 0.1–10 mM H₂O₂ for 30 minutes and endocytosis measured and cytoskeletal structure examined. H₂O₂ exposure (0.5 and 1 mM) promoted heightened EC endocytosis, which similarly occurs with high LDL exposure. Likewise, cytoskeletal examination of EC perturbed with 1 mM H₂O₂ reveals structural remodeling with a marked increase in stress fibers, which similarly happens with high LDL levels. The above observations that high LDL levels cause increased EC H₂O₂ production, and direct H₂O₂ exposure promotes cellular functional changes similar to those induced by high LDL concentrations, suggest a modulatory role for reactive oxygen species. Thus LDL-induced reactive oxygen species generation may contribute mechanistically to endothelial perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis. © 1996 Wiley-Liss, Inc.     

Tryptic amaranth glutelin digests induce endothelial nitric oxide production through inhibition of ACE: Antihypertensive role of amaranth peptides

Amaranth seed proteins have a better balance of essential amino acids than cereals and legumes. In addition, the tryptic hydrolysis of amaranth proteins generates, among other peptides, angiotensin converting enzyme (ACE) inhibitory (ACEi) peptides. ACE converts angiotensin I (Ang I) into Ang II, but is also responsible for the degradation of bradykinin (BK). In contrast to Ang II, BK stimulates vasodilation modulated through endothelial nitric oxide (NO) production. The aim of the present study was to characterize the ACEi activity of amaranth trypsin-digested glutelins (TDGs) and their ability to induce endothelial NO production. An IC₅₀ value of 200 μg ml^?1 was measured for TDG inhibition of ACE. TDGs stimulated endothelial NO production in coronary endothelial cells (CEC) by 52% compared to control. The effects of TDGs were comparable to those of BK and Captopril, both used as positive controls of NO production. Consistent with these effects, TDGs induced, in a dose-dependent manner, endothelial NO-dependent vasodilation in isolated rat aortic rings. These results suggest that TDGs induce endothelial NO production and consequent vasodilation through their ACEi activity. Amaranth TDGs have a high potential as a nutraceutical food in prevention of cardiovascular diseases. Further molecular, cellular and physiological studies are currently under way and the results may contribute to a better understanding and control of cardiovascular disorders.