Canwen Jiang

Endothelium‐independent relaxation of rabbit coronary artery by 17β‐oestradiol in vitro

1 We assessed the relaxant effect of 17β‐oestradiol (10−7, 10−6 and 10−5 m) on rabbit isolated coronary arteries precontracted with prostaglandin F2α (3 × 10−6 m), high extracellular potassium (30 mm) and Bay K 8644 (10−6 m) plus high extracellular potassium (15 mm) by measuring isometric tension. 17β‐Oestradiol (10−6 and 10−5 m) induced significant relaxation in coronary arteries from male and female rabbits. No differences were seen between arteries with or without endothelium. There were also no differences between coronary arteries isolated from male and female rabbits. 2 Inhibitors of endothelium‐derived relaxing factor and vasodilator prostanoids, namely, reduced haemoglobin, Nω‐nitro‐l‐arginine methyl ester and indomethacin, did not affect the relaxation induced by 17β‐oestradiol in endothelium‐intact coronary arteries. 3 Methylene blue, an inhibitor of guanylate cyclase, did not affect the coronary artery relaxation induced by 17β‐oestradiol. 4 The calcium concentration‐dependent contraction curve in potassium‐depolarization medium was shifted to the right by 17β‐oestradiol (10−6 and 10−5 m) in the rabbit coronary artery and rat aorta. The −log EC50 s of calcium in control and after incubation with 17β‐oestradiol (10−6 and 10−5 m) were 3.7 ± 0.09, 3.1 ± 0.10 and 2.8 ± 0.08 respectively in rabbit coronary arteries and 3.8 ± 0.11, 3.3 ± 0.14 and 2.9 ±0.15 in rat aorta. 5 The results indicate that 17β‐oestradiol induces rabbit coronary artery relaxation by an endothelium‐independent mechanism in vitro. A calcium antagonistic property may be involved in the mechanism of rabbit coronary arterial relaxation by 17β‐oestradiol.;

Hypoxia-Inducible Factor-1 Mediates Activation of Cultured Vascular Endothelial Cells by Inducing Multiple Angiogenic Factors

Abstract— Hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of vascular endothelial growth factor (VEGF) and other hypoxia-responsive genes. Transgenic expression of a constitutively stable HIF-1&agr; mutant increases the number of vascular vessels without vascular leakage, tissue edema, or inflammation. This study aimed to investigate the molecular basis by which HIF-1 mediates the angiogenic response to hypoxia. In primary human endothelial cells, hypoxia, desferrioxamine, or infection with Ad2/HIF-1&agr;/VP16, an adenoviral vector encoding a constitutively stable hybrid form of HIF-1&agr;, increased the mRNA and protein levels of VEGF, angiopoietin-2 (Ang-2), and angiopoietin-4 (Ang-4). Infection with Ad2/CMVEV (a control vector expressing no transgene) had no effect. Angiopoietin-1 (Ang-1) expression was not detected in human endothelial cells. Ang-4 was also induced by hypoxia or Ad2/HIF-1&agr;/VP16 in human cardiac cells, whereas Ang-1 expression remained unchanged. Recombinant Ang-4 protein protected endothelial cells against serum starvation-induced apoptosis and increased cultured endothelial cell migration and tube formation. Ad2/HIF-1&agr;/VP16 stimulated endothelial cell proliferation and tube formation. Hypoxia- or Ad2/HIF-1&agr;/VP16-induced tube formation was significantly reduced by a Tie-2 inhibitor. These results suggest that HIF-1 mediates the angiogenic response to hypoxia by upregulating the expression of multiple angiogenic factors. Ang-4 can function similarly as Ang-1 and substitute for Ang-1 to participate in hypoxia-induced angiogenesis. Activation of the angiopoietin/Tie-2 system may play a role in the ability of HIF-1 to induce hypervascularity without excessive permeability.

Cardiovascular protection by oestrogen-a calcium antagonist effect?

During their premenopausal years, women have a lower risk than men of getting cardiovascular disease. This protection continues after the menopause if women receive oestrogen replacement. Based on new experimental evidence we propose that some of the cardiovascular benefits of oestrogen replacement therapy may be due to a long-term calcium antagonist effect of oestrogen.

Effect of 17β‐oestradiol on contraction, Ca2+ current and intracellular free Ca2+ in guinea‐pig isolated cardiac myocytes

1 The effect of 17β‐oestradiol on cardiac cell contraction, inward Ca2+ current and intracellular free Ca2+ ([free Ca2+]i) was investigated in guinea‐pig single, isolated ventricular myocytes. The changes of cell length were measured by use of a photodiode array, the voltage‐clamp experiments were performed with a switch clamp system and [free Ca2+]i was measured with the Ca2+ indicator, Fura‐2. 2 17β‐Oestradiol (10, 30 μm) caused a decrease in cell shortening at both 22 and 35°C. This negative inotropic effect was accompanied by a decrease in action potential duration mainly brought about by a shortening of the plateau region of the action potential. 17β‐Oestradiol (10, 30 μm) induced a similar decrease in cell shortening in voltage‐clamped and current‐clamped cells. 3 In Fura‐2 loaded cells, 17β‐oestradiol (10 and 30 μm) decreased systolic Fura‐2 fluorescence to 72 ± 7% and 47 ± 4% (n = 6, P < 0.001) of control respectively. 17β‐Oestradiol (10 μm) had no significant effect on diastolic Fura‐2 fluorescence, but at higher concentration (30 μm) induced a slight decrease in resting Fura‐2 fluorescence. The effect of 17β‐oestradiol was reversible after 1–2 min of washout of the steroid. 4 17β‐Oestradiol (10 and 30 μm) decreased the peak inward Ca2+ current (ICa), which was sensitive to [Ca2+]o, dihydropyridines and isoprenaline, to 59 ± 3% and 39 ± 5% (n = 7∼9, P < 0.01) respectively, without producing any significant change in the shape of the current‐voltage relationship. 5 The recovery time of ICa from inactivation was delayed by 17β‐oestradiol (10 μm). The inhibitory effect of 17β‐oestradiol on ICa was less at a holding potential of −80 mV than at −40mV. 6 We conclude that 17β‐oestradiol has a negative inotropic effect on guinea‐pig single ventricular myocytes by inhibiting ICa and so reducing systolic [Ca2+]i. 17β‐Oestradiol may therefore have a Ca2+ channel blocking property in guinea‐pig isolated ventricular myocytes.

Progesterone induces endothelium-independent relaxation of rabbit coronary artery in vitro

The effect of progesterone on isolated rabbit coronary arteries and its possible mechanism was investigated by measuring changes of isometric tension. Progesterone (1, 3, 10 and 30 microM) induced significant coronary relaxation in K+ (30 mM)-, prostaglandin F2 alpha (3 microM)- or Bay K 8644 (1 microM plus 15 mM K+)- precontracted arteries. There was no difference between endothelium-intact and -denuded coronary arteries from both male and female rabbits, precontracted with these three agents. Haemoglobin, indomethacin, methylene blue, glibenclamide or barium chloride did not affect the relaxation. In endothelium-denuded rabbit coronary arteries, progesterone shifted calcium concentration-dependent constrictor-response curves to the right, the maximal contraction was also reduced. The -log ED50s were 3.6 in control, and 3.3 and 2.9 after incubation with progesterone (3 and 30 microM), respectively. Similar results were obtained in rat aorta. We conclude that progesterone induces significant endothelium-independent relaxation in rabbit coronary arteries in vitro, possibly by affecting calcium influx.

Oestrogen relaxes human epicardial coronary arteries through non-endothelium-dependent mechanisms

BackgroundOestrogen-replacement therapy is associated with a reduced incidence of cardiovascular disease. The acute administration of oestrogen improves myocardial ischemia in women with coronary heart disease. In this study we investigated the relaxing effect of oestradiol-17β on human coronary arteries in vitro and determined the role of endothelial modulation in this relaxation by using isolated human coronary arteries. MethodsAtherosclerosis-free epicardial arteries from men and women were removed from patients undergoing heart or combined heart and lung transplantation. The arteries were cut into ring segments and placed into organ baths containing Tyrodes solution. Changes in isometric tension were measured. The relaxing response to oestradiol-17β (10-10-10-5mol/l) was investigated and the effects of endothelium, NGmonomethyl-L-arginine and indomethacin on the response of oestradiol-17β were assessed. ResultsOestradiol-17β (10-10-10-5mol/l) induced significant relaxation in coronary arteries pre-contracted with the thromboxane A2 analog (U46619; 3 × 10-8mol/l). Relaxation was significantly greater in coronary arteries from female patients. No significant differences were observed between arteries with or without endothelium nor after nitric oxide synthase or cyclo-oxygenase inhibition. These results indicate that oestradiol-17β induces human coronary artery relaxation via an endothelium-independent mechanism in vitro. The sex of the patients significantly affects sensitivity of the coronary arterial rings to oestrogen. ConclusionOestradiol-17β-induced coronary relaxation may play an important role in the regulation of coronary tone, and may partly explain why oestrogen improves myocardial ischemia in women and why it protects postmenopausal women from the risk of developing coronary heart disease.

A Constitutively Active Hypoxia-Inducible Factor-1α/VP16 Hybrid Factor Activates Expression of the Human B-Type Natriuretic Peptide Gene

Hypoxia-inducible factor-1 (HIF-1) is a primary regulator of the physiological response to hypoxia. A recombinant adenovirus expressing a constitutively active hybrid form of the HIF-1α subunit (Ad2/HIF-1α/VP16) is being evaluated as a gene therapy for the treatment of peripheral vascular disease. Ad2/HIF-1α/VP16 up-regulates known HIF-1-responsive genes, including those involved in angiogenesis. Expression profile analysis revealed that the brain natriuretic peptide (BNP) gene was significantly up-regulated in response to HIF-1α/VP16 in human fetal cardiac cells. Real-time reverse transcription-polymerase chain reaction analyses confirmed transcriptional activation of the BNP gene by HIF-1α/VP16 in human but not rat cardiac cells. Because hypoxia itself did not increase human BNP gene expression in these analyses, the mechanism of the HIF-1α/VP16 effect was determined. Analyses of promoter deletion mutants suggested that the cis-acting sequence in the human BNP promoter mediating activation by HIF-1α/VP16 was a putative HIF-1 responsive element (HRE) located at -466. An SV40 basal promoter-luciferase plasmid containing a minimal BNP HRE was up-regulated by HIF-1α/VP16, whereas a similar construct carrying a mutation within the HIF-1 binding site was not. Mutation of an E-box motif within the BNP HRE reduced HIF-1α/VP16-mediated transcriptional activation by 50%. Gel-shift analyses showed that both the native HIF-1α and HIF-1α/VP16 are able to bind to a probe containing the HIF-1 binding site. These experiments demonstrate the existence of a functional HRE in the BNP promoter and further define the scope and mechanism of action of Ad2/HIF-1α/VP16.

Inhibition of hypoxia-induced relaxation of rabbit isolated coronary arteries by NG-monomethyl-l-arginine but not glibenclamide

1 The effects of NG‐monomethyl‐l‐arginine, tetrodotoxin and glibenclamide on hypoxia‐induced coronary artery relaxation, induced by bubbling Krebs solution with 95% N2 and 5% CO2 instead of 95% O2 and 5% CO2, were assessed by measuring the changes in isometric tension in isolated epicardial coronary artery rings of the rabbit. In addition, the effects of glibenclamide on the relaxation induced by adenosine were investigated. 2 Hypoxia caused a transient relaxation of 38 ± 3% (P < 0.01) and 17 ± 2% (P < 0.01) in endothelium‐intact or ‐denuded arteries respectively. NG‐monomethyl‐l‐arginine (30 and 100 μm) inhibited the relaxation in endothelium‐intact rings to 31 ± 2% (P < 0.05) and 16 ± 2% (P < 0.01) respectively and slightly but significantly attenuated the relaxation in endothelium‐denuded rings to 15 ± 1% and 13 ± 1% (P < 0.05) respectively. 3 Glibenclamide, a potassium channel inhibitor, did not significantly affect the hypoxia‐induced relaxation. 4 Incubation with tetrodotoxin (3 and 10 μm) for 30 min reduced the relaxation to 31 ± 3% (P < 0.05) and 14 ± 2% (P < 0.01), and 14 ± 2% (P < 0.05) and 11 ± 1% (P < 0.05) in endothelium‐intact and ‐denuded rings respectively. However, indomethacin (10 μm), atropine (1 μm), propranolol (10 μm) and phentolamine (10 μm) did not significantly affect the relaxation. 5 Adenosine (1, 10 and 100 μm) caused relaxation of 6 ± 1%, 52 ± 3% and 97 ± 2% respectively in endothelium‐denuded rings precontracted with prostaglandin F2α (PGF2α, 3 μm) and the relaxation was markedly inhibited by 8‐phenyltheophylline. Furthermore, glibenclamide (1 and 10 μm) reduced the relaxation induced by adenosine (1, 10 and 100 μm) to 2 ± 1% (P < 0.05), 38 ± 3% (P < 0.05) and 85 ± 2% (P < 0.05), and 0.6 ± 0.4% (P < 0.05), 27 ± 4% (P < 0.05) and 72 ± 4% (P < 0.01) respectively, in these endothelium‐denuded preparations. 6 These data suggest that hypoxia‐induced relaxation is mediated by the release of nitric oxide rather than by the activation of glibenclamide‐sensitive potassium channels in rabbit isolated coronary arteries. A neurogenic mechanism partially modulates the relaxation, possibly by activating non‐adrenergic and non‐cholinergic nerve endings. The inhibition by glibenclamide on adenosine‐induced relaxation in isolated coronary arteries may help to explain the fact that glibenclamide inhibits hypoxic coronary relaxation in perfused hearts but not in isolated coronary preparations.

Myocardial Expression of Baculoviral p35 Alleviates Doxorubicin-Induced Cardiomyopathy in Rats

The clinical use of doxorubicin, one of the most effective antitumor drugs, is limited by its cardiotoxicity, which results in irreversible cardiomyopathy and congestive heart failure. This study aimed to evaluate a gene therapy approach using adenovirus-mediated expression of p35, a baculoviral antiapoptotic gene, for alleviating doxorubicin-induced cardiomyopathy. In cultured neonatal rat cardiomyocytes, transduction with a recombinant adenoviral vector expressing p35 (Ad2/CMVp35) but not a control adenoviral vector expressing no transgene (Ad2/CMVEV) significantly inhibited doxorubicin-induced increase in cellular reactive oxygen species (ROS), the activity of caspases 8 and 3, cytochrome c release, and apoptosis. Direct injection of Ad2/CMVp35 into the left ventricular wall inhibited myocardial caspase 3 activity and apoptosis and improved left ventricular performance in rats treated with doxorubicin, whereas the same dose of Ad2/CMV beta gal encoding beta-galactosidase had no effect. These results suggest that adenovirus-mediated expression of p35 protects cardiomyocytes against doxorubicin cardiotoxicity, possibly by inhibiting caspase activity and by reducing cellular ROS levels. Localized delivery of gene transfer vectors expressing an antiapoptotic protein such as p35 to the myocardium may represent a therapeutic approach to alleviate doxorubicin-induced cardiomyopathy.

Potential therapeutic gene for the treatment of ischemic disease: Ad2/HIF-1α/VP16 enhances BNP gene expression via an HIF-1 responsive element.

In this issue of Molecular Pharmacology, Luo et al. (p. 1953) present a study employing a HIF-1α/VP16 chimera to investigate the mechanism by which this constitutively active transcription factor activates expression of brain natriuretic peptide (BNP). The results define a functional hypoxia responsive element (HRE) in the promoter of the human BNP gene and demonstrate that this HRE is necessary for HIF-1α/VP16-induced gene expression in human cardiomyocytes grown under normoxic conditions. Luo et al. also show that a consensus E-box DNA binding sequence is necessary for appropriate BNP regulation. Because HIF-1 is known to elicit protective and beneficial gene expression programs in many scenarios and because BNP is known to be cardioprotective, this study provides support for the therapeutic use of the chimeric HIF-1α/VP16 protein in coronary heart disease. However, because HIF-1α is a key regulatory molecule that acts upon a large number of downstream gene networks, there remains a need for further investigation. Particularly useful would be comprehensive gene expression profiling coupled with functional analysis of HIF-1α/VP16-regulated genes. The results of such studies will elucidate the mechanism of beneficial effects and address concerns regarding potential adverse effects of activating specific HIF-1α/VP16-dependent gene programs.