Chi Ming Wong

Raloxifene Relaxes Rat Cerebral Arteries In Vitro and Inhibits L-Type Voltage-Sensitive Ca2+ Channels

Background and Purpose— Because of their mixed estrogen-agonist and estrogen-antagonist properties, selective estrogen receptor modulators (SERMs) are considered promising substitutes for hormone replacement therapy. Raloxifene and other SERMs confer estrogen-like cardiovascular protective effects but lack the carcinogenic activity of exogenous estrogen. However, little is known about the cerebrovascular action of raloxifene. Therefore, we studied the effects of raloxifene on the mechanisms regulating rat cerebral artery tone. Methods and Results— Ring segments of the isolated rat posterior communicating cerebral arteries were mounted in a microvessel myograph for measurement of isometric tension. Whole-cell L-type voltage-sensitive Ca2+ currents were recorded using the perforated patch-clamp technique. Raloxifene (0.1 to 10 μmol/L) reduced the contractile responses to U46619, phenylephrine, and endothelin-1 in normal Krebs solution or to CaCl2 in Ca2+-free, high K+-containing solution. Raloxifene-induced relaxation was identical in endothelium-intact and endothelium-denuded rings. ICI 182780 had no effect on raloxifene-induced relaxation. Raloxifene reduced L-type Ca2+ currents with a p D2 of 5.98±0.06, close to that (6.44±0.09) for raloxifene-induced relaxation of 60 mmol/L K+-contracted rings. Conclusions— This study demonstrates that raloxifene acutely relaxes rat cerebral arteries largely via an endothelium-independent mechanism, involving inhibition of Ca2+ influx through L-type Ca2+ channels.

Estrogen and Tamoxifen Modulate Cerebrovascular Tone in Ovariectomized Female Rats

Postmenopausal estrogen deficiency increases the incidence of cerebrovascular disease. However, hormone replacement therapy is associated with an increased cardiovascular risk. Tamoxifen is a selective estrogen receptor modulator with estrogenic effects on cardiovascular risk factors, but its long-term impacts on cerebral vasculature are unknown. We hypothesized that chronic 17β-estradiol or tamoxifen treatment exerted similar effects in reducing cerebrovascular tension in ovariectomized rats. We therefore determine whether (1) chronic 17β-estradiol treatment could influence vasomotor activities, (2) chronic tamoxifen therapy could exert an estrogen-like or estrogen-antagonistic effect, and (3) acute exposure to estrogen could mimic the effect of 17β-estradiol. Isometric tension was measured in cerebral arteries from female rat groups: control, ovariectomy, ovariectomy plus 17β-estradiol treatment, ovariectomy plus tamoxifen treatment, and ovariectomized rats treated with tamoxifen and 17β-estradiol. Ovariectomy enhanced cerebrovascular contractions to endothelin-1 or CaCl2, but not to U46619 or phenylephrine. 17β-Estradiol therapy reversed these effects. Chronic tamoxifen treatment exerted estrogen-like actions by reversing ovariectomy-induced enhancement of vessel tone without antagonizing the effect of chronic 17β-estradiol treatment. Ovariectomy enhanced the relaxing potency of nicardipine, and 17β-estradiol treatment prevented this effect. Acute exposure to 10−9 mol/L 17β-estradiol or 10−8 mol/L tamoxifen did not modulate contractions in rings from nonoperated female rats. In conclusion, ovariectomy differentially enhances agonist-induced cerebrovascular tone, an effect that was reversed by estrogen therapy. Tamoxifen does not act as an estrogen antagonist; instead, it functions as an estrogen agonist during estrogen deficiency. Thus, tamoxifen may confer beneficial effects similar to estrogen in cerebrovascular vessels.

Raloxifene protects endothelial cell function against oxidative stress

Maintaining a delicate balance between the generation of nitric oxide (NO) and removal of reactive oxygen species (ROS) within the vascular wall is crucial to the physiological regulation of vascular tone. Increased production of ROS reduces the effect and/or bioavailability of NO, leading to an impaired endothelial function. This study tested the hypothesis that raloxifene, a selective oestrogen receptor modulator, can prevent endothelial dysfunction under oxidative stress.

Contribution of K+ channels to relaxation induced by 17beta-estradiol but not by progesterone in isolated rat mesenteric artery rings.

17&bgr;-Estradiol and progesterone were found to relax various vascular beds through multiple mechanisms. However, the exact ionic mechanisms underlying the acute relaxant responses to both hormones are incompletely understood. This study was aimed to examine the possible role of K+ channel activation in the relaxation induced by both hormones in isolated rat mesenteric artery rings. Isometric tension of each ring was measured with Grass force displacement transducers. In rat endothelium–denuded rings preconstricted by 9,11-dideoxy-11&agr;,9&agr;-epoxy-methanoprostaglandin F2&agr; (U46619), the relaxation induced by 17&bgr;-estradiol was partially inhibited by tetrapentylammonium, 4-aminopyridine, iberiotoxin, BaCl2, and tertiapin-Q but not by tetraethylammonium, charybdotoxin, apamin, or glibenclamide. In contrast, these putative K+ channel blockers, except for glibenclamide, did not affect the relaxant response to progesterone. In 4 × 10−2M K+-preconstricted rings, the K+ channel blockers lost their inhibitory effects on 17&bgr;-estradiol–induced relaxation. Endothelium did not seem to be involved in the effects of K+ channel blockers on 17&bgr;-estradiol–mediated relaxation. Nifedipine-induced relaxation was not inhibited but was instead enhanced by tetrapentylammonium, iberiotoxin, 4-aminopyridine, and BaCl2. The above results indicate that in rat mesenteric artery rings, nonselective activation of K+ channels contributes partially to the relaxation induced by 17&bgr;-estradiol. These K+ channels involved in the estrogen response appeared to be sensitive to inhibition by KCa, KV, and KIR channel blockers. Lack of effect of K+ channel blockers on progesterone-induced relaxation suggests that these K+ channels play little or no role. The present findings provide pharmacological evidence for an additional mechanism contributing to acute vasorelaxation induced by 17&bgr;-estradiol.

Upregulation of Angiotensin (1-7)-Mediated Signaling Preserves Endothelial Function Through Reducing Oxidative Stress in Diabetes

AIMSnAngiotensin-converting enzyme 2 (ACE2)-angiotensin (1-7) [Ang (1-7)]-Mas constitutes the vasoprotective axis and is demonstrated to antagonize the vascular pathophysiological effects of the classical renin-angiotensin system. We sought to study the hypothesis that upregulation of ACE2-Ang (1-7) signaling protects endothelial function through reducing oxidative stress that would result in beneficial outcome in diabetes.nnnRESULTSnEx vivo treatment with Ang (1-7) enhanced endothelium-dependent relaxation (EDR) in renal arteries from diabetic patients. Both Ang (1-7) infusion via osmotic pump (500u2009ng/kg/min) for 2 weeks and exogenous ACE2 overexpression mediated by adenoviral ACE2 via tail vein injection (10(9) pfu/mouse) rescued the impaired EDR and flow-mediated dilatation (FMD) in db/db mice. Diminazene aceturate treatment (15u2009mg/kg/day) activated ACE2, increased the circulating Ang (1-7) level, and augmented EDR and FMD in db/db mouse arteries. In addition, activation of the ACE2-Ang (1-7) axis reduced reactive oxygen species (ROS) overproduction determined by dihydroethidium staining, CM-H2DCFDA fluorescence imaging, and chemiluminescence assay in db/db mouse aortas and also in high-glucose-treated endothelial cells. Pharmacological benefits of ACE2-Ang (1-7) upregulation on endothelial function were confirmed in ACE2 knockout (ACE2 KO) mice both ex vivo and in vitro.nnnINNOVATIONnWe elucidate that the ACE2-Ang (1-7)-Mas axis serves as an important signal pathway in endothelial cell protection in diabetic mice, especially in diabetic human arteries.nnnCONCLUSIONnEndogenous ACE2-Ang (1-7) activation or ACE2 overexpression preserves endothelial function in diabetic mice through increasing nitric oxide bioavailability and inhibiting oxidative stress, suggesting the therapeutic potential of ACE2-Ang(1-7) axis activation against diabetic vasculopathy. Antioxid.

RALOXIFENE, TAMOXIFEN AND VASCULAR TONE

1 Oestrogen deficiency causes progressive reduction in endothelial function. Despite the benefits of hormone‐replacement therapy (HRT) evident in earlier epidemiological studies, recent randomized trials of HRT for the prevention of heart disease found no overall benefit. Instead, HRT users had higher incidences of stroke and heart attack. Most women discontinue HRT because of its many side‐effects and/or the increased risk of breast and uterine cancer. This has contributed to the development of selective oestrogen receptor modulators (SERMs), such as tamoxifen and raloxifene, as alternative oestrogenic agents. 2 A SERM is a molecule that binds with high affinity to oestrogen receptors but has tissue‐specific effects distinct from oestrogen, acting as an oestrogen agonist in some tissues and as an antagonist in others. Clinical and animal studies suggest multiple cardiovascular effects of SERMs. For example, raloxifene lowers serum levels of cholesterol and homocysteine, attenuates oxidation of low‐density lipoprotein, inhibits endothelial–leucocyte interaction, improves endothelial function and reduces vascular smooth muscle tone. 3 Available evidence suggests that raloxifene and tamoxifen are capable of acting directly on both endothelial cells and the underlying vascular smooth muscle cells and cause a multitude of favourable modifications of the vascular wall, which jointly contribute to improved local blood flow. The outcome of the Raloxifene Use for the Heart (RUTH) trial will determine whether raloxifene, currently approved for the treatment of post‐menopausal osteoporosis, could substitute for HRT in alleviating cardiovascular symptoms in post‐menopausal women.

Contribution of Na+‐Ca2+ exchanger to pinacidil‐induced relaxation in the rat mesenteric artery

Pinacidil relaxes blood vessels through opening the KATP channels with a resultant membrane hyperpolarization and inhibition of Ca2+ influx. The aim of this study was to examine the mechanisms thereby pinacidil induces K+ channel‐independent relaxation in isolated endothelium‐denuded rat mesenteric artery. Pinacidil‐induced relaxation was inhibited by glibenclamide (1–10 μM) in phenylephrine‐preconstricted rings, but was unaffected by glibenclamide after inhibition of K+ channels and VGCCs. Pinacidil‐induced K+ channel‐independent relaxation remained unchanged after treatment with cyclopiazonic acid (10 μM), thapsigargin (1 μM), ouabain (100 μM), propranolol (10 μM), Rp‐cAMPS triethylamine (30 μM), L‐NNA (100 μM), or ODQ (10 μM). Pinacidil induced more relaxant effect in the presence of nifedipine than in the presence of 60 mM K+ plus nifedipine. Pretreatment with Na+‐Ca2+ exchanger inhibitors, nickel (30–300 μM) or benzamil (20 μM) attenuated pinacidil‐induced relaxation in normal or in nifedipine‐containing solution. Pinacidil (1 μM) produced less relaxant effect with decreasing extracellular Na+ concentration. Na+‐free condition abolished the inhibitory effect of benzamil. Both nickel and benzamil inhibited pinacidil‐induced relaxation in the presence of glibenclamide (10 μM). Nickel (300 μM) did not affect the relaxant response to sodium nitroprusside. Pinacidil relaxed the rings preconstricted by active phorbol and U46619 with similar potency. The present results indicate that stimulation of the forward mode Na+‐Ca2+ exchange pathway is in part responsible for pinacidil‐induced K+ channel‐independent vasorelaxation. Pinacidil also induces K+ channel‐dependent but VGCCs‐independent relaxation. The PKC‐mediated cellular pathway may be a target site for pinacidil only in higher concentrations.

Differential effects of estrogen and progesterone on potassium channels expressed in Xenopus oocytes.

HYPOTHESISnPotassium (K(+)) channel activation contributes in part to estrogen-mediated vasorelaxation. However, the underlying mechanism is still unclear. We hypothesize that estrogen increases K(+) currents via membrane-associated, non-genomic interaction and that steroid hormones have differential effects on different types of K(+) channels.nnnEXPERIMENTALnHuman large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) and human voltage-gated K(+) channels (K(V1.5)) were expressed in Xenopus oocytes, and K(+) currents elicited by voltage clamp were measured.nnnRESULTSnBoth 17beta-estradiol and BSA-conjugated 17beta-estradiol increased the BK(Ca) current in a concentration-dependent manner and this effect was abolished by tetraethylammonium ions and iberiotoxin (putative BK(Ca) channel blockers). 17beta-estradiol-stimulated increase in the BK(Ca) current was unaffected by treatment with ICI 182,780 (classic estrogen receptor antagonist), tamoxifen (estrogen receptor agonist/antagonist), actinomycin D (RNA synthesis inhibitor), or cycloheximide (protein synthesis inhibitor). In contrast, progesterone reduced the BK(Ca) current in the absence or presence of NS 1619 (BK(Ca) channel activator). Progesterone also inhibited 17beta-estradiol-stimulated increase in the BK(Ca) current. Finally, progesterone but not 17beta-estradiol reduced the K(V1.5) current.nnnCONCLUSIONSnThe present results show that 17beta-estradiol stimulates BK(Ca) channels without affecting K(V1.5) channels. This effect is ICI 182,780-insensitive and is likely mediated via a membrane-bound binding site. Progesterone inhibits both BK(Ca)- and K(V1.5)-encoded currents. The present results suggest that inhibition of K(+) channels may contribute in part to its reported antagonism against 17beta-estradiol-mediated vascular relaxation via BK(Ca) channels.

Bone morphogenic protein-4-induced oxidant signaling via protein carbonylation for endothelial dysfunction.

The increased expression of bone morphogenic protein-4 (BMP-4) under hyperglycemic and diabetic conditions mediates the overgeneration of reactive oxygen species to cause endothelial cell dysfunction and apoptosis. Protein carbonylation plays an important role in oxidant signaling through ligand-receptor interactions in vascular smooth muscle cells, cardiac cells, and bronchial smooth muscle cells to trigger different diseases. However, the role of oxidant signaling via protein carbonylation in endothelial dysfunction is unclear. The level of protein carbonylation was higher in renal arteries from diabetic patients than those from nondiabetic subjects. BMP-4 promoted protein carbonylation, which was followed by decarbonylation or degradation in primary rat aortic endothelial cells. Organ culture of normal C57BL/6J mouse aortas treated with either hydralazine or deferoxamine inhibited the effect of BMP-4 on impairment of acetylcholine-induced endothelium-dependent relaxation (EDR). In isolated diabetic db/db mouse aortas, treatment with hydralazine improved the impaired EDR while deferoxamine had no effect. BMP-4-induced carbonylated proteins in aortic endothelial cells were successfully identified by a proteomic approach. These proteins have important cellular functions and include glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, alpha-enolase, protein disulfide-isomerase A3, annexin II, 26S protease regulatory subunit, integrin-linked protein kinase, and vimentin. Protein carbonylation induced by BMP-4 was inhibited by BMP-4 antagonist while protein decarbonylation induced by BMP-4 was thiol dependent. The carbonyl signals did not involve 4-hydrononenal and malondialdehyde. The present results suggest that BMP-4- or diabetes-mediated endothelial dysfunction is partly triggered through protein carbonylation and blockade of this metal-catalyzed protein oxidation can be considered as an alternative therapeutic strategy to alleviate diabetic vasculopathy.

Tetramethylpyrazine Protects against Hydrogen Peroxide-Provoked Endothelial Dysfunction in Isolated Rat Aortic Rings: Implications for Antioxidant Therapy of Vascular Diseases.

Background and Objectives. Oxidative stress can initiate endothelial dysfunction and atherosclerosis. This study evaluated whether tetramethylpyrazine (TMP), the predominant active ingredient in Rhizoma Ligustici Wallichii (chuanxiong), prevents endothelial dysfunction in a rat model of oxidative stress. Methods. Isolated rat aortic rings were pretreated with various drugs before the induction of endothelial dysfunction by hydrogen peroxide (H2O2). Changes in isometric tension were then measured in acetylcholine- (ACh-) relaxed rings. Endothelial nitric oxide synthase (eNOS) expression was evaluated in the rings by Western blotting, and superoxide anion (O2 ∙−) content was assessed in primary rat aortic endothelial cells by dihydroethidium- (DHE-) mediated fluorescence microscopy. Results. ACh-induced endothelium-dependent relaxation (EDR) was disrupted by H2O2 in endothelium-intact aortic rings. H2O2-impaired relaxation was ameliorated by acute pretreatment with low concentrations of TMP, as well as by pretreatment with catalase and the NADPH oxidase inhibitors, apocynin and diphenyleneiodonium (DPI). TMP, apocynin, and DPI also reduced O2 ∙− accumulation in endothelial cells,but TMP failed to alter eNOS expression in aortic rings incubated with H2O2. Conclusions. TMP safeguards against oxidative stress-induced endothelial dysfunction, suggesting that the agent might find therapeutic utility in the management of vascular diseases. However, TMPs role in inhibiting NADPH oxidase and its vascular-protective mechanism of action requires further investigation.