Michel Y. Farhat

17β-Estradiol Prevents Dysfunction of Canine Coronary Endothelium and Myocardium and Reperfusion Arrhythmias After Brief Ischemia/Reperfusion

BACKGROUND Brief myocardial ischemia is associated with myocardial and coronary endothelial dysfunction caused by oxygen free radicals released during reperfusion. Estrogen, known to have antioxidant activity, may prevent these complications. METHODS AND RESULTS We assessed the effect of 2 weeks of treatment with 17 beta-estradiol (E, 100 micrograms.kg-1.d-1, n = 12) or placebo (P, n = 15) on myocardial and coronary endothelial function during the first 2 hours of reperfusion in dogs subjected to 15 minutes of ischemia induced by occlusion of the left anterior descending coronary artery (LAD). Our results show that the incidence of ventricular arrhythmias significantly decreased in E (3 of 12) compared with P (11 of 15). Systolic shortening, significantly depressed in P during early reperfusion, was maintained at preischemic levels in E. During reperfusion, the increase in LAD flow to acetylcholine, attenuated in P (60 +/- 6%), was preserved in E endothelium. (151 +/- 28%) and was associated with increased serum nitrite/nitrate concentration. n-Pentane in exhaled gas in vivo, an index of lipid peroxidation, increased significantly during early reperfusion in P (from 9.1 +/- 1.9 to 41.6 +/- 13.0 ppb, P < .05) but not in E (23.0 +/- 6.9 ppb). In vitro, arterial segments from E generated significantly less superoxide anion after hypoxia/reoxygenation than those from P. Ischemic/reperfused LAD segments from E also revealed a better preservation of endothelium-dependent relaxation in vitro (maximum relaxation, 42 +/- 4% versus 24 +/- 4% in P; P < .05). CONCLUSIONS Estrogen protects against endothelial and myocardial dysfunction resulting from brief ischemia/reperfusion. This protection may relate to an antioxidant effect of estrogen.

l-arginine reverses the adverse pregnancy changes induced by nitric oxide synthase inhibition in the rat

OBJECTIVE Inhibition of nitric oxide synthase with N omega-nitro-L-arginine methyl ester (L-NAME) induces a preeclampsia-like syndrome of hypertension, proteinuria, intrauterine growth restriction, and renal glomerular capillary endothelial lesions in pregnant rats. We attempted to reverse these changes with late-pregnancy administration of L-arginine. STUDY DESIGN Sprague Dawley rats with timed pregnancies received infusions of either saline solution (n = 12) (group SC) or L-NAME (n = 12) (group LC) (160 mg/kg per day) on gestational day 10 through term. On gestational day 16 half of the saline solution group (group SA) and half of the L-NAME group (group LA) received L-arginine (21 mg/kg per day) through delivery. Systolic blood pressures were determined via tail cuff on days 10, 16, and 21. Pup weights were assessed at delivery, serum and urine were collected and analyzed for nitrites and nitrates, and renal tissue was processed for histologic examination. Data were analyzed with the one-way analysis of variance and the Newman-Keuls test for multiple comparisons. RESULTS In the L-NAME-treated animals L-arginine significantly lowered systolic blood pressure at late pregnancy (125 +/- 2.42 vs 153 +/- 3.0 mm Hg) (p < 0.01), increased mean pup weight (5.6 +/- 0.11 gm in group LA vs. 5.0 +/- 0.02 gm in group LC) (p < 0.001), decreased the degree of proteinuria (2+ vs trace), and decreased the proportion of injured glomeruli (7% vs 64%) (p < 0.001). CONCLUSIONS Lesions induced by chronic inhibition of endothelium-derived nitric oxide synthesis (hypertension, intrauterine growth restriction, proteinuria, renal glomerulus injury) are reversed by treatment with L-arginine. These findings lend support to the potential for use of nitric oxide donors in the treatment and prevention of preeclampsia.

Protection by oestradiol against the development of cardiovascular changes associated with monocrotaline pulmonary hypertension in rats

1 We studied the effects of oestradiol 17β on the development of pulmonary vascular changes and right ventricular (RV) hypertrophy in response to monocrotaline in male Sprague‐Dawley rats. 2 Rats were treated with either placebo or oestradiol 17β (10 mg) in the form of slow release pellets implanted subcutaneously 48 h before monocrotaline administration. Rats were injected with either saline or a single dose of monocrotaline (60 mg kg−1, i.m.). Pulmonary vascular changes and RV hypertrophy were studied at 4 weeks following monocrotaline administration. 3 Monocrotaline induced a significant increase in the ratio of right ventricle (RV) to left ventricle‐plus‐septum (LV + S) weights. Monocrotaline‐treated rats also showed significant myointimal proliferation in small pulmonary arteries, decrease of arterial numbers and increase in the number of abnormal alveolar macrophages. 4 Oestradiol 17β attenuated myointimal hyperplasia in pulmonary vessels, decreased the RV/(LV + S) ratio in monocrotaline‐treated rats. Oestradiol 17β had no significant effect on control animals. 5 Oestradiol treatment prevented the increase in lung wet to dry weight ratio, observed 7 days post monocrotaline administration. 6 These results suggest that oestradiol 17β protects against the pulmonary vascular remodelling and RV hypertrophy associated with monocrotaline‐induced pulmonary hypertension in the rat. Oestradiol also protects against microvascular leak observed in the early days of lesion.

Release of immunoreactive-neuropeptide Y by rat platelets

Neuropeptide Y, a potent vasoconstrictor and cardiac depressant, is re-leased from sympathetic nerve endings. Its presence in megakaryocytes suggests this peptide might be stored in platelet granules and released during aggregation. Immunoreactive-neuropeptide Y was measured in platelet rich and platelet poor plasma, and was substantially greater in the former. Addition of collagen to platelets resulted in release of neuropeptide Y which paralleled, in a concentration-dependent manner, the degree of platelet aggregation. Adenosine diphosphate, at concentrations which induce only the first phase of aggregation and not the release reaction, caused only a minor release of neuropeptide Y. These results suggest that platelet release could be a major source of circulating neuropeptide Y and could contribute to hemodynamics of pathophysiological states involving platelet activation.

In vitro effect of oestradiol on thymidine uptake in pulmonary vascular smooth muscle cell : role of the endothelium

1 The effect of different concentrations of oestradiol‐17β (3–300 nm) on [3H]‐thymidine uptake was studied in segments from canine pulmonary artery, and cultures of rat pulmonary vascular smooth muscle cells (VSMC). 2 Incubation with oestradiol‐17β for 24 h, potentiated in a concentration‐dependent manner [3H]‐thymidine uptake in VSMC cultures. 3 Oestradiol‐17β potentiated thymidine uptake by pulmonary arterial segments but only when the endothelium had been removed. Autoradiography showed dense incorporation of radioactive thymidine in the vascular smooth muscle cells of the media. 4 The non‐steroidal oestrogen, stilboestrol (300 nm), also significantly potentiated [3H]‐thymidine uptake, in both VSMC cultures and pulmonary artery segments. Testosterone was ineffective at a similar concentration. 5 Pre‐incubation of the pulmonary VSMC with the anti‐oestrogen tamoxifen (1 μm) antagonized the potentiating effect of oestradiol‐17β on [3H]‐thymidine incorporation. The effect of tamoxifen was less pronounced in pulmonary arterial segments. 6 These data suggest that oestrogen may promote proliferation of pulmonary VSMC. Endothelial injury or dysfunction may be an important factor in the expression of the oestrogenic effect. 7 We speculate that plasma oestrogen may be a contributing factor to the proliferative lesion observed in certain forms of pulmonary vascular injury in women.

Effect of testosterone treatment on vasoconstrictor response of left anterior descending coronary artery in male and female pigs.

Summary Androgens may he risk factors in the pathogenesis of coronary artery disease by promoting coronary vasoconstriction. We studied the effect of testosterone treatment on coronary vascular reactivity of male and female domestic pigs treated for 2 weeks with either placebo or testosterone 10 mg/kg subcutaneous (s.c.) pellets. Vascular reactivity was studied in ring segments (4–5 mm) isolated from the left anterior descending coronary artery (LAD). No significant sex difference was noted in the response of LAD segments from placebo-treated male and female animals to KCI and prostaglandin F2α (PGF2α). Androgen treatment increased the maximum response (Tmax) of intact vessels to KC1 from 3,647 ± 689 mg in controls to 8.939 ± 1,284 mg in testosterone-treated males (p < 0.01) and from 3,405 ± 669 to 10.524 ± 1.663 mg in testosterone-treated female pigs (p < 0.01 ). Testosterone similarly increased the response to PGF2α 10 6M from a mean of 2.149 ± 1.036 to 3,163 ± 867 mg in males (p < 0.05) and from 2.076 ± 810 to 3.565 ± 578 mg in female segments (p < 0.05). Endothelial denudation significantly decreased the potentiating effect of testosterone treatment in males to both KC1 and PGF2α (p < 0.05). but not in segments from females. Our data show that testosterone treatment potentiates contractility of porcine LAD segments to both receptor- and nonreceptor-mediated agonists. In male pigs, this effect may be mediated by an effect on endothelium.

17-β estradiol regulation of myocardial glutathione and its role in protection against myocardial stunning in dogs

We studied the effect of 2-week treatment with estradiol 17beta on myocardial glutathione concentration in dogs and isolated perfused rat heart subjected to brief coronary ischemia and reperfusion. Estradiol protected against ischemia/reperfusion-induced myocardial systolic shortening and malonylaldehyde production and increased myocardial glutathione concentration and glucose-6-phosphate dehydrogenase enzyme activity. Reduction of myocardial glutathione with buthionine sulfoximine to levels seen in the absence of estrogen reversed the protective effect of estradiol against myocardial dysfunction and lipid peroxidation associated with ischemia/reperfusion. These results suggest that the antioxidant effect of estradiol in ischemia/reperfusion may be mediated by regulation of myocardial glutathione metabolism.

Effect of estradiol 17β on pressor responses of rat mesenteric bed to norepinephrine, K+, and U-46619

Summary We reinvestigated the effect of estradiol 17β on the responses of adrenergic and nonadrenergic vasoconstrictors characterized it in terms of steroid specificity, time course, and the role of classic estrogen receptors. We evaluated the effect of estradiol 17β on the pressor responses of isolated perfused rat mesenteric vascular bed (McGregors preparation). Estradiol 17β (7–700 nM) significantly increased the pressor response to bolus applications of norepinephrine (NE) (p < 0.05). However, estradiol 17β did not significantly increase the responses to endogenous NE release induced by electrical field stimulation. Other steroids, testosterone, and the 17α isomer of estradiol (7 and 700 nM) were ineffective. Estradiol 17β (700 nM) also significantly increased the maximum pressor response of rat mesenteric preparation to both the prostaglandin endoperoxide analogue U-46619 and to K+. The potentiation by estradiol 17β of mesenteric vasoconstriction elicited by NE, K+, and U-46619 was rapid (2–8 min), suggesting that a nuclear receptor may not be involved. This notion received further support in that significant potentiation of the NE-induced pressor response was also observed with estradiol 17β conjugated to albumin (700 nM), but not when electrical field stimulation was used. The conjugate increased the effect of all NE concentrations. Its effect was also more consistent (p < 0.01) than that elicited by free estradiol 17β. The dose-response curve was shifted to the left, and the maximum effect was increased. These data suggest that estradiol 17β may possess rapid nongenomic actions unrelated to nuclear receptor binding and gene transcription.

Estradiol effect on rate of proliferation of rat carotid segments: effect of gender and tamoxifen.

Using organ culture of carotid artery segments from sexually mature male and female rats, we examined the effect of estradiol 17 beta on proliferation. The index of cell proliferation was [3H]thymidine uptake. Estradiol 17 beta (0.18-0.36 microM) inhibited the uptake of thymidine in a concentration-dependent manner (p < 0.05). Estradiol 17 beta inhibited [3H]thymidine uptake only in the absence of the weak estrogen receptor agonist phenol red and in carotid artery segments from sexually mature female (p < 0.01) but not male rats. Tamoxifen (0.1 and 1 microM), a partial agonist of estrogen receptors, significantly inhibited thymidine uptake (p < 0.01). However, preincubation of the segments with tamoxifen (0.1 and 1.0 microM) for 4 h before the exposure to estradiol, blocked estradiol 17 beta-induced inhibition of thymidine uptake (p < 0.05 and p < 0.01 for 0.1 and 1.0 microM, respectively). The cyclooxygenase inhibitor indomethacin (5 microM) did not affect either the basal [3H]thymidine uptake or the estradiol 17 beta-induced inhibition of that uptake. This latter finding suggests that prostacyclin or prostaglandin E2 does not mediate the inhibitory response to estradiol 17 beta. The results of these experiments suggest that estradiol 17 beta-induced inhibition of proliferation of rat carotid artery segments is mediated through activation of estrogen receptors.

Vascular Non-genomic Effects of Estrogen

Studies of the cellular mechanisms of action of steroids lead to extensive investigation of DNA-binding and gene regulatory proteins. Steroids are thought to passively diffuse into the cell and bind to their nuclear receptor protein. Each receptor is both ligand and cell specific, and binds to its respective steroid with high affinity (K d: 0.1–1.0 nM). The ligand-receptor complex becomes an activated transcription factor, which binds gene regulatory elements on DNA to enhance transcription of several target genes. Protein synthesis and processing follows (Moudgil 1987).