Clot stability as a determinant of effective factor VIII replacement in hemophilia A.

BACKGROUND: Factor VIII (FVIII) replacement is standard of care for patients with hemophilia A (HemA); however, patient response does not always correlate with FVIII levels. We hypothesize this may be in part due to the physical properties of clots and contributions of fibrin, platelets, and erythrocytes, which may be important for hemostasis. OBJECTIVE: To understand how FVIII contributes to effective hemostasis in terms of clot structure and mechanical properties. PATIENTS/METHODS: In vitro HemA clots in human plasma or whole blood were analyzed using turbidity waveform analysis, confocal microscopy, and rheometry with or without added FVIII. In vivo clots from saphenous vein puncture in wild-type and HemA mice with varying FVIII levels were examined using scanning electron microscopy. RESULTS: FVIII profoundly affected HemA clot structure and physical properties; added FVIII converted the open and porous fibrin meshwork and low stiffness of HemA clots to a highly branched and dense meshwork with higher stiffness. Platelets and erythrocytes incorporated into clots modulated clot properties. The clots formed in the mouse saphenous vein model contained variable amounts of compressed erythrocytes (polyhedrocytes), fibrin, and platelets depending on the levels of FVIII, correlating with bleeding times. FVIII effects on clot characteristics were dose-dependent and reached a maximum at ~25% FVIII, such that HemA clots formed with this level of FVIII resembled clots from unaffected controls. CONCLUSIONS: Effective clot formation can be achieved in HemA by replacement therapy, which alters the architecture of the fibrin network and associated cells, thus increasing clot stiffness and decreasing clot permeability.

Identification and function probing of an antithrombin IIIß conformation-specific antibody.

UNLABELLED: ESSENTIALS: Antithrombin III (AT)ß binds heparin with higher affinity than ATα. A conformation-specific antibody against ATß, TPP2009, was made to investigate ATß in hemostasis. TPP2009 bound specifically to heparin-ATß and greatly reduced the anticoagulant effect of AT. This antibody was effective in elucidating the importance of ATß in hemostasis. BACKGROUND: Antithrombin III (AT)ß is an isoform of AT that lacks the post-translational carbohydrate modification at Asn135. This isoform binds heparin with greater affinity than ATα, and has been shown to target antithrombotic function to the extracellular vascular endothelial injury site. OBJECTIVES: To characterize a conformation-specific antibody against ATß and begin to investigate the role of ATß in maintaining hemostasis. METHODS: Surface plasmon resonance (SPR), antigen binding and functional assays were conducted to characterize the mode of action of antibodies generated against heparin-bound ATß (ATß*H) by the use of phage display. RESULTS: SPR and binding studies showed that one of the antibodies, TPP2009, bound specifically to ATß*H and glycosaminoglycan-associated ATß on endothelial cells. In diluted prothrombin and activated factor X (FXa)-induced clotting assays, TPP2009 dose-dependently reduced the anticoagulant effect of heparin in non-hemophilic and FVIII-deficient human plasma, with half-maximal effective concentrations (EC50 ) of 10.5 nm and 4.7 nm, respectively. In AT-deficient human plasma, TPP2009 dose-dependently inhibited the effects of exogenously added ATß and heparin. In purified systems with ATß and pentasaccharide, TPP2009 restored > 91% of FXa activity. TPP2009 dose-dependently reversed the effects of heparin in rabbit (EC50 , 25.7 nm) and cynomolgus monkey (EC50 , 21.5 nm) plasma, but not in mouse plasma. TPP2009 was also effective in partially restoring FXa activity in rabbit and cynomolgus monkey plasma treated with FVIII function-neutralizing antibodies. CONCLUSIONS: TPP2009 specifically targets a unique conformational epitope on ATß*H and blocks ATß-mediated anticoagulation. It effectively promotes coagulation in plasma, indicating the importance of ATß in hemostasis.

Overexpression of PAI-1 prevents the development of abdominal aortic aneurysm in mice.

Vessel wall inflammation and matrix destruction are critical to abdominal aortic aneurysm (AAA) formation and rupture. We have previously shown that urokinase plasminogen activator (uPA) is highly expressed in experimental AAA and is essential for AAA formation and expansion. In this study, we examined the effects of overexpression of a natural inhibitor of uPA, plasminogen activator inhibitor-1 (PAI-1), on the development of angiotensin (Ang) II-induced AAA in ApoE-deficient (ApoE(-/-)) mice. Mice were treated with recombinant adenovirus containing either the human PAI-1 gene (Ad5.CMV.PAI-1) or the luciferase gene (Ad5.CMV.Luc) delivered either locally by intra-adventitial injection or systemically by tail vein injection. Our results show that local delivery of the PAI-1 gene completely prevented AAA formation (0 vs 55.6% in Ad5.CMV.Luc controls, P<0.05). In contrast, systemic delivery of the PAI-1 gene did not affect AAA incidence (78 vs 90% in Ad5.CMV.Luc controls, P=0.125). Local delivery of the PAI-1 gene 2 weeks after Ang II infusion prevented further expansion of small aneurysms, but had no significant effect on the progression of larger aneurysms. These data suggest that local PAI-1 gene transfer could be used to stabilize small AAA and reduce the rate of expansion and risk of rupture.

Role of endothelial nitric oxide in bone marrow-derived progenitor cell mobilization.

Mobilization and recruitment of bone marrow-derived progenitor cells (BMDPCs) play an important role in postischemic tissue repair. Patients with coronary artery disease (CAD) or peripheral vascular disease (PVD) exhibit endothelial dysfunction, and as a result are likely to have a reduced number of progenitor cells mobilized in their peripheral circulation following ischemic injury. Identification of eNOS independent pathways for BMDPC mobilization may have important therapeutic value in this patient population. To identify such mechanisms we investigated the effect of granulocyte-colony stimulating factor (GCSF) and stem cell factor (SCF) in eNOS-KO mice with and without surgical hind-limb ischemia. Our results suggest that BMDPC mobilization can be achieved via activation of NO-independent pathways.

Effective treatment of vascular endothelial growth factor refractory hindlimb ischemia by a mutant endothelial nitric oxide synthase gene.

Gene delivery of angiogenic growth factors is a promising approach for the treatment of ischemic cardiovascular diseases. However, success of this new therapeutic principle is hindered by the lack of critical understanding as to how disease pathology affects the efficiency of gene delivery and/or the downstream signaling pathways of angiogenesis. Critical limb ischemia occurs in patients with advanced atherosclerosis often exhibiting deficiency in endothelial nitric oxide production. Similar to these patients, segmental femoral artery resection progresses into severe ischemic necrosis in mice deficient in endothelial nitric oxide synthase (ecNOS-KO) as well as in balb/c mice. We used these models to evaluate the influence of severe ischemia on transfection efficiency and duration of transgene expression in the skeletal muscle following plasmid injection in combination with electroporation. Subsequently, we also explored the potential therapeutic effect of the phosphomimetic mutant of ecNOS gene (NOS1177D) using optimized delivery parameters, and found significant benefit both in ecNOS-KO and balb/c mice. Our results indicate that NOS1177D gene delivery to the ischemic skeletal muscle can be efficient to reverse critical limb ischemia in pathological settings, which are refractory to treatments with a single growth factor, such as vascular endothelial growth factor.

Increased nitric oxide accounts for decreased basal vascular tone and responsiveness in the resistance vessels of high-cholesterol-fed rabbits.

The objective of this study was to determine the effects of hypercholesterolemia on basal vascular tone and vascular responses to pharmacologic agents in hindquarter resistance vessels. Blood pressure and hindquarter blood flow were measured in conscious rabbits fed a high cholesterol diet (1%) for 17 weeks (HC) compared to age-matched rabbits fed a normal diet (control). Basal hindquarter blood flow and vascular conductance were significantly higher in HC than in control rabbits. Administration of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME (100 mg/kg) decreased basal hindquarter blood flow and vascular conductance in a greater magnitude in HC than in control rabbits, thus, abolished the differences in both the flow and conductance between 2 groups, indicating that increased NO was responsible for reduced basal vascular tone in the HC rabbits. L-NIL (30 mg/kg), a selective inducible NOS (iNOS) inhibitor had no effects on either flow or conductance. This result does not support the involvement of iNOS. In separate experiments, animals were anesthetized and instrumented with an extracorporeal circuit to measure perfusion pressure under constant blood flow to the hindquarter vascular bed. In the HC group, vascular responses to acetylcholine, S-nitroso-N-acetyl-penicillamine and phenylephrine were all attenuated when compared to the responses in the control rabbits. These results indicate that local overproduction of NO due to hypercholesteremia could desensitize smooth muscle reactivity, thus causing general vascular hyporesponsiveness to vasoactive agents.

Advanced atherosclerotic lesions in the innominate artery of the ApoE knockout mouse.

Most previous studies of atherosclerosis in hyperlipidemic mouse models have focused their investigations on lesions within the aorta or aortic sinus in young animals. None of these studies has demonstrated clinically significant advanced lesions. We previously mapped the distribution of lesions throughout the arterial tree of apolipoprotein E knockout (apoE(-/-)) mice between the ages of 24 and 60 weeks. We found that the innominate artery, a small vessel connecting the aortic arch to the right subclavian and right carotid artery, exhibits a highly consistent rate of lesion progression and develops a narrowed vessel characterized by atrophic media and perivascular inflammation. The present study reports the characteristics of advanced lesions in the innominate artery of apoE(-/-) mice aged 42 to 60 weeks. In animals aged 42 to 54 weeks, there is a very high frequency of intraplaque hemorrhage and a fibrotic conversion of necrotic zones accompanied by loss of the fibrous cap. By 60 weeks of age, the lesions are characterized by the presence of collagen-rich fibrofatty nodules often flanked by lateral xanthomas. The processes underlying these changes in the innominate artery of older apoE(-/-) mice could well be a model for the critical processes leading to the breakdown and healing of the human atherosclerotic plaque.

Identification of selective estrogen receptor modulators by their gene expression fingerprints.

Clinical studies have shown that estrogen replacement therapy (ERT) reduces the incidence and severity of osteoporosis and cardiovascular disease in postmenopausal women. However, long term estrogen treatment also increases the risk of endometrial and breast cancer. The selective estrogen receptor (ER) modulators (SERMs) tamoxifen and raloxifene, cause antagonistic and agonistic responses when bound to the ER. Their predominantly antagonistic actions in the mammary gland form the rationale for their therapeutic utility in estrogen-responsive breast cancer, while their agonistic estrogen-like effects in bone and the cardiovascular system make them candidates for ERT regimens. Of these two SERMs, raloxifene is preferred because it has markedly less uterine-stimulatory activity than either estrogen or tamoxifen. To identify additional SERMs, a method to classify compounds based on differential gene expression modulation was developed. By analysis of 24 different combinations of genes and cells, a selected set of assays that permitted discrimination between estrogen, tamoxifen, raloxifene, and the pure ER antagonist ICI164384 was generated. This assay panel was employed to measure the activity of 38 compounds, and the gene expression fingerprints (GEFs) obtained for each compound were used to classify all compounds into eight groups. The compound's GEF predicted its uterine-stimulatory activity. One group of compounds was evaluated for activity in attenuating bone loss in ovariectomized rats. Most compounds with similar GEFs had similar in vivo activities, thereby suggesting that GEF-based screens could be useful in predicting a compound's in vivo pharmacological profile.

Role of endogenous nitric oxide in progression of atherosclerosis in apolipoprotein E-deficient mice.

This study investigated the role of endogenous nitric oxide (NO) in the progression of atherosclerosis in apolipoprotein E-deficient [apoE-knockout (KO)] mice. Mice were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME) an inhibitor of nitric oxide synthase (NOS) or with the NOS substrate L-arginine for 8 wk. L-NAME treatment resulted in a significant inhibition of NO-mediated vascular responses and a significant increase in the atherosclerotic plaque/surface area in the aorta of apoE-KO mice. L-arginine treatment had no influence on endothelial function and did not alter lesion size. Mean arterial blood pressure and serum lipid levels were not altered by the treatments. At the beginning of the study impairment in endothelial function was only apparent in the case of N(G)-nitro-L-arginine-induced, NO-mediated contraction, whereas ACh-induced, NO-mediated relaxation was not different between age-matched apoE-KO and C57Bl/6J mice. After the 8-wk treatment with the NOS inhibitor, both NO-mediated responses were significantly inhibited. The acceleration in lesion size concomitant to the severely impaired NO-mediated responses indicates that lack of endogenous NO is an important progression factor of atherosclerosis in the apoE-KO mouse.

Increased aortic stiffness assessed by pulse wave velocity in apolipoprotein E-deficient mice.

Atherosclerosis develops and progresses spontaneously in apolipoprotein E-knockout (apoE-KO) mice. A direct consequence of atherosclerosis is an increase in vascular stiffness. Pulse wave velocity (PWV) has been used to assess the stiffness of large vessels and was found to be increased in patients with atherosclerosis. In the present study, aortic stiffness was assessed by PWV in 4- and 13-mo-old apoE-KO mice and age-matched controls (C57BL/6J). In 13-mo-old apoE-KO mice with extensive atherosclerotic lesions in the aorta (61 +/- 4%), PWV increased significantly (3.8 +/- 0.2 m/s) compared with controls (2.9 +/- 0.2 m/s). Endothelial nitric oxide (EDNO)-mediated vasorelaxation in response to ACh was markedly diminished in the aortic rings isolated from 13-mo-old apoE-KO mice compared with age-matched controls. In contrast, in 4-mo-old apoE-KO mice with only moderate atherosclerotic lesions in the aorta (23 +/- 5%), there were no significant changes in PWV and EDNO-mediated relaxation compared with controls. Blood pressure was not different among the four groups of mice. There were no significant differences in endothelium-independent vascular responses to sodium nitroprusside among different groups investigated. Histological evaluation revealed focal fragmentation of the elastic laminae in the aortic walls of 13-mo-old apoE-KO mice. These results demonstrate for the first time that aortic stiffness determined by PWV increases in 13-mo-old apoE-KO mice. Endothelial dysfunction and elastic destruction in vascular wall caused by atherosclerosis may have contributed.

Accelerated atherosclerosis and premature calcified cartilaginous metaplasia in the aorta of diabetic male Apo E knockout mice can be prevented by chronic treatment with 17 beta-estradiol.

Epidemiological data indicate that estrogens significantly reduce the risk of morbidity and mortality due to cardiovascular diseases in postmenopausal women. Although numerous animal studies demonstrated inhibition of early atheromatous lesion formation by estrogen treatment in several species, information about the potential benefits of estrogens on complex, advanced atherosclerotic lesions is still lacking. The present study was designed to test whether chronic treatment with 17 beta-estradiol affects hyperglycemia-induced premature advanced lesion formation in 40-week-old male apolipoprotein E-deficient (Apo E-KO) mice. In order to accelerate advanced lesion formation, we treated male Apo E-KO mice with streptozotocin (STZ) at the age of 6 weeks. Two weeks later the STZ-treated mice received a slow release pellet containing either 17 beta-estradiol or placebo. STZ treatment caused sustained hyperglycemia without changes in serum total cholesterol or triglyceride levels compared to citrate control mice. STZ-treated Apo E-KO mice developed significantly more lesions in some (but not all) parts of the aorta and its main branches, and caused premature calcified cartilaginous metaplasia in the lesions of the proximal aorta. Chronic treatment with 17 beta-estradiol lead to a significant decrease in blood glucose and triglyceride levels, reduced the lesion area in all vascular segments studied and prevented cartilaginous metaplasia in STZ-treated Apo E-KO mice. The results of this study show that STZ treatment leads to significant acceleration of atherosclerotic lesion formation and premature occurrence of calcified cartilaginous areas in Apo E-KO mice, which could be effectively prevented by chronic estrogen treatment.

Effect of 17beta-estradiol in hypercholesterolemic rabbits with severe endothelial dysfunction.

17beta-Estradiol prevents early vascular lesion development and may also affect advanced atherosclerosis. To test the antiatherosclerotic effect of estrogen under conditions that resemble more advanced human atherosclerosis with severe endothelial dysfunction, we have investigated the effect of 17beta-estradiol in hypercholesterolemic rabbits treated with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME). Chronic L-NAME administration attenuated endothelial nitric oxide (EDNO)-mediated vascular responses leading to significantly accelerated atherosclerotic plaque development. 17beta-Estradiol treatment alone inhibited aortic lesion formation with concurrent increase in EDNO-mediated responses. The beneficial effect of estrogen persisted in the L-NAME-treated rabbits, suggesting that the antiatherogenic action of 17beta-estradiol involves NO-independent mechanisms as well. Serum cholesterol levels were not altered by any of the treatments. 17beta-Estradiol treatment significantly increased EDNO production under these conditions as well. The reduction in plaque size by 17beta-estradiol was always accompanied by increased EDNO production, suggesting a strong association between these two events. The results demonstrate that estrogen treatment may exert protection against atherosclerosis even in patients with severe endothelial dysfunction.

Expression of interleukin-6 in atherosclerotic lesions of male ApoE-knockout mice: inhibition by 17beta-estradiol.

Increased levels of interleukin-6 (IL-6) have been proposed to contribute to a number of pathological disorders, including osteoporosis and Alzheimer's disease. In human atherosclerotic lesions, IL-6 protein and mRNA have been detected, although the role of IL-6 in plaque formation is unknown. We have examined the expression pattern of IL-6 mRNA and secreted protein in male apolipoprotein E-knockout (apoE-KO) mice aortas. Furthermore, we have evaluated the effects of 17beta-estradiol (E2), a vasculoprotective sex steroid hormone, on the secretion of this inflammatory cytokine from isolated male apoE-KO mice aortas. The expression of IL-6 mRNA was detected by reverse transcription-polymerase chain reaction in the apoE-KO mouse aortas but not in the aortas of age-matched control mice. Similarly, the secretion of IL-6 protein from isolated apoE-KO aortic segments was significantly greater than that from aortas of age-matched control animals. The secretion of IL-6 from isolated aortic rings of apoE-KO mice ranging in age from 6 to 48 weeks showed a significant, positive correlation with percent lesion area measured in the same tissue. Immunohistochemical staining of apoE-KO mouse aortic tissue sections demonstrated colocalization of IL-6 expression with macrophages. Treatment of male apoE-KO mice with E2 for 3 weeks resulted in a statistically significant 50% reduction in IL-6 secretion from ex vivo aortic tissue segments. There was no significant change in total serum cholesterol and triglyceride levels in the E2-treated group compared with placebo-treated controls. These data demonstrate that (1) IL-6 mRNA and protein are expressed in the atherosclerotic plaques of apoE-KO mice aortas and (2) IL-6 production is suppressed by E2 treatment, which may contribute to the antiatherosclerotic effects of E2 in the apoE-KO mouse model of atherosclerosis.

Effect of 17beta-oestradiol on cytokine-induced nitric oxide production in rat isolated aorta.

1. Studies were performed on isolated aortic rings without endothelium to investigate the effect of 17beta-oestradiol on cytokine-induced nitric oxide production by the inducible nitric oxide synthase (iNOS). 2. Treatment of the isolated aortic rings with interleukin-1beta (IL-1beta, 20 micro ml(-1)) led to the expression of iNOS mRNA and protein, as well as significant nitrite accumulation in the incubation media and suppression of phenylephrine (1 nM-10 microM)-evoked contraction. 3. Cycloheximide (1 microM), a protein synthesis inhibitor, prevented iNOS protein expression, nitrite accumulation and the suppression of contractility by IL-1beta on the isolated aortic rings. 17Beta-oestradiol (1 nM-10 microM) and the partial oestrogen receptor agonist 4-OH-tamoxifen (1 nM-10 microM) produced concentration-dependent inhibition of IL-1beta-induced nitrite accumulation and restored vasoconstrictor responsiveness to phenylephrine, similar to the iNOS inhibitor aminoguanidine (100 microM). 4. Semiquantitative PCR demonstrated decreased iNOS mRNA in the IL-1beta-induced and 17beta-oestradiol-treated rings. Western blot analysis of rat aorta homogenates revealed that 17beta-oestradiol treatment resulted in a reduction in IL-1beta-induced iNOS protein level. 5. Incubation with tumour necrosis factor alpha (TNF alpha, 1 ng ml(-1)) resulted in significant nitrite accumulation in the incubation media and suppression of the smooth muscle contractile response to phenylephrine, similar to IL-1beta. The effects of TNF alpha were also inhibited by co-incubation of the rings with 17beta-oestradiol and 4-OH-tamoxifen (1 microM). 6. The anti-transforming growth factor-beta1 (TGF-beta1) antibody, which inhibited TGF-beta1-induced suppression of nitrite production from IL-1beta-treated vascular rings, did not affect the inhibitory action of 17beta-oestradiol, suggesting that the effect of oestrogen on iNOS inhibition was not mediated by TGF-beta1. 7. These results show that the ovarian sex steroid, 17beta-oestradiol is a modulator of cytokine-induced iNOS activity in rat vascular smooth muscle and its mechanism of action involves decrease of iNOS mRNA and protein.

Effects of estrus cycle, ovariectomy, and treatment with estrogen, tamoxifen, and progesterone on apolipoprotein(a) gene expression in transgenic mice.

Plasma levels of lipoprotein(a) (Lp(a)), are regulated by the synthetic rate of apolipoprotein(a) (apo(a)), a major protein component of this atherogenic lipoprotein. Exogenously administered sex steroid hormones are potent regulators of plasma Lp(a) concentrations. We utilized a recently developed apo(a) yeast artificial chromosome (YAC) transgenic mouse model to study the effects of ovariectomy, estrus cycle, and exogenous administration of ethinyl-estradiol, the partial estrogen receptor agonist, tamoxifen, and progesterone on circulating apo(a) plasma levels. Analysis of liver RNA revealed that estrogen and tamoxifen exerts their plasma apo(a) lowering effect at the level of apo(a) mRNA. This action of estrogen and tamoxifen may contribute to their antiatherosclerotic and cardiovascular protective effect.

17 beta-Estradiol attenuates endotoxin-induced excessive nitric oxide production in ovariectomized rats in vivo.

Experiments were designed to examine the effect of 17 beta-estradiol on lipopolysaccharide (LPS)-induced excessive nitric oxide production in vivo. Ovariectomized and sham-operated female rats were injected with LPS (5 mg/kg ip), and different groups of ovariectomized, LPS-injected animals were treated with either 17 beta-estradiol, dexamethasone, or aminoguanidine. Nitric oxide generation was estimated by measuring plasma nitrite levels 12 h after LPS injection. LPS treatment of the rats resulted in a four- to fivefold increase in circulating plasma nitrite level, significantly higher in the ovariectomized animals compared with the sham-operated animals. The LPS-induced plasma nitrite increase could be prevented by dexamethasone (3 mg/kg ip) injected 1 h before LPS treatment. 17 beta-Estradiol (3 micrograms/rat sc), administered 48 h before LPS treatment, significantly attenuated the LPS-induced elevation in plasma nitrite levels. This effect was comparable to that achieved by aminoguanidine (200 microM/kg), an inhibitor of inducible nitric oxide synthase, injected 1 h after LPS treatment. The present findings suggest that estrogens may be beneficial in pathological conditions associated with excessive nitric oxide generation.

Quantitative measurement for endothelial constitutive nitric oxide synthase in cultured human endothelial cells.

Constitutively expressed endothelial nitric oxide synthase (ecNOS) produces nitric oxide (NO) from L-arginine and is important for the maintenance of cardiovascular homeostasis. We report the development of a capture ELISA which is specific for ecNOS. The assay detection limit is 0.5 ng/ml ecNOS protein, allowing the measurement of ecNOS from as few as 6000 human endothelial cells cultured in 96-well microtiter plates. This ELISA has been used to measure a downregulation of ecNOS with 24-h TNFalpha treatment, consistent with results obtained by Western blot analysis. Quantitation of ecNOS in human endothelial cells showed a higher expression of ecNOS in human aortic endothelial cells (18.3+/-1.35 ng ecNOS per 10(6) cells, n = 3 donors) than in human umbilical vein endothelial cells (10.4+/-0.48 ng ecNOS per 10(6) cells, n = 3 donors). These studies demonstrate that this convenient, quantitative assay is currently the most sensitive method for investigating ecNOS protein regulation.

Vascular estrogen receptors and endothelium-derived nitric oxide production in the mouse aorta. Gender difference and effect of estrogen receptor gene disruption.

The present study was designed to test the hypothesis that estrogen receptors (ER) in the blood vessel wall play a role in the modulation of the release of endothelium-derived nitric oxide (EDNO). Both basal and stimulated release of EDNO were determined in aortic rings isolated from female and male wild-type and male homozygous estrogen receptor knock-out (ERKO) mice. 125I-17beta-estradiol binding in aortic tissue showed significantly more high affinity cytosolic- nuclear-binding sites in male compared with female wildtype mice. Estrogen receptor transcripts were present in the aorta of male wild-type mice, but they were absent in male ERKO animals. Basal release of EDNO (determined by endothelium-dependent contraction caused by NG-nitro-arginine) was significantly higher in aorta of wild-type male mice compared with wild-type female mice, and significantly lower in the aorta of male ERKO compared with male wild-type mice. Acetylcholine-induced endothelium-dependent relaxation was similar in all groups studied. No difference was observed in the activity of calcium-dependent nitric oxide synthase in homogenates of lungs and brain taken from male wild-type and ERKO mice. These studies show a significant association between the number of estrogen receptors and basal release of EDNO in the aorta of mice, and suggest that decreased vascular estrogen receptor number may represent a novel risk factor for cardiovascular diseases.

Potential cellular signaling mechanisms mediating upregulation of endothelial nitric oxide production by estrogen.

Experimental and clinical studies have provided ample evidence that estrogens exert a significant antiatherosclerotic effect and reduce morbidity and mortality due to cardiovascular diseases. The exact cellular mechanism of this vasculoprotective action of estrogen is not known, but recent work in our and other laboratories suggests that upregulation of endothelial nitric oxide (NO) production may significantly contribute to the mechanism. The vascular endothelium of female animals and humans produces more NO than that of males. Estrogen treatment significantly increases endothelial NO generation in ovariectomized animals and in postmenopausal women. Reduced endothelial NO production in the aorta of estrogen-receptor-deficient mice indicates that the nuclear estrogen receptor mediates the effect of estrogen. The most probable mechanism of estrogen-induced upregulation of endothelial NO production is the transcriptional stimulation of NOS III gene expression. However, the following alternative mechanisms may be involved as well: (1) inhibition of cytokine-induced downregulation of NOS III gene expression, (2) posttranslational modification of NOS III protein, (3) increased cofactor or L-arginine availability, (4) nongenomic activation of second messengers (e.g., Ca2+, cAMP) and tyrosine kinase, and (5) modulation of NO degrading systems (e.g., reactive oxygen radical generation and antioxidants). This paper reviews current data supporting these potential mechanisms.

Gender difference in endothelial dysfunction in the aorta of spontaneously hypertensive rats.

We investigated endothelium-dependent responses of thoracic aorta isolated from age-matched male and female spontaneously hypertensive rats (SHR) to explore gender differences in endothelial dysfunction that may contribute to the sexual dimorphism observed in the development of hypertension in this strain. Endothelium-dependent relaxation in response to acetylcholine (10(-9) to 10(-4) mol/L) was significantly greater in female rats than in male rats, although impaired responses were seen in both sexes compared with normotensive controls. Inhibition of cyclooxygenase by indomethacin (10(-5) mol/L) improved endothelium-dependent relaxation, but it did not abolish the gender difference. Relaxations in response to sodium nitroprusside were identical in denuded aortic rings from male and female SHR. Acetylcholine at higher concentrations (10(-6) to 10(-4) mol/L) induced endothelium-dependent contraction in intact, quiescent aortic rings from male SHR but not in those from female SHR. After incubation with NG-nitro-L-arginine (10(-4) mol/L), contraction in response to acetylcholine became apparent in rings from female SHR, but it was still significantly less pronounced than in similarly treated rings from male SHR. Endothelium-dependent contraction was prevented by indomethacin in both sexes, suggesting that a cyclooxygenase product such as endoperoxide may be mediating this effect. Because responses evoked by the thromboxane/endoperoxide receptor agonist U46619 (10(-10) to 10(-6) mol/L) were not greater in rings from male SHR than those from female SHR, increased smooth muscle responsiveness or higher thromboxane/endoperoxide receptor density in the males could not account for the differences in endothelium-dependent contraction. These results suggest that sex steroid hormones may control endothelium-dependent vascular reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)