Differential effects of 17beta-estradiol and raloxifene on VSMC phenotype and expression of osteoblast-associated proteins

Several studies demonstrate an association between osteoporosis and arterial calcific disease, both of which being common in elderly women. Estradiol and raloxifene, a selective estrogen receptor modulator, prevent bone loss in postmenopausal women. Little is known regarding how these agents affect arterial calcification. The aim of this study was to determine whether or not 17beta-estradiol and raloxifene reduced vascular smooth muscle cell (VSMC) differentiation and expression of bone-associated proteins during phosphate-induced calcification in vitro. Aortic VSMC were cultured from adult, gonadally intact, and ovariectomized (OVX) female pigs. Calcifying medium was added, and cells were treated with solvent (control), 17beta-estradiol (E(2)), or raloxifene. Extent of calcification and phenotypic expression of bone-associated proteins [matrix gla protein (MGP), osteoprotegerin (OPG), and bone sialoprotein (BSP)] were examined at 3-day intervals over 2 wk. Calcium content increased in all groups but was greater in VSMC derived from intact compared with OVX animals. E(2) reduced calcification and preserved a contractile phenotype. Expression of OPG significantly decreased with time; this decrease was significantly greater in VSMC derived from OVX compared with gonadally intact pigs. E(2) and raloxifene preserved expression of OPG only in VSMC from intact pigs. Expression of MGP increased significantly with time and was not affected by E(2) or raloxifene treatments. E(2) treatment significantly inhibited synthesis of BSP in cells from both groups. In conclusion, E(2) slows differentiation of VSMC induced by excess phosphate. Effectiveness of raloxifene to preserve expression of bone cell-associated proteins depends on the hormonal status of the tissue donor.     

Differential effects of estrogens and progestins on the anticoagulant tissue factor pathway inhibitor in the rat

Oral contraceptives (OC) and postmenopausal hormone therapy (HT) modulate plasma levels of proteins that regulate blood coagulation. It remains unclear whether the progestin component contributes to these changes. The present study was designed to determine whether progestins modulate two essential plasma anticoagulants, antithrombin (AT) and tissue factor pathway inhibitor (TFPI), in an animal model. Ovariectomized rats were treated orally with three progestins, norethindrone acetate (NETA), trimegestone (TMG), or drospirenone (DSP), either alone or combined with 17alpha-ethyinylestradiol (EE). Plasma AT levels were unchanged. However, TFPI activity was reduced by EE alone (10-100 microg/kg/day) in a dose-dependent manner; NETA (3 or 10 mg/kg/day) reduced TFPI by approximately 40 or approximately 80%, respectively, while TMG and DSP had no effect. NETA and EE effects were blocked by co-administration of ICI-182,780, an estrogen receptor antagonist, suggesting that both responses were likely estrogen receptor-mediated. Reduced TFPI after NETA or EE treatment was not accompanied by changes in TFPI mRNA levels in tissues that express TFPI, but there was a positive correlation between plasma TFPI and total cholesterol. Sex hormone effects on TFPI in this model and as reported in women may help to shift the coagulation balance to a more prothrombotic state. Progestins such as TMG and DSP that lack estrogenic activity could potentially have an improved clinical profile.     

Catabolism of factor VIIa bound to tissue factor in fibroblasts in the presence and absence of tissue factor pathway inhibitor

Vascular injury leads to the exposure of blood to fibroblasts and smooth muscle cells within the vessel wall. These cells constitutively express tissue factor (TF), the cellular receptor for plasma clotting factor VIIa (FVIIa). Formation of TF.FVIIa complexes on cell surfaces triggers the blood coagulation cascade. In the present study, we have investigated the fate of TF.FVIIa complexes formed on the cell surface of fibroblasts in the presence and absence of plasma inhibitor, tissue factor pathway inhibitor (TFPI). FVIIa bound to TF on the cell surface was internalized and degraded without depleting the cell surface TF antigen and activity. TFPI significantly enhanced the TF-specific internalization and degradation of FVIIa. TFPI-enhanced internalization and degradation of FVIIa requires the C-terminal domain of TFPI and factor Xa. TFPI. Xa-mediated internalization of FVIIa was associated with the depletion of TF from the cell surface. A majority of the internalized FVIIa was degraded, but a small portion of the internalized FVIIa recycles back to the cell surface as an intact protein. In addition to TF, other cell surface components, such as low density lipoprotein receptor-related protein (LRP) and heparan sulfates, are essential for TFPI.Xa-induced internalization of FVIIa. Acidification of cytosol, which selectively inhibits the endocytotic pathway via coated pits, inhibited TFPI.Xa-mediated internalization but not the basal internalization of FVIIa. Overall, our data support the concept that FVIIa bound to cell surface TF was endocytosed by two different pathways. FVIIa complexed with TF in the absence of the inhibitor was internalized via a LRP-independent and probably noncoated pit pathway, whereas FVIIa complexed with TF along with the inhibitor was internalized via LRP-dependent coated pit pathway.     

Estrogen modulates ghrelin expression in the female rat stomach

Ghrelin was recently identified as an endogenous ligand for GH secretagogue receptor. In this study, we investigated the effects of ovariectomy on the numbers of ghrelin-immunopositive and -expressing cells, ghrelin mRNA levels, and plasma ghrelin concentrations in 4- and 9-week-old female rats. Three days after ovariectomy, the number of ghrelin cells and plasma ghrelin level significantly increased in both 4- and 9-week-old rats and the ghrelin mRNA level also increased in 4-week-old rats. These responses were reversed by 17beta-estradiol replacement. We also found that ghrelin-immunopositive cells express estrogen receptor alpha. These results suggested that estrogen is involved in the regulation of ghrelin expression.     

Differential expression of tissue factor and tissue factor pathway inhibitor in metastatic melanoma lesions

Tissue factor (TF) is involved in tumor progression and metastatic potency in some malignant tumors and its function is regulated by tissue factor pathway inhibitor (TFPI) therefore the interaction of both molecules is crucial for their functional role. We evaluated the clinical relevance of TF and TFPI expression in benign and malignant melanocytic lesions. Expression of both was examined by immunoperoxidase staining using serial tissue sections in 16 nevi, 34 primary and 15 metastatic melanoma lesions. TF and TFPI were ubiquitously expressed in benign and malignant melanocytic lesions. This finding was confirmed by Western blot analysis using cultured human melanocytes, nevi cells (NCN) and melanoma cell lines. Although TF expression was not associated with malignant transformation and disease progression, TFPI expression in primary and metastatic melanoma lesions was significantly lower and weaker than that in nevi lesions in terms of intensity and percentage of stained cells. In addition, TFPI expression in metastatic lesions was significantly lower and weaker than that of TF. These results suggest that the relative expression of TF to TFPI may play a crucial role in the malignant transformation and metastatic potency in melanocytic cells.     

Progesterone utilizes distinct membrane pools of tissue factor to increase coagulation and invasion and these effects are inhibited by TFPI

Tissue factor (TF) serving as the receptor for coagulation factor VII (FVII) initiates the extrinsic coagulation pathway. We previously demonstrated that progesterone increases TF, coagulation and invasion in breast cancer cell lines. Herein, we investigated if tissue factor pathway inhibitor (TFPI) could down-regulate progesterone-increased TF activity in these cells. Classically, TFPI redistributes TF-FVII-FX-TFPI in an inactive quaternary complex to membrane associated lipid raft regions. Herein, we demonstrate that TF increased by progesterone is localized to the heavy membrane fraction, despite progesterone-increased coagulation originating almost exclusively from lipid raft domains, where TF levels are extremely low. The progesterone increase in coagulation is not a rapid effect, but is progesterone receptor (PR) dependent and requires protein synthesis. Although a partial relocalization of TF occurs, TFPI does not require the redistribution to lipid rafts to inhibit coagulation or invasion. Inhibition by TFPI and anti-TF antibodies in lipid raft membrane fractions confirmed the dependence on TF for progesterone-mediated coagulation. Through the use of pathway inhibitors, we further demonstrate that the TF up-regulated by progesterone is not coupled to the progesterone increase in TF-mediated coagulation. However, the progesterone up-regulated TF protein may be involved in progesterone-mediated breast cancer cell invasion, which TFPI also inhibits.     

Caveolae optimize tissue factor-Factor VIIa inhibitory activity of cell-surface-associated tissue factor pathway inhibitor

TFPI (tissue factor pathway inhibitor) is an anticoagulant protein that prevents intravascular coagulation through inhibition of fXa (Factor Xa) and the TF (tissue factor)-fVIIa (Factor VIIa) complex. Localization of TFPI within caveolae enhances its anticoagulant activity. To define further how caveolae contribute to TFPI anticoagulant activity, CHO (Chinese-hamster ovary) cells were co-transfected with TF and membrane-associated TFPI targeted to either caveolae [TFPI-GPI (TFPI-glycosylphosphatidylinositol anchor chimaera)] or to bulk plasma membrane [TFPI-TM (TFPI-transmembrane anchor chimaera)]. Stable clones had equal expression of surface TF and TFPI. TX-114 cellular lysis confirmed localization of TFPI-GPI to detergent-insoluble membrane fractions, whereas TFPI-TM localized to the aqueous phase. TFPI-GPI and TFPI-TM were equally effective direct inhibitors of fXa in amidolytic assays. However, TFPI-GPI was a significantly better inhibitor of TF-fVIIa than TFPI-TM, as measured in both amidolytic and plasma-clotting assays. Disrupting caveolae by removing membrane cholesterol from EA.hy926 cells, which make TFPIα, CHO cells transfected with TFPIβ and HUVECs (human umbilical vein endothelial cells) did not affect their fXa inhibition, but significantly decreased their inhibition of TF-fVIIa. These studies confirm and quantify the enhanced anticoagulant activity of TFPI localized within caveolae, demonstrate that caveolae enhance the inhibitory activity of both TFPI isoforms and define the effect of caveolae as specifically enhancing the anti-TF activity of TFPI.     

Inhibition of tissue factor-factor VIIa-catalyzed factor X activation by factor Xa-tissue factor pathway inhibitor. A rotating disc study on the effect of phospholipid membrane composition.

The physiological inhibitor of tissue factor (TF).factor VIIa (FVIIa), full-length tissue factor pathway inhibitor (TFPI(FL)) in complex with factor Xa (FXa), has a high affinity for anionic phospholipid membranes. The role of anionic phospholipids in the inhibition of TF.FVIIa-catalyzed FX activation was investigated. FXa generation at a rotating disc coated with TF embedded in a membrane composed of pure phosphatidylcholine (TF.PC) or 25% phosphatidylserine and 75% phosphatidylcholine (TF.PSPC) was measured in the presence of preformed complexes of FXa.TFPI(FL) or FXa.TFPI(1-161) (TFPI lacking the third Kunitz domain and C terminus). At TF.PC, FXa.TFPI(FL) and FXa.TFPI(1-161) showed similar rate constants of inhibition (0.07 x 10(8) M(-1) s(-1) and 0.1 x 10(8) M(-1) s(-1), respectively). With phosphatidylserine present, the rate constant of inhibition for FXa.TFPI(FL) increased 3-fold compared with a 9-fold increase in the rate constant for FXa. TFPI(1-161). Incubation of TF.PSPC with FXa.TFPI(FL) in the absence of FVIIa followed by depletion of solution FXa.TFPI(FL) showed that FXa.TFPI(FL) remained bound at the membrane and pursued its inhibitory activity. This was not observed with FXa.TFPI(1-161) or at TF.PC membranes. These data suggest that the membrane-bound pool of FXa.TFPI(FL) may be of physiological importance in an on-site regulation of TF.FVIIa activity.     

Platelet-associated tissue factor contributes to the collagen-triggered activation of blood coagulation

The extravascular localization of tissue factor (TF), the central initiator of coagulation, is thought to ensure that thrombus formation is prevented in the intact vessel. We observed that during a 5-min stimulation of human blood with collagen (type I), TF antigen appeared on the surface of platelets adhering to leukocytes. The rapidly presented intravascular TF was competent to start the coagulation cascade. The isolated platelets from healthy donors contained appreciable amounts of the TF protein, while no TF antigen was detected in the neutrophils and rapidly isolated monocytes. Direct interactions with the neutrophils and monocytes were apparently necessary to activate the platelet-associated TF. This was most likely mediated by inactivation of tissue factor pathway inhibitor through leukocyte elastase. In summary, the leukocyte-elicited activation of the platelet TF participates in the rapid initiation of coagulation by collagen.     

Selective estrogen receptor modulators protect hippocampal neurons from kainic acid excitotoxicity: differences with the effect of estradiol

Neuroprotective effects of estradiol are well characterized in animal experimental models. However, in humans, the outcome of estrogen treatment for cognitive function and neurological diseases is very controversial. Selective estrogen receptor modulators (SERMs) may represent an alternative to estrogen for the treatment or the prevention of neurodegenerative disorders. SERMs interact with the estrogen receptors and have tissue-specific effects distinct from those of estradiol, acting as estrogen agonists in some tissues and as antagonists in others. In this study we have assessed the effect of tamoxifen, raloxifene, lasofoxifene (CP-336,156), bazedoxifene (TSE-424), and 17beta-estradiol on the hippocampus of adult ovariectomized rats, after the administration of the excitotoxin kainic acid. Administration of kainic acid induced the expression of vimentin in reactive astroglia and a significant neuronal loss in the hilus. SERMs did not affect vimentin immunoreactivity in the hilus, while 17beta-estradiol significantly reduced the surface density of vimentin immunoreactive profiles. Estradiol, tamoxifen (0.4-2 mg/kg), raloxifene (0.4-2 mg/kg), and bazedoxifene (2 mg/kg) prevented neuronal loss in the hilus after the administration of kainic acid. Lasofoxifene (0.4-2 mg/kg) was not neuroprotective. These findings indicate that SERMs present different dose-dependent neuroprotective effects. Furthermore, the mechanisms of neuroprotection by SERMs and estradiol are not identical, because SERMs do not significantly affect reactive gliosis while neuroprotection by estradiol is associated with a strong down-regulation of reactive astroglia.     

Matrix metalloproteinases cleave tissue factor pathway inhibitor. Effects on coagulation

The capacity of inflammatory cell-derived matrix metalloproteinases (MMPs) to cleave tissue factor pathway inhibitor (TFPI) and alter its activity was investigated. MMP-7 (matrilysin) rapidly cleaved TFPI to a major 35-kDa product. In contrast, MMP-1 (collagenase-1), MMP-9 (gelatinase B), and MMP-12 (macrophage elastase) cleaved TFPI into several fragments including the 35-kDa band. However, rates of cleavage were most rapid for MMP-7 and MMP-9. NH(2)-terminal amino acid sequencing revealed that MMP-12 cleaved TFPI at Lys(20)-Leu(21)(close to Kunitz I domain and producing a 35-kDa band), Arg(83)-Ile(84) (between Kunitz I and II domains), and Ser(174)-Thr(175) (between Kunitz II and III domains). MMP-7 and MMP-9 cleaved TFPI at Lys(20)-Leu(21) with additional COOH-terminal processing. These MMPs did not cleave tissue factor (TF), factor VII, and factor Xa. Proteolytic cleavage by MMP-1, MMP-7, MMP-9, and MMP-12 resulted in considerable loss of TFPI activity. These observations indicate specific cleavage of TFPI by MMPs, which broadens their substrate profile. Co-localization of MMPs, TF, and TFPI in atherosclerotic tissues suggests that release of MMPs from inflammatory cell leukocytes may effect TF-mediated coagulation.     

Ovariectomy upregulates expression of estrogen receptors,NOS, and HSPs in porcine platelets

Platelets participate in normal and pathological thrombotic processes. Hormone replacement in postmenopausal women is associated with increase risk for thrombosis. However, little is known regarding how platelets are affected by hormonal status. Nitric oxide (NO) modulates platelet functions and is modulated by hormones. Therefore, the present study was designed to determine how loss of ovarian hormones changes expression of estrogen receptors and regulatory proteins for NO synthase (NOS) in platelets. Estrogen receptors (ER alpha and ER beta), NOS, heat shock proteins 70 and 90 (HSP70 and HSP90), caveolin-1, -2, and -3, calmodulin, NOS activity, and cGMP were analyzed in a lysate of platelets from gonadally intact and ovariectomized female pigs. Expression of ER beta and ER alpha receptors, endothelial NOS (eNOS), HSP70, and HSP90 increased with ovariectomy. NOS activity and cGMP also increased; calmodulin was unchanged. Caveolins were not detected. These results suggest that ovarian hormones influence expression of estrogen receptors and eNOS in platelets. Changes in estrogen receptors and NOS could affect platelet aggregation in response to hormone replacement.     

Estrogen,inflammation, and platelet phenotype

Background: Although exogenous estrogenic therapies increase the risk of thrombosis, the effects of estrogen on formed elements of blood are uncertain.Objective: This article examines the genomic and nongenomic actions of estrogen on platelet phenotype that may contribute to increased thrombotic risk.Methods: To determine aggregation, secretion, protein expression, and thrombin generation, platelets were collected from experimental animals of varying hormonal status and from women enrolled in the Kronos Early Estrogen Prevention Study.Results: Estrogen receptor β predominates in circulating platelets. Estrogenic treatment in ovariectomized animals decreased platelet aggregation and adenosine triphosphate (ATP) secretion. However, acute exposure to 17β-estradiol did not reverse decreases in platelet ATP secretion invoked by lipopolysaccharide. Thrombin generation was positively correlated to the number of circulating microvesicles expressing phosphatidylserine.Conclusion: Assessing the effect of estrogen treatments on blood platelets may lead to new ways of identifying women at risk for adverse thrombotic events with such therapies.     

Additional effects of postpuberal estrogen injections on the vaginal epithelium in neonatally estrogenized mice

In the ovariectomized mice given 10 injections of 100 micrograms 17 beta-estradiol at intervals of 2 weeks from 60 days of age, the vaginal epithelium was atrophic when killed more than 2 months after the last injection. If mice given 3 daily injections of 20 micrograms 17 beta-estradiol from the day of birth were similarly treated with estradiol after postpuberal ovariectomy, the vaginal epithelium was stratified and hyperplastic at autopsy performed more than 2 months later. These changes in the epithelium persisted for at least 30 days after transplantation of the vaginae to normal ovariectomized hosts. Neonatal treatments only did not produce such persistent vaginal changes. In view of these results, additional effects of neonatal and postpuberal injections of estrogen on the vaginal epithelium are evident. However, effects of such neonatal and postpuberal injections of estrogen might be transient on the uterine epithelium, since abnormal proliferation was not observed in it.     

Regulation of leptin by steroid hormones in rat adipose tissue.

We investigated if steroid hormones regulate the secretion and the expression of leptin in female and male rat adipose tissue fragments in vitro. Dexamethasone time and dose-dependently increased the secretion and mRNA expression of leptin with a half-maximal stimulation of approximately 1 nM. A time-course revealed a maximal stimulatory effect of 17 beta-estradiol after 24 hours. In male adipose tissue 17 beta-estradiol increased leptin secretion (32% by 50 nM 17 beta-estradiol, P = 0.07 and 34% by 500 nM 17 beta-estradiol, P < 1780.05) after 24 hours. An additional effect of estrogen was seen in the dexamethasone (50 nM) stimulated cells (38% with 50 nM 17 beta-estradiol, P < 0.05 and 48% by 500 nM 17 beta-estradiol, P < 0.05). Basal secretion of leptin was equal in female and male adipose tissue, whereas the effects of 17 beta-estradiol (50 nM) and dexamethasone were significantly increased in female as compared with male adipose tissue. Progesterone, testosterone, dihydrotestosterone and dehydroepiandrostendione-sulfate neither affected leptin secretion in male nor female adipose tissue in vitro. Furthermore, to investigate the effect of estrogen female rats were ovariectomized (OVX) and the adipose tissue was incubated in vitro and compared with adipose tissue leptin secretion from sham operated rats (SHAM), and with ovariectomized rats treated with 17 beta-estradiol (EST). A decreased basal and dexamethasone-stimulated leptin secretion from OVX rats compared with SHAM rats was found (P < 0.005) whereas 17 beta-estradiol treatment of ovariectomized rats maintained a normal leptin secretion. However, the dexamethasone stimulation was equally increased above basal levels in SHAM, OVX and EST rats (3.7 +/- 1.2, 2.9 +/- 0.8, 4.2 +/- 1.4, NS, ANOVA) respectively.