Marinella Rosselli

Circulating Nitric Oxide (Nitrite/Nitrate) Levels in Postmenopausal Women Substituted With 17β-Estradiol and Norethisterone Acetate A Two-Year Follow-up Study

Postmenopausal women (PMW) have an increased risk of cardiovascular disease that is attenuated by hormone replacement therapy (HRT). Inasmuch as hypertension and atherosclerosis are associated with diminished endothelium-derived nitric oxide (NO), we investigated whether HRT augments NO release in PMW. We determined serum levels of nitrite/nitrate (NO2 + NO3) at baseline and during the 6th, 12th, and 24th months of the study in two groups of PMW. One group (HRT-PMW, n = 13) received continuous transdermal administration of 17 beta-estradiol (Estraderm-TTS-50) supplemented with oral norethisterone acetate (NETA) on days 1 through 12 of each month, and the other group (control PMW, n = 13) did not receive HRT. Blood samples in the HRT-PMW group were collected without regard to whether subjects were taking NETA at the time of blood sampling. Serum NO2 + NO3 levels increased in HRT-PMW for the duration of the study, whereas serum NO2 + NO3 levels remained unchanged in control PMW. When all samples regardless of timing of collection with respect to NETA treatment were included in the statistical analysis, the change in NO2 + NO3 levels in HRT-PMW was significantly greater compared with the change in control PMW (P = .037). Likewise, when only those samples collected when estradiol-treated subjects were not taking oral NETA were included in the statistical analysis, the change in NO2 + NO3 levels in the HRT-PMW group remained significant (P = .047) compared with control PMW.(ABSTRACT TRUNCATED AT 250 WORDS)

Phytoestrogens Inhibit Growth and MAP Kinase Activity in Human Aortic Smooth Muscle Cells

-Estrogens are known to induce cardioprotective effects by inhibiting smooth muscle cell (SMC) growth and neointima formation. However, the use of estrogens as cardioprotective agents is limited by carcinogenic effects in women and feminizing effects in men. If noncarcinogenic and nonfeminizing estrogenlike compounds, such as natural phytoestrogens, afford cardioprotection, this would provide a safe method for prevention of cardiovascular disease in both men and women. Therefore, we evaluated and compared in human aortic SMCs the effects of phytoestrogens (formononetin, genistein, biochanin A, daidzein, and equol) on 2.5% fetal calf serum-induced proliferation (3H-thymidine incorporation and cell number), collagen synthesis (3H-proline incorporation), and total protein synthesis (3H-leucine incorporation) and on PDGF-BB (25 ng/mL)-induced migration (modified Boydens chambers). Moreover, the effects of phytoestrogens on PDGF-BB (25 ng/mL)-induced mitogen-activated protein kinase (MAP kinase) activity in SMCs was also studied. Phytoestrogens inhibited proliferation, collagen and total protein synthesis, migration, and MAP kinase activity in a concentration-dependent manner and in the following order of potency: biochanin A>genistein>equol>daidzein>formononetin. In conclusion, our studies provide the first evidence that in human aortic SMCs phytoestrogens inhibit mitogen-induced proliferation, migration and extracellular matrix synthesis and inhibit/downregulate MAP kinase activity. Thus, phytoestrogens may confer protective effects on the cardiovascular system by inhibiting vascular remodeling and neointima formation and may be clinically useful as a safer substitute for feminizing estrogens in preventing cardiovascular disease in both women and men.

Impairment of the developmental potential of frozen-thawed human zygotes obtained after intracytoplasmic sperm injection

OBJECTIVE To evaluate the effects of cryopreservation on the survival, cleavage, and morphology of embryos and on the implantation and embryonic loss rates of human zygotes obtained after ICSI compared with frozen-thawed zygotes obtained after traditional IVF. A further objective was to evaluate the same parameters in nonfrozen sibling ICSI and IVF zygotes and to compare them with corresponding frozen-thawed zygotes. DESIGN Open, retrospective, comparative study. SETTING University-associated assisted reproductive program. PATIENT(S) Couples with severe male factor infertility and couples undergoing IVF during the same period. INTERVENTION(S) A cohort of 408 ICSI zygotes and 299 IVF zygotes was frozen in 1,2 propanediol and sucrose using a slow-freezing protocol. Both groups of zygotes were frozen at approximately the same time after microassisted or conventional insemination. One hundred and eighty-seven ICSI and 110 IVF frozen zygotes were rapidly thawed during 44 ICSI cycles and 24 IVF cycles. Zygotes that appeared to have survived were cultured for 24 hours, and most of these embryos that were morphologically normal were transferred into patients. MAIN OUTCOME MEASURE(S) Survival rate (morphologically intact after thawing), cleavage rate and morphology of embryos, implantation rate, and the incidence of embryonic losses. RESULT(S) Except for survival rates, for which both ICSI and IVF frozen-thawed zygotes showed similar and relatively high values (87.7% and 89.1%), the outcomes of other parameters evaluated were significantly different. Thus, from a total of 128 ICSI and 68 IVF embryos transferred, 14 (10.9%) and 17 (25.0%) implanted in 44 ICSI and 24 IVF frozen-thawed cycles, respectively. This difference in implantation corresponded with the rate of cleavage and morphology of the replaced embryos; the embryos that developed from frozen-thawed IVF zygotes cleaved faster and were more regular compared to the frozen-thawed ICSI zygotes. The embryonic loss rate was 57.1% for cryopreserved ICSI zygotes and 11.8% for IVF zygotes. On the other hand, no difference in cleavage pattern, embryo morphology, implantation, and embryonic loss rates was found between nonfrozen sibling ICSI and IVF zygotes. CONCLUSION(S) The zygotes arising from ICSI cycles survived cryopreservation at a rate similar to IVF zygotes, but their ability to implant and develop further was probably affected by the cryopreservation procedure. The timing of zygote freezing was considered to be the principal reason for the lower developmental potential of frozen-thawed ICSI zygotes in the present study.

Candidate Genes and Mechanisms for 2-Methoxyestradiol–Mediated Vasoprotection

2-Methoxyestradiol (2-ME; estradiol metabolite) inhibits vascular smooth muscle cell (VSMC) growth and protects against atherosclerosis and vascular injury; however, the mechanisms by which 2-ME induces these actions remain obscure. To assess the impact of 2-ME on biochemical pathways regulating VSMC biology, we used high-density oligonucleotide microarrays to identify differentially expressed genes in cultured human female aortic VSMCs treated with 2-ME acutely (4 hours) or long term (30 hours). Both single gene analysis and Gene Set Enrichment Analysis revealed 2-ME–induced downregulation of genes involved in mitotic spindle assembly and function in VSMCs. Also, Gene Set Enrichment Analysis identified effects of 2-ME on genes regulating cell-cycle progression, cell migration/adhesion, vasorelaxation, inflammation, and cholesterol metabolism. Transcriptional changes were associated with changes in protein expression, including inhibition of cyclin D1, cyclin B1, cyclin-dependent kinase 6, cyclin-dependent kinase 4, tubulin polymerization, cholesterol and steroid synthesis, and upregulation of cyclooxygenase 2 and matrix metalloproteinase 1. Microarray data suggested that 2-ME may activate peroxisome proliferator-activated receptors (PPARs) in VSMCs, and 2-ME has structural similarities with rosiglitazone (PPAR&ggr; agonist). However, our finding of weak activation and lack of binding of 2-ME to PPARs suggests that 2-ME may modulate PPAR-associated genes via indirect mechanisms, potentially involving cyclooxygenase 2. Indeed, the antimitogenic effects of 2-ME at concentrations that do not inhibit tubulin polymerization were blocked by the PPAR antagonist GW9662 and the cyclooxygenase 2 inhibitor NS398. Finally, we demonstrated that 2-ME inhibited hypoxia-inducible factor 1&agr;. Identification of candidate genes that are positively or negatively regulated by 2-ME provides important leads to investigate and better understand the mechanisms by which 2-ME induces its vasoprotective actions.

Oviduct cells express the cyclic AMP-adenosine pathway.

Abstract The extracellular cAMP-adenosine pathway refers to the local production of adenosine mediated by cAMP egress into the extracellular space, conversion of cAMP to AMP by ectophosphodiesterase (PDE), and the metabolism of AMP to adenosine by ecto-5′-nucleotidase. The goal of this study was to assess whether the cAMP-adenosine pathway is expressed in oviduct cells. Studies were conducted in cultured bovine oviduct cells (mixed cultures of fibroblasts and epithelial cells, 1:1 ratio). Confluent monolayers of oviduct cells were exposed to cAMP (0.01–100 μmol/L) in the presence and absence of 3-isobutyl-1-methylxanthine (IBMX, 1 mmol/L, an inhibitor of both extracellular and intracellular PDE activity), 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX, 100 μmol/L, a xanthine that can inhibit extracellular or ecto-PDE activity at high concentrations), or α,β-methylene-adenosine-5′-diphosphate (AMPCP, 100 μmol/L, an ecto-5′-nucleotidase inhibitor) for 0–60 min. The medium was then sampled and assayed for AMP, adenosine, and inosine. Addition of exogenous cAMP to oviduct cells increased extracellular levels of AMP, adenosine, and inosine in a concentration- and time-dependent manner. This effect was attenuated by blockade of total (extracellular and intracellular) PDE activity (IBMX), ecto-PDE activity (DPSPX), or ecto-5′-nucleotidase (AMPCP). The functional relevance of the cAMP-adenosine pathway is supported by the findings that treatment with adenylyl cyclase stimulants (forskolin plus isoproterenol) resulted in the egress of cAMP (97% extracellular) into the extracellular space and its conversion into adenosine. The extracellular cAMP-adenosine pathway exists in oviduct cells and may play an important role in regulating the biology and physiology of the oviduct. This pathway also may play a critical role in regulating sperm function, fertilization, and early embryo development.

Extracellular 3′,5′-cAMP-Adenosine Pathway Inhibits Glomerular Mesangial Cell Growth

Abnormal growth of glomerular mesangial cells (GMCs) contributes to the pathophysiology of many types of nephropathy. Because adenosine is an autocrine/paracrine factor that potentially could regulate GMC proliferation and because the extracellular 3′,5′-cAMP-adenosine pathway (i.e., the conversion of extracellular 3′,5′-cAMP to 5′-AMP and adenosine on the cell surface) could generate adenosine in the biophase of GMC receptors, we investigated the role of the 3′,5′-cAMP-adenosine pathway in modulating growth [cell proliferation, DNA synthesis ([3H]thymidine incorporation), collagen synthesis ([3H]proline incorporation), and mitogen-activated protein kinase activity] of GMCs. The addition of exogenous 3′,5′-cAMP to human GMCs increased extracellular levels of 5′-AMP, adenosine, and inosine, and 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor), 1,3-dipropyl-8-p-sulfophenylxanthine (ecto-phosphodiesterase inhibitor), and α,β-methylene-adenosine-5′-diphosphate (ecto-5′-nucleotidase inhibitor) attenuated the increases in adenosine and inosine. Forskolin augmented extracellular 3′,5′-cAMP and adenosine concentrations, and 2′,5′-dideoxyadenosine (adenylyl cyclase inhibitor) blocked these increases. Exogenous 3′,5′-cAMP and forskolin inhibited all indices of cell growth, and antagonism of A2 [(E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine, KF17837] or A1/A2 (1,3-dipropyl-8-p-sulfophenylxanthine, DPSPX), but not A1 (8-cyclopentyl-1,3-dipropylxanthine), or A3{N-(2-methoxyphenyl)-N′-[2-(3-pyridinyl)-4-quinazolinyl]-urea, VUF5574}, adenosine receptors blocked the growth-inhibitory actions of exogenous 3′,5′-cAMP, but not the effects of 8-bromo-3′,5′-cAMP (stable 3′,5′-cAMP analog). Erythro-9-(2-hydroxy-3-nonyl)adenine (adenosine deaminase inhibitor) plus 5-iodotubercidin (adenosine kinase inhibitor) enhanced the growth inhibition by exogenous 3′,5′-cAMP and forskolin, and A2 receptor antagonism blocked this effect. In rat GMCs, down-regulation of A2B receptors with antisense, but not sense or scrambled, oligonucleotides abrogated the inhibitory effects of 3′,5′-cAMP and forskolin on cell growth. The extracellular 3′,5′-cAMP-adenosine pathway exists in GMCs and attenuates cell growth via A2B receptors. Pharmacological augmentation of this pathway could abate pathological glomerular remodeling.

Serum concentrations of high–molecular weight adiponectin and their association with sex steroids in premenopausal women

At present, the association between adiponectin and sex hormones in women is controversial. Recent studies suggest that it is high-molecular weight (HMW) adiponectin and the HMW to total adiponectin ratio rather than total adiponectin that are associated with antiatherogenic activities, insulin sensitivity, metabolic syndrome, and prediction of cardiovascular events. The present study aimed to investigate whether measuring HMW adiponectin and the HMW to total adiponectin ratio rather than total adiponectin might be more useful to detect an association between circulating female sex steroids and adipocytokines. In a clinical trial, we investigated the associations of total adiponectin, HMW adiponectin, and the HMW to adiponectin ratio with several androgens and estradiol in 36 healthy premenopausal women with regular cycles. No association between the investigated sex hormones and adiponectin was observed. The HMW adiponectin was negatively correlated with estradiol after adjustment for age and body mass index. The HMW to total adiponectin ratio was significantly negatively associated with testosterone, free testosterone, and androstenedione. The testosterone to estradiol ratio, as a parameter for the estrogen-androgen balance, was not associated with adiponectin or the HMW isoform. In conclusion, there is a negative association between estradiol and HMW adiponectin, and between testosterone, free testosterone, and androstenedione and the HMW to adiponectin ratio. Thus, one mechanism whereby female sex steroids may influence the cardiovascular risk of women could be alteration of the relationship between HMW and total adiponectin concentrations in plasma.

Differential effects of natural and environmental estrogens on endothelin synthesis in bovine oviduct cells

Abstract Endothelin-1 (ET-1), a vasoconstrictor and mitogenic peptide that plays an important role within the endocrine/reproductive system, is synthesized by oviduct cells and regulates tubal contractility. Because 17β-estradiol (estradiol) regulates oviduct function by influencing the synthesis of autocrine/paracrine factors, estradiol may also regulate ET-1 synthesis. Furthermore, environmental estrogens (EEs; phytoestrogens and xenoestrogens), which structurally resemble estradiol and possess estrogenic activity, may mimic the effects of estradiol on ET-1 synthesis and may influence the reproductive system. Using cultures of bovine oviduct cells (epithelial cells:fibroblasts, 1:1), we investigated and compared the modulatory effects of estradiol, phytoestrogens, and xenoestrogens on ET-1 synthesis and determined whether these effects were estrogen receptor (ER) mediated. A quantitative ELISA for ET-1 in the culture medium revealed that 17β-estradiol inhibits ET-1 synthesis in a concentration-dependent manner (4–400 nmol/L). In contrast to estradiol, ET-1 synthesis was induced in cell cultures treated with xenoestrogens in the following order of potency (0.1 μmol/L): 4-hydroxy-trichlorobiphenyl > 4-hydroxy-dichlorobiphenyl > trichlorobiphenyl. The stimulatory effects of xenoestrogens on ET-1 production were mimicked by the phytoestrogens biochanin-A and genistein but not by formononetin, equol, and daidzein. The oviduct cells expressed both ERs (α and β), but the modulatory effects of estradiol, but not EEs, on ET-1 synthesis were blocked by ICI-182 780 (1 μM), a pure ER antagonist. Our results provide evidence that estradiol inhibits ET-1 synthesis in oviduct cells via an ER-dependent mechanism, whereas, EEs induce ET-1 synthesis via an ER-independent mechanism. The contrasting effects of EEs on ET-1 synthesis suggests that EEs may act as endocrine modulators/disruptors and may have deleterious effects on the reproductive system by adversely influencing the biology and physiology of the oviduct.

Estradiol Stimulates Capillary Formation by Human Endothelial Progenitor Cells: Role of Estrogen Receptor-α/β, Heme Oxygenase 1, and Tyrosine Kinase

Endothelial progenitor cells (EPCs) repair damaged endothelium and promote capillary formation, processes involving receptor tyrosine kinases (RTKs) and heme oxygenase 1 (HO-1). Because estradiol augments vascular repair, we hypothesize that estradiol increases EPC proliferation and capillary formation via RTK activation and induction of HO-1. Physiological concentrations of estradiol (10 nmol/L) increased EPC-induced capillary sprout and lumen formation in matrigel/fibrin/collagen systems. Propyl-pyrazole-triol (PPT; 100 nmol/L; estrogen receptor [ER]-&agr; agonist), but not diarylpropionitrile (ER-&bgr; agonist), mimicked the stimulatory effects of estradiol on capillary formation, and methyl-piperidino-pyrazole (ER-&agr; antagonist) abolished the effects of estradiol and PPT. Three different RTK activators (vascular endothelial growth factor, hepatocyte growth factor, and stromal derived growth factor 1) mimicked the capillary-stimulating effects of estradiol and PPT. SU5416 (RTK inhibitor) blocked the stimulatory effects of estradiol and PPT on capillary formation. Estradiol increased HO-1 expression by 2- to 3-fold, an effect blocked by SU5416, and PPT mimicked the effects of estradiol on HO-1. The ability of estradiol to enhance capillary formation, increase expression of HO-1, and augment phosphorylation of extracellular signal–regulated kinase 1/2, Akt, and vascular endothelial growth factor receptor 2 was mimicked by its cell-impermeable analog BSA estradiol. Actinomycin (transcription inhibitor) did not alter the effects of estradiol on RTK activity or vascular endothelial growth factor secretion. We conclude that estradiol via ER-&agr; promotes EPC-mediated capillary formation by a mechanism that involves nongenomic activation of RTKs and HO-1 activation. Estradiol in particular and ER-&agr; agonists in general may promote healing of injured vascular beds by promoting EPC activity leading to more rapid endothelial recovery and capillary formation after injury.

Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D

The goal of this study was to determine whether and how adenosine affects the proliferation of human coronary artery smooth muscle cells (HCASMCs). In HCASMCs, 2-chloroadenosine (stable adenosine analogue), but not N6-cyclopentyladenosine, CGS21680, or N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide, inhibited HCASMC proliferation (A2B receptor profile). 2-Chloroadenosine increased cAMP, reduced phosphorylation (activation) of ERK and Akt (protein kinases known to increase cyclin D expression and activity, respectively), and reduced levels of cyclin D1 (cyclin that promotes cell-cycle progression in G1). Moreover, 2-chloroadenosine inhibited expression of S-phase kinase–associated protein-2 (Skp2; promotes proteolysis of p27Kip1) and upregulated levels of p27Kip1 (cell-cycle regulator that impairs cyclin D function). 2-Chloroadenosine also inhibited signaling downstream of cyclin D, including hyperphosphorylation of retinoblastoma protein and expression of cyclin A (S phase cyclin). Knockdown of A2B receptors prevented the effects of 2-chloroadenosine on ERK1/2, Akt, Skp2, p27Kip1, cyclin D1, cyclin A, and proliferation. Likewise, inhibition of adenylyl cyclase and protein kinase A abrogated 2-chloroadenosine’s inhibitory effects on Skp2 and stimulatory effects on p27Kip1 and rescued HCASMCs from 2-chloroadenosine–mediated inhibition. Knockdown of p27Kip1 also reversed the inhibitory effects of 2-chloroadenosine on HCASMC proliferation. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 &mgr;mol/L for 7 days) downregulated vascular expression of Skp2, upregulated vascular expression of p27Kip1, and reduced neointima hyperplasia by 71% (P<0.05; neointimal thickness: control, 37 424±18 371 pixels; treated, 10 352±2824 pixels). In conclusion, the adenosine/A2B receptor/cAMP/protein kinase A axis inhibits HCASMC proliferation by blocking multiple signaling pathways (ERK1/2, Akt, and Skp2) that converge at cyclin D, a key G1 cyclin that controls cell-cycle progression.