Michele Bonaiuto

Effects of the Phytoestrogen Genistein on Bone Metabolism in Osteopenic Postmenopausal Women: A Randomized Trial

Context Women seeking alternative treatments to preserve bone often use phytoestrogens, but evidence of their effectiveness is lacking. Phytoestrogens are found in soy products. Genistein is a phytoestrogen with a structure similar to that of 17-estradiol. Contribution This randomized trial compared genistein, 54 mg/d, with placebo for 24 months in 389 osteopenic postmenopausal women. Increases in bone mineral density were greater with genistein than with placebo. Genistein also had favorable effects on markers of bone metabolism. Genistein did not increase endometrial thickness, but it did cause gastrointestinal side effects. Implications Genistein appears to have a favorable effect on markers of bone health in osteopenic postmenopausal women. Studies of its effect on fractures are needed. The Editors Postmenopausal osteoporosis is caused by a sharp decrease in estrogen levels that leads to an increased rate of bone remodeling (13). Currently available treatments for postmenopausal osteoporosis include hormone replacement therapy; calcitonin; bisphosphonates; and selective estrogen receptor modulators, such as raloxifene (4, 5). Although hormone replacement therapy is effective in reducing postmenopausal bone loss (68), it is associated with a higher risk for breast, endometrial, and ovarian cancer; cardiovascular disease; venous thromboembolism; and stroke (810). Epidemiologic data indicate that women who ingest high amounts of phytoestrogens, particularly isoflavones in soy products, have less risk for osteoporosis than do those who consume a typical Western diet (1113). Consequently, many women use phytoestrogens to maintain bone density. Genistein, an isoflavone phytoestrogen that is abundant in soybean products, structurally resembles 17-estradiol (14). As a natural selective estrogen receptor modulator, genistein may positively regulate bone cell metabolism without harmful estrogenic activity in the breast and uterus. This safe profile results from the greater affinity of genistein for estrogen receptor-, which is more abundant in bone, than for estrogen receptor-, which is abundant in reproductive tissue. Observational studies suggest that postmenopausal Asian women who consume diets high in isoflavones have a lower rate of fracture than that in other groups (15, 16). However, the mechanism of action of genistein on bone is not yet fully understood. In postmenopausal women, treatment with genistein (54 mg/d) increased bone mineral density (BMD) at the lumbar spine and femoral neck with no clinically significant adverse effects on the breast and uterus (17). In the same cohort, genistein decreased the ratio of soluble receptor activator of nuclear factor-B ligand to osteoprotegerin, which may partly account for its positive effects on BMD (18). These investigations were the first to evaluate the effects of purified, standardized genistein on bone health, but they were short in duration and included only 90 patients. One published trial assessed the effect of isoflavones on BMD in postmenopausal women, but it compared isoflavone-rich soy milk (containing only a small amount of genistein) with natural transdermal progesterone (19). Other studies of pure genistein from our research group have focused on cardiovascular outcomes or menopausal symptoms rather than on bone health (2022). We performed a randomized, placebo-controlled trial of the effects of pure genistein on bone density and bone metabolism over 24 months in a larger cohort of osteopenic postmenopausal women. Methods Design and Setting The study protocol is consistent with the principles of the Declaration of Helsinki, and participants gave written informed consent. Participants were recruited from women reporting to the Center for Osteoporosis in the Department of Internal Medicine and the Center for Menopause in the Department of Obstetrical and Gynecological Sciences, University of Messina (Messina, Italy), and to the Department of Medical Physiopathology, University La Sapienza (Rome, Italy). Three hundred eighty-nine women met the inclusion criteria and agreed to participate (Figure 1). Figure 1. Study flow diagram. Participants Participants were women 49 to 67 years of age who had been postmenopausal for at least 12 months at baseline, were in good general health, had not had a menstrual period in the preceding year, had not undergone surgically induced menopause, and had a follicle-stimulating hormone level greater than 50 IU/L and a serum 17-estradiol level of 100 pmol/L or less (27 pg/mL). At the start of the study, a complete family history was obtained, physical examination and laboratory evaluation (chemical analytes and hematologic measurements) were performed, and BMD was measured at the lumbar spine and femoral neck. Exclusion criteria were clinical or laboratory evidence of confounding systemic diseases, such as cardiovascular, hepatic, or renal disorders; coagulopathy; use of oral or transdermal estrogen, progestin, androgens, selective estrogen receptor modulators, or other steroids; use of biphosphonates, cholesterol-lowering therapy, or cardiovascular medications (including antihypertensive drugs) in the preceding 6 months; smoking habit of more than 2 cigarettes daily; treatment in the preceding year with any drug that could have affected the skeleton; family history of estrogen-dependent cancer; and BMD at the femoral neck greater than 0.795 g/cm2 (which corresponds to a T-score of 1.0 SD). Randomization and Intervention We assigned patients to groups by using a computer-generated randomization sequence with a permuted block size of 4, stratified by center. After a 4-week stabilization period during which participants received a standard low-soy, reduced-fat diet, participants were assigned to receive genistein (n= 198), 54 mg/d in 2 tablets (Laboratori Plants, Messina, Italy), or placebo (n= 191) (Figure 1). The biological effects of phytoestrogen intake are described elsewhere (23). No patient withdrew from the study during the stabilization period. The purity of genistein was 98%. Placebo and genistein tablets were identical in appearance and taste. Both genistein and placebo tablets contained calcium carbonate (500 mg) and vitamin D (400 IU). All participants were counseled on an isocaloric, reduced-fat diet composed of 25% to 30% energy from fat, less than 10% energy from saturated fatty acids, 55% to 60% energy from carbohydrates, and 15% energy from protein, with a cholesterol intake less than 300 mg/d and fiber intake of 35 g/d or greater. Recommended daily caloric intake was based on body size and was calculated by using the HarrisBenedict equation. We used this diet during the stabilization period to ensure that all participants had the same energy intake and to avoid interference with the lipid profile. The intake of soy products, legumes, or other nutrient supplements was prohibited. The isoflavone intake before randomization, as assessed by using a food-frequency questionnaire, was 1 to 2 mg/d. This intake has been shown to be typical in Western populations (24). Participants used this diet throughout the study, and adherence was reinforced by a nutritionist. Diet and body mass index were evaluated in all participants during follow-up. Primary Outcome The BMD at the anteroposterior lumbar spine and femoral neck was measured by using dual-energy x-ray absorptiometry (Hologic QDR 4500 W, Technologic, Turin, Italy) at baseline and after 12 and 24 months of treatment. The instrument was calibrated daily according to the manufacturers instructions. Reproducibility was calculated as a coefficient of variation obtained by weekly measurements of a standard phantom on the instrument and by repeated measurements obtained in 3 patients of different ages. The coefficient of variation of our instrument is 0.5% with the standard phantom; in vivo, we calculated a coefficient of variation of 1.1% for the lumbar spine and 1.5% for the femoral neck. Secondary Outcomes Bone Resorption Markers At baseline, 12 months, and 24 months, a 2-hour fasting morning urine sample was collected at the same time of day to assess urinary excretion of pyridinium crosslinks (pyridinoline and deoxypyridinoline). Pyridinoline (normal range, 26 to 91 pmol/mol creatinine) and deoxypyridinoline (normal range, 3 to 21 pmol/mol creatinine) were measured by using high-performance liquid chromatography (Bio-Rad Laboratories, Hercules, California). Bone Formation and Bone Growth Markers and Other Variables After an overnight fast, venous blood samples were collected between 8 a.m. and 9 a.m. through a polyethylene catheter inserted in a forearm vein. The serum was separated from the blood corpuscles by centrifugation and kept frozen at 70C until analysis for bone formation and bone growth markers, calcium, intact parathyroid hormone, 25-hydroxyvitamin D3, 17-estradiol, follicle-stimulating hormone, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides. Serum bone-specific alkaline phosphatase (normal range, 8.5 to 17.9 g/L) and insulin-like growth factor I (normal range, 9.6 to 21.2 nmol/L) were measured by using an immunoenzymatic assay (Pantec, Turin, Italy). Serum calcium (normal range, 2.25 to 2.75 mmol/L [9 to 11 mg/dL]), serum phosphorus (normal range, 1.13 to 1.45 mmol/L [3.5 to 4.5 mg/dL]), and urinary creatinine (130 to 220 molkg1d1 [14.71 to 24.89 mg/kg of body weight per day]) were measured by using automated routine procedures. Parathyroid hormone (normal range, 12 to 100 pg/dL), 25-hydroxyvitamin D3 (normal range, 1.25 to 7.5 nmol/L), and follicle-stimulating hormone (normal range, 21 to 153 IU/L in the postmenopausal phase) were measured by using high-performance liquid chromatography (Bio-Rad Laboratories). 17-Estradiol (normal range, 37 to 110 pmol/L in the postmenopausal phase) was evaluated by using a solid-phase immunoassay (Roche Diagnostics, Monza, Italy). Total cholesterol,

Simvastatin enhances VEGF production and ameliorates impaired wound healing in experimental diabetes

Statins have different effects beyond cholesterol reduction and stimulate angiogenesis. We investigated the effect of simvastatin in diabetes-related healing defects. An incisional skin wound model produced on the back of female diabetic mice (db(+)/db(+)) and their normoglycemic littermates (db(+)/(+)m) was used. Animals were treated daily either with simvastatin (5 mg/(kgi.p.)) or vehicle. Mice were killed on different days (3, 6 and 12 after skin injury) for measurement of vascular endothelial growth factor (VEGF) mRNA and protein expression, to assess histologically the healing process and to evaluate wound breaking strength and angiogenesis by CD31 immunostaining. Simvastatin administration in diabetic mice increased VEGF mRNA (simvastatin=4.8+/-0.6n-fold/beta-actin; vehicle=2.3+/-0.4n-fold/beta-actin) and protein expression (simvastatin=5+/-0.7 integrated intensity; vehicle=2.2+/-0.3 integrated intensity) and enhanced nitric oxide wound content at day 6. Additionally, the statin augmented breaking strength and PECAM-1 immunostaining at day 12. Finally, simvastatin administration restored the impaired wound healing process in diabetic mice. Similar results were obtained in normoglycaemic mice. Passive immunization with anti-VEGF antibody (10 microg/mouse) completely abrogated the beneficial effects of simvastatin on healing in diabetic mice. Simvastatin has potential application in diabetes-related wound healing disorders.

OPG and sRANKL Serum Concentrations in Osteopenic, Postmenopausal Women After 2‐Year Genistein Administration

Introduction: RANKL and its decoy receptor osteoprotegerin (OPG) constitute a complex physiological mediator system involved in the regulation of bone resorption and may be responsible for the homeostatic mechanism of normal bone remodeling. Genistein, an isoflavone representing 1–5% of total phytoestrogen content in soybean products, may positively regulate cellular bone metabolism, but its mechanism of action on bone is not yet fully understood.

Antioxidant effect of atorvastatin is independent of PON1 gene T(-107)C, Q192R and L55M polymorphisms in hypercholesterolaemic patients.

ABSTRACT Background: Serum paraoxonase (PON1), a high density lipoprotein (HDL)-bound antioxidant enzyme, plays a role in atherosclerosis. An increase in PON1 activity has been reported following statin treatment. Objective: In the present study the following factors were evaluated: the influence of PON1 gene Q192R, L55M and T(–107)C polymorphisms on the response of LDL oxidisability and PON1 activity to atorvastatin treatment. Research design and methods: 205 Sicilian subjects with primary hypercholesterolaemia (HCh) and 69 healthy subjects as controls were concurrently enrolled. Hypercholesterolaemic patients were randomly divided into two groups: an atorvastatin group (10 mg/day atorvastatin) and a placebo group. Lipid profile, markers of LDL resistance to in vitro oxidation (lag-phase, oxidation rate and thiobarbituric acid-reactive substances), vitamin E content in LDL, PON1 activity and genotypes in both HCh and control subjects were determined at baseline. The same parameters were measured again after 3 weeks of treatment in both the atorvastatin and placebo groups. Results: HCh subjects showed significantly lower LDL resistance to oxidation, vitamin E content and PON1 activity levels than controls. A strong association was found among PON1 T(–107)C genotypes, LDL susceptibility to oxidation, vitamin E content and PON1 activity. After treatment, the atorvastatin group displayed a significant decrease in total cholesterol, LDL-cholesterol levels, and LDL susceptibility to oxidation, and an increase in vitamin E content and PON1 activity, compared with baseline values. Unlike PON1 activity levels, no difference among PON1 gene polymorphisms and reduction in markers of LDL oxidisability was observed. Conclusions: These results show, for the first time, that atorvastatin is able to improve the resistance to LDL oxidation independently of PON1 gene polymorphism.

Effects of simvastatin treatment on sICAM-1 and sE-selectin levels in hypercholesterolemic subjects

This study was performed to determine whether the levels of soluble intercellular adhesion molecule-1 (sICAM-l) and soluble endothelial molecule-1 (sE-selectin) were elevated in subjects with hypercholesterolemia who presented with no other risk factors or evidence of atherosclerosis. The effects of administration of an HMG-CoA reductase inhibitor on the serum levels of these molecules were also examined. Forty hypercholesterolemic subjects (HCh) (19 males and 21 females), without hypertension or cardiovascular disease, received placebo for 4 weeks. The patients were then randomized in two groups; 20 of them (simvastatin group) were treated with simvastatin (20 mg/day) and the other 20 (placebo group) continued placebo administration. After 12 and 24 weeks of either simvastatin or placebo treatment, sICAM-1 and sE-selectin levels were measured. The same parameters were measured in 20 control subjects (C) with normal cholesterol levels, matched for sex and age. HCh had sICAM-1 basal values higher than C (352.4+/-57.9 ng/ml versus 114.9+/-89.6 ng/ml; P<0.001); however, sE-selectin basal values were not different in the two groups. No correlation was observed between HCh sICAM-1 levels and cholesterol levels (total and low-density lipoprotein). Furthermore, cholesterol-lowering treatment with simvastatin did not significantly diminish sICAM-1 levels. Our findings would support the hypothesis that patients with isolated hypercholesterolemia and without clinical atherosclerosis may be silent carriers of arterial subendothelial inflammation, expressed as an increase of sICAM-1.

Effects of AT1 Receptor Antagonist Losartan on sICAM-1 and TNF-a Levels in Uncomplicated Hypertensive Patients

This study was designed to determine whether the levels of soluble intercellular adhesion molecule-1 (sICAM-1) and tumor necrosis factor-a (TNF-a) were elevated in subjects with uncomplicated hypertension who presented with no other risk factors or evidence of atherosclerosis. The effects of administration of an angiotensin type-1 antagonist (losartan) on the serum concentrations of these molecules were also examined. Twenty hypertensive (HT) subjects (12 men and 8 women, mean age 49.1 ±7.2 years) without other risk factors or cardiovascular disease received placebo for 4 weeks. The patients were then treated with losartan (50 mg/day) for 24 weeks. After 4, 12, and 24 weeks of losartan treatment, sICAM-1 and TNF-a levels were measured. The same parameters were measured in 20 normotensive control subjects (C), matched for sex and age. HT had sICAM-1 and TNF-a basal values higher than C (respectively 351.7 ±97.4 vs 201.6 ±32.3 ng/mL, p<0.001 and 31.8 ±2.4 vs 15.3 ±2.2 pg/mL, p<0.001). There was a positive correlation between sICAM-1 and TNF-a levels, but no correlation in HT between the average diastolic and systolic blood pressure (clinic and ambulatory monitoring) and the sICAM-1 or TNF-a levels was observed. Losartan treatment caused a significant decrease of sICAM-1 levels at the end of the first month of treatment (300.2 ±64.4 ng/mL, p<0.05), but the values reverted to the basal levels at the following time points. No variation of TNF-a levels during losartan treatment was observed. These results show that patients with uncomplicated mild essential hypertension presented with high plasma ICAM-1 and TNF-a concentrations. Although all the patients were responsive to the antihypertensive treatment with losartan, their plasma concentrations of TNF-a were not modified, and sICAM-1 concentrations decreased only for a short period of time. This suggests that in uncomplicated hypertension other factors besides the blood pressure modulate the endothelial inflammation.

Effects of atorvastatin treatment on sICAM-1 and plasma nitric oxide levels in hypercholesterolemic subjects.

This study investigated the behavior of soluble intercellular adhesion molecule-1 (sICAM-1) and serum nitric oxide (NO) products, nitrite/nitrate (NO 2-NO,-), in subjects with primary hypercholesterolemia (HCh) without other risk factors and atherosclerosis. The effect of a short-term cholesterol-lowering treatment with atorvastatin, an HMG-CoA reductase inhibitor, on the levels of sICAM-1 and NO2-/NO3 were also investigated. After 4 weeks of placebo administration, 40 HCh (15 males and 25 females) were randomized in 2 groups: 20 subjects (atorvastatin group) received 10 mg/day of atorvastatin and the remaining 20 (placebo group) continued to take placebo. At baseline and after 4 and 12 weeks of atorvastatin or placebo administration, serum sICAM-1 and NO2-/NO3-levels were evaluated. The basal levels of these parameters were compared with those of 20 healthy subjects (C), matched for sex and age. Hypercholesterolemic subjects showed sICAM-1 and NO2-/NO3-basal values that were higher (331.7 ± 60.3 ng/mL vs. 202.3 ± 32.3 ng/mL, p<0.001) and lower (10.4 ± 2.5 μmol/L vs. 20.7 ± 4.4 μmol/L, p<0.01) than controls. No correlation between sICAM-1 or NO products and plasma cholesterol values was found, whereas there was an inverse correlation between sICAM-1 and NO2-/NO3-levels. Atorvastatin administration significantly decreased sICAM-1 and increased NO2-/NO3-levels, however these changes were not correlated with the reduction of plasma cholesterol. These data support the hypothesize that patients with HCh with no signs of arterial lesions, may have latent atherosclerosis, expressed as an increase of sICAM-1 and decrease in NO product levels. An improvement in the levels of these parameters after a short-time treatment with atorvastatin was also demonstrated.

Circulating progenitor cells and the elderly: A seven-year observational study

Cardiovascular (CV) diseases and related complications are the main causes of morbidity and mortality in the elderly. CV progenitor cells, including CD34+ cells, play a role in delaying the progression of atherosclerosis. In the present study we observed 100 octogenarians for seven years, in order to address the question of whether CD34+ cell number is a predictor of longevity in selected survivors. We also checked for associations of cell expression of manganese superoxide dismutase (Mn-SOD), catalase (CAT), and glutathione peroxidase type-1 (GPx-1) antioxidative enzymes, with number of CD34+ progenitor cells and mortality. We found that in very old subjects the number of CD34+ cells at baseline were higher in subjects who reached older age at death or were still living at the end of observation period, with respect to subjects who died from all causes, including CV deaths. On the other hand, HDL-C plasma levels and, with the exception of diabetes, the classic CV risk factors (hypertension, smoking, hypercholesterolemia) showed a loss of their predictive power. A significant association between the redox system of CD34+ cells and mortality was also observed. These data suggest that, even in the elderly, CD34+ cells maintain their role in predicting mortality. CD34+ cells could thus be considered as a biomarker of longevity.

Platelet activating factor-acetylhydrolase (PAF-AH) activity and HDL levels, but not PAF-AH gene polymorphisms, are associated with successful aging in Sicilian octogenarians

Background and aims: Aging is associated with an increased risk of developing atherosclerosis. Subjects over 80 years of age without cardiovascular disease provide a model to investigate the protective factors increasing their resistance to atherosclerotic disease. Platelet-activating factor acetylhydrolase (PAF-AH) is an enzyme associated with low density lipoprotein (LDL) and high density lipoprotein (HDL) inactivating platelet-activating factor (PAF) and preventing LDL oxidation by hydrolysis of oxidized phospholipids. The aim of the present study was to evaluate the contribution of the PAF-AH gene Arg92His, Ile198Thr and Ala379Val polymorphisms to resistance toward developing cardiovascular events in healthy Sicilian octogenarians. Methods: Distribution of PAF-AH genotypes and activity, and biochemical parameters, were compared between 100 octogenarians and 200 healthy adults. Results: The individuals in the elderly group displayed significantly higher levels of HDL-C (p<0.001) and plasma (p<0.001) and HDL (p<0.001) PAF-AH activity. Analysis of PAF-AH genotype distributions showed no significant differences between octogenarians and controls. No differences among PAF-AH genotypes with respect to plasma and HDL PAF-AH activity were found in either group. Conclusions: Our results provide no evidence of a significant association between the PAF-AH gene Arg92His, Ile198Thr and Ala379Val polymorphisms and successful aging in Sicilians. They also emphasize that, in these subjects, aging is characterized by increased levels of PAF-AH activity and HDL-C.

Effects of fluvastatin treatment on red blood cell Na+ transport systems in hypercholesterolemic subjects.

This study was performed to ascertain the effects of short-term cholesterol-lowering therapy with fluvastatin on red blood cells Na+ transport systems. Forty familial hypercholesterolemic subjects (FH; 19 men and 21 women) without hypertension or cardiovascular disease were given a placebo for 4 weeks, and then randomized in two groups. Twenty (fluvastatin group) were given fluvastatin (40 mg/day), and the other 20 (placebo group) continued placebo administration. After the placebo period and after 4 and 12 weeks of placebo or fluvastatin treatment, we measured Na+/K+ pump activity, Na+/K+ cotransport (Na+/K+ Ct), Na+/Li+ countertransport (Na+/Li+ Cnt), passive Na+ permeability (Na+PP), and internal Na+ content (Na+i). The same parameters were measured in 23 control subjects (C) with normal cholesterolemic values, who were matched for sex and age. FH had higher Na+/Li+ Cnt values than C (193.2 +/- 59.4 vs. 139.8 +/- 48.7 microM cells/h; p < 0.01), an increase in Na(+)PP (0.034 +/- 0.012/h vs. 0.018 +/- 0.004/h; p < 0.001), and higher Na(+)i (7.5 +/- 1.5 vs. 6.2 +/- 0.9 mM cells; p < 0.001). In hypercholesterolemic subjects, Na(+)i values were correlated with cholesterol (total and LDL) and apo B levels, whereas an inverse correlation was found for HDL-c and apo AI levels. Reduced total and LDL cholesterol and apo B levels after fluvastatin treatment caused a decrease in both Na(+)/Li(+) Cnt (from 186.1 +/- 60.5 to 125.1 +/- 34.0 microM cells/h; p < 0.001) and Na(+) PP (from 0.035 +/- 0.013/h to 0.02 +/- 0.016/h; p < 0.01), and an increase in Na+/K+ pump activity (from 1,549.0 +/- 507.7 to 1,894.2 +/- 536.2 microM cells/h; p < 0.04), with a significant reduction in the internal Na+ content (from 7.5 +/- 1.6 to 5.8 +/- 2.4 mM cells; p < 0.001). Our findings show that hypercholesterolemia affects red blood cell Na+ transport systems, with an increase in Na+/Li+Cnt, Na+PP, and the internal Na+ content. Cholesterol-lowering treatment with fluvastatin influences Na+ transport systems and reduces the internal Na+ content. This might also be responsible for the greater vascular reactivity observed in hypercholesterolemic patients, and its amelioration after a reduction in cholesterol levels.