Platelet vitamin D receptor is reduced in osteoporotic patients.

AIM: It is well known that vitamin D plays an important role in maintaining bone homeostasis and in regulating calcium absorption. The active form of vitamin D interacts with its receptor the VDR that is expressed in multiple tissues and it is involved in platelets (PLTs) function. In the present study we evaluate PLTs' VDR expression in osteoporotic as opposed to healthy subjects. METHODS: We enrolled in the study 77 women with postmenopausal osteoporosis, 33 healthy women of childbearing age, 49 healthy men, and 11 healthy women matched with patients for age and postmenopausal period. Thirty-nine patients had had one femoral fracture occurred after the age of fifty and attributable to primary osteoporosis. Bone mineral density, markers of bone metabolism and VDR levels were measured in all the subjects. RESULTS: Our data show that VDR level is lower in patients as respect to controls and is positively correlated with bone density, but not with markers of bone metabolism. We also found a decrease in the phosphorus levels in patients without differences in vitamin D levels and in the dietary calcium intake. CONCLUSION: The lower VDR expression in osteoporotic could indicate a lower ability to respond to vitamin D, and could be the explanation of the increase in the PTH and decrease in the phosphorus levels in patients with respect to controls.

Teriparatide increases the maturation of circulating osteoblast precursors.

UNLABELLED: This study shows that teriparatide promotes the circulating osteoblast (OB) precursor degree of maturation in patients affected by postmenopausal osteoporosis. INTRODUCTION: Anabolic treatment with teriparatide has proven effective for the therapy of postmenopausal osteoporosis and significantly reduces the risk of non-vertebral fragility fractures. The aim of this study was to investigate the effect of teriparatide on circulating OB precursors. METHODS: We evaluated by flow cytometry and real-time PCR the expression of OBs typical markers in peripheral blood mononuclear cells during treatment with teriparatide plus calcium and vitamin D, raloxifene plus calcium and vitamin D or calcium and vitamin D alone at various time points. Serum bone alkaline phosphatase and osteocalcin (OC) were measured as markers of bone turnover. RESULTS: Our results show that circulating OB precursors are more numerous and more immature in patients affected by fragility fractures than in osteoporotic patients without fractures. We also show that teriparatide treatment increases the expression of alkaline phosphatase and of OC in OB precursors; thus, it increases their degree of maturation. CONCLUSIONS: We suggest that teriparatide acts as anabolic agents also by promoting the maturation of OB precursors.

Bone and bone marrow pro-osteoclastogenic cytokines are up-regulated in osteoporosis fragility fractures.

UNLABELLED: This study evaluates cytokines production in bone and bone marrow of patients with an osteoporotic fracture or with osteoarthritis by real time PCR, Western blot and immunohistochemistry. We demonstrate that the cytokine pattern is shifted towards osteoclast activation and osteoblast inhibition in patients with osteoporotic fractures. INTRODUCTION: Fragility fractures are the resultant of low bone mass and poor bone architecture typical of osteoporosis. Cytokines involved in the control of bone cell maturation and function are produced by both bone itself and bone marrow cells, but the roles of these two sources in its control and the amounts they produce are not clear. This study compares their production in patients with an osteoporotic fracture and those with osteoarthritis. METHODS: We evaluated 52 femoral heads from women subjected to hip-joint replacement surgery for femoral neck fractures due to low-energy trauma (37), or for osteoarthritis (15). Total RNA was extracted from both bone and bone marrow, and quantitative PCR was used to identify the receptor activator of nuclear factor kB Ligand (RANKL), osteoprotegerin (OPG), macrophage colony stimulating factor (M-CSF), transforming growth factor ß (TGFß), Dickoppf-1 (DKK-1) and sclerostin (SOST) expression. Immunohistochemistry and Western blot were performed in order to quantify and localize in bone and bone marrow the cytokines. RESULTS: We found an increase of RANKL/OPG ratio, M-CSF, SOST and DKK-1 in fractured patients, whereas TGFß was increased in osteoarthritic bone. Bone marrow produced greater amounts of RANKL, M-CSF and TGFß compared to bone, whereas the production of DKK-1 and SOST was higher in bone. CONCLUSIONS: We show that bone marrow cells produced the greater amount of pro-osteoclastogenic cytokines, whereas bone cells produced higher amount of osteoblast inhibitors in patients with fragility fracture, thus the cytokine pattern is shifted towards osteoclast activation and osteoblast inhibition in these patients.

Alendronate reduces osteoclast precursors in osteoporosis.

UNLABELLED: This study evaluates the effect of alendronate on osteoclastogenesis, cytokine production, and bone resorption in postmenopausal women. We suggest that it acts on mature bone resorbing osteoclasts after 3 months of treatment, whereas, after 1 year, it diminishes their formation by reducing their precursors and serum RANKL. INTRODUCTION: Osteoclasts are the target cells of bisphosphonates, though the most drug-sensitive steps of their formation and activity have not been determined. The present study evaluates the effect of alendronate on osteoclastogenesis, cytokine production, and bone resorption in postmenopausal women. METHODS: The study was conducted on 35 osteoporotic women; 15 were pretreated with alendronate 70 mg/week, whereas, 20 were treated with calcium 1 g/day and vitamin D 800 IU/day. After 3 months, 30 received alendonate 70/mg, vitamin D 2800 IU/week, and calcium 1 g/day for 12 months (combined therapy), whereas, the other five patients remained on calcium 1 g/day and vitamin D 800 IU/day. The following parameters were assessed before and after therapy: changes in bone resorption markers, circulating osteoclast precursors, formation of osteoclasts in peripheral blood mononuclear cell cultures, their viability, and variations in cytokines production. RESULTS: After 3 months of alendronate, there was no significant reduction in the number of osteoclast precursors, osteoclast formation and viability, and cytokine levels, whereas, there was a significant reduction of bone resorption markers. One year of the combined therapy, on the other hand, reduced osteoclast precursors, osteoclast formation, and serum RANKL, whereas, calcium plus vitamin D alone had no effect. CONCLUSIONS: We suggest that alendronate mainly acts on mature bone resorbing osteoclasts in the short term, whereas, its long-term administration diminishes their formation by reducing their precursors and serum RANKL.

Geranylgeraniol prevents the cytotoxic effects of mevastatin in THP-1 cells, without decreasing the beneficial effects on cholesterol synthesis.

BACKGROUND AND PURPOSE: Statins, inhibitors of hydroxymethylglutaryl-CoA reductase, reduce the intracellular synthesis of cholesterol and prevent the onset of atherosclerosis. They also decrease the synthesis of isoprenoid molecules, such as the side chain of ubiquinone and geranylgeranyl pyrophosphate. As a consequence, statins impair mitochondrial metabolism and the activation of small monomeric GTPases (such as Rho and Ras), causing toxic effects. To date, a successful strategy to prevent statin toxicity is lacking. EXPERIMENTAL APPROACH: In human monocytic THP-1 cells, we measured the synthesis of cholesterol and isoprenoids, mitochondrial electron flow, the activity of RhoA and Rac, cell death and proliferation. KEY RESULTS: Mevastatin reduced the synthesis of cholesterol, geranylgeranyl pyrophosphate and ubiquinone, mitochondrial electron transport, activity of RhoA and Rac, and cell proliferation, accompanied by increased cell death. Geranylgeraniol, a cell-permeable analogue of geranylgeranyl pyrophosphate, reversed all these effects of mevastatin, without affecting its ability to reduce cholesterol synthesis. Notably, geranylgeraniol was more effective than the addition of exogenous ubiquinone, which rescued mitochondrial respiratory activity and reversed mevastatin cytotoxicity, but did not alter the decrease in cell proliferation. The same results were obtained in human liver HepG2 cells. CONCLUSIONS AND IMPLICATIONS: Geranylgeraniol had a broader protective effect against the cytotoxicity of statins than exogenous ubiquinone. Therefore, geranylgeraniol may be a more useful and practical means of limiting the toxicities of statins, without reducing their efficacy as cholesterol lowering agents.

Digoxin and ouabain increase the synthesis of cholesterol in human liver cells.

Digoxin and ouabain are steroid drugs that inhibit the Na(+)/K(+)-ATPase, and are widely used in the treatment of heart diseases. They may also have additional effects, such as on metabolism of steroid hormones, although until now no evidence has been provided about the effects of these cardioactive glycosides on the synthesis of cholesterol. Here we report that digoxin and ouabain increased the synthesis of cholesterol in human liver HepG2 cells, enhancing the activity and the expression of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), the rate-limiting enzyme of the cholesterol synthesis. This effect was mediated by the binding of the sterol regulatory element binding protein-2 (SREBP-2) to the HMGCR promoter, and was lost in cells silenced for SREBP-2 or loaded with increasing amounts of cholesterol. Digoxin and ouabain competed with cholesterol for binding to the SREBP-cleavage-activating protein, and are critical regulators of cholesterol synthesis in human liver cells.

Artemisinin induces doxorubicin resistance in human colon cancer cells via calcium-dependent activation of HIF-1alpha and P-glycoprotein overexpression.

BACKGROUND AND PURPOSE: Artemisinin is an antimalarial drug exerting pleiotropic effects, such as the inhibition of the transcription factor nuclear factor-kappa B and of the sarcoplasmic/endoplasmic reticulum Ca(++)-ATPase (SERCA) of P. falciparum. As the sesquiterpene lactone thapsigargin, a known inhibitor of mammalian SERCA, enhances the expression of P-glycoprotein (Pgp) by increasing the intracellular Ca(++) ([Ca(++)](i)) level, we investigated whether artemisinin and its structural homologue parthenolide could inhibit SERCA in human colon carcinoma HT29 cells and induce a resistance to doxorubicin. EXPERIMENTAL APPROACH: HT29 cells were incubated with artemisinin or parthenolide and assessed for SERCA activity, [Ca(++)](i) levels, Pgp expression, doxorubicin accumulation and toxicity, and translocation of the hypoxia-inducible factor, HIF-1alpha. KEY RESULTS: Artemisinin and parthenolide, like the specific SERCA inhibitors thapsigargin and cyclopiazonic acid, reduced the activity of SERCA. They also increased intracellular calcium concentration ([Ca(++)](i)) and Pgp expression and decreased doxorubicin accumulation and cytotoxicity. The intracellular Ca(++) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, and the inhibitor of calmodulin-dependent kinase II (CaMKII) KN93 prevented these effects. CaMKII is known to promote the phosphorylation and the activation of HIF-1alpha, which may induce Pgp. In HT29 cells, artemisinin and parthenolide induced the phosphorylation of HIF-1alpha, which was inhibited by KN93. CONCLUSIONS AND IMPLICATIONS: Our results suggest that artemisinin and parthenolide may act as SERCA inhibitors and, like other SERCA inhibitors, induce resistance to doxorubicin in human colon cancer cells, via the CaMKII-dependent activation of HIF-1alpha and the induction of Pgp.

Glutathione levels in patients with erectile dysfunction, with or without diabetes mellitus.

The reduced form of glutathione (GSH) is the most important cell antioxidant and is also an essential cofactor for nitric oxide (NO) synthase that synthesizes NO from l-arginine. Reduced levels of GSH, due both to a hyperglycaemia-induced increase of free radical production and to a decrease of NADPH levels [like in diabetes mellitus (DM)], can hamper the endothelial cell functions. This condition may play an important role in the aetiology of some clinical signs, like erectile dysfunction (ED). The aim of this study was to test the hypothesis that GSH concentration is reduced in patients with ED and type 2 diabetes mellitus. We studied 111 male patients with ED: 64 with diabetes (ED/DM) and 47 without diabetes (ED/wDM); 20 patients with diabetes but without ED (DM) and 26 male normal subjects as a control group (C). The GSH red blood cell concentration was significantly lower in ED than in C (X +/- SD; 1782.12 +/- 518.02 vs. 2269.20 +/- 231.56 mumol/L, p < 0.001). In particular, GSH was significantly reduced in ED/DM vs. ED/wDM (1670.74 +/- 437.68 vs. 1930.63 +/- 581.01 micromol/L, p < 0.01). In DM, GSH was significantly lower than in C and significantly higher than in ED/DM (2084.20 +/- 118.14 vs. 2269.20 +/- 231.56 and vs. 1670.74 +/- 437.68 micromol/L, p < 0.002 and p < 0.001 respectively). GSH showed a negative correlation with fasting glucose concentrations (r = -0.34, p < 0.01) and with the duration of DM (r = -0.25, p < 0.05). A GSH depletion can lead to a reduction of NO synthesis, thus impairing vasodilation in the corpora cavernosa.

Platelet cGMP inversely correlates with age in healthy subjects.

Biochemical modifications associated with the increase in platelet activity with age are not well defined. Furthermore it is well known that the nitric oxide/cyclic 3', 5'-guanosine monophosphate (cGMP) pathway regulates platelet aggregation. The aim of the present study was to examine the relationship between platelet content of cGMP and age. 120 normal subjects, evaluating the cGMP platelet concentration, 17betaE2, IGF-I, dehydroepiandrosterone sulphate (DHEAS), insulin, plasma glucose, lipid pattern, homocysteine and PAI-I antigen, were studied. The multivariate analysis in a model with cGMP as dependent variable and with age, LDL, apolipoprotein B (ApoB), DHEAS, E2 and insulin-like growth factor (IGF)-I as independent variables shows a negative correlation between cGMP and age (p<0.01, beta=-0.388). In conclusion our data suggest that the reduced efficiency of the system constitutive nitric oxide synthase (cNOS)/guanylate cyclase represents at least one of the reasons of the increased platelet aggregability with age.

Platelet cyclic guanosine monophosphate production during menstrual cycle in healthy women.

UNLABELLED: The incidence of cardiovascular disease among women during their reproductive years is considerably less than in men and this difference decreases after menopause. Since in cultured endothelial cells and in platelets E2 increases nitric oxide (NO) production, it is possible that their cardioprotective effect may be mediated by NO. The aim of this study was to evaluate platelet cyclic guanosine monophosphate (cGMP), as a marker of NO production, during menstrual cycle. Fifteen women aged 26-40 yr were studied to evaluate: LH, FSH, E2, P and cGMP on the 5th follicular and 22nd luteal day of the cycle and during the ovulatory period. Platelet cGMP was evaluated in basal condition (3-isobuthyl 1-methylxanthine-IBMX) and with ionomycine (IONO) and sodium nitroprusside (SNP). RESULTS: LH, FSH, E2 and P demonstrated the typical patterns of ovulatory cycle. During follicular and luteal IBMX, SNP and IONO phase were homogeneous while they increased during the ovulatory period. A correlation between IBMX cGMP and E2 (p<0.002, rs=0.456) was found. In conclusion the data show an increase in platelet cGMP during the ovulatory period and a correlation between E2 and cGMP suggesting that E2 modulates NO production. The cardioprotective effect of E2 may be, at least in part, mediated by the increase in NO production.

Serum citrate levels, haptoglobin haplotypes and transferrin receptor (CD71) in patients with HIV-1 infection.

BACKGROUND: The progression of HIV-1 infection towards its more advanced stages is accompanied by changes in iron metabolism and increased body iron stores. PATIENTS AND METHODS: Given the ability of HIV to alter iron metabolism, we studied the principal (transferrin system) and alternative (citrate system) iron pathways in a group of 65 HIV-infected patients (symptomatic stage B1-B3) and in a group of 36 healthy seronegative individuals. We determined serum citrate levels, haptoglobin (Hp) haplotypes, expression of transferrin receptor (CD71) on cell lines infected with HIV-1 as well as iron markers including blood iron, transferrin and ferritin. RESULTS: Our data showed decreased serum citrate levels in the HIV-infected patients compared to controls (92.9 +/- 22.4 microM/l vs 126.2 +/- 29.2 microM/L; p < 0.01). In particular, the serum citrate levels negatively correlated with HIV-1 RNA copy number (mean: 2.53 +/- 1.88 x 10(5)/ml, r(s) = 0.70, p < 0.01) and positively correlated with CD4+ T-lymphocyte count (mean: 241 +/- 168/ml, r(s) = 0.64, p > 0.05). Accordingly, blood iron, transferrin and red cell concentrations were lower in HIV-infected patients compared to the controls, whereas serum ferritin levels were higher in HIV-infected patients. Moreover, the Hp haplotype distribution showed significant differences only in the group of HIV-infected patients (p = 0.02; chi2 test). CONCLUSION: Our results show that iron metabolism is altered in patients with HIV-1 infection. The alternative pathway (citrate system) is particularly affected, since when citrate levels are low, both aconitase activity and HIV-1 replication need iron.

High density lipoproteins (HDL) in women with postmenopausal osteoporosis: a preliminary study.

OBJECTIVE: Since a previous study showed an inverse correlation between high density lipoproteins (HDL) and bone mineral density (BMD), we searched for a possible relationship between HDL level and the presence of postmenopausal osteoporosis. DESIGN: We measured HDL levels in 37 women with postmenopausal osteoporosis, and compared them with a control group of 43 healthy postmenopausal women. The HDL levels were compared between the two groups using Student's t test and were correlated with BMD by Pearson's coefficient. To avoid possible selection bias, we compared patients and controls for body mass index by chi 2 test. The sensitivity and specificity of HDL level higher than 65 mg% (positive test) or lower than 45 mg% (negative test) was compared with double emission x-ray absorptiometry (considered the gold standard in the measurement of BMD). RESULTS: The level of HDL was significantly higher in the osteoporotic patients than in the controls (67.7 +/- 15.5 mg% vs 58.3 +/- 11.6 mg%, p = 0.0039). HDL was inversely correlated with BMD (r = -0.29, p = 0.0083). HDL higher than 65 mg% has a high specificity (77%) for patients with osteoporosis, while HDL lower than 45 mg% has a high sensitivity (97%) in detecting subject without osteoporosis. CONCLUSIONS: Our preliminary data suggest an interesting, as yet unexplained association between HDL and bone mineral density in postmenopausal women.

Iron inhibits the nitric oxide synthesis elicited by asbestos in murine macrophages.

Crocidolite fibers stimulated nitric oxide synthase (NOS) activity and expression in glial and alveolar murine macrophages: this effect was inhibited by iron supplementation and enhanced by iron chelation. We suggest that in these cells crocidolite stimulates NOS expression by decreasing the iron bioavailability and activating an iron-sensitive transcription factor.

Local interaction simulation approach for the response of the vascular system to metabolic changes of cell behavior.

The self-regulatory interactions between cells and the vascular system are mediated by signals propagating at a finite speed. In order to build up a physical model of these processes, several features, such as storing of internal energy, nonclassical nonlinear behavior, and delay and threshold effects, have to be taken into account. Considering cells as particles in different metabolic states according to their internal energy, we have developed a model based on the local interaction simulation approach. Several numerical results, in qualitative agreement with biological observations, illustrate the applicability of the model and the method to implement it.

Administration of glutathione in patients with type 2 diabetes mellitus increases the platelet constitutive nitric oxide synthase activity and reduces PAI-1.

Several studies suggest that nitric oxide (NO) production is impaired in diabetes mellitus. Reduced levels of NO could contribute to cardiovascular mortality. Furthermore, NO synthesis is impaired in glutathione (GSH)-depleted human umbilical vein endothelial cells and GSH is reduced in patients with type 2 diabetes mellitus (T2DM). We tested the hypothesis that treatment with GSH may improve platelet constitutive NO sinthase (cNOS) activity in patients with T2DM. Fifteen patients with T2DM underwent a treatment with GSH 600 mg/day i.m. for 10 days. With respect to the basal values on the 10th day of treatment, the red blood cell GSH concentration and platelets cNOS increased (1.4+/-0.1 vs 1.9+/-0.1 micromol/10(10) RBC, p<0.001 and 0.7+/-0.1 vs 2.9+/-0.2 fmol x min(-1) x 10(-9) PLTs, p<0.001, respectively) and the plasma PAI-1 levels diminished (81.4+/-3.7 vs 68.7+/-4.0 ng/ml, p<0.002). A negative correlation between the cNOS and the PAI-1 was found on the basal values. After a wash-out of 30 days the values of red blood cell GSH concentration, platelet cNOS activity and PAI-1 Ag returned to the basal levels. These data suggest that the administration of GSH, in patients with T2DM, is able to improve platelet cNOS activity together with a reduction of PAI-1.

Serum DHEAS levels correlate with platelet cGMP in normal women.

Several studies suggest that DHEAS is a protective factor against atherosclerosis and coronary artery disease in man, but the mechanism of its biological role is unclear. Recently, it has been suggested that DHEAS can retard atherosclerosis formation through an increase in nitric oxide (NO) production by increasing E2 synthesis. The aim of the study was to evaluate the platelet cGMP concentrations (i.e. a marker of NO production) and the serum levels of DHEAS and E2 in normal females. Blood samples were taken from 51 normal women (age 42.3+/-1.9 yr, range: 22-67 yr, BMI 23.0+/-0.6 kg/m2) to evaluate platelet cGMP concentrations and serum levels of DHEAS and E2. To determine the platelet cGMP content, platelet rich plasma (PRP) was incubated at 37 C (30 min) in the presence of IBMX. The amount of platelet cGMP was measured by a cGMP (3H) assay kit. In all subjects the mean of platelet cGMP was 536.2+/-45.3 fmol/10(6) platelets and the mean of serum DHEAS and E2 was 151.4+/-13.9 microg/dl and 34.7+/-6.1 pg/ml, respectively. In all subjects DHEAS positively correlates with cGMP (p<0.001, r=0.513) and with E2 (p<0.001, r=0.650); furthermore E2 positively correlates with cGMP (p<0.001, r=0.663). In conclusion our data support the hypothesis that DHEAS exerts its antiatherogenic effect by increasing the NO production directly and/or by increasing the E2 synthesis.

Analysis of a "phase transition" from tumor growth to latency.

A mathematical model, based on the local interaction simulation approach, is developed in order to allow simulations of the spatiotemporal evolution of neoplasies. The model consists of a set of rules, which govern the interaction of cancerous cells among themselves and in competition with other cell populations for the acquisition of essential nutrients. As a result of small variations in the basic parameters, it leads to four different outcomes: indefinite growth, metastasis, latency, and complete regression. In the present contribution a detailed analysis of the dormant phase is carried on and the critical parameters for the transition to other phases are computed. Interesting chaotic behaviors can also be observed, with different attractors in the parameters space. Interest in the latency phase has been aroused by therapeutical strategies aiming to reduce a growing tumor to dormancy. The effect of such strategies may be simulated with our approach.

A physical-based model for the simulation of neoplastic growth and metastasis.

BACKGROUND AND OBJECTIVES: It is possible to formulate models capable of reproducing the main details of the physical processes involved in the evolution of biological systems. The complexity of the problem requires to begin with a simple and universal model for the description of the cellular growth, to be adapted successively to the local conditions found in clinically observed neoplastic growths. METHODS: A model based on the Local Interaction Simulation Approach (LISA) has been formulated for the simulation of growth, diffusion, and metastasis of neoplasms. The vascularization is described by a blood vessel located on one edge of the specimen in which a constant and homogeneous flow is assumed. A nutrient density is defined to mimic the blood flow within the tissue. RESULTS: Photograms taken at proper times may identify the main characteristics of the tumor evolution and describe its volume variations in a transversal section. Furthermore, it is possible to monitor constantly the volume of the neoplasm and of the necrotic tissue as a function of time, as well as the portion of cells that have migrated in the blood vessel. CONCLUSIONS: In spite of strong simplifying assumptions, the model presents good qualitative agreement with clinical data, which may be further improved by more detailed information about cancer cells properties or local vascular system patterns.