The PPAR-gamma agonist rosiglitazone facilitates Akt rephosphorylation and inhibits apoptosis in cardiomyocytes during hypoxia/reoxygenation.

BACKGROUND AND AIM: Results on the cardiovascular effects of PPAR-gamma agonists are conflicting. On one hand, it was suggested that the PPAR-gamma agonist rosiglitazone may increase the risk of cardiovascular events. On the other hand, PPAR-gamma agonists reduce myocardial infarct size and improve myocardial function during ischemia/reperfusion in animal studies in vivo. However, the mechanism of this effect is unclear, and it is open if PPAR-gamma agonists have a direct effect on cardiac myocyte survival in ischemia/reperfusion. The aim of this study was to determine the effect of the PPAR-gamma agonist rosiglitazone on hypoxia/reoxygenation-induced apoptosis of isolated cardiomyocytes. METHODS: Isolated rat cardiac myocytes were pretreated with rosiglitazone or vehicle for 30 min before they were subjected to hypoxia for 4 h followed by different times of reoxygenation (5 min to 12 h). Apoptosis was determined by in situ hybridization for DNA fragmentation (TUNEL) as well as detection of cytoplasmic accumulation of histone-associated DNA fragments by enzyme-linked immunosorbent assay (ELISA). Activation of apoptosis regulating intracellular signalling pathways was studied by immunoblotting using phosphospecific antibodies. RESULTS: Rosiglitazone significantly reduced apoptosis of isolated cardiomyocytes subjected to hypoxia/reoxygenation, independently determined with two methods. After 4 h of hypoxia and 12 h of reoxygenation, 34 +/- 3.6% of the vehicle treated cardiac myocytes stained positive for DNA fragmentation in the TUNEL staining. Rosiglitazone treatment reduced this effect by 23% (p < 0.01). Even more pronounced, cytoplasmic accumulation of histone-associated DNA fragments detected by ELISA was reduced by 35% (p < 0.05) in the presence of rosiglitazone. This inhibition of hypoxia/reoxygenation-induced apoptosis was associated with an increased reoxygenation-induced rephosphorylation of the protein kinase Akt, a crucial mediator of cardiomyocyte survival in ischemia/reperfusion of the heart. This effect was reversed by GW-9662, an irreversible PPAR-gamma antagonist. However, rosiglitazone did not alter phosphorylation of the MAP kinases ERK1/2 and c-Jun N-terminal kinase (JNK). CONCLUSION: It can be concluded that cardiac myocytes are direct targets of PPAR-gamma agonists promoting its survival in ischemia/reperfusion, at least in part by facilitating Akt rephosphorylation. This effect may be of clinical relevance inhibiting the reperfusion-induced injury in patients suffering from myocardial infarction or undergoing cardiac surgery.

Role of TNF-alpha producing T-cells in bone loss induced by estrogen deficiency.

Many study in literature have suggested a possible role of T cells and tumor necrosis factor-alpha (TNF-alpha) in the pathogenesis of bone loss that occurs in pathological conditions, such as systemic inflammatory diseases; the molecular bases through which this phenomenon occurs and the relevance of this mechanism also in estrogen deficiency induced bone loss remain unclear. In our study we observed that TNF-alpha knock-out mice (TNF-/-), as well as transgenic mice without thymus (and therefore without mature T cell), do not lose bone after ovariectomy like observed for mice of normal genetic background (wild type, WT). Moreover, after transfer into athymic mice of T cell isolated from WT ovariectomized animals (and so stimulated by estrogen deficiency to proliferate and to produce TNF-alpha), ovariectomy recovers its ability to induce bone loss; whereas there is no change in bone density after injection into athymic mice of T-cell purified from TNF-/- animals which, even if mature, are unable to produce TNF-alpha. Therefore the presence of TNF-alpha producing T-cell is essential for estrogen deficiency to influence bone metabolism. In the following study of the research group of Prof. Pacifici it has been shown that the increased activation of TNF-alpha producing T-cell in the ovariectomized mice is due to increased INF-gamma levels, resulting from ovariectomy-induced enhanced secretion of IL-12 and IL-18 by macrophages. INF-gamma promotes expression in immunocompetent cells of class II transactivator (CIITA), that, up-regulating expression of the major system of histocompatibility of class II, makes the macrophages more active in antigen presentation to T-cells, which in turn start producing TNF. For the first time an immune mechanism is involved in the pathogenesis of post-menopausal osteoporosis; nevertheless the applicability of these conclusions also in humans remains still to be proved.

Bone turnover in children and adolescents with McCune-Albright syndrome treated with pamidronate for bone fibrous dysplasia.

Bone fibrous dysplasia is one of the main features of McCune-Albright syndrome, a rare genetic condition caused by constitutive activating mutations of Gs-protein and defined by skin dysplasia, bone fibrous dysplasia, and autonomous multiple endocrinopathies. Raised serum alkaline phosphatase (ALP) and urinary hydroxyproline levels indicating bone metabolic hyperactivity have been reported in these patients. Encouraging therapeutic results have been achieved, mainly in adults, with pamidronate, an aminobisphosphonate. In this study we investigate newer bone metabolic indices in a cohort of 11 children and adolescents treated with pamidronate. Tenfold increases of bone ALP and urinary pyridinoline cross-links were found and osteocalcin levels were twofold higher compared with reference values. After treatment, significant decreases in bone ALP and cross-links (Wilcoxon test P < 0.06) were found. Bone mineral density (BMD) significantly increased during treatment. There were signs of radiological healing as thickening of the cortical bone was found in some cases.

Up-regulation of TNF-producing T cells in the bone marrow: a key mechanism by which estrogen deficiency induces bone loss in vivo.

In vivo studies have shown T cells to be central to the mechanism by which estrogen deficiency induces bone loss, but the mechanism involved remains, in part, undefined. In vitro, T cells from ovariectomized mice produce increased amounts of tumor necrosis factor (TNF), which augments receptor activator of NF-kappa B ligand (RANKL)-induced osteoclastogenesis. However, both the mechanism and the relevance of this phenomenon in vivo remain to be established. In this study, we found that ovariectomy increased the number of bone marrow T cell-producing TNF without altering production of TNF per T cell. Attesting to the essential contribution of TNF, ovariectomy induced rapid bone loss in wild type (wt) mice but failed to do so in TNF-deficient (TNF(-/-)) mice. Furthermore, ovariectomy induced bone loss, which was absent in T cell-deficient nude mice, was restored by adoptive transfer of wt T cells, but not by reconstitution with T cells from TNF(-/-) mice. These findings demonstrate the key causal role of T cell-produced TNF in the bone loss after estrogen withdrawal. Finally, ovariectomy caused bone loss in wt mice and in mice lacking p75 TNF receptor but failed to do so in mice lacking the p55 TNF receptor. These findings demonstrate that enhanced T cell production of TNF resulting from increased bone marrow T cell number is a key mechanism by which estrogen deficiency induces bone loss in vivo. The data also demonstrate that the bone-wasting effect of TNF in vivo is mediated by the p55 TNF receptor.

Estrogen deficiency induces bone loss by enhancing T-cell production of TNF-alpha.

Estrogen deficiency induces bone loss by upregulating osteoclastogenesis by mechanisms not completely defined. We found that ovariectomy-enhanced T-cell production of TNF-alpha, which, acting through the TNF-alpha receptor p55, augments macrophage colony-stimulating factor-induced (M-CSF-induced) and RANKL-induced osteoclastogenesis. Ovariectomy failed to induce bone loss, stimulate bone resorption, or increase M-CSF- and RANKL-dependent osteoclastogenesis in T-cell deficient mice, establishing T cells as essential mediators of the bone-wasting effects of estrogen deficiency in vivo. These findings demonstrate that the ability of estrogen to target T cells, suppressing their production of TNF-alpha, is a key mechanism by which estrogen prevents osteoclastic bone resorption and bone loss.

Bone turnover in hyperthyroidism before and after thyrostatic management.

Hyperthyroidism is associated with enhanced osteoblastic and osteoclastic activity, and patients frequently have low bone mineral density and high bone turnover. The aim of this study was to examine the bone formation and resorption markers trend in 12 female patients, before and after normalization of thyroid activity. The following measurements were made at baseline and 1 and 6 months after hormone normalization induced by methimazole treatment: total alkaline phosphatase (ALP), bone alkaline phosphatase (BALP), collagen type C-terminal propeptide (PICP), osteocalcin (BGP), telopeptide (ICTP), urinary-hydroxyproline/urinary creatinine (uOHP/uCreat), urinary calcium/urinary creatinine (uCa/uCreat) and deoxypyridinoline crosslinks (D-Pyr). Compared with controls, all of these parameters were significantly increased (ALP p = 0.014; BALP p = 0.0001; PICP p = 0.013; BGP p = 0.009; ICTP p = 0.0001; uOHP/uCreat p = 0.002; uCa/uCreat p = 0.044; crosslinks p = 0.0001). After treatment the values of ALP, BALP and PICP in hyperthyroid patients showed an initial slight increase and then a significant downwards trend (ALP p = 0.008, BAP p = 0.001, PICP p = 0.026). Furthermore, resorption markers showed a significant decrease (uOHP/ uCreat p < 0.005 and D-Pyr p < 0.008). As regards lumbar BMD patients, measurements were significantly reduced in comparison with the control group (p = 0.005). Six months after serum thyroid hormones level normalization, we observed a significant increase (p=0.014 vs baseline). Both neoformation and resorption markers are useful to assess pathological bone turnover and bone involvement in hyperthyroidism. They could also be employed to monitor the effect of antithyroid treatment on bone and to indicate if bone antiresorption therapy should be considered.

Bone disorders in cholestatic liver diseases.

Osteopenia is a recognised complication of cholestatic liver diseases (CLD), usually ascribed to metabolic bone diseases such as osteomalacia or osteoporosis, with a prevalence from 10 to 56%, depending on the nature of liver disease. Primary biliary cirrhosis (PBC) is the condition causing osteopenia more frequently, but other cholestatic liver diseases like primary sclerosing cholangitis (PSC), haemochromatosis and alcoholic liver disease are also frequently associated with this disorder. The pathogenesis of bone disease in both adults and children with chronic cholestasis is not completely understood. There has been considerable disagreement regarding the relative importance of osteomalacia versus osteoporosis as the factors leading to osteopenia of liver disease. Osteopenia predisposes to atraumatic fractures, particularly in PBC patients undergoing orthotopic liver transplantation and treated with high corticosteroid doses. Bone mineral density measurement is the best way to assess the presence and severity of osteopenia in CLD patients, while laboratory tests give important information about the metabolic status of the bone. In this review prevalence data, diagnostic tools, pathophysiology and treatment of osteopenia in CLD are discussed.