Juan Ignacio Felice

Opposing effects of bisphosphonates and advanced glycation end-products on osteoblastic cells

Patients with long-standing Diabetes mellitus can develop osteopenia and osteoporosis. We have previously shown that advanced glycation endproducts reduce the bone-forming activity of osteoblasts. Bisphosphonates are used for the treatment of various bone disorders, since they reduce osteoclastic function and survival, and stimulate osteoblastic bone-forming capacity. In this work we have investigated whether bisphosphonates are able to revert advanced glycation endproducts-induced deleterious effects in osteoblasts. MC3T3E1 and UMR106 osteoblastic cells were incubated with control or advanced glycation endproducts-modified bovine serum albumin, in the presence or absence of different doses of the bisphosphonates Alendronate, Pamidronate or Zoledronate. After 24-72 h of culture, we evaluated their effects on cell proliferation and apoptosis, type-1 collagen production, alkaline and neutral phosphatase activity, and intracellular reactive oxygen species production. Advanced glycation endproducts significantly decreased osteoblast proliferation, alkaline phosphatase activity and type 1 collagen production, while increasing osteoblastic apoptosis and reactive oxygen species production. These effects were completely reverted by low doses (10(-8) M) of bisphosphonates. High doses of bisphosphonates (10(-4)-10(-5) M) were toxic for osteoblasts. Nifedipine (L-type calcium channel blocker) did not affect the advanced glycation endproducts-induced decrease in osteoblastic proliferation, although it blocked the reversion of this effect by 10(-8) M Alendronate. Both advanced glycation endproducts and Alendronate inhibited the activity of intracellular neutral phosphatases. In conclusion, we show that bisphosphonates revert the deleterious actions of advanced glycation endproducts on osteoblastic cells, and that these effects of bisphosphonates depend on: (a) Ca(2+) influx through L-type voltage-sensitive channels, and (b) blockage of advanced glycation endproducts-induced reactive oxygen species generation.

Metformin prevents anti-osteogenic in vivo and ex vivo effects of rosiglitazone in rats

Long-term treatment with the insulin-sensitizer rosiglitazone reduces bone mass and increases fracture risk. We have recently shown that orally administered metformin stimulates bone reossification and increases the osteogenic potential of bone marrow progenitor cells (BMPC). In the present study we investigated the effect of a 2-week metformin and/or rosiglitazone treatment on bone repair, trabecular bone microarchitecture and BMPC osteogenic potential, in young male Sprague-Dawley rats. Compared to untreated controls, rosiglitazone monotherapy decreased bone regeneration, femoral metaphysis trabecular area, osteoblastic and osteocytic density, and TRAP activity associated with epiphyseal growth plates. It also decreased the ex vivo osteogenic commitment of BMPC, inducing an increase in PPARγ expression, and a decrease in Runx2/Cbfa1 expression, in AMP-kinase phosphorylation, and in osteoblastic differentiation and mineralization. After monotherapy with metformin, with the exception of PPARγ expression which was blunted, all of the above parameters were significantly increased (compared to untreated controls). Metformin/rosiglitazone co-treatment prevented all the in vivo and ex vivo anti-osteogenic effects of rosiglitazone monotherapy, with a reversion back to control levels of PPARγ, Runx2/Cbfa1 and AMP-kinase phosphorylation of BMPC. In vitro co-incubation of BMPC with metformin and compound C-an inhibitor of AMPK phosphorylation-abrogated the metformin-induced increase in type-1 collagen production, a marker of osteoblastic differentiation. In conclusion, in rodent models metformin not only induces direct osteogenic in vivo and ex vivo actions, but when it is administered orally in combination with rosiglitazone it can prevent several of the adverse effects that this thiazolidenedione shows on bone tissue.

Effects of a metabolic syndrome induced by a fructose-rich diet on bone metabolism in rats

OBJECTIVE The aims of this study were: first, to evaluate the possible effects of a fructose rich diet (FRD)-induced metabolic syndrome (MS) on different aspects of long bone histomorphometry in young male rats; second, to investigate the effects of this diet on bone tissue regeneration; and third, to correlate these morphometric alterations with changes in the osteogenic/adipogenic potential and expression of specific transcription factors, of marrow stromal cells (MSC) isolated from rats with fructose-induced MS. MATERIALS/METHODS MS was induced in rats by treatment with a FRD for 28 days. Halfway through treatment, a parietal wound was made and bone healing was evaluated 14 days later. After treatments, histomorphometric analysis was performed in dissected femoral and parietal bones. MSC were isolated from the femora of control or fructose-treated rats and differentiated either to osteoblasts (evaluated by type 1 collagen, Alkaline phosphatase and extracellular nodule mineralization) or to adipocytes (evaluated by intracellular triglyceride accumulation). Expression of Runx2 and PPARγ was assessed by Western blot. RESULTS Fructose-induced MS induced deleterious effects on femoral metaphysis microarchitecture and impaired bone regeneration. Fructose treatment decreased the osteogenic potential of MSC and Runx2 expression. In addition, it increased the adipogenic commitment of MSC and PPARγ expression. CONCLUSIONS Fructose-induced MS is associated with deleterious effects on bone microarchitecture and with a decrease in bone repair. These alterations could be due to a deviation in the adipogenic/osteogenic commitment of MSC, probably by modulation of the Runx2/PPARγ ratio.

Effects of fructose-induced metabolic syndrome on rat skeletal cells and tissue, and their responses to metformin treatment

AIMS Deleterious effects of metabolic syndrome (MS) on bone are still controversial. In this study we evaluated the effects of a fructose-induced MS, and/or an oral treatment with metformin on the osteogenic potential of bone marrow mesenchymal stromal cells (MSC), as well as on bone formation and architecture. METHODS 32 male 8week-old Wistar rats were assigned to four groups: control (C), control plus oral metformin (CM), rats receiving 10% fructose in drinking water (FRD), and FRD plus metformin (FRDM). Samples were collected to measure blood parameters, and to perform pQCT analysis and static and dynamic histomorphometry. MSC were isolated to determine their osteogenic potential. RESULTS Metformin improved blood parameters in FRDM rats. pQCT and static and dynamic histomorphometry showed no significant differences in trabecular and cortical bone parameters among groups. FRD reduced TRAP expression and osteocyte density in trabecular bone and metformin only normalized osteocyte density. FRD decreased the osteogenic potential of MSC and metformin administration could revert some of these parameters. CONCLUSIONS FRD-induced MS shows reduction in MSC osteogenic potential, in osteocyte density and in TRAP activity. Oral metformin treatment was able to prevent trabecular osteocyte loss and the reduction in extracellular mineralization induced by FRD-induced MS.

Experimental assessment of a myocyte-based multiscale model of cardiac contractile dysfunction

Cardiac contractile dysfunction (CD) is a multifactorial syndrome caused by different acute or progressive diseases which hamper assessing the role of the underlying mechanisms characterizing a defined pathological condition. Mathematical modeling can help to understand the processes involved in CD and analyze their relative impact in the overall response. The aim of this study was thus to use a myocyte-based multiscale model of the circulatory system to simulate the effects of halothane, a volatile anesthetic which at high doses elicits significant acute CD both in isolated myocytes and intact animals. Ventricular chambers built using a human myocyte model were incorporated into a whole circulatory system represented by resistances and capacitances. Halothane-induced decreased sarco(endo)plasmic reticulum Ca2+ (SERCA2a) reuptake pump, transient outward K+ (Ito), Na+-Ca2+ exchanger (INCX) and L-type Ca2+ channel (ICaL) currents, together with ryanodine receptor (RyR2) increased open probability (Po) and reduced myofilament Ca2+ sensitivity, reproduced equivalent decreased action potential duration at 90% repolarization and intracellular Ca2+ concentration at the myocyte level reported in the literature. In the whole circulatory system, model reduction in mean arterial pressure, cardiac output and regional wall thickening fraction was similar to experimental results in open-chest sheep subjected to acute halothane overdose. Effective model performance indicates that the model structure could be used to study other changes in myocyte targets eliciting CD.