Mirko Zanatta

Circulating Mesenchymal Stem Cells With Abnormal Osteogenic Differentiation in Patients With Osteoporosis

OBJECTIVE While the role of osteoclasts in bone loss has been well investigated, the involvement of osteoblast-lineage cells has not been completely elucidated. Several genes contribute to normal osteoblastic differentiation from mesenchymal stem cells (MSCs), but an understanding of their role in the pathogenesis of osteoporosis is still lacking. The present study was undertaken to evaluate a possible alteration of osteogenic gene expression as a mechanism contributing to bone loss. METHODS We studied the osteogenic differentiation process in MSCs obtained from the peripheral blood of 31 patients with osteoporosis and 20 normal donors. The cells were evaluated by colony-forming unit-fibroblastic assay and cultured in osteogenic medium to analyze the transcription factors runt-related transcription factor 2 (RUNX-2) and Sp7 and the bone-related genes COL1A1, SPARC, and SPP1 after 3, 8, and 15 days of differentiation. In addition, to determine possible differences between the 2 groups in terms of osteoclastic and osteoblastic activation, we quantified the osteoprotegerin (OPG) and RANKL levels in the supernatants of osteoblastic culture. RESULTS Circulating MSCs were increased in osteoporosis patients compared with normal donors. In contrast, gene expression analysis revealed down-regulation of RUNX2, Sp7, COL1A1, SPARC, and SPP1 in patients with osteoporosis, associated with a lower OPG:RANKL ratio. CONCLUSION These results suggest that an alteration of osteoblastic differentiation may contribute to the pathogenesis of osteoporosis. The noninvasive approach used in the present study could be proposed as a useful tool for studying mesenchymal involvement in bone diseases.

Serum 25-hydroxyvitamin D levels modulate the acute-phase response associated with the first nitrogen-containing bisphosphonate infusion

The acute‐phase response (APR) is the most frequent side effect after the first dose of intravenous nitrogen‐containing bisphosphonates (N‐BPs). It has been demonstrated in vitro that N‐BPs stimulate γδ T‐cell proliferation and production of cytokines and that vitamin D is able to modulate them. Therefore, we have studied the relationship between bone metabolism parameters, particularly for 25‐hydroxyvitamin D [25(OH)D], and APR in patients treated with 5 mg zoledronic acid intravenously. Ninety N‐BP‐naive osteoporotic women (63.7 ± 10.6 years of age) were stratified for the occurrence of APR (APR+) or not (APR–) and quantified by body temperature and C‐reactive protein (CRP). The APR+ women had significantly lower 25(OH)D levels than the APR– women. Levels of 25(OH)D were normal (>30 ng/mL) in 31% of APR+ women and in 76% of APR– women. The odds ratio (OR) to have APR in 25(OH)D‐depleted women was 5.8 [95% confidence interval (CI) 5.30–6.29; p < .0002] unadjusted and 2.38 (95% CI 1.85–2.81; p < .028) after multiple adjustments (for age, body mass index, CRP, calcium, parathyroid hormone, and C‐telopeptide of type I collagen). Levels of 25(OH)D were negatively correlated with postdose body temperature (r = −0.64, p < .0001) and CRP (r = −0.79, p < .001). An exponential increase in fever and CRP has been found with 25(OH)D levels lower than 30 ng/mL and body temperature less than 37 °C, whereas normal CRP was associated with 25(OH)D levels above 40 ng/mL. The association between post‐N‐BPs APR and 25(OH)D suggests an interesting interplay among N‐BPs, 25(OH)D, and the immune system, but a causal role of 25(OH)D in APR has to be proven by a randomized, controlled trial. However, if confirmed, it should have some practical implications in preventing APR.

Gene expression analysis in osteoblastic differentiation from peripheral blood mesenchymal stem cells

MSCs are known to have an extensive proliferative potential and ability to differentiate in various cell types. Osteoblastic differentiation from mesenchymal progenitor cells is an important step of bone formation, though the pattern of gene expression during differentiation is not yet well understood. Here, to investigate the possibility to obtain a model for in vitro bone differentiation using mesenchymal stem cells (hMSCs) from human subjects non-invasively, we developed a method to obtain hMSCs-like cells from peripheral blood by a two step method that included an enrichment of mononuclear cells followed by depletion of unwanted cells. Using these cells, we analyzed the expression of transcription factor genes (runt-related transcription factor 2 (RUNX2) and osterix (SP7)) and bone related genes (osteopontin (SPP1), osteonectin (SPARC) and collagen, type I, alpha 1 (COLIA1)) during osteoblastic differentiation. Our results demonstrated that hMSCs can be obtained from peripheral blood and that they are able to generate CFU-F and to differentiate in osteoblast and adipocyte; in this study, we also identified a possible gene expression timing during osteoblastic differentiation that provided a powerful tool to study bone physiology.

The effect of bisphosphonates on gene expression: GAPDH as a housekeeping or a new target gene?

BackgroundRT-PCR has been widely used for the analysis of gene expression in many systems, including tumor samples. GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) has been frequently considered as a constitutive housekeeping gene and used to normalize changes in specific gene expression. However, GAPDH has been shown to be up-regulated in many cancers and down-regulated by chemotherapic drugs. Bisphosphonates, potent inhibitors of bone resorption, have recently shown a direct and indirect antitumor effect in vitro and in animal models. They exert their effects mainly by inhibiting the mevalonate pathway but also by modulating the expression of many genes not only in osteoclasts but also in cancer cells.MethodsWe evaluated GAPDH gene expression by real time RT PCR in breast (MCF-7 and T47D) and prostate (PC3 and DU-145) cancer cell lines treated with amino and non-amino bisphosphonates.ResultsOur results showed that amino-bisphosphonates significantly decrease in a dose-dependent manner the expression of GAPDH gene.ConclusionTherefore, GAPDH is inaccurate to normalize mRNA levels in studies investigating the effect of bisphosphonates on gene expression and it should be avoided. On the other hand, this gene could be considered a potential target to observe the effects of bisphosphonates on cancer cells.

Role of Ox-PAPCs in the Differentiation of Mesenchymal Stem Cells (MSCs) and Runx2 and PPARγ2 Expression in MSCs-Like of Osteoporotic Patients

Background Mesenchymal stem cells (MSCs) can differentiate into osteoblasts and adipocytes and conditions causing bone loss may induce a switch from the osteoblast to adipocyte lineage. In addition, the expression of Runx2 and the PPARγ2 transcription factor genes is essential for cellular commitment to an osteogenic and adipogenic differentiation, respectively. Modified lipoproteins derived from the oxidation of arachidonate-containing phospholipids (ox-PAPCs: POVPC, PGPC and PEIPC) are considered important factors in atherogenesis. Methodology We investigated the effect of ox-PAPCs on osteogenesis and adipogenesis in human mesenchymal stem cells (hMSCs). In particular, we analyzed the transcription factor Runx2 and the PPARγ2 gene expression during osteogenic and adipogenic differentiation in absence and in presence of ox-PAPCs. We also analyzed gene expression level in a panel of osteoblastic and adipogenic differentiation markers. In addition, as circulating blood cells can be used as a “sentinel” that responds to changes in the macro- or micro-environment, we analyzed the Runx2 and the PPARγ2 gene expression in MSCs-like and ox-PAPC levels in serum of osteoporotic patients (OPs). Finally, we examined the effects of sera obtained from OPs in hMSCs comparing the results with age-matched normal donors (NDs). Principal findings Quantitative RT-PCR demonstrated that ox-PAPCs enhanced PPARγ2 and adipogenic gene expression and reduced Runx2 and osteoblast differentiation marker gene expression in differentiating hMSCs. In OPs, ox-PAPC levels and PPARγ2 expression were higher than in NDs, whereas Runx2 was lower than in ND circulant MSCs-like. Conclusions Ox-PAPCs affect the osteogenic differentiation by promoting adipogenic differentiation and this effect may appear involved in bone loss in OPs.

Safety and tolerability of zoledronic acid and other bisphosphonates in osteoporosis management

Bisphosphonates (BPs) are widely used in the treatment of postmenopausal osteoporosis and other metabolic bone diseases. They bind strongly to bone matrix and reduce bone loss through inhibition of osteoclast activity. They are classified as nitrogen- and non-nitrogen-containing bisphosphonates (NBPs and NNBPs, respectively). The former inhibit farnesyl diphosphate synthase while the latter induce the production of toxic analogs of adenosine triphosphate. These mechanisms of action are associated with different antifracture efficacy, and NBPs show the most powerful action. Moreover, recent evidence indicates that NBPs can also stimulate osteoblast activity and differentiation. Several randomized control trials have demonstrated that NBPs significantly improve bone mineral density, suppress bone turnover, and reduce the incidence of both vertebral and nonvertebral fragility fractures. Although they are generally considered safe, some side effects are reported (esophagitis, acute phase reaction, hypocalcemia, uveitis), and compliance with therapy is often inadequate. In particular, gastrointestinal discomfort is frequent with the older daily oral administrations and is responsible for a high proportion of discontinuation. The most recent weekly and monthly formulations, and in particular the yearly infusion of zoledronate, significantly improve persistence with treatment, and optimize clinical, densitometric, and antifracture outcomes.

Densitometric Threshold and Vertebral Fractures in Heart Transplant Patients

Background. Bone disease is one of the major complications of solid organ transplantation, causes considerable morbidity, and most patients are treated with immunosuppressant drugs after graft. The majority of studies reported rapid bone loss and an increased incidence of fractures after transplantation. The aim of our study was to evaluate osteoporosis and fracture prevalence, bone metabolism, and the effect of immunosuppressant agents on bone after heart transplantation. Methods. We planned a cross-sectional study in 180 heart transplant patients recruited from 3 different centers with a less than 10 years from graft. Each patient underwent a densitometric scan, and in 157 of them, an x-ray of the spine was performed to evaluate fractures. Biochemical assessment of bone metabolism was made at the time of the visit. Physical activity, diet, and calcium intake were evaluated using a specific questionnaire. Results. Vertebral fractures were diagnosed in 40% of subjects, but densitometric osteoporosis was observed only in 13% of spine and in 25% of hip scans. Interestingly, increasing T-score threshold up to −1.5 standard deviation, the prevalence of fractured patient improved significantly, reaching 60% in both genders. Bone content was inversely correlated with glucocorticoids, while a positive correlation was found with cyclosporine A. Almost all subjects had vitamin D deficiency. Conclusions. Standard densitometric criteria are unreliable to identify bone fragility after transplantation, and a different threshold (−1.5 standard deviation) should be considered. Transplanted patients should be adequately supplemented with vitamin D, and the effects of immunosuppressant agents on bone need further investigation.

Runx-2 gene expression is associated with age-related changes of bone mineral density in the healthy young-adult population

Bone mineral density (BMD) and peak bone mass (PBM) are important determinants of skeletal resistance. The development of bone densitometry improved the possibility of studying BMD and the influence of genetic and environmental factors on bone. Heredity factors are important for BMD, and Runx-2 is accepted as a regulator of osteoblasts and bone formation. The aim of our study was to evaluate the behavior of Runx-2 during skeletal maturity in the healthy young-adult population. We analyzed spine and hip BMD in 153 volunteers, 98 women and 55 men, using dual-energy X-ray absorptiometry. In a subgroup of these volunteers, a sample of peripheral blood was taken to perform gene expression analysis of Runx-2 both in peripheral mesenchymal stem cells (MSCs; 28 subjects) and in peripheral mononuclear cells (PBMCs; 140 subjects). In our work BMD was comparable in both genders after puberty, then became higher in men than women during the third and fourth decades. PBM was achieved in the third decade in women and in the fourth in men. More interestingly, Runx-2 gene expression highly correlated with BMD in both genders. MSCs and PBMCs showed the same gene expression profile of Runx-2. In conclusion, PBM is reached earlier in females, BMD becomes higher in males later in life, and BMD and PBM are strictly associated with Runx-2. In addition, PBMC should be considered an important source for gene expression analysis in bone diseases.

The effect of risedronate on osteogenic lineage is mediated by cyclooxygenase-2 gene upregulation.

IntroductionThe purpose of this study was to evaluate the effects of risedronate (Ris) in the modulation of bone formation in rats with glucocorticoid (GC)-induced osteoporosis by histomorphometric, immunohistochemical and gene expression analyses.MethodsWe analyzed structure, turnover and microarchitecture, cyclooxygenase 2 (COX-2) levels and osteocyte apoptosis in 40 female rats divided as follows: 1) vehicle of methylprednisolone (vGC) + vehicle of risedronate (vRis); 2) Ris 5 μg/Kg + vGC; 3) methylprednisolone (GC) 7 mg/Kg + vRis; 4) GC 7 mg/Kg +Ris 5 μg/Kg. In addition, we evaluated cell proliferation and expression of COX-2 and bone alkaline phosphatase (b-ALP) genes in bone marrow cells and MLO-y4 osteocytes treated with Ris alone or in co-treatment with the selective COX-2 inhibitor NS-398 or with dexametasone.ResultsRis reduced apoptosis induced by GC of osteocytes (41% vs 86%, P < 0.0001) and increased COX-2 expression with respect to controls (Immuno-Hystochemical Score (IHS): 8.75 vs 1.00, P < 0.0001). These positive effects of Ris in bone formation were confirmed by in vitro data as the viability and expression of b-ALP gene in bone marrow cells resulted increased in a dose dependent manner.ConclusionsThese findings suggest a positive effect of Ris in bone formation and support the hypothesis that the up-regulation of COX-2 could be an additional mechanism of anabolic effect of Ris.