Daniel Rivas

Alendronate has an anabolic effect on bone through the differentiation of mesenchymal stem cells.

We committed MSCs to differentiate into either osteoblasts or adipocytes and examined the effect of ALN on both adipogenesis and osteoblastogenesis. ALN inhibited adipogenesis while promoting osteoblast differentiation and activity. Our results reveal a new anabolic effect of ALN in differentiating bone marrow cells.

Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro.

Although increased bone marrow fat in age‐related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two‐chamber system to co‐culture normal human osteoblasts (NHOst) with differentiating pre‐adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell–cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co‐culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS‐formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte‐conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age‐related changes in bone mass and can be prevented by the inhibition of FA synthase.

Interferon-γ plays a role in bone formation in vivo and rescues osteoporosis in ovariectomized mice

Interferon γ (IFN‐γ) is a cytokine produced locally in the bone microenvironment by cells of immune origin as well as mesenchymal stem cells. However, its role in normal bone remodeling is still poorly understood. In this study we first examined the consequences of IFN‐γ ablation in vivo in C57BL/6 mice expressing the IFN‐γ receptor knockout phenotype (IFNγR1−/−). Compared with their wild‐type littermates (IFNγR1+/+), IFNγR1−/− mice exhibit a reduction in bone volume associated with significant changes in cortical and trabecular structural parameters characteristic of an osteoporotic phenotype. Bone histomorphometry of IFNγR1−/− mice showed a low‐bone‐turnover pattern with a decrease in bone formation, a significant reduction in osteoblast and osteoclast numbers, and a reduction in circulating levels of bone‐formation and bone‐resorption markers. Furthermore, administration of IFN‐γ (2000 and 10,000 units) to wild‐type C57BL/6 sham‐operated (SHAM) and ovariectomized (OVX) female mice significantly improved bone mass and microarchitecture, mechanical properties of bone, and the ratio between bone formation and bone resorption in SHAM mice and rescued osteoporosis in OVX mice. These data therefore support an important physiologic role for IFN‐γ signaling as a potential new anabolic therapeutic target for osteoporosis.

Effect of Lamin A/C Knockdown on Osteoblast Differentiation and Function†

Recent studies have associated mutations in lamin A/C, a component of the nuclear lamina, with premature aging and severe bone loss. In this study, we hypothesized that reduced expression of lamin A/C has a negative impact on osteoblastogenesis and bone formation in vitro. We inhibited lamin A/C using increasing doses of lamin A/C siRNA in normal human osteoblasts and differentiating mesenchymal stem cells (MSCs). Untreated cells and cells treated with vehicle but without the siRNA‐oligo were used as control. The level of effectiveness of siRNA was determined by RT‐PCR, Western blot, and immunofluorescence. Nuclear blebbing, a typical finding of lamin A/C inhibition, was quantified using propidium iodine staining, and its effect on cell survival was determined using MTS‐formazan. Furthermore, alizarin red and alkaline phosphatase staining were correlated with osteocalcin secretion and levels of expression of osteocalcin, osterix, bone sialoprotein, and Runx2. Finally, the nuclear binding activity of Runx2, an essential transcription factor for osteoblast differentiation, was assessed using ELISA and EMSA. A successful inhibitory effect on the lamin A/C gene at doses of 400–800 nM oligo was obtained without affecting cell survival. Whereas osteoblast function was significantly affected by lamin A/C inhibition, siRNA‐treated MSC showed a higher incidence of nuclear changes, lower osteoblast differentiation, and enhanced adipocyte differentiation. Finally, lamin A/C knockdown reduced Runx2 nuclear binding activity without affecting Runx2 expression. In summary, our results indicate that lamin A/C is a new factor needed for osteoblast differentiation that plays an important role in the cellular mechanisms of age‐related bone loss.

Autocrine Regulation of Interferon γ in Mesenchymal Stem Cells plays a Role in Early Osteoblastogenesis

Interferon (IFN)γ is a strong inhibitor of osteoclast differentiation and activity. However, its role in osteoblastogenesis has not been carefully examined. Using microarray expression analysis, we found that several IFNγ‐inducible genes were upregulated during early phases of osteoblast differentiation of human mesenchymal stem cells (hMSCs). We therefore hypothesized that IFNγ may play a role in this process. We first observed a strong and transient increase in IFNγ production following hMSC induction to differentiate into osteoblasts. We next blocked this endogenous production using a knockdown approach with small interfering RNA and observed a strong inhibition of hMSC differentiation into osteoblasts with a concomitant decrease in Runx2, a factor indispensable for osteoblast development. Additionally, exogenous addition of IFNγ accelerated hMSC differentiation into osteoblasts in a dose‐dependent manner and induced higher levels of Runx2 expression during the early phase of differentiation. We next examined IFNγ signaling in vivo in IFNγ receptor 1 knockout (IFNγR1−/−) mice. Compared with their wild‐type littermates, IFNγR1−/− mice exhibited a reduction in bone mineral density. As in the in vitro experiments, MSCs obtained from IFNγR1−/− mice showed a lower capacity to differentiate into osteoblasts. In summary, we demonstrate that the presence of IFNγ plays an important role during the commitment of MSCs into the osteoblastic lineage both in vitro and in vivo, and that this process can be accelerated by exogenous addition of IFNγ. These data therefore support a new role for IFNγ as an autocrine regulator of hMSC differentiation and as a potential new target of bone‐forming cells in vivo. STEM CELLS 2009;27:550–558

Age-related changes in lamin A/C expression in the osteoarticular system : Laminopathies as a potential new aging mechanism

BACKGROUND Mutations in lamin A/C have been described as associated to severe changes in bone and joints. In this study we hypothesize that the expression of lamin A/C may play a role in the pathogenesis of age-related diseases in the osteoarticular system. METHODOLOGY C57BL/6 young and old mice (4 months; n=10 and 24 months; n=10) were sacrificed. Limbs were isolated for histopathological and Western blot analysis. The proportion of cells (osteoblasts and chondrocytes) positive for lamin A/C was quantified by immunohistochemistry. Lamin B1 was used as control. Finally, lamin A/C expression in bone marrow cells was quantified by Western blot. RESULTS A significant reduction in lamin A/C was found in osteoblasts of old as compared to young mice (42% versus 76%, p<0.001). Interestingly, lamin A/C but not lamin B1 expression was found in bone matrix with higher levels in young bone. Additionally, a significant reduction in the number of lamin A/C expressing chondrocytes was seen in old mice as compared to young mice (32% versus 84%, p<0.001). Finally, a reduction in lamin A/C expression was found in bone marrow cells obtained from old mice as compared to young mice. CONCLUSION This is the first assessment of the age-related changes in lamin A/C expression in the osteoarticular system. We conclude that with aging there is a reduction in lamin A/C expression which could have a significance on osteoarticular cells function and viability.

Myocardial Infarction–Associated SNP at 6p24 Interferes With MEF2 Binding and Associates With PHACTR1 Expression Levels in Human Coronary Arteries

Objective—Coronary artery disease (CAD), including myocardial infarction (MI), is the main cause of death in the world. Genome-wide association studies have identified dozens of single nucleotide polymorphisms (SNPs) associated with CAD/MI. One of the most robust CAD/MI genetic associations is with intronic SNPs in the gene PHACTR1 on chromosome 6p24. How these PHACTR1 SNPs influence CAD/MI risk, and whether PHACTR1 itself is the causal gene at the locus, is currently unknown. Approach and Results—Using genetic fine-mapping and DNA resequencing experiments, we prioritized an intronic SNP (rs9349379) in PHACTR1 as causal variant. We showed that this variant is an expression quantitative trait locus for PHACTR1 expression in human coronary arteries. Experiments in endothelial cell extracts confirmed that alleles at rs9349379 are differentially bound by the transcription factors myocyte enhancer factor-2. We engineered a deletion of this myocyte enhancer factor-2–binding site using CRISPR/Cas9 genome-editing methodology. Heterozygous endothelial cells carrying this deletion express 35% less PHACTR1. Finally, we found no evidence that PHACTR1 expression levels are induced when stimulating human endothelial cells with vascular endothelial growth factor, tumor necrosis factor-&agr;, or shear stress. Conclusions—Our results establish a link between intronic SNPs in PHACTR1, myocyte enhancer factor-2 binding, and transcriptional functions at the locus, PHACTR1 expression levels in coronary arteries and CAD/MI risk. Because PHACTR1 SNPs are not associated with the traditional risk factors for CAD/MI (eg, blood lipids or pressure, diabetes mellitus), our results suggest that PHACTR1 may influence CAD/MI risk through as yet unknown mechanisms in the vascular endothelium.

Pharmacological inhibition of PPARγ increases osteoblastogenesis and bone mass in male C57BL/6 mice

Infiltration of bone marrow with fat is a prevalent feature in people with age‐related bone loss and osteoporosis, which correlates inversely with bone formation and positively with high expression levels of peroxisomal proliferator‐activated receptor gamma (PPARγ). Inhibition of PPARγ thus represents a potential therapeutic approach for age‐related bone loss. In this study, we examined the effect of PPARγ inhibition on bone in skeletally mature C57BL/6 male mice. Nine‐month‐old mice were treated with a PPARγ antagonist, bisphenol‐A‐diglycidyl ether (BADGE), alone or in combination with active vitamin D (1,25[OH]2D3) for 6 weeks. Micro‐computed tomography and bone histomorphometry indicated that mice treated with either BADGE or BADGE + 1,25(OH)2D3 had significantly increased bone volume and improved bone quality compared with vehicle‐treated mice. This phenotype occurred in the absence of alterations in osteoclast number. Furthermore, the BADGE + 1,25(OH)2D3‐treated mice exhibited higher levels of unmineralized osteoid. All of the treated groups showed a significant increase in circulating levels of bone formation markers without changes in bone resorption markers, while blood glucose, parathyroid hormone, and Ca+ remained normal. Furthermore, treatment with BADGE induced higher levels of expression of vitamin D receptor within the bone marrow. Overall, treated mice showed higher levels of osteoblastogenesis and bone formation concomitant with decreased marrow adiposity and ex vivo adipogenesis. Taken together, these observations demonstrate that pharmacological inhibition of PPARγ may represent an effective anabolic therapy for osteoporosis in the near future.

Shear Stress Regulates Angiotensin Type 1 Receptor Expression in Endothelial Cells

Rationale: Shear stress (SS) has an established role in atherosclerotic plaque localization, but how it exerts its protective effect is not fully understood. Objective: To test the hypothesis that SS may downregulate angiotensin type 1 receptors (AT1Rs). Angiotensin II has been shown to be proinflammatory and to promote atherosclerosis. Methods and Results: Using immunohistochemistry, we found a pronounced expression of AT1R in the inner, atheroprone regions of the aortic arch of C57BL/6 and endothelial NO synthase–deficient (eNOS−/−) mice but not eNOS-overexpressing mice. In human umbilical vein endothelial cells (HUVECs), laminar SS (15 dyn/cm2) induced a biphasic decrease in AT1R protein expression characterized by a first reduction at 1 hour (31±4% of static control, P<0.01), partial recovery at 3 hours (65±9%), and a second more prolonged decline at 6, 12, and 24 hours (48±9%, 36±9%, 33±5%, respectively, P<0.05). One and 24 hours of SS significantly reduced fluorescent angiotensin binding compared to static HUVECs. Shear-induced downregulation of AT1R was abolished by treatment with protein kinase A and G inhibitors or NG-nitro-l-arginine methyl ester (L-NAME). Fittingly, stimulating static HUVECs with an NO donor decreased AT1R protein levels. RT-PCR revealed a significant (P<0.05) decrease of AT1R mRNA in HUVECs exposed to SS during 3 (6±2% of static control), 6 (4±1%), 12 (4±1%), and 24 hours (15±4%), suggesting a transcriptional downregulation of AT1R at length. Finally, angiotensin-induced vascular cell adhesion molecule was abated in HUVECs exposed to SS and in the outer aortic arch of mice. Conclusions: Our results demonstrate that SS may convey some of its atheroprotective effects through downregulation of AT1R in endothelial cells.

Accelerated Features of Age-Related Bone Loss in Zmpste24 Metalloproteinase-Deficient Mice

Age-related bone loss is associated with changes in bone cellularity, which include marrow fat infiltration and decreasing levels of osteoblastogenesis. The mechanisms that explain these changes remain unclear. Although nuclear lamina alterations occur in premature aging syndromes that include changes in body fat and severe osteoporosis, the role of proteins of the nuclear lamina in age-related bone loss remains unknown. Using the Zmpste24-null progeroid mice (Zmpste24(-/-)), which exhibit nuclear lamina defects and accumulate unprocessed prelamin A, we identified several alterations in bone cellularity in vivo. We found that defective prelamin A processing induced accelerated features of age-related bone loss including lower osteoblast and osteocyte numbers and higher levels of marrow adipogenesis. In summary, processing of prelamin A could become a new approach to regulate osteoblastogenesis and bone turnover and thus for the prevention and treatment of senile osteoporosis.