The balance between bone forming cells (osteoblasts/osteocytes) and bone resorbing cells (osteoclasts) plays a crucial role in tissue homeostasis and bone repair. Several hormones, cytokines, and growth factors—in particular the members of the TGF-β superfamily such as the bone morphogenetic proteins—not only regulate the proliferation, differentiation, and functioning of these cells, but also coordinate the communication between them to ensure an appropriate response. Therefore, this review focuses on TGF-β superfamily and its influence on bone formation and repair, through the regulation of osteoclastogenesis, osteogenic differentiation of stem cells, and osteoblasts/osteoclasts balance. After introducing the main types of bone cells, their differentiation and cooperation during bone remodeling and fracture healing processes are discussed. Then, the TGF-β superfamily, its signaling via canonical and non-canonical pathways, as well as its regulation by Wnt/Notch or microRNAs are described and discussed. Its important role in bone homeostasis, repair, or disease is also highlighted. Finally, the clinical therapeutic uses of members of the TGF-β superfamily and their associated complications are debated.

Background-Aortic valve regurgitation (AR) is a volume-overload disease causing severe eccentric left ventricular (LV) hypertrophy and eventually heart failure. There is currently no approved drug to treat patients with AR. Many vasodilators including angiotensin-converting enzyme inhibitors have been evaluated in clinical trials, but although some results were promising, others were inconclusive. Overall, no drug has yet been able to improve clinical outcome in AR and the controversy remains. We have previously shown in an animal model that captopril (Cpt) reduced LV hypertrophy and protected LV systolic function, but we had not evaluated the clinical outcome. This protocol was designed to evaluate the effects of a long-term Cpt treatment on survival in the same animal model of severe aortic valve regurgitation. Methods and Results-Forty Wistar rats with AR were treated or untreated with Cpt (1 g/L in drinking water) for a period of 7 months to evaluate survival, myocardial remodeling, and function by echocardiography as well as myocardial metabolism by µ positron emission tomography scan. Survival was significantly improved in Cpt-treated animals with a survival benefit visible as soon as after 4 months of treatment. Cpt reduced LV dilatation and LV hypertrophy. It also significantly improved the myocardial metabolic profile by restoring the level of fatty acids metabolic enzymes and use. Conclusions-In a controlled animal model of pure severe aortic valve regurgitation, Cpt treatment reduced LV remodeling and LV hypertrophy and improved myocardial metabolic profile and survival. These results support the need to reevaluate the role of angiotensin-converting enzyme inhibitors in humans with AR in a large, carefully designed prospective clinical trial. (Circ Heart Fail. 2013;6:1021-1028.)

Sunitinib is a tyrosine kinase inhibitor approved for the treatment of multiple solid tumors. However, cardiotoxicity is of increasing concern, with a need to develop rational mechanism driven approaches for the early detection of cardiac dysfunction. We sought to interrogate changes in cardiac energy substrate usage during sunitinib treatment, hypothesising that these changes could represent a strategy for the early detection of cardiotoxicity. Balb/CJ mice or Sprague-Dawley rats were treated orally for 4 weeks with 40 or 20 mg/kg/day sunitinib. Cardiac positron emission tomography (PET) was implemented to investigate alterations in myocardial glucose and oxidative metabolism. Following treatment, blood pressure increased, and left ventricular ejection fraction decreased. Cardiac [18F]-fluorodeoxyglucose (FDG)-PET revealed increased glucose uptake after 48 hours. [11C]Acetate-PET showed decreased myocardial perfusion following treatment. Electron microscopy revealed significant lipid accumulation in the myocardium. Proteomic analyses indicated that oxidative metabolism, fatty acid β-oxidation and mitochondrial dysfunction were among the top myocardial signalling pathways perturbed. Sunitinib treatment results in an increased reliance on glycolysis, increased myocardial lipid deposition and perturbed mitochondrial function, indicative of a fundamental energy crisis resulting in compromised myocardial energy metabolism and function. Our findings suggest that a cardiac PET strategy may represent a rational approach to non-invasively monitor metabolic pathway remodeling following sunitinib treatment.

Schizosaccharomyces pombe Rmt3 is a member of the proteinarginine methyltransferase (PRMT) family and is the homolog of human PRMT3. We previously characterized Rmt3 as a ribosomal protein methyltransferase based on the identification of the 40 S Rps2 (ribosomal protein S2) as a substrate of Rmt3. RMT3-null cells produce nonmethylated Rps2 and show misregulation of the 40 S/60 S ribosomal subunit ratio due to a small subunit deficit. For this study, we have generated a series of RMT3 alleles that express various amino acid substitutions to characterize the functional domains of Rmt3 in Rps2 binding, Rps2 arginine methylation, and small ribosomal subunit production. Notably, catalytically inactive versions of Rmt3 restored the ribosomal subunit imbalance detected in RMT3-null cells. Consistent with a methyltransferase-independent function for Rmt3 in small ribosomal subunit production, the expression of an Rps2 variant in which the identified methylarginine residues were substituted with lysines showed normal levels of 40 S subunit. Importantly, substitutions within the zinc finger domain of Rmt3 that abolished Rps2 binding did not rescue the 40 S ribosomal subunit deficit of RMT3-null cells. Our findings suggest that the Rmt3-Rps2 interaction, rather than Rps2 methylation, is important for the function of Rmt3 in the regulation of small ribosomal subunit production.Protein arginine methylation is a posttranslational modification catalyzed by a family of enzymes known as protein-arginine methyltransferases (PRMTs).2 Although protein-arginine methyltransferase activity has never been demonstrated in prokaryotic organisms, genes encoding PRMTs have been identified in a variety of unicellular and multicellular eukaryotes (1, 2). In humans, 10 PRMTs have so far been identified (3). Most PRMTs are divided in two major classes, depending of the type of dimethylarginine they produce. Whereas both type I and II PRMTs use S-adenosyl-L-methionine as a cofactor for the monomethylation of specific arginines within substrate proteins, type I and type II enzymes can also produce asymmetricarginine, respectively (1). Interestingly, protein arginine methylation is often found within arginine-glycine (RG)-rich regions of nucleic acid-binding proteins (4). The functional role of PRMTs is likely to be mediated by the modification of substrate proteins. Accordingly, proteins involved in specific steps of gene expression, including transcription (5, 6), splicing (7), polyadenylation (8, 9), mRNA export (10), and translation (11-13), are modified by arginine methylation. Methylation of specific arginine residues within the N-terminal tails of nucleosomal histones is also important for gene regulation and chromatin remodeling (14, 15), thereby influencing biological processes, such as cell fate determination (16) and oncogenesis (17). As yet, however, the biological role of most PRMTs remains poorly understood.The ribosome is the macromolecular complex responsible for protein synthesis in all living cells. In eukaryotes, the 80 S ribos...

This study describes an [ 11 C]acetate rest/stress method to obtain an indirect estimate of myocardial blood flow (MBF) and myocardial oxygen consumption (MVO 2 ) in rats. Doxorubicin cardiotoxicity was used to test the usefulness of this approach for the assessment of congestive heart failure. Methods-[11 C]acetate rest-stress have been used in clinical research to assess the capacity of the coronary arteries to respond to stress. In this article we used this approach to assess the cardiotoxicity of doxorubicin in a rat model. The method was first validated in a group of healthy rats and then used to follow the effect of doxorubicin chemotherapy on cardiac function. The effect of doxorubicin on myocardial perfusion and oxygen consumption reserve was measured at rest and under dobutamine stimulation.Results-Validation of the protocol showed a good correlation between the MBF and MVO 2 (r 2 = 0.68). The doxorubicin treated group showed a significant (p=0.04) decrease in cardiovascular perfusion reserve at 1.3 ± 0.2 compared to the control animals at 1.6 ± 0.2. Similar results were obtained for the myocardial oxygen consumption reserve (treated 1.8 ± 0.4 vs controls 2.3 ± 0.3; p=0.02).Conclusions-We describe an [ 11 C]acetate PET rest/stress protocol for the assessment of cardiotoxicity in rat and its application to the follow-up of doxorubicin chemotherapy. This is a CIHR Author ManuscriptCIHR Author Manuscript CIHR Author Manuscript rapid and reliable approach to the measurement of cardiac perfusion and oxygen consumption reserve that could be applied to the development of new strategies to reduce the cardiotoxicity of anthracycline.

Chitosan (Chit) currently used to prepare nanoparticles (NPs) for brain application can be complexed with negatively charged polymers such as alginate (Alg) to better entrap positively charged molecules such as CXCL12. A sustained CXCL12 gradient created by a delivery system can be used, as a therapeutic approach, to control the migration of cancerous cells infiltrated in peri-tumoral tissues similar to those of glioblastoma multiforme (GBM). For this purpose, we prepared Alg/Chit NPs entrapping CXCL12 and characterized them. We demonstrated that Alg/Chit NPs, with an average size of ~250 nm, entrapped CXCL12 with ~98% efficiency for initial mass loadings varying from 0.372 to 1.490 µg/mg NPs. The release kinetic profiles of CXCL12 were dependent on the initial mass loading, and the released chemokine from NPs after seven days reached 12.6%, 32.3%, and 59.9% of cumulative release for initial contents of 0.372, 0.744, and 1.490 µg CXCL12/mg NPs, respectively. Mathematical modeling of released kinetics showed a predominant diffusive process with strong interactions between Alg and CXCL12. The CXCL12-NPs were not toxic and did not promote F98 GBM cell proliferation, while the released CXCL12 kept its chemotaxis effect. Thus, we developed an efficient and tunable CXCL12 delivery system as a promising therapeutic strategy that aims to be injected into a hydrogel used to fill the cavity after surgical tumor resection. This system will be used to attract infiltrated GBM cells prior to their elimination by conventional treatment without affecting a large zone of healthy brain tissue.

Small-animal nuclear imaging modalities have become essential tools in the development process of new drugs, diagnostic procedures, and therapies. Quantification of metabolic or physiologic parameters is based on pharmacokinetic modeling of radiotracer biodistribution, which requires the blood input function in addition to tissue images. Such measurements are challenging in small animals because of their small blood volume. In this work, we propose a microfluidic counting system to monitor rodent blood radioactivity in real time, with high efficiency and small detection volume (∼1 μL). Methods: A microfluidic channel is built directly above unpackaged p-i-n photodiodes to detect β-particles with maximum efficiency. The device is embedded in a compact system comprising dedicated electronics, shielding, and pumping unit controlled by custom firmware to enable measurements next to small-animal scanners. Data corrections required to use the input function in pharmacokinetic models were established using calibrated solutions of the most common PET and SPECT radiotracers. Sensitivity, dead time, propagation delay, dispersion, background sensitivity, and the effect of sample temperature were characterized. The system was tested for pharmacokinetic studies in mice by quantifying myocardial perfusion and oxygen consumption with 11 C-acetate (PET) and by measuring the arterial input function using 99m TcO 4 − (SPECT). Results: Sensitivity for PET isotopes reached 20%-47%, a 2-to 10-fold improvement relative to conventional catheter-based geometries. Furthermore, the system detected 99m Tc-based SPECT tracers with an efficiency of 4%, an outcome not possible through a catheter. Correction for dead time was found to be unnecessary for small-animal experiments, whereas propagation delay and dispersion within the microfluidic channel were accurately corrected. Background activity and sample temperature were shown to have no influence on measurements. Finally, the system was successfully used in animal studies. Conclusion: A fully operational microfluidic blood-counting system for preclinical pharmacokinetic studies was developed. Microfluidics enabled reliable and high-efficiency measurement of the blood concentration of most common PET and SPECT radiotracers with high temporal resolution in small blood volume. Radi onuclide-based molecular imaging using PET and SPECT is a leading diagnostic tool in oncology, cardiology, and neurology (1). In research applications, small-animal models are needed to facilitate the development of new drugs, radiotracers, and therapies that can eventually be translated to humans (2). Quantification of metabolic or physiologic disorders using radiotracer pharmacokinetic models requires dynamic blood analysis in addition to tissue imaging data (3). Whole-blood radioactivity as a function of time, the so-called arterial input function (AIF), can be extracted from PET images (4,5), using an intravascular b-microprobe (6,7) or an external blood counter connected to an artery via a catheter (8-10). Fo...

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