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Osteoporosis is a progressive bone disease due to low osteoblast activity and/or high osteoclast activity. NELL-1 is a potential therapy for osteoporosis because it specifically increases osteoblast differentiation. However, similar to other protein drugs, the bioavailability of NELL-1 may be limited by its in vivo half-life and rapid clearance from body. The purpose of the present study is to prolong NELL-1 circulation time in vivo by PEGylation with three monomeric PEG sizes (5, 20, 40 kDa). While linear PEG 5k yielded the most efficient PEGylation and the most thermally stable conjugate, linear PEG 20k resulted in the conjugate with the highest Mw and longest in vivo circulation. Compared to non-modified NELL-1, all three PEGylated conjugates showed enhanced thermal stability and each prolonged the in vivo circulation time significantly. Furthermore, PEGylated NELL-1 retained its osteoblastic activity without any appreciable cytotoxicity. These findings motivate further studies to evaluate the efficacy of PEGylated NELL-1 on the prevention and treatment of osteoporosis.

Introduction: Skeletal aging is associated not only with alterations in osteoblast (OB) and osteoclast (OC) number and activity within the basic metabolic unit, but also with increased marrow adiposity. Peroxisome proliferatoractivated receptor gamma (PPARg) is commonly considered the master transcriptional regulator of adipogenesis, however, it has known roles in osteoblast and osteoclast function as well. Here, we designed a lentiviral delivery system for PPARg shRNA, and examined its effects in vitro on bone marrow stromal cells (BMSC) and in a mouse intramedullary injection model. Methods: PPARg shRNA was delivered by a replication-deficient lentiviral vector, after in vitro testing to confirm purity, concentration, and efficacy for Pparg transcript reduction. Next, control green fluorescent protein lentivirus or PPARg shRNA expressing lentivirus were delivered by intramedullary injection into the femoral bone marrow of male SCID mice. Analyses included daily monitoring of animal health, and postmortem analysis at 4 weeks. Postmortem analyses included high resolution microcomputed tomography (microCT) reconstructions and analysis, routine histology and histomorphometric analysis, quantitative real time polymerase chain reaction analysis of Pparg transcript levels, and immunohistochemical analysis for markers of adipocytes (PPARg, fatty acid binding protein 4 [FABP4]), osteoblasts (alkaline phosphatase [ALP], osteocalcin [OCN]), and osteoclasts (tartrateresistant acid phosphatase [TRAP], Cathepsin K).Results: In vitro, PPARg shRNA delivery significantly reduced Pparg expression in mouse BMSC, accompanied by a significant reduction in lipid droplet accumulation. In vivo, a near total reduction in mature marrow adipocytes was observed at 4 weeks postinjection. This was accompanied by significant reductions in adipocytespecific markers. Parameters of trabecular bone were significantly increased by both microCT and histomorphometric analysis. By immunohistochemical staining and semi-quantification, a significant increase in OCN + osteoblasts and decrease in TRAP + multinucleated osteoclasts was observed with PPARg shRNA treatment. Discussion: These findings suggest that acute loss of PPARg in the bone marrow compartment has a significant role beyond anti-adipose effects. Specifically, we found pro-osteoblastogenic, anti-osteoclastic effects after PPARg shRNA treatment, resulting in improved trabecular bone architecture. Future studies will examine the isolated and direct effects of PPARg shRNA on OB and OC cell types, and it may help determine whether PPARg antagonists are potential therapeutic agents for osteoporotic bone loss.

Our review of real-time three-dimensional echocardiography (RT3DE) discusses the diagnostic utility of RT3DE and provides a comparison with two-dimensional echocardiography (2DE) in clinical cardiology. A Pubmed literature search on RT3DE was performed using the following key words: transthoracic, two-dimensional, three-dimensional, real-time, and left ventricular (LV) function. Articles included perspective clinical studies and meta-analyses in the English language, and focused on the role of RT3DE in human subjects. Application of RT3DE includes analysis of the pericardium, right ventricular (RV) and LV cavities, wall motion, valvular disease, great vessels, congenital anomalies, and traumatic injury, such as myocardial contusion. RT3DE, through a transthoracic echocardiography (TTE), allows for increasingly accurate volume and valve motion assessment, estimated LV ejection fraction, and volume measurements. Chamber motion and LV mass approximation have been more accurately evaluated by RT3DE by improved inclusion of the third dimension and quantification of volumetric movement. Moreover, RT3DE was shown to have no statistical significance when comparing the ejection fractions of RT3DE to cardiac magnetic resonance (CMR). Analysis of RT3DE data sets of the LV endocardial exterior allows for the volume to be directly quantified for specific phases of the cardiac cycle, ranging from end systole to end diastole, eliminating error from wall motion abnormalities and asymmetrical left ventricles. RT3DE through TTE measures cardiac function with superior diagnostic accuracy in predicting LV mass, systolic function, along with LV and RV volume when compared with 2DE with comparable results to CMR.