Exposure to microgravity causes bone loss in humans, and the underlying mechanism is thought to be at least partially due to a decrease in bone formation by osteoblasts. In the present study, we examined the hypothesis that microgravity changes osteoblast gene expression profiles, resulting in bone loss. For this study, we developed an in vitro system that simulates microgravity using the Random Positioning Machine (RPM) to study the effects of microgravity on 2T3 preosteoblast cells grown in gas-permeable culture disks. Exposure of 2T3 cells to simulated microgravity using the RPM for up to 9 days significantly inhibited alkaline phosphatase activity, recapitulating a bone loss response that occurs in real microgravity conditions without altering cell proliferation and shape. Next, we performed DNA microarray analysis to determine the gene expression profile of 2T3 cells exposed to 3 days of simulated microgravity. Among 10,000 genes examined using the microarray, 88 were downregulated and 52 were upregulated significantly more than twofold using simulated microgravity compared with the static 1-g condition. We then verified the microarray data for some of the genes relevant in bone biology using real-time PCR assays and immunoblotting. We confirmed that microgravity downregulated levels of alkaline phosphatase, runt-related transcription factor 2, osteomodulin, and parathyroid hormone receptor 1 mRNA; upregulated cathepsin K mRNA; and did not significantly affect bone morphogenic protein 4 and cystatin C protein levels. The identification of gravisensitive genes provides useful insight that may lead to further hypotheses regarding their roles in not only microgravity-induced bone loss but also the general patient population with similar pathological conditions, such as osteoporosis.

Rationale: The aromatase inhibitor anastrozole blocks the conversion of androgens to estrogen and blunts pulmonary hypertension in animals, but its efficacy in treating patients with pulmonary arterial hypertension (PAH) is unknown.Objectives: We aimed to determine the safety and efficacy of anastrozole in PAH.Methods: We performed a randomized, double-blind, placebocontrolled trial of anastrozole in patients with PAH who received background therapy at two centers. Measurements and Main Results:A total of 18 patients with PAH were randomized to anastrozole 1 mg or matching placebo in a 2:1 ratio. The two co-primary outcomes were percent change from baseline in 17b-estradiol levels (E2) and tricuspid annular plane systolic excursion (TAPSE) at 3 months. Anastrozole significantly reduced E2 levels compared with placebo (percent change: 240%; interquartile range [IQR], 261 to 226% vs. 24%; IQR, 214 to 14%; P = 0.003), but there was no difference in TAPSE. Anastrozole significantly increased the 6-minute-walk distance (median change = 126 m) compared with placebo (median change = 212 m) (median percent change: anastrozole group, 8%; IQR, 2 to 17% vs. placebo 22%; IQR, 27 to 11%; P = 0.042). Anastrozole had no effect on circulating biomarkers, functional class, or health-related quality of life. There was no difference in adverse events.Conclusions: Anastrozole significantly reduced E2 levels in patients with PAH but had no effect on TAPSE. Anastrozole was safe, well tolerated, and improved 6-minute-walk distance in this small "proof-of-principle" study. Larger and longer phase II clinical trials of anastrozole may be warranted in patients with PAH.Clinical trial registered with www.clinicaltrials.gov (NCT 1545336).

Weightlessness or microgravity of spaceflight induces bone loss due in part to decreased bone formation by unknown mechanisms. Due to difficulty in performing experiments in space, several ground-based simulators such as the Rotating Wall Vessel (RWV) and Random Positioning Machine (RPM) have become critical venues to continue studying space biology. However, these simulators have not been systematically compared to each other or to mechanical stimulating models. Here, we hypothesized that exposure to RWV inhibits differentiation and alters gene expression profiles of 2T3 cells, and a subset of these mechanosensitive genes behaves in a manner consistent to the RPM and opposite to the trends incurred by mechanical stimulation of mouse tibiae. Exposure of 2T3 preosteoblast cells to the RWV for 3 days inhibited alkaline phosphatase activity, a marker of differentiation, and downregulated 61 and upregulated 45 genes by more than twofold compared to static 1 g controls, as shown by microarray analysis. The microarray results were confirmed by real-time PCR and/or Western blots for seven separate genes and proteins including osteomodulin, runx2, and osteoglycin. Comparison of the RWV data to the RPM microarray study that we previously published showed 14 mechanosensitive genes that changed in the same direction. Further comparison of the RWV and RPM results to microarray data from mechanically loaded mouse tibiae reported by an independent group revealed that three genes including osteoglycin were upregulated by the loading and downregulated by our simulators. These mechanosensitive genes may provide novel insights into understanding the mechanisms regulating bone formation and potential targets for countermeasures against decreased bone formation during space flight and in pathologies associated with lack of bone formation.

Bone loss due to osteoporosis or disuse such as in paraplegia or microgravity is a significant health problem. As a treatment for osteoporosis, brief exposure of intact animals or humans to low magnitude and high frequency (LMHF) mechanical loading has been shown to normalize and prevent bone loss. However, the underlying molecular changes and the target cells by which LMHF mechanical loading alleviate bone loss are not known. Here, we hypothesized that direct application of LMHF mechanical loading to osteoblasts alters their cell responses, preventing decreased bone formation induced by disuse or microgravity conditions. To test our hypothesis, preosteoblast 2T3 cells were exposed to a disuse condition using the Random Positioning Machine (RPM) and intervened with an LMHF mechanical load (0.1-0.4g at 30Hz for 10-60 min/day). Exposure of 2T3 cells to the RPM decreased bone formation responses as determined by alkaline phosphatase (ALP) activity and mineralization even in the presence of a submaximal dose of BMP4 (20ng/ml). However, LMHF mechanical loading prevented the RPM-induced decrease in ALP activity and mineralization. Mineralization induced by LMHF mechanical loading was enhanced by treatment with bone morphogenic protein 4 (BMP4) and blocked by the BMP antagonist noggin, suggesting a role for BMPs in this response. In addition, LMHF mechanical loading rescued the RPM-induced decrease in gene expression of ALP, runx2, osteomodulin, parathyroid hormone receptor 1, and osteoglycin. These findings suggest that preosteoblasts may directly respond to LMHF mechanical loading to induce differentiation responses. The mechanosensitive genes identified here provide potential targets for pharmaceutical treatments that may be used in combination with low level mechanical loading to better treat osteoporosis or disuse-induced bone loss.