Toshitaka Nakamura

Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation.

Basic fibroblast growth factor (FGF-2), an important modulator of cartilage and bone growth and differentiation, is expressed and regulated in osteoblastic cells. To investigate the role of FGF-2 in bone, we examined mice with a disruption of the Fgf2 gene. Measurement of trabecular bone architecture of the femoral metaphysis of Fgf2(+/+) and Fgf2(-/-) adult mice by micro-CT revealed that the platelike trabecular structures were markedly reduced and many of the connecting rods of trabecular bone were lost in the Fgf2(-/-) mice. Dynamic histomorphometry confirmed a significant decrease in trabecular bone volume, mineral apposition, and bone formation rates. In addition, there was a profound decreased mineralization of bone marrow stromal cultures from Fgf2(-/-) mice. This study provides strong evidence that FGF-2 helps determine bone mass as well as bone formation.

Alfacalcidol Inhibits Bone Resorption and Stimulates Formation in an Ovariectomized Rat Model of Osteoporosis: Distinct Actions from Estrogen

Although alfacalcidol has been widely used for the treatment of osteoporosis in certain countries, its mechanism of action in bone, especially in the vitamin D–replete state, remains unclear. Here we provide histomorphometric as well as biochemical evidence that alfacalcidol suppresses osteoclastic bone resorption in an ovariectomized rat model of osteoporosis. Furthermore, when compared with 17β‐estradiol, a representative antiresorptive drug, it is evident that alfacalcidol causes a dose‐dependent suppression of bone resorption, and yet maintains or even stimulates bone formation, as reflected in increases in serum osteocalcin levels and bone formation rate at both trabecular and cortical sites. 17β‐Estradiol, which suppresses bone resorption to the same extent as alfacalcidol, causes a parallel reduction in the biochemical and histomorphometric markers of bone formation. As a final outcome, treatment with alfacalcidol increases bone mineral density and improves mechanical strength more effectively than 17β‐estradiol, with a more pronounced difference in cortical bone. We conclude that estrogens depress bone turnover primarily by suppressing bone resorption and, as a consequence, bone formation as well, whereas alfacalcidol “supercouples” these processes, in that it suppresses bone resorption while maintaining or stimulating bone formation.

Vascular endothelial growth factor is expressed along with its receptors during the healing process of bone and bone marrow after drill-hole injury in rats

In this study, we investigated the expression of vascular endothelial growth factor (VEGF) mRNA along with its receptors in the healing process following rat femoral drill-hole injury. The cellular events involved in the differential expression of VEGF were studied by reverse transcription-polymerase chain reaction, immunocytochemistry, and in situ hybridization. Abundant alkaline phosphatase-positive osteoprogenitor cells were present in the bone marrow cavity surrounding the wound region at day 3. Some of the cells were immunoreactive for Flk-1, a marker of angioblasts. At day 5, osteoblasts expressing osteocalcin mRNA actively participated in bone formation. After day 11, medullary bone gradually decreased and hematopoietic cells covered the wound region. The expressions of the VEGF splice variants VEGF120 and VEGF164 were detected at days 1 and 3, and VEGF188 mRNA began to appear from day 5. The expressions of the three VEGF splice variants gradually decreased after day 11. VEGF immunoreactivity and mRNA expression were strongly detected in angioblasts, osteoprogenitor cells, and osteoblasts between days 3 and 7, but gradually decreased after day 11. Immunoreactivity for Flt-1 was also detected in endothelial cells, osteoprogenitor cells, and osteoblasts between days 3 and 7. However, immunoreactivity for Flk-1 was not detected on osteoblasts but rather on endothelial cells. These findings indicate that the differential expression of VEGF splicing isoforms along with its receptors may play an important role in the healing process after rat femoral drill-hole injury.

Randomized Teriparatide [Human Parathyroid Hormone (PTH) 1–34] Once-Weekly Efficacy Research (TOWER) Trial for Examining the Reduction in New Vertebral Fractures in Subjects with Primary Osteoporosis and High Fracture Risk

CONTEXT Weekly teriparatide injection at a dose of 56.5 μg has been shown to increase bone mineral density. OBJECTIVE A phase 3 study was conducted to determine the efficacy of once-weekly teriparatide injection for reducing the incidence of vertebral fractures in patients with osteoporosis. DESIGN AND SETTING In this randomized, multicenter, double-blind, placebo-controlled trial conducted in Japan, the incidence of morphological vertebral fractures by radiographs was assessed. PATIENTS Subjects were 578 Japanese patients between the ages of 65 and 95 yr who had prevalent vertebral fracture. INTERVENTION Subjects were randomly assigned to receive once-weekly s.c. injections of teriparatide (56.5 μg) or placebo for 72 wk. MAIN OUTCOME MEASURE The primary endpoint was the incidence of new vertebral fracture. RESULTS Once-weekly injections of teriparatide reduced the risk of new vertebral fracture with a cumulative incidence of 3.1% in the teriparatide group, compared with 14.5% in the placebo group (P < 0.01), and a relative risk of 0.20 (95% confidence interval, 0.09 to 0.45). At 72 wk, teriparatide administration increased bone mineral density by 6.4, 3.0, and 2.3% at the lumbar spine, the total hip, and the femoral neck, respectively, compared with the placebo (P < 0.01). Adverse events (AE) and the dropout rates by AE were more frequently experienced in the teriparatide group, but AE were generally mild and tolerable. CONCLUSION Weekly s.c. administration of teriparatide at a dose of 56.5 μg may provide another option of anabolic treatments in patients with osteoporosis at higher fracture risk.

Guidelines for the use of biochemical markers of bone turnover in osteoporosis (2004)

A definition of osteoporosis was agreed upon at the 1993 consensus conference held in Hong Kong. It states that osteoporosis is “characterized by low bone mass and the microarchitectural deterioration of bony tissue, with a consequent increase in bone fragility and susceptibility to fracture.” This definition had been internationally used without revision until recently, when the definition was significantly changed at a National Institutes of Health (NIH) consensus conference in 2000 [1]. According to the consensus statement, osteoporosis is defined as “a skeletal disorder characterized by compromised bone strength predisposing a person to an increased risk of fracture.” Bone strength is determined by integrating bone mineral density (BMD) and bone quality. BMD is expressed as grams of mineral per area or volume, and, currently, BMD is defined by the individual peak bone density and the resorption rate from the peak. Bone quality is determined by characteristics of the bone matrix, such as microarchitecture, bone turnover, microdamage accumulation, the degree of calcification, and collagen [2,3]. Currently, it is thought that bone quality may not be clinically assessed by measures other than the determination of bone metabolism with biochemical markers of bone turnover. The change in definition may be the result of more recent findings [4], one that demonstrates bone fractures routinely occur despite patients having modest BMD levels, and another that has shown no significant reduction in the risk of a fracture occurring in patients taking one of the two standard medications, one that significantly increases BMD and the other that moderately increases it.

Intravenous pamidronate prevents femoral bone loss and renal stone formation during 90-day bed rest

Long‐term bed rest has potential risks of bone loss and renal stone formation. We examined the effects of resistive exercise and intravenous pamidronate on BMD, bone turnover, urinary calcium, and renal stone formation in 25 healthy males during 90‐day bed rest. Pamidronate prevented femoral bone loss and renal stone formation, but resistive exercise showed little effects.

Elevated expression of vacuolar proton pump genes and cellular PH in cisplatin resistance.

V‐ATPases are proton‐translocating enzymes, which are found not only in numerous intracellular organelles but also in the plasma membranes of many eukaryotic cells. Using differential display, we have identified one of the proton pump subunit genes, ATP6C, as a cisplatin‐inducible gene. Northern blot analysis demonstrated that expression of other members of the subunit is inducible by cisplatin treatment. Proton pump gene expression is also upregulated in 3 independent cisplatin‐resistant cell lines but not in vincristine‐ or etoposide‐resistant cell lines. Cellular pH was significantly higher in cisplatin‐resistant cells than in sensitive parental cells. In vitro DNA‐binding activity of cisplatin was markedly increased in acidic conditions, suggesting that the cytotoxicity of cisplatin is modulated by cellular pH. Furthermore, the proton pump inhibitor bafilomycin can synergistically potentiate the cytotoxicity of cisplatin but not of etoposide or camptothecin. These results indicate that cellular pH is one of the critical parameters for effective cancer chemotherapy with cisplatin.

A new active vitamin D3 analog, eldecalcitol, prevents the risk of osteoporotic fractures--a randomized, active comparator, double-blind study.

BACKGROUND Eldecalcitol is an analog of 1,25-dihydroxyvitamin D(3) that improves bone mineral density; however, the effect of eldecalcitol on the risk of fractures is unclear. The objective of this study is to examine whether eldecalcitol is superior to alfacalcidol in preventing osteoporotic fractures. This trial is registered with ClinicalTrials.gov, number NCT00144456. METHODS AND RESULTS This 3 year randomized, double-blind, active comparator, superiority trial tested the efficacy of daily oral 0.75 μg eldecalcitol versus 1.0 μg alfacalcidol for prevention of osteoporotic fractures. 1054 osteoporotic patients 46 to 92 years old were randomly assigned 1:1 to receive eldecalcitol (n=528) or alfacalcidol (n=526). Patients were stratified by study site and serum 25-hydroxyvitamin D level. Patients with low serum 25-hydroxyvitamin D levels (<50 nmol/L) were supplemented with 400 IU/day vitamin D(3). Primary end point was incident vertebral fractures. Secondary end points included any non-vertebral fractures and change in bone mineral density and bone turnover markers. Compared with the alfacalcidol group, the incidence of vertebral fractures was lower in eldecalcitol group after 36 months of treatment (13.4 vs. 17.5%; hazard ratio, 0.74; predefined 90% confidence interval [CI], 0.56-0.97). Eldecalcitol reduced turnover markers and increased bone mineral density more strongly than alfacalcidol. Eldecalcitol reduced the incidence of three major non-vertebral fractures, which was due to a marked reduction in wrist fractures by a post-hoc analysis (1.1 vs. 3.6%; hazard ratio, 0.29; 95% CI, 0.11-0.77). Among the adverse events, the incidence of increase in serum and urinary calcium was higher in the eldecalcitol group, without any difference in glomerular filtration rate between the two groups. CONCLUSIONS Eldecalcitol is more efficacious than alfacalcidol in preventing vertebral and wrist fractures in osteoporotic patients with vitamin D sufficiency, with a safety profile similar to alfacalcidol.

Nonenzymatic collagen cross-links induced by glycoxidation (pentosidine) predicts vertebral fractures.

Advanced glycation end products (AGE) in collagen have been reported to decrease the mechanical property of bone. However, there are no available data on the relation between fracture risk and levels of glycoxidative (nonenzymatic) cross-links of collagen in clinical samples. A total of 432 Japanese elderly women who were not receiving any drug treatment for osteoporosis were selected and followed for 5.2 ± 3.3 (mean ± SD) years for this observational study. Vertebral fractures and bone mineral density were assessed at baseline and then at 1- to 2-year intervals or at indication of any symptom. Two types of collagen metabolites were measured at baseline: urinary N-terminal telopeptide of type I collagen (NTX), a marker of pyridinium cross-link, and urinary pentosidine, a nonenzymatic collagen cross-link produced by AGEs. A total of 97 incident vertebral fractures on 72 subjects were observed. Simple regression analysis using Coxs hazards model showed that log-transformed urinary NTX and pentosidine are significant risk factors for time-dependent incidence of vertebral fractures, in addition to the traditional risk factors (age, lumbar bone mineral density, and number of prevalent vertebral fractures). However, urinary excretion of pentosidine (hazard ratio, 1.33; 95% CI, 1.01–1.76, P = 0.04) was a significant predictor of incident vertebral fracture after adjustment for other traditional risk factors. The present data suggest that AGE-related collagen cross-link is a novel risk for vertebral fracture.

Effects of teriparatide on bone mineral density and bone turnover markers in Japanese subjects with osteoporosis at high risk of fracture in a 24-month clinical study: 12-Month, randomized, placebo-controlled, double-blind and 12-month open-label phases

This multicenter study assessed the safety and efficacy of teriparatide 20 microg/day in Japanese men and women with osteoporosis at high risk of fracture during a 12-month, randomized, double-blind, placebo-controlled treatment period followed by second and third treatment periods (to 18 and 24 months, respectively,) in which all subjects received open-label teriparatide. Subjects (93% female; median age 70 years) were randomized 2:1 to teriparatide versus placebo (randomized at baseline, teriparatide n=137, placebo-teriparatide n=70; entering the second period, teriparatide n=119, placebo-teriparatide n=59; entering the third period, teriparatide n=102, placebo-teriparatide n=50). For subjects with measurements at 12 months, teriparatide significantly increased bone mineral density (BMD) at the lumbar spine L2-L4 (mean percent change+/-SD, teriparatide 10.04+/-5.23% versus placebo-teriparatide 0.19+/-4.33%), the femoral neck (teriparatide 2.01+/-4.63% versus placebo-teriparatide 0.44+/-3.97%), and the total hip (teriparatide 2.72+/-4.04% versus placebo-teriparatide -0.26+/-3.42%). In the placebo-teriparatide group at 24 months (12-month teriparatide dosing) BMD increased by 9.11+/-5.14% at the lumbar spine, 2.19+/-4.81% at the femoral neck and 2.46+/-3.54% at the total hip. In the teriparatide group at 18 and 24 months, BMD increased from baseline at the lumbar spine by 11.93+/-5.79% and 13.42+/-6.12%, respectively; at the femoral neck by 2.68+/-4.45% and 3.26+/-4.25%, respectively; and at the total hip by 3.02+/-3.79% and 3.67+/-3.98%, respectively. Serum procollagen I N-terminal pro-peptide (PINP) increased rapidly with teriparatide treatment (P<0.001 versus placebo at 1 month) and changed from baseline in the teriparatide and placebo-teriparatide groups at 12 months by a median of 78.95% and -17.23%, respectively, (P<0.001) and at 24 months by 49.24% and 76.12%, respectively. The incidence of treatment-emergent adverse events (TEAEs), serious TEAEs, and discontinuations due to TEAEs were comparable in the teriparatide and placebo-teriparatide groups. These data show that teriparatide 20 microg/day was well tolerated and stimulated bone formation in Japanese subjects with osteoporosis at high risk of fracture during 18 and 24 months of treatment.