Victor Shen

Strontium Ranelate Improves Bone Resistance by Increasing Bone Mass and Improving Architecture in Intact Female Rats

Strontium ranelate given to intact rats at doses up to 900 mg/kg/day increases bone resistance, cortical and trabecular bone volume, micro‐architecture, bone mass, and total ALP activity, thus indicating a bone‐forming activity and an improvement of overall bone tissue quality.

A Novel Tetracycline Labeling Schedule for Longitudinal Evaluation of the Short‐Term Effects of Anabolic Therapy With a Single Iliac Crest Bone Biopsy: Early Actions of Teriparatide

We describe a quadruple tetracycline labeling method that allows longitudinal assessment of short‐term changes in bone formation in a single biopsy. We show that 1 month of hPTH(1‐34) treatment extends the bone‐forming surface, increases mineral apposition rate, and initiates modeling‐based formation.

Long‐Term Protective Effects of Zoledronic Acid on Cancellous and Cortical Bone in the Ovariectomized Rat

Current bisphosphonate therapies effectively prevent bone loss in postmenopausal women. We studied the effect of a single intravenous dose of ZOL in ovariectomized rats. Protection from bone loss was dose dependent, lasting for up to 32 weeks, supporting the rationale for an annual intravenous dosing regimen of ZOL for treatment of postmenopausal osteoporosis.

Effects of Cyclic Versus Daily hPTH(1-34) Regimens on Bone Strength in Association With BMD, Biochemical Markers, and Bone Structure in Mice

We developed a cyclic PTH regimen with repeated cycles of 1‐week on and off daily PTH injection and explored its effects on bone strength, BMD, bone markers, and bone structure in mice. Cyclic protocols produced 60–85% of the effects achieved by daily protocols with 57% of the total PTH given, indicating more economic use of PTH. The study supports further exploration of cyclic PTH regimens for the treatment of osteoporosis.

Infrequent Delivery of a Long-Acting PTH-Fc Fusion Protein Has Potent Anabolic Effects on Cortical and Cancellous Bone

Skeletal anabolism with PTH is achieved through daily injections that result in brief exposure to the peptide. We hypothesized that similar anabolic effects could be achieved with less frequent but more sustained exposures to PTH. A PTH‐Fc fusion protein with a longer half‐life than PTH(1–34) increased cortical and cancellous BMD and bone strength with once‐ or twice‐weekly injections.

Cyclic AMP-dependent and calcium-dependent signals in parathyroid hormone function.

Previous work demonstrated that parathyroid hormone (PTH) activates the Ca2+/protein kinase C (PKC) system in addition to cAMP production. Therefore, the authors explored the role of cAMP-dependent and Ca2(+)-dependent signals in the regulation of osteoblastic growth and bone resorption. In exponentially growing UMR 106-01 osteogenic sarcoma cells, PTH (10(-7) M) inhibited [3H] thymidine incorporation by 80%. This effect was reproduced by maximal doses of both dibutyryl-cAMP (dbcAMP) and forskolin. The Ca2+ ionophore ionomycin (10(-7) M) had no effect, whereas phorbol 12-myristate 13-acetate (PMA) was slightly mitogenic. The antimitogenic action of dbcAMP was dose-dependent, with ED0.5 at about 3 X 10(-5) M. Ionomycin enhanced this dbcAMP effect at submaximal doses of the cAMP analog. PMA used in combination with both dbcAMP and ionomycin induced further depression of cell proliferation, indicating synergism with cAMP. Both dbcAMP (10(-4) M) and ionomycin (10(-7) M) stimulated 45Ca release from fetal rat limb bones after five days in culture, although the Ca2+ ionophore was less potent. 1-Oleoyl 2-acetyl-glycerol (2 X 10(-6) M) was ineffective alone, and slightly inhibited the 45Ca release produced by the other second messenger analogs in all combinations. The combination of dbcAMP and ionomycin showed a synergistic effect, and fully reproduced PTH effect. In conclusion, PTH signal transduction for control of cell proliferation and bone resorption is mediated mainly by cAMP. Activation of the Ca2+/PKC message system is nevertheless necessary to express a full hormonal response in both cell and organ culture systems.

An acidic fibroblast growth factor stimulates DNA synthesis, inhibits collagen and alkaline phosphatase synthesis and induces resorption in bone

We have examined the effects of an acidic fibroblast growth factor (aFGF) on bone cell growth and differentiation and on osteoclastic resorption using rat fetus organ cultured calvariae and long bones, respectively. Low concentrations of this aFGF stimulated DNA synthesis (1.28 ng/ml) and inhibited collagen formation (1.28 ng/ml) and alkaline phosphatase activity (0.64 ng/ml) in the isolated calvariae. The inhibition of collagen synthesis was independent of the aFGFs mitogenic effect, and was evident in periosteum-containing and periosteum-free bones, pointing to osteoblasts as the aFGF-responsive cells. Our preparation of aFGF enhanced resorption in the long bones by a calcitonin-inhibitable mechanism. PGE2 release accompanied and indomethacin prevented the enhancement of resorption. By contrast, indomethacin did not block the stimulation of DNA and collagen synthesis caused by our aFGF. These results indicate that our aFGF exerts a PGE2-independent effect on DNA synthesis and collagen synthesis and a PGE2-dependent effect on resorption in bone tissue in vitro.

Regulation of plasminogen activator and plasminogen activator inhibitor production by growth factors and cytokines in rat calvarial cells

SummaryFetal rat osteoblast-enriched calvarial cells were used to study the effects of various growth factors and cytokines on plasminogen activator (PA) and plasminogen activator inhibitor (PAI) activities and the possible relationship of these effects to bone resorption. Confluent cultures were exposed to various factors under serum-free conditions, and levels of PA and PAI activities were examined in both conditioned medium (CM) and cell layer using the125I-fibrin plate assay, fibrin zymogram, and reverse fibrin zymogram. According to the125I-fibrin plate assay or zymogram, incubation of cells with acidic fibroblast growth factor (aFGF), basic FGF (bFGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) elevated the PA activity in the CM as well as in the cell layer extract. Incubation with interleukin 1α (IL-1α), tumor necrosis factor α (TNFα), and insulin-like growth factor I (IGF-I) produced no change in PA activity in either CM or cell layer. Addition of transforming growth factor β (TGFβ) to calvarial cells resulted in nearly undetectable PA activity in CM with the fibrin plate assay but increased PA activity on the fibrin zymogram after PAI was separated from PA by SDS-PAGE. A reverse fibrin zymogram indicated that PAI activity was greatly enhanced in TGFβ-treated CM. TGFβ treatment also increased PA activity in the cell layer of calvarial cells. Treatment of calvarial cells with bFGF and PDGF slightly increased PAI secretion into medium. This increase, however, was not as dramatic as the increase of PA induced by these two agents. IL-1α and TNFα did not change PAI concentration in CM. No detectable PAI activity was found in the cell layer in control and treated groups. The PA found in the CM and cell layer of rat calvarial cells was the urokinase type; the PAI stimulated by TGFβ was the endothelial cell type, PAI-1. The regulation of PA activity by growth factors and cytokines did not correlate with their resorption-stimulating activities. Thus, PA secreted by osteoblasts may not be the only factor involved in the initiation of bone resorption. Delineation of the function of PA and PAI in the physiology of bone tissue awaits further studies.

Monokines produced by macrophages stimulate the growth of osteoblasts.

We have previously reported that the J774A.1 macrophage-like tumor cell line produces two potent monokines which stimulate the growth of osteoblasts and chondrocytes. These growth factors, which have an affinity for heparin-agarose, have been termed HEP I (a 30 Kd PDGF-like molecule) and HEP II (an approximately 20 Kd molecule), respectively, based on their elution profile. Unlike HEP I, HEP II does not stimulate the growth of fibroblasts. Extensive biological and chromatographic studies disclosed that HEP II appears to be a unique bone cell mitogen unlike any known growth factor, including the FGFs, IL-1s, and TNFs, EGF, IGF-I and -II, TGF-beta, beta 2 microglobulin, G-CSF, CSF-1 and GM-CSF. To characterize more fully the effects of the macrophage-derived monokines on osteoblast growth and function, clones were derived from calvaria explant cultures. Two clones, SDFRC-2.05 and SDFRC-3, were developed and found to exhibit osteoblastic characteristics, including high levels of alkaline phosphatase, synthesis of type I but not type III collagen, and an increased intracellular cAMP production in response to PTH. The SDFRC-3 cells exhibited a polygonal morphology like that of the explant-derived cells while SDFRC-2.05 cells exhibited a more fibroblastic morphology. When tested on the explant cultures and clones, HEP I and HEP II were found to stimulate DNA synthesis and increase protein per culture, but decreased alkaline phosphatase activity. Clone SDFRC-3 was found to be more responsive to HEP II than clone SDFRC-2.05. Both monokines were found to be more potent mitogens for bone cells than TGF-beta. HEP II, but not HEP I or TGF-beta, induced a transformation of bone cells from a polygonal to a fibroblastic morphology, suggesting the induction of migration prior to proliferation. Thus, macrophages may be responsible not only for bone repair but also for ensuring the linkage of bone formation to resorption during physiological remodeling.

Characterization of a (Ca2+ +Mg2+)-ATPase system in the osteoblast plasma membrane

A high affinity, calmodulin-sensitive (Ca2 + Mg2+)-ATPase was demonstrated in the plasma membrane preparation of three different osteosarcoma cell lines previously demonstrated to respond to parathyroid hormone with an increase in cytosolic calcium and a decrease in pH. The maximal velocity of the enzyme activity in the membrane preparations ranged from 0.83 to 2.42 nmol Pi released per min per mg protein with half-saturation constants of 26 nM of free Ca. The enzyme activity was not affected by Na+, K+, ouabain and azide, and exhibited an absolute requirement for Mg2+ ions. These results suggest a possible role for a membrane Ca2 + Mg2+-ATPase in initiating and perpetuating the ionic control of osteoblastic function.