Effects of Vitamin K2 and Risedronate on Bone Formation and Resorption, Osteocyte Lacunar System, and Porosity in the Cortical Bone of Glucocorticoid-Treated Rats

The purpose of the present study was to examine the effects of vitamin K(2) and risedronate on bone formation and resorption, the osteocyte lacunar system, and porosity in the cortical bone of glucocorticoid (GC)-treated rats. Forty-nine female Sprague-Dawley rats, 3 months of age, were randomized into five groups according to the following treatment schedule: age-matched control, GC administration, and GC administration with concomitant administration of vitamin K(2), risedronate, or vitamin K(2) + risedronate. At the end of the 8-week experiment, classical bone histomorphometric analysis was performed, and the osteocyte lacunar system and porosity were evaluated on the cortical bone of the tibial diaphysis. GC administration decreased percent cortical bone area and increased percent marrow area as a result of decreased periosteal bone formation, and increased endocortical bone erosion, and increased cortical porosity. Vitamin K(2) prevented a reduction in periosteal bone formation but did not affect percent cortical bone and marrow areas. Risedronate prevented a reduction in periosteal bone formation and an increase in endocortical bone erosion, resulting in prevention of alterations in percent cortical bone and marrow areas. Both vitamin K(2) and risedronate increased osteocyte density and lacunar occupancy and prevented a GC-induced increase in cortical porosity. Vitamin K(2) and risedronate had additive effects on osteocyte density and lacunar occupancy and a synergistic effect on cortical porosity. The present study showed the efficacy of vitamin K(2) and risedronate for bone formation and resorption, the osteocyte lacunar system, and porosity in the cortical bone of GC-treated rats.     

Prostaglandin E2 binding and cyclic AMP production in isolated bone cells

Summary The binding of prostaglandin E₂ (PGE₂) to bone cells was studied to provide direct evidence for the existence of specific receptors in bone. Bone cells were isolated by collagenase digestion of fetal and newborn rat calvaria. Isolated cells were incubated with³H-PGE₂ and collected on Millipore filters. Specific binding was determined by subtracting the binding that occurred with 10⁻⁶ M non-radioactive PGE₂ and³H-PGE₂ from that with³H-PGE₂ alone. With heterogeneous cell preparations and at PGE₂ concentrations from 10⁻⁹ − 1.7 × 10⁻⁸ M at 37°C, specific binding reached steady state within 10 min. Bound³H-PGE₂ was displaced by the addition of increasing amounts of unlabeled PGE₂. Inhibition of PGE₂ binding was observed with PGE₁ and the endoperoxide analog, U44069, but not with PGE_2α, a lipopolysaccharide, or 13,14-dihydro 15-keto PGE₂. Studies with bone cell populations, obtained by sequential digestions, indicated that an osteoclastic population binds 30-fold more PGE₂ than osteoblastic cells. Scatchard analyses revealed that the osteoclastic cells have an affinity constant for PGE₂ binding similar to that obtained with heterogeneous populations. However, the PGE₂ binding capacity in this osteoclastic population was fivefold greater than in the heterogeneous population. The osteoclastic population responded with an increase in cyclic AMP to lower concentrations of PGE₂ than the osteoblastic populations. These studies suggest that differences in the binding capacity of PGE₂ receptors exist among bone celltypes and that these differences are reflected in the cellular cyclic AMP response.     

Anabolic Effect of Prostaglandin E2 in Bone Is not Dependent on Pituitary Hormones in Rats

Prostaglandin E₂ (PGE₂) is an anabolic agent of bone in vivo but the mechanism of its action still remains unclear. The aim of this study was to determine whether the effect of PGE₂ on skeleton is mediated by pituitary hormones. Forty female, Sprague-Dawley rats were divided into four groups: baseline control (basal), age-matched intact control (CON), hypophysectomy (HX), and HX + PGE₂ (2 mg/kg/day) with 10 animals in each group. The basal group was sacrified at 2 months of age, and the remaining groups after 6 weeks of treatment. Cancellous and cortical bone histomorphometry was performed on double fluorescent-labeled 40 μm-thick sections of the proximal tibia and tibial shaft. Our results show that HX resulted in a cessation of bone growth, a decrease in cancellous bone volume, and cortical bone gain compared with the age-matched, intact CON rats. Compared with the HX group, the HX + PGE₂ group had a significantly greater tibial bone density (mean ± SE, HX + PGE₂:1.595 ± 0.007 versus HX:1.545 ± 0.013), percent cancellous bone volume (21.4 ± 2.0 versus 8.41 ± 1.70), percent cortical bone area (87.2 ± 0.85 versus 81.7 ± 0.7), and ratio of cortical area to marrow area (7.14 ± 0.56 versus 4.52 ± 0.21). Increased bone masses by PGE₂ in the HX animals were accompanied by an increase in the trabecular and endosteal-labeled surface and bone formation rate. The trabecular number and width were increased whereas trabecular separation was decreased in the HX + PGE₂ group compared with the HX group (P < 0.05). PGE₂ treatment also caused a decrease in the tibial endosteal eroded surface and medullar cavity of the HX animals. In conclusion, this study clearly demonstrates that PGE₂ (2 mg/kg/day) in the HX rats increases both cortical and cancellous bones and improves trabecular architecture in the tibia after 6 weeks of treatment. These skeletal alterations are due to a stimulation of bone formation and a suppression of bone resorption activity. These findings suggest that the anabolic effect of PGE₂ in bone is independent of pituitary hormones.     

Increased bone growth by local prostaglandin E2 in rats

Summary The effects of prostaglandin E₂ (PGE₂) on bone growth were investigated in rats. Daily injection of PGE₂ (1, 10, and 100 pmol) was given via local intraosseous route into the metaphysis of the left tibia for 14 days. The contralateral right tibia injected with vehicle and saline was for the control. The rats receiving no injection provided as normal control. The results obtained indicated that PGE₂ slightly but significantly decreased the body weight increment without effect on tibial length. The most prominent effect of PGE₂ was the increase of metaphyseal bone trabeculae by 45–81% in a dose-dependent manner. The microscopic examination revealed that PGE₂ unequivocally increased the new woven bone formation. The bone cell population study showed no difference between the number of osteoblasts and osteoclasts in primary spongiosa of the PGE₂-injected limbs and those of contralateral limbs. However, the numbers of osteoblasts and osteoclasts were markedly increased in secondary spongiosa in the PGE₂-injected limbs. This finding confirmed a stimulatory role of PGE₂ in the bone formation. The local intraosseous injection of PGE₂ was proven to be a good model for the study of local growth factors on bone metabolism with a lower effective dose which eliminates the systemic side effects.     

Prostaglandin E1 reduces blood loss during and after resection of lumbar herniated disc

Controlled hypotension was employed during resection of lumbar herniated disc on 10 patients. Prostaglandin E₁ (PG) was used as a hypotensive agent. The systolic blood pressure was lowered less than 100mmHg in the hypotensive group. The average blood loss during surgery was 95 ± 41ml for the hypotensive group compared with 154 ± 81ml for the normotensive group (P 0.05). The blood loss after surgery was also significantly less in the hypotensive group than in the normotensive group (P 0.05). We conclude that PG is an effective hypotensive agent on blood loss during and after surgery.(Kashimoto S, Nakamura T, Yamaguchi T: Prostaglandin E₁ reduces blood loss during and after resection of lumbar herniated disc. J Anesth 6: 294–296, 1992)     

Prostaglandin E1 increased cardiac contractility in cardiac arrest during open-heart surgery

Key words  Prostaglandin E₁  - Cardiac Arrest - Open-Heart surgery     

Phorbol ester (TPA) reduces prostaglandin E2-stimulated cAMP production by desensitization of prostaglandin E2 receptors in a clonal osteoblast-like cell line,MOB 3-4

Summary A clonal osteoblast-like cell line, MOB 3-4, increased cAMP production in response to prostaglandin E₂ (PGE₂) (5–500 ng/ml). The purpose of this study was to show the effects of tumor-promoting phorbol ester (e.g., 12-O-tetradecanoylphorbol 13-acetate, TPA) on basal and PGE₂-stimulated cAMP production and the affinity of PGE₂ receptors in the cells. Pretreatment with TPA (1 nM–10 μM) for 30 minutes increased basal cAMP production, whereas it markedly reduced the PGE₂-stimulated cAMP production in the presence of 0.1 mM isobuthylmethyl xanthine. Both the TPA increase and reduction were dose- and time-dependent. However, TPA exerted no effect on forskolinor cholera toxin-stimulated cAMP production. Copretreatment with TPA and H-7, an inhibitor of protein kinase C (PKC), prevented the TPA-induced increase in basal cAMP production, whereas it did not prevent the reduction of the PGE₂-stimulated cAMP production. On the other hand, TPA (0.1–10 μM) decreased³H-PGE₂ binding in a dose- and time-dependent manner. Scatchard analysis revealed that TPA decreased the apparent affinity of PGE₂ receptors without effect on their apparent number. In addition, 1-oleoyl-2-acetylglycerol (12.6 μM), a synthetic diacylglycerol analog, did not mimic the TPA action on³H-PGE₂ binding. Thus, TPA at relatively high concentrations appeared to increase basal cAMP production by a PKC-mediated mechanism, and it appeared to directly act on PGE₂ receptors to decrease their apparent affinity and thereby reduce the PGE₂-stimulated cAMP production in the clonal osteoblast-like MOB 3-4 cell line.     

Systemic effects of prostaglandin E2 on vertebral trabecular remodeling in beagles used in a healing study

Summary Prostaglandin E₂ (PGE₂) at a dose of 10 mg/kg was administered orally to beagles used in a study of rib fracture and drill hole defect healing. Double fluochrome labels were given prior to surgical manipulation and before necropsy at 30 days. Bone remodeling was evaluated in trabecular bone of the fourth lumbar vertebra. There was a decrease in the number and extent of posttreatment labels (P<0.05) in the controls, with decreased mineral apposition rate (P<0.05) and decreased active bone formation rate (P<0.01). In dogs given PGE₂ for 30 days following surgery, the extent of posttreatment labels (P<0.05) and bone formation rate (P<0.01) were increased. There was no difference found, however, in static morphometric parameters, including osteoid and osteoblast-covered surface, indicating that the stimulation of bone formation may have been transitory and matrix synthesis had declined. In another group of dogs given PGE₂ for 5 days prior to surgical manipulation and between the first and second pretreatment labels, the extent of the double-labeled surface was increased (P<0.05) indicating an acute PG effect to sustain formation at remodeling sites. These studies show that PGE₂ given orally has a systemic effect on bone remodeling in vertebral trabeculae that involves the stimulation of formation activity.     

Effect of prostaglandin E2 on mineralization of bone nodules formed by fetal rat calvarial cells

The effects of PGE₂ on mineralized bone nodule formation were studied in fetal rat calvarial (RC) cells in vitro. Continuous exposure of RC cells to 3x10^-8M PGE₂ induced a twofold increase in mineralized bone nodule formation and a 1.5-fold increase in alkaline phosphatase (ALPase) activity without affecting RC cell growth. These stimulatory effects were evoked by concentrations of 3x 10^-9-3x10^-6 M PGE₂ and the maximal effect was observed with 3x10^-8 M PGE₂. The in vitro effects of PGE₂ were evident when RC cells were exposed to it on days 8–14 and 8–21, which correspond to the post-confluent culture stage, but no effects were observed when the cells were exposed on days 1–7, the growth stage. The ALPase activity was also higher (1.2–1.4-fold) when 3x10^-8 M PGE₂ was added during the post-confluent stage. In order to determine the effect of PGE₂ during the mineralization phase of bone nodules in the presence of a large population of osteoprogenitor cells, RC cells were exposed to dexamethasone for 7 days before PGE₂ was added during the post-confluent stage. A significantly higher percentage of nodules mineralized were observed with 3x10^-8-3x10^-9 M PGE₂ (1.6-and 1.4-fold, respectively), than in control cultures. Analysis of the mineral-related proteins by EDTA extraction of bone nodules followed by electrophoresis and Stains-All staining revealed an increased total amount of osteopontin extracted from the mineralized matrix after PGE₂ treatment. The osteopontin content was highest after 3x10^-8 M PGE₂, with a 73% increase of the densitometric intensity of the bands, although this increase, reflected the increased number of mineralized bone nodules due to PGE₂. These findings suggest that PGE₂ may increase the proportion of functional osteoblasts able to produce mineralized bone nodules in the population by stimulating differentiation during the postconfluent stage of RC cell culture.     

The effects of prostaglandin E2 on DNA and collagen synthesis in osteoblastsIn Vitro

Summary The effects of prostaglandin E₂(PGE₂) on DNA and collagen synthesis in two separate cell populations were investigated. In view of their morphology, ALPase activity, DNA and collagen synthesis, and response to PGE₂, one population was in an undifferentiated state consisting of preosteoblast-like (PL) cells and the other was in a differentiated state consisting predominantly of osteoblastlike (OB) cells. As parameters of bone-forming activity, the incorporation of³H-thymidine into DNA and the incorporation of³H-proline into collagenase digestible protein were measured to assess DNA and collagen synthesis. The cells were treated with PGE₂ in the presence of indomethacin (IM) to avoid the influence of endogenous prostaglandins. At 24 hours, IM stimulated the DNA synthesis in both cell populations. Furthermore, a greater stimulation was found in the PL cells than in the OB cells. On the other hand, exogenously supplemented PGE₂ reversed the IM-induced stimulation of DNA synthesis. In contrast, high concentrations of PGE₂ alone increased the DNA synthesis. With respect to collagen synthesis, IM showed an inhibitory effect, especially in the PL cells. This inhibitory effect was also reversed by the addition of PGE₂. In addition to the stimulation of collagen synthesis, PGE₂ enhanced the proportion of protein synthesized as collagen. In the PL cells, the percentage of collagen synthesis was markedly decreased when cultured with IM for 48 hours. These results suggested that the effects of IM were mediated in part via its ability to reduce biosynthesis of prostaglandins, and that PGE₂ is a multifunctional autocrine regulator of bone formation.     

Reduced T-Cell and Dendritic Cell Function Is Related to Cyclooxygenase-2 Overexpression and Prostaglandin E<Subscript>2</Subscript> Secretion in Patients With Breast Cancer

Background: In several neoplastic diseases, including breast cancer, immunosuppression correlates with disease stage, progression, and outcome. Thus, thorough analysis of immune parameters in breast cancer patients may be beneficial in designing effective anticancer immune-based therapies.Methods: We investigated dendritic cell and T-cell function in breast cancer patients at various stages of the disease and in age-matched controls. We also evaluated cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE₂) levels within the tumor milieu and in the circulation.Results: T cells from cancer patients showed decreased proliferation in response to CD3 antibody stimulation. Analysis of T-cell helper type 1 and 2 cytokines revealed reduced levels of interferon-, tumor necrosis factor-, interleukin (IL)-12, and IL-2 and increased levels of IL-10 and IL-4. Dendritic cells from these patients showed significantly reduced expression of co-stimulatory molecules (B7 and CD40) and demonstrated reduced phagocytic ability, reduced antigen presentation to T cells, and reduced ability to mature in response to lipopolysaccharide. Data revealed increased synthesis of PGE₂, an immune suppressor, along with increased expression of COX-2, a key regulator of PGE₂ synthesis.Conclusions: COX-2–induced PGE₂ may contribute to immunosuppression and may directly block antitumor immunity while promoting tumor growth, providing us with the rationale for using COX-2 inhibition combined with immunotherapy.     

Effect of low-dose infusion of prostaglandin E1 on vecuronium-induced neuromuscular blockade

The effect of low-dose (20 ng·kg⁻¹·min⁻¹) infusion of prostaglandin E₁ (PGE₁) on vecuronium-induced neuromuscular blockade was studied. The study population consisted of 24 elderly patients (65–75 years old) and 24 younger adult patients (25–56 years old). They were randomly assigned to the control and PGE₁ groups. The steady-state dose requirement (SSDR) of vecuronium was derived from ondemand infusion of the drug which produced a stable twitch height of 20% of its baseline reading, and recovery time after steady-state infusion was defined as the time for recovery from twitch height from 25% to 75%. The patients in the PGE₁ group received an infusion of PGE₁ 20 ng·kg⁻¹·min⁻¹, while those in the control group received an infusion of normal saline. The SSDR (23.2±9.1 and 34.2±5.9 μg·kg⁻¹. hr⁻¹, respectively;P=0.02) was significantly less and the recovery time (35.0±9.5 and 19.9±4.2 min, respectively;P=0.01) was significantly longer in the elderly than in the younger patients. However, low-dose infusion of PGE₁ significantly increased the SSDR (23.2±9.1 to 37.4±3.7 μg· kg⁻¹·hr⁻¹;P=0.01) and shortened the recovery time (35.0±9.5 to 23.5±4.0 min;P=0.02) in elderly patients. We concluded that low-dose infusion of PGE₁ is effective in preventing the prolonged action of vecuronium in elderly patients.     

The effect of vitamin K2 on bone metabolism in aged female rats

Reactive oxygen species (ROS) may contribute to aging and osteoporosis resulting from marked decreases in plasma antioxidants in aged osteoporotic women. On the other hand, high-dose vitamin K₂ (menaquinone-4: menatrenone, MK-4) supplementation has been reported to reduce ovariectomy-induced bone loss in rats and to decrease osteoporotic fracture in postmenopausal women. However, the mechanism by which vitamin K₂ prevents osteoporosis is unclear. Recently, vitamin K₂ has been suggested to preserve antioxidant activity as a novel function. Therefore, we investigated the effect of vitamin K₂ on the osteoporosis of aged rats by evaluating the relationships between serum antioxidant levels and bone metabolism. Aged female rats exhibited significantly lower serum alkaline phosphatase activity and osteocalcin level, together with lower serum levels of antioxidants such as 17-estradiol, macrophage migration inhibitory factor (MIF) and glutathione peroxidase (GPx) activity, as compared with young female rats. On the other hand, vitamin K₂ supplementation (500 mg/kg, food intake) for 98 days led to a significantly increased serum vitamin K₂ level (3,045±915 ng/ml in the vitamin K₂ supplemented group vs. 4.6±3.4 ng/ml in the control diet group; P <0.0001) with increased serum alkaline phosphatase activity and MIF level ( P <0.05). Unexpectedly, however, it failed to increase the serum level of antioxidants such as GPx. Nor did it affect bone metabolism markers such as oteocalcin and osteopontin, which were significantly lower than in the young female rats ( P <0.05). Finally, the histomorphometric properties of the proximal tibia in the femur were not altered by vitamin K₂. These results suggest that high-dose vitamin K₂ supplementation neither improves lowered antioxidant levels nor stimulates bone formation in aged rats.     

Effect of cyclooxygenase-2 inhibitor pretreatment on gas exchange after hydrochloric acid aspiration in rats

The present study was carried out to determine the effect of cyclooxygenase-2 (COX-2) inhibitor on acid aspiration-induced lung injury in rats. Rats were allocated into one of four groups. Group H received intratracheal instillation of HCl. Group S received saline intratracheally. Group HC received COX-2 inhibitor (celecoxib) 10 mg/kg intravenously 30 min before intratracheal instillation of HCl. Group C underwent bronchoalveolar lavage (BAL) only. All rats were mechanically ventilated for 30 min before BAL. Arterial blood gas analysis was done immediately before BAL. Groups H, S, and HC were subdivided to each two groups. Groups H-1, S-1, and HC-1 underwent BAL 1 h after instillation, whereas groups H-8, S-8, and HC-8 underwent BAL 8 h after instillation. The BAL fluid was used to measure the prostaglandin E₂ (PGE₂) concentration. Intratracheal HCl resulted in impaired oxygenation. COX-2 inhibitor attenuated the impairment of oxygenation 8 h after instillation but not after 1 h. Intratracheal HCl caused an increase in PGE₂ concentration. COX-2 inhibitor attenuated an increase in PGE₂ concentration 8 h after instillation but not after 1 h. The results show that COX-2 inhibitor attenuates the oxygenation impairment and the increase in alveolar PGE2 concentration during the inflammatory phase of acid aspiration-induced lung injury in rats.     

Effect of 1α-Hydroxyvitamin D3 on Cancellous Bone Matrix: An Experimental Study on Adult Rats

Two groups of adult male rats were treated perorally for 6 weeks with 0.1 μg and 1.0 μg of 1a-hydroxyvitamin D₃ (la-OH-D₃), respectively. The effect of the treatment on cancellous bone matrix was studied by chemical analysis and morphometric measurements. The effect of the 1.0 μg dose on the inorganic composition, and on the calcification of the cancellous bone matrix, was significantly more pronounced, decreasing the amount of glycosaminoglycans. The lower dose level, 0.1 μg of la-OH-D₃, increased the collagen metabolism, whereas the higher dose level did not. The amount of cancellous bone determined morphometrically increased significantly during treatment with both dose levels. 1a-OH-D₃, converted in the organism to the hormonal form 1.25 (OH)₂D₃, induces new bone formation, probably by direct influence on the cancellous bone tissue itself.     

Effects of long-term daily administration of prostaglandin-E2 on maintaining elevated proximal tibial metaphyseal cancellous bone mass in male rats

Summary The effects of long-term prostaglandin E₂ (PGE₂) on cancellous bone in proximal tibial metaphysis were studied in 7-month-old male Sprague-Dawley rats given daily subcutaneous injections of 0, 1, 3, and 6 mg PGE₂/kg/day and sacrificed after 60, 120, and 180 days. Histomorphometric analyses were performed on double fluorescent-labeled undecalcified bone specimens. After 60 days of treatment, PGE₂ produced diffusely labeled trabecular bone area, increased trabecular bone area, eroded and labeled trabecular perimeter, mineral apposition rate, and bone formation rate at all dose levels when compared with age-matched controls. In rats given PGE₂ for longer time periods (120 and 180 days), trabecular bone area, diffusely labeled trabecular bone area, labeled perimeter, mineral apposition, and bone formation rates were sustained at the elevated levels achieved earlier at 60-day treatment. The eroded perimeter continued to increase until 120 days, then plateau. The observation that continuous systemic PGE₂ administration to adult male rats elevated metaphyseal cancellous bone mass to 3.5-fold of the control level within 60 days and maintained it for another 120 days indicates that the powerful skeletal anabolic effects of PGE₂ can be sustained with continuous administration.     

Development of the cyclic AMP response to parathyroid hormone and prostaglandin E2 in the embryonic chick limb

Summary The developing chick limb was studied to determine the ability of parathyroid hormone (PTH) and prostaglandin E₂ (PGE₂) to increase intracellular cyclic AMP (cAMP) during various stages of development. All developmental stages examined (stages 20–21, 24–25, and 26–28) responded to PGE₂ when the cells were assayed immediately following the removal of the limbs from the embryos. In contrast, only stage 26–28 limb cells responded to PTH when assayed in a similar manner. The response to PTH was temporally correlated with the appearance of cartilage matrix in vivo. Undifferentiated limb cells were also cultured and assayed at various times for hormone responsiveness. Stage 24–25 high-density cell cultures responded initially to PGE₂ but not to PTH. However, by 36 h and in all subsequent itme intervals tested, the response to PTH was significantly greater than that to PGE₂. The PTH receptor, in contrast to that of PGE₂, was shown to be sensitive to trypsin treatment, but could be regenerated during subsequent cell culture. The majority of the hormoneresponsive cells were found in cartilaginous regions of the limb, and were shown to respond to both hormones in a dose-dependent manner. The PTH-induced cAMP response was affected by low cell density and mouse serum, both of which significantly inhibit the chondrogenic potential of cultured limb cells. These findings are consistent with a temporal correlation between the development of the PTH response and chondrogenesis in vivo.     

Inhibitory effect of prostaglandin E1 on gastric secretion during general anesthesia in humans

The present study was undertaken to clarify the effects of prostaglandin E₁ (PGE₁) on gastric secretion during general anesthesia. Thirty-three patients, 16 with (PGE₁ group) and 17 without (control group) PGE₁ administration, scheduled for selective surgery were studied during general anesthesia with nitrous oxide (67%) and enflurane (1%–2% inspired). PGE₁ was administered at a rate of 50–200 ng·kg⁻¹·min⁻¹ when hypotensive medication was required. In the PGE₁ group, gastric juice was collected serially three times before and during administration and 60 min after discontinuation of PGE₁. In the control group, it was collected three times corresponding to those in the PGE₁ group. The pH of gastric juice increased significantly, and the acidity and pepsin activity decreased after the beginning of the administration of PGE₁, and these changes were observed even 1h after discontinuation. There was significant differences in the pH, acidity, and pepsin activity between the two groups after administration of PGE₁. The results indicate that PGE₁ inhibits gastric secretion at doses that produce a sufficient hypotensive effect under general anesthesia.