Osteoporosis in experimental postmenopausal polyarthritis: the relative contributions of estrogen deficiency and inflammation

Generalized osteoporosis in postmenopausal rheumatoid arthritis (RA) is caused both by estrogen deficiency and by the inflammatory disease. The relative importance of each of these factors is unknown. The aim of this study was to establish a murine model of osteoporosis in postmenopausal RA, and to evaluate the relative importance and mechanisms of menopause and arthritis-related osteoporosis. To mimic postmenopausal RA, DBA/1 mice were ovariectomized, followed by the induction of type II collagen-induced arthritis. After the mice had been killed, paws were collected for histology, one femur for bone mineral density (BMD) and sera for analyses of markers of bone resorption (RatLaps; type I collagen cross-links, bone formation (osteocalcin) and cartilage destruction (cartilage oligomeric matrix protein), and for the evaluation of antigen-specific and innate immune responsiveness. Ovariectomized mice displayed more severe arthritis than sham-operated controls. At termination of the experiment, arthritic control mice and non-arthritic ovariectomized mice displayed trabecular bone losses of 26% and 22%, respectively. Ovariectomized mice with arthritis had as much as 58% decrease in trabecular BMD. Interestingly, cortical BMD was decreased by arthritis but was not affected by hormonal status. In addition, markers of bone resorption and cartilage destruction were increased in arthritic mice, whereas markers of bone formation were increased in ovariectomized mice. This study demonstrates that the loss of endogenous estrogen and inflammation contribute additively and equally to osteoporosis in experimental postmenopausal polyarthritis. Markers of bone remodeling and bone marrow lymphocyte phenotypes indicate different mechanisms for the development of osteoporosis caused by ovariectomy and arthritis in this model.     

Female reproductive system and bone

The female reproductive system plays a major role in regulating the acquisition and loss of bone by the skeleton from menarche through senescence. Onset of gonadal sex steroid secretion at puberty is the major factor responsible for skeletal longitudinal and radial growth, as well as significant gain in bone density, until peak bone density is achieved in third decade of life. Gonadal sex steroids then help maintain peak bone density until menopause, including during the transient changes in skeletal mineral content associated with pregnancy and lactation. At menopause, decreased gonadal sex steroid production normally leads to rapid bone loss. The most rapid bone loss associated with decreased estrogen levels occurs in the first 8-10 years after menopause, with slower age-related bone loss occurring during later life. Age-related bone loss in women after the early menopausal phase of bone loss is caused by ongoing gonadal sex steroid deficiency, vitamin D deficiency, and secondary hyperparathyroidism. Other factors also contribute to age-related bone loss, including intrinsic defects in osteoblast function, impairment of the GH/IGF axis, reduced peak bone mass, age-associated sarcopenia, and various sporadic secondary causes. Further understanding of the relative contributions of the female reproductive system and each of the other factors to development and maintenance of the female skeleton, bone loss, and fracture risk will lead to improved approaches for prevention and treatment of osteoporosis.     

Effects of reproduction on sexual dimorphisms in rat bone mechanics

Osteoporosis most commonly affects postmenopausal women. Although men are also affected, women over 65 are 6 times more likely to develop osteoporosis than men of the same age. This is largely due to accelerated bone remodeling after menopause; however, the peak bone mass attained during young adulthood also plays an important role in osteoporosis risk. Multiple studies have demonstrated sexual dimorphisms in peak bone mass, and additionally, the female skeleton is significantly altered during pregnancy/lactation. Although clinical studies suggest that a reproductive history does not increase the risk of developing postmenopausal osteoporosis, reproduction has been shown to induce long-lasting alterations in maternal bone structure and mechanics, and the effects of pregnancy and lactation on maternal peak bone quality are not well understood. This study compared the structural and mechanical properties of male, virgin female, and post-reproductive female rat bone at multiple skeletal sites and at three different ages. We found that virgin females had a larger quantity of trabecular bone with greater trabecular number and more plate-like morphology, and, relative to their body weight, had a greater cortical bone size and greater bone strength than males. Post-reproductive females had altered trabecular microarchitecture relative to virgins, which was highly similar to that of male rats, and showed similar cortical bone size and bone mechanics to virgin females. This suggests that, to compensate for future reproductive bone losses, females may start off with more trabecular bone than is mechanically necessary, which may explain the paradox that reproduction induces long-lasting changes in maternal bone without increasing postmenopausal fracture risk.     

Systemically administered rhBMP-2 promotes MSC activity and reverses bone and cartilage loss in osteopenic mice

Osteoporosis is a disease manifested in drastic bone loss resulting in osteopenia and high risk for fractures. This disease is generally divided into two subtypes. The first, post-menopausal (type I) osteoporosis, is primarily related to estrogen deficiency. The second, senile (type II) osteoporosis, is mostly related to aging. Decreased bone formation, as well as increased bone resorption and turnover, are thought to play roles in the pathophysiology of both types of osteoporosis. In this study, we demonstrate in murine models for both type I (estrogen deficiency) and type II (senile) osteopenia/osteoporosis that reduced bone formation is related to a decrease in adult mesenchymal stem cell (AMSC) number, osteogenic activity, and proliferation. Decreased proliferation is coupled with increased apoptosis in AMSC cultures obtained from osteopenic mice. Recombinant human bone morphogenetic protein (rhBMP-2) is a highly osteoinductive protein, promoting osteogenic differentiation of AMSCs. Systemic intra-peritoneal (i.p.) injections of rhBMP-2 into osteopenic mice were able to reverse this phenotype in the bones of these animals. Moreover, this change in bone mass was coupled to an increase in AMSCs numbers, osteogenic activity, and proliferation as well as a decrease in apoptosis. Bone formation activity was increased as well. However, the magnitude of this response to rhBMP-2 varied among different stains of mice. In old osteopenic BALB/c male mice (type II osteoporosis model), rhBMP-2 systemic treatment also restored both articular and epiphyseal cartilage width to the levels seen in young mice. In summary, our study shows that AMSCs are a good target for systemically active anabolic compounds like rhBMP-2.     

Measurement of bone mineral density (BMD) with quantitative computed tomography (QCT) in postmenopausal osteoporosis: effect of estrogen.

To determine the efficacy of the estrogen replacement therapy (ERT) on the bone mineral density (BMD) measured with quantitative computed tomography (QCT) in postmenopausal osteoporosis 16 women aged 46-72 were examined. They were divided into two groups: 8 women treated with conjugated estrogens (Group I) and 8 who did not received ERT (Group II). In all 16 patients the serum hormonal concentrations (LH, FSH and estradiol) were measured with radioimmunological methods. The bone densitometry was performed in all of them using the single-energy computed tomography (QCT) with the computer Picker 1200. Bone mineral density was measured in three lumbar vertebra (L1-L3) and expressed in milligrams K2HPO4 per ml. The bone mineral density (BMD) was statistically significantly higher in the estrogen treated group (Group I) in every vertebra compared with that of controls (Group II). The serum FSH concentration was statistically significantly lower in the ERT group (Group I) and a statistically significant correlation between FSH level and average BMD (Lmean) was present. In conclusion: 1. the ERT is very efficacious in preventing bone loss in postmenopausal women; 2. measurement of BMD in lumbar vertebra L1 or L3 may be a sufficiently reliable and accurate, cost-effective and time-saving method of screening for osteoporosis; 3. the serum FSH determination seems to be useful in monitoring of the estrogen therapy for postmenopausal osteoporosis.     

Clinical Significance and Pathogenesis of Osteoporosis

The development of osteoporosis with advancing age in man is a widespread if not a universal phenomenon. The average loss between youth and old age amounts to about 15% of the skeleton but involves a much larger proportion of trabecular than of cortical bone.The principal clinical manifestation of osteoporosis is fracture, and three osteoporotic fracture syndromes can be defined: the lower forearm fracture, which predominantly affects women between the ages of 50 and 65; the fracture of the proximal femur, which affects both sexes over the age of 70; and the relatively rare vertebral crush fracture syndrome, which may present at any age but is most common in elderly women.The lower forearm fracture rate is inversely related to the mean normal lower forearm x-ray “density” of the wrist, which falls by about 30% in the 15 years following the menopause. This process, which is associated with corresponding trabecular bone loss elsewhere in the skeleton, is associated with a corresponding rise in the fasting urinary calcium excretion. Some degree of negative calcium balance, and consequent bone resorption, probably occurs in everyone during the later part of the night because calcium absorption is completed within about three to five hours of a meal. In postmenopausal women, however, the sensitivity of the bone to parathyroid hormone appears to be increased, and their nocturnal negative calcium balance therefore comes to exceed the positive balance which can be achieved during the waking hours.Femoral neck fractures in old people reflect the further progression of osteoporosis with advancing age since the fracture rate is inversely correlated with the mean thickness of the metacarpal cortex in the normal population. This progressive osteoporosis is associated with and could well result from a steady decline in calcium absorption which is at least partially attributable to vitamin-D deficiency and reversible on vitamin-D treatment.The vertebral crush fracture syndrome represents a severe degree of spinal osteoporosis which may be associated with relatively normal peripheral bones. It probably results from an accelerated negative calcium balance which mobilizes trabecular bone preferentially. Some of the factors which may contribute to this accelerated negative balance have been identified and include a reduced rate of bone turnover, impaired calcium absorption, and low oestrogen activity as judged by vaginal smears, but there may well be others as yet unidentified.     

In vivo anti-osteoporotic activity of isotaxiresinol, a lignan from wood of Taxus yunnanensis

Isotaxiresinol, the main lignan isolated from the water extract of wood of Taxus yunnanensis, was investigated for its effect on bone loss, on serum biochemical markers for bone remodeling and on uterine tissue, using ovariectomized (OVX) rats as the model of postmenopausal osteoporosis. After oral administration of isotaxiresinol (50 and 100mg/kg/d) for 6 weeks, bone mineral content (BMC) and bone mineral density (BMD) in total and cortical bones were increased as compared to those of OVX control rats, and decreases of three bone strength indexes induced by OVX surgery were prevented. Serum biochemical markers for bone remodeling revealed that isotaxiresinol slightly increased bone formation and significantly inhibited bone resorption without side effect on uterine tissue. These results suggest that isotaxiresinol may be useful for treatment of postmenopausal osteoporosis, especially for prevention of bone fracture induced by estrogen deficiency.     

Clinical problems related to postmenopausal osteoporosis]

Authors discuss current hypotheses related to the causes of osteoporosis and its possible therapy. One of the frequent menopausal disorders is osteoporosis, i.e. decrease in bone density, which may eventually lead to the pathological fractures and marked deformities of the skeleton. Etiology of the postmenopausal osteoporosis is not clearly explained. However, one may assume that hormonal disturbances, especially estrogen deficit, plays an important causative role. The treatment is difficult. It is usually aimed at preventing of bone loss.     

Age-associated Iron Accumulation in Bone: Implications for Postmenopausal Osteoporosis and a New Target for Prevention and Treatment by Chelation

Iron accumulation in tissues is believed to be a characteristic of aged humans and a risk factor for some chronic diseases. However, it is not known whether age-associated iron accumulation is part of the pathogenesis of postmenopausal osteoporosis that affects approximately one out three women worldwide. Here, we confirmed that this accumulation of iron was associated with osteopenia in ovariectomized (OVX) rats (a model of peri- and postmenopausal osteoporosis due to estrogen deficiency). To further investigate whether the increased iron level plays a causal role in the onset of bone loss, we treated OVX rats with an orally active and bone targeted chelator that prevented iron accumulation in their skeletal tissues. The results showed that this treatment mitigated the loss of bone mass and the deterioration of bone micro-architecture. We also found that one possible mechanism of the protective action of iron chelation was to significantly reduce bone resorption. Thus, these findings provide a novel target and a potentially useful therapeutic strategy for the prevention and treatment of postmenopausal osteoporosis and perhaps other age-related diseases.     

Bone mineral density in Addison's disease: evidence for an effect of adrenal androgens on bone mass.

It is unknown whether replacement doses of cortisone acetate and the absence of the small amounts of androgens secreted by the adrenal cortex may cause osteoporosis. This was studied in 35 patients (12 men and 23 women) suffering from primary adrenocortical failure and taking cortisone acetate 25-37.5 mg and fludrocortisone 50-100 micrograms daily. Bone mineral density was measured by single photon absorptiometry at the midshaft of the radius, representing cortical bone, and at the distal part of the radius, a site with a significant trabecular component. The bone mineral density was normal in premenopausal female patients as well as in male patients, showing that replacement doses of cortisone acetate do not affect bone mass. By contrast, in postmenopausal patients there was a dramatic bone loss in addition to the physiological postmenopausal decrease in bone mass. This loss, combined with the low plasma concentrations of androstenedione, dehydroepiandrosterone, and testosterone (and low concentrations of oestrone of adrenal origin), indicates that adrenal androgens may be essential for the maintenance of bone mass in postmenopausal women with Addison''s disease. In addition, these data indicate that the small amounts of androgens secreted by the adrenal cortex have a role in the maintenance of bone mass in normal postmenopausal women.     

Perspectives on using nonhuman primates to understand the etiology and treatment of postmenopausal osteoporosis

The reproductive physiology and skeletal anatomy of nonhuman primates are very similar to those of women and these similarities have prompted studies of the effects of ovariectomy in monkeys on bone metabolism. Following ovariectomy, monkey bone exhibits increases in remodeling activity resulting in bone loss. Since similar bone changes occur after menopause in women, ovariectomized monkeys provide an excellent model of the early skeletal events following menopause and have been employed to study the skeletal actions of drugs designed to treat postmenopausal osteoporosis. This review describes the motivations for examining monkeys, practical aspects of working with monkeys, comparisons of human and monkey bone anatomy, endocrinological aspects of monkey bone metabolism, and the available data obtained in monkeys related to postmenopausal and other forms of osteoporosis.     

Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts

Estrogen prevents osteoporotic bone loss by attenuating bone resorption; however, the molecular basis for this is unknown. Here, we report a critical role for the osteoclastic estrogen receptor alpha (ERalpha) in mediating estrogen-dependent bone maintenance in female mice. We selectively ablated ERalpha in differentiated osteoclasts (ERalpha(DeltaOc/DeltaOc)) and found that ERalpha(DeltaOc/DeltaOc) females, but not males, exhibited trabecular bone loss, similar to the osteoporotic bone phenotype in postmenopausal women. Further, we show that estrogen induced apoptosis and upregulation of Fas ligand (FasL) expression in osteoclasts of the trabecular bones of WT but not ERalpha(DeltaOc/DeltaOc) mice. The expression of ERalpha was also required for the induction of apoptosis by tamoxifen and estrogen in cultured osteoclasts. Our results support a model in which estrogen regulates the life span of mature osteoclasts via the induction of the Fas/FasL system, thereby providing an explanation for the osteoprotective function of estrogen as well as SERMs.     

Evidence of type II estrogen receptor in human osteoblast-like cells

Osteoblast-like cells isolated from human bone bioptic specimens were characterized and analysed for the presence of type II estrogen receptor (type II EBS). The amount of type II EBS was measured by a whole-cell assay at 4 degrees C for 2.5 h using [(3)H]-estradiol as tracer. Saturation analysis, used to investigate the binding characteristic of type II EBS, resulted in a sigmoid curve. Scatchard analysis showed the binding affinity of the estrogen receptor, yielding a concave plot. The dissociation constant (K(d)), determined from the [(3)H]-estradiol concentration required for half saturation was about 12+/-2 nM (SD). The number of type II EBS, estimated at maximum binding, was 197,000+/-8800 sites per cell. If the regulation of the receptor by flavonoids would be confirmed, the evidence of type II EBS in osteoblast-like cells could suggest a direct action of ipriflavone and others flavonoids on bone density in postmenopausal osteoporosis.     

Evaluation of the relative rates of bone mineral content loss in postmenopause due to both estrogen deficiency and ageing

To evaluate the relative rates of bone mineral content loss in postmenopause due to both estrogen deficiency and ageing, three groups of women were studied by computerized bone densitometry at the radius mid-point and at the distal point, modified according to the Abwrey technique. All women were in apparent good health and never had estrogen therapy. In the first group there were 64 women aged between 30 and 50 who were ovariectomized between 25 and 35 years of age. The second group was made up of 309 women between 50 and 55 years. In the third group there were 136 women aged 30-50 with normal ovaric function. The ordinary functions of linear polynomial regression were used to describe the variations in density with age. The percentage of postmenopausal bone loss was determined by calculating the BMC value at the start of the menopause and again twenty years later, according to the linear regression equation of postmenopausal period of each group of women in the study. The women who had natural menopause showed an average bone loss per year of 1.63% at the mid radius and 1.0% at the distal point. The ovariectomized women had an average loss of 0.85% at the mid point and 0.66% at the distal point. No significant decrease of bone mass was found before menopause. From a comparison between the two groups of women with analogous periods of menopause, it comes out that, during the first 20 years of natural menopause, estrogen deficiency is responsible for 52.5%-66.4% of the bone mineral loss, the remaining amount being attributable to other causes, connected with ageing. Estrogen deficiency is therefore, the principal factor causing bone mineral loss in natural menopause.     

New approaches to the treatment of osteoporosis

Under physiological conditions, maintenance of skeletal mass is the result of a tightly coupled process of bone formation and bone resorption. Disease states, osteoporosis included, arise when this delicate balance is disrupted such as in menopause, when estrogen levels decrease dramatically corresponding with the cessation of ovarian function. Current therapies for the treatment of osteoporosis, including estrogen replacement therapy, selective estrogen receptor modulators and bisphosphonates, are primarily based on blunting the resorption component of bone homeostasis. Although selective estrogen receptor modulators offer bone protection without the side effects of estrogen replacement therapy, there are some areas of improvement for the current generation of selective estrogen receptor modulators; particularly in reducing their antagonistic properties in the central nervous system that lead to vasomotor symptoms. There are few therapies that are focused on increasing bone formation, but they offer promising avenues in which to expand the repertoire of drugs to restore bone mass. Selective androgen receptor modulators, parathyroid hormone analogs, oxytocin analogs and statins, all with improved pharmacological properties in bone, are among the potential approaches to eliciting anabolic effects in the skeleton.     

Overview: animal models of osteopenia and osteoporosis

Prior to initiating a clinical trial in a post-menopausal osteoporosis study, it is reasonable to recommence the evaluation of treatment in the 9-month-old ovariectomized female rat. A female rat of this age has reached peak bone mass and can be manipulated to simulate clinical findings of post-menopausal osteoporosis. Ample time exists for experimental protocols that either prevent estrogen depletion osteopenia or restore bone loss after estrogen depletion. More time can be saved by acceleration of the development of the osteopenia by combining ovariectomized (OVX) plus immobilization (IM) models. Methods like serum biochemistry, histomorphometry and densitometry used in humans are applicable in rats. Like most animal models of osteopenia, the rat develops no fragility fractures, but mechanical testing of rat bones substitutes as a predictor of bone fragility. Recent studies have shown that the prevailing activity in cancellous and cortical bone of the sampling sites in rats is remodeling. The problems of dealing with a growing skeleton, the site specificity of the OVX and IM models, the lack of trabecular and Haversian remodeling and the slow developing cortical bone loss have been and can be overcome by adding beginning and pre-treatment controls and muscle mass measurements in all experimental designs, selecting cancellous bone sampling sites that are remodeling, concentrating the analysis of cortical bone loss to the peri-medullary bone and combining OVX and IM in a model to accelerate the development of both cancellous and cortical bone osteopenia. Not to be forgotten is the distal tibia site, an adult bone site with growth plate closure at 3 months and low trabecular bone turnover and architecture similar to human spongiosa. This site would be most challenging to the action of bone anabolic agents. Data about estrogen-deplete mice are encouraging, but the ovariectomized rat model suggests that developing an ovariectomized mouse model as an alternative is not urgent. Nevertheless, the mouse model has a place in drug development and skeletal research. In dealing with drug development, it could be a useful model because it is a much smaller animal requiring fewer drugs for screening. In skeletal research mice are useful in revealing genetic markers for peak bone mass and gene manipulations that affect bone mass, structure and strength. When the exciting mouse glucocorticoid-induced bone loss model of Weinstein and Manolagas is confirmed by others, it could be a significant breakthrough for that area of research. Lastly, we find that the information generated from skeletal studies of nonhuman primates has been most disappointing and recommend that these expensive skeletal studies be curtailed unless it is required by a regulatory agency for safety studies.     

Reduced bone turnover in mice lacking the P2Y(13) receptor of ADP

Osteoporosis is a condition of excessive and uncoupled bone turnover, in which osteoclastic resorption exceeds osteoblastic bone formation, resulting in an overall net bone loss, bone fragility, and morbidity. Although numerous treatments have been developed to inhibit bone loss by blocking osteoclastic bone resorption, understanding of the mechanisms behind bone loss is incomplete. The purinergic signaling system is emerging to be a pivotal regulator of bone homeostasis, and extracellular ADP has previously been shown to be a powerful osteolytic agent in vitro. We report here that deletion of the P2Y(13) receptor, a G protein-coupled receptor for extracellular ADP, leads to a 40% reduction in trabecular bone mass, 50% reduction in osteoblast and osteoclast numbers in vivo, as well as activity in vitro, and an overall 50% reduction in the rate of bone remodeling in mice in vivo. Down-regulation of RhoA/ROCK I signaling and a reduced ratio of receptor activator of nuclear factor κB ligand/osteoprotegerin observed in osteoblasts from P2Y(13)R(-/-) mice might explain this bone phenotype. Furthermore, because one of the main causes of osteoporosis in older women is lack of estrogen, we examined the effect of ovariectomy of the P2Y(13)R(-/-) mice and found them to be protected from ovariectomy-induced bone loss by up to 65%. These data confirm a role of purinergic ADP signaling in the skeleton, whereby deletion of the P2Y(13) receptor leads to reduced bone turnover rates, which provide a protective advantage in conditions of accelerated bone turnover such as oestrogen deficiency-induced osteoporosis.     

Estrogen improves the proliferation and differentiation of hBMSCs derived from postmenopausal osteoporosis through notch signaling pathway

Estrogen deficiency is the main reason of bone loss, leading to postmenopausal osteoporosis, and estrogen replacement therapy (ERT) has been demonstrated to protect bone loss efficiently. Notch signaling controls proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Moreover, imperfect estrogen-responsive elements (EREs) were found in the 5′-untranslated region of Notch1 and Jagged1. Thus, we examined the molecular and biological links between estrogen and the Notch signaling in postmenopausal osteoporosis in vitro. hBMSCs were obtained from healthy women and patients with postmenopausal osteoporosis. Notch signaling molecules were quantified using real-time polymerase chain reaction (real-time PCR) and Western Blot. Luciferase reporter constructs with putative EREs were transfected into hBMSCs and analyzed. hBMSCs were transduced with lentiviral vectors containing human Notch1 intracellular domain (NICD1). We also used N-[N-(3, 5-diflurophenylacetate)-l-alanyl]-(S)-phenylglycine t-butyl ester, a γ-secretase inhibitor, to suppress the Notch signaling. We found that estrogen enhanced the Notch signaling in hBMSCs by promoting the expression of Jagged1. hBMSCs cultured with estrogen resulted in the up-regulation of Notch signaling and increased proliferation and differentiation. Enhanced Notch signaling could enhance the proliferation and differentiation of hBMSCs from patients with postmenopausal osteoporosis (OP-hBMSCs). Our results demonstrated that estrogen preserved bone mass partly by activating the Notch signaling. Because long-term ERT has been associated with several side effects, the Notch signaling could be a potential target for treating postmenopausal osteoporosis.