Effects of ovariectomy and estrogen therapy on type II collagen degradation and structural integrity of articular cartilage in rats: implications of the time of initiation.

OBJECTIVE: To investigate how the time of initiation influences the effects of estrogen therapy on type II collagen (CII) turnover and the structural integrity of articular cartilage in ovariectomized rats and to determine whether estrogen exerts direct effects on the catabolic function of chondrocytes ex vivo. METHODS: A total of 46 Sprague-Dawley rats were distributed into 1 of the following treatment groups: 1) ovariectomy, 2) ovariectomy plus early estrogen therapy, 3) ovariectomy plus delayed estrogen therapy, or 4) sham operation. Cartilage turnover was estimated by measuring the serum levels of C-telopeptide of type II collagen (CTX-II). Cartilage lesions at week 9 were quantified using a published scoring technique. The presence of the CTX-II epitope in articular cartilage was assessed by immunohistochemistry. The effects of estrogen (1-100 nM) on chondrocytes were investigated in bovine cartilage explants subjected to catabolic cytokines (tumor necrosis factor alpha [TNFalpha] and oncostatin M [OSM]). RESULTS: In ovariectomized rats, estrogen therapy evoked significant decreases in serum CTX-II independently of the time of initiation; yet, delayed initiation resulted in diminished efficacy in terms of preventing cartilage lesions. CTX-II fragments were present in articular cartilage, colocalizing with early lesions at the cartilage surface. In untreated animals, the early relative increases in serum CTX-II were proportional to the severity of cartilage lesions at week 9 (r = 0.73, P < 0.01). Estrogen significantly and dose-dependently countered CTX-II release from TNFalpha plus OSM-stimulated cartilage explants ex vivo. CONCLUSION: Our results suggest that estrogen counters the acceleration of CII degradation and related structural alterations, and these benefits can be maximized by early initiation after menopause. The protective effect of estrogen seems to involve direct inhibition of the catabolic function of chondrocytes.

Osteoclasts from patients with autosomal dominant osteopetrosis type I caused by a T253I mutation in low-density lipoprotein receptor-related protein 5 are normal in vitro, but have decreased resorption capacity in vivo.

Autosomal dominant osteopetrosis type I (ADOI) is presumably caused by gain-of-function mutations in the LRP5 gene. Patients with a T253I mutation in LRP5 have a high bone mass phenotype, characterized by increased mineralizing surface index but abnormally low numbers of small osteoclasts. To investigate the effect of the T253I mutation in LRP5 on osteoclasts, we isolated CD14+ monocytes from ADOI patients and assessed their ability to generate osteoclasts when treated with RANKL and M-CSF compared to that of age- and sex-matched control osteoclasts. We found normal osteoclastogenesis, expression of osteoclast markers, morphology, and localization of proteins involved in bone resorption, such as ClC-7 and cathepsin K. The ability to resorb bone was also normal. In vivo, we compared the bone resorption and bone formation response to T3 in ADOI patients and age- and sex-matched controls. We found attenuated resorptive response to T3 stimulation, despite a normal bone formation response, in alignment with the reduced number of osteoclasts in vivo. These data demonstrate that ADOI osteoclasts are normal with respect to all aspects investigated in vitro. We speculate that the mutations causing ADOI alter the osteoblastic phenotype toward a smaller potential for supporting osteoclastogenesis.

Suppression of elevated cartilage turnover in postmenopausal women and in ovariectomized rats by estrogen and a selective estrogen-receptor modulator (SERM).

OBJECTIVE: Several observational studies indicate that estrogen deficiency increases the incidence of osteoarthritis in postmenopausal women. To validate this observation, we investigated the effects of ovariectomy (OVX) on cartilage erosion in rats using histology and an established bio-assay of cartilage-specific collagen type II degradation products (CTX-II). Furthermore, we investigated whether estrogen and levormeloxifene, a selective estrogen-receptor modulator (SERM), can prevent the OVX-induced changes in cartilage degradation. The clinical relevance was assessed in postmenopausal women by measuring the changes in CTX-II during 12-month treatment with levormeloxifene versus placebo. DESIGN: Sixty 6-month-old rats were divided in five groups. One group was subjected to sham and the others to OVX, followed by treatment with vehicle alone, estradiol or 0.2 mg/kg/day or 5 mg/kg/day of levormeloxifene. The rats were treated for 9 weeks with biweekly blood and urine sampling for measurement of bone resorption and cartilage turnover. After study termination, hind knees were removed for histological analysis of erosions. The effect of levormeloxifene in post-menopausal women was assessed by measuring CTX-II in samples from 301 women who were participating in a phase II study of this SERM. RESULTS: OVX rats showed significant increases in the urinary excretion of CTX-II. After 9 weeks this was manifested as increased surface erosion of knee articular cartilage compared with sham-operated rats. Treatment with estrogen or levormeloxifene prevented the OVX-induced changes. There was a significant correlation between the 4-week changes in CTX-II and cartilage erosion at week 9 (r = 0.64, P < 0.001). In postmenopausal women treated with levormeloxifene, the urinary excretion of CTX-II was decreased by approximately 50% and restored CTX-II levels to the premenopausal range. CONCLUSIONS: This study is the first to demonstrate that a SERM suppresses cartilage degradation in both rodents and humans, suggesting potential therapeutical benefits in the prevention of destructive joint diseases such as osteoarthritis.

Ovariectomized rats as a model of postmenopausal osteoarthritis: validation and application.

We aimed to assess the effect of ovariectomy on cartilage turnover and degradation, to evaluate whether ovariectomized (OVX) rats could form an experimental model of postmenopausal osteoarthritis. The effect of ovariectomy on cartilage was studied using two cohorts of female Sprague-Dawley rats, aged 5 and 7 months. In a third cohort, the effect of exogenous estrogen and a selective estrogen receptor modulator was analyzed. Knee joints were assessed by histological analysis of the articular cartilage after 9 weeks. Cartilage turnover was measured in urine by an immunoassay specific for collagen type II degradation products (CTX-II), and bone resorption was quantified in serum using an assay for bone collagen type I fragments (CTX-I). Surface erosion in the cartilage of the knee was more severe in OVX rats than in sham-operated animals, particularly in the 7-month-old cohort (P = 0.008). Ovariectomy also significant increased CTX-I and CTX-II. Both the absolute levels of CTX-II and the relative changes from baseline seen at week 4 correlated strongly with the severity of cartilage surface erosion at termination (r = 0.74, P < 0.01). Both estrogen and the selective estrogen receptor modulator inhibited the ovariectomy-induced acceleration of cartilage and bone turnover and significantly suppressed cartilage degradation and erosion seen in vehicle-treated OVX rats. The study indicates that estrogen deficiency accelerates cartilage turnover and increases cartilage surface erosion. OVX rats provide a useful experimental model for the evaluation of the chondroprotective effects of estrogens and estrogen-like substances and the model may be an in vivo representation of osteoarthritis in postmenopausal women.

Estradiol enhances osteolytic lesions in mice inoculated with human estrogen receptor-negative MDA-231 breast cancer cells in vivo.

The effect of 17-beta-estradiol (E2) on the induction of osteolytic lesions by estrogen receptor (ER)-negative breast cancer cells was investigated in 4-week-old female nude mice. Exposure to exogenous E2 was found to increase osteolytic areas on radiographs up to 5.3 times in mice inoculated intracardially with MDA-231 human breast cancer cells. The MDA-231 cells were ER-negative, both before inoculation, and after isolation from osteolytic lesions, and the corresponding cell cultures were insensitive to E2. The induction of skeletal lesions by E2 in this mouse model was mainly effectuated at the early development of bone metastases, since exposure to E2 for 8 days around MDA-231 inoculation increased osteolysis to the same level, as did E2 given throughout the entire 31-day experimental period, and because E2-exposure for just the final 14 days had no effect. Independently of exposure to E2, histology revealed cancer cells in hind limp long bones of approximately 80% of the mice, and tumors were absent in non-skeletal organs. In vitro studies showed that the number and activity of osteoclasts generated from mouse bone marrow cells were increased 5-6 times when co-cultured with MDA-231 cells. Addition of 0.1-10 nM E2 further dose-dependently increased the osteoclastogenesis and associated bone resorption in these co-cultures. In conclusion, E2 was found to increase the morbidity in mice inoculated with ER-negative MDA-231 cells, and to stimulate osteoclast formation and bone resorption in co-cultures of bone marrow cells and MDA-231, suggesting that the progression of osteolytic metastases by ER-negative breast cancer cells can be induced by E2 due to stimulation of osteoclastogenesis.

Synthetic matrix metalloproteinase inhibitors inhibit growth of established breast cancer osteolytic lesions and prolong survival in mice.

PURPOSE: Breast cancer frequently leads to incurable bone metastasis. Essential requirements for the development of bone metastasis are cell-cell and cell-matrix interactions, release of bioactive growth factors and cytokines, and removal of large amounts of bone matrix. Matrix metalloproteinases (MMPs) play an important role in all of these processes, but the possibility of using synthetic MMP inhibitors to decrease bone metastasis has received little attention. EXPERIMENTAL DESIGN: In the present study, we tested two general MMP inhibitors, BB-94 and GM6001, in a mouse model of breast cancer-induced bone metastasis. RESULTS: In a simulation of intervention therapy, mice were inoculated with breast cancer cells, and at the time of diagnosis of osteolytic lesions, the mice were treated for 10 or 15 consecutive days with BB-94 or GM6001, respectively. Both inhibitors reduced the growth of osteolytic lesions by >55% compared with control mice. Next, we simulated prevention therapy by initiating treatment with GM6001 at time of inoculation with cancer cells or 3 days earlier. Assessment of osteolytic lesions 28 days after inoculation showed that, in both cases, the treatment reduced the size of the osteolytic lesions by 60%, compared with that of control mice. Importantly, MMP inhibition also resulted in extension of symptom-free survival in the mice, whether the treatment was initiated at the time of diagnosis of osteolytic lesions or of cancer cell inoculation. CONCLUSIONS: The present study suggests the potential of synthetic MMP inhibitors as intervention or prevention treatments of breast cancer-induced osteolysis.