Extracellular matrix of cultured bovine aortic endothelial cells contains functionally active type 1 plasminogen activator inhibitor

The extracellular matrix (ECM) of cultured bovine aortic endothelial cells (BAEs) was analyzed by immunoblotting and reverse fibrin autography and shown to contain type 1 plasminogen activator inhibitor (PAI-1). Most PAI-1 in the ECM formed complexes with exogenously added tissue-type plasminogen activator (tPA), demonstrating that this PAI-1 was functionally active. The resulting tPA/PAI-1 complexes were recovered in the reaction solution, indicating that the PAI-1 in such complexes no longer bound to ECM. The PAI-1 could not be removed by incubating ECM in high salt (2 mol/L NaCl), sugars (1 mol/L galactose, 1 mol/L mannose), glycosaminoglycans (10 mmol/L heparin, 10 mmol/L dermatan sulfate), or epsilon-aminocaproic acid (0.1 mol/L). However, PAI-1 could be extracted from ECM by treatment with either arginine (0.5 mol/L) or potassium thiocyanate (2 mol/L), or by incubation under acidic conditions (pH 2.5). ECM depleted of PAI-1 by acid extraction was able to bind both the active and latent forms of PAI-1. In this instance, most of the bound PAI-1 did not form complexes with tPA, indicating that the latent form was not activated as a consequence of binding to ECM. Although the PAI-1 activity in conditioned medium decayed with a half-life (t 1/2) of less than 3 hours, the t 1/2 of ECM- associated PAI-1 was greater than 24 hours. These data suggest that PAI- 1 is produced by cultured BAEs in an active form and is then either released into the medium where it is rapidly inactivated or into the subendothelium where it binds to ECM. The specific binding of PAI-1 to ECM protects it from this inactivation.     

Skeletal Histomorphometry in Subjects on Teriparatide or Zoledronic Acid Therapy (SHOTZ) Study: A Randomized Controlled Trial

Context: Recent studies on the mechanism of action (MOA) of bone-active drugs have rekindled interest in how to present and interpret dynamic histomorphometric parameters of bone remodeling. Objective: We compared the effects of an established anabolic agent, teriparatide (TPTD), with those of a prototypical antiresorptive agent, zoledronic acid (ZOL). Design: This was a 12-month, randomized, double-blind, active-comparator controlled, cross-sectional biopsy study. Setting: The study was conducted at 12 U.S. and Canadian centers. Subjects: Healthy postmenopausal women with osteoporosis participated in the study. Interventions: Subjects received TPTD 20 μg once daily by sc injection (n = 34) or ZOL 5 mg by iv infusion at baseline (n = 35). Main Outcome Measures: The primary end point was mineralizing surface/bone surface (MS/BS), a dynamic measure of bone formation, at month 6. A standard panel of dynamic and static histomorphometric indices was also assessed. When specimens with missing labels were encountered, several methods were used to calculate mineral apposition rate (MAR). Serum markers of bone turnover were also measured. Results: Among 58 subjects with evaluable biopsies (TPTD = 28; ZOL = 30), MS/BS was significantly higher in the TPTD group (median: 5.60 vs. 0.16%, P < 0.001). Other bone formation indices, including MAR, were also higher in the TPTD group (P < 0.05). TPTD significantly increased procollagen type 1 N-terminal propeptide (PINP) at months 1, 3, 6, and 12 and carboxyterminal cross-linking telopeptide of collagen type 1 (CTX) from months 3 to 12. ZOL significantly decreased PINP and CTX below baseline at all time points. Conclusions: TPTD and ZOL possess fundamentally different mechanisms of action with opposite effects on bone formation based on this analysis of both histomorphometric data and serum markers of bone formation and resorption. An important mechanistic difference was a substantially higher MS/BS in the TPTD group. Overall, these results define the dynamic histomorphometric characteristics of anabolic activity relative to antiresorptive activity after treatment with these two drugs.     

A Novel Monthly Dosing Regimen of Risedronate for the Treatment of Postmenopausal Osteoporosis: 2-Year Data

This 2-year trial evaluated the efficacy and tolerability of a monthly oral regimen of risedronate. Postmenopausal women with osteoporosis were randomly assigned to double-blind treatment with risedronate 75 mg on 2 consecutive days each month (2CDM) or 5 mg daily. The primary end point was the percentage change from baseline in lumbar spine bone mineral density (BMD) at 12 months. Secondary end points included the change in BMD of the lumbar spine and proximal femur and in bone turnover markers as well as the number of subjects with at least one new vertebral fracture over 24 months. Among 1,229 patients who were randomized and received at least one dose of risedronate, lumbar spine BMD was increased in both treatment groups: mean percentage change from baseline was 4.2 ± 0.19 and 4.3 ± 0.19 % in the 75 mg 2CDM and 5 mg daily groups, respectively, at month 24. The treatment difference was 0.17 (95 % confidence interval ?0.35 to 0.68). There were no statistically significant differences between treatment groups on any secondary efficacy parameters. Both treatment regimens were well tolerated. Risedronate 75 mg 2CDM was noninferior in BMD efficacy and did not show a difference in tolerability compared to 5 mg daily after 24 months of treatment in women with postmenopausal osteoporosis. This monthly regimen may provide a more convenient dosing schedule to some patients with postmenopausal osteoporosis.     

Oral Ibandronate in Postmenopausal Osteoporotic Women Alters Micromechanical Properties Independently of Changes in Mineralization

Postmenopausal osteoporotic (PMOP) women treated with ibandronate had higher bone mineral density, lower bone turnover, and decreased incidence of new vertebral fractures. The aim of this study was to investigate the effect of daily or intermittent oral ibandronate on the degree of mineralization (DMB) of bone and microhardness (Hv) at the bone tissue and bone structural unit (BSU) levels. A total of 110 iliac biopsies were taken from patients treated for 22 or 34 months with an oral placebo (n = 36), 2.5 mg daily oral ibandronate (n = 40), or 20 mg intermittent oral ibandronate (n = 34). These regimens provide annual cumulative exposures (ACEs) that are about half of the therapeutic doses currently licensed for PMOP women. DMB and Hv were measured at the global level (i.e., cortical or cancellous) and the focal level (i.e., BSU). At the global level, DMB and its distribution were not significantly different from placebo after 22 and 34 months of treatment. Hv was significantly higher in the cortical, cancellous, and total bone after 22 and 34 months of ibandronate versus placebo for both regimens. At the focal level, DMB and Hv, measured simultaneously in 3,760 BSUs, were significantly and positively correlated in all groups (r = 0.59–0.65, p < 0.0001). However, analysis of covariance highlighted the differences in the y intercepts of the linear regressions of the placebo- and ibandronate-treated groups. We infer that a low ACE of oral ibandronate altered the bone micromechanical properties irrespective of changes in secondary mineralization.     

Effects of Odanacatib on Bone Structure and Quality in Postmenopausal Women With Osteoporosis: 5-Year Data From the Phase 3 Long-Term Odanacatib Fracture Trial (LOFT) and its Extension

Odanacatib (ODN), a selective oral inhibitor of cathepsin K, was an investigational agent previously in development for the treatment of osteoporosis. In this analysis, the effects of ODN on bone remodeling/modeling and structure were examined in the randomized, double-blind, placebo-controlled, event-driven, Phase 3, Long-term Odanacatib Fracture Trial (LOFT; NCT00529373) and planned double-blind extension in postmenopausal women with osteoporosis. A total of 386 transilial bone biopsies, obtained from consenting patients at baseline (ODN n = 17, placebo n = 23), month 24 (ODN n = 112, placebo n = 104), month 36 (ODN n = 42, placebo n = 41), and month 60 (ODN n = 27, placebo n = 20) were assessed by dynamic and static bone histomorphometry. Patient characteristics at baseline and BMD changes over 5 years for this subset were comparable to the overall LOFT population. Qualitative assessment of biopsies revealed no abnormalities. Consistent with the mechanism of ODN, osteoclast number was higher with ODN versus placebo over time. Regarding bone remodeling, dynamic bone formation indices in trabecular, intracortical, and endocortical surfaces were generally similar in ODN-treated versus placebo-treated patients after 2 years of treatment. Regarding periosteal modeling, the proportion of patients with periosteal double labels and the bone formation indices increased over time in the ODN-treated patients compared with placebo. This finding supported the observed numerical increase in cortical thickness at month 60 versus placebo. In conclusion, ODN treatment for 5 years did not reduce bone remodeling and increased the proportion of patients with periosteal bone formation. These results are consistent with the mechanism of action of ODN, and are associated with continued BMD increases and reduced risk of fractures compared with placebo in the LOFT Phase 3 fracture trial. © 2020 American Society for Bone and Mineral Research.     

Bone microstructure in osteoporosis: transilial biopsy and histomorphometry

The bone fragility of osteoporosis is not fully explained by a deficit in bone mass. Histomorphometric examination of transilial bone biopsies has identified microstructural defects that-in light of what is known about the mechanical properties of structural materials-further compromise bone strength. Histomorphometric measures describe the biopsy specimen, the configuration of its trabeculae in space, and the extent to which its trabecular lattice is intact. In postmenopausal women with established osteoporosis, a deficit of both cortical and cancellous bone is typical, i.e., both cortical thickness and cancellous bone volume tend to be substantially reduced. Much of the cancellous bone deficit can be attributed to loss of entire trabecular elements rather than to generalized thinning of trabeculae. Direct measures of trabecular connectivity confirm this impression: women with established osteoporosis have fewer trabecular nodes and more termini than healthy women, even at the same cancellous bone volume. Evidence for accumulated microdamage in transilial biopsies is circumstantial, and the phenomenon itself may well be localized to fracture sites. Histomorphometric data from transilial biopsies comprise a large body of information about the structural and functional character of osteoporosis and provide valuable information about the effects of new treatments on bone microstructure.     

Relationship of Bone Mineralization Density Distribution (BMDD) in Cortical and Cancellous Bone Within the Iliac Crest of Healthy Premenopausal Women

Bone mineralization density distribution (BMDD) is an important determinant of bone mechanical properties. The most available skeletal site for access to the BMDD is the iliac crest. Compared to cancellous bone much less information on BMDD is available for cortical bone. Hence, we analyzed complete transiliac crest bone biopsy samples from premenopausal women (n = 73) aged 25–48 years, clinically classified as healthy, by quantitative backscattered electron imaging for cortical (Ct.) and cancellous (Cn.) BMDD. The Ct.BMDD was characterized by the arithmetic mean of the BMDD of the cortical plates. We found correlations between Ct. and Cn. BMDD variables with correlation coefficients r between 0.42 and 0.73 (all p < 0.001). Additionally to this synchronous behavior of cortical and cancellous compartments, we found that the heterogeneity of mineralization densities (Ct.Ca_Width), as well as the cortical porosity (Ct.Po) was larger for a lower average degree of mineralization (Ct.Ca_Mean). Moreover, Ct.Po correlated negatively with the percentage of highly mineralized bone areas (Ct.Ca_High) and positively with the percentage of lowly mineralized bone areas (Ct.Ca_Low). In conclusion, the correlation of cortical with cancellous BMDD in the iliac crest of the study cohort suggests coordinated regulation of bone turnover between both bone compartments. Only in a few cases, there was a difference in the degree of mineralization of >1wt % between both cortices suggesting a possible modeling situation. This normative dataset of healthy premenopausal women will provide a reference standard by which disease- and treatment-specific effects can be assessed at the level of cortical bone BMDD.