Loncastuximab tesirine in relapsed/refractory diffuse large B-cell lymphoma: long-term efficacy and safety from the phase II LOTIS-2 study.

Therapies that demonstrate durable, long-term responses with manageable safety and tolerability are needed for patients with relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL). Loncastuximab tesirine (loncastuximab tesirine-lpyl [Lonca]), an anti-CD19 antibody conjugated to a potent pyrrolobenzodiazepine dimer, demonstrated single-agent antitumor activity in the pivotal phase II LOTIS-2 study in heavily pretreated patients with R/R DLBCL. Here we present updated efficacy and safety analyses from LOTIS-2, performed for all patients and in subsets of patients with a complete response (CR), including patients with CR who were event-free (no progressive disease or death) for ≥1 year and ≥2 years from cycle 1, day 1 of treatment. Lonca was administered every 3 weeks (0.15 mg/kg for 2 cycles; 0.075 mg/kg for subsequent cycles). As of the final data cutoff (September 15, 2022; median follow-up: 7.8 months [range, 0.3-42.6]), 70 of 145 (48.3%) patients achieved an overall response. Thirty-six (24.8%) patients achieved CR, of which 16 (44%) and 11 (31%) were event-free for ≥1 year and ≥2 years, respectively. In the all-treated population, the median overall survival was 9.5 months; the median progression-free survival was 4.9 months. Among patients with CR, median overall survival and progression-free survival were not reached, with 24-month overall and progression-free survival rates of 68.2% (95% CI: 50.0-81.0) and 72.5% (95% CI: 48.2-86.8), respectively. No new safety concerns were detected. With additional follow-up, Lonca continued to demonstrate durable, long-term responses with manageable safety and tolerability in patients with CR (clinicaltrials gov. Identifier: NCT03589469).

Activin A marks a novel progenitor cell population during fracture healing and reveals a therapeutic strategy.

Insufficient bone fracture repair represents a major clinical and societal burden and novel strategies are needed to address it. Our data reveal that the transforming growth factor-ß superfamily member Activin A became very abundant during mouse and human bone fracture healing but was minimally detectable in intact bones. Single-cell RNA-sequencing revealed that the Activin A-encoding gene Inhba was highly expressed in a unique, highly proliferative progenitor cell (PPC) population with a myofibroblast character that quickly emerged after fracture and represented the center of a developmental trajectory bifurcation producing cartilage and bone cells within callus. Systemic administration of neutralizing Activin A antibody inhibited bone healing. In contrast, a single recombinant Activin A implantation at fracture site in young and aged mice boosted: PPC numbers; phosphorylated SMAD2 signaling levels; and bone repair and mechanical properties in endochondral and intramembranous healing models. Activin A directly stimulated myofibroblastic differentiation, chondrogenesis and osteogenesis in periosteal mesenchymal progenitor culture. Our data identify a distinct population of Activin A-expressing PPCs central to fracture healing and establish Activin A as a potential new therapeutic tool.

Risk factors for pulmonary cement embolism after percutaneous vertebroplasty and radiofrequency ablation for spinal metastases.

Objective: Pulmonary cement embolism is a rare but underestimated complication of vertebroplasty due to the relative lack of study and examination. This study aims to investigate the incidence of pulmonary cement embolism in patients with spinal metastasis who undergo PVP with RFA and to analyze the relative risk factors. Methods: A total of 47 patients were retrospectively included and classified into pulmonary cement embolism (PCE) group and non-pulmonary cement embolism (NPCE) group by comparing pre- and postoperative pulmonary CT scan images. The demographic and clinical information of the patients was obtained. Demographic data in the two groups were compared using the chi-square test for qualitative data and the unpaired t test for quantitative data. Multiple logistic regression analysis was used to identify risk factors related to pulmonary cement embolism. Results: Pulmonary cement embolism was detected in 11 patients (23.4%), and all patients were asymptomatic and followed up regularly. Risk analysis showed that multiple segments (≥3, p=0.022), thoracic vertebrae (p=0.0008), and unipedicular puncture approach (p=0.0059) were risk factors for pulmonary cement embolism. There was a high incidence of pulmonary cement embolism if bone cement leaked into the para vertebral venous plexus in the thoracic vertebra (p<0.0001). Vein leakage of cement was related to the integrity of the vertebral cortex. Conclusion: The number of involved vertebrae, lesion location, and puncture approach are independent risk factors for pulmonary cement embolism. There was a high incidence of pulmonary cement embolism if bone cement leaked into the para vertebral venous plexus in the thoracic vertebra. Surgeons should consider these factors when formulating therapeutic strategies.

Melanoma Molecular Subtypes and Development of Prognostic and Immunotherapy-Related Genetic Characteristics by Ferroptosis Gene Analysis.

The dissimilarity is a major problem in clinical therapy of skin cutaneous melanoma (SKCM). Objective and reproducible classification systems may help decode SKCM heterogeneity. ConsensusClusterPlus was used to establish a stable immune molecular classification based on ferroptosis-related genes that had been acquired from FerrDb. Moreover, the prognosis, somatic mutations, immune microenvironment characteristics, functional enrichment, and clinical responsiveness to the immune checkpoint blockade of different subtypes in two independent melanin datasets were compared. Kaplan-Meier curves, univariate, multivariate, least absolute contraction, and selection operator (LASSO) Cox regression analysis were used to develop a molecular model for predicting survival, which was verified by a nomogram on the basis of independent prognostic indicators. Two molecular subtypes (C1 and C2) for SKCM were first identified according to ferroptosis-related genes; C1 showed a poor prognosis, with lower infiltration degree of immune cells and TIED score and higher homologous recombination defects, fraction altered, the number of segments, and copy number amplification and deletion. These characteristics of C2 were the opposite of C1. A ferroptosis-related prognosis risk score (FPRS) model was constructed using 6 of 463 genes with differential expression between C1 and C2. This model splits patients into low- and high-risk cohorts. There were significant differences in the infiltration and proportion of immune cells, immune checkpoint gene expression, responsiveness to immune checkpoint therapy, and sensitivity to chemotherapeutic medications between low- and high-risk cohorts. This model was an independent prognostic marker for SKCM and has a high AUC. In summary, we have identified two subtypes of SKCM with different molecular and immune characteristics on the basis of ferroptosis-related genes and further developed and verified an FPRS model, which might independently serve as a prognostic marker for SKCM.

MiR-21 participates in LPS-induced myocardial injury by targeting Bcl-2 and CDK6.

OBJECTIVE: This study aimed to investigate the relationship between miR-21 and lipopolysaccharide (LPS)-induced myocardial injury and its molecular and regulatory mechanisms. METHODS: We constructed LPS-mediated myocardial injury model using C57BL/6J mice and H9c2 cells. In-vivo, in-vitro, RIP and dual-luciferase reporter assays were used to determine the effect of miR-21 on myocardial injury. RESULTS: In-vivo and in-vitro results showed that the expression of miR-21 was increased in LPS-treated H9c2 cells and myocardial tissues of mice, and the pro-inflammatory cytokines (IL-1ß, IL-6, IL-8 and TNF-α) and NF-κB pathway were activated in LPS-treated H9c2 cells. Besides, the B-cell lymphoma-2 (Bcl-2) and cyclin-dependent kinase 6 (CDK6) expression levels decreased, while Bax and cleaved caspase 9 levels increased in LPS-treated H9c2 cells. Inhibition of miR-21 could suppress LPS-induced apoptosis, inflammatory reactions and NF-κB activation to attenuate LPS-induced myocardial injury in H9c2 cells, and effectively improve survival of mice with sepsis. Most importantly, Bcl-2 and CDK6 were found to be the direct target of miR-21 using dual-luciferase reporter and RNA immunoprecipitation assays. Further gain-of-function assay demonstrated that Bcl-2 or CDK6 over-expression promoted the protective effects of miR-21 inhibitor on LPS-mediated myocardial cells. CONCLUSION: Our findings revealed that the down-regulation or antagonism of miR-21 protects myocardial cells against LPS-induced apoptosis and inflammation through up-regulating Bcl-2 and CDK6 expression, which provided a new insight for prevention and treatment of myocardial injury.

Health-Related Quality of Life, Symptoms, and Tolerability of Loncastuximab Tesirine in Patients With Relapsed or Refractory Diffuse Large B-Cell Lymphoma.

BACKGROUND: Loncastuximab tesirine has shown antitumor activity with an acceptable toxicity profile in patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) who were relapsed or refractory after ≥2 prior therapies, including activity in patients with high-risk disease characteristics. This analysis examined health-related quality of life (HRQoL), symptoms, and tolerability in patients receiving loncastuximab tesirine for relapsed or refractory DLBCL. PATIENTS AND METHODS: The single-arm, open-label phase II LOTIS-2 study (ADCT-402-201; NCT03589469) enrolled 145 patients aged ≥18 years. Patients received loncastuximab tesirine as a 30-minute intravenous infusion on day 1 of each 3-week treatment cycle. Patient-reported outcomes were measured using EQ-5D and FACT-Lym at baseline, day 1 of each cycle, and the end-of-treatment visit. RESULTS: During the course of treatment, EQ VAS overall health score was improved over time. The adjusted improvement was 0.65 per cycle (95% CI, 0.26-1.04; P = .001), and the adjusted mean change from baseline score was 5.00 (95% CI, 1.75-8.25; P = .003) at cycle 9, day 1. FACT-Lym total scores remained stable during treatment. More patients reported improvement compared to baseline in pain, lumps/swelling, and losing weight for a majority of visits. More than 60% of patients reported being "not at all" or "a little bit" bothered by treatment side effects for all treatment visits. Findings in elderly patients were similar to the population as whole. CONCLUSION: The findings on HRQoL, symptoms, and tolerability further support the clinical use of loncastuximab tesirine for the treatment of relapsed or refractory DLBCL. FUNDING: This work was funded by ADC Therapeutics SA. Authors affiliated with ADC Therapeutics SA participated in designing the study; in collecting, analyzing, and interpreting the data; in writing the report; and in the decision to submit the article for publication.

Short Cyclic Regimen With Parathyroid Hormone (PTH) Results in Prolonged Anabolic Effect Relative to Continuous Treatment Followed by Discontinuation in Ovariectomized Rats.

Despite the potent effect of intermittent parathyroid hormone (PTH) treatment on promoting new bone formation, bone mineral density (BMD) rapidly decreases upon discontinuation of PTH administration. To uncover the mechanisms behind this adverse phenomenon, we investigated the immediate responses in bone microstructure and bone cell activities to PTH treatment withdrawal and the associated long-term consequences. Unexpectedly, intact female and estrogen-deficient female rats had distinct responses to the discontinuation of PTH treatment. Significant tibial bone loss and bone microarchitecture deterioration occurred in estrogen-deficient rats, with the treatment benefits of PTH completely lost 9 weeks after discontinuation. In contrast, no adverse effect was observed in intact rats, with sustained treatment benefit 9 weeks after discontinuation. Intriguingly, there is an extended anabolic period during the first week of treatment withdrawal in estrogen-deficient rats, during which no significant change occurred in the number of osteoclasts, whereas the number of osteoblasts remained elevated compared with vehicle-treated rats. However, increases in number of osteoclasts and decreases in number of osteoblasts occurred 2 weeks after discontinuation of PTH treatment, leading to significant reduction in bone mass and bone microarchitecture. To leverage the extended anabolic period upon early withdrawal from PTH, a cyclic administration regimen with repeated cycles of on and off PTH treatment was explored. We demonstrated that the cyclic treatment regimen efficiently alleviated the PTH withdrawal-induced bone loss, improved bone mass, bone microarchitecture, and whole-bone mechanical properties, and extended the treatment duration. © 2021 American Society for Bone and Mineral Research (ASBMR).

[Effects of miR-155-3p on the degradation rate of EAF1 mRNA and malignant proliferation in HANK1 cells].

Objective: To investigate the effects and mechanisms of miR-155-3p on the malignant behavior of human NK/T cell lymphoma cell line HANK1. Methods: Targetscan database was used to predict the target gene of miR-155-3p. HANK1 cells in logarithmic growth period were cultured, and the cells were divided into blank group, over-expressed group, control group and interference group, which were transfected with pENTER-puro vector, pENTER-miR-155-3p vector, GV248 control vector and GV248-miR-155-3p siRNA interference vector, respectively. Meanwhile, actinomycin D (ActD) was used to treat each group of cells, and the expressions of miR-155-3p, EAf1, ß-catenin and c-Myc in each group were detected by real-time fluorescence quantitative PCR (n=5). The degradation rate of EAF1 mRNA, the expressions of EAF1, ß-catenin and c-Myc protein were detected by Western blot (n=3), and the malignant proliferation abilities of cells were detected by CCK-8 (n=5). Results: Compared with the blank group, the expression levels of miR-155-3p, ß-catenin and c-Myc in the over-expressed group were significantly higher, the expression level of EAF1 was lower, the half-life of EAF1 mRNA was shortened, and the malignant proliferation ability of the cells was strengthened (P＜0.05). Compared with the control group, the expression levels of miR-155-3p, ß-catenin and c-Myc in the interference group were significantly lower, and the expression level of EAF1 was increased, the half-life of mRNA was prolonged and the ability of cell proliferation was decreased (P＜0.05). Conclusion: miR-155-3p can promote EAF1 mRNA degradation and proliferation in HANK1 cells.

Plasminogen Regulates Fracture Repair by Promoting the Functions of Periosteal Mesenchymal Progenitors.

Defective or insufficient bone repair and regeneration are common in patients as a result of major trauma or severe disease. Cell therapy with periosteal mesenchymal progenitors, which can be limited in severe injury, serves as a promising approach; however, its efficacy is limited due to a repair-hostile ischemic tissue microenvironment after traumatic fracture. Here we report that plasminogen (Plg), a factor that is upregulated in these environments, is critical for fracture healing. Plg knockout mice had impaired trabecular and cortical bone structure and exhibited delayed and incomplete fracture healing. Interestingly, Plg deficiency greatly reduced the thickness of expanded periosteum, suggesting a role of Plg in periosteal mesenchymal progenitor-mediated bone repair. In culture, Plg increased cell proliferation and migration in periosteal mesenchymal progenitors and inhibited cell death under ischemic conditions. Mechanistically, we revealed that Plg cleaved and activated Cyr61 to regulate periosteal progenitor function. Thus, our study uncovers a cellular mechanism underlying fracture healing, by which Plg activates Cyr61 to promote periosteal progenitor proliferation, survival, and migration and improves bone repair after fracture. Targeting Plg may offer a rational and effective therapeutic opportunity for improving fracture healing. © 2021 American Society for Bone and Mineral Research (ASBMR).

Gli1 Defines a Subset of Fibro-adipogenic Progenitors that Promote Skeletal Muscle Regeneration With Less Fat Accumulation.

Skeletal muscle has remarkable regenerative ability after injury. Mesenchymal fibro-adipogenic progenitors (FAPs) are necessary, active participants during this repair process, but the molecular signatures of these cells and their functional relevance remain largely unexplored. Here, using a lineage tracing mouse model (Gli1-CreER Tomato), we demonstrate that Gli1 marks a small subset of muscle-resident FAPs with elevated Hedgehog (Hh) signaling. Upon notexin muscle injury, these cells preferentially and rapidly expanded within FAPs. Ablation of Gli1+ cells using a DTA mouse model drastically reduced fibroblastic colony-forming unit (CFU-F) colonies generated by muscle cells and impaired muscle repair at 28 days. Pharmacologic manipulation revealed that Gli1+ FAPs rely on Hh signaling to increase the size of regenerating myofiber. Sorted Gli1+ FAPs displayed superior clonogenicity and reduced adipogenic differentiation ability in culture compared to sorted Gli1- FAPs. In a glycerol injury model, Gli1+ FAPs were less likely to give rise to muscle adipocytes compared to other FAPs. Further cell ablation and Hh activator/inhibitor treatments demonstrated their dual actions in enhancing myogenesis and reducing adipogenesis after injury. Examining single-cell RNA-sequencing dataset of FAPs from normal mice indicated that Gli1+ FAPs with increased Hh signaling provide trophic signals to myogenic cells while restrict their own adipogenic differentiation. Collectively, our findings identified a subpopulation of FAPs that play an essential role in skeletal muscle repair. © 2021 American Society for Bone and Mineral Research (ASBMR).

Final results of a phase 1 study of loncastuximab tesirine in relapsed/refractory B-cell non-Hodgkin lymphoma.

The prognosis for patients with relapsed or refractory (R/R) B-cell non-Hodgkin lymphoma (B-NHL) remains poor, with a need for alternatives to current salvage therapies. Loncastuximab tesirine (ADCT-402) is an antibody-drug conjugate comprising a humanized anti-CD19 monoclonal antibody conjugated to a pyrrolobenzodiazepine dimer toxin. Presented here are final results of a phase 1 dose-escalation and dose-expansion study in patients with R/R B-NHL. Objectives were to determine the maximum tolerated dose (MTD) and recommended dose(s) for expansion and evaluate safety, clinical activity, pharmacokinetics, and immunogenicity of loncastuximab tesirine. Overall, 183 patients received loncastuximab tesirine, with 3 + 3 dose escalation at 15 to 200 µg/kg and dose expansion at 120 and 150 µg/kg. Dose-limiting toxicities (all hematologic) were reported in 4 patients. The MTD was not reached, although cumulative toxicity was higher at 200 µg/kg. Hematologic treatment-emergent adverse events were most common, followed by fatigue, nausea, edema, and liver enzyme abnormalities. Overall response rate (ORR) in evaluable patients was 45.6%, including 26.7% complete responses (CRs). ORRs in patients with diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, and follicular lymphoma were 42.3%, 46.7%, and 78.6%, respectively. Median duration of response in all patients was 5.4 months and not reached in patients with DLBCL (doses ≥120 µg/kg) who achieved a CR. Loncastuximab tesirine had good stability in serum, notable antitumor activity, and an acceptable safety profile, warranting continued study in B-NHL. The recommended dose for phase 2 was determined as 150 µg/kg every 3 weeks for 2 doses followed by 75 µg/kg every 3 weeks. This trial was registered at www.clinicaltrials.gov as #NCT02669017.

Single cell transcriptomics identifies a unique adipose lineage cell population that regulates bone marrow environment.

Bone marrow mesenchymal lineage cells are a heterogeneous cell population involved in bone homeostasis and diseases such as osteoporosis. While it is long postulated that they originate from mesenchymal stem cells, the true identity of progenitors and their in vivo bifurcated differentiation routes into osteoblasts and adipocytes remain poorly understood. Here, by employing large scale single cell transcriptome analysis, we computationally defined mesenchymal progenitors at different stages and delineated their bi-lineage differentiation paths in young, adult and aging mice. One identified subpopulation is a unique cell type that expresses adipocyte markers but contains no lipid droplets. As non-proliferative precursors for adipocytes, they exist abundantly as pericytes and stromal cells that form a ubiquitous 3D network inside the marrow cavity. Functionally they play critical roles in maintaining marrow vasculature and suppressing bone formation. Therefore, we name them marrow adipogenic lineage precursors (MALPs) and conclude that they are a newly identified component of marrow adipose tissue.

Periosteal Mesenchymal Progenitor Dysfunction and Extraskeletally-Derived Fibrosis Contribute to Atrophic Fracture Nonunion.

Atrophic nonunion represents an extremely challenging clinical dilemma for both physicians and fracture patients alike, but its underlying mechanisms are still largely unknown. Here, we established a mouse model that recapitulates clinical atrophic nonunion through the administration of focal radiation to the long bone midshaft 2 weeks before a closed, semistabilized, transverse fracture. Strikingly, fractures in previously irradiated bone showed no bony bridging with a 100% nonunion rate. Radiation triggered distinct repair responses, separated by the fracture line: a less robust callus formation at the proximal side (close to the knee) and bony atrophy at the distal side (close to the ankle) characterized by sustained fibrotic cells and type I collagen-rich matrix. These fibrotic cells, similar to human nonunion samples, lacked osteogenic and chondrogenic differentiation and exhibited impaired blood vessel infiltration. Mechanistically, focal radiation reduced the numbers of periosteal mesenchymal progenitors and blood vessels and blunted injury-induced proliferation of mesenchymal progenitors shortly after fracture, with greater damage particularly at the distal side. In culture, radiation drastically suppressed proliferation of periosteal mesenchymal progenitors. Radiation did not affect hypoxia-induced periosteal cell chondrogenesis but greatly reduced osteogenic differentiation. Lineage tracing using multiple reporter mouse models revealed that mesenchymal progenitors within the bone marrow or along the periosteal bone surface did not contribute to nonunion fibrosis. Therefore, we conclude that atrophic nonunion fractures are caused by severe damage to the periosteal mesenchymal progenitors and are accompanied by an extraskeletal, fibro-cellular response. In addition, we present this radiation-induced periosteal damage model as a new, clinically relevant tool to study the biologic basis of therapies for atrophic nonunion. © 2018 American Society for Bone and Mineral Research.

Periarticular Mesenchymal Progenitors Initiate and Contribute to Secondary Ossification Center Formation During Mouse Long Bone Development.

Long bone development involves the embryonic formation of a primary ossification center (POC) in the incipient diaphysis followed by postnatal development of a secondary ossification center (SOC) at each epiphysis. Studies have elucidated major basic mechanisms of POC development, but relatively little is known about SOC development. To gain insights into SOC formation, we used Col2-Cre Rosa-tdTomato (Col2/Tomato) reporter mice and found that their periarticular region contained numerous Tomato-positive lineage cells expressing much higher Tomato fluorescence (termed TomatoH ) than underlying epiphyseal chondrocytes (termed TomatoL ). With time, the TomatoH cells became evident at the SOC invagination site and cartilage canal, increased in number in the expanding SOC, and were present as mesenchymal lineage cells in the subchondral bone. These data were verified in two mouse lineage tracing models, Col2-CreER Rosa-tdTomato and Gli1-CreER Rosa-tdTomato. In vitro tests showed that the periarticular TomatoH cells from Col2/Tomato mice contained mesenchymal progenitors with multidifferentiation abilities. During canal initiation, the cells expressed vascular endothelial growth factor (VEGF) and migrated into epiphyseal cartilage ahead of individual or clusters of endothelial cells, suggesting a unique role in promoting vasculogenesis. Later during SOC expansion, chondrocytes in epiphyseal cartilage expressed VEGF, and angiogenic blood vessels preceded TomatoH cells. Gene expression analyses of microdissected samples revealed upregulation of MMPs in periarticular cells at the invagination site and suggested potential roles for novel kinase and growth factor signaling pathways in regulating SOC canal initiation. In summary, our data indicate that the periarticular region surrounding epiphyseal cartilage contains mesenchymal progenitors that initiate SOC development and form subchondral bone. Stem Cells 2019;37:677-689.

Extracellular Matrix and Adhesion Molecule Gene Expression in the Normal and Injured Murine Intervertebral Disc.

OBJECTIVE: The aim of the study was to determine the transcription profile of the mouse nucleus pulposus and annulus fibrosus with an unbiased method. Furthermore, pathophysiological relevance of selected genes was demonstrated in the mouse tail intervertebral disc injury model. DESIGN: Paired normal mouse nucleus pulposus and annulus fibrosus tissue from C57BL/6j mice was examined by a polymerase chain reaction array. Key gene expression in the normal and injured intervertebral discs was confirmed by real-time polymerase chain reaction. RESULTS: Among the 84 genes studied, 63 were expressed higher in annulus fibrosus than in nucleus pulposus; only four genes were expressed higher in nucleus pulposus than in annulus fibrosus (n = 4, P ≤ 0.05). Real-time polymerase chain reaction confirmed that cadherin (cdh) 2 gene expression was higher in nucleus pulposus than in annulus fibrosus, and type I collagen (col1) gene expression was higher in the annulus fibrosus than in nucleus pulposus (n = 8, P < 0.01). One week after tail intervertebral disc injury, cdh2 gene expression decreased, while col1 expression increased (n = 8, P < 0.01). CONCLUSIONS: This is the first study to examine the relative expression of 84 genes in normal mouse nucleus pulposus and annulus fibrosus. Key genes in the normal and injured mouse intervertebral discs were confirmed with real-time polymerase chain reaction. This information should be useful for studying the mouse model of intervertebral disc degeneration and guide future cell therapy approaches.

Analysis of short-term treatment with the phosphodiesterase type 5 inhibitor tadalafil on long bone development in young rats.

Cyclic GMP (cGMP) is an important intracellular regulator of endochondral bone growth and skeletal remodeling. Tadalafil, an inhibitor of the phosphodiesterase (PDE) type 5 (PDE5) that specifically hydrolyzes cGMP, is increasingly used to treat children with pulmonary arterial hypertension (PAH), but the effect of tadalafil on bone growth and strength has not been previously investigated. In this study, we first analyzed the expression of transcripts encoding PDEs in primary cultures of chondrocytes from newborn rat epiphyses. We detected robust expression of PDE5 as the major phosphodiesterase hydrolyzing cGMP. Time-course experiments showed that C-type natriuretic peptide increased intracellular levels of cGMP in primary chondrocytes with a peak at 2 min, and in the presence of tadalafil the peak level of intracellular cGMP was 37% greater ( P < 0.01) and the decline was significantly attenuated. Next, we treated 1-mo-old Sprague Dawley rats with vehicle or tadalafil for 3 wk. Although 10 mg·kg-1·day-1 tadalafil led to a significant 52% ( P < 0.01) increase in tissue levels of cGMP and a 9% reduction ( P < 0.01) in bodyweight gain, it did not alter long bone length, cortical or trabecular bone properties, and histological features. In conclusion, our results indicate that PDE5 is highly expressed in growth plate chondrocytes, and short-term tadalafil treatment of growing rats at doses comparable to those used in children with PAH has neither obvious beneficial effect on long bone growth nor any observable adverse effect on growth plate structure and trabecular and cortical bone structure.

Proteasome inhibitor bortezomib is a novel therapeutic agent for focal radiation-induced osteoporosis.

Bone atrophy and its related fragility fractures are frequent, late side effects of radiotherapy in cancer survivors and have a detrimental impact on their quality of life. In another study, we showed that parathyroid hormone 1-34 and anti-sclerostin antibody attenuates radiation-induced bone damage by accelerating DNA repair in osteoblasts. DNA damage responses are partially regulated by the ubiquitin proteasome pathway. In the current study, we examined whether proteasome inhibitors have similar bone-protective effects against radiation damage. MG132 treatment greatly reduced radiation-induced apoptosis in cultured osteoblastic cells. This survival effect was owing to accelerated DNA repair as revealed by γH2AX foci and comet assays and to the up-regulation of Ku70 and DNA-dependent protein kinase, catalytic subunit, essential DNA repair proteins in the nonhomologous end-joining pathway. Administration of bortezomib (Bzb) reversed the loss of trabecular bone structure and strength in mice at 4 wk after focal radiation. Histomorphometry revealed that Bzb significantly increased the number of osteoblasts and activity in the irradiated area and suppressed the number and activity of osteoclasts, regardless of irradiation. Two weeks of Bzb treatment accelerated DNA repair in bone-lining osteoblasts and thus promoted their survival. Meanwhile, it also inhibited bone marrow adiposity. Taken together, we demonstrate a novel role of proteasome inhibitors in treating radiation-induced osteoporosis.-Chandra, A., Wang, L., Young, T., Zhong, L., Tseng, W.-J., Levine, M. A., Cengel, K., Liu, X. S., Zhang, Y., Pignolo, R. J., Qin, L. Proteasome inhibitor bortezomib is a novel therapeutic agent for focal radiation-induced osteoporosis.

Loading-Induced Reduction in Sclerostin as a Mechanism of Subchondral Bone Plate Sclerosis in Mouse Knee Joints During Late-Stage Osteoarthritis.

OBJECTIVE: To establish an unbiased, 3-dimensional (3-D) approach that quantifies subchondral bone plate (SBP) changes in mouse joints, and to investigate the mechanism that mediates SBP sclerosis at a late stage of osteoarthritis (OA). METHODS: A new micro-computed tomography (micro-CT) protocol was developed to characterize the entire thickness of the SBP in the distal femur of a normal mouse knee. Four mouse models of severe joint OA were generated: cartilage-specific Egfr-knockout (Egfr-CKO) mice at 2 months after surgical destabilization of the medial meniscus (DMM), Egfr-CKO mice with aging-related spontaneous OA, wild-type (WT) mice at 10 months after DMM, and WT mice at 14 weeks after DMM plus hemisectomy of the meniscus (DMMH) surgery. As an additional model, mice with knockout of the sclerostin gene (Sost-KO) were subjected to DMMH surgery. Knee joints were examined by micro-CT, histology, and immunohistochemical analyses. RESULTS: Examination of the mouse distal femur by 3-D micro-CT revealed a positive correlation between SBP thickness and the loading status in normal knees. In all 4 mouse models of late-stage OA, SBP sclerosis was restricted to the areas under severely eroded articular cartilage. This was accompanied by elevated bone formation at the bone marrow side of the SBP and a drastic reduction in the levels of sclerostin in osteocytes within the SBP. Unlike in WT mice, no further increase in the thickness of the SBP was observed in response to DMMH in Sost-KO mice. CONCLUSION: Since focal stress on the SBP underlying sites of cartilage damage increases during late stages of OA, these findings establish mechanical loading-induced attenuation of sclerostin expression and elevation of bone formation along the SBP surface as the major mechanisms characterizing subchondral bone phenotypes associated with severe late-stage OA in mice.

EGFR signaling is critical for maintaining the superficial layer of articular cartilage and preventing osteoarthritis initiation.

Osteoarthritis (OA) is the most common joint disease, characterized by progressive destruction of the articular cartilage. The surface of joint cartilage is the first defensive and affected site of OA, but our knowledge of genesis and homeostasis of this superficial zone is scarce. EGFR signaling is important for tissue homeostasis. Immunostaining revealed that its activity is mostly dominant in the superficial layer of healthy cartilage but greatly diminished when OA initiates. To evaluate the role of EGFR signaling in the articular cartilage, we studied a cartilage-specific Egfr-deficient (CKO) mouse model (Col2-Cre EgfrWa5/flox). These mice developed early cartilage degeneration at 6 mo of age. By 2 mo of age, although their gross cartilage morphology appears normal, CKO mice had a drastically reduced number of superficial chondrocytes and decreased lubricant secretion at the surface. Using superficial chondrocyte and cartilage explant cultures, we demonstrated that EGFR signaling is critical for maintaining the number and properties of superficial chondrocytes, promoting chondrogenic proteoglycan 4 (Prg4) expression, and stimulating the lubrication function of the cartilage surface. In addition, EGFR deficiency greatly disorganized collagen fibrils in articular cartilage and strikingly reduced cartilage surface modulus. After surgical induction of OA at 3 mo of age, CKO mice quickly developed the most severe OA phenotype, including a complete loss of cartilage, extremely high surface modulus, subchondral bone plate thickening, and elevated joint pain. Taken together, our studies establish EGFR signaling as an important regulator of the superficial layer during articular cartilage development and OA initiation.

Skeletal lesions in primary hyperparathyroidism.

BACKGROUND: Osteitis fibrosa cystica (OFC), a relatively rare skeletal disorder caused by excess parathyroid hormone, is often misdiagnosed as a neoplasm. A summary of the diagnostic procedures and treatment protocols, especially the indications for orthopedic surgery, is helpful to avoid overtreatment. METHODS: Eight patients from the Orthopedic Department of Qilu Hospital of Shandong University diagnosed with OFC caused by primary hyperparathyroidism were treated, and the clinical manifestations, biochemical and radiography examination findings, surgeries and prognoses were recorded. RESULTS: All cases (5 female and 3 male) were admitted to our department with the complaint of bone pain (5/8) or fracture after mild trauma (3/8). Biochemical screening revealed hypercalcemia and high parathyroid hormone of varying levels. Two cases were misdiagnosed as primary bone lesion and metastasis. All cases were treated with parathyroidectomy and experienced spontaneous and progressive regression of the boney disease. Four cases underwent orthopedic surgery. Bone biopsies were necessary to exclude malignant tumors, especially with orthopedic procedures. CONCLUSIONS: OFC can easily be misdiagnosed in orthopedic patients because of a lack of radiological and histologic specificity. Reaching the correct diagnosis requires a combination of clinical manifestations, routine biochemical screenings, radiographic examinations of bone and parathyroid and bone biopsy. It is generally acknowledged that parathyroidectomy is effective, but orthopedic surgery is sometimes necessary after a correct diagnosis and parathyroidectomy. However, the indications for orthopedic surgery must be strictly controlled to avoid overtreatment.