Mesenchymal stromal cell chondrogenesis under ALK1/2/3-specific BMP inhibition: a revision of the prohypertrophic signalling network concept.

BACKGROUND: In vitro chondrogenesis of mesenchymal stromal cells (MSCs) driven by the essential chondro-inducer transforming growth factor (TGF)-ß is instable and yields undesired hypertrophic cartilage predisposed to bone formation in vivo. TGF-ß can non-canonically activate bone morphogenetic protein-associated ALK1/2/3 receptors. These have been accused of driving hypertrophic MSC misdifferentiation, but data remained conflicting. We here tested the antihypertrophic capacity of two highly specific ALK1/2/3 inhibitors - compound A (CompA) and LDN-212854 (LDN21) - in order to reveal potential prohypertrophic contributions of these BMP/non-canonical TGF-ß receptors during MSC in vitro chondrogenesis. METHODS: Standard chondrogenic pellet cultures of human bone marrow-derived MSCs were treated with TGF-ß and CompA (500 nM) or LDN21 (500 nM). Daily 6-hour pulses of parathyroid hormone-related peptide (PTHrP[1-34], 2.5 nM, from day 7) served as potent antihypertrophic control treatment. Day 28 samples were subcutaneously implanted into immunodeficient mice. RESULTS: All groups underwent strong chondrogenesis, but GAG/DNA deposition and ACAN expression were slightly but significantly reduced by ALK inhibition compared to solvent controls along with a mild decrease of the hypertrophy markers IHH-, SPP1-mRNA, and Alkaline phosphatase (ALP) activity. When corrected for the degree of chondrogenesis (COL2A1 expression), only pulsed PTHrP but not ALK1/2/3 inhibition qualified as antihypertrophic treatment. In vivo, all subcutaneous cartilaginous implants mineralized within 8 weeks, but PTHrP pretreated samples formed less bone and attracted significantly less haematopoietic marrow than ALK1/2/3 inhibitor groups. CONCLUSIONS: Overall, our data show that BMP-ALK1/2/3 inhibition cannot program mesenchymal stromal cells toward stable chondrogenesis. BMP-ALK1/2/3 signalling is no driver of hypertrophic MSC misdifferentiation and BMP receptor induction is not an adverse prohypertrophic side effect of TGF-ß that leads to endochondral MSC misdifferentiation. Instead, the prohypertrophic network comprises misregulated PTHrP/hedgehog signalling and WNT activity, and a potential contribution of TGF-ß-ALK4/5-mediated SMAD1/5/9 signalling should be further investigated to decide about its postulated prohypertrophic activity. This will help to successfully engineer cartilage replacement tissues from MSCs in vitro and translate these into clinical cartilage regenerative therapies.

Enriched mesoporous bioactive glass scaffolds as bone substitutes in critical diaphyseal bone defects in rabbits.

In the field of orthopedic surgery, there is an increasing need for the development of bone replacement materials for the treatment of bone defects. One of the main focuses of biomaterials engineering are advanced bioceramics like mesoporous bioactive glasses (MBG´s). The present study compared the new bone formation after 12 weeks of implantation of MBG scaffolds with composition 82,5SiO2-10CaO-5P2O5-x 2.5SrO alone (MBGA), enriched with osteostatin, an osteoinductive peptide, (MBGO) or enriched with bone marrow aspirate (MBGB) in a long bone critical defect in radius bone of adult New Zealand rabbits. New bone formation from the MBG scaffold groups was compared to the gold standard defect filled with iliac crest autograft and to the unfilled defect. Radiographic follow-up was performed at 2, 6, and 12 weeks, and microCT and histologic examination were performed at 12 weeks. X-Ray study showed the highest bone formation scores in the group with the defect filled with autograft, followed by the MBGB group, in addition, the microCT study showed that bone within defect scores (BV/TV) were higher in the MBGO group. This difference could be explained by the higher density of newly formed bone in the osteostatin enriched MBG scaffold group. Therefore, MBG scaffold alone and enriched with osteostatin or bone marrow aspirate increase bone formation compared to defect unfilled, being higher in the osteostatin group. The present results showed the potential to treat critical bone defects by combining MBGs with osteogenic peptides such as osteostatin, with good prospects for translation into clinical practice. STATEMENT OF SIGNIFICANCE: Treatment of bone defects without the capacity for self-repair is a global problem in the field of Orthopedic Surgery, as evidenced by the fact that in the U.S alone it affects approximately 100,000 patients per year. The gold standard of treatment in these cases is the autograft, but its use has limitations both in the amount of graft to be obtained and in the morbidity produced in the donor site. In the field of materials engineering, there is a growing interest in the development of a bone substitute equivalent. Mesoporous bioactive glass (MBG´s) scaffolds with three-dimensional architecture have shown great potential for use as a bone substitutes. The osteostatin-enriched Sr-MBG used in this long bone defect in rabbit radius bone in vivo study showed an increase in bone formation close to autograft, which makes us think that it may be an option to consider as bone substitute.

Osteostatin, a peptide for the future treatment of musculoskeletal diseases.

Nowadays, the treatment of musculoskeletal diseases represents a major challenge in the developed world. Diseases such as osteoporosis, osteoarthritis and arthritis have a high incidence and prevalence as a consequence of population aging, and they are also associated with a socioeconomic burden. Many efforts have been made to find a treatment for these diseases with various levels of success, but new approaches are still needed to deal with these pathologies. In this context, one peptide derived for the C-terminal extreme of the Parathormone related Peptide (PTHrP) called Osteostatin can be useful to treat musculoskeletal diseases. This pentapeptide (TRSAW) has demonstrated both in different in vitro and in vivo models, its role as a molecule with anti-resorptive, anabolic, anti-inflammatory, and anti-antioxidant properties. Our aim with this work is to review the Osteostatin main features, the knowledge of its mechanisms of action as well as its possible use for the treatment of osteoporosis, bone regeneration and fractures and against arthritis given its anti-inflammatory properties.

Abaloparatide is more potent than teriparatide in restoring bone mass and strength in type 1 diabetic male mice.

This study investigated the efficacy of the two FDA-approved bone anabolic ligands of the parathyroid hormone receptor 1 (PTH1R), teriparatide or human parathyroid hormone 1-34 (PTH) and abaloparatide (ABL), to restoring skeletal health using a preclinical murine model of streptozotocin-induced T1-DM. Intermittent daily subcutaneous injections of equal molar doses (12 pmoles/g/day) of PTH (50 ng/g/day), ABL (47.5 ng/g/day), or vehicle, were administered for 28 days to 5-month-old C57Bl/6 J male mice with established T1-DM or control (C) mice. ABL was superior to PTH in increasing or restoring bone mass in control or T1-MD mice, respectively, which was associated with superior stimulation of trabecular and periosteal bone formation, upregulation of osteoclastic/osteoblastic gene expression, and increased circulating bone remodeling markers. Only ABL corrected the reduction in ultimate load, which is a measure of bone strength, induced by T1-DM, and it also increased energy to ultimate load. In addition, bones from T1-DM mice treated with PTH or ABL exhibited increased ultimate stress, a material index, compared to T1-DM mice administered with vehicle. And both PTH and ABL prevented the increased expression of the Wnt antagonist Sost/sclerostin displayed by T1-DM mice. Further, PTH and ABL increased to a similar extent the circulating bone resorption marker CTX and the bone formation marker P1NP in T1-DM after 2 weeks of treatment; however, only ABL sustained these increases after 4 weeks of treatment. We conclude that at equal molar doses, ABL is more effective than PTH in increasing bone mass and restoring the cortical and trabecular bone lost with T1-DM, due to higher and longer-lasting increases in bone remodeling.

PTHrP attenuates spontaneous contractions in detrusor smooth muscle of the rat bladder by activating spontaneous transient outward potassium currents.

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) cells upon bladder distension attenuates spontaneous phasic contractions (SPCs) in DSM and associated afferent firing to facilitate urine storage. Here, we investigate the mechanisms underlying PTHrP-induced inhibition of SPCs, focusing on large-conductance Ca2+-activated K+ channels (BK channels) that play a central role in stabilizing DSM excitability. Perforated patch-clamp techniques were applied to DSM cells of the rat bladder dispersed using collagenase. Isometric tension changes were recorded from DSM strips, while intracellular Ca2+ dynamics were visualized using Cal520 AM -loaded DSM bundles. DSM cells developed spontaneous transient outward potassium currents (STOCs) arising from the opening of BK channels. PTHrP (10 nM) increased the frequency of STOCs without affecting their amplitude at a holding potential of - 30 mV but not - 40 mV. PTHrP enlarged depolarization-induced, BK-mediated outward currents at membrane potentials positive to + 20 mV in a manner sensitive to iberiotoxin (100 nM), the BK channel blocker. The PTHrP-induced increases in BK currents were also prevented by inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (CPA 10 µM), L-type voltage-dependent Ca2+ channel (LVDCC) (nifedipine 3 µM) or adenylyl cyclase (SQ22536 100 µM). PTHrP had no effect on depolarization-induced LVDCC currents. PTHrP suppressed and slowed SPCs in an iberiotoxin (100 nM)-sensitive manner. PTHrP also reduced the number of Ca2+ spikes during each burst of spontaneous Ca2+ transients. In conclusion, PTHrP accelerates STOCs discharge presumably by facilitating SR Ca2+ release which prematurely terminates Ca2+ transient bursts resulting in the attenuation of SPCs.

Effects of stepwise administration of osteoprotegerin and parathyroid hormone-related peptide DNA vectors on bone formation in ovariectomized rat model.

Osteoporosis is a metabolic bone disease that impairs bone mineral density, microarchitecture, and strength. It requires continuous management, and further research into new treatment options is necessary. Osteoprotegerin (OPG) inhibits bone resorption and osteoclast activity. The objective of this study was to investigate the effects of stepwise administration of OPG-encoded minicircles (mcOPG) and a bone formation regulator, parathyroid hormone-related peptide (PTHrP)-encoded minicircles (mcPTHrP) in osteoporosis. The combined treatment with mcOPG and mcPTHrP significantly increased osteogenic marker expression in osteoblast differentiation compared with the single treatment groups. A model of postmenopausal osteoporosis was established in 12-week-old female rats through ovariectomy (OVX). After 8 weeks of OVX, mcOPG (80 µg/kg) was administered via intravenous injection. After 16 weeks of OVX, mcPTHrP (80 µg/kg) was injected once a week for 3 weeks. The bone microstructure in the femur was evaluated 24 weeks after OVX using micro-CT. In a proof-of-concept study, stepwise treatment with mcOPG and mcPTHrP on an OVX rat model significantly improved bone microstructure compared to treatment with mcOPG or mcPTHrP alone. These results suggest that stepwise treatment with mcOPG and mcPTHrP may be a potential treatment for osteoporosis.

Local injection of abaloparatide promotes mandibular condyle lengthening in adolescent rats via enhancing chondrogenesis and ossification.

BACKGROUND: Mandibular condylar hypoplasia negatively affects patient's facial appearance and dentofacial function. OBJECTIVE: To investigate the effect of local injection of the drug abaloparatide (ABL), an analogue of parathyroid hormone related protein (PTHrP), on promoting lengthening of the mandibular condyle. METHODS: Thirty adolescent male Sprague-Dawley rats were randomly divided into two groups, which received the injection of ABL or normal saline (the control) every 3 days in the temporomandibular joint (TMJ) cavity. Cone-beam computed tomography and immunohistochemistry assays were performed at 2, 4 and 6 weeks since the injection. Mandibular condylar chondrocytes (MCC) and pre-osteoblasts were treated with ABL or PBS, followed by the CCK-8 detection, IC50, real-time PCR assay, Western Blot and immunofluorescence staining. RESULTS: In vivo, compared with the control, the ABL group significantly increased the mandibular condylar process length (by 1.34 ± 0.59 mm at 6 weeks), the thickness of the cartilage layer, and enhanced the matrix synthesis. The ABL group had significant up-regulation of SOX 9, COL II, PTHrP and PTH1R, down-regulation of COL X in the cartilage, up-regulation of RUNX 2, and unchanged osteoclastogenesis in the subchondral bone. In vitro, the intra-TMJ injection of ABL promoted the MCC proliferation, with up-regulated expression of chondrogenic genes, and enhanced osteogenic differentiation of the pre-osteoblasts. CONCLUSIONS: Intra-TMJ injection of abaloparatide promotes mandibular condyle lengthening in the adolescent rats via enhancing chondrogenesis in the mandibular condylar cartilage and ossification in the subchondral bone.

Exploring dual effects of dinutuximab beta on cell death and proliferation of insulinoma.

Insulinoma INS-1 cells are pancreatic beta cell tumors. Dinutuximab beta (DB) is a monoclonal antibody used in the treatment of neuroblastoma. The aim of this study is to investigate the effects of DB on pancreatic beta cell tumors at the molecular level. DB (Qarziba®) was available from EUSA Pharma. Streptozotocin (STZ) was used induce to cell cytotoxicity. DB was applied to the cells before or after the STZ application. KCND3, KCNN4, KCNK1, and PTHrP gene expression levels were analyzed by q-RT-PCR, and protein levels were analyzed by Western blotting. Analysis of glucose-stimulated insulin secretion was performed. Ca+2 and CA19-9 levels were determined by the ELISA kit. PERK, CHOP, HSP90, p-c-Jun, p-Atf2, and p-Elk1 protein levels were analyzed by simple WES. Decreased KCND3, KCNK1, and PTHrP protein levels and increased KCND3, KCNN4, KCNK1, and PTHrP gene expression levels were observed with DB applied after STZ application. Cell dysfunction was detected with DB applied before and after STZ application. Ca19-9 and Ca+2 levels were increased with DB applied after STZ application. PERK, CHOP, and p-Elk1 levels decreased, while HSP90 levels increased with DB applied after STZ application. CHOP, p-Akt-2, and p-c-Jun levels increased in the DB group. As a result, INS-1 cells go to cell death via the ERK signaling pathway without ER stress and release insulin with the decrease of K+ channels and an increase in Ca+2 levels with DB applied after STZ application. Moreover, the cells proliferate via JNK signaling with DB application. DB holds promise for the treatment of insulinoma. The study should be supported by in vivo studies.

Comparative study in estrogen-depleted mice identifies skeletal and osteocyte transcriptomic responses to abaloparatide and teriparatide.

Osteocytes express parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptors and respond to the PTHrP analog abaloparatide (ABL) and to the PTH 1-34 fragment teriparatide (TPTD), which are used to treat osteoporosis. Several studies indicate overlapping but distinct skeletal responses to ABL or TPTD, but their effects on cortical bone may differ. Little is known about their differential effects on osteocytes. We compared cortical osteocyte and skeletal responses to ABL and TPTD in sham-operated and ovariectomized mice. Administered 7 weeks after ovariectomy for 4 weeks at a dose of 40 µg/kg/d, TPTD and ABL had similar effects on trabecular bone, but ABL showed stronger effects in cortical bone. In cortical osteocytes, both treatments decreased lacunar area, reflecting altered peri-lacunar remodeling favoring matrix accumulation. Osteocyte RNA-Seq revealed that several genes and pathways were altered by ovariectomy and affected similarly by TPTD and ABL. Notwithstanding, several signaling pathways were uniquely regulated by ABL. Thus, in mice, TPTD and ABL induced a positive osteocyte peri-lacunar remodeling balance, but ABL induced stronger cortical responses and affected the osteocyte transcriptome differently. We concluded that ABL affected the cortical osteocyte transcriptome in a manner subtly different from TPTD, resulting in more beneficial remodeling/modeling changes and homeostasis of the cortex.

A Novel PTH-Related Peptide Combined With 3D Printed Macroporous Titanium Alloy Scaffold Enhances Osteoporotic Osseointegration.

Previous parathyroid hormone (PTH)-related peptides (PTHrPs) cannot be used to prevent implant loosening in osteoporosis patients due to the catabolic effect of local sustained release. A novel PTHrP (PTHrP-2) that can be used locally to promote osseointegration of macroporous titanium alloy scaffold (mTAS) and counteract implant slippage in osteoporosis patients is designed. In vitro, PTHrP-2 enhances the proliferation, adhesion, and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) within the mTAS. Further, it promotes proliferation, migration, angiogenesis-related protein expression, and angiogenesis in human umbilical vein endothelial cells (HUVECs). Compared to PTH(1-34), PTHrP-2 can partially weaken the osteoclast differentiation of RAW 264.7 cells. Even in an oxidative stress microenvironment, PTHrP-2 safeguards the proliferation and migration of BMSCs and HUVECs, reduces reactive oxygen species generation and mitochondrial damage, and partially preserves the angiogenesis of HUVECs. In the Sprague-Dawley (SD) rat osteoporosis model, the therapeutic benefits of PTHrP-2-releasing mTAS (mTASP2 ) and ordinary mTAS implanted for 12 weeks via micro-CT, sequential fluorescent labeling, and histology are compared. The results demonstrate that mTASP2 exhibits high bone growth rate, without osteophyte formation. Consequently, PTHrP-2 exhibits unique local synthesis properties and holds the potential for assisting the osseointegration of alloy implants in osteoporosis patients.

A Recombinant Parathyroid Hormone-Related Peptide Locally Applied in Osteoporotic Bone Defect.

The local application of drug-loaded bioactive scaffold materials is one of the important directions to solve the clinical problem of osteoporotic (OP) bone defects. This study retains the advantages of drug loading and mechanical properties of natural 3D bioactive scaffolds. The scaffolds are functionally modified through chemical and self-assembly approaches with application of polydopamine (PDA) nanoparticles and parathyroid hormone-related peptide-1 (PTHrP-1) for efficient local drug loading. This study investigates the effects of the novel bioactive scaffolds on ossification, osteoclastogenesis, and macrophage polarization. This work elucidates the effects of the scaffolds in regulating osteoclastic activity and new bone formation in vitro. Further studies on the establishment and repair of OP bone defects in small animals are conducted, and the potential of natural bioactive porous scaffold materials to promote the repair of OP bone defects is initially verified. The preparation of safe and economical anti-OP bone repair material provides a theoretical basis for clinical translational applications.

Hypoxia-mimicking scaffolds with controlled release of DMOG and PTHrP to promote cartilage regeneration via the HIF-1α/YAP signaling pathway.

Efficiently driving chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) while avoiding undesired hypertrophy remains a challenge in the field of cartilage tissue engineering. Here, we report the sequential combined application of dimethyloxalylglycine (DMOG) and parathyroid hormone-related protein (PTHrP) to facilitate chondrogenesis and prevent hypertrophy. To support their delivery, poly(lactic-co-glycolic acid) (PLGA) microspheres were fabricated using a double emulsion method. Subsequently, these microspheres were incorporated onto a poly(l-lactic acid) (PLLA) scaffold with a highly porous structure, high interconnectivity and collagen-like nanofiber architecture to construct a microsphere-based scaffold delivery system. These functional constructs demonstrated that the spatiotemporally controlled release of DMOG and PTHrP effectively mimicked the hypoxic microenvironment to promote chondrogenic differentiation with phenotypic stability in a 3D culture system, which had a certain correlation with the interaction between hypoxia-inducible Factor 1 alpha (HIF-1α) and yes-associated protein (YAP). Subcutaneous implantation in nude mice revealed that the constructs were able to maintain cartilage formation in vivo at 4 and 8 weeks. Overall, this study indicated that DMOG and PTHrP controlled-release PLGA microspheres incorporated with PLLA nanofibrous scaffolds provided an advantageous 3D hypoxic microenvironment for efficacious and clinically relevant cartilage regeneration and is a promising treatment for cartilage injury.

Differential effects of parathyroid hormone, parathyroid hormone-related protein, and abaloparatide on collagen 1 expression by mouse cementoblasts and mouse tooth root density.

INTRODUCTION: Parathyroid hormone (PTH) plays an important role in maintaining mineral homeostasis by regulating calcium and phosphate levels. Clinical trials have shown that peptides of PTH (1-34), PTH-related protein (PTHrP 1-36), and the new peptide modeled on PTHrP, abaloparatide, can have different anabolic effects on osteoporotic subjects, but the underlying mechanisms are still unclear. The prevalence of moderate and major gingival recession has been shown to be higher in postmenopausal women with osteoporosis. In addition, there is a significant association between osteoporosis and tooth loss. METHODS: We investigated the actions of these peptides on the cementoblasts and teeth of mice. The murine cementoblast line, OCCM-30, known to express collagen I (Col1a1), was treated with intermittent PTH (1-34), PTHrP (1-36), or abaloparatide for 6 h/d for 3 days. Microcomputed tomography was performed on the teeth of mice receiving daily injections of phosphate-buffered saline, PTH (1-34), or abaloparatide. Statistical differences were analyzed by a 2-way or 1-way analysis of variance followed by a Tukey's post-hoc test. Results are expressed as mean ± standard deviation, and P <0.05 was considered significant. RESULTS: Gene expression showed regulation of Bsp, Col1a1, Opg, Rankl, and Mmp13 by the 3 peptides in these cells. Western blots revealed that after intermittent treatment for 3 days, PTH (1-34) caused an increase in COL1A1 protein immediately after treatment. In contrast, abaloparatide showed a latent effect in increasing COL1A1 protein 18 hours after treatment. PTHrP had no effect on COL1A1 expression. Immunofluorescence confirmed the same result as the Western blots. Microcomputed tomography of teeth showed PTH (1-34) injections increased molar root mineral density in mice, whereas abaloparatide increased density in roots of incisors and molars. CONCLUSIONS: This study reveals the differential anabolic effects of intermittent PTH (1-34), PTHrP (1-36), and abaloparatide on cementoblasts, as revealed by COL1A1 expression and root mineral density. Abaloparatide may be a potential therapeutic approach for achieving improved cementogenesis.

Abaloparatide and teriparatide enhance mandibular growth in adolescent rats with site-specific and mechano-related effects.

OBJECTIVE: Teriparatide (TPTD) and abaloparatide (ABL) are two osteoanabolic drugs targeting parathyroid hormone (PTH)1R signalling. This study aimed to investigate the effects of TPTD and ABL on the adolescent mandibular growth. METHOD: In total, 70 4-week-old male Sprague-Dawley rats were randomly divided into 14 groups, treated with intermittent TPDT or ABL at various doses, accompanied by mandibular advancement (MA) or not. 3D printing was used to fabricate an innovative splint for MA. After a 4-week treatment, morphological measurement, histological and immunohistochemical analysis were performed. Mandibular condylar chondrocytes (MCCs) were treated with TPTD or ABL, followed by CCK-8 assay, alcian blue staining, real time-PCR and immunofluorescent staining. RESULT: In vivo, TPTD or ABL alone increased the condylar length and cartilage thickness, with up-regulated SOX9 and COL II, whilst down-regulated COL X; however, when combined with MA, the promotive effects were attenuated. TPTD or ABL alone increased the mandibular body height and mandibular angle width, whilst increased the mandibular body length and alveolar bone width when combined with MA. In vitro, TPTD or ABL enhanced the MCC proliferation, glycosaminoglycan synthesis, COL II and SOX9 expression, whilst down-regulated COL X, Ihh and PTH1R expression. CONCLUSION: Both ABL and TPTD enhance mandibular growth in adolescent rats with site-specific and mechano-related effects, including propelling chondrogenesis at the condylar cartilage and promoting bone apposition at other mechano-responsive sites. They behave as promising drugs for mandibular growth modification, and in general ABL seems more potent than TPTD in this context.

Molecular recognition of two endogenous hormones by the human parathyroid hormone receptor-1.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) are two endogenous hormones recognized by PTH receptor-1 (PTH1R), a member of class B G protein- coupled receptors (GPCRs). Both PTH and PTHrP analogs including teriparatide and abaloparatide are approved drugs for osteoporosis, but they exhibit distinct pharmacology. Here we report two cryo-EM structures of human PTH1R bound to PTH and PTHrP in the G protein-bound state at resolutions of 2.62 Å and 3.25 Å, respectively. Detailed analysis of these structures uncovers both common and unique features for the agonism of PTH and PTHrP. Molecular dynamics (MD) simulation together with site-directed mutagenesis studies reveal the molecular basis of endogenous hormones recognition specificity and selectivity to PTH1R. These results provide a rational template for the clinical use of PTH and PTHrP analogs as an anabolic therapy for osteoporosis and other disorders.

The Efficacy and Safety of Abaloparatide-SC in Men With Osteoporosis: A Randomized Clinical Trial.

Abaloparatide significantly increased bone mineral density (BMD) in women with postmenopausal osteoporosis and decreased risk of vertebral, nonvertebral, and clinical fractures compared with placebo. The Abaloparatide for the Treatment of Men with Osteoporosis (ATOM; NCT03512262) study evaluated the efficacy and safety of abaloparatide compared with placebo in men. Eligible men aged 40 to 85 years with osteoporosis were randomized 2:1 to daily subcutaneous injections of abaloparatide 80 µg or placebo for 12 months. The primary endpoint was change from baseline in lumbar spine BMD. Key secondary endpoints included BMD change from baseline at the total hip and femoral neck. A total of 228 men were randomized (abaloparatide, n = 149; placebo, n = 79). Baseline characteristics were similar across treatment groups (mean age, 68.3 years; mean lumbar spine BMD T-score, -2.1). At 12 months, BMD gains were greater with abaloparatide compared with placebo at the lumbar spine (least squares mean percentage change [standard error]: 8.48 [0.54] versus 1.17 [0.72]), total hip (2.14 [0.27] versus 0.01 [0.35]), and femoral neck (2.98 [0.34] versus 0.15 [0.45]) (all p < 0.0001). The most common (≥5%) treatment-emergent adverse events were injection site reaction, dizziness, nasopharyngitis, arthralgia, bronchitis, hypertension, and headache. During 12 months of abaloparatide treatment, men with osteoporosis exhibited rapid and significant improvements in BMD with a safety profile consistent with previous studies. These results suggest abaloparatide can be considered as an effective anabolic treatment option for men with osteoporosis. © 2022 Radius Health Inc and The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide.

BACKGROUND: Parathyroid hormone-related peptide (PTHrP) plays a key role in the development and progression of many tumors. We found that in colorectal cancer (CRC) HCT116 cells, the binding of PTHrP to its receptor PTHR type 1 (PTHR1) activates events associated with an aggressive phenotype. In HCT116 cell xenografts, PTHrP modulates the expression of molecular markers linked to tumor progression. Empirical evidence suggests that the Met receptor is involved in the development and evolution of CRC. Based on these data, we hypothesized that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells. AIM: To elucidate the relationship among PTHR1, PTHrP, and Met in CRC models. METHODS: For in vitro assays, HCT116 and Caco-2 cells derived from human CRC were incubated in the absence or presence of PTHrP (1-34) (10-8 M). Where indicated, cells were pre-incubated with specific kinase inhibitors or dimethylsulfoxide, the vehicle of the inhibitors. The protein levels were evaluated by Western blot technique. Real-time polymerase chain reaction (RT-qPCR) was carried out to determine the changes in gene expression. Wound healing assay and morphological monitoring were performed to evaluate cell migration and changes related to the epithelial-mesenchymal transition (EMT), respectively. The number of viable HCT116 cells was counted by trypan blue dye exclusion test to evaluate the effects of irinotecan (CPT-11), oxaliplatin (OXA), or doxorubicin (DOXO) with or without PTHrP. For in vivo tests, HCT116 cell xenografts on 6-wk-old male N:NIH (S)_nu mice received daily intratumoral injections of PTHrP (40 µg/kg) in 100 µL phosphate-buffered saline (PBS) or the vehicle (PBS) as a control during 20 d. Humanitarian slaughter was carried out and the tumors were removed, weighed, and fixed in a 4% formaldehyde solution for subsequent treatment by immunoassays. To evaluate the expression of molecular markers in human tumor samples, we studied 23 specimens obtained from CRC patients which were treated at the Hospital Interzonal de Graves y Agudos Dr. José Penna (Bahía Blanca, Buenos Aires, Argentina) and the Hospital Provincial de Neuquén (Neuquén, Neuquén, Argentina) from January 1990 to December 2007. Seven cases with normal colorectal tissues were assigned to the control group. Tumor tissue samples and clinical histories of patients were analyzed. Paraffin-embedded blocks from primary tumors were reviewed by hematoxylin-eosin staining technique; subsequently, representative histological samples were selected from each patient. From each paraffin block, tumor sections were stained for immunohistochemical detection. The statistical significance of differences was analyzed using proper statistical analysis. The results were considered statistically significant at P < 0.05. RESULTS: By Western blot analysis and using total Met antibody, we found that PTHrP regulated Met expression in HCT116 cells but not in Caco-2 cells. In HCT116 cells, Met protein levels increased at 30 min (P < 0.01) and at 20 h (P < 0.01) whereas the levels diminished at 3 min (P < 0.05), 10 min (P < 0.01), and 1 h to 5 h (P < 0.01) of PTHrP treatment. Using an active Met antibody, we found that where the protein levels of total Met decreased (3 min, 10 min, and 60 min of PTHrP exposure), the status of phosphorylated/activated Met increased (P < 0.01) at the same time, suggesting that Met undergoes proteasomal degradation after its phosphorylation/activation by PTHrP. The increment of its protein level after these decreases (at 30 min and 20 h) suggests a modulation of Met expression by PTHrP in order to improve Met levels and this idea is supported by our observation that the cytokine increased Met mRNA levels at least at 15 min in HCT116 cells as revealed by RT-qPCR analysis (P < 0.05). We then proceeded to evaluate the signaling pathways that mediate the phosphorylation/ activation of Met induced by PTHrP in HCT116 cells. By Western blot technique, we observed that PP1, a specific inhibitor of the activation of the proto-oncogene protein tyrosine kinase Src, blocked the effect of PTHrP on Met phosphorylation (P < 0.05). Furthermore, the selective inhibition of the ERK 1/2 mitogen-activated protein kinase (ERK 1/2 MAPK) using PD98059 and the p38 MAPK using SB203580 diminished the effect of PTHrP on Met phosphorylation/activation (P < 0.05). Using SU11274, the specific inhibitor of Met activation, and trypan blue dye exclusion test, Western blot, wound healing assay, and morphological analysis with a microscope, we observed the reversal of cell events induced by PTHrP such as cell proliferation (P < 0.05), migration (P < 0.05), and the EMT program (P < 0.01) in HCT116 cells. Also, PTHrP favored the chemoresistance to CPT-11 (P < 0.001), OXA (P < 0.01), and DOXO (P < 0.01) through the Met pathway. Taken together, these findings suggest that Met activated by PTHrP participates in events associated with the aggressive phenotype of CRC cells. By immunohistochemical analysis, we found that PTHrP in HCT116 cell xenografts enhanced the protein expression of Met (0.190 ± 0.014) compared to tumors from control mice (0.110 ± 0.012; P < 0.05) and of its own receptor (2.27 ± 0.20) compared to tumors from control mice (1.98 ± 0.14; P < 0.01). Finally, assuming that the changes in the expression of PTHrP and its receptor are directly correlated, we investigated the expression of both Met and PTHR1 in biopsies of CRC patients by immunohistochemical analysis. Comparing histologically differentiated tumors with respect to those less differentiated, we found that the labeling intensity for Met and PTHR1 increased and diminished in a gradual manner, respectively (P < 0.05). CONCLUSION: PTHrP acts through the Met pathway in CRC cells and regulates Met expression in a CRC animal model. More basic and clinical studies are needed to further evaluate the PTHrP/Met relationship.

miR-200b-3p/ERG/PTHrP axis mediates the inhibitory effect of ethanol on the differentiation of fetal cartilage into articular cartilage.

PURPOSE: This study aims to further explore cartilage development in prenatal ethanol exposure (PEE) offspring at different times to explore the specific time points and mechanism of ethanol-induced fetal cartilage dysplasia. METHODS: On gestational day (GD)14, GD17, and GD20, PEE fetal cartilage was evaluated by morphological analysis. RT-qPCR, immunohistochemistry, and immunofluorescence were used to detect the expression of cartilage marker genes and their regulatory factors. Bone marrow mesenchymal stem cells (BMSCs) were used to explore the effect of ethanol on the differentiation of chondrocytes. Additionally, we used inhibitors, overexpression plasmids and a luciferase reporter assay on GD17 chondrocytes to verify the mechanism. RESULTS: PEE significantly reduced cartilage matrix content and the expression of marker genes on GD17 and GD20 but had no effect on GD14. The inhibition of chondrogenic differentiation by PEE mainly occurred on GD14-17. Furthermore, the expression of miR-200b-3p was increased, while that of ERG and PTHrP was markedly reduced in PEE fetal cartilage. In vitro, ethanol (30-120 mM) inhibited the differentiation of BMSCs into chondrocytes in a concentration-dependent manner, accompanied by strong expression of miR-200b-3p and low expression of ERG and PTHrP. Moreover, PTHLH and ERG overexpressed, as well as a miR-200b-3p inhibitor reversed the inhibitory effect of ethanol on the differentiation of fetal chondrocytes. Furthermore, miR-200b-3p could target and negatively regulate ERG. CONCLUSIONS: PEE can significantly inhibit the development of articular cartilage, especially during articular cartilage formation. The mechanism is related to the decreased differentiation of fetal cartilage into articular cartilage mediated by the miR-200b-3p/ERG/PTHrP axis.

Structural pharmacology of PTH and PTHrP.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) regulate extracellular phosphate and calcium homeostasis as well as bone remodeling. PTH is a classic endocrine peptide hormone whose synthesis and negative feedback by multiple factors control release from the parathyroid glands. PTHrP is ubiquitously expressed (pre- and postnatally) and acts in an autocrine/paracrine manner. This review considers the structural pharmacology and actions of PTH and PTHrP, biological consequences of inherited mutations, engineered analogs that illuminate similarities and differences in physiologic actions, and targeted therapeutic opportunities.

Abaloparatide Enhances Fusion and Bone Formation in a Rabbit Spinal Arthrodesis Model.

STUDY DESIGN: Prospective randomized placebo controlled animal trial. OBJECTIVE: Determine the effect of daily subcutaneous abaloparatide injection on the intervertebral fusion rate in rabbits undergoing posterolateral fusion. STUDY OF BACKGROUND DATA: Despite the wide utilization of spine fusion, pseudarthrosis remains prevalent, and results in increased morbidity. Abaloparatide is a novel analog of parathyroid hormone-related peptide (1-34) and has shown efficacy in a rat posterolateral spine fusion model to increase fusion rates. The effect of abaloparatide on the fusion rate in a larger animal model remains unknown. MATERIALS AND METHODS: A total of 24 skeletally mature New Zealand White male rabbits underwent bilateral posterolateral spine fusion. Following surgery, the rabbits were randomized to receive either saline as control or abaloparatide subcutaneous injection daily. Specimens underwent manual assessment of fusion, radiographic analysis with both x-ray and high-resolution peripheral quantitative computed tomography, and biomechanical assessment. RESULTS: Rabbits that received abaloparatide had a 100% (10/10) fusion rate compared with 45% (5/11) for controls ( P <0.02) as assessed by manual palpation. Radiographic analysis determined an overall mean fusion score of 4.17±1.03 in the abaloparatide group versus 3.39±1.21 for controls ( P <0.001). The abaloparatide group also had a greater volume of bone formed with a bone volume of 1209±543 mm 3 compared with 551±152 mm 3 ( P <0.001) for controls. The abaloparatide group had significantly greater trabecular bone volume fraction and trabecular thickness and lower specific bone surface and connectivity density in the adjacent levels when compared with controls. Abaloparatide treatment did not impact trabecular number or separation. There were no differences in biomechanical testing in flexion, extension, or lateral bending ( P >0.05) between groups. CONCLUSIONS: Abaloparatide significantly increased the fusion rate in a rabbit posterolateral fusion model as assessed by manual palpation. In addition, there were marked increases in the radiographic evaluation of fusion.