Stimulator Of Endochondral Bone Growth Research Articles

OP021: Vosoritide for children with achondroplasia: Growth velocity and pubertal milestones

Vosoritide is an analogue of C-type natriuretic peptide (CNP), a potent stimulator of endochondral bone growth. Like any growth agent used in children, efficacy as indicated by annualized growth velocity (AGV) should be evaluated in the context of pubertal milestone timing. We report on the growth velocity during puberty and pubertal milestones in children with achondroplasia (ACH) treated with vosoritide.

Persistent and Stable Growth Promoting Effects of Vosoritide in Children With Achondroplasia for up to 2 Years: Results From the Ongoing Phase 3 Extension Study

Objectives: Vosoritide is a potent stimulator of endochondral bone growth and is in development for the treatment of achondroplasia, the most common form of disproportionate short stature. We previously reported on a 52-week, phase 3, pivotal study that demonstrated a highly statistically significant improvement in annualized growth velocity (AGV) when vosoritide was compared to placebo in children with achondroplasia aged 5-18 years (Savarirayan et al, Lancet, 2020). This is an analysis of data after an additional 52 weeks of treatment in the ongoing phase 3 extension study. Methods: After completion of the phase 3 placebo-controlled study, 119 children were enrolled into the extension study, where they all receive open label 15 μg/kg/day vosoritide. AGV, height Z-score and body proportion ratio were analyzed to assess efficacy of vosoritide in children who were treated with vosoritide for up to 2 years. Fifty-eight continued treatment with vosoritide and 61 switched from placebo to vosoritide. Two participants on continuous vosoritide treatment discontinued before the Week 52 timepoint. Four participants on continuous vosoritide treatment and 7 participants who switched from placebo to vosoritide missed the Week 52 assessment due to Covid-19. Results: In children randomized to receive daily vosoritide, baseline mean (SD) AGV was 4.26 (1.53) cm/year. After the first 52 weeks of treatment, mean (SD) AGV was 5.67 (0.98) cm/year. Mean (SD) AGV over the second year was 5.57 (1.10) cm/year. Mean (SD) change from baseline in height Z-score improved by +0.24 (0.31) at Week 52 in the pivotal study and +0.45 (0.56) at Week 52 in the extension study. Mean (SD) upper-to-lower body segment ratio improved with a change from baseline of -0.03 (0.11) at Week 52 in the pivotal study and -0.09 (0.11) at Week 52 in the extension study. In children who switched from placebo to vosoritide after 52 weeks, baseline AGV was 4.06 (1.20) cm/year and 3.94 (1.07) cm/year after 52 weeks on placebo. In the second year, after receiving 52 weeks of vosoritide, mean AGV was 5.65 (1.47) cm/year, the mean (SD) change in height Z-score was +0.24 (0.34), and the change in upper-to-lower body segment ratio was -0.03 (0.08). No new adverse events associated with vosoritide treatment were detected with up to 2 years of continuous daily, subcutaneous treatment. Most adverse events were mild and no serious adverse events were attributed to vosoritide. The most common adverse event remains mild and transient injection site reactions. Conclusions: The effect of vosoritide administration on growth as measured through AGV and height Z-score was maintained for up to 2-years in children with achondroplasia aged 5 to 18 years, with an improvement of body proportions.

Exogenous C-type natriuretic peptide restores normal growth and prevents early growth plate closure in its deficient rats.

Signaling by C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B, is a pivotal stimulator of endochondral bone growth. We recently developed CNP knockout (KO) rats that exhibit impaired skeletal growth with early growth plate closure. In the current study, we further characterized the phenotype and growth plate morphology in CNP-KO rats, and the effects of exogenous CNP in rats. We used CNP-53, an endogenous form of CNP consisting of 53 amino acids, and administered it for four weeks by continuous subcutaneous infusion at 0.15 or 0.5 mg/kg/day to four-week old CNP-KO and littermate wild type (WT) rats. We demonstrated that CNP-KO rats were useful as a reproducible animal model for skeletal dysplasia, due to their impairment in endochondral bone growth. There was no significant difference in plasma bone-turnover markers between the CNP-KO and WT rats. At eight weeks of age, growth plate closure was observed in the distal end of the tibia and the calcaneus of CNP-KO rats. Continuous subcutaneous infusion of CNP-53 significantly, and in a dose-dependent manner, stimulated skeletal growth in CNP-KO and WT rats, with CNP-KO rats being more sensitive to the treatment. CNP-53 also normalized the length of long bones and the growth plate thickness, and prevented growth plate closure in the CNP-KO rats. Using organ culture experiment of fetal rat tibia, gene set enrichment analysis indicated that CNP might have a negative influence on mitogen activated protein kinase signaling cascades in chondrocyte. Our results indicated that CNP-KO rats might be a valuable animal model for investigating growth plate physiology and the mechanism of growth plate closure, and that CNP-53, or its analog, may have the potential to promote growth and to prevent early growth plate closure in the short stature.

C-type natriuretic peptide improves growth retardation in a mouse model of cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous (CFC) syndrome, a genetic disorder caused by germline mutations in BRAF, KRAS, MAP2K1 and MAP2K2, is characterized by growth retardation, heart defects, dysmorphic facial appearance and dermatologic abnormalities. We have previously reported that knock-in mice expressing the CFC syndrome-associated mutation, Braf Q241R, showed growth retardation because of gastrointestinal dysfunction. However, other factors associated with growth retardation, including chondrogenesis and endocrinological profile, have not been examined. Here, we show that 3- and 4-week-old BrafQ241R/+ mice have decreased body weight and length, as well as reduced growth plate width in the proximal tibiae. Furthermore, proliferative and hypertrophic chondrocyte zones of the growth plate were reduced in BrafQ241R/+ mice compared with Braf+/+ mice. Immunohistological analysis revealed that extracellular signal-regulated kinase (ERK) activation was enhanced in hypertrophic chondrocytes in BrafQ241R/+ mice. In accordance with growth retardation and reduced growth plate width, decreased serum levels of insulin-like growth factor 1 (IGF-1) and IGF binding protein 3 (IGFBP-3) were observed in BrafQ241R/+ mice at 3 and 4 weeks of age. Treatment with C-type natriuretic peptide (CNP), a stimulator of endochondral bone growth and a potent inhibitor of the FGFR3-RAF1-MEK/ERK signaling, increased body and tail lengths in Braf+/+ and BrafQ241R/+ mice. In conclusion, ERK activation in chondrocytes and low serum IGF-1/IGFBP-3 levels could be associated with the growth retardation observed in BrafQ241R/+ mice. Our data also suggest that CNP is a potential therapeutic target in CFC syndrome.

Live imaging analysis of the growth plate in a murine long bone explanted culture system

Skeletal growth in mammals, which owes the growth of an individual, occurs at the growth plate and to observe and analyze its dynamic growth is of high interest. Here we performed live imaging analysis of the growth plate of a fetal murine long bone organ culture using two-photon excitation microscopy. We could observe a dynamic growth of the growth plate of explanted fetal murine ulna, as well as the resultant linear elongation of the explants. As for the factors contributing to the elongation of the growth plate, the displacement length of each chondrocyte was larger in the prehypertrophic or hypertrophic zone than in the proliferative zone. The segmented area and its extracellular component were increased in both the proliferative and prehypertrophic-hypertrophic zones, whereas an increase in cellular components was only seen in the prehypertrophic-hypertrophic zone. C-type natriuretic peptide, a known positive stimulator of endochondral bone growth mainly targeting prehypertrophic-hypertrophic zone, augmented all of the factors affecting growth plate elongation, whereas it had little effect on the proliferation of chondrocytes. Collectively, the axial trajectory of each chondrocyte mainly owes cellular or extracellular expansion especially in prehypertrophic-hypertrophic zone and results in growth plate elongation, which might finally result in endochondral bone elongation.

Circulatory CNP Rescues Craniofacial Hypoplasia in Achondroplasia

Achondroplasia is the most common genetic form of human dwarfism, characterized by midfacial hypoplasia resulting in occlusal abnormality and foramen magnum stenosis, leading to serious neurologic complications and hydrocephalus. Currently, surgery is the only way to manage jaw deformity, neurologic complications, and hydrocephalus in patients with achondroplasia. We previously showed that C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth of long bones and vertebrae and is also a potent stimulator in the craniofacial region, which is crucial for midfacial skeletogenesis. In this study, we analyzed craniofacial morphology in a mouse model of achondroplasia, in which fibroblast growth factor receptor 3 (FGFR3) is specifically activated in cartilage (Fgfr3ach mice), and investigated the mechanisms of jaw deformities caused by this mutation. Furthermore, we analyzed the effect of CNP on the maxillofacial area in these animals. Fgfr3ach mice exhibited midfacial hypoplasia, especially in the sagittal direction, caused by impaired endochondral ossification in craniofacial cartilage and by premature closure of the spheno-occipital synchondrosis, an important growth center in craniomaxillofacial skeletogenesis. We crossed Fgfr3ach mice with transgenic mice in which CNP is expressed in the liver under the control of the human serum amyloid-P component promoter, resulting in elevated levels of circulatory CNP (Fgfr3ach/SAP-Nppc-Tg mice). In the progeny, midfacial hypoplasia in the sagittal direction observed in Fgfr3ach mice was improved significantly by restoring the thickness of synchondrosis and promoting proliferation of chondrocytes in the craniofacial cartilage. In addition, the foramen magnum stenosis observed in Fgfr3ach mice was significantly ameliorated in Fgfr3ach/SAP-Nppc-Tg mice due to enhanced endochondral bone growth of the anterior intraoccipital synchondrosis. These results clearly demonstrate the therapeutic potential of CNP for treatment of midfacial hypoplasia and foramen magnum stenosis in achondroplasia.

C-type natriuretic peptide restores impaired skeletal growth in a murine model of glucocorticoid-induced growth retardation

Glucocorticoids are widely used for treating autoimmune conditions or inflammatory disorders. Long-term use of glucocorticoids causes impaired skeletal growth, a serious side effect when they are used in children. We have previously demonstrated that C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth. In this study, we investigated the effect of CNP on impaired bone growth caused by glucocorticoids by using a transgenic mouse model with an increased circulating CNP level. Daily administration of a high dose of dexamethasone (DEX) to 4-week-old male wild-type mice for 4weeks significantly shortened their naso-anal length, which was restored completely in DEX-treated CNP transgenic mice. Impaired growth of the long bones and vertebrae by DEX was restored to a large extent in the CNP transgenic background, with recovery in the narrowed growth plate by increased cell volume, whereas the decreased proliferation and increased apoptosis of the growth plate chondrocytes were unaffected. Trabecular bone volume was not changed by DEX treatment, but decreased significantly in a CNP transgenic background. In young male rats, the administration of high doses of DEX greatly decreased N-terminal proCNP concentrations, a marker of CNP production. In organ culture experiments using fetal wild-type murine tibias, longitudinal growth of tibial explants was inhibited by DEX but reversed by CNP. These findings now warrant further study of the therapeutic potency of CNP in glucocorticoid-induced bone growth impairment.

Increased Bone Turnover and Possible Accelerated Fracture Healing in a Murine Model With an Increased Circulating C-Type Natriuretic Peptide.

Recent studies have revealed that C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth. Nevertheless, the effect of CNP on bone turnover has not yet been well studied. To elucidate this issue, we investigated the bone phenotype of a mouse model with elevated plasma CNP concentrations (SAP-CNP-Tg mice) in the present study. Microcomputed tomography (CT) analysis revealed less bone in femurs, but not in lumber vertebrae, of young adult SAP-CNP-Tg mice than that of wild-type mice. Bone histomorphometry of the tibiae from 8-week-old SAP-CNP-Tg mice showed enhanced osteoblastic and osteoclastic activities, in accordance with elevated serum levels of osteocalcin and tartrate-resistant acid phosphatase-5b, respectively. Next we performed an open and stabilized femoral fracture using 8-week-old SAP-CNP-Tg mice and compared the healing process with age-matched wild-type mice. An immunohistochemical study revealed that CNP and its receptors, natriuretic peptide receptor-B and natriuretic peptide clearance receptor, are expressed in hard calluses of wild-type mice, suggesting a possible role of CNP/natriuretic peptide receptor-B signaling in fracture repair, especially in bone remodeling stage. On micro-CT analysis, a rapid decrease in callus volume was observed in SAP-CNP-Tg mice, followed by a generation of significantly higher new bone volume with a tendency of increased bone strength. In addition, a micro-CT analysis also showed that bone remodeling was accelerated in SAP-CNP-Tg mice, which was also evident from increased serum osteocalcin and tartrate-resistant acid phosphatase-5b levels in SAP-CNP-Tg mice at the remodeling stage of fracture repair. These results indicate that CNP activates bone turnover and remodeling in vivo and possibly accelerates fracture healing in our mouse model.

A novel method for the large-scale production of PG-CNP37, a C-type natriuretic peptide analogue

Achondroplasia is the most common form of human dwarfism caused by a mutation in the fibroblast growth factor receptor 3 (FGFR3), resulting in abnormal endochondral bone formation. C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth and represents a potential therapy for achondroplasia. We have developed a novel, simple and cost effective method to produce a CNP analogue, PG-CNP37, at a large scale from Escherichia coli. A PG-CNP37 fusion protein was over-expressed as inclusion bodies in E. coli, which were purified then cleaved by formic acid to release the PG-CNP37 peptide. Approximately 0.5g of 95% pure, soluble and active PG-CNP37 peptide was produced from 1L of culture using this method and may represent a viable means for large-scale production of other therapeutic peptides.

Genetic basis for skeletal disease. CNP therapy for achondroplasia

By using transgenic and knockout mice, we have elucidated that C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth. Furthermore, loss-of-function mutations in the gene coding for guanylyl cyclase-B (GC-B) , the specific receptor for CNP, have been proved to cause impaired skeletal growth in humans. Following these results, we have started to translate the stimulatory effect of CNP on endochondral bone growth into the therapy for patients with achondroplasia, and have shown that targeted overexpression of CNP in cartilage or systemic administration of CNP reverses the impaired skeletal growth of mice model of achondroplasia.

Circulating C-Type Natriuretic Peptide (CNP) Rescues Chondrodysplastic CNP Knockout Mice from Their Impaired Skeletal Growth and Early Death

C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth through a subtype of membranous guanylyl cyclase receptor, GC-B. Although its two cognate natriuretic peptides, ANP and BNP, are cardiac hormones produced from heart, CNP is thought to act as an autocrine/paracrine regulator. To elucidate whether systemic administration of CNP would be a novel medical treatment for chondrodysplasias, for which no drug therapy has yet been developed, we investigated the effect of circulating CNP by using the CNP transgenic mice with an increased circulating CNP under the control of human serum amyloid P component promoter (SAP-Nppc-Tg mice). SAP-Nppc-Tg mice developed prominent overgrowth of bones formed through endochondral ossification. In organ culture experiments, the growth of tibial explants of SAP-Nppc-Tg mice was not changed from that of their wild-type littermates, exhibiting that the stimulatory effect on endochondral bone growth observed in SAP-Nppc-Tg mice is humoral. Then we crossed chondrodysplastic CNP-depleted mice with SAP-Nppc-Tg mice. Impaired endochondral bone growth in CNP knockout mice were considerably and significantly recovered by increased circulating CNP, followed by the improvement in not only their longitudinal growth but also their body weight. In addition, the mortality of CNP knockout mice was greatly decreased by circulating CNP. Systemic administration of CNP might have therapeutic potential against not only impaired skeletal growth but also other aspects of impaired growth including impaired body weight gain in patients suffering from chondrodysplasias and might resultantly protect them from their early death.

Translational research of C-type natriuretic peptide (CNP) into skeletal dysplasias

By using transgenic and knockout mice, we have elucidated that C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth. In humans, loss-of-function mutations in the gene coding for guanylyl cyclase-B (GC-B), the specific receptor for CNP, have been proved to be the cause of acromesomelic dysplasia, type Maroteaux, one form of human skeletal dysplasias. Following these results, we have started to translate the stimulatory effect of CNP on endochondral bone growth into the therapy for patients with skeletal dysplasias. We have shown that targeted overexpression of CNP in cartilage or systemic administration of CNP reverses the impaired skeletal growth of mice model of achondroplasia, the most common form of human skeletal dysplasias.

Therapeutic potential of CNP for skeletal dysplasias

Background We had revealed that C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth by using transgenic and knockout mice: transgenic mice with targeted overexpression of CNP in cartilage develop prominent skeletal overgrowth [1], whereas mice depleted with CNP exhibit impaired endochondral bone growth [2]. We planned to translate this effect of CNP into the therapy for skeletal dysplasias, of which the effective drug therapy is not established yet. Here we investigated the effect of CNP on the impaired bone growth of mice model of achondroplasia, the most common form of skeletal dysplasias.

α-MSH Counteracts the Suppressive Effect of Ultraviolet B on Nrf1/2 and Nrf-Dependent Gene Expression in Human Skin

α-MSH Counteracts the Suppressive Effect of Ultraviolet B on Nrf1/2 and Nrf-Dependent Gene Expression in Human Skin

Systemic Administration of C-Type Natriuretic Peptide as a Novel Therapeutic Strategy for Skeletal Dysplasias

Skeletal dysplasias are a group of genetic disorders characterized by severe impairment of bone growth. Various forms of them add to produce a significant morbidity and mortality, yet no efficient drug therapy has been developed to date. We previously demonstrated that C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, is a potent stimulator of endochondral bone growth. Furthermore, we exhibited that targeted overexpression of a CNP transgene in the growth plate rescued the impaired bone growth observed in a mouse model of achondroplasia (Ach), the most frequent form of human skeletal dysplasias, leading us to propose that CNP may prove to be an effective treatment for this disorder. In the present study, to elucidate whether or not the systemic administration of CNP is a novel drug therapy for skeletal dysplasias, we have investigated the effects of plasma CNP on impaired bone growth in Ach mice that specifically overexpress CNP in the liver under the control of human serum amyloid P component promoter or in those treated with a continuous CNP infusion system. Our results demonstrated that increased plasma CNP from the liver or by iv administration of synthetic CNP-22 rescued the impaired bone growth phenotype of Ach mice without significant adverse effects. These results indicate that treatment with systemic CNP is a potential therapeutic strategy for skeletal dysplasias, including Ach, in humans.

Systemic Administration of C-Type Natriuretic Peptide as a Novel Therapeutic Strategy for Skeletal Dysplasias

Skeletal dysplasias are a group of genetic disorders characterized by severe impairment of bone growth. Various forms of them add to produce a significant morbidity and mortality, yet no efficient drug therapy has been developed to date. We previously demonstrated that C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, is a potent stimulator of endochondral bone growth. Further we exhibited that targeted overexpression of a CNP transgene in the growth plate rescued the impaired bone growth observed in mice model of achondroplasia (Ach), the most frequent form of human skeletal dysplasias, leading us to propose that CNP may prove to be an effective treatment for this disorder. In the present study, in order to elucidate whether or not the systemic administration of CNP is the novel drug therapy for skeletal dysplasias, we have investigated the effects of plasma CNP on impaired bone growth in Ach mice that specifically overexpress CNP in the liver under the control of human serum amyloid P component promoter, or in those treated with a continuous CNP infusion system. Our results demonstrated that increased plasma CNP from the liver or by intravenous administration of synthetic CNP-22 rescued the impaired bone growth phenotype of Ach mice without significant adverse effects. These results indicate that treatment with systemic CNP is a potential therapeutic strategy for skeletal dysplasias, including Ach, in humans.