Adalbert Raimann

Fibroblast growth factor 23 and Klotho are present in the growth plate.

Introduction: Regulation of phosphate homeostasis is essential for mineralization and enchondral ossification. Fibroblast growth factor 23 (FGF23) and its obligatory co-receptor Klotho (KL) play a key role in this process by influencing both renal phosphate reabsorption and vitamin D metabolism. In disease, excessive action of FGF23 leads to hypophosphatemic rickets, while its deficiency causes tumoral calcinosis. Although osteocytes and osteoblasts are widely seen as the primary source of FGF23 under physiological conditions, the origin of systemic FGF23 remains controversial. In this study, we investigated the expression of FGF23 and KL in porcine growth plate cartilage, adjacent tissues, and parenchymal tissues. Materials and Methods: Tissue samples were obtained from 4- to 6-week-old piglets. mRNA expression was quantified by real-time PCR and normalized to 18S rRNA. Immunohistochemical staining was performed for FGF23, KL, collagen type X, and FGF receptor 1. Growth plate chondrocyte subpopulations were acquired by collagenase digestion of growth plate explants and subsequent density gradient centrifugation. Results: We could detect both FGF23 and KL mRNA and protein in growth plate chondrocytes. FGF23 expression was mainly found in hypertrophic and resting chondrocytes. Furthermore, significant expression of both genes was observed in bone, liver, and spleen. Conclusion: These data challenge previous expression analyses, in particular theories of bone as the exclusive source of FGF23. Moreover, significant expression of FGF23 and KL within the growth plate and adjacent tissues imply a potential local role of FGF23 in chondrocyte differentiation and tissue mineralization.

1,25-Dihydroxyvitamin D Alone Improves Skeletal Growth, Microarchitecture, and Strength in a Murine Model of XLH, Despite Enhanced FGF23 Expression

X‐linked hypophosphatemia (XLH) is characterized by impaired renal tubular reabsorption of phosphate owing to increased circulating FGF23 levels, resulting in rickets in growing children and impaired bone mineralization. Increased FGF23 decreases renal brush border membrane sodium‐dependent phosphate transporter IIa (Npt2a) causing renal phosphate wasting, impairs 1‐α hydroxylation of 25‐hydroxyvitamin D, and induces the vitamin D 24‐hydroxylase, leading to inappropriately low circulating levels of 1,25‐dihydroxyvitamin D (1,25D). The goal of therapy is prevention of rickets and improvement of growth in children by phosphate and 1,25D supplementation. However, this therapy is often complicated by hypercalcemia and nephrocalcinosis and does not always prevent hyperparathyroidism. To determine if 1,25D or blocking FGF23 action can improve the skeletal phenotype without phosphate supplementation, mice with XLH (Hyp) were treated with daily 1,25D repletion, FGF23 antibodies (FGF23Ab), or biweekly high‐dose 1,25D from d2 to d75 without supplemental phosphate. All treatments maintained normocalcemia, increased serum phosphate, and normalized parathyroid hormone levels. They also prevented the loss of Npt2a, α‐Klotho, and pERK1/2 immunoreactivity observed in the kidneys of untreated Hyp mice. Daily treatment with 1,25D decreased urine phosphate losses despite a marked increase in bone FGF23 mRNA and in circulating FGF23 levels. Daily 1,25D was more effective than other treatments in normalizing the growth plate and metaphyseal organization. In addition to being the only therapy that normalized lumbar vertebral height and body weight, daily 1,25D therapy normalized bone geometry and was more effective than FGF23Ab in improving trabecular bone structure. Daily 1,25D and FGF23Ab improved cortical microarchitecture and whole‐bone biomechanical properties more so than biweekly 1,25D. Thus, monotherapy with 1,25D improves growth, skeletal microarchitecture, and bone strength in the absence of phosphate supplementation despite enhancing FGF23 expression, demonstrating that 1,25D has direct beneficial effects on the skeleton in XLH, independent of its role in phosphate homeostasis.

A novel homozygous mutation in the parathyroid hormone gene (PTH) in a girl with isolated hypoparathyroidism

CASE REPORT A female patient with consanguineous parents presented with severe symptomatic hypocalcemia (1.62mmol/l) at the age of 4 months. Treatment with oral 1,25-(OH)2-vitamin D and calcium carbonate was started and serum calcium concentrations were stabilized at the lower end of the normal range. Subsequently she developed normally and had no evidence for additional abnormalities. Over the next 6 years of observation, serum levels of PTH were always low but detectable (5.3-2.5pg/ml; normal: 15-65pg/ml) resulting in the diagnosis of isolated hypoparathyroidism. Disturbances in the vitamin-D metabolism, autoimmune polyendocrine syndrome (APS), chromosomal anomalies or mutations in the calcium-sensing receptor gene (CaSR) were excluded. Nucleotide sequence analysis of PTH revealed the presence of a homozygous point mutation (c.68C>A) in exon 2 that introduces a premature termination codon (p.Ser23X in the Pre- sequence of PTH) resulting in a non-functional PTH-precursor. CONCLUSION A novel, homozygous PTH mutations was identified, which is obviously a very rare cause of isolated hypoparathyroidism (IHP). Although activating CaSR mutations are the most common cause of hypoparathyroidism, analysis of the PTH gene should be considered in those IHP patients in whom a CaSR has been excluded, particularly if the parents are likely to be consanguineous.

Insulin-like growth factor I (IGF-1) Ec/Mechano Growth factor--a splice variant of IGF-1 within the growth plate.

Human insulin-like growth factor 1 Ec (IGF-1Ec), also called mechano growth factor (MGF), is a splice variant of insulin-like growth factor 1 (IGF-1), which has been shown in vitro as well as in vivo to induce growth and hypertrophy in mechanically stimulated or damaged muscle. Growth, hypertrophy and responses to mechanical stimulation are important reactions of cartilaginous tissues, especially those in growth plates. Therefore, we wanted to ascertain if MGF is expressed in growth plate cartilage and if it influences proliferation of chondrocytes, as it does in musculoskeletal tissues. MGF expression was analyzed in growth plate and control tissue samples from piglets aged 3 to 6 weeks. Furthermore, growth plate chondrocyte cell culture was used to evaluate the effects of the MGF peptide on proliferation. We showed that MGF is expressed in considerable amounts in the tissues evaluated. We found the MGF peptide to be primarily located in the cytoplasm, and in some instances, it was also found in the nucleus of the cells. Addition of MGF peptides was not associated with growth plate chondrocyte proliferation.

IGF expression patterns and regulation in growth plate chondrocytes

IGF-I and IGF-II are key regulators of growth and metabolism. Still, data about their expression and distribution within the growth plate in different animal models remain contradictory. Inferences drawn from rodent animal models can only be applied to human conditions to a limited extent as the rodents growth plate never fuses. In this study, we compared the expression of IGF-I and IGF-II in native growth plates of prepubertal piglets and under different cell culture conditions. We detected IGF-I mRNA expression and abundantly expressed IGF-II within the growth plate. IGF-I expression increased during monolayer cell culture while IGF-II expression dramatically decreased. Our studies revealed that these expression patterns remained unaffected by growth hormone stimulation in vitro. The abundant expression of IGF-II in porcine growth plate tissue, both on the mRNA and on the protein level, suggests that IGF-II also has a role in growth regulation at the early postnatal stage.

c-Raf promotes angiogenesis during normal growth plate maturation

Extracellular phosphate plays a key role in growth plate maturation by inducing Erk1/2 (Mapk3/1) phosphorylation, leading to hypertrophic chondrocyte apoptosis. The Raf kinases induce Mek1/2 (Map2k1/2) and Erk1/2 phosphorylation; however, a role for Raf kinases in endochondral bone formation has not been identified. Ablation of both A-Raf (Araf) and B-Raf (Braf) in chondrocytes does not alter growth plate maturation. Because c-Raf (Raf1) phosphorylation is increased by extracellular phosphate and c-Raf is the predominant isoform expressed in hypertrophic chondrocytes, chondrocyte-specific c-Raf knockout mice (c-Raff/f;ColII-Cre+) were generated to define a role for c-Raf in growth plate maturation. In vivo studies demonstrated that loss of c-Raf in chondrocytes leads to expansion of the hypertrophic layer of the growth plate, with decreased phospho-Erk1/2 immunoreactivity and impaired hypertrophic chondrocyte apoptosis. However, cultured hypertrophic chondrocytes from these mice did not exhibit impairment of phosphate-induced Erk1/2 phosphorylation. Studies performed to reconcile the discrepancy between the in vitro and in vivo hypertrophic chondrocyte phenotypes revealed normal chondrocyte differentiation in c-Raff/f;ColII-Cre+ mice and lack of compensatory increase in the expression of A-Raf and B-Raf. However, VEGF (Vegfa) immunoreactivity in the hypertrophic chondrocytes of c-Raff/f;ColII-Cre+ mice was significantly reduced, associated with increased ubiquitylation of VEGF protein. Thus, c-Raf plays an important role in growth plate maturation by regulating vascular invasion, which is crucial for replacement of terminally differentiated hypertrophic chondrocytes by bone. Summary: The chondrocyte-specific ablation of c-Raf in mice results in a delay in vascular invasion and growth plate maturation due to increased ubiquitin-dependent degradation of VEGF.

A journey through growth plates: tracking differences in morphology and regulation between the spine and the long bones in a pig model

BACKGROUND CONTEXT The process of linear growth is driven by axial elongation of both long bones and vertebral bodies and is accomplished by enchondral ossification. Differences in regulation between the two skeletal sites are mirrored clinically by the age course in body proportions. Whereas long bone growth plates (GPs) can easily be discriminated, vertebral GPs are part of the cartilaginous end plate, which typically shows important species differences. PURPOSE The objective of this study was to describe and compare histologic, histomorphometric, and regulatory characteristics in the GPs of the spine and the long bones in a porcine model. MATERIALS AND METHODS Two- and six-week-old piglet GPs of three vertebral segments (cervical, thoracic, and lumbar) and eight long bones (proximal and distal radius, humerus, tibia, and femur) were analyzed morphometrically. Further, estrogen receptors, proliferation markers, and growth factor expressions were examined by immunohistochemistry. RESULTS Individual vertebral GPs were smaller in width and contained fewer chondrocytes than long bone GPs, although their proliferation activity was similar. Whereas the expression pattern of growth hormone-associated factors such as insulin-like growth factor (IGF)-1 and IGF-1 receptor (IGF-1R) was similar, estrogen receptor (ER)-ß and IGF-2 were distinctly expressed in the vertebral samples. CONCLUSIONS Vertebral GPs display differential growth, with measurements similar to the slowest-growing GPs of long bones. Further investigation is needed to decipher the molecular basis of the differential growth of the spine and the long bones. Knowledge on the distinct mechanism will ultimately improve the assessment of clinically essential characteristics of spinal growth, such as vertebral elongation potential and GP fusion.

Growth Plate Research

Growth in mammals is a unique and fascinating process which takes place in the growth plates (GP) of long bones and vertebrae.

Bilateral Infiltrative Dacryoadenitis and Granulomatous Pneumonia in an 11-Year-Old Boy: A Case Report

Bilateral dacryoadenitis is a rare inflammatory condition of the lacrimal gland, which, in case of insufficient treatment, might lead to cellulitis and orbital abscess formation (Derr C et al., J Emerg Trauma Shock 2012, 5(1):92–94). Infections are the most common cause of an acute dacryoadenitis in children and are either viral (mumps, measles, influenza, Epstein Barr, cytomegaly or herpes virus), or bacterial (Streptococcus pyogenes, Haemophilus influenza, Neisseria gonorrhea, Chlamydia trachomatis and Treponema pallidum) (Rai P et al., Med Channel 2009, 15:71–76, Srivastava V. Med J Armed Forces India 2000, 56:151–152). Dacryoadenitis may also originate from autoimmune diseases, such as granulomatosis with polyangitis, Graves’ disease and Sjogren’s syndrome or malignant diseases, such as lymphoma. There is need to exclude these underlying diseases in particular in cases of bilateral dacryoadenitis (Belanger C et al. Am J Ophthalmol 2010, 150(4):460–463).

A Pediatric Patient with a CYP24A1 Mutation: Four Years of Clinical, Biochemical, and Imaging Follow-Up

Background: A female infant was admitted to hospital due to failure to thrive. She presented hypercalcemia (4.09 mmol/L, normal range: 2.2-2.65 mmol/L), high 25-hydroxyvitamin D (283 nmol/L, normal range: 75-250 nmol/L), 1,25-dihydroxyvitamin D in the upper normal range, and low parathyroid hormone. Vitamin D intoxication was suspected. The patient had received routine rickets prophylaxis. Methods: Williams-Beuren syndrome was genetically excluded. Sequencing of CYP24A1 showed 2 mutations: c.443T>C and c.1186C>T. Results: The patients clinical status improved after intravenous rehydration, cessation of supplementation, and on a low-calcium diet. 25-Hydroxyvitamin D concentrations normalized within days, while 1,25-dihydroxyvitamin D remained in the upper normal range. We also investigated our patients bone health. Conclusion: The patient was hospitalized initially on suspicion of vitamin D intoxication but proved to be a case of compound heterozygosity. Data on the long-term clinical and biochemical evolution of patients with idiopathic infantile hypercalcemia are sparse. Our follow-up showed seasonal variations of vitamin D and calcium parameters, with no influence on kidney function or bone health for the investigated period.