Density-Dependent Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Parathyroid-Hormone-Releasing Cells.

Mesenchymal stem cells (MSCs) can differentiate into endoderm lineages, especially parathyroid-hormone (PTH)-releasing cells. We have previously reported that tonsil-derived MSC (T-MSC) can differentiate into PTH-releasing cells (T-MSC-PTHCs), which restored the parathyroid functions in parathyroidectomy (PTX) rats. In this study, we demonstrate quality optimization by standardizing the differentiation rate for a better clinical application of T-MSC-PTHCs to overcome donor-dependent variation of T-MSCs. Quantitation results of PTH mRNA copy number in the differentiated cells and the PTH concentration in the conditioned medium confirmed that the differentiation efficiency largely varied depending on the cells from each donor. In addition, the differentiation rate of the cells from all the donors greatly improved when differentiation was started at a high cell density (100% confluence). The large-scale expression profiling of T-MSC-PTHCs by RNA sequencing indicated that those genes involved in exiting the differentiation and the cell cycle were the major pathways for the differentiation of T-MSC-PTHCs. Furthermore, the implantation of the T-MSC-PTHCs, which were differentiated at a high cell density embedded in hyaluronic acid, resulted in a higher serum PTH in the PTX model. This standardized efficiency of differentiation into PTHC was achieved by initiating differentiation at a high cell density. Our findings provide a potential solution to overcome the limitations due to donor-dependent variation by establishing a standardized differentiation protocol for the clinical application of T-MSC therapy in treating hypoparathyroidism.

Polymeric RNAi Constructs Tailored with Appreciable Transcellular Trafficking Functions for Potential Suppression of Parathyroid Hormone Production.

Polymeric small interfering RNA (siRNA) conjugate was elaborated to sequentially circumvent the predefined biological barriers encountered in the journey of transcellular delivery of siRNA into cytosol. Herein, classic ring-opening polymerization was employed for synthesis of well-defined poly(amino acid) derivatives possessing an array of carboxyl groups in an attempt to resemble the structural characteristics of hyaluronan. Furthermore, the hyaluronan-like synthetic was conjugated with a multiple of siRNA through a glutathione (GSH)-responsive disulfide linkage. The siRNA conjugate appeared to utilize the hyaluronan-specific receptors of CD44 for cell internalization, indicating similar functionalities to our hyaluronan-mimicking synthetic. Furthermore, the carboxyl groups of hyaluronan-like synthetics were designed to be selectively detached in subcellular acidic endosomes/lysosomes and transform into the cytomembrane-disruptive flanking ethylenediamine moieties, which appeared to be crucial in facilitating translocation of siRNA payloads from entrapment and degradation in lysosomes toward the cytosol. Eventually, active siRNA could be smoothly released from the synthetic due to the GSH cleavage disulfide linkage (disulfide), consequently accounting for potent RNA knockdown activities (>90%) toward cancerous cells. In addition, appreciable knockdown of parathyroid hormone was also achieved from our proposed siRNA conjugates in parathyroid cells. Hence, the elaborated siRNA conjugate showed tremendous potential in treatment of hyperparathyroidism, and could be developed further for systemic RNA interference (RNAi) therapeutics. Moreover, this study could also be the first example of a synthetic mimic to hyaluronan acquiring its functionalities, which could have important implications for further development of biomimic materials in pursuit of biomedical applications.

Local NF-κB Activation Promotes Parathyroid Hormone Synthesis and Secretion in Uremic Patients.

Secondary hyperparathyroidism (SHPT) in uremic patients is characterized by parathyroid gland (PTG) hyperplasia and parathyroid hormone (PTH) elevation. Previously, we demonstrated that NF-κB activation contributed to parathyroid cell proliferation in rats with chronic kidney disease. Although vitamin D inhibits inflammation and ameliorates SHPT, the contribution of vitamin D deficiency to SHPT via local NF-κB activation remains to be clarified. PTGs collected from 10 uremic patients with advanced SHPT were used to test the expressions of vitamin D receptor (VDR), NF-κB, and proliferating cell nuclear antigen (PCNA). Freshly excised PTG tissues were incubated for 24 hours in vitro with VDR activator (VDRA) calcitriol or NF-κB inhibitor pyrrolidine thiocarbamate (PDTC). Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to investigate the regulation of PTH transcription by NF-κB. We found higher levels of activated NF-κB and lower expression of VDR in nodular hyperplastic PTGs than in diffuse hyperplasia. In cultured PTG tissues, treatment with VDRA or PDTC inhibited NF-κB activation and PCNA expression, and downregulated preproPTH mRNA and intact PTH levels. ChIP assays demonstrated the presence of NF-κB binding sites in PTH promoter. Furthermore, in luciferase reporter assays, addition of exogenous p65 significantly increased PTH luciferase activity by 2.4-fold (P < 0.01), while mutation of NF-κB binding site at position -908 of the PTH promoter suppressed p65-induced PTH reporter activity (P < 0.01). In summary, local NF-κB activation contributes to SHPT and mediates the transcriptional activation of PTH directly in uremic patients. Vitamin D deficiency may be involved in SHPT via the activation of NF-κB pathway.

Gender Predilection in Sporadic Parathyroid Adenomas.

Primary hyperparathyroidism is a common endocrinopathy that is mainly caused by benign parathyroid adenomas. The frequency, clinical presentation and complications of the disease show significant differences between genders, with the majority of cases being reported in postmenopausal women. Due to this gender predilection, several studies have investigated the role of sex hormones in the pathogenesis of the disease and their potential use as targets for optimal and gender-specific management. Epigenetic mechanisms that regulate gene transcription may also contribute to these differences between genders. In this review, we outline what is currently known regarding the role of sex hormones and the recent data on the role of non-coding RNAs in the differences between genders in primary hyperparathyroidism due to sporadic parathyroid adenomas.

Embryology of the Parathyroid Glands.

The parathyroid glands are essential for regulating calcium homeostasis in the body. The genetic programs that control parathyroid fate specification, morphogenesis, differentiation, and survival are only beginning to be delineated, but are all centered around a key transcription factor, GCM2. Mutations in the Gcm2 gene as well as in several other genes involved in parathyroid organogenesis have been found to cause parathyroid disorders in humans. Therefore, understanding the normal development of the parathyroid will provide insight into the origins of parathyroid disorders.

Genetic Disorders of Parathyroid Development and Function.

Hypoparathyroidism is characterized by hypocalcemia and hyperphosphatemia and is due to insufficient levels of circulating parathyroid hormone. Hypoparathyroidism may be an isolated condition or a component of a complex syndrome. Although genetic disorders are not the most common cause of hypoparathyroidism, molecular analyses have identified a growing number of genes that when defective result in impaired formation of the parathyroid glands, disordered synthesis or secretion of parathyroid hormone, or postnatal destruction of the parathyroid glands.

[New scenarios in secondary hyperparathyroidism: etelcalcetide. Position paper of Nephrologists form Lombardy].

Bone mineral abnormalities (defined as Chronic Kidney Disease Mineral Bone Disorder; CKD-MBD) are prevalent and associated with a substantial risk burden and poor prognosis in CKD population. Several lines of evidence support the notion that a large proportion of patients receiving maintenance dialysis experience a suboptimal biochemical control of CKD-MBD. Although no study has ever demonstrated conclusively that CKD-MBD control is associated with improved survival, an expanding therapeutic armamentarium is available to correct bone mineral abnormalities. In this position paper of Lombardy Nephrologists, a summary of the state of art of CKD-MBD as well as a summary of the unmet clinical needs will be provided. Furthermore, this position paper will focus on the potential and drawbacks of a new injectable calcimimetic, etelcalcetide, a drug available in Italy since few months ago.

Interrelated role of Klotho and calcium-sensing receptor in parathyroid hormone synthesis and parathyroid hyperplasia.

The pathogenesis of parathyroid gland hyperplasia is poorly understood, and a better understanding is essential if there is to be improvement over the current strategies for prevention and treatment of secondary hyperparathyroidism. Here we investigate the specific role of Klotho expressed in the parathyroid glands (PTGs) in mediating parathyroid hormone (PTH) and serum calcium homeostasis, as well as the potential interaction between calcium-sensing receptor (CaSR) and Klotho. We generated mouse strains with PTG-specific deletion of Klotho and CaSR and dual deletion of both genes. We show that ablating CaSR in the PTGs increases PTH synthesis, that Klotho has a pivotal role in suppressing PTH in the absence of CaSR, and that CaSR together with Klotho regulates PTH biosynthesis and PTG growth. We utilized the tdTomato gene in our mice to visualize and collect PTGs to reveal an inhibitory function of Klotho on PTG cell proliferation. Chronic hypocalcemia and ex vivo PTG culture demonstrated an independent role for Klotho in mediating PTH secretion. Moreover, we identify an interaction between PTG-expressed CaSR and Klotho. These findings reveal essential and interrelated functions for CaSR and Klotho during parathyroid hyperplasia.

Hypercalcaemia secondary to ectopic parathyroid hormone expression in an adolescent with metastatic alveolar rhabdomyosarcoma.

We report the case of a 14-year-old male with metastatic alveolar rhabdomyosarcoma, presenting with hypercalcaemia (3.89 mmol/l) and elevated parathyroid hormone (PTH) level (10.2 pmol/l). Imaging demonstrated extensive bony lytic damage, with "floating teeth" in the mandible. Normalisation of calcium levels and bony reformation of the mandible occurred following chemotherapy; PTH levels decreased initially but remained above normal levels. Imaging did not demonstrate any evidence of parathyroid abnormality. Tumour ectopic PTH secretion is a very rare cause of hypercalcaemia of malignancy in children. Hypercalcaemia with an elevated PTH, in the absence of parathyroid-related cause, should prompt investigation for underlying malignancy.

Endogenous Parathyroid Hormone Promotes Fracture Healing by Increasing Expression of BMPR2 through cAMP/PKA/CREB Pathway in Mice.

BACKGROUND/AIMS: Endogenous parathyroid hormone (PTH) plays an important role in fracture healing. This study investigated whether endogenous PTH regulates fracture healing by bone morphogenetic protein (BMP) and/or the transforming growth factor-ß (TGF-ß) signaling pathway. METHODS: Eight-week-old wild-type (WT) and PTH-knockout (PTH KO) male mice were selected, and models of open right-femoral fracture were constructed. Fracture healing and callus characteristics of mice in the two groups were compared by X-ray, micro-computed tomography, histological, and immunohistochemical examinations. Bone marrow mesenchymal stem cells (BMMSCs) of 8-week-old WT and PTHKO male mice were obtained and induced into osteoblasts and chondrocytes. RESULTS: We found that expression levels of Runt-related transcription factor (RUNX2), bone morphogenetic protein-receptor-type Ⅱ (BMPR2), phosphorylated Smad 1/5/8, and phosphorylated cyclic adenosine monophosphate-responsive element binding protein (CREB) in the callus of PTHKO mice were significantly decreased, whereas no significant difference in expression of SOX9, TGF-ßR2,or pSMAD2/3 was observed between PTHKO and WT mice. Additionally, the activity of osteoblast alkaline phosphatase was low at 7 days post-induction, and was upregulated by addition of PTH or dibutyryl cyclic adenosine monophosphate (dbcAMP) to the cell culture. Furthermore, H89 (protein kinase A inhibitor)eliminated the simulating effects of PTH and dbcAMP, and a low concentration of cyclic adenosine monophosphate (cAMP) was observed in PTHKO mouse BMMSCs. CONCLUSION: These results suggested that endogenous PTH enhanced BMPR2 expression by a cAMP/PKA/CREB pathway in osteoblasts, and increased RUNX2 expression through transduction of the BMP/pSMAD1/5/8 signaling pathway.

Parathyroid reimplantation with PR-FaST technique in unselected patients during thyroidectomy. A case series with long term follow up confirming graft vitality and parathormone production.

INTRODUCTION: Parathyroid damage or unintentional excision still affect thyroid surgery and may cause permanent hypoparathyroidism. The only way to recover the excised or ischemic gland functionality is still reimplantation. Many sites of reimplantation have been described, each of one showing both advantages and drawbacks. The aim of this study is to verify results of a new procedure called PR-FaST: Parathyroid Reimplantation in Forearm Subcutaneous Tissue, in a series of unselected patients after long-term follow-up. MATERIALS AND METHODS: From January 2013 to October 2015, 296 consecutive total thyroidectomies have been performed) to treat both benign and malignant thyroid diseases. in 42 cases (14.1%), due to an insufficient blood supply or accidental removal, one parathyroid gland was reimplanted with the PR-FaST technique. Post-operative evaluation was carried out by: total serum calcium (Ca), magnesium (Mg) and phosphorus (P) analysis in the 1st and 2nd postoperative days; Ca, Mg, P and serum iPTH from both arms analysis one week after surgery; Ca and iPTH measurement from both arms 1 months, 3, 6 and 12 months after surgery. RESULTS: We observed transient hypocalcemia requiring calcium replacement therapy in 5 on 42 (11.9%) patients submitted to PR-FaST. No case of permanent hypoparathyroidism was reported. At 1 week after surgery, only 20 patients (47.6%) showed graft vitality, while the number of patients showing graft vitality arised to 33 (79%) after 1 month and to 39 (92.8%) after three and six months. At 1 year 38 (90.5%) patients showed good graft functionality. Considering levels of serum iPTH from both arms, we observed that in case of graft functionality, samples from reimplanted arm revealed in almost all cases values at least 2-3 folds higher than in non reimplanted arm. CONCLUSIONS: Results from this prospective evaluation suggest that PR-FaST is a safe and effective procedure, with potential advantages when compared to other techniques of parathyroid reimplantation, that are mainly the possibility to evaluate graft functionality in the follow-up and the easy and well reproducible technique. Furthermore, it can be applied, when needed, to potentially all patients undergoing thyroidectomy.

Vitamin D: Recent Recommendations and Discoveries.

The evidence continues to mount for the important role of vitamin D as a hormone that provides protective benefits to the immune system. Guidelines have been developed recommending routine vitamin D supplementation in order to optimize bone health, including aiming for a goal 25(OH)D concentration greater than 30 ng/mL among select patient groups who are at risk for skeletal deficits or vitamin D deficiency itself. Whether this level is the optimal one that confers the extraskeletal benefits of vitamin D remains unknown. Whether healthy children and adolescents may benefit from maintenance of 25(OH)D levels at higher thresholds also is unknown and under study. Adolescence is a critical time for the accrual of peak bone mass, and optimizing vitamin D during this important developmental period and throughout the life cycle may be beneficial for and beyond the skeleton. Although routine screening of 25(OH)D levels is not currently recommended, those individuals at higher risk for insufficiency/deficiency should be screened and appropriately supplemented.

Recombinant production of TEV cleaved human parathyroid hormone.

The parathyroid hormone, PTH, is responsible for calcium and phosphate ion homeostasis in the body. The first 34 amino acids of the peptide maintain the biological activity of the hormone and is currently marketed for calcium imbalance disorders. Although several methods for the production of recombinant PTH(1-34) have been reported, most involve the use of cleavage conditions that result in a modified peptide or unfavorable side products. Herein, we detail the recombinant production of (15) N-enriched human parathyroid hormone, (15) N PTH(1-34), generated via a plasmid vector that gives reasonable yield, low-cost protease cleavage (leaving the native N-terminal serine in its amino form), and purification by affinity and size exclusion chromatography. We characterize the product by multidimensional, heteronuclear NMR, circular dichroism, and LC/MS.

Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115.

Human serum albumin (HSA) and human parathyroid hormone (1-34) [PTH (1-34)] fusion protein [HSA/PTH (1-34)] is a promising long-acting form of PTH (1-34) for osteoporosis treatment. Secretory expression of intact HSA/PTH (1-34) in Pichia pastoris GS115 was accompanied by two degradation fragments, with molecular weights around 66 kDa, in addition to the well-known ~45 kDa HSA-truncated fragment, resulting in a low yield of intact protein. In this study, two internal cleavage sites were identified in the PTH (1-34) portion of the fusion protein by Western Blot analysis. To minimize proteolytic cleavages, several protease genes including PEP4 (encoding proteinase A), PRB1 (proteinase B) and seven YPSs genes (yapsin family members) were knocked out respectively by disruption of the individual genes and the selective combinations. Reduced degradation was observed by single disruption of either PEP4 gene or YPS1 gene, and the lowest level of degradation was observed in a pep4△yps1△ double disruptant. After 72 h of induction, more than 80 % of the HSA/PTH (1-34) secreted by the pep4△yps1△ double disruptant remained intact, in comparison to only 30 % with the wild-type strain.

Magnesium incorporation into ß-TCP reduced its in vivo resorption by decreasing parathormone production.

Beta-tricalcium phosphate (ß-TCP), one of the most widely used bioresorbable materials for bone therapy, can be doped with magnesium ions, generating ß-TCMP. The objectives of this work were to evaluate, on a murine dental alveolus grafting model, the biocompatibility of ß-TCP and ß-TMCP granules by histomorphometric analysis, as well as the impact on plasmatic levels of receptor activator of nuclear factor κB ligand (RANK-L), osteoprotegerin (OPG), osteocalcin, osteopontin, and parathormone (PTH) during bone repair, using Luminex multiplexing technology. After grafting for 42 days, ß-TCP grafted group presented higher bioresorption and induced more newly formed bone than ß-TCMP (p < 0.05). ß-TCP grafting also induced higher plasmatic levels of RANK-L, compared to ß-TCMP and control (blood clot) groups at 21st day (p < 0.05). PTH, which remained at low levels in control group, presented a time-dependent increase in grafted groups, attaining significantly higher levels with ß-TCP by the 42nd day (p < 0.05). RANK-L/OPG ratio increased on ß-TCP group and attained a peak on the 21st day. In conclusion, ß-TCP granules were more bioresorbable and osteogenic than ß-TCMP granules, and the resorption of both materials might have been affected by osteoclastogenesis modulated by changes in the plasmatic levels of PTH and RANK-L.

Potential role of high mobility group box protein 1 and intermittent PTH (1-34) in periodontal tissue repair following orthodontic tooth movement in rats.

OBJECTIVES: Recent studies indicate that high mobility group box protein 1 (HMGB1) can be released by necrotic and damaged cells and functions as an alarmin that is recognized by the innate immune system. Little is known about the role of HMGB1 within the periodontal ligament (PDL). Therefore, we examined HMGB1 expression by PDL cells in vitro and compared the findings to an in vivo model of orthodontically induced tooth root resorption. In addition, we addressed the question of whether a potentially anabolic intermittent administration of parathyroid hormone (iPTH) would modulate the expression of HMGB1. MATERIALS AND METHODS: In confluent PDL cell cultures, HMGB1 messenger RNA (mRNA) expression was quantified by real-time polymerase chain reaction. In a rat model comprising 25 animals, mechanical loading for 5 days was followed by administration of either iPTH (1-34) systemically or sham injections for up to 56 days. HMGB1 expression was determined by means of immunohistochemistry and histomorphometry. RESULTS: The in vitro experiments revealed an inhibitory effect of iPTH on basal HMGB1 mRNA expression in confluent PDL cells. In vivo, the mechanical force-induced enhanced HMGB1 protein expression declined time dependently. Intermittent PTH further inhibited HMGB1 expression. The significantly higher basal HMGB1 protein expression in the former compression side was followed by a more pronounced time- and iPTH-dependent decline in the same area. CONCLUSIONS: These data indicate a major role for HMGB1 in the regulation of PDL wound healing following mechanical load-induced tissue injury. CLINICAL RELEVANCE: The findings point to the potential benefit of iPTH in the attempt to support these immune-associated reparative processes.

One-step purification of recombinant human amelogenin and use of amelogenin as a fusion partner.

Amelogenin is an extracellular protein first identified as a matrix component important for formation of dental enamel during tooth development. Lately, amelogenin has also been found to have positive effects on clinical important areas, such as treatment of periodontal defects, wound healing, and bone regeneration. Here we present a simple method for purification of recombinant human amelogenin expressed in Escherichia coli, based on the solubility properties of amelogenin. The method combines cell lysis with recovery/purification of the protein and generates a >95% pure amelogenin in one step using intact harvested cells as starting material. By using amelogenin as a fusion partner we could further demonstrate that the same method also be can explored to purify other target proteins/peptides in an effective manner. For instance, a fusion between the clinically used protein PTH (parathyroid hormone) and amelogenin was successfully expressed and purified, and the amelogenin part could be removed from PTH by using a site-specific protease.

Effects of embryonic exposure to polychlorinated biphenyls on zebrafish skeletal development.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that affect embryonic development. The purpose of this study was to examine the effects of embryonic exposure to PCBs on early skeletal development in zebrafish (Danio rerio). Zebrafish embryos were immediately exposed to various concentrations (0, 0.125, 0.25, 0.5 and 1.0 mg/l) of PCBs (Aroclor 1254) after fertilization. Embryos were assessed at 24, 48, 72, 96 and 120 h post-fertilization (hpf) for changes in embryonic survival and malformation rates. Calcium content and vitamin D receptor (VDR), parathyroid hormone (PTH) and TRVP6 mRNA expressions were assessed at 120 hpf. The results showed that PCBs exposure decreased the survival rate of the embryos in a time-and dose-dependent manner. The embryos exposed to the higher concentrations of PCBs (0.5 and 1.0 mg/l) displayed obvious skeletal morphological deformities. At 120 hpf, the calcium content of the zebrafish was downregulated in all the PCB-treated groups. VDR, PTH and TRVP6 mRNA expressions were all affected by PCBs. By 120 hpf, the mRNA expressions of VDR, PTH and TRVP6 from the PCB-treated larvae were all upregulated. The expressions of PTH and TRVP6 positively correlated with the level of PCBs to which the embryos were exposed. These results suggest that embryonic exposure to PCBs induces developmental deficits in the zebrafish skeleton.

Nasal administration of a novel recombinant human parathyroid hormone (1-34) analog for the treatment of osteoporosis of ovariectomized rats.

Synthetic human parathyroid (1-34) (hPTH (1-34)) is known to have the full biological activity of the holohormone for osteoporosis. This study is about designing a novel analog of hPTH (1-34) which is more suitable for intranasal administration. We likewise evaluate effectiveness of the nasal drops against osteoroporosis. Through fusion expression of combining gene, cell disruption, inclusion body washing, ethanol fraction precipitation, acid hydrolysis, and CM-52 ion exchange column chromatography Pro-Pro-[Arg¹¹] hPTH (1-34)-Pro-Pro was designed and produced. Nasal drops of Pro-Pro-[Arg¹¹] hPTH (1-34)-Pro-Pro were prepared and administrated to ovariectomized rats. After 12 weeks of raising, Bone Material Densities (BMD) of vertebrae were examined by Dual Energy X-Ray Absorptiometry (DEXA). The average BMD of these groups treated with nasal drops of the peptide were 28.0%-47.2% (P<0.01) higher than that of the group treated with normal saline (NS). The subchondral bone plates of the femoral heads were examined by scanning electron microscopy and a defined planar section was photographed. Percentage of the area of the cancellous bone was calculated. Percentages of the groups treated with nasal drops of the peptide increased; values were significantly different to that of the group treated with NS (P<0.001) and were even equivalent to that of normal groups. These results show that nasal drops of Pro-Pro-[Arg¹¹] hPTH (1-34)-Pro-Pro are effective against osteoporosis.