Mice Lacking Wnt9a or Wnt4 Are Prone to Develop Spontaneous Osteoarthritis With Age and Display Alteration in Either the Trabecular or Cortical Bone Compartment

Osteoarthritis (OA) is a common degenerative disease of the joint, with a complex multifactorial not yet fully understood etiology. Over the past years, the Wnt signaling pathway has been implicated in osteoarthritis. In a recent genomewide association study (GWAS), the chromosomal location on chromosome 1, linked to the Wnt3a-Wnt9a gene locus, was identified as the most significant locus associated with a thumb osteoarthritis endophenotype. Previously, it was shown that WNT9a is involved in maintaining synovial cell identity in the elbow joint during embryogenesis. Here, we report that the conditional loss of Wnt9a in the Prx1-Cre expressing limb mesenchyme or Prg4-CreER expressing cells predispositions the mice to develop spontaneous OA-like changes with age. In addition, the trabecular bone volume is altered in these mice. Similarly, mice with a conditional loss of Wnt4 in the limb mesenchyme are also more prone to develop spontaneously OA-like joint alterations with age. These mice display additional alterations in their cortical bone. The combined loss of Wnt9a and Wnt4 increased the likelihood of the mice developing osteoarthritis-like changes and enhanced disease severity in the affected mice. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Biomarkers in WNT1 and PLS3 Osteoporosis: Altered Concentrations of DKK1 and FGF23

Recent advancements in genetic research have uncovered new forms of monogenic osteoporosis, expanding our understanding of the molecular pathways regulating bone health. Despite active research, knowledge on the pathomechanisms, disease-specific biomarkers, and optimal treatment in these disorders is still limited. Mutations in WNT1, encoding a WNT/β-catenin pathway ligand WNT1, and PLS3, encoding X chromosomally inherited plastin 3 (PLS3), both result in early-onset osteoporosis with prevalent fractures and disrupted bone metabolism. However, despite marked skeletal pathology, conventional bone markers are usually normal in both diseases. Our study aimed to identify novel bone markers in PLS3 and WNT1 osteoporosis that could offer diagnostic potential and shed light on the mechanisms behind these skeletal pathologies. We measured several parameters of bone metabolism, including serum dickkopf-1 (DKK1), sclerostin, and intact and C-terminal fibroblast growth factor 23 (FGF23) concentrations in 17 WNT1 and 14 PLS3 mutation-positive subjects. Findings were compared with 34 healthy mutation-negative subjects from the same families. Results confirmed normal concentrations of conventional metabolic bone markers in both groups. DKK1 concentrations were significantly elevated in PLS3 mutation-positive subjects compared with WNT1 mutation-positive subjects (p < .001) or the mutation-negative subjects (p = .002). Similar differences were not seen in WNT1 subjects. Sclerostin concentrations did not differ between any groups. Both intact and C-terminal FGF23 were significantly elevated in WNT1 mutation-positive subjects (p = .039 and p = .027, respectively) and normal in PLS3 subjects. Our results indicate a link between PLS3 and DKK1 and WNT1 and FGF23 in bone metabolism. The normal sclerostin and DKK1 levels in patients with impaired WNT signaling suggest another parallel regulatory mechanism. These findings provide novel information on the molecular networks in bone. Extended studies are needed to investigate whether these biomarkers offer diagnostic value or potential as treatment targets in osteoporosis. © 2020 American Society for Bone and Mineral Research.     

Sclerostin Antibody Treatment Increases Bone Mass and Normalizes Circulating Phosphate Levels in Growing Hyp Mice

X-linked hypophosphatemia (XLH), caused by a loss-of-function mutation in the phosphate regulating gene with homology to endopeptidase located on the X chromosome (PHEX), is the most common form of vitamin D-resistant rickets. Loss of functional PHEX results in elevated fibroblast growth factor 23 (FGF23) levels, impaired phosphate reabsorption, and inhibited skeletal mineralization. Sclerostin, a protein produced primarily in osteocytes, suppresses bone formation by antagonizing Wnt signaling and is reported to be elevated in XLH patients. This study used the Hyp mouse model to investigate sclerostin's role in the pathophysiology of XLH by evaluating the use of a monoclonal antibody to sclerostin in a mouse model of XLH, the Hyp mouse. Male and female wild-type and Hyp littermates were injected with 25 mg/kg of vehicle or sclerostin antibody (Scl-Ab) twice weekly, beginning at 4 weeks of age and euthanized at 8 weeks of age. Scl-Ab treatment increased serum phosphate levels and suppressed circulating levels of intact FGF23 in treated wild-type and Hyp mice of both sexes. Cortical area, trabecular bone volume fraction (BV/TV), metaphyseal apparent density, and the peak load increased with Scl-Ab treatment in both sexes. This short-term treatment study suggests that Scl-Ab treatment can effectively improve some of the pathologies associated with XLH, including normalization of phosphate, and that sclerostin may play a role in regulating FGF23 and phosphate metabolism in XLH. © 2019 American Society for Bone and Mineral Research.     

Pharmacological inhibition of fibroblast growth factor (FGF) receptor signaling ameliorates FGF23‐mediated hypophosphatemic rickets

Fibroblast growth factor 23 (FGF23) is a circulating factor secreted by osteocytes that is essential for phosphate homeostasis. In kidney proximal tubular cells FGF23 inhibits phosphate reabsorption and leads to decreased synthesis and enhanced catabolism of 1,25‐dihydroxyvitamin D₃ (1,25[OH]₂D₃). Excess levels of FGF23 cause renal phosphate wasting and suppression of circulating 1,25(OH)₂D₃ levels and are associated with several hereditary hypophosphatemic disorders with skeletal abnormalities, including X‐linked hypophosphatemic rickets (XLH) and autosomal recessive hypophosphatemic rickets (ARHR). Currently, therapeutic approaches to these diseases are limited to treatment with activated vitamin D analogues and phosphate supplementation, often merely resulting in partial correction of the skeletal aberrations. In this study, we evaluate the use of FGFR inhibitors for the treatment of FGF23‐mediated hypophosphatemic disorders using NVP‐BGJ398, a novel selective, pan‐specific FGFR inhibitor currently in Phase I clinical trials for cancer therapy. In two different hypophosphatemic mouse models, Hyp and Dmp1‐null mice, resembling the human diseases XLH and ARHR, we find that pharmacological inhibition of FGFRs efficiently abrogates aberrant FGF23 signaling and normalizes the hypophosphatemic and hypocalcemic conditions of these mice. Correspondingly, long‐term FGFR inhibition in Hyp mice leads to enhanced bone growth, increased mineralization, and reorganization of the disturbed growth plate structure. We therefore propose NVP‐BGJ398 treatment as a novel approach for the therapy of FGF23‐mediated hypophosphatemic diseases. © 2013 American Society for Bone and Mineral Research.     

Affinity evaluation of gelatin for hepatocyte growth factor of different types to design the release carrier

The objective of this study was to investigate the physicochemical interaction of hepatocyte growth factor (HGF) and its variant with 5 amino-acid residues deleted (dHGF) with an acidic gelatin for the design of factors release from the gelatin hydrogel. When the interaction of HGF or dHGF with gelatin-immobilized agarose beads was evaluated by Scatchard binding assay, the dissociation constant of dHGF was higher than that of HGF, although the two proteins had a similar binding ratio. dHGF was released more rapidly from the hydrogel of acidic gelatin than HGF. In vivo release study with ¹²⁵I-labeled HGF or dHGF in mice subcutis showed that HGF was released from the gelatin hydrogel as a result of hydrogel degradation. In contrast, dHGF was rapidly released by a simple diffusion from the gelatin hydrogel. From electrophoresis experiments, mixing with the acidic gelatin enabled HGF to complex and suppressing the trypsin-digested molecular weight loss, in marked contrast to that of dHGF. In addition, the percentage of HGF recognized by the antibody was reduced by the gelatin complexation, but that of dHGF was not. We conclude that unlike dHGF, HGF has a strong affinity for the acidic gelatin, resulting in the controlled release of HGF accompanied with hydrogel degradation of the release carrier.     

Interaction of hepatocyte growth factor with gelatin as the carrier material

The objective of this study was to physicochemically investigate the interaction between hepatocyte growth factor (HGF) and acidic gelatin compared with that between HGF and basic gelatin or heparin. Gelatin- or heparin-immobilized agarose beads were prepared and HGF interaction with them was evaluated by Scatchard binding assay. The dissociation constant of HGF with the acidic gelatin was about 2–3 orders of magnitude higher than that of heparin. The cell proliferation assay revealed that the proliferation promotion activity of HGF complexed with the acidic gelatin was detected, although it was lower than that of original HGF. The ability of HGF to enhance the cell proliferation was reduced by the trypsin treatment, although the extent of the reduction was significantly suppressed by HGF complexation with acidic gelatin. Electrophoresis experimentally confirmed enhanced resistance to the molecular mass loss of HGF by gelatin complexation. Moreover, the recognized level of an antibody to HGF was reduced by the complexation with the acidic gelatin, indicating that the acidic gelatin is present around HGF molecules. It is possible that the HGF molecule is covered with the acidic gelatin, resulting in protection from enzymatic digestion.     

Glutathione peroxidase levels throughout normal pregnancy and in pre-eclampsia

Objective: Evidence suggests that hemoglobin, in addition to its function as a carrier of oxygen, also serves to transport nitric oxide, as S-nitroso cysteine, from the lungs to the peripheral circulation, where it can be released. Glutathione peroxidase, besides being an important antioxidant, is known to catalyze the release of nitric oxide from smaller carrier molecules, and may play a role in the distribution of nitric oxide throughout the body. In light of these findings, we sought to determine whether glutathione peroxidase levels differed throughout gestation, and specifically between pre-eclamptic and normal women. Methods: A nested case-control study of women receiving routine prenatal care was conducted. Pre-eclampsia was defined by a blood pressure of at least 140 mmHg systolic and/or 90 mmHg diastolic as well as proteinuria > 300 mg/24 h or > 2+ by dipstick, both occurring on two occasions at least 6 h apart. Blood was collected in heparinized tubes and was then centrifuged in a clinical centrifuge for 10 min. Plasma was frozen promptly at -80°C for later enzyme-linked immunosorbent assay (ELISA), with which plasma glutathione peroxidase was determined. Results: The maternal demographics of the pre-eclamptic and non-pre-eclamptic study groups did not significantly vary with respect to mean maternal age, gravidity, parity and gestational age at the time of delivery. The median maternal ages were 33 and 34 years, and the median gestational ages at the time of birth were 37.5 and 38.1 weeks, respectively. In evaluating the glutathione peroxidase levels of all patients across the three trimesters, we found that there was essentially no difference in mean levels (83.7, 81.0 and 89.5 ng/ml, respectively). There was no difference between the pre-eclamptic and non-pre-eclamptic patients, again stratified by trimester. A linear regression analysis indicated that the plasma glutathione peroxidase concentration did not correlate with gestational age or the presence of pre-eclampsia. Conclusions: Plasma glutathione peroxidase expression is similar across all trimesters. There is no change in the glutathione peroxidase levels in pre-eclamptic patients.     

Attenuation of pain perception after transposition of the greater omentum to the cauda equina region of rats—a preliminary observation

Abstract

Background: This paper addresses a specific experimental design to suggest the possible role of the greater omentum in the modulation of pain in rats.

Methods: Fifteen male Sprague–Dawley rats weighing between 275 and 325 g were selected. The animals were randomized and then anesthetized with pentobarbital (35 mg/kg) and divided into three groups: (1) sham: laparotomy followed by laminectomy with exposure of the spinal epidural space (n=5); (2) transposition of pedicled omentum (n=5) to the cauda equina epidural space; and (3) transposition of pedicled omentum (n=5) to the cauda equina intradural space. The animals were operated upon and once more randomized by an independent investigator, so that the groups were thought to be similar during post-operative testing. The latency of paw withdrawal to noxious heat stimulation was tested and the values (seconds) plotted for 1, 3, 6, 11, 14 and 30 days after surgery. Randomization codes were open after the animals were euthanized. The analysis of variance (ANOVA) without replication was applied for each of the dataset and comparisons established among the different study groups involved. The omenta were removed and standard immunohistochemistry was performed for gamma-amino-butyric acid (GABA), serotonin, calcitonin-gene related protein (CGRP), vascular intestinal peptide (VIP) and Met-enkephalin.

Results: The response to high heating rates of stimulation favored intradural versus sham and epidural omental transpositions. High and low noxious heat stimulation suggested an increased threshold to noxious stimulation after the 3 and 30 days of omental transposition. In the low heat stimulation series, responses were comparatively higher than in the sham animals.

Conclusions: The suggested increased threshold of response to noxious stimulation after transposition of the greater omentum onto the spinal cord of rats suggested a novel role of the omentum and a potential future application in the clinical arena.     

Multiple roles for protease-activated receptor-2 in gastric mucosa

Protease-activated receptor-2 (PAR-2), a G-protein-coupled receptor, is activated by proteolytic unmasking of the N-terminal cryptic tethered ligand. In gastric mucosa, activation of PAR-2 expressed by sensory neurons triggers release of CGRP and tachykinins, leading to mucus secretion via activation of CGRP₁ and NK₂ receptors, respectively. The PAR-2 agonist reveals mucosal cytoprotective effects in several gastric injury models. Further, PAR-2 activation inhibits gastric acid secretion, independently of sensory neurons or prostanoid formation. The PAR-2 agonist also induces increase in gastric mucosal blood flow, an effect being independent of endogenous CGRP or NO. Endothelium-derived hyperpolarizing factor (EDHF) appears to be involved in the PAR-2-mediated enhancement of mucosal blood flow. In contrast, mucosal chief cells are abundant in immunoreactive PAR-2, and PAR-2 stimulation triggers pepsinogen secretion. Taken together, primarily, PAR-2 plays protective roles in gastric mucosa through multiple mechanisms. Considering PAR-2-mediated pepsinogen secretion, PAR-2 might function as a double-edged sword in gastric mucosa.     

湿热证缠绵难愈的病理机制探讨

目的：探讨湿热病湿热证病邪易滞留气分，缠绵难愈的病理机制。方法：以肥甘饮食造成高脂血症模型，观察注射内毒素后模型动物新西兰白兔的α－肿瘤坏死因子、白细胞介素－1的分泌情况。结果：肥甘饮食使新西兰白兔形成高脂血症后，致内毒素诱导的α－肿瘤坏死因子、白细胞介素－1分泌峰值降低，但消退延缓。结论：提示温病湿热病邪致病，不仅仅是湿邪和热邪病理性质的简单相加，还存在湿邪和热邪相互影响、蕴蒸合化的过程。