Monika Egerbacher

FGF23 promotes renal calcium reabsorption through the TRPV5 channel

αKlotho is thought to activate the epithelial calcium channel Transient Receptor Potential Vanilloid‐5 (TRPV5) in distal renal tubules through its putative glucuronidase/sialidase activity, thereby preventing renal calcium loss. However, αKlotho also functions as the obligatory co‐receptor for fibroblast growth factor‐23 (FGF23), a bone‐derived phosphaturic hormone. Here, we show that renal calcium reabsorption and renal membrane abundance of TRPV5 are reduced in Fgf23 knockout mice, similar to what is seen in αKlotho knockout mice. We further demonstrate that αKlotho neither co‐localizes with TRPV5 nor is regulated by FGF23. Rather, apical membrane abundance of TRPV5 in renal distal tubules and thus renal calcium reabsorption are regulated by FGF23, which binds the FGF receptor‐αKlotho complex and activates a signaling cascade involving ERK1/2, SGK1, and WNK4. Our data thereby identify FGF23, not αKlotho, as a calcium‐conserving hormone in the kidney.

Localization of Matrix Metalloproteinases, (MMPs) Their Tissue Inhibitors, and Vascular Endothelial Growth Factor (VEGF) in Growth Plates of Children and Adolescents Indicates a Role for MMPs in Human Postnatal Growth and Skeletal Maturation

Numerous studies have focused on the expression, regulation, and biological significance of matrix metalloproteinases (MMPs) in the growth plate. Findings in mouse knockout models and in vitro data from various species indicate that MMPs not only degrade extracellular matrix components but may regulate the activity of local growth factors. In this study we investigated the presence, distribution, and activity of various MMPs and inhibitors, tissue transglutaminase (tTG or TG2) and vascular endothelial growth factor (VEGF) in the human child and adolescent growth plates by means of immunohistochemistry and gelatin zymography. Tissue was derived during orthopedic surgery (epiphysiodesis) in two prepubertal and four pubertal patients.MMP-2 and MMP-14 were present in reserve cell chondrocytes. MMP-14 was the most prominent MMP within all zones of the growth plate including proliferating chondrocytes. MMP-1 and MMP-13 (collagenases 1 and 3), MMP-9 (gelatinases B), MMP-10, and MMP-11 (stromelysins) and VEGF were positive in hypertrophic chondrocytes and osteoblasts. MMP-2 showed the same expression pattern but was negative in osteoblasts. Osteoclasts stained positive for MMP-9, MMP-2, and TG2. Tissue inhibitor of MMP (TIMP)-1 was present in all zones of the growth plate, osteoblasts, and osteoclasts; TIMP-2 was found in hypertrophic chondrocytes and osteoblasts. In summary, the presence of MMPs, TIMPs, TG2, and VEGF in our study indicated that the MMPs are relevant in growth plate physiology during the postnatal period in humans. The specific location of MMP expression within the growth plate may be the basis for further studies on the role of MMPs in the local regulation of chondrocyte differentiation, proliferation, and ossification at the chondroosseus junction.

Platelet-rich fibrin constructs elute higher concentrations of transforming growth factor-β1 and increase tendon cell proliferation over time when compared to blood clots: A comparative in vitro analysis

OBJECTIVE To compare the concentration of a representative growth factor (transforming growth factor-beta [TGF-β]1) eluted from a platelet-rich fibrin matrix (PRFMatrix), a platelet-rich fibrin membrane (PRFMembrane), and a whole blood clot (BC) over time, and to compare the mitogenic effect of the eluents from each construct. STUDY DESIGN In vitro study. SAMPLE POPULATION PRFMatrix, PRFMembrane, and BC (n=4/construct/time point). METHODS Each construct was placed in tissue culture wells containing media for 7 days. The media was collected and replenished on days 1, 3, 5, and 7 and the concentration of eluted TGF-β1 was measured by enzyme-linked immunosorbent assay. Canine tendon cells were subjected to additional aliquots of the conditioned media and the amount of cell proliferation compared. RESULTS The media from both PRFM (PRFMatrix and PRFMembrane) constructs contained significantly more (P≤.026) TGF-β1 at days 1 and 3 and produced a significant increase (P≤.044) in cell proliferation at all time points compared with the BC. The PRFMembrane media contained significantly more (P≤.05) TGF-β1 at days 1 and 3 and produced a significant increase (P≤.002) in cell proliferation at all time points compared with the PRFMatrix. CONCLUSIONS Both PRFM constructs are comprised of a dense fibrin scaffold that contains increased concentrations of TGF-β1 and are capable of increasing tendon cell proliferation over time when compared with a BC. CLINICAL RELEVANCE The sustained increase in growth factor availability in PRFM constructs may be beneficial in the healing of biologically compromised tissues.

Integrins and extracellular matrix proteins in the human childhood and adolescent growth plate

AbstractInteraction of chondrocytes with the surrounding matrix significantly influences differentiation and growth. These processes involve cell surface proteins, particularly integrins. The aim of this study was to compare the expression of integrins (a1, a2, a3, a5, a6, av, b1, b3, and b5 subunits) together with matching binding proteins in human childhood and adolescent growth plate cartilage using immunohistochemistry. Integrin b1 was detected in all chondrocytes of the growth plate cartilage, b3 only in osteoclasts of the opening zone, and b5 in hypertrophic chondrocytes and osteoblasts. Integrin a1, a2, and a5 subunits were expressed by chondrocytes in the proliferative and hypertrophic zone as well as in osteoblasts and osteoclasts. Integrin av and a6 subunits were present in chondrocytes of all zones, a3 only in osteoclasts. Collagen type II and fibronectin were seen throughout the growth plate, collagen type X in the hypertrophic zone, collagen type I in the ossifying trabecules. Laminin was expressed by chondrocytes in the resting zone and more weakly in the proliferative zone, collagen VI was present in the pericellular and interterritorial matrix in all zones of the growth plate. These results differ from previous reports on the distribution of integrins in the fetal growth plate. However, there was no difference in integrin expression in children before and during puberty. Our results indicate that integrin expression is not influenced by endocrine factors during sexual maturation and suggest that the process of skeletal maturation is not regulated via altered integrin expression.

Morphology of the pancreatic duct system in mammals

The morphology of pancreatic excretory duct segments was reviewed in mammals. The fine structure of the epithelial lining was described in intercalated ducts, intra‐ and extralobular ducts, and in major pancreatic ducts. Morphological characteristics of the various cell types comprising to the duct epithelium were detailed. Principal cells in the epithelial linings of interlobular and major pancreatic ducts (“Wirsungiocytes”) were discussed with respect to their appearance as either clear or dark variety. In addition, the capacity of both these cell types in elaborating mucoid glycoprotein secretions was considered and intra‐ and extraepithelial mucoid glands of major pancreatic ducts (ductular glands, accessory glands) was described. Finally, the wall composition of the various excretory duct segments was described. The presence of smooth muscle cells, myofibroblasts, and a peculiar periductal vascular plexus in major interlobular ducts and in main pancreatic ducts was emphasized. Microsc. Res. Tech. 37:407–417, 1997.

Expression of vascular endothelial growth factor and its receptors in canine lymphoma.

Vascular endothelial growth factor (VEGF) stimulates endothelial cell proliferation and has a pivotal role in tumour angiogenesis. The expression of VEGF and its receptors VEGFR-1 and VEGFR-2 was examined immunohistochemically in 43 specimens of canine lymphoma and in six normal lymph nodes. Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to detect VEGF protein and mRNA, respectively. VEGF protein was expressed by 60% of the tumours with diffuse cytoplasmic labelling of the neoplastic cells. Endothelial cells, macrophages and plasma cells were also immunolabelled. VEGFR-1 was expressed by variable numbers of neoplastic cells in 54% of lymphoma specimens. VEGFR-1 was also expressed by macrophages, plasma cells, reticulum cells, and vascular endothelial cells. Macrophages and lymphocytes in germinal centres of normal lymph nodes were also immunoreactive with anti-VEGF and VEGFR-1. Most tumours did not express VEGFR-2 but in 7% of sections there was focal labelling of neoplastic and endothelial cells, with a cytoplasmic and perinuclear pattern. The observed variability in expression of VEGF and its receptors probably relates to the fact that lymphoma is a heterogeneous lymphoproliferative tumour. Individual differences in VEGF and VEGFR expression must be taken into account when VEGF and VEGFR-targeted approaches for anti-angiogenic therapy are considered in dogs.

Ciprofloxacin causes cytoskeletal changes and detachment of human and rat chondrocytes in vitro

Abstract Quinolones cause damage of articular cartilage in different species by forming chelate complexes with divalent cations and inducing magnesium deficiency. Cations are important for regular function of integrins, a group of transmembrane proteins which connect extracellular matrix proteins with the intracellular cytoskeleton. We have shown that cultivation of rat chondrocytes in ciprofloxacin (CFX)-supplemented and Mg2+-free medium led to pronounced changes in the cytoskeleton and decreased adhesion of cells to the culture dish. In order to test whether or not these effects are species-specific, we extended our studies on human chondrocytes. Human chondrocytes cultivated in CFX-supplemented medium (10, 40, 80 and 160 μg/ml) or Mg2+-free medium showed decreased ability to adhere to growth support, cell shape changes, and alterations in actin and vimentin cytoskeleton in a concentration dependent manner. Attachment of human chondrocytes to collagen type II coated cover slips was reduced to 90% in CFX group and 75% in Mg2+-free group on day 1. This effect even increased after 4 days of culture in the respective medium (32% in CFX and 58% in Mg2+-free group). We concluded that Mg2+ deficiency is exerted via integrins, resulting in decreased ability to attach to extracellular matrix proteins and cytoskeletal changes. These effects are not species-specific. The attachment assay proves to be an easy to use experimental set-up to test ciprofloxacin and other quinolones for their chondrotoxic effects.

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.

Re-establishment of cytoskeletal tensional homeostasis in lax tendons occurs through an actin-mediated cellular contraction of the extracellular matrix.

Cytoskeletal tensional homeostasis is known to be an important factor in controlling catabolic gene expression in tendon cells. Loss of cell tension in lax rat tail tendon fascicles (RTTfs) has been associated with an upregulation of MMP‐13 gene expression and protein synthesis. To determine the role of the actin cytoskeleton in re‐establishing tensional homeostasis in lax tendons, RTTfs were allowed to freely contract in vitro for 8 days. The cultured RTTfs contracted rapidly, reaching 50% of their initial length by 3 days. This contraction was associated with the presence of α‐smooth muscle actin positive cells within the tendon. Disruption of the actin network by cytochalasian D caused an immediate and significant elongation of the contracted RTTfs. Subsequent removal of the cytochalasian D re‐initiated the contraction process. When lax RTTfs were allowed to contract between fixed clamps in culture and become taut, they demonstrated a marked decrease in MMP‐13 staining intensity when compared to freely contracting RTTfs. The ability of native tendon cells to contract lax tendons and re‐establish their homeostatic “set point” with respect to collagenase production may be an important mechanism in the recovery of tendons elongated by injury, surgical positioning, or cyclic, viscoelastic creep secondary to repetitive exercise.

Development of pancreas.

Pancreatic development is reviewed in man, mammals, and birds. Anatomical differences and differing topography of pancreatic excretory ducts are described in a series of mammalian species. Species differences are discussed with respect to their embryological significance. The developmental potency of the hepatopancreatic ring is stressed. Cytodifferentiation of exocrine and endocrine cells is considered. Microsc. Res. Tech. 37:374–383, 1997.