M. A. Oosterlaken-Dijksterhuis

Lipid mixing is mediated by the hydrophobic surfactant protein SP-B but not by SP-C.

Pulmonary surfactant contains two families of hydrophobic proteins, SP-B and SP-C. Both proteins are thought to promote the formation of the phospholipid monolayer at the air/fluid interface of the lung. The excimer/monomer ratio of pyrene-labeled PC fluorescence intensities was used to investigate the capacity of the hydrophobic surfactant proteins, SP-B and SP-C, to induce lipid mixing between protein-containing small unilamellar vesicles and pyrene-PC-labeled small unilamellar vesicles. At 37 degrees C SP-B induced lipid mixing between protein-containing vesicles and pyrene-PC-labeled vesicles. In the presence of negatively charged phospholipids (PG or PI) the SP-B-induced lipid mixing was enhanced, and dependent on the presence of (divalent) cations. The extent of lipid mixing was maximal at a protein concentration of 0.2 mol%. SP-C was not capable of inducing lipid mixing at 37 degrees C not even at protein concentrations of 1 mol%. The SP-B-induced lipid mixing may occur during the Ca(2+)-dependent transformation of lamellar bodies into tubular myelin and the subsequent formation of the phospholipid monolayer.

Expression of the insulin-like growth factor (IGF) system and steroidogenic enzymes in canine testis tumors

Testis tumors occur frequently in dogs. The main types of tumors are Sertoli cell tumors, seminomas, and Leydig cell tumors. Mixed tumors and bilateral occurrence of tumors may be encountered frequently. To elucidate the possible relationship between the insulin-like growth factor (IGF) system and the development of different types of testis tumors in dogs, the expression of insulin-like growth factor-I and II (IGF-I and IGF-II), their type I receptor (IGF-IR), and their binding proteins (IGFBPs) was examined. In addition the expression of the steroidogenic enzymes p450-aromatase and 5α-reductase type I and type II, and the androgen receptor (AR) was investigated by a semiquantitative reverse-transcriptase PCR (RT-PCR). Both normal testes and testes with tumors were studied. In normal testes a clear expression of IGF-I, IGF-II, IGF-IR, IGFBP2, IGFBP4 and IGFBP5 was found. Expression of IGFBP1 and IGFBP3 was weak. There was also clear expression of the steroidogenic enzymes 5α-reductase, aromatase, and the AR. Quantification of RT-PCR products revealed significantly less expression of IGFBP1, IGF-I, and 5α-reductase type I in Sertoli cell tumors and seminomas. Leydig cell tumors and mixed tumors had a significantly higher expression of IGFBP4 and IGF-IR than normal testes. The expression of aromatase was lower in seminomas and in mixed tumors. The expression of AR, IGF-II and IGFBP2, IGFBP3, IGFBP5, and 5α-reductase type II did not differ among the different types of tumors. It was concluded that Sertoli cell tumors and seminomas have a comparable expression of the IGF system while Leydig cell tumors have a different pattern, suggesting difference in pathobiology among these types of tumors.

Dietary 135-fold cholecalciferol supplementation severely disturbs the endochondral ossification in growing dogs

The effects of excessive non-toxic dietary Vitamin D(3) supplementation on Ca homeostasis with specific effects on endochondral ossification and skeletal remodeling were investigated in a group of growing Great Dane dogs supplemented with cholecalciferol (Vitamin D(3); HVitD) versus a control group (CVitD) (1350 microg versus 11.4 microg Vitamin D(3) per kilogram diet) from 6 to 21 weeks of age. There were no differences between groups in plasma concentrations of total Ca, inorganic phosphate, growth hormone, and insulin-like growth factor I and no signs of Vitamin D(3) intoxication in HVitD. For the duration of the study in HVitD compared to CVitD, plasma levels of parathyroid hormone (PTH) decreased, calcitonin (CT) increased, 25-hydroxycholecalciferol [25(OH)D(3)] increased 30- to 75-fold, 24,25-dihydroxycholecalciferol [24,25(OH)(2)D(3)] increased 12- to 16-fold, and 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)] decreased by approximately 40%. The latter was attributed to the two-fold increased metabolic clearance rate in the HVitD versus CVitD accompanied by the absence of the anabolic effect of PTH on the production of 1,25(OH)(2)D(3). Fractional Ca absorption (alpha) did not differ between groups at 8 and 14 weeks of age, whereas at 20 weeks of age alpha increased by only 16.4% in HVitD compared to CVitD. Excessive non-toxic Vitamin D(3) supplementation resulted in decreased bone remodeling and focal enlargement of the growth plate with morphology resembling those induced by administration of CT. Hypercalcitoninemia and the imbalanced relationship between 1,25(OH)(2)D(3) and 24,25(OH)(2)D(3) are potent candidates for the disturbed endochondral ossification.

IGF-I and retinoic acid regulate the distribution pattern of IGFBPs synthesized by the canine mammary tumor cell line CMT-U335.

Stromal-epithelial interactions modulate growth and development in normal and neoplastic mammary gland. The release of IGF binding proteins (IGFBPs) by the stromal compartment of the mammary gland may play a modulating role in the IGF-mediated proliferation of mammary epithelium. Therefore, the IGFBP-expression pattern of the canine mammary tumor cell line U335 (CMT-U335), which has a mesenchymal phenotype, was determined. In addition, the effects of IGFs and all trans retinoic acid (RA) on DNA synthesis, and IGFBP secretion and distribution were examined. The IGFBPs secreted by CMT-U335 were characterized as IGFBP-2, -4, -5, and -6. Moreover, CMT-U335 appeared to be a suitable mammary mesenchymal cell line for study of the regulatory factors of IGFBP expression and the mechanism(s) involved. IGFs and RA enhanced IGFBP concentrations in cell-conditioned medium with IGF-I and RA having an additive effect. The IGF-I-stimulated DNA synthesis, however, was inhibited by RA. The difference between IGF-I and RA was an enhanced IGFBP-5 binding to the extracellular matrix (ECM) by RA, whereas IGF-I reduced binding to the ECM. Because high doses of insulin had no significant effects on IGFBP concentrations in the medium, it is concluded that IGF-I-induced changes in IGFBP concentrations are not mediated by type-I IGF receptors and may be the consequence of IGFBP redistribution.

Growth hormone modulates cholecalciferol metabolism with moderate effects on intestinal mineral absorption and specific effects on bone formation in growing dogs raised on balanced food.

The aim of the study was to investigate the influence of growth hormone (GH) on Vitamin D3 metabolism and the subsequent effects on calcium (Ca) homeostasis and skeletal growth in growing dogs. A group of Miniature Poodles received supraphysiological doses of GH (GH group; n = 6; 0.5 IU GH per kg body per day) from 12 to 21 weeks of age and was compared with a control placebo-treated group (n = 8). Biologic activity of GH in the GH compared to the control group was indicated by (a) the 2.5- to 3.5-fold increase in the plasma concentrations of insulin-like growth factor I (IGF-I), (b) the increased production of 1,25-dihydroxycholecalciferol as indicated by the significantly increased plasma 1,25-dihydroxycholecalciferol concentrations and the 12.9-fold increase in renal 1alpha-hydroxylase gene expression, and (c) the inhibited production of 24,25-dihydroxycholecalciferol as indicated by the significantly lower plasma 24,25-dihydroxycholecalciferol concentrations and the similar levels of renal 24-hydroxylase gene expression. Despite the distinct effects on Vitamin D(3) metabolism in the GH group, there were only moderate effects on the intestine, i.e. at 20 weeks of age there was a significant increase of 14.4 and 5.6% in fractional absorption of Ca and phosphate (Pi), respectively, compared to the control group. GH administration resulted in significantly elevated glomerular filtration rate, with no differences in Pi urine excretion as a result of a concomitant increase in the tubular reabsorption of Pi. GH had only limited disturbing effects on endochondral ossification as indicated by the maintenance of the regularity of the growth plates. However, GH had specific anabolic effects on bone formation without concomitant effect on bone resorption that may result in disorders of skeletal remodeling and manifestation of enostosis.

Perinatal expression of IGFBPs in rat lung and its hormonal regulation in fetal lung explants.

To gain more insight into the regulation of the expression of insulin-like growth factor (IGF) binding proteins (IGFBPs) in the lung, the developmental patterns of the abundance of the mRNAs encoding IGFBPs were measured in the perinatal rat lung and in explant cultures of fetal rat lung. In hormone-free explant cultures, the levels of the mRNAs encoding IGFBP-2 through -5 changed with a pattern similar to that occurring in vivo (although in the case of IGFBP-3 to -5 at a faster rate), indicating that the developmental regulation of the expression of these IGFBPs in perinatal lung is mimicked in the explants. For the IGFBP-6 mRNA level, the pattern in vitro differed from that in vivo. In the explant cultures, dexamethasone decreased the production of IGFBP-3 and -4 and decreased the abundance of the mRNAs encoding IGFBP-2 to -5 but increased the abundance of IGFBP-6 mRNA. These observations indicate that glucocorticoids may be involved in the developmental regulation of the expression of these components of the IGF system and that the IGF system may be involved in the physiological effects of glucocorticoids on lung development. No appreciable effects of 3,3,5-triiodothyronine on the expression of the IGFBPs were observed.

Expression of osteotropic growth factors and growth hormone receptor in a canine distraction osteogenesis model.

Osteotropic growth factors play an important role in bone metabolism. Nevertheless, knowledge about their expression in relation to distraction osteogenesis remains limited. The aim of the present study was to determine the expression of growth hormone (GH), growth hormone receptor (GHR), insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II), and bone morphogenetic protein 2 (BMP-2) in distraction-induced bone regeneration. Expression of these factors was assessed during the consolidation phase, comparing distraction osteogenesis with osteotomy-induced bone formation. Real-time PCR was performed as a semiquantitative measurement of mRNA, and the relative expression levels of these factors were determined. In addition, plasma GH profiles and plasma concentrations of IGF-I, IGF-II, and insulin-like growth factor-binding protein 4 and -6 (IGFBP-4 and -6) were measured to assess their potential systemic role during bone formation. Expression of GHR, IGF-I, and BMP-2 had significantly increased in comparison with the expression of these factors in mature bone. Expression of GHR was significantly higher in distraction-induced bone regenerate than in osteotomy-induced bone. No significant differences were found for the expression of IGF-I and BMP-2 between distraction and osteotomy. Plasma concentrations of GH, IGF-I, IGF-II, IGFBP-4, and IGFBP-6 did not demonstrate any significant differences between treatment groups and controls. Upregulation of GHR expression in distraction osteogenesis may enhance sensitivity to endogenous systemic GH and thus promote consolidation of the regenerated bone. Changes in the systemic osteotropic growth factors GH, IGF-I, IGF-II, IGFBP-4, and IGFBP-6 do not seem to be of importance during distraction osteogenesis.

Growth hormone stimulates bone healing in a critical-sized bone defect model.

Growth hormone plays an important role in bone metabolism. Treating bone deficits is a major topic in orthopaedic surgery. Our hypothesis was that local continuous growth hormone administration stimulates bone healing in a canine critical-sized bone defect model. Bone formation in the defects was quantified using densitometric image analysis and histomorphometry. After growth hormone treatment, expression levels of insulin-like growth factors-I and II, and growth hormone receptor were determined in the bone regenerate of the original defects. Circulating plasma concentrations of insulin-like growth factors-I and II, and insulin- like growth factor binding proteins-4, and 6 were measured during treatment. Growth hormone administration resulted in healing of bone defects but without an additional effect of local infusion. Expression of insulin-like growth factor-I in the bone regenerate was lower in the growth hormonetreated dogs, whereas insulin-like growth factor-II and growth hormone receptor expression were not increased. Growth hormone increased circulating insulin-like growth factor-I and growth factor-II plasma concentrations. Continuous infusion of growth hormone stimulated bone healing in a canine critical-sized bone defect model. Local delivery of growth hormone did not additionally enhance bone healing. Increased circulating plasma concentrations of insulin-like growth factors-I and II most likely induced bone formation.