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Growth Factors (general) (BS)
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GROWTH FACTORS OR36-4 SYSTEMIC ADMINISTRATION OF IGF BINDING PROTEIN-4 (IGFBP-4) INCREASES BONE FORMATION PARAMETERS IN MICE BY INCREASING IGF BIOAVAILABILITY VIA AN IGFBP-4 PROTEASE DEPENDENT MECHANISM IGFBP-4, one of the most abundant IGFBPs produced by bone cells, is a potent inhibitor of IGF actions in vitro. Accordingly, local administration of IGFBP-4 inhibited IGF-I-induced increase in bone formation (BF) parameters in mice. Surprisingly systemic administration of IGFBP-4 at pharmacological doses caused a significant increase in BF parameters. Based on the findings that IGFBP-4 administration caused a shift in circulating IGF-I from the 150 kDa fraction which cannot cross the vascular endothelium into 50 kDa IGFBP-4+IGF complex and that mouse serum contains an IGF-dependent IGFBP-4 protease capable of degrading IGFBP-4 to release IGFs, we proposed that the mechanism by which IGFBP-4 increases BF may involve increased IGF bioavailability via proteolysis of IGFBP-4. To evaluate this hypothesis, we produced wild-type (WT), protease-resistant (PR) and IGFBP-4 proteolytic fragments and determined their effects on BF in mice using biochemical markers. PR IGFBP-4 was more potent than WT IGFBP-4 in inhibiting IGF-I-induced mouse osteoblast cell proliferation in vitro and in inhibiting IGF-I-induced increase in ALP activity in vivo. IGFBP-4 proteolytic fragments were inactive. Systemic administration of WT but not PR IGFBP-4 increased serum osteocalcin, ALP and ALP in skeletal extracts in a dose-dependent manner (40% ? at 1.25 nmol/mouse, p<0.05). Systemic administration of WT but not PR IGFBP-4 increased free IGF-I levels (50% ? after 30 min treatment, P<0.05) in serum. If IGFBP-4 increases BF by a mechanism involving increased IGF-I availability in the circulation, we predict IGFBP-4 to be ineffective in mice deficient in circulating levels of IGF-I. Accordingly, IGF-I but not WT IGFBP-4 increased BF parameters in IGF-I midi mice which exhibit 70% reduced IGF-I levels compared to control mice. Conclusions: 1) Systemic administration of IGFBP-4 increases BF parameters by increasing IGF bioavailability via an IGFBP-4 protease dependent mechanism; and 2) We predict that physiological regulation of IGF-I in the IGFBP-4+IGF complex may influence the levels of free IGF-I and, thereby, the endocrine actions of IGF-I. Basic Science Oral: Growth Factors (General) (11:00 AM-12:30 PM) Presentation Date: Saturday, June 23, 2001; Time: 11:45 AM; Location: A 108 GROWTH FACTORS OR36-4 Lay explanation of abstract: Osteoporosis is the most common bone disease that incurs large medical expenses. It is characterized by thinning of the bones, which become fragile and break more easily. One out of every two white women will have a osteoporotic fracture at some point in her lifetime. Osteoporosis occurs mainly because of : 1) lower peak bone mass than normal at the end of sexual maturity; and 2) increased bone destruction rate that can occur, for example, because of estrogen deficiency, without a corresponding increase in the formation of new bone. Most available treatments for osteoporosis prevent bone loss by preventing bone destruction. Because many osteoporotic patients who come for treatment have already lost a substantial amount of bone, there is a need to develop treatments that increase bone mass by stimulating new bone formation. Our basic research presented in this abstract explored the mechanism by which insulin-like growth factor binding protein-4 (IGFBP-4) increases bone formation parameters in mice, when given systemically. Insulin-like growth factors (IGFs) resemble insulin in structure and are very important in controlling the amount of new bone formed both during development and during adulthood. IGFs are produced within bone, where they act as local growth factors. In addition, IGFs circulate in blood in large amounts and thereby act as endocrine hormones. The action of IGFs in blood is controlled by a set of proteins called IGF binding proteins (IGFBPs) that bind IGFs and prevent them from being active. About 80% of IGFs in the blood are present as a large complex by binding to IGFBP-3 and another protein (acid labile subunit, ALS), which cannot go through the small pores of the blood vessel. The remaining 20% of IGFs are present as small complex (e.g., IGFBP-4+IGF) that can freely move through the pores of the blood vessel and thereby become available to bone and other tissues. In the present study, we administered IGFBP-4 to mice so that more of the IGFs are bound to the smaller complex (IGFBP-4+IGF) and less is bound to the larger complex (ALS+IGFBP-3+IGF). There are two advantages with the smaller IGFBP-4+IGF complex. One, IGFBP-4 in this complex can be broken down by proteases in the blood, thereby releasing IGFs to go through the pores of the blood vessel to bone and other tissues to act as a growth factor. Second, the smaller IGFBP-4+IGF complex is able to go through the pores of the blood vessel to bone where a protease can degrade IGFBP-4 to release IGF for action. We confirmed this mechanism by using a form of IGFBP-4 that is resistant to degradation by proteases in the blood. Our basic research is significant. Based on our data, we predict that mechanisms that increase the level of free IGF-I in serum could be used as therapy to increase bone formation and treat osteoporosis. This work was supported by funds from the National Institutes of Health and the Veterans Administration. GROWTH FACTORS OR36-6 MORPHOLOGIC RECAPITULATION OF HUMAN PITUITARY TUMORIGENESIS BY A NOVEL TRANSFORMING ISOFORM OF FIBROBLAST GROWTH FACTOR RECEPTOR 4 Fibroblast growth factors (FGFs) and altered FGF receptors (FGFRs) have been implicated in human tumorigenesis. We have previously reported the identification of a novel pituitary tumor-derived N-terminally truncated isoform of FGFR4 (ptd-FGFR4) that we isolated from primary human pituitaries. ptd-FGFR4 transcription initiates in exon 5, resulting in an mRNA that encodes a polypeptide containing the intact third Ig-like domain, a transmembrane domain, and an intact kinase region of FGFR4 but lacks a signal peptide. ptd-FGFR4 is constitutively phosphorylated; it is transforming in vitro and in vivo. We now demonstrate convincing evidence of a direct role for ptd-FGFR4 in pituitary tumorigenesis in transgenic mice. Using a pituitary specific prolactin PRL promoter, we generated transgenic mice selectively expressing ptd-FGFR4 in the pituitary. At 7 months, lactotrophs were numerous with intense cytoplasmic FGFR4 expression. By the age of 11 months, 90% of the animals developed pituitary tumors that exhibited disruption of the normal acinar architecture of the adenohypophysis and prominent vascularity. The tumor cells contained cytoplasmic immunoreactivity for PRL as well as FGFR4. Other cell types of the adenohypophysis were unaffected. Tumors were found equally in males and females, but the tumors were significantly larger in females, with some exhibiting intracranial extension and invasion of brain. Males consistently developed microadenomas that were found on microscopic examination. Age- and sex-matched non-transgenic littermates had no pituitary pathology. Circulating PRL levels of transgenic animals were higher than those of non-transgenic littermates and correlated with tumor size. These findings demonstrate that a constitutively activated, cytoplasmic, truncated FGFR4 identified from primary human pituitary tumors can morphologically recapitulate human pituitary tumorigenesis. Basic Science Oral: Growth Factors (General) (11:00 AM-12:30 PM) Presentation Date: Saturday, June 23, 2001; Time: 12:15 PM; Location: A 108 GROWTH FACTORS OR36-6 Lay explanation of abstract: The pituitary gland is a pea-sized structure that lies under the brain, encased within the bone of the base of the skull. It is the master gland that regulates the function of many hormone-producing glands. Tumors of this gland can be seen on scans of the brain in up to 20% of adults. Some of these grow and function in a manner that can have serious consequences on fertility, sexual function, heart function, and ultimately longevity. The mechanisms that lead to the development of these tumors are largely unknown. In this study, a specific defect in the processing of a gene responsible for a receptor that transmits growth signals was identified in nearly 40% of human pituitary tumors. Re-constitution of this genetic defect in living cells, immunologically tolerant mice, and mice genetically engineered to produce this defect only in the pituitary resulted in tumorous cell growth. Perhaps the most remarkable aspect of this study is that it was based on a genetic defect that is unique to this gland and whose re-capitulation in experimental models faithfully reproduced the pathology of this gland as it is seen in patients. The results of this study represent the blueprints required to devise more selective approaches to the diagnosis and treatment of tumors of this gland. Moreover, this study provides a framework to characterize generic defects in the various endocrine glands, which appear to behave distinctly from non-hormone producing malignancies of the body. This research was funded by the Medical Research Council of Canada/Canadian Institutes for Health Research. GROWTH FACTORS P3-380 EFFECTS OF RETINOIC ACID AND INSULIN-LIKE GROWTH FACTORS IN PRIMARY HYPERPARATHYROIDISM Background: The precise cause of primary hyperparathyroidism (1HPT) is uncertain. The insulin-like growth factor (IGF) system is multifunctional and ubiquitous in normal and neoplastic tissues. Retinoic acid (RA), a derivative of vitamin A and a ligand for the IGF II receptor, is an important regulatory factor in growth and differentiation of epithelial tissue and has been shown to inhibit cancer growth. Aims: To assess the inhibitory actions of RA and its interactions with the IGF system in a 1HPT primary cell culture model. Methods: Fresh human parathyroid glands were obtained from 9 patients undergoing surgical treatment for primary hyperparathyroidism. Glands were bisected for histology (frozen section) and immediate cell culture. Tissue was dispersed by enzyme digestion and cells cultured in 24-well plates. Following adhesion, the cells were transferred to serum free medium and dosed once with growth factors ± RA for 96hrs. Proliferation was assessed by measuring tritiated thymidine incorporation. Results: Compared with controls (n=5) IGF I and IGF II increased DNA synthesis by 55.9% ± 6.4 (sem) and 51.1% ± 7.9 respectively (100ng/ml, P0.05) compared with wild-type IGF II. Conclusion: These data implicate a novel antiproliferative role for RA in enhancing the pericellular clearance of IGF II via the IGF II receptor thus preventing ligand activation of the IGF I receptor. This may have broader implications for RA effects in other tumours. Basic Science Poster: Growth Factors - 06 (General) (Poster Session) (11:00 AM-12:00 PM and 2:30 PM-3:30 PM) Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall GROWTH FACTORS P3-380 Lay explanation of abstract: The parathyroid glands are four pea-sized glands behind the thyroid gland in the neck. These glands produce a hormone (parathyroid hormone) that controls the serum calcium. Excess production of this hormone causes excess calcium in the blood (hyperparathyroidism) that may cause osteoporosis, bone fractures, and kidney stones. Imbalances of vitamins and growth factors have been suggested as causes of this condition. To date, surgery remains the only definitive treatment for hyperparathyroidism. This study presents novel data on the mechanism and potential use of retinoic acid, a vitamin A derivative, as treatment for hyperparathyroidism. We have proposed a mechanism: retinoic acid may modulate insulin-like growth factor II availability. (This mechanism may also be important in cancer treatments.) The insulin-like growth factor axis consists of growth factors I and II (IGF I and II) and corresponding receptors I and II. Insulin-like growth factors are important growth factors in both normal and cancer growth. Insulin-like growth factor receptor II (IGF receptor II) is a multi-functional receptor that binds retinoic acid, mannose-6-phosphate, and IGF II for elimination, by transporting them to sites in the cell for degradation. IGF receptor I binds both IGF I and IGF II to mediate their growth effects. Our study evaluated the role of retinoic acid and insulin-like growth factors in primary hyperparathyroidism. Parathyroid glands from hyperparathyroid patients undergoing surgery were used in this study. Glands were dispersed and processed for cell culture experiments using retinoic acid and IGF I and II. Nine glands from nine patients were included in this study. On its own, retinoic acid inhibited the growth of parathyroid cells. IGF I and II stimulated the growth of parathyroid cells. These data indicate a novel anti-proliferative role for retinoic acid in enhancing the clearance of IGF II through the IGF receptor II, which prevents ligand activation of IGF receptor I. This may have broader implications for retinoic acid effects in other tumors. Further background information: When retinoic acid was given in combination with IGF I and II, the growth response of IGF II was completely abolished. To evaluate the role of IGF receptor II in mediating the actions of retinoic acid, we used the IGF II analogues [Leu27]IGF II (10 to 20-fold reduced affinity for insulin-like growth factor receptor I). The IGF II inhibitory effect of retinoic acid was enhanced in the presence of analogues [Leu27]IGF II compared with wild-type IGF II. This project was completed at the Bristol Royal Infirmary, U.K. The project was sponsored by the Royal College of Surgeons Research Fellowship, Mason's Medical Research Foundation, Hamamelis Trust. GROWTH FACTORS P3-389 GROWTH HORMONE (GH) HAS POTENT ENDOTHELIUM-INDEPENDENT VASODILATING EFFECTS IN VITRO GH has been reported to exert acute beneficial effects in heart failure. The mechanisms of this action are still unknown. We have hypothesised that GH may act via a reduction in peripheral vascular resistances. The aim of our study was to evaluate these GH vasoactive properties and investigate the mechanisms involved. Rabbit aortic strips were superfused in vitro with different vasoconstrictive agents (10-7M noradrenaline N, 10-6 M PGF2 and 50mM KCl) in the presence of human rGH (0.1-5 mg/L). We observed that: 1) GH potently counteracted the NA and PGF2-mediated contraction (maximal inhibition 36.6 ± 3.5 and 57.2 ± 9.1%, respectively); 2) GH activity was not dependent on endothelium since both NOS (10-4M L-NAME) and COX (10-6M indomethacin) inhibitions did not reverse its effect; in addition the mechanical removal of endothelium did not affect GH action; 3) GH depressed responsiveness of vascular smooth muscle to KCl-induced contraction. In fact, in the presence of GH at 5 mg/L, we observed a shift to the right of the KCl contraction curve (EC50 increased from 18.1 ± 1.39 to 28.8 ± 1.62; p<0.01); 4) GH vasodilation did not depend on inhibition of calcium influx through L-type calcium channel; in fact, L-type calcium blocker diltiazem (at 10-6M) did not affect GH activity. In conclusion, GH exerts potent endothelium independent vasodilating action on rabbit aortic strips in vitro. We hypothesise that vascular smooth muscle potassium channels may be involved in the GH-mediated vasodilatation. Basic Science Poster: P3: Growth Factors - 06 (General) (Poster Session) (11:00 AM-12:00 PM and 2:30 PM-3:30 PM) Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall GROWTH FACTORS P3-389 Lay explanation of abstract: We have made a new discovery: growth hormone (GH) has a potent vasodilating effect on rabbit aortic strips in-vitro. Moreover, for the first time we have obtained some mechanistic insights into this effect, which shows that this action is independent of endothelium and involves vascular smooth muscle relation via potassium channels. The aim of our study was to conduct an in-depth evaluation of the peripheral vasodilating properties of growth hormone and the underlying mechanisms. In detail, we superinfused rabbit aortic strips with agents that are known to induce potent vasoconstriction in this in vitro system. GH was able to reverse this vasoconstriction independent of the presence of arterial endothelium. The interesting part of this research is that it provides an explanation for the beneficial effects of GH in patients with heart failure. Because this disease is one of the major causes of morbidity and mortality in the western population and treatment for this disease is not yet very effective, our study suggests that there is a role for GH as an adjunctive medical treatment of heart failure. Therefore, this basic study has potential impact in the treatment of millions of patients with heart failure. Additional background information: Human studies have shown that acute prolonged infusion with recombinant human GH may have beneficial effects in patients with heart failure, by decreasing pulmonary pressure and increasing cardiac output. To allow a correct clinical use of growth hormone in patients with heart failure, it is crucial that we learn more about the mechanism of this hormone action. In fact, both a direct inotropic action at the heart level and a peripheral systemic vasodilating action have been hypothesized to be involved in the beneficial effect of growth hormone in heart failure. Research was funded by Fondazione Maugeri of Gussago, Italy and by Eli Lilly-Sesto Fiorentino, Italy. GROWTH FACTORS P3-396 LEPTIN BONE MARROW CONCENTRATIONS DURING INITIAL CHEMOTHERAPY IN CHILDREN SUFFERING FROM ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) Leptin is a secretory protein mainly produced by adipocytes. Leptin is not only critical for the regulation of body weight but part of various endocrine axes, e.g. immune response, reproduction and hematopoesis. Beside adipocytes other cells had been reported to secrete leptin (e.g. stroma cells). Leptin stimulates normal myeloid and erythroid cell development. Hematopoetic progenitor cells express the leptin receptor, implying a physiological role of leptin during expansion and differentiation of hematopoetic cells. In order to investigate the role of leptin in childhood leukemia plasma levels of leptin were measured by ELISA in 38 children at diagnosis of ALL. The tumour clones generally represents 50-90% of leucocytes. At the time of diagnosis leptin concentrations were 0.92 ±0.79 ng/ml. After 33 days of chemotherapy (Prednisone, Virncristin, Daunorubicin and Asparaginase) all children had reached hematological remission. At day 33 leptin levels of 2.6 ±2.4 ng/ml were detected. In 32 of 38 patients an increase of plasma leptin was found to the 2.8 fold of the initial levels (range: 0.14-3.39 at diagnosis, 0.65-11.62 at day 33; p < 0.01). The underlying mechanism for the observed variability of plasma leptin levels are still unknown. On the one hand, the presence of the tumour clone and the subsequent relative decrease of other cell types may represent a direct cause for the low leptin levels at the time of diagnosis. On the other hand, indirect regulatory effects such as the repression of leptin expression by cytokines secreted by the tumour clone may play a role as well. Interestingly, glucocorticoids causes a 3 fold rise of leptin levels, which might explain the 2.8 fold increase found after 33 days of chemotherapy. In conclusion, we found changes in bone marrow plasma leptin concentrations in children with ALL before and during chemotherapy. The potential usefulness of leptin as a prognostic indicator needs to be further evaluated. Basic Science Poster: Growth Factors - 06 (General) (Poster Session) (11:00 AM-12:00 PM and 2:30 PM-3:30 PM) Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall GROWTH FACTORS P3-396 Lay explanation of abstract: The term 'leptin' originates from the Greek word 'leptos', which means 'thin.' Leptin represents an endogenous hormone that is a major regulator of body fat. During recent years, much information has been collected to explain the role of leptin in weight regulation. Leptin is predominantly produced by fat cells and released into the bloodstream. Leptin concentration increases after food intake, which leads to decreasing appetite. In general, the basal level of leptin depends on body mass. A high body mass is usually accompanied by an increase of body fat that leads to elevated leptin levels compared to a person with normal body mass index. Due to its regulatory ability to decrease appetite, the hormone leptin has generated much interest in the past decade. In 1994, the gene encoding for leptin became a 'celebrity gene' and physicians began to regard obesity as a chronic disease. Physicians looked forward to treating obesity with a natural and safe weight-reducing drug. Meanwhile, current research has revealed that leptin is not only involved in fat regulation. Unexpectedly, leptin receptors (discovered in 1998) were not found only in cells known to be involved in metabolic functions, but also in capillaries, bone cells, brain, and cells of the immune system. Based on this tissue distribution of leptin receptors, scientists suggested more complex functions for leptin. This new view on leptin has been confirmed by numerous recent publications that prove that the hormone leptin is functionally involved in diverse immune processes, in inflammatory processes, and in angiogenesis, bone metabolism, and hematopoesis. Thus, leptin possesses proliferating effects on different cancer cells. We found that cells from children suffering from leukemia express the receptor for leptin, which led us to hypothesize that leptin present in the bloodstream of these children might function as 'positive growth factor' for the tumor cells. In this study the leptin concentration in blood plasma of these children was measured by a commercially available enzyme kit. All together, we analyzed 38 blood samples at the time of diagnosis of the leukemia, when usually 50-90% of all white blood cells are tumor cells, and at day 33 of the chemotherapy, when the tumor cells have widely disappeared and normal healthy blood cells started to come back. The leptin concentration at day 33 of chemotherapy was 2.8times the initial level. The underlying mechanism for the observed changes in plasma leptin levels are still unknown. A plausible explanation might be the enrichment of the tumor clone at the time of diagnosis, which leads subsequently to a relative decrease of other cells that express and secret leptin. In conclusion, we found changes in plasma leptin concentrations in children with leukemia before and during chemotherapy. The increase of leptin is probably a consequence of a regeneration process of normal blood and bone marrow cells. The potential usefulness of leptin as a prognostic indicator needs to be further evaluated. Funding was provided by W.A. Drenckmann Stiftung, Germany, and Magdeburger F–rderkreis krebskranker Kinder e.V., Germany.
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