The Association of Early Dietary Supplementation with Vitamin E with the Incidence of Ulcerative Dermatitis in Mice on a C57BL/6 Background: Diet and Ulcerative Dermatitis in Mice.

The purpose of this study was to ascertain if prophylactic ingestion of a diet rich in vitamin E would prevent or impede the development of ulcerative dermatitis in mice on a C57BL/6 background. Mice were fed either a standard mouse diet, vitamin E (99 IU/kg), or a mouse diet fortified with vitamin E (3000 IU/kg) after weaning. Cases of ulcerative dermatitis were recorded by individuals unmasked to the diet assignment. The incidence of ulcerative dermatitis in a retrospective cohort of mice on standard diet was compared with the group on the diet fortified with vitamin E. Age was associated with ulcerative dermatitis in standard diet and vitamin E fortified diet groups, r = 0.43, p-value < 0.0001 and r = 0.18, p-value < 0.02, respectively. The average age of incidence for ulcerative dermatitis in the mice fed the standard diet was 89 weeks and for the mice fed the vitamin E diet it was 41 weeks. The unadjusted odds ratio comparing the incidence of ulcerative dermatitis between the two diet groups was 4.6 with a 95% confidence interval of (2.44, 8.58), χ(2) p-value < 0.0001. Therefore, there was an association between the diets and ulcerative dermatitis, with the mice on the vitamin E fortified diet having almost five times the odds of having ulcerative dermatitis compared with mice on the standard diet. Incidence of ulcerative dermatitis was not influenced by sex or genotype. Our study results show that a diet fortified in vitamin E initiated at weaning does not prevent or impede the development of ulcerative dermatitis in mice on a C57BL/6 background and may accelerate development when administered to young mice.

Regulation of gonadotropin releasing hormone release by neuropeptide Y at the median eminence during the preovulatory period in ewes.

The median eminence (ME) of the hypothalamus is known to be an important brain site where hypophysiotropic release might be regulated by excitatory and inhibitory signals impinging on their neuronal terminals. Since a role for neuropeptide Y (NPY) on preovulatory luteinizing hormone (LH) release has been suggested, we hypothesized that NPY might act at the ME to control preovulatory gonadotropin-releasing hormone (GnRH) release and thus the onset of the preovulatory surge of LH. To examine this possibility, we used the ewe as an animal model to determine: (a) immunocytochemical distribution of GnRH and NPY in the ewe ME; (b) changes in in vivo release of NPY and GnRH using ME push-pull cannula (PPC) perfusate samples, as well as in plasma LH, during the luteal, follicular and preovulatory phases of a synchronized estrous cycle, and (c) effects of ME perfusion of NPY or a Y1-NPY antagonist, or an NPY antiserum on in vivo release of ME-GnRH and plasma LH during a synchronized follicular phase. Immunolocalization reveals a dense plexus of beaded GnRH-containing neurites in the arcuate nucleus and in its vicinity, the pituitary stalk and the palisade. In contrast, a dense plexus of NPY-containing neurites occurs in the internal layer, with occasional fibers found in the intermediate and lateral external zone of the ME. In the area between the lateral internal and lateral external layers, both NPY and GnRH-containing processes were found, thus providing opportunities for synaptic and/or paracrine interactions between NPY- and GnRH-containing neurons. Hormonal analysis indicated that a synchronized preovulatory surge of LH is elicited within a 2-hour window by the sequential implantation and removal of silastic-encased estradiol (E2) or progesterone (P4) implants. In this paradigm, there was a parallel increase in ME release of both NPY and GnRH preceding the synchronized LH surge. The onset of this synchronized LH surge was advanced by ME perfusion of exogenous NPY and was both delayed and blunted by ME perfusion with the NPY antagonist (both were perfused through the PPC probe for 2 h, starting 2-3 h before the expected onset of the LH surge). In addition, NPY perfusion in the ME increases, while perfusion of the Y1-NPY antagonist or of the NPY antiserum decreases ME-PPC GnRH content and plasma levels of LH in early follicular ewes. Finally, perfusion of NPY antiserum during an ongoing LH surge disrupted LH release. These results suggest that interactions between NPY and GnRH neurons are important in controlling the timing, magnitude and maintenance of the preovulatory LH surge.

Combined effects of insulin-like growth factor-1 and transforming growth factor-beta1 on periosteal mesenchymal cells during chondrogenesis in vitro.

OBJECTIVE: Periosteum contains undifferentiated mesenchymal stem cells that have both chondrogenic and osteogenic potential, and has been used to repair articular cartilage defects. During this process, the role of growth factors that stimulate the periosteal mesenchymal cells toward chondrogenesis to regenerate articular cartilage and maintain its phenotype is not yet fully understood. In this study, we examined the effects of insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta1 (TGF-beta1), alone and in combination, on periosteal chondrogenesis using an in vitro organ culture model. METHODS: Periosteal explants from the medial proximal tibia of 2-month-old rabbits were cultured in agarose under serum free conditions for up to 6 weeks. After culture the explants were weighed, assayed for cartilage production via Safranin O staining and histomorphometry, assessed for proliferation via proliferative cell nuclear antigen (PCNA) immunostaining, and assessed for type II collagen mRNA expression via in situ hybridization. RESULTS: IGF-1 significantly increased chondrogenesis in a dose-dependent manner when administered continuously throughout the culture period. Continuous IGF-1, in combination with TGF-beta1 for the first 2 days, further enhanced overall total cartilage growth. Immunohistochemistry for PCNA revealed that combining IGF-1 with TGF-beta1 gave the strongest proliferative stimulus early during chondrogenesis. In situ hybridization for type II collagen showed that continuous IGF-1 maintained type II collagen mRNA expression throughout the cambium layer from 2 to 6 weeks. CONCLUSION: The results of this study demonstrate that IGF-1 and TGF-beta1 can act in combination to regulate proliferation and differentiation of periosteal mesenchymal cells during chondrogenesis.

Identification and regulation of the IGFBP-4 protease and its physiological inhibitor in human trophoblasts and endometrial stroma: evidence for paracrine regulation of IGF-II bioavailability in the placental bed during human implantation.

The IGF family plays an important role in implantation and placental physiology. IGF-II is abundantly expressed by placental trophoblasts, and IGF binding protein (IGFBP)-4, a potent inhibitor of IGF actions, is the second most abundant IGFBP in the placental bed, expressed exclusively by the maternal decidua. Proteolysis of IGFBP-4 results in decreased affinity for IGF peptides, thereby enhancing IGF actions. In the current study, we have identified the IGFBP-4 protease and its inhibitor in human trophoblast and decidualized endometrial stromal cell cultures, and we have investigated their regulation in an effort to understand control of IGF-II bioavailability at the placental-decidual interface in human implantation. IGFBP-4 protease activity was detected in conditioned media (CM) from human trophoblasts and decidualized endometrial stromal cells using (125)I-IGFBP-4 substrate. Identification of the IGFBP-4 protease as pregnancy-associated plasma protein-A (PAPP-A) was confirmed by specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with specific PAPP-A antibodies. The IGFBP-4 protease activity was IGF-II-dependent in trophoblast CM. In decidualized stromal CM, PAPP-A/IGFBP-4 protease activity was also IGF-II-dependent, but was evident only when IGF-II was added in molar excess of the predominant IGFBP in decidualized stromal cell CM, IGFBP-1, supporting bioavailable IGF-II as a key cofactor of IGFBP-4 proteolysis by PAPP-A. Cultured first and second trimester human trophoblasts (n = 5) secreted PAPP-A into CM with mean +/- SEM levels of 172.4 +/- 32.8 mIU/liter.10(5) cells, determined by specific ELISA. PAPP-A in trophoblast CM (n = 3) and did not change in the presence of IGF-II (1-100 ng/ml). Cultured human endometrial stromal cells (n = 4) secreted low levels of PAPP-A (6.25 +/- 3.6 mIU/liter.10(5) cells). A physiological inhibitor of PAPP-A, the proform of eosinophil major basic protein (proMBP), was detected in trophoblast CM at levels of 1853 +/- 308 mIU/liter.10(5) cells, determined by specific ELISA, and was nearly undetectable in CM of human endometrial stromal cells. Upon in vitro decidualization of endometrial stromal cells with progesterone, PAPP-A levels in CM increased nearly 9-fold without a concomitant change in proMBP. In contrast to the experiments with trophoblasts, IGF-II and the IGF analogues, Leu(27) IGF-II, and Des (1-6) IGF-II, resulted in a dose-dependent decrease of PAPP-A levels in decidualized endometrial stromal CM by 70-90%, and a dose-dependent increase in proMBP of 14- to 41-fold. The data demonstrate conclusively that the IGF-II-dependent IGFBP-4 protease of human trophoblast and decidual origin is PAPP-A. Furthermore, the differential regulation of decidual PAPP-A and proMBP by insulin-like peptides supports a role for trophoblast-derived IGF-II as a paracrine regulator of these maternal decidual products that have the potential to regulate IGF-II bioavailability at the trophoblast-decidual interface. Overall, the data underscore potential roles for a complex family of enzyme (PAPP-A), substrate (IGFBP-4), inhibitor (proMBP), and cofactor (IGF-II) in the placental bed during human implantation.

Pregnancy-associated plasma protein-A (PAPP-A) in ovine, bovine, porcine, and equine ovarian follicles: involvement in IGF binding protein-4 proteolytic degradation and mRNA expression during follicular development.

IGF binding protein-4 (IGFBP-4) proteolytic degradation is a common feature of preovulatory follicles from human, ovine, bovine, porcine, and equine ovary. In all these species, the protease is a zinc-dependent metalloprotease and its ability to degrade IGFBP-4 is IGF dependent. The human intrafollicular IGFBP-4-degrading protease has recently been identified as pregnancy-associated plasma protein-A (PAPP-A). The aim of this study was to investigate whether PAPP-A is also involved in IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles and to study the expression of PAPP-A mRNA in bovine and porcine granulosa cells from different classes of follicles. Immunoneutralization and immunoprecipitation with polyclonal antibodies raised against human PAPP-A inhibited IGFBP-4 proteolytic degradation in preovulatory follicular fluid from the four species studied. As previously reported for the intrafollicular proteolytic activity degrading IGFBP-4, recombinant human PAPP-A generated in vitro 17- and 10-kDa IGFBP-4-proteolytic fragments. Recombinant PAPP-A activity was also shown to be IGF dependent and was inhibited by heparin-binding domain-containing peptides. In all mammalian species studied, the PAPP-A sequences showed high degree of identity. Moreover, the PAPP-A gene was localized on porcine chromosome 1 (1q29-1q213), in agreement with the localization of human PAPP-A gene on human chromosome 9q33.1. In bovine and porcine ovaries, real-time quantitative RT-PCR showed that PAPP-A mRNA expression in granulosa cells was maximal in fully differentiated follicles and was positively correlated with expression of P450 aromatase and LH receptor mRNAs. Overall, these data show that PAPP-A is responsible for IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles. The high expression of PAPP-A mRNA in granulosa cells from large, differentiated follicles suggest that it is a new functional marker of follicular development.

Pregnancy-associated plasma protein A as a marker of acute coronary syndromes.

BACKGROUND: Circulating markers indicating the instability of atherosclerotic plaques could have diagnostic value in unstable angina or acute myocardial infarction. We evaluated pregnancy-associated plasma protein A (PAPP-A), a potentially proatherosclerotic metalloproteinase, as a marker of acute coronary syndromes. METHODS: We examined the level of expression of PAPP-A in eight culprit unstable coronary plaques and four stable plaques from eight patients who had died suddenly of cardiac causes. We also measured circulating levels of PAPP-A, C-reactive protein, and insulin-like growth factor I (IGF-I) in 17 patients with acute myocardial infarction, 20 with unstable angina, 19 with stable angina, and 13 controls without atherosclerosis. RESULTS: PAPP-A was abundantly expressed in plaque cells and extracellular matrix of ruptured and eroded unstable plaques, but not in stable plaques. Circulating PAPP-A levels were significantly higher in patients with unstable angina or acute myocardial infarction than in patients with stable angina and controls (P<0.001). A PAPP-A threshold value of 10 mlU per liter identified patients who had acute coronary syndromes with a sensitivity of 89.2 percent and a specificity of 81.3 percent. PAPP-A levels correlated with levels of C-reactive protein and free IGF-I, but not with markers of myocardial injury (troponin I and the MB isoform of creatine kinase). CONCLUSIONS: PAPP-A is present in unstable plaques, and circulating levels are elevated in acute coronary syndromes; these increased levels may reflect the instability of atherosclerotic plaques. PAPP-A is a new candidate marker of unstable angina and acute myocardial infarction.

Pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor binding protein (IGFBP)-5 independent of IGF: implications for the mechanism of IGFBP-4 proteolysis by PAPP-A.

Pregnancy-associated plasma protein-A (PAPP-A) has recently been identified as the proteinase responsible for cleavage of insulin-like growth factor binding protein (IGFBP)-4, an inhibitor of IGF action, in several biological fluids. Cleavage of IGFBP-4 by PAPP-A is believed to occur only in the presence of IGF. We here report that in addition to IGFBP-4, PAPP-A also cleaves IGFBP-5. Cleavage occurs at one site, between Ser-143 and Lys-144 of IGFBP-5. In the presence of IGF, IGFBP-4 and -5 are cleaved with similar rates by PAPP-A. Interestingly, cleavage of IGFBP-5 by PAPP-A does not require the presence of IGF, but is slightly inhibited by IGF. These findings have implications for the mechanism of proteolysis of IGFBP-4 by PAPP-A, suggesting that IGFBP-4 binds IGF, which then becomes a PAPP-A substrate. Using highly purified, recombinant proteins, we establish that (1) PAPP-A cleavage of IGFBP-4 can occur in the absence of IGF, although the rate of hydrolysis is very slow, and (2) IGF is unable to bind to PAPP-A. We thus conclude that IGF enhances proteolysis by binding to IGFBP-4, not by interaction with PAPP-A, which could not previously be ruled out.

Pregnancy-associated plasma protein-a is the insulin-like growth factor binding protein-4 protease secreted by human ovarian granulosa cells and is a marker of dominant follicle selection and the corpus luteum.

Insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and IGFBP proteases are important in ovarian function. IGFs stimulate granulosa steroidogenesis, an effect that is inhibited by IGFBP-4 and augmented by IGFBP-4 proteolysis. We have recently identified the IGFBP-4 protease in human ovarian follicular fluid (FF) as pregnancy-associated plasma protein-A (PAPP-A). In the current study, we identify the IGFBP-4 protease secreted by cultured human ovarian granulosa cells as PAPP-A, based on specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with PAPP-A polyclonal antibodies and immunorecognition by PAPP-A monoclonal antibodies in ELISA. PAPP-A was barely detectable in conditioned media (CM) from granulosa derived from /=9 mm, coincident with dominant follicle selection, and by luteinizing granulosa. PAPP-A levels in CM from the latter did not change in response to IGF-II or hCG (100 ng/mL). A naturally occurring inhibitor of PAPP-A, proform of eosinophil major basic protein (proMBP), was detected by ELISA in estrogen-dominant follicular fluid FF, but not in CM from granulosa or luteinizing granulosa cells treated with IGF-II (0-200 ng/mL), FSH (0-100 ng/mL) or hCG (0-100 ng/mL), suggesting an alternative source (other than granulosa) for proMBP, compared to PAPP-A. The data demonstrate granulosa cells as a source of PAPP-A in human ovary and suggest that PAPP-A is a marker of ovarian follicle selection and corpus luteum formation. In addition the data suggest complex regulation of this system in human ovary.

Pregnancy-associated plasma protein-A2 (PAPP-A2), a novel insulin-like growth factor-binding protein-5 proteinase.

A novel metalloproteinase with similarity to pregnancy-associated plasma protein-A (PAPP-A), which we denoted PAPP-A2, has been identified. Through expression in mammalian cells we showed that recombinant PAPP-A2 polypeptide of 1558 residues resulted from processing of a 1791-residue prepro-protein. Unlike PAPP-A, PAPP-A2 migrated as a monomer (of 220 kDa) in non-reducing SDS-polyacrylamide gel electrophoresis. The prepro-parts of PAPP-A2 and PAPP-A are not homologous, but mature PAPP-A2 shares 45% of its residues with PAPP-A. Because PAPP-A specifically cleaves insulin-like growth factor-binding protein (IGFBP)-4, one of six known modulators of IGF-I and -II, we looked for a possible PAPP-A2 substrate among the members of this family. We showed that PAPP-A2 specifically cleaved IGFBP-5 at one site, between Ser-143 and Lys-144. In contrast to the cleavage of IGFBP-4 by PAPP-A that strictly requires the presence of IGF, the cleavage of IGFBP-5 by PAPP-A2 was IGF-independent. Recent data firmly establish PAPP-A and IGFBP-4 as an important functional pair in several systems. Because of its close relationship with PAPP-A, both structurally and functionally, PAPP-A2 is a likely candidate IGFBP-5 proteinase in many tissues and conditioned media where IGFBP-5 proteolysis has been reported.

Insulin-like growth factor binding protein-4 protease produced by smooth muscle cells increases in the coronary artery after angioplasty.

Insulin-like growth factor (IGF)-I stimulates vascular smooth muscle cell (VSMC) migration and proliferation, which are fundamental to neointimal hyperplasia in postangioplasty restenosis. IGF-I action is modulated by several high-affinity IGF binding proteins (IGFBPs). IGFBP-4 is the predominant IGFBP produced by VSMCs and is a potent inhibitor of IGF-I action. However, specific IGFBP-4 proteases can cleave IGFBP-4 and liberate active IGF-I. In this study, we document IGFBP-4 protease produced by human and porcine coronary artery VSMCs in culture as pregnancy-associated plasma protein-A (PAPP-A). This was shown by a distinctive IGFBP-4 cleavage pattern, specific inhibition of IGFBP-4 protease activity with PAPP-A polyclonal antibodies, and immunorecognition of PAPP-A by monoclonal antibodies. Furthermore, we found a 2-fold increase in IGFBP-4 protease activity in injured porcine VSMC cultures in vitro (P<0.05). We also evaluated IGFBP-4 protease/PAPP-A expression in vivo after coronary artery balloon injury. Twenty-five immature female pigs underwent coronary overstretch balloon injury, and vessels were examined at defined time points after the procedure. Abundant PAPP-A expression was observed in the cytoplasm of medial and neointimal cells 7, 14, and 28 days after angioplasty (P<0.01 vs control). The highest PAPP-A labeling indices were located in the neointima (36.1+/-2.1%) and the media (31.7+/-1.2%) 28 days after injury. Western blot analysis confirmed increased PAPP-A in injured vessels. PAPP-A, a regulator of IGF-I bioavailability through proteolysis of IGFBP-4, is thus expressed by VSMCs in vitro and in restenotic lesions in vivo. These results suggest a possible role for PAPP-A in neointimal hyperplasia.

In vitro studies of insulin-like growth factor I and bone.

Insulin-like growth factor I (IGF-I) stimulates osteoblastic cells in culture to proliferate and to synthesize bone matrix proteins. However, local IGF-I action is modulated by a family of IGF-binding proteins (IGFBPs) that, in turn, are modified by specific IGFBP proteases. All these components of the IGF-I regulatory system are present and operative in bone cells in vitro.

Insulin-like growth factor (IGF) binding protein-3 potentiation of IGF action is mediated through the phosphatidylinositol-3-kinase pathway and is associated with alteration in protein kinase B/AKT sensitivity.

Cell-association and processing of insulin-like growth factor binding protein-3 (IGFBP-3) by cultured bovine fibroblasts results in markedly enhanced type I IGF receptor signaling at a step distal to ligand binding. The purpose of the present study was to determine the intracellular mediators of IGFBP-3's potentiating effect. Preincubation of cultured bovine fibroblasts with 50 nM IGFBP-3 had no effect alone, but enhanced by 3- to 4-fold IGF-I-stimulated 3H-aminoisobutryric acid (AIB) uptake. IGFBP-3-induced potentiation was specifically prevented if an inhibitor of phosphatidylinositol 3 (PI3)-kinase activation (LY294002), but not an inhibitor of mitogen-activated protein kinase activation (PD98059), was present during the preincubation period. IGFBP-3 did not directly activate the downstream effector of PI3-kinase, protein kinase B (PKB)/Akt. However, the sensitivity of PKB/Akt to activation by IGF-I was increased by 2- to 4-fold with IGFBP-3 pretreatment. This increased sensitivity was accompanied by altered mobility of PKB/Akt on SDS-polyacrylamide gels, suggestive of a diminished phosphorylation state. Consistent with this, okadaic acid, a potent serine/threonine phosphatase inhibitor, was able to block the potentiation effect of IGFBP-3 and prevent the altered mobility of the PKB/Akt molecule in response to IGFBP-3 treatment. PKB/Akt immunoprecipitated from IGFBP-3-pretreated cells was no longer recognized by an antibody specific for phosphorylated threonine followed by proline. These data indicate that IGFBP-3 modulates type I IGF receptor signaling through an effect on PI-3-kinase pathway substrates and suggest a novel mechanism of dephosphorylation whereby PKB/Akt is transformed into a more sensitive substrate of type I IGF receptor signaling.

Expression of recombinant human pregnancy-associated plasma protein-A and identification of the proform of eosinophil major basic protein as its physiological inhibitor.

Pregnancy-associated plasma protein-A (PAPP-A), originally known from human pregnancy serum, has recently been demonstrated to be a metzincin superfamily metalloproteinase involved in normal and pathological insulin-like growth factor (IGF) physiology. PAPP-A specifically cleaves IGF-binding protein (IGFBP)-4, one of six antagonists of IGF action, which results in release of IGF bound to IGFBP-4. IGFBP-4 is the only known PAPP-A substrate. Its cleavage by PAPP-A uniquely depends on the presence of IGF. We here report mammalian expression and purification of recombinant 1547-residue PAPP-A (rPAPP-A). The recombinant protein is secreted as a homodimer of about 400 kDa composed of two 200-kDa disulfide-bound subunits. Antigenically and functionally, rPAPP-A behaves like the native protein. In human pregnancy, PAPP-A is known to circulate as a 500-kDa disulfide-bound 2:2 complex with the proform of eosinophil major basic protein (proMBP), PAPP-A/proMBP. A comparison between rPAPP-A and pregnancy serum PAPP-A/proMBP complex surprisingly reveals a difference greater than 100-fold in proteolytic activity, showing that proMBP functions as a proteinase inhibitor in vivo. We find that polyclonal antibodies against PAPP-A abrogate all detectable IGFBP-4 proteolytic activity in pregnancy serum, pointing at PAPP-A as the dominating, if not the only, IGFBP-4 proteinase present in the circulation. We further show that pregnancy serum and plasma contain traces (<1%) of uncomplexed PAPP-A with a much higher specific activity than the PAPP-A/proMBP complex. The measurable activity of the PAPP-A/proMBP complex probably results from the presence of a minor subpopulation of partly inhibited PAPP-A that exists in a 2:1 complex with proMBP. Inhibition of PAPP-A by proMBP represents a novel inhibitory mechanism with the enzyme irreversibly bound to its inhibitor by disulfide bonds.

Mechanisms of lysolipid phosphate effects on cellular survival and proliferation.

The specificity of cellular effects of lysolipid phosphate (LLP) growth factors is determined by binding to endothelial differentiation gene-encoded G protein-coupled receptors (EDG Rs), which transduce diverse proliferative and effector signals. The primary determinants of cellular responses to LLPs are the generative and biodegradative events, which establish steady-state concentrations of each LLP at cell surfaces, and the relative frequency of expression of each EDG R. There are major differences among types of cells in the net effective generation of the LLPs, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), and in their profile of expression of EDG Rs. The less well characterized secondary determinants of cellular specificity of LLPs are high-affinity binding proteins with carrier and cell-presentation functions, cell-selective regulators of expression of EDG Rs, and cellular factors that govern coupling of EDG Rs to G protein transductional pathways. The roles of components of the LLP-EDG R system in normal physiology and disease processes will be definitively elucidated only after development of animal models with biologically meaningful alterations in genes encoding EDG Rs and the discovery of potent and selective pharmacological probes.

The insulin-like growth factor axis: A review of atherosclerosis and restenosis.

Insulin-like growth factors I and II (IGF-I and -II) and their regulatory proteins are secreted by cells of the cardiovascular system. They are growth promoters for arterial cells and mediators of cardiovascular disease. IGFs are bound to IGF binding proteins (IGFBPs), which modulate IGF ligand-receptor interaction and consequently to IGF action. IGFBPs are in turn posttranslationally modulated by specific proteases. This dynamic balance (IGFs, IGFBPs, and IGFBP proteases) constitutes the IGF axis and ultimately determines the extent of IGF-dependent cellular effects. Dysregulated actions of this axis influence coronary atherosclerosis through effects on vascular smooth muscle cell growth, migration, and extracellular matrix synthesis in the atherosclerotic plaque. IGF-I promotes macrophage chemotaxis, excess LDL cholesterol uptake, and release of proinflammatory cytokines. Endothelial cells also receive the effects of IGFs stimulating their migration and organization forming capillary networks. Neointimal hyperplasia of restenosis after coronary artery injury is also modulated by the IGF axis. IGFs stimulate vascular smooth muscle cell proliferation and migration to form the neointima and upregulate tropoelastin synthesis after disruption of the elastic layer. Understanding IGF axis regulation establishes a scientific basis for strategies directed to limit or reverse plaque growth and vulnerability in atherosclerosis and in the neointimal hyperplasia of restenosis.

Characterization of the enzymatic specificity of the IGF-dependent insulin-like growth factor binding protein-4 (IGFBP-4) protease.

The insulin-like growth factor (IGF)-dependent IGF binding protein-4 protease secreted by cultured adult human fibroblasts was recently identified as pregnancy-associated plasma protein-A (PAPP-A). In this study we showed that in addition to human IGFBP-4 the IGF-dependent IGFBP-4 protease also digests recombinant rat IGFBP-4 into two fragments by specifically cleaving at the carboxyl-terminal side of methionine at position 131 for rat IGFBP-4. Thus the cleavage site is at the KMKV site, which is not represented in other IGFBPs. While kallikrein may cleave at this site, its action is not specific.

Evidence that the insulin-like growth factor binding protein-4 protease in human ovarian follicular fluid is pregnancy associated plasma protein-A.

Ovarian insulin-like growth factor binding protein-4 (IGFBP-4) proteolysis is involved in the regulation of follicular development, but until now the identity of the responsible enzyme was unknown. In this study, we identify the IGFBP4 protease in human follicular fluid as pregnancy associated plasma protein-A (PAPP-A) based on distinctive IGFBP-4 cleavage pattern, the same protease inhibitor profile, specific inhibition and immunodepletion of IGFBP-4 protease activity with PAPP-A polyclonal antibodies, and immunorecognition by PAPP-A monoclonal antibodies in ELISA. Furthermore, PAPP-A levels in estrogen-dominant and androgen-dominant follicular fluids reflect their IGFBP-4 proteolytic activity. PAPP-A was also secreted by human granulosa cells, the reputed source of IGFBP-4 protease activity in follicular fluid. We have the molecular and biochemical tools to begin to delineate the regulation and biological function of PAPP-A in normal and dysregulated follicular development and atresia.

Effects of androgens on the insulin-like growth factor system in an androgen-responsive human osteoblastic cell line.

Although androgens have significant effects on bone metabolism, the mediators of their effects are still unclear. As the insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) have important effects on osteoblast proliferation and differentiation, we examined androgen effects on the IGF system in a conditionally immortalized human fetal osteoblastic cell line, hFOB/AR-6, which displays a mature osteoblastic phenotype and physiological levels of functional androgen receptors. The nonaromatizable androgen, 5alpha-dihydrotestosterone (5alphaDHT), and testosterone, but not dehydroepiandrosterone, increased IGF-I messenger RNA (mRNA) levels up to 4-fold in a dose (10(-12)-10(-6) M)- and time (2-72 h)-dependent fashion. These changes were prevented by the specific androgen receptor antagonist, hydroxyflutamide. In addition, 5alpha-DHT decreased IGFBP-4 mRNA and protein levels by 2- and 4-fold, respectively, and increased IGFBP-2 and -3 mRNA and protein levels by 6- and 7-fold (for mRNA) and 3- and 5-fold (for protein), respectively. hFOB/AR-6 cells expressed the type-I IGF receptor, but this was not regulated by 5alphaDHT. 5alphaDHT and IGFBP-3 specifically increased hFOB/AR-6 cell proliferation, and a monoclonal antibody specific for IGF-I blocked this effect. Thus, androgens increase the expression of IGF-I, IGFBP-2, and IGFBP-3, but decrease levels of the inhibitory IGFBP-4 in an androgen-responsive human osteoblastic cell line. Our data are consistent with the hypothesis that the effects of androgen on bone cells may be mediated at least in part by increases in IGF-I production and by differential regulation of IGFBPs.

Response of bipotential human marrow stromal cells to insulin-like growth factors: effect on binding protein production, proliferation, and commitment to osteoblasts and adipocytes.

Insulin-like growth factors (IGFs) are important regulators of the activity of mature osteoblasts, but their effects on osteoprogenitor cells in human bone marrow stroma are unclear. In this study, we assessed the effects of IGFs on a conditionally immortalized human marrow stromal cell line, hMS(3-4), which has the ability to differentiate to either mature osteoblasts or adipocytes. hMS(3-4) cells expressed functional receptors for IGFs as well as specific IGF-binding proteins (IGFBP-3, -4, -5, and -6). IGF treatment of hMS(3-4) cells did not alter IGFBP expression, but resulted in distinct posttranslational modifications of secreted IGFBP-3 and IGFBP-4 proteins. IGF-I, IGF-II, and their receptor-activating analogs significantly increased by 2-fold the proliferation rate of the hMS(3-4) cells, but had a more complex effect on hMS(3-4) cell differentiation. Treatment with IGFs did not affect gene expression of Cbfa1 or peroxisome proliferator-activated receptor gamma2 (transcription factors involved in commitment to osteoblast and adipocyte pathways, respectively), alkaline phosphatase, type I collagen, and osteocalcin (markers of the osteoblast lineage), or lipoprotein lipase and adipsin (markers of the adipocyte lineage) and did not change alkaline phosphatase activity or type I collagen and osteocalcin protein relative to total protein production. In contrast, IGFs significantly increased type I collagen expression in differentiated hMS(3-4) cells as well as mature osteoblasts and promoted lipid accumulation in differentiated adipocytes. In summary, hMS(3-4) cells express essential components of the IGF system and respond to IGF treatment with increased proliferation. There was no evidence for IGFs directly modulating the commitment of hMS(3-4) cells to either osteoblast or adipocyte pathways, and their effects on differentiation within these lineages were dependent on the stage of cell maturation.

Distinctive expression and functions of the type 4 endothelial differentiation gene-encoded G protein-coupled receptor for lysophosphatidic acid in ovarian cancer.

Endothelial differentiation gene (edg)-encoded G protein-coupled receptors (Edg Rs)-1, -3, and -5 bind sphingosine 1-phosphate (S1P), and Edg-2 and -4 bind lysophosphatidic acid (LPA). Edg Rs transduce signals from LPA and S1P that stimulate ras- and rho-dependent cellular proliferation, enhance cellular survival, and suppress apoptosis. That high levels of LPA in plasma and ascitic fluid of patients with ovarian cancer correlate with widespread invasion suggested the importance of investigating expression and functions of Edg Rs in ovarian cancer cells (OCCs) as compared with nonmalignant ovarian surface epithelial cells (OSEs). Analyses of Edg Rs by semiquantitative reverse transcription-PCR, a radioactively quantified variant of PCR, and Western blots developed with monoclonal antibodies showed prominent expression of Edg-4 R in primary cultures and established lines of OCCs but none in OSEs. In contrast, levels of Edg-2, -3, and -5 were higher in OSEs than OCCs. LPA stimulated proliferation and signaled a serum response element-luciferase reporter of immediate-early gene activation in OCCs but not OSEs, whereas S1P evoked similar responses in both OSEs and OCCs. Pharmacological inhibitors of Edg R signaling suppressed OCC responses to LPA. A combination of monoclonal anti-Edg-4 R antibody and phorbol myristate acetate, which were inactive separately, evoked proliferative and serum response element-luciferase responses of OCCs but not OSEs. Thus the Edg-4 R may represent a distinctive marker of OCC that transduces growth-promoting signals from the high local concentrations of LPA characteristic of aggressive ovarian cancer.