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The IGF-I pathway and renin-angiotensin-aldosterone axis are both involved in the pathogenesis of hypertension and atherosclerosis, but no information is available about IGF-I and aldosterone interaction or their potential synergistic effects in vascular smooth muscle cells (VSMCs). The aims of this study were to investigate whether aldosterone influences IGF-I signaling and to determine the mechanism(s) by which aldosterone affects IGF-I function. Aldosterone resulted in significant increases in the Akt (1.87 ± 0.24, P < 0.001), MAPK (1.78 ± 0.13, P < 0.001), p70S6kinase (1.92 ± 0.15, P < 0.001), IGF-I receptor (1.69 ± 0.05, P < 0.01), and insulin receptor substrate-1 (1.7 ± 0.04, P < 0.01) (fold increase, mean ± SEM, n = 3) phosphorylation responses to IGF-I compared with IGF-I treatment alone. There were also significant increases in VSMC proliferation, migration, and protein synthesis (1.63 ± 0.03-, 1.56 ± 0.08-, and 1.51 ± 0.04-fold increases compared with IGF-I alone, respectively, n = 3, P < 0.001). Aldosterone induced osteopontin (OPN) mRNA expression and activation of αVβ3-integrin as well as an increase in the synthesis of IGF-I receptor. The enhancing effects of aldosterone were inhibited by eplerenone (10 μmol/liter), actinomycin-D (20 nmol/liter), and an anti-αVβ3-integrin antibody that blocks OPN binding. The antioxidant N-acetylcysteine (2 mmol/liter) completely inhibited the ability of aldosterone to induce any of these changes. In conclusion, our results show that aldosterone enhances IGF-I signaling and biological actions in VSMCs through induction of OPN followed by its subsequent activation of the αVβ3-integrin and by increasing IGF-I receptor. These changes are mediated in part through increased oxidative stress. The findings suggest a new mechanism by which aldosterone could accelerate the development of atherosclerosis.

IGF-I has been shown to play a role in the progression of atherosclerosis in experimental animal models. IGF-binding protein-4 (IGFBP-4) binds to IGF-I and prevents its association with receptors. Overexpression of a protease-resistant form of IGFBP-4 has been shown to inhibit the ability of IGF-I to stimulate normal smooth muscle cell growth in mice. Based on these observations, we prepared a protease-resistant form of IGFBP-4 and infused it into hypercholesterolemic pigs. Infusion of the protease-resistant mutant inhibited lesion development by 53.3 +/- 6.1% (n = 6; P < 0.01). Control vessels that received an equimolar concentration of IGF-I and the protease-resistant IGFBP-4 showed no reduction in lesion size compared with control lesions that were infused with vehicle. Infusion of a nonmutated form of IGFBP-4 did not significantly inhibit lesion development. Proliferating cell nuclear antigen analysis showed that the mutant IGFBP-4 appeared to inhibit cell proliferation. The area occupied by extracellular matrix was also reduced proportionally compared with total lesion area. Immunoblotting revealed that the mutant IGFBP-4 remained intact, whereas the wild-type IGFBP-4 that was infused was proteolytically cleaved. Further analysis of the lesions revealed that a marker protein, IGFBP-5, whose synthesis is stimulated by IGF-I, was decreased in the lesions that received the protease-resistant, IGFBP-4 mutant, whereas there was no change in lesions that received wild-type IGFBP-4 or the mutant protein plus IGF-I. These findings clearly illustrate that infusion of protease-resistant IGFBP-4 into the perilesion environment results in inhibition of cell proliferation and attenuation of the development of neointima. The findings support the hypothesis that inhibiting IGFBP-4 proteolysis in the lesion microenvironment could be an effective means for regulating neointimal expansion.

IGF binding protein-5 (IGFBP-5) is an important trophic factor for controlling the actions of IGF-I in human dermal fibroblasts and porcine aortic smooth muscle cells. When IGFBP-5 is associated with extracellular matrix, it acts to enhance the cell growth response to IGF-I. The amount of IGFBP-5 within the extracellular matrix is related in part to the amount that is present in conditioned medium, which is related to its rate of synthesis and degradation. A serine protease that degrades IGFBP-5 is present in the conditioned medium of both of these cell types. Because the IGFBP-5 protease activity that is secreted by fibroblasts has been shown to be due to the complement components C1r and C1s, these studies were undertaken to determine whether smooth muscle cells also secreted these proteases and to identify some of the factors that regulate their secretion by both cell types. Both smooth muscle cells and human fibroblasts were shown to release C1r and C1s into conditioned medium. Both C1r and C1s were detected as activated forms, as determined by SDS-PAGE using reducing conditions. The addition of increasing concentrations of either IL-1beta or TNFalpha resulted in increased synthesis of C1s by fibroblasts and smooth muscle cells, and they each increased C1r release. TNFalpha (50 ng/ml) and IL-1beta (20 ng/ml) resulted in maximum stimulation of release of both proteases. In contrast dexamethasone (10(-7) M) had no effect on C1s release and stimulated C1r release only by smooth muscle cells. To determine the physiological significance of this increase in C1r and C1s, the amount of IGFBP-5 protease activity that was present in conditioned medium was determined before and after exposure to TNFalpha, IL-beta, and dexamethasone. All three compounds resulted in an increase in the amount of IGFBP-5 proteolytic activity. Dexamethasone inhibited the release of C(1) inhibitor from fibroblasts, and this contributed to the net increase in proteolytic activity. TNFalpha inhibited the smooth muscle cell DNA synthesis response to IGF-I, but the effect of IGF-I was partially restored by the addition of C1 inhibitor. In conclusion, both C1r and C1s are released by cultured fibroblasts, and the release of each into fibroblast or porcine smooth muscle cells medium is stimulated by TNFalpha and IL-1beta. This increase results in a net increase in IGFBP-5 proteolysis, which has the potential to modify IGF-I and IGFBP-5 actions.

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) has been shown to bind to extracellular matrix (ECM) with relatively high affinity, but the ECM components that mediate this interaction have not been identified. These studies show that radiolabeled IGFBP-5 specifically coprecipitates with two ECM proteins, thrombospondin-1 (TSP-1) and osteopontin (OPN). As TSP-1 binds avidly to heparin, as does IGFBP-5, the effect of glycosaminoglycans on the TSP-1/IGFBP-5 interaction was analyzed. Heparan and dermatan sulfate inhibited binding, whereas heparin increased binding. Chondroitin sulfate A and B had no effect. In contrast, both heparin and heparan sulfate significantly inhibited the OPN-IGFBP-5 interaction and chondroitin sulfate A, B, and C had no effect. To determine the region of IGFBP-5 that was involved in each interaction, synthetic peptides that spanned several regions of IGFBP-5 were tested for their capacity to competitively inhibit coprecipitation. A peptide that contained the amino acids between positions 201 and 218 resulted in 76% and 86% inhibition of binding to TSP-1 and OPN, respectively. Three other synthetic peptides that spanned regions ofIGFBP-5 with several charged residues had no effect. IGFBP-5 mutants that contained substitutions for basic residues in the 201-218 region were tested for their ability to bind to TSP-1 or OPN. A mutant with substitutions for amino acids at positions R201 and K202 and a mutant with substitutions for K211, R214, K217, and R218 had the greatest reduction in binding to TSP-1. Mutants containing substitutions for R214 alone and the combined K217A, R218A mutant had the greatest reductions in OPN binding. When the smooth muscle cell growth response to these components was assessed, IGF-I plus IGFBP-5 or the combination of TSP-1 or OPN with IGF-I potentiated the IGF-I effect. The addition of IGFBP-5 to these combinations resulted in further significant growth stimulation. Both OPN and TSP-1 specifically bind to IGFBP-5 with high affinity. These interactions may be important for concentrating intact IGFBP-5 in extracellular matrix and for modulating the cooperative interaction between the IGF-I receptor and integrin receptor signaling pathways.

We previously reported that human fibroblasts secrete a protease into their conditioned medium that cleaves insulin-like growth factor binding protein-5 (IGFBP-5) into non-IGF-I binding fragments. Because the protease activity in the fibroblast medium has characteristics of both serine and metalloproteases, the activity was purified and analyzed to determine whether it retained serine or metalloprotease properties. The protease was purified by heparin Sepharose affinity chromatography followed by alpha1 antichymotrypsin affinity or gelatin agarose chromatography. The heparin Sepharose purified material degraded IGFBP-5 into 22-, 17-, and 16-kDa fragments. Amino acid sequencing showed that the 22-kDa fragment contained the amino-terminus of the protein. The protease activity in the fibroblast conditioned medium that was purified by heparin Sepharose was inhibited by both serine and metalloprotease inhibitors. To attempt to separate these activities, the heparin Sepharose purified activity was further purified by gelatin agarose chromatography. The IGFBP-5 protease activity that did not bind to gelatin agarose was inhibited by serine protease inhibitors, such as 3,4 dichloroisocoumain (3,4 DCI), whereas tissue inhibitor metalloprotease-1 (TIMP-1) had minimal activity. When this same pool of protease activity that had been eluted from heparin Sepharose was applied to an alpha1 antichymotrypsin peptide affinity column, the protease activity that bound to the column was inhibited by 3,4 dichloroisocoumain, but was not inhibited by TIMP-1. In contrast, the activity that did not adhere to this column was inhibited by TIMP-1. IGFBP-5 zymography showed that the Mr estimate of the protease that was inhibited by serine protease inhibitors was 92 kDa, whereas gelatin zymography showed that the metalloproteases had Mr estimates of 72, 69, and 55 kDa. When the protease activity in the crude conditioned medium was analyzed by zymography, almost all of the detectable protease had an Mr estimate of 92 kDa, suggesting that the metalloproteases that were detected in the partially purified fractions were inactive in the medium. In summary, fibroblasts secrete a 92-kDa protease that cleaves IGFBP-5 into 22-, 17-, and 16-kDa fragments. The protease inhibitor specificity results, chromatographic characteristics, and zymographic analyses suggest that this is a serine protease. Although metalloproteases are secreted by these cells, the 92-kDa serine protease is the predominate form of activity in the conditioned medium that cleaves IGFBP-5.

Human dermal fibroblasts secrete insulin-like growth factor-binding protein-3 (IGFBP-3), -4, and -5. Fibroblast-conditioned medium contains minimal intact IGFBP-5, and this form of IGFBP is predominantely a 23-kilodalton fragment, suggesting that the IGFBP-5 fragment is derived from intact IGFBP-5 by proteolysis. In this study we investigated the effects of glycosaminoglycans on IGFBP-5 degradation in fibroblast-conditioned medium. The addition of heparin, heparan sulfate, and dermatan sulfate (100 micrograms/ml) to the medium of fibroblast monolayer cultures inhibited IGFBP-5 degradation, as determined by the conversion of intact IGFBP-5 to a 23-kilodalton fragment. In contrast, hyaluronic acid, keratan sulfate, and chondroitin sulfate-A and -C had no effect. Heparin and heparan sulfate inhibited IGFBP-5 degradation at concentrations of 1 or 2.5 micrograms/ml, but 100 micrograms/ml dermatan sulfate were required. Heparin was also inhibitory in vitro, that is when conditioned medium and heparin were incubated without cells. Experiments with modified forms of heparin showed that O-sulfate groups in the 2 or 3 carbon position were required for heparin to be inhibitory. Completely desulfated heparin had no activity, and N-resulfation of desulfated heparin had only a minimal effect. Dextran sulfate, pentosan polysulfate, and fucoidan, which are composed of different saccharide units but contain O-sulfate groups in the 2 or 3 carbon positions, also inhibited IGFBP-5 degradation. These results demonstrate that heparin-like molecules are important regulators of IGFBP-5 degradation. O-Sulfation of the 2 or 3 position of the saccharide ring is required for inhibitory activity. As glycosaminoglycan side-chains are present in proteoglycans that are present in extracellular matrix and on cell surfaces, these side-chains represent a potential mechanism for regulating IGFBP-5 proteolysis in vivo.

We have previously reported the presence of proteolytic activity in conditioned medium from human fibroblast cultures that cleaves insulin-like growth factor-binding protein-5 (IGFBP-5) into non-IGF-I-binding fragments. Coincubation of IGF-I or IGF-II and IGFBP-5 with fibroblast cultures decreased proteolysis. The protease was purified by heparin-Sepharose affinity chromatography. The purified protease cleaved IGFBP-5 into 22-, 20-, and 17-kilodalton non-IGF-I-binding fragments. Protease inhibitor profiles obtained using partially purified enzyme showed that it was a calcium-dependent serine protease. After chelation with EDTA, the activity could only be partially restored with zinc, indicating that it was probably not a metalloprotease. The protease was specific for IGFBP-5 and did not cleave pure IGFBP-1, -2, -3, or -4. IGF-I and IGF-II caused minimal inhibition of proteolysis in vitro. This suggests that the IGF-I-induced increase in IGFBP-5 in fibroblast medium is only partially due to direct protease inhibition. Heparin, antithrombin-III (AT-III), and heparin cofactor-II had inhibitory activity, and heparin potentiated the activity of AT-III. Synthetic peptides, that contained the active sites of AT-III and alpha 1-antichymotrypsin, were also inhibitory. Peptides containing sequences found in two basic regions of IGFBP-5 were tested, and one had inhibitory activity. In summary, fibroblasts secrete a serine protease that cleaves IGFBP-5 and is specific for this form of IGFBP. The protease has properties that are similar to kallikreins, a family of serine proteases that is known to cleave epidermal and nerve growth factor-binding proteins.

Recent studies have shown that human decidual explants synthesize a 25,272 dalton form of insulin-like growth factor binding protein (IGFBP-1) that is identical to the most abundant form of IGFBP detected in human amniotic fluid. To determine whether decidual cells also secrete other structurally distinct forms of IGFBP, conditioned medium obtained from human decidual cells was analyzed by ligand blotting and immunoblotting using antisera that were specific for IGFBP-1 or 2. IGFBP-2 is a form of binding protein that is structurally distinct from IGFBP-1. Ligand blotting analysis (which detects all forms of IGFBPs) showed that 3 forms of IGFBP with Mr estimates of 34,000, 30,000 and 24,000 were present in basal, unstimulated, conditioned media. Immunoblotting showed that the 30,000 Mr form reacted with an antibody that is specific for IGFBP-1, whereas the 34,000 Mr form reacted with an antibody that is specific for IGFBP-2. The 24,000 Mr band did not react with antisera to either IGFBP-1 or 2. Basal IGFBP-1 release from human decidual cultures, as measured by RIA, showed a significant decrease during the 5 day period from 34 +/- 1.6 on day 1 to 12 +/- 1.9 ng/ml on day 5. Exposure of the cells to (Bu)2cAMP (1.0 mM) for 5 days had no significant effect on IGFBP-1 release during the first day of exposure, but caused a progressive stimulation of release during the subsequent 4 days. By day 5, exposure to (Bu)2cAMP induced a 5- to 6-fold increase in IGFBP-1 release that was completely inhibited by exposure to cycloheximide. Exposure of the cells to cholera toxin (0.1 micrograms/ml) for 4 days caused a 3.7-fold increase in IGFBP-1 release. The stimulation of IGFBP-1 release by (Bu)2cAMP was completely inhibited by simultaneous exposure to insulin (100 ng/ml) and/or IGF-I (100 ng/ml) (day 5 control = 35 ng/ml, (Bu)2cAMP alone = 248 ng/ml, insulin + (Bu)2cAMP = 36 ng/ml, or IGF-I + (Bu)2cAMP = 10.1 ng/ml). Hydrocortisone and the phorbol ester (phorbol myristate acetate) caused no significant changes in basal IGFBP-1 release but prevented the decrease in the basal rate of release that occurred between day 1 and 5. Ligand blotting studies showed that the 24,000 Mr form of binding protein was also stimulated by (Bu)2cAMP (3.3-fold increase); however, cholera toxin and phorbol myristate acetate were without effect. In contrast, the release of the 34,000 Mr form was unaffected by exposure to any of these agents, suggesting that regulation of this form of IGFBP is distinct from the other 2 forms.(ABSTRACT TRUNCATED AT 400 WORDS)

A peptide alpha-amidating enzyme was purified to apparent homogeneity from porcine pituitary. This enzyme is a glycoprotein with a mol wt of 64,000, a metal prosthetic group, and a dependence upon ascorbate and molecular oxygen. The purified enzyme has a strong preference for peptides ending in glycine. It also catalyzes the oxidation of valylglycine bonds more rapidly than prolylglycine bonds, and demonstrates a primary isotope effect greater than 5 when the alpha-hydrogens of glycine are replaced by deuterium. Kinetic analysis is consistent with a ping-pong or double displacement catalytic mechanism in which both the peptide substrate and ascorbate are competitive inhibitors with respect to each other. With respect to its kinetic properties, catalytic mechanism, and cofactor requirements, the purified amidating enzyme is very similar to dopamine beta-hydroxylase, a finding which supports the previous suggestion that the peptide alpha-amidating enzyme be classified as a peptidyl glycine monooxygenase.