Structure-function relationships of insulin-like growth factor binding protein 6 (IGFBP-6) and its chimeras.

Insulin-like growth factor binding protein 6 (IGFBP-6) is a high-affinity IGFBP with substantially greater affinity for insulin-like growth factor-II (IGF-II) than IGF-I. IGFBP-6(3) is a chimera which has a 20 amino acidC -terminal portion of IGFBP-6 switched with the homologous area of IGFBP-3, P3. Unlike IGFBP-4(3), in which the P3 region was exchanged for the homologous region of IGFBP-4 (P4), IGFBP-6(3) does not bind to endothelial cells. Double mutations were made with the P3 region exchanged as well as a second area differing from IGFBP-3 to form IGFBP-6(3)A and IGFBP-6(3)B, by replacing this area with the homologous region of IGFBP-3. Neither [(125)I]IGFBP-6(3)A nor IGFBP-6(3)B specifically bound to endothelial cells. However, each double mutant competed for [(125)I]IGFBP-3 binding to cultured cells. In the perfused heart, transendothelial transport of IGFBP-6 and IGFBP-6(3) was only 25% of similar transendothelial transport of perfused IGFBP-3. We conclude that chimeras of IGFBP-6 and IGFBP-3(6) clearly differ from IGFBP-4(3) in their ability to bind specifically to endothelial cells and in their capacity to undergo transendothelial transportation in the perfused heart.

IGFBP-3 binding to endothelial cells inhibits plasmin and thrombin proteolysis.

Insulin-like growth factor-binding protein (IGFBP)-3 contains a highly basic COOH-terminal heparin-binding region, the P3 region, which is thought to be important in the binding of IGFBP-3 to endothelial cells. IGFBP-3 and IGFBP-4, and their chimeras IGFBP-3(4) and IGFBP-4(3), were treated with plasmin and with thrombin, proteases known to cleave IGFBP-3. IGFBP-3 was highly susceptible to plasmin, whereas IGFBP-4 was less so. Substitution of the P3 region for the P4 region in IGFBP-4 (IGFBP-4(3)) increased the ability of the protease to digest IGFBP-4(3); substitution of the P4 region for the P3 region in IGFBP-3 (IGFBP-3(4)) decreased the digestion of IGFBP-3(4). When 125I-labeled IGFBP-3 or 125I-IGFBP-4(3) was first bound to vascular endothelial cells, subsequent proteolysis by either plasmin or thrombin was substantially inhibited. Proteolysis of 125I-IGFBP-3(4) was not inhibited in the presence of endothelial cells. The P3 peptide was cleaved by plasmin but not by thrombin. We conclude that the P3 region is central to proteolysis of IGFBP-3 by plasmin and thrombin, processes which were inhibited by association of IGFBP-3 with endothelial cells.

Distribution of chimeric IGF binding protein (IGFBP)-3 and IGFBP-4 in the rat heart: importance of C-terminal basic region.

IGF binding proteins-3 and -4, whether given in the perfused rat heart or given iv in the intact animal, cross the microvascular endothelium of the heart and distribute in subendothelial tissues. IGF binding protein-3, like IGF-I/II, localizes in cardiac muscle, with lesser concentrations in CT elements. In contrast, IGFBP-4 preferentially localizes in CT. In this study, chimeric IGF binding proteins were prepared in which a basic 20-amino-acid C-terminal region of IGF binding protein-3 was switched with the homologous region of IGF binding protein-4, and vice-versa, to create IGF binding protein-3(4) and IGF binding protein-4(3). Perfused IGF binding protein-3(4) behaved like IGF binding protein-4, localizing in connective tissue elements, whereas IGF binding protein-4(3) now localized in cardiac muscle at concentrations identical to perfused IGF binding protein-3. To determine whether these small mutations altered the affinity of the chimera for cells, the ability of (125)I-IGF binding protein-3(4) and (125)I-IGF binding protein-4(3) to bind to microvascular endothelial cells was determined and compared with IGF binding protein-3. IGF binding protein-3(4) retained 15% of the binding capacity of IGF binding protein-3, whereas IGF binding protein-4(3) bound to microvessel endothelial cells with higher affinity and greater total binding than that of IGF binding protein-3. We conclude that small changes in the C-terminal basic domain of IGF binding protein-3 and the corresponding region of IGF binding protein-4 can alter their affinity for cultured cells and influence their tissue distribution in the rat heart.

Effect of IGFBP-derived peptides on incorporation of(35)SO(4)into proteoglycans.

18 amino acid peptides from the C-terminal region of IGFBP-3, -5 (P3, P5), increased the incorporation of(35)SO(4)into proteoglycans in endothelial cells with greater stimulation in large vessel than microvessel cells. The homologous region of IGFBP-6 (P6) also stimulated sulfate uptake, but less potently than P3 and P5. P6 variants were synthesized with one or two amino acids changed to the basic amino acid in the equivalent position of P3. The P6 variants with one additional basic amino acid behaved similarly to P6. The P6 mutant with two altered amino acids was equipotent to P3. P3F, a scrambled version of P3 was less effective than P3. P3, P5, P6, P3F and all P6 variants all stimulated glucose uptake, which occurred only in microvessel cells. P1, P2, P4, and equimolar intact IGFBP-3 stimulated neither glucose uptake nor sulfate incorporation. Thus, C-terminal basic portions of IGFBP-3, -5 and -6 alter two specific functions of endothelial cells with sufficient differences to suggest mediation by distinct mechanisms.

Connective tissue growth factor (IGFBP-rP2) expression and regulation in cultured bovine endothelial cells.

Media from large vessel endothelial cells (pulmonary artery, aorta) contained intact connective tissue growth factor (CTGF) and a dominant 19-kDa band. N-terminal analysis of the 19-kDa band showed sequence corresponding to CTGF amino acid 181-190, suggesting that the 19-kDa band represented a proteolytic fragment of CTGF. Intact CTGF was increased by cAMP but not by transforming growth factor-beta (TGFbeta). CTGF messenger RNA (mRNA) was not changed by cAMP nor TGFbeta. In two microvessel endothelial cells, mRNA was found at low levels by PCR and Northern analysis, but no CTGF protein was seen on Western analysis. In the microvessel cells, TGFbeta increased and cAMP did not change CTGF mRNA levels, with neither TGFbeta nor cAMP increasing CTGF protein. The discordance between protein and mRNA levels in large vessel and microvessel endothelial cells was mostly explained by the effects of cAMP and TGFbeta on media proteolytic activity; in large vessel cells, cAMP inhibited degradation of CTGF, whereas in microvessel cells, TGFbeta and cAMP stimulated proteolytic activity against CTGF. We conclude that in large vessel endothelial cells, cAMP increased intact CTGF protein by inhibiting degradation of CTGF, whereas TGFbeta stimulated neither CTGF mRNA nor protein; in microvessel cells, TGFbeta increased CTGF mRNA, while both TGFbeta and cAMP stimulated CTGF degradation.

Isolation and characterization of plasmin-generated bioactive fragments of IGFBP-3.

Insulin-like growth factor-binding protein-3 (IGFBP-3) was digested with plasmin, and the proteolytic fragments were isolated by HPLC and tested for bioactivity as measured by stimulation of glucose uptake in microvessel endothelial cells. Two of the pooled fractions of the digest stimulated glucose uptake. The major bioactive pool, at an estimated protein concentration <50 ng/ml, stimulated glucose uptake to 150% of control with greater stimulation and 220% of control at approximately 250 ng/ml. Two fragments were present in the bioactive fraction, the dominant one migrating at approximately 20,000 and the other at approximately 8,000. Both fragments bound 125I-labeled insulin-like growth factor and [3H]heparin. NH2-terminal amino acid analysis of the bioactive peak yielded two sequences. One, representing the majority of the material, had an NH2-terminal sequence identical to IGFBP-3; the second fragment began at amino acid 202 of IGFBP-3. In contrast to the bioactive fragments, intact IGFBP-3, at concentrations up to 130 microgram/ml, had no bioactivity. These findings demonstrate that IGFBP-3 can be degraded into fragments that have potent bioactivities that are not present in the intact IGFBP-3 molecule.

Infused IGF-I/IGFBP-3 complex causes glomerular localization of IGF-I in the rat kidney.

Insulin-like growth factor I (IGF-I) increases renal blood flow, glomerular filtration rate (GFR), and proximal tubule reabsorption of phosphate in humans and rodents. The biological effects of IGF-I are likely to be influenced by cellular localization of IGF-I within the kidney. We therefore tested whether the renal localization of infused IGF-I could be altered if given with selected IGF-binding proteins (IGFBPs). Rats were treated with intravenous injections of 125I-labeled IGF-I, 125I-IGFBP-3, or 125I-IGFBP-4 alone or with complexes of 125I-IGF-I and IGFBP-3 or IGFBP-4. The cellular localization of IGF and the IGFBP within the kidney was then determined. 125I-IGF-I, 125I-IGFBP-4, and 125I-IGF-I/IGFBP-4 complexes were found almost exclusively in vacuolar structures (endosomes) of proximal renal tubules. In contrast, about one-third of renal 125I-IGFBP-3 and 125I-IGF-I/IGFBP-3 was localized to glomeruli. When 125I-IGF-I was given alone, 3% was found in glomeruli and 89% in proximal tubules. When given as 125I-IGF-I/IGFBP-3, 29% was in glomeruli and 65% in proximal tubules. We conclude that the cellular localization of IGF-I within the kidney can be directed to glomerular elements if the IGF-I is given with IGFBP-3.

Regulations of IGF binding proteins in human aorta vascular smooth muscle cells by cAMP, dexamethasone and IGF-I.

Human vascular smooth muscle cells produce IGFBP-3, IGFBP-4, IGFBP-6 and proteases specific for IGFBP-3 and IGFBP-4. This study evaluated the regulation of IGFBPs in human aorta smooth muscle cells by cyclic AMP, dexamethasone and IGF-I. cAMP decreased IGFBP-3, increased IGFBP-4 and increased IGFBP-6. Dexamethasone decreased IGFBP-3, slightly increased IGFBP-4 and increased IGFBP-6. IGF-I increased IGFBP-3 and IGFBP-6 while decreasing IGFBP-4. Co-incubation with IGF-I and dexamethasone or cAMP increased media IGFBP-3, despite a decrease in IGFBP-3 mRNA, due to the dominant effect of IGF-I-induced dissociation of cell surface-bound IGFBP-3. In cells incubated with cAMP and IGF-I, media IGFBP-4 was decreased, despite increased IGFBP-4 mRNA, in this case secondary to the dominant effect of IGF-I-stimulated IGFBP-4 protease. These findings suggest that cAMP, dexamethasone and IGF-I regulate IGFBP production in human aorta smooth muscle cells via a complex interplay of changes in transcription, protease activation and dissociation of cell surface-bound IGFBPs.

Insulin-like growth factor binding protein production by bovine and human vascular smooth muscle cells: production of insulin-like growth factor binding protein-6 by human smooth muscle.

Insulin-like growth factor binding protein (IGFBP) secretory profiles were determined for vascular smooth muscle cells (VSMC) derived from bovine aorta and human aorta, pulmonary artery, and coronary artery. The bovine cells produced IGFBP-4, IGFBP-3, and an IGFBP-3 protease. IGF-I stimulated messenger RNA (mRNA) and media levels of IGFBP-3. The human cells produced IGFBP-3, IGFBP-4, and IGFBP-3 and IGFBP-4 proteases. The three human cells also produced a 30K IGFBP, shown to be IGFBP-6, based on increased affinity for IGF-II vs. IGF-I, size decrease when treated with O-glycanase, but not N-glycanase, reactivity with IGFBP-6 antiserum, presence of a 1.3-kilobase pair mRNA that hybridized to IGFBP-6 specific complementary DNA, and N-terminal amino acid sequence corresponding to IGFBP-6. In the human cells, IGF-I increased media levels of IGFBP-3 through stimulation of IGFBP-3 mRNA and dissociation of cell bound IGFBP-3, and decreased IGFBP-4 via potentiation of IGFBP-4 proteolysis. Neither the bovine nor the human aorta VSMC produced sufficient IGFBP-2 or IGFBP-2 mRNA to be detected by ligand blot and Northern analysis, as previously reported for porcine and rat aorta smooth muscle cells. The variable expression of IGFBPs and IGFBP proteases by VSMC are likely to contribute to differential vascular reactivity to the IGFs in larger arterial blood vessels.

Structure-function relationships in the heparin-binding C-terminal region of insulin-like growth factor binding protein-3.

IGFBP-3 contains a carboxyterminal basic region which, when present as an isolated 18 amino acid peptide (P3), binds heparin, associates with cultured endothelial cells and stimulates glucose uptake. The P3 molecule has now been modified relative to charge, amino acid sequence and size to determine structure-function relationships relative to four properties of P3: affinity for heparin; inhibition of IGFBP-3 binding; stimulation of glucose uptake; and displacement of bFGF from the extracellular matrix of endothelial cells. Results indicate: (1) the presence or absence of heparin binding was concordant with the presence/absence of the other three properties; (2) the number of basic amino acids was an important, if not limiting, factor for each property; (3) the order of potency of the basic amino acids was arginine = lysine > > histidine; (4) the unrelated, basic protein, protamine, mimics all properties of P3; and (5) the putative consensus heparin-binding sequence of P3 was not essential for any of the P3 activities.

Regulation of endothelial IGFBP-3 synthesis and secretion by IGF-I and TGF-beta.

We have examined the regulation of endothelial IGFBP-3 production by IGF-I and TGF-beta, two growth factors thought to play a major roles in the complications of diabetes mellitus. In addition, we developed a sensitive method for IGFBP-3 mRNA quantitation by adapting the fluorescent modification of the competitive PCR strategy. Our results using both Northern analysis and the fluorescent competitive PCR method indicate that: (1) IGFBP-3 mRNA is increased 2- to 10-fold by IGF-I and maximally reduced to 20% of control by TGF-beta; (2) the changes in mRNA levels correlate with the levels of IGFBP-3 protein secreted into the media by these cells; (3) the induction of IGFBP-3 mRNA and protein by IGF-I analogs was directly related to their ability to bind to the type I IGF receptor, reflecting an IGF-I receptor-mediated process; and (4) steady state IGFBP-3 mRNA levels did not change significantly after a 6 h incubation with actinomycin D in the presence or absence of the growth factors suggesting that the observed IGF-I/TGF-beta effects occur at the level of gene transcription rather than mRNA stability.

IGFBP-3 and IGFBP-5 association with endothelial cells: role of C-terminal heparin binding domain.

IGFBP-3 and IGFBP-5, but not the other 4 IGF binding proteins, specifically bound to endothelial cell (EC) monolayers. Charged compounds, such as heparin and heparan sulfate, competed for this binding. Of the 6 IGFBPs, IGFBP-3 and IGFBP-5 had the greatest heparin affinity. Peptides of 18 amino acids were synthesized, corresponding to a common basic region of IGFBP-3 (P3), IGFBP-5 and IGFBP-6 (P6) which contained a heparin binding sequence. P3 and P6 inhibited IGFBP-3 and -5 binding to endothelial cell monolayers and the peptides bound directly to EC extracellular matrix. This suggested that the C-terminal basic segment of IGFBP-3/-5 is important for the association of the binding protein with the EC monolayer.

Endothelial cells express insulin-like growth factor-binding proteins 2 to 6.

Cultured endothelial cells have been shown to produce insulin-like growth factor-binding proteins (IGFBPs); however, the identity of these BPs has not been defined. We now demonstrate that cultured bovine endothelial cells produce IGFBP2, IGFBP3, and IGFBP4 and have mRNA specific for IGFBP2, -3, -4, -5 and -6. DNA probes for bovine IGFBP2-6 were obtained by polymerase chain reaction (PCR) amplification of cDNA from bovine large vessel pulmonary artery and aortic endothelial cells as well as omental and periaortic fat microvessel cells, using oligonucleotide primers whose sequences were based on the reported cDNA sequences of IGFBP2-6. The PCR-derived probes were labeled with 32P and used for Northern blot analysis of RNAs obtained from the four bovine endothelial cell types. Transcripts corresponding to IGFBP2-6 were found in RNA from large vessel endothelial cells (bovine pulmonary artery and bovine aorta) and microvessel cells (periaortic and omental fat). The PCR-derived probe for IGFBP4 was used to screen a bovine pulmonary artery cDNA library for a full-length bovine IGFBP4 cDNA clone. One positive clone, containing a single EcoRI insert of approximately 2.0 kilobases, was selected for further characterization by DNA sequence analysis. This clone contained an open reading frame encoding a 258-amino acid protein that was 97% identical to human IGFBP4, 268 basepairs of 5'-untranslated region, and a longer 1044 basepairs of 3'-untranslated region. IGFBP4 protein was purified from bovine pulmonary artery-conditioned medium, shown to have N-terminal amino acid sequence DEAIHCPPCSEEKLARCR (identical to human IGFBP4) and to be secreted in glycosylated and nonglycosylated forms. Immunoblots further demonstrated that microvessel cells, at early passage, secrete predominantly IGFBP2 and IGFBP3, while large vessel cells, at early and late passages, secrete IGFBP3 and IGFBP4. Thus, cultured bovine endothelial cells synthesize and secrete IGFBP2, IGFBP3, and IGFBP4 and have mRNA encoding IGFBP2-6. The production of specific IGFBPs by endothelial cells raises the interesting possibility that the vascular endothelium contributes to circulating and tissue levels of specific IGFBPs in vivo.

Insulin-like growth factor binding protein (IGFBP)4 accounts for the connective tissue distribution of endothelial cell IGFBPs perfused through the isolated heart.

Insulin-like growth factor binding protein 4 (IGFBP4) was purified to homogeneity from conditioned media of bovine pulmonary artery endothelial cells and shown to have the N-terminal amino acid sequence DEAIHCPPCS, a sequence unique to IGFBP4. The IGFBP4 was separated into predominantly glycosylated and nonglycosylated fractions, with each fraction separately perfused through isolated, beating rat hearts. Both forms of IGFBP4 crossed the capillary boundary of the heart and distributed primarily in subendothelial connective tissue components with a connective tissue/cardiac muscle distribution ratio of 20:1 for the glycosylated fraction and 27:1 for the nonglycosylated fraction. Perfused IGFBP1, 2, 3, and IGF-I also crossed the capillary boundary but in contrast to IGFBP4, preferentially localized in cardiac muscle with a connective tissue/muscle ratio of approximately 1:3. We conclude that the connective tissue distribution previously reported for IGFBPs in conditioned media of pulmonary artery endothelial cells is due to IGFBP4.

Interactions of cultured endothelial cells with TGF-beta, bFGF, PDGF and IGF-I.

Endothelial cells in culture synthesize the growth factors transforming growth factor beta (TGF-beta), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF) and, perhaps, insulin like growth factor I (IGF-I). We have previously demonstrated that IGF-I and PDGF have both high affinity receptors and stimulate glucose and AIB uptake in the microvessel cells under study and that IGF-I, but not PDGF, has similar high affinity receptors in cultured large vessel endothelial cells. In the present study, cultured bovine endothelial cells were exposed to these four growth factors to determine a) their effects on the acute metabolic processes of neutral amino acid (AIB) and glucose uptake and b) their interactions at the endothelial cell surface. In microvessel endothelial cells, each growth factor stimulated AIB and glucose uptake 2-4 fold whereas in large vessel endothelial cells only bFGF stimulated glucose uptake. Each growth factor had specific high affinity binding to the microvessel cells that was not influenced by the presence of the other growth factors. In large vessel endothelial cells, similar high affinity binding was present only for IGF-I and to a lesser degree TGF-beta. When cells were exposed to a given growth factor for 18 hours, homologous receptor downregulation was observed, with a maximal 60-95% decrease in surface binding. These findings suggest several potential levels of interaction of the growth factors TGF-beta, bFGF, PDGF and IGF-I in cultured vascular endothelial cells.

Intrinsic bioactivity of insulin-like growth factor-binding proteins from vascular endothelial cells.

Conditioned medium from cultured vascular endothelial cells contains material capable of stimulating acute metabolic processes in endothelial cells. The bioactivity of the conditioned medium is not caused by the copurification of known growth factors produced by the cells, in particular platelet-derived growth factor, basic fibroblast growth factor, or insulin-like growth factor (IGF)-I/II. We now demonstrate that the bioactivity is directly due to an IGF-binding protein(s) (ECBP) and, further, that the bioactive domain of the binding protein differs from the IGF-binding domain. Binding proteins (BPs) from cultured pulmonary artery endothelial cells were purified by sequential passage over sizing, multiplication-stimulating activity affinity, and hydrophobic columns. BP fractions were separated into those with and those without biological activity. The bioactive binding protein(s) was cross-linked with disuccinimidyl suberate to IGF-I or the recombinant IGF analog [1-27,Gly4,38-70]IGF-I (Analog). The IGF-I Analog, by itself, had minimal interaction with the type I IGF receptor in cultured microvessel endothelial cells and no intrinsic bioactivity, but did bind with high affinity to ECBP. All free BP and free IGF-I/Analog were removed from the cross-linked mixture by passage over gel filtration and IGF affinity columns. The cross-linked BP-IGF-I complex did not bind to the type I receptor of cultured endothelial cells, but did stimulate glucose and alpha-aminoisobutyric acid uptake in endothelial cells (approximately 2-fold increase); the magnitude of the response was nearly equal to the effect of ECBP or IGF-I alone. The BP-Analog complex also stimulated glucose and alpha-aminoisobutyric acid uptake, with the magnitude of the response approaching the effect of ECBP alone. The BP-Analog complex also did not react with type I IGF receptors on the cultured endothelial cells. We conclude 1) IGF-BP produced by endothelial cells possess intrinsic biological activity; 2) bioactivity of the BP(s) is retained when the IGF-binding domain of the BP is occupied by IGF-I or an inactive IGF-I analog; and 3) IGF-I bound to the bioactive BP does not react with its receptor and possesses minimal, if any, bioactivity in vitro.

Tissue localization of perfused endothelial cell IGF binding protein is markedly altered by association with IGF-I.

Perfused endothelial cell IGF binding proteins (ECBP) have been previously demonstrated to leave the microcirculation of the rat heart and distribute primarily in connective tissue elements of the heart. In the present study, ECBP have been crosslinked to IGF-I and the biologically inactive [1-27,gly4,38-70]-hlGF-I, an analog of IGF-I lacking the type I IGF receptor domain. The crosslinked ECBPs were perfused through the isolated rat heart and their tissue distributions determined. Both [ECBP-Analog] and [ECBP-IGF-I] left the microcirculation of the heart. [ECBP-Analog] preferentially localized in connective tissue elements with a muscle:connective tissue ratio of approximately 1:6, similar to the tissue distribution of perfused ECBP. In contrast, the [ECBP-IGF-I] complexes localized in cardiac muscle with a muscle to connective tissue ratio of approximately 3:1, virtually identical to the tissue distribution of IGF-I when the IGF-I is perfused through the heart in the absence of any binding proteins. We conclude that 1) ECBP in the presence of IGF will cross capillary boundaries and 2) the tissue distribution of [ECBP-IGF-I] is dictated by the IGF-I molecule.

Transcapillary permeability and subendothelial distribution of endothelial and amniotic fluid insulin-like growth factor binding proteins in the rat heart.

Insulin-like growth factor (IGF) binding proteins (IGFBP) were purified from conditioned media of cultured bovine endothelial cells (ECBP) and from human amniotic fluid (IGFBP-1), and then labeled by radioiodination. 125I-ECBP and 125I-IGFBP-1 were perfused through isolated beating rat hearts for 1 and 5 min, and the hearts fixed and analyzed for 125I-BP content and distribution. One to 4% of the perfused 125I-ECBP and 125I-IGFBP-1 crossed the capillary boundary. The ECBPs predominantly localized as intact 125I-BP in connective tissue elements of the heart with less 125I-BP in cardiac muscle. The ratio of 125I-ECBP in connective tissue: muscle (normalized to percent vol of these compartments) was greater than or equal to 10:1. In contrast, the IGFBP-1 had a greater affinity for cardiac muscle with ratios of 125I-IGFBP-1 in connective tissue:muscle of approximately 1:2. When 125I-IGF-I, in the absence of any BPs, was perfused through the hearts approximately 3-5% left the microcirculation and was found in subendothelial tissues. 125I-IGF-I localized primarily to cardiac muscle with a distribution of connective tissue:cardiac muscle of about 1:3. The findings in the isolated perfused heart were confirmed in intact animals. After 125I-IGFBP-1 was injected into anesthetized rats and allowed to circulate for 5 min, substantial radioactivity was associated with the heart. As in the isolated heart, the IGFBP-1 preferentially localized to cardiac muscle with a connective tissue:cardiac muscle ratio of 1:3. We conclude that IGFBPs produced by endothelial cells and the IGFBP-1 contained in amniotic fluid can cross the capillary boundaries of the rat heart, and that the ECBPs preferentially localize in connective tissue elements of the myocardium, whereas IGFBP-1 predominantly localizes in cardiac muscle.

Insulin differentially alters transcapillary movement of intravascular IGFBP-1, IGFBP-2 and endothelial cell IGF-binding proteins in the rat heart.

Insulin-like growth factor binding-proteins 1 and 2 (IGFBP-1, IGFBP-2) and endothelial cell IGF binding proteins (ECBP) were individually perfused through isolated beating rat hearts in the absence and presence of insulin. Insulin caused an increased movement of IGFBP-1 from the vascular space to tissues of the heart. Subendothelial content of IGFBP-1 was 110%, 126% (p less than .01) and 132% (p less than 0.05) of control hearts when perfused with 1, 10 and 100 ng/ml insulin, respectively. . In contrast, insulin treatment was associated with a decrease in ECBP content in cardiac tissue, being 83%, 62% (p less than 0.005) and 73% (p less than 0.05) of control when perfused with 1, 10 and 100 ng/ml insulin. The efflux of IGFBP-2 from the intravascular space was unaffected by insulin. The subendothelial tissue distribution of the transported binding proteins was not changed by insulin perfusion, with IGFBP-1 and IGFBP-2 localizing predominantly in cardiac muscle and ECBP having greater affinity for connective tissue elements. We conclude that in the perfused rat heart, insulin can differentially alter transcapillary movement of IGFBP-1, IGFBP-2 and endothelial cell IGF-binding proteins. Such insulin-facilitated changes could potentially mediate nutrient-dependent transport of IGF-I and IGF-II to peripheral tissues.

Insulin-like growth factor-binding proteins from vascular endothelial cells: purification, characterization, and intrinsic biological activities.

Insulin-like growth factor (IGF)-binding proteins are produced by several cell types, including vascular endothelial cells. The production of IGF-binding proteins by endothelial cells is of particular interest, since these cells are directly bathed by the circulating IGFs and form the initial barrier to the passage of circulating IGFs from the bloodstream to subendothelial tissues. We have purified IGF-binding proteins from medium conditioned by cultured bovine endothelial cells by sequential passage over Bio-Gel P-60, multiplication-stimulating activity affinity, anion exchange (DEAE cellulose) and/or hydrophobic (phenyl-Sepharose) chromatography. Two peaks of IGF-binding activity were eluted from the phenyl-Sepharose column. After cross-linking, each peak contained two to five protein bands on gels that specifically bound IGF-I and -II with mol wt ranging from about 28-44K. Material in peak 1 bound IGF-I congruent to IGF-II and had no affinity for insulin and proinsulin. Peak 2 IGF-binding proteins bound IGF-II with substantially higher affinity than IGF-I and did not recognize insulin or proinsulin. Peak 1 material from phenyl-Sepharose chromatography was a potent stimulator of both glucose transport and aminoisobutyric acid (AIB) uptake in microvessel endothelial cells, with maximal stimulation of both processes being 300-400% of control values. In contrast, peak 2 material either had no intrinsic bioactivity or was slightly inhibitory or stimulatory, depending on the concentration of peak 2 material that was added. The bioactivity in peak 1 was not due to copurification of other endothelial proteins capable of stimulating glucose and AIB uptake, such as IGF-I/-II, platelet-derived growth factor, and basic fibroblast growth factor, since bioactivity was retained after acid treatment, antibody neutralization, and selective affinity chromatography to deplete these other factors. When peak 1 material was added to IGF-I the bioeffects (glucose and AIB uptake) of IGF and binding proteins were additive, and in some experiments the binding proteins potentiated the effect of IGF on endothelial cells, suggesting that the binding protein-IGF complex may retain the bioactivity of both the binding protein(s) and the IGF.