Regenerating endothelial cells express insulin-like growth factor-I immunoreactivity after arterial injury

Summary In the present study the expression of insulin-like growth factor I (IGF-I; somatomedin C) immunoreactivity was examined in endothelial cells during repair after injury to the intima in the femoral artery of adult rats. Two types of injury were examined: (1) endothelial denudation induced by the use of a catheter, and (2) vessel compression by short-term ligation. In untreated rats, arterial endothelial cells showed no or, only infrequently, low IGF-I immunoreactivity in their cytoplasm. Endothelial cells at the border to the denuded area showed increased IGF-I immunoreactivity one day after injury to the intima of the femoral artery. Thrombocytes and fibrin deposits as well as vital endothelial cells, covered by clots, were immunonegative. The maximal intensity of IGF-I immunoreactivity was reached within 3 days after insult. The IGF-I immunoreactivity in the endothelial cells remained elevated for at least 4 weeks, compared to the controls. Intimai thickenings appeared within a week after injury and many cells in these thickenings showed intense IGF-I immunoreactivity as did the covering endothelial cells. Smooth muscle cells in the media were generally immunonegative during control conditions and after endothelial denudation. Spontaneously hypertensive rats (SHR) showed, similarly to their matched controls (WKY), approximately the same patterns of IGF-I immunoreactivity in their endothelial cells both under normal conditions and after injury. It is concluded that IGF-I is likely to be involved in the repair of the intima in injured arteries.     

Hormonal regulation of insulin-like growth factor I secretion by bovine adrenal cells

The role of insulin-like growth factor I (IGF-I) on the specific function of several steroidogenic cells has been recently reported. Since IGF-I is produced by several tissues, we have investigated whether bovine adrenal cells secrete this peptide. Purification of conditioned medium from adrenal cells incubated with [35S]methionine through affinity chromatography (monoclonal anti-IGF-I antibody), high pressure liquid chromatography, and polyacrylamide gel electrophoresis revealed a single band of similar Mr as pure recombinant IGF-I. Moreover, the purified adrenal-secreted IGF-I displaced bound 125I-IGF-I to its adrenal receptors, and pretreatment of adrenal cells with the purified peptide enhanced the acute corticotropin (ACTH)-induced cAMP production as recombinant IGF-I. The basal secretion of IGF-I (6 +/- 1 ng/48 h/10(6) cells) was stimulated 3-, 4.5-, and 9.5-fold by fibroblast growth factor, angiotensin II (A-II), and ACTH, respectively, but not by growth hormone. The stimulatory effects of A-II and ACTH were dose-dependent (ED50 congruent to 2.5 x 10(-8) and 1.5 x 10(-10) M, respectively), and the effects of both hormones were additive. Glucocorticoids were not the mediators of the effect of the two hormones on IGF-I secretion, since inhibition of their steroidogenic action by aminoglutethimide did not significantly modify IGF-I secretion. An immunoreactive IGF-I material was also secreted by mouse adrenal tumor cell line Y-1, but the stimulatory effect of ACTH was only 2-fold, and there was no effect of A-II. Since bovine adrenal cells contain specific IGF-I receptors and this peptide is required for the maintenance of some adrenal cell-specific function, the present data suggest that IGF-I may act in an autocrine fashion to stimulate adrenal cell differentiation stimulated by ACTH and A-II.     

Lipid synthesis and lipoprotein secretion by chick liver cells in culture: influence of growth hormone and insulin-like growth factor-I

1. Chick liver cells were incubated in unsupplemented medium (control), or medium supplanted with either 1 microgram/ml pituitary derived chicken growth hormone (GH), 50 ng/ml recombinant human insulin like growth factor-I (IGF-I), or 1 microgram growth hormone/ml and 50 ng insulin like growth factor-I/ml (GH + IGF-I). 2. GH supplementation stimulated acetate incorporation into liver cell lipid. Low density lipoprotein (LDL) lipid secretion was increased quantitatively by GH. 3. Cells incubated with IGF-I incorporated more acetate into lipid and secreted more lipid as VLDL and HDL than controls. 4. A metabolic antagonism between GH and IGF-I was evident with respect to lipogenesis. 5. Neither GH nor IGF-I altered, quantitatively, cell protein synthesis or apoprotein secretion.     

Enhanced secretion of endothelin-1 by elevated glucose levels from cultured bovine aortic endothelial cells

We have investigated the effect of glucose on the release of endothelin-1-like immunoreactivity (ET-1-LI) from cultured bovine aortic endothelial cells. Elevation of glucose concentrations in cultured media from 5.5 to 11.1 or 22.2 mM significantly stimulated ET-1-LI release from cultured endothelial cells. An aldose reductase inhibitor did not affect the high glucose-induced ET-1-LI release. These findings suggest the possibility that hyperglycemia in diabetic patients enhances ET-1-LI release at the local site of vascular endothelium, which might be involved in the developments of vascular complications and atherosclerosis.     

Internalization and degradation of heparin binding growth factor-I by endothelial cells

The fate of 125I-labeled heparin binding growth factor I (125I-HBGF-I) after binding to its cell surface receptor has been studied using murine lung capillary endothelial cells (LEII). Binding of 125I-HBGF-I to its receptor at 4 degrees C shows pH dependence with optimal binding at pH 6.5-7.5. The majority (approximately 80%) of 125I-HBGF-I bound to cells at 4 degrees C can be removed by washing with low pH medium, but rapidly becomes acid resistant upon shifting cells to 37 degrees C, with 50% of the 125I-HBGF-I becoming acid resistant after 20 minutes. Electrophoretic analysis of internalized 125I-HBGF-I shows that degradation begins approximately 2 hours after internalization with the appearance of two major labeled fragments of Mr 15,000 and Mr 10,000. Degradation of internalized 125I-HBGF-I is inhibited by the lysosomotropic agent chloroquine. These data suggest that cell-associated 125I-HBGF-I is rapidly internalized and directed to a lysosomal cellular compartment where it is slowly degraded.     

Overexpression of ovine insulin-like growth factor-I stimulates autonomous autocrine or paracrine growth in bovine mammary-derived epithelial cells.

To test the hypothesis that insulin-like growth factor-I (IGF-I) affects the growth of bovine mammary epithelial cells through an autocrine and/or paracrine pathway, a cell line (MD-IGF-I) was originated from MAC-T cells by cotransfection with a construct containing the cDNA for an ovine exon 2-encoded prepro-IGF-I under control of the mouse mammary tumor virus-long terminal repeat promoter. Clone MD-IGF-I contained multiple copies of the plasmid integrated into the genome, expressed the highest level of IGF-I mRNA, and secreted radioimmunoactive IGF-I into the medium. The mitogenic activity of MD-IGF-I cells was stimulated 80% by dexamethasone (DEX). The total DNA in MD-IGF-I cells was 2.5-fold higher than that in parental MAC-T cells in the presence of DEX. Conditioned medium from MD-IGF-I cells, induced with DEX, stimulated [3H]thymidine incorporation into DNA of MAC-T cells and uninduced MD-IGF-I cells. These data provide evidence that IGF-I was secreted into medium by MD-IGF-I cells. It is suggested that IGF-I can stimulate the growth of mammary epithelial cells by an autocrine and/or paracrine mode of action. The MD-IGF-I cell line may be a suitable system to study translational and posttranslational modifications of IGF-I peptides.     

Treatment with recombinant bovine somatotrophin enhances ovarian follicle development and increases the secretion of insulin-like growth factor-I by ovarian follicles in ewes

To investigate the effect of recombinant bovine somatotrophin (rGH) on ovarian folliculogenesis in sheep, 18 mature Scottish Blackface ewes were assigned randomly to two treatment groups. Starting from day 5 of the synchronised oestrous cycle, animals were injected daily with either vehicle (control group) or 12.5 mg rGH (rGH-treated group) for 7 days. Blood samples were collected once daily during the experimental period for the measurement of growth hormone (GH), insulin-like growth factor-I (IGF-I), insulin, follicle-stimulating hormone (FSH), luteinising hormone (LH) and progesterone. At the end of treatment animals were killed and ovaries collected. All follicles at least 1.0 mm in diameter were dissected out and diameters measured to assess follicular populations for individual animals. Five small follicles (1.0–3.4 mm in diameter) and all the large follicles (at least 3.5 mm) from each animal were incubated in 1 ml of Medium 199 for 1 h. Medium was then changed and incubation continued for a further hour. All medium samples were assayed for IGF-I, oestradiol, testosterone and progesterone.Treatment of ewes with rGH had no effect on the total number of follicles at least 1.0 mm in diameter (control, 34.4 ± 2.6; rGH-treated, 31.3 ± 1.4; P > 0.2). However, when follicles were further classified into different size categories (1.0–2.0, 2.1–3.0, 3.1–4.0, 4.1–5.0, 5.1–6.0 and over 6.0 mm in diameter), the population of follicles 2.1–3.0 mm in diameter was significantly increased by rGH treatment (control, 9.2 ± 0.7; rGH-treated, 13.8 ± 1.1; P = 0.02). The number of follicles of 3.1–4.0 mm diameter in the rGH-treated group tended to be increased (P = 0.09), whilst the population of follicles 1.0–2.0 mm in diameter was reduced (P = 0.07). Treatment of ewes with rGH significantly increased peripheral concentrations of GH (P < 0.01), IGF-I (P < 0.01), insulin (P < 0.01) and progesterone (P < 0.05). There was no effect of rGH treatment on circulating concentrations of FSH and LH. Both large and small follicles from rGH-treated ewes secreted significantly (P < 0.001) more IGF-I (37.8 ± 2.2 ng ml h⁻¹, n = 50) than follicles from the control group (26.7 ± 1.6 ng ml⁻¹ h⁻¹, n = 73). However, there was no significant effect of rGH treatment on the secretion of oestradiol, testosterone and progesterone by either large or small follicles.It is concluded that treatment of mature ewes with rGH can enhance the development of ovarian follicles to the gonadotrophin-dependent stages. Furthermore, rGH appears to act through increased secretion of ovarian IGF-I, as well as increased peripheral concentrations of IGF-I and insulin.     

Plasminogen activator activity by cultured bovine aortic endothelial cells

This communication reports the observations that bovine aortic endothelial cells (EC) in culture during their log phase of growth secrete plasminogen activator. Hydrocortisone, dibutyryl cAMP, theophylline, colchicine and cycloheximide, dependent upon concentration, inhibit plasminogen activator activity. Several substances associated with inflammation and thrombosis, such as thrombin, serotonin,catecholomines, histamine, vasopressin, endotoxin, and indomethacin, at the concentrations tested, did not significantly alter plasminogen activator activity when compared with controls.     

Influences of epidermal growth factor and insulin-like growth factor-I on bovine blastocyst development in vitro

Experiments were carried out to investigate putative beneficial effects of adding epidermal growth factor (EGF) or insulin-like growth factor-I (IGF-I) for bovine embryo culture in chemically defined media. Presumptive zygotes (18 h post-insemination) were randomly assigned to culture treatments. In experiment 1, treatments involved additions of recombinant human EGF to provide concentrations of 0 ng (control), 1, 5, and 25 ng/ml. No differences were seen in numbers of 4-cell stage embryos between groups. A concentration of 5 ng/ml EGF but not 1 or 25 ng/ml during embryo culture improved percentages of 4-cell stage embryos reaching blastocysts compared to the control (P<0.05). Numbers of inner cell mass (ICM) cells and trophoblast cells of day 8 blastocysts were similar for the control and 5 ng/ml EGF-treated groups. In experiment 2, culture with recombinant human IGF-I in concentrations of 0 ng (control), 2, 10, and 50 ng/ml resulted in no differences in numbers of 4-cell stage embryos between groups. When compared to controls, IGF-I treatments at 10 and 50 ng/ml improved proportions of 4-cell stage embryos that reached blastocysts (P<0.05). In experiment 3, numbers of ICM cells of day 8 blastocysts were significantly higher after being cultured with 50 ng/ml of IGF-I compared to those of the controls (P<0.05). No additive effect of combining EGF (5 ng/ml) and IGF-I (50 ng/ml) was seen when results were compared to those following supplementation of the media with either EGF or IGF-I alone. In conclusion, both EGF and IGF-I could independently enhance bovine preimplantational development in chemically defined media and IGF-I but not EGF may play a mitogenic role during early bovine development.     

Stimulation of proteoglycan biosynthesis by serum and insulin-like growth factor-I in cultured bovine articular cartilage.

The addition of foetal calf serum to explant cultures of adult bovine articular cartilage is known to stimulate proteoglycan synthesis in a dose-dependent manner. We have now shown the activity in serum responsible for this effect to be heat- and acid-stable, to be associated with a high-Mr complex in normal serum but converted to a low-Mr form under acid conditions. The activity has an apparent Mr approximately 10,000 and isoelectric points similar to those reported for insulin-like growth factors (IGFs). Addition of a monoclonal antibody against insulin-like growth factor-I (IGF-I) prevented foetal calf serum from stimulating proteoglycan synthesis. Physiological concentrations of recombinant IGF-I or pharmacological levels of insulin when added to cartilage cultures mimicked the proteoglycan-stimulatory activity of serum. IGF-I appeared to act by increasing the rate of proteoglycan synthesis and did not change the nature of the proteoglycan synthesized nor the rate of proteoglycan catabolism by the tissue, suggesting that IGF-I may be important in the regulation of proteoglycan metabolism in adult articular cartilage. Furthermore, IGF-I can replace foetal calf serum in the culture medium, thereby allowing the use of a fully-defined medium which will maintain the synthesis and tissue levels of proteoglycan in adult articular cartilage explants for up to 5 days.     

The effect of insulin-like growth factor-I, relaxin and luteinizing hormone on vascular endothelial growth factor secretion by cultured endometrial stromal cells on different days of early pregnancy in pigs

The effect of insulin-like growth factor-I (IGF-I), relaxin (RLX) and luteinizing hormone (LH) on vascular endothelial growth factor (VEGF) in vitro secretion by endometrial stromal cells in pigs was investigated on days 10-12 and 20-22 of gestation. LH-stimulated stromal cell secretion of VEGF did not differ among tested days of early pregnancy. However, IGF-I- and RLX-mediated release of VEGF depended on the day of pregnancy. It seems that IGF-I and RLX may be considered as potent activators of VEGF-mediated angiogenesis in porcine endometrium, and their action may be more pronounced during maternal recognition of pregnancy.     

Selection and characterization of bovine aortic endothelial cells

This paper reports techniques for isolation, selection and long-term passage of bovine aortic endothelium (BAE). A [3H]thymidine-selection technique was developed to limit overgrowth of cultures by contaminating smooth-muscle cells. The resulting cultures could be passaged for a replicative life span of 35 to 40 doublings and maintained a stable, normal karyotpye throughout this period. Despite the fact that these cultures reached a stable monolayer with density-inhibited growth state, postconfluent cells showed focal areas of a second growth pattern called "sprouting." This was seen only when cultures were maintained at high densities for periods of 1 to 2 weeks. Ultrastructural analysis, as well as immunofluorescence studies with markers for endothelial cells (factor VIII) and smooth-muscle cells (actin), indicates that this phenomenon is not due to overgrowth of a residual population of smooth-muscle cells, but may represent a second growth pattern of the endothelial cells themselves.     

Regulation of production of a platelet-derived growth factor-like protein by cultured bovine aortic endothelial cells

Platelet-derived growth factor (PDGF) is a potent mitogen for cultured cells of mesenchymal origin. Known sources of PDGF or PDGF-like protein are blood platelets, several transformed cell lines, and cultured endothelial cells (EC). We have examined the regulation of production of a PDGF-like protein in cultures of bovine aortic EC using a specific radioreceptor assay for PDGF. EC constitutively secreted PDGF-like protein into serum-containing or serum-free medium. The rate of production of PDGF-like protein was constant for at least 3 weeks and was not due to release of an internal store, since cell lysis by repeated freeze/thaw cycles did not relase significant amounts of the protein. Synthesis of PDGF-like protein was sensitive to changes in the pH of the media and was maximal at pH 8.5. Production of PDGF-like protein was independent of EC growth rate: rapidly dividing cells and confluent, quiescent cells produced equal amounts per cell. However, sparse, quiescent EC produced more PDGF-like protein per cell than did confluent, quiescent cells. Several phorbol esters stimulated production of PDGF-like protein. At a concentration of 10^?6 M, a twofold stimulation was observed upon addition of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and nearly a fourfold stimulation upon addition of the nonpromoting analog, methyl TPA. Incubation of EC with endotoxin (10 μ/ml) resulted in a twofold stimulation of PDGF-like protein production. In all experiments with endotoxin and phorbol esters, an increase in the production of PDGF-like protein was accompanied by morphological changes in the EC cultures. The cells appeared elongated and fibroblastic and exhibited low viability. A mathematical model was developed in which PDGF-like protein production was shown to consist of two separate components—production at a constant rate by healthy cells and a large burst of synthesis and secretion by dying cells. These results suggest that injurious agents may be capable of stimulating production of a growth factor by the endothelium.     

Growth regulation by insulin-like growth factor-I in fish

Insulin-like growth factor-I (IGF-I) is a mitogenic polypeptide that plays an essential role in the regulation of development and somatic growth of vertebrates, mainly by mediating growth hormone actions. It has clearly been established that the structure of IGF-I and its biological function has been highly conserved among vertebrates. In this paper, we review the recent developments in the molecular, biochemical, and physiological properties of IGF-I in fish.     

Selection and characterization of bovine aortic endothelial cells

Summary This paper reports techniques for isolation, selection and long-term passage of bovine aortic endothelium (BAE). A [³H]thymidine-selection technique was developed to limit overgrowth of cultures by contaminating smooth-muscle cells. The resulting cultures could be passaged for a replicative life span of 35 to 40 doublings and maintained a stable, normal karyotype throughout this period. Despite the fact that these cultures reached a stable monolayer with density-inhibited growth state, postconfluent cells showed focal areas of a second growth pattern called “sprouting.” This was seen only when cultures were maintained at high densities for periods of 1 to 2 weeks. Ultrastructural analysis, as well as immunofluorescence studies with markers for endothelial cells (factor VIII) and smooth-muscle cells (actin), indicates that this phenomenon is not due to overgrowth of a residual population of smooth-muscle cells, but may represent a second growth pattern of the endothelial cells themselves. This research was supported by NIH Grant HL 18645. This work was done during the tenure of an Established Investigationship of the American Heart Association.     

Effects of growth hormone and insulin-like growth factor-I on development of in vitro derived bovine embryos

The objectives of this study were to determine whether the addition of growth hormone (GH) to maturation medium and GH or insulin-like growth factor-I (IGF-I) to culture medium affects development of cultured bovine embryos. We matured groups of 10 cumulus-oocyte complexes (COCs) in serum-free TCM-199 medium containing FSH and estradiol with or without 100 ng/ml GH. After fertilization, we transferred groups of 10 putative zygotes to 25 microl drops of a modified KSOM medium containing the following treatments: non-specific IgG (a control antibody, 10 microg/ml); GH (100 ng/ml) + IgG (10 microg/ml, GH/IgG); IGF-I (100 ng/ml) + IgG (10 microg/ml, IGF/IgG); antibody to IGF-I (10 microg/ml, anti-IGF); GH (100 ng/ml) + anti-IGF (10 microg/ml GH/anti-IGF); IGF-I (100 ng/ml) + anti-IGF (10 microg/ml, IGF/anti-IGF); no further additions (control). We repeated the experiment six times. Adding GH to the maturation medium increased cleavage rates at Day 3 compared to control (87.3 +/- 1.2% > 83.9 +/- 1.2%; P < 0.05) but had no effects on blastocyst development at Day 8. At Day 8, blastocyst development was greater (P < 0.01) for GH/IgG (24.8 +/- 2.5%) and IGF/IgG (33.7 +/- 2.5%) than for IgG (16.1 +/- 2.1%) and greater for IGF/IgG than for GH/IgG (P < 0.02). Blastocyst development at Day 8 did not differ between anti-IGF (20.4 +/- 1.8%) and GH/anti-IGF (24.1 +/- 1.9%) or IGF/anti-IGF (17.7 +/- 1.9%), but it was greater for GH/anti-IGF than for IGF/anti-IGF (P < 0.05). The Day 8 blastocysts of GH/IgG and IGF-I/IgG groups had a higher (P < 0.01) number of cells than the IgG group. The addition of anti-IGF-I eliminated the effects of IGF-I on cell number but did not alter GH effects. In conclusion, both GH and IGF-I stimulate embryonic development in cattle and GH effects may likely involve IGF-I-independent mechanisms.     

Binding of insulin-like growth factor-I by glomerular endothelial and epithelial cells: further evidence for IGF-I action in the renal glomerulus

The renal glomerulus is both a site of action and synthesis of IGF-I. We previously demonstrated the presence of IGF-I receptor and synthesis in glomerular mesangial cells. In this study we investigated the presence of specific IGF-I receptors on mouse glomerular endothelial and epithelial cells in culture. [125I]IGF-I specifically bound to the cell surface of both cell types. Maximum specific binding, 0.141 B/F for endothelial cells and 0.301 B/F for epithelial cells, was obtained at 22 degrees C after 150 min incubation. The estimated Kd values were 2.25 x 10(-9) for endothelial cells and 1.5 x 10(-9) for epithelial cells. Cross-linking studies showed a single band of radioactivity with an estimated mol wt of 145K, consistent with the alpha-subunit of the IGF-I receptor. Radiolabelled IGF-I was not degraded by either cell types. These findings suggest a possible paracrine action of IGF-I in the renal glomerulus.     

Biosynthesis and release of oxytocin by granulosa cells derived from preovulatory bovine follicles: effects of forskolin and insulin-like growth factor-I.

Granulosa cells derived from preovulatory bovine follicles were cultured in the presence of insulin-like growth factor-I (IGF-I, 10-100 ng/ml), forskolin (10 microM), or a combination of the two agents. Forskolin alone was the most potent stimulator of both oxytocin (OT) and progesterone (P4) secretion. The two hormones had different patterns of secretion during the course of incubation. OT production peaked on Day 5 of culture and declined thereafter, whereas P4 rose gradually to a peak between Days 7 and 9. The addition of IGF-I to forskolin did not augment OT release beyond that achieved with forskolin alone, but it did maintain higher levels of OT secretion beyond the Day-5 peak. Two antisera, (antiserum I and antiserum II) directed against OT and its C-terminally extended forms, respectively, were used to identify the OT forms in culture media and granulosa cell and corpus luteum extracts. Fully processed OT was detected only in small amounts (0.43 ng/mg protein) in granulosa cell extracts, whereas the corpus luteum extracts contained 6 ng/mg protein. However, granulosa cells that had been incubated with forskolin contained stores of the OT precursor oxytocin-neurophysin, which is found in young corpora lutea. These data indicate that forskolin (whose action probably mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release in cultured bovine granulosa cells.