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Endocrinology Vol. 141, No. 6 2062-2067
Copyright © 2000 by The Endocrine Society

ARTICLES

Vascular Endothelial Growth Factor and Transforming Growth Factor-ß1 Regulate the Expression of Insulin-Like Growth Factor-Binding Protein-3, -4, and -5 in Large Vessel Endothelial Cells1

Gunilla Dahlfors and Hans J. Arnqvist

Departments of Cell Biology (G.D., H.J.A.) and Internal Medicine (H.J.A.), Faculty of Health Science, University of Linkoping, S-581855 Linkoping, Sweden

Address all correspondence and requests for reprints to: Dr. Gunilla Dahlfors, Department of Biomedicine and Surgery, Division of Cell Biology, Floor 10, Faculty of Health Sciences, Linkoping University, S-58185 Linkoping, Sweden. E-mail: gunda{at}mcb.liu.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the effect of diabetes-associated growth factors on the expression of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs) in cultured endothelial cells from bovine aorta. Gene expression was measured by solution hybridization, and proteins were measured by enzyme-linked immunosorbent assay, RIA, or Western blot. The cells expressed messenger RNA (mRNA) for IGFBP-2 through -6 and IGFBP-2 through -5 proteins were detected in conditioned medium. Vascular endothelial growth factor inhibited IGFBP-3 mRNA (P < 0.01) and protein expression and increased IGFBP-5 mRNA (P < 0.001) and protein. Transforming growth factor-ß1 inhibited IGFBP-3 (P < 0.01), IGFBP-4 (P < 0.01), and IGF-I mRNA expression, whereas at the protein level only IGFBP-3 was significantly decreased. IGF-I, insulin, or angiotensin II did not affect IGF-I or IGFBP mRNA expression. At the protein level, IGF-I clearly increased IGFBP-5 levels in conditioned medium. In conclusion, vascular endothelial growth factor and transforming growth factor-ß1 regulate IGFBP expression in bovine aortic endothelial cells. These observations provide a new aspect of regulation for the IGF-system in macrovascular endothelium, with possible implications for subendothelial smooth muscle cells and development of diabetic angiopathy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ENDOTHELIUM possesses a unique position in the vascular wall by constituting the border between the arterial wall and the components in the blood. By producing growth factors and other molecules such as nitric oxide, the endothelium regulates different processes in the underlying smooth muscle cells, including metabolism, migration, proliferation, apoptosis, and production of extracellular matrix (1). Disorders of endothelial function are closely associated with vascular diseases (2). In diabetes mellitus both endothelial dysfunction and an increased prevalence of vascular disease are found (3). Different growth factors and hormones, such as vascular endothelial growth factor (VEGF), transforming growth factor-ß (TGFß), angiotensin II, insulin-like growth factor-I (IGF-I), and insulin, have been suggested to be involved in the development of diabetic angiopathy (1, 4, 5).

IGF-I is a known mitogen and regulator of smooth muscle cells (6). The bioactivity of IGF-I is modulated by six binding proteins, IGFBP-1 through -6 (7). Tissue levels of IGF-I and IGFBPs are dependent on local production and may also be derived from the circulation. Studies with targeted overexpression of IGF-I and IGFBP-4 suggest that local production of components in the IGF system is of importance in the regulation of vascular smooth muscle cells (8, 9). Expression of IGFBP-2 through -6 has been reported in endothelial cells (10), although large species and tissue differences are seen (10, 11, 12, 13). The expression of IGFBPs is influenced by various conditions and growth factors. In endothelial cells, IGFBP expression has been reported to be regulated by cell density (14), glucose (12), hypoxia (13), TGFß1, and IGF-I (15). Of these, only the studies on cell density and hypoxia were made on large vessel endothelial cells. To investigate a possible role for large vessel endothelium in regulating the vascular IGF system in diabetes, we studied the effects of different growth factors associated with diabetic complications on IGF-I and IGFBP expression in bovine aortic endothelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Bovine aortic endothelial cells were isolated and cultured according to a modified method of Liu and colleagues (16). Fresh bovine aortas were obtained from a local slaughterhouse and placed in PBS with penicillin (100 U/ml), streptomycin (100 µg/ml), and fungizone (2 µg/ml). Within 1 h, the endothelial cells were isolated by a single gentle scraping of the endothelial surface with a scalpel blade. After isolation, the cells were seeded in 75-cm2 culturing flasks in culture medium consisting of medium 199 with 50 µg/ml gentamicin sulfate, 2 µg/ml fungizone, and 20% FCS. The cells were cultured in a humidified atmosphere of 5% CO2 in air, and medium was changed twice a week. The cells were harvested for passaging at confluence with a trypsin-EDTA (0.05% trypsin and 0.02% EDTA) solution. Cultured cells were characterized as endothelial cells by morphological criteria and by indirect immunofluorescence microscopy after staining for factor VIII-related antigen. Experiments were performed with cells at passages 14–20. Before experiments, cells were made quiescent by a 24-h incubation in serum-free medium containing 50 µg/ml gentamicin sulfate in medium 199.

Measurements of messenger RNA (mRNA) by solution hybridization
Endothelial cells were plated on petri dishes and grown until confluent. Quiescent cells were incubated with different substances as indicated for 18 h. The incubation time was based on a report by Erondu and co-workers in which the time course for the regulation of IGFBP-3 expression in microvessel endothelial cells was studied (15). The cells were then solubilized with 1 x SET buffer (1% SDS, 20 mM Tris, and 10 mM EDTA, pH 7.5) and homogenized with a Polytron (Ultra Turrax T25, Janke & Kunkel, Staufen, Germany). Nucleic acids were extracted as described previously (17). Samples were digested with proteinase K and extracted with phenol and chloroform. Nucleic acids were precipitated with ethanol. Total nucleic acids were measured by spectrophotometry. DNA content was measured by fluorometry according to a method described previously (17).

The mRNA levels for IGF-I and IGFBP-1 through -6 were determined using [35S]UTP-labeled RNA probes. The IGFBP-1 probe was synthesized from 350 bp of a human complementary DNA (cDNA) (18), the IGFBP-2 probe was synthesized from 446 bp of a human cDNA (19), the IGFBP-3 probe was synthesized from 475 bp of a human cDNA (20), the IGFBP-4 probe was synthesized from 505 bp of a human cDNA (21), the IGFBP-5 probe was synthesized from 317 bp of a human cDNA (22), and the IGFBP-6 probe was synthesized from 267 bp of a human cDNA (23). The IGF-I probe was synthesized from 775 bp of a human cDNA (from Dr. Peter Rotwein, Portland, OR). The probes were prepared as described previously (17) and were hybridized to total nucleic acid samples at 70 C for 20 h. Hybridization was performed in 40 µl 0.6 M NaCl, 20 mM Tris-HCl (pH 7.5), 4 mM EDTA, 0.1% SDS, 0.75 mM dithiothreitol, 25% formamide, and 20,000 cpm [35S]UTP-labeled probe/incubation. The samples were exposed to ribonucleases, the hybrids were precipitated with 100 µl trichloroacetic acid (TCA; 6 M) and collected on glass microfiber filters, and radioactivity was measured in a liquid scintillation counter (1214 Rackbeta, LKB, Gaithersburg, MD). The radioactivity of each sample was then compared with a standard curve constructed from a sample with a known amount of in vitro synthesized sense RNA complementary to the probe. A standard curve was included in each assay, and samples were analyzed in triplicate. Tubes including only hybridization buffer and probe served as blanks.

Detection of proteins in conditioned medium
Endothelial cells were cultured as described above and stimulated for 18 h with the indicated substances in serum-free medium. Proteins secreted into the medium, conditioned for 18 h, were then precipitated with TCA. Fifty microliters of 0.1% BSA and 500 µl 100% TCA were added to 10 ml conditioned medium to precipitate the proteins. Samples were incubated at 4 C overnight and centrifuged at 11,000 rpm for 30 min. The pellet was dissolved in 600 µl electrode buffer. For Western blot, the proteins were separated on a 10–15% SDS-PAGE and then electrotransferred onto a polyvinylidene difluoride membrane and blocked overnight with 0.2% polyvenyl alcohol dissolved in Tris-buffered saline. The membrane was washed in TBS-T (0.1% Tween in Tris-buffered saline) and immunoblotted for 1 h with specific primary antibodies using a 1:2,000 dilution for the IGFBP-2 and -4 antibody and a 1:1,000 dilution for the IGFBP-5 antibody. The membrane was then washed and incubated with horseradish peroxidase-linked anti-IgG and then washed again before detection with an enhanced chemiluminescence detection system.

For detection and quantitation of IGF-I, samples were measured with a commercially available RIA for IGF-I (Nichols Institute Diagnostics, San Juan Capistrano, CA). Before assay, IGF-I was separated from binding proteins by acid-ethanol precipitation and measured according to the manufacturer’s instructions.

IGFBP-3 was measured with a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Diagnostics Systems Laboratories, Inc., Webster, TX) according to the manufacturer’s instructions.

Chemicals and solutions
Recombinant, human TGFß1 and VEGF165 were purchased from R\|[amp ]\|D Systems (Abingdon, UK), and human recombinant IGFBP-4 and -5 were obtained from Austral Biologicals (San Ramon, CA). Angiotensin II was obtained from Sigma (St. Louis, MO). IGF-I was purchased from Pharmacia & Upjohn, Inc. (Stockholm, Sweden), and human regular insulin was obtained from Novo Industries A/S (Copenhagen, Denmark). Antibodies for IGFBP-2 were purchased from Upstate Biotechnology, Inc. (Lake placid, NY), and antibodies for IGFBP-4 and -5 were obtained from Austral Biologicals (San Ramon, CA). Factor VIII-related antigen was purchased from Dakopatts (Dakopatts A/S, Glostrup, Denmark). The RIA for IGF-I was purchased from Nichols Institute Diagnostics, and the ELISA kit for IGFBP-3 was obtained from Diagnostics Systems Laboratories, Inc.. [35S]UTP was purchased from Amersham International (Aylesbury, UK), and chemicals for probe synthesis were obtained from Promega Corp. (Madison, WI). Ribonucleases, proteinase K, and herring sperm DNA were purchased from Roche (Mannheim, Germany); and phenol was purchased from Fisher Scientific (Fairlawn, NJ). Chemicals and solutions for cell culture were obtained from Life Technologies, Inc. (Taby, Sweden).

Statistics
Values are given as the mean ± SE. Statistical analysis of the data was performed using ANOVA Scheffe’s F test or Fisher’s protected least significant difference. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The gene expression of IGFBP-1–6 was measured in bovine endothelial cells (Fig. 1Go). IGFBP-3, -4, and -5 mRNA were excessively expressed: 1.9 ± 0.3, 7.1 ± 0.7, and 8.4 ± 1.3 amol/µg DNA, respectively. IGFBP-2 and -6 expression were at the detection limit, and IGFBP-1 mRNA could not be detected. The corresponding proteins, IGFBP-2 (Fig 2Go), IGFBP-3 (Fig. 3bGo), IGFBP-4 (Fig. 4bGo), and IGFBP-5 (Fig. 5bGo), could be detected in conditioned medium. IGFBP-1 and -6 proteins were not measured.



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  		Figure 1. Basal expression of IGFBP-1 through IGFBP-6 (bars 1–6) mRNA in endothelial cells from bovine aorta. Levels of mRNA were measured by solution hybridization. Bars are the mean ± SE of duplicate measurements from six experiments. N. D., Not detected; #, expression was at the limit of detection (~0.1 amol/µg DNA).

 


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  		Figure 2. IGFBP-2 in conditioned medium from confluent quiescent bovine aortic endothelial cells measured by Western immunoblot. Two control samples are shown.

 


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  		Figure 3. A, Expression of IGFBP-3 mRNA in control cells (a) and cells stimulated with VEGF (b), TGFß1 (c), angiotensin II (d), IGF-I (e), or insulin (f). Endothelial cells from bovine aorta were starved for 24 h before the addition of growth factors for 18 h. Levels of mRNA were measured by solution hybridization. Values are given as the mean ± SE of duplicate measurements from three experiments. Statistical comparisons were made using the ANOVA Scheffe’s F test. **, P < 0.01 compared with control. B, IGFBP-3 in conditioned medium of control cells (a) and cells stimulated with VEGF (b), TGFß1 (c), angiotensin II (d), IGF-I (e), or insulin (f). Bovine aortic endothelial cells were starved for 24 h before the addition of growth factors for 18 h. Protein levels were measured by an enzyme-linked immunoassay. Values are given as the mean ± SE from three experiments. Statistical comparisons were made using ANOVA Fisher’s protected least significant difference. {Delta}, P < 0.05; {Delta}{Delta}, P < 0.01 (compared with control). No significant differences were found using ANOVA Scheffe’s F test.

 


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  		Figure 4. A, IGFBP-4 mRNA in control cells (a) and cells stimulated with VEGF (b), TGFß1 (c), angiotensin II (d), IGF-I (e), or insulin (f). Confluent endothelial cells from bovine aorta were starved for 24 h before the addition of growth factors for 18 h. Levels of mRNA were measured by solution hybridization. Values are given as the mean ± SE of duplicate measurements from three experiments. Statistical comparisons were made using ANOVA Scheffe’s F test. **, P < 0.01 compared with control. B, Western immunoblot analysis of IGFBP-4 in conditioned medium of control cells (lane 2) and bovine aortic endothelial cells stimulated with angiotensin II (lane 3), IGF-I (lane 4), insulin (lane 5), TGFß1 (lane 6), or VEGF (lane 7). Human recombinant IGFBP-4 served as a positive control (lane 1). Cells were starved for 24 h before the addition of growth factors for 18 h. Conditioned medium was collected and prepared as described in Materials and Methods.

 


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  		Figure 5. A, Expression of IGFBP-5 mRNA in control cells (a) and cells stimulated with VEGF (b), TGFß1 (c), angiotensin II (d), IGF-I (e), or insulin (f). Endothelial cells from bovine aorta were starved for 24 h before the addition of growth factors for 18 h. Levels of mRNA were measured by solution hybridization. Values are given as the mean ± SE of duplicate measurements from three experiments. Statistical comparisons were made using ANOVA Scheffe’s F test. ***, P < 0.001 compared with control. B, IGFBP-5 protein levels in conditioned medium of control cells (lane 2) and bovine aortic endothelial cells stimulated with VEGF (lane 3), TGFß1 (lane 4), angiotensin II (lane 5), IGF-I (lane 6), or insulin (lane 7). Human recombinant IGFBP-5 served as a positive control (lane 1). Protein levels were measured by Western immunoblot as described in Materials and Methods. One representative experiment is shown (n = 3).

 
The expression of IGFBP-3 mRNA was, after 18-h incubation, inhibited to 43% of the control value by VEGF and to 40% of the control value by TGFß1 (Fig. 3aGo). Angiotensin II, IGF-I, or insulin did not affect IGFBP-3 mRNA levels. The inhibition of IGFBP-3 mRNA expression by VEGF and TGFß1 was associated with a lowered concentration of IGFBP-3 protein in conditioned medium after 18 h, as measured by ELISA (Fig. 3bGo).

IGFBP-4 mRNA expression was inhibited to 61% of the control value by TGFß1, whereas VEGF, angiotensin II, IGF-I, and insulin did not have any effect (Fig. 4aGo). IGFBP-4 in conditioned medium was detected by Western blot. However, no consistent effect on IGFBP-4 expression (by any of the growth factors) could be seen (Fig. 4bGo). In only one of three experiments were IGFBP-4 levels in conditioned medium clearly decreased by TGFß1 (data not shown in figure).

Levels of IGFBP-5 mRNA were markedly increased by VEGF (Fig. 5aGo). TGFß1, angiotensin II, IGF-I, or insulin did not significantly alter IGFBP-5 mRNA expression, although a tendency to inhibition was seen for TGFß1. Detection of IGFBP-5 protein levels by Western immunoblot indicated that the increased expression of IGFBP-5 mRNA by VEGF was correlated with increased levels of IGFBP-5 protein secreted into conditioned medium (Fig. 5bGo). In addition, high levels of IGFBP-5 were found in conditioned medium from IGF-I-stimulated cells.

The expression and regulation of IGF-I were measured in bovine aortic endothelial cells (Fig. 6Go, a and b). IGF-I mRNA was not affected at 18 h of incubation with VEGF, angiotensin II, IGF-I, or insulin, but was inhibited by TGFß1 (Fig. 6aGo). IGF-I in conditioned medium tended to be slightly higher in VEGF-treated cells and slightly lower in TGFß1-treated cells, but no significant differences were found (Fig. 6bGo). Expression of IGF-I in conditioned medium was approximately 2 µg/liter (~10-10 M). The secretion of endogenously produced IGF-I into conditioned medium of IGF-I-stimulated cells was not measured because of interference from exogenously added IGF-I.



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  		Figure 6. A, IGF-I mRNA expression in control cells (a) and cells stimulated with VEGF (b), TGFß1 (c), angiotensin II (d), IGF-I (e), or insulin (f). Bovine aortic endothelial cells were starved for 24 h before the addition of growth factors for 18 h. Levels of mRNA were measured by solution hybridization. Values are given as the mean ± SE of duplicate measurements from three experiments. Statistical comparisons were made using ANOVA Fisher’s protected least significant difference. {Delta}{Delta}, P < 0.01 compared with control. No significant differences were found using ANOVA Scheffe’s F test. B, Levels of IGF-I in conditioned medium in control cells (a) and bovine aortic endothelial cells stimulated with VEGF (b), TGFß1 (c), angiotensin II (d), or insulin (e). Cells were starved for 24 h before the addition of growth factors for 18 h. Conditioned medium was collected and prepared as described in Materials and Methods. Protein levels were measured using a radioisotopic assay. Values are given as the mean ± SE from three experiments. Statistical comparisons were made using ANOVA Scheffe’s F test and Fisher’s protected least significant difference. No significant differences were found.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have shown for the first time that VEGF, a growth factor associated with diabetic retinopathy (4) and atherosclerosis (24, 25, 26), regulates the expression of IGFBPs. VEGF inhibited IGFBP-3 expression, had no effect on IGFBP-4, and augmented the expression of IGFBP-5 in bovine aortic endothelial cells. There was no effect of VEGF on IGF-I expression. The differential regulation of IGFBPs noted in this report suggests that the IGFBPs play different roles in the regulation of IGF-I. Indeed, large differences in the effects of the IGFBPs have been found. IGFBP-3 and IGFBP-4 bind IGF-I with high affinity and are reported to inhibit IGF-I-induced effects in smooth muscle cells (17, 27). IGFBP-5 has been shown to have a stimulatory effect on the bioactivity of IGF-I in smooth muscle cells (28). This stimulatory effect of IGFBP-5 seems to be dependent on binding to extracellular matrix (29). It is possible that both the inhibited IGFBP-3 expression and the stimulated IGFBP-5 expression by VEGF in endothelial cells might act concomitantly to increase the bioavailability of IGF-I in the subintimal space, thereby stimulating migration and proliferation of smooth muscle cells adjacent to the endothelial lining.

IGF-I has been reported to stimulate VEGF mRNA and protein expression in human retinal pigment epithelial cells, and a weak stimulation of VEGF mRNA in bovine smooth muscle cells was also noticed (30). There is to our knowledge no report on the effect of VEGF on the expression of IGF-I. We found no effect of VEGF on IGF-I expression in bovine aortic endothelial cells, but, as mentioned above, VEGF might stimulate IGF-I bioavailability by modulating the expression of IGFBPs. VEGF is known as a highly specific mitogen for endothelial cells, and receptors for VEGF are almost exclusively expressed in endothelial cells (31). Our results suggest that VEGF might indirectly regulate smooth muscle cell functions by modulating the expression of regulatory molecules from the endothelium. A few recent studies report on VEGF receptor expression and direct effects of VEGF in smooth muscle cells (32, 33, 34). The significance of direct VEGF effects on smooth muscle cells, however, remains unclear.

TGFß1, another growth factor associated with diabetic complications (5, 35), also regulated the expression of IGFBPs from bovine aortic endothelial cells. We found that TGFß1 inhibited the expression of IGFBP-3 and –4, but did not have a significant effect on the expression of IGFBP-5. IGFBP-3 and -4 are potent inhibitors of IGF-I action in smooth muscle cells (17, 27). TGFß1-induced inhibition of IGFBP-3 and -4 expression by the endothelium might thus increase the amount of free IGF-I locally in the subintimal space. However, TGFß1 also inhibited IGF-I mRNA expression from the endothelium. This inhibition was not accompanied by an inhibited expression of IGF-I in conditioned medium at 18 h of incubation, but it is possible that an inhibition could be seen after a longer incubation. The effect of the inhibited IGFBP-3 and -4 expression might thus be counteracted by an inhibited expression of IGF-I. It should also be noted that changes in IGFBP expression can have effects other than regulating IGF-bioavailability, as IGFBPs have been shown to have effects of their own (7). This is especially reported for IGFBP-3, but has also been shown for IGFBP-5 (36).

In agreement with this study, a previous report by Erondu and colleagues (15) showed that TGFß1 inhibited IGFBP-3 expression in microvascular endothelial cells. In various other cells TGFß has consistently been shown to stimulate the expression of IGFBP-3 (37, 38, 39, 40, 41, 42). This opposite effect of TGFß1 on IGFBP-3 expression in endothelial cells compared with that in other cells studied accentuates the unique role and properties of the endothelium.

In addition to the inhibitory effect by TGFß1, it was reported by Erondu and co-workers that IGF-I stimulated IGFBP-3 expression in microvascular endothelial cells (15). We could not, however, see any effect of IGF-I on IGFBP-3 expression in this study. This could be due to the different cells used, large vessel endothelial cells in our study and microvessel endothelial cells in the previous study. It has been shown that insulin and IGF-I have differential effects on microvascular and macrovascular endothelial cells (43, 44). IGF-I and insulin were shown to stimulate DNA synthesis in microvessel endothelial cells, but had no effect on large vessel endothelial cells, as also confirmed by us (Dahlfors, G., unpublished data), although receptors for IGF-I and insulin were found in both cell types (43, 44). In line with this it is plausible that IGF-I might stimulate IGFBP-3 expression in microvessel, but not in large vessel, endothelial cells. This might also explain why we found no effect of insulin on IGF-I or IGFBP expression in large vessel endothelial cells. No effect of IGF-I was seen on IGFBP-4 or -5 mRNA expression in bovine aortic endothelial cells in this study. However, protein levels of IGFBP-5 were clearly increased by IGF-I. Previous reports have shown that IGF-I increases IGFBP-5 levels in articular chondrocytes by protecting IGFBP-5 from being degraded by proteases (45, 46). Consistent with our results, one of these reports showed that IGF-I increased IGFBP-5 levels without affecting IGFBP-5 mRNA expression (46). It has been suggested that IGF-I-induced IGFBP-5 gene expression might be cell type specific, because IGF-I was shown to increase IGFBP-5 mRNA levels in aortic smooth muscle cells, but not in fibroblasts, glioblastoma cells, or intestinal smooth muscle cells (47).

We studied the effect of angiotensin II on IGF-I and IGFBP-3, -4, and -5 expression in bovine aortic endothelial cells, but no effect was seen. Angiotensin II regulates various properties of the endothelium (1), but the effect of angiotensin II on IGF-I and IGFBP expression in endothelial cells has not previously been studied. In contrast to the absence of angiotensin II effect on IGFBP expression in endothelial cells seen in this study, we have previously shown that angiotensin II inhibits IGFBP-2 and -4 expression in rat smooth muscle cells (48). Angiotensin II has also been shown to affect IGF-I mRNA expression in vascular smooth muscle cells (48, 49), whereas we found no effect in aortic endothelial cells in this study. These differences between endothelial cells and vascular smooth muscle cells in responsiveness to angiotensin II further emphasize the special characteristics of the endothelium.

In conclusion, we have shown that the expression of IGFBP-3, -4, and -5 is regulated by VEGF and TGFß1 in bovine aortic endothelial cells. IGF-I did not regulate IGFBP mRNA expression, but increased IGFBP-5 protein levels, probably by inhibiting proteolysis of IGFBP-5. No effect on IGFBP expression was seen by angiotensin II or insulin. These observations provide a new aspect of regulation for the IGF system in macrovascular endothelium, with possible implications for subendothelial smooth muscle cells and development of diabetic angiopathy.


   Acknowledgments
 
We thank Associate Prof. T. Sundqvist for kindly providing bovine aortic endothelial cells for this study, and Mrs. M. Karlsson and S. Thunholm for excellent technical assistance.


   Footnotes
 
1 This work was supported by Landstinget i Östergötland (98/123), the Swedish Medical Research Council (19x-4952), the Swedish Diabetes Association, and Barndiabetes fonden; and JDF-Wallenberg Diabetes Research Program (K200099JD-12813-O2C). Back

Received August 27, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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