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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1022
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 1 372-378
Copyright © 2005 by The Endocrine Society

Effects of Insulin-Like Growth Factor I and II on Prostaglandin Synthesis and Plasminogen Activator Activity in Human Umbilical Vein Endothelial Cells

Fiorella Miceli1, Anna Tropea1, Francesca Minici, Mariateresa Orlando, Giuseppina Lamanna, Maria Francesca Gangale, Barbara Panetta, Federica Tiberi, Sergio Vaccari, Rita Canipari, Antonio Lanzone and Rosanna Apa

Cattedra di Fisiopatologia della Riproduzione Umana (F.Mic., F.Min., M.O., G.L., M.F.G., B.P., R.A.) and Istituto Scientifico Internazionale "Paolo VI" (F.T.), Università Cattolica del Sacro Cuore, 00168 Rome, Italy; Dipartimento di Istologia ed Embriologia Medica (S.V., R.C.), Universitá degli Studi di Roma "La Sapienza", 00161 Rome, Italy; and Istituto di Ricerca "Associazione OASI Maria SS ONLUS" (A.T., A.L.), 94018 Troina (EN), Italy

Address all correspondence and requests for reprints to: Rosanna Apa, M.D., Cattedra di Fisiopatologia della Riproduzione Umana, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Roma, Italia. E-mail: krimisa{at}libero.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGFs seem to contribute to the endothelial dysfunction observed in some vascular diseases. Because locally increased IGFs levels were detected in the preeclamptic fetoplacental unit, we hypothesized their involvement in the dysregulation of fibrinolysis and vascular tone typically observed in the fetoplacental compartment in this pregnancy disease. Therefore, in human umbilical vein endothelial cells (HUVECs), the potential effect of IGFs on the synthesis of plasminogen activators (PAs), PA inibitor-1 (PAI-1), and vasodilator and vasoconstrictor prostaglandins (PGs) was investigated. Moreover, in HUVECs treated with IGFs, the expression of cyclooxygenase (COX)-2, the rate-limiting enzyme in PG synthesis, was evaluated.

HUVECs were treated for 24 h with IGFs (1–100 ng/ml) or IL-1ß (0.1 ng/ml). PA, PAI-1, and COX-2 mRNA was determined by RT-PCR and PG release and PA activity by RIA and colorimetric assay, respectively.

We demonstrated an inhibition of urokinase-type PA activity and a 50% reduction of urokinase-type PA mRNA in HUVECs treated with IGFs. No effect was seen on PAI-1. Finally, both IGFs significantly decreased all PGs tested and COX-2 mRNA, whereas, as expected, IL-1ß had an opposite effect.

In conclusion, our results suggest for IGFs a potential involvement in the endothelial dysfunction observed in preeclamptic fetoplacental unit.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGFs, STRUCTURALLY PROINSULIN-like polypeptides, are potent stimulators of cell division and differentiation and play a central role in regulating both placental and fetal development (1, 2). Their biological actions are regulated through association with high-affinity IGF binding proteins (IGFBPs) that determine their bioavailability (3). Both IGF-I and IGF-II are key growth factors during fetal life (1), and particularly IGF-II, in association with the decidual-derived IGFBP-1, also participates in placenta development (4).

However, the IGF family seems to be also involved in the pathophysiology of some pregnancy diseases mainly characterized by vascular disorders. Locally increased IGF-I and IGF-II levels have been detected in the preeclamptic fetoplacental unit (5, 6). In addition, IGF-I seems to alter the umbilical cord structure and its responsiveness to vasoactive substances (7). Generally, IGFs have been described as factors causing endothelium dysfunction. They can induce endothelial injury by enhancing proinflammatory cytokine signal transduction (8), and finally they can also attenuate fibrinolytic activity (9). Furthermore, in animal and in vitro studies, both IGFs have been demonstrated to promote atherosclerotic process (10, 11, 12).

Frequently in many diseases characterized by vascular disorders, the endothelial injury also results in a dysregulation of the fibrinolytic system. Among the other factors, in normal conditions the plasminogen activators (PAs), tissue-type (tPA) and urokinase-type (uPA), convert plasminogen into plasmin, the active fibrinolytic enzyme, whereas specific PA inhibitors (PAIs), PAI-1 and PAI-2, balance PA activity (13). In some vascular diseases, this reciprocal control among the different factors is lost; in fact, for instance in preeclampsia, a significant reduction of uPA concentrations in maternal plasma has been described (14, 15, 16), whereas PAI-1 levels were significantly increased (17, 18). Similar alterations have been found in umbilical cord circulation (19).

In addition to the endothelial integrity and a normal fibrinolytic activity, substances influencing the vascular tone are important to assure a normal vascular function. In this sense, prostaglandins (PGs) are classically known as key factors involved in this delicate function. An imbalance of their levels can alter this equilibrium; in fact, increased levels of vasoconstrictor and reduced levels of vasodilator PGs, such as prostacyclin (PGI2) and PGE2, have been observed in preeclampsia both in maternal (20) and fetoplacental unit circulation (21, 22, 23, 24, 25).

Based on these data, the aim of this study was to investigate the possibility for IGFs to affect PG synthesis and PA and PAI expression in endothelial cells obtained from human umbilical vein endothelial cells (HUVECs) and therefore their possible involvement in the dysregulation of fibrinolysis and vascular tone observed in the preeclamptic fetoplacental unit. In addition, we evaluated the gene expression of cyclooxygenase (COX)-2, the rate-limiting enzyme in PG synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals

Human recombinant IGF-I and IGF-II, type IA collagenase, antibiotics, and Hank’s balanced salt solution (HBSS) were purchased from Sigma Chemical Co., Inc. (St. Louis, MO). IL-1ß and endothelial cell growth factor were obtained from Roche Molecular Biochemicals (Indianapolis, IN), whereas heparin was obtained from Parke-Davis SpA (Ann Arbor, MI). Medium 199 and fetal bovine serum were purchased from Life Technologies, Inc. (Gaithersburg, MD) and HyClone Laboratories, Inc. (Logan, UT), respectively. TRIzol and RT-PCR system were obtained from Invitrogen, Life Technologies. [3H]PGE2, [3H]PGF2{alpha}, and [3H]6-ketoPGF1{alpha} were obtained from NEN Life Science Products (Milan, Italy). The chromogenic plasmin substrate D-val-leu-lys-p-nitroanilide 2HCl was obtained from Bachem Feinchemikalien AG (Basel, Switzerland).

Cell cultures

Human umbilical cords were obtained from healthy women who underwent uncomplicated term pregnancies. After collection, the umbilical cord was rapidly immersed in sterile saline solution (0.9% NaCl) and immediately processed for endothelial cell isolation. Under a laminar flow sterile hood, the umbilical vein was cannulated and thoroughly rinsed with sterile saline solution. After clamping the other extremity, the vein was filled for 10 min with 0.2% type IA collagenase solution (in HBSS without Ca2+ and Mg2+) prewarmed at 37 C. The collagenase solution was then discarded, and the vein was gently washed with 40 ml HBSS (Ca2+ and Mg2+ free) that was collected in a 50-ml sterile polypropylene tube. The cells were pelleted by centrifugation at 4 C for 10 min at 1300 rpm, the supernatant was discarded, and the cell pellet was gently resuspended in medium 199 containing 10% heat-inactivated fetal bovine serum, antibiotics, endothelial cell growth factor (20 µg/ml), and heparin (50 µg/ml). The cells were then plated on tissue culture flasks and cultured in 5% CO2-95% air at 37 C. The endothelial cell identity was confirmed by the typical cobblestone aspect and immunostaining with an antibody vs. factor VIII-related antigen in representative dishes.

Once grown to confluence, the cells were replated on tissue culture flasks at 20,000 cells/cm2 and grown up to the moment of the experiments.

Informed consent was obtained from each subject.

PG assays

To measure PGs secreted in culture medium, cells were plated in 48-well culture dishes (75,000 cells/well), grown to monolayers, and then incubated for 24 h with fresh serum-free medium alone (controls) or containing IL-1ß (0.1 ng/ml) or with different doses of IGF-I and IGF-II (1, 10, 100 ng/ml). At the end of each experiment, culture media were collected in tubes and then immediately frozen at –20 C, in which they remained until the assay. Four different wells were used for each experimental condition, and culture media were separately collected and assayed for PG detection.

The RIAs for PGE2, PGF2{alpha}, and 6-ketoPGF1{alpha} (a stable metabolite of prostacyclin) used in this study were first characterized for measurement of prostanoids in human urine (26) and later used successfully to measure PGs produced and released by several cell types in vitro (27).

For each assay, incubation mixtures of 1.5 ml were prepared in disposable plastic tubes in which 50 µl (for PGE2, PGF2{alpha}, and 6-ketoPGF1{alpha}) incubation medium was diluted to 250 µl with 0.025 mol/liter phosphate buffer (pH 7.5). Tritiated PGE2, or PGF2{alpha} or 6-ketoPGF1{alpha} (2500–3500 cpm) and appropriately diluted antisera were added together to a final volume of 1.5 ml. The antisera (provided by Prof. G. Ciabattoni, Istituto di Farmacologia Universitá Catholica del Sacro Cuore, Rome, Italy) were employed at a final dilution of 1:120,000. A duplicated standard curve ranging from 2 to 400 pg/tube was run for each assay. All tubes were incubated for 24 h at 4 C. Separation of antibody-bound PGs was obtained with 2.5 mg charcoal (Norit-A), which absorbs 95–98% of free PGs; a charcoal suspension (2.5 mg/50 µl) in 0.025 mol/liter phosphate buffer (pH 7.5) was added to each tube after the addition of 100 µl 5% BSA. The tubes were briefly shaken and then centrifuged for 10 min at 4 C. Supernatants were decanted into 10 ml scintillation liquid. Radioactivity was measured by liquid scintillation counting. The detection limit of the assay was 2 pg/tube in all cases. The inter- and intraassay variability coefficients were 2.7 and 2.9% for PGE2, 3.2 and 2.8% for PGF2{alpha}, and 5% for 6-ketoPGF1{alpha}.

PA assay

Enzymatic activity of PA was assayed according to the method of Shimada et al. (28) using a chromogenic substrate (substrate D-val-leu-lys-p-nitroanilide) assay. Samples were incubated with plasminogen, and the absorbance generated at 405 nm is related to PA activity.

Gel electrophoresis and zymography

For the zymography of PA, aliquots of conditioned media and cell homogenates were separated by electrophoresis in 8% polyacrylamide slab gels in the presence of sodium dodecyl sulfate (SDS-PAGE) under nonreducing conditions according to the procedure of Laemmli (29). PA was then visualized by placing the Triton-X-washed gel on a casein-agar-plasminogen underlay as previously described (30). Molecular weights were calculated from the position of prestained markers that were subjected to electrophoresis in parallel lines. The lytic zones were plasminogen dependent.

Total RNA extraction and quantification

To evaluate COX-2, tPA, uPA, and PAI-1 expression, HUVECs were plated in 25-cm2 tissue culture flasks. Once grown to confluence, the cells were incubated with fresh serum-free medium alone (controls) or containing IL-1ß (0.1 ng/ml) or with IGF-I and IGF-II (100 ng/ml). After 24 h, HUVECs were treated for total RNA extraction for RT-PCR. To this end, the standard TRIzol extraction method was used, according to the instructions provided by the manufacturer. The purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis.

RT-PCR

For mRNA analysis, semiquantitative RT-PCR was carried out according to the instructions provided by the manufacturer.

Total RNA (1–2 µg) was reverse transcribed to cDNA by a reaction containing 0.5 mmol/liter each deoxynucleoside triphosphate, 0.75 µg oligo(dT)12–18 primer, 0.2 µmol dithiothreitol, and 200 U murine Moloney virus reverse transcriptase. The reaction was run at 65 C for 5 min and quickly chilled on ice for 1 min at 37 C for 52 min and then at 70 C for 15 min.

The PCRs were carried out using Taq DNA polymerase according to the manufacturer’s suggestions. All PCR amplifications were carried out for 24, 26, 30, or 35 cycles, using annealing temperature provided for each primer set. For each sample 10 ml of the PCR product was submitted to electrophoresis on agarose gel (1.5%) and stained with ethidium bromide. Quantification of each gene product was obtained from the cycles in a linear range of amplification. COX-2, tPA, uPA, and PAI-1 PCR products remained in a linear range of amplification even after 30–35 cycles. Amplified products were quantified by means of computer analyses, and mRNA levels were normalized against the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or ß-actin mRNA. Primers used to amplify COX-2, tPA, uPA, PAI-1, GAPDH, and ß-actin are shown in Table 1Go.


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  		TABLE 1. Sequence of selected oligonucleotides used as RT-PCR primers

 
Data analysis

Statistical analysis was performed using ANOVA followed by the Tukey-Kramer test for comparisons of multiple groups or paired Student’s t test for comparison of data derived from two groups. Values with P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of IGF-I, IGF-II, and IL-1ß on PG release from HUVECs

HUVECs were incubated for 24 h in the presence of increasing concentrations of IGF-I or IGF-II (1–100 ng/ml) or with 0.l ng/ml of IL-1ß. Prostanoids production was estimated by measuring the amounts of 6-ketoPGF1{alpha} (a stable metabolite of prostacyclin), PGE2, and PGF2{alpha} into the conditioned medium.

As shown in Fig. 1AGo, all tested doses of both IGFs were able to significantly decrease 6-keto PGF1{alpha} release. The mean concentration of 6-keto PGF1{alpha} in the control was 155.2 ± 11.7 pg/ml.



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  		FIG. 1. Effect of IGF-I and IGF-II on 6-ketoPGF1{alpha}, PGE2, and PGF2{alpha} secretion by HUVECs. HUVECs were cultured for 24 h in medium alone (C) or with IGF-I or IGF-II (1–100 ng/ml) or IL-1ß (0.1ng/ml). Each value represents the mean ± SEM of six independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. ***, P < 0.001, **, P < 0.01, *, P < 0.05 vs. C values.

 
The next step was to investigate the effect of both IGFs on the production of PGE2, another vasodilator prostanoid. As shown in Fig. 1BGo, IGF-I was able to significantly decrease PGE2 production at all tested doses, except for the lowest one (1 ng/ml), whereas only the highest used dose (l00 ng/ml) of IGF-II exerted this effect in a significant manner. The mean concentration of PGE2 in the control was 28.9 ± 2.8 pg/ml.

The vasoconstrictor PGF2{alpha} was the latest prostanoid assayed in the culture medium. As can be observed in Fig. 1CGo, both IGFs were able to significantly reduce PGF2{alpha} release at all tested doses. The mean concentration of PGF2{alpha} in the control was 110.4 ± 12.6 pg/ml.

As expected, IL-1ß increased all PGs in a very significant manner (31, 32), and for this reason it was used as positive control.

Effect of IGF-I, IGF-II, and IL-1ß on COX-2 mRNA expression from HUVECs

To investigate whether COX-2 was involved in IGFs effect on PG synthesis, RT-PCR experiments were performed on total RNA extracted from HUVECs cultured with medium alone (control), IGF-I or IGF-II (l00 ng/ml), or IL-1ß (0.1 ng/ml). As expected, IL-l ß enhanced COX-2 expression (32, 33). IGF-I and IGF-II were both able to significantly decrease COX-2 mRNA (Fig. 2Go).



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  		FIG. 2. Effect of IGF-I and IGF-II on COX-2 mRNA levels in HUVECs. Total RNA was prepared from HUVECs cultured for 24 h with either medium alone (C), IL-1ß 0.1 ng/ml, or IGF-I or IGF-II 100 ng/ml. A, Total RNA was subjected to RT-PCR using a specific set of primers for COX-2 gene (see Table 1Go). The expression of GAPDH was used as an internal standard. DNA molecular mass standards (MW) appear in the far right lane. B, The intensities of bands were quantified by densitometry and normalized by respective GAPDH values. Each bar represents the mean ± SEM of six separate experiments using total RNA preparations from different cords. Results are expressed as percentage of control set equal to 100. *, P < 0.001 vs. C values.

 
Effect of IGF-I on uPA, tPA, and PAI-1 production by HUVECs

Finally, to investigate whether IGFs could modulate PA activity in HUVECs, the cells were cultured for 24 h in medium alone (control) or in the presence of increasing concentrations of IGF-I and IGF-II (ranging from 1 to 100 ng/ml). PA activity was assayed in the conditioned medium and cell lysate by colorimetric assay. The majority of the activity was found to be cell associated. The mean concentration of uPA in the control in cell lysate and conditioned medium was 68.7 ± 2.1 and 1.9 ± 0.7 mIU x 10–4/ml, respectively. Both IGFs inhibited secreted uPA, whereas only IGF-I caused a dose-dependent inhibition of cell-associated PA (Fig. 3Go). To characterize the type of PA produced by HUVECs, aliquots of conditioned media and cell lysates were processed for SDS-PAGE followed by zymography. As can be observed in Fig. 4AGo, two bands were observed in conditioned media: one, barely detectable, corresponding to the molecular weight of uPA, and a higher-molecular-weight band that suggested the presence of a PA-PAI complex. Only the uPA band was present in cell lysates (Fig. 4BGo). No tPA activity was present in both medium and cell lysate.



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  		FIG. 3. Effect of IGF-I and IGF-II on PA secretion by HUVECs. HUVECs were cultured for 24 h in medium alone (C) or with IGF-II or IGF-I (1–100 ng/ml). PA activity was analyzed by chromogenic substrate assay in conditioned medium and cell lysate. Each value represents the mean ± SEM of five independent experiments each done in triplicate. Results are expressed as percentage of control set equal to 100. ***, P < 0.001; **, P < 0.01; *, P < 0.05 vs. C values.

 


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  		FIG. 4. Effect of IGF-I on HUVEC PA and PAI production. HUVECs were cultured for 24 h with medium alone (C) or IGF-I (1–100 ng/ml). Aliquots of conditioned media (20 ml) (A) and cell lysate (15 ml) (B) were analyzed by zymography. The photographs were taken after 24 h of incubation at 37 C. Shown are representative zymographies of two independent experiments.

 
Effect of IGF-I on uPA, tPA, and PAI-1 expression from HUVECs

To determine whether the effects of IGFs on PA and PAI activity were associated with a parallel modulation of mRNA levels, RT-PCR experiments were performed on total RNA extracted from HUVECs cultured for 24 h with medium alone (control) or 100 ng/ml IGF-I. As shown in Fig. 5Go, treatment with IGF-I caused an approximately 50% decrease in uPA transcript expression. Conversely, no significant effect was observed on PAI-1 gene expression. RT-PCR analysis confirmed the absence of tPA (data not shown).



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  		FIG. 5. Effect of IGF-I on uPA and PAI-1 mRNA levels in HUVECs. Total RNA was prepared from HUVECs cultured for 24 h with either medium alone (C) or IGF-I 100 ng/ml. A, Total RNA was subjected to RT-PCR using specific sets of primers for uPA and PAI-1 genes (see Table 1Go). The expression of ß-actin was used as an internal standard. DNA molecular mass standards appear in the middle lane. B, The intensities of bands were quantified by densitometry and normalized by respective ß-actin values. Each bar represents the mean ± SEM of three separate experiments using total RNA preparations of different cords. Results are expressed as percentage of control set equal to 100. *, P < 0.001 vs. C values.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we investigated the potential role(s) of IGFs in the pathogenesis of the fetoplacental vascular dysfunction frequently described in preeclampsia. In particular, we aimed to evaluate whether they could play a role in the endothelial vasoconstrictor/vasodilator PG imbalance and the coagulation/fibrinolysis disequilibrium typically observed in preeclamptic fetoplacental unit.

An involvement of IGFs in hypertensive disorders complicating pregnancy, i.e. the preeclampsia, has been suggested by many published data. In preeclamptic placentas, increased IGF-II mRNA levels have been observed in the intermediate trophoblast of periinfarct regions (5), and recent evidence suggests that locally produced IGF-II may be involved in vascular injury (11). Therefore, it is possible that a local overexpression of IGF-II could have a share in the well-known preeclampsia-induced endothelial dysfunction characterized by fibrin deposition and vasoconstriction. These alterations could result in reduced blood flow that may be responsible for placental infarction and fetal growth restriction. Interestingly, IGF-I also is likely to be involved in this pathological pathway. In fact, its ability to play a role in vascular injury, by increasing proinflammatory cytokines able to injure endothelial cells (8, 11, 34) and also attenuating fibrinolytic activity (9), has been reported. In addition, IGF-I seems to act also at the umbilical cord level. In fact, it has been well described that in preeclampsia a remodeling of funis structure occurs. This remodeling, characterized by an accumulation of collagen (35) and sulfated glycosaminoglycans (GAGs) (36), induces a reduction of the umbilical cord arteries elasticity with a consequent increase of peripheral resistance. The ability of IGF-I to stimulate both collagen and GAG biosynthesis (37) and its increased levels in preeclamptic funis (6, 38) strongly support a role for this growth factor in the impairment of umbilical cord arteries.

Based on these data, our aim was to better define the possible mechanism(s) through which IGFs could participate to the impairment of fetoplacental vascular function(s) described in some pregnancy-related diseases, such as preeclampsia. To this end, we used endothelial cells obtained from human umbilical cord as an in vitro model to investigate the pathophysiology of placenta-fetal circulation.

We demonstrated that both IGF-I and IGF-II were able to significantly decrease PG release from HUVECs. These results are in accord with those of Rosenthal and Ocasio (39) showing that insulin, which shares homologous structure and receptors with IGFs, inhibits both basal and arachidonic acid-stimulated production of PGI2 from HUVECs in a dose-dependent manner. It is important to remember the classical actions of PGs, especially prostacyclin (PGI2), on both vascular tone and coagulation. In fact, PGI2 and PGE2 cause vasodilatation (40), whereas PGF2{alpha} is a vasoconstrictor factor (41, 42); furthermore, PGI2 is an important antithrombotic factor for its effects on platelets (43). The ability of IGFs to inhibit PG release by HUVECs suggest a possible double role for these growth factors: they could facilitate thrombosis by reducing the production of thrombo-resistant substances, such us PGI2, from the endothelium, and they could participate to the imbalance between increased vasoconstrictor and decreased vasodilator prostanoids by decreasing the synthesis of PGI2 and PGE2, both vasodilator substances. In this assumption, the meaning of the IGF-induced reduction of the vasoconstrictor PGF2{alpha} remains to be elucidated. It is likely that other cytokines in vivo prevail to cause the vasoconstrictor PG increase observed, i.e. in preeclampsia. To investigate the route through which IGFs affected PGs synthesis, we evaluated their effect on COX-2 expression in HUVECs. COX-2 is the inducible isoform of COX that catalyzes the rate-limiting step in PG synthesis. In HUVECs COX-2 isoform is linked to PGI2 and PGE2 production, whereas COX-1, the constitutive isoform, favors tromboxane production (44). In IGF-treated HUVECs, our observation of a significant reduction in COX-2 expression suggests that an IGF-negative effect on PG synthesis occurs via the COX-2 pathway.

As previously stated, in addition to altered vascular reactivity, coagulatory disorders are frequently observed in hypertensive disease complicating pregnancy (45, 46). In fact, preeclampsia is associated with maternal endothelial cell dysfunction and altered expression of PA system components. In particular, higher levels of tPA and PAI-1 and lower levels of uPA and PAI-2 have been detected in plasma of women with severe preeclampsia (14, 15, 16, 17, 19). Roes et al. (19) found that in preeclampsia the fetal fibrinolytic system was also affected. In fact, they found a very high increase of PAI-1 levels and a decrease of uPA levels in the umbilical cord of preeclamptic patients that suggest a decreased fibrinolysis in the fetal circulation. Our data demonstrated that IGFs were able to significantly decrease uPA production by HUVECs; however, we did not find any effect on PAI-1 expression. It should be noted that PAI-1 is produced by not only endothelial cells but also activated platelets and trophoblasts (47). Therefore, the high levels of PAI-1 found by Roes et al. (19) may not be of endothelial origin. Our experiments demonstrated that IGFs control the decrease in uPA production via modulation of the steady-state level of uPA-mRNA. In addition, the fact that the majority of uPA activity was found in cell lysate suggests that uPA is associated with cells via uPA receptors, which have been demonstrated to be present on endothelial cells (48). IGF-I has already been shown to decrease uPA activity in a human osteosarcoma cell line (49). It has also been suggested that the PA/PAI system may be involved in the regulation of IGF activity. In fact, IGF-I controls its own bioavailability via stimulation of IGFBP-3 production and inhibition of the PA/PAI system, which regulates IGFBP-3 proteolysis (49, 50). A limited proteolysis of IGFBP-3 promotes dissociation of IGFs bound to it and increases IGFs availability to the cells. Therefore, a decrease in uPA activity favors a decrement in IGFs bioavailability. However, IGF activity is under control of other binding proteins. During preeclampsia, a dramatic dephosphorylation of IGFBP-1 has been observed (50). This dephosphorylation causes a decreased IGFBP affinity for IGF-I and increases IGF-I bioavailability. Because IGF-I stimulates collagen and GAG production, the decrease in uPA activity in the umbilical cord arteries may reduce collagen breakdown and promote its accumulation as observed in preeclampsia.

In conclusion, we demonstrated that IGFs were able to significantly decrease PG synthesis from HUVECs and that this effect was mediated by COX-2. Furthermore, IGF-I significantly inhibited uPA expression from the same cells, whereas it was ineffective on PAI-1.

To our knowledge, this is the first work that defines the mechanism(s) through which the increased IGF levels described by several authors in preeclamptic fetoplacental unit can be involved in the pathogenesis of this disease. In fact, our data suggest that, in the fetoplacental unit, the locally increased levels of IGFs can participate in vascular injury through two different mechanisms: by altering the synthesis of substances that influence vascular hemodynamics in the physiologic state, such as PGs, and disrupting the coagulation/fibrinolysis equilibrium by affecting PGI2 and uPA levels. New experiments are currently ongoing in our laboratory to further explore the role of IGFs as disturbing vascular factors.


   Acknowledgments
 
The authors thank Stefania Catino for her excellent technical assistance.


   Footnotes
 
This work was supported by grants from the Ministero per l’Università e la Ricerca [Cofinanziamento Ministero dell’Instruzione dell’Universita e della Ricerca (COFIN) 2002–2003 to A.L., 60% and COFIN 2003–2004 to R.C.] and from the Ministero della Salute (Current Research: 2003; title project: "La prevenzione dell’handicap mentale: modelli di studio biologici e clinici in epoca preconcezionale, riproduttiva e prenatale").

First Published Online October 26, 2004

1 F.M. and A.T. contributed equally to this work. Back

Abbreviations: COX, Cyclooxygenase; GAG, glycosaminoglycan; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HBSS, Hank’s balanced salt solution; HUVEC, human umbilical vein endothelial cell; IGFBP, IGF binding protein; PA, plasminogen activator; PAI-1, PA inibitor-1; PG, prostaglandin; tPA, tissue-type PA; uPA, urokinase-type PA.

Received June 1, 2004.

Accepted October 19, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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