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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 6 2303-2309
Copyright © 2006 by The Endocrine Society

Regulation of Vascular Endothelial Growth Factor Synthesis and Release by Human Luteal Cells in Vitro

Anna Tropea1, Fiorella Miceli1, Francesca Minici, Federica Tiberi, Mariateresa Orlando, Maria Francesca Gangale, Federica Romani, Stefania Catino, Salvatore Mancuso, Pierluigi Navarra, Antonio Lanzone and Rosanna Apa

Cattedra di Fisiopatologia della Riproduzione Umana (A.T., F.Mic., F.Min., M.O., M.F.G., F.R., S.M., R.A.), Istituto Scientifico Internazionale "Paolo VI" (F.T.), and Istituto di Farmacologia (P.N.), Università Cattolica del Sacro Cuore, 00168 Roma, Italy; and Istituto di Ricerca "Associazione OASI Maria SS ONLUS" (S.C., 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
 
Context: Vascular endothelial growth factor (VEGF) is essential for normal luteal development and function, but little is still known about the regulation of its production by human midluteal phase luteal cells.

Objective: We investigated whether human chorionic gonadotropin (hCG) or local factors, including chemical hypoxia, IGF-I and IGF-II, prostaglandin (PG)E2, and PGF2{alpha} prevail in modulating VEGF mRNA and protein production in human midluteal phase luteal cells. The effect of progesterone (P) on luteal VEGF mRNA expression and protein secretion was also evaluated. Finally, we investigated whether VEGF could directly affect luteal P secretion.

Interventions: In human midluteal phase luteal cells, VEGF mRNA expression was evaluated by semiquantitative RT-PCR, whereas VEGF and P release was evaluated by ELISA and RIA, respectively.

Results: hCG was unable to significantly affect luteal VEGF mRNA and protein synthesis, which in turn was significantly increased by both chemical hypoxia and IGFs. Conversely, VEGF mRNA and protein production was reduced by PGs and P. Finally, VEGF did not affect P luteal secretion.

Conclusions: Our results suggest that local ovarian factors, rather than hCG, predominate in regulating VEGF mRNA and protein production by human midluteal phase luteal cells. For VEGF, a lack of a direct luteal steroidogenic effect was also demonstrated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEVERAL FACTORS DEMONSTRATE that angiogenesis is essential for normal luteal development and function (1), the suppression of this process being associated with inhibition of luteal function (2). Vascular endothelial growth factor (VEGF), the well-known regulator of endothelial proliferation, vasculogenesis, and vascular permeability (1), plays a critical role in corpus luteum angiogenesis (2, 3, 4). Indeed, in nonhuman primates, the inhibition of VEGF in luteal phase is able to prevent luteal vasculogenesis and decrease progesterone (P) plasma levels (2, 5, 6). VEGF is expressed in both luteinizing granulosa cells (LGCs) (7) and differentiated luteal cells (8). In particular, in luteal cells Sugino et al. (8) demonstrated that VEGF expression remains constant from early to late luteal phase, becomes higher if pregnancy occurs, or decreases in the regression phase. Nevertheless, little is known about the regulation of VEGF luteal synthesis. Although a decline in local oxygen concentration (hypoxia) is believed the primary stimulator of VEGF production in most tissues (9), human chorionic gonadotropin (hCG) has been demonstrated to significantly stimulate VEGF synthesis in LGCs (10, 11, 12, 13). Conversely, in monkey luteal cells, recent in vitro data failed to confirm a stimulatory effect for hCG on VEGF expression, the latter being enhanced by local hypoxia (14). Even if species-dependent differences cannot be ruled out, these results suggest that local factors, rather than gonadotropin, could prevail in regulating VEGF production by differentiated luteal cells. To better define this aspect, in highly purified cultures of human midluteal phase luteal cells, the effect of hCG or CoCl2 (chemical hypoxia) (15) on VEGF mRNA expression and protein secretion was examined. Furthermore, because P, the main luteal hormone, is able to affect VEGF synthesis in different human tissues (16, 17), we investigated its potential role in regulating VEGF expression and production in midluteal phase luteal cells. Finally, the effect of IGF-I, IGF-II, prostaglandin (PG) E2, classical local luteotropic factors (18, 19), and PGF2{alpha}, luteolytic modulator (18, 19), on VEGF expression and production was examined in midluteal phase luteal cells.

In fully functional corpus luteum, when luteal vasculature is largely complete (20), VEGF seems to act as both endothelial cell survival and vascular permeability factor (13). The inhibition of vessel leakiness has been proposed by Dickson et al. (20) to justify the rapid decrease of P plasma levels observed in primates after anti-VEGF treatment in midluteal phase. To better define the role of VEGF in fully functional corpus luteum, in the present study, we investigated whether this protein could directly affect luteal P production by midluteal phase luteal cells. In fact, both VEGF receptors, the fms-like tyrosine kinase and the kinase insert domain-containing region, were immunohistochemically detected in corpus luteum on not only endothelial cells but also steroidogenic cells (8, 21, 22), suggesting for this growth factor a possible paracrine-autocrine role in regulating luteal function.


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

VEGF, PGE2, PGF2{alpha}, P, IGF-I, IGF-II, collagenase type IV, antibiotics, and HEPES were purchased from Sigma Chemical Co. (St. Louis, MO). hCG was obtained from Serono (Milano, Italy), nutrient mixture F-12 (Ham) from Flow Laboratories (Milano, Italy), and fetal calf serum from Biological Industries (Kibbutz Beit Hemeek, Israel). TRIzol and RT-PCR kit reagents were purchased from Invitrogen, Life Technologies (Carlsbad, CA). The VEGF and human 18S rRNA primers were obtained from Sigma-Proligo (Sigma-Aldrich S.r.l., Milano, Italy). The P RIA kits were obtained by Radim (Roma, Italy) and VEGF ELISA kits by R&D Systems (Minneapolis, MN).

Cell cultures

Corpora lutea (CL) were obtained at the time of hysterectomy or myomectomy performed for nonendocrine gynecological disease (leiomyomatosis) in the midluteal phase of the menstrual cycle (d 5–6 from ovulation). A total of 27 patients (aged 28–41 yr) were included in the present study. All had a history of regular menstrual cycles. The present protocol was approved by the Institutional Review Board of Università Cattolica del Sacro Cuore; all patients provided written informed consent.

The age of CL was determined as follows. All patients were monitored until ovulation by daily measurement of basal body temperature and ultrasound examination of follicular growth. Once the maximal follicular diameter reached 18 mm, daily determination of plasma P values was made. The time of ovulation (d 0) was detected by the biphasic pattern of basal body temperature, the typical ultrasound disappearance of the dominant follicle or the ultrasound detection of CL, and the rise in plasma P concentrations. At the time of surgery, immediately before anesthesia, plasma samples were collected to determine plasma P concentrations.

The removed luteal tissue was immediately freed from blood vessels and ovarian stroma under a dissecting microscope, dissected, and minced. Human CL cultures were performed as previously described (23). The luteal cell identity was confirmed by their positive staining for lipids with oil red O14 (24). At the end of the isolation procedure and 24 h after all the treatments mentioned in the next paragraphs, cells were counted in a hemocytometer, and viability was determined by the trypan blue exclusion test.

Preliminary experiments were performed to exclude that any solvent for dissolving the tested substances could affect luteal cell function and/or viability.

VEGF assay

To measure VEGF secreted in culture medium, luteal cells were plated on 48-well dishes (250,000 cells/ml) and cultured for 24 h in 5% CO2-95% air at 37 C. Cells were incubated for 24 h with fresh serum-free medium alone [control (C)] or CoCl2 (10 µM) (25), IGF-I or -II (1–100 ng/ml), or P (1, 10, 50, 100 ng/ml). Moreover, cells were incubated for 24, 48, and 72 h with fresh serum-free medium alone (C) or containing hCG (100, 500, 1000 ng/ml). Three different wells were used for each experimental condition, and culture media were separately collected and assayed for VEGF detection. To this aim, commercial VEGF ELISA kits were carried out according to the instructions provided by the manufacturer. In particular, the Quantikine VEGF immunoassay is a 4.5-h solid-phase ELISA designed to measure VEGF165 levels in cell culture supernates, serum, and plasma. It contains Sf21-expressed, recombinant human VEGF165 and antibodies raised against the recombinant protein. Results obtained for naturally occurring human VEGF and recombinant human VEGF121 showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for natural human VEGF. The intra- and interassay coefficients of variation were 3.5 and 6.7%, respectively. The ELISA sensitivity regarding VEGF was 5.0 pg/ml.

P assay

To measure P secreted in culture medium, luteal cells were plated on 48-well culture dishes (250,000 cells/ml) and incubated for 24 h with fresh serum-free medium alone (C) or with hCG (100 ng/ml), VEGF (100 ng/ml), or CoCl2 (10 µM). For each experimental condition, three different wells were performed, and culture media were separately collected and assayed for P detection. To this aim, according to the manufacturer’s instructions, commercial P RIA kits were used. The intra- and interassay coefficients of variation were 4 and 10%, respectively. The RIA sensitivity regarding P was 5 pg/tube.

Total RNA extraction and quantification

To evaluate VEGF expression, luteal cells were plated in 25-cm2 tissue culture flasks. Cells were incubated for 24 h with fresh serum-free medium alone (C) or containing CoCl2 (10 µM), IGF-I or -II (100 ng/ml), P (10 ng/ml), PGE2, or PGF2{alpha} (10–7 M). Moreover, cells were cultured for 24, 48, and 72 h with fresh serum-free medium alone (C) or containing hCG (100 ng/ml). After incubation, luteal cells 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.

Human VEGF gene is composed of eight exons, and different isoforms arise from the same gene by alternative splicing. To date, six human VEGF splice variants, ranging in length from 121 to 206 amino acids, have been identified (26). These isoforms appear to have similar biological activities in vitro. VEGF isoforms are distinguished by the presence or absence of two distinct heparin-binding domains encoded by exons 6 and 7 of the VEGF gene (22). The presence or absence of these domains influences solubility and receptor binding; thus, VEGF 121 isoform, lacking the domains encoded by exons 6 and 7, and VEGF 165 isoform, lacking the domain encoded by exon 6, are highly and moderately diffusible isoforms, respectively (27).

The expression of VEGF was first analyzed by using a set of primers to amplify mRNA encoding all the VEGF isoforms expressed in human CL (22). Four cDNA products were obtained by RT-PCR migrating at 517, 589, 649, and 721 bp, which corresponded to human VEGF 121, 145, 165, and 189, respectively.

Moreover, a common sense primer and two specific antisense primers were designed to separately amplify mRNAs encoding VEGF 121 and VEGF 165 splice variants, which are the most abundant VEGF isoforms expressed in human healthy tissues (27) as well as human CL (8, 22). One cDNA product was obtained by each RT-PCR migrating at 245 bp for both VEGF 121 and VEGF 165 isoforms.

The identity of each PCR product was confirmed by sequence analysis.

PCR conditions were the following: 33 cycles consisting of 95 C (60 sec), 64 C (60 sec), and 72 C (90 sec) using the unique set of primers for all the VEGF isoforms (5'-tcgggcctccgaaaccatga-3' and 5'-cctggtgagagatctggttc-3'); 33 cycles consisting of 95 C (20 sec), 58 C (20 sec), and 72 C (30 sec) using the common sense VEGF primer (5'-ccctgatgagatcgagtacatctt-3') and the VEGF 121-specific antisense primer (5'-agcaaggcccacagggattt-3') or the VEGF-specific 165 antisense primer (5'-gcctcggcttgtcacatttt-3'); 30 cycles consisting of 95 C (25 sec), 58 C (25 sec), and 72 C (25 sec) using the two 18S rRNA-specific primers (5'-ctgccctatcaactttcgatggtag-3' and 5'-ccgtttctcaggctccctctc-3').

PCR products were separated on a 2.0 or 3.0% agarose gel containing ethidium bromide.

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
 
In the first group of experiments, human luteal cells were incubated for 24, 48, and 72 h with medium alone (C) or increasing concentrations of hCG (100, 500, 1000 ng/ml). As shown in Fig. 1AGo, after 24 h hCG was not able to significantly affect VEGF luteal secretion at any tested doses. Moreover, incubation with hCG (100, 500, 1000 ng/ml) failed to affect VEGF secretion even after 48 and 72 h (data not shown).


Figure 1
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  		FIG. 1. A, Effect of CoCl2 and hCG on VEGF secretion by human midluteal phase luteal cells. Luteal cells were cultured for 24 h in medium alone (C) or with hCG (100, 500, 1000 ng/ml) or CoCl2 (10 µM). 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 vs. C values. B, Effect of IGF-I and IGF-II on VEGF secretion by human midluteal phase luteal cells. Luteal cells were cultured for 24 h in medium alone (C) or with IGF-I or IGF-II (1–100 ng/ml). Each value represents the mean ± SEM of seven independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. **, P < 0.01, *, P < 0.05 vs. C values.

 
Moreover, we investigated whether VEGF production could be influenced by local hypoxia, a well-known stimulator of VEGF release in most tissues. To this aim, chemical hypoxia was induced by adding CoCl2 (10 µM) to the culture media. We observed that incubation of luteal cells for 24 h with CoCl2 was able to significantly enhance VEGF production (Fig. 1AGo).

Another set of experiments was performed to investigate whether IGFs could affect VEGF production. Luteal cells were cultured for 24 h in medium alone (C) or in presence of increasing concentrations of IGF-I or IGF-II (ranging from 1 to 100 ng/ml). As shown in Fig. 1BGo, IGF-I was able to significantly enhance VEGF release at all tested doses, whereas only the highest IGF-II dose (100 ng/ml) exerted this effect in a significant manner.

Finally, to evaluate whether luteal VEGF production could be influenced by PGs or P, luteal cells were incubated for 24 h with medium alone (C) or in presence of increasing PGE2 or PGF2{alpha} concentrations (10–8 to 10–6 M) or with P (1, 10, 50, 100 ng/ml). Our results demonstrated that both PGs were able to significantly decrease VEGF release at all tested doses, except for the lowest PGF2{alpha} concentration (10–8 M) (Fig. 2AGo). Similarly, VEGF levels were significantly reduced when luteal cells were cultured with all the tested doses of P (Fig. 2BGo). The mean concentration of VEGF in the control was 0.25 ± 0.02 pg/ml.


Figure 2
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  		FIG. 2. A, Effect of PGE2 and PGF2{alpha} on VEGF secretion by human midluteal phase luteal cells. Luteal cells were cultured for 24 h in medium alone (C) or with PGE2 or PGF2{alpha} (10–8 to 10–6 M). Each value represents the mean ± SEM of eight independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. ***, P < 0.001, *, P < 0.05 vs. C values. B, Effect of P on VEGF secretion by human midluteal phase luteal cells. Luteal cells were cultured for 24 h in medium alone (C) or with increasing concentrations of P (1, 10, 50, 100 ng/ml). Each value represents the mean ± SEM of seven independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. ***, P < 0.001, *, P < 0.05 vs. C values.

 
To investigate whether all the above-mentioned substances could also influence VEGF luteal synthesis, RT-PCR experiments were performed on total RNA extracted from luteal cells cultured for 24 h with medium alone (C) or CoCl2 (10 µM), IGF-I or IGF-II (100 ng/ml), PGE2 or PGF2{alpha} (10–7 M), or P (10 ng/ml). Moreover, cells were incubated for 24, 48, and 72 h with medium alone (C) or hCG (100 ng/ml).

We formerly demonstrated the expression of VEGF 121, 145, 165, and 189 in human luteal cells by using a unique set of primers to amplify all VEGF isoforms (Fig. 3Go). Nevertheless, definitive statements concerning the relative quantities of the different splice variants obtained by competitive PCR cannot be affirmed.


Figure 3
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  		FIG. 3. Detection of all the VEGF isoforms expressed in human luteal cells. Total RNA was subjected to RT-PCR using a unique set of primers for all VEGF isoforms mRNA amplification, as described in Materials and Methods. The figure shows the 517-, 589-, 649-, and 721-bp PCR products, which corresponds to VEGF 121, VEGF 145, VEGF 165, and VEGF 189, respectively. MW, DNA molecular mass standards.

 
Lastly, to analyze VEGF mRNA modulation in human luteal cells, attention has been focused on diffusible VEGF 121 and VEGF 165 isoforms mRNA expression, which are the most abundant VEGF isoforms expressed in human healthy tissues (27) as well as human CL (8, 22). CoCl2 (10 µM) and IGFs (100 ng/ml) were both able to significantly increase the expression of both VEGF 121 and VEGF 165 isoforms; moreover, P (10 ng/ml), PGE2, and PGF2{alpha} (10–7 M) were all able to decrease VEGF 121 and VEGF 165 isoforms (Figs. 4Go and 5Go). Conversely, in our in vitro system, VEGF mRNA levels were not affected by hCG, after 24 h incubation (Figs. 4Go and 5Go) or 48 and 72 h (data not shown), suggesting that in midluteal phase luteal cells, the gonadotropin is unable to influence either VEGF secretion or VEGF expression.


Figure 4
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  		FIG. 4. Effect of hCG, CoCl2, IGF-I, IGF-II, PGE2, PGF2{alpha}, and P on VEGF 121 isoform mRNA levels in human midluteal phase luteal cells. Total RNA was prepared from luteal cells cultured for 24 h with medium alone (C) or CoCl2 (10 µM), hCG, IGF-I or IGF-II (100 ng/ml), P (10 ng/ml), or PGE2 and PGF2{alpha} (10–7 M). A, Total RNA was subjected to RT-PCR using a common sense primer and a specific antisense primer for VEGF 121 isoform, as described in Materials and Methods. The figure shows the 245-bp fragment corresponding to VEGF 121. The expression of 18S rRNA was used as an internal standard. DNA molecular mass standards (MW) appear in the far left lane. B, The intensities of VEGF 121 bands were quantified by densitometry and normalized by respective 18S rRNA values. Each bar represents the mean ± SEM of five separate experiments using total RNA preparations from different CL. Results are expressed as percentage of control set equal to 100. ***, P < 0.001, **, P < 0.01 vs. C values.

 

Figure 5
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  		FIG. 5. Effect of hCG, CoCl2, IGF-I and IGF-II, PGE2, PGF2{alpha}, and P on VEGF 165 isoform mRNA levels in human midluteal phase luteal cells. Total RNA was prepared from luteal cells cultured for 24 h with medium alone (C) or CoCl2 (10 µM), hCG or IGF-I or IGF-II (100 ng/ml), P (10 ng/ml), or PGE2 and PGF2{alpha} (10–7 M). A, Total RNA was subjected to RT-PCR using a common sense primer and a specific antisense primer for VEGF 165 isoform, as described in Materials and Methods. The figure shows the 245-bp fragment corresponding to VEGF 165. The expression of 18S rRNA was used as an internal standard. DNA molecular mass standards (MW) appear in the far left lane. B, The intensities of VEGF 165 bands were quantified by densitometry and normalized by respective 18S rRNA values. Each bar represents the mean ± SEM of five separate experiments using total RNA preparations from different CL. Results are expressed as percentage of control set equal to 100. ***, P < 0.001; **, P < 0.01; * P < 0.05 vs. C values.

 
Finally, experiments were performed to evaluate whether in midluteal phase VEGF could directly affect luteal steroidogenesis. To this aim luteal cells were cultured for 24 h with medium alone (C) or in presence of VEGF (100 ng/ml). As shown in Fig. 6Go, our results demonstrated the lack of a direct VEGF effect on P production. Moreover, we wanted to evaluate whether local hypoxia could negatively affect human luteal steroidogenesis, as previously reported in nonhuman primate luteal cells (14). Consistently we demonstrated that CoCl2 (10 µM) was able to significantly decrease P production in our cell culture system (data not shown). The mean concentration of P in the control was 14.5 ± 1.1 ng/ml.


Figure 6
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  		FIG. 6. Effect of VEGF on P production in human midluteal phase luteal cells. Luteal cells were cultured for 24 h in medium alone (C) or with VEGF (100 ng/ml) or hCG (100 ng/ml). 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 vs. C values.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the current study, our aim was to investigate the regulation of VEGF synthesis in human differentiated luteal cells isolated from midluteal phase CL. So far, all data regarding VEGF and human luteal physiology have been obtained using the in vitro model of LGCs (10, 11, 12, 13). After gonadotropin surge, LGCs result from follicular steroidogenic cells during the early stage of luteinization. Indeed, mature luteal cell formation occurs at the end of this process of differentiation, in which a morphological and biochemical remodeling converts follicular cells into highly steroidogenic luteal cells synthesizing large amounts of P (28).

In human ovary, LH/hCG seems to play a critical role in enhancing VEGF (29). Many authors (10, 11, 12, 13) demonstrated the stimulation exerted by hCG on VEGF production by LCG; this effect was independent of the gonadotropin-stimulated P synthesis (12). In our system we did not observe a stimulatory role for hCG on VEGF mRNA and protein production. In fact, hCG was not able to significantly increase VEGF synthesis, whereas, as expected, a pronounced enhancement in P secretion was observed. Our current data are consistent with the hypothesis, very recently proposed in monkey CL by Tesone et al. (14) that in differentiated luteal cells other factors, rather than hCG, are likely to prevail in VEGF regulation.

Interestingly, in nonhuman primate differentiated luteal cells, Tesone et al. (14) reported that VEGF production is primarily regulated by local O2 milieu, which in turn is unable to influence VEGF levels in LGCs (30). Similarly, in the current study, our data clearly demonstrated the ability for chemical hypoxia to significantly increase VEGF mRNA expression and protein production by midluteal phase human luteal cells.

Likewise, as previously reported in animal studies (14, 31), in hypoxia-treated luteal cells, a pronounced inhibition of P production was observed.

In contrast to tumor vessel neoformation, the transient luteal vasculogenesis is a tightly regulated process, which is driven by proangiogenic and tempered by antiangiogenic factors (9, 32). Then, given the pivotal role of VEGF in this process, physiological factors involved in ovarian function are likely to influence luteal VEGF production. In addition to hCG/LH, P is the other key factor characterizing the luteal phase. Because receptors for P, the primary secretory product of CL, are known to be expressed in luteal cells (33), we analyzed whether this hormone could affect luteal VEGF synthesis. Our data clearly indicate that P is able to decrease VEGF mRNA and protein levels in differentiated luteal cells.

As mentioned above, in human midluteal phase luteal cells, hCG was not able to affect VEGF mRNA and protein production, whereas this gonadotropin highly stimulated P secretion. On the basis of these results, we cannot rule out that after the hCG-mediated P increase, the potent inhibitory effect of P on luteal VEGF synthesis could mask a possible direct stimulatory effect of hCG.

Interestingly, immunohistochemistry technique demonstrated specific receptors for VEGF in human luteal cells (8, 21, 22). These data prompted us to evaluate whether, in addition to its angiogenic action, VEGF could directly affect steroidogenesis in midluteal phase luteal cells. In the present study, the lack of a direct VEGF effect on P luteal secretion was demonstrated.

In fully functional CL, VEGF has been previously suggested to influence luteal steroidogenesis by maintaining vascular permeability. Indeed, vessel leakiness seems to be crucial for both the delivery of P precursors to luteal cells and P secretion into the bloodstream (6, 20). Then even if in the present study VEGF did not exert a direct steroidogenic effect in human luteal cells, we can speculate that in midluteal phase, when the vasculature is already highly developed (29) and P maximally secreted (20), P may exert a negative autocrine-paracrine control on its own luteal production by decreasing VEGF-dependent vessel leakiness.

In the past years, in addition to the classical LH/hCG, other factors have been found to be important regulators of luteal function. For this reason we tested their possible influence on VEGF mRNA and protein production. Both IGF-I and -II are known to be important effectors of luteal function (19, 34) and key modulators of ovarian angiogenesis (30, 35). Our current results confirm and extend the important luteotropic role for these growth factors in human CL. Indeed, besides directly promoting luteal steroidogenesis (24, 36), in midluteal phase both IGFs were able to directly enhance VEGF mRNA and protein production. Because both IGFs and their receptors are expressed in luteal cells (37), it is tempting to hypothesize that IGF system can influence VEGF synthesis by an autocrine or paracrine manner.

A role in modulating vascular permeability and angiogenesis has been proposed also for PGs (38, 39), well-known local regulators of luteal function (18). In differentiated luteal cells, we demonstrated the ability for the luteolytic PGF2{alpha} to significantly decrease VEGF mRNA and protein levels. Our current data are consistent with recent in vivo evidence reporting a marked suppression of luteal VEGF expression in PGF2{alpha}-treated animals (40). We can speculate that this effect could partially explain the PGF2{alpha} role in luteal regression. In this assumption, the meaning of the reduced VEGF mRNA and protein production induced by the luteotropic PGE2 remains to be elucidated. However, because throughout the CL luteum life span, the expression of specific PG receptors changes (18), the same prostanoid could exert different effects in mature rather than developing CL. Indeed, in regressing CL, the ability for PGE2 to increase PGF2{alpha} luteolytic effect has been postulated (18). Therefore, even if PGE2 is able to enhance VEGF expression in human LGCs (11), it is tempting to speculate that in differentiated luteal cells, PGE2, as well as PGF2{alpha}, negatively modulates VEGF production.

In conclusion, as very recently suggested in nonhuman primate models (14), we demonstrated that VEGF regulation becomes different when human LGCs differentiate in mature luteal cells. Rather than hCG, local factors seem to prevail in modulating VEGF synthesis in isolated luteal cells from midluteal phase human CL. In particular, we demonstrated that local hypoxia and both IGFs significantly increased VEGF mRNA and protein production. On the contrary PGs and P negatively influenced VEGF both mRNA expression and protein release. Finally, we demonstrated that VEGF was not able to directly affect P release in human midluteal phase luteal cells.


   Acknowledgments
 
The authors thank Dr. Maurizio Guido and Dr. Giovanna Salerno for their help in sample collection.


   Footnotes
 
This work was supported by the Ministero della Salute (current research: 2004; project title: "La prevenzione dell’handicap mentale: modeli di studio biologici e clinici in epoca preconcezionale, riproduttiva e prenatale").

First Published Online April 4, 2006

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

Abbreviations: C, Control; CL, corpora lutea; hCG, human chorionic gonadotropin; LGC, luteinizing granulosa cell; P, progesterone; PG, prostaglandin; VEGF, vascular endothelial growth factor.

Received November 9, 2005.

Accepted March 29, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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