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Endocrinology, doi:10.1210/en.2006-1127
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Endocrinology Vol. 148, No. 3 1296-1305
Copyright © 2007 by The Endocrine Society

Single-Chain Bifunctional Vascular Endothelial Growth Factor (VEGF)-Follicle-Stimulating Hormone (FSH)-C-Terminal Peptide (CTP) Is Superior to the Combination Therapy of Recombinant VEGF plus FSH-CTP in Stimulating Angiogenesis during Ovarian Folliculogenesis

Rhonda K. Trousdale, Susan V. Pollak, Jeffrey Klein, Leslie Lobel, Yasuhiro Funahashi, Nikki Feirt and Joyce W. Lustbader

Departments of Obstetrics and Gynecology (R.K.T., S.V.P., J.K., L.L., Y.F., J.W.L.) and Pathology (N.F.) and Center for Reproductive Sciences (J.W.L.), Columbia University, College of Physicians and Surgeons, New York, New York 10032

Address all correspondence and requests for reprints to: Joyce W. Lustbader, Department of Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York City, New York 10032. E-mail: jwl2{at}columbia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Infertility technologies often employ exogenous gonadotropin therapy to increase antral follicle production. In an effort to enhance ovarian response, several long-acting FSH therapies have been developed including an FSH-C-terminal peptide (CTP), where the FSH subunits are linked by the CTP moiety from human chorionic gonadotropin, which is responsible for the increased half-life of human chorionic gonadotropin. We found that administration of FSH-CTP for ovarian hyperstimulation in rats blunted ovarian follicle vascular development. In women, reduced ovarian vasculature has been associated with lower pregnancy rates. We were interested in determining whether vascular endothelial growth factor (VEGF) therapy could enhance ovarian angiogenesis in FSH-CTP-treated rats. Coadministration of systemic FSH-CTP plus recombinant VEGF was compared with treatment with a novel, single-chain bifunctional VEGF-FSH-CTP (VFC) analog. For VFC, the FSH portion targets the protein to the ovary and stimulates follicle growth, whereas VEGF enhances local vascular development. Both in vitro and in vivo studies confirm the dual FSH and VEGF action of the VFC protein. Evaluation of ovarian follicle development demonstrates that administration of combination therapy using VEGF and FSH-CTP failed to increase follicle vasculature above levels seen with FSH-CTP monotherapy. However, treatment with VFC significantly increased follicle vascular development while concurrently increasing the number of large antral follicles produced. In conclusion, we report the production and characterization of a long-acting, bifunctional VEGF-FSH-CTP protein that is superior to combination therapy for enhancing VEGF activity in the ovary and stimulating follicular angiogenesis in rats.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVARIAN FOLLICULOGENESIS IS a complex process with the ultimate goal of producing and releasing a mature oocyte from the ovary. This process requires the co-development of an extensive perifollicular vascular network to facilitate the transport of essential nutrients, growth hormones, and oxygen to the growing and metabolically dynamic follicle. Administration of agents that block angiogenesis, such as a vascular endothelial growth factor (VEGF) antagonist, limit follicle vascular development, slow follicle growth, and impede the formation of large, antral follicles in mice and nonhuman primates (see Ref. 1 for review). In infertile women, decreased ovarian vascularity has been associated with lower pregnancy rates in women of all age groups (2). In turn, several studies show statistically higher pregnancy rates when oocytes isolated from well vascularized follicles were chosen for fertilization and embryo transfer (3, 4, 5). Thus, ovarian vascular development during ovarian stimulation is a potential novel target for developing new infertility therapies.

During antral follicle growth, there is a strong correlation between increased follicle area and both theca and perifollicular vascular development. Specifically, theca cell proliferation has been shown to occur before increased angiogenesis within the theca compartment (6). Although LH/human chorionic gonadotropin (hCG) supplementation is the most potent stimulator of theca development, our studies demonstrate that recombinant human (rh)FSH monotherapy was still able to enhance both theca area and vascular development throughout all stages of antral follicle development in rats. Recently, several laboratories have developed long-acting FSH analogs by altering the carbohydrate content of the protein (7, 8, 9, 10). Our laboratory previously reported the development of a single-chain rhFSH-CTP analog capable of increasing the number of follicles progressing to the large antral follicle stage with a single injection (11). However, careful histological examination revealed that whereas rhFSH monotherapy enhances vascular development in rodents, treatment with rhFSH-CTP without LH/hCG supplementation limited both theca development and perifollicular angiogenesis in large antral follicles.

We were interested in determining whether the reduced vascular development observed with rhFSH-CTP monotherapy could be enhanced with VEGF therapy. Two approaches were examined. First, recombinant human VEGF-A165 (rec-VEGF) was systemically coadministered with rhFSH-CTP. VEGF-A has been identified as a potent stimulator of angiogenesis in several organ systems (12, 13). Direct injection of VEGF DNA gene fragments into the ovaries of miniature gilts was found to increase the capillary density of the theca interna by at least 50% at all follicle stages (14, 15). There are problems, however, associated with systemic rec-VEGF protein therapy including the short half-life (approximately 30 min) (16, 17), the ubiquitous expression of VEGF receptors making protein targeting difficult, and significant deleterious side effects observed in humans and other large mammals (17, 18, 19, 20).

Our second approach was to develop a long-acting bifunctional VEGF-FSH-CTP analog (rhVFC) exhibiting both biological FSH and VEGF activities. By tethering VEGF-A to long-acting FSH-CTP, our goal was to enhance the half-life of VEGF. The second goal was to limit systemic VEGF activity. Because VEGF is only active as a homodimer (21), the rhVFC protein included a single VEGF monomer, and thus a solitary rhVFC protein would be unable to activate VEGF receptors. We hypothesized that rhVFC proteins would migrate into the ovary with the FSH moiety binding and activating local ovarian FSH receptors. Once the concentration of rhVFC proteins accumulated to a high enough concentration, the VEGF monomers present on rhVFC would be able to dimerize with neighboring monomers and subsequently activate local VEGF receptors within the ovary. Thus, rhVFC therapy would limit systemic activation of VEGF receptors and enhance local angiogenesis only in tissues where a high concentration of local FSH receptors was present, such as growing follicles within the ovary.

We report here the development and characterization of the rhVFC protein, which was found to have a longer half-life, exhibit both FSH and VEGF activity in vitro, and also enhance in vivo ovarian follicle development in female rats. In evaluating follicle vascular development, we demonstrate that systemic administration of rec-VEGF in combination with rhFSH-CTP failed to stimulate follicular angiogenesis above rhFSH-CTP monotherapy. However, treatment with bifunctional rhVFC stimulated a significant increase in perifollicular angiogenesis while concurrently increasing the number of large antral follicles produced. In conclusion, tissue targeting of VEGF as part of a long-acting, bifunctional VEGF-FSH protein enhanced local ovarian follicle angiogenesis without producing adverse side effects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Approval was obtained from the Institutional Animal Care and Use Committee at Columbia University in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animal experiments were conducted in accord with accepted standards of humane animal care.

Construction of fusion proteins
The construction of rhFSH-CTP has been described previously (7). The rhVFC construct was developed through modification of the rhFSH-CTP construct. VEGF-A165 including the upstream signal peptide sequence was cloned from a cDNA library of human umbilical vein endothelial cells (HUVEC) (generously provided by Dr. Jan Kitajewski, Columbia University). At the 5' end of the VEGF PCR primer, a HindIII restriction site was introduced, and at the 3' end, the VEGF termination codon was replaced with an XbaI restriction site. Both the VEGF PCR product and the rhFSH-CTP construct were digested with HindIII and XbaI followed by ligation of the products to create VEGF-A165-ß-hFSH-CTP-{alpha}-hFSH (rhVFC). This arrangement facilitated expression of the nascent protein to begin with the VEGF leader sequence and culminated with the translation of the {alpha}-subunit of FSH as a single-chain protein product.

Production of fusion protein
The rhVFC construct was inserted into an SV40 expression vector and cotransfected into CHO-K1 cells together with a SV2neo plasmid encoding resistance to G418. The Chinese hamster ovary (CHO) cell transformation was performed using a standard calcium phosphate precipitate technique. Selectable medium containing G418 (Gemini Bioproducts, Woodland, CA) was used to isolate a high-secreting clone as previously described (8). To further increase yields, cells were grown in suspension cultures with spinner bottles seeded at 105 cells/ml in CHO-SFM II (Life Technologies, Rockville, MD) containing 400 µg/ml G418. Cultures generally reached a density of 1.5 x 106 cells/ml on d 6 or 7, and the cell supernatant was harvested on d 7 or 8. Supernatants received 0.2 mmol/liter phenylmethylsulfonyl fluoride and were filtered through a 0.2-µm membrane and stored at 4 C until the day of purification.

For affinity purification of protein analogs, a Sepharose column was prepared by coupling purified A103 (22), a monoclonal antibody specific for the {alpha}-subunit of human gonadotropins, to cyanogen bromide (CNBr)-Sepharose-4B according to the manufacturer’s instruction (Amersham Biosciences, Piscataway, NJ) at a concentration of 5 mg antibody/ml Sepharose. The CHO cell supernatant was passed over the Sepharose column and then washed with 50 bed volumes of PBS followed by 2 bed volumes of distilled water. Protein was eluted with 3–4 bed volumes of 1 M acetic acid and immediately dried on a Speed-Vac concentrator (ThermoForma, Marietta, OH). The purified protein was then resuspended in dH2O.

Western blot analysis
The rhFSH, single-chain rhFSH-CTP, rec-VEGF, and rhVFC were electrophoresed through an SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membrane using standard techniques. Samples were denatured by boiling before electrophoresis. The membrane was blocked in 5% BSA overnight at 4 C and then incubated for 2 h in a 1:8000 dilution of either rabbit antihuman antibody R116 (23), which recognizes the {alpha}-subunit of gonadotropin hormones including FSH, or a 1:400 dilution of rabbit antihuman VEGF antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The membrane was subsequently washed five times in Tris-buffered saline and incubated in a 1:40,000 dilution of peroxidase-conjugated polyclonal antibody to rabbit Ig (Amersham) for 1 h. Bands were visualized, after additional washes in Tris-buffered saline, by incubation in chemiluminescent detection reagent (Pierce, Rockford, IL) and exposed to x-ray film.

Evaluation of protein concentration
The rhFSH-CTP and rhVFC protein concentrations were determined by an FSH RIA using an antibody to FSH ß-subunit (Biodesign International, Saco, ME). The concentration of rhVFC was expressed as a weight per volume ratio of the VEGF moiety, using a molecular mass of 22.5 kDa for a single chain of VEGF.

In vitro studies of FSH activity
FSH activity of the FSH analogs rhFSH-CTP and rhVFC were evaluated by receptor-binding activity in CHO cells transfected with the FSH receptor (CHO-FSH-R) (generously provided by Serono Laboratories, Rockland, MA). CHO-FSH-R cells were aliquoted at 2.5 x 104 cells per tube, and then commercial rhFSH (Serono), rhFSH-CTP, rhVFC, and rec-VEGF (BD Biosciences, Bedford, MA) were added to the cells at amounts of 0, 0.3, 1, 3, 10, 30, 100, 300, and 1000 fmol and allowed to incubate at 37 C for 15 min and then transferred to a 75 C water bath for 3 min. Supernatant was collected and evaluated for cAMP activity using an RIA kit according to the manufacturer’s instructions (Perkin-Elmer Life Sciences, Boston, MA).

In vitro studies of VEGF activity
VEGF activity was detected by two methods. First, we evaluated the ability of the rhVFC analog to stimulate VEGF II receptor (KDR) autophosphorylation. Microvascular endothelial cells isolated from human foreskin (24) were plated at 100,000 cells per well in 12-well plates and cultured for 5 h in endothelial basal medium (Clonetics, San Diego, CA) containing the supplier’s endothelial growth medium growth factor supplements. The cells were then starved overnight in endothelial basal medium containing only 0.2% BSA without endothelial growth medium supplement. The next morning, 1 ml of the following samples was added to each well: 1) media only, 2) commercial rec-VEGF (10 ng/ml), 3) rhFSH-CTP at (0.67 pmol/ml), or 4) rhVFC (0.67 pmol/ml, which equals 15 ng/ml VEGF moiety). All wells were incubated for 1 h at 4 C. Cells were subsequently washed twice in cold PBS containing calcium and magnesium and then lysed in 250 µl RIPA buffer. The lysate was filtered by centrifugation in a 0.65µm Durapor PVDF filter (Millipore, Bedford, MA) at 3000 rpm for 30 min. The filtrate was incubated in 2 µg/ml anti-KDR monoclonal antibody (25) for 2 h. Fifty microliters of water-washed protein A-Sepharose (Amersham) was added, and the mixture was incubated overnight at 4 C with gentle mixing. The Sepharose was washed twice in RIPA buffer and then boiled for 5 min in 25 µl of 2x SDS-PAGE sample buffer. Supernatants were electrophoresed in a gradient gel of 4–15% acrylamide (Bio-Rad, Hercules, CA) and electroblotted onto a PVDF membrane. After blocking, the membrane was washed three times in PBS containing 0.05% Tween 20 (PBS-T). The membrane was incubated overnight in horseradish-peroxidase-conjugated anti-phosphotyrosine (10 ng/ml) monoclonal antibody (clone 4G10; Upstate Biotechnology, Lake Placid, NY) in PBS-T containing 3% BSA. The membrane was washed three times in PBS-T and developed using a chemiluminescent system (ECL; Amersham).

The second method of detection was the sandwich tubal formation assay. A 400-µl aliquot of collagen gel was added to each well of a 24-well plate and allowed to gel for at least 1 h at 37 C. After gelation, HUVEC (26) were plated on the gel (1–1.2 x 105 cells per well) in human endothelial serum-free medium (SFM) basal growth medium (Invitrogen, Carlsbad, CA) containing 20 ng/ml of epidermal growth factor (EGF) (Invitrogen) and incubated at 37 C for 3 h. HUVEC were subsequently covered with 400 µl collagen gel and further incubated for 3 h at 37 C. The gel was then covered with SFM supplemented with 20 ng/ml EGF and either 1) rhFSH, 2) rhFSH-CTP, 3) rec-VEGF at 20 ng/ml, or 4) rhVFC. All FSH analogs were added at a concentration of 889 fmol/ml, which is equivalent to 20 ng/ml VEGF in the rhVFC analog. Cells were allowed to grow for 5 d and then evaluated for tubal formation using a phase-contrast microscope. Tubal length was quantified using ImagePro Plus version 4.01 software (Media Cybernetics, Silver Spring, MD).

Half-life evaluation of rhVFC
Sprague Dawley rats were ordered from Harlan Co. (Indianapolis, IN). For half-life determination, postnatal d-21 female animals were randomized to receive a single ip injection of one of four treatments (n = 5 per group): 1) saline, 2) rhFSH (42 IU), 3) rhFSH-CTP (42 IU), or 4) rhVFC (42 IU). Animals were anesthetized with isoflurane, and blood was drawn at 3, 6, 9, 12, 25, 30, and 72 h. Blood levels of FSH were determined by an immunoassay specific for human FSH (Immulite, Diagnostics Products Corp., Los Angeles, CA). Pharmacokinetic analysis was performed using PK Solutions 2.0 software (Summit Research Services, Montrose, CO).

Evaluation of ovarian follicle development
Postnatal d-21 Sprague Dawley female rats (n = 3) were given a single ip injection of 1) saline, 2) 42 IU rhFSH, or 3) 42 IU rhFSH-CTP. Additionally d-21 Sprague Dawley female rats (n = 5) were given daily ip injections of 1) rhFSH-CTP (21 IU/d), 2) rhVFC (21 IU/d = 1 µg VEGF/d), or 3) rhFSH-CTP (21 IU/d) plus rec-VEGF (1 µg/d) (Peprotec, Rocky Hill, NJ) for 2 d. Forty-eight hours after the first injection, animals were euthanized with inhaled isoflurane, blood collected by cardiac puncture, and the following organs removed for pathological evaluation: ovary, uterus, kidney, liver, lung, heart, and pituitary. Ovaries were trimmed and weighed after removal. One ovary and portions of each organ were fixed in 4% paraformaldehyde, and the contralateral ovary and portions of each organ were also frozen in OCT media (TissueTek, Sakura Finetek USA, Torrance, CA).

Paraformaldehyde-fixed tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin. All tissues were evaluated by a pathologist for evidence of abnormal bleeding. For ovarian follicle analysis, the entire ovary was serial sectioned at 8 µm, and all sections were examined. The section in which both the oocyte and the germinal vesicle were present was used for follicle staging. Follicles with clear antrum development [stage 6–8 of the Pedersen and Peters grading system (27)] were included in the follicle count, and atretic follicles as determined by greater than 10 apoptotic granulosa cells present in the follicle were excluded from the count.

Frozen ovarian tissue was sectioned every 8 µm for immunohistochemistry, and at least four sections from each ovary were analyzed. The sections were first immersed in cold acetone, and then endogenous biotinylated activity was inhibited using the avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA). To decrease nonspecific binding, the tissues were blocked in normal horse serum. Next, the mouse antirat platelet endothelial cell adhesion molecule-1 (PECAM-1) antibody (BD PharMingen, San Diego, CA) was added at a concentration of 1:100 and allowed to incubate overnight at 4 C. After being washed in PBS, a biotinylated horse antimouse IgG (rat-adsorbed) antibody (BD PharMingen) was added at a concentration of 1:800 and allowed to incubate for 30 min. The sample was washed again in PBS, and a brown signal was generated with the peroxidase/diaminobenzidine system (Vectastain ABC Kit; Vector) followed by counterstaining with hematoxylin.

PECAM-stained ovarian sections were examined under the microscope, and a computer imaging system linked to Nikon Eclipse E800 camera was used to evaluate ovarian follicle area, theca area, and area of follicle vasculature. All follicles were analyzed at x100, and data were processed using the ImagePro Plus version 4.01 software (Media Cybernetics).

Statistical analysis
Data are expressed as mean ± SE. Unpaired t test with two-tailed P value was used for comparison of different treatment groups. The Mann-Whitney U test was used for analysis of nonparametric data. All analysis calculations were computed using the PRISM software (GraphPad, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Histological evaluation of ovarian follicles with FSH monotherapy
Previously we described the production of a single-chain, long-acting FSH protein that included FSH {alpha}- and ß-subunits linked by the CTP moiety from the hCG ß-subunit (7). CTP, with four O-linked oligosaccharides, is responsible for decreased metabolic breakdown and increased half-life activity of hCG compared with the other gonadotropin family members (LH, FSH, and TSH) (28). A single injection of the rhFSH-CTP analog without concomitant LH or hCG treatment was sufficient to enhance ovarian weight and increase the number of large antral follicles present within the ovary after 72 h (8). On initial assessment, the ovarian follicles from rhFSH-CTP-treated animals were found to have normal granulosa cell organization, standard antrum development, and appropriate oocyte growth (8).

To study theca and vascular development more precisely, 21-d-old Sprague Dawley female rats (n = 3 per group) were randomized to receive a single ip injection of either 1) saline, 2) rhFSH (42 IU), or 3) rhFSH-CTP (42 IU). The dose of 42 IU was chosen to maintain consistency with our previously reported experiments. Animals were killed 48 h later when an hCG injection would normally be administered in rodents for ovulation induction. Immunohistochemistry using PECAM as a marker of perifollicular blood vessels was performed for histological evaluation. Using the ImagePro Analysis system, several measurements of individual antral follicles were obtained as diagrammed in Fig. 1AGo. The inner area included only the granulosa cells, the antrum, and the oocyte. The outer area included the inner area plus the surrounding theca cells. Theca area was calculated as the difference between the inner area and the outer area. Perifollicular vasculature was measured as the PECAM-positive areas within the theca layer.


Figure 1
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  		FIG. 1. Evaluation of ovarian follicle areas. A, Schematic of antral follicle area measurements. Red circle delineates inner area, black circle defines the outer area, and the light blue area represents the theca layer. Theca area is calculated as inner area subtracted from outer area. B, Linear regression analysis correlating theca area to inner follicle area. C, Linear regression analysis correlating vascular area to outer follicle area. In B and C, black represents saline; green, rhFSH; and blue, rhFSH-CTP.

 
As outlined in Table 1Go, treatment with a single dose of rhFSH or rhFSH-CTP increased both antral follicle production and ovarian weight. The rhFSH-CTP therapy stimulated a significantly higher mean antral follicle size compared with rhFSH therapy (198,753 vs. 148,726 µm2, respectively). Despite the increased follicle size, treatment with rhFSH-CTP ovaries had a total mean theca area similar to the rhFSH-treated ovaries (43,442 and 46,163 µm2, respectively). To confirm that theca development lagged behind follicle growth with rhFSH-CTP treatment, a ratio of theca area to inner area was calculated [(theca area/inner follicle area) x 100]. Treatment with rhFSH-CTP produced a significantly lower theca to inner follicle area ratio of 31.80 compared with the saline and rhFSH-treated animals (43.36 and 43.13, respectively). Additionally, the reduction in theca development was present at all stages of antral follicle growth as determined by linear regression analysis (Fig. 1BGo).


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  		TABLE 1. Ovarian parameters with FSH single-dose therapy

 
In assessing ovarian follicle vascular development, it is common to report vascular density as vasculature area per theca area. However, this measurement assumes all hormonal therapies stimulate theca development equally. In rhFSH-CTP animals, vascular density would be artificially elevated compared with saline- and rhFSH-treated animals because the theca area is reduced. To identify a more accurate measurement of vascular development, we compared the total vascular area to total follicle area for all treatment groups. Linear correlation analysis revealed the strongest correlation between total vascular area and outer follicle area (r = 0.91; P < 0.0001). Again, despite the larger mean antral follicle size after rhFSH-CTP treatment, there was no significant increase in total vasculature area compared with saline and rhFSH (Table 1Go). Calculation of the vascular area to outer follicle ratio confirmed reduced angiogenesis with rhFSH-CTP (6.39) compared with rhFSH (7.92). Linear regression analysis demonstrates a lower vascular development at all stages of antral follicle growth (Fig. 1CGo). Thus, although rhFSH-CTP therapy increased the number of large antral follicles present in the ovary, this was associated with reduced theca development and blunted vascular development compared with rhFSH therapy.

Synthesis of VEGF-FSH fusion protein
The previously described rhFSH-CTP protein was chosen for the FSH moiety (11). For the VEGF portion, we chose the VEGF-A165 isoform, because it is the predominant circulating VEGF isoform in the ovary (13). In determining which orientation VEGF could be tethered to FSH-CTP, evaluation of crystallographic and activity analyses of VEGF indicated the carboxy terminus was more flexible and appeared to be less directly involved in receptor binding (20, 29, 30). Hence, nucleotides encoding the ß-hFSH signal peptide were removed and VEGF-A165 was tethered by its carboxy terminus to the amino terminus of rhFSH-CTP. The final construct encoded a single polypeptide, rhVFC, consisting of VEGF-A165 monomer-ßFSH-CTP-{alpha}FSH.

After protein purification, immunoblotting of rhVFC using an FSH antibody specific for the {alpha}-subunit identified a band at approximately 80 kDa, a protein with a larger mass than the rhFSH-CTP protein (55 kDa). An antibody against VEGF also detected purified rhVFC protein at 80 kDa, confirming that the fusion protein contained both VEGF and FSH epitopes (Fig. 2Go).


Figure 2
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  		FIG. 2. Western blot analysis of purified FSH, VEGF, and FSH analogs. A, Anti-FSH-{alpha} antibody R116: rhFSH ({alpha}-subunit) (25 kD), rhFSH-CTP (55 kD), and rhVFC (80 kD); B, anti-VEGF antibody: rhFSH (no signal); rhFSH-CTP (no signal); purified VEGF (22.5 kD), and rhVFC (80 kD). All proteins were denatured before immunoblotting.

 
In vitro FSH bioactivity of rhVFC
In vitro FSH bioactivity was evaluated by exposing a CHO cell line, CHO-K1, ectopically expressing the FSH receptor, to increasing concentrations of rhFSH, rhFSH-CTP, rhVFC, or rec-VEGF. The rhVFC-mediated signal transduction through the FSH receptor was detected by the increase of cAMP. We have previously shown that rhFSH and rhFSH-CTP increase CHO-FSH-R-stimulated cAMP formation (7, 8). Here, rhVFC stimulated cAMP production at levels comparable to rhFSH and rhFSH-CTP. Stimulation with rec-VEGF-A was negligible (Fig. 3Go).


Figure 3
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  		FIG. 3. FSH in vitro analysis. Each purified protein was evaluated for its ability to bind and activate the FSH receptor as determined by elevation in intracellular cAMP signaling. The x-axis correlates to increasing concentrations of each protein.

 
In vitro VEGF bioactivity of rhVFC
We confirmed VEGF bioactivity of rhVFC using two different in vitro assay systems. The VEGF receptor autophosphorylation assay demonstrated the ability of rhVFC to bind and activate the VEGF receptor II (KDR). Briefly, human foreskin microvascular endothelial cells were serum-starved overnight and subsequently stimulated by medium, rec-VEGF (10 ng/ml), rhFSH-CTP (0.67 pmol/ml), or rhVFC (0.67 pmol/ml, which is equivalent to 15 ng/ml of VEGF moiety). Cell lysate was analyzed for the presence of phosphorylated KDR. The rec-VEGF and rhVFC stimulated KDR phosphorylation, whereas nonsupplemented medium or rhFSH-CTP lacked specific activity (Fig. 4AGo).


Figure 4
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  		FIG. 4. VEGF in vitro activity. A, KDR phosphorylation assay: lane 1, medium control; lane 2, rec-VEGF (10 ng/ml); lane 3, rhFSH-CTP (0.67 pmol/ml); lane 4, rhVFC (0.67 pmol/ml equivalent to 15 ng/ml VEGF). Experiments were performed in triplicate, and a single representative sample from each group is presented. B, Sandwich tubal formation assay: panel 1, EGF only (20 ng/ml); panel 2, EGF plus rhFSH (889 fmol/ml); panel 3, EGF plus rhFSH-CTP (889 fmol/ml); panel 4, EGF plus rhVFC (889 fmol/ml equivalent to 20 ng/ml VEGF); panel 5, EGF plus rec-VEGF (20 ng/ml). Scale bar, 50 µm. Panel 6, Bar graph of tubal formation length in response to hormone treatments. Rec-VEGF was standardized as 100%. Error bars represent SEM. *, P < 0.01 compared with EGF, EGF plus rhFSH, and EGF plus rhFSH-CTP.

 
The sandwich tubal formation assay evaluated in vitro stimulation of blood vessel formation (31). Briefly, HUVEC were cultured on collagen in SFM supplemented with EGF (20 ng/ml) alone or a combination of EGF and FSH (889 fmol/ml), EGF and rhFSH-CTP (889 fmol/ml), EGF and rec-VEGF (20 ng/ml), or EGF and rhVFC (889 fmol/ml, equivalent to 20 ng/ml of VEGF moiety). Although HUVEC lack FSH receptors, VEGF monomer dimerization was still able to occur with subsequent activation of the VEGF receptor because rhVFC was present at a high enough concentration within the enclosed culture system. After 5 d of incubation, tubal formation was analytically assessed using the microscope. Three random areas of each culture well were photographed, and tubal length was quantified using the ImagePro Plus analysis system. Recombinant VEGF tubal formation was standardized at 100%. Neither rhFSH (6.24 ± 1.24%) nor rhFSH-CTP (6.78 ± 0.36%) was able to promote tubal formation above EGF media (5.36 ± 1.49%), whereas rhVFC stimulated tubal formation (84.79 ± 1.99%) with statistical equivalence to rec-VEGF treatment (Fig. 4BGo).

Half-life of rhVFC
For pharmacokinetic analysis of rhVFC, a single dose of 42 IU rhVFC was administered by ip injection to 21-d-old Sprague Dawley rats, blood was collected at multiple time points, and rhVFC measured by FSH Immulite assay. We previously reported the serum elimination half-life of rhFSH as 6.3 h, whereas rhFSH-CTP was 12.1 h after ip injection of Sprague Dawley rats (8). The elimination half-life of rhVFC was 9.5 h with levels declining to baseline by 72 h. This half-life is shorter than rhFSH-CTP, suggesting that the VEGF portion of the protein accelerates clearance of the rhVFC protein; nonetheless, it is a significantly greater half-life than rec-VEGF (30 min).

In vivo comparison of rhVFC vs. combination rhFSH-CTP plus rec-VEGF therapy
Given the decreased half-life of rhVFC compared with rhFSH-CTP, there was concern that a single injection of rhVFC would not be effective for the entire 48 h of the study. Therefore, rats were randomized to receive a split dose of 1) rhFSH-CTP, 21 IU at 0 and 24 h for a total dose 42 IU (rhFSH-CTPspd); 2) rhVFC, 21 IU ip at 0 and 24 h for a total dose of 42 IU, which equals 2 µg VEGF moiety; or 3) rhFSH-CTP, 21 IU ip plus rec-VEGF 1 µg at 0 and 24 h for a total 42 IU and 2 µg VEGF moiety. Blood and tissue samples were collected 48 h after the first injection.

When comparing rhFSH-CTP as a single dose vs. split dose, the split-dose therapy was not as effective as single-dose therapy in augmenting ovarian weight and antral follicle production (Tables 1Go and 2Go); however, the split dose showed similar effects on follicle development in terms of reduced theca to inner follicle area ratio (31.88, Table 2Go) compared with rhFSH-CTP single dose (31.80, Table 1Go) as well as a reduced vascular to outer follicle area ratio (5.77 and 6.39, respectively). This result confirmed that rhFSH-CTP given in divided doses represented a reproducible model of limited follicle vascular development.


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  		TABLE 2. Ovarian parameters with FSH split-dose therapy (spd)

 
Evaluation of rhVFC revealed an increase in both ovarian weight and antral follicle count which verified the in vivo FSH bioactivity of the rhVFC protein (Table 2Go). Mean antral follicle size and theca areas were not significantly different between the rhVFC and rhFSH-CTPspd groups. Yet, rhVFC therapy produced a statistically significant increase in follicular angiogenesis with the highest mean total vasculature per follicle (13,608 µm2) for all treatment groups and a high ratio of vasculature per outer area (7.49) compared with rhFSH-CTPspd therapy (5.77) (Table 2Go). Linear regression analysis illustrates the increase in both theca and vascular development stimulated by rhVFC therapy (Fig. 5Go). Figure 6Go compares PECAM staining of similarly sized large antral follicles from the ovaries of rhFSH-CTPspd, combination rhFSH-CTPspd plus r-VEGF, and rhVFC-treated animals. These data confirmed enhanced in vivo VEGF activity in the ovary after rhVFC treatment.


Figure 5
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  		FIG. 5. Evaluation of ovarian follicle areas after split-dose therapy. A, Linear regression correlating theca area to inner follicle area; B, linear regression correlating vascular area to outer follicle area. Blue represents FSH-CTPspd; pink, FSH-CTPspd plus rec-VEGF; and red, rhVFC.

 

Figure 6
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  		FIG. 6. Comparison of large antral follicle vasculature by immunohistochemisty with anti-PECAM antibody. A, rhFSH-CTPspd ovary; B, rhFSH-CTPspd plus rec-VEGF; C, rhVFC. Magnification (all pictures), x150.

 
In contrast, combination rhFSH-CTPspd plus VEGF treatment did not enhance follicle angiogenesis above levels obtained with rhFSH-CTPspd monotherapy (Table 2Go). The vascular area as well as the ratio of vasculature to follicle area was statistically equivalent between combination therapy and rhFSH-CTPspd therapy groups. The ratio of vascular to follicle area was significantly lower for combination therapy (5.87) compared with rhVFC (7.49). Interestingly, mean antral follicle size was significantly greater with rec-VEGF combination therapy compared with the other split-dose therapies (Table 2Go). In concert with bigger antral follicles, a higher ovarian weight was found in combination therapy rats compared with rhFSH-CTP. The number of large antral follicles present did not increase with combination therapy, suggesting that rec-VEGF did not enhance the number of follicles progressing to the large antral follicle phase, but it was stimulating greater growth of the antral follicles that were present. Evaluation of the theca to follicle area ratio was also quite low in combination therapy ovaries. Here, regression analysis revealed that theca development slowed considerably in the larger antral follicles (Fig. 5Go).

Pathological evaluation
Ovaries from rhVFC-treated animals were indistinguishable from animals treated with saline, rhFSH, or rhFSH-CTP. However, ovaries from animals receiving concomitant rhFSH-CTP and rec-VEGF were found to have earlier initiation of antrum development (Fig. 7Go). Small preantral follicles through stage 5a classification were similar among all therapies (Fig. 7Go, A and D). However, early follicular fluid collection could be seen in stage 5b follicles of combination rhFSH-CTP with rec-VEGF-treated rats but not in similar sized follicles from the rhFSH-CTP-treated animals (Fig. 7Go, B and E). Similarly the antrum appeared more fully developed in the combination ovaries compared with similarly sized follicles in rhFSH-CTP ovaries (Fig. 7Go, C and F). There was no evidence of abnormal bleeding or endothelial damage in these ovaries.


Figure 7
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  		FIG. 7. Histological comparison of ovarian follicle development. Comparison of rhFSH-CTPspd rats (A–C) with combination therapy of rhFSH-CTPspd plus rec-VEGF rats (D–F). A and D, Small preantral follicles (stage 5a) with no significant difference observed. Magnification, x400. B and E, Medium-size follicles (stage 5b) with multiple layers of granulosa cells (GC). Early follicular fluid accumulation is seen in the combination-treated follicles. Magnification, x400. C and F, Early antrum (A) formation (stage 6). For two similarly sized follicles, the rhFSH-CTPspd ovaries show pockets of follicular fluid accumulation, whereas combination-treated follicles exhibit full antrum development. Magnification, x200.

 
To evaluate for the presence of harmful systemic side effects, animals were closely monitored during both the pharmacokinetic studies and all ovarian stimulation studies. The behavior of rhVFC-treated animals was indistinguishable from other treatment groups. Rats moved freely about the cage, eating and drinking ad libitum, and exhibited no obvious adverse affects. Both gross and histological pathology examination of the extraovarian tissues (liver, lung, kidney, heart, and pituitary) in animals of all treatment groups revealed no evidence of VEGF activity in nonovarian tissues. There was no evidence of bleeding, vascular proliferation, endothelial damage, or changes in organ architecture after administration of rhVFC (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During the ovarian reproductive cycle, a cohort of arrested primordial follicles are stimulated to reenter the growth cycle and develop into large antral follicles in preparation for ovulation. Studies in mice show FSH is essential for the transition from a preantral follicle to a large antral follicle (32). More recently, VEGF-A has been identified as a key modulator in antral follicle production; blocking VEGF-A results in slow antrum formation and limited progression to the large antral follicle phase (1). In humans, the systemic production of VEGF significantly decreases with age (33, 34), and reduced ovarian vascular development has been proposed as a cause for infertility, especially in older women (2). Thus, increasing ovarian VEGF levels to enhance follicle angiogenesis provides a promising novel target for infertility therapies.

There are no naturally occurring mouse models exhibiting reduced ovarian vascular development. However, we report here that after administration of the novel, long-acting rhFSH-CTP analog without LH/hCG supplementation to rats, theca development and perifollicular angiogenesis lagged behind follicle growth during ovarian hyperstimulation. We were subsequently interested in determining whether VEGF could enhance ovarian follicle angiogenesis in rhFSH-CTP-treated animals.

Systemic therapy with recombinant VEGF has been shown to enhance anigiogenesis in the heart after myocardial infarction and in skeletal muscle after limb ischemia (35, 36). Thus, one obvious method of increasing ovarian vascularization would be systemic coadministration of rec-VEGF with rhFSH-CTP. However, several obstacles have been associated with systemic VEGF protein therapy such as the relatively short half-life of VEGF (30 min), thereby requiring medication to be delivered by iv infusion (17, 37). Furthermore, because VEGF receptors are expressed ubiquitously, VEGF can activate receptors in multiple organ systems leading to profound hypotension, increased bleeding, and greater blood vessel permeability with edema formation (17, 18, 19).

As an alternative to systemic rec-VEGF therapy, we developed a bifunctional VEGF-FSH protein that would stimulate ovarian follicle production via FSH activity and enhance local angiogenesis via VEGF activity. The bifunctional protein was constructed by tethering VEGF-A165, a potent stimulator of angiogenesis, to the long-acting rhFSH-CTP protein to form rhVFC. Pharmacokinetic studies confirmed an increased half-life for the entire bifunctional fusion protein of 9.5 h, which is significantly longer than the 30-min half-life of recombinant VEGF.

In an effort to prevent systemic VEGF activity, each individual rhVFC fusion protein contained a single VEGF monomer. Because VEGF bioactivity requires the covalent dimerization of two identical VEGF monomers (38), a single rhVFC protein in isolation would be unable to activate a VEGF receptor. We hypothesized that the VEGF-FSH fusion protein would aggregate within the ovary by FSH binding to local FSH receptors on granulosa cells and perifollicular endothelial cells (39, 40, 41). Once the VEGF-FSH protein was present at a high enough concentration in the ovary, VEGF monomers on neighboring rhVFC proteins could dimerize, activate local VEGF receptors, and stimulate angiogenesis.

Initially, an LH-VEGF protein was considered because follicle vasculature develops in the theca where LH receptors predominate; however, LH overexpression in mice has been reported to cause ovarian cyst formation, ovarian tumor development, and ovulation defects (42). Additionally, LH receptors have been located in numerous nongonadal tissues, e.g. brain, mammary gland, spinal cord, and skin (43). High concentrations of FSH receptors, however, have been reported only in the ovary; thus, the rhVFC protein would theoretically aggregate within the ovary. Because of a recent report of FSH receptor expression on osteoclast cells in the bone (44), additional studies into the tissue-specific localization of the rhVFC protein are ongoing in our laboratory.

In rats, we found that coadministration of rec-VEGF with rhFSH-CTP was unable to enhance ovarian follicle angiogenesis. Specifically, there was no significant difference between rhFSH-CTP monotherapy compared with combination treatment with rec-VEGF and rhFSH-CTP with regard to either the total vascular area development or the vasculature to follicle area ratio. In contrast, rhVFC therapy significantly stimulated follicle angiogenesis with a significant increase in the total vasculature compared with rhFSH-CTP monotherapy. Additionally, rhVFC treatment produced a higher vasculature to outer area ratio of 7.49 compared with both rhFSH-CTPspd (5.77) and rhFSH-CTP plus rec-VEGF (5.87). Furthermore, more antral follicles were present after rhVFC therapy compared with other therapies, suggesting that by enhancing vascular development, more follicles were able to continue progressing to the large antral follicle stage.

One possible explanation for the minimal angiogenic effect after combination rec-VEGF and rhFSH-CTP therapy was the relatively low dose of VEGF administered in these rat experiments, a total of 2 µg VEGF, or 40 µg/kg. Previous studies of VEGF activity in rats reported doses ranging from 250-1300 µg/kg to stimulate angiogenesis (16, 37). Yet, for clinical trials, only 12 µg/kg of rec-VEGF was slowly infused to avoid systemic side effects (17). This low rec-VEGF dose was chosen because it is equivalent to the amount of VEGF moiety present in 42 IU of rhVFC. Furthermore, for a therapy to be safe in larger mammals, it would need to be efficacious at a relatively low dose of VEGF moiety.

One reason for the significant proangiogenic effect of rhVFC specifically in the ovary could be an increased concentration of active dimerized VEGF present locally. Hypothetically, after the rhVFC protein enters the ovary to bind and activate local FSH receptors, the VEGF moiety would have two possible fates. First, the rhVFC protein could bind FSH receptors to stimulate follicle development but remain isolated such that the VEGF monomer would be ineffective in activating the local VEGF receptor. Second, in locations where the rhVFC protein is present in high concentrations, two adjacent VEGF monomers from rhVFC proteins could form a dimer to activate local VEGF receptors. Our in vitro tubal formation studies confirmed that when rhVFC proteins were present in high enough concentration in the enclosed in vitro system, the VEGF monomers were capable of dimerization and activation of local VEGF receptors, irregardless that rhVFC was unable to bind local FSH receptors because HUVEC do not express FSH receptors. Thus, the aggregation of rhVFC within the ovary would significantly enhance the concentration of VEGF dimers capable of forming in the ovary. Furthermore, as reported above, tethering VEGF to a long-acting hyperglycosylated FSH-CTP prolonged the protein half-life, thus providing an extended period of receptor activation for each VEGF dimer present, further enhancing local VEGF activity. The studies presented here confirm that rhVFC is capable of enhancing ovarian angiogenesis; however, additional studies are required to define the exact molecular mechanisms involved with this phenomenon.

Finally, one surprising difference between rhVFC and combination therapy was the effect on antrum development. Rats treated with combination rec-VEGF plus rhFSH-CTP therapy were found to have accelerated antrum expansion. When rhFSH-CTP and rhVFC follicles were starting to have small pockets of follicular fluid collecting between granulosa cells, similar sized follicles in combination-treated ovaries exhibited a fully developed cavity of follicular fluid around the oocyte. Interestingly, combination therapy did not increase the number of large stage 7–8 antral follicles present, but treatment with rhVFC leading to enhanced follicle anigiogenesis did increase the number of large antral follicles present. This suggests that it is the proangiogenic effects of VEGF that aid in promoting large follicles to progress to the antral follicle phase, whereas other nonangiogenic activities of VEGF also play an important role in antrum development.

In conclusion, we report the production and characterization of a long-acting, bifunctional VEGF-FSH protein that exhibits both FSH and VEGF bioactivity in vitro as well as in vivo in female rats. Administration of rhVFC enhanced ovarian follicle vascular development and increased the number of large antral follicles that developed compared with treatment with rhFSH-CTP alone. The coadminstration of rec-VEGF with rhFSH-CTP was unable to increase vascular development and follicle production above montherapy with rhFSH-CTP. For women with poor ovarian vascular development, use of rhVFC may potentially enhance their response to ovarian hyperstimulation. Furthermore, the bifunctional VEGF protein design provides a potential template for therapeutic targeting of angiogenesis in other disease states such as myocardial infarction, stroke, and wound healing while limiting adverse side effects.


   Acknowledgments
 
We thank Karl-Heinz Thierauch, and M. Fenten of AG, Research Laboratories, Berlin, Germany, for technical assistance. We also thank Dr. Stephen Palmer, Serono Reproductive Biology Institute, for cell lines.


   Footnotes
 
This work was supported by the Jakob Tulczinsky Foundation (R.K.T.) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK063224 (to J.W.L.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 22, 2006

Abbreviations: CHO, Chinese hamster ovary; CHO-FSH-R, CHO cells transfected with the FSH receptor; EGF, epidermal growth factor; hCG, human chorionic gonadotropin; HUVEC, human umbilical vein endothelial cells; KDR, VEGF II receptor; PBS-T, PBS containing 0.05% Tween 20; PECAM, platelet endothelial cell adhesion molecule; PVDF, polyvinylidene fluoride; rec-VEGF, recombinant human VEGF-A165; rh, recombinant human; rhFSH-CTPspd, rhFSH-CTP split dose; SFM, serum-free medium; VEGF, vascular endothelial growth factor; VFC, VEGF-FSH-CTP.

Received August 15, 2006.

Accepted for publication November 14, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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