Signaling through FSH receptors on human umbilical vein endothelial cells promotes angiogenesis.

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Signaling Through FSH Receptors on Human Umbilical Vein Endothelial Cells Promotes Angiogenesis Julie A. Stilley, Rongbin Guan, Diane M. Duffy, and Deborah L. Segaloff Department of Molecular Biophysics and Physiology (J.A.S., R.G., D.L.S.), The University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242; and Department of Physiological Sciences (D.M.D.), Eastern Virginia Medical School, Norfolk, Virginia 23508

Context: The FSH receptor (FSHR) is traditionally thought to play a role in female reproductive physiology solely within the context of ovarian FSHR. However, FSHR is also expressed in endothelial cells of the placental vasculature and human umbilical cord vessels, suggesting additional facets of female reproduction regulated by extragonadal FSHR. Objective: We sought to determine the functional role of FSHR on human umbilical cord endothelial cells (HUVECs), hypothesizing that activation of the FSHR would stimulate angiogenesis. Design: The ability of FSH to stimulate several angiogenic processes in HUVECs was determined. Setting: This was a laboratory-based study using commercially prepared HUVECs. Results: Tube formation, wound healing, cell migration, cell proliferation, nitric oxide production, and cell survival were stimulated in response to FSH. Quantitative comparisons between HUVECs incubated with maximally stimulatory concentrations of FSH vs vascular endothelial growth factor (VEGF), a well-characterized angiogenic factor, revealed that FSH is as efficacious as VEGF in promoting angiogenic processes. FSH did not provoke increased secretion of VEGF by HUVECs, suggesting the direct stimulation of angiogenic processes by FSH in endothelial cells. In contrast to gonadal cells, the FSHR on HUVECs did not mediate an FSH-stimulated increase in cAMP. However, increased phosphorylation of AKT in response to FSH was observed, suggesting that FSH stimulation of HUVEC FSHR stimulates the PI3K/AKT signaling pathway. Conclusions: Our studies reveal a novel role for FSHR in female reproductive physiology. Its ability to promote angiogenesis in placental endothelial cells suggests that the FSHR may have an influential role in pregnancy. (J Clin Endocrinol Metab 99: E813–E820, 2014)

he successful delivery of a healthy newborn, while still maintaining the health of the mother, is the goal of all pregnancies. The appropriate development and maintenance of the placental vasculature is one of many processes that are required for the normal development of the fetus. Disturbances in placental vasculature can lead to complications of pregnancy including preterm delivery, pre-eclampsia, and intrauterine growth restriction (1). These complications arise, in part, by a failure of signaling in a tightly controlled environment of growth factors and angiogenic stimuli

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that exert either stimulatory or restraining effects on angiogenesis (2). The FSH receptor (FSHR) plays a well-known and critical role in reproductive physiology by promoting follicular growth and estrogen synthesis in the ovary and spermatogenesis in the testes. Studies in recent years have shown that the FSHR is expressed in extragonadal tissues as well (3, 4), suggesting additional physiological roles for FSHR-mediated signaling. In particular, Radu et al (3) demonstrated FSHR expression on endothelial cells of the vasculature on the periphery of several types of human

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received August 16, 2013. Accepted February 3, 2014. First Published Online February 14, 2014

Abbreviations: FSHR, FSH receptor; HUVEC, human umbilical cord endothelial cell; LSGS, low serum growth supplement; VEGF, vascular endothelial growth factor.

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J Clin Endocrinol Metab, May 2014, 99(5):E813–E820

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HUVEC FSHR Promotes Angiogenesis

tumors but not in adjacent normal tissue. Due to similarities in tumor and placental growth, these investigators also examined human term placenta, where they observed FSHR on endothelial cells in all vessels of the chorionic villi. We hypothesized that FSH signaling through the FSHR on endothelial cells of the placenta vasculature would stimulate angiogenesis. We show herein that human umbilical vein endothelial cells (HUVECs) express FSHR and, using primary cultures of these cells, show that FSH stimulates angiogenic processes with the same efficacy (ie, maximal response) as vascular endothelial growth factor (VEGF). Our results suggest that signaling through placental endothelial cell FSHR may be a heretofore unrecognized mediator of placental vascular development and function.

Materials and Methods Hormones and supplies Highly purified recombinant human FSH (preparation AFP8468A; specific activity, 6650 IU/mg) was purchased from Dr A. Parlow and the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health (NIH). It was iodinated as described (5). Biologically active recombinant human VEGF165, a form of VEGF-A (referred to as VEGF), was purchased from R&D Systems. FSHR-323 hybridoma cells, which express IgG2a recognizing the extracellular domain of the human FSHR, were created and characterized by E. Milgrom and colleagues (3, 6) and deposited with the American Type Tissue Collection. We had previously obtained the cells there and had ascites prepared. Purified IgGs were isolated using the NAb Protein G Spin Kit (Thermo Fisher Scientific Inc).


for 1 hour at room temperature. The ABC Standard Kit (Vector Laboratories, Inc) was used per the manufacturer’s instructions, and the immunoreactivity was visualized using 3,3-diaminobenzidine (Dako North America, Inc) developed for 30 seconds. Tissues were counterstained using 10% Harris’s hematoxylin (Leica Microsystems Inc) before dehydration and coverslipping. Images were captured using an Olympus BX61 Light Microscope (Olympus Inc).

Tissue culture of HUVECs Pooled HUVECs were obtained from Invitrogen Life Technologies and cultured according to supplier specifications in media (Medium 200PRF; Invitrogen) with low serum growth supplement (LSGS; Invitrogen), providing final concentrations of 2% fetal bovine serum supplemented with 2% vol/vol hydrocortisone, 1 ␮g/mL human epidermal growth factor, 3 ng/mL basic fibroblast growth factor, and 10 ␮g/mL heparin. All experiments were performed using HUVECs in passage four.

Immunofluorescence microscopy, FSH binding, PCR, tube formation, wound healing, Boyden chamber cell migration, proliferation, cell survival, AKT, VEGF, and nitric oxide assays These methods are provided in the Supplemental Data (published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org).

Statistical analyses Data were analyzed statistically using Prism (GraphPad Software Inc) by one-way ANOVA and Tukey’s test for post hoc pairwise multiple comparisons unless otherwise noted. Statistically significant differences were defined as those with a P value ⬍.05 and are denoted by an asterisk.

Results Sources of tissues Whole ovaries were obtained from adult female cynomolgus macaques at the Eastern Virginia Medical School (Norfolk, VA), as previously described (7). All animal protocols were approved by the Eastern Virginia Medical School Animal Care and Use Committee and were conducted in accordance with the NIH’s Guide for the Care and Use of Laboratory Animals. Portions of ovary were paraffin-embedded and sectioned onto slides. Slides containing de-identified umbilical cord from term pregnancies were obtained from the University of Iowa Department of Pathology.

Immunohistochemistry Slides containing tissues were deparaffinized in xylenes and rehydrated in an ethanol series. Antigen retrieval was performed using citrate buffer at 95°C for 15 minutes. Nonspecific binding was blocked using filtered PBS (137 mM NaCl, 2.7 mM KCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4; pH 7.4) containing 10% normal goat serum (Sigma Aldrich) for 2 hours at room temperature. FSHR-323 IgG2a or non-immune IgG2a (R&D Systems) were added at 5 ␮g/mL in blocking buffer overnight at 4°C. After washing, biotinylated goat antimouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc) (4.2 ␮g/mL) was added

Previous studies by Radu et al (3) using a well-characterized and specific monoclonal antibody to the extracellular domain of the human FSHR demonstrated FSHR on endothelial cells of the chorionic villi of human term placenta. Using the same anti-FSHR, we sought to determine by immunohistochemistry whether FSHR is expressed in human umbilical cord endothelial cells. Using nonhuman primate ovary, we first established immunohistochemistry conditions yielding specific staining of FSHR on granulosa cells (Supplemental Figure 1). Using these conditions, endothelial cells of human umbilical cord vein were shown to express FSHR (Figure 1, A and B). Specific immunohistochemical staining for FSHR was observed in the tunica intima (containing endothelial cells) and, to a lesser extent, the tunic media. Primary cultures of HUVECs similarly express FSHR, as evidenced by specific immunofluorescence staining for FSHR (Figure 1, C and D) and by relatively low, but highly reproducible, cell surface-specific binding of 125I-FSH (0.85 ⫾ 0.20 ng 125I-FSH bound/


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Figure 1. FSHR is detected immunohistochemically on endothelial cells of human umbilical cord vein and on HUVECs. The vein of human umbilical cord from term pregnancy was stained with anti-FSHR323 (A) or with isotype- and concentration-matched nonimmune IgG (B). A and B, Magnification ⫻200. Labeled are the tunica interna (TI), tunica media (TM), and Whartson’s Jelly (WJ). Immunofluorescence of HUVECs stained with anti-FSHR323 and counterstained with DAPI (blue) (C) or with isotype- and concentration-matched nonimmune IgG and counterstained with DAPI (blue) (D). The immunohistochemical staining of FSHR in human umbilical cord is representative of that observed in at least six different cords. The immunofluorescence imaging of FSHR in HUVECs is representative of three independent experiments.

106 cells; n ⫽ 6). The binding of 125I-FSH was too low to permit an accurate determination of the binding affinity. Using routine PCR, FSHR mRNA was detected in HUVECs (Figure 2A). Thus, primers spanning exon 2 through exon 3 yielded the expected 120-bp product in both human ovary and HUVECs. A semiquantitative comparison suggests a much lower amount of FSHR mRNA in HUVECs than in ovaries. Specific PCR conditions (genespecific reverse transcription) and primer sets described by Blair and colleagues (8, 9) were then used to distinguish between the full-length FSHR transcript and a splice variant lacking exon 9. Primers spanning exons 8 through 10 yielded two transcripts in the ovary, consistent with sizes predicted for a full-length transcript and a transcript lacking exon 9, whereas only the transcript consistent with the absence of exon 9 was detected in HUVECs (Figure 2B). To confirm the presence of human FSHR mRNA lacking exon 9, a forward primer designed to hybridize to contiguous nucleotides from the 3⬘ end of exon 8 and the 5⬘ end of exon 10 was used in conjunction with a reverse primer hybridizing to a more 3⬘ region of exon 10. The expected 134-bp product was observed in HUVECs and ovaries (Figure 2C). These data suggest that, in contrast to ovaries, which express both full-length FSHR mRNA and FSHR mRNA lacking exon 9, HUVECs only express FSHR mRNA lacking exon 9. We hypothesized that FSH would exert proangiogenic effects on HUVECs and tested this by evaluating FSH addition on several processes associated with angiogenesis. HUVECs are a particularly good model system for the in

Figure 2. FSHR mRNA in HUVECs. cDNA was extended using endpoint PCR. Traditional PCR amplifying exons 2 though 3 (A), genespecific PCR amplifying exons 8 through 10 (B), and gene-specific PCR amplifying an FSHR mRNA splice variant lacking exon 9 (C) were performed as described in the Supplemental Data. Shown are the results using three different cultures of pooled HUVECs and from pooled human ovaries. Data shown are representative of three independent experiments.

vitro investigation of angiogenesis because, when plated on a three-dimensional gel matrix containing extracellular matrix components in the presence of angiogenic stimuli, the cells form tube-like structures, and this process is amenable to quantification (10). Under these conditions, FSH was observed to stimulate tube formation similar to that induced by the addition of LSGS, a commercial low serum growth supplement that provides full support for cultured endothelial cells (Figure 3, A–C). Quantification of FSHstimulated tube length over a range of FSH concentrations showed that the maximally effective concentration of FSH is 600 ng/mL, and concentrations lower as well as higher than that are less stimulatory (Figure 3D). To enable us to compare the relative efficacy of FSH in stimulating proangiogenic processes in the HUVECs relative to VEGF, a well-characterized angiogenic factor (11), we quantified tube formation as a function of VEGF concentration as well (Figure 3E). These data similarly show a nonmonotonic bell-shaped dose response curve, with the maximally effective concentration of VEGF being 50 ng/mL (Figure


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case, VEGF was readily detected (Table 1). Therefore, we conclude that the proangiogenic effects of FSH on HUVECs as described herein are not due to FSH-stimulated secretion of VEGF. VEGF stimulates the synthesis of nitric oxide, a potent vasodilator and a positive mediator of angiogenesis. To determine whether FSH has similar properties, HUVECs were incubated with no additions or with FSH or VEGF, and nitric oxide was measured in the conditioned media. As shown in Figure 5A, there was a significant increase in nitric oxide in reFigure 3. FSH stimulates tube formation of HUVECs. Serum-starved HUVECs plated on a sponse to FSH, and this increase was reduced growth factor basement membrane matrix were incubated with no additions (A), FSH (600 ng/mL final concentration) (B), or LSGS (C). Data are representative of three independent similar to that elicited by VEGF. experiments. Dose response curves quantifying tube length as a function of final concentrations VEGF is also known to exert protecof FSH (D) or VEGF (E) are shown. Data shown in D and E are each the mean ⫾ SEM of triplicate tive, antiapoptotic effects on endodeterminations of a single experiment and each are representative of two individual experiments. thelial cells. To evaluate the potential protective effects of FSH, 3E). Experiments were then performed in which tube forapoptosis was induced in HUVECs by incubation with a mation was quantitatively compared between cells within reduced concentration of serum. In the presence of FSH or the same experiment that were incubated with media supVEGF, the percentage of apoptotic cells was significantly plemented with vehicle, FSH (600 ng/mL), VEGF (50 ng/ reduced (Figure 5B). Again, FSH showed efficacy similar mL), or the commercial growth factor and serum suppleto VEGF with respect to promoting antiapoptotic effects. ment LSGS. As shown in Figure 4A, tube formation in Interestingly, although one of the primary signaling pathresponse to the maximally stimulatory concentrations of inFSH and VEGF was similar. We then analyzed the ability ways stimulated by FSH in ovarian granulosa cells is125 creased cAMP synthesis (12), using a highly sensitive Iof FSH relative to VEGF to stimulate other angiogenic processes in HUVECs. Cell migration as determined by cAMP-based RIA (sensitivity of 60 fmol cAMP/mL) (5), wound-healing assays (Figure 4B), cell migration as de- we did not observe a detectable increase in cAMP in FSHtermined by Boyden chamber assays (Figure 4C), and cell treated HUVECs (data not shown). FSH does, however, proliferation (Figure 4D and Supplemental Figure 2) were increase phosphorylation of Akt in HUVECs (Figure 6), all stimulated by FSH. Importantly, in all the different again with similar efficacy as VEGF. (Although Akt phosassays, the response to FSH was comparable to that phorylation by VEGF appears to be greater than by FSH, achieved with VEGF. These data suggest that FSH is as the responses are not statistically different.) These results efficacious as VEGF in stimulating proangiogenic pro- suggest that, similar to VEGF, FSH stimulates the PI3K/ Akt signaling pathway in HUVECs, a pathway that processes in HUVECs. To determine whether the angiogenic responses of motes cell survival (11). HUVECs to FSH are a result of direct actions of FSH on HUVECs or indirect effects caused by FSH-stimulated VEGF secretion by the cells, we measured VEGF in con- Discussion ditioned media of cells incubated with media containing FSH for 4 hours (the maximal time period used in the In the context of female reproductive physiology, the above experiments). Conditioned media was collected, FSHR has traditionally been thought to be restricted in it concentrated, and assayed for VEGF using an ELISA as- expression, and therefore function, to the ovary. Howsay. In two independent experiments, no detectable VEGF ever, recent studies have shown that FSHR is also exwas observed in the conditioned media (Supplemental Ta- pressed in osteoclasts, where FSH signaling promotes ble 1). To rule out the potential loss of any secreted VEGF bone loss (4). It has also been shown by Radu et al (3) that during the concentration steps, conditioned media from FSHR is expressed on endothelial cells of the vasculature VEGF-treated cells was similarly processed. In the latter on the perimeter of many different types of tumors and in


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Figure 5. FSH stimulates nitric oxide production and promotes cell survival in HUVECs. Serum-starved HUVECs were incubated with vehicle (no additions [NA]), FSH at 600 ng/mL final concentration, or VEGF at 50 ng/mL final concentration. Nitric oxide production (A) and antiapoptotic effects (B) were quantified. Data shown are the mean ⫾ SEM of four and three independent experiments in A and B, respectively. Asterisks denote differences with P ⬍ .5.

tures of HUVECs. Using HUVECs as a model system, we demonstrate that FSH stimulates several proangiogenic processes on endothelial cells. Thus, FSH stimulates the formation of tube-like structures, cell migration, wound healing, cell proliferation, inhibition of apoptosis, and increased nitric oxide production. It is well known that

Figure 4. FSH stimulates several angiogenic processes in HUVECs with a similar efficacy as VEGF. Within a given experiment in each assay, serum-starved HUVECs were incubated with vehicle (no additions [NA]), FSH at 600 ng/mL final concentration, VEGF at 50 ng/mL final concentration, or LSGS, and the following assays were performed and quantified: tube formation (A), wound healing (B), cell migration (C), and cell proliferation as determined by Click-iT EdU (D). Data shown are the mean ⫾ SEM of five, three, three, and four independent experiments in A, B, C, and D, respectively. Asterisks denote differences with P ⬍ .5.

the chorionic villi of term placenta. To elucidate the role of FSHR in endothelial cells of the placental vasculature, we first confirmed that FSHR is indeed expressed on endothelial cells of umbilical cord vein and on primary cul-

Figure 6. FSH stimulates Akt phosphorylation in HUVECs. Serumstarved HUVECs were incubated for the indicated times with FSH at 600 ng/mL final concentration or VEGF at 50 ng/mL final concentration. Total Akt (t-Akt) and phosphorylated Akt (p-Akt) were determined by Western blotting. A, Western blots from one representative experiment. B, The ratio of p-Akt/t-Akt was determined and plotted as the fold-increase relative to time zero. Data shown are the mean ⫾ SEM of three independent experiments.


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VEGF is a potent stimulator of angiogenesis and that it serves a critical role in placental angiogenesis (11). Significantly, our data reveal that FSH is as efficacious as VEGF in the stimulation of angiogenic processes in HUVECs. However, no detectable effect of FSH on VEGF secretion was observed, suggesting that the effects of FSH on HUVECs are direct. These findings are in contrast to those observed in the ovary, where FSH stimulates angiogenesis by inducing VEGF production in granulosa cells (13, 14). Interestingly, it has been reported that LH receptors and TSH receptors are expressed on endothelial cells and stimulate angiogenic processes, suggesting that the glycoprotein hormones share a common feature of promoting angiogenesis (15–17). The dose-response curve for FSH-stimulated tube formation in HUVECs is biphasic. As such, rather than displaying saturation, concentrations higher than the maximal stimulatory concentration of 600 ng/mL evoked submaximal degrees of tube formation. Although the physiological consequences of signaling through placental FSHR remain to be elucidated, these results suggest that concentrations of FSH available to the placental FSHR that are either too high or too low may be deleterious. A maximally stimulatory concentration of 600 ng/mL of FSH is not inordinately high when considering the great variability in concentrations of FSH required to evoke maximal cellular responses, which vary depending upon the signaling pathway stimulated by FSH as well as the cell type expressing the FSHR. For example, although maximal stimulation of cAMP by FSH in granulosa cells is typically observed at approximately 100 ng/mL, much higher concentrations are required to stimulate inositol phosphate production (18, 19). Similarly, higher concentrations of FSH are required to stimulate G␣h than G␣s in rat Sertoli cells (20). It should be noted that, when examining dose response curves for FSH-stimulated G␣h, Lin et al (20) used 450 ng/mL as the highest concentration, and this was not yet saturating. Similarly, in osteoclasts, where FSH stimulation has been shown to activate Gi, the highest concentration tested was 300 ng/mL, and this too was not yet saturating (21). In these contexts, the concentration of FSH required to be maximally effective in stimulating angiogenic processes in HUVECs (600 ng/mL) is not inordinately high. Although we have demonstrated that endothelial cells of human umbilical vein express functional cell surface FSHR, the levels of FSHR as determined by cell surface 125 I-FSH binding to HUVECs is extremely low (⬍1 ng 125 I-FSH bound/106 cells), precluding an accurate determination of binding affinity. It is not possible to compare the 125I-FSH binding capacity of HUVECs to human granulosa cells because the latter cells can only be procured as


granulosa-lutein cells after gonadotropin treatments for ooctye retrieval, which results in the down-regulation of the FSHR (22). However, a semiquantitative comparison of FSHR mRNA levels in HUVECs compared to human ovary suggests a much lower expression of FSHR mRNA in the HUVECs. Our PCR results further suggest that the primary FSHR mRNA expressed in HUVECs is an alternatively spliced variant lacking exon 9. Interestingly, this variant is also the predominant FSHR transcript present in osteoclasts where, similar to HUVECs, it is expressed at relatively low levels (8, 9). Importantly, the FSHR in osteoclasts has been demonstrated to play a role in mediating FSH-stimulated bone loss (4, 21), providing precedence for the physiological relevance of low abundance extragonadal FSHR. Although macrovascular cells such as HUVECs are not identical to microvascular cells (23), they do share several properties, and HUVECs have served as a widely used model system for studying placental angiogenesis in vitro (11). Within this context, our data demonstrating the FSH-dependent stimulation of angiogenic processes in HUVECs suggest that the FSHR in endothelial cells of the placental vasculature would be predicted to stimulate angiogenesis in response to FSH and, therefore, potentially play a significant role in placental development and the maintenance of a healthy pregnancy (1). A role for FSH/ FSHR signaling has not previously been considered for two reasons. First, women homozygous for loss-of-function mutations of the FSHR or mice homozygous for knockout of the Fshr are infertile due to the impairment or loss, respectively, of ovarian FSHR and consequent absence of follicular and ooctye development (24). Therefore, any subsequent potential role that placental FSHR might play in pregnancy could not be ascertained. It should be noted, however, that when mice heterozygous for global deletion of Fshr were crossed with each other, there was a 14% reduction in the number of pups born homozygous for Fshr knockout compared to wild-type pups. Considering that the placental vasculature is determined by the fetal genome, these data suggest greater fetal demise when FSHR is absent from placental endothelial cells (25). Mouse models with conditional deletions of Fshr will be necessary to evaluate postovulatory effects of the FSHR on pregnancy. Secondly, serum levels of maternal FSH during pregnancy are quite low due to suppression of pituitary FSH secretion by the elevated circulating steroid hormones (26, 27). However, it should be considered that the placenta synthesizes several hormones and growth factors that act by paracrine and/or autocrine mechanisms. VEGF, essential for placental development, is quite low in maternal serum during pregnancy and is thought to be synthesized locally (28). Supporting the pos-


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sible local synthesis of FSH in placenta are microarray data deposited in the NCBI Gene Expression Omnibus that indicate expression of placental FSH␤ mRNA (as well as mRNA for the common glycoprotein hormone ␣-subunit, required for human chorionic gonadotropin synthesis) (29 –32). Notably, two studies show FSH␤ mRNA to be present in placenta in all three trimesters and at levels 33% or greater than other mRNAs in the samples (30, 32). It has been reported that singletons conceived after assisted reproductive intervention are at increased risk for low birth weight, premature birth, and perinatal death (33–36). A recent study comparing singleton siblings born to a given woman by assisted fertilization and subsequently with no interventions found no differences in the rates of adverse outcomes in the two groups, suggesting that adverse fetal outcomes are attributable to factors contributing to the infertility and not to assisted fertilization (37). Given that ovarian FSHR is known to be essential for fertility and is a target for enhancing fertility, the sibling study begs the question whether women with impaired fertility due to alterations in FSHR expression and/or activity may be at increased risk for adverse fetal outcomes due to impaired FSHR expression and/or activity in the placenta and/or extragonadal reproductive tract. Related to this, Muglia and colleagues (38) identified the FSHR as a gene associated with premature birth. Our data herein document FSHR in placental endothelial cells, where it mediates angiogenic processes. Studies on tissues from extragonadal reproductive tissues of nonpregnant and pregnant women have revealed FSHR on endothelial cells of blood vessels in other tissues as well as FSHR expression in several other cell types (manuscript in preparation). Taken together, it is reasonable to postulate that the FSHR may play an integral role in pregnancy and parturition. Finally, in light of studies by Ghinea and colleagues documenting FSHR expression on endothelial cells of blood vessels in many types of human tumors (3) and our present studies demonstrating FSH-provoked stimulation of angiogenic processes in FSHR-expressing endothelial cells, further studies examining the potential roles of signaling through endothelial FSHR on tumor growth are warranted.

Acknowledgments We thank Dr Patricia Kirby (Department of Pathology, University of Iowa Carver College of Medicine), for providing slides containing samples of term umbilical cord used in these studies, and Dr Harry Blair (University of Pittsburgh) for helpful discussions on PCR amplification of FSHR mRNA from HUVECs.


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Address all correspondence and requests for reprints to: Deborah L. Segaloff, PhD, Department of Molecular Physiology and Biophysics, 5-470 Bowen Science Building, The University of Iowa, Iowa City, IA 52242. E-mail: [email protected]. This work was supported in part by National Institutes of Health (NIH) Grants HD022196 (to D.L.S.) and HD054691 (to D.M.D.). J.A.S. was supported by NIH Grant T32DK007690. Disclosure Summary: The authors have nothing to disclose.

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CXCL12 enhances angiogenesis through CXCR7 activation in human umbilical vein endothelial cells.

Arnebin-1 promotes the angiogenesis of human umbilical vein endothelial cells and accelerates the wound healing process in diabetic rats.

Irisin promotes human umbilical vein endothelial cell proliferation through the ERK signaling pathway and partly suppresses high glucose-induced apoptosis.

Up-regulation of FGFBP1 signaling contributes to miR-146a-induced angiogenesis in human umbilical vein endothelial cells.

Adrenergic signaling promotes angiogenesis through endothelial cell-tumor cell crosstalk.

Extract of Artemisia lavandulaefolia Inhibits In Vitro Angiogenesis in Human Umbilical Vein Endothelial Cells.

Lipopolysaccharide-induced microRNA-146a targets CARD10 and regulates angiogenesis in human umbilical vein endothelial cells.

miR-155 Promotes ox-LDL-Induced Autophagy in Human Umbilical Vein Endothelial Cells.

Autocrine receptors for endothelins in the primary culture of endothelial cells of human umbilical vein.

Propofol attenuates hydrogenperoxide-induced apoptosis in human umbilical vein endothelial cells via multiple signaling pathways.

Berberine Protects Human Umbilical Vein Endothelial Cells against LPS-Induced Apoptosis by Blocking JNK-Mediated Signaling.

Retinoic acid receptor transcripts in human umbilical vein endothelial cells.

SIRT4 Suppresses Inflammatory Responses in Human Umbilical Vein Endothelial Cells.

KNDC1 knockdown protects human umbilical vein endothelial cells from senescence.

Effects of gintonin on the proliferation, migration, and tube formation of human umbilical-vein endothelial cells: involvement of lysophosphatidic-acid receptors and vascular-endothelial-growth-factor signaling.

Donepezil attenuates high glucose-accelerated senescence in human umbilical vein endothelial cells through SIRT1 activation.

Morphological analysis of human umbilical vein endothelial cells co-cultured with ovarian cancer cells in 3D: An oncogenic angiogenesis assay.

KLF4 Promotes Angiogenesis by Activating VEGF Signaling in Human Retinal Microvascular Endothelial Cells.

The seeding of human aortic endothelial cells on the extra-cellular matrix of human umbilical vein endothelial cells.

MicroRNA-217 promotes angiogenesis of human cytomegalovirus-infected endothelial cells through downregulation of SIRT1 and FOXO3A.

Exosomal transfer of vasorin expressed in hepatocellular carcinoma cells promotes migration of human umbilical vein endothelial cells.

Extracellular Signal-Regulated Kinase 5 is Required for Low-Concentration H2O2-Induced Angiogenesis of Human Umbilical Vein Endothelial Cells.

Ginsenoside compound K inhibits angiogenesis via regulation of sphingosine kinase-1 in human umbilical vein endothelial cells.

Proteome profiling in IL-1β and VEGF-activated human umbilical vein endothelial cells delineates the interlink between inflammation and angiogenesis.