| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Departments of Obstetrics and Gynecology3 and Human Biological Chemistry and Genetics,4 University of Texas Medical Branch, Galveston, Texas 77555-1062
ABSTRACT
Recent studies have shown that homozygous knockout of gene for calcitonin gene-related peptide (CALCA) receptor component, calcitonin receptor-like receptor (CALCRL), led to extreme hydrops fetalis and embryonic death, underlining the critical role of CALCA in embryonic development and fetal growth. The present study was designed to determine the cellular localization of CALCA and its receptor components, CALCRL and receptor activity modifying protein 1 (RAMP1), at the human implantation site during early pregnancy; to assess whether CALCA regulates in vitro angiogenesis of human endothelial cells; and to examine whether CALCA can improve angiogenic imbalance in preeclamptic placental explants. Our studies demonstrated that both protein and mRNA for CALCA were expressed by the villous and extravillous trophoblasts and decidual cells in the first-trimester villous tissues. CALCA receptor components, CALCRL and RAMP1, were expressed by both villous and extravillous trophoblast cells, as well as vascular endothelial cells. CALCA induced both endothelial proliferation and migration in a dose- and time-dependent manner, and it promoted capillarylike tube formation of human umbilical vein endothelial cells (HUVECs) on Matrigel. CALCA-induced angiogenesis of human endothelial cells was completely blocked by CALCA antagonist CALCA8–37. Further, conditioned medium from preeclamptic placental explants significantly inhibited HUVEC capillarylike tube formation compared with gestational age-matched controls, and conditioned medium from preeclamptic placental explants incubated with CALCA significantly improved capillarylike tube formation. We conclude that CALCA induces in vitro angiogenesis by stimulating endothelial cell proliferation, migration, and capillarylike tube formation; thus, CALCA at the human implantation site may constitute a potential autocrine or paracrine mechanism that could modify placental angiogenesis and neovascularization.
angiogenesis,, CALCA, decidua, placenta, trophoblast
Normal pregnancy depends on the formation of placenta. Successful placentation requires sufficient angiogenesis and neovascularization to establish feto-placental vasculature [1]. In the human, the vascularizaton of placental villi starts at Day 21 after conception, resulting from the local de novo formation of capillaries [2]. As gestation progresses, cytotrophoblasts from the anchoring villi invade the maternal spiral arteries and initiate a epithelial-to-endothelial phenotypic transformation [3], and replace the endothelial lining of the spiral arteries [4], resulting in the completion of the physical connection between the fetus and the mother [5].
Numerous angiogenic proteins synthesized in the placenta are thought to be involved during the stage of placental vascularization and development; however, the molecular mechanisms modulating angiogenic process in early pregnancy remain elusive.
Calcitonin gene-related peptide (CALCA) is a 37-amino acid multifunctional peptide resulting from alternative splicing of the primary transcript of the calcitonin gene [6]. The peptide was initially demonstrated to be distributed mainly in the central and peripheral nervous system and have a potent vasodilatory effect on vascular tone [7]. Since then, it has become apparent that it is expressed by a variety of cells, including decidual cells [8], epithelial cells [9], and trophoblast cells [10], with a biologic effect on cell growth and differentiation [11], migration [12], and adhesion [13]. Thus, the biologic activities of CALCA are not restricted to the vascular system; it may be a ubiquitous peptide with various actions on cell proliferation and differentiation.
CALCA exerts its effect through a 7-transmembrane G protein-coupled receptor, calcitonin receptor-like receptor (CALCRL). It has been reported that CALCRL functions as a receptor for three ligands, CALCA, adrenomedullin (ADM), and intermedin (ADM2), in the presence of its receptor activity modifying proteins (RAMP1, RAMP2, and RAMP3) [14]. Coexpression of CALCRL with RAMP1 forms a CALCA receptor, whereas RAMP2 or RAMP3 produces an ADM receptor, and CALCRL with either of the three RAMPs mediates ADM2 signaling [15]. Although the three ligands are capable of interacting with CALCRL, optimal regulation by this G protein-coupled receptor-signaling pathway likely depends on a integrated release of different endocrine/paracrine ligands in a tissue-specific manner.
It has been recently reported that homozygous knockout of gene for CALCA receptor component, calcitonin receptor-like receptor (CALCRL), led to extreme hydrops fetalis and cardiovascular defects [16], and CALCRL–/– embryos die between Embryonic Day 13.5 (E13.5) and E14.5 of gestation, underlining the critical role of CALCA in embryonic development and fetal growth. The present studies were designed to determine: 1) the expression of CALCA and its receptor components, CALCRL and RAMP1, at the human implantation site during early pregnancy; 2) whether CALCA regulates in vitro angiogenesis of human endothelial cells; and 3) whether CALCA can improve angiogenic imbalance in preeclamptic placental explants. In this study we found that CALCA is localized primarily in the villous and extravillous trophoblast and decidual cells in first-trimester placenta. CALCA receptor components, CALCRL and RAMP1, are expressed mainly by trophoblasts and vascular endothelial cells. CALCA stimulated vascular endothelial cell proliferation, migration, and capillarylike tube formation, and these actions were completely blocked by CALCA antagonist CALCA8–37. Further, conditioned medium from preeclamptic placental explants significantly inhibited endothelial cell capillarylike tube formation compared with gestational age-matched controls. Culture medium from preeclamptic placental explants incubated with CALCA significantly improved placental angiogenesis, as evidenced by enhanced capillary-like tube formation. Thus, characterization of the angiogenic properties of CALCA in our study has further confirmed an essential role for this peptide in placental angiogenesis and fetal growth.
Patients and Tissue Collection
Placental tissues were obtained either during legal first-trimester pregnancy termination (7~12 wk) or during the cesarean delivery or vaginal delivery in normal-term pregnancies and those with preeclampsia. The population for study on placental explants consisted of normotensive pregnant women and women with preeclampsia who were admitted to the Department of Obstetrics and Gynecology at the University of Texas Medical Branch (UTMB; Galveston, TX) from June 2005 to February 2006. These studies were approved by the Institutional Review Board at the UTMB. Informed consent was obtained from all patients, and studies were conducted according to the Declaration of Helsinki Principles. Patients with preeclampsia were diagnosed according to the following criteria [17]: 1) no history of hypertension prior to pregnancy; systolic pressure 
140 mm Hg or diastolic pressure 90 mm Hg; 2) proteinuria 0.05 g/24 h or 30 mg/dl in a catheterized specimen; 3) hyperuricemia >5.5 mg/dl; and 4) return to normal blood pressure and resolution of proteinuria by 12 wk postpartum. Normotensive parturients were selected using the following criteria: normal blood pressure before, during, and after parturition, and no proteinuria. Normotensive patients with idiopathic preterm labor were included in the control group to match placental samples from patients with preeclampsia for gestational age. The clinical characteristics of the patients for study on placental explants are shown in Table 1.
|
Immunofluorescent Confocal Imaging Analysis
Buffered formalin-fixed, paraffin-embedded tissue sections (4 µm) were deparaffinized and rehydrated by passage through xylene and graded ethanol solutions. Slides were incubated in 0.05% casein (Sigma, St. Louis, MO)/0.05% Tween-20 (DAKO, Glostrup, Denmark)/PBS for 30 min to block nonspecific protein binding. The slides then were treated with Image-It Enhancer (Molecular Probes, Eugene, OR). The first primary antibody, rabbit anti-CALCA antibody (Peninsula, San Carlos, CA) was applied at a 1:160 dilution. Slides were incubated with goat anti-rabbit IgG Alexa 488 followed by donkey anti-goat IgG Alexa 488. Rabbit IgG Ready-to-Use (DAKO) was used as negative control. After thoroug rinsing with Tween/PBS and the application of casein, the second primary antibody, monoclonal anti-cytokeratin-7 antibody (DAKO), was applied to sections at a 1:750 dilution for 60 min. Mouse IgG1 (DAKO) was applied as a negative control. Chicken anti-mouse IgG-Alexa Fluor 594 (Molecular Probes) was used for detection of the second primary antibody. Slides then were counterstained with DAPI (Vector Laboratories Inc., Burlingame, CA), treated with Autofluorescence Eliminator Reagent (Chemicon International, Temecula, CA), and viewed under an Olympus BX51 microscope, and the images were recorded by a DP70 Digital Camera (Olympus Optical Co. Ltd., Tokyo, Japan).
The probes were designed in our laboratory per the published sequence of human alpha CALCA (accession no. M12667). Complete exon 5 of alpha CALCA was amplified and subcloned in pBluescript11 SK vector. The cloned plasmid was linearized with BamHI or SalI restriction enzyme to create sense and antisense probes, respectively. In the presence of digoxigenin-labeled UTP, the linearized plasmid was transcribed in vitro with T7 RNA polymerase for the sense probe and with T3 RNA polymerase for the antisense probe. Paraffin sections of human villous tissues were rehydrated and incubated with 0.25% pepsin/0.1 N HCl for 30 min at 30°C. The slides were dehydrated in a series of graded ethanol and then air dried. A total of 30 µl DIG-labeled sense and antisense oligo-probes (Roche Diagnostic Co., Indianapolis, IN) at the same concentration of 80 ng/ml was applied and sealed with Hybri-well press-seal in hybridization chambers (Sigma). Hybridization was performed at 45°C for 2 h in the Hybrite Kit (Vysis Inc., Downers Groove, IL). Posthybridization washing was completed by incubation with 2x SSC, 1x SSC, and 0.5x SSC. After having quenched endogenous avidin/biotin and protein blocking, a detection system of biotinylated mouse anti-DIG antibody and strepavidin-Alexa Fluor 594 was used for probe detection. Slides then were counterstained with DAPI and viewed under an Olympus BX51 fluorescence microscope, and images were recorded by DP70 Digital Canon (Olympus Optical Co. Ltd.).
Buffered formalin-fixed, paraffin-embedded human villous tissue sections (5 µm) were deparaffinized and rehydrated by passage through xylene and graded ethanol solutions. Following sequential 15-min incubations with 0.1% avidin and 0.01% biotin (Vector Laboratories Inc., Burlingame, CA), slides were incubated in 0.05% casein in 0.05% Tween-20/PBS for 30 min to block nonspecific binding. Primary antibodies to N-terminal domains of CALCRL and RAMP1 (produced and well characterized by Dr. Yallampalli's laboratory at UTMB) were applied to sections for 60 min. Preimmune serum was applied as a negative control. Biotinylated F(ab')2 fragment of swine anti-rabbit immunoglobulins (DAKO) served as the secondary antibody, were detected by streptavidin-HRP, and were visualized by DAB (DAKO). Slides were counterstained with Mayer modified hematoxylin (Poly Scientific, Bay Shore, NY) and viewed under an Olympus microscope with Image-ProPlus software (Olympus Optical Co. Ltd.).
Human umbilical vein endothelial cells (HUVECs; American Type Culture Collection, Manassas, VA) were cultured in Ham F12K medium with L-glutamine (2 mM; Gibco, Grand Island, NY) and supplemented with sodium bicarbonate (1.5 g/l), heparin (0.1 mg/ml), endothelial cell growth supplement (ECGS; 0.04 mg/ml), and fetal bovine serum (10%; Sigma). Cell proliferation was determined using a kit for methylthiazoltetrazolium (MTT) assay (Promega, San Luis Obispo, CA). MTT is a water-soluble tetrozolium salt that metabolically active cells are capable of converting to the water-insoluble dark blue formazan by reduction of cleavage of the tetrazolium ring [18]. Cells were seeded onto gelatin-coated 96-well plates at a density of 4 x 105 cells/ml in Ham F12K medium. After serum starvation for 12 h, cells were treated with varying does of CALCA (10–9 to 10–7 M; Sigma) with or without CALCA antagonist CALCA8–37 (10–7 M; Sigma) over time courses of 24, 48, and 72 h at 37°C in 5% CO2 and 95% air. At the end of the treatment, MTT-containing medium (0.5 mg/ml) was added to the wells and incubated at 37°C for 3 h. DMSO (200 µl) was added to each well and cultured at 37°C for 5 min. Colored formazan was measured at 510 nM with a kinetic microplate reader (Molecular Devices Co., Sunnyvale, CA).
HUVEC migration was measured with 24-well Matrigel migration chamber plates (BD Biosciences, Bedford, MA). Cells were suspended in the Ham F12K nutrient mixture medium (Gibco) with 0.2% BSA (Sigma). A total of 250 µl of the cell suspension (4 x 105 cells/ml) was added to the upper compartment. The lower wells were filled with medium containing CALCA at various concentrations (10–9 to 10–7 M) with or without CALCA8–37 (10–7 M). After 4, 12, and 24 h of incubation at 37°C in 5% CO2 and 95% air, fluorescent dye calcein AM (8 µg/ml) was added to the wells. After incubating plates for 90 min at 37°C in 5% CO2 and 95% air, the fluorescence of migrated HUVECs was read using a fluorescence plate reader with bottom reading capabilities at excitation/emission wavelengths of 485/530 nm (PEI200-TE200-IUC Quantitative Fluorescence Live-Cell and Multi-dimensional Imaging System; Nikon, Tokyo, Japan). The cell migration was presented as the ratio of fluorescence in treated cells compared to the untreated controls.
Capillary like Tube Formation Assay
The capillarylike tube formation assay was performed using Endothelial Cell Tube Formation System (BD Biosciences) per the manufacturer's instruction. A total of 50 µl of the HUVEC suspension (4 x 105 cells/ml) was added to the wells of 96-well plates in the presence or absence of CALCA (10–8 M) or CALCA (10–8 M) plus CALCA8–37 (10–7 M). The plates were incubated for 4, 12, and 24 h at 37°C in 5% CO2 and 95% air and then were viewed under a microscope with a digital microscope camera system (Olympus Optical Co. Ltd.). Quantitation of HUVEC capillarylike tube formation was made by measuring the lengths of tubes in three randomly chosen fields from each well using Image-Pro Plus software (Olympus Optical Co. Ltd.) and calculating them against untreated groups.
Villous Tissue Explant Culture
Placental tissues were obtained after cesarean delivery or vaginal delivery immediately after removal of the placentas from the patients. In our studies, four full-depth columns of villous tissue were taken from each quadrant of the placenta. Tissue columns then were diced to produce pieces of roughly equal size, and the control parts of the tissue were examined at light microscopy levels to make sure that only villous tissues free of maternal vessels from the basal plate were used for the incubation. Villous tissues were rinsed in sterile PBS (0.01 M, pH 7.4) and aseptically dissected to remove endometrial tissues and fetal membranes. The tissues were then dissected into 15- to 20-mg pieces, and five to six pieces were placed into the wells of 24-well plates (Corning Inc., Corning, NY) containing 1 ml RPMI 1640 (Cellgro, Herndon, VA) with or without CALCA (10–8 M) or CALCA8–37 (10–7 M). Explants were incubated at 37°C under standard culture conditions of 5% CO2 and 95% air. After 48 h of culture, the culture medium was collected, and 50 µl of medium from each treatment was added to wells in the Endothelial Cell Tube Formation System containing 50 µl of HUVEC suspension (4 x 105 cells/ml) in each well, and the analysis of capillarylike tube formation was performed as mentioned above.
Data are presented as mean ± SEM. Comparisons between groups were performed by one-way analysis of variance (ANOVA) followed by Bonferroni/Dunn posthoc test where appropriate. Unless specified, we determined six to eight samples per group in all experiments. A value of P < 0.05 was considered significant.
First-trimester (7~12 wk) villous tissues were collected from patients undergoing elective abortion at the Department of Obstetrics and Gynecology at UTMB. Placental tissues for studies on villous explant culture were collected from patients listed in Table 1. There were no significant differences for maternal age, gestational age, and the rate for cesarean delivery between groups. However, the patients with preeclampsia had significantly lower birth weights and parity but higher primiparity when compared to normotensive controls. This is consistent with the clinical features in preeclamptic patients in a recent report [19].
CALCA Proteins Were Expressed by Both Villous and Extravillous Trophoblasts
The cellular localization of CALCA in the first-trimester villous tissues was determined by immunofluorescent confocal imaging. The first primary antibody, rabbit anti-CALCA polyclonal antibody, was applied to sections and detected by Alexa 488 with color green. The second primary antibody, monoclonal anti-cytokeratin-7 (a trophoblast marker), was applied to sections and was detected by Fluor 594 with color red. As shown in Figure 1, diffuse CALCA staining could be recognized in the cytoplasm of cytotrophoblasts and syncytiotrophoblasts in the villi and its discrete labeling in the trophoblasts in the interstitial tissues, suggesting that CALCA is present at the human implantation site in both villous and extravillous trophoblast cells.
|
CALCA mRNA Was Present in Trophoblasts and Decidual Cells at the Implantation Site
In situ hybridization was performed to determine CALCA mRNA expression in the first-trimester placental tissues. As shown in Figure 2, CALCA mRNA expression was localized predominantly in the cytotrophoblasts and syncytiotrophoblasts in the chorionic villi and decidualis cells in the basal plate deciduals, suggesting the de novo generation of CALCA at the human implantation site during early pregnancy.
|
CALCA Receptors Were Detected on Trophoblasts and Endothelial Cells
Immunohistochemistry using polyclonal antibodies to N-terminal domains of CALCRL and RAMP1 was performed on paraffin sections of first-trimester villous tissues. As shown in Figure 3, CALCA receptor components, CALCRL and RAMP1, are demonstrated on both villous syncytiotrophoblasts and cytotrophoblasts, as well as vascular endothelial cells identified by CD34 antibody, implying that an autocrine/paracrine effect of CALCA on trophoblasts and endothelial cells may exist.
|
CALCA Induced Endothelial Cell Growth
To determine the effects of CALCA on endothelial cell proliferation in vitro, we performed MTT assays on HUVECs. As shown in Figure 4A, CALCA dose-dependently stimulated HUVEC proliferation (P < 0.05), and this stimulation was totally blocked by CALCA antagonist CALCA8–37, suggesting the involvement of CALCA receptors in endothelial cell proliferation. Further, as shown in Figure 4B, the endothelial cell growth in HUVECs (48 and 72 h of culture) treated with CALCA (10–8 M) was three times greater than in controls. Thus, CALCA stimulated endothelial cell growth in a time- and dose-dependent manner.
|
CALCA Stimulated Endothelial Cell Migration
To determine whether CALCA affects endothelial cell migration, we cultured the cells in a 24-well Matrigel migration chamber plate. The cells that migrated to the lower side of the filter were stained with Calcein AM, and migrated cells were viewed and analyzed under a fluorescence live-cell and multidimensional imaging system. As shown in Figure 5, quantitative analyses reveal that CALCA (10–9 to 10–7 M) increased HUVEC migration in a dose-dependent manner (P < 0.05; Fig. 5A). Further, we performed a time course study at 4, 12, and 24 h of culture (Fig. 5B). A significant increase in cell migration was detected at 12 h after CALCA treatment (150.2 ± 12.3 vs. 116.7 ± 7.4 in controls, P < 0.05), and further increase was noted at 24 h after CALCA treatment (205.2 ± 16.6 vs. 120.7 ± 8.9 in controls, P < 0.01). These results indicate that CALCA has the bioactivity to stimulate endothelial cell migration, one of the critical steps of angiogenesis, and this action is mediated via CALCA receptors.
|
CALCA Promotes Endothelial Cell Capillarylike Tube Formation
To investigate whether CALCA could affect the angiogenic properties of endothelial cells, we performed a capillarylike tube formation assay, a commonly used approach for in vitro angiogenesis assessment. HUVECs (4 x 105 cells/ml) were cultured in the wells of Endothelial Cell Tube Formation System in the presence or absence of CALCA (10–8 M), or CALCA (10–8 M) + CALCA8–37 (10–7 M). After 4, 12, and 24 h of culture in standard conditions, the capillarylike tube formations were viewed under a microscope and evaluated using Image Pro-Plus Software. As shown in Figure 6A, CALCA (10–8 M, 12 h) promotes endothelial cell capillarylike tube formation (A2) compared with control (A1), and this effect is completely blocked by CALCA8–37 (A3). Quantitative analysis revealed that the length of capillarylike tubes induced by CALCA (10–8 M) was significantly increased over that of the untreated controls (Fig. 6B). The significant increase was observed at 12 h of culture (242.7 ± 15.9 vs. 121.5 ± 5.9 in controls, P < 0.01), and further increase was noted at 24 h (274.0 ± 18.6 vs. 143.8 ± 11.6 in controls, P < 0.01). CALCA induced capillarylike tube formations were blocked by CALCA8–37 at each time point. These data support the hypothesis that CALCA promotes angiogenesis of human endothelial cells via binding to its receptors on the cells.
|
In our pre-experiments on the cell migration and capillarylike tube formation assay we noted a significant increase in cell migration and tube formation after 12 and 24 h of exposure to CALCA (10–8 M), but no further changes were detected thereafter. Thus, we conducted our cell migration and capillarylike tube formation assays over 4~24 h, and this is consistent with the manufacturer's instructions for the Matrigel plate (BD Biosciences).
CALCA Induces Capillarylike Tube Formation in Conditioned Medium from Normal or Preeclamptic Placental Explants
Human placental tissues were obtained from pregnancies complicated by preeclampsia and gestational age-matched, uncomplicated controls. Villous fragments were cultured in RPMI-1640 containing 0.2% BSA in the presence or absence of CALCA (10–8 M) or CALCA8–37 (10–7 M) in standard culture conditions. After 48 h of culture, the conditioned media were collected, and 50 µl of the media was added to HUVECs cultured in the Endothelial Cell Tube Formation System. After 10 h of incubation, capillarylike tube formation was quantified under a microscope using Image Pro-Plus Software. As shown in Figure 7, a significant increase was noted in the length of tube formation when HUVECs were stimulated with conditioned medium from normal placental explants compared with preeclamptic placentas. Culture medium from normal placenta treated with CALCA8–37 caused a significant reduction in tube formation, suggesting that endogenous CALCA produced by placenta may act as a proangiogenic growth factor during pregnancy. Further, culture medium from preeclamptic placental explants incubated with CALCA significantly improved in vitro angiogenesis, as evidenced by increased capillarylike tube formation, suggesting that reduced levels of CALCA in preeclampsia are likely to be responsible, at least in part, for the poorly developed placenta vasculature associated with this disorder.
|
Angiogenesis is critical for successful implantation and placentation. Three different vascular processes are thought to be involved in the human placental development [20]: 1) adequate uterine angiogenesis and vascularity at the time of implantation; 2) development and expansion of the villous vasculature; and 3) remodeling of the maternal uterine arteriols at the feto-maternal interface. Although many factors may modulate these vascular processes, the mechanism regulating placenta angiogenesis in the human remains unclear. In this study we found that CALCA, a multifunctional peptide, is highly expressed by decidual cells and trophoblast cells at the human implantation site. CALCA receptor components, CALCRL and RAMP1, are abundantly expressed by trophoblast and vascular endothelial cells. In vitro, CALCA stimulates endothelial cell growth and migration and promotes capillarylike tube formation. Further, CALCA improves angiogenesis by conditioned medium from preeclamptic placental explants. Taken together, these results indicate that CALCA at the human implantation site may constitute potential autocrine or paracrine mechanisms that could modify placental angiogenesis and neovascularization.
Despite the absence of innervation at the feto-maternal interface in the human, several neuropeptides have been demonstrated within decidua and placenta, including basoactive intestinal polypeptide, neuropeptide tyrosine, endothelin-1, and CALCA [9]. Immunohistologic analysis of first-trimester placental villi showed that CALCA protein was expressed in decidual and glandular cells [9], and isolated decidual cells produced CALCA peptide in vitro [8]. CALCA content in the supernatants of placental extracts has been detected using a specific radioimmunoassay [21]. Transcripts of CALCA also were detected in the term placental villi and chorionic plate using quantitative RT-PCR [10]. In addition, CALCA receptors have been identified and purified from human term placenta using lectin and beta CALCA-affinity chromatography [22], and Scatchard analysis of 125I-
-CALCA binding revealed a single-affinity biding site for human -CALCA at the syncytiotrophoblast brush-border and basal plasma membranes [23]. In this study we confirmed by immunofluorescent confocal imaging and in situ hybridization that both protein and mRNA for CALCA are abundantly expressed by villous and extravillous trophoblast cells in first-trimester placental tissues, suggesting the de novo generation of CALCA at the human implantation site during early pregnancy. Further, immunohistochemistry using polyclonal antibodies to N-terminal domains of CALCRL and RAMP1 revealed that CALCA receptors are present on both syncytiotrophoblasts and cytotrophoblasts, as well as vascular endothelial cells, implying that an autocrine/paracrine effect of CALCA on trophoblasts and endothelial cells may exist during early gestation.
In addition to being a neuropeptide acting principally on the nervous and cardiovascular system [7], CALCA has been demonstrated to be a growth factor that may be involved in several key steps of angiogenesis. CALCA stimulates proliferation of various cell types, including T lymphocytes, Schwann cells, and tracheal epithelial cells [24–26]. CALCA stimulates the growth of prostate cancer cells [27] and enhances the prostate cancer cell invasion to Matrigel through activation of cell motility [12]. In vitro, CALCA increases both cell number and DNA synthesis in cultured endothelial cells [28]. In vivo, both systemic and local administration of CALCA accelerates the ulcer healing in animal models by stimulating the growth factor expression, epithelial cell proliferation, and angiogenesis [29]. Here we show that CALCA induces both endothelial proliferation and migration in a dose- and time-dependent manner. Further, CALCA promotes capillarylike tube formation of HUVECs on Matrigel, and these actions are blocked by CALCA antagonist. These results suggested that CALCA induces human endothelial cell proliferation and migration and promotes capillarylike tube formation, the critical steps in angiogenesis and neovascularization. Therefore, our data support the hypothesis that CALCA is a proangiogenic growth factor and plays a role in the human placental development.
In this study, we found that trophoblast cells expressed both CALCA and CALCA receptor components, CALCRL and RAMP1, indicating that CALCA may play a role in the regulation of trophoblast functions. It is well known that during early pregnancy, trophoblast cells act as the leading edge of embryo invasion of the maternal endometrium. Trophoblast cells exert endocrine functions, synthesizing and secreting steroid and peptide hormones [30]. Further, trophoblast cells have been proposed as contributing to the establishment of the feto-placental circulation via production of vascular endothelial growth factors (VEGF) and placental growth factor (PGF), and their antagonist, soluble fms-like tyrosine kinase 1 (FLT1) [31]. Apparently, additional studies determining the effect of CALCA on trophoblast angiogenic functions are required to fully understand the role of CALCA in human implantation.
Preeclampsia is a multiorgan disorder that complicates approximately 7%–10% of all pregnancies [32]. It is associated with significant morbidity and mortality for the mother and the baby. Despite extensive effort to elucidate the cause of preeclampsia, its pathogenesis and pathophysiology remain unclear. Numerous angiogenic proteins made in the placenta are thought to be involved during the stage of placental vascularization and development [1], and the imbalanced angiogenesis has been linked to the pathogenesis of preeclampsia, including the aberrant regulation of VEGF, PGF, soluble FLT1, and CALCA [31]. Evidence from others and our laboratory has shown that CALCA:β-actin and CALCA:GAPDH mRNA ratios were significantly lower in placental villi in preeclampsia than in gestational age-matched controls [10]. In addition, CALCA is detectable in peripheral blood, and the alteration of plasma CALCA concentration has been reported by several groups. Villous CALCA production and plasma CALCA concentrations in both maternal and fetal circulation were reduced in preeclampsia [33–35], indicating a reduction of CALCA in preeclampsia at both transcriptional and translational levels. The present study adds to the previous findings showing that CALCA is a proangiogenic growth factor; thus, insufficient CALCA angiogenic bioactivity might be involved in the pathophysiology of preeclampsia. In addition, we have shown in this study a significant decrease in capillarylike tube formation when HUVECs were stimulated with conditioned medium from preeclamptic placental explants compared with normal controls. Culture medium from normal placenta treated with CALCA8–37 caused a significant reduction in tube formation, suggesting that endogenous CALCA produced by villous tissues may act as a proangiogenic factor in placental vascular development. Further, culture medium from preeclamptic placental explants incubated with CALCA significantly improved in vitro angiogenesis, as evidenced by increased tube formation, implying that reduced levels of CALCA in the placenta of preeclamptic patients is likely to be responsible, at least in part, for the poorly developed placental vasculature associated with this disorder.
Recent studies have revealed that late-onset preeclampsia (>34 wk) had a minimal influence on placental villous and vascular morphology [36] compared with gestational age-matched controls, but early-onset preeclampsia (<34 wk) was associated with a reduction in placental weights, volume of the intervillous space, terminal villous volumes, and surface areas of terminal villi, indicating that the abnormal feto-placental angiogenesis is much more evident in the early-onset cases. Therefore, the use of villous tissues from patients with term preeclampsia is a limitation of the study, and the effect of CALCA on villous tissues from patients with early-onset preeclampsia needs further evaluation.
In summary, our study suggests that CALCA and its receptors are expressed at the human implantation site, and that CALCA functions as a proangiogenic growth factor through direct stimulation of endothelial cell proliferation and migration, as well as promotion of capillarylike tube formation. Thus, we propose that locally produced CALCA at the feto-maternal interface may have critical autocrine/paracrine effects on vasculogenic-angiogenic transformation in placental development and fetal growth. The effect of CALCA on endothelial cells suggests a role in angiogenesis; however, regulation of CALCA and its receptor at the human implantation site during early pregnancies and the mechanisms of insufficient CALCA production by villous tissue from preeclamptic pregnancies warrant further investigation.
ACKNOWLEDGMENTS
We thank Ms. Cheryl R. Welch for administrative assistance and John Helms for graphic support with this article.
FOOTNOTES
1Supported by National Institutes of Health grants HL70883, HD50266, and HL58144. 
Correspondence: 2Yuan-Lin Dong, Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Blvd., MRB, 11.138, Rt. 1062, Galveston, TX 77555-1062. FAX: 409 747 0475; e-mail: ydong{at}utmb.edu
Received: 20 November 2006.
First decision: 18 December 2006.
Accepted: 29 January 2007.
REFERENCES
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
