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,25-Dihydroxycholecalciferol Increases the Expression of Vascular Endothelial Growth Factor in C3H10T
Mouse Embryo Fibroblasts1,2
Interdepartmental Nutrition Program, Department of Foods and Nutrition, Purdue University, West Lafayette, IN 47907
3To whom correspondence should be addressed. E-mail: teegarden{at}cfs.purdue.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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mouse fibroblasts and their Harvey ras-oncogene transfected counterparts (rasneo11a cells) induced human umbilical vein endothelial cell (HUVEC) proliferation (1.3 and 0.3 times, respectively, P < 0.05), suggesting that 1,25(OH)2D3 altered the angiogenic phenotype of the cells. Although rasneo11a cells secreted less VEGF than C3H10T cells (97%, P < 0.005), 1,25(OH)2D3 induced C3H10T and rasneo11a cells to secrete 2 and 3 times, respectively, more VEGF than controls (P < 0.05). Similar effects on VEGF release occurred after 1,25(OH)2D3 treatment of MCF10A and MCF10Aras cells, a human breast epithelial cell model for multistage carcinogenesis. In C3H10T cells, 1,25(OH)2D3 activated the VEGF promoter in a dose-dependent (5–100 nmol/L) manner (maximum 60%) and all doses induced VEGF secretion (P < 0.05). 1,25(OH)2D3 induced VEGF mRNA expression (
50%) from 2 through 24 h; VEGF release was significantly increased at 8 h and sustained for 24 h. VEGF mRNA expression and release declined as C3H10T cells grew more confluent, whereas the magnitude of 1,25(OH)2D3-stimulated changes in VEGF was greater in confluent (3.3 times RNA; 3.5 times release) than in subconfluent (50% RNA; 100% release) cultures (P < 0.05). Thus, 1,25(OH)2D3 increases VEGF secretion, and in C3H10T cells, this is likely through activation of the VEGF promoter and induction of gene expression. These data contribute to understanding the role 1,25(OH)2D3 plays in regulation of angiogenesis in normal compared with disease states.
KEY WORDS: • 1,25 dihydroxyvitamin D • C3H10T • ras • vascular endothelial growth factor • angiogenesis
Epidemiologic and experimental evidence demonstrates that poor vitamin D status or impaired vitamin D metabolism are associated with cancers of the breast, prostate, and colon (1–3). Thus, in addition to its role in maintaining calcium homeostasis, the active metabolite of cholecalciferol, 1
,25 dihydroxycholecalciferol [1,25(OH)2D3],4 may play a role in cancer prevention. Angiogenesis, the formation of new blood vessels from preexisting vasculature, is essential in normal development, growth and maintenance of bone structure, reproduction, and wound healing (4–8). It is increasingly clear that angiogenesis also plays an important role in the progression of pathological states such as cancer (9,10). Because 1,25(OH)2D3 affects multiple cellular signaling pathways to modulate a variety of cellular processes including proliferation, differentiation, and apoptosis in multiple cell types (2,3), it was hypothesized that such complex signaling also influences angiogenesis (10).
Angiogenesis is a highly regulated, multistep process involving the coordination of many signaling pathways that control the balance of pro- and antiangiogenic factors (5,11,12) to influence the proliferation and migration of vascular endothelial cells to ultimately form new capillaries. Vascular endothelial growth factor (VEGF) A is one of the most potent and well-characterized proangiogenic factors. This family of 46-kDa proteins targets primarily vascular endothelial cells to increase proliferation, promote chemotaxis, enhance vascular permeability, and induce capillary sprouting. The VEGF gene can be alternatively spliced and processed to form 4 major human isoforms: VEGF121, VEGF165, VEGF189, and VEGF206. VEGF121 and VEGF165 (VEGF120 and VEGF164 in mice) are freely diffusible upon secretion into the extracellular matrix, and VEGF165/164 has a combination of properties that make it the most biologically potent (7,8,13).
Although hypoxia is a strong inducer of VEGF transcription and secretion in vivo and in vitro (14,15), VEGF is also regulated in normoxic conditions by cytokines, hormones, and oncogenes such as ras (7,8,16,17). Mutations of the ras gene, which increase the proportion of Ras in the GTP-bound state relative to Ras-GDP, activate Ras signaling and promote mitogenesis (18). Grugel et al. (19) and Rak et al. (20) were among the first to suggest that tumor growth was augmented by oncogene-driven tumor-induced angiogenesis. For example, v-H-ras–transfected NIH3T3 cells have greater cellular VEGF protein levels than control NIH3T3 cells (20). Ras also increased NIH3T3 VEGF gene expression and protein secretion in vitro and in vivo (20). Indeed, oncogenic ras induces transcription, synthesis, and release of VEGF both in vitro and in vivo, contributing toward carcinogenesis and angiogenesis (7,17,21).
1,25(OH)2D3 influences angiogenesis in a variety of target tissues by regulating 1 or more components of the angiogenic process. In a study of 1,25(OH)2D3-induced reversal of rickets, Hunter et al. (22) observed restoration of normal bone vascularization compared with rachitic animals. Research in chondrocytes and osteoblasts demonstrated that in addition to inducing proliferation and growth, 1,25(OH)2D3 increases VEGF mRNA and secretion, subsequently increasing angiogenesis in bone, which is critical to normal bone development, maintenance, and fracture healing (23–27). VEGF secretion was also enhanced by 1,25(OH)2D3 treatment of vascular smooth muscle cells, the primary source of circulating VEGF (28). Conversely, 1,25(OH)2D3 suppresses angiogenesis, tumor size and number, and VEGF staining in several animal models of cancer (29–31). In addition, 1,25(OH)2D3 suppresses endothelial cell migration (32) and inhibits VEGF-induced endothelial cell elongation by inducing apoptosis (33). 1,25(OH)2D3 also induces apoptosis and growth arrest of several transformed cells (2,3). Therefore, the suppression of angiogenesis in some animal models of cancer may be partially explained by reduction of the proliferating tumor cell population, thus decreasing the source of VEGF. Furthermore, there may be fewer cells to respond to VEGF because 1,25(OH)2D3 induces apoptosis of vascular endothelial cells (33). There is evidence that 1,25(OH)2D3 regulates angiogenesis and VEGF production, although the exact targets of 1,25(OH)2D3 remain unclear.
The effects of 1,25(OH)2D3 on VEGF, and therefore angiogenesis, may depend on the cell type and model system studied. Therefore, we undertook this study to determine how 1,25(OH)2D3 and the H-ras oncogene influence VEGF120/164 release from the C3H10T mouse fibroblast cell model of multistage carcinogenesis (34–36). Additionally, we characterized the effects of 1,25(OH)2D3 on VEGF promoter activity, mRNA expression, and release in C3H10T cells at varying degrees of confluence. These studies contribute to clarifying the molecular targets by which 1,25(OH)2D3 regulates VEGF. This knowledge will lead to understanding the effects of 1,25(OH)2D3 on angiogenesis in normal compared with cancerous states.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cell culture conditions.
The C3H10T mouse embryo fibroblast cell (clone 8) and human umbilical vein endothelial cell (HUVEC) lines were obtained from American Type Culture Collection (ATCC, CCL-226 and CRL-1730, respectively). C3H10T cells and C3H10T cells stably transfected with the Harvey ras oncogene (rasneo11a cells) (34–36) were grown in DMEM supplemented with 10% heat inactivated fetal bovine serum (HI-FBS), 1 x 105 units/L penicillin and 100 mg/L streptomycin. Cells (confluence studies) were harvested on d 3, 4, and 5 after seeding. All other experiments were harvested on d 3 after seeding, when cells were no >75% confluent. HUVECs were grown to confluence in F12K Nutrient Mixture (Kaighn’s Modification) supplemented with 100 mg/L heparin, 30 mg/L endothelial cell growth supplement, 10% HI-FBS, 1 x 105 units/L penicillin and 100 mg/L streptomycin. MCF10A human epithelial breast cells and MCF10A cells stably transfected with the Harvey ras oncogene (MCF10Aras cells) were obtained from Dr. Michael Kinch, Purdue University (37,38). MCF10A and MCF10Aras were cultured to 70% confluence in DMEM/Ham’s F-12 (1:1, v:v), containing 5% horse serum, and supplemented with 2.5 mmol/L L-glutamine, 10 mg/L insulin, 20 µg/L epidermal growth factor, 50 µg/L cholera toxin, and 50 mg/L hydrocortisone. All cell lines were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. 1,25(OH)2D3 was delivered to cells in 100% ethanol at a final ethanol concentration < 1%.
Quantification of VEGF secretion.
The conditioned media (CM) from C3H10T and rasneo11a cells were collected and frozen at –20°C until analysis. Secreted murine VEGF (mVEGF) concentrations in these samples were determined in duplicate using the quantitative mVEGF Duo-Set ELISA development kit (R&D Systems) which detects both VEGF120 and VEGF164. Optical densities were measured at 450 and 540 nm (plate correction) using a Powerwave X spectrophotometer microplate reader (Biotek Instruments). VEGF concentrations were calculated with KC4 software (Biotek Instruments) using a 7-point standard curve created from recombinant mouse VEGF standards present on each 96-well plate by plotting a 4-PL curve fit of the logarithm of the mean absorbance of each standard vs. the logarithm of the VEGF concentration. The amount of mVEGF (ng/L) per sample was normalized to total cellular protein (mg) from the same sample. For MCF10A and MCF10Aras cells, human VEGF165 concentrations from stored CM samples were determined with the human VEGF Duo-Set ELISA (R&D Systems), and total hVEGF (fg) was normalized to cell number, as assessed by trypan blue exclusion. In some instances, data are reported as VEGF release relative to vehicle-treated controls.
Analysis of mVEGF promoter activity.
The mouse VEGF promoter-luciferase construct, obtained from Dr. Patricia D’Amore, Department of Pathology, Harvard Medical School (39), was used as a reporter gene for VEGF promoter activation. Briefly, the construct is a 1.6-kb fragment of mouse VEGF genomic DNA, which includes 1.2 kb of the 5'-flanking sequence, the transcription start site, and 0.4 kb of the corresponding 5'-UTR ligated upstream of a promoterless firefly luciferase gene in the pGL3-basic plasmid. pTK-Renilla luciferase (pTK-RL, Promega) was used to verify transfection efficiency. C3H10T cells were transiently cotransfected with VEGF-luciferase and pTK-Renilla luciferase (a ratio of 20:1 VEGF-luciferase: pTK-RL) employing Lipofectamine Plus (Gibco-BRL) according to the manufacturer’s instructions. After transfection, cells were treated for 24 h with 100 nmol/L 1,25(OH)2D3 or vehicle. Activity of the VEGF-luciferase reporter gene was determined by the Dual Luciferase Reporter (DLR) Assay system (Promega) according to the manufacturer’s instructions. Luminescence was determined for both the VEGF firefly luciferase and the Renilla luciferase in a Turner TD-20/20 luminometer (Turner Designs). The data are expressed as the firefly/Renilla luminescence (Relative Luminescence Units, RLU) of treated cells divided by the RLU of vehicle controls.
Semiquantitative RT-PCR analysis of mVEGF mRNA levels. Total RNA was isolated using Tri-Zol reagent (Gibco-BRL). Reverse transcription to generate first-strand cDNA was performed on 2 µg RNA employing random decamers provided with Ambion’s RETROscript Kit (Ambion) according to the manufacturer’s directions. cDNA solution (1 µL) served as the template for PCR amplification, which was performed with SuperTaq DNA Polymerase (Ambion) and the proprietary components of the mouse VEGF Relative RT-PCR Kit (Ambion) including primers specific for mVEGF (322 bp product) and 18S rRNA (495 bp product) as an internal standard. To verify the absence of genomic DNA contamination, PCR reactions without template and on total RNA without reverse transcriptase were routinely performed and were negative. Separate PCR reactions were carried out for mVEGF and 18S primers in a Gene Amp PCRsystem 9700 thermocycler (Applied Biosystems) using the following parameters: 94°C, 3 min; followed by 94°C, 30 s; 59°C, 30 s; 72°C, 30 s for 29 (mVEGF) or 15 (18S) cycles with a final extension at 72°C for 10 min. Equal volumes of PCR products were subjected to electrophoresis on 2% agarose gels containing ethidium bromide. Bands were visualized under UV light using the Fluor-S MultiImager system and quantified with QuantityOne software (BioRad). For each sample, the adjusted optical density of the VEGF band was divided by the adjusted optical density of the corresponding 18S band and ratios were normalized to the vehicle control. Data are expressed as VEGF mRNA levels relative to controls.
Proliferation of HUVECs.
HUVEC cells were seeded in F12K growth media in the wells of 96-well plates. After 48 h, the medium was replaced with direct HUVEC treatment consisting of a 1:1 mixture of complete F12K to DMEM containing either vehicle or 100 nmol/L 1,25(OH)2D3 [making the final 1,25(OH)2D3 concentration 50 nmol/L], or a 1:1 mixture of F12K medium and the CM from either vehicle- or 100 nmol/L 1,25(OH)2D3-treated C3H10T or rasneo11a cells. After 24 h, proliferation was assessed by the CellTiter 96 AQqueous Non-Radioactive Proliferation Assay (Promega) according to the manufacturer’s instructions. Data are reported normalized to values resulting from vehicle treatment of HUVECs, and results from CM treatment are also adjusted to total protein from the source cells (C3H10T or rasneo11a) to control for the influence of cell number on factors in the CM.
Cell harvest conditions for total protein quantification.
C3H10T cells were washed with CMF-PBS and harvested on ice into a buffer containing 25 mmol/L HEPES, 150 mmol/L NaCl, 5 mmol/L EDTA, 0.1% Triton, and 1% protease inhibitor cocktail-I [containing 4-(2-aminoethyl) benzene-sulfonyl fluoride, 104 mmol/L; aprotinin, 0.08 mmol/L; leupeptin, 2 mmol/L; bestatin, 4 mmol/L; pepstatin A, 1.5 mmol/L; and E-64, 1.4 mmol/L] and phosphatase inhibitor cocktail-II [containing sodium vanadate, sodium molybdate, sodium tartrate, and imidazole]. The lysate was incubated on ice for 15 min, and centrifuged at 12,000 x g for 10 min before total protein analysis of the supernatant. Total cellular protein was determined by the bicinchoninic acid assay (Pierce).
Design and statistics. All experiments were performed at least in duplicate, and at least 2 experiments were completed at least 3 times. Comparisons between 2 groups were done with Student’s t tests and multiple mean comparisons were performed after 1- or 2-way ANOVA using Newman-Keuls and Dunnett’s post-hoc analyses with SAS, version 8.0. Differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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or rasneo11a cells had no effect on HUVEC proliferation compared with HUVECs treated directly with vehicle. However, CM from 1,25(OH)2D3-treated C3H10T and rasneo11a cells significantly increased HUVEC proliferation 1.3 and 0.3 times, respectively, compared with CM from the corresponding vehicle controls (Fig. 1). These results indicate that 1,25(OH)2D3 enhances the angiogenic potential of both C3H10T and rasneo11a cells.
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cells (Fig. 2A). Similar to the rasneo11a cells, MCF10Aras cells released significantly less (37%) hVEGF than MCF10A cells (Fig. 2B). Thus, stable transfection of the ras oncogene reduced VEGF release from cells belonging to 2 models of multistage carcinogenesis.
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and rasneo11a cells treated with 1,25(OH)2D3 produced CM that increased HUVEC proliferation (Fig. 1), the effect of 1,25(OH)2D3 on VEGF release was assessed. C3H10T cells secreted nearly 2 times more VEGF in response to 24 h of treatment with 1,25(OH)2D3 compared with vehicle controls (P < 0.05) (Fig. 3A). 1,25(OH)2D3 also stimulated the release of VEGF from rasneo11a cells approximately 3-fold (P < 0.05) (Fig. 3A). Similarly, both MCF10A and MCF10Aras cells treated with 1,25(OH)2D3 released more VEGF than vehicle-treated controls (P < 0.05) (Fig. 3B). Therefore, in 2 cell models of multistage carcinogenesis, 1,25(OH)2D3 stimulated the release of VEGF, even in the presence of the ras oncogene.
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cells treated with doses of 1,25(OH)2D3 ranging from 5 to 100 nmol/L increased VEGF release at 24 h compared with vehicle-treated control (Fig. 4A). Although there is no known traditional vitamin D response element within the VEGF promoter (27), 1,25(OH)2D3 also stimulated VEGF promoter activation in a dose-dependent manner (maximum 60%, P < 0.05) as assessed by the VEGF reporter gene assay (Fig. 4B). 1,25(OH)2D3 stimulated VEGF mRNA expression (60%) after 2 h (Fig. 5A) and induced VEGF release relative to the vehicle control (80%) after 8 h (Fig. 5B). These effects were still significant at 24 h.
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cells grown in culture for 2, 3, or 4 d and treated with vehicle or 1,25(OH)2D3 for an additional 24 h, both constitutive VEGF mRNA levels (Fig. 6A) and secreted VEGF (Fig. 6B) decreased significantly as confluence increased. However, the magnitude of the 1,25(OH)2D3-induced change in VEGF mRNA expression and release increased with each day in culture (Fig. 6) from 50% (mRNA) and 100% (release) on d 3 to 3.3- and 3.5-fold on d 5. These results were not due to differences in cell number because the protein content per dish did not differ between vehicle- and 1,25(OH)2D3-treated cells (Fig. 6C). Thus, 1,25(OH)2D3 stimulates VEGF expression and release in C3H10T cells, and the more confluent the cells, the greater the magnitude of stimulation.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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cells with the ras oncogene provided a cellular model of multistage carcinogenesis to compare normal cellular responses with those of cells representing the initiation stage of cancer, rasneo11a (34,35). This cell model was used to investigate whether oncogenic ras and 1,25(OH)2D3 altered the C3H10T or rasneo11a cells’ angiogenic potential as assessed by HUVEC proliferation assays and to characterize the effects of 1,25(OH)2D3 on VEGF release and gene expression from C3H10T cells. Further, this system can be used to investigate specific molecular targets of 1,25(OH)2D3 with respect to VEGF regulation in normal vs. transformed cells.
In the present study, stable transfection with the ras oncogene did not affect the angiogenic potential of C3H10T cells. Neither the CM from vehicle-treated C3H10T or rasneo11a cells induced the proliferation of HUVEC cells. This appears to disagree with data demonstrating that the ras oncogene contributes to the angiogenic phenotype of transformed cells, in part, by increasing production of proangiogenic endothelial mitogens, such as VEGF (17,19,20,40).
Further, rasneo11a cells secreted constitutively less VEGF than the C3H10T cells (Fig. 2A). Therefore, the absence of an increase in HUVEC proliferation by the CM from rasneo11a cells compared with the CM from C3H10T cells is not likely due to VEGF release, but probably due to other, as yet unexamined, factors regulated by activated Ras signaling. Similarly, in a second model of multistage carcinogenesis, MCF10Aras cells secreted less VEGF than MCF10A cells (Fig. 2B). Although contradictory to published evidence that oncogenic ras contributes to a proangiogenic phenotype by increasing VEGF transcription and secretion in both NIH3T3 fibroblasts and IES epithelial cells (20,40), our results may be explained by differences in cell line properties. Both the H-ras-transfected counterparts of the C3H10T and MCF10A models of carcinogenesis represent early stages of transformation, as evidenced by rasneo11a cells’ weak growth in soft agar and inability to form tumors in nude mice or the formation of primarily benign ductal aggregate-nodules by MCF10Aras cells (34–38). In comparison, the NIH3T3-ras cells used in other studies represent fully transformed cells because they do grow in soft agar and readily form tumors in nude mice (35). Also, the studies of ras-transformed NIH3T3 cells were performed with serum-starved confluent cells (40), whereas the present studies employing rasneo11a and MCF10Aras cells that were not serum starved and were conducted when cells were subconfluent. Thus, regulation of VEGF release by the ras gene product may depend on cell type, cell status, and/or cell culture conditions.
The CM from 1,25(OH)2D3-treated C3H10T and rasneo11a cells stimulated more HUVEC proliferation than CM from vehicle-treated cells (Fig. 1). Thus 1,25(OH)2D3 enhanced the angiogenic potential of both the C3H10T and rasneo11a cells. The effects on HUVECs were not likely due to unmetabolized 1,25(OH)2D3 in the CM because direct treatment of HUVECs with 1,25(OH)2D3 caused a significant reduction in HUVEC proliferation. This is consistent with results obtained by Mantell et al. (33) who found that 1,25(OH)2D3 decreased vascular endothelial cell number by stimulating apoptotic cell death. However, although 1,25(OH)2D3 induced C3H10T and rasneo11a cells to secrete 2 and 3 times more VEGF, respectively, (Fig. 3A), this translated into smaller increases in HUVEC proliferation, i.e., 1.3-fold by CM from 1,25(OH)2D3 treated C3H10T and 0.3-fold by CM from rasneo11a cells. Although this appears to be a discrepancy, this result demonstrates that VEGF is not the only factor regulated by 1,25(OH)2D3 or ras that could alter the angiogenic potential of cells. Other growth factors secreted by the cells, or unmetabolized 1,25(OH)2D3, may potentially oppose the effects of newly secreted VEGF; however, even in the potential presence of these or other currently unknown factors, significant increases in HUVEC proliferation were detected. Therefore, 1,25(OH)2D3 treatment causes C3H10T and rasneo11a cells to alter the balance of secretory factors in favor of HUVEC proliferation.
1,25(OH)2D3 increased VEGF secretion by cells from both murine and human models of multistage carcinogenesis (Fig. 3). It is likely that 1,25(OH)2D3 induction of VEGF release is at least part of the mechanism explaining 1,25(OH)2D3 enhancement of the angiogenic potential of C3H10T and rasneo11a cells described in Figure 1. The fact that 1,25(OH)2D3 increases VEGF release in the presence of the ras oncogene is interesting because we previously showed that rasneo11a cells exhibit reduced 1,25(OH)2D3-mediated vitamin D receptor transcriptional activity compared with C3H10T cells, although vitamin D receptor levels are similar (41). This implies that the cellular machinery for 1,25(OH)2D3 to induce VEGF synthesis is not dependent on vitamin D receptor–mediated transcription and is not altered by signaling through oncogenic ras pathways. Potential mechanisms for 1,25(OH)2D3 induction of VEGF are being explored in C3H10T cells.
To study how 1,25(OH)2D3 increases VEGF in untransformed cells, further characterization of this effect was conducted in C3H10T cells. All doses of 1,25(OH)2D3 increased VEGF promoter activity and secretion by C3H10T cells (Fig. 4). This is interesting because the VEGF promoter contains no known traditional vitamin D response elements (27). 1,25(OH)2D3-induced VEGF mRNA levels were elevated within 2 h after treatment; increased secretion was significant by 8 h and levels remained elevated for 24 h (Fig. 5). Additionally, in vitro, changes in VEGF expression and secretion may also be related to cell density. In this study, as the cells became more confluent, constitutive VEGF mRNA and secretion decreased (Fig. 6). This is in contrast to results published by Koura et al. (42) and Mukhopadhyay et al. (43), who showed that VEGF mRNA and secretion increase as density increases, and with Rak et al. (19,40), who observed no significant change in VEGF mRNA in dense compared with sparse rat intestinal epithelial cell cultures. We also demonstrated that with increasing confluence, C3H10T cells not only retained their ability to respond to a 24-h dose of 1,25(OH)2D3, but exhibited enhanced VEGF mRNA levels and secretion, as indicated by the fact that the fold induction of VEGF by 1,25(OH)2D3 was greater with each subsequent day in culture.
Induction of VEGF by 1,25(OH)2D3 was previously demonstrated in other cell types. For example, Yamamoto et al. (28) reported that 1,25(OH)2D3 increased VEGF release from aortic smooth muscle cells. Also, studies employing cells from osteoblast and chondrocyte lineages showed an increase in synthesis of VEGF in response to 1,25(OH)2D3 (24–27). However, this is the first report of 1,25(OH)2D3 increasing VEGF secretion in models of multistage carcinogenesis, in the presence of the ras oncogene. In cells of normal tissues, represented in these studies by C3H10T and MCF10A cells, enhanced VEGF release in response to 1,25(OH)2D3 treatment may be related to promoting angiogenesis to enhance tissue development or maintenance. Future research is warranted to elucidate the mechanism of 1,25(OH)2D3 induction of VEGF in vitro and to clarify the role of the ras oncogene in this process. Understanding the pathways responsible for vitamin D regulation of VEGF and angiogenesis during normal growth compared with disease states will contribute to new dietary recommendations to enhance optimal tissue growth, such as during development, bone remodeling, and tissue repair.
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FOOTNOTES |
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2 Supported by American Cancer Society-RPG-00–038-01-CNE.
4 Abbreviations used: CM, conditioned media; CMF-PBS, calcium magnesium free-PBS; 1,25(OH)2D3, 1,25 dihydroxycholecalciferol; DLR, dual luciferase reporter; HI-FBS, heat inactivated fetal bovine serum; HUVEC, human umbilical vein endothelial cell; hVEGF, human VEGF; mVEGF, murine VEGF; pTK-RL, pTK-Renilla luciferase reporter gene; RLU, relative luminescence units; VEGF, vascular endothelial growth factor.
Manuscript received 5 March 2004. Initial review completed 14 April 2004. Revision accepted 30 June 2004.
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5; *different from C3H10T
