Cancer Research Cancer Epigenetics Telomeres
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martínez, A.
Right arrow Articles by Cuttitta, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martínez, A.
Right arrow Articles by Cuttitta, F.
[Cancer Research 64, 6489-6494, September 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Proadrenomedullin NH2-Terminal 20 Peptide Is a Potent Angiogenic Factor, and Its Inhibition Results in Reduction of Tumor Growth

Alfredo Martínez1,3, Enrique Zudaire1,3, Sergio Portal-Núñez1,3, Liliana Guédez1,3, Steven K. Libutti2,3, William G. Stetler-Stevenson1,3 and Frank Cuttitta1,3

1 Cell and Cancer Biology Branch, 2 Surgery Branch, 3 Vascular Biology Faculty, National Cancer Institute, NIH, Bethesda, Maryland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have found through ex vivo and in vivo angiogenesis models that the adrenomedullin gene-related peptide, proadrenomedullin NH2-terminal 20 peptide (PAMP), exhibits a potent angiogenic potential at femtomolar concentrations, whereas classic angiogenic factors such as vascular endothelial growth factor and adrenomedullin mediate a comparable effect at nanomolar concentrations. We found that human microvascular endothelial cells express PAMP receptors and respond to exogenous addition of PAMP by increasing migration and cord formation. Exposure of endothelial cells to PAMP increases gene expression of other angiogenic factors such as adrenomedullin, vascular endothelial growth factor, basic fibroblast growth factor, and platelet-derived growth factor C. In addition, the peptide fragment PAMP(12-20) inhibits tumor cell–induced angiogenesis in vivo and reduces tumor growth in xenograft models. Together, our data demonstrate PAMP to be an extremely potent angiogenic factor and implicate this peptide as an attractive molecular target for angiogenesis-based antitumor therapy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The formation of new blood vessels from preexisting ones, or angiogenesis, is necessary for normal physiology in processes such as embryogenesis, growth, wound healing, and the endometrial cycle. However, when the balance between proangiogenic and antiangiogenic regulators gets distorted, the resulting lack or excess of blood vessels can lead to numerous pathological conditions (1) . Angiogenesis is a multistep process that requires at least endothelial cell proliferation, production of extracellular proteases, migration of endothelial cells, tube formation, development of loops, and recruitment of pericytes and smooth muscle cells for larger vessels (2) .

Like normal tissues, tumors require an adequate blood supply to maintain their metabolic needs. As the tumor grows, the cells located at the center of the tissue mass experience hypoxia, a physiologic stimulus for the expression and secretion of numerous proangiogenic factors, which in turn will promote enough blood vessel formation to support additional tumor growth (3) . Hence the hypothesis that depriving tumors from their proangiogenic factors would stop tumor progression (4 , 5) . In the last few years, antiangiogenic therapies have been found to successfully delay tumor growth in animals, and several antiangiogenic factors are currently undergoing clinical trials (6 , 7) .

The regulatory peptide adrenomedullin is a multifunctional molecule (8) that has been recently characterized as a proangiogenic factor with the help of ex vivo and in vivo animal models (9, 10, 11, 12 ; among others). In addition to inducing angiogenesis, adrenomedullin functions in cancer cells as an autocrine growth factor, enhances thymidine incorporation, reduces apoptosis, and is induced by hypoxia, therefore suggesting that this peptide may be an important tumor cell survival factor and a potential target for antitumor therapy (13) .

Despite the growing interest in the angiogenic effects of adrenomedullin and its influence in tumor biology, no attention has been paid to the actions of proadrenomedullin NH2-terminal 20 peptide (PAMP) in this field, and we decided to investigate them. Here, we report that PAMP is a very potent angiogenic factor, able to induce neovascularization in animal models at concentrations 6 orders of magnitude lower than other classic proangiogenic factors such as vascular endothelial growth factor (VEGF) and adrenomedullin. We demonstrate that human microvascular endothelial cells have receptors for PAMP and respond to it by increasing migration and cord formation in Matrigel assays. In addition, PAMP stimulation induces expression of classic angiogenic factors in endothelial cells. We use a fragment of PAMP that specifically inhibits PAMP-induced effects as a tool to define for the first time the important role of PAMP in angiogenesis. This fragment acts as an inhibitor of tumor cell–induced angiogenesis and is able to delay tumor growth in xenograft models of tumor progression.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Synthetic human adrenomedullin, PAMP, and PAMP(12-20) were purchased from Bachem (King of Prussia, PA). Recombinant human VEGF and basic fibroblast growth factor (bFGF) were obtained from R&D Systems (Minneapolis, MN).

Chick Embryo Aortic Arch Assay.
The chick embryo aortic arch assay is an ex vivo angiogenesis assay that was performed as described previously (14 , 15) . In brief, aortic rings of approximately 0.8 mm in length were prepared from the five aortic arches of 13-day-old chicken embryos (CBT Farms, Chestertown, MD), and the soft connective tissue of the adventitia layer was carefully removed with tweezers. Each aortic ring was placed in the center of a well in a 48-well plate and covered with 10 µL of Matrigel (BD Biosciences, San Jose, CA). After the Matrigel solidified, 300 µL of growth factor–free human endothelial-SFM basal growth medium (Invitrogen, Carlsbad, CA) containing the proper concentration of the test substances were added to each well. The plates were kept in a humid incubator at 37°C in 5% CO2 for 24 to 36 hours. Microvessels sprouting from each aortic ring were photographed in an inverted microscope, and the area covered by the newly formed capillaries was estimated as reported previously (14) .

Directed In vivo Angiogenesis Assay.
Analysis and quantitation of angiogenesis was done using directed in vivo angiogenesis assay as described previously (10 , 16) . In brief, 10-mm-long surgical-grade silicone tubes with only one end open (angioreactors) were filled with 20 µL of Matrigel alone or mixed with adrenomedullin, bFGF, VEGF, PAMP, and/or PAMP(12-20) at the indicated concentrations. Human lung cancer cell lines (see below) were also premixed with Matrigel alone or in combination with PAMP(12-20) at 10,000 cells per angioreactor. After the Matrigel solidified, the angioreactors were implanted into the dorsal flanks of athymic nude mice (NCI colony). After 11 days, the mice received i.v. injections of 25 mg/mL FITC-dextran (100 µL/mouse; Sigma, St. Louis, MO) 20 minutes before removing angioreactors. Photographs of the implants were taken for visual examination of angiogenic response. Quantitation of neovascularization in the angioreactors was determined as the amount of fluorescence trapped in the implants and was measured in a HP Spectrophotometer (Perkin-Elmer, Boston, MA). This protocol was approved by the internal NIH animal committee.

The human cancer cell lines used, A549 and H1299, were obtained from the American Tissue Culture Collection (Manassas, VA) and fed with RPMI 1640 containing 10% fetal bovine serum (Invitrogen). Before they were used in animals, both cell lines were tested for a panel of human and murine pathogens and found to be pathogen-free.

Ca2+ Measurements.
Human dermal microvascular endothelial cells were cultured in 96-well plates at 1.0 x 105 cells per well. The cells were loaded for 60 minutes at room temperature with the fluorescent dye FLIPR (Molecular Devices, Sunnyvale, CA) and then transferred to the FlexStation II (Molecular Devices) for analysis. The test compounds were prepared in another plate at 5x concentration and added to the proper wells by the robotic arm of the FlexStation II. Fluorescence was measured every 5 seconds in each well and recorded. ATP (1 mmol/L; Sigma) was used as a Ca2+ influx agonist (17) .

Proliferation Assay.
The same microvascular endothelial cells were seeded in 96-well plates at a density of 2.0 x 105 cells per well in serum-free medium containing different concentrations of the test peptides. After 3 days in culture, the number of viable cells per well was estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as reported previously (10) . Results are represented as the percentage of growth over the untreated control.

Migration Assay.
Cell motility was measured as described previously (10) . Test peptides were placed at various concentrations at the bottom of a ChemoTx chamber (NeuroProbe, Inc., Gaithersburg, MD). The intermediate membrane was coated with 10 µg/mL fibronectin, and in the upper chamber, 5.0 x 105 human endothelial cells were added. After a 4-hour incubation at 37°C, the membrane was fixed and stained (Protocol Hema3; Biochemical Sciences, Inc., Bridgeport, NJ). The cells trapped in the porous membrane were photographed through a x25 microscope objective, and the number of cells per photographic field was counted.

Cord Formation Assay.
Human endothelial cells were seeded at 2.0 x 105 cells per well over a solid layer of Matrigel covering the bottom of a 24-well plate in the presence or absence of the test peptides, as described previously (18) . After an overnight incubation, the tubular structures were photographed, and the number of knots per photographic field were counted as a measure of lattice complexity.

Real-Time Polymerase Chain Reaction Quantification of Gene Expression.
Human endothelial cells were cultured in T-75 flasks until they reached a density of approximately 2.5 x 106 cells per flask. Cells were treated with 10 nmol/L PAMP in serum-free medium for 24 hours. Total RNA was extracted using the RNeasy Mini kit from Qiagen (Valencia, CA) and reverse transcribed using the SuperScript First-Strand Synthesis system (Invitrogen). Quantification of gene expression was performed by real-time PCR as described previously (19) . The PCR reaction was run in an Opticon cycler (MJ Research, Waltham, MA) using Sybr Green PCR master mix (Applied Biosystems, Foster City, CA). Thermocycling was performed in a final volume of 25 µL containing 2 µL of cDNA (1:10 dilution) and 400 nmol/L of primers (see below). All targets were amplified in triplicate in the same run as the house-keeping gene, using the following cycle scheme: After initial denaturation of the samples at 95°C for 2 minutes, 46 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds were performed. Fluorescence was measured in every cycle, and mRNA levels were normalized by the 18S RNA values in all samples. A melting curve was run after PCR by increasing the temperature from 60°C to 96°C (0.5°C increments). A single peak was obtained for all amplicons, thus confirming the specificity of the reaction.

Primers were as follows: adrenomedullin forward, ACA TGA AGG GTG CCT CTC GAA; adrenomedullin reverse, AGG CCC TGG AAG TTG TTC ATG; VEGF forward, TCA GAG CGG AGA AAG CAT TTG T; VEGF reverse, TCG GCT TGT CAC ATC TGC AA; bFGF forward, CGA CCC TCA CAT CAA GCT ACA AC; bFGF reverse, CCA GTT CGT TTC AGT GCC ACA T; platelet-derived growth factor (PDGF) A forward, TTC GGA GGA AGA GAA GCA TCG; PDGF A reverse, GCA CTT GAC ACT GCT CGT GTT G; PDGF B forward, AAC AAC CGC AAC GTG CAG T; PDGF B reverse, TCT CGA TCT TTC TCA CCT GGA C; PDGF C forward, TTG AGG AAC CCA GTG ATG GAA C; PDGF C reverse, CAG CTT CTG TGA ATT GTG GCA T; 18 S forward, ATG CTC TTA GCT GAG TGT CCC G; and 18 S reverse, ATT CCT AGC TGC GGT ATC CAG G.

Xenograft Experiments.
For the first xenograft experiment, 30 female athymic nude mice from the NIH colony in Frederick, Maryland, received s.c. injections of 1.0 x 107 A549 cells per mouse. Two weeks later, all of the mice had developed palpable tumors under the skin, and at this time, they were randomly divided in three groups. Three times a week, each individual tumor was measured (length, height, and thickness), and every mouse received an intratumoral injection, according to their group. Group 1 (control) received 100 µL of PBS; group 2 received 100 µL of 10 nmol/L PAMP(12-20) in PBS; and group 3 received 100 µL of 1 µmol/L PAMP(12-20) in PBS. When the tumor burden became unbearable (larger than 2000 mm3), the mice were sacrificed. This schedule was chosen based on previous antiangiogenic treatments in xenograft studies (20) . In a second experiment, another 30 mice that received injections of A549 cells were divided in three groups. Group 1 (control) received 100 µL of PBS; group 2 received 100 µL of 100 nmol/L PAMP in PBS; and group 3 received 100 µL of 1 nmol/L PAMP in PBS. As with the other animal experiments, these studies were done under a NIH-approved protocol.

Statistical Analysis.
When appropriate, data were compared by two-tailed Student’s t test. P values lower than 0.05 were considered statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Angiogenic Potential of Proadrenomedullin NH2-Terminal 20 Peptide in Ex vivo and In vivo Assays.
There are many ways of testing angiogenesis (15) , and we chose a chick embryo aortic arch assay for a preliminary assessment of the angiogenic properties of PAMP. Angiogenic factors such as adrenomedullin and VEGF induced a statistically significant increase of the number of sprouting blood vessels over untreated controls at concentrations of 100 nmol/L and higher (results not shown). PAMP induced comparable growth of newly formed blood vessels at concentrations as low as 1 nmol/L (Fig. 1D)Citation . At this concentration (1 nmol/L), neither adrenomedullin (Fig. 1B)Citation nor VEGF (Fig. 1C)Citation caused any significant proliferation over the control (Fig. 1A)Citation . The sprouting new vessels covered a surface of 2,579,500 ± 378,300 µm2 after treatment with PAMP, which is about 10 times the surface covered by the primitive capillaries formed in the untreated control (261,500 ± 60,800 µm2; P < 0.001). This initial observation suggests that PAMP may be a more potent proangiogenic factor than previously described molecules.



View larger version (108K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Comparative angiogenic potential of adrenomedullin, VEGF, and PAMP in the chick embryo aortic ring assay. Aortic rings were embedded in Matrigel and exposed to serum-free medium containing either PBS as a negative control (A) or 1 nmol/L of the peptides adrenomedullin (B), VEGF (C), or PAMP (D). Only in the case of PAMP is the crown of sprouting new vessels significantly larger than the control. Representative examples of four repeats. Bar = 0.5 mm.

 
To further characterize this intriguing observation, we used an in vivo assay to more precisely quantitate the angiogenic properties of PAMP. This approach is called directed in vivo angiogenesis assay (10 , 16) . In this assay, small silicone capsules carrying the test substances are implanted under the skin of nude mice. After 11 days, the mice received injections of a specific amount of FITC-dextran, and the volume of blood circulating through the implant is quantified by measuring the fluorescence in the capsule. In addition, the new blood vessels growing into the silicone tube can be seen directly by transparency (Fig. 2, A–F)Citation . PAMP elicited a measurable angiogenic response at concentrations as low as 1 fmol/L (Fig. 2C)Citation , which was clearly distinct from the negative control (Fig. 2A)Citation . The angiogenic response elicited by PAMP was dose-dependent (Fig. 2, C–G)Citation . When compared with adrenomedullin and VEGF responses at equimolar concentrations, a clearly significant difference was observed over a wide concentration range. In this animal model, adrenomedullin and VEGF induce angiogenesis at nanomolar concentrations, whereas PAMP was already active in the femtomolar range (Fig. 2G)Citation .



View larger version (52K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Angiogenic potential in vivo of adrenomedullin (AM), VEGF, and PAMP as compared by the directed in vivo angiogenesis assay. Silicone capsules containing different concentrations of the peptides in Matrigel were implanted under the skin of nude mice for 11 days. A to F, photographs of the angioreactors still attached to the skin at the end of the experiment. The new blood vessels can be seen growing from the tube opening. The capsules contain PBS as a negative control (A), 7 µmol/L bFGF as a positive control (B), 10–15 mol/L PAMP (C), 10–13 mol/L PAMP (D), 10–11 mol/L PAMP (E), and 10–9 mol/L PAMP (F). Bar = 2.0 mm. G. The Matrigel plugs were digested with dispase, and the FITC-dextran contents were measured to quantify the volume of blood circulating through the implants. Each bar represents the mean and SD of six independent values. Statistically significant differences between implants treated with PAMP and VEGF at the same concentration are represented by *. *, P < 0.05; **, 0.01 > P > 0.001; ***, P < 0.001.

 
Proadrenomedullin NH2-Terminal 20 Peptide Reduces Ca2+ Flux in Endothelial Cells.
Although the adrenomedullin receptor has been well characterized at the molecular level (21) , the structure of the PAMP receptor is not yet available; but it has been shown that exposure of adrenal medulla cells to PAMP results in a decrease of carbachol-induced Ca2+ influx (22) . To demonstrate whether a similar biological response takes place in endothelial cells, we stimulated them with 1 mmol/L ATP to induce a Ca2+ influx (Fig. 3Citation , squares) as reported previously (17) . This response was greatly reduced (>50%) by the presence of 10 nmol/L PAMP in the medium (Fig. 3Citation , diamonds). The peptide fragment PAMP(12-20) has been shown to have opposite actions to full-length PAMP (23) , suggesting its specific effect as a PAMP antagonist. To demonstrate the specificity of the PAMP-induced inhibition in Ca2+ influx, we added an excess of PAMP(12-20) and were able to recover the initial response (Fig. 3Citation , circles). Taken together, these data show that there is a functional PAMP receptor in the membrane of the endothelial cells, and therefore this peptide may activate directly the angiogenic response described above.



View larger version (14K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. PAMP reduces Ca2+ flux in human microvascular endothelial cells. Addition of 1 mmol/L ATP induces an increase in Ca2+ flux in endothelial cells ({square}), whereas addition of 10 nmol/L full-length PAMP greatly reduces this effect ({diamond}). This blocking effect was reversed by 100 nmol/L PAMP(12-20) ({circ}). Representative example of five repeats. R.F.U., relative fluorescence units.

 
Physiologic Effects of Proadrenomedullin NH2-Terminal 20 Peptide on Endothelial Cells.
For angiogenesis to occur, endothelial cells have to proliferate, migrate into new locations, and organize themselves into solid cords that eventually will develop into hollow tubes (2) . These processes are promoted by proangiogenic substances, and all proangiogenic molecules must elicit at least one of these physiologic actions. To investigate which of these phenomena are induced by PAMP, we exposed human dermal microvascular endothelial cells to increasing concentrations of PAMP, adrenomedullin, and VEGF and compared their effects on growth (Fig. 4A)Citation , migration (Fig. 4B)Citation , and cord formation (Fig. 4C)Citation .



View larger version (32K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Influence of adrenomedullin (AM), VEGF, and PAMP in the physiology of endothelial cells. A. Human microvascular endothelial cells were seeded in 96-well plates in the presence of increasing concentrations of the angiogenic peptides in serum-free medium. After 3 days in culture, the number of viable cells was estimated by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and expressed as percent growth over untreated controls. Each point represents the mean and SD of eight independent measurements. B. The same cells were seeded in the top chamber of a ChemoTx microplate coated with fibronectin in which different concentrations of the peptides had been placed in the bottom chambers. Each point represents the mean and SD of four independent measurements. C. Endothelial cells were seeded over a layer of solidified Matrigel in the presence of different concentrations of angiogenic peptides. The complexity of the cord network was estimated by the number of knots per microscopic field. Each bar represents the mean and SD of three independent wells. *, statistically significant differences with the untreated cells. *, P < 0.05; **, 0.01 > P > 0.001; ***, P < 0.001. D. The same cells were exposed to 10 nmol/L PAMP overnight, and their contents in mRNA of angiogenic molecules were measured by real-time PCR. Each bar represents the ratio between the gene of interest in the treated cells and the contents in the untreated cells. The horizontal line indicates no change over untreated conditions.

 
Endothelial cell growth analysis showed that adrenomedullin and VEGF significantly increase proliferation over the control at a concentration of 10–8 mol/L (P < 0.001 for both). However, PAMP did not significantly enhance cell growth at the concentrations tested (Fig. 4A)Citation .

We tested the migratory potential of endothelial cells over a range of peptide concentrations. Adrenomedullin did not modify cell migration significantly on the concentration range tested, whereas both VEGF and PAMP produced a 4-fold increase in migration at a concentration of 10–11 mol/L when compared with untreated controls (P < 0.001 for both). As previously reported for VEGF (24) , both VEGF and PAMP showed a peak of migration stimulation at 10–11 mol/L. The shape of the peak was different for both peptides: more steep for PAMP and more gradual for VEGF (Fig. 4B)Citation .

We also tested the ability of adrenomedullin, VEGF, and PAMP to induce cord formation in a Matrigel assay. All three factors were able to induce statistically higher cord formation in a dose-dependent manner when compared with untreated controls. VEGF was the most efficient mediator, followed by adrenomedullin, whereas PAMP had a modest effect on cord formation (Fig. 4C)Citation .

We also studied the effects of exogenously added PAMP on the gene expression for other proangiogenic molecules (Fig. 4D)Citation . Real-time PCR experiments showed that PAMP modestly induces the expression of its own gene (adrenomedullin/PAMP), about 50% over basal levels. PAMP was also capable of elevating the expression of VEGF, bFGF, and PDGF C by 80, 300, and 300%, respectively. Conversely, no significant modification in the expression of PDGF A or PDGF B was observed (Fig. 4D)Citation . The addition of 10 nmol/L adrenomedullin or VEGF to endothelial cells had no effect on the expression of the adrenomedullin/PAMP gene (results not shown).

A Proadrenomedullin NH2-Terminal 20 Peptide Antagonist Inhibits Angiogenesis In vivo.
To further evaluate the role of PAMP in angiogenesis, we evaluated its influence in tumor growth, using PAMP(12-20) as a PAMP-receptor antagonist. First, we studied the competition between synthetic full-length PAMP at 1 nmol/L concentration and increasing doses of PAMP(12-20) in the directed in vivo angiogenesis assay. We observed a dose-dependent inhibition of the angiogenic response elicited by PAMP (Fig. 5A)Citation . A 100-fold excess of the peptide fragment (100 nmol/L) inhibited angiogenesis to the basal levels (P > 0.05, comparing 100 nmol/L of the peptide fragment with untreated control). The peptide fragment by itself did not modify basal angiogenesis (last bar in Fig. 5ACitation ).



View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Characterization of PAMP(12-20) as an inhibitor of PAMP-induced angiogenesis. A. Different concentrations of full-length PAMP and PAMP(12-20) were added to angioreactors and inserted under the skin of nude mice following the directed in vivo angiogenesis assay protocol as indicated. Each bar represents the mean and SD of six independent measurements. Statistical differences with PAMP alone (first bar) are represented as * (P < 0.05) or ** (P < 0.01). B. PAMP(12-20) was also able to inhibit the angiogenesis induced by two human lung cancer tumor cells embedded in the Matrigel capsules. PAMP(12-20) was added at 100 nmol/L. Each bar represents the mean and SD of six independent measurements. Statistical differences with untreated cells are represented as * (P < 0.05).

 
Because tumor cells produce many angiogenic factors (25) , we evaluated the contribution of PAMP to the total angiogenic response. Two human non-small cell lung cancer cell lines (A549 and H1299) were embedded in Matrigel and placed in the directed in vivo angiogenesis assay. Both cell lines induced a significant angiogenic response (Fig. 5B)Citation that was completely blocked by 100 nmol/L PAMP(12-20) (P > 0.05, comparing tumor cells plus PAMP(12-20) with Matrigel only control), indicating that PAMP signaling is somehow necessary for initiating angiogenesis.

For further evaluation of in vivo effects of PAMP, we designed a xenograft experiment. The human lung cancer cell line A549 was injected under the skin of 30 athymic nude mice, and after 2 weeks, all animals developed palpable tumor masses at the injection site. These mice were divided into three groups, and each set received a different treatment three times a week. The control group was treated with the vehicle (PBS), and the tumor mass kept increasing until the mice had to be sacrificed 18 days after treatment began (Fig. 6Citation , squares). The group of animals whose tumors received injections of 10 nmol/L PAMP(12-20) did not show any significant difference with the PBS-treated group (results not shown). In contrast, the mice that received 1 µmol/L PAMP(12-20) showed a slower rate of tumor growth (Fig. 6Citation , diamonds). Statistical differences in tumor size between the groups were observed after 9 days of treatment and continued for the rest of the experiment.



View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Antitumoral effect of PAMP(12-20) in a xenograft model. Athymic nude mice carrying A549 tumors were treated with either PBS ({square}) or 1 µmol/L PAMP(12-20) ({diamond}), three times a week. Each point represents the mean and SD of 10 mice. *, statistical differences of tumor volume between treatments. *, P < 0.05; **, P < 0.01.

 
We also investigated whether injection of full-length PAMP had any impact in xenograft tumor growth. At the concentrations tested (1 and 100 nmol/L PAMP), no difference was observed between tumors treated with peptide or vehicle (results not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we have demonstrated that the adrenomedullin gene-related peptide PAMP is a potent angiogenic factor that is active at concentrations 6 orders of magnitude lower than previously recognized angiogenic molecules such as adrenomedullin and VEGF. In addition, we have shown that a receptor for PAMP is present in human endothelial cells and that these cells react to the presence of PAMP by increasing their migration and cord formation potential, at the same time that expression for other angiogenic factors is boosted. We also found that the peptide fragment PAMP(12-20) acts as an angiogenesis antagonist for either neovascularization induced by synthetic PAMP or by tumor cells and reduces tumor growth in a xenograft model.

Tissue Regulation of Proadrenomedullin NH2-Terminal 20 Peptide Levels.
An obvious question raised by our observations is how a peptide with such extraordinary angiogenic effects can be produced normally without causing an excessive amount of blood vessel formation. The expression of the adrenomedullin/PAMP gene during development is tightly regulated in a spatio-temporal manner (26) , underscoring the need for a controlled liberation of these molecules. In addition, PAMP is rapidly degraded by neutral endopeptidases (27 , 28) , resulting in extremely low levels of circulating PAMP (29) . The silicone implant used in the directed in vivo angiogenesis assay may provide a secluded environment protecting PAMP from protease degradation, thus contributing to the angiogenic response observed at very low concentrations of PAMP. These data suggest a scenario in which PAMP will be secreted at a low concentration in the proximity of the target cells (endothelial cells), and after effecting its paracrine action, it will be rapidly cleaved by neutral endopeptidases to avoid excessive proliferation of blood vessels. In a way, this biology will not be too different from the mechanisms exhibited by other potent biological substances that exert their function locally, such as neurotransmitters.

The rapid and efficient cleavage of PAMP by resident endopeptidases may also explain the lack of effect observed in the xenograft experiment when we added full-length peptide. Alternatively, we can speculate that the tumor cells may be producing enough PAMP to saturate the receptors present in the endothelial cell surface, thus any additional peptide would not modify the angiogenic response. Similar results have been reported for adrenomedullin in breast cancer cells (30) .

There are many substances that regulate the expression of the adrenomedullin/PAMP gene, including cytokines, other hormones, growth factors, interferons, lipopolysaccharide, etc. (8) . Hypoxia is a common inducer of angiogenesis in tumors (31) , and the adrenomedullin/PAMP gene is one of the many genes that is induced by low oxygen tension through a mechanism involving the transcription factor HIF-1 (32) . Under hypoxic conditions, the alternative splicing mechanism of the gene favors the production of the longer mRNA species that produces PAMP but not adrenomedullin, thus increasing the relative amount of PAMP that is secreted from the cell (27) . This regulatory mechanism is consistent with the angiogenic activity that we have described for PAMP.

How Many Angiogenic Factors Are Necessary to Induce Neovascularization?
Although tumor cells and their environment produce more than one angiogenic factor at a time, it is surprising that the inhibition of a single factor can result in complete abrogation of angiogenesis (33 , 34) . Angiogenesis may be composed of an interdependent chain of events that when one of those individual steps is blocked, the whole process has to stop. The profound effects of PAMP(12-20) on human lung tumor cells in the directed in vivo angiogenesis assay and the xenograft experiments is consistent with this proposed hypothesis. Our finding that PAMP induces the expression of several proangiogenic genes in endothelial cells is also in agreement with that hypothesis. This would suggest that PAMP has both a direct effect on endothelial cells by binding to its receptor and lowering Ca2+ flux and an indirect effect by inducing production of other angiogenic promoters.

In summary, our experimental findings have revealed PAMP to be an extremely potent angiogenic factor with activity several orders of magnitude higher than the previously established "gold standard," VEGF. We have shown that the peptide fragment PAMP(12-20) functions as an antagonist that can suppress in vivo angiogenesis induced by the intact ligand or by tumor cell–derived peptide. Finally, PAMP(12-20) treatment partially blocked the in vivo growth of human tumors as assessed in a nude mouse xenograft model. In light of significant recent clinical benefits of antiangiogenic therapies based on VEGF blockade (7) , PAMP antagonists provide a conceptually attractive tool to further explore this strategy.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the expert technical assistance of Vickie Keck with the xenograft studies.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Alfredo Martínez, Cell and Cancer Biology Branch, National Cancer Institute, NIH, Building 10, Room 13N262, Bethesda, MD 20892. Phone: 301-402-3308; Fax: 301-435-8036; E-mail: martinea{at}mail.nih.gov

Received 1/13/04. Revised 6/28/04. Accepted 7/12/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Carmeliet P. Angiogenesis in health and disease. Nat Med, 2003;9:653-60, [CrossRef][Medline]
  2. Distler J, Hirth A, Kurowska-Stolarska M, Gay RE, Gay S, Distler O. Angiogenic and angiostatic factors in the molecular control of angiogenesis. Q J Nucl Med, 2003;47:149-61, [Medline]
  3. Shannon AM, Bouchier-Hayes DJ, Condron CM, Toomey D. Tumor hypoxia, chemotherapeutic resistance and hypoxia-related therapies. Cancer Treat Rev, 2003;29:297-307, [CrossRef][Medline]
  4. Folkman J, Watson K, Ingber D, Hanahan D. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature, 1989;339:58-61, [CrossRef][Medline]
  5. Cavallaro U, Christofori G. Molecular mechanisms of tumor angiogenesis and tumor progression. J Neurooncol, 2000;50:63-70, [CrossRef][Medline]
  6. Scappaticci FA. The therapeutic potential of novel antiangiogenic therapies. Expert Opin Investig Drugs, 2003;12:923-32, [CrossRef][Medline]
  7. Yang JC, Haworth L, Sherry RM, et al A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med, 2003;349:427-34, [Abstract/Free Full Text]
  8. López J, Martínez A. Cell and molecular biology of the multifunctional peptide, adrenomedullin. Int Rev Cytol, 2002;221:1-92, [Medline]
  9. Zhao Y, Hague S, Manek S, Zhang L, Bicknell R, Rees MC. PCR display identifies tamoxifen induction of the novel angiogenic factor adrenomedullin by a non estrogenic mechanism in the human endometrium. Oncogene, 1998;16:409-15, [CrossRef][Medline]
  10. Martínez A, Vos M, Guédez L, et al The effects of adrenomedullin overexpression in breast tumor cells. J Natl Cancer Inst (Bethesda), 2002;94:1226-37, [Abstract/Free Full Text]
  11. Oehler MK, Hague S, Rees MC, Bicknell R. Adrenomedullin promotes formation of xenografted endometrial tumors by stimulation of autocrine growth and angiogenesis. Oncogene, 2002;21:2815-21, [CrossRef][Medline]
  12. Ishikawa T, Chen J, Wang J, et al Adrenomedullin antagonist suppresses in vivo growth of human pancreatic cancer cells in SCID mice by suppressing angiogenesis. Oncogene, 2003;22:1238-42, [CrossRef][Medline]
  13. Cuttitta F, Pío R, Garayoa M, et al Adrenomedullin functions as an important tumor survival factor in human carcinogenesis. Microsc Res Tech, 2002;57:110-9, [CrossRef][Medline]
  14. Isaacs JS, Jung Y, Mimnaugh EG, Martínez A, Cuttitta F, Neckers LM. Hsp90 regulates a VHL-independent HIF-1{alpha} degradative pathway. J Biol Chem, 2002;277:29936-44, [Abstract/Free Full Text]
  15. Auerbach R, Lewis R, Shinners B, Kubai L, Akhtar N. Angiogenesis assays: a critical overview. Clin Chem, 2003;49:32-40, [Abstract/Free Full Text]
  16. Guédez L, Rivera AM, Salloum R, et al Quantitative assessment of angiogenic responses by the directed in vivo angiogenesis assay. Am J Pathol, 2003;162:1431-9, [Abstract/Free Full Text]
  17. Lau KL, Kong SK, Ko WH, Kwan HY, Huang Y, Yao X. cGMP stimulates endoplasmic reticulum Ca(2+)-ATPase in vascular endothelial cells. Life Sci, 2003;73:2019-28, [CrossRef][Medline]
  18. Macpherson GR, Ng SS, Forbes SL, et al Anti-angiogenic activity of human endostatin is HIF-1-independent in vitro and sensitive to timing of treatment in a human saphenous vein assay. Mol Cancer Ther, 2003;2:845-54, [Abstract/Free Full Text]
  19. Martínez A, Saldise L, Ramírez MJ, et al Adrenomedullin expression and function in the rat carotid body. J Endocrinol, 2003;176:95-102, [Abstract]
  20. Ouafik L, Sauze S, Boudouresque F, et al Neutralization of adrenomedullin inhibits the growth of human glioblastoma cell lines in vitro and suppresses tumor xenograft growth in vivo. Am J Pathol, 2002;160:1279-92, [Abstract/Free Full Text]
  21. McLatchie LM, Fraser NJ, Main MJ, et al RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature, 1998;393:333-9, [CrossRef][Medline]
  22. Katoh F, Kitamura K, Niina H, et al Proadrenomedullin N-terminal 20 peptide (PAMP), an endogenous anticholinergic peptide: its exocytotic secretion and inhibition of catecholamine secretion in adrenal medulla. J Neurochem, 1995;64:459-61, [Medline]
  23. Belloni AS, Rossi GP, Andreis PG, et al Proadrenomedullin N-terminal 20 peptide (PAMP), acting through PAMP(12-20)-sensitive receptors, inhibits Ca2+-dependent, agonist-stimulated secretion of human adrenal glands. Hypertension, 1999;33:1185-9, [Abstract/Free Full Text]
  24. Yahata Y, Shirakata Y, Tokumaru S, et al Nuclear translocation of phosphorylated STAT3 is essential for VEGF-induced human dermal microvascular endothelial cell migration and tube formation. J Biol Chem, 2003;278:40026-31, [Abstract/Free Full Text]
  25. Chlenski A, Liu S, Cohn SL. The regulation of angiogenesis in neuroblastoma. Cancer Lett, 2003;197:47-52, [CrossRef][Medline]
  26. Montuenga LM, Martínez A, Miller MJ, Unsworth EJ, Cuttitta F. Expression of adrenomedullin and its receptor during embryogenesis suggests autocrine or paracrine modes of action. Endocrinology, 1997;138:440-51, [Abstract/Free Full Text]
  27. Martínez A, Hodge DL, Garayoa M, Young HA, Cuttitta F. Alternative splicing of the proadrenomedullin gene results in differential expression of gene products. J Mol Endocrinol, 2001;27:31-41, [Abstract]
  28. Nagatomo Y, Kitamura K, Kangawa K, Fujimoto Y, Eto T. Proadrenomedullin N-terminal 20 peptide is rapidly cleaved by neutral endopeptidase. Biochem Biophys Res Commun, 1996;223:539-43, [CrossRef][Medline]
  29. Washimine H, Kitamura K, Ichiki Y, et al Immunoreactive proadrenomedullin N-terminal 20 peptide in human tissue, plasma and urine. Biochem Biophys Res Commun, 1994;202:1081-7, [CrossRef][Medline]
  30. Miller MJ, Martínez A, Unsworth EJ, et al Adrenomedullin expression in human tumor cell lines: its potential role as an autocrine growth factor. J Biol Chem, 1996;271:23345-51, [Abstract/Free Full Text]
  31. Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med, 2003;9:677-84, [CrossRef][Medline]
  32. Garayoa M, Martínez A, Lee S, et al Hypoxia-inducible factor-1 (HIF-1) up-regulates adrenomedullin expression in human tumor cell lines during oxygen deprivation: a possible promotion mechanism of carcinogenesis. Mol Endocrinol, 2000;14:848-62, [Abstract/Free Full Text]
  33. Giavazzi R, Sennino B, Coltrini D, et al Distinct role of fibroblast growth factor-2 and vascular endothelial growth factor on tumor growth and angiogenesis. Am J Pathol, 2003;162:1913-26, [Abstract/Free Full Text]
  34. Takano S, Tsuboi K, Matsumura A, Nose T. Anti-vascular endothelial growth factor antibody and nimustine as combined therapy: effects on tumour growth and angiogenesis in human glioblastoma xenografts. Neuro-oncol, 2003;5:1-7, [Abstract]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
D. L. Sackett, L. Ozbun, E. Zudaire, L. Wessner, J. M. Chirgwin, F. Cuttitta, and A. Martinez
Intracellular Proadrenomedullin-Derived Peptides Decorate the Microtubules and Contribute to Cytoskeleton Function
Endocrinology, June 1, 2008; 149(6): 2888 - 2898.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
D. Ribatti, M. T. Conconi, and G. G. Nussdorfer
Nonclassic Endogenous Novel Regulators of Angiogenesis
Pharmacol. Rev., June 1, 2007; 59(2): 185 - 205.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
A. Martinez, J. A. Bengoechea, and F. Cuttitta
Molecular Evolution of Proadrenomedullin N-Terminal 20 Peptide (PAMP): Evidence for Gene Co-Option
Endocrinology, July 1, 2006; 147(7): 3457 - 3461.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
L. Notari, A. Miller, A. Martinez, J. Amaral, M. Ju, G. Robinson, L. E. H. Smith, and S. P. Becerra
Pigment Epithelium-Derived Factor Is a Substrate for Matrix Metalloproteinase Type 2 and Type 9: Implications for Downregulation in Hypoxia
Invest. Ophthalmol. Vis. Sci., August 1, 2005; 46(8): 2736 - 2747.
[Abstract] [Full Text] [PDF]

Home page
 Neuro Oncol DukeHome page
B. Kaur, F. W. Khwaja, E. A. Severson, S. L. Matheny, D. J. Brat, and E. G. Van Meir
Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis
Neuro-oncol, April 1, 2005; 7(2): 134 - 153.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martínez, A.
Right arrow Articles by Cuttitta, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martínez, A.
Right arrow Articles by Cuttitta, F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online