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Adrenomedullin as an Autocrine/Paracrine Apoptosis Survival Factor for Rat Endothelial Cells¹

Endocrine-Hypertension Division, Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo 113, Japan

Address all correspondence and requests for reprints to: Masayoshi Shichiri, M.D., Second Department of Internal Medicine, Tokyo Medical and Dental University, Yushima 1–5-45, Bunkyo-ku, Tokyo 113, Japan. (Masayoshi Shichiri). E-mail address: mshichiri.med2{at}med.tmd.ac.jp

	Abstract

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Adrenomedullin is a potent vasorelaxant/hypotensive peptide recently isolated from human pheochromocytoma. We demonstrate here a novel role of this peptide as an apoptosis survival factor for rat endothelial cells. When rendered quiescent by serum deprivation, a fraction of endothelial cell cultures showed morphological and biochemical features characteristic of apoptosis. Adrenomedullin significantly suppressed apoptosis without inducing cell proliferation. Rat endothelial cells that contained high affinity binding sites for adrenomedullin expressed adrenomedullin gene and released the peptide into culture media. Addition of preimmune rabbit serum prevented apoptosis, whereas rabbit antiadrenomedullin antiserum partially, but significantly, abrogated the protective effect of the preimmune serum, suggesting its autocrine/paracrine role. Although adrenomedullin induced intracellular cAMP formation, other cAMP-elevating agonists, such as prostaglandin I₂ and forskolin, did not affect apoptosis. Furthermore, adenosine 3',5'-cyclicmonophosphothioate Rp-isomer, a cAMP antagonist, did not block the cell survival effect of adrenomedullin. Adrenomedullin neither increased intracellular Ca²⁺ concentrations nor inositol-1,4,5-trisphosphate levels in rat endothelial cells. These results demonstrate that adrenomedullin suppresses serum deprivation-induced apoptosis of rat endothelial cells via cAMP-independent mechanism.

	Introduction

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APOPTOSIS, a genetically controlled response to eliminate unwanted cells, is involved in the regulation of cell number in several physiological and pathological conditions. Apoptosis is associated with distinctive morphological and biological events, such as cellular shrinkage, membrane blebbing, and chromatin condensation and fragmentation (1). Diverse stimuli, such as serum deprivation, radiation, chemotherapeutic agents, and antioxidants, induce apoptosis in many cell types (2, 3, 4, 5), whereas growth factors and cytokines are known to modulate apoptosis triggered by such environmental signals (6, 7, 8, 9, 10, 11). Several gene products have been characterized to activate or suppress genetic program of apoptosis (12). Although endothelial cells have been demonstrated to undergo apoptosis (13, 14, 15), its regulation in normal cellular physiology as well as in pathophysiological conditions remains largely unknown.

Adrenomedullin, a potent vasorelaxant/hypotensive peptide with 52 amino acid residues, was recently isolated from human pheochromocytoma by monitoring its activity to induce cAMP formation in platelets (16). Adrenomedullin has a conserved structure among mammals such as rat (17) and shows a partial homology with calcitonin gene-related peptide (16). Intravenous bolus injection of adrenomedullin causes a potent and long-lasting hypotensive effect in anesthetized rats in vivo (18). Recently, it has been reported that adrenomedullin messenger RNA (mRNA) is expressed not only in adrenal glands, but in a variety of tissues, including vascular smooth muscle cells (19) and endothelial cells (20) of various species. Furthermore, it has been shown that adrenomedullin receptor is functionally coupled to adenylate cyclase in vascular smooth muscle cells (21). A recent preliminary report suggests that adrenomedullin suppresses mitogenesis in rat mesangial cells (22). Although the above results suggest an autocrine/paracrine role for adrenomedullin not only to regulate vascular tonus, but also cell growth, its role as an apoptosis modulator has not been described to date.

Here we have studied the effect of adrenomedullin on apoptosis in cultured rat endothelial cells. Our data indicate that adrenomedullin acts on endothelial cells to protect them from apoptotic death in an autocrine/paracrine manner, and that the antiapoptotic action appears to be mediated by mechanism(s) not involving adenylate cyclase.

	Materials and Methods

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Cell culture
Rat endothelial cells were prepared from 15-week-old male Wistar rat aorta by collagenase and elastase digestion, as described (23). The endothelial origin of the cultures was confirmed by the presence of Factor VIII by immunohistochemical detection. All cells (8–13th passages) were cultured in DMEM in a 5% CO₂ atmosphere at 37 C, supplemented with 10% FBS.

Reagents
Synthetic rat adrenomedullin was purchased from Peptide Institute, Inc. (Osaka, Japan), DMEM from Life Technologies (Grand Island, NY), FBS from Hyclone Laboratories (Logan, UT), forskolin and prostaglandin I₂ sodium salt (PGI₂) from Wako Pure Chemical Industries (Osaka, Japan), and adenosine-3':5'-monophosphothionate, adenosine 3',5'-cyclicmonophosphothioate Rp-isomer (Rp-cAMPS) from Boehringer Mannheim Biochemicals (Bremen, Germany). All other reagents were of analytical grade.

Quantitative determination of apoptosis
Rat endothelial cells were plated in 24-well dishes in serum-containing medium at a density of 1.5 x 10⁴ cells per well and incubated for 24 h. The wells were extensively washed with PBS, and the medium was changed to serum-free DMEM containing adrenomedullin, PGI₂, or forskolin as indicated in the text. Control cultures received serum-free DMEM with no additives. After 24 h, all floating cells were collected with two PBS washes. The number and size distribution of the floating cells were determined with a Coulter Counter model ZM (Coulter Electronics, Hialeah). To demonstrate nucleosome laddering, apoptotic DNA fragments were extracted with NP-40 lysis method that eliminates intact chromatin (24), and fractionated on 1.6% agarose gels.

Immunohistochemical staining using IgG specific for single-stranded DNA
Rat endothelial cells were cultured, serum-deprived for 8 h, and stained with polyclonal antibody raised against single-stranded DNA (25, 26) and/or with hematoxylin. Immunohistochemical staining was carried out using an avidin-biotin-peroxidase (ABC) kit (Vector Laboratories, Burlingame, CA). In brief, cells grown on coverslips were washed with PBS and then fixed for 15 min in ice-cold 70% acetone. After fixation, the cells were sequentially exposed to normal goat serum (1:60 dilution) for 20 min, IgG specific for single-stranded DNA (dilution 1:500) for 16 h, and biotinylated goat antirabbit IgG (dilution 1:400) for 30 min. The cells were then incubated for 60 min in ABC solution prepared by adding avidin DH and biotinylated peroxidase to dilution buffer. Peroxidase activity was visualized by incubation with 3–3'-diaminobenzidine tetrahydrochloride and 0.03% H₂O₂ in 0.1 M Tris-HCl (pH 7.6). The stained specimens were dehydrated with ethanol and xylene and mounted under glass coverslips with HSR mounting solution.

Iodination of rat adrenomedullin
Radiolabeled rat adrenomedullin was prepared by the lactoperoxidase method (21) and purified by a reverse-phase HPLC using an octadecylsilica column (0.46 x 25 cm, Tosoh, Tokyo, Japan) eluted with a linear gradient (10–60%) of acetonitrile in 0.1% trifluoroacetic acid (TFA) for 1 h (flow rate: 1 ml/min). Monoiodinated [¹²⁵I]-rat adrenomedullin (specific activity: 7.4 x 10⁷ MBq/mmol) was used for RIA and the binding experiments.

RIA of adrenomedullin
Rat adrenomedullin-like immunoreactivity (LI) was determined by the double antibody RIA, essentially in the same manner as recently reported for human adrenomedullin (27). Culture media were acidified with 0.1% TFA, centrifuged, and the supernatant applied to the preactivated Sep Pak C₁₈ cartridge (Waters Associates, MA), which was then eluted with 70% acetonitrile/0.1% TFA. The eluates were evaporated, reconstituted, and subjected to RIA. The antibody used recognizes the C-terminal region (22–50) of rat adrenomedullin and does not cross-react with other polypeptide hormones; the final dilution of antiserum was 1:6000. The sensitivity was 10 fmol/tube and 50% intercept was 60 fmol/tube. The intra- and interassay variations were less than 10%.

Binding experiments
Confluent cells (10⁶ cells/well) were washed with PBS and incubated with [¹²⁵I]-rat adrenomedullin (5.9 x 10^-15 mol) for 4 h at 4 C in 0.5 ml DMEM in the absence and presence of unlabeled rat adrenomedullin, as reported (21). After completion, cells were extensively washed with HBSS, solubilized in 0.5 N NaOH, and the cell-bound radioactivity was determined. Specific binding was obtained by subtracting nonspecific binding in the presence of excess (10^-6 M) unlabeled rat adrenomedullin from total binding.

Northern hybridization analysis
RNA was extracted from rat endothelial cells by the guanidinium thiocyanate method, as described (28). Total RNA (20 µg per lane) was electrophoresed on formaldehyde-agarose gels. Blotting was onto MagnaGraph nylon membranes (Micron Separations, Inc.). Complementary DNA (cDNA) probes for rat adrenomedullin gene were labeled with -[³²P]-dCTP using the random-priming method. After UV cross-linking, membranes were hybridized at 42 C in the presence of 50% formamide. Washing was with 0.1 x SSPE and 0.5% SDS at 37 C for 15 min, and signals were quantitated using a BAS2000 Imaging Analyzer (Fuji Photo Film, Inc.).

Determination of DNA synthesis
Subconfluent cells in 24-well plates (10⁶ cells per well) were serum-deprived for 24 h, incubated with or without rat adrenomedullin for 24 h, and further incubated for 4 h with 0.5 µCi of [³H]-thymidine (Amersham International, Inc., Tokyo, Japan). At the end of the labeling period, cultures were rinsed twice with ice-cold PBS and incubated with 5% trichloroacetic acid on ice for 20 min. After washing twice with ice-cold 5% trichloroacetic acid, the cells were solubilized in 0.5 N NaOH, and the radioactivity was determined with a liquid scintillation counter.

Determination of growth rates
Subconfluent cultures were seeded at an approximate density of 500 cells/cm² in 24-well plates, grown in 10% FBS for 24 h, replaced with DMEM containing 0.25% FBS for 24 h, and further incubated with and without rat adrenomedullin. Starvation with 0.25% FBS for 24 h minimized apoptotic death: less than 1/10 cell death compared with that of serum starvation. All adherent cells were trypsinized and cell number was counted.

Determination of cAMP
Confluent cells were incubated at 37 C for 10 min in 0.2 ml DMEM containing 0.5 mM IBMX in the absence and presence of rat adrenomedullin, PGI₂ and forskolin as reported (21). Incubation was terminated by the addition of HCl, and intracellular cAMP was measured by a RIA kit (Yamasa, Chiba, Japan).

Measurement of inositol 1,4,5-trisphosphate (Ins-1, 4, 5-P₃)
Confluent cells were incubated with or without 10^-6 M rat adrenomedullin in 2 ml HBSS, pH 7.4, containing 10 mM LiCl at 37 C for 30 sec as reported (23). Incubation was terminated by the addition of trichloroacetic acid, and Ins-1,4,5-P₃ was measured using a protein binding assay kit (Amersham International).

Measurements of intracellular free calcium concentration, [Ca²⁺]_i
Confluent cultures that had been serum-deprived for 24 h were dispersed with 0.05% trypsin and 0.02% EDTA, and incubated with 5 µM fura-2 acetoxymethylester (Dojin Chemical Inc., Kumamoto, Japan) at 37 C for 20 min in HBSS. Fluorescence of fura-2 loaded suspended cells (5 x 10⁶ cells/ml) was measured at 37 C using continuous rapid alternating excitation from dual monochromators (340 and 380 nm), and emission at 505 nm (CAF-100, Japan Spectroscopic Co. Ltd., Tokyo, Japan) as reported (28).

	Results

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Suppression of apoptosis by adrenomedullin
Cultured human endothelial cells have been demonstrated to undergo apoptosis when detached from culture plates (29). We confirmed that rat endothelial cells underwent apoptosis upon deprivation of growth factors. When medium was deprived of serum, rat endothelial cell cultures always contained a fraction of floating cells; almost all floating and many adherent endothelial cells showed morphological features characteristic of apoptosis, such as membrane blebbing, cellular shrinkage, nuclear condensation, and fragmentation. Electrophoresis of DNA samples from total culture obtained with NP-40 lysis method showed a nucleosomal ladder (Fig. 1A, left panel). Many adherent cultured cells also showed nuclear and cellular fragments when deprived of serum. When the cells were stained with antibody against single-stranded DNA and with hematoxylin, immunohistochemically positive cells coincided very well with those showing cellular and nuclear fragments (Fig. 1B, upper panel). Addition of adrenomedullin (10^-8 M) markedly suppressed apoptotic cell death of both total culture (Fig. 1A, right panel) and of adherent culture (Fig. 1B, lower panel). The onset of apoptosis was rapid and significant number of cells (approximately 4%) started to float as early as 1 h after serum-deprivation, which increased markedly (40%) after 24 h: the addition of adrenomedullin (10^-7 M) prevented the apoptosis even 1 h after serum deprivation, which persisted during 24 h (Fig. 2A). Adrenomedullin dose dependently (10^-10–10^-6 M) suppressed rat endothelial cell apoptosis induced after 24-h serum deprivation (Fig. 2B); the minimum inhibitory effect was induced with 10^-10 M and 50% inhibition was induced with 10^-6 M.

View larger version (68K): [in this window] [in a new window]   Figure 1. Inhibition of serum-deprivation-induced apoptosis by adrenomedullin in endothelial cells. A, Demonstration of nucleosomal ladders from serum-starved endothelial cells and its suppression by 10-8 M adrenomedullin. Confluent culture of rat endothelial cells in 10 cm dishes were replaced with serum-free DMEM, incubated in the absence (left lane) and presence (right lane) of 10-8 M adrenomedullin for 4 h, and all floating and adherent cells were collected. Fragmented DNA was extracted using NP-40 lysis method and was separated by electrophoresis in 1.6% agarose gel. B, Immunohistochemical detection of apoptotic endothelial cells. Endothelial cells, deprived of serum and incubated for 8 h with or without adrenomedullin, were stained simultanously with antibody against single-stranded DNA and with hematoxylin. The number of adherent live cells with homogenous, lightly stained nucleus reduced after serum-deprivation, and immunohistochemically positive cells showed apoptotic bodies and fragmented nuclei (upper panel). Addition of adrenomedullin (10-8 M) increased the number of adherent intact cells and reduced apoptosis (lower panel).

View larger version (20K): [in this window] [in a new window]   Figure 2. Adrenomedullin suppresses apoptosis in endothelial cells. A, Time course of endothelial cell death after serum deprivation in the absence () and presence (•) of adrenomedullin (10-7 M). B, Dose-response effect of adrenomedullin on rat endothelial cell death. Exponentially growing cells were changed into serum-free medium, and all floating cells after 24 h were counted with a Coulter Counter. Each point and bar represents mean ± SEM (n = 6); values were normalized to the total number of floating dead cells 24 h after serum deprivation in the absence of adrenomedullin, which was set at 100%. *, P < 0.05, **, P < 0.01, treated cells vs. nontreated cells.

View larger version (16K): [in this window] [in a new window]   Figure 3. Adrenomedullin gene and its receptors are expressed by rat endothelial cells. A, Northern blot analysis of adrenomedullin mRNA. Total cellular RNA (20 µg) prepared from the cells was subjected to Northern hybridization with rat adrenomedullin cDNA as a probe. B, Competitive binding study of [125I]-rat adrenomedullin. Confluent cells (106 cells/well) were incubated at 4 C for 4 h with [125I]-rat adrenomedullin in the absence or presence of indicated concentrations of unlabeled peptide. Results are expressed as the percentage to the total specific bindings; each point is the mean of duplicate experiments. Inset, Scatchard plot of binding data.

Cell proliferation
We examined whether adrenomedullin has any effect on endothelial cell proliferation. Adrenomedullin (10^-11-10^-6 M) neither induced cell proliferation, nor stimulated [³H]-thymidine incorporation of rat endothelial cells (data not shown).

Effect of antiadrenomedullin antibody on apoptosis
Because rat endothelial cells synthesize and secrete adrenomedullin into culture media, and express adrenomedullin receptors, we reasoned that the cells could influence their own apoptotic death in an autocrine/paracrine fashion. To address this question, the effect of polyclonal antiadrenomedullin antibody on apoptosis was examined (Fig. 4). After 24-h serum deprivation of growing rat endothelial cells, approximately 40% of cells underwent apoptosis. Addition of nonimmune rabbit serum to serum-deprived rat endothelial cells suppressed apoptosis in a concentration-dependent manner: serum at dilution of 1:100 (1%) markedly (70%) prevented apoptosis. In contrast, addition of rabbit antiadrenomedullin antiserum at the same dilution partially but significantly (P < 0.01) abrogated the apoptosis protective effect by the normal serum.

View larger version (22K): [in this window] [in a new window]   Figure 4. Effects of antiadrenomedullin antiserum and preimmune serum on apoptosis of endothelial cells. Apoptosis assays with exponentially growing rat endothelial cell cultures were performed as described in Fig. 2. During the 24-h serum deprivation period, cells were exposed to either rabbit nonimmune serum () or antiadrenomedullin antiserum () at the indicated dilutions. Each column with bar represents mean ± SEM (n = 6); values were normalized to the total number of floating dead cells in the absence of adrenomedullin, which was set at 100%. **, P < 0.01, nonimmune serum vs. adrenomedullin antiserum values.

View larger version (32K): [in this window] [in a new window]   Figure 5. Comparison of intracellular cAMP increase and apoptosis protective responses by adrenomedullin and other cAMP-elevating agonists. A, Confluent cells (105 cells/well) were incubated at 37 C for 10 min with rat adrenomedullin (•), PGI2 (), or forskolin () at the indicated concentrations and intracellular cAMP concentrations were measured. Each point represents the mean of six wells; bars show SEM. B, Cells were treated with or without adrenomedullin (10-6 M), adrenomedullin plus Rp-cAMPS (10-3 M), PGI2 (10-6 M), and forskolin (10-5 M) after 24-h serum deprivation: the number of floating apoptotic cells were counted as described in Fig. 2. Each column with bar represents mean ± SEM (n = 6); values were normalized to the total number of floating dead cells in the absence of adrenomedullin, which was set at 100%. *, P < 0.05, treated cells vs. nontreated cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We demonstrate here that a novel vasoactive peptide, adrenomedullin, markedly inhibited serum-deprivation-induced apoptosis of rat endothelial cells. Apoptosis of endothelial cells after serum deprivation was supported by two lines of evidence. First, floating dead cells had morphological and biochemical features characteristic of apoptosis, such as membrane blebbing, cellular shrinkage, nuclear condensation and fragmentation, and nucleosomal laddering on agarose gel electrophoresis. Second, adherent cells were stained positively with anti-single-stranded DNA antiserum after serum deprivation.

Adrenomedullin prevented apoptosis as early as 1 h after serum deprivation, whose effect was time-dependent during 24-h incubation period. Furthermore, the protective effect of adrenomedullin on serum-deprived apoptosis was concentration-dependent. The minimum effective concentration to induce apoptotic protection by adrenomedullin (10^-10 M) is almost comparable with that (3 x 10^-11 M) secreted into cultured media by endothelial cells. These levels are also far lower than those reported with other growth factors and cytokines to block apoptosis in fibroblasts (9), or in human endothelial cells (14).

Our findings also suggest that rat endothelial cells not only express adrenomedullin gene and release the peptide, but contain receptors for adrenomedullin. However, there appears to be discrepancy between the low-affinity of the receptor (K_d, 7.7 x 10^-8 M) as estimated from Scatchard analysis of the binding study and the low concentration of adrenomedullin secreted into media (3 x 10^-11 M) as determined by RIA. This may be accounted for by the underestimation of the accurate values because endogenous adrenomedullin constitutively secreted from endothelial cells may readily bind to its receptors in an autocrine/paracrine fashion and subsequently be degraded, thereby masking the high affinity binding sites and decreasing its concentration detected in media.

To address the question whether adrenomedullin molecule released from the cells control their own apoptotic cell death, neutralization experiments using polyclonal antiadrenomedullin antibody were performed. Control rabbit serum markedly (70%) suppressed apoptosis, whereas rabbit antiadrenomedullin antiserum partially but significantly abrogated the apoptotic protective effect by the control serum at the same dilution (1:100). The difference (40%) between the apoptosis protective effects by the preimmune serum and the antiserum may be most likely due to endogenous adrenomedullin constitutively secreted. However, the remaining serum-induced protective effect (30%) not reversed by the antiadrenomedullin antiserum may be due to factors derived from serum and/or endothelial cells other than adrenomedullin. Taken together, our data are consistent with the notion that adrenomedullin produced by and released from the cells could act on themselves to block apoptosis, thus functioning as self-protection by an autocrine/paracrine mechanism.

Because adrenomedullin stimulates cAMP formation in platelets and vascular smooth muscle cells, cAMP has been suggested as a second messenger for its vasorelaxation. The present study shows that adrenomedullin also induces cAMP generation in rat endothelial cells. However, cAMP-elevating agonists (PGI₂ and forskolin) induced cAMP levels comparable to those by adrenomedullin, but did not inhibit apoptosis. Moreover, cAMP antagonist (Rp-cAMPS) did not antagonize the antiapoptosis effect by adrenomedullin. These findings support the view that the antiapoptotic action of adrenomedullin does not appear to involve cAMP-dependent mechanism.

The precise mechanisms of apoptosis protection by adrenomedullin observed in the present study remain unknown. It has been reported that apoptosis of vascular endothelial cells is prevented by basic fibroblast growth factor and dexamethasone (14, 15) but aggravated by tumor necrosis factor- (13) and transforming growth factor-ß (30). Possible involvement of protein kinase C has been suggested in the mediation of basic fibroblast growth factor’s protection of endothelial cells against apoptosis induced by serum starvation and radiation (15, 31). Adrenomedullin has recently been reported to induce Ca²⁺ mobilization from intracellular storage sites in bovine endothelial cells (32). However, our cultured rat endothelial cells showed neither transient rise of [Ca²⁺]_i, nor increase in Ins-1,4,5-P₃ formation in response to adrenomedullin. Therefore, neither protein kinase C nor intracellular Ca²⁺ appear to be involved in the mechanism of adrenomedullin-induced protection from apoptosis in rat endothelial cells. Inhibition of anchorage-dependent cell spreading and acquisition of round shape have been demonstrated to trigger apoptosis in human endothelial cells (29). However, the present study revealed that rat endothelial cells that remained flattened shape and firmly attached to culture plates, exhibited distinct morphological and biochemical evidence of apoptosis, thus arguing against the involvement of the inhibition of anchorage-dependent cell spreading.

In summary, we have shown a hitherto undescribed role of a novel hypotensive peptide, adrenomedullin, as an apoptosis survival factor for endothelial cells in an autocrine/paracrine manner. The antiapoptotic action of adrenomedullin does not appear to be mediated via cAMP, protein kinase C or intracellular Ca²⁺. The physiological significance of adrenomedullin as an apoptosis survival factor for endothelial cells as well as its intracellular signal(s) remains to be determined.

	Footnotes

 
¹ This study was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture [07671117, 08671140 (to M.S.), and 07457121 (to Y.H.)], and the Ministry of Health and Welfare (8A1) (to Y. H.) of Japan, by the Chiiki-Igaku Research Fund (to M. S.), and by the Tanabe Medical Frontier Conference (to M. S.).

Received December 9, 1996.

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				R. Norregaard, T. Bodker, B. L. Jensen, L. Stodkilde, S. Nielsen, and J. Frokiaer Increased renal adrenomedullin expression in rats with ureteral obstruction Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2009; 296(1): R185 - R192. [Abstract] [Full Text] [PDF]

				M. Miksa, R. Wu, X. Cui, W. Dong, P. Das, H. H. Simms, T. S. Ravikumar, and P. Wang Vasoactive Hormone Adrenomedullin and Its Binding Protein: Anti-Inflammatory Effects by Up-Regulating Peroxisome Proliferator-Activated Receptor-{gamma} J. Immunol., November 1, 2007; 179(9): 6263 - 6272. [Abstract] [Full Text] [PDF]

				T. Itoh, H. Obata, S. Murakami, K. Hamada, K. Kangawa, H. Kimura, and N. Nagaya Adrenomedullin ameliorates lipopolysaccharide-induced acute lung injury in rats Am J Physiol Lung Cell Mol Physiol, August 1, 2007; 293(2): L446 - L452. [Abstract] [Full Text] [PDF]

				M. Nowicki, D. Ostalska-Nowicka, B. Kondraciuk, and B. Miskowiak The significance of substance P in physiological and malignant haematopoiesis J. Clin. Pathol., July 1, 2007; 60(7): 749 - 755. [Abstract] [Full Text] [PDF]

				A. P. Gomez, M. J. Moreno, A. Iglesias, P. X. Coral, and A. Hernandez Endothelin 1, its Endothelin Type A Receptor, Connective Tissue Growth Factor, Platelet-Derived Growth Factor, and Adrenomedullin Expression in Lungs of Pulmonary Hypertensive and Nonhypertensive Chickens Poult. Sci., May 1, 2007; 86(5): 909 - 916. [Abstract] [Full Text] [PDF]

				V. Ramachandran, T. Arumugam, R. F. Hwang, J. K. Greenson, D. M. Simeone, and C. D. Logsdon Adrenomedullin Is Expressed in Pancreatic Cancer and Stimulates Cell Proliferation and Invasion in an Autocrine Manner via the Adrenomedullin Receptor, ADMR Cancer Res., March 15, 2007; 67(6): 2666 - 2675. [Abstract] [Full Text] [PDF]

				Q. Xu, N. Ohara, W. Chen, J. Liu, H. Sasaki, A. Morikawa, R. Sitruk-Ware, E. D.B. Johansson, and T. Maruo Progesterone receptor modulator CDB-2914 down-regulates vascular endothelial growth factor, adrenomedullin and their receptors and modulates progesterone receptor content in cultured human uterine leiomyoma cells Hum. Reprod., September 1, 2006; 21(9): 2408 - 2416. [Abstract] [Full Text] [PDF]

				B. Uzan, H.-K. Ea, J.-M. Launay, J.-M. Garel, R. Champy, M. Cressent, and F. Liote A Critical Role for Adrenomedullin-Calcitonin Receptor-Like Receptor in Regulating Rheumatoid Fibroblast-Like Synoviocyte Apoptosis J. Immunol., May 1, 2006; 176(9): 5548 - 5558. [Abstract] [Full Text] [PDF]

				L. L. Nikitenko, N. Blucher, S. B. Fox, R. Bicknell, D. M. Smith, and M. C. P. Rees Adrenomedullin and CGRP interact with endogenous calcitonin-receptor-like receptor in endothelial cells and induce its desensitisation by different mechanisms. J. Cell Sci., March 1, 2006; 119(Pt 5): 910 - 922. [Abstract] [Full Text] [PDF]

				H Watanabe, E Takahashi, M Kobayashi, M Goto, A Krust, P Chambon, and T Iguchi The estrogen-responsive adrenomedullin and receptor-modifying protein 3 gene identified by DNA microarray analysis are directly regulated by estrogen receptor J. Mol. Endocrinol., February 1, 2006; 36(1): 81 - 89. [Abstract] [Full Text] [PDF]

				J. Kato, T. Tsuruda, T. Kita, K. Kitamura, and T. Eto Adrenomedullin: A Protective Factor for Blood Vessels Arterioscler. Thromb. Vasc. Biol., December 1, 2005; 25(12): 2480 - 2487. [Abstract] [Full Text] [PDF]

				X. Zhang, K. E. Green, C. Yallampalli, and Y. L. Dong Adrenomedullin Enhances Invasion by Trophoblast Cell Lines Biol Reprod, October 1, 2005; 73(4): 619 - 626. [Abstract] [Full Text] [PDF]

				S. Murakami, N. Nagaya, T. Itoh, T. Iwase, T. Fujisato, K. Nishioka, K. Hamada, K. Kangawa, and H. Kimura Adrenomedullin Regenerates Alveoli and Vasculature in Elastase-induced Pulmonary Emphysema in Mice Am. J. Respir. Crit. Care Med., September 1, 2005; 172(5): 581 - 589. [Abstract] [Full Text] [PDF]

				S. Frede, P. Freitag, T. Otto, C. Heilmaier, and J. Fandrey The Proinflammatory Cytokine Interleukin 1{beta} and Hypoxia Cooperatively Induce the Expression of Adrenomedullin in Ovarian Carcinoma Cells through Hypoxia Inducible Factor 1 Activation Cancer Res., June 1, 2005; 65(11): 4690 - 4697. [Abstract] [Full Text] [PDF]

				N. Nagaya, H. Mori, S. Murakami, K. Kangawa, and S. Kitamura Adrenomedullin: angiogenesis and gene therapy Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2005; 288(6): R1432 - R1437. [Abstract] [Full Text] [PDF]

				K. Hanabusa, N. Nagaya, T. Iwase, T. Itoh, S. Murakami, Y. Shimizu, W. Taki, K. Miyatake, and K. Kangawa Adrenomedullin Enhances Therapeutic Potency of Mesenchymal Stem Cells After Experimental Stroke in Rats Stroke, April 1, 2005; 36(4): 853 - 858. [Abstract] [Full Text] [PDF]

				N. Fukai, T. Yoshimoto, T. Sugiyama, N. Ozawa, R. Sato, M. Shichiri, and Y. Hirata Concomitant expression of adrenomedullin and its receptor components in rat adipose tissues Am J Physiol Endocrinol Metab, January 1, 2005; 288(1): E56 - E62. [Abstract] [Full Text] [PDF]

				J. Penchalaneni, S. J. Wimalawansa, and C. Yallampalli Adrenomedullin Antagonist Treatment During Early Gestation in Rats Causes Fetoplacental Growth Restriction Through Apoptosis Biol Reprod, November 1, 2004; 71(5): 1475 - 1483. [Abstract] [Full Text] [PDF]

				N. Ozawa, M. Shichiri, N. Fukai, T. Yoshimoto, and Y. Hirata Regulation of Adrenomedullin Gene Transcription and Degradation by the c-myc Gene Endocrinology, September 1, 2004; 145(9): 4244 - 4250. [Abstract] [Full Text] [PDF]

				T. Yoshimoto, N. Fukai, R. Sato, T. Sugiyama, N. Ozawa, M. Shichiri, and Y. Hirata Antioxidant Effect of Adrenomedullin on Angiotensin II-Induced Reactive Oxygen Species Generation in Vascular Smooth Muscle Cells Endocrinology, July 1, 2004; 145(7): 3331 - 3337. [Abstract] [Full Text] [PDF]

				P. Sandner, K. H. Hofbauer, H. Tinel, A. Kurtz, H. C. Thiesson, P. D. Ottosen, S. Walter, O. Skott, and B. L. Jensen Expression of adrenomedullin in hypoxic and ischemic rat kidneys and human kidneys with arterial stenosis Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2004; 286(5): R942 - R951. [Abstract] [Full Text] [PDF]

				N. Tokunaga, N. Nagaya, M. Shirai, E. Tanaka, H. Ishibashi-Ueda, M. Harada-Shiba, M. Kanda, T. Ito, W. Shimizu, Y. Tabata, et al. Adrenomedullin Gene Transfer Induces Therapeutic Angiogenesis in a Rabbit Model of Chronic Hind Limb Ischemia: Benefits of a Novel Nonviral Vector, Gelatin Circulation, February 3, 2004; 109(4): 526 - 531. [Abstract] [Full Text] [PDF]

				H. Okumura, N. Nagaya, T. Itoh, I. Okano, J. Hino, K. Mori, Y. Tsukamoto, H. Ishibashi-Ueda, S. Miwa, K. Tambara, et al. Adrenomedullin Infusion Attenuates Myocardial Ischemia/Reperfusion Injury Through the Phosphatidylinositol 3-Kinase/Akt-Dependent Pathway Circulation, January 20, 2004; 109(2): 242 - 248. [Abstract] [Full Text] [PDF]

				N. Fukai, M. Shichiri, N. Ozawa, M. Matsushita, and Y. Hirata Coexpression of Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 2 or 3 Mediates the Antimigratory Effect of Adrenomedullin Endocrinology, February 1, 2003; 144(2): 447 - 453. [Abstract] [Full Text] [PDF]

				A. Martinez, M. Vos, L. Guedez, G. Kaur, Z. Chen, M. Garayoa, R. Pio, T. Moody, W. G. Stetler-Stevenson, H. K. Kleinman, et al. The Effects of Adrenomedullin Overexpression in Breast Tumor Cells J Natl Cancer Inst, August 21, 2002; 94(16): 1226 - 1237. [Abstract] [Full Text] [PDF]