Adrecizumab: an investigational agent for the biomarker-guided treatment of sepsis.

Introduction: Sepsis is a major health problem with a high incidence and mortality. ADM, a free-circulating peptide mainly expressed and secreted by vascular endothelial cells, shows vasodilatory properties and causes hypotension when present in higher concentrations during sepsis. Areas covered: Adrecizumab (ADZ) (HAM 8101) is a humanized targeted therapy directed against the N-terminus of adrenomedullin (ADM). ADZ inhibits excessive circulating sepsis-induced ADM and stimulates protective effects on the endothelial barrier, and decreases interstitial vasodilatory effects. ADZ demonstrated a promising safety profile in healthy subjects in phase I studies. According to these results, a phase II proof of concept study enrolling 300 septic patients is currently in course (NCT03085758). Expert opinion: ADZ is the first humanized antibody directed against ADM. The main interest of ADZ is its potential use as a 'biomarker-guided therapy' in septic patients with high circulating ADM. ADZ is increasingly seen as a potential adjunct therapy to restore endothelial function in septic shock. A positive pivotal phase III trial is indeed needed to convince the intensive care community to prescribe ADZ in septic shock patients. Further, it would be of interest to see whether ADZ might also benefit other critical diseases such as cardiogenic shock where endothelial dysfunction has also been described.

Inhibition of Intermedin (Adrenomedullin 2) Suppresses the Growth of Glioblastoma and Increases the Antitumor Activity of Temozolomide.

Glioblastoma multiforme (GBM; grade IV glioma) is the most malignant type of primary brain tumor and is characterized by rapid proliferation and invasive growth. Intermedin (IMD) is an endogenous peptide belonging to the calcitonin gene-related peptide family and has been reported to play an important role in cell survival and invasiveness in several types of cancers. In this study, we found that the expression level of IMD was positively related to the malignancy grade of gliomas. The highest expression of IMD was found in GBM, indicating that IMD may play an important role in glioma malignancy. IMD increased the invasive ability of glioma cells by promoting filopodia formation, which is dependent on ERK1/2 activation. IMD-induced ERK1/2 phosphorylation also promoted GBM cell proliferation. In addition, IMD enhanced mitochondrial function and hypoxia-induced responses in GBM cells. Treatment with anti-IMD monoclonal antibodies not only inhibited tumor growth in both ectopic and orthotopic models of GBM but also significantly enhanced the antitumor activity of temozolomide. Our study may provide novel insights into the mechanism of GBM cell invasion and proliferation and provide an effective strategy to improve the therapeutic effect of GBM treatments.

Impact of adrenomedullin blockage on lipid metabolism in female mice exposed to high-fat diet.

PURPOSE: Adrenomedullin (ADM) levels are elevated in gestational and type 2 diabetic patients. ADM also stimulates lipolysis in vitro. Disturbed lipid metabolism has been implicated in the pathogenesis of diabetes. Here, we explore whether blockade of ADM is beneficial for metabolic homeostasis in a diabetic mouse model. METHODS: C57BL/6J female mice were placed on either a control or a high fat high sucrose (HFHS) diet for 8 weeks. At week 4, osmotic mini-pumps were implanted for constant infusion of either saline or ADM antagonist, ADM22-52. Glucose tolerance tests were performed prior to infusion and 4 weeks after infusion began. Animals were then sacrificed and visceral adipose tissue collected for further analysis. RESULTS: Mice fed HFHS displayed glucose intolerance, increased mRNA expressions in VAT for Adm and its receptor components, Crlr. HFHS fed mice also had increased basal and isoprenaline-induced glycerol release by VAT explants. ADM22-52 did not significantly affect glucose intolerance. ADM22-52 did suppress basal and isoprenaline-induced glycerol release by VAT explants. This alteration was associated with enhanced mRNA expression of insulin signaling factors Insr and Glut4, and adipogenic factor Pck1. CONCLUSIONS: HFHS diet induces glucose intolerance and enhances ADM and its receptor expressions in VAT in female mice. ADM22-52 treatment did not affect glucose intolerance in HFHS mice, but reduced both basal and isoprenaline-induced lipolysis, which is associated with enhanced expression of genes involved in adipogenesis. These results warrant further research on the effects of ADM blockade in improving lipid homeostasis in diabetic patients.

Preclinical safety evaluation of the adrenomedullin-binding antibody Adrecizumab in rodents, dogs and non-human primates.

Adrenomedullin (ADM) is a vasoactive peptide in sepsis. The non-neutralizing ADM-binding antibody Adrecizumab improved outcome in animal models of systemic inflammation and sepsis. Herein, we evaluated the preclinical safety of Adrecizumab in various animal species. First, Wistar rats received vehicle, 100, 200 or 400 mg/kg/day of Adrecizumab intravenously (n = 20 each) on days 1, 4, 8 and 14. An additional set of rats received vehicle or 400 mg/kg/day (n = 10 each) on the same days and were followed for 42 days. For toxicokinetics, satellite animals received vehicle (n = 6), 100, 200, or 400 mg/kg/day Adrecizumab intravenously (n = 18 each). A hemodynamic study was performed in Beagle dogs (n = 3) receiving vehicle (day 1), 2 mg/kg (day 3), 10 mg/kg (day 5), 50 mg/kg (day 8) and 10 mg/kg Adrecizumab intravenously (day 29). In final experiments, cynomolgus monkeys received vehicle, 25, 50 or 100 mg/kg/day Adrecizumab intravenously (n = 6 each) on days 1, 4, 8 and 14. Additional groups of monkeys received vehicle or 100 mg/kg/day Adrecizumab intravenously (n = 4 each) on the same days and were followed for 42 days. No mortality or moribund conditions occurred and no toxicologically relevant effects were attributed to Adrecizumab. Adrecizumab significantly increased circulating concentrations of its target peptide ADM, consistent with previous studies and mechanistically relevant. Toxicokinetic analyses showed immediate and dose-dependent peak concentrations, slow elimination and no gender differences. In conclusion, intravenous, repeated administration of high doses of Adrecizumab appeared well-tolerated across species. These results pave the way for further investigation of Adrecizumab in humans (intended dose of 2 mg/kg).

Circulating Adrenomedullin Is Elevated in Gestational Diabetes and Its Role in Impaired Insulin Production by ß-Cells.

Context: Defective pancreatic ß-cell adaptation in pregnancy plays an important role in the pathophysiology of gestational diabetes mellitus (GDM), but the molecular basis remains unclear. Objectives of this study were to determine if circulating levels of adrenomedullin (ADM) in women with GDM are elevated and to assess the effects of ADM on insulin synthesis and secretion by human pancreatic ß-cells. Design: A stable gene product of ADM precursor, midregional pro-adrenomedullin (MR-proADM), was measured in plasma of pregnant women with normal glucose tolerance (NGT, n = 10) or GDM (n = 11). The ß-Lox5 cell line, derived from human pancreatic ß-cells, was transduced with homeodomain transcription factor pancreatic-duodenal homeobox (PDX) factor 1 (PDX1) encoding lentiviral vector and treated with different doses of ADM. mRNA for insulin, ADM, and its receptor components in ß-Lox5 cells and insulin in media were measured. Results: Plasma MR-proADM levels were significantly higher in GDM compared with patients with NGT. Pancreatic ß-Lox5 cells express mRNA for insulin, ADM, and its receptor components. PDX1 transduction and cell-cell contact synergistically promote ß-Lox5 cells insulin mRNA and secretion. Furthermore, ADM dose-dependently inhibited mRNA and secretion of insulin in ß-Lox5 cell aggregates. These inhibitory effects were blocked by ADM antagonist ADM22-52, cAMP-dependent protein kinase A inhibitor KT5720, and Erk inhibitor PD98059, but not by PI-3K the inhibitor wortmannin. Conclusions: Circulating ADM concentrations were elevated in pregnant women with GDM. ADM suppresses insulin synthesis and secretion by pancreatic ß-cells in vitro. Thus, increased circulating ADM may contribute to the defective adaptation of ß-cells in diabetic pregnancies, and blockade of ADM actions with its antagonists may improve ß-cell functions.

Intrathecal administration of adrenomedullin induces mechanical allodynia and neurochemical changes in spinal cord and DRG.

This study investigated the effect of adrenomedullin (AM) on mechanical pain sensitivity and its possible mechanisms. Intrathecal injection of AM receptor agonist AM1-50 (20 µg) once per day briefly reduced mechanical pain threshold on days 1 and 2 but induced prolonged mechanical allodynia on day 3. However, AM1-50 did not change mechanical pain sensation when the AM receptor antagonist AM22-52 (20 µg) was intrathecally co-administered. Daily administration of AM1-50 (20 µg) for 3 days increased expression of phosphorylated extracellular signal-regulated protein kinase (pERK) and neuronal nitric oxide synthase (nNOS) in the spinal dorsal horn. The AM-induced increase in pERK and nNOS was inhibited by the co-administration of AM22-52. The chronic administration of AM1-50 also increased expression of microglial maker Iba1 and astrocytic marker GFAP (glial fibrillary acidic protein) in the spinal dorsal horn in an AM22-52-sensitive manner. Furthermore, the application of AM1-50 (10 nM, 3 h) to dorsal root ganglion (DRG) explant cultures induced an increase in the expression of transient receptor potential vanilloid 1 (TRPV1). The treatment with AM1-50 did not change TRPV1 expression in DRG in the presence of AM22-52 (2 µM). These results suggest that the increased AM bioactivity induced mechanical allodynia and may contribute to the mechanical pain hypersensitivity under pathological conditions. The mechanisms may involve the activation of ERK signaling pathway and spinal glia as well as the recruitment of nNOS and TRPV1 in the spinal dorsal horn or DRG. The present study indicates that inhibition of the activation AM receptor might provide a fruitful strategy to relieving chronic pain.

Safety, tolerability and pharmacokinetics/pharmacodynamics of the adrenomedullin antibody adrecizumab in a first-in-human study and during experimental human endotoxaemia in healthy subjects.

AIMS: Adrenomedullin (ADM) is an important regulator of endothelial barrier function and vascular tone, and may represent a novel treatment target in sepsis. The non-neutralizing ADM antibody adrecizumab has shown promising results in preclinical sepsis models. In the present study, we investigated the safety, tolerability and pharmacokinetics (PK)/pharmacodynamics of adrecizumab in a first-in-man study and in a second study during experimental human endotoxaemia. METHODS: Forty-eight healthy male volunteers were enrolled in two randomized, double-blind, placebo-controlled phase I studies. In both studies, subjects received placebo or one of three doses of adrecizumab (n = 6 per group). In the second study, a bolus of 1 ng kg-1 endotoxin was followed by infusion of 1 ng kg-1 h-1 endotoxin for 3 h to induce systemic inflammation, and the study medication infusion started 1 h after endotoxin bolus administration. RESULTS: Adrecizumab showed an excellent safety profile in both studies. PK analyses showed proportional increases in the maximum plasma concentration of adrecizumab with increasing doses, a small volume of distribution, a low clearance rate and a terminal half-life of ~14 days. adrecizumab elicited a pronounced increase in plasma ADM levels, whereas levels of mid-regional pro-adrenomedullin remained unchanged, indicating that de novo synthesis of ADM was not influenced. In the second study, no effects of adrecizumab on cytokine clearance were observed, whereas endotoxin-induced flu-like symptoms resolved more rapidly. CONCLUSIONS: Administration of adrecizumab is safe and well tolerated in humans, both in the absence and presence of systemic inflammation. These findings pave the way for further investigation of adrecizumab in sepsis patients.

Adrenomedullin, a Novel Target for Neurodegenerative Diseases.

Neurodegenerative diseases represent a heterogeneous group of disorders whose common characteristic is the progressive degeneration of neuronal structure and function. Although much knowledge has been accumulated on the pathophysiology of neurodegenerative diseases over the years, more efforts are needed to understand the processes that underlie these diseases and hence to propose new treatments. Adrenomedullin (AM) is a multifunctional peptide involved in vasodilation, hormone secretion, antimicrobial defense, cellular growth, and angiogenesis. In neurons, AM and related peptides are associated with some structural and functional cytoskeletal proteins that interfere with microtubule dynamics. Furthermore, AM may intervene in neuronal dysfunction through other mechanisms such as immune and inflammatory response, apoptosis, or calcium dyshomeostasis. Alterations in AM expression have been described in neurodegenerative processes such as Alzheimer's disease or vascular dementia. This review addresses the current state of knowledge on AM and its possible implication in neurodegenerative diseases.

Mechanism of adrenomedullin 2/intermedin mediated vasorelaxation in rat main pulmonary artery.

Adrenomedullin 2/intermedin (AM2/IMD) is a member of calcitonin related gene peptide family and an important nitric oxide mediated vasorelaxant in various vascular beds. However, the mechanism of post receptor-interaction is not clear and may differ depending on tissue type and species. In this study, we aimed to investigate the exact mechanism and the role of BKCa and calcium channels on the vasorelaxant effect of AM2/IMD in rat PA. Changes in the AM2/IMD-mediated vasorelaxation were evaluated in the presence of various inhibitors. CGRP(8-37) (10-6 M), L-NAME (10-4 M), ODQ (10-5 M), SQ22536 (10-4 M), H89 (10-6 M), TEA (10-2 M), iberiotoxin (3 × 10-7 M), and verapamil (10-5 M), all partly or completely inhibited the vasorelaxation. The relaxation was also abolished by removal of the endothelium, or in KCl precontracted PAs. AM2/IMD did not elicit vasorelaxation in the Ca2+-free conditions. However, the vasorelaxation was not inhibited with AM(22-52) (10-6 M), 4-AP (3 × 10-3 M), glibenclamide (10-5 M), apamin (3 × 10-7 M), TRAM-34 (10-5 M), and La+3 (10-4 M). AM2/IMD -induced changes in intracellular calcium levels and isometric force were monitored simultaneously in fura-2-loaded, endothelium-intact PAs. The AM2/IMD-induced increase in intracellular Ca2+ concentration was inhibited in the presence of iberiotoxin and verapamil, whereas no change was observed with La3+ incubation. Our data suggest that the cAMP/PKA pathway is one of the important pathways AM2/IMD-induced vasorelaxation. AM2/IMD acts through activation of endothelial BKCa and subsequently causes hyperpolarization of the endothelial cell membrane. The hyperpolarization induces Ca2+ influx, which leads to NO production and subsequent vasorelaxation.

Effects of the Humanized Anti-Adrenomedullin Antibody Adrecizumab (HAM8101) on Vascular Barrier Function and Survival in Rodent Models of Systemic Inflammation and Sepsis.

PURPOSE: Adrenomedullin (ADM) is an important regulator of endothelial barrier function during sepsis. Administration of a murine antibody targeted against the N-terminus of ADM (HAM1101) resulted in improved outcome in models of murine sepsis. We studied the effects of a humanized form of this antibody (HAM8101, also known as Adrecizumab) on vascular barrier dysfunction and survival in rodent models of systemic inflammation and sepsis. METHODS: Rats (n=48) received different dosages of HAM8101 or placebo (n = 8 per group), directly followed by administration of lipopolysaccharide (5 mg/kg). Twenty-four hours later, Evans Blue dye was administered to assess vascular leakage in kidney and liver tissue. Furthermore, mice (n = 24) were administered different dosages of HAM8101 or placebo (n = 6 per group), immediately followed by cecal ligation and puncture (CLP). Eighteen hours later, albumin, vascular endothelial growth factor (VEGF), and angiopoietin-1 were analyzed in the kidney. Finally, effects of single and repeated dose administration of HAM1101, HAM8101 and placebo on survival were assessed in CLP-induced murine sepsis (n = 60, n = 10 per group). RESULTS: Dosages of 0.1 and 2.5 mg/kg HAM8101 attenuated renal albumin leakage in endotoxemic rats. Dosages of 0.1, 2.0, and 20 mg/kg HAM8101 reduced renal concentrations of albumin and the detrimental protein VEGF in septic mice, whereas concentrations of the protective protein angiopoietin-1 were augmented. Both single and repeated administration of both HAM1101 and HAM8101 resulted in improved survival during murine sepsis. CONCLUSIONS: Pretreatment with the humanized anti-ADM antibody HAM8101 improved vascular barrier function and survival in rodent models of systemic inflammation and sepsis.

Adrenomedullin blockade suppresses sunitinib-resistant renal cell carcinoma growth by targeting the ERK/MAPK pathway.

PURPOSE: To evaluate the mechanisms underlying sunitinib resistance in RCC and to identify targets that may be used to overcome this resistance. RESULTS: Reanalysis of transcriptome microarray datasets (GSE64052 and GSE76068) showed that adrenomedullin expression was increased in sunitinib-resistant tumors. And adrenomedullin expression was increased in sunitinib-resistant tumor xenografts, accompanied by upregulation of phospho-ERK levels. However, blocking adrenomedullin inhibited sunitinib-resistant tumor growth. Treatment of RCC cells with sunitinib and ADM22-52 was superior to monotherapy with either agent. Additionally, adrenomedullin upregulated cAMP and activated the ERK/MAPK pathway, promoting cell proliferation, while knockdown of adrenomedullin inhibited RCC cell growth and invasion in vitro. MATERIALS AND METHODS: We searched the Gene Expression Omnibus (GEO) database to find data regarding sunitinib-resistant RCC. These data were subsequently reanalyzed to identify targets that contribute to sunitinib resistance, and adrenomedullin upregulation was found to mediate sunitinib resistance in RCC. Then, we created an RCC mouse xenograft model. Mice were treated with sunitinib, an adrenomedullin receptor antagonist (ADM22-52), a MEK inhibitor (PD98059) and different combinations of these three drugs to investigate their effects on tumor growth. RCC cells (786-0) were cultured in vitro and treated with an ADM22-52 or PD98059 to determine whether adrenomedullin activates the ERK/MAPK pathway. Adrenomedullin was knocked down in 786-0 cells via siRNA, and the effects of this knockdown on cell were subsequently investigated. CONCLUSIONS: Adrenomedullin plays an important role in RCC resistance to sunitinib treatment. The combination of sunitinib and an adrenomedullin receptor antagonist may result in better outcomes in advanced RCC patients.

Tumor-expressed adrenomedullin accelerates breast cancer bone metastasis.

INTRODUCTION: Adrenomedullin (AM) is secreted by breast cancer cells and increased by hypoxia. It is a multifunctional peptide that stimulates angiogenesis and proliferation. The peptide is also a potent paracrine stimulator of osteoblasts and bone formation, suggesting a role in skeletal metastases-a major site of treatment-refractory tumor growth in patients with advanced disease. METHODS: The role of adrenomedullin in bone metastases was tested by stable overexpression in MDA-MB-231 breast cancer cells, which cause osteolytic bone metastases in a standard animal model. Cells with fivefold increased expression of AM were characterized in vitro, inoculated into immunodeficient mice and compared for their ability to form bone metastases versus control subclones. Bone destruction was monitored by X-ray, and tumor burden and osteoclast numbers were determined by quantitative histomorphometry. The effects of AM overexpression on tumor growth and angiogenesis in the mammary fat pad were determined. The effects of AM peptide on osteoclast-like multinucleated cell formation were tested in vitro. A small-molecule AM antagonist was tested for its effects on AM-stimulated ex vivo bone cell cultures and co-cultures with tumor cells, where responses of tumor and bone were distinguished by species-specific real-time PCR. RESULTS: Overexpression of AM mRNA did not alter cell proliferation in vitro, expression of tumor-secreted factors or cell cycle progression. AM-overexpressing cells caused osteolytic bone metastases to develop more rapidly, which was accompanied by decreased survival. In the mammary fat pad, tumors grew more rapidly with unchanged blood vessel formation. Tumor growth in the bone was also more rapid, and osteoclasts were increased. AM peptide potently stimulated bone cultures ex vivo; responses that were blocked by small-molecule adrenomedullin antagonists in the absence of cellular toxicity. Antagonist treatment dramatically suppressed tumor growth in bone and decreased markers of osteoclast activity. CONCLUSIONS: The results identify AM as a target for therapeutic intervention against bone metastases. Adrenomedullin potentiates osteolytic responses in bone to metastatic breast cancer cells. Small-molecule antagonists can effectively block bone-mediated responses to tumor-secreted adrenomedullin, and such agents warrant development for testing in vivo.

Adrenomedullin promotes rat trophoblast stem cell differentiation.

Accumulating data suggest that adrenomedullin (ADM) regulates the trophoblast cell growth, migration, and invasion. However, the effect of ADM on trophoblast differentiation is poorly understood. In this study, we hypothesized that ADM promotes the differentiation of trophoblast stem cells (TSCs) into trophoblast giant cells (TGCs). Using rat TSCs, Rcho-1 cells, we investigated the effect of ADM on TSC differentiation into TGCs in differentiation or stem cell media, respectively, and explored the effect of ADM on the mechanistic target of rapamycin (MTOR) signaling in trophoblast cell differentiation. The results include: 1) in the presence of differentiation medium, 10⁻7 M ADM, but not lower doses, elevated (P < 0.05) Prl3b1/Esrrb (i.e., the ratio of mRNA levels) by 1.7-fold compared to that in control; 2) the supplementation of ADM antagonist, regardless of the concentration of ADM, reduced (P < 0.05) Prl3b1/Esrrb by 2-fold, compared to control group, while the supplementation of CGRP antagonist, regardless of the concentration of ADM, did not change Prl3b1/Esrrb; 3) in the presence of stem cell medium, ADM did not alter the expression of TSC and TGC marker genes, however, the ratio of Prl3b1/Esrrb was reduced (P < 0.05) by ADM antagonist compared to that in control; and 4) ADM increased (P < 0.05) phosphorylated MTOR proteins and the ratio of phosphorylated to total MTOR proteins by 2.0- and 1.7-fold, respectively. The results indicate that ADM promotes but does not induce the differentiation of TSCs to TGCs in a dose-dependent manner and MTOR signaling may play a role in this process.

Cigarette smoke-induced placental adrenomedullin expression and trophoblast cell invasion.

Smoking in pregnancy reduces preeclampsia risk, but the mechanism of this effect is unknown. Prior studies have demonstrated that women with preeclampsia have lower placental adrenomedullin (AM) expression, and cigarette smoke extract (CSE) treatment of placental trophoblast cells in culture increases AM cellular production. We hypothesized that CSE alters trophoblast invasion through an AM-mediated mechanism, and that placental AM expression is greater among smokers. HTR-8/SVneo trophoblast cells were incubated for 24 hours in Matrigel-invasion chambers with 6 treatment groups: nonstimulated (NS), AM, AM inhibitor (AM22-52), 1% CSE, AM + AM22-52, and 1% CSE + AM22-52. Cells that penetrated the lower surface of the chambers were quantified, invasion indices were calculated, and compared using a 1-way analysis of variance with Bonferroni corrections for multiple comparisons. Trophoblast cells treated with both AM and 1% CSE demonstrated increased cellular invasion compared to NS controls (1.5-fold [P < .01] and 1.45-fold [P < .01], respectively). Cotreatment with the AM inhibitor significantly attenuated the increased invasion seen with both AM and CSE alone. Next, the placental tissue was obtained from 11 smokers and 11 nonsmokers at term and processed for immunohistochemistry (IHC) and real-time quantitative polymerase chain reaction (PCR) for AM. Placentas from smokers demonstrated more intense AM staining and increased AM gene (ADM) expression compared to placentas from nonsmokers (P = .004 for IHC, P = .022 for PCR). The CSE increases trophoblast cell invasion through an AM-mediated process, and placental AM expression is increased among term smokers compared to nonsmokers. These findings provide evidence that the AM pathway may play a role in the protection from preeclampsia seen in smokers.

Adrenomedullin blockade suppresses growth of human hormone-independent prostate tumor xenograft in mice.

PURPOSE: To study the role of the adrenomedullin system [adrenomedullin and its receptors (AMR), CLR, RAMP2, and RAMP3] in prostate cancer androgen-independent growth. EXPERIMENTAL DESIGN: Androgen-dependent and -independent prostate cancer models were used to investigate the role and mechanisms of adrenomedullin in prostate cancer hormone-independent growth and tumor-associated angiogenesis and lymphangiogenesis. RESULTS: Adrenomedullin and AMR were immunohistochemically localized in the carcinomatous epithelial compartment of prostate cancer specimens of high grade (Gleason score >7), suggesting a role of the adrenomedullin system in prostate cancer growth. We used the androgen-independent Du145 cells, for which we demonstrate that adrenomedullin stimulated cell proliferation in vitro through the cAMP/CRAF/MEK/ERK pathway. The proliferation of Du145 and PC3 cells is decreased by anti-adrenomedullin antibody (αAM), supporting the fact that adrenomedullin may function as a potent autocrine/paracrine growth factor for prostate cancer androgen-independent cells. In vivo, αAM therapy inhibits the growth of Du145 androgen-independent xenografts and interestingly of LNCaP androgen-dependent xenografts only in castrated animals, suggesting strongly that adrenomedullin might play an important role in tumor regrowth following androgen ablation. Histologic examination of αAM-treated tumors showed evidence of disruption of tumor vascularity, with depletion of vascular as well as lymphatic endothelial cells and pericytes, and increased lymphatic endothelial cell apoptosis. Importantly, αAM potently blocks tumor-associated lymphangiogenesis, but does not affect established vasculature and lymphatic vessels in normal adult mice. CONCLUSIONS: We conclude that expression of adrenomedullin upon androgen ablation in prostate cancer plays an important role in hormone-independent tumor growth and in neovascularization by supplying/amplifying signals essential for pathologic neoangiogenesis and lymphangiogenesis. Clin Cancer Res; 19(22); 6138-50. ©2013 AACR.

Adrenomedullin-RAMP2 system is crucially involved in retinal angiogenesis.

Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilating molecule. We previously showed that in mice, homozygous knockout of ADM (ADM(-/-)) or its receptor regulating protein, RAMP2 (RAMP2(-/-)), is embryonically lethal due to abnormal vascular development, thereby demonstrating the importance of ADM and its receptor signaling to vascular development. ADM expression in the retina is strongly induced by ischemia; however, its role in retinal pathophysiology remains unknown. Here, we analyzed oxygen-induced retinopathy (OIR) using heterozygous ADM and RAMP2 knockout mice models (ADM(+/-) or RAMP2(+/-), respectively). In addition, we analyzed the role of the ADM-RAMP2 system during earlier stages of retinal angiogenesis using an inducible endothelial cell-specific RAMP2 knockout mouse line (DI-E-RAMP2(-/-)). Finally, we assessed the ability of antibody-induced ADM blockade to control pathological retinal angiogenesis in OIR. In OIR, neovascular tufts, avascular zones, and hypoxic areas were all smaller in ADM(+/-) retinas compared with wild-type mice. ADM(+/-) retinas also exhibited reduced levels of VEGF and eNOS expression. DI-E-RAMP2(-/-) showed abnormal retinal vascular patterns in the early stages of development. However, ADM enhanced the proliferation and migration of retinal endothelial cells. Finally, we found intravitreal injection of anti-ADM antibody reduced pathological retinal angiogenesis. In conclusion, the ADM-RAMP2 system is crucially involved in retinal angiogenesis. ADM and its receptor system are potential therapeutic targets for controlling pathological retinal angiogenesis.

An angiogenic role for adrenomedullin in choroidal neovascularization.

PURPOSE: Adrenomedullin (ADM) has been shown to take part in physiological and pathological angiogenesis. The purpose of this study was to investigate whether ADM signaling is involved in choroidal neovascularization (CNV) using a mouse model. METHODS AND RESULTS: CNV was induced by laser photocoagulation in 8-week-old C57BL/6 mice. ADM mRNA expression significantly increased following treatment, peaking 4 days thereafter. The expression of ADM receptor (ADM-R) components (CRLR, RAMP2 and RAMP 3) was higher in CD31(+)CD45(-) endothelial cells (ECs) than CD31(-)CD45(-) non-ECs. Inflammatory stimulation upregulated the expression of ADM not only in cell lines but also in cells in primary cultures of the choroid/retinal pigment epithelium complex. Supernatants from TNFα-treated macrophage cell lines potentiated the proliferation of ECs and this was partially suppressed by an ADM antagonist, ADM (22-52). Intravitreous injection of ADM (22-52) or ADM neutralizing monoclonal antibody (mAb) after laser treatment significantly reduced the size of CNV compared with vehicle-treated controls (p<0.01). CONCLUSIONS: ADM signaling is involved in laser-induced CNV formation, because both an ADM antagonist and ADM mAb significantly inhibited it. Suppression of ADM signaling might be a valuable alternative treatment for CNV associated with age-related macular degeneration.

Adrenomedullin gene dosage correlates with tumor and lymph node lymphangiogenesis.

Adrenomedullin (AM) is a potent lymphangiogenic factor that promotes lymphatic endothelial cell (LEC) proliferation through a pharmacologically tractable G-protein-coupled receptor. Numerous types of human cancers have increased levels of AM; however, the functional consequences of this fact have not been characterized. Therefore, we evaluated whether modulating adrenomedullin (Adm) gene dosage within tumor cells affects lymphangiogenesis. Murine Lewis lung carcinoma (LLC) cells that overexpress or underexpress Adm were injected subcutaneously into C57BL/6 mice, and tumors were evaluated for growth and vascularization. A dosage range from ∼10 to 200% of wild-type Adm expression did not affect LLC proliferation in vitro or in vivo, nor did it affect angiogenesis. Notably, the dosage of Adm markedly and significantly influenced tumor lymphangiogenesis. Reduced Adm expression in tumors decreased the proliferation of LECs and the number of lymphatic vessels, while elevated tumor Adm expression led to enlarged lymphatic vessels. Moreover, overexpression of Adm in tumors induced sentinel lymph node lymphangiogenesis and led to an increased incidence of Ki67-positive foci within the lung. These data show that tumor-secreted AM is a critical factor for driving both tumor and lymph node lymphangiogenesis. Thus, pharmacological targeting of AM signaling may provide a new avenue for inhibition of tumor lymphangiogenesis.

Preimplantation antagonism of adrenomedullin action compromises fetoplacental development and reduces litter size.

Concentrations of adrenomedullin (ADM) in circulation, the uterus, and corpora lutea (CL) increase during pregnancy. We previously reported a temporal-spatial pattern of ADM level and gene expression of Adm and its receptor components, from early pregnancy through midpregnancy to late pregnancy in rats. Two earlier reports using an in vivo model of ADM antagonism demonstrated the important roles of ADM in the post-implantation period. Treatment with ADM receptor blocker hADM22-52 starting from gestation Day 8 or Day 14 resulted in fetal-placental growth restriction and reduction in litter size. In this study, the endogenous ADM actions were abolished in the preimplantation period by infusing the antagonist for the ADM receptor (hADM22-52) with the osmotic (Alzet) pump from Days 1-4 of pregnancy. We inferred that ADM, acting through the ADM receptor, had critical roles during preimplantation, as brief inhibition of ADM action by hADM22-52 during this period reduced litter size by restricting placental growth and increasing fetal resorption in midpregnancy.

The effect of cigarette smoke extract on trophoblast cell viability and migration: the role of adrenomedullin.

We tested the hypothesis that cigarette smoke extract (CSE) leads to differences in expression of genes involved in angiogenesis and affects cell viability and migration in a first-trimester cytotrophoblast cell line (HTR-8/SVneo). HTR-8/SVneo cells were treated with 1% CSE, and gene expression for adrenomedullin (ADM), placental growth factor (PlGF), soluble fms-like tyrosine kinase 1 (sFLT-1), and vascular endothelia growth factor (VEGF) and protein content for ADM, PlGF, and sFlt-1 determined. A cell viability assay and a cell migration scratch assay were utilized following treatment with CSE with and without ADM inhibitor. Adrenomedullin, PlGF, and VEGF gene transcripts were significantly upregulated by 1% CSE treatment compared with unstimulated cells or cells treated with nicotine alone. Neither 1% CSE nor nicotine treatment alone affected sFlt-1 gene expression. There was a significant increase in secreted ADM protein from cells treated with 1% CSE detected by enzyme-linked immunosorbant assay, though no differences in PlGF or sFlt-1 production were seen. Treatment with 1% CSE increased cell viability and cell migration compared with unstimulated cells and was inhibited by co-treatment with ADM inhibitor. Treatment of a first-trimester trophoblast cell line with CSE increases cell viability and cell migration that are reversed by co-treatment with ADM inhibitor, suggesting that ADM at least partially mediates cell growth and viability following CSE treatment.