Adrenomedullin insufficiency alters macrophage activities in fallopian tube: a pathophysiologic explanation of tubal ectopic pregnancy.

Ectopic pregnancy is the major cause of maternal morbidity and mortality in the first trimester of pregnancy. Tubal ectopic pregnancy (TEP) accounts for nearly 98% of all ectopic pregnancies. TEP is usually associated with salpingitis but the underlying mechanism in salpingitis leading to TEP remains unclear. Adrenomedullin (ADM) is a peptide hormone abundantly expressed in the fallopian tube with potent anti-inflammatory activities. Its expression peaks at the early luteal phase when the developing embryo is being transported through the fallopian tube. In the present study, we demonstrated reduced expression of ADM in fallopian tubes of patients with salpingitis and TEP. Using macrophages isolated from the fallopian tubes of these women, our data revealed that the salpingistis-associated ADM reduction contributed to aggravated pro-inflammatory responses of the tubal macrophages resulting in production of pro-inflammatory and pro-implantation cytokines IL-6 and IL-8. These cytokines activated the expression of implantation-associated molecules and Wnt signaling pathway predisposing the tubal epithelium to an adhesive and receptive state for embryo implantation. In conclusion, this study provided evidence for the role of ADM in the pathogenesis of TEP through regulating the functions of tubal macrophages.

Deficiency of the adrenomedullin-RAMP3 system suppresses metastasis through the modification of cancer-associated fibroblasts.

Tumor metastasis is a primary source of morbidity and mortality in cancer. Adrenomedullin (AM) is a multifunctional peptide regulated by receptor activity-modifying proteins (RAMPs). We previously reported that the AM-RAMP2 system is involved in tumor angiogenesis, but the function of the AM-RAMP3 system remains largely unknown. Here, we investigated the actions of the AM-RAMP2 and 3 systems in the tumor microenvironment and their impact on metastasis. PAN02 pancreatic cancer cells were injected into the spleens of mice, leading to spontaneous liver metastasis. Tumor metastasis was enhanced in vascular endothelial cell-specific RAMP2 knockout mice (DI-E-RAMP2-/-). By contrast, metastasis was suppressed in RAMP3-/- mice, where the number of podoplanin (PDPN)-positive cancer-associated fibroblasts (CAFs) was reduced in the periphery of tumors at metastatic sites. Because PDPN-positive CAFs are a hallmark of tumor malignancy, we assessed the regulation of PDPN and found that Src/Cas/PDPN signaling is mediated by RAMP3. In fact, RAMP3 deficiency CAFs suppressed migration, proliferation, and metastasis in co-cultures with tumor cells in vitro and in vivo. Moreover, the activation of RAMP2 in RAMP3-/- mice suppressed both tumor growth and metastasis. Based on these results, we suggest that the upregulation of PDPN in DI-E-RAMP2-/- mice increases malignancy, while the downregulation of PDPN in RAMP3-/- mice reduces it. Selective activation of RAMP2 and inhibition of RAMP3 would therefore be expected to suppress tumor metastasis. This study provides the first evidence that understanding and targeting to AM-RAMP systems could contribute to the development of novel therapeutics against metastasis.

Lack of adrenomedullin in mouse endothelial cells results in defective angiogenesis, enhanced vascular permeability, less metastasis, and more brain damage.

Adrenomedullin (AM) is a vasodilating peptide involved in the regulation of circulatory homeostasis and in the pathophysiology of certain cardiovascular diseases. AM plays critical roles in blood vessels, including regulation of vascular stability and permeability. To elucidate the autocrine/paracrine function of AM in endothelial cells (EC) in vivo, a conditional knockout of AM in EC (AM(EC-KO)) was used. The amount of vascularization of the matrigel implants was lower in AM(EC-KO) mice indicating a defective angiogenesis. Moreover, ablation of AM in EC revealed increased vascular permeability in comparison with wild type (WT) littermates. In addition, AM(EC-KO) lungs exhibited significantly less tumor growth than littermate WT mice using a syngeneic model of metastasis. Furthermore, following middle cerebral artery permanent occlusion, there was a significant infarct size decrease in animals lacking endothelial AM when compared to their WT counterparts. AM is an important regulator of EC function, angiogenesis, tumorigenesis, and brain response to ischemia. Studies of AM should bring novel approaches to the treatment of vascular diseases.

Adrenomedullin deficiency potentiates hyperoxic injury in fetal human pulmonary microvascular endothelial cells.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of premature infants that is characterized by alveolar simplification and decreased lung angiogenesis. Hyperoxia-induced oxidative stress and inflammation contributes to the development of BPD in premature infants. Adrenomedullin (AM) is an endogenous peptide with potent angiogenic, anti-oxidant, and anti-inflammatory properties. Whether AM regulates hyperoxic injury in fetal primary human lung cells is unknown. Therefore, we tested the hypothesis that AM-deficient fetal primary human pulmonary microvascular endothelial cells (HPMEC) will have increased oxidative stress, inflammation, and cytotoxicity compared to AM-sufficient HPMEC upon exposure to hyperoxia. Adrenomedullin gene (Adm) was knocked down in HPMEC by siRNA-mediated transfection and the resultant AM-sufficient and -deficient cells were evaluated for hyperoxia-induced oxidative stress, inflammation, cytotoxicity, and Akt activation. AM-deficient HPMEC had significantly increased hyperoxia-induced reactive oxygen species (ROS) generation and cytotoxicity compared to AM-sufficient HPMEC. Additionally, AM-deficient cell culture supernatants had increased macrophage inflammatory protein 1α and 1ß, indicating a heightened inflammatory state. Interestingly, AM deficiency was associated with an abrogated Akt activation upon exposure to hyperoxia. These findings support the hypothesis that AM deficiency potentiates hyperoxic injury in primary human fetal HPMEC via mechanisms entailing Akt activation.

Adrenomedullin deficiency and aging exacerbate ischemic white matter injury after prolonged cerebral hypoperfusion in mice.

Adrenomedullin was originally isolated from pheochromocytoma cells and reduces insulin resistance by decreasing oxidative stress. White matter lesions induced by aging and hyperglycemia play a crucial role in cognitive impairment in poststroke patients. Here, we examine whether adrenomedullin deficiency and aging exacerbate ischemic white matter injury after prolonged cerebral hypoperfusion. Adrenomedullin heterozygous, wild-type young/aged mice were subjected to prolonged hypoperfusion. Prolonged cerebral hypoperfusion followed by immunohistochemical analysis was used to evaluate white matter injury. After prolonged hypoperfusion, white matter damage progressed in a time-dependent manner in AM(+/-) group compared with the wild-type group. The number of oligodendrocyte progenitor cells gradually increased after prolonged hypoperfusion, whereas oligodendrocytes decreased following a transient increase, but the ratio of increase was mild in the AM(+/-) group (P < 0.05). Oxidative stress was detected in oligodendrocytes, with a larger increase in the AM(+/-) group (P < 0.05). Aged mice showed the same tendency, but white matter damage was worse, especially in the aged AM(+/-) group. Our results demonstrated that white matter injury was increased in adrenomedullin deficiency, which induced oxidative stress. White matter injury was more exacerbated because of hyperglycemia in aged AM(+/-) group. Adrenomedullin may be an important target in the control of ischemic white matter injury.

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.

Proadrenomedullin N-terminal 20 peptide increases kinesin's velocity both in vitro and in vivo.

Intracellular cargo transport relies on microtubules and motor proteins such as kinesins and dyneins. Currently we have ample knowledge of the mechanisms by which motor proteins propel themselves along the microtubules, but little is known about intracellular factors that regulate motor speed. Here we show that proadrenomedullin N-terminal 20 peptide (PAMP) increases kinesin velocity and ATP consumption in a dose-dependent manner, using a variety of human kinesins. Structure-activity studies found that the terminal amide of PAMP is required for modulating kinesin activity and that the smallest peptide fragment retaining this role is PAMP12₋20. On the other hand, peptide fragments as small as PAMP18₋20 maintained the ability of delaying tubulin polymerization, another function previously described for PAMP, indicating that these two activities depend on different regions of the molecule. To demonstrate that these observations are also relevant in vivo, hippocampal neurons were isolated from mice lacking the gene coding for PAMP and from wild type littermates. Intravital stains followed by time-lapse microscopy analysis revealed that mitochondrial speed inside neurons lacking PAMP was significantly slower than in cells expressing the peptide. External addition of synthetic PAMP reversed this phenotype in PAMP-null neurons. Besides the obvious implications for better understanding cell biology, these results may be also relevant for the rapidly evolving discipline of nanotechnology because PAMP may be used as an accelerator of nanodevices based on microtubules and motor proteins.

Lack of adrenomedullin in the central nervous system results in apparently paradoxical alterations on pain sensitivity.

Adrenomedullin (AM) is a regulatory peptide, coded by the adm gene, which is involved in numerous physiological processes, including pain sensitivity. Previous studies have shown that intrathecal injection of AM induced hyperalgesia in the rat. Here, we explore pain sensitivity in a mouse conditional knockout for adm in neurons of the central nervous system, including the spinal cord and dorsal root ganglia. Double immunofluorescence in wild-type (WT) animals shows that AM immunoreactivity is found in calcitonin gene-related peptide-positive neurons of the dorsal root ganglia but not in neurons that bind isolectin B4. Mice lacking adm had modified expression of canonical sensorial neuropeptides, having significantly more calcitonin gene-related peptide and less substance P and enkephalin than their WT littermates. Furthermore, the spinal cord of adm knockout mice expressed higher levels of the AM receptor components, suggesting a compensation attempt to deal with the lack of afferent AM signaling. Behavioral nociceptive tests also found differences between genotypes. In the tail-flick test, which measures mostly spinal reflexes, the adm-null animals had a longer latency than their WT counterparts. On the other hand, in the hotplate test, which requires encephalic processing, mice lacking adm had shorter latencies than normal littermates. These results suggest that AM acts as a nociceptive modulator in spinal reflexes, whereas it may have an analgesic function at higher cognitive levels. This study confirms the important role of AM in pain sensitivity processing but unveils a more complex scenario than previously surmised.

Lack of adrenomedullin, but not complement factor H, results in larger infarct size and more extensive brain damage in a focal ischemia model.

Adrenomedullin (AM) and its binding protein, complement factor H (FH), are expressed throughout the brain. In this study we used a brain-specific conditional knockout for AM and a complete knockout for FH to investigate the effect of these molecules on the pathophysiology of stroke. Following 48 h of middle cerebral artery permanent occlusion, there was a statistically significant infarct size increase in animals lacking AM when compared to their wild type littermates. In contrast, lack of FH did not affect infarct volume. To investigate some of the mechanisms by which lack of AM may augment brain damage, markers of nitrosative stress, apoptosis, and autophagy were studied at the mRNA and protein levels. There was a significant increase of inducible nitric oxide synthase (iNOS), matrix metalloproteinase-9 (MMP9), fractin, and Beclin-1 in the peri-infarct area of AM-deficient mice when compared to their wild type counterparts and to contralateral and sham-operated controls. These data suggest that AM exerts a neuroprotective action in the brain and that this protection may be mediated by regulation of iNOS, matrix metalloproteases, and inflammatory mediators. In the future, substances that increase AM actions in the central nervous system may be used as potential neuroprotective agents in stroke.

Protein kinase A-dependent suppression of reactive oxygen species in transient focal ischemia in adrenomedullin-deficient mice.

This study was designed to examine the effect of adrenomedullin deficiency on cerebral infarction and the relationship between adrenomedullin and cyclic AMP-protein kinase A pathway in regulating reactive oxygen species (ROS). Adrenomedullin heterozygous and wild-type mice were subjected to 60-mins focal ischemia. We used adrenomedullin heterozygous mice because adrenomedullin homozygotes die in utero. Infarct volume, neurologic deficit scores, and immunohistochemical analyses were evaluated at several time points after ischemia. The infarct volume and neurologic deficit scores were significantly worse in adrenomedullin heterozygous mice. Significant accumulation of inducible nitric oxide, oxidative DNA damage, and lipid peroxidation was noted after reperfusion in adrenomedullin heterozygous mice. Treatment of wild-type mice with H89, a protein kinase A inhibitor, resulted in increased infarct size, and worsening of neurologic deficit score and other parameters to levels comparable to those of adrenomedullin heterozygous mice. In contrast, cilostazol, which increases cyclic AMP, rescued neurologic deficit and ROS accumulation in adrenomedullin heterozygous mice. This study showed that adrenomedullin downregulation results in increase in ROS after transient focal ischemia in mice. The results also indicated that adrenomedullin has an important function against ischemic injury through the cyclic AMP-protein kinase A pathway.

Lack of adrenomedullin in the mouse brain results in behavioral changes, anxiety, and lower survival under stress conditions.

The adrenomedullin (AM) gene, adm, is widely expressed in the central nervous system (CNS) and several functions have been suggested for brain AM. Until now, a formal confirmation of these actions using genetic models has been elusive since the systemic adm knockout results in embryo lethality. We have built a conditional knockout mouse model using the Cre/loxP approach. When crossed with transgenic mice expressing the Cre recombinase under the tubulin Talpha-1 promoter, we obtained animals with no AM expression in the CNS but normal levels in other organs. These animals lead normal lives and do not present any gross morphological defect. Specific areas of the brain of animals lacking CNS AM contain hyperpolymerized tubulin, a consequence of AM downregulation. Behavioral analysis shows that mice with no AM in their brain have impaired motor coordination and are hyperactive and overanxious when compared to their wild-type littermates. Treatment with methylphenidate, haloperidol, and diazepam did not show differences between genotypes. Circulating levels of adrenocorticotropic hormone and corticosterone were similar in knockout and wild-type mice. Animals with no brain AM were less resistant to hypobaric hypoxia than wild-type mice, demonstrating the neuroprotective function of AM in the CNS. In conclusion, AM exerts a beneficial action in the brain by maintaining homeostasis both under normal and stress conditions.