Up-regulation Of Adrenomedullin Research Articles

Single-cell transcriptomics identifies aberrant glomerular angiogenic signalling in the early stages of WT1 kidney disease.

WT1 encodes a podocyte transcription factor whose variants can cause an untreatable glomerular disease in early childhood. Although WT1 regulates many podocyte genes, it is poorly understood which of them are initiators in disease and how they subsequently influence other cell-types in the glomerulus. We hypothesised that this could be resolved using single-cell RNA sequencing (scRNA-seq) and ligand-receptor analysis to profile glomerular cell-cell communication during the early stages of disease in mice harbouring an orthologous human mutation in WT1 (Wt1R394W/+). Podocytes were the most dysregulated cell-type in the early stages of Wt1R394W/+ disease, with disrupted angiogenic signalling between podocytes and the endothelium, including the significant downregulation of transcripts for the vascular factors Vegfa and Nrp1. These signalling changes preceded glomerular endothelial cell loss in advancing disease, a feature also observed in biopsy samples from human WT1 glomerulopathies. Addition of conditioned medium from murine Wt1R394W/+ primary podocytes to wild-type glomerular endothelial cells resulted in impaired endothelial looping and reduced vascular complexity. Despite the loss of key angiogenic molecules in Wt1R394W/+ podocytes, the pro-vascular molecule adrenomedullin was upregulated in Wt1R394W/+ podocytes and plasma and its further administration was able to rescue the impaired looping observed when glomerular endothelium was exposed to Wt1R394W/+ podocyte medium. In comparative analyses, adrenomedullin upregulation was part of a common injury signature across multiple murine and human glomerular disease datasets, whilst other gene changes were unique to WT1 disease. Collectively, our study describes a novel role for altered angiogenic signalling in the initiation of WT1 glomerulopathy. We also identify adrenomedullin as a proangiogenic factor, which despite being upregulated in early injury, offers an insufficient protective response due to the wider milieu of dampened vascular signalling that results in endothelial cell loss in later disease. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Extracellular elevation of adrenomedullin, a gene associated with schizophrenia, suppresses heat shock protein 1A/1B mRNA.

Several recent gene expression studies on schizophrenia, including one using monozygotic twins discordant for the disease, have reported the upregulation of adrenomedullin (ADM), which was initially identified as a vasodilator hormone. It has been hypothesized that upregulation of ADM may be a susceptibility factor for schizophrenia, although the exact role of ADM in the central nervous system remains unclear. In this study, we used a microarray analysis to investigate the changes in global gene expression induced by the administration of exogenous ADM in SK-N-SH cells, which allowed us to evaluate the effects of elevated ADM on the central nervous system. A quantitative reverse-transcription PCR study showed that the levels of HSPA1A/1B mRNA, another gene that has been associated with schizophrenia, were significantly suppressed after exogenous ADM treatment. These results indicate that elevated ADM may be involved in the etiology of schizophrenia through the regulation of heat shock protein signaling.

Adrenomedullin Expression in Alzheimer's Brain.

Adrenomedullin (AM) is a potent vasodilator peptide highly expressed throughout the brain and originally isolated from pheochromocytoma cells. In addition to its vasoactive properties, AM is considered a neuromodulator that possesses antiapoptotic and antioxidant properties that suggest that this peptide can protect the brain from damage. In a previous study, we found that AM exerts a neuroprotective action in the brain and that this effect may be mediated by regulation of nitric oxide synthases, matrix metalloproteases, and inflammatory mediators. AM upregulation contributes to neuroprotection, but understanding the precise roles played by AM and its receptor (RAMP2) in neurodegenerative diseases including Alzheimer's disease (AD), awaits further research. In search of Alzheimer's biomarkers, the expression levels of peptides with endothelial vasodilatory action, including AM, were found to be significantly altered in mild AD or during pre-dementia stage of mild cognitive impairment. These studies concluded that ratio of AM or its precursor fragment mid-regional proAM in blood hold promise as diagnostic marker for AD. We are now presenting a study regarding the hypothesis that the AMRAMP2 system might be implicated in the pathophysiology of AD.

Regulation of adrenomedullin and nitric oxide production by periodontal bacteria.

In periodontitis the host response to bacterial challenge includes activity of the multifunctional molecules adrenomedullin (AM) and nitric oxide (NO). The aim of this study was to investigate the role of periodontal bacteria in regulating the production of these molecules from cultured cells. Regulation of AM and NO production from oral keratinocytes when challenged with culture supernatants from Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Veillonella atypica, Streptococcus salivarius and Candida albicans was examined. AM and NO were measured in cell culture supernatants using an enzyme-linked immunosorbent assay and the nitrate/nitrite (NO metabolites) Griess assay respectively. Cellular production of AM and inducible NO synthase was also analysed in target cells by immunofluorescence and Western blot analysis. The inter-relationship of AM and NO production were further investigated with macrophages. A.actinomycetemcomitans and C.rectus induced maximal levels of both AM and NO after 6 and 48h respectively from oral keratinocytes. AM production in macrophages was upregulated in response to the NO donor S-nitrosoglutathione and partially blocked by the inducible NO synthase inhibitor, N(ω) -Nitro-l-arginine methyl ester hydrochloride. Likewise, NO production was increased upon exposure to AM, while the AM receptor antagonist AM 22-52 reduced the release of NO. Pathogens associated with aggressive periodontitis, A.actinomycetemcomitans and C.rectus, were more effective than those associated with chronic periodontitis, P.gingivalis and Prev.intermedia, and commensals, S.salivarius and V.atypica, as regards the upregulation of AM and NO production from oral keratinocytes. Interaction between these molecules was also demonstrated with macrophages. Understanding the coordinated regulation of AM and NO production in response to periodontal bacteria may identify ways to promote their protective effects and minimize destructive potential.

Adrenomedullin and intermedin gene transcription is increased in leukocytes of patients with chronic heart failure at different stages of the disease

Adrenomedullin (ADM) is a vasodilatory peptide expressed in many tissues. Its levels are elevated in various diseases including chronic heart failure (CHF) and it has been suggested that the up-regulation of ADM in cardiac disease represents a protective mechanism. Similarly, intermedin (IMD), a novel member of the calcitonin/calcitonin gene-related peptide family, is considered a potential endogenous protector of the heart. Previous studies demonstrated that in CHF patients, elevated plasma concentrations of ADM and IMD reflect the patient's disease severity and prognosis, while the behavior of mRNA expression is not known. The aim of this study was to evaluate ADM/IDM transcriptomic profiling in human leukocytes of CHF patients as a function of clinical severity, assessing possible changes with respect to healthy subjects (C). mRNA expression was evaluated by Real-Time PCR and total RNA was extracted from leukocytes of C (n=8) and from CHF patients (NYHA I–II n=10; NYHA III–IV n=14) with PAXgene Blood RNA Kit. Significantly higher levels of ADM and IMD mRNA were found in CHF as a function of clinical severity (ADM: C=0.03±0.013, NYHA I–II=0.11±0.084, NYHA III–IV=11.46±4.72, p=0.037 C vs NYHA III–IV, p=0.028 NYHA I–II vs NYHA III–IV; IMD: C=0.158±0.041, NYHA I–II=0.93±0.40, NYHA III–IV=2.6±0.67, p=0.014 C vs NYHA III–IV, p=0.014 NYHA I–II vs NYHA III–IV). This study highlights, for the first time, the possibility of evaluating ADM and IMD mRNA expression in human whole blood samples by Real-Time PCR study providing further relevant information and providing a more complete interpretation of the pathophysiology of the disease.

Repression of miR-126 and upregulation of adrenomedullin in the stromal endothelium by cancer-stromal cross talks confers angiogenesis of cervical cancer

miR-126 is an endothelial-specific microRNA essential for maintaining vessel integrity during development. Its role of tumor angiogenesis in cancer stroma is unclear. This study investigated the temporal and spatial expression and the role of miR-126 in the course of cervical carcinogenesis. miR-126 was found to be mainly expressed in the stromal endothelium of the uterine cervix. This downregulation was recapitulated in a cell coculture model, wherein cross talk of cervical cancer cells and fibroblasts induced a downregulation of miR-126 in human umbilical vein endothelial cells, with consequent increase of tube formation. Coinjection of cancer-associated fibroblasts of human cervix enhanced tumorigenesis of cervical cancer cells, with an increase of microvessel density and dye retention in the tumor vasculature. In association with angiogenesis, host-originated miR-126 in these xenograft tumors was progressively downregulated, whereas supplement of the miR-126 precursor in the coinjection suppressed angiogenesis and tumor growth. A proangiogenic gene adrenomedullin (ADM), which was found to be upregulated in the stroma of cervical cancer and which localized mainly in the blood and lymphatic vessels, was identified as a target of inhibition by miR-126 at the carcinoma in situ-to-invasion stage. The study suggests a cancer stroma cross talk induced repression of miR-126 and upregulation of ADM, and probably other proangiogenic factors, to facilitate angiogenesis and invasion growth of cervical cancer.

A Role for Protein Kinase C-Dependent Upregulation of Adrenomedullin in the Development of Morphine Tolerance in Male Rats

Adrenomedullin (AM) belongs to calcitonin gene-related peptide (CGRP) family and is a pronociceptive mediator. This study investigated whether AM plays a role in the development of tolerance to morphine-induced analgesia. Repetitive intrathecal injection of morphine increased the expression of AM-like immunoreactivity (AM-IR) in the spinal dorsal horn and dorsal root ganglion (DRG) neurons. Ganglion explant culture study showed that this upregulation of AM-IR was μ-opioid receptor dependent through the use of another agonist, fentanyl, and a selective antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)). The coadministration of the selective AM receptor antagonist AM(22-52) markedly attenuated the development of morphine tolerance, associated thermal hyperalgesia, and increase in AM-IR. A likely autocrine mechanism is supported by the finding that AM-IR is colocalized with AM receptor components in DRG neurons. Furthermore, opiate-induced increase in AM content was blocked by protein kinase C (PKC) inhibitors, whereas a PKC activator increased AM synthesis and release. A treatment with AM(22-52) also inhibited increases in the expression of CGRP-IR in the spinal cord and DRGs as well as in culture ganglion explants, whereas exposure to CGRP failed to alter AM content. Together, these results reveal that a sustained opiate treatment induces an upregulation of AM through the activation of μ-opioid receptors and the PKC signaling pathway. This phenomenon contributes to the development of tolerance to the antinociceptive effects of opiates at least partially via the upregulation of CGRP. Targeting AM and its receptors should be considered as a novel approach to preserve the analgesic potency of opiates during their chronic use.

Upregulation of adrenomedullin in the spinal cord and dorsal root ganglia in the early phase of CFA-induced inflammation in rats

Adrenomedullin (AM), a member of calcitonin gene-related peptide (CGRP) family, has been demonstrated to be a pronociceptive mediator [28]. This study was undertaken to investigate the role of AM in a model of complete Freund’s adjuvant (CFA)-induced inflammatory pain. Injection of CFA, but not of saline, in the unilateral hindpaw produced an increase in the expression of AM-like immunoreactivity (AM-IR) in laminae I–II of the spinal cord as well as in small- and medium-sized dorsal root ganglion (DRG) neurons at 48 h. The content of AM in DRG on the side ipsilateral to CFA injection started to increase at 4 h and remained at high levels at 24 and 48 h. The selective antagonist of AM receptors, AM 22–52, administered intrathecally (i.t.) 24 h after CFA injection inhibited inflammation-associated hyperalgesia in a dose-dependent manner (2, 5 and 10 nmol). Impressively, this anti-hyperalgesic effect lasted for at least 24 h. I.t. administration of AM 22–52 (10 nmol) also reversed CFA-induced increase in AM-IR in the spinal dorsal horn and DRG. Furthermore, blockade of AM receptors abolished CFA-induced changes in the expression and content of CGRP-like immunoreactivity in these regions. Taken together, our results suggest that the upregulation of AM in DRG neurons contributes to the development of inflammatory pain, and this effect is mediated, at least in part, by enhancing the expression and release of CGRP. Blocking AM receptor downstream signaling effects using antagonists has the potential of relieving pain following the induction of inflammation.

Lipopolysaccharides stimulate adrenomedullin synthesis in intestinal epithelial cells: Release kinetics and secretion polarity

Adrenomedullin (AM), a potent vasodilator peptide initially isolated from a human pheochromocytoma, functions as an antimicrobial peptide in host defense. In this study, we investigated changes in AM levels in intestinal epithelial cells and the mechanism of its secretion and cellular polarity after exposure to lipopolysaccharides (LPS). When a rat small intestinal cell line (IEC-18 cells) was exposed to LPS, enzyme-linked immunosorbent assay revealed a dose-dependent increase in AM together with an increase in AM mRNA expression, as determined by real-time polymerase chain reaction. Up-regulation of AM by LPS was dose-dependently inhibited by LY294002, PD98059, SP600125 and calphostin-C, suggesting the involvement of the phosphatidylinositol 3 kinase, extracellular signal-regulated kinase, c-Jun NH2-terminal kinase and protein kinase C pathways, respectively, in this process. When polarized IEC-18 cells in a Transwell chamber were stimulated with LPS, AM secretion was directed primarily toward the side of LPS administration (either the apical or basolateral side). In situ hybridization revealed that AM mRNA was expressed in epithelial cells and in the connective tissue in the lamina propria of the jejunum after intraperitoneal or oral administration of LPS. Higher levels of AM mRNA expression were observed in rats treated with LPS via the intraperitoneal route, compared with those treated via the oral route. These findings suggest that intestinal AM plays an important role in mucosal defense in the case of intestinal luminal infection, as well as in the modulation of hemodynamics in endotoxemia.

Adrenomedullin Expression is Up‐regulated by Acute Hypobaric Hypoxia in the Cerebral Cortex of the Adult Rat

Hypobaric hypoxia can produce neuropsychological disorders such as insomnia, dizziness, memory deficiencies, headache and nausea. Here we report the changes in adrenomedullin (AM) expression observed in rats exposed to hypobaric hypoxia and different times of reoxygenation. AM immunoreactivity was transiently elevated in the cerebral cortex after 7 h of exposure to a simulated altitude of 8325 m (27 000 ft). This higher expression was seen in all pyramidal cells and in a subset of small interneurons. AM-positive nonpyramidal neurons contained also calbindin and calretinin, but no parvalbumin immunoreactivity, thus identifying them as bipolar and double bouquet cells. Small blood vessels and related astroglia also became immunoreactive following the hypobaric insult. AM up-regulation decreased progressively with the time of reoxygenation, reaching almost control levels after 5 days. Real-time PCR quantification of AM mRNA and Western blotting confirmed the up-regulation of AM expression following hypobaria. In addition, hypobaria modulates alternative splicing of the AM gene resulting in a higher production of AM. Our data show that AM expression regulation constitutes a cortical response to hypobaria, suggesting that AM modulation may provide new therapeutic avenues to prevent and/or treat the symptoms produced by hypobaria.

Adrenomedullin protects against oxidative stress-induced podocyte injury as an endogenous antioxidant

We previously reported that puromycin aminonucleoside (PAN) increased adrenomedullin (AM) secretion and AM mRNA expression in podocytes, through overproduction of oxidative stress. To clarify the cytoprotective role of AM as antioxidative and antiapoptotic substance in podocytes, we investigated the effect of exogenous AM and AM antagonist on PAN-induced apoptosis in conditionally immortalized murine podocytes. The expression of AM, RAMP 2 and RAMP 3 was investigated using real-time PCR, western blotting analysis and immunofluorescence microscopy. Reactive oxygen species (ROS) production was measured by CM-H(2)DCFDA fluorescence intensity method. The percentage of apoptotic cells was measured by Hoechst 33342 staining. PAN (100 microg/ml) significantly (P < 0.01) increased ROS production, associated with an increase in apoptosis; the percentage of apoptotic cells is 5.3% + 0.05% (P < 0.01) with 36 h treatment of PAN compared to 0.24 + 0.16% with no treatment. Several antioxidants could markedly reduce PAN-induced apoptosis in cultured podocytes, suggesting that PAN-induced apoptosis might be attributable to the overproduction of ROS. Accordingly, the administration of exogenous AM (10(-6) M) could significantly reduce not only ROS production via a PKA-dependent pathway, but also the resultant apoptosis induced by PAN. AM antagonists, CGRP8-37, augmented PAN-induced apoptosis, associated with increased ROS production, 2.2- and 2.3-Fold, respectively. RAMP 2 and RAMP 3 could be detected in podocytes and glomeruli. This suggests that ROS-induced up-regulation of AM with PAN could counteract ROS-induced apoptosis, by the suppression of ROS production. Therefore, AM might have the endogenous antioxidant potential to protect against ROS-induced podocyte injury.

Differential regulation of components of the AM/IMD receptor system in nitric oxide-deficient cardiomyocytes

In myocardial ischemia and remodeling produced by NO synthase inhibition, upregulation of adrenomedullin (AM) and receptor components, RAMP2 and 3, in hypertrophied cardiomyocytes is prevented by blood pressure (BP) lowering (hydralazine+thiazide); that of RAMP1 and intermedin (IMD) is not. The hypothesis is that IMD/RAMP1 are regulated by hypoxia. Effects were examined of (i) anti-oxidant intervention; (ii) intervention to alleviate pressure loading and ischemic injury concurrently. l-NAME (35 mg/kg/day) was given to rats for 8 weeks±(in mg/kg/day) (i) vitamin C (25)+Tempol (25) [VT] or (ii) atenolol (25)+nifedipine (20) [AN].

The Role of Adrenomedullin in Obstructive Sleep Apnea

Obstructive sleep apnea (OSA) is associated with increased cardiovascular morbidity and mortality (1). Although cardiovascular morbidity in patients who have OSA is not only due to atherosclerosis, atherosclerosis may play important roles in OSA-associated cardiovascular mortality because deaths from cardiovascular diseases in patients with untreated sleep apnea are due mainly to acute myocardial infarction and stroke (2). Recent studies suggest that ongoing inflammatory responses are involved in the pathogenesis of atherosclerosis. Several inflammatory markers, including high sensitivity Creactive protein (CRP), tumor necrosis factor-α, interleukin6, and matrix metalloproteinase-9, are reportedly elevated in patients with OSA (3‐5). Moreover, carotid intima-media thickness and the percentage of silent brain infarction are also increased in patients with OSA (6, 7). These findings suggest that OSA may increase the risks of atherosclerosis and cardiovascular diseases. OSA is characterized by repetitive periods of upper airway collapse and results in cyclic periods of hypoxia/reoxygeneration that cause the increased generation of oxygen species by oxidative stress (8). In fact, production of oxygen species from neutrophils and monocytes obtained from patients with OSA has been shown to be enhanced, and treatment with nasal continuous positive airway pressure (nCPAP) significantly decreased the production of oxygen species from these cells (9). Because repeated apnea-related hypoxia is present in patients with OSA, oxidative stress may promote both oxidative stress and inflammation (10). Therefore, oxidative stress may contribute to the cardiovascular risk profile in patients with OSA. Adrenomedullin (AM) can be produced by a variety of cell types, including endothelial cells and vascular smooth muscle cells (11, 12). AM acts as a potent vasodilator. Several stimuli, such as hypoxia, shear stress, and inflammatory cytokines, can induce AM production. Because these stimuli are increased in patients with OSA, a few investigators have tried to measure the circulating levels of AM in patients with OSA and to evaluate the effect of nCPAP on the levels of AM. However, the results of these reports have been inconsistent (13, 14). In this issue of Hypertension Research, Yamamoto and colleagues provide the further important observation that circulating levels of AM were elevated and were associated with the magnitude of oxyhemoglobin desaturation in highly selected patients with OSA by excluding several comorbid conditions that may have influence on the levels of AM (15). Furthermore, they also demonstrated that oxidative stress as measured by reactive oxygen species (ROS) production by leukocytes was associated with the levels of AM. Moreover, treatment with nCPAP reduced all these parameters in patients with OSA. Therefore, in addition to hypoxia, shear stress, and inflammatory cytokines, ROS may induce the production of AM. The upregulation of AM presented in this study constitutes an adaptive counteractive mechanism to protect against cardiovascular diseases in patients with OSA. Further studies are necessary to elucidate the precise role of circulating AM on hypertension as well as atherosclerosis in patients with OSA.

Differential Expression of Components of the Cardiomyocyte Adrenomedullin/Intermedin Receptor System following Blood Pressure Reduction in Nitric Oxide-Deficient Hypertension

Adrenomedullin (AM) and intermedin (IMD; adrenomedulln-2) are vasodilator peptides related to calcitonin gene-related peptide (CGRP). The actions of these peptides are mediated by the calcitonin receptor-like receptor (CLR) in association with one of three receptor activity-modifying proteins. CGRP is selective for CLR/receptor activity modifying protein (RAMP)1, AM for CLR/RAMP2 and -3, and IMD acts at both CGRP and AM receptors. In a model of pressure overload induced by inhibition of nitric-oxide synthase, up-regulation of AM was observed previously in cardiomyocytes demonstrating a hypertrophic phenotype. The current objective was to examine the effects of blood pressure reduction on cardiomyocyte expression of AM and IMD and their receptor components. Nomega-nitro-L-arginine methyl ester (L-NAME) (35 mg/kg/day) was administered to rats for 8 weeks, with or without concurrent administration of hydralazine (50 mg/kg/day) and hydrochlorothiazide (7.5 mg/kg/day). In left ventricular cardiomyocytes from L-NAME-treated rats, increases (-fold) in mRNA expression were 1.6 (preproAM), 8.4 (preproIMD), 3.4 (CLR), 4.1 (RAMP1), 2.8 (RAMP2), and 4.4 (RAMP3). Hydralazine/hydrochlorothiazide normalized systolic blood pressure (BP) and abolished mRNA up-regulation of hypertrophic markers sk-alpha-actin and BNP and of preproAM, CLR, RAMP2, and RAMP3 but did not normalize cardiomyocyte width nor preproIMD or RAMP1 mRNA expression. The robust increase in IMD expression indicates an important role for this peptide in the cardiac pathology of this model but, unlike AM, IMD is not associated with pressure overload upon the myocardium. The concordance of IMD and RAMP1 up-regulation indicates a CGRP-type receptor action; considering also a lack of response to BP reduction, IMD may, like CGRP, have an anti-ischemic function.

Upregulation of adrenomedullin and its receptor components during cardiomyocyte hypertrophy induced by chronic inhibition of nitric oxide synthesis in rats

Adrenomedullin may provide a compensatory mechanism to attenuate left ventricular hypertrophy (LVH). Nitric oxide synthase inhibition, induced by chronic administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) to rats, induces cardiac hypertrophy in some, but not all cases; there are few reports of direct assessment of cardiomyocyte parameters. The objective was to characterize hypertrophic parameters in left (LV) and right ventricular (RV) cardiomyocytes after administration of L-NAME to rats for 8 wk and to determine whether adrenomedullin and its receptor components were upregulated. After treatment with L-NAME (20 and 50 mg x kg(-1) x day(-1)), compared with nontreated animals, 1) systolic blood pressure increased (by 34.2 and 104.9 mmHg), 2) heart weight-to-body wt ratio increased 24.1% at the higher dose (P < 0.05), 3) cardiomyocyte protein mass increased (P = NS), 4) cardiomyocyte protein synthesis ([14C]phenylalanine incorporation) increased (P < 0.05), 5) expression of skeletal alpha-actin, atrial natriuretic peptide, brain natriuretic peptide, and ET-1 mRNAs was enhanced (P < 0.05) in LV but not RV cardiomyocytes at 20 and 50 mg x kg(-1) x day(-1), respectively, and 6) expression of adrenomedullin, receptor activity-modifying protein 3 (RAMP3), and RAMP2 (but not calcitonin receptor-like receptor and RAMP1) mRNAs was increased by L-NAME (20 mg x kg(-1) x day(-1)) in LV. In conclusion, L-NAME enhanced protein synthesis in both LV and RV cardiomyocytes but elicited a hypertrophic phenotype accompanied by altered expression of the counterregulatory peptide adrenomedullin and receptor components (RAMP2, RAMP3) in LV only, indicating that the former is due to impaired nitric oxide synthesis, whereas the phenotypic changes are due to pressure overload.

Gene expression profiling of pulpal tissue reveals the molecular complexity of dental caries

High-throughput characterisation of the molecular response of pulpal tissue under carious lesions may contribute to improved future diagnosis and treatment. To identify genes associated with this process, oligonucleotide microarrays containing approximately 15,000 human sequences were screened using pooled total RNA isolated from pulpal tissue from both healthy and carious teeth. Data analysis identified 445 genes with 2-fold or greater difference in expression level, with 85 more abundant in health and 360 more abundant in disease. Subsequent gene ontological grouping identified a variety of processes and functions potentially activated or down-modulated during caries. Validation of microarray results was obtained by a combination of real-time and semi-quantitative PCR for selected genes, confirming down-regulation of Dentin Matrix Protein-1 (DMP-1), SLIT 2, Period-2 (PER 2), Period-3 (PER 3), osteoadherin, Glypican-3, Midkine, activin receptor interacting protein-1 (AIP 1), osteoadherin and growth hormone receptor (GHR), and up-regulation of Adrenomedullin (ADM), Interleukin-11 (IL-11), Bone sialoprotein (BSP), matrix Gla protein (MGP), endothelial cell growth factor-1 (ECGF 1), inhibin beta A and orosomucoid-1 (ORM 1), in diseased pulp. Real-time PCR analyses of ADM and DMP-1 in a panel of healthy and carious pulpal tissue and also in immune system cells highlighted the heterogeneity of caries and indicated increased expression of ADM in neutrophils activated by bacterial products. In contrast, DMP-1 was predominantly expressed by cells native to healthy pulpal tissue. This study has greatly extended our molecular knowledge of dental tissue disease and identified involvement of genes previously unassociated with this process.

Adrenomedullin Is Highly Expressed in Blood Monocytes Associated with Acute Kawasaki Disease: A Microarray Gene Expression Study

Kawasaki disease (KD) is an acute inflammatory disorder of children frequently associated with the development of coronary artery abnormalities. Although a great deal is known about inflammatory and immune responses in acute KD, the mechanisms linking the immune response to vascular changes are not known. To gain further insight into this process, we performed a microarray gene expression analysis on RNA isolated from the peripheral blood mononuclear cells of four patients with KD during both their acute and convalescent phases. Forty-seven genes of 7129 genes examined showed an increased expression in three or all four patients in the acute compared with the convalescent phase of KD. Fourteen of these genes were significantly (p < 0.05) up-regulated, including several inflammatory response genes (e.g. S-100 A9 protein) and also anti-inflammatory genes (e.g. TSG-6). Of greatest interest, the adrenomedullin (ADM) gene, known to be associated with coronary artery vasodilation, was up-regulated in the acute phase of KD (p = 0.024). Up-regulation of ADM in the acute phase of KD was confirmed in peripheral blood mononuclear cells of 11 additional KD patients by reverse transcriptase-PCR (p < 0.01). Isolated blood monocytes but not lymphocytes were demonstrated by real-time PCR to have increased ADM mRNA (p = 0.01). Plasma ADM protein level in 32 additional KD patients was also confirmed to be higher in acute KD compared with convalescent KD (p < 0.032). It is interesting that from microarray results, other molecules known to be associated with coronary dilation, including nitric oxide, prostacyclin, acetylcholine, bradykinin, substance P, and serotonin, were not elevated in acute KD. Our current study suggests that ADM-expressing monocytes that infiltrate the coronary vascular wall may be the cause of coronary dilation in the acute phase of KD.

Changes of adrenomedullin and receptor activity modifying protein 2 (RAMP2) in myocardium and aorta in rats with isoproterenol-induced myocardial ischemia

Adrenomedullin is a potent vasodilator peptide originally isolated from a pheochromocytoma. Recently, a novel adrenomedullin receptor has been identified as a complex of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 2 (RAMP2). To explore the pathophysiological roles of adrenomedullin and its receptor component RAMP2 in ischemic cardiovascular diseases, we studied the changes of adrenomedullin and RAMP2 mRNA in myocardium and aorta in rats with isoproterenol (ISO)-induced myocardial impairment. In ISO-treated rats, heart became enlarged markedly, the ratio of heart to body weight was increased by 54% ( P<0.01), and myocardial malondialdehyde content and plasma lactate dehydrogenase activity was elevated by 43% ( P<0.01) and 138% ( P<0.01), respectively. Immunoreactive adrenomedullin (ADM) in plasma, myocardium and aorta was augmented by 116.7% ( P<0.01), 50.8% ( P<0.01) and 12.5% ( P>0.05), respectively. ADM mRNA in myocardium and aorta was increased by 96.8% ( P<0.01) and 38.5% ( P<0.01), respectively. RAMP2 mRNA in myocardium and aorta was increased by 19.6% ( P<0.05) and 15.8% ( P<0.01), respectively. These results suggest that the increase of ADM level and the up-regulation of ADM and RAMP2 gene in myocardium and aorta may be significant in the pathogenesis of ischemic myocardiopathy.

Adrenomedullin has multiple roles in disease stress: development and remission of the inflammatory response.

The upregulation of adrenomedullin (AM) gene expression and increases in systemic circulatory as well as localized tissue AM concentrations is well coordinated with the onset and progression of trauma, infection, and sepsis. As such, the coordinated change in AM suggests a key role for this peptide in the inflammatory response. By clinical definition, the process of inflammation constitutes an orchestrated cascade of localized tissue and systemic responses to immunological challenges. Classical responses to the onset of disease stresses are manifested in the timely elaboration of humoral, blood-borne signal effectors (such as adrenocortical and locally produced tissue hormones, immune cytokines, and inorganic signals such as nitric oxide) as well as patterned migration and infiltration of circulating bone marrow-derived cells (mononuclear cells such as monocyte-macrophages and polymorphonuclear cells like neutrophils) largely associated with or delivered through the vascular system. The body's attempts to combat acute infection to restore homeostatic equilibrium are further compromised by underlying disease situations. Atherosclerosis, diabetes, and cardiovascular disease, as well as nutritional metabolic derangements and persistent subclinical infection perturb the regulatory feedback loops necessary for proper control of response effectors like hormones and cytokines. When imbalances occur, tissue necrosis can ensue as driven by free radical damage to cell components. A true appreciation of the inflammatory response can only be grasped through an integrative approach in which the relationship between the different physiological systems is viewed in terms of a changing, dynamic interaction. In essence, the inflammatory response can be thought of in three phases: a period of severity assessment, a period of remediation, and a period of homeostatic restoration. Indeed, AM has differential effects on cellular metabolism, immune function, endocrine function, and cardiovascular function. This peptide appears to play a pivotal role in both reprioritizing the biological needs of tissues and organs during the three phases of inflammatory response as well as a role in restoring homeostatic equilibrium to the body.

Adrenomedullin, an endogenous peptide, counteracts cardiovascular damage.

Adrenomedullin (AM), a potent vasodilator peptide, is produced by posttranslational splicing of pro-adrenomedullin together with proadrenomedullin N-terminal 20 peptide (PAMP), another hypotensive peptide. Although both AM and PAMP have the potential not only to decrease blood pressure but also to protect organs from damage, there is no direct evidence for their individual physiological roles in vivo. Using knockout mice with the disruption of AM peptide alone, we investigated the organ-protective effect of AM. Although the AM(-/-) mutation in mice was embryonic lethal without any apparent phenotypic changes, AM(+/-) mice were viable and fertile; plasma and organ AM concentrations were almost half of those in AM(+/+) mice. With the administration of angiotensin II (Ang II) on a high-salt diet for 12 days, marked perivascular fibrosis and intimal hyperplasia were found in coronary arteries of Ang II/salt-treated AM(+/-) mice, without the AM upregulation that was observed in Ang II/salt-treated AM(+/+) mice. In AM(+/-) mice, Ang II/salt loading increased both urinary excretion of 8-hydroxydeoxyguanosine and isoprostane, markers of oxidative stress. Consistently, immunostaining of both p67phox and gp91phox, subunits of NAD(P)H oxidase and 3-nitrotyrosine, the metabolites of reactive oxygen species (ROS), and the generation of ROS measured by electron spin resonance spectroscopy apparently increased in the Ang II/salt-treated heart. These data suggested that the overproduction of oxidative stress might be involved in the cardiovascular changes induced by Ang II/salt loading. The evidence presented supports the hypothesis that endogenous AM possesses a protective action against cardiovascular damage, possibly through the inhibition of oxidative stress production.