Human Adrenomedullin Gene Research Articles

Expression of human adrenomedullin gene in gastric adenocarcinoma and construction and identification of adrenomedullin overexpression vector and adrenomedullin-shRNA vector

Expression of human adrenomedullin gene in gastric adenocarcinoma and construction and identification of adrenomedullin overexpression vector and adrenomedullin-shRNA vector

The Krüppel-like factor KLF15 inhibits transcription of the adrenomedullin gene in adipocytes

KLF15 (Krüppel-like factor 15) plays a key role in adipocyte differentiation and glucose transport in adipocytes through activation of its target genes. We have now identified six target genes regulated directly by KLF15 in 3T3-L1 mouse adipocytes with the use of a combination of microarray-based chromatin immunoprecipitation and gene expression analyses. We confirmed the direct regulation by KLF15 of one of these genes, that for adrenomedullin, with the use of a luciferase reporter assay in 3T3-L1 preadipocytes and adipocytes. Such analysis revealed that the most proximal CACCC element in the promoter of the human adrenomedullin gene (located in the region spanning nucleotides −70 and −29) is required for trans-inhibition by KLF15. Furthermore, chromatin immunoprecipitation showed that KLF15 binds to this region of the human adrenomedullin gene promoter in cultured human adipocytes. These results thus implicate KLF15 in the regulation of adrenomedullin expression in adipose tissue.

Synergistic activation of the human adrenomedullin gene promoter by Sp1 and AP-2α

Adrenomedullin (AM) is a potent vasodilator peptide, which is ubiquitously expressed and has various biological actions, such as proliferative action and anti-oxidative stress action. AM expression is induced by various stresses, such as hypoxia and inflammatory cytokines, and during cell differentiation. The human AM gene promoter region (−70/−29) contains binding sites for stimulatory protein 1 (Sp1) and activator protein-2α (AP-2α), and has been shown to be important for the AM gene expression during cell differentiation to macrophages or adipocytes. We here show that Sp1 and AP-2α synergistically activate the AM gene promoter. Co-transfection of the reporter plasmid containing the AM promoter region (−103/−29) with Sp1 and AP-2α expression plasmids showed that Sp1 and AP-2α synergistically increased the promoter activity in HeLa cells. Sp1 or AP-2α alone caused only small increases in the promoter activity. EMSA showed that Sp1 bound to the promoter region (−70/−29), whereas AP-2α bound to a more upstream promoter region (−103/−71). Thus, the synergistic activation of the human AM gene promoter by Sp1 and AP-2α may be mediated by the binding of Sp1 to the promoter region (−70/−29) and the interaction with AP-2α, which binds to the promoter region (−103/−71).

Adrenomedullin in adipocyte differentiation of human mesenchymal stem cells

Expression of adrenomedullin (AM), a potent vasodilator peptide, was studied during adipocyte differentiation of human mesenchymal stem cells (hMSCs). Immunoreactive AM levels in the medium were increased at day 4 and 8 of the adipocyte differentiation. Northern blot analysis showed increased expression of AM mRNA in hMSCs-derived adipocytes at day 4, 8, 12, and 18. Transient transfection assay showed that the promoter activity was higher in hMSCs-derived adipocytes than in hMSCs, when cells were transfected with plasmids containing a cis-acting region (−70/−29) of the human AM gene. Electrophoretic mobility shift assay showed that specific bands bound to the region (−70/−29) in hMSCs-derived adipocytes but not in hMSCs, and were abolished by the stimulatory protein 1 (Sp1) antibody. The present study has shown that AM expression is up-regulated during adipocyte differentiation of hMSCs probably via the interaction between Sp1 or Sp1-related factor(s) and the AM promoter region (−70/−29).

Potential of Gene Therapy Strategy for the Treatment of Hypertension

Hypertension is one of the most important risk factors for cardiovascular diseases (CVDs). CVD extends across all populations and represents the leading cause of morbidity and mortality in industrialized countries. Hypertension increases the risk of peripheral arterial disease, cardiomyopathy, stroke, and renal failure. The disease affects &50 million Americans and goes undetected in at least one third of the population. In spite of the tremendous progress made by pharmacotherapy in the management and control of hypertension, there has been a steady escalation in its prevalence during the last decade. This has led many to conclude that traditional pharmacotherapy has reached an intellectual plateau and that novel and innovative approaches must be sought for the treatment, control, and possible cure of hypertension. As a result, efforts of our group and those of many others have been diverted to explore the use of gene transfer and gene therapy strategies for a long-term control of hypertension. We have argued that gene therapy offers potentially significant improvements and benefits over the use of traditional pharmacotherapy: (1) noncompliance by patients can be significantly reduced or even eliminated because of the fact that a single treatment involving gene transfer could remain effective for months or even years; (2) side effects associated with pharmacotherapy can be minimized as a result of specific targeting of a therapeutic gene in a given tissue and optimally influencing cardiovascular pathophysiology on an individual patient basis; and (3) with its potential to produce long-term beneficial outcomes in end-organ damage, the gene therapy strategy could lead to a cure of hypertension and its related pathophysiology. It is noteworthy to mention that current pharmacotherapies are often unable to reverse end-organ damage associated with hypertension. In order for the gene therapy for hypertension to be successful, one must provide conceptual support of its efficacy in …

Human adrenomedullin gene silencing by short interfering RNAs: A preliminary study

Adrenomedullin (AM) is a regulatory peptide widely expressed, along its receptors, in cells and tissues, of which it controls many basic and specific functions acting in an autocrine-paracrine manner. However, the unequivocal demonstration of the physiological relevance of the regulatory role of AM would require the study of cells where endogenous AM system had been suppressed. Hence, we developed a protocol to silence the human AM gene by transfection with short interfering RNAs (siRNAs). Eight possible AM-siRNA sequences were designed: six siRNAs were synthesized in our laboratory and two were provided by Ambion. As positive control the suppression of the glyceraldehyde-3-phosphate dehydrogenase (GADPH) gene was tested using the Ambion Silencer GADPH siRNA kit. Cultured human embryonal kidney cell line HEK-293 and human umbilical vein endothelial cells (HUVECs) were transfected using either the Qiagen or the Ambion transfection reagent, and transfection visualization, carried out using Cy3-labeled siRNA and examining red fluorescence within the cells, showed that the former reagent was the most efficient. AM-gene silencing was determined in HUVECs by measuring AM mRNA levels in transfected and control cells by real-time polymerase chain reaction. Only Ambion siRNAs were effective, and the best AM-gene silencing (about 80%) was observed 48 or 72 h after transfection with 3 or 6 microg of siRNAs. The conclusion is drawn that siRNA technology can be useful in the investigations on AM functions, but that the complete suppression of the AM-gene transcription is very difficult to obtain.

Haplotype-Based Case-Control Study Revealing an Association between the Adrenomedullin Gene and Proteinuria in Subjects with Essential Hypertension

Adrenomedullin (AM) has various physiological actions on the cardiovascular system, including vasodilatation, diuresis, natriuresis, inhibition of aldosterone secretion, and increases of the cardiac output, all of which cause hypotension. Since AM plays a role in the pathophysiology of vascular diseases, genes controlling AM might be involved in the development and etiology of essential hypertension (EH). However, there have been few studies examining the relationship between the AM gene and hypertension. The aims of this study were to genotype some of the genetic markers for the human AM gene in Japanese subjects, and via a haplotype-based case-control study, assess the association between and the AM gene and EH or its risk factors, such as hyperlipidemia, renal damage, and proteinuria. We genotyped 205 EH patients and 210 age-matched normotensive (NT) individuals for two single nucleotide polymorphisms of rs4399321, rs7944706 and a microsatellite polymorphism located approximately 5,400 base pairs downstream of the 3' end of the human AM gene. The overall distribution in each variant and haplotype did not significantly differ between the two groups. However, after dividing the groups into those subjects with and without proteinuria, the haplotype analysis revealed a positive association. In conclusion, a possible mutation linked to the haplotype may indicate a genetic predisposition for proteinuria in EH.

Adrenomedullin inhibits prostate cancer cell proliferation through a cAMP-independent autocrine mechanism

Adrenomedullin (AM) is a multifunctional peptide expressed in the normal and malignant prostate, and in prostate cancer cells. To elucidate the potential role of AM in prostate cancer, we have transfected the human AM gene into PC-3, DU 145, and LNCaP prostate cancer cells. Northern blot, Western blot, and radioimmunoassay techniques confirmed an increase in the synthesis and secretion of the 6 kDa mature peptide, in the AM-transfected clones. Proliferation and cell cycle assays demonstrated that AM overexpression inhibited cell proliferation in PC-3 and LNCaP cells through a G0/G1 cell cycle arrest, but not in DU 145 cells. In vivo growth assays also confirmed that, at least in PC-3, AM produced a very significant reduction of tumor volume. In addition, the three cell lines expressed the CL/RCP/RAMP-2 receptor complex by RT-PCR, which suggests that AM peptide acts through an autocrine loop in prostate cancer cells. Although cAMP elevation is the most common pathway involved in AM signalling, stimulation of PC-3, DU 145, and LNCaP with synthetic AM did not increase intracellular cAMP. However, short-term stimulation of PC-3 cells with synthetic AM increased ERK1/2 activation. On the contrary, long-term stimulation, or AM overexpression, caused a reduction in the basal activation of ERK1/2. In summary, our results demonstrate that AM (either overexpressed or exogenously added) causes an inhibition of prostate cancer cell growth. This inhibition does not depend on changes in intracellular cAMP levels, but may be related to ERK1/2 activation.

Regulation of adrenomedullin gene transcription and degradation by the c-myc gene.

Adrenomedullin, a vasodilatory peptide originally isolated from pheochromocytoma, is known to regulate cell growth, apoptosis, and migration. Overexpression of the c-myc oncogene has been shown to suppress the mouse adrenomedullin gene via the initiator element. We investigated whether c-myc regulates rat and human adrenomedullin genes because there appears to be no initiator elements in their promoter regions. Transactivation of the human adrenomedullin gene by c-myc was demonstrated using a luciferase reporter construct containing an upstream sequence. Using a panel of isogenic rat fibroblast cell lines with differential c-myc expression obtained by targeted homologous recombination, we found markedly elevated adrenomedullin transcript levels in cells stably overexpressing c-myc but a minimal decrease in two independent cell lines containing a homozygous null deletion of c-myc. Degradation of adrenomedullin mRNA was enhanced by a c-myc transgene, resulting in a relatively reserved increase in cellular secretion of adrenomedullin-like immunoreactivity. These results indicate that c-myc transactivates rat and human adrenomedullin genes and accelerates the degradation rate of adrenomedullin mRNA. However, c-myc is not essential for basal expression of the adrenomedullin gene.

428. Adrenomedullin Gene Delivery Attenuates Renal Damage and Hypertension in Spontaneous Hypertension Rats

Background: Human adrenomedullin (AM) is a potent vasodilating peptide with natriuetic activity originally discovered in human pheochromocytoma. AM has been implicated as an important regulator in the renal and cardiovascular systems. By using a new double stranded adeno-associated virus vector (dsAAV), human AM gene can get more rapid and highly efficient transduction than before. In this study, we employed a somatic gene delivery approach to explore its potential hypotensive and protective roles in spontaneous hypertension rats (SHR). Method: The dsAAV was used to harbor human AM gene (or GFP gene) cDNA, and dsAAV-AM and dsAAV-GFP was administered intravenously to two group (n=8 for each) of male SHR (2 × 1010 p.f.u. /rat) respectively. The caudal arterial pressure was measured using tail cuff device once a week, and 24-hour urine and fasting blood were collected to determine levels urinary trace of albumin (Alb) and AM concentrations every 2 weeks. By inserting a catheter into the left ventricle, the heart rate (HR), the left ventricle systolic pressure (LVP) and the maximal/minimum rate of LVP (±LV dp/dtmax) were measured before animals were sacrificed (16 weeks after gene delivery). The main organs were obtained for histological analysis. Results: The dsAAV-AM delivery significantly decreased blood pressure in SHR (up to 28mmHg), which lasted for all experimental duration, compared with the control group (dsAAV-GFP). Expression of human AM mRNA was detected in the heart, renal and liver after human AM gene delivery. Analysis of blood by enzyme-linked immunosorbent assay (ELISA) displayed stable AM gene expression after gene deliveries until experiments ended in dsAAV-AM group, but AM was undetectable in dsAAV-GFP group. In dsAAV-AM group, the level of Alb was much more higher than the dsAAV-GFP group. After human AM gene delivery, the hemodynamic variables deterioration was prevented, compared with the dsAAV-GFP group. Human AM gene delivery significantly attenuated increases fibrosis in the heart and glomerular sclerosis, tubular injuries and protein casts in the kidney. Conclusions: DsAAV-AM can result in significant decrease of blood pressure in SHR, improve renal function and prevent cardiovascular remodeling due to hypertension. These results suggest that dsAAV-AM may have significance in therapeutic applications for hypertension.

Microsatellite DNA polymorphism of human adrenomedullin gene in type 2 diabetic patients with renal failure

Adrenomedullin (AM) is a hypotensive peptide widely produced in the cardiovascular organs and tissues such as the heart, kidney, and the vascular cells. We have previously cloned and sequenced the genomic DNA encoding human AM gene, and determined that the gene is located in the short arm of chromosome 11. The 3'-end of the gene is flanked by the microsatellite marker of cytosine adenine (CA) repeats. In this study, we investigated the association between DNA variations in AM gene and the predisposition to develop nephropathy in type 2 diabetes mellitus. Genomic DNA was obtained from the peripheral leukocytes of 233 normal healthy subjects (NH), 139 type 2 diabetic patients on hemodialysis (DM-HD), 106 control patients with type 2 diabetes without nephropathy (DM-C) and 318 hemodialysis patients due to chronic glomerulonephritis (CGN-HD). The genomic DNA was subject to polymerase chain reaction (PCR) using a fluorescence-labeled primer, and the number of CA repeats were determined by polyacrylamide gel electrophoresis (PAGE). In our Japanese subjects, there existed four types of alleles with different CA-repeat number; 11, 13, 14, and 19. The frequencies of these alleles were 11: 27.7%, 13: 32.8%, 14: 35.6%, and 19: 3.9% in NH. These allele frequencies were not significantly different in DM-C and CGN-HD. However, DM-HD showed significantly different distribution of allele frequency from other groups (chi 2 = 18.9, P = 0.026). Namely, the frequency of 19-repeat allele in DM-HD was higher (9.0%) than NH, DM-C, and CGN-HD (P = 0.005, 0.041, and 0.004, respectively). The microsatellite DNA polymorphism of AM gene may be associated with the genetic predisposition to develop nephropathy in Japanese patients with type 2 diabetes mellitus.

Variations of human adrenomedullin gene and its relation to cardiovascular diseases.

The studies concerning the structure and variations of the human adrenomedullin (AM) gene are reviewed, and their relations to the gene function and genetic predisposition to cardiovascular diseases are discussed. The genomic human AM gene is composed of four exons, and the whole nucleotide sequence corresponding to mature AM resides in the fourth exon. In chromosomal sublocalization, the AM gene is located in the distal portion of the short arm of chromosome 11 (11p15.1-3). Analysis of the promoter region of the AM gene has revealed that two transcription factors, nuclear factor for interleukin-6 expression (NF-IL6) and activator protein 2 (AP-2), participate in the regulation of AM gene expression. It is surmised that NF-IL6 mediates inflammatory stimuli and AP-2 mediates signals of phospholipase C and protein kinase C activation. In addition to these factors, hypoxia induces AM gene expression via the hypoxia inducible factor-1 (HIF-1) binding site. The 3'-end of the AM gene is flanked by a microsatellite marker of cytosine adenine (CA) repeats. In Japanese, there are four types of alleles with different CA-repeat numbers: 11, 13, 14 and 19. It is suggested that existence of the 19-repeat allele is associated with genetic predispositions to develop essential hypertension and diabetic nephropathy.

Adrenomedullin improves cardiac function and prevents renal damage in streptozotocin-induced diabetic rats.

Adrenomedullin (AM) is a potent vasodilating peptide and is involved in cardiovascular and renal disease. In the present study, we investigated the role of AM in cardiac and renal function in streptozotocin (STZ)-induced diabetic rats. A single tail-vein injection of adenoviral vectors harboring the human AM gene (Ad.CMV-AM) was administered to the rats 1-wk post-STZ treatment (65 mg/kg iv). Immunoreactive human AM was detected in the plasma and urine of STZ-diabetic rats treated with Ad.CMV-AM. Morphological and chemical examination showed that AM gene delivery significantly reduced glycogen accumulation within the hearts of STZ-diabetic rats. AM gene delivery improved cardiac function compared with STZ-diabetic rats injected with control virus, as observed by decreased left ventricular end-diastolic pressure, increased cardiac output, cardiac index, and heart rate. AM gene transfer significantly increased left ventricular long axis (11.69 +/- 0.46 vs. 10.31 +/- 0.70 mm, n = 10, P < 0.05) and rate of pressure rise and fall (+6,090.1 +/- 597.3 vs. +4,648.5 +/- 807.1 mmHg/s), (-4,902.6 +/- 644.2 vs. -3,915.5 +/- 805.8 mmHg/s, n = 11, P < 0.05). AM also significantly attenuated renal glycogen accumulation and tubular damage in STZ-diabetic rats as well as increased urinary cAMP and cGMP levels, along with increased cardiac cAMP and Akt phosphorylation. We also observed that delivery of the AM gene caused an increase in body weight along with phospho-Akt and membrane-bound GLUT4 levels in skeletal muscle. These results suggest that AM plays a protective role in hyperglycemia-induced glycogen accumulation and cardiac and renal dysfunction via Akt signal transduction pathways.

The effects of adrenomedullin overexpression in breast tumor cells.

Adrenomedullin is a secreted peptide hormone with multiple activities. Several reports have indicated that adrenomedullin may be involved in tumor survival, but this has not been directly shown. Here we evaluate the in vitro and in vivo effects of adrenomedullin overexpression in human breast cancer cells. The human breast cancer cell lines T47D and MCF7, both of which express low basal levels of adrenomedullin, were stably transfected with an expression construct that contained the coding region of the human adrenomedullin gene or with empty expression vector. Properties of the transfected cells were assessed by proliferation and apoptosis assays, in vitro and in vivo angiogenesis assays, cell migration experiments, and xenograft implants. The effect of synthetic adrenomedullin on human ovarian (ECV) cancer cell motility was also tested. Western blot analysis was used to compare expression levels of several genes whose products are associated with cell growth and regulation of apoptosis. T47D and MCF7 cells transfected with the adrenomedullin construct both expressed high levels of adrenomedullin mRNA and protein. Compared with cells transfected with empty vector, cells that overexpressed adrenomedullin displayed a more pleiotropic morphology, an increased angiogenic potential both in vitro and in vivo, and less apoptosis after serum deprivation. T47D and MCF7 cells did not display measurable motility, but ECV ovarian cancer cells treated with synthetic adrenomedullin were more motile than saline-treated ECV cells. Adrenomedullin-overexpressing T47D cells had higher levels of proteins involved in oncogenic signal transduction pathways (such as Ras, Raf, PKC, and MAPKp49) and lower levels of pro-apoptotic proteins (such as Bax, Bid, and caspase 8) than T47D cells transfected with empty vector. In a preliminary in vivo experiment, three of 10 nude mice injected with adrenomedullin-overexpressing T47D cells developed xenograft tumors, whereas none of the 10 nude mice injected with cells carrying the empty plasmid developed tumors. These results further support the role of adrenomedullin as a survival factor for tumors. Development of physiologically efficient inhibitors of adrenomedullin may prove useful in the clinical management of cancer.

Human adrenomedullin gene delivery protects against cardiovascular remodeling and renal injury

We investigated the potential roles of adrenomedullin (AM) in cardiovascular and renal function by somatic gene delivery. We showed that a single intravenous injection of the human AM gene under the control of cytomegalovirus promoter/enhancer induces a prolonged delay in blood pressure rise for several weeks in spontaneously hypertensive rats, Dahl salt-sensitive, DOCA-salt, and two-kidney one-clip hypertensive rats as compared to their respective controls injected with a reporter gene. Expression of the human AM transcript was identified in the heart, kidney, lung, liver and aorta of the rat after adenovirus-mediated AM gene delivery by RT-PCR followed by Southern blot analysis. Immunoreactive human AM levels were measured in rat plasma and urine following AM gene delivery. AM gene delivery induced significant reduction of left ventricular mass in these hypertensive animal models. It also reduces urinary protein excretion and increases glomerular filtration rate, renal blood flow and urinary cAMP levels. AM gene transfer attenuated cardiomyocyte diameter and interstitial fibrosis in the heart, and reduced glomerular sclerosis, tubular disruption, protein cast accumulation and renal cell proliferation in the kidney. In the rat model with myocardial ischmia/reperfusion injury, AM gene delivery significantly reduced myocardial infarction, apoptosis, and superoxide production. Furthermore, local AM gene delivery significantly inhibited arterial thickening, promoted re-endothelialization and increased vascular cGMP levels in rat artery after balloon angioplasty. Collectively, these results indicate that human AM gene delivery attenuates hypertension, myocardial infarction, renal injury and cardiovascular remodeling in animal models via cAMP and cGMP signaling pathways. These findings provide new insights into the role of AM in cardiovascular and renal function.

A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive and vasodilating peptides

Adrenomedullin (AM), identified from pheochromocytoma and having 52 amino acids, elicits a long-lasting vasodilatation and diuresis. AM is mainly mediated by the intracellular adenylate cyclase coupled with cyclic adenosine monophosphate (cAMP) and nitric oxide (NO) -cyclic guanosine monophosphate (cGMP) pathway through its specific receptor. The calcitonin receptor-like receptor (CLCR) and receptor-activity modifying protein (RAMP) 2 or RAMP3 models have been proposed as the candidate receptor. AM is produced mainly in cardiovascular tissues in response to stimuli such as shear stress and stretch, hormonal factors and cytokines. Recently established AM knockout mice lines revealed that AM is essential for development of vitelline vessels of embryo. Plasma AM levels elevate in cardiovascular diseases such as heart failure, hypertension and septic shock, where AM may play protective roles through its characteristic biological activities. Human AM gene delivery improves hypertension, renal function, cardiac hypertrophy and nephrosclerosis in the hypertensive rats. AM decreases cardiac preload and afterload and improves cardiac contractility and diuresis in patients with heart failure and hypertension. Advances in gene engineering and receptor studies may contribute to further understandings of biological implication and therapeutic availability of AM.

Chromosomal sublocalization and microsatellite DNA polymorphism of human adrenomedullin gene

Adrenomedullin (AM) is a hypotensive peptide widely produced in the cardiovascular organs and tissues such as the heart, kidney and vascular cells. We have cloned and sequenced genomic DNA encoding the human AM gene. In this study, we determined that the AM gene was located in the short arm of chromosome 11 (p15.1–3). The 3′-end of the gene is flanked by a microsatellite marker of cytosine adenine (CA) repeats. Moreover, we analyzed this DNA variation in the AM gene in the general Japanese population. Genomic DNA was obtained from the peripheral leukocytes of healthy normotensive subjects, 327 men and 149 women, aged 51 ± 8 years (mean ± SD). The genomic DNA was subject to PCR using a fluorescence-labeled primer, and the number of CA repeats were determined via polyacrylamide gel electrophoresis (PAGE). Plasma AM concentration was measured by RIA and compared with respect to the number of CA repeats adjacent to the AM gene. In Japanese, four types of alleles with different CA-repeat numbers; 11, 13, 14 and 19, appear to exist. The frequencies of these alleles were as follows: 11 repeats, 28.8%; 13 repeats, 33.1%; 14 repeats, 35.0% and 19 repeats, 3.1%. This DNA variation does not seem to affect the transcription of the AM gene, because plasma concentrations of AM were not significantly different between the genotypes.

Microsatellite DNA polymorphism of human adrenomedullin gene in normotensive subjects and patients with essential hypertension.

Adrenomedullin (AM) is a hypotensive peptide widely produced in the cardiovascular organs and tissues. We have cloned and sequenced the genomic DNA encoding the human AM gene and have determined that the gene is located in the short arm of chromosome 11. The 3'-end of the gene is flanked by the microsatellite marker of cytosine adenine (CA) repeats. In this study, we investigated the association between DNA variations in AM gene and the predisposition to hypertension. Genomic DNA was obtained from 272 healthy normotensive subjects (NT) age 57+/-5 years and 266 patients with essential hypertension (EH) age 53+/-11 years. The DNA was subject to PCR using a fluorescence-labeled primer, and the number of CA repeats were determined by poly-acrylamide gel electrophoresis. The averaged blood pressure was 117+/-13/73+/-9 mm Hg in NT and 170+/-23/104+/-12 mm Hg in EH. In Japanese, there existed 4 types of alleles with different CA-repeat numbers: 11, 13, 14, and 19. The frequencies of these alleles were significantly different between NT and EH (chi(2)=9.43, P=0.024). Namely, 13.5% of EH carried the 19-repeat allele, whereas the frequency was 6.2% in NT (chi(2)=7.62, P=0.007). In NT, plasma AM concentrations were not significantly different between the genotypes. In conclusion, microsatellite DNA polymorphism of AM gene may be associated with the genetic predisposition to EH, although the gene expression is not likely to be affected by the genotypes.

Adrenomedullin gene delivery attenuates renal damage and cardiac hypertrophy in Goldblatt hypertensive rats.

Adrenomedullin (AM) is a potent vasodilator and natriuretic peptide that plays an important role in cardiovascular function. In this study, we employed a somatic gene delivery approach to explore its potential protective role in renovascular hypertension. A single tail vein injection of adenovirus harboring the human AM gene significantly blunted a blood pressure increase that lasted for more than 3 wk in two-kidney one-clip (2K1C) hypertensive rats. The expression of human AM mRNA was detected in the kidney, adrenal gland, heart, lung, and liver, and immunoreactive human AM was detected in the plasma and urine of 2K1C rats after human AM gene delivery. A maximal blood pressure difference of 28 mmHg was observed 10 days after AM gene delivery, compared with that in rats injected with the control virus carrying the LacZ gene. Human AM gene delivery significantly attenuated increases in the ratio of left ventricular weight to heart weight, cardiomyocyte diameter, and fibrosis in the heart, as well as glomerular sclerosis, tubular injuries, and protein casts in the kidney. The beneficial effects of AM gene delivery were accompanied by increased urinary cAMP levels, indicating activation of AM receptors. These findings provide new insights into the role of AM in renovascular hypertension and may have significance in therapeutic applications in cardiovascular diseases.

Interaction between monocytes and vascular endothelial cells induces adrenomedullin production

Adrenomedullin (AM), a potent vasodilator peptide, has natriuretic effects, and its plasma concentration is elevated in cardiovascular diseases. In the present study, we investigated the induction of AM expression due to interactions between THP-1 cells (human monocytic cell line) and human umbilical cord vein endothelial cells (HUVECs). AM levels in the culture medium were measured by radioimmunoassay. The luciferase vector containing the 5′-flanking region of the human AM gene was transfected into either HUVECs or THP-1 cells. Addition of THP-1 cells to HUVECs for 48 h induced marked increases in AM levels, which were 16-fold higher than those of HUVECs alone. Luciferase vectors containing the 5′-flanking region of human AM gene (pLCF-1534) were transferred into THP-1 cells or HUVECs. Addition of THP-1 cells to pLCF-1534-transfected HUVECs induced an increase in luciferase activity in cell lysates, which was 5-fold higher than that of the transfected HUVECs alone. In contrast, the luciferase activity of lysates from pLCF-1534-transfected THP-1 cells was not affected by coculture with HUVECs. A separate coculture experiment revealed that direct contact of THP-1 cells and HUVECs contributed to enhanced AM production in the cocoulture. Co-incubation of the cell membrane fraction from THP-1 cells augmented AM production by HUVECs. Both anti-interleukin (IL)-1α antibody and IL-1 receptor antagonist significantly inhibited AM production in the cocultures. The cell-to-cell interaction between monocytes and HUVECs induces AM production by HUVECs, which may play an important role in the pathogenesis of vascular disorders.